Contents and Abstracts of Latest Issue of BBB

(Vol.62 No.8 1998)

Binding of Lactoferrin to Bacterial Cells of the Clostridium Species and Their Agglutination
Shinichi TOMITA, Kenji HAGIWARA, Jun MATSUYAMA, and Isao KIYOSAWA 1471

Binding Characteristics of Bovine Lactoferrin to the Cell Surface of Clostridium Species and Identification of the Lactoferrin-binding Protein
Shinichi TOMITA, Nami SHIRASAKI, Hideshi HAYASHIZAKI, Jun MATSUYAMA, Yoshimi BENNO,＊ and Isao KIYOSAWA 1476

Proanthocyanidins from Barley Bran Potentiate Retinoic Acid-induced
Granulocytic and Sodium Butyrate-induced Monocytic
Differentiation of HL60 Cells
Koji TAMAGAWA,＊ Sei FUKUSHIMA,＊ Masuko KOBORI,＊＊ Hiroshi SHINMOTO,＊＊ and Tojiro TSUSHIDA＊＊ 1483


Novel Histamine Measurement by HPLC Analysis Used to Assay
Histidine Decarboxylase Inhibitory Activity of Shoyuflavones
from Soy Sauce
Emiko KINOSHITA・ and Makoto SAITO 1488


Structure-Function Relationship of T-2 Toxin and Its Metabolites
in Inducing Thymic Apoptosis in Vivo in Mice
Zahidul ISLAM, Masahiro NAGASE, Akemi OTA,＊ Shigeharu UEDA,＊Takumi YOSHIZAWA, and Nobuo SAKATO・ 1492


Lipoxygenase-1 from Soybean Seed Inhibiting the Activity of Pancreatic Lipase
Kiyoshi SATOUCHI,a＊ Kaoru HIRANO,a Osamu FUJINO,a Masakazu IKOMA,a Tamotsu TANAKA,a
and Keisuke KITAMURAb 1498


Effect of Indigestible Oligosaccharides on the Hepatotoxic Action of D-Galactosamine in Rats
Binbin WANG, Yukari EGASHIRA, Takeo OHTA, and Hiroo SANADA・ 1504


Effects of hydroxylysine on the growth and morphology of Rhizobium leguminosarum bv. phaseoli
Keiko IGAWA, Kaoru KIKUCHI, Tetsuya SATO, and Takuji OHWADA 1510


Construction of a BAC Library of the Rice Blast Fungus Magnaporthe grisea and Finding Specific Genome Regions in which Its Transposons Tend to Cluster
Marie NISHIMURA,1,＊ Shingo NAKAMURA,1,・ Nagao HAYASHI,2,・・ Shuichi ASAKAWA,3 Nobuyoshi SHIMIZU,3
Hisatoshi KAKU,1 Akira HASEBE,1 and Shinji KAWASAKI1 1515


Production of a Mixture of Antimicrobial Organic Acids from Lactose by Co-Culture of Bifidobacterium longum
and Propionibacterium freudenreichii
Masayuki TANIGUCHI,1,・ Hideya NAKAZAWA,1 Osamu TAKEDA,1 Tsutomu KANEKO,2 Kazuhiro HOSHINO,3
and Takaaki TANAKA1 1522


Purification and Characterization of Recombinant Human Granulocyte Colony-Stimulating
Factor (rhG-CSF) Derivatives: KW-2228 and Other Derivatives
Motoo YAMASAKI,＊,1 Noboru KONISHI,＊,2 Kazuo YAMAGUCHI,＊＊ Seiga ITOH,＊＊
and Yoshiharu YOKOO＊,2 1528


Effects of Polyphenol Substances Derived from Theobroma cacao on Gastric Mucosal Lesion Induced by Ethanol
Naomi OSAKABE,1 Chiaki SANBONGI,1 Megumi YAMAGISHI,1 Toshio TAKIZAWA,1 and Toshihiko OSAWA2 1535


Thermostable β-Galactosidase from an Extreme Thermophile, Thermus sp. A4: Enzyme Purification
and Characterization, and Gene Cloning and Sequencing
Naomi OHTSU,・ Hidemasa MOTOSHIMA, Kenji GOTO, Fuji TSUKASAKI, and Hiroshi MATSUZAWA＊ 1539


Insecticidal Component in Thunberg Spiraea, Spiraea thunbergii, against Thrips palmi
Chul-Sa KIM,・ Tetsuro HARA, Probal Kanti DATTA, Eiji ITOH, and Michio HORIIKE 1546


Enantioselective Synthesis of a (＋)-(2R, 3R)-1,4-Benzodioxane-7-carbaldehyde Derivative, a Key Intermediate
in the Total Synthesis of Haedoxan Analogs
Yuji NAKAMURA, Makoto HIRATA, Eiichi KUWANO,・ and Eiji TANIGUCHI 1550


Xylanase Induction by L-Sorbose in a Fungus, Trichoderma reesei PC-3-7
Jianping XU, Masahiro NOGAWA, Hirofumi OKADA, Yasushi MORIKAWA＊ 1555


Molecular Properties and Activity of a Carboxyl-Terminal Truncated Form of Xylanase 3
from Aeromonas caviae W-61
Naoko OKAI, Masashi FUKASAKU, Jun KANEKO, Toshio TOMITA, Koji MURAMOTO, and Yoshiyuki KAMIO＊ 1560


Synthesis and Biological Activities of (－)-6-n-Octyl-indolactam-V, a New Potent Analogue
of the Tumor Promoter (－)-Indolactam-V
Yu NAKAGAWA, Kazuhiro IRIE,・ Yoshimasa NAKAMURA, Hajime OHIGASHI, and Hideo HAYASHI＊ 1568


Characterization of a Mutant of Lactococcus lactis with Reduced Membrane-bound ATPase Activity
under Acidic Conditions
Seigo AMACHI,1 Kohei ISHIKAWA,1 Shuji TOYODA,2 Yasuo KAGAWA,3 Atsushi YOKOTA,1,
＊and Fusao TOMITA1 1574


Degradation of Derivatives of N-Acetyl-D-glucosamine by Rhodococcus rhodochrous IFO 15564: Substrate Specificity and Its Application to the Synthesis of Allyl α-N-Acetyl-D-glucosaminide
Atsuhito KUBOKI, Ryosuke KOMIYA, Takahiro SEKIGUCHI, Kenji KATSURAGI, Takeshi SUGAI,・ and Hiromichi OHTA 1581


Note
Distribution of Threonine Aldolase Activity with Different Stereospecificities in Aerobic Bacteria
Masaru WADA,・・ Mitsuru SAKAMOTO, Michihiko KATAOKA, Ji-Quan LIU,＊ Hideaki YAMADA,＊
and Sakayu SHIMIZU・ 1586


Note
Glycolaldehyde Production from Ethylene Glycol with Immobilized Alcohol Oxidase and Catalase
Hiroyuki UKEDA,・ Tohru ISHII, Masayoshi SAWAMURA, and Kimiyasu ISOBE＊ 1589


Note
Changes in the Level of Sialic Acid in Plasma, Brain and Liver of Inherently Scorbutic Rats
during Vitamin C and E Deficiencies
Kyoko TANAKA, Sadako TOKUMARU,・ and Shosuke KOJO＊ 1592


Note
Use of Escherichia coli Polyphosphate Kinase for Oligosaccharide Synthesis
Toshitada NOGUCHI and Toshikazu SHIBA＊ 1594


Note
Whole Sequence of spoIIIE-Like, Sporulation-inhibitory, and Transfer Gene (spi) in a Conjugative Plasmid, pSA1.1, of Streptomyces azureus and Detection of spi-Like Gene in the Actinomycete Chromosome
Katsumi DOI, Yasunori ONO, Eiji YOKOYAMA, Yuki TSUKAGOE, and Seiya OGATA・ 1597　

Note
Aspartate Decarboxylation Encoded on the Plasmid in the Soy Sauce Lactic Acid Bacterium,
Tetragenococcus halophila D10
Takeshi HIGUCHI,・,・・ Kinji UCHIDA, and Keietsu ABE 1601


