Contents and Abstracts of Latest Issue of BBB

(Vol.65 No.8 2001)

Spin-Trapping Study on the Hydroxyl Radical Formed
from a Tea Catechin-Cu(II) System
Hisashi YOSHIOKA,1,† Yasushi SENBA,1 Kieko SAITO,1 Takahide KIMURA,2
and Fumiko HAYAKAWA3　p.1697

Identification of 3,4-Dihydroxy-2-oxo-butanal (L-threosone) as an Intermediate
Compound in Oxidative Degradation of Dehydro-L-ascorbic Acid
and 2,3-Diketo-L-gulonic Acid in a Deuterium Oxide Phosphate Buffer
Yoko NISHIKAWA,† Yuka TOYOSHIMA, and Tadao KURATA p.1707

Influence of α-Helices on the Emulsifying Properties of Proteins
Simon POON, Adrienne CLARKE, Graeme CURRIE,＊ and Carolyn SCHULTZ p.1713

Aggregation Pheromone Activity of the Female Sex Pheromone, β-Acaridial,
in Caloglyphus polyphyllae (Acari: Acaridae)†
Nobuhiro SHIMIZU, Naoki MORI, and Yasumasa KUWAHARA†† p.1724

Consumption of Hypoallergenic Flour Prevents Gluten-induced Airway
Inflammation in Brown Norway Rats
Jun WATANABE,1,2,† Soichi TANABE,3 Michiko WATANABE,3 Takanori KASAI,1
and Kei SONOYAMA1 p.1729

Novel Method for Enzymatic Synthesis of CMP-NeuAc
Kazuya ISHIGE,† Tomoki HAMAMOTO, Toshikazu SHIBA,＊ and Toshitada NOGUCHI
p.1736

Apoptosis Induction by T-2 Toxin: Activation of Caspase-9, Caspase-3,
and DFF-40/CAD through Cytosolic Release
of Cytochrome c in HL-60 Cells
Masahiro NAGASE,＊＊ M. Murshedul ALAM, Akiko TSUSHIMA, Takumi YOSHIZAWA,＊
and Nobuo SAKATO p.1741

Comparative Effects of Dietary Fat Types on Hepatic Enzyme Activities Related
to the Synthesis and Oxidation of Fatty Acid and to Lipogenesis in Rats
Hisanao TAKEUCHI,† Takeshi NAKAMOTO, Yoshiaki MORI, Masako KAWAKAMI,
Hiroyuki MABUCHI, Yoshinori OHISHI, Natsuko ICHIKAWA,
Akiko KOIKE, and Keiko MASUDA p.1748

The Third Naturally Occurring Attractant toward Zoospores
of Phytopathogenic Aphanomyces cochlioides
from the Spinacia oleracea Host Plant
Satoshi TAHARA,1,2,† Kaori OHKAWA,1 Tomohiko TAKAYAMA,1 and Yuko OGAWA1 p.1755

Effects of Amino Acids on the Amidation of Polyaromatic
Carboxylic Acids by Bacillus cereus
Reiji MARUYAMA, Akiko KAWATA, Shin ONO, Mikio NISHIZAWA†,
Seiji ITO†, and Masami INOUE＊ p.1761

Binding of a Highly De-N-acetylated Chitosan to Japanese Pheasan
Lysozyme as Measured by 1H-NMR Spectroscopy
Tamo FUKAMIZO,1,† Tsugihisa YAMAGUCHI,1 Tomohiro ARAKI,2 Takao TORIKATA,2
Are KRISTIANSEN,3,†† and Kjell M. V<0197>RUM3 p.1766

Biotransformation of Phenanthrene and 1-Methoxynaphthalene
with Streptomyces lividans Cells Expressing a Marine Bacterial
Phenanthrene Dioxygenase Gene Cluster
Hyo-Kon CHUN,1,＃ Yasuo OHNISHI,1 Norihiko MISAWA,2,† Kazutoshi SHINDO,3
Miki HAYASHI,2 Shigeaki HARAYAMA,4 and Sueharu HORINOUCHI1 p.1774

Comparison of the ω-Transaminases from Different Microorganisms
and Application to Production of Chiral Amines
Jong-Shik SHIN† and Byung-Gee KIM＊ p.1782

Production of Pyridoxal Phosphate by a Mutant Strain
of Schizosaccharomyces pombe
Ruamsub CHUMNANTANA, Kumi HIROSE, Hiromichi BABA, and Toshiharu YAGI p.1789

Improvement of Freezing Tolerance in Transgenic Tobacco Leaves
by Expressing the hiC6 Gene
Ken-ichi HONJOH,1,† Hideyuki SHIMIZU,1 Noriko NAGAISHI,1 Hiroko MATSUMOTO,1 Koushirou SUGA,1,2 Takahisa MIYAMOTO,1 Masayoshi IIO,1 and Shoji HATANO3 p.1796

Structural Confirmation of 15-Norlubiminol and 15-Norepilubiminol,
Isolated from Solanum aethiopicum, by Chemical Conversion
from Lubimin and Epilubimin, and their Antifungal Activity
Ayako WATANABE,1,2 Hiroaki TOSHIMA,1 Hiroshi NAGASE,1 Toshinori NAGAOKA,3
and Teruhiko YOSHIHARA1,2,† p.1805

Characterization of Clofibrate-induced Retrograde Golgi Membrane Movement
to the Endoplasmic Reticulum: Clofibrate Distinguishes the Golgi
from the Trans Golgi Network
Machiko NAKAMURA,＊,¶ Natsuko KUROIWA,＊,‡ Yoshiki KONO,¶ and Akira TAKATSUKI＊,† p.1812

Cloning, Sequencing, and Expression of the Gene Encoding an Intracellular
β-D-Xylosidase from Streptomyces thermoviolaceus OPC-520
Hiroshi TSUJIBO,† Chiaki TAKADA, Akihiko TSUJI, Mitsuo KOSAKA,
Katsushiro MIYAMOTO, and Yoshihiko INAMORI p.1824

A Recombinant Molt-inhibiting Hormone of the Kuruma Prawn Has a Similar
Secondary Structure to a Native Hormone: Determination of Disulfide Bond
Arrangement and Measurements of Circular Dichroism Spectra
Hidekazu KATAYAMA,1 Tsuyoshi OHIRA,1 Koji NAGATA,2 and Hiromichi NAGASAWA1
p.1832

Purification and Structural Determination of a Phosphorylated Peptide
with Anti-calcification and Chitin-binding Activities in the Exoskeleton
of the Crayfish, Procambarus clarkii
Hirotaka INOUE, Noriaki OZAKI, and Hiromichi NAGASAWA† p.1840

Note
Electron Transport Inhibitor in Cyperus javanicus
Masanori MORIMOTO,† Yoshiyuki SHIMOMURA, Ryoko MIZUNO, and Koichiro KOMAI
p.1849

Note
Active Oxygen Radicals Induce Peroxidase Activity in Rice Blade Tissues
Arisa HIGA, Tomoe HIDAKA, Yuji MINAI, Yoshitaka MATSUOKA, and Minoru HAGA
p.1852

Note
Isolation and Structure Elucidation of a New Antifungal and Antibacterial
Antibiotic Produced by Streptomyces sp. 201†
Gajen N. BORDOLOI, Babita KUMARI, Arijit GUHA, Manobjyoti BORDOLOI,＊
R. N. S. YADAV,＃ Monoj K. ROY, and Tarun C. BORA p.1856

Note
Inhibitory Effects of Capsaicinoids on Fatty Acid Desaturation
in a Rat Liver Cell Line
Nobuhiro NAKANO, Norifumi SHIRASAKA, Kazuki MASUOKA, Tetsuo MURAKAMI,
Tatsuo WATANABE,1 Kenji KOBATA,1 Sakayu SHIMIZU,2 and Hajime YOSHIZUMI† p.1859

Note
Prevention by Lactic Acid Bacteria of the Oxidation of Human LDL
Masaki TERAHARA,† Saori KURAMA, and Naoki TAKEMOTO p.1864

Note
Ellagic Acid Formation from Galloylglucoses by a Crude Enzyme
of Cornus capitata Adventitious Roots
Norie TANAKA＊,†, Koichiro SHIMOMURA,＊＊ and Kanji ISHIMARU＊ p.1869

Note
Role of Activity of Gastrointestinal Microflora in Absorption of Calcium
and Magnesium in Rats Fed β1--4 Linked Galactooligosaccharides
Osamu CHONAN, Rie TAKAHASHI, and Masaaki WATANUKI p.1872

Note
Purification and Characterization of L-2,3-Butanediol Dehydrogenase
of Brevibacterium saccharolyticum C-1012 Expressed in Escherichia coli
Yusuke TAKUSAGAWA,1,† Masato OTAGIRI,1 Sadaharu UI,1 Takashi OHTSUKI,1
Akio MIMURA,1 Moriya OHKUMA,2 and Toshiaki KUDO2 p.1876

Note
Synthesis of the L-Enantiomer of 4′-C-Ethynyl-2′-deoxycytidine
Satoru KOHGO,1 Hiroaki MITSUYA,2,3 and Hiroshi OHRUI1,† p.1879

Note
Chitinase in Cucumber Xylem Sap
Susumu MASUDA,1,† Hiroshi KAMADA,2 and Shinobu SATOH2 p.1883

Note
Specific Antimicrobial Synergism of Synthetic Hydroxy Isothiocyanates
with Aminoglycoside Antibiotics
Hirokuni TAJIMA, Hisashi KIMOTO,1 and Akira TAKETO2 p.1886

Note
Increased Transglycosylation Activity of Rhodotorula glutinis
Endo-β-Glucanase in Media Containing Organic Solvent
Tadao OIKAWA,1,2 Yasuyuki TSUKAGAWA,1 Masashi CHINO,1 and Kenji SODA1,2,†
p.1889

Note
Effect of Germanium-132 on Low-density Lipoprotein Oxidation
and Atherosclerosis in Kurosawa and Kusanagi Hypercholesterolemic Rabbits
Yasuhisa WAKABAYASHI† p.1893

Note
Enzymatic Glycosylation of Lincomycin
Lenka WEIGNEROV<0193>A, Jaroslav SP<0193>I<0189>ZEK, Lucie NAJMANOV<0193>A, and Vladim<0225><0131>r K<0189>REN† p.1897

Note
Influences of Nitrogen Sources on Usnic Acid Production in a Cultured
Mycobiont of the Lichen Usnea hirta (L.) Wigg.
Yasuhiro KINOSHITA,1 Yoshikazu YAMAMOTO,2,† Teiko KUROKAWA,3 and Isao YOSHIMURA3
p.1900

Note
Enantioselective Synthesis of Ancepsenolide and its Analogs
Katsuki TAKAI and Ryozo IRIYE† p.1903

Note
Multiplicity of Aromatic Ring Hydroxylation Dioxygenase Genes
in a Strong PCB Degrader, Rhodococcus sp. Strain RHA1 Demonstrated
by Denaturing Gradient Gel Electrophoresis
Wataru KITAGAWA,1 Asaka SUZUKI,2 Toshihiro HOAKI,2 Eiji MASAI,1 and Masao FUKUDA1
p.1907

Note
Mutational Analyses of Dictyostelium IQGAP-Related Protein GAPA:
Possible Interaction with Small GTPases in Cytokinesis
Masao SAKURAI, Hiroyuki ADACHI,＃,† and Kazuo SUTOH p.1912

Note
A New Double Coupling System: Synthesis of Citronellyl Acetate via
Transacetylation to Citronellol from Acetyl Coenzyme A Produced
from Glucose and Free Fatty Acids
Shinobu ODA1,† and Hiromichi OHTA2 p.1917

Note
NMR Determination of the Absolute Configuration of a Macrophomate
Synthase Inhibitor by Using an Axial Chiral Reagent
Hiroki FUKUI,1 Yukiharu FUKUSHI,1,2,† Satoshi OHASHI,1 Hideaki OIKAWA,1
and Satoshi TAHARA1,2 p.1920

Note
Suppressive Effect of Coffee on Lipopolysaccharide-induced Hepatitis
in D-Galactosamine-sensitized Rats
Puming HE, Yasuhiro NODA, and Kimio SUGIYAMA† p.1924

Note
A Transformation System for an Ectomycorrhizal Basidiomycete,
Lyophyllum shimeji
Takeshi SAITO,1,2 Norio TANAKA,2 and Takao SHINOZAWA1,† p.1928

-1-
Spin-Trapping Study on the Hydroxyl Radical Formed
from a Tea Catechin-Cu(II) System

Hisashi YOSHIOKA,1,† Yasushi SENBA,1 Kieko SAITO,1 Takahide KIMURA,2
and Fumiko HAYAKAWA3

1Institute for Environmental Sciences, University of Shizuoka, Shizuoka-shi 422-8529, Japan
2Department of Chemistry, Shiga University of Medical Science, Seta, Otsu, Shiga 520-2192, Japan
3Department of Life Style Studies, School of Human Cultures, The University of Shiga Prefecture,
Hassaka-cho, Hikone, Shiga 522-8533, Japan

Received September 25, 2000; Accepted March 28, 2001
A spin-trapping method was applied to examine the formation of the hydroxyl (OH) radical from a tea catechin-Cu(II) system to elucidate a previous result that some tea catechin-Cu(II) systems induced DNA scission. Three tea catechins, (-)-epigallocatechin (EGC), (-)-epigallocatechin gallate (EGCg) and (-)-epicatechin (EC), were used. The spin-trapping agent, 5,5′-dimethyl-pyrroline-1-oxide (DMPO), was dissolved in a pH 9 phosphate buffer solution, then a catechin and Cu(II) were added in that order, and the ESR spectral change was monitored for one hour. The order of adding the catechin and Cu(II) was then reversed, and the ESR spectral change was again monitored to examine the coordinating activity of each catechin toward the Cu(II) ion and the effect on OH radical generation. The intensity changes of the spin adducts, DMPO-OH, DMPO-CH3 and DMPO-H, were analyzed, the results suggesting that the OH radical generated in the system decomposed DMPO, resulting in the formation of DMPO-CH3 and DMPO-H. The results show that EGC formed a stable complex with Cu(II) and generated the OH radical. EGCg seemed to have this activity, but the OH radical that was generated was scavenged by the gallate group existing in the complex. EC did not show strong coordinating and OH-generating activities. These characteristics of the three catechins are consistent with the results shown for DNA scission.
Key words: spin trapping method; hydroxyl radical; 5,5′-Dimethyl-1-pyrroline N-oxide (DMPO); tea catechin; cupric ion

-2-
Identification of 3,4-Dihydroxy-2-oxo-butanal (L-threosone) as an Intermediate
Compound in Oxidative Degradation of Dehydro-L-ascorbic Acid
and 2,3-Diketo-L-gulonic Acid in a Deuterium Oxide Phosphate Buffer

Yoko NISHIKAWA,† Yuka TOYOSHIMA, and Tadao KURATA

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

Received October 4, 2000; Accepted March 28, 2001
Dehydro-L-ascorbic acid (DAA), an oxidation product of L-ascorbic acid (vitamin C), is unstable in the neutral and basic pH regions. When DAA was incubated in a phosphate buffer with deuterium oxide (pH 7.4), it was degraded to form the main degradation compound, which was identified as 3,4-dihydroxy-2-oxo-butanal (L-threosone). This compound was also formed from diketo-L-gulonic acid (DKG) in a phosphate buffer with deuterium oxide. L-threosone had reducing activity, probably due to its enolization, and is likely to have been involved in the formation of the reducing activity that was observed in aqueous DAA and DKG solutions. As a reactive dicarbonyl compound, L-threosone might also take some role in the cross-linking of tissue proteins that are formed in vivo in the Maillard reaction.
Key words: L-ascorbic acid; degradation; L-threosone; diketo-L-gulonic acid

-3-
Influence of α-Helices on the Emulsifying Properties of Proteins

Simon POON, Adrienne CLARKE, Graeme CURRIE,＊ and Carolyn SCHULTZ

Cooperative Research Centre for Bioproducts, School of Botany, University of Melbourne, Vic. 3010, Australia
＊Plant Cell Biology Research Centre, School of Botany, University of Melbourne, Vic. 3010, Australia

Received November 27, 2000; Accepted March 30, 2001
A peptide derived from apomyoglobin by cyanogen bromide cleavage was found to be an active emulsifier. This molecule, peptide 1-55, has two potential amphipathic α-helices and a hydrophilic C-terminal domain. The importance of each of these domains to the emulsifying properties of this molecule was investigated by testing the products of gene constructs based on the sequence of peptide 1-55, but lacking one of the three domains. The emulsifying activity of the peptides lacking either of the α-helices was correlated with the hydrophobic moments of their respective helices. The hydrophobic moment is a measure of the amphipathicity of α-helices; a hydrophobic moment analysis of other emulsifying peptides supports the hypothesis that a high hydrophobic moment contributes to good emulsifying properties in a molecule which contains α-helices.
Key words: protein emulsifier; α-helices; hydrophobic moment; apomyoglobin; melittin

-4-
Aggregation Pheromone Activity of the Female Sex Pheromone, β-Acaridial,
in Caloglyphus polyphyllae (Acari: Acaridae)†

Nobuhiro SHIMIZU, Naoki MORI, and Yasumasa KUWAHARA††

Laboratory of Chemical Ecology, Division of Applied Life Sciences, Graduate School of Agriculture,
Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan

Received January 9, 2001; Accepted April 12, 2001
Caloglyphus (＝Sancasania) polyphyllae discharges from a pair of opisthonotal glands a characteristic set of volatiles, i.e. three monoterpenes and seven hydrocarbons. Among them, β-acaridial, which is known as the female sex pheromone of the species and has antifungal activity, was newly identified as the aggregation pheromone for unfeeding and unmating mites. Feeding mites, however, exhibited sexually aroused behavior instead of the tendency to cluster when exposed to β-acaridial. This is the first example of the compound demonstrating two pheromone functions depending upon the circumstances faced by the mites.
Key words: mite; Caloglyphus polyphyllae; β-acaridial; aggregation pheromone; sex pheromone

-5-
Consumption of Hypoallergenic Flour Prevents Gluten-induced Airway
Inflammation in Brown Norway Rats

Jun WATANABE,1,2,† Soichi TANABE,3 Michiko WATANABE,3 Takanori KASAI,1
and Kei SONOYAMA1

1Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan
2Japan Society for the Promotion of Science, Chiyoda-ku, Tokyo 102-8471, Japan
3Faculty of Education, Tokyo Gakugei University, Koganei-shi, Tokyo 184-8501, Japan

Received January 9, 2001; Accepted March 24, 2001
Brown Norway rats were immunized with gluten, and then fed a diet containing hypoallergenic flour or an amino acid mixture. The rats were then made to inhale a solubilized gluten to induce gluten-specific bronchial asthma. The antibody levels in the serum of rats were measured by ELISA, and cell counts were done on cytospin preparations of bronchoalveolar lavage fluid. Body weight was decreased after allergen challenge in rats fed the amino acid mixture but not in rats fed the hypoallergenic flour. Antibody levels in the serum were significantly lower in rats fed hypoallergenic flour than in those fed the amino acid mixture. Differential cell counts in the bronchoalveolar lavage fluid showed that the numbers of eosinophils, lymphocytes, and neutrophils were significantly lower in rats fed the hypoallergenic flour than in those fed the amino acid mixture. These results suggest that hypoallergenic flour actively suppresses the allergic reactions, probably by inducing oral tolerance.
Key words: hypoallergenic flour; wheat-specific allergy; allergy prevention; Brown Norway rats; IgE

-6-
Novel Method for Enzymatic Synthesis of CMP-NeuAc

Kazuya ISHIGE,† Tomoki HAMAMOTO, Toshikazu SHIBA,＊ and Toshitada NOGUCHI

Biochemicals Division, YAMASA Corporation, Choshi, Chiba 288-0056, Japan
＊Division of Molecular Chemistry, Graduate School of Engineering, Hokkaido University,
Sapporo 060-0810, Japan

Received January 9, 2001; Accepted March 21, 2001
A novel method for synthesizing CMP-NeuAc was established. We first confirmed that the putative neuA gene of Haemophilus influenzae, identified by its whole genome sequence project, indeed encodes CMP-NeuAc synthetase (EC 2.7.7.43). The enzyme requires CTP as a cytidylyl donor for cytidylylation of NeuAc. The enzyme was coupled with an enzymatic CTP-generating system from CMP and inorganic polyphosphate as a sole phospho-donor driven by the combination of polyphosphate kinase and CMP kinase, where phosphorylation of CMP is done by the combined activity expressed by both enzymes, and subsequent phosphorylation of CDP by polyphosphate kinase itself occurred efficiently. When CMP-NeuAc synthetase of H. influenzae, polyphosphate kinase, and CMP kinase were added to the reaction mixture containing equimolar concentrations (15 mM) of CMP and NeuAc, and polyphosphate (150 mM in terms of phosphate), CMP-NeuAc was synthesized up to 10 mM in 67％ yield.
Key words: CMP-NeuAc; Haemophilus influenzae; inorganic polyphosphate; polyphosphate kinase; CMP kinase

-7-
Apoptosis Induction by T-2 Toxin: Activation of Caspase-9, Caspase-3,
and DFF-40/CAD through Cytosolic Release
of Cytochrome c in HL-60 Cells

Masahiro NAGASE,＊＊ M. Murshedul ALAM, Akiko TSUSHIMA, Takumi YOSHIZAWA,＊
and Nobuo SAKATO

Department of Life Sciences, ＊Department of Biochemistry and Food Science, Faculty of Agriculture,
Kagawa University, 2393 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0795, Japan

Received January 9, 2001; Accepted April 4, 2001
The molecules participating in apoptosis induced by T-2 toxin in human leukemia HL-60 cells were investigated. The rank order of the potency of trichothecene mycotoxins to induce internucleosomal DNA fragmentation was found to be T-2, satratoxin G, roridin
A＞<0192>diacetoxyscirpenol＞baccharin B-5＞<0192>nivalenol, deoxynivalenol, 3-acetyldeoxynivalenol, fusarenon-X, baccharin B-4＝vehicle control. Western blot analysis of caspase-3 in T-2-treated cells clearly indicated the appearance of its catalytically active fragment of 17-kDa. Increased caspase-3 activity was also detected by using a fluorogenic substrate, DEVD-AMC. Next, cells exposed to T-2 led to cleavage of PARP from its native 116-kDa form to the 85-kDa product. Moreover, DFF-45/ICAD were cleaved to give a 12.5-kDa fragment via T-2 treatment. T-2 caused the release of cytochrome c from mitochondria into the cytosol. Increased enzymic activity of caspase-9 on LEHD-AMC was shown. These data indicate that T-2-induced apoptosis involves activation of caspase-3 and DFF-40/CAD through cytosolic accumulation of cytochrome c along with caspase-9 activation.
Key words: trichothecene mycotoxins; apoptosis; caspase-3; DFF-45/ICAD; cytochrome c

-8-
Comparative Effects of Dietary Fat Types on Hepatic Enzyme Activities Related
to the Synthesis and Oxidation of Fatty Acid and to Lipogenesis in Rats

Hisanao TAKEUCHI,† Takeshi NAKAMOTO, Yoshiaki MORI, Masako KAWAKAMI,
Hiroyuki MABUCHI, Yoshinori OHISHI, Natsuko ICHIKAWA,
Akiko KOIKE, and Keiko MASUDA

Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya,
Shizuoka 422-8529, Japan

Received January 10, 2001; Accepted March 16, 2001
The effects of different types of dietary fat on the activities of hepatic enzymes related to fatty acid synthesis ｛glucose-6-phosphate dehydrogenase (G6PDH) and acetyl-CoA carboxylase (ACC)｝, oxidation ｛acyl-CoA synthetase (AST), carnitine palmitoyl transferase (CPT), and peroxisomal β-oxidation (PβOX)｝, and lipogenesis ｛phosphatidate phosphohydrolase (PAP), diacylglycerol acyltransferase (DGAT), and phosphocholine diacylglycerol transferase (PCDGT)｝, and plasma and liver lipid levels were investigated in male Wistar rats. The animals were 6 weeks old and about 120 g of body weight, and were fed on test diets containing 20％ of a mixture of tripalmitin, tristearin and corn oil (SFA), olive oil (OLI), sunflower oil (SUN), linseed oil (LIS), and sardine oil (SAR) for 2 weeks. The concentrations of plasma total cholesterol (T-CHOL), high-density lipoprotein-cholesterol (HDL-CHOL), triacylglycerol (TG) and phospholipid (PL) were generally higher in the rats fed on SFA and OLI than in those given SUN, LIS and SAR. The rats fed on OLI had a higher level of liver T-CHOL than those fed on the other fats. The liver TG content was nearly higher from the intake of SFA and OLI than from SUN, LIS and SAR, although the liver PL level was not affected by the type of dietary fat. The SFA and OLI groups had the highest activities of hepatic G6PDH and ACC, and the SAR group, the lowest activities. The activities of AST and CPT, and peroxisomal PβOX in the liver were higher in the rats fed on the LIS and SAR diets than in those given the other diets. The hepatic PAP activity was higher from the intake of OLI and SUN, and tended to be higher from SFA than from LIS and SAR. The activity of liver DGAT was higher from SFA and inclined to be higher from OLI, SUN, and LIS than from SAR, while the PCDGT activity in the liver was not effected by the type of dietary fat. The concentrations of plasma and liver TG were generally positively correlated with the activities of liver enzymes related to the synthesis of fatty acids and lipids, and negatively with those involved in fatty acid oxidation. Based on these results, it is suggested that the levels of plasma and liver TG were controlled by different types of dietary fat through changes in the hepatic enzyme activities related to fatty acid synthesis, lipogenesis, and fatty acid oxidation.
Key words: dietary fat; enzymes for fatty acid synthesis; enzymes for lipogenesis; enzymes for β-oxidation

-9-
The Third Naturally Occurring Attractant toward Zoospores
of Phytopathogenic Aphanomyces cochlioides
from the Spinacia oleracea Host Plant

Satoshi TAHARA,1,2,† Kaori OHKAWA,1 Tomohiko TAKAYAMA,1 and Yuko OGAWA1

1Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Kita-ku,
Sapporo 060-8589, Japan
2CREST, Japan Science and Technology Corporation, 4-1-8 Honmachi, Kawaguchi 332-0012, Saitama, Japan

Received January 12, 2001; Accepted March 12, 2001
A bioassay-guided survey of spinach leaf constituents resulted in 5,4′-dihydroxy-3,3′-dimethoxy-6,7-methylenedioxyflavone being identified as the third naturally-occurring attractant in the host plant toward the zoospores of its pathogen, Aphanomyces cochlioides. The isolate showed attracting activity around Chromosorb W AW particles (60--80 mesh) coated with a 10-5 M solution in a zoospore suspension. However, this activity was 1/100--1/1000 less than that of cochliophilin A, an attractant in the roots of spinach. Bioassays with the present isolate and related compounds revealed that 5,3′,4′-trihydroxy-3-methoxy-6,7-methylenedioxyflavone did not possess attractant activity, but rather weak antagonistic activity toward the former two attractants from spinach.
Key words: Aphanomyces cochlioides; zoosporeattractant; spinach; Spinacia oleracea; 5,4′-dihydroxy-3,3′-dimethoxy-6,7-methylenedioxyflavone

-10-
Effects of Amino Acids on the Amidation of Polyaromatic
Carboxylic Acids by Bacillus cereus

Reiji MARUYAMA, Akiko KAWATA, Shin ONO, Mikio NISHIZAWA†,
Seiji ITO†, and Masami INOUE＊

Department of Cell Engineering, Faculty of Engineering, Toyama University, 3190 Gofuku,
Toyama 930-8555, Japan
†Department of Medical Chemistry, Kansai Medical University, 10-15 Fumizono, Moriguchi,
Osaka 570-8506, Japan

Received January 18, 2001; Accepted March 30, 2001
The soil bacterium Bacillus cereus Tim-r01 efficiently transformed polyaromatic carboxylic acids (PACA) such as 4-biphenylcarboxylic acid (4-BPCA), 4-biphenylacetic acid, and 4-phenoxybenzoic acid into their corresponding amides. The amidation activity was expressed at 37°C (pH 7--8) in the presence of grown cells in nutrients under an aerobic atmosphere. Other strains of B. cereus, IFO 3001 and IAM 1229, also gave the amide from 4-BPCA. In phosphate-buffered saline (PBS), the addition of normal amino acids was essential, while sulfur-containing amino acids such as methionine and cysteine drastically inhibited the amidation. Tracer experiments using N-15-isoleucine and N-15-alanine showed that the nitrogen atom of the amide came from an amino group of amino acids but not from ammonia or alkylamines.
Key words: Bacillus cereus; biotransformation; amidation; 4-biphenylcarboxylic acid; polyaromatic carboxylic acid

-11-
Binding of a Highly De-N-acetylated Chitosan to Japanese Pheasan
Lysozyme as Measured by 1H-NMR Spectroscopy

Tamo FUKAMIZO,1,† Tsugihisa YAMAGUCHI,1 Tomohiro ARAKI,2 Takao TORIKATA,2
Are KRISTIANSEN,3,†† and Kjell M. V<0197>RUM3

1Laboratory of Enzyme System Science, Department of Food and Nutrition, Kinki University,
3327-204 Nakamachi, Nara 631-8505, Japan
2Laboratory of Biochemistry, Faculty of Agriculture, Kyushu Tokai University, Choyo-mura, Aso,
Kumamoto 869-1404, Japan
3Norwegian Biopolymer Laboratory (NOBIPOL), Department of Biotechnology, The Norwegian
University of Science and Technology, N-7491 Trondheim, Norway

Received January 22, 2001; Accepted March 23, 2001
Binding of a highly de-N-acetylated chitosan to Japanese pheasant lysozyme (JPL), which differs from hen egg white lysozyme (HEWL) by nine amino acid substitutions (including Arg114→His), was investigated by 1H-NMR spectroscopy. The profile of the one-dimensional spectrum of JPL is essentially identical to that of HEWL. Using two-dimensional spectra of JPL and HEWL, several aromatic and aliphatic proton resonances of JPL were assigned by comparison. When a highly de-N-acetylated chitosan (number-average degree of polymerization, about 18; degree of acetylation, 0.04), where the N-acetylated units are predominantly surrounded by de-N-acetylated units (a monoacetylated chitosan), was added to the JPL solution, the NMR signals were clearly affected in Trp28 C5H and Ile98 γCH, as in the case of binding to HEWL. The dissociation constant of the monoacetylated chitosan evaluated from the NMR signal responses was calculated to be 0.23±0.05 mM (-31.5 kJ/mol), which is similar to that of HEWL (0.11±0.02 mM, -33.3 kJ/mol). Thus, the Arg→His substitution of the 114th amino acid, which participates in sugar residue binding at the right-sided subsite F, did not significantly affect the chitosan binding. In addition, the C2H signal of His114 of JPL was not affected by the chitosan binding. These results suggest that the monoacetylated chitosan binds to subsites E and F through the left-sided binding mode.
Key words: lysozyme; chitosan; ligand binding; 1H-NMR

-12-
Biotransformation of Phenanthrene and 1-Methoxynaphthalene
with Streptomyces lividans Cells Expressing a Marine Bacterial
Phenanthrene Dioxygenase Gene Cluster

Hyo-Kon CHUN,1,＃ Yasuo OHNISHI,1 Norihiko MISAWA,2,† Kazutoshi SHINDO,3
Miki HAYASHI,2 Shigeaki HARAYAMA,4 and Sueharu HORINOUCHI1

1Department of Biotechnology, Graduate School of Agriculture and Life Sciences, The University of Tokyo,
Bunkyo-ku, Tokyo 113-8657, Japan
2Central Laboratories for Key Technology, Kirin Brewery Co. Ltd., 1-13-5, Fukuura, Kanazawa-ku,
Yokohama 236-0004, Japan
3Pharmaceutical Research Laboratory, Kirin Brewery Co., Ltd., 3, Miyahara-cho, Takasaki 370-1295, Japan
4Marine Biotechnology Institute, Heita, Kamaishi 026-0001, Japan

Received January 23, 2001; Accepted April 6, 2001
The phdABCD gene cluster in a marine bacterium Nocardioides sp. strain KP7 codes for the multicomponent enzyme phenanthrene dioxygenase. phdA encoding an iron-sulfur protein large subunit α, phdB encoding its small subunit β, phdC encoding ferredoxin, and phdD encoding ferredoxin reductase, were replaced in such a way that the termination codons of the preceding open reading frames were overlapped with the initiation codons of the following genes. This manipulated phdABCD gene cluster was positioned downstream of the thiostrepton-inducible promoter PtipA in a high-copy-number vector pIJ6021, and introduced into the gram-positive, soil-inhabiting, filamentous bacterium Streptomyces lividans. The recombinant S. lividans cells converted phenanthrene into a cis-diol form, which was determined to be cis-3,4-dihydroxy-3,4-dihydrophenanthrene by its UV spectral data as well as HPLC property, using the authentic sample for comparison. This biotransformation proceeded very efficiently; 200 μMKey words: and 2 mMKey words: of phenanthrene were almost completely converted to its cis-diol form in 6 h and 32 h, respectively. In addition, the S. lividans cells carrying the phdABCD gene cluster were found to transform 1-methoxynaphthalene to two products, which were identified to be 8-methoxy-2-naphthol in addition to 8-methoxy-1,2-dihydro-1,2-naphthalenediol by their EI-MS, ＾{1}H- and ＾{13}C-NMR spectral data.
Key words: arene dioxygenase; phenanthrene; 1-methoxynaphthalene; 8-methoxy-2-naphthol; Streptomyces lividans

-13-
Comparison of the ω-Transaminases from Different Microorganisms
and Application to Production of Chiral Amines

Jong-Shik SHIN† and Byung-Gee KIM＊

School of Chemical Engineering and Institute for Molecular Biology and Genetics, Seoul National University,
Seoul, Korea

Received February 2, 2001; Accepted April 2, 2001
Microorganisms that are capable of (S)-enantioselective transamination of chiral amines were isolated from soil samples by selective enrichment using (S)-α-methylbenzylamine ((S)-α-MBA) as a sole nitrogen source. Among them, Klebsiella pneumoniae JS2F, Bacillus thuringiensis JS64, and Vibrio fluvialis JS17 showed good ω-transaminase (ω-TA) activities and the properties of the ω-TAs were investigated. The induction level of the enzyme was strongly dependent on the nitrogen source for the strains, except for V. fluvialis JS17. All the ω-TAs showed high enantioselectivity (E＞50) toward (S)-α-MBA and broad amino donor specificities for arylic and aliphatic chiral amines. Besides pyruvate, aldehydes such as propionaldehyde and butyraldehyde showed good amino acceptor reactivities. All the ω-TAs showed substrate inhibition by (S)-α-MBA above 200 mM. Moreover, substrate inhibition by pyruvate above 10 mM was observed for ω-TA from V. fluvialis JS17. In the case of product inhibition, acetophenone showed much greater inhibitions than L-alanine for all ω-TAs. Comparison of the enzyme properties indicates that ω-transaminase from V. fluvialis JS17 is the best one for both kinetic resolution and asymmetric synthesis to produce enantiomerically pure chiral amines. Kinetic resolution of sec-butylamine (20 mM) was done under reduced pressure (150 Torr) to selectively remove an inhibitory product (2-butanone) using the enzyme from V. fluvialis JS17. Enantiomeric excess of (R)-sec-butylamine reached 94.7％ after 12 h of reaction.
Key words: ω-transaminase; enrichment culture; chiral amine; kinetic resolution; asymmetric synthesis

-14-
Production of Pyridoxal Phosphate by a Mutant Strain
of Schizosaccharomyces pombe

Ruamsub CHUMNANTANA, Kumi HIROSE, Hiromichi BABA, and Toshiharu YAGI

Department of Bioresources Science, Faculty of Agriculture, Kochi University, Monobe-Otsu 200,
Nankoku, Kochi 783-8502, Japan

Received February 9, 2001; Accepted March 26, 2001
Conditions for extracellular production of vitamin B6 compounds (B6), especially pyridoxal 5′-phosphate (PLP) by Schizosaccharomyces pombe leu1 strain were examined. The productivity was dependent on concentration of L-leucine in the culture medium: 30 mg/l gave the highest concentrations of total B6 and PLP. The viable cells harvested at different growth phases showed different productivity: middle and late exponential phase cells showed the highest productivity of total B6 and PLP, respectively. D-Glucose (1％, w/v) among other sugars gave the best productivity. Supplementation of air and ammonium sulfate significantly increased extracellular production of PLP. Superoxide anion producers, menadione and plumbagin, and H2O2 increased the productivity of PLP. Cycloheximide inhibited the increase of PLP by the oxidative stress and, in contrast, increased pyridoxine.
Key words: pyridoxal phosphate-production; oxidative stress; yeast; Schizosaccharomyces pombe

-15-
Improvement of Freezing Tolerance in Transgenic Tobacco Leaves
by Expressing the hiC6 Gene

Ken-ichi HONJOH,1,† Hideyuki SHIMIZU,1 Noriko NAGAISHI,1 Hiroko MATSUMOTO,1 Koushirou SUGA,1,2 Takahisa MIYAMOTO,1 Masayoshi IIO,1 and Shoji HATANO3

1Division of Food Biotechnology, Department of Bioscience and Biotechnology, Faculty of Agriculture,
Graduate School, Kyushu University, Fukuoka 812-8581, Japan
2Chlorella Industries Co. Ltd., 1343 Hisatomi, Chikugo, Fukuoka 833-0056, Japan
3Graduate School of Health and Social Welfare Science, Nishikyushu University, 4490-9 Ooazaozaki,
Kanzaki-machi, Kanzaki-gun, Saga 842-8585, Japan

Received February 13, 2001; Accepted April 2, 2001
A cryoprotective protein, HIC6, was expressed transgenically in tobacco, a cold-sensitive plant, and the localization of the protein within the cell as well as freezing tolerance of the transgenic tobacco was investigated. For constitutive expression of HIC6 in tobacco, its corresponding gene was subcloned into pBI121. Through the transformation with pBI121/hiC6, fifteen transgenic tobacco lines were acquired, out of which twelve lines expressed the HIC6 protein. None of the transgenic tobacco lines, however, showed significant differences in freezing tolerance from the control plants (wild-type and transformed with pBI121) at -1, -3, and -4°C, with the exception that their freezing temperature was -2°C. In order to increase the accumulation level of HIC6, pBE2113 with a stronger promoter was used. Eight lines expressed the protein out of thirteen lines transformed with pBE2113/hiC6. The accumulation levels of the protein were clearly higher in the tobacco plants transformed with pBE2113/hiC6 than in those with pBI121/hiC6. The HIC6 protein seemed to be localized in mitochondria of the transgenic tobacco plants. Freezing-tolerance tests at -1---4°C showed that the degree of electrolyte leakage was significantly lower in the plants with pBE2113/hiC6 than in the control plants. A leaf browning observation also showed that high accumulation of HIC6 significantly suppressed injury caused by freezing to the transgenic tobacco at -3°C.
Key words: Chlorella vulgaris C-27; freezing tolerance; LEA protein; Nicotiana tabacum; transgenic plants

-16-
Structural Confirmation of 15-Norlubiminol and 15-Norepilubiminol,
Isolated from Solanum aethiopicum, by Chemical Conversion
from Lubimin and Epilubimin, and their Antifungal Activity

Ayako WATANABE,1,2 Hiroaki TOSHIMA,1 Hiroshi NAGASE,1 Toshinori NAGAOKA,3
and Teruhiko YOSHIHARA1,2,†

1Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University,
Sapporo 060-8589, Japan
2CREST, Japan Science and Technology Corporation, Honcho 4-1-8, Kawaguchi 332-0012, Japan
3Faculty of Applied Biological Science, Hiroshima University, Kagamiyama 1-4-4,
Higashi-Hiroshima 739-8528, Japan

Received February 22, 2001; Accepted April 17, 2001
15-Norlubiminol and 15-norepilubiminol were obtained from Solanum aethiopicum as an inseparable 1:1 mixture in a relatively poor yield to that of the major phytoalexins, lubimin and epilubimin. Their structures were confirmed by chemical conversion starting from lubimin and epilubimin. Baeyer-Villiger oxidation of the protected lubimins with m-chloroperoxybenzoic acid provided the desired formates. Deoxygenation with triphenylphosphine selenide and subsequent methanolysis provided 15-norlubiminols, whose 1H-NMR spectra were respectively identical with that of the corresponding isomer in the natural 15-norlubiminol mixture. The antifungal activity of 15-norlubiminols would be weaker than that of lubimins.
Key words: lubimin; epilubimin; sesquiterpene; 15-norlubiminol; 15-norepilubiminol

-17-
Characterization of Clofibrate-induced Retrograde Golgi Membrane Movement
to the Endoplasmic Reticulum: Clofibrate Distinguishes the Golgi
from the Trans Golgi Network

Machiko NAKAMURA,＊,¶ Natsuko KUROIWA,＊,‡ Yoshiki KONO,¶ and Akira TAKATSUKI＊,†

＊Animal and Cellular Systems Laboratory, RIKEN (The Institute of Physical and Chemical Research),
Hirosawa 2-1, Wako-shi, Saitama 351-0198, Japan
¶School of Agriculture, Ibaraki University, Ami, Ibaraki 300-0332, Japan
‡School of Life Science, Tokyo University of Pharmacy and Life Science, Hachioji, Tokyo 192-0392, Japan

Received February 23, 2001; Accepted April 7, 2001
Clofibrate-induced retrograde Golgi membrane movement was blocked or retarded when NRK cells were treated with sodium azide/2-deoxyglucose, nocodazole, taxol, and destruxin B, indicating that it depends on energy, and the dynamic state of microtubules, and being acidic or vacuolar-type ATPase function. PDMP and phospholipase A_{2} inhibitors also blocked it. These characteristics are similar to those of brefeldin A (BFA) and nordihydroguaiaretic acid (NDGA), inducers of retrograde Golgi membrane movement. However, clofibrate was distinguished from BFA in that BFA action was insensitive to phospholipase A_{2} inhibitors and from NDGA in that NDGA stabilized microtubules against nocodazole and its action was almost insensitive to taxol. The trans Golgi network (TGN) was resistant to clofibrate, while BFA and NDGA dispersed it. To our knowledge, clofibrate is the first drug to show such different effects on the Golgi and TGN and, therefore, is expected to be a useful tool to distinguish their architecture and/or membrane dynamics.
Key words: clofibrate; brefeldin A; nordihydroguaiaretic acid; retrograde trafficking; trans Golgi network

-18-
Cloning, Sequencing, and Expression of the Gene Encoding an Intracellular
β-D-Xylosidase from Streptomyces thermoviolaceus OPC-520

Hiroshi TSUJIBO,† Chiaki TAKADA, Akihiko TSUJI, Mitsuo KOSAKA,
Katsushiro MIYAMOTO, and Yoshihiko INAMORI

Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan

Received March 1, 2001; Accepted April 18, 2001
The intracellular β-xylosidase was induced when Streptomyces thermoviolaceus OPC-520 was grown at 50°C in a minimal medium containing xylan or xylo-oligosaccharides. The 82-kDa protein with β-xylosidase activity was partially purified and its N-terminal amino acid sequence was analyzed. The gene encoding the enzyme was cloned, sequenced, and expressed in Escherichia coli. The bxlA gene consists of a 2,100-bp open reading frame encoding 770 amino acids. The deduced amino acid sequence of the bxlA gene product had significant similarity with β-xylosidases classified into family 3 of glycosyl hydrolases. The bxlA gene was expressed in E. coli, and the recombinant protein was purified to homogeneity. The enzyme was a monomer with a molecular mass of 82 kDa. The purified enzyme showed hydrolytic activity towards only p-nitrophenyl-β-D-xylopyranoside among the synthetic glycosides tested. Thin-layer chromatography analysis showed that the enzyme is an exo-type enzyme that hydrolyze xylo-oligosaccharides, but had no activity toward xylan. High activity against pNPX occurred in the pH range 6.0--7.0 and temperature range 40--50°C.
Key words: β-xylosidase; xylan degradation; Streptomyces thermoviolaceus

-19-
A Recombinant Molt-inhibiting Hormone of the Kuruma Prawn Has a Similar
Secondary Structure to a Native Hormone: Determination of Disulfide Bond
Arrangement and Measurements of Circular Dichroism Spectra

Hidekazu KATAYAMA,1 Tsuyoshi OHIRA,1 Koji NAGATA,2 and Hiromichi NAGASAWA1

1Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences,
The University of Tokyo, Yayoi, Bunkyo, Tokyo 113-8657, Japan
2Biotechnology Research Center, The University of Tokyo, Yayoi, Bunkyo, Tokyo 113-8657, Japan

Received March 22, 2001; Accepted May 7, 2001
In crustaceans, molt-inhibiting hormone (MIH) is presumed to regulate molting through suppressing synthesis and/or secretion of ecdysteroids by the Y-organ. Recently, a recombinant MIH of the kuruma prawn Penaeus japonicus was produced in E. coli. To approximate the secondary structure of native and recombinant MIH of P. japonicus containing six cysteine residues, the arrangements of disulfide bridges in both MIHs were determined by characterizing their enzymatic digests, and their circular dichroism spectra were measured. The arrangements of disulfide bonds in both MIHs were determined to be identical, and they were linked between Cys7 and Cys44, Cys24 and Cys40, and Cys27 and Cys53. The circular dichroism spectra of both MIHs were very close, and demonstrated that they were rich in α-helix. α-Helix contents in native and recombinant MIHs were calculated to be 49.3％ and 46.0％, respectively. All these results strongly suggested that the recombinant MIH was folded in the same manner as the native MIH.
Key words: molt-inhibiting hormone; crustacean hyperglycemic hormone; Penaeus japonicus; disulfide bond arrangement; secondary structure
js8-302Biosci. Biotechnol. Biochem., 65 (8), 1840--1848, 2001Cuticle Phosphorylated Peptide of Procambarus clarkiiH. INOUE et al.

-20-
Purification and Structural Determination of a Phosphorylated Peptide
with Anti-calcification and Chitin-binding Activities in the Exoskeleton
of the Crayfish, Procambarus clarkii

Hirotaka INOUE, Noriaki OZAKI, and Hiromichi NAGASAWA†

Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences,
The University of Tokyo, Yayoi, Bunkyo, Tokyo 113-8657, Japan

Received March 22, 2001; Accepted May 7, 2001
Organic matrices in calcified hard tissues have been considered to control calcification. A matrix peptide, designated CAP-1, was extracted and purified by anion-exchange and reverse-phase high performance liquid chromatographies from the exoskeleton of the crayfish, Procambarus clarkii. The amino acid sequence of CAP-1 was determined by mass spectral and sequence analyses of the intact peptide and its enzymatically digested peptides. CAP-1 consisted of 78 amino acid residues, including a phosphoserine residue, and was rich in acidic amino acid residues. CAP-1 had a Rebers-Riddiford consensus sequence, which is conserved in cuticle proteins from many arthropods. CAP-1 inhibited precipitation of calcium carbonate in an in vitro anti-calcification assay dose-dependently, and completely inhibited it at 3×10-7 M. CAP-1 also showed chitin-binding ability, indicating that this molecule was bifunctional and played an important role in formation of the exoskeleton.
Key words: Procambarus clarkii; calcification; cuticle peptide; phosphorylated peptide

-21-
Note
Electron Transport Inhibitor in Cyperus javanicus

Masanori MORIMOTO,† Yoshiyuki SHIMOMURA, Ryoko MIZUNO, and Koichiro KOMAI

Department of Agricultural Chemistry, Faculty of Agriculture, Kinki University, Nakamachi,
Nara 631-8505, Japan

Received July 28, 2000; Accepted April 13, 2001
The natural quinone, hydroxydietrichequinone (3-
heptadec-8-enyl-2-hydroxy-5-methoxy-[1,4]benzoqui-
none) is a secondary metabolite of Cyperus javanicus. We found that this quinone inhibited both mitochondrial respiration and photosynthesis in their electron transportation systems. The quinone was found to have a mode of action against the ubiquinone reductase site from the results of different electron donor experiments on intact mitochondria from rat liver. The electron transport system, photosystem-II (PS-II), in chroloplast from spinach leaves was inhibited by the quinone in a similar way to that of the triazin sires herbicide, atrazin, with its mode of action against PS II. This natural quinone has a long aliphatic chain (C17) including an unsaturated bond at its midpoint. We recognized 8-9 unsaturated bonds in the aliphatic chain from an MS analysis of the methylthio-addact, and spectral data presumed a configuration of cis. form.
Key words: hydroxydietrichequinone; Cyperus javanicus; electron transport inhibitor; respiration; photosynthesis
Biosci. Biotechnol. Biochem., 65 (8), 1852--1855, 2001Peroxidase Induction by Active Oxygen Radicals in Rice BladesA. HIGA et al.Note

-22-
Note
Active Oxygen Radicals Induce Peroxidase Activity in Rice Blade Tissues

Arisa HIGA, Tomoe HIDAKA, Yuji MINAI, Yoshitaka MATSUOKA, and Minoru HAGA

Department of Applied Biological Chemistry, Faculty of Agriculture, Tamagawa University,
6-1-1 Tamagawagakuen, Machida-shi, Tokyo 194-8610, Japan

Received November 6, 2000; Accepted April 9, 2001
The relationship between active oxygen radicals and peroxidase induction on disease resistance in rice blades was investigated. Nitric oxide was produced in the whole blade stimulated by blast fungus elicitor. The induction of peroxidase activity was detected in active oxygen radical-treated rice blades 1 hour after treatment and thereafter. These results suggest that active oxygen radicals produced by stimulation with the elicitor could trigger peroxidase induction.
Key words: active oxygen radicals; nitric oxide; peroxidase; rice blade

-23-
Note
Isolation and Structure Elucidation of a New Antifungal and Antibacterial
Antibiotic Produced by Streptomyces sp. 201†

Gajen N. BORDOLOI, Babita KUMARI, Arijit GUHA, Manobjyoti BORDOLOI,＊
R. N. S. YADAV,＃ Monoj K. ROY, and Tarun C. BORA

Biochemistry Division and Natural Product Chemistry,＊ Regional Research Laboratory (CSIR),
Jorhat-785006, Assam, India
＃Department of Life Sciences, Dibrugarh University, Dibrugarh, Assam, India

Received November 6, 2000; Accepted March 26, 2001
An antibacterial and antifungal antibiotic was isolated from the culture filtrate of Streptomyces sp. 201, and its structure was determined as 2-methyl-heptyl isonicotinate by extensive use of NMR spectroscopy. The compound exhibited marked antimicrobial activity against Bacillus subtilis, Shigella sp., Klebsiella sp., E. coli, Proteus mirabilis, and the pathogenic fungi, Fusarium moniliforme, F. semitectum, F. oxysporum, F. solani and Rhizoctonia solani.
Key words: Streptomyces sp. 201; antibacterial; antifungal; antibiotic; 2-methylheptyl isonicotinate

-24-
Note
Inhibitory Effects of Capsaicinoids on Fatty Acid Desaturation
in a Rat Liver Cell Line

Nobuhiro NAKANO, Norifumi SHIRASAKA, Kazuki MASUOKA, Tetsuo MURAKAMI,
Tatsuo WATANABE,1 Kenji KOBATA,1 Sakayu SHIMIZU,2 and Hajime YOSHIZUMI†

Department of Food Science and Nutrition, Faculty of Agriculture, Kinki University,
3327-204 Naka-machi, Nara 631-8505, Japan
1School of Food and Nutritional Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan
1Division of Applied Life Science, Graduate School of Agriculture, Kyoto University, Sakyo-ku,
Kyoto 606-8502, Japan

Received November 20, 2000; Accepted March 29, 2001
The inhibitory effects of such vanillylamides as capsaicin and nine capsaicinoids on fatty acid desaturation in liver cells were investigated by using the cultured rat liver cell line, BRL-3A. When capsaicin was added to the medium, it had a relatively strong inhibitory effect on Δ6 desaturation and clear inhibitory effects on Δ5 and C24Δ6 desaturation (Δ6 desaturation of C24-polyunsaturated fatty acids). Capsaicinoids with side carbon chain lengths of C10:0 and C12:0 expressed the maximum inhibitory effects of the nine capsaicinoids on fatty acid desaturation in the BRL-3A cells. The inhibitory effects of the capsaicinoids were not correlated with their pungency.
Key words: desaturation; polyunsaturated fatty acid; capsaicin; capsaicinoid

-25-
Note
Prevention by Lactic Acid Bacteria of the Oxidation of Human LDL

Masaki TERAHARA,† Saori KURAMA, and Naoki TAKEMOTO

Food Functionality Research Institute, Meiji Milk Products Co., Ltd., 1-21-3 Sakae-Cho, Higashimurayama,
Tokyo 189-8530, Japan

Received November 22, 2000; Accepted April 13, 2001
Ether extracts of lactic acid bacteria were analyzed for prevention of the oxidation of erythrocyte membrane and human low-density lipoprotein in vivo.
Streptococcus thermophilus 1131 and Lactobacillus delbrueckii subsp. bulgaricus 2038, yogurt starters, were chosen as test-strains, and ether extracts of these cultures were used as samples. Both strain 1131 and strain 2038 produced radical scavengers and inhibited oxidation of erythrocyte membranes and low-density lipoproteins. The antioxidative activity of strain 2038 was higher than that of strain 1131.
Key words: erythrocyte membrane; thiobarbituric acid-reactive substances; low-density lipoprotein

-26-
Note
Ellagic Acid Formation from Galloylglucoses by a Crude Enzyme
of Cornus capitata Adventitious Roots

Norie TANAKA＊,†, Koichiro SHIMOMURA,＊＊ and Kanji ISHIMARU

＊Department of Applied Biological Sciences, Faculty of Agriculture, Saga University, 1 Honjo,
Saga 840-8502, Japan
＊＊Tsukuba Medicinal Plant Research Station, National Institute of Health Sciences, 1 Hachimandai,
Tsukuba, Ibaraki 305-0843, Japan

Received December 4, 2000; Accepted February 9, 2001
The aqueous extract of acetone powder, which had been prepared from Cornus capitata <0192>Mountain Moon′ adventitious roots, cultured in MS medium with a high concentration of Cu2+(10 μM), showed strong oxidative activity toward galloylglucoses. A compound formed from galloyglucoses, such as 1,2,3,4,6-penta-O-galloyl-β-D-glucose and tannic acid, by the reaction with the crude enzyme solution of the adventitious roots was isolated and characterized as ellagic acid by spectrometric analyses.
Key words: Cornus capitata; adventitious root culture; tannic acid; 1,2,3,4,6-penta-O-galloyl-β-D-glucose; ellagic acid

-27-
Note
Role of Activity of Gastrointestinal Microflora in Absorption of Calcium
and Magnesium in Rats Fed β1--4 Linked Galactooligosaccharides

Osamu CHONAN, Rie TAKAHASHI, and Masaaki WATANUKI

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

Received December 13, 2000; Accepted April 9, 2001
Rats fed a diet containing β1--4 linked galactooligosaccharides (GOS) (5 g/100 g of diet) absorbed calcium and magnesium more efficiently than those fed the control diet. However, the increment obtained through GOS-feeding was reduced by neomycin sulfate (0.67 g/100 g of diet). Since the decrease in cecal pH in rats fed GOS was suppressed by neomycin-feeding, bacterial action in the digestive tract was considered to be reduced by neomycin-feeding. Our findings suggest that the action of intestinal bacteria is necessary for the effects of GOS.
Key words: galactooligosaccharides; calcium; magnesium; absorption; neomycin

-28-
Note
Purification and Characterization of L-2,3-Butanediol Dehydrogenase
of Brevibacterium saccharolyticum C-1012 Expressed in Escherichia coli

Yusuke TAKUSAGAWA,1,† Masato OTAGIRI,1 Sadaharu UI,1 Takashi OHTSUKI,1
Akio MIMURA,1 Moriya OHKUMA,2 and Toshiaki KUDO2

1Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Yamanashi University,
Takeda, Kofu, Yamanