Contents and Abstracts of Latest Issue of BBB

(Vol.63 No.9 1999)

Jun TSUNEHIRO,・ Katsuyuki OKAMOTO,＊ Yasuo FURUYAMA, Tsuneya YATAKE,＊
and Toshiyuki KANEKO＊

Research and Development Center, Kashima Laboratory, Showa Sangyo Co. Ltd., 6 Higashi Fukashiba,
Kamisu, Kashima-gun, Ibaraki 314-0194, Japan
＊Research and Development Center, Showa Sangyo Co. Ltd., 2-20-2 Hinode, Funabashi, Chiba 273-0015, Japan

Received December 28, 1998; Accepted May 25, 1999
The digestibility of the hydrogenated derivative of an isomaltooligosaccharide mixture (IMO-H) was investigated. In an in vitro experiment, the digestibility of IMO-H was examined by models of the digestive system. IMO-H was resistant to two types of α-amylase and to artificial gastric juice. Enzymes in the rat small intestinal mucosa hydrolyzed tri-, tetra- and higher saccharide alcohols to disaccharide alcohol, removing successive glucose units from the non-reducing ends of the chains. The hydrolysis ratio for IMO-H was intermediate between the values for maltose and maltitol. In an in vivo study, growing rats were fed on an experimental diet containing IMO-H, maltitol, or hydrogenated palatinose in the range from 5％ to 20％. The growth parameters of the rats fed on the test sugar show that the availability of IMO-H was about 1.2 to 1.25 times that of maltitol or hydrogenated palatinose.
digestibility; hydrogenated isomaltooligosaccharide; mucosal enzyme; availability; sugar alcohol

-2-
Responses of Mentha Suspension-Cultured Cells to 2,4-Dichlorophenoxyacetic
Acid and Accumulation of Esterified Phenolic Acids in Their Cell Walls

Jian-Gang YANG, Syunsuke MIYAO,＊ and Takeo UCHIYAMA＊,・

Graduate School of Science and Technology, Niigata University, Ikarashi, Niigata 950-2101, Japan
＊Faculty of Agriculture, Niigata University, Ikarashi, Niigata 950-2181, Japan

Received February 12, 1999; Accepted June 1, 1999
Two distinct types of cell growth of suspension-cultured Mentha were formed when the cells maintained in the medium containing 1000 μgl−1 2,4-D were subcultured into different 2,4-D concentrations. Few cell elongation of Mentha (average cell length: 34~40 μm) was observed after division in the medium containing 1~200 μgl−1 2,4-D; and significant cell elongation (average cell length: 95~130 μm) was observed after cell division in the medium containing 500~2000 μgl−1 2,4-D. A close correlation between culture medium and water content in the cells indicated that 2,4-D promoted cell elongation by water uptake. Amounts of phenolic acid in cell walls were much higher in unelongated cell walls than in elongated ones during the cultivation, and there was a close correlation between the amounts and the level of PAL activity in elongated and unelongated cells. However, there was no significant difference in cell wall components and its neutral sugar composition between elongated and unelongated cells.
Mentha suspension culture; 2,4-dichlorophenoxyacetic acid; cell elongation; plant cell wall; phenolic acids

-3-
Characterization of Bacillus subtilis ExoA Protein: a Multifunctional DNA-repair
Enzyme Similar to the Escherichia coli Exonuclease III

Toshio SHIDA,＋,・ Tomoyoshi OGAWA,＋ Naotake OGASAWARA,＃ and Junichi SEKIGUCHI＋

＋Department of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, 3-15-1 Tokida, Ueda, Nagano 386-8567, Japan
＃Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5, Takayama-cho, Ikoma, Nara 630-0101, Japan

Received February 19, 1999; Accepted April 19, 1999
To discover the physiological role of the Bacillus subtilis ExoA protein, which is similar in amino acid sequence to Escherichia coli exonuclease III, an exoA::Cm disruption was constructed in the chromosomal DNA of B. subtilis. There was no clear difference in tolerance to hydrogen peroxide and alkylating agents between the disruptant and the wild type strain. An expression plasmid of the ExoA in E. coli was constructed by inserting the exoA gene into the expression vector pKP1500. The purified ExoA was used to clarify enzymatic characterizations using synthetic DNA oligomers as substrates. A DNA oligomer containing a 1′, 2′-dideoxyribose residue as an AP site, a DNA-RNA chimera oligomer, and a 3′ end 32P-labeled oligomer were synthesized. It has been shown that the ExoA has AP endonuclease, 3′--5′ exonuclease, ribonuclease H, and 3′-phosphomonoesterase activities. Thus, it has been confirmed that ExoA is a multifunctional DNA-repair enzyme in B. subtilis that is very similar to E. coli exonuclease III except that ExoA has lower 3′--5′ exonuclease activity than that of E. coli exonuclease III.
Bacillus subtilis; ExoA; AP endonuclease; synthetic DNA; multifunctional DNA-repair enzyme

-4-
Thermostabilization by Proline Substitution in an Alkaline,
Liquefying α-Amylase from Bacillus sp. Strain KSM-1378

Kazuaki IGARASHI,・ Tadahiro OZAWA, Kaori IKAWA-KITAYAMA, Yasuhiro HAYASHI,
Hiroyuki ARAKI, Keiji ENDO, Hiroshi HAGIHARA, Katsuya OZAKI,
Shuji KAWAI, and Susumu ITO

Tochigi Research Laboratories of Kao Corporation, 2606 Akabane, Ichikai, Haga, Tochigi 321-3497, Japan

