Contents and Abstracts of Latest Issue of BBB

(Vol.64 No.4 2000)

Eiji ICHISHIMA・

Department of Bioengineering, Graduate School of Engineering, Soka University, Hachioji,
Tokyo 192-8577, Japan

This review covers the unique catalytic and molecular properties of three proteolytic enzymes and a glycosidase from Aspergillus. An aspartic proteinase from A. saitoi, aspergillopepsin I (EC 3.4.23.18), favors hydrophobic amino acids at P1 and P1? like gastric pepsin. However, aspergillopepsin I accommodates a Lys residue at P1, which leads to activation of trypsinogens like duodenum enteropeptidase. Substitution of Asp76 to Ser or Thr and deletion of Ser78, corresponding to the mammalian aspartic proteinases, cathepsin D and pepsin, caused drastic decreases in the activities towards substrates containing a basic amino acid residue at P1. In addition, the double mutant T77D/G78(S)G79 of porcine pepsin was able to activate bovine trypsinogen to trypsin by the selective cleavage of the K6-I7 bond of trypsinogen.
Deuterolysin (EC 3.4.24.39) from A. oryzae, which contains 1 g atom of zinc/mol of enzyme, is a single chain of 177 amino acid residues, includes three disulfide bonds, and has a molecular mass of 19,018 Da. It was concluded that His128, His132, and Asp164 provide the Zn2+ ligands of the enzyme according to a 65Zn binding assay. Deuterolysin is a member of a family of metalloendopeptidases with a new zinc-binding motif, aspzincin, defined by the ``HEXXH+D′′ motif and an aspartic acid as the third zinc ligand.
Acid carboxypeptidase (EC 3.4.16.1) from A. saitoi is a glycoprotein that contains both N- and O-linked sugar chains. Site-directed mutagenesis of the cpdS, cDNA encoding A. saitoi carboxypeptidase, was cloned and expressed. A. saitoi carboxypeptidase indicated that Ser153, Asp357, and His436 residues were essential for the enzymic catalysis. The N-glycanase released high-mannose type oligosaccharides that were separated on HPLC. Two, which had unique structures of Man10GlcNAc2 and Man11GlcNAc2, were characterized.
An acidic 1,2-α-mannosidase (EC 3.2.1.113) was isolated from the culture of A. saitoi. A highly efficient overexpression system of 1,2-α-mannosidase fusion gene (f-msdS) in A. oryzae was made. A yeast mutant capable of producing Man5GlcNAc2 human-compatible sugar chains on glycoproteins was constructed. An expression vector for 1,2-α-mannosidase with the ``HDEL′′ endoplasmic reticulum retention/retrieval tag was designed and expressed in Saccharomyces cerevisiae. The first report of production of human-compatible high mannose-type (Man5GlcNAc2) sugar chains in S. cerevisiae was described.
Key words: aspergillopepsin I; aspzincin; carboxypeptidase; deuterolysin; α-mannosidase

-2-
Autolysis of Calpain Large Subunit Inducing Irreversible Dissociation
of Stoichiometric Heterodimer of Calpain

Hiroshi KITAGAKI,1,2 Shigeo TOMIOKA,1 Toshio YOSHIZAWA,1 Hiroyuki SORIMACHI,1,3
Takaomi C. SAIDO,4 Shoichi ISHIURA,1,5 and Koichi SUZUKI1

1Department of Molecular Biology, Institute of Molecular and Cellular Biosciences, University of Tokyo,
1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
2Office of Brewing Technology, Osaka Regional Taxation Bureau, 1-5-63, Otemae, Chuo-ku, Osaka City,
Osaka 540-0008, Japan
3Department of Applied Biological Chemistry, and Department of Applied Biological Engineering, Graduate School of Agricultural and Life Science, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
4Laboratory for Proteolytic Neuroscience, RIKEN Brain Science Institute, 2-1, Hirosawa, Wako,
Saitama 351-0198, Japan
5Department of Life Science, Graduate School of Arts and Science, University of Tokyo, Meguro-ku,
Tokyo 153-8902, Japan

Received July 26, 1999; Accepted December 13, 1999
Calpain, a calcium dependent cysteine protease, consists of a catalytic large subunit and a regulatory small subunit. Two models have been proposed to explain calpain activation: an autolysis model and a dissociation model. In the autolysis model, the autolyzed form is the active species, which is sensitized to Ca2+. In the dissociation model, dissociated large subunit is the active species. We have reported that the Ca2+ concentration regulates reversible dissociation of subunits. We found further that in chicken μ/m-calpain autolysis of the large subunit induces irreversible dissociation from the small subunit as well as activation. So we could propose a new mechanism for activation of the calpain by combining our findings. Our model insists that autolyzed large subunit remains dissociated from the small subunit even after the removal of Ca2+ to keep it sensitized to Ca2+. This model could be expanded to other calpains and give a new perspective on calpain activation.
Key words: calpain; activation; autolysis; dissociation of subunits; autolysis-induced irreversible dissociation model

-3-
Enzyme-Linked Immunosorbent Assay for Detection of Chitooligosaccharides

Soon-Young KIM, Dong-Hwa SHON,＊,・ and Ke-Ho LEE

Department of Food Science and Technology, College of Agricultural Life Sciences, Seoul National University, Suwon-si, Kyonggi-do 441-744, Korea
＊Food Chemistry and Biotechnology Division, Korea Food Research Institute, Seongnam-si, Kyonggi-do 463-420, Korea

Received July 26, 1999; Accepted November 26, 1999
In order to detect chitooligosaccharides (COS), an enzyme-linked immunosorbent assay (ELISA) was developed. A chitooligosaccharide mixture (COSM) conjugated to bovine serum albumin was used to immunize rabbits to produce an anti-COS polyclonal antibody. By use of specific antibody and COSM-horseradish peroxidase conjugate, we established a competitive direct ELISA (cdELISA) the detection limit of which was about 0.1 μg/ml. In the cdELISA, the cross-reactivities of the specific antibody toward glucosamine, chitobiose, chitotriose, chitotetraose, chitopentaose, and chitohexaose were 0.27, 27, 75, 75, 144, and 100％, respectively, and those toward N-acetylchitobiose, N-acetylchitotriose, N-acetylchitotetraose, N-acetylchitopentaose, and N-acetylchitohexaose were 1.58, 0.005, 1.08, 0.05, and 0.40％, respectively.
Key words: enzyme-linked immunosorbent assay; chitooligosaccharides; N-acetylchitooligosaccharides; cross-reactivity

-4-
Teasterone-3-O-β-D-glucopyranoside, A New Conjugated Brassinosteroid
Metabolite from Lily Cell Suspension Cultures and Its Identification
in Lily Anthers

Kazuo SOENO, Yoshimasa KYOKAWA,・ Masahiro NATSUME, and Hiroshi ABE・・

Department of Applied Biological Science, Faculty of Agriculture, Tokyo University of Agriculture
and Technology, Saiwai-cho, Fuchu, Tokyo 183-8509, Japan

Received September 2, 1999; Accepted December 3, 1999
The new brassinosteroid conjugate, teasterone-3-O-β-D-glucopyranoside, was found as a metabolite of teasterone in lily cell suspension cultures. Its structure was determined by means of FAB-MS and 1H-NMR upon comparison with the authentic compound. Furthermore, its presence in lily anthers was confirmed by FAB-MS and LC-APCI-SIM data. This is the first natural brassinosteroid conjugate glucosylated at a hydroxyl group in ring A.
Key words: brassinosteroid; conjugated brassinosteroid; teasterone; teasterone glucoside; Lilium longiflorum

-5-
Monitoring the Irradiation-induced Conformational Changes of Ovalbumin
by Using Monoclonal Antibodies and Surface Plasmon Resonance

Tetsuya MASUDA,1,・ Kyoden YASUMOTO,2 and Naofumi KITABATAKE1

1Research Institute for Food Science, Kyoto University Gokasho, Uji, Kyoto 611-0011, Japan
2Department of Food and Nutrition, Sugiyama Jogakuen University, Hoshigaokamotomachi, Chikusa-ku, Nagoya 464-8662, Japan