Note
Characterization of Ribonucleases from Culture Medium of Lentinus edodes
Hiroko KOBAYASI, Michie IMANAKA, Norio INOKUCHI, Takashi KOYAMA, and Masachika IRIE・ 1604


Note
Cationic Peroxidases Secreted from Cultured Cells May Localize to Apoplasts in Tobacco Plant Roots
Nozomu KOIZUMI, Yoko OKUSHIMA, and Hiroshi NARITA1 1609


Note
Effects of Glycosylation of the Residue at Position 14 in Ovine Angiotensinogen on the Human Renin Reaction
Yoshito INUI, Takenori ORIHASHI,＊ Tsutomu NAKAGAWA,＊ Akio EBIHARA,＊＊ Fumiaki SUZUKI,＊＊＊
and Yukio NAKAMURA＊,・ 1612


Note
Expression of Aspergillus aculeatus No. F-50 Cellobiohydrolase I (cbhI)and β-glucosidase 1 (bgl1)
genes by Saccharomyces cerevisiae
Goro TAKADA, Takashi KAWAGUCHI, Jun-ichi SUMITANI, and Motoo ARAI＊ 1615


Note
Effects of Batatasin III and Its Analogs on Gibberellic Acid-Dependent α-Amylase Induction
in Embryoless Barley Seeds and on Cress Growth
Hiroshi ASAHINA, Hiromichi YOSHIKAWA,＊ and Yoshihiro SHUTO 1619


Note
Biological and Structural Properties of Cyclic Peptides Derived from the α-Amylase Inhibitor Tendamistat
Shin ONO,＊ Tomiya HIRANO, Hiroki YASUTAKE,・ Toshihiko MATSUMOTO,・Izumi YAMAURA,・
Tetsuo KATO,・ Hiroyuki MORITA, Takayoshi FUJII,Isao YAMAZAKI,・・ Choichiro SHIMASAKI,
and Toshiaki YOSHIMURA 1621


Note
Pesthetoxin, a New Phytotoxin Produced by the Gray Blight Fungus, Pestalotiopsis theae
Yasuo KIMURA,・ Ayumi KOUGE, Kazuto NAKAMURA, Hiroyuki KOSHINO,＊
Jun UZAWA,＊ Shozo FUJIOKA,＊ and Tsuyoshi KAWANO 1624


Note
Practical Preparation of K-252a from a Fermentation Solution
Mitsutaka KINO, Kenzo SHONO, Tetsuo NISHIMURA, and Satoru NAGAMURA＊ 1627


Note
Formation of N-(1-Oxo-2,4,5,6-hydroxyhexyl)-2′-deoxyguanosine by the Reaction of 2′-Deoxyguanosine with 3-Deoxyglucosone
Fumitaka HAYASE＃ and Koji KANEKO 1630


Note
Phospholipid Deacylating Activities Included in Yeast
Takahiro MORIMOTO, Hideki OISHI, Yasuo WATANABE, and Youichi TAMAI・ 1633


Note
Nucleotide Sequence of the Gene Encoding the Precursor Protein of Pepstatin insensitive Acid Protease B, Scytalidopepsin B, from Scytalidium lignicolum
Naoko ODA, Yoshikazu GOTOH, Hiroshi OYAMA, Sawao MURAO, Kohei ODA,＊ and Daisuke TSURU＃ 1637


Preliminary Communication
Mapping of Human DNA-binding Nuclear Protein (NP220) toChromosome Band 2p13.1-p13.2
and Its Relation to Matrin 3
Katsuzumi OKUMURA,＊＊ Masahiro NOGAMI, Yuichi MATSUSHIMA,＊ Kiyoshi MATSUMURA,＊ Kazuyasu NAKAMURA,＊ Hiroshi TAGUCHI, and Yasuo KITAGAWA＊ 1640


Preliminary Communication
HPLC Analysis of Anomeric Formation and Cleavage Pattern by Chitinolytic Enzyme
Daizo KOGA,1＊ Takanori YOSHIOKA,1 and Yasuyuki ARAKANE2 1643


-1-
Binding of Lactoferrin to Bacterial Cells of the Clostridium Species and Their Agglutination

Shinichi TOMITA, Kenji HAGIWARA, Jun MATSUYAMA, and Isao KIYOSAWA

Department of Agricultural Chemistry, Tamagawa University, 6-1-1 Tamagawa-Gakuen, Machida-shi,
Tokyo 194-8610, Japan

Received October 1, 1997
Cell agglutination in cell suspensions of 10 strains of Clostridium by lactoferrin (Lf) was observed by obtaining the ratio of increased absorbance (RIA) at 450 nm. The RIA values were very different among the species, being higher in the cell suspensions with bovine Lf (bLf) than in those with human Lf. The binding ability of bLf to the bacterial cells was also observed by an enzyme-linked ligand-binding assay, using the conjugate of iron-free or iron-saturated bLf with horseradish peroxidase (HRPO). The binding ability of bLf was very different among the 10 species, and showed a significant correlation with the cell agglutination of each strain. bLf formed a complex with the cells of C. perfringens, did not dissociate in 2 M NaCl or 4 M urea, but did dissociate in 1 M KSCN. These results suggest that the agglutination of cells of the Clostridium species by bLf is probably caused by the cooperative action of at least electrostatic and hydrophobic interactions between bLf and certain components of the cell surface.
lactoferrin; Clostridium; cell agglutination; binding behavior

-2-
Binding Characteristics of Bovine Lactoferrin to the Cell Surface of Clostridium Species and Identification of the Lactoferrin-binding Protein

Shinichi TOMITA, Nami SHIRASAKI, Hideshi HAYASHIZAKI, Jun MATSUYAMA, Yoshimi BENNO,＊ and Isao KIYOSAWA

Department of Agricultural Chemistry, Tamagawa University, 6-1-1 Tamagawa-Gakuen, Machida-shi, Tokyo 194-8610, Japan
＊The Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan

Received November 4, 1997
The binding characteristics of bovine lactoferrin (bLf) to cells of the Clostridium species were observed by using a horseradish peroxidase-bLf conjugate. A bLf-binding protein (BP) having a relative molecular mass of about 33 kDa was confirmed in the surface layer components from 7 strains of the Clostridium species. The binding of the conjugate to bLf-BP of C. perfringens was strongly blocked by intact Lfs, lysine or arginine residues modified bLf, and deglycosylated bLf, but was not by other milk proteins or by the constituent sugars of glycan. Bacterial growth was inhibited by bLf, but was slightly inhibited by lysine residues modified bLf or deglycosylated bLf. Lactoferricin B did not block the binding of the conjugate, but strongly inhibited the bacterial growth. This suggests that the lysine or arginine residues and glycan of bLf hardly participated in binding bLf to the bacterial cells, but that the amino acid residues and glycan played an important role in inhibiting the growth of bacteria.
lactoferrin; Clostridium; lactoferrin-binding protein; binding characteristics; bacterial growth inhibition

-3-
Proanthocyanidins from Barley Bran Potentiate Retinoic Acid-induced Granulocytic and Sodium Butyrate-induced Monocytic Differentiation of HL60 Cells

Koji TAMAGAWA,＊ Sei FUKUSHIMA,＊ Masuko KOBORI,＊＊ Hiroshi SHINMOTO,＊＊ and Tojiro TSUSHIDA＊＊

＊Research and Development Center, Hakubaku Co., Ltd., 3492 Aoyagi, Masuho-cho, Yamanashi 400-0501, Japan
＊＊National Food and Research Institute, Ministry of Agriculture, Forestry and Fisheries, 2-1-2 Kannondai,
Tukuba, Ibaraki 305-0856, Japan

Received January 9, 1998
Polyphenol extract from barley bran (BPE) induced nitro blue tetrazolium (NBT) reducing activity and alpha-naphthyl butyrate esterase activity in HL60 human myeloid leukemia cells. Because BPE induced the biochemical markers of HL60 cell differentiation, we investigated the effects of proanthocyanidins isolated from BPE on the HL60 cell differentiation of HL60 cells. Prodelphinidin B-3, T1, T2, and T3 induced 26--40％ NBT-positive cells and 22--32％ alpha-naphthyl butyrate esterase-positive cells. Proanthocyanidins potentiated retinoic acid (all-trans-retinoic acid)-induced granulocytic and sodium butyrate-induced monocytic differentiation in HL60 cells.
proanthocyanidins; barley bran; differentiation; HL60 cells

