Contents and Abstracts of Latest Issue of BBB

(Vol.64 No.3 2000)

Kuniho NAKATA,・,＊ Naoko HARADA,＊＊ Kunihito SUMITOMO,＊＊ and Kazuo YONEDA＊＊

＊Central Research Laboratories, Mercian Corporation, 4-9-1 Johnan, Fujisawa 251-0057, Japan
＊＊Department of Floriculture and Ornamental Horticulture, College of Bioresource Sciences,
Nihon University, 1866 Kameino, Fujisawa 252-0813, Japan

Received June 28, 1999; Accepted November 5, 1999
The antibiotic-producing bacterium, Pseudomonas fluorescens, is assumed to be important in protecting plants from soilborne diseases. S. fluorescens S272, a hyper-producing strain of pyoluteorin (PT) and 2,4-diacetylphloroglucinol (DG), had previously been isolated from soil. The present paper reported that the growth of water-cultivated Kaiware radish was promoted to 120--140％ of its normal level by the coaddition of an S272 culture broth (0.01--1％ v/v) and a polysaccharide flocculant (1--100 ppm) from Klebsiella pneumoniae H12. Tight adhesion of S272 cells to the root tissue was microscopically observed. The growth
promotion is assumed to have been caused by antibiotic
effects for the following two reasons: 1) PT (4 mg/l) and DG (24 mg/l) addition to a radish culture enhanced stem growth to 130％ of the normal level; 2) a culture solution containing the S272 culture broth (0.01--1％ v/v) markedly inhibited the decomposition of hypersensitive chrysanthemum leaves. A soil-cultivation experiment with Gomphrena globosa under natural conditions also exhibited enhanced stem length (160％) by coaddition of the S272 culture broth and H12 polysaccharide. These results suggest that polysaccharide-enhanced adhesion of P. fluorescens S272 cells might be useful for promoting plant growth through the increased antibiotic effect.
symbiosis; flocculant; polysaccharide; Pseudomonas; antibiotic

-2-
Lower Plasma Triglyceride Level in Syrian Hamsters Fed on Skim Milk
Fermented with Lactobacillus casei strain Shirota

Hiroko KIKUCHI-HAYAKAWA,・ Harue SHIBAHARA-SONE, Kuniko OSADA,
Norie ONODERA-MASUOKA, Fumiyasu ISHIKAWA, and Masaaki WATANUKI

Yakult Central Institute for Microbiological Research, Yaho 1796, Kunitachi-shi, Tokyo 186-8650, Japan

Received July 12, 1999; Accepted November 2, 1999
The effect of fermented skim milk (FSM) by Lactobacillus casei strain Shirota on plasma lipids in hamsters was examined. Hamsters fed on cholesterol-free and -enriched diets containing 30％ FSM had lower levels of plasma triglyceride than those fed on the control diet. In the experiment with the cholesterol-enriched diet-fed hamsters, the plasma triglyceride level was suppressed by FSM at concentrations of 10％ to 30％. Unfermented milk tended to lower the level of triglyceride, but not significantly. The plasma cholesterol concentration was not affected by an FSM and unfermented skim milk supplement to the diet. L. casei strain Shirota grew well in the presence of mixed lipid micelles containing bile acid, but did not have the ability to remove cholesterol from the culture broth. These results indicate that FSM lowered the plasma triglyceride level in hamsters.
fermented skim milk; lactic acid bacteria; triglyceride; hamster; hyperlipidemia

-3-
Interconversion between Dehydro-L-Ascorbic Acid and L-Ascorbic Acid

Yoko NISHIKAWA and Tadao KURATA

Institute of Environmental Science for Human Life, Ochanomizu University, 2-1-1 Otsuka, Bunkyo-ku,
Tokyo 112-8610, Japan

Received August 9, 1999; Accepted November 30, 1999
L-Ascorbic acid (AA) plays an important role in biological systems as an electron donor. Erythorbic acid (EA) is the epimer of AA and has chemical characteristics very similar to those of AA. It is demonstrated in the present study by 1H-NMR that dehydro-L-ascorbic acid (DAA) was reduced by EA under neutral conditions but not acidic, and that dehydroerythorbic acid
・(DEA) was also reduced by AA under the same condi-・
・tions. These reactions also occurred at a low concen-・
tration close to the concentration of AA in such
biological tissue as the liver. Furthermore, the interconversion of DAA and AA at neutral pH and low concentration was also confirmed by radioluminography. These results suggest the interconversion between DAA and AA in vivo.
dehydro-L-ascorbic acid; L-ascorbic acid; erythorbic acid; dehydroerythorbic acid; interconversion

-4-
Role of the DNA Sequence Downstream of the Bacillus subtilis hut Promoter in Regulation of the hut Operon

Shima EDA,1,2 Takayuki HOSHINO,1 and Masanao ODA2・

1Institute of Applied Biochemistry, University of Tsukuba, Tsukuba City, Ibaraki 305-8572, Japan
2National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, MITI, Tsukuba 305-8566, Japan

Received August 9, 1999; Accepted November 18, 1999
To identify the role of the downstream region of a hut promoter in regulation of the Bacillus subtilis hut operon, three single-base substitutions (＋9G→A,
＋14C→T, and ＋23T→G) were introduced into the hut operon. Analysis of expression of the hut operon containing each of these three single-base substitutions and the hut-lacZ fusions with the single-base substitutions at position ＋14 showed that the position at ＋14 and probably the position at ＋23 were required for amino acid repression at the hut promoter, while the position at ＋14 was not required for catabolite repression at the hut promoter. The position at ＋9 was required for a histidine-dependent increase of activity of the hut promoter. Analysis of expression of the hut-lacZ fusions and the hut operon in the codY mutant indicated that the position at ＋14 and probably the position at ＋23 were involved in CodY-mediated amino acid repression at the hut promoter and that CodY was not required for catabolite repression at the hut promoter.
catabolite repression; amino acid repression; Bacillus subtilis; hut operon; CodY

-5-
Purification and Characterization of Membrane-bound Hydrogenase
from Hydrogenobacter thermophilus Strain TK-6, an Obligately
Autotrophic, Thermophilic, Hydrogen-oxidizing Bacterium

Masaharu ISHII,1 Seiichi TAKISHITA,1 Toshio IWASAKI,2 Yuwadee PEERAPORNPISAL,3
Jun-ichiro YOSHINO,1 Tohru KODAMA,4 and Yasuo IGARASHI1,・

1Department of Biotechnology, the University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan
2Department of Biochemistry and Molecular Biology, Nippon Medical School, Bunkyo-ku,
Tokyo 113-8602, Japan
3Department of Biology, Chiangmai University, Chiangmai 50200, Thailand
4Faculty of Textile Science and Technology, Shinshu University, Ueda, Nagano 386-0018, Japan

Received August 23, 1999; Accepted November 26, 1999
A membrane-bound hydrogenase was purified to electrophoretic homogeneity from the cells of Hydrogenobacter thermophilus strain TK-6, an obligately autotrophic, thermophilic, hydrogen-oxidizing bacterium. Solubilization and purification were done aerobically in the presence of Triton X-100. Three chromatography steps were done for purification; Butyl-Sepharose, Mono-Q, and Superose 6, in this order. Purification was completed with 6.73％ yield of total activity and with 21.4-fold increase of specific activity when compared with the values for the membrane fraction. The purified hydrogenase was shown to be a tetramer with α2β2 structure, with a molecular mass of 60,000 Da for the large subunit and 38,000 Da for the small subunit. The purified hydrogenase directly reduced methionaquinone with an apparent Km of around 300 μM and with a turnover number around 2900 (min−1). Metal analysis and EPR properties of the hydrogenase have shown that the enzyme is one of the [NiFe]-hydrogenases. Also, optimum pH and temperature for reaction, thermal stability, and electron acceptor specificity were reported. Finally, a model is presented for energy and central metabolism of H. thermophilus strain TK-6.
membrane-bound hydrogenase; quinone reduction; EPR; hydrogen bacterium; Hydrogenobacter

-6-
Inhibition of Radiation-Induced Lipid Peroxidation by Tetrahydrocurcumin: Possible Mechanisms by Pulse Radiolysis

・Sujata M. KHOPDE,＊ K. Indira PRIYADARSINI,＊ Shambhu Nath GUHA,＊ Jagganath G. SATAV,＃・

Parameswaran VENKATESAN,$ and Mysore N. Aswathanarana RAO$
＊Radiation Chemistry and Chemical Dynamics Division, ＃Radiation Biology Division, Bhabha Atomic Research Centre, Trombay, Mumbai-400085, India
$College of Pharmaceutical Sciences, Kasturba Medical College, Manipal-576119, India

Received August 26, 1999; Accepted November 25, 1999
The antioxidant property of tetrahydrocurcumin (THC), a reduced derivative of curcumin, was examined by its ability to inhibit radiation-induced lipid peroxidation in rat liver microsomes and compared with curcumin. The lipid peroxidation caused by irradiation of N2O-purged and aerated buffered aqueous solutions was found to be inhibited by THC in a dose- and concentration-dependent manner. In order to understand the actual reaction mechanisms involved in the inhibition process, pulse radiolysis investigation of THC with radiolytically produced radicals like hydroxyl, model peroxyl radicals, and azide radicals were done and the transients were detected by kinetic spectrophotometry. The reaction of THC with hydroxyl and azide radicals gave rise to transient absorption in the region 200--400 nm with two peaks at 310 nm and 390 nm. From the spectral properties and kinetics of these radicals, a suitable mechanism is discussed to explain the antioxidant actions of THC.
curcumin; tetrahydrocurcumin (THC); γ-radiolysis; lipid peroxidation; pulse radiolysis

-7-
A Simple and Rapid Method for the Detection of Poly(ADP-ribose)
by Flow Cytometry

Shin OGATA, Katsuzumi OKUMURA, and Hiroshi TAGUCHI・

Laboratory of Biological Chemistry, Faculty of Bioresources, Mie University, Tsu, Mie 514-8507, Japan

Received September 6, 1999; Accepted November 16, 1999
Measurement of poly(ADP-ribose) levels was performed by a new method using a monoclonal antibody against poly(ADP-ribose) and flow cytometry from small amount of cultured cells without the need for isolation of poly(ADP-ribose) polymer. The increase of poly(ADP-ribose)-associated fluorescence intensity was observed in individual human leukemic HL-60 cells when treated with the carcinogen, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), and was blocked by the treatment with 3-aminobenzamide before MNNG treatment. It is easy and rapid to detect the time-dependent change of poly(ADP-ribose) levels in HL-60 cells after MNNG treatment. We easily found that the increase of the poly(ADP-ribose) level in nicotinic acid-treated lymphocytes after MNNG treatment was observed, but not in nicotinamide-treated lymphocytes. We investigated the change of poly(ADP-ribose) levels especially in the early phase of apoptosis. Our method is simple and rapid. It is suggested that the investigation of poly(ADP-ribosyl)ation in various fields is possible by using this new method.
poly(ADP-ribose); niacin; flow cytometry; DNA repair; N-methyl-N′-nitro-N-nitrosoguanidine

-8-
Detection of the Cholera Toxin-binding Activity of κ-Casein Macropeptide and Optimization of Its Production by the Response Surface Methodology

・Sejong OH,1 Randy W. WOROBO,1 Byoung-chul KIM,2 Sungsue RHEEM,3 and Saehun KIM2,＊・・

1Department of Food Science and Technology, New York State Agricultural Experiment Station, Cornell University, Geneva, NY 14456, U.S.A.
2Department of Animal Science and 3Department of Informational Statistics, Korea University,
5-1 Anam-dong, Sungbuk-gu, Seoul 136-701, Korea

Received September 13, 1999; Accepted October 29, 1999
The cholera toxin (CT)-binding activity of purified
κ-casein macropeptide (CMP) from bovine κ-casein was detected. In addition, a statistical model was developed to optimize the production of CMP. CMP was prepared by chymosin hydrolysis of κ-casein and a subsequent 3％ trichloroacetic acid treatment. CMP was further fractionated in an ion-exchange column by FPLC. CT binding activity was eluted at 0.18 M NaCl and was a single 8.9 kDa peptide without tyrosine and arginine residues. The CT binding activity was rapidly lost by a carbohydrase treatment. The conditions for CMP production with chymosin were optimized by using the response surface methodology (RSM). The estimated optimum levels of the factors were as follows: reaction temperature, 38.5°C; pH, 6.44; and time, 35.9 min. A validation experiment was performed in which CMP was prepared under the predicted parameters, and it was ascertained that the estimated optimum conditions gave better production of CMP than any other conditions.
κ-casein macropeptide; cholera toxin; response surface methodology

-9-
Cloning, Sequencing, and Expression of UDP-Glucose Pyrophosphorylase
Gene from Acetobacter xylinum BRC5

Hyun Min KOO, Seok-Won YIM, Chang-Seung LEE, Yu Ryang PYUN, and Yu Sam KIM＊

Department of Biochemistry, College of Science, Bioproducts Research Center, Yonsei University,
Seoul 120-749, Korea

Received September 17, 1999; Accepted November 9, 1999
A UDP-glucose pyrophosphorylase (UGPase) gene from Acetobacter xylinum BRC5 has been cloned, sequenced, and expressed in Escherichia coli. The gene consists of 867 nucleotides and encodes a polypeptide of 289 amino acid residues with a calculated molecular mass of 31,493 Da. The amino acid sequences of the enzyme showed an 85.8％ identity to those of an enzyme from A. xilinum ATCC 23768. A polyhistidine-UGPase fusion enzyme was expressed and purified from the transformed E. coli. The enzyme showed a 35,620-Da single protein band on SDS/PAGE and an about 160,000-Da protein band on 8-16％ pore-gradient polyacrylamide gel, indicating the enzyme may be a tetramer or pentamer composed of four or five identical subunits. Kinetic analysis of the enzyme showed a typical Michaelis-Menten substrate saturation pattern, from which Km and Vmax were calculated to be 3.22 mM and 175.4 μmol・min−1・mg−1 for UDP-glucose and 0.24 mM and 69.4 μmol・min−1・mg−1 for PPi, respectively, required Mg2＋ for maximal activity, and was inhibited by free pyrophosphate. Computer-aided comparison of the Acetobacter enzyme sequence with those of other bacterial enzymes found significant similarities among them and predicted that Lys84 is a catalytically important residue. Lys84 in the enzyme, which was also conserved in other bacterial enzyme sequences, was replaced by arginine or leucine. The K84R mutant enzyme was successfully expressed in E. coli and showed enzyme activity (63％ of the wild-type enzyme activity), but K84L was not isolated in stable form. These results suggest that Lys84 is significant in not only catalysis but also maintenance of the active structure.
Acetobacter xylinum BRC5; UDP-glucose pyrophosphorylase; Lys84

-10-
Substrate Diversity of Macrophomate Synthase Catalyzing an Unusual
Multistep Transformation from 2-Pyrones to Benzoates

Kenji WATANABE, Takashi MIE, Akitami ICHIHARA, Hideaki OIKAWA,・ and Mamoru HONMA

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

Received September 22, 1999; Accepted November 10, 1999
Macrophomate synthase, which we have recently purified, catalyzes an unusual multistep transformation from 5-acetyl-4-methoxy-6-methyl-2-pyrone to 4-acetyl-3-methoxy-5-methyl-benzoic acid (macrophomic acid). To investigate the substrate diversity of the enzyme, 40 analogs of 2-pyrone were prepared and their relative efficiency was examined in the enzymatic conversions. The experimental results reveal the structural requirements of the substrates and the rough size of the enzyme active site, and eliminate the ambiguity caused by contamination by other enzymes in the whole-cell experiments.
macrophomate synthase; Macrophoma
commelinae; 2-pyrone; oxalacetate

-11-
Biosynthesis of Violacein: Intact Incorporation of the Tryptophan Molecule
on the Oxindole Side, with Intramolecular Rearrangement of the Indole Ring
on the 5-Hydroxyindole Side＊

A. Z. M. Ruhul MOMEN and Tsutomu HOSHINO・

Department of Applied Biological Chemistry, Faculty of Agriculture, and Graduate School of Science and Technology, Niigata University, Ikarashi, Niigata 950-2181, Japan

Received September 27, 1999; Accepted November 10, 1999
Feeding experiments with a mixture of [2-13C]- and [indole-3-13C]tryptophans, of [3-13C]- and [indole-3-13C]tryptophans (1:1 molar ratio) and of others have proved that the 1,2-shift of the indole ring occurred via an intramolecular process for formation of the left part (5-hydroxyindole side) of the violacein skeleton and demonstrated that the C--C bond from C2 of the indole ring to C2 of the side chain was completely retained for formation of the right part (oxindole side) during the entire biosynthetic process. Due to the involvement of transaminase, it has remained unresolved whether indolylpyruvic acid is the biosynthetic intermediate and/or from where the nitrogen atom of the pyrrolidone ring originates. An incorporation experiment with a mixture of [2-13C]- and [α-15N]tryptophans (1:1 molar ratio) verified that the nitrogen atom in the central ring was exclusively derived from the right-side tryptophan. Thus, all the carbon and nitrogen atoms in the right part of the violacein skeleton were constructed by intact incorporation of the tryptophan molecule, with decarboxylation probably occurring at a later biosynthetic stage.
violacein; Chromobacterium violaceum; tryptophan; biosynthesis; stable isotope

-12-
Characteristics of the Biotin Enhancement of Glucose-induced Insulin Release
in Pancreatic Islets of the Rat

Hideyuki SONE, Michiko ITO, Muneshige SHIMIZU, Yuka SASAKI, Michio KOMAI,
and Yuji FURUKAWA・

Laboratory of Nutrition, Department of Applied Biological Chemistry, Faculty of Agriculture,
Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan

Received September 27, 1999; Accepted November 29, 1999
Perifused isolated rat islets were used to show that biotin plus 16.5 mM glucose evoked more insulin secretion than 16.5 mM glucose alone. Whether or not this reinforcement of glucose-induced insulin secretion by biotin is unique was studied by using perifused islets stimulated with 16.5 mM glucose plus 100 μM of one of various components of the vitamin B group. No effect of any of these vitamins was found on glucose-induced insulin secretion. These results indicate that biotin is unique among the members of the vitamin B group in enhancing glucose-induced insulin secretion.
Static incubation experiments showed that biotin did not potentiate insulin release when the islets were incubated with an experimental solution containing either no or 2.8 mM glucose. The addition of biotin to 27.7 mM glucose, which is the maximal concentration for stimulating insulin release, did not significantly enhance the effect of the glucose on insulin release (although it did at 16.5 mM glucose). These findings indicate that biotin, by itself, does not stimulate insulin secretion, and does not enhance glucose-induced insulin secretion beyond the ability of glucose itself to stimulate insulin secretion.
biotin; insulin release; pancreatic islet; vitamin B group

-13-
Increased Hepatic Activity of Inducible Nitric Oxide Synthase in Rats Fed
on a High-Fat Diet

Guanghua WAN, Shoko OHNOMI, and Norihisa KATO・

Department of Applied Biochemistry, Faculty of Applied Biological Science, Hiroshima University,
Higashi-Hiroshima 739-8528, Japan

Received September 27, 1999; Accepted November 17, 1999
The effects were examined of the dietary level of fat on the activity of inducible nitric oxide synthase (iNOS) in the liver of rats. In experiment 1, rats were fed on a diet containing 5％ or 20％ beef tallow or safflower oil for 32 d. The animals were given a subcutaneous injection of the carcinogen, 1,2-dimethylhydrazine (DMH), on d 4. The activity of hepatic iNOS was significantly elevated by the high-fat diet, but was unaffected by the dietary source of the fat examined. In experiment 2, rats were fed on a 5％ or 20％ beef tallow diet for 11 d or 32 d with or without the DMH treatment. Feeding the high-fat diet and DMH treatment caused higher activity of hepatic iNOS. In experiment 3, the high-fat diet elevated hepatic iNOS activity and the amount of its protein in the lipopolysaccharide-treated rats. The results suggest that hepatic NO production is enhanced by a high-fat diet.
nitric oxide; inducible nitric oxide synthase; high-fat diet; 1,2-dimethylhydrazine; tumor

-14-
Changes in N-Linked Oligosaccharides during Seed Development
of Ginkgo biloba＊

Yoshinobu KIMURA・ and Sayuri MATSUO

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

Received September 29, 1999; Accepted November 11, 1999
Structural changes in N-linked oligosaccharides of glycoproteins during seed development of Ginkgo biloba have been explored to discover possible endogenous substrate(s) for the Ginko endo-β-N-acetylglucosaminidase (endo-GB; Kimura, Y., et al. (1998) Biosci. Biotechnol. Biochem., 62, 253--261), which should be involved in the production of high-mannose type free N-glycans.
The structural analysis of the pyridylaminated oligosaccharides with a 2D sugar chain map, by ESI-MS/MS spectroscopy, showed that all N-glycans expressed on glycoproteins through the developmental stage of the Ginkgo seeds have the xylose-containing type (GlcNAc2~0Man3Xyl1Fuc1~0GlcNAc2) but no high-mannose type structure. Man3Xyl1Fuc1GlcNAc2, a typical plant complex type structure especially found in vacuolar glycoproteins, was a dominant structure through the seed development, while the amount of expression of GlcNAc2Man3Xyl1Fuc1GlcNAc2 and GlcNAc1Man3Xyl1Fuc1GlcNAc2 decreased as the seeds developed. The dominantly occurrence of xylose-containing type structures and the absence of the high-mannose type structures on Ginkgo glycoproteins were also shown by lectin-blotting and immunoblotting of SDS-soluble glycoproteins extracted from the developing seeds at various developmental stages.
Concerning the endogenous substrates for plant endo-β-N-acetylglucosaminidase, these results suggested that the endogenous substrates might be the dolicol-oligosaccharide intermediates or some glycopeptides with the high-mannose type N-glycan(s) derived from misfolded glycoproteins in the quality control system for newly synthesized glycoproteins.
plant N-glycan; storage glycoprotein; endo-β-N-acetylglucosaminidase; seed development; Ginkgo biloba

-15-
Molecular Analysis of Maleate cis-trans Isomerase from Thermophilic Bacteria

Kazuhisa HATAKEYAMA, Makoto GOTO, Yasukazu UCHIDA, Miki KOBAYASHI,・
Masato TERASAWA, and Hideaki YUKAWA＊

Biochemicals Laboratory, Mitsubishi Chemical Corporation, 8-3-1 Chuo, Ami, Inashiki, Ibaraki 300-0332, Japan

Received September 30, 1999; Accepted November 17, 1999
Several strains of thermophilic bacteria containing maleate cis-trans isomerase were isolated from soil samples and identified as Bacillus stearothermophilus, Bacillus circulans, Bacillus brevis, and Deleya halophila. The maleate cis-trans isomerase was purified and characterized from one of the isolated strains, B. stearothermophilus MI-102. The purified enzyme of strain MI-102 showed higher thermal stability than the enzyme of a mesophile, Alcaligenes faecalis IFO13111. The seven maleate cis-trans isomerase genes (maiA)
of thermophile were cloned and sequenced. B. stearothemophilus MI-102 MaiA has 67％ amino acid identity with A. faecalis MaiA. All eight amino acid sequences of maiA gene products had significant conserved regions containing cysteine residues, which were previously suggested to be involved in an active site of the enzyme. To probe the catalytic mechanism, three cysteine residues in the conserved regions of A. faecalis MaiA were replaced with serine by site-directed mutagenesis. The results suggest that Cys80 and Cys198 play important roles in the enzyme activity.
maleate cis-trans isomerase; Bacillus stearothermophilus

-16-
Methionine-induced Phytoalexin Production in Rice Leaves

Yumiko NAKAZATO,＊,＊＊ Shigeru TAMOGAMI,＊＊＊ Hiroshi KAWAI,＊＊
Morifumi HASEGAWA,＊,＊＊＊ and Osamu KODAMA＊,＊＊＊,・

＊Laboratory of Phytochemical Ecology, School of Agriculture, Ibaraki University, 3-21-1 Chuo,
Ami, Ibaraki 300-0393, Japan
＊＊VISTA Co., Ltd. 2-5-10 Kohoku-ku, Yokohama, Kanagawa 222-0033, Japan
＊＊＊Core Research of Evolutional Science and Technology of Japan Science and Technology Corporation, School of Agriculture, Ibaraki University, 3-21-1 Chuo, Ami, Ibaraki 300-0393, Japan

Received October 4, 1999; Accepted November 20, 1999
The application of methionine on wounded rice
leaves induced the production of rice phytoalexins,
sakuranetin and momilactone A. This induction resulted from stimulation of phenylalanine ammonia-lyase and naringenin 7-O-methyltransferase activity. Jasmonic acid, ethylene, and active oxygen species are important as signal transducers in disease resistance mechanisms. However, although the endogenous level of jasmonic acid rapidly increased in reaction to
・wound, methionine treatment could not induced en-・
dogenous JA production. Ethylene induced the production of the flavonoid phytoalexin, sakuranetin,
but did not induce the production of a terpenoid
phytoalexin, momilactone A. On the other hand, a free radical scavenger, Tiron, counteracted the induction of both sakuranetin and momilactone A production in methionine-treated leaves. Active oxygen species may be important in methionine-induced production of phytoalexins.
methionine; phytoalexin; rice

-17-
Note
Glycosidic Fraction of Flue-cured Tobacco Leaves: Its Separation
and Component Analysis

Kenji ITO,・ Yoko TANABE, Shuji KATO, Takeshi YAMAMOTO, Akira SAITO,
and Masataka MORI

Tobacco Science Research Laboratory, Japan Tobacco Inc., 6-2 Umegaoka, Aoba-ku, Yokohama, Kanagawa 227-8512, Japan

Received July 19, 1999; Accepted November 24, 1999
The fraction containing glycosidic components was separated from flue-cured tobacco (Nicotiana tabacum L.) leaves by a facile method. Some components of the fraction were isolated and elucidated to be syringin, coniferin, cichoriin, benzyl-β-D-glucoside, Blumenol A-β-D-glucoside, and 5,6-epoxy-5,6-dihydro-3-hydroxy-β-ionyl-β-D-glucoside. Syringin and coniferin were detected in the Nicotiana species for the first time.
fraction; glycoside; flue-cured tobacco leaf; flavor precursor

-18-
Note
Suppressive Effect of Excess Dietary Histidine on the Expression of Hepatic
Metallothionein-1 in Rats

Yoritaka AOYAMA・ and Chie KATO

Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Nishi-9, Kita-9,
Kita-ku, Sapporo 060-8589, Japan

Received July 26, 1999; Accepted November 8, 1999
The gene expression of liver metallothionein-1 in excess dietary histidine was investigated by feeding rats ad libitum on either a basal or histidine-excess (50 g of L-histidine per kg of diet) diet for 5 d. The copper content of the liver and zinc level in the serum of the rats fed on the histidine-excess diet were lower by 21％ and 61％, respectively of the figures for the rats fed on the basal diet, but the zinc content of the liver and copper level in the serum were not affected. Excess dietary histidine caused an increase in the urinary output of both copper and zinc. The level of liver metallothionine-1 mRNA was markedly lower at 19％ in the rats fed on the a histidine-excess diet compared to the level with the basal diet. It thus appears that such a response by the level of liver metallothionein-1 mRNA might have been be due to the lower content of liver copper.
histidine; metallothionein; copper; zinc

-19-
Note
Detection of a Spontaneous High Expression of Heat Shock Protein 70
in Developing Zebrafish (Danio rerio)

Fu-Lung YEH and Todd HSU・

Institute of Marine Biotechnology, National Taiwan Ocean University, Keelung 20224, Taiwan,
Republic of China

Received August 16, 1999; Accepted November 16, 1999
A spontaneous high expression of heat shock protein 70 (HSP 70) was detected in zebrafish (Danio rerio) at early larval stage (84 h after fertilization), but the HSP 70 level was either low or barely detectable in 12, 24, 36, 60, and 108 h after fertilization. The extracts of zebrafish at 80 and 84 h after fertilization formed a clear protein-DNA complex with a probe containing heat shock elements (HSEs), suggesting that this spontaneous expression of HSP 70 may be turned on via the binding of stage-specific HSE-binding factors to HSP 70 gene promotor. The protein-HSE complexes produced by the spontaneous binding, however, were found to be different from those formed by the extracts of heat-treated zebrafish in electrophoretic mobility.
Danio rerio; development; heat shock element; heat shock factor; heat shock protein 70

-20-
Note
Alkylated Benzothiophene Desulfurization by Rhodococcus sp. Strain T09

Toru MATSUI,＊,・ Toshimitsu ONAKA,＊＊＊ Yasuhiro TANAKA,＊ Toshiyuki TEZUKA,＊＊
Masanori SUZUKI,＊＊＊ and Ryuichiro KURANE＊＊

＊Tsukuba branch of Bio-Refining Process Laboratory, Advanced Technology and Research Institute, Petroleum Energy Center, 1-1 Higashi Tsukuba-shi, Ibaraki, 305-8566, Japan
＊＊National Institute of Bioscience and Human-Technology (NIBH), AIST, MITI, 1-1 Higashi Tsukuba-shi, Ibaraki, 305-8566, Japan
＊＊＊Bio-Refining Process Laboratory, Advanced Technology and Research Institute, Petroleum Energy Center, 1900 Sodeshi-cho Shimizu-shi, Shizuoka 424-0037, Japan

Received August 16, 1999; Accepted November 17, 1999
A benzothiophene desulfurizing bacterium was isolated and identified as Rhodococcus sp. strain T09. Growth assays revealed that this strain assimilated, as the sole sulfur source, various organosulfur compounds
・that cannot be assimilated by the well-studied diben-・
zothiophene-desulfurizing Rhodococcus sp. IGTS8. The cellular growth rate of strain T09 for the alkylated benzothiophenes depended on the alkylated position and the length of the alkyl moiety.
desulfurization; Rhodococcus sp.; benzothiophene

-21-
Note
Preparation of (−)-Periplanone D and Its Physical and Spectroscopic
Properties

Shigefumi KUWAHARA,・ Izuru NAGASHIMA, Walter Soares LEAL,＃ Jiro ISHIKAWA,
and Osamu KODAMA

Laboratory of Agricultural Chemicals, Faculty of Agriculture, Ibaraki University, Ami-machi, Inashiki-gun, Ibaraki 300-0393, Japan
＃Laboratory of Chemical Prospecting, National Institute of Sericultural and Entomological Science,
1-2 Ohwashi, Tsukuba 305-8634, Japan

Received August 24, 1999; Accepted November 4, 1999
(−)-Periplanones C and D were obtained in reproducible yields by modifying reported procedures. Our synthetic sample of (−)-periplanone D showed somewhat different physical and spectroscopic properties from those reported in the literature.
sex pheromone; periplanone; Periplaneta fuliginosa

-22-
Note
Expression and Mutational Analysis of Amino Acid Residues Involved in Catalytic Activity in a Ribonuclease MC1 from the Seeds of Bitter Gourd

・Tomoyuki NUMATA,1 Tohru KASHIBA,2 Madoka HINO,1 Gunki FUNATSU,2 Masatune ISHIGURO,2・
Nobuyuki YAMASAKI,1 and Makoto KIMURA1,・

1Laboratory of Biochemistry, Faculty of Agriculture, and 2Laboratory of Protein Chemistry and Engineering, Graduate School of Genetic Resources Technology, Kyushu University, Fukuoka 812-8581, Japan

Received September 2, 1999; Accepted November 24, 1999
The ribonuclease MC1 (RNase MC1) from seeds of bitter gourd (Momordica charantia) consists of 190 amino acids and belongs to the RNase T2 family, including fungal RNases typified by RNase Rh from Rhizopus niveus. We expressed RNase MC1 in
Escherichia coli cells and made use of site-directed mutagenesis to identify essential amino acid residues for catalytic activity. Mutations of His34 and His88 to Ala completely abolished the enzymatic activity, and considerable decreases in the enzymatic activity were observed in cases of mutations of His83, Glu84, and Lys87, when yeast RNA was used as a substrate. Kinetic parameters for the enzymatic activity of the mutants of His83, Glu84, and Lys87 were analyzed using a dinucleoside monophosphate CpU. Km values for the mutants were approximately like that for wild-type, while kcat values were decreased by about 6 to 25-fold. These results suggest that His34, His83, Glu84, Lys87, and His88 in RNase MC1 may be involved in the catalytic function. These observation suggests that RNase MC1 from a plant catalyzes RNA degradation in a similar manner to that of fungal RNases.
bitter gourd; Momordica charantia; RNase MC1; site-directed mutagenesis

-23-
Note
Group I Intron Located in PR Protein Homologue Gene in Youngia japonica

Hiromi NISHIDA,・ Atsushi OGURA, Akira YOKOTA, Isamu YAMAGUCHI,＊ and Junta SUGIYAMA

Institute of Molecular and Cellular Biosciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-0032
＊The Institute of Physical and Chemical Research (RIKEN), Hirosawa 2-1, Wako-shi, Saitama 351-0198

Received September 10, 1999; Accepted November 10, 1999
A Youngia japonica strain had a group I intron that was suggested to have been transferred from Protomyces inouyei, a pathogenic fungus of Y. japonica. It was located in the miraculin homologue coding gene by reverse complementation. The deduced amino acid sequence of this miraculin homologue of Y. japonica was similar to the amino acid sequences of tobacco and tomato pathogenesis-related proteins.
Youngia japonica; Protomyces inouyei; group I intron; horizontal transfer; miraculin homologue

-24-
Note
Synthesis of 2,2-Diphenylpropionate Derivatives and Their Effects
on Larval Growth of the Silkworm

Nozomu TOYOMURA, In-Hae KIM, Tetsuya YOSHIDA, Takahiro SHIOTSUKI,＊
and Eiichi KUWANO・

Laboratory of Pesticide Chemistry, Division of Bioresource and Bioenvironmental Sciences, Graduate School, Kyushu University, Fukuoka 812-8581, Japan
＊Department of Insect Physiology and Behavior, National Institute of Sericultural and Entomological Science, Tsukuba, Ibaraki 305-8634, Japan

Received September 13, 1999; Accepted November 15, 1999
A variety of 2,2-diphenylpropionate derivatives with an amino substituent were synthesized and their effects on larval growth of the silkworm, Bombyx mori, were examined by dietary administration. Of the compounds tested, 3-(4-ethylpiperazin-1-yl)propyl 2,2-diphenylpropionate (3) caused significant prolongation of the larval period. Studies on the structure-activity relationship indicate that a piperazine ring and the bond distances between the nitrogen atom and the ester group were important for this activity. Treatment of compound 3 delayed the increase of ecdysteroid titers in the hemolymph by 3 days compared with that of the control, which correlates with the delay in molting.
2,2-diphenylpropionates; ecdysteroids; antimuscarinic agent

-25-
Note
Increased Intestinal Calcium Absorption from the Ingestion
of a Phosphorylated Guar Gum Hydrolysate Independent
of Cecal Fermentation in Rats

Osamu WATANABE,1,2,・ Hiroshi HARA,1 Yoritaka AOYAMA,1 and Takanori KASAI1

1Department of Bioscience and Chemistry, Faculty of Agriculture, Hokkaido University, Kita 9, Nishi 9, Kita-ku, Sapporo 060-8589, Japan
2Hokkaido Food Processing Research Center, Midorimachi 589-4, Bunkyodai, Ebetsu 069-0836, Japan

Received September 13, 1999; Accepted Norvember 8, 1999
The Apparent calcium absorption was increased in rats fed on P-GGH and GGH. However, this increase in calcium absorption from GGH feeding was cancelled by a cecectomy, whereas the corresponding increase from P-GGH feeding was not. The change in femoral calcium content was similar to that in calcium absorption. The calcium solubility in the ileum was increased in those rats fed on P-GGH. We conclude that cecal fermentation did not contribute to the increased calcium absorption by the rats fed on P-GGH.
calcium; rat; absorption; guar gum; cecal fermentation

-26-
Note
Changes in γ-Aminobutyric Acid Content during beni-koji Making

Isato KONO and Kunio HIMENO＊

Industrial Technology Center of Okayama Prefecture, 5301 Haga, Okayama-shi, Okayama 701-1296, Japan
＊Himeno Fermented Food Office, 1-11-22 Kyoyama, Okayama-shi, Okayama 700-0015, Japan

Received September 17, 1999; Accepted November 16, 1999
The changes in the γ-aminobutyric acid (GABA) content during the making of beni-koji prepared with Monascus pilosus IFO 4520 vs. the difference in the rate of tomo koji (10％, 30％, and 50％) were examined. The increased proportion of tomo koji would increase the GABA production and the productions of GABA peaked on the fifth day and thereafter declined. The glutamate decarboxylase activity during beni-koji making with 50％ tomo koji steadily increased after the start of the koji making, reaching its peak on the fifth day. The succinic acid content increased after the sixth day. The mycelial growth was in the stationary phase after the sixth day. Therefore, the GABA content increases with an increase in the proportion of tomo koji. It is presumed that the maximum amount of GABA reached on the fifth day was the cause of the increasing amount of conversion of GABA into succinic acid, in addition to the decline in the GAD activity after the fifth day of koji making.
γ-aminobutyric acid; beni-koji; Monascus pilosus; tomo koji

-27-
Note
Novel Fungal Metabolites, Demethylsorbicillin and Oxosorbicillinol,
Isolated from Trichoderma sp. USF-2690

Naoki ABE,・ Kazunobu YAMAMOTO, and Akira HIROTA

Laboratory of Applied Microbiology, School of Food and Nutritional Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan

Received September 20, 1999; Accepted November 3, 1999
The novel compounds, demethylsorbicillin (1) and oxosorbicillinol (2), were isolated from a fermentation broth of Trichoderma sp. USF-2690. The structures of these compounds, which were determined from spectroscopic evidence, suggest the possibility that methylation at C-6 and oxidation at C-1 and C-6 of sorbicillin were controlled in the early polyketide stage before the formation of oxidized sorbicillin dimers. In a 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging assay, 2 gave an ED50 value of 87.7 μM.
demethylsorbicillin; oxosorbicillinol; Trichoderma sp. USF-2690; DPPH radical scavenger

-28-
Note
Isolation and Some Properties of a Serine Protease from the Fruits
of Cudrania cochinchinensis (Lour.) Kudo et Masam.

Tetsuya UCHIKOBA,・ Kazunari ARIMA,＊ Masayuki SHIMADA, Hiroo YONEZAWA,
and Mokoto KANEDA

Department of Chemistry, Faculty of Science, Kagoshima University, Korimoto 1-21-35, Kagoshima 890-0065, Japan
＊Plant Gene Expression Center, Department of Plant and Microbial Biology, University of California,
Berkeley, 800 Buchanan St. Albany, CA 94710 U.S.A.

Received September 24, 1999; Accepted November 22, 1999
An endopeptidase (Cudrania protease) with a molecular mass of 76 kDa has been purified from the fruits of Cudrania cochinchinensis (Lour.) Kudo et Masam. The enzyme was stable between pH 6 and 10 at 30°C for 60 min. The enzyme activity was inhibited by diisopropyl fluorophosphate, chymostatin, and aprotinin, but not by EDTA or pepstatin. These results indicated that the enzyme was a serine protease.
Cudrania cochinchinensis; fruits; Moraceae; serine protease

-29-
Note
Absolute Structure of N-p-Coumaroyloctopamine in Elicitor-treated Potato
Tuber Tissue

Fumio MATSUDA,・ Hisashi MIYAGAWA, and Tamio UENO

Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan

Received September 24, 1999; Accepted November 21, 1999
Treatment of potato tuber tissue with β-1,3-oligoglucosaccharide causes an accumulation of N-p-coumaroyloctopamine (1). In order to determine the absolute structure of 1 in potato, optically active 1 was synthesized from (R)-octopamine which had been obtained from the racemic mixture by the fractional crystallization. By comparing the chromatographic behavior of synthetic and naturally-occurring samples with a chiral HPLC analysis, the absolute configuration of 1 in potato was determined to be S. This indicates that the absolute configuration of the octopamine moiety of 1 is opposite to that of octopamine formed in animal tissues.
Solanum tuberosum; octopamine; N-p-coumaroyloctopamine

-30-
Note
Characterization and Amino Acid Sequences of Cytochromes c6
from Two Strains of the Green Alga Chlorella vulgaris

Seiji YAMADA,1 Toshio NAKAMURA,2 Yoshimasa TANAKA,2 Yasuhiro ISOGAI,3
Toshiyuki NISHIO,1 and Tadatake OKU1,・

1Department of Biological Chemistry, College of Bioresource Sciences, Nihon University, Shimouma 3-34-1, Setagaya-ku, Tokyo 154-8513, Japan
2Chlorella Industry, Co., Ltd., Hisatomi 1343, Chikugo, Fukuoka 833-0056, Japan
3The Institute of Physical and Chemical Research (RIKEN), Hirosawa 2-1, Wako, Saitama 351-0198, Japan

Received September 30, 1999; Accepted November 13, 1999
Cytochromes c6 from the green algae Chlorella vulgaris CK-5 (CK5cyc6) and C. vulgaris CK-22 (CK22cyc6) were characterized and their amino acid sequences were analyzed. CK5cyc6 had a molecular mass of 9.3 kDa, isoelectric points of 3.0 (reduced) and 3.6 (oxidized), and a redox potential of ＋362 mV at pH 7.0. CK22cyc6 had a molecular mass of 9.5 kDa, isoelectric points of 2.9 (reduced) and 3.5 (oxidized), and a redox potential of ＋355 mV at pH 7.0. The absorption spectra of both cytochromes c6 showed 4 maxima in reduced form, and 2 maxima and a weak peak at 695 nm in oxidized form. The pyridine ferrohemochrome spectra indicated that their prosthetic group was heme c. These physicochemical properties were similar to those of other algal cytochromes c6. The amino acids (88 residues) of CK5cyc6 and CK22cyc6 were sequenced and the sequence motif --CXXCH--, which is typical of the heme-binding site of c-type cytochrome, was clearly confirmed in both cytochromes. Twenty-six amino acid residues were substituted, and the similarity score of each of them was 70.45％.
cytochrome c6; amino acid sequence; Chlorella vulgaris CK-5; Chlorella vulgaris CK-22

-31-
Note
Purification of Membrane-bound Lactoferrin from the Human Milk
Fat Globule Membrane

Jin-Kook CHO1,2 Norihiro AZUMA,1 Chi-Ho LEE,3 Jae-Hyeun YU,3 and Choemon KANNO1,・

1Department of Applied Biochemistry, Utsunomiya University, 350 Mine-machi, Utsunomiya 321-8505, Japan
2Animal Resources Research Center, and 3Faculty of Animal Life Science, Kon-kuk University, 93-1, Mojin-Dong, Kwangjin-Ku, Seoul 143-701, Korea

Received September 30, 1999; Accepted November 17, 1999
Although lactoferrin is known as a basic soluble glycoprotein, the presence of the membrane-bound form of this protein has also been demonstrated in human milk. Membrane-bound lactoferrin was extracted from the human milk fat globule membrane with a detergent mixture of 1％ Tween-20, 0.5％ C12E8, and 0.5 M KCl in 20 mM Tris-HCl (pH 7.4). Lactoferrin in the detergent-soluble fraction was purified by affinity chromatography with Concanavalin A and by hydrophobic chromatography with phenyl-Superose. The purified protein gave a single band of 80 kDa by SDS-PAGE. Its N-terminal amino acid sequence was consistent with that of human lactoferrin.
lactoferrin; human milk; milk fat globule membrane; purification

-32-
Note
A Homologue of EGL1 Encoding Endo-1,4-β-glucanase in Elongating
Pea Stems

Takumi TAKEDA, Fukumi SAKAI, and Takahisa HAYASHI・

Wood Research Institute, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan

Received October 6, 1999; Accepted November 11, 1999
A gene (EGL2) encoding an endo-1,4-β-glucanase in peas has been cloned as a homologue of EGL1. EGL2 encodes a polypeptide of 506 amino acids, including a 24-mer putative signal polypeptide. The gene product contains a domain conserved in endo-1,4-β-glucanase (family 9) showing 60％ amino acid identity to EGL1. EGL2 mRNA was accumulated only in the elongating regions of pea stems, although EGL1 mRNA was abundant in both elongating and non-elongating
tissues. However, the level of EGL2 mRNA was not increased by the treatment with sucrose and auxin in pea segments. These results suggest that the expression of EGL2 either requires the presence of other factors related to the auxin effect or occurs independent of auxin in the elongating pea stems.
Pisum sativum; endo-1,4-β-glucanase; elongating stems

-33-
Note
Molecular Cloning and Characterization of rpbA Encoding RNA Polymerase II
Largest Subunit from a Filamentous Fungus, Aspergillus oryzae

Keiichi NAKAJIMA, Young-Chae CHANG,＊ Tomoo SUZUKI,＊＊ Yoshifumi JIGAMI,＊ and Masayuki MACHIDA＊

United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan
＊Molecular Biology Department, National Institute of Bioscience and Human-Technology, Tsukuba, Ibaraki 305-8566, Japan
＊＊Department of Bioproductive Science, Faculty of Agriculture, Utsunomiya University, Utsunomiya, Tochigi 321-8505, Japan

Received October 6, 1999; Accepted November 16, 1999
We have cloned rpbA encoding the RNA polymerase II largest subunit (polIIL) from a filamentous fungus, Aspergillus oryzae. The rpbA product included eight highly conserved regions and the carboxyl-terminal domain (CTD). A. oryzae polIIL CTD with 184 amino acids was composed of 25 CTD consensus repeats, which was a similar number to those of lower eukaryotes. The amino acids in each repeat of A. oryzae polIIL, however, conformed less to the CTD consensus than those of polIILs from other lower eukaryotes.
RNA polymerase II largest subunit; Aspergillus oryzae; carboxyl-terminal domain (CTD)

-34-
Note
Synthesis of 2,2,4,4-Tetramethyl-N,N′-bis(2,6-dimethylphenyl)cyclobutane-1,3-diimine, a Unique Compound from Arundo donax, and Its Analogues to Test Their Antifeedant Activity Against the Boll Weevil, Anthonomus grandis

Kiminori MOCHIZUKI, Hirosato TAKIKAWA, and Kenji MORI・

Department of Chemistry, Faculty of Science, Science University of Tokyo, Kagurazaka 1-3, Shinjuku-ku, Tokyo 162-8601, Japan

Received October 6, 1999; Accepted November 12, 1999
2,2,4,4 - Tetramethyl - N,N′ - bis(2,6 - dimethylphenyl)
cyclobutane-1,3-diimine (1), which was isolated from the Thai plant Arundo donax as an antifeedant against the boll weevil (Anthonomus grandis), and its analogues (9--13) were synthesized and shown to possess no remarkable antifeedant activity of practical interest.
Arundo donax; cyclobutanes; imines; NMR spectroscopy; phytochemistry

-35-
Note
Establishment of Monoclonal Antibodies Specific for Bacillus subtilis DB9011

Yoshiya ASANO,＊ Emi AKAISHI,＊ Kentaro TAJIMA,＊＊ and Takao SHINOZAWA＊

＊Department of Biological and Chemical Engineering, Faculty of Engineering, Gunma University,
Kiryu 376-8515, Japan
＊＊AHC Inc., Maebashi 371-0831, Japan

Received October 6, 1999; Accepted November 26, 1999
Bacillus subtilis DB9011 is a strain with useful functions for agriculture. To establish a method for the discrimination of this strain from others, monoclonal antibodies (MAbs) were prepared. Although two established MAbs (MAb9B6 and MAb14D2) cross-react with some other Bacillus strains in ELISA, only B. subtilis DB9011 vegetative cells are recognized by both MAbs. MAb14D2 recognizes flagellin, a 34-kDa unit protein of flagella. The two MAbs established will provide powerful tools with which detailed analysis of this bacterial strain can be obtained under environmental conditions.
Bacillus subtilis DB9011; ELISA; flagellin; monoclonal antibody

-36-
Note
GTP, an Inhibitor of Transglutaminases, is Hydrolyzed by Tissue-type
Transglutaminase (TGase 2) but not by Epidermal-type
Transglutaminase (TGase 3)

Kiyotaka HITOMI,1 Koji IKURA,＊ and Masatoshi MAKI

Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences,
Nagoya University, Chikusa, Nagoya 464-8601, Japan
＊Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology,
Kyoto 606-8585, Japan

Recieved October 6, 1999; Accepted November 19, 1999
Epidermal-type transglutaminase (TGase 3) is devoid of GTPase activity, but its TGase activity is inhibited by GTP as in the case of tissue-type TGase (TGase 2). In addition, the inhibition was not affected by the presence of higher concentrations of Ca ion. These results indicate that GTP interacts with TGase 3 in a manner different from its action on TGase 2.
transglutaminase; GTP; GTPase; calcium

-37-
Note
Cloning of a Full-length cDNA Encoding ent-Kaurene Synthase
from Gibberella fujikuroi: Functional Analysis of a Bifunctional
Diterpene Cyclase

Tomonobu TOYOMASU,・ Hiroshi KAWAIDE,＊ Atsuko ISHIZAKI, Shoko SHINODA,
Minoru OTSUKA, Wataru MITSUHASHI, and Takeshi SASSA

Department of Bioresource Engineering, Yamagata University, Tsuruoka-shi, Yamagata 997-8555, Japan
＊Frontier Research Program, The Institute of Physical and Chemical Research (RIKEN), Wako-shi, Saitama 351-0198, Japan

Received October 12, 1999; Accepted November 15, 1999
We report here the nucleotide sequence of a full-length cDNA encoding ent-kaurene synthase that was isolated by a reverse-transcription polymerase chain reaction from Gibberella fujikuroi (Gcps/ks). This cDNA encodes 952 amino acid residues with a relative molecular mass of 107 kDa. The sequence similarity between Gcps/ks and ent-kaurene synthase of the gibberellin A1-producing fungus, Phaeosphaeria sp. L487, is very high, suggesting that Gcps/ks is also a bifunctional diterpene cyclase. Its recombinant protein expressed in Escherichia coli converted geranylgeranyl diphosphate to copalyl diphosphate and ent-kaurene.
diterpene cyclase; ent-kaurene synthase; Gibberella fujikuroi; gibberellin biosynthesis

-38-
Note
Efficient Production of N-terminally Truncated Biologically Active Human Interleukin-6 by Bacillus brevis

Yasuhiro SHIGA,1 Makiko MAKI,2 Toshihiro OHTA,1 Shin-ichi TOKISHITA,1
Akou OKAMOTO,1 Norihiro TSUKAGOSHI,2 Shigezo UDAKA,3 Atsushi KONISHI,4
Yuko KODAMA,4 Daisuke EJIMA,4 Hiroshi MATSUI,4 and Hideo YAMAGATA1,・

1School of Life Science, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
2Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan
3Faculty of Applied Bio-Science, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan
4Pharmaceutical Research Laboratories, Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki-shi, Kanagawa 210-8681, Japan

Received October 21, 1999; Accepted November 26, 1999
cDNAs encoding human interleukin 6 (hIL-6) and its variants lacking the N-terminal Pro and Pro-Val-Pro-Pro, respectively, were expressed in Bacillus brevis by using the signal peptide fusion approach. The presence of Pro at the N-terminus of the mature protein hindered the action of the Bacillus brevis signal peptidase. hIL-6 lacking the N-terminal Pro-Val-Pro-Pro was most efficiently secreted in a biologically active form and accumulated in the culture medium to a level of 200 mg per liter, which is the highest level reported for the bacterial secretion of hIL-6.
Bacillus brevis; human interleukin-6; N-terminal truncation; secretion; signal peptide

-39-
Preliminary Communication
An Automated System for Genome Analysis to Support Microbial
Whole-genome Shotgun Sequencing

Tokuki SAKIYAMA,a Hideto TAKAMI,a,? Naotake OGASAWARA,b Satoru KUHARA,c
Tokio KOZUKI,d Kosuke DOGA,d Akira OHYAMA,d and Koki HORIKOSHIa

aDeep-sea Microorganisms Research Group, Japan Marine Science and Technology Center,
2-15 Natsushima-cho, Yokosuka, Kanagawa 237-0061, Japan
bGraduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama,
Ikoma, Nara 630-0101, Japan
cGraduate School of Genetic Resources Technology, Kyushu University, Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan
dBiotechnology Systems Department, Mitsui Knowledge Inc. Ltd., 2-7-14 Higashinakano, Nakano-ku,
Tokyo 164-8555, Japan

Recieved October 29, 1999; Accepted December 17, 1999
We developed a semi-automated genome analysis system called GAMBLER in order to support the current whole-genome sequencing project focusing on alkaliphilic Bacillus halodurans C-125. GAMBLER was designed to reduce the human intervention required and to reduce the complications in annotating thousands of ORFs in the microbial genome. GAMBLER automates three major routines: analyzing assembly results provided by genome assembler software, assigning ORFs, and homology searching. GAMBLER is equipped with an interface for convenience of annotation. All processes and options are manipulatable through a WWW browser that enables scientists to share their genome analysis results without choosing computer platforms.
software; genome analysis; whole-genome shotgun method; Bacillus halodurans C-125