Received September 13, 1999; Accepted December 17, 1999
Two types of conformationally specific anti-irradiated ovalbumin monoclonal antibodies were prepared in order to study and monitor irradiation-induced structural changes in the ovalbumin molecule. Surface plasmon resonance (SPR) detection was used to investigate the kinetic parameters of the reaction between antibodies and ovalbumin which had been administered with different doses of irradiation (0, 1.5, 2.0, 5.0, 10, 20, 50, and 100 kGy). The results demonstrate that the combination of monoclonal antibodies and the SPR method can be used to characterize the irradiation-induced conformational change with an unlabelled reagent.
Key words: monoclonal antibody; surface plasmon resonance; antigen-antibody interaction; ovalbumin; irradiation

-6-
Molecular Cloning and Heterologous Expression of Pea Seedling Copper
Amine Oxidase

Tomoyoshi KOYANAGI, Kiyoyuki MATSUMURA, Shun′ichi KURODA, and Katsuyuki TANIZAWA・

Department of Structural Molecular Biology, The Institute of Scientific and Industrial Research,
Osaka University, Ibaraki, Osaka 567-0047, Japan

Received September 16, 1999; Accepted December 2, 1999
The cDNA coding for copper amine oxidase has been cloned from etiolated pea seedlings (Pisum sativum). The deduced amino acid sequence, consisting of 674 residues including the signal peptide, agreed well with those reported for the enzymes from a different cultivar of P. sativum and other plant sources, except for several evolutionary replacements located mostly on the molecular surface. A heterologous expression system for the cloned pea enzyme was constructed with the yeast Pichia pastoris, using the AOX1 promoter and the yeast α-factor secretion signal. Adding copper to the culture medium increased the secretion of an active, quinone-containing enzyme. Furthermore, the inactive enzyme produced in a copper-deficient medium was activated considerably by subsequent incubation with excess cupric ions. These results strongly suggest that the Tyr-derived redox cofactor, 2,4,5-trihydroxyphenylalanylquinone (topa quinone, TPQ), is produced in the plant enzyme by post-translational modification that proceeds through the copper-dependent, self-processing mechanism, as in the enzymes from bacteria and yeast.
Key words: copper amine oxidase; pea seedling; quinoprotein; topa quinone

-7-
Comparison of Chitinase Isozymes from Yam Tuber---Enzymatic Factor
Controlling the Lytic Activity of Chitinases

Yasuyuki ARAKANE, Hironori HOSHIKA, Natsumi KAWASHIMA, Chika FUJIYA-TSUJIMOTO,
Yuji SASAKI, and Daizo KOGA・

Laboratory of Biochemistry, Department of Biological Science, Faculty of Agriculture, Yamaguchi University,
Yamaguchi 753-8515, Japan

Received October 1, 1999; Accepted December 15, 1999
To evaluate the anti-pathogen activity of chitinases, we developed a new method for measuring the lytic activity, and investigated the correlation of the lytic activity with the enzymatic properties by using four chitinase isozymes, Chitinases E, F, H1 and G, which had been purified from yam tubers by column chromatography. Chitinases E, F and H1 had high lytic activity against the plant pathogen, Fusarium oxysporum, but Chitinase G did not. Chitinase E, which is the family 19 chitinase, was similar to Chitinases F and G in its antigenecity, but not to Chitinase H1 or H2. Chitinases H1 and H2 were recognized by the anti-Bombyx mori chitinase antibody, suggesting that Chitinases H1 and H2 are family 18 chitinases like B. mori chitinases. Chitinases E, F and H1 had two optimum pH ranges of 3--4 and 7.5--9 toward glycolchitin, but Chitinase G had only one optimum pH value of 5. Chitinases E, F and H1 had higher affinity to the polymer substrate, glycolchitin, than Chitinase G. These results suggest that the lytic activity of plant chitinases may be related to the chitin affinity and probably to the characteristic optimum pH value, or two values, but not related to its classification. The correlation of the lytic activity of a chitinase isozyme with its elicitor specificity is also discussed.
Key words: chitinases; yam (Diosscorea opposita THUNB); Fusarium oxysporum; plant self-defense; PR-proteins

-8-
Effects of Branched Cyclodextrins on the Solubility and Stability of Terpenes

Noriko AJISAKA,＊ Koji HARA, Katsuhiko MIKUNI, Kozo HARA,
and Hitoshi HASHIMOTO

Bio Research Corporation of Yokohama, 13-46 Daikoku-cho, Tsurumi-ku, Yokohama 230-0053, Japan

Received October 6, 1999; Accepted December 15, 1999
In order to investigate the application potential of branched CDs, the solubilizing ability and the stabilizing ability of G2-βCD and GUG-βCD were investigated by using twelve terpenes (d-limonene, myrcene, terpinolene, geraniol, l-menthol, nerol, α-terpineol, citral, d-citronellal, l-perillaldehyde, (R)-l-carvone, and menthone) as guest compounds. G2-βCD and GUG-βCD showed more solubilizing ability for these twelve terpenes than βCD, and the ability of GUG-βCD was almost the same as that of G2-βCD. The stabilizing ability of terpene-GUG-βCD complexes was different from that of G2-βCD. GUG-βCD was superior to G2-βCD, especially in the solid state. This result may have been caused by the difference in structure of side chain, namely the hydroxymethyl group in G2-βCD and the carboxyl group in GUG-βCD.
Key words: cyclodextrin; terpene

-9-
Galactosylation of Thiol Group by β-Galactosidase

Hirofumi NAKANO, Motohiro SHIZUMA, Taro KISO, and Sumio KITAHATA

Osaka Municipal Technical Research Institute, 6-50 Morinomiya 1-chome, Joto-ku, Osaka 536-8553, Japan

Received October 12, 1999; Accepted November 24, 1999
β-Galactosidase catalyzed β-galactosylation not only of a hydroxyl group but also of a thiol group in the condensation reaction of D-galactose and 2-mercaptoethanol. The thio-galactosylation product was confirmed as 2-hydroxyethyl S-β-D-galactoside on the bases of fast atom bombardment mass spectrometry, infrared spectroscopy, and nuclear magnetic resonance spectorometry. Aspergillus oryzae β-galactosidase hydrolyzed p-nitrophenyl S-β-D-galactoside most rapidly among several β-galactosidases and produced the thio-galactosylation product most efficiently. The Penicillim multicolor enzyme was as effective as the A. oryzae enzyme. However the enzymes from Escherichia coli, Saccharomyces fragilis, Kluyveromyces lactis, and Bacillus circulans galactosylated hydroxyl groups predominantly to produce O-galactoside. The thio-galactoside was synthesized most effectively at a 2-mercaptoethanol concentration of about 1.25 M. Galactose concentration at 0.8--2.8 M did not affect the synthetic yield of the thio-galactoside so greatly.
Key words: β-galactosidase; Aspergillus oryzae; condensation reaction; thiol compoud; galactosylation

-10-
Syntheses of 4-Methylumbelliferyl-β-D-Xylobioside and 5-Bromo-3-Indolyl-
β-D-Xylobioside for Sensitive Detection of Xylanase Activity on Agar Plates

Satoshi KANEKO,1 Motomitsu KITAOKA,1 Atsushi KUNO,2 and Kiyoshi HAYASHI1,・

1National Food Research Institute, Ministry of Agriculture, Forestry, and Fisheries, 2-1-2 Kannondai, Tsukuba,
Ibaraki 305-8642, Japan
2Department of Material and Biological Chemistry, Faculty of Science, Yamagata University, Yamagata 990-8560, Japan

Received October 14, 1999; Accepted November 24, 1999
4-Methylumbelliferyl-β-D-xylobioside (MU-X2) and 5-bromo-3-indolyl-β-D-xylobioside (BI-X2) were synthesized as substrates for the detection of xylanase activity on agar plates. A family F/10 xylanase from Streptomyces olivaceoviridis E-86 (FXYN) was able to be more sensitively detected than RBB-xylan by using MU-X2 as a substrate. A mutant xylanase E128H/FXYN having only 1/1000 of the activity of FXYN was also able to be detected on the MU-X2 plate but was not detected on the RBB-xylan plate. A family G/11 xylanase from Streptomyces lividans 66 (Xyn B) was not detected on the MU-X2 plate, but it was able to be detected on the RBB-xylan plate, suggesting that the MU-X2 substrate is specific to family F/10 xylanases. However, none of the xylanases were detected effectively by using BI-X2 as a substrate.
Key words: 4-methylumbelliferyl-β-D-xylobioside; 5-bromo-3-indolyl-β-D-xylobioside; xylanase; family F/10 xylanase; fluorescent substrate

-11-
Enzymatic Production of trans-4-Hydroxy-L-proline by Regio- and Stereospecific Hydroxylation of L-Proline

Takeshi SHIBASAKI,＊,・ Hideo MORI, and Akio OZAKI・

Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 3-6-6, Asahimachi, Machida,
Tokyo 194-8533, Japan

Received October 15, 1999; Accepted November 24, 1999
A proline 4-hydroxylase gene, which was cloned from Dactylosporangium sp. RH1, was overexpressed in Escherichia coli W1485 on a plasmid under a tryptophan tandem promoter after the codon usage of the 5? end of the gene was optimized. The proline 4-hydroxylase activity was1600-fold higher than that in Dactylosporangium sp. RH1. trans-4-Hydroxy-L-proline(Hyp) was produced and accumulated to 41 g/L (87％ yield from L-proline) in 100 h when the recombinant E. coli was cultivated in a medium containing L-proline and glucose. 2-Oxoglutarate, which is necessary for the hydroxylation of L-proline by proline 4-hydroxylase, was apparently supplied from glucose through the cellular metabolic pathway. The putA mutant of W1485, which is not able to degrade L-proline, has allowed the quantitative conversion of L-proline to Hyp. The formation of other isomers of hydroxyproline was not observed. Productivity of Hyp was almost the same in a larger-scale culture. The method of manufacturing Hyp from L-proline was established.
Key words: hydroxyproline; proline; bioconversion; proline 4-hydroxylase; 2-oxoglutarate-dependent dioxygenase

-12-
Characterization of a Phage Resistance Plasmid, pLKS, of Silage-Making
Lactobacillus plantarum NGRI0101

・Tomoko EGUCHI,1 Katsumi DOI,1 Kanako NISHIYAMA,1 Sadahiro OHMOMO,2 and Seiya OGATA1・・

1Microbial Genetics Division, Institute of Genetic Resources, Faculty of Agriculture, Kyushu University, Hakozaki, Fukuoka 812-8581, Japan
2National Institute of Animal Industry, Ibaraki 305-0901, Japan

Received October 18,1999 ; Accepted December 17, 1999
Phage contamination has resulted in abnormal fermentation in silage. We isolated a phage-resistant strain, Lactobacillus plantarum NGRI0101 from silage. The strain carried two plasmids, pLKL (6.8 kb) and pLKS (2.0 kb). By curing and retransformation of the plasmids, we clarified that pLKS has phage resistant activity, characterized as no adsorption inhibition. pLKS has 2,025 bp and three orfs, orf123, orf132, and orf918. The predicted amino acid sequence of the orf918 product showed high similarity to those of Rep proteins of Pediococcus halophilus plasmid pUCL287 and Lactobacillus acidophilus plasmid pLA103. The replication origin (ori) was upstream from orf918. There was no gene similar to typical phage resistant genes encoded by known plasmids. The phage resistance of L. plantarum NGRI0101 may possibly be due to a plasmid-encoded abortive infection.
Key words: phage resistance; plasmid; silage-making Lactobacillus

-13-
Genetic Characteristics of Cellulose-Forming Acetic Acid Bacteria Identified Phenotypically as Gluconacetobacter xylinus

Machiko TANAKA,・ Shuichiro MURAKAMI,＊ Ryu SHINKE, and Kenji AOKI

Laboratory of Bio-functional Chemistry, Division of Bioscience, The Graduate School of Science
and Technology, Kobe University, 1 Rokko, Kobe 657-8501, Japan
＊Laboratory of Applied Microbiology, Department of Biofunctional Chemistry, Faculty of Agriculture,
Kobe University, 1 Rokko, Kobe 657-8501, Japan

Received October 22, 1999; Accepted December 6, 1999
Gluconacetobacter xylinus (＝Acetobacter xylinum) shows variety in acid formation from sugars and sugar-alcohols. Toyosaki et al. proposed new subspecies of G. xylinus (＝Acetobacter xylinum) subsp. sucrofermentans in point of acid formation from sucrose and a homology index of 58.2％ with the type strain of G. xylinus subsp. xylinus in DNA-DNA hybridization experiments. We tried DNA-DNA hybridization to clarify relationship between acid formation from sugars and classification of G. xylinus. The G+C contents of G. xylinus showed 60.1--62.4 mol％ with a range of 2.3 mol％. When type strains of G. xylinus subsp. xylinus, G. xylinus subsp. sucrofermentans, and IFO 3288 forming acid from sucrose, were used as probes, the DNAs from three strains showed 67--100％, 64--89％, and 60--100％ similarity to those from sixteen strains including bacteria that form acid from sucrose or not. These results show that homology indexes do not reflect differences of acid formation from sucrose. As a results, the species G. xylinus was proved to be genetically homogeneous.
Key words: cellulose-forming acetic acid bacteria; Acetobacter xylinum; Gluconacetobacter xylinus

-14-
Development of an Improved Assay for Purine Nucleoside Kinase Activity
in Cell Extracts and Detection of Inosine Kinase Activity in Brevibacterium
acetylicum ATCC 953, Related Species, and Corynebacterium
flaccumfaciens ATCC 6887

Hisashi KAWASAKI,・ Yoshihiro USUDA, Megumi SHIMAOKA, and Takashi UTAGAWA

Applied Microbiology Laboratory, Fermentation and Biotechnology Laboratories, Ajinomoto Co., Inc., 1-1
Suzuki-cho, Kawasaki-ku, Kawasaki-shi 210-8681, Japan

Received October 25, 1999; Accepted December 7, 1999
An improved assay was developed to detect direct purine nucleoside phosphorylating activity in cell-free extracts. Direct inosine phosphorylating activity was detected in 2 of 70 species tested. Both activities, which depended on magnesium ion and ATP, phosphorylated a hydroxyl group at the 5? position of inosine. The new assay was shown to be useful for screening of direct purine nucleoside phosphorylating activity and have the potential to detect inosine kinase in the presence of a background of nucleoside phosphorylase and purine phosphoribosyltransferase activities. Previously, the latter two activities made it difficult to correctly detect direct phosphorylation of inosine by inosine kinase.
Key words: inosine-guanosine kinase; purine nucleoside; purine nucleotide; Brevibacterium; Corynebacterium

-15-
Some Properties of a Macromolecular Conjugate of Lysozyme Prepared
by Modification with a Monomethoxypolyethylene Glycol Derivative

Yuichi NODAKE and Nobuyuki YAMASAKI・

Laboratory of Biochemistry, Division of Bioresource and Bioenvironmental Sciences, Graduate School
of Kyushu University, Hakozaki, Fukuoka 812-8581, Japan

Received October 25, 1999; Accepted November 25, 1999
Hen egg-white lysozyme was modified with a succinyl ester derivative of monomethoxypolyethylene glycol (mPEG-COONSu), and some properties of the resulting conjugate (mPEG-lysozyme) were studied. The conjugate was prepared by modification of lysozyme with mPEG-COONSu and purified with use of columns of CM-Toyopearl 650M and Sephadex G-75. Analytical data indicated that in the conjugate, 1.05 moles of mPEG with an average molecular weight of 5,000 were covalently attached to the lysozyme molecule. Tryptic peptide analysis of the conjugate showed that Lys 33 in lysozyme is the residue mainly modified with mPEG-COONSu. Covalent attachment of the mPEG-derivative to amino groups greatly increased the thermostability of lysozyme without any conformational change of the protein molecule. mPEG-lysozyme retained full enzyme activity for glycol chitin, but lytic activity for Micrococcus luteus cells in neutral media was 75％ of that of native lysozyme and its optimal pH was at pH 5.0. It was also found that the reactivity of lysozyme with anti-lysozyme antibody from BALB/c mice or human lymphocytes was decreased by modification with mPEG-COONSu. From these findings, it was suggested that mPEG-COONSu can be advantageously used for protein tailoring of lysozyme.
Key words: hen egg-white lysozyme; monomethoxypolyethylene glycol (mPEG); protein-polymer conjugate; thermostability; immunogenicity

-16-
Isolation of a 41-kDa Protein with Cell Adhesion Activity for Animal Tumor
Cells from the Mushroom Hypsizigus marmoreus by Affinity Chromatography
with Type IV Collagen Immobilized on Agarose

Yutaka SHOJI, Mamoru ISEMURA,・ Haruhiko MUTO,＊ Satoko ISEMURA,＊＊
and Yutaka AOYAGI＊＊＊