-4-
Novel Histamine Measurement by HPLC Analysis Used to Assay Histidine Decarboxylase Inhibitory Activity of Shoyuflavones from Soy Sauce

Emiko KINOSHITA・ and Makoto SAITO

Research ＆ Development Division, Kikkoman Corporation, 399 Noda, Noda, Chiba 278-0037, Japan

Received January 13, 1998
An easy and highly sensitive method for measuring histamine by HPLC analysis coupled with precolumn derivatization was established. The amino group of histamine was completely colorimetrically labelled with 4-N,N-dimethylamino-azobenzene-4′-isothiocyanate (DABITC) in the presence of sodium bicarbonate at 90°C for 5 min. The derivative was sensitively and easily analyzed by HPLC on a Cosmosil 5SL column using CHCl3/N,N-dimethylformamide/H2O (210:90:4) containing 0.4％ acetic acid. Using the established method, histidine decarboxylase (HDC) inhibitory activities of three tartaric acid isoflavone derivatives, named shoyuflavones, isolated from soy sauce were examined in vitro by measuring the histamine produced by HDC. They showed intense inhibition of the activities of HDC from both mouse mastocytoma P-815 cells and Clostridium perfringens.
isoflavone; histamine; histidine decarboxylase; 4-N,N-dimethylamino-azobenzene-4′-isothiocyanate; histamine synthesis inhibition

-5-
Structure-Function Relationship of T-2 Toxin and Its Metabolites in Inducing Thymic Apoptosis in Vivo in Mice

Zahidul ISLAM, Masahiro NAGASE, Akemi OTA,＊ Shigeharu UEDA,＊ Takumi YOSHIZAWA, and Nobuo SAKATO・

Faculty of Agriculture, Kagawa University, Ikenobe 2393, Miki-cho, Kita-gun, Kagawa 761-0795, Japan
＊Research Institute for Microbial Diseases, Osaka University, Yamada-Oka, Suita 565-0871, Japan

Received January 13, 1998
Recently we found that a single administration of T-2 toxin (T-2), a trichothecene mycotoxin, into mice induced DNA fragmentation, a biochemical hallmark of apoptosis, in the thymus.1) In this study, we investigated the effective chemical structure(s) of T-2-derived metabolites capable ofinducing thymic apoptosis in vivo in mice. Metabolic||conversion of T-2 to 3′-hydroxy-T-2 toxin (3′-OH-T-2) (Fig. 1)||did not diminish the apoptosis-inducing activity, since||essentially the same level of fragmented DNA was detected in||the thymus taken from mice injected with either T-2 or 3′-OH-T-2. In contrast, hydrolysis of T-2 and 3′-OH-T-2 at the carbon-4 (C-4) position to HT-2 toxin (HT-2) and 3′-hydroxy-HT-2 toxin (3′-OH-HT-2), respectively, greatly decreased the level of DNA fragmentation. Similarly,hydrolysis of T-2 at the carbon-8 (C-8) position to||neosolaniol strongly diminished its ability to induce DNA||fragmentation. T-2 tetraol, having no ester groups, was||unable to induce apoptosis. Based on the data presented in||this study, we concluded that both the acetyl group at the C-4 position and the isovaleryl or 3′-hydroxyisovaleryl group at the C-8 position of the T-2 molecule are important for inducing cell death through apoptosis in the thymus.
apoptosis; T-2 toxin; DNA fragmentation; trichothecene mycotoxin; mouse thymus

-6-
Lipoxygenase-1 from Soybean Seed Inhibiting the Activity of Pancreatic Lipase

Kiyoshi SATOUCHI,a＊ Kaoru HIRANO,a Osamu FUJINO,a Masakazu IKOMA,a Tamotsu TANAKA,a and Keisuke KITAMURAb

aDepartment of Food Science and Technology, Fukuyama University, Gakuen-cho, Fukuyama 729-0292, Japan
bMinistry of Agriculture, Forestry, and Fisheries, Kasumigaseki, Tokyo 100-8950, Japan

Received January 19, 1998
There are some similar characteristics in protein nature between the lipase inhibitor from soybean seed and soybean lipoxygenase-1 (LOX-1). Thus, the inhibiting protein for pancreatic lipase was prepared from defatted soybean meal by the procedure for the isolation of LOX-1 [Axelrod et al., Methods in Enzymology, 71, 441--451 (1981)]. The LOX-1 from soybean seed dose-dependently inhibited the release of fatty acid from a soybean oil emulsion, and the concentration of LOX-1 to cause half inhibition of the lipase activity was 3.2×10＾{－7} M. The LOX-1 obtained from E. coli transfected with a plasmid carrying the soybean LOX-1 gene also inhibited the lipase activity. However, the lipase-inhibiting activity by the LOX-1 was not affected by the presence of nordihydroguaiaretic acid, an inhibitor for LOX, in the reaction mixture.
These results show that the soybean LOX-1 inhibits lipase activity regardless of its lipoxygenase activity.
lipase-inhibiting protein; lipoxygenase-1; soybean seed

-7-
Effect of Indigestible Oligosaccharides on the Hepatotoxic Action of D-Galactosamine in Rats

Binbin WANG, Yukari EGASHIRA, Takeo OHTA, and Hiroo SANADA・

Graduate School of Science and Technology, Chiba University, 648 Matsudo, Matsudo-Shi, Chiba 271-8510, Japan

Received January 23, 1998
The effects of dietary oligosaccharides on the hepatotoxic action of D-galactosamine (GalN) were investigated in this study. Male Wistar rats fed with 20％ casein diets containing 10％ oligosaccharide or D-galactose (Gal) for 2 weeks were injected with GalN (1,900 mg/kg of body weight), and the plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities and the hepatic glycogen concentration were examined 20 hours after the injection. The plasma AST and ALT activities in experiment 1 for the Gal＋neomycin (NEO) group were significantly lower than those for the control (C), NEO, raffinose (RAF)＋NEO and galacto-oligosaccharide (GALO)＋NEO groups. In experiment 2, these activities were significantly lower in the Gal, Gal＋NEO and RAF groups than in the RAF＋NEO group when the groups were treated with GalN. On the other hand, in respect of the hepatic glycogen concentration in experiment 1, that of the Gal＋NEO group was higher than that of the C, NEO, RAF＋NEO or GALO＋NEO groups. In experiment 2, this parameter was significantly higher in the Gal, Gal＋NEO and RAF groups than in the RAF＋NEO group after the GalN treatment. As a result, it is suggested that the GalN-hepatitis-suppressive effects of indigestible oligosaccharides such as RAF or GALO is mediated by the action of intestinal bacteria.
D-galactosamine; raffinose; galactose; hepatic injury; hepatitis

-8-
Effects of hydroxylysine on the growth and morphology of Rhizobium leguminosarum bv. phaseoli

Keiko IGAWA, Kaoru KIKUCHI, Tetsuya SATO, and Takuji OHWADA

Department of Bioresource Chemistry, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro City, Hokkaido 080-8555, Japan

Received February 4, 1998
It was found that both bacteroids and free-living cells of Rhizobium leguminosarum bv. phaseoli were highly susceptible to hydroxylysine (Hyl) and the inhibition of RNA and/or protein synthesis caused by the hydroxyl residue of Hyl appeared to be responsible for the growth inhibition. The size of free-living cells was enlarged by the addition of Hyl and some cells reached around 5 μm, which were close to the length of bacteroids. Under the same condition, the polyhydroxybutyrate (PHB) content in the cells was conspicuously increased. These results suggest that Hyl is not only a notable growth inhibitor of Rhizobium bacteria but also plays a role in a differentiation to bacteroids.
hydroxylysine (Hyl); Rhizobium; root nodule bacteria

-9-
Construction of a BAC Library of the Rice Blast Fungus Magnaporthe grisea and Finding Specific Genome Regions in which Its Transposons Tend to Cluster