Received February 24, 1999; Accepted May 24, 1999
α-Amylase (LAMY) from alkaliphilic Bacillus sp. strain KSM-1378 is a novel semi-alkaline enzyme which has 5-fold higher specific activity than that of a Bacillus licheniformis enzyme. The Arg124 in LAMY was replaced with proline by site-directed mutagenesis to increase thermostability of the enzyme. The wild-type and engineered LAMYs were very similar with respect to specific activity, kinetic values, pH-activity curve, and degree of inhibition by chelating reagents. Thermostability and structure stiffness of LAMYs as measured by fluorescence were increased by the proline substitution. The change of Arg124 to proline is assumed to stabilize the loop region involving aminio acid residues from 122 to 134. This is the first report that thermostability of an α-amylase is improved by proline substitution.
alkaliphile; Bacillus; α-amylase; thermostability; site-directed mutagenesis

-5-
Involvement of Cytochrome a in Iron Oxidation of a Moderately Thermophilic
Iron-Oxidizing Bacterium, Strain TI-1

Masaki TAKAI, Kazuo KAMIMURA, and Tsuyoshi SUGIO・

Department of Biological Function and Genetic Resources Science, Faculty of Agriculture, Okayama University,
1-1-1 Tsushima Naka, Okayama 700-8530, Japan

Received March 4, 1999; Accepted May 17, 1999
The iron-oxidizing activity of a moderately thermophilic iron-oxidizing bacterium, strain TI-1, was located in the plasma membrane. When the strain was grown in Fe2＋ (60 mM)-salts medium containing yeast extract (0.03％), the plasma membrane had iron-oxidizing activity of 0.129 μmol O2 uptake/mg/min. Iron oxidase was solubilized from the plasma membrane with 1.0％ n-octyl-β-D-glucopyranoside (OGL) containing 25％ (v/v) glycerol (pH 3.0) and purified 37-fold by a SP Sepharose FF column chromatography. Iron oxidase solubilized from the plasma membrane was stable at pH 3.0, but quite unstable in the buffer with the pH above 6.0 or below 1.0. The optimum pH and temperature for iron oxidation were 3.0 and 55°C, respectively. Solubilized enzyme from the membrane showed absorption peaks characteristic of cytochromes a and b. Cyanide and azide, inhibitors of cytochrome c oxidase, completely inhibited iron-oxidizing activity at 100 μM, but antimycin A, 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO) and myxothiazol, inhibitors of electron transport systems involved with cytochrome b, did not inhibit enzyme activity at 10 μM. The absorption spectrum of the most active enzyme fraction from SP Sepharose FF column chromatography (4.76 μmol O2 uptake/mg/min) compared with lower active fractions from the chromatography (0.009 and 2.10 μmol O2 uptake/mg/min) showed a large α-peak of cytochrome a at 602 nm and a smaller α-peak of cytochrome b at 560 nm. The absorption spectrum of pyridine ferrohemochrome prepared from the most highly purified enzyme showed an α-peak characteristic of heme a at 587 nm, but not the α-peak characteristic of heme c at 550 nm. The cytochrome a, but not cytochrome b, in the most highly purified enzyme fraction was reduced by the addition of ferrous iron at pH 3.0, indicating that electrons from Fe2＋ were transported to cytochrome a, but not cytochrome b. These results strongly suggest that cytochrome a, but not cytochromes b and c, is involved in iron oxidation of strain TI-1.
iron oxidase; moderately thermophile; iron-oxidizing bacterium; cytochrome a

-6-
Steady-State Inhibitory Kinetic Studies on the Ligand Binding Modes
of Aspergillus niger Glucoamylase

Akiyoshi TANAKA,＊,・ Mayumi OHYA,＊＊ Takehito YAMAMOTO,＊＊ Chika NAKAGAWA,＊＊
Takashi TSUJI,＊ Keishi SENOO,＊ and Hitoshi OBATA＊

＊Faculty of Bioresources, ＊＊Faculty of Education, Mie University, Tsu, Mie 514-8507, Japan

Received March 4, 1999; Accepted May 14, 1999
Inhibitory activities of 1-deoxynojirimycin and gluconolactone on Aspergillus niger glucoamylase were studied in relation to the subsite structure of the enzyme. Although both of these inhibitors are considered to bind at subsite 1 of the enzyme active site, 1-deoxynojirimycin showed competitive type inhibition but gluconolactone was a mixed type (or noncompetitive type) inhibitor for the hydrolysis of p-nitrophenyl α-D-glucoside. The former type of inhibition suggested that the main binding mode of the substrate was productive, but the latter, nonproductive. A possible way of explaining these apparent inconsistent results is to assume that the main binding mode of the substrate is productive and gluconolactone forms a nonproductive ternary complex with the enzyme and the substrate.
glucoamylase; ligand binding; mixed-type inhibition; steady-state kinetics; subsite structure

-7-
Role of Olfaction in Food Preference as Evaluated in an Animal Model

Cheng-Wen LIEN, Tatsuhiro HISATSUNE,・ and Shuichi KAMINOGAWA

Department of Applied Biological Chemistry, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan

Received March 15, 1999; Accepted May 15, 1999
Food preference in individual animals is regulated by brain activity. Two murine model systems for investigating food preference were developed by focusing on fruit juices. In a home-cage, two-bottle test, the volume of apple juice consumed was found to be much larger than that of orange juice. In a two-nozzle ``Drinkometer′′ test, by which each mouse was kept in a 38 cm (W)×32 cm (D) cage and each drinking event was recorded by an electronic ``Drinkometer′′ device, it was again found that the mice preferred drinking apple juice to orange juice. To elucidate the role of olfaction in this food preference, mice were subjected to an olfactory bulbectomy to remove the olfaction capability. In the home-cage two-bottle test, the preference for apple juice over orange juice was apparent even after the olfactory bulbectomy, indicating that olfaction was not essential for the formation of food preference behavior. In contrast, in the two-nozzle ``Drinkometer′′ test, the preference for apple juice over orange juice was found to be abrogated by this surgery, implying the involvement of olfaction-based memory on food preference behavior.
food preference; fruit juice; animal behavior; olfaction; olfactory bulbectomy