School of Food and Nutritional Sciences, University of Shizuoka, Yada, Shizuoka 422-8526, Japan
＊Shizuoka Prefecture Forestry and Forest Products Research Institute, Negata, Hamakita,
Shizuoka 434-0016, Japan
＊＊Nippon Dental University Junior Collage at Niigata, Niigata 951-8151, Japan
＊＊＊Department of Third Internal Medicine, Niigata University School of Medicine, Niigata 951-8122, Japan

Received October 29, 1999; Accepted December 22, 1999
A type IV collagen-binding protein of 41 kDa was isolated from the mushroom Hypsizigus marmoreus and the protein was designated as HM41. The Western blotting analysis with anti-HM41 antibodies demonstrated that HM41 was unrelated to HM23, which had been shown to have an affinity for type IV collagen. The microsequence analysis of the membrane-blotted peptides generated by fragmentation with cyanogen bromide showed no homologous proteins reported. HM41 had cell adhesion-promoting activity for murine Lewis lung carcinoma LL2 cells and human fibrosarcoma HT1080 cells. These results indicate that HM41 is a hitherto undescribed fungus protein that can interact both with animal extracellular matrix protein type IV collagen and with animal tumor cells.
Key words: type IV collagen; mushroom; cell adhesion; cancer cells; extracellular matrix

-17-
Resolution and Synthesis of Optically Active Alcohols
with Immobilized Water-soluble Proteins from Green Pea,
Soybean and Buckwheat as New Bio-catalysts

Hiroyuki NAGAOKA1,3 and Hiroshi KAYAHARA2

1The United Graduate School of Agricultural Science, Gifu University, 1-1 Yanagido,
Gifu 501-1193, Japan
2Department of Bioscience and Biotechnology, Shinshu University, 8304 Minamiminowa,
Kamiina, Nagano Japan
3Sanyo Shokuhin Co., Ltd., 555-4 Asakura, Maebashi Gunma 371-0811, Japan

Received November 11, 1999; Accept December 1, 1999
Kinetic resolution of racemic alcohols, (±)-1-(4-substituted phenyl)ethanol and (±)-1-(2-naphthyl)ethanol, was done with immobilized green pea, soybean, or buckwheat proteins. The resolution was done stereoselectively by oxidizing only one enantiomer of a racemic alcohol to leave an optically active alcohol with a high purity. In addition, each protein could be reused consecutively at least three times without any decrease of yield or optical purity.
Key words: immobilized green pea protein; immobilized soybean protein; immobilized buckwheat protein; (±)-1-(4-substituted phenyl)ethanol; (±)-1-(2-naphthyl)ethanol

-18-
An ELISA-Based Assay for Detergent-solubilized Cellular
β1,4-Galactosyltransferase Activity.
Use of a Polyacrylamide Derivative with GlcNAc-β Side Chains
as a Solid Phase Acceptor Substrate

Mohamed OUBIHI, Kenji OSHIMA, Naohito AOKI, Kazukiyo KOBAYASHI,＊
Ken KITAJIMA, and Tsukasa MATSUDA・

Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences,
Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan
＊Department of Molecular Design and Biotechnology, Graduate School of Engineering,
Nagoya University, Nagoya 464-8601, Japan

Received November 12, 1999; Accepted December 20, 1999
In our previous paper [Oubihi et al. (1998) Anal. Biochem., 257, 169--175], we have shown that a polyacrylamide-derived synthetic glycopolymer with GlcNAcβ side chains, termed PAP(GlcNAcβ), is useful as a solid phase acceptor substrate for the ELISA-based analyses of soluble β1,4(--)galactosyltransferase (GalT) activity in milk. This method is now used to assay detergent-solubilized cellular GalT. The glycopolymer coated on polystyrene plates was shown to be highly stable against the non-ionic and ionic detergents tested (0~5％ solutions of Triton X-100 and SDS). Such stability made it possible to incubate the ELISA plate with detergent-solubilized GalT and to wash the ELISA plate with SDS solution after the GalT reaction, leading to high accuracy and sensitivity of this assay. The GalT activity was assayed using this method for 1％ Triton X-100 extracts of various tissue samples of mice and several cultured cell lines. The results showed that the specific GalT activity of tissue extracts was low in brain and intestine, and high in ovary, muscle, and kidney. As for the cultured cell lines, COS7, COMMA-1D and C2C12 cells showed high specific activity, while CHO and MDCK cells showed low activity. The myoblast C2C12 had a slight increase in GalT activity during starvation-induced cell differentiation. On the other hand, GalT-I transcript estimated by RT-PCR rather decreased during C2C12 cell differentiation, suggesting a differentiation-dependent switch in GalT isozymes. Taken all together, the ELISA-based assay using PAP(GlcNAcβ) as a solid phase acceptor substrate was demonstrated to be a useful method for the assay of membrane-bound galactosyltransferases.
Key words: galactosyltransferase; glycopolymer; ELISA; detergent; RCA1 lectin

-19-
Structured Triacylglycerol Containing Medium-Chain Fatty Acids in sn-1(3)
Facilitates the Absorption of Dietary Long-Chain Fatty Acids in Rats

Octavio CARVAJAL, Masanobu SAKONO,＊ Hirofumi SONOKI, Masahiro NAKAYAMA, Taiji KISHI,
Masao SATO, Ikuo IKEDA, Michihiro SUGANO,＊＊ and Katsumi IMAIZUMI・

Laboratory of Nutrition Chemistry, Division of Bioresource and Bioenvironmental Sciences,
Graduate School, Kyushu University, Fukuoka 812-8581, Japan
＊Laboratory of Nutrition Chemistry, Department of Biological Resource Sciences,
Faculty of Agriculture, Miyazaki University, Miyazaki 889-2192, Japan
＊＊Kumamoto Prefecture University, Kumamoto 862-0920, Japan

Received November 12, 1999; Accepted December 24, 1999
A study was carried out to examine if the positional distribution of medium chain fatty acids (MCF) in triacylglycerol influences dietary fat absorption in rats. Two types of structure-specific fats, one predominantly composed of MCF in sn-1(3) and linoleic acid in sn-2 [sn-1(3)MCF-structured] and the others of MCF in sn-2 and linoleic acid in sn-1(3) [sn-2MCF-structured], were initially prepared, and the two structure-specific fats were interesterified and designated as sn-1(3)MCF-interesterified and sn-2MCF-interesterified. Synthetic fat was mixed with an equal amount of cocoa butter (103 g/kg of diet) and was supplemented to the AIN93G-based diet. Rats were fed on the diets for 4 wk. Long-chain saturated fatty acids were the predominant fatty acids excreted into the feces, and the positional distribution of MCF resulted in an altered fat absorption rate (％) of 81.8, 82.5, 84.2 and 86.3 for the rats fed on the diets containing sn-2MCF-structured, sn-1(3)MCF-interesterified, sn-2MCF-interesterified and sn-1(3)MCF-structured fats, respectively. The proportion of MCF in the serum, liver and adipose tissue triacylglycerols was not affected by the MCF distribution of the dietary fats. These results indicate that the distribution of MCF in dietary triacylglycerol is a determinant of intestinal fat absorption.
Key words: fecal fatty acids; medium-chain fatty acids; structured triacylglycerol

-20-
Deletion of the yhhP Gene Results in Filamentous Cell Morphology
in Escherichia coli

Yuko ISHII,＊ Hisami YAMADA,＊ Takafumi YAMASHINO,＊ Kenji OHASHI,＊ Etsuko KATOH,＊＊
Heisaburo SHINDO,＊＊＊ Toshimasa YAMAZAKI,＊＊ and Takeshi MIZUNO＊

＊Laboratory of Molecular Microbiology, School of Agriculture, Nagoya University, Chikusa-ku,
Nagoya 464-86-1, Japan
＊＊Structural Biology Unit, National Institute of Agrobiological Resources, Tsukuba,
Ibaraki 305-862, Japan
＊＊＊Tokyo University of Pharmacy and Life Science, Horinouchi, Hachioji, Tokyo 192-0003, Japan