Marie NISHIMURA,1,＊ Shingo NAKAMURA,1,・ Nagao HAYASHI,2,・・ Shuichi ASAKAWA,3 Nobuyoshi SHIMIZU,3 Hisatoshi KAKU,1 Akira HASEBE,1 and Shinji KAWASAKI1

1National Institute of Agrobiological Resources (NIAR), Kannon-dai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan
2National Agricultural Research Center (NARC), Kannon-dai 3-1-1, Tsukuba, Ibaraki 305-8666, Japan
3Dept. of Molecular Biology, Keio University, School of Medicine, Shinanomachi 35, Shinjuku, Tokyo 160-0016, Japan

Received February 9, 1998
We have constructed a BAC library of the rice blast fungus Magnaporthe grisea consisting of 5760 clones. The insert size ranged from 35 to 175 kbp, with an average of 120 kbp. The library is about 18 genomes equivalent, therefore covering more than 99.999％ of the genome. This library is the first to be constructed using a rice pathogenic wild type isolate. Improved high molecular weight DNA size fractionating helped to construct the library with high efficiency. Total library clones were arranged onto two nylon membranes for efficient screening. Test hybridization with a single-copy RFLP marker showed ten positive clones, of which restriction patterns indicated no chimerality or deletions. As a model case of application of this library, the distribution of the well-studied fungal retrotransposons MGSR1, MGR583, and MAGGY and DNA transposons MGR586 and Pot2 was analyzed. Of all the BAC clones, 10％, 13％, 18％, 12％, and 23％ contained MGSR1, MGR583, MAGGY, MGR586 and Pot2, respectively. The percentage of clones possessing more than five kinds of transposons was 1.4％, 215 times greater than the expected number. The results show that these transposons were distributed in clusters in the M. grisea genome
.Magnaporthe grisea; BAC library; transposon distribution

-10-
Production of a Mixture of Antimicrobial Organic Acids from Lactose by Co-Culture of Bifidobacterium longum and Propionibacterium freudenreichii

Masayuki TANIGUCHI,1,・ Hideya NAKAZAWA,1 Osamu TAKEDA,1 Tsutomu KANEKO,2
Kazuhiro HOSHINO,3 and Takaaki TANAKA1

1Department of Materials Science and Technology, Faculty of Engineering, Niigata University,Ikarashi 2,
Niigata 950-2181, Japan
2Central Research Institute, Meiji Milk Products Co., Ltd., 1-21-3 Sakae-Cho, Higashimurayama,
Tokyo 189-0013, Japan
3Department of Chemical and Biochemical Engineering, Faculty of Engineering, Toyama University,Gokufu,
Toyama 930-8555, Japan

Received February 9, 1998
The antimicrobial activities of standard solutions of three organic acids (lactic, acetic, and propionic acids) were compared using Micrococcus luteus, Pseudomonas sp. and Staphylococcus aureus as test microorganisms. At the same concentrations of the undissociated form, the antimicrobial activities of acetic and propionic acids were higher than that of lactic acid, irrespective of test microorganisms. In a single cultivation of Bifidobacterium longum, a mixture of lactic (17 g/l) and acetic (20 g/l) acids was produced from 50 g/l lactose and its antimicrobial activities against M. luteus, Pseudomonas sp., and S. aureus correspond to that of 32, 19, and 25 g/l of acetic acid, respectively. To increase the total antimicrobial activity, a co-culture of B. longum and Propionibacterium freudenreichii, in which lactic acid produced once from lactose by B. longum was converted to acetic and propionic acids by P. freudenreichii, was done using TPY medium containing commercially available peptones as a nitrogen source. By the sequential conversion of lactose using the two microorganisms, the culture supernatant containing a mixture of acetic (27 g/l) and propionic (13 g/l) acids without lactic acid was produced. The antimicrobial activities of the mixture against M. luteus, Pseudomonas sp., and S. aureus were 35, 30, and 26 g/l as a concentration of acetic acid, respectively, higher than that obtained in the cultivation of B. longum alone. When the medium containing an enzymatic hydrolyzate of whey proteins with a protease was used in the co-culture of B. longum and P. freudenreichii, the culture supernatant containing the mixture of organic acids was also obtained in the same manner as the co-culture using TPY medium and the activities were 43, 29, and 29 g/l as a concentration of acetic acid for M. luteus, Pseudomonas sp. and S. aureus, respectively.
co-culture; antimicrobial culture; food||preservative; Bifidobacterium longum;||Propionibacterium freudenreichii

-11-
Purification and Characterization of Recombinant Human Granulocyte Colony-Stimulating Factor (rhG-CSF) Derivatives: KW-2228 and Other Derivatives

Motoo YAMASAKI,＊,1 Noboru KONISHI,＊,2 Kazuo YAMAGUCHI,＊＊ Seiga ITOH,＊＊ and Yoshiharu YOKOO＊,2

＊Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 3-6-6 Asahi-machi, Machidashi, Tokyo 194-8533, Japan
＊＊Pharmaceutical Research Institute, Kyowa Hakko Kogyo Co., Ltd., 1188 Shimotogari, Nagaizumi-chou, Suntou-gun, Shizuoka 411-8731, Japan

Received February 17, 1998
Various derivatives of recombinant human granulocyte colony-stimulating factor (rhG-CSF) have been overproduced in Escherichia coli with the strong, inducible trp promoter. A derivative designated as KW-2228 in which the amino acids were replaced at five positions showed more potent granulopoietic activity and stability than those of wild-type both in vitro and in vivo. The purification involved a sequential renaturation process and three-step chromatography. Refolding succeeded in very high yield using a urea system. The purity of KW-2228 was greater than 99％ as measured by SDS-PAGE and HPLC analysis. According to circular dichroism and nuclear magnetic resonance spectroscopy, rhG-CSF and KW-2228 have very similar conformations. This suggests that the substitution of five amino acids does not appreciably change the conformation of hG-CSF. KW-2228 ([Ala1, Thr3, Tyr4, Arg5, and Ser17]-hG-CSF) and derivative A ([Ala1, Thr3, Tyr4, Arg5]-hG-CSF) are easily crystallized and they show similar in vitro activity. On the other hand, neither rhG-CSF nor derivative B ([Ser17]-hG-CSF) are crystallized under the same conditions. Thus, the four amino acid substitution (Ala1, Thr3, Tyr4, Arg5) of the N-terminal sequence may facilitate crystallization. The change of Cys17 to Ser may not influence the stability and activity of hG-CSF.
human granulocyte colony-stimulating factor; protein engineering; physicochemical properties

-12-
Effects of Polyphenol Substances Derived from Theobroma cacao on Gastric Mucosal Lesion Induced by Ethanol

Naomi OSAKABE,1 Chiaki SANBONGI,1 Megumi YAMAGISHI,1 Toshio TAKIZAWA,1 and Toshihiko OSAWA2

1Functional Food Research and Development Laboratories, Meiji Seika Kaisha, Ltd., 5-3-1, Chiyoda, Sakadoshi, Saitama 350-0289, Japan
2Department of Applied Biological Science, Nagoya University, Chikusa, Nagoya, Aichi 464-8601, Japan

Received February 18, 1998
The antiulcer activity of cacao liquor water-soluble crude polyphenols (CWSP) was examined.
CWSP, α-tocopherol, sucralfate (500 mg/kg), and cimetidine (250 mg/kg) were orally administerted to male SD rats 30 minutes before ethanol treatment. 5 ml/kg of ethanol given intragastrically caused lesions in mucosa of the glandular stomach. CWSP caused a reduction of such hemorrhagic lesions as well as cimetidine and sucralfate which are typical antiulcer drugs, but α-tocopherol was less effective. Thiobarbituric acid reactive substances in gastric mucosa significantly increased with ethanol administration. CWSP treatment significantly reduced this change. The administration of ethanol extensively increased myeloperoxidase (MPO) but not xanthine oxidase (XOD) activity. CWSP reduced the activities of both enzymes; they were considered the main sources of oxygen radicals. According to an in vitro study, CWSP directly reducted XOD but not MPO. These results suggest that the antiulcer mechanism of CWSP was not only radical scavenging but also modulation of leukocyte function.
cacao liquor; polyphenols; antioxidant; gastric mucosal injury; ethanol　

-13-
Thermostable β-Galactosidase from an Extreme Thermophile, Thermus sp. A4: Enzyme Purification and Characterization, and Gene Cloning and Sequencing

Naomi OHTSU,・ Hidemasa MOTOSHIMA, Kenji GOTO, Fuji TSUKASAKI, and Hiroshi MATSUZAWA＊

Research Center, Yotsuba Milk Products Co., Ltd., 465-1 Wattsu, Kitahiroshima, Hokkaido 061-1264, Japan
＊Department of Biotechnology and Biotechnology Research Center, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan

Received February 19, 1998
We purified and characterized a thermophilic β-galactosidase from Thermus sp. A4 isolated from the Atagawa hot spring (Shizuoka, Japan). The enzyme was monomeric, and its molecular mass was estimated to be 75 kDa by SDS-polyacrylamide gel electrophoresis. The enzyme was extremely thermostable and retained its full activity after incubation at 70°C for 20 h. The Km observed were 5.9 mM for o-nitrophenyl β-D-galactopyranoside and 19 mM for lactose. We cloned and analyzed the complete sequence of the gene encoding this enzyme. It was found to consist of 645 amino acid residues. We propose that this enzyme and seven other unclassified β-galactosidases are new members of family 42 of the glycosyl hydrolases.
thermostable β-galactosidase; extreme thermophile; Thermus; family 42 glycosyl hydrolase

-14-
Insecticidal Component in Thunberg Spiraea, Spiraea thunbergii, against Thrips palmi

Chul-Sa KIM,・ Tetsuro HARA, Probal Kanti DATTA, Eiji ITOH, and Michio HORIIKE

Department of Bioresources Science, Faculty of Agriculture, Kochi University, B200 Monobe, Nankoku 783-8502, Japan

Received February 25, 1998
α-Methylene-γ-butyrolactone (tulipalin A) was isolated and identified as an insecticidal component from thunberg spiraea, Spiraea thunbergii. This compound showed high insecticidal activity against Thrips palmi.
insecticidal activity; thunberg spiraea (Spiraea thunbergii); α-methylene-γ-butyrolactone (tulipalin A)

-15-
Enantioselective Synthesis of a (＋)-(2R, 3R)-1,4-Benzodioxane-7-carbaldehyde Derivative, a Key Intermediate in the Total Synthesis of Haedoxan Analogs

Yuji NAKAMURA, Makoto HIRATA, Eiichi KUWANO,・ and Eiji TANIGUCHI

Department of Agricultural Chemistry, Faculty of Agriculture, Kyushu University, Hakozaki 6-10-1,Higashi-ku, Fukuoka 812-8581, Japan

Received March 9, 1998
||(＋)-(2R, 3R)-7-Formyl-6-methoxy-2-methoxymethyl-3-||||(3,4 - methylenedioxyphenyl) - 2,3 - dihydro - 1,4 - benzodioxin||(2), a key building block for the total synthesis of haedoxan A, was synthesized from (4R)-4-(phenylmethyl)-2-oxazolidinone (3) in ten steps with a 12％ overall yield.
benzodioxane; haedoxan A; sesquilignan

-16-
Xylanase Induction by L-Sorbose in a Fungus, Trichoderma reesei PC-3-7

Jianping XU, Masahiro NOGAWA, Hirofumi OKADA, Yasushi MORIKAWA＊

Department of Bioengineering, Nagaoka University of Technology,Kamitomioka 1603-1, Nagaoka, Niigata, 940-2188, Japan

Received March 12, 1998
Xylanase induction by L-sorbose was studied in a resting cell system of a filamentous fungus, Trichoderma reesei PC-3-7, a hypercellulolytic mutant, and compared with that by other inducers. L-Sorbose induced xylanase activity as well as cellulase. It induced a higher level of xylanase activity than sophorose and xylose did. Three main xylanases, xylanase I (Xyn I), xylanase II (Xyn II), and a non-specific endoglucanase I (EG I), were separated using cation-exchange chromatography, and their activity were measured. Xyn II was induced in about the same proportion (60--80％ of the total xylanase activity) by all inducers used. On the other hand, Xyn I was apparently induced by L-sorbose, xylose, and xylooligosaccharides, but only a little by sophorose. Northern blot analysis showed that L-sorbose induced Xyn I and Xyn II at the transcriptional level, and more xyn1 mRNA was transcribed after L-sorbose addition than after sophorose. These results suggested that the expressions of both Xyn I and Xyn II are regulated, at least in part, in a different manner. Furthermore, the Xyn I induction by L-sorbose indicated that an unknown common regulatory mechanism may exist between Xyn I and cellulase inductions.
xylanase; cellulase; L-sorbose; Trichoderma reesei

-17-
Molecular Properties and Activity of a Carboxyl-Terminal Truncated Form of Xylanase 3 from Aeromonas caviae W-61

Naoko OKAI, Masashi FUKASAKU, Jun KANEKO, Toshio TOMITA, Koji MURAMOTO, and Yoshiyuki KAMIO＊

Department of Applied Biological Chemistry, Faculty of Agriculture, Tohoku University, 1-1 Tsutsumi-dori Amamiya-machi, Aoba-ku, Sendai 981-8555, Japan

Received March 19, 1998
Aeromonas caviae W-61 produces five species of||xylanases, xylanases 1, 2, 3, 4, and 5 [Nguyen, V.D. et al.,||Biosci. Biotechnol. Biochem., 56, 1708--1712 (1993) and Appl. Environ. Microbiol., 57, 445--449 (1991)]. While preserving a purified xylanase 3 preparation from A. caviae in solution at 4°C, the xylanase 3 was found to be proteolyzed to give a truncated form with a smaller molecular mass than that of the intact one. It appears likely that the truncated form of xylanase 3 was produced in this particular purification experiment by the action of a contaminating protease. We isolated the truncated form of xylanase 3 (Xyn3tr), of which the C-terminal 102-residue segment is missing. By the chemical analysis of the N- and C-terminal amino acid residues of Xyn3tr and the DNA sequencing analysis of the xylanase 3 gene (xyn3), the N-terminal 398th proline residue of xylanase 3 was found to be the C-terminus of Xyn3tr. Xyn3tr had the activity to form xylotriose (X3), xylotetraose (X4), xylopentaose (X5), and xylohexaose (X6) as main final products from oat spelt xylan. In contrast, intact xylanase 3 released X6 and higher xylo-oligosaccharides as main products. Xylanase 3 hydrolysed X4 through X6. However, Xyn3tr had no activity towards X4 and X5. The recombinant Xyn3tr (XYN3tr) and recombinant xylanase 3 (XYN3) were purified homogeneously from the periplasmic space of E. coli harboring the plasmids pXYN3 and pXYN3tr, which include xyn3 and xyn3tr genes, respectively, and their enzymatic activities were measured. The cleavage patterns of oat spelt and xylo-oligosaccharides by XYN3tr were identical with that by intact Xyn3tr. Thus, we conclude that the C-terminal region comprising a 102-residue segment in xylanase 3 is involved in governing the molecular size of xylo-oligosaccharides cleaved from β-1,4-xylan by the enzyme and in the hydrolytic activity towards X4 and X5.
Aeromonas caviae; xylanase 3; carboxyl-terminal region; truncated mutant; xyn3 gene; xyn3 expression

-18-
Synthesis and Biological Activities of (－)-6-n-Octyl-indolactam-V, a New Potent Analogue of the Tumor Promoter (－)-Indolactam-V

Yu NAKAGAWA, Kazuhiro IRIE,・ Yoshimasa NAKAMURA, Hajime OHIGASHI, and Hideo HAYASHI＊

Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
＊Department of Applied Biological Chemistry, Osaka Prefecture University, Sakai 599-8531, Japan

Received March 23, 1998
(－)-Indolactam-V (1) without a hydrophobic chain at positions 6 and 7 of the indole ring is a weak tumor promoter compared with teleocidin Bs. To investigate the effects of the hydrophobic substituent at position 6 of teleocidin Bs, we synthesized (－)-6-n-octyl-indolactam-V (2) by a palladium-catalyzed coupling reaction from (－)-6-bromo-indolactam-V (7) which had been obtained by microbial conversion with Streptoverticillium blastmyceticum NA34-17 as the key step. (－)-7-n-Octyl-indolactam-V (3) with potent biological activities comparable to those of teleocidin Bs was similarly synthesized from (－)-7-bromo-indolactam-V as a positive control. Compound 2 showed similar biological activities to those of 3, indicating that the effect of the hydrophobic substituent at position 6 of 1 was identical to that at position 7.
Epstein-Barr virus; (－)-6-n-octyl-indolactam-V; protein kinase C; teleocidin; tumor promoter

-19-
Characterization of a Mutant of Lactococcus lactis with Reduced Membrane-bound ATPase Activity under Acidic Conditions

Seigo AMACHI,1 Kohei ISHIKAWA,1 Shuji TOYODA,2 Yasuo KAGAWA,3 Atsushi YOKOTA,1,＊ and Fusao TOMITA1

1Laboratory of Applied Microbiology, Department of Bioscience and Chemistry, Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Japan
2Sapporo Research Laboratory, Snow Brand Milk Products Co., Ltd., Sapporo 065-0043, Japan
3Department of Biochemistry, Jichi Medical School, Minamikawachimachi, Tochigi 329-0498, Japan

Received April 6, 1998
A mutant of Lactococcus lactis subsp. lactis C2 with reduced membrane-bound ATPase activity was characterized to clarify its acid sensitivity. The cytoplasmic pH of the mutant was measured in reference to the parental strain under various pH conditions. At low pH, the mutant could not maintain its cytoplasmic pH near neutral, and lost its viability faster than the parental strain. The ATPase activities of cells cultured under neutral and acidic conditions using pH-controlled jar fermentors were measured. The relative ATPase activity of the mutant at pH 7.0 was 42％ of the parental strain. At pH 4.5, the parental strain showed an ATPase activity 2.8-fold higher than that at pH 7.0, while the level of increase in the mutant was only 1.6. Northern and Western blot analyses found that at pH 7.0 the transcriptional level and the amount of F1β subunit were similar in both strains, suggesting that the mutant has a defective ATPase structural gene. On the other hand, at pH 4.5 the transcriptional level and the amount of F1β subunit were found to be significantly higher in both strains than those at pH 7.0. From these results, it was suggested that the mutant has a normal regulation system for ATPase gene expression. It was concluded that the mutant is acid sensitive due to its inability to extrude protons out of the cell with defective ATPase under acidic conditions.
Lactococcus lactis; ATPase; cytoplasmic pH; acid sensitivity; gene expression

-20-
Degradation of Derivatives of N-Acetyl-D-glucosamine by Rhodococcus rhodochrous IFO 15564: Substrate Specificity and Its Application to the Synthesis of Allyl α-N-Acetyl-D-glucosaminide

Atsuhito KUBOKI, Ryosuke KOMIYA, Takahiro SEKIGUCHI, Kenji KATSURAGI, Takeshi SUGAI,・ and Hiromichi OHTA

Department of Chemistry, Keio University 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan

Received April 7, 1998
The substrate specificity was studied for the metabolic degradation of N-acetyl-D-glucosamine (GlcNAc) derivatives by Rhodococcus rhodochrous IFO 15564 which possesses N-acetyl-D-glucosamine deacetylase as a key-step enzyme. This microorganism degraded a wide range of substrates with modified N-acyl groups. The metabolizing activity of this strain became low to the substrates substituted at 1,3,4,6-positions of GlcNAc, and GlcNAc itself was suggested to be metabolized via an open-chain aldehyde form. Based on these results, a simplified procedure for the isolation of allyl α-N-acetyl-D-glucosaminide from an α, β-anomeric mixture was developed by selectively hydrolyzing the β-anomer with Jackbean β-N-acetyl-D-glucosaminidase and subsequently degrading the resulting N-acetyl-D-glucosamine in the reaction mixture with this microorganism.
Rhodococcus rhodochrous IFO 15564; N-acetyl-D-glucosamine; N-acetyl-D-glucosamine deacetylase; β-N-acetyl-D-glucosaminidase; allyl α-N-acetyl-D-glucosaminide

-21-
Distribution of Threonine Aldolase Activity with Different Stereospecificities in Aerobic Bacteria

Masaru WADA,・・ Mitsuru SAKAMOTO, Michihiko KATAOKA, Ji-Quan LIU,＊ Hideaki YAMADA,＊ and Sakayu SHIMIZU・

Division of Applied Life Science, Graduate School of Agriculture, Kyoto University, Kitashirakawa, Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan
＊Laboratory of Biocatalytic Chemistry, Biotechnology Research Center, Toyama Prefectural University, Toyama 939-0398, Japan

Received January 14, 1998
Threonine aldolase activities (threonine acetaldehyde-lyase activity) with different stereospecificities toward 4 isomers of threonine were found in a variety of aerobic bacteria. The strains could be divided into three groups on the basis of the substrate specificity of a cell-free extract. The enzyme activities toward D-allo- and D-threonine were inhibited by EDTA, which suggested that a metal ion participates in the D-specific aldolase reaction.
threonine aldolase; L-allo-threonine; D-allo-threonin

-22-
Note
Glycolaldehyde Production from Ethylene Glycol with Immobilized Alcohol Oxidase and Catalase

Hiroyuki UKEDA,・ Tohru ISHII, Masayoshi SAWAMURA, and Kimiyasu ISOBE＊

Department of Bioresources Science, Faculty of Agriculture, Kochi University, Monobe B-200, Nankoku 783-8502, Japan
＊Research and Development Division, Amano Pharmaceutical Co., Ltd, Kunotsubo, Nihiharu-cho, Nishikasugai-gun, Aichi 481-0041, Japan

Received January 21, 1998
An enzymatic method for glycolaldehyde production from ethylene glycol was investigated using immobilized alcohol oxidase and catalase. Those enzymes were immobilized onto Chitopearl BCW 3501. When only alcohol oxidase was immobilized onto it, the apparent activity was 190 units/g in wet gel using methanol as the substrate. Tris-HCl buffer (1.5 M; pH 9.0) was selected based on a high stability of glycolaldehyde and a low production of glyoxal as a by-product. Under the optimum conditions, 0.97 M glycolaldehyde was formed from 1.0 M ethylene glycol and the ratio of glyoxal to glycolaldehyde was less than 1％.
glycolaldehyde; ethylene glycol; alcohol oxidase; catalase; immobilized enzyme　

-23-
Note
Changes in the Level of Sialic Acid in Plasma, Brain and Liver of Inherently Scorbutic Rats during Vitamin C and E Deficiencies

Kyoko TANAKA, Sadako TOKUMARU,・ and Shosuke KOJO＊

Department of Food Science and Nutrition, Nara Women′s University, Nara 630-8506, Japan
・Department of Life and Health Sciences, Joetsu University of Education, Joetsu, Niigata 943-8512, Japan

Received January 23, 1998
The plasma level of sialic acid (NeuAc) in inherently scorbutic [Osteogenic Disorder Shionogi (ODS)] rats was increased by 21 days of vitamin C deficiency and simultaneous vitamins C and E deficiency. The brain content of NeuAc was decreased by deficiencies of these vitamins. The NeuAc level in the liver was not affected significantly by these deficiencies.
sialic acid; N-acetylneuraminic acid; vitamin C; vitamin E; ODS rat

-24-
Note
Use of Escherichia coli Polyphosphate Kinase for Oligosaccharide Synthesis

Toshitada NOGUCHI and Toshikazu SHIBA＊

Biochemicals Division, Yamasa Corporation, Choshi, Chiba 288-0056, Japan
＊Graduate School of Engineering, Hokkaido University, Sapporo, Hokkaido 060-0813, Japan

Received January 26, 1998
The Escherichia coli polyphosphate kinase (PPK) has been known to catalyze the reversible transfer of phosphate molecules between ATP and polyphosphate (poly(P)). It has also been found that the PPK catalyzes the kination of not only ADP but also other nucleoside diphosphates (NDPs) using poly(P) as a phosphate donor, yielding nucleotide triphosphates (NTPs). We used the PPK and poly(P) in place of pyruvate kinase and phosphoenol pyruvate for NTP regeneration followed by synthesis of sugar nucleotides in a cyclic synthesis system for oligosaccharides. It was confirmed that the PPK efficiently catalyzed the UTP regeneration in the cyclic system of N-acetyllactosamine synthesis. This novel activity of PPK enables us to perform the practical synthesis of oligosaccharides.
polyphosphate; polyphosphate kinase; glycosyltransferase; oligosaccharides; N-acetyllactosamine

-25-
Note
Whole Sequence of spoIIIE-Like, Sporulation-inhibitory, and Transfer Gene (spi) in a Conjugative Plasmid, pSA1.1, of Streptomyces azureus and Detection of spi-Like Gene in the Actinomycete Chromosome

Katsumi DOI, Yasunori ONO, Eiji YOKOYAMA, Yuki TSUKAGOE, and Seiya OGATA・

Microbial Genetics Division, Institute of Genetic Resources, Faculty of Agriculture, Kyushu University, Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan

Received February 2, 1998
The nucleotide sequence of spoIIIE-like and the sporulation-inhibitory and transfer gene (spi) in a conjugative plasmid, pSA1.1, of Streptomyces azureus were examined to detect the promoter region. Using S￣outhern blotting and a spi fragment as probe, spi-like genes were detected in chromosomes of the host and other actinomycetes. These results suggest that there is a spi- and spoIIIE-like gene in chromosomes of some actinomycetes.
Streptomyces azureus; plasmid; sporulation inhibitory gene; spoIIIE-like gene; promoter region

-26-
Note
Aspartate Decarboxylation Encoded on the Plasmid in the Soy Sauce Lactic Acid Bacterium, Tetragenococcus halophila D10

Takeshi HIGUCHI,・,・・ Kinji UCHIDA, and Keietsu ABE

Soy Sauce Research Laboratory, R＆D Division, Kikkoman Corporation, 399 Noda, Noda City, Chiba 278-0037, Japan

Received February 12, 1998
Tetragenococcus halophila D10 decarboxylates aspartate to alanine, but T. halophila D10 derivatives generated by a curing treatment could not (Asd－ derivatives). We observed by electrophoresis three plasmid bands in T. halophila D10; all Asd－ derivatives lost the largest of these bands. This plasmid, pD1, has two SalI sites. We cloned and sequenced the 10 kb SalI fragment. The DNA sequence suggests that this fragment contains the aspartate decarboxylating trait.
Tetragenococcus halophila; plasmid; aspartate decarboxylation; soy sauce; curing

-27-
Note
Characterization of Ribonucleases from Culture Medium of Lentinus edodes

-28-
Note
Cationic Peroxidases Secreted from Cultured Cells May Localize to Apoplasts in Tobacco Plant Roots

Nozomu KOIZUMI, Yoko OKUSHIMA, and Hiroshi NARITA1

Research and Education Center for Genetic Information, Nara Institute of Science and Technology, 8916-5 Takayama-cho, Ikoma 630-0101, Japan
1Department of Food Science, Kyoto Women′s University, Kitahiyoshi-cho, Imakumano, Higashiyama-ku, Kyoto 605-0926, Japan

Received March 2, 1998
The transcripts for 38-kDa and 40-kDa cationic peroxidases of tobacco plants are accumulated in cultured cells, cultured roots, and roots of intact plants. In roots, 38-kDa and 40-kDa peroxidases appear to be localized to apoplasts, where peroxidase proteins may possibly be bound to the cell wall.
Nicotiana tabacum; peroxidase

-29-
Note
Effects of Glycosylation of the Residue at Position 14 in Ovine Angiotensinogen on the Human Renin Reaction

Yoshito INUI, Takenori ORIHASHI,＊ Tsutomu NAKAGAWA,＊ Akio EBIHARA,＊＊ Fumiaki SUZUKI,＊＊＊ and Yukio NAKAMURA＊,・

United Graduate School of Agricultural Science,
＊Department of Biotechnology, Faculty of Agriculture, and
＊＊＊Molecular Genetic Research Center, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
＊＊Institute of Applied Biochemistry, University of Tsukuba, Ibaraki 305-0006, Japan

Received March 4, 1998
A mutant angiotensinogen, S14N, in which Ser14 of ovine angiotensinogen was replaced by Asn to form a N-glycosylation site, was produced in CHO cells. The molecular weight was about 3,000 larger than that of wild-type ovine angiotensinogen, indicating that S14N angiotensinogen was glycosylated at Asn14. In the reaction with human renin, the Km of mutant angiotensinogen was 3 times increased, but the Vmax was not affected by the mutation.
sheep; ovine angiotensinogen; human renin; glycosylation of angiotensinogen

-30-
Note
Expression of Aspergillus aculeatus No. F-50 Cellobiohydrolase I (cbhI) and β-glucosidase 1 (bgl1) genes by Saccharomyces cerevisiae

Goro TAKADA, Takashi KAWAGUCHI, Jun-ichi SUMITANI, and Motoo ARAI＊

Department of Applied Biological Chemistry, College of Agriculture, Osaka Prefecture University, Sakai 599-8531, Japan

Received March 4, 1998
A cellobiohydrolase I (cbhI) and a β-glucosidase 1 (bgl1) gene of Aspergillus aculeatus were expressed in Saccharomyces cerevisiae. The transformed cells secreted the enzymes efficiently in an active form. The recombinant CBHI gave two bands of different molecular mass (110 and 90 kDa) and the recombinant BGL1 gave one band (180 kDa) by SDS-PAGE. The recombinant CBHI and BGL1 had the same enzymatical properties as the native enzyme except for the specific activity toward cellulosic substrates. By the combination of three different types of cellulases, FI-CMCase, CBHI, and BGL1, we could hydrolyze Avicel up to 59％ under our experimental conditions.
Aspergillus aculeatus; cellobiohydrolase; β-glucosidase; synergism

-31-
Note
Effects of Batatasin III and Its Analogs on Gibberellic Acid-Dependent α-Amylase Induction in Embryoless Barley Seeds and on Cress Growth

Hiroshi ASAHINA, Hiromichi YOSHIKAWA,＊ and Yoshihiro SHUTO

Faculty of Agriculture, Ehime University, Matsuyama 790-8566, Japan
＊Department of Environmental Chemistry, Kyushu Kyoritsu University, Yahatanishi-ku, Kitakyushu 807-8585, Japan

Received March 6, 1998The effects of batatasin III and its analogs on gibberellic acid (GA3)-dependent α-amylase induction in embryoless barley seeds and on cress root-growth were examined. Batatasin III was most effective and caused 68％ inhibition of α-amylase induction at 4×10－4 M, but its potency was low compared with that of abscisic acid. In the cress test, p-hydroxybibenzyl had high activity.
batatasin III; dihydrostilbene derivatives; GA3-dependent α-amylase induction; abscisic acid-like activity

-32-
Note
Biological and Structural Properties of Cyclic Peptides Derived from the α-Amylase Inhibitor Tendamistat

Shin ONO,＊ Tomiya HIRANO, Hiroki YASUTAKE,・ Toshihiko MATSUMOTO,・ Izumi YAMAURA,・ Tetsuo KATO,・ Hiroyuki MORITA, Takayoshi FUJII, Isao YAMAZAKI,・・ Choichiro SHIMASAKI, and Toshiaki YOSHIMURA

Department of Chemical and Biochemical Engineering, Toyama University, Toyama 930-8555, Japan
・Department of Applied Microbial Technology, Kumamoto Institute of Technology, Kumamoto 860-0082, Japan
・・Yayoi Kagaku Kogyo Co. Ltd., Fukuoka-machi, Nishitonami-gun, Toyama 939-0135, Japan

Received March 11, 1998
Six cyclic peptides with 5, 7, 9, 11, 13, and 15 amino acids, with the inhibitory sequence of the α-amylase inhibitor tendamistat, were synthesized. The 11-residue peptide inhibited porcine pancreatic α-amylase most potently (Ki 0.29±0.09 μM). For the 11-residue peptide, the circular dichroism study suggested a preliminary relationship between its inhibitory activity and structural property.
tendamistat; cyclic peptides; circular dichroism; inhibitory activity; porcine pancreatic α-amylase

-33-
Note
Pesthetoxin, a New Phytotoxin Produced by the Gray Blight Fungus, Pestalotiopsis theae

Yasuo KIMURA,・ Ayumi KOUGE, Kazuto NAKAMURA, Hiroyuki KOSHINO,＊ Jun UZAWA,＊ Shozo FUJIOKA,＊ and Tsuyoshi KAWANO

Department of Agricultural Chemistry, Faculty of Agriculture, Tottori University, Koyama, Tottori 680-0945, Japan
＊The Institute of Physical and Chemical Research (RIKEN), Wako-shi, Saitama 351-0198, Japan

Received March 16, 1998
Pestalotiopsis theae is known to be a causal fungus for tea gray blight disease. Pesthetoxin was isolated from P. theae as a potent leaf-necrosis substance against tea. The structure of pesthetoxin was established principally by NMR studies to be of four different enolic forms, viz the pairs of internal tautomers (1a)/(1b) and (1c)/(1d) with external tautomerism between them.
pesthetoxin; tea gray blight; Pestalotiopsis theae; leaf-necrosis assay; tautomerism.

-34-
Note
Practical Preparation of K-252a from a Fermentation Solution

Mitsutaka KINO, Kenzo SHONO, Tetsuo NISHIMURA, and Satoru NAGAMURA＊

Kyowa Hakko Kogyo Co. Ltd., Technical Research Laboratories, 1-1 Kyowa-machi, Hofu, Yamaguchi 747-8522, Japan

Received March 19, 1998
We developed a practical preparation procedure for K-252a by methylating K-252b on an industrial scale. The water-insoluble K-252a, which was present in the cell mass, was converted to the water-soluble K-252b Na salt in an alkaline solution. The obtained K-252b was methylated with dimethylsulfate in the presence of potassium carbonate in dimethylacetamide. We have already used this method to manufacture 90 kg of K-252b from the fermentation broth, and regenerated 65 kg of K-252a from K-252b.
K-252a; purification; methylation

-35-
Note
Formation of N-(1-Oxo-2,4,5,6-hydroxyhexyl)-2′-deoxyguanosine by the Reaction of 2′-Deoxyguanosine with 3-Deoxyglucosone

Fumitaka HAYASE＃ and Koji KANEKO

Department of Agricultural Chemistry, Meiji University, Tama-ku, Kawasaki, Kanagawa 214-8571, Japan

Received March 19, 1998
3-Deoxyglucosone (3DG) has weaker mutagenicity than methylglyoxal by the Ames test. 3DG reacted readily with 2′-deoxyguanosine (dG) in nucleosides. Two major products (G-A and G-B) were isolated and purified from the reaction mixture of 50 mM 3DG and 50 mM dG at 50°C and pH 7.4 for 6d. G-A was identified as N-(1-oxo-2,4,5,6-hydroxyhexyl)-2′-deoxyguanosine. G-B was identified as a diastereomer of G-A.
Maillard reaction; 3-deoxyglucosone; 2′-deoxyguanosine; methylglyoxal; nucleosides

-36-
Note
Phospholipid Deacylating Activities Included in Yeast

Takahiro MORIMOTO, Hideki OISHI, Yasuo WATANABE, and Youichi TAMAI・

Department of Bioresources, Faculty of Agriculture, Ehime University, Matsuyama, Ehime 790-8566, Japan

Received March 20, 1998
Yeast had been known to contain only one kind of phospholipid deacylating enzyme with an optimal pH in the acidic range. However, among 8 genera and 25 strains, 7 genera and 13 strains had phospholipid deacylating activity at pH 8.0, when screening of enzyme activity was done with micellar and liposomal substrates. The enzymatic properties of phospholipid deacylating enzyme existing in yeast was not related to genetic identity based on 18S rRNA gene sequence. The results suggest that yeast contains enzymes showing a variety of properties depending on the species of yeast, and further studies on this enzyme with more varieties of yeasts are necessary for understanding the physiological roles of the enzyme in yeast.
phospholipase B; yeast; phospholipid deacylating enzyme

-37-
Note
Nucleotide Sequence of the Gene Encoding the Precursor Protein of Pepstatin insensitive Acid Protease B, Scytalidopepsin B, from Scytalidium lignicolum

Naoko ODA, Yoshikazu GOTOH, Hiroshi OYAMA, Sawao MURAO, Kohei ODA,＊ and Daisuke TSURU＃

Department of Applied Microbial Technology, Kumamoto Institute of Technology, Ikeda 4-22-1, Kumamoto 860-0082, Japan
＊Faculty of Textile Science, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-0962, Japan

Received March 25, 1998
A chromosomal DNA of Scytalidium lignicolum was digested with Sau3AI. The digest was self-ligated and amplified by inverse PCR procedure using primers designed based on the nucleotide sequences of up- and down-stream regions of an intron present in the scytalidopepsin B gene. Analysis of the nucleotide sequence of PCR product (700 bp) showed that the enzyme is synthesized as a precursor protein consisting of the prepro- and mature enzyme regions. The deduced amino acid sequence was highly similar to those of aspergillopepsin A and recently reported endothiapepsins B and C, but quite different from those of pepstatin-insensitive bacterial acid proteases and the pepstatin-sensitive aspartic protease family.
pepstatin-insensitive acid protease; scytalidopepsin B

-38-
Preliminary Communication
Mapping of Human DNA-binding Nuclear Protein (NP220) to Chromosome Band 2p13.1-p13.2 and Its Relation to Matrin 3

Katsuzumi OKUMURA,＊＊ Masahiro NOGAMI, Yuichi MATSUSHIMA,＊ Kiyoshi MATSUMURA,＊ Kazuyasu NAKAMURA,＊ Hiroshi TAGUCHI, and Yasuo KITAGAWA＊

Laboratory of Biological Chemistry, Faculty of Bioresources, Mie University, Tsu, Mie 514-8507, Japan
＊Graduate Program of Biochemical Regulation, School of Agriculture, Nagoya University and
・Nagoya University BioSciences Center, Chikusa, Nagoya 464-8601, Japan

Received April 16, 1998
Human NP220 (hNP220) is a novel DNA-binding nuclear protein, which has an arginine/serine-rich motif and polypyrimidine tract-binding motif, and NP220s and matrin 3 are thought to form a novel family of nuclear proteins. We have determined a chromosomal localization of the cDNA encoding human NP220 to 2p13.1-p13.2 by using fluorescence in situ hybridization. Human matrin 3 cDNA was mapped to chromosomes 1p13.1-p21.1 and 5q31.3, demonstrating that these novel nuclear proteins with similar functions are on different chromosomes.
gene mapping; nuclear protein; FISH

-39-
Preliminary Communication
HPLC Analysis of Anomeric Formation and Cleavage Pattern by Chitinolytic Enzyme

Daizo KOGA,1＊ Takanori YOSHIOKA,1 and Yasuyuki ARAKANE2

1Laboratory of Biochemistry, Faculty of Agriculture, Yamaguchi University, Yoshida, Yamaguchi 753-8515, Japan
2Research Laboratory, Bankaku So-honpo, Tokai, Aichi 476-0003, Japan

Received april 16, 1998
The reactions of N-acetylchitooligosaccharides with chitinolytic enzyme were analyzed by HPLC using a Tosoh||TSK-Gel amide-80 column with 70％ acetonitrile as an el||uent. We separated α and β anomeric forms of N-acetylchitooligosaccharides, and obtain the following advantages of this HPLC method.
1. We can easily identify the reaction mechanism of||chitinolytic enzymes by this method, distinguishing the in||||verting mechanism showing α anomer formation from the||retaining mechanism showing β anomer formation.
2. We can also estimate the cleavage patterns of N-acetylchitooligosaccharides by chitinolytic enzymes by using natural substrates.
chitinase; lysozyme; chitinolytic enzyme; N-acetylchitooligosaccharide

Back