-8-
Inhibition of Candida rugosa Lipase by Berberine and Structurally Related
Alkaloids, Evaluated by High-Performance Liquid Chromatography

Eleonora GRIPPA, Roberto VALLA, Lucia BATTINELLI, Gabriela MAZZANTI,
Luciano SASO and Bruno SILVESTRINI・

Department of Pharmacology of Natural Substances and General Physiology, University of Rome ``La Sapienza′′, P.le Aldo Moro 5, 00185 Rome, Italy

Received March 19, 1999; Accepted May 17, 1999
It is known that certain microorganisms produce extracellular lipase to better colonize the skin and mucosal surfaces. Since different extracts from medicinal plants have anti-lipase activity (Shimura et al., Biosci. Biotechnol. Biochem., 56: 1478--1479, 1992), we examined the effects of selected natural substances on Candida rugosa lipase. In the presence of the compounds under examination, the enzyme was incubated with β-naphthyl laurate, and β-naphthol, produced by the enzymatic reaction, was extracted with ethyl acetate and analyzed by reversed phase HPLC, using a C-18 column. Thus, the inhibitory activity was calculated by a proper formula based on the variations of the area under the chromatographic peak of β-naphthol. The method was validated by analyzing substances with known anti-lipase activity such as saturated fatty acids (C10--16) and tetracycline. Berberine and a number of structurally related alkaloids such as chelidonine, chelerythrine, and sanguinarine appeared active. This property of berberine and sanguinarine is of interest because they are used in pathological conditions in which microbial lipases could play a pathogenic role.
Candida rugosa lipase; inhibition; berberine; sanguinarine; HPLC

-9-
Selective and Continuous Degradation of Carbazole Contained in Petroleum Oil
by Resting Cells of Sphingomonas sp. CDH-7

Kohtaro KIRIMURA,・ Hiroyuki NAKAGAWA, Kenji TSUJI, Kazuya MATSUDA, Ryuichiro KURANE,＊
and Shoji USAMI

Department of Applied Chemistry, School of Science and Engineering, Waseda University, Ohkubo 3-4-1,
Shinjuku-ku, Tokyo 169-8555, Japan
＊National Institute of Bioscience and Human-Technology (NIBH), Higashi 1-1, Tsukuba-shi, Ibaraki 305-0046, Japan

Received March 23, 1999; Accepted May 18, 1999
Microbial degradation of carbazole (CA), a model of hard-removal heterocyclic nitrogen compounds contained in petroleum oil, was examined using Sphingomonas sp. CDH-7 isolated from a soil sample by screening for CA-assimilating microorganisms. CDH-7 used CA as a sole source of carbon and nitrogen, and metabolized CA to ammonia via anthranilic acid as an intermediate product. When CDH-7 was cultivated in the medium containing CA at the concentration of 500 mg/l (2.99 mM), CA was completely degraded within 50 h. By the reaction with the resting cells of CDH-7, 500 mg/l of CA was completely degraded within 4 h, with 1.64 mM of ammonia accumulated in the reaction mixture. When CA was added at the concentration of 100 mg/l (0.599 mM) periodically to the reaction mixture ten times, 925 mg/l (5.54 mM) of CA was degraded within 48 h by the resting cells, and 4.50 mM of ammonia was accumulated in the reaction mixture with a 75.1％ molar conversion yield based on total CA added. The resting cells could almost completely degrade CA in a two-liquid-phase system which consists of water and organic solvent, even in the presence of 20％ (v/v) isooctane, n-hexane, cyclohexane, and kerosene as a model petroleum oil. In the presence of an organic solvent system such as 20％ (v/v) p-xylene, toluene, and heptanol, however, CA degradation yields decreased.
biodegradation; carbazole; heterocyclic nitrogen compound; resting cell reaction; Sphingomonas sp.

-10-
Effects of a Probiotic on the Lipid Metabolism of Cocks Fed
on a Cholesterol-enriched Diet

Tsuyoshi ENDO,1,2 Masuo NAKANO,3,・ Satoru SHIMIZU,2 Michihiro FUKUSHIMA,3
and Shunzo MIYOSHI4

1The United Graduate School of Agriculture Sciences, Iwate University, Morioka, Iwate 020-8550, Japan
2Total Science Institute, Zukohsha Co. Ltd., Obihiro, Hokkaido 080-0048, Japan
3Department of Bioresource Science, Obihiro University of Agriculture and Veterinary Medicine, Obihiro,
Hokkaido 080-8555, Japan
4Department of Animal Production and Agricultural Economics, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan

Received April 5, 1999; Accepted May 24, 1999
The effects of a probiotic (a mixture of Bacillus, Lactobacillus, Streptococcus, Clostridium, Saccharomyces and Candida) on the lipid metabolism, and caecal flora and metabolites of cocks were studied. The cholesterol level of the liver and serum was significantly decreased in the cocks fed on the cholesterol-enriched diet containing the probiotic. The distribution and frequency of occurrence of flora, and the chemical characteristics of the metabolites in the caecal content of the cocks were also affected by the inclusion of the probiotic in the basal and cholesterol-enriched diets. The Enterobacteriaceae species were significantly decreased in number, while the Bacillus, Streptococcus, Bifidobacterium and Lactobacillus species were significantly increased. The presence of yeast was observed, and the ammonia level was significantly reduced. The pH value, however, was not affected. The concentration of short-chain fatty acids in the caecal content of the cocks fed on the cholesterol-enriched diet supplemented with the probiotic was increased. It is, therefore, suggested that the incorporation of a probiotic in the diet would improve the balance of the intestinal flora and metabolites. Furthermore, it would also suppress the serum and liver cholesterol levels of cocks fed on the cholesterol-enriched diet.
probiotic; cholesterol; caecal flora; cock

-11-
Expression of a Recombinant Molt-Inhibiting Hormone of the Kuruma Prawn
Penaeus japonicus in Escherichia coli

Tsuyoshi OHIRA,1 Takayuki NISHIMURA,2 Haruyuki SONOBE,2 Atsuro OKUNO,3
Toshiki WATANABE,4 Hiromichi NAGASAWA,3,・ Ichiro KAWAZOE,1 and Katsumi AIDA1

1Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi, Bunkyo, Tokyo 113-8657, Japan
2Department of Biology, Faculty of Science, Konan University, Higashinada, Kobe 658-0072, Japan
3Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi, Bunkyo, Tokyo 113-8657, Japan
4Ocean Research Institute, The University of Tokyo,1-15-1 Minamidai, Nakano-ku, Tokyo 164-0014, Japan

Received April 5, 1999; accepted May 13,1999
The crustacean molt-inhibiting hormone (MIH) suppresses ecdysteroid synthesis by the Y-organ. The MIH of the kuruma prawn Penaeus japonicus has recently been isolated and its cDNA cloned. In this study, we expressed the MIH in Escherichia coli to obtain a large quantity of this hormone with biological activity. The MIH cDNA was processed and ligated into an expression plasmid. E. coli was transformed with this plasmid, and then the recombinant MIH (r-MIH) was expressed. The r-MIH was put through the refolding reaction and was purified by reverse-phase HPLC. N-terminal amino acid sequence and time-of-flight mass spectral analyses supported the idea that the r-MIH had the entire sequence. By in vitro bioassay using the Y-organ of the crayfish, the r-MIH was found to be comparable to natural MIH in inhibiting ecdysteroid synthesis.
molt-inhibiting hormone; molting; crustacean; Penaeus japonicus; bacterial expression

-12-
Molecular Cloning of Isomaltotrio-dextranase Gene from Brevibacterium fuscum
var. dextranlyticum strain 0407 and Its Expression in Escherichia coli

Takafumi MIZUNO, Haruhide MORI, Hiroyuki ITO, Hirokazu MATSUI,・
Atsuo KIMURA, and Seiya CHIBA

Department of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan

Received April 6, 1999; Accepted May 24, 1999
The gene encoding an extracellular isomaltotrio-dextranase (IMTD), designed dexT, was cloned from the chromosomal DNA of Brevibacterium fuscum var. dextranlyticum strain 0407, and expressed in Escherichia coli. A single open reading frame consisting of 1923 base pairs that encoded a polypeptide composed of a signal peptide of 37 amino acids and a mature protein of 604 amino acids (Mr, 68,300) was found. The primary structure had no significant similarity with the structure of two other reported exo-type dextranases (glucodextranase and isomalto-dextranase), but had high similarity with that of an endo-dextranase isolated from Arthrobacter sp. Transformed E. coli cells carrying the gene encoding mature protein of IMTD overproduced IMTD under the control of the T7 phage promoter induced by IPTG. The purified recombinant enzyme showed the same optimum pH, lower specific activity, and similar hydrolytic pattern, as to those of native IMTD.
isomaltotrio-dextranase; Brevibacterium fuscum var. dextranlyticum; gene structure; over-expression

-13-
Crystallization and Properties of NAD-Dependent D-Sorbitol Dehydrogenase
from Gluconobacter suboxydans IFO 3257＃

Osao ADACHI,・ Hirohide TOYAMA, Gunjana THEERAGOOL,＊ Napha LOTONG,＊
and Kazunobu MATSUSHITA

Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan
＊Department of Microbiology, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand

Received April 6, 1999; Accepted April 30, 1999
NAD-dependent D-sorbitol dehydrogenase (EC 1.1.1.14) was crystallized from the cytosolic fraction of Gluconobacter suboxydans IFO 3257. This is the first example of the enzyme crystallized from acetic acid bacteria. The enzyme catalyzed oxidoreduction between D-sorbitol and D-fructose in the presence of NAD or NADH. The crystalline enzyme showed a single sedimentation peak in analytical ultracentrifugation, giving an apparent sedimentation constant of 5.1s. Gel filtration on a Sephadex G-200 column gave the molecular mass of 98 kDa for the enzyme, which dissociated into 26-kDa subunits on SDS-PAGE, indicating that the enzyme is composed of four identical subunits. Oxidation of D-sorbitol to D-fructose and xylitol to D-xylulose predominated in the presence of NAD at the optimum pH of 9.5--10.0. Reductions of D-fructose to D-sorbitol and D-xylulose to xylitol were also observed in the presence of NADH with the optimum pH around 6.0. The relative rate of D-fructose reduction was about one-fourth of that of D-sorbitol oxidation. NADP and NADPH were inert for the both reactions. Since the reation rate in D-sorbitol oxidation predominated over D-fructose reduction at some alkaline pH, the enzyme could be available for direct enzymatic measurement of D-sorbitol. Even in the presence of a large excess of D-glucose and other substances, reduction of NAD to NADH was highly specific and stoichiometric to the D-sorbitol oxidized.
acetic acid bacteria; NAD-dependent D-sorbitol dehydrogenase; fructose reductase; Gluconobacter suboxydans; sorbitol dehydrogenase

-14-
Cloning, Sequencing, and Expression of the Gene Encoding
the Clostridium stercorarium Xylanase C in Escherichia coli

Mursheda K. ALI, Masayuki FUKUMURA, Katsushi SAKANO, Shuichi KARITA,＊
Tetsuya KIMURA, Kazuo SAKKA,・ and Kunio OHMIYA

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

Received April 8, 1999; Accepted June 7, 1999
The nucleotide sequence of the Clostridium stercorarium F-9 xynC gene, encoding a xylanase XynC, consists of 3,093 bp and encodes a 1,031-amino acids with a molecular weight of 115,322. XynC is a multidomain enzyme composed of an N-terminal signal peptide and six domains in the following order: two thermostabilizing domains, a family 10 xylanase domain, a family IX cellulose-binding domain, and two S-layer homologous domains. Immunological analysis indicated the presence of XynC in the culture supernatant of C. stercorarium F-9 and in the cells, most likely on the cell surface. XynC purified from a recombinant E. coli was highly active toward xylan and slightly active toward p-nitrophenyl-β-D-xylopyranoside, p-nitrophenyl-β-D-cellobioside, p-nitrophenyl-β-D-glucopyranoside, and carboxymethylcellulose. XynC hydrolyzed xylan and xylooligosaccharides larger than xylotriose to produce xylose and xylobiose. This enzyme was optimally active at 85°C and was stable up to 75°C at pH 5.0 and over the pH range of 4 to 7 at 25°C.
xylanase; Clostridium stercorarium; cellulose-binding dom

-15-
Stereoselective Synthesis of Optically Active Methylene Lactone, Key Intermediate
for the Synthesis of 1,2-Oxidized Furofuran 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 April 22, 1999; Accepted May 17, 1999
||(4R) - [(1S) - 1 - [(tert - Butyldimethylsilyl)oxy] - 1 - (2 - meth||||oxy - 4,5 - methylenedioxyphenyl)methyl] - 3 - methylenedihydro-||||2(3H)-furanone and (4R)-[(1R)-1-[(tert-butyldimethylsilyl)||||oxy] - 1 - (2 - methoxy - 4,5 - methylenedioxyphenyl)methyl]-||3-methylenedihydro-2(3H)-furanone, which are key intermediates for the synthesis of 1,2-oxidized furofuran lignan, were each stereoselectively synthesized from L-glutamic acid.
lignan; furofuran lignan; stereoselective synthesis

-16-
Occurrence and Characterization of 2-Hydroxy-1,4-benzoxazin-3-one and Indole
Hydroxylases in Juvenile Wheat

Junya TANABE, Masayuki SUE, Atsushi ISHIHARA, and Hajime IWAMURA・

Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
CREST, Japan Science and Technology Corporation (JST), Japan

Received April 28, 1999; Accepted June 7, 1999
Cyclic hydroxamic acids, 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) and its 7-methoxy analogue (DIMBOA), occur transiently in high amounts in wheat and maize during the juvenile, non-autotrophic stage of growth. To elucidate the biosynthetic enzymes operating for the transient production of these compounds, we examined the hydroxylating activities for 2-hydroxy-1,4-benzoxazin-3-one (HBOA), the immediate precursor of DIBOA, and indole, the first intermediate of the biosynthetic pathway that branches off from the tryptophan pathway, by using microsomes prepared from wheat seedlings. Both hydroxylases occurred soon after germination, reached a maximum 48 h after germination, and decreased to finally disappear as the plants grew into the autotrophic growth stage. The mode of appearance and disappearance similar to that of hydroxamic acids, suggesting that elevated expression of the whole set of enzymes involved in the biosynthesis after indole was responsible for the transient occurrence of hydroxamic acids. The hydroxylating activity was also observed for 1,4-benzoxazin-3-one, a putative precursor of HBOA, but to significantly less extent than that for HBOA and indole.
cyclic hydroxamic acid; biosynthesis; Triticum aestivum; hydroxylase; microsomes

-17-
Note
Induction of Isoflavonoid and Retrochalcone Branches of the Flavonoid Pathway
in Cultured Glycyrrhiza echinata Cells Treated with Yeast Extract

Kazuhiko NAKAMURA, Tomoyoshi AKASHI, Toshio AOKI, Kiichiro KAWAGUCHI,＊
and Shin-ichi AYABE・

Department of Applied Biological Sciences, Nihon University, Fujisawa, Kanagawa 252-8510, Japan
＊Medicinal Plant Garden, School of Pharmaceutical Sciences, Kitasato University, Sagamihara,
Kanagawa 228-8555, Japan

Received February 8, 1999; Accepted May 26, 1999
Yeast extract-treated suspension cultures of a new cell line, AK-1, of Glycyrrhiza echinata were induced to produce an isoflavonoid phytoalexin (medicarpin) and metabolites of retrochalcone/flavone pathway (echinatin, licodione, and 7,4′-dihydroxyflavone). From these cells, putative full-length cDNAs encoding cytochrome P450s,||(2S)-flavanone 2-hydroxylase and isoflavone 2′-hydrox||ylase, were cloned.
Glycyrrhiza echinata; Fabaceae; flavonoids; elicitor; cytochrome P450

-18-
Note
Radical Scavenging Activity of Tea Catechins and Their Related Compounds

Fumio NANJO,・ Masao MORI, Keiichi GOTO, and Yukihiko HARA

Food Research Institute, Mitsui Norin Co. Ltd., 223-1 Miyabara, Fujieda-shi, Shizuoka 426-0133, Japan

Received February 12; 1999 Accepted May 14; 1999
(−)-Epigallocatechin gallate was found to be the most effective scavenger among tea catechins for the superoxide anion, hydroxyl radical, and 1,1-diphenyl-2-picrylhydrazyl radical. Examination of the scavenging effects of tea catechins and their glucosides on superoxide anion showed that the presence of at least an ortho-dihydroxyl group in the B ring and a galloyl moiety at the 3 position was important in maintaining the effectiveness of the radical scavenging ability. Stoichiometric factors of tea catechins were estimated to be 2 for (＋)-catechin and (−)-epicatechin, 5 for (−)-epigallocatechin, 7 for (−)-epicatechin gallate, and 10 for (−)-epigallocatechin gallate.
tea; catechin; radical scavenging; superoxide anion; stoichiometric factor

-19-
Note
Efficient Production of Recombinant Human Erythropoietin
by Replenishment of Microcarriers in the Hollow Fiber Culture Cassette

Yuichi INOUE,＊ Nobuhisa ARITA, Kiichiro TERUYA, Yoshinori KATAKURA,
and Sanetaka SHIRAHATA

＊Department of Biochemical Science and Technology, Faculty of Agriculture, Kagoshima University,
1-21-24 Korimoto, Kagoshima 890-0065, Japan
Graduate School of Genetic Resources Technology, Kyushu University, 6-10-1 Hakozaki, Higashi-ku,
Fukuoka 812-0053, Japan

Received February 24, 1999; Accepted May 12, 1999
||Human erythropoietin (EPO)-producing recombinant||BHK cells were cultured in culture medium containing microcarriers, and then microcarriers attached with cells were replenished in the hollow fiber culture cassette. By culture for 14 days, it was possible to produce 450 μg of the recombinant EPO, which corresponded to over two-fold of the recombinant EPO production by control hollow fiber culture without microcarriers.
BHK-21 cells; hollow fiber bioreactor; microcarrier; human erythropoietin; ras oncogene

-20-
Note
Biochemical Characterization of a Putative Cytokinin-Responsive His-kinase,
CKI1, from Arabidopsis thaliana

Ayako NAKAMURA, Tatsuo KAKIMOTO,＃ Aya IMAMURA, Tomomi SUZUKI,
Chiharu UEGUCHI, and Takeshi MIZUNO・

Department of Biological Mechanisms and Functions, Graduate School of Bioagricultural Science,
Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan
＃Department of Biology, Graduate School of Science, Osaka University, Toyanaka, Osaka 560-0043, Japan

Received March 4, 1999; Accepted April 27, 1999
His-Asp phosphorelays are evolutionary-conserved powerful biological tactics for intracellular signal transduction. Such a phosphorelay is generally made up of ``sensor histidine (His)-kinases′′, ``response regulators′′, and ``histidine-containing (HPt) phosphotransmitters′′. Results from recent intensive studies suggested that, in the higher plant Arabidopsis thaliana, His-Asp phosphorelays may be widely used for propagating environmental stimuli, such as phytohormones (e.g., ethylene and cytokinin). In this study, we characterized, in vitro, the putative cytokinin-responsive CKI1 His-kinase, in terms of His-Asp phosphorelays. It was demonstrated for the first time that the receiver domain in this sensor exhibits a strong phosphohistidine phosphatase activity toward some Arabidopsis HPt phosphotransmitters (AHP1 and AHP2), suggesting the functional importance of the receiver domain for a presumed interaction of the sensor His-kinase with other His-Asp phosphorelay components.
Arabidopsis thaliana; His-Asp phosphorelay; signal transduction; sensor His-kinase; receiver

-21-
Note
(Z)-3-Hexenyl and trans-Linalool 3,7-oxide β-Primeverosides Isolated as Aroma
Precursors from Leaves of a Green Tea Cultivar

Mariko NISHIKITANI, Dongmei WANG, Kikue KUBOTA, Akio KOBAYASHI,・ and Fumio SUGAWARA＊

Laboratory of Food Chemistry, Department of Nutrition and Food Science, Ochanomizu University, 2-1-1 Otsuka, Bunkyo-ku, Tokyo 112-8610, Japan
＊Department of Applied Biological Science, Science University of Tokyo, Noda, Chiba 278-0022, Japan

Received March 5, 1999; Accepted April 21, 1999
6-O-β-D-Xylopyranosyl-β-D-glucopyranosides (β-primeverosides) of (Z)-3-hexenol and trans-linalool 3,7-oxide were newly isolated from fresh leaves of a tea cultivar (Camellia sinensis var. sinensis cv. Yabukita). In addition, the already identified β-primeverosides of benzyl alcohol, methyl salicylate, and trans-linalool 3,6-oxide from an oolong tea cultivar were isolated from the Yabukita cultivar. It was confirmed that all aglycones of the linalool oxide glycosides isolated here were of the optically active S-form by chiral GC after enzymatic hydrolysis with glycosidase.
green tea; Camellia sinensis; linalool oxide (trans-pyranoid and franoid); glycosides; (Z)-3-hexenyl β-primeveroside

-22-
Note
Hypertriglyceridemia in Rats Induced by Consumption of a Food-derived
Carcinogen, 2-Amino-1-methyl-phenylimidazo[4,5b]pyridine (PhIP)

Yoshimasa ISIMURA,＊ Hiromitsu WATANABE,＊ Norihisa KATO,＊＊,・ Teruyoshi YANAGITA,＃
and Keiji WAKABAYASHI＃＃

＊Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553, Japan
＊＊Department of Applied Biochemistry, Faculty of Applied Biological Science, Hiroshima University,
Higashi-Hiroshima 739-8528, Japan
＃Department of Applied Biological Sciences, Saga University, Saga 840-8502, Japan
＃＃Biochemistry Division, National Cancer Center Research Institute, Tokyo 104-0045, Japan

Received March 18, 1999; Accepted April 26, 1999
2-Amino-1-methyl-phenylimidazo[4,5b]pyridine (PhIP), the most abundant mutagenic heterocyclic amine produced in cooked meat and fish, is known to be a carcinogen for rats and mice. This study provides the first evidence for hypertriglyceridemia in rats exposed to PhIP, suggesting its potential risk to induce not only carcinogenesis, but also atherosclerosis, and highlighting the potential importance of PhIP for humans.
||carcinogen; 2-amino-1-methyl-phenylimidazo||[4,5b]pyridine (PhIP); hypertriglyceridemia; rats

-23-
Note
New Potent Antioxidative o-Dihydroxyisoflavones in Fermented Japanese
Soybean Products

Hideo ESAKI,・ Shunro KAWAKISHI, Yasujiro MORIMITSU,＊ and Toshihiko OSAWA＊

Department of Food and Nutrition, Sugiyama Jogakuen University, 17-3 Hoshigaoka-motomachi, Chikusa-ku, Nagoya 464-8662, Japan
＊Department of Applied Biological Sciences, Nagoya University, Nagoya 464-8601, Japan

Received March 23, 1999; Accepted May 26, 1999
A potent antioxidative 6-hydroxydaidzein (6-OHD) was newly isolated from soybean koji fermented with Aspergillus oryzae. 6-OHD, in addition to 8-hydroxydaidzein and 8-hydroxygenistein, were found to be present in various fermented soybean products, including their koji. Considering that these o-dihydroxyisoflavones had strong antioxidative activities, they may contribute to protecting from oxidative deterioration during the processing of fermented soybean products.
antioxidant; 6-hydroxydaidzein; 8-hydroxydaidzein; 8-hydoxygenistein; fermented soybean products

-24-
Note
Contribution of Histidine Residues to Oligomerization of θ-Toxin
(Perfringolysin O), a Cholesterol-Binding Cytolysin

Megumi NAKAMURA,・ Naoko SEKINO-SUZUKI,・・ Yukiko SHIMADA, and Yoshiko OHNO-IWASHITA

Protein Biochemistry, Tokyo Metropolitan Institute of Gerontology, Tokyo 173-0015, Japan

Received March 23, 1999; Accepted May 13, 1999
θ-Toxin (perfringolysin O) modified by diethyl pyrocarbonate, a histidine-specific reagent, lost its hemolytic activity. The modified toxin retains the activities of binding to and insertion into cholesterol-containing membranes but lacks the ability to form oligomers. These results suggest that histidine residues of θ-toxin contribute their share to cytolysis, especially the oligomerization process.
θ-toxin; histidine; cytolysis; oligomerization

-25-
Note
Purification and Properties of Chitinase from Arthrobacter sp. NHB-10

Katsuichiro OKAZAKI,1,・ Toshiyuki KAWABATA,1 Masahito NAKANO,1 and Shigeru HAYAKAWA2

Department of Life Sciences1 and Department of Biochemistry and Food Science,2 Faculty of Agriculture, Kagawa University, Miki, Kagawa 761-0795, Japan

Received March 24, 1999; Accepted May 12, 1999
A chitinase was purified from the culture filtrate of nigeran-degrading Arthrobacter sp. NHB-10 by precipitation with ammonium sulfate and column chromatographies on DEAE-Sephadex A-50 and Superose 12. The final preparation was homogenous in polyacrylamide gel electrophoresis. The molecular weight of the purified enzyme was 30,000 and its isoelectric point was 6.8. The optimum pH and temperature for the enzyme activity were 5.0 and 45°C, respectively. The enzyme was stable from pH 3 to 7 and up to 55°C. The enzyme activity was inhibited by Hg2＋ and p-chloromercuribenzoic acid. Two internal amino acid sequences of the enzyme were AGPQLLTGYY and IGGVMT.
chitinase; Arthrobacter; chitin

-26-
Note
Coenzyme Activity of NAD Analogs for 3-Isopropylmalate Dehydrogenase
from Thermus thermophilus HB8

Akira CHIBA, Tadashi EGUCHI,・ Tairo OSHIMA,・ and Katsumi KAKINUMA＊

Department of Chemistry, Tokyo Institute of Technology, O-okayama, Meguro-ku, Tokyo 152-8551, Japan
・Department of Chemistry and Materials Science, Tokyo Institute of Technology
・School of Life Science, Tokyo University of Pharmacy and Life Science, Horinouchi, Hachioji-shi,
Tokyo 192-0392, Japan

Received April 5, 1999; Accepted June 2, 1999
In order to elucidate the enzyme-substrate-cofactor interaction in 3-isopropylmalate dehydrogenase, the coenzyme activity of NAD analogs which have a 3-substituted pyridine ring was examined. Analogs 3--5 showed diminished kcat values compared with those of NAD＋, whereas thiocarboxamide 2 was almost as equally active as NAD＋. This suggests that the NH2 functionality of NAD＋ is more important for the catalysis of IPMDH than a carbonyl group.
Thermus thermophilus HB8; 3-isopropylmalate dehydrogenase; NAD analog; coenzyme activity

-27-
Note
3-Geranyl-4-hydroxy-5-(3′-methyl-2′-butenyl)benzoic Acid
as an Anti-inflammatory Compound from Myrsine seguinii

Mei DONG, Maki NAGAOKA,＊ Shintaro MIYAZAKI,＊ Ryozo IRIYE,＊ 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 April 5, 1999; Accepted May 24, 1999
Bioassay-guided isolation of anti-inflammatory compounds from the methanol extract of Myrsine seguinii yielded an anti-inflammatory compound (1). The structure of compound 1 was elucidated to be 3-geranyl-4-hydroxy-5-(3′-methyl-2′-butenyl)benzoic acid on the basis of its spectroscopic data. Compound 1 strongly suppressed 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation on mouse ears at a dose of 500 μg (inhibitory effect (IE): 65％). The acetate and the methyl ether of 1 showed moderate activity at a 500-μg application, with IE 38％ and 27％, respectively. However, the methyl ester and the dimethyl derivative of 1 did not show activity at the same dose. The related compounds of 1, o-, m- and p-hydroxybenzoic acids also did not exhibit notable activity. These results indicate that the carboxylic acid and lipophilic terpene moieties of 1 were significant structural features for anti-inflammatory activity.
anti-inflammatory; Myrsine seguinii; TPA-induced edema

-28-
Note
DNA Cleavage Activities of (−)-Epigallocatechin, (−)-Epicatechin,
(＋)-Catechin, and (−)-Epigallocatechin Gallate with Various Kind of Metal Ions

Fumiko HAYAKAWA,1 Takahide KIMURA,2 Nobuo HOSHINO,3 and Takashi ANDO2

1Department of Life Style Studies, School of Human Cultures, The University of Shiga Prefecture, Hassaka-cho, Hikone, Shiga 522-8533, Japan
2Department of Chemistry and 3Department of Pharmacy, Shiga University of Medical Science, Seta, Otsu, Shiga 520-2192, Japan

Received April 6, 1999; Accepted June 4, 1999
The DNA cleavage activities of (＋)-catechin (C), (−)-epicatechin (EC), (−)-epigallocatechin (EGC), and (−)-epigallocatechin gallate (EGCg) were examined with 16 different metal ions. Cu2＋ with all the catechins facilitated DNA cleavage, while Ag＋ with EGC and EC showed a strong repressive effect. The other metal ions examined showed little effect.
catechin; metal ion; DNA cleavage; antioxidant; prooxidant

-29-
Note
Inhibition of Plant Transformation by Phytolaccoside B from Phytolacca
americana Callus

Hiroshi KANZAKI,・ Toshihiko KAGEMORI, Yoko YAMACHIKA, Teruhiko NITODA,
and Kazuyoshi KAWAZU

Laboratory of Bioresources Chemistry, Faculty of Agriculture, Okayama University, Okayama 700-8530, Japan

Received April 13, 1999, Accepted May 18, 1999
The newly established GUS expression bioassay on the callus extracts of 22 species of plants revealed that the methanol extract of Phytolacca americana callus had the most potent inhibitory activity against agrobacterial plant transformation. A triterpene glycoside phytolaccoside B was isolated from the extract as a genuine plant transformation inhibitor having neither antiagrobacterial nor phytotoxic activity. This compound is promising for use as a biochemical probe for studies on the plant transformation mechanism.
Agrobacterium tumefaciens; GUS expression assay; Ageratum conyzoides; Phytolacca americana

-30-
Note
A Novel Rearranged Taxoid from Needles of the Japanese Yew, Taxus cuspidata
Sieb. et Zucc.

Ryo MURAKAMI,・ Qing-Wen SHI, Tohru HORIGUCHI, and Takayuki ORITANI＊

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

Received April 14, 1999; Accepted June 7, 1999
Eleven taxoids were isolated from needles of the Japanese yew, Taxus cuspidata Sieb. et Zucc., one of them being a new compound whose structure was established as||5α - cinnamoyl - 9α,10β,13α - triacetoxy - 11(15→1)abeotaxa-||4(20),11-dien-15-ol (7-deactoxytaxuspine J) on the basis of a spectroscopic analysis. Its relative stereochemistry is proposed from the results of a NOESY experiment.
Taxus cuspidata; needle; taxane diterpenoid; Taxaceae; Taxus

-31-
Note
Preparative-scale Enzyme-catalyzed Synthesis of (R)-α-Fluorophenylacetic Acid

Yasuaki FUKUYAMA,＊ Kaori MATOISHI,＊ Masakazu IWASAKI,＊ Eiji TAKIZAWA,＊
Mamoru MIYAZAKI,＊ Hiromichi OHTA,＊ Satoshi HANZAWA,＊＊ Hitoshi KAKIDANI,＊＊
and Takeshi SUGAI＊,・

＊Department of Chemistry, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan
＊＊Tokyo Research Center, Tosoh Corp., 2743-1 Hayakawa, Ayase 252-1123, Japan

Received April 15, 1999; Accepted May 17, 1999
A preparative-scale asymmetric synthesis of (R)-α-fluorophenylacetic acid, a useful chiral derivatizing reagent, is described. Starting from ethyl α-bromophenylacetate, α-fluorophenylmalonic acid dipotassium salt was prepared in three steps (54％ yield), including nucleophilic substitution by the fluoride ion as the keystep. Both the purified form and crude preparation of arylmalonate decarboxylase in E. coli worked well on this substrate, and (R)-α-flurophenylacetic acid (>99％ e.e.) was prepared in a quantitative yield.
(R)-α-fluorophenylacetic acid; arylmalonate decarboxylase; α-fluorophenylmalonic acid; asymmetric decarboxylation

-32-
Note
1,2-Hydride Shift in the Biosynthesis of Pinguisane-type Sesquiterpenes

Hiroyuki TAZAKI,1,・ Takeshi IWASAKI,1 Yukihiro KAWAKAMI,2 and Kensuke NABETA1

1Department of Bioresource Science, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho,
Obihiro 080-8555, Japan
2Basic Research Div. 3, Takasago International Corporation, 4-11, 1-chome, Nishi-Yawata, Hiratsuka city,
Kanagawa 254-0073, Japan

Received April 16, 1999; Accepted May 16, 1999
A 2H-NMR analysis of 6α-hydroxy-3-oxo-pinguis-5(10)-ene-11,6-olide produced by axenic culture of liverwort Aneura pinguis in the presence of [4-2H2]-labeled mevalonate clarified the presence of a 1,2-hydride shift and retention of deuterium at the C-4 position in the biosynthesis of pinguisane-type sesquiterpenes from FPP.
Aneura pinguis; Hepaticae; pinguisone; sesquiterpene; biosynthesis

-33-
Note
Isolation of (−)-14-O-Malonylindolactam-V as a Possible Precursor
of (−)-Indolactam-V and (−)-14-O-Acetylindolactam-V
from Streptomyces blastmyceticum

Kazuhiro IRIE,・ Satoru TOMIMATSU, Yu NAKAGAWA, Hajime OHIGASHI, and Hideo HAYASHI＃

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

Received April 28, 1999; Accepted June 4, 1999
(−)-Indolactam-V (1) has the fundamental structure of potent tumor-promoting teleocidins. A new teleocidin-related metabolite, (−)-14-O-malonylindolactam-V (2), was isolated from the culture broth of Streptomyces blastmyceticum NA34-17 which produced a large quantity of 1 along with a small amount of (−)-14-O-acetylindolactam-V (3). Heat treatment of 2 in methanol readily produced 1 and 3, suggesting that S. blastmyceticum NA34-17 excreted 1 as a malonic acid conjugate to accumulate 1 and 3 in the culture broth during cultivation.
(−)-indolactam-V; malonic acid conjugate; Streptomyces blastmyceticum; teleocidin; tumor promote