Received November 17, 1999; Accepted December 16, 1999
The Escherichia coli yhhP gene was predicted to encode a small hypothetical protein of 81 amino acids, the cellular function of which is not known. To gain insight into the function of this uncharacterized YhhP protein, genetic and biochemical studies were done. We first tried to express and purify the YhhP protein to prepare an anti-YhhP antiserum. Western blotting showed that the hypothetical yhhP gene is indeed transcribed and translated as a minor cytoplasmic protein. YhhP-deficient (ΔyhhP) cells formed colonies poorly on a rich medium (e.g., Luria-Bertani medium) containing a relatively low concentration of NaCl, while they can grow normally either in LB containing 3％ NaCl or in a synthetic medium (e.g., M9-glucose). During exponential growth in rich medium, an early step of cell division was inhibited in ΔyhhP cells, forming filaments. For the YhhP-deficient filamentous cells, the FtsZ-ring formation was analyzed with immunofluorescence microscopy. The FtsZ-ring formation did not occur normally in the ΔyhhP filaments, although the filamentous cells contained the FtsZ protein at a certain level comparable to that in the wild-type cells. The ftsZ gene was found to function as a multicopy suppressor of the ΔyhhP mutant. Another multicopy suppressor gene was identified as the dksA gene. Provided that either the ftsZ or dksA gene was introduced into the mutant cells with its multicopy state, the resulting transformants were capable of growing in rich medium, formed wild-type short rods. These results are discussed with regard to the presumed function of this ubiquitous protein.
Key words: Escherichia coli; YhhP protein; yhhP mutant; cell division; FtsZ protein

-21-
Synthesis and Biological Activities of 4-Chloroindole-3-acetic Acid
and Its Esters

Masato KATAYAMA・

Laboratory of Bioorganic Chemistry, Department of Chemistry, National Industrial Research Institute
of Nagoya, 1-1 Hirate-cho, Kita-ku, Nagoya 462-8510, Japan

Received November 18, 1999; Accepted December 15, 1999
4-Chloroindole-3-acetic acid (4-Cl-IAA) and its esters were synthesized from 2-chloro-6-nitrotoluene as the starting material. The biological activities of 4-Cl-IAA and its esters were determined by four bioassays. Except for the tert-butyl ester, 4-Cl-IAA and its esters had stronger elongation activity toward Avena coleoptiles than had indole-3-acetic acid. The biological activities of the methyl, ethyl and allyl esters were as strong as the activity of the free acid. All the esters, except for the tert-butyl, inhibited Chinese cabbage hypocotyl growth more than the free acid did, and all the esters induced severe swelling and formation of numerous lateral roots in black gram seedlings even at a low concentration. Furthermore, adventitious root formation was strongly promoted in Serissa japonica cuttings by all the esters. The root formation-promoting activities of the ethyl and allyl esters were about three times the value for indole-3-butyric acid which is used to promote and accelerate root formation in plant cuttings.
Key words: 4-chloroindole-3-acetic acid esters; chlorinated auxins; Avena coleoptile elongation; hypocotyl growth inhibition; adventitious root formation

-22-
Molecular Cloning and Characterization of a Transcriptional Activator Gene, amyR, Involved in the Amylolytic Gene Expression in Aspergillus oryzae

Katsuya GOMI,1,・ Terumi AKENO,1 Toshitaka MINETOKI,2 Kenji OZEKI,2 Chieko KUMAGAI,2 Naoto OKAZAKI,1 and Yuzuru IIMURA1,＃

1National Research Institute of Brewing, 3-7-1, Kagamiyama, Higashi-Hiroshima, Hiroshima 739-0046, Japan
2General Research Laboratories, Ozeki Corp., 4-9, Imazu Dezaike-cho, Nishinomiya, Hyogo 663-8227, Japan

Received November 19, 1999; Accepted December 20, 1999
A gene, designated amyR, coding for a transcriptional activator involved in amylolytic gene expression has been cloned from Aspergillus oryzae by screening for a clone that enabled to reverse the reduced expression of the α-amylase gene (amyB) promoter. amyR encodes 604 amino acid residues of a putative DNA-binding protein carrying a zinc binuclear cluster motif (Zn(II)2Cys6) belonging to the GAL4 family of transcription factors. The amyR gene disruptants showed a significant restricted growth on starch medium and produced little of the amylolytic enzymes including α-amylase and glucoamylase compared with a non-disruptant, indicating that amyR is a transcriptional activator gene involved in starch/maltose-induced efficient expression of the amylolytic genes in A. oryzae. In addition, sequencing analysis found that amyR, agdA (encoding α-glucosidase), and amyA (encoding α-amylase), are clustered on a 12-kb DNA fragment of the largest chromosome in A. oryzae, and that amyR is about 1.5 kb upstream of agdA and transcribed in the opposite direction. Furthermore, transcriptional analysis revealed that the amyR gene was expressed in the presence of glucose comparable to the level in the presence of maltose, while the amylolytic genes were transcribed at high levels only in the presence of maltose.
Key words: Aspergillus oryzae; transcriptional activator; amylolytic enzyme; zinc finger motif; gene cluster

-23-
Mutational Evidence Supporting the Involvement of Tripartite Residues His183,
Asp185, and His243 in Streptomyces clavuligerus Deacetoxycephalosporin
C Synthase for Catalysis

Janet SIM and Tiow-Suan SIM

Department of Microbiology, Faculty of Medicine, National University of Singapore, 5 Science Drive 2,
Singapore 117597

Received November 22, 1999; Accepted December 7, 1999
Deacetoxycephalosporin C synthase (DAOCS) is a non-heme iron-binding and α-ketoglutarate dependent enzyme involved in catalyzing the biosynthesis of cephalosporins and cephamycins, antibiotics more potent than penicillins. In the crystal structure complex of Streptomyces clavuligerus DAOCS (scDAOCS), it was proposed that histidine-183, aspartate-185, and histidine-243 are putative iron-binding ligands. However, coordinates proposed for crystal structures of proteins may not definitely comply with catalysis. Hence, site-directed mutagenesis was done to replace each of these amino acid residues with leucine. The constructed expression vectors bearing the mutations were found to express the respective scDAOCS mutant enzymes at high levels in Escherichia coli BL21(DE3). Through enzymatic assays, it was shown that while the wildtype enzyme could convert penicillin to a more active cephalosporin, the substitution of the three proposed iron-binding sites of scDAOCS completely abolished the same activity in the respective mutant enzymes. Thus, these results clearly indicate that histidine-183, aspartate-185, and histidine-243 of scDAOCS are essential for the ring expansion activity.
Key words: deacetoxycephalosporin C synthase; Streptomyces clavuligerus; site-directed mutagenesis; iron-binding; histidine

-24-
Note
Hybridization and Breeding of the Benomyl Resistant Mutant, Trichoderma
harzianum Antagonized to Phytopathogenic Fungi by Protoplast Fusion

Kihachiro OGAWA,・ Naoto YOSHIDA, Wanwilai GESNARA,＊ Crispinus A. OMUMASABA,
and Chiradej CHAMUSWARNG＊

Department of Biological Resource Sciences, Faculty of Agriculture, Miyazaki University, Miyazaki 889-2192,
Japan
＊Department of Pathology, Kasetsart University, Kamphaeng Saen, Nakhon Pathom, 73140, Thailand

Received June 7, 1999; Accepted December 3, 1999
A diploid strain obtained from heterokaryons of Trichoderma harzianum by protoplast fusion grew on minimal medium containing 100 ppm benomyl. This strain inhibited the growth of the phytopathogenic fungus Fusarium oxysporum f. sp. raphani on paired cultures and also protected against radish yellows and a drop in germination induced by F. oxysporum f. sp. raphani.
Key words: protoplast fusion; antagonistic fungus; Trichoderma harzianum; phytopathogenic fungi

-25-
Note
Superoxide-Scavenging and Prolyl Endopeptidase Inhibitory Activities
of Bangladeshi Indigenous Medicinal Plants

Firoza KHANOM, Hiroshi KAYAHARA,1,・ and Koji TADASA1

The United Graduate School of Agricultural Science, Gifu University, 1-1 Yanagido, Gifushi,
Gifu 501-1193, Japan
1Division of Bio-organic Chemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture,
Shinshu University, 8304 Minamiminowa-mura, Kamiina-gun, Nagano-ken 399-4598, Japan

Received June 16, 1999; Accepted December 21, 1999
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The superoxide-scavenging and prolyl endopeptidase (PEP) inhibitory activities of 15 different kinds of Bangladeshi medicinal plants were evaluated. Methanol extraction was performed for the screening tests. Swertia chirata, Emblica officinalis, Zingiber officinale and Myristica malabarica were screened as superoxide-scavenging samples. Similarly, E. officinalis was identified as one of the strongest PEP inhibitory samples. The 50％ O−2-scavenging and PEP-inhibitory concentrations from E. officinalis methanol extracts were found to be 13.17 and 26.10 μg/ml, respectively.
Key words: Bangladeshi indigenous medicinal plants; superoxide-scavenging activity; prolyl endopeptidase inhibitory activity

-26-
Note
Secreted Phytase Activities of Yeasts

Yoshihiro NAKAMURA,1,・ Hiroshi FUKUHARA,2 and Konosuke SANO3

1Ajinomoto Co., Inc., 1-15-1 Kyobashi, Chuo-ku, Tokyo 104-8315, Japan
2Institut Curie Section de Recherche, Centre Universitaire Paris XI, Orsay 91405, France
3Ajinomoto Co. Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki-shi 210-8681, Japan

Received July 16, 1999; Accepted December 8, 1999
The enzyme phytase dephosphorylates phytin (inositol hexaphosphate), a major phosphate reserve in plants. We found that a large number of yeast species secreted a phytase. Several species were identified as high phytase producers. The yeast enzymes had an optimal activity at pH 4--5 and generally a very high optimal temperature, ranging from 60°C to 80°C.
Key words: yeast screening; Pichia rhodanensis; Pichia spartinae; secreted phytase; thermo-resistance

-27-
Note
Development of a Simple and Efficient Method for Transformation
of Buckwheat Plants (Fagopyrum esculentum)
Using Agrobacterium tumefaciens

Mineo KOJIMA, Yukie ARAI, Narumi IWASE, Kimiko SHIROTORI,
Hidenari SHIOIRI, and Masayuki NOZUE

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

Received August 11, 1999; Accepted December 25, 1999
Apical meristems of seedlings of buckwheat (Fagopyrum esculentum var. Shinano No. 1) were pricked with a needle and inoculated with Agrobacterium tumefaciens (LBA4404, pBI121). The inoculated seedlings were grown to maturation and allowed to pollinate randomly to set the seeds (T1 plants). The transformation efficiency of the T1 plants was estimated by germination in the presence of geneticin (20 μg/ml) and by detection of β-glucuronidase (GUS) gene with PCR, indicating that 36％ and 70％ of the T1 plants were transformed, respectively. Four plants taking on a mutated morphology were selected from T1 plants which were transformed with the method using A. tumefaciens harboring a modified pBI121 for plasmid rescue. Southern blot analysis of plasmids rescued from the 4 T1 plants demonstrated that each plasmid contained a different flanking DNA of the buckwheat genome, an evidence that T-DNA was integrated in different sites of the genomic DNA among the 4 T1 plants.
Key words: Agrobacterium tumefaciens; apical meristem; Fagopyrum esculentum; plasmid rescue; transformation

-28-
Note
Dependence of Rat Spot14 Promoter Activity on NF-Y Binding
to the Inverted CCAAT-element at --100

Karim RODER,・ Siegmund S. WOLF,・ and Michael SCHWEIZER・

Department of Biological Sciences, Heriot-Watt University, Edinburgh EH14 4AS, UK

Received August 17, 1999; Accepted November 12, 1999
・・・E・lectrophoretic ・・・m・obility ・・・s・hift ・・・a・ssay (EMSA) and in vitro transcription/translation show that NF-Y binds to the inverted CCAAT-element in the promoter of the rodent spot14 gene. The NF-Y-binding sequence has been shown to be responsible for basal activity in H4IIE. Given the similar role found for the inverted CCAAT-element in the promoter of the FAS gene, NF-Y may have an important function in the control of lipogenesis.
Key words: spot14 promoter; fatty acid synthase; NF-Y; inverted CCAAT-element

-29-
Note
Gene Organization for Nitric Oxide Reduction in Alcaligenes faecalis S-6

Mutsuko KUKIMOTO,1 Makoto NISHIYAMA,2,・ Masaru TANOKURA,2 and Sueharu HORINOUCHI1

1Department of Biotechnology and 2Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi,
Bunkyo-ku, Tokyo 113-8657, Japan

Received August 30, 1999; Accepted December 3, 1999
norB and norC encoding the cytochrome b-containing subunit and the cytochrome c-containing subunit, respectively, of the nitric oxide reductase (NOR) in Alcaligenes faecalis S-6 were cloned and sequenced. Both NorB and NorC showed more than 40％ sequence identity to the corresponding subunits of cytochrome bc-type NORs in other denitrifying bacteria. norCB was in a gene cluster containing seven other genes; these were named dnr, orf2, orf3, norE, norF, norQ, and norD on the basis of their similarity with NOR systems in other bacteria. Potential FNR-binding sites were present in front of norCB, norEF, and/or orf2/orf3, suggesting that most of these genes are regulated simultaneously by an FNR-related protein. NorB and NorC proteins produced in the membrane fraction in Escherichia coli showed no enzyme activity, probably due to lack of NorQ and NorD, which appear to perform some essential function for activation of the NorB-NorC complex in the recombinant E. coli.
Key words: Alcaligenes faecalis; denitrification; nitric oxide reductase; cytochrome bc complex

-30-
Note
Distribution of Sarcophytol A in Soft Coral of the Sarcophyton Genus

Mamoru KOH, Tetsuo IWANAGA,＊ Masahiro HATANAKA,＊＊ Akio NAKANO,
Kazuyuki MORIHARA, and Kaoru TAKEMURA＊＊,・

Institute for Applied Life Science, University of East Asia, Graduate School, 2-1 Ichinomiya-Gakuen cho,
Shimonoseki-shi, Yamaguchi 751-0807, Japan
＊Department of Chemistry, Faculty of Science, Kagoshima University, 1-21-24 Kohrimoto, Kagoshima-shi,
Kagoshima 890-0065, Japan
＊＊Research Laboratory, Sankei Chemical Co., 2-9 Nanei, Kagoshima-shi, Kagoshima 891-0122, Japan

Received September 6, 1999; Accepted December 24, 1999
The distribution of sarcophytol-A in the Sarcophyton genus was investigated in seven samples belonging to S. glaucum (3 samples), S. infundibulifurme (2 samples), S. crassocaule (1 sample) and S. trocheliophorum (1 sample) that were collected on Ishigaki Island in Okinawa Prefecture. Sarcophytol-A was present in one sample each of S. glaucum and S. infundibulifurme. This study indicates that the composition of cembranoids in the Sarcophyton genus is not related with the respective species, but with the individual samples collected.
Key words: sarcophytol-A; sarcophytonin-A; 2-[(E, E, E) - 7?,8? - epoxy - 4?,8?,12? - trimethylcyclotetradeca - 1?, 3?, 11? - trienyl]propan-2-ol; Sarcophyton glaucum; anti-tumor-
promoter

-31-
Note
Dehydrodimers of Caffeic Acid in the Cell Walls
of Suspension-Cultured Mentha

Jian-Gang YANG and Takeo UCHIYAMA＊,・

Graduate School of Science and Technology, Niigata University, Ikarashi, Niigata 950-2102, Japan
＊Faculty of Agriculture, Niigata University, Ikarashi, Niigata 950-2181, Japan

Received September 13, 1999; Accepeted December 25, 1999
Dehydrodicaffeic acid derivatives were found in the cell walls of suspension-cultured cells of Mentha. Using gas chromatography/mass spectrometry (GC-MS) in a single ion chromatography at m/z 790 and m/z 718, eleven peaks of trimethylsilylated dehydrodimers of caffeic acid were detected in the extracts from the cell walls of suspension-cultured cells of Mentha using sodium hydroxide. The result suggests that dehydrodicaffeates are formed in the cell walls from two molecules of caffeate, probably formed through C--C, and C--O--C coupling processes.
Key words: Mentha suspension-cultured cells; cell wall; caffeic acid; C--C and C--O--C couplings; dehydrodicaffeic acid

-32-
Note
Identification of L-Inositol and Scyllitol and Their Distribution
in Various Organs in Chrysanthemum

Kazuo ICHIMURA,・ Katsunori KOHATA, Yuichi YAMAGUCHI, Mitsuru DOUZONO,
Hiroshi IKEDA, and Mamoru KOKETSU1

National Research Institute of Vegetables, Ornamental Plants and Tea, Ano, Mie 514-2392, Japan
1Faculty of Engineering, Gifu University, Gifu 501-1193, Japan

Received September 16, 1999; Accepted December 24, 1999
Two unidentified soluble carbohydrates were isolated from chrysanthemum (Dendranthema×grandiflorum (Ramat.) Kitamura) leaves using HPLC. The compounds were identified as 1 L-chiro-inositol, called L-inositol (1) and scyllo-inositol, called scyllitol (2) from the results of 1H-NMR, 13C-NMR, and CI-MS spectra. L-Inositol and scyllitol were distributed in four cultivars tested. L-Inositol concentration of petals gradually decreased during the flower bud development, but the L-inositol content increased by about 7 times. Scyllitol was detected only at an early stage of flower bud.
Key words: chrysanthemum; cut flower; L-inositol; scyllitol; soluble carbohydrate

-33-
Note
New Taxane Diterpenoid from Seed of the Chinese Yew, Taxus yunnanensis＊

Qing-wen SHI, Takayuki ORITANI,・ and Ding ZHAO・＃

Laboratory of Applied Bioorganic Chemistry, Division of Life Science, Graduate School of Agricultural
Sciences, Tohoku University, 1-1 Tsutsumidori-Amamiya, Aoba-ku, Sendai 981-8555, Japan
・＃Department of Chemistry of Medicinal Natural Products, Faculty of Pharmaceutal Sciences,
Hebei Medical University, 361 Zhongshan East Street, Shijiazhuang 050017, P.R.China

Received September 22, 1999; Accepted December 21, 1999
A novel taxane diterpenoid with a rearranged 5/7/6-membered ring system was isolated from seeds of the Chinese yew, Taxus yunnanensis. Its structure was established as 9α,13α-diacetoxy-10β-benzoxy-5α-cinnamoyl-11(15→1)-abeotaxa-4(20),11-dien-15-ol on the basis of a spectroscopic analysis. Its relative stereochemistry is proposed from the results of NOESY experiments.
Key words: Taxus yunnanensis; taxoid; isolation; Taxaceae; Taxus

-34-
Note
A New Chlorinated Red Naphthoquinone from Roots of Sesamum indicum

A. F. M. Feroj HASAN, Setara BEGUM, Toshio FURUMOTO, and Hiroshi FUKUI・

Department of Biochemistry and Food Science, Faculty of Agriculture, Kagawa University,
Kagawa 761-0795, Japan

Received October 4, 1999; Accepted December 8, 1999
A new chlorinated red naphthoquinone pigment having antifungal activity, named chlorosesamone, was isolated from the roots of Sesamum indicum. Its structure was characterized as 2-chloro-5,8-dihydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinone on the basis of spectral evidence.
Key words: Sesamum indicum; Pedaliaceae; chlorinated naphthoquinone; chlorosesamone; antifungal activity

-35-
Note
Steroid 5α-Reductase Inhibitory Activity and Hair Regrowth Effects
of an Extract from Boehmeria nipononivea

Kuniyoshi SHIMIZU,1 Ryuichiro KONDO,1＊ Kokki SAKAI,1 Yoshihiro SHOYAMA,2
Hiroaki SATO,3 and Tetsuya UENO3

1Department of Forest Products, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan
2Faculty of Pharmaceutical Science, Kyushu University, Fukuoka 812-8582, Japan
3Kansai Koso Co. Ltd., Fukuoka 816-0921, Japan

Received October 12, 1999; Accepted December 16, 1999
The acetone extract of Boehmeria nipononivea showed both potent 5α-reductase inhibitory activity and hair regrowth promotion effects on mice. 5α-Reductase inhibitory activity-guided fractionation led to six active fatty acids: α-linolenic, linoleic, palmitic, elaidic, oleic and stearic acids. The extract of B. nipononivea, and α-linolenic, elaidic and stearic acids exhibited a hair regrowth effect.
Key words: Boehmeria nipononivea; hair regrowth; testosterone 5α-reductase inhibitor; fatty acid; Urticaceae

-36-
Note
Stereoselective Synthesis of the Optically Active Samin Type of Lignan
from L-Glutamic Acid

Satoshi YAMAUCHI,・ Nobuyuki YAMAMOTO, and Yoshiro KINOSHITA

College of Agriculture, Ehime University, Tarumi 3-5-7, Matsuyama, Ehime 790-8566, Japan

Received October13, 1999; Accepted December 20, 1999
The optically active samin type of lignan, (1R,2S,5R,
6S) - 6 - (2 - methoxy - 4,5 - methylenedioxyphenyl) - 3,7 - dioxabicyclo[3.3.0]octan-2-ol, was stereoselectively synthesized from L-glutamic acid via (2R,3R)-2-[(1S and R)-
1 - [(tert - butyldimethylsilyl)oxy] - 1 - (2 - methoxy - 4,5-methylenedioxyphenyl)methyl] - 3 - [(tert - butyldiphenylsilyl)oxy]methyl-1,4-butanediol.
Key words: lignan; furofuran lignan; samin

-37-
Note
(10E,12Z,15Z)-9-Hydroxy-10,12,15-octadecatrienoic Acid Methyl Ester
as an Anti-inflammatory Compound from Ehretia dicksonii

Mei DONG, Yukiko ODA,＊ and Mitsuru HIROTA＊・

The United Graduate School of Agricultural Science, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
＊Department of Bioscience and Biotechnology, Faculty of Agriculture, Shinshu University,
8304 Minami-minowa, Kami-ina, Nagano 399-4598, Japan

Received October 18, 1999; Accepted December 6, 1999
The methanol extract of Ehretia dicksonii provided (10E, 12Z, 15Z) - 9 - hydroxy - 10, 12, 15 - octadecatrienoic acid methyl ester (1) which was isolated as an anti-inflammatory compound. Compound 1 suppressed 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced inflammation on mouse ears at a dose of 500 μg (the inhibitory effect (IE) was 43％). Linolenic acid methyl ester did not inhibit this inflammation at the same dose. However, the related compounds of 1, (9Z,11E)-13-hydroxy-9,11-octadecadienoic acid (5) and (9Z,11E)-
13-oxo-9,11-octadecadienoic acid (6), showed potent activity (IE_{500 μg} of 63％ and 79％, respectively). Compounds 1, 4 ((9Z,12Z,14E)-16-hydroxy-9,12,14-octadecatrienoic acid), 5 and 6 also showed inhibitory activity toward soybean lipoxygenase at a concentration of 10 μg/ml.
Key words: anti-inflammatory; Ehretia dicksonii; TPA-induced edema; linolenic acid; linoleic acid

-38-
Note
Purification and Partial Characterization of a Basic Xylanase Produced
by Thermoalkaliphilic Bacillus sp. Strain TAR-1

Hidenori TAKAHASHI, Ryuichiro NAKAI, and Satoshi NAKAMURA・

Department of Bioengineering, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku,
Yokohama 226-8501, Japan

Received October 18, 1999; Accepted November 24, 1999
A basic xylanase was purified from the culture supernatant of thermoalkaliphilic Bacillus sp. strain TAR-1. Its molecular mass and isoelectric point were 23 kDa and >pH 9.3, respectively. The enzyme showed a broad pH profile and was optimally active at 70°C. Analyses of xylan-degradation products and N-terminal amino acid sequence revealed that the enzyme would be a family 11/G endoxylanase.
Key words: xylanase; thermoalkaliphile; Bacillus sp.; family 11/G; pulp bleaching

-39-
Note
Effects of Soybean Saponin on Protease Hydrolyses of β-Lactoglobulin
and α-Lactalbumin

Makoto SHIMOYAMADA,・ Ryoji OOTSUBO, Taichi NARUSE, and Kenji WATANABE

Laboratory of Food Material Engineering, Faculty of Agriculture, Gifu University, 1-1 Yanagido,
Gifu 501-1193, Japan

Received October 18, 1999; Accepted December 27, 1999
The effects of soybean saponin on tryptic and chymotryptic hydrolyses of whey proteins were evaluated. β-lactoglobulin and α-lactalbumin became more sensitive to both trypsin and chymotrypsin by interacting with saponin in contrast to serum albumin. Soybean saponin was shown to have different effects on various proteins according to their nature.
Key words: soybean saponin; milk whey protein; β-lactoglobulin; α-lactalbumin; proteases

-40-
Note
Two Novel Taxane Diterpenoids From the Needles of Japanese Yew,
Taxus Cuspidata

Qian CHENG,・ Takayuki ORITANI,・ and Tohru HORIGUCHI

Laboratory of Applied Bioorganic Chemistry, Division of Life Science, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan

Received October 21, 1999; Accepted December 7, 1999
Two novel taxane diterpenoids were isolated from the needles of Japanese yew, Taxus cuspidata, and their structures were determined to be 1β-hydroxy-7β-acetoxytaxinine (1) and 1β,7β-dihydroxytaxinine (2) on the basis of spectral analyses including 2D-NMR studies.
Key words: antitumour compound; Taxus cuspidata; taxaceae; diterpenoid; taxane

-41-
Note
Efficient Selection for Thermostable Protease in Thermus thermophilus

Hiroshi TAKAGI,＊ Akitoshi SUZUMURA, Yoshiyuki HASUURA, Takayuki HOSHINO,・
and Shigeru NAKAMORI

Department of Bioscience, Fukui Prefectural University, Matsuoka-cho, Fukui 910-1195, Japan
・Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan

Received October 26, 1999; Accepted December 13, 1999
An efficient procedure was established to select for thermostable proteases in an extreme thermophile, Thermus thermophilus. A non-protease-secreting mutant derived from T. thermophilus TH125 was used as host and the expression plasmid for aqualysin I from T. aquaticus YT-1 was constructed as a source of thermostable protease. T. thermophilus cells harboring the recombinant plasmid produced active aqualysin I into the medium and were able to grow on a minimal medium containing milk casein as the sole source of carbon and nitrogen.
Key words: aqualysin I; screening; thermostable protease; Thermus thermophilus

-42-
Note
The Tyrosine Kinase Activity of the Chicken Insulin Receptor Is Similar
to That of the Human Insulin Receptor

Hisanori KATO,・ Yumiko OKUBO,＊ Yuki MATSUMURA,＊＊ Charles T. ROBERTS, Jr.,＊＊＊
Kunio SUGAHARA,＊＊ and Derek LEROITH＊

Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences,
The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan
＊Clinical Endocrine Branch, National Institute of Diabetes and Digestive and Kidney Diseases,
National Institutes of Health, Bethesda, Maryland 20892-1758, U.S.A.
＊＊Department of Animal Science, Faculty of Agriculture, Utsunomiya University, 350 Mine-machi,
Utsunomiya 321-8505, Japan
＊＊＊Department of Pediatrics, Oregon Health Sciences University, Portland, Oregon 97201-3042, U.S.A.

Received October 28, 1999; Accepted December 27, 1999
The tyrosine kinase activity of a chimeric insulin receptor composed of the extracellular domain of the human insulin receptor (IR) and the intracellular domain of the chicken IR was compared with wild-type human IR. The degrees of autophosphorylation, phosphorylation of IRS-1, and in vitro phosphorylation of an exogenous substrate after stimulation by human insulin were similar to that seen with the human IR. We conclude that the insulin resistance of chickens is not attributable to a lower level of intrinsic tyrosine kinase activity of IR.
Key words: insulin receptor; chicken; tyrosine kinase; IRS-1

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Note
(+)-4-epi-α-Bisabolol as a Major Sesquiterepene Constituent in the Leaves
of Two Rosa rugosa Hybrids, Martin Frobisher and Vanguard

Yasuyuki HASHIDOKO,1,2 Keiko ENDOH,1 Toshihiro KUDO,3 and Satoshi TAHARA1,2

1Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Kita-ku,
Sapporo 060-8589, Japan
2CREST, Japan Science and Technology Corporation, Honmachi 4-1-8, Kawaguchi 332-0012, Japan
3Yurigahara Park, Sapporo Parks Green Development Association, 210 Shinorocho-Taihei, Kita-ku,
Sapporo 002-8051, Japan

Received October 29, 1999; Accepted December 21, 1999
During an investigation of the sesquiterpene phases and contents in leaves of several Rosa rugosa hybrids (hybrid rugosas), Martin Frobisher and Vanguard were found to accumulate a large amount of (+)-4-epi-α-bisabolol (1) as a single constituent. Although glandular trichomes of Martin Frobisher on the leaves are dense, this R. rugosa hybrid produces none of the carota-1,4-dienaldehyde (2) or bisaborosaol A (3) that are both found as representative sesquiterpenes of the carotane and bisabolane classes, respectively, in a glandular trichome exudate of wild-type R. rugosa. Compound 1 was also apparent as a nearly single constituent detectable by GC in the leaf constituents of Vanguard possesses sparse glandular trichomes on the leaf. Martin Frobisher and Vanguard had likely lost their capability to form carotane-type sesquiterpenes and had also lost their activity to oxygenate the C-7 allyl methyl carbon of compound 1 to convert 3. The presence of (+)-4-epi-α-bisabolol-accumulating R. rugosa hybrids is significant when considering the sesquiterpene biogenesis of Rosa rugosa.
Key words: Martin Frobisher; Vanguard; Rosa rugosa hybrid; glandular trichome; (+)-4-epi-α-bisabolol

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Note
Galacturonic-acid-induced Increase of Superoxide Production in Red Tide Phytoplankton Chattonella marina and Heterosigma akashiwo

Daekyung KIM,1 Tatsuya ODA,1,・ Atsushi ISHIMATSU,2 and Tsuyoshi MURAMATSU1

1Division of Biochemistry, Faculty of Fisheries, Nagasaki University, Bunkyo-machi, Nagasaki 852-8521,
Japan
2Marine Research Institute, Nagasaki University, Taira-cho, Nagasaki 851-2213, Japan

Received October 29; Accepted December 14, 1999
Red tide phytoplankton, Chattonella marina and Heterosigma akashiwo, are known to generate superoxide anion (O--2). We found that galacturonic acid (GalUA) stimulated C. marina and H. akashiwo to generate increased amounts of O--2. Since such effect was not observed in any other monosaccharides tested, our results suggest that the binding of GalUA to specific sites on the flagellate cell surface may induce the increase of O--2 production.
Key words: red tide plankton; Chattonella marina; Heterosigma akashiwo; galacturonic acid; reactive oxygen species

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Note
Structural Analysis of the ntrBC Genes of Deep-sea Piezophilic
Shewanella violacea

Akihiko IKEGAMI,＊,＊＊ Kaoru NAKASONE,＊＊,・ Chiaki KATO,＊＊ Ron USAMI,＊
and Koki HORIKOSHI＊,＊＊

＊Department of Applied Chemistry, Faculty of Engineering, Toyo University, 2100 Kujirai, Kawagoe,
Saitama 350-0852, Japan
＊＊The DEEP STAR Group, Japan Marine Science and Technology Center, 2-15 Natsushima-cho,
Yokosuka, Kanagawa 237-0061, Japan

Received November 8, 1999; Accepted December 16, 1999
The ntrBC genes coding for the bacterial signal-transducing protein NtrB and the bacterial enhancer-binding protein NtrC of deep-sea piezophilic Shewanella violacea were cloned and their nucleotide sequences were analyzed. The conserved regions of NtrB and those of NtrC are well conserved in the case of the ntrBC products of S. violacea.
Key words: Shewanella violacea; two-component system; transcription

-46-
Note
Identification and Characterization of a Potent Antibacterial Agent,
NH125 against Drug-resistant Bacteria

Kaneyoshi YAMAMOTO, Takashi KITAYAMA, Noriyasu ISHIDA, Takafumi WATANABE,
Hiroyuki TANABE, Masahiro TAKATANI, Tadashi OKAMOTO, and Ryutaro UTSUMI・

Department of Agricultural Chemistry, Kinki University 3327-204 Nakamachi Nara 631-8505, Japan

Received November 22, 1999; Accepted December 27, 1999
New imidazole compounds were synthesized to develop a novel and effective antibacterial agent (1-benzyl-3-cetyl-2-methylimidazolium iodide, NH125). In vitro experiments demonstrated that NH125 effectively inhibited a number of different histidine protein kinases. Furthermore, oxacillin-resistant Staphylococcus aureus (ORSA), vancomycin-resistant Enterococcus faecalis (VRE), penicillin-resistant Streptococcus pneumoniae (PRS), and other Gram-positive and Gram-negative bacteria were found to be very sensitive to NH125.
Key words: drug-resistant pathogens; histidine protein kinase; two-component system; bacterial signal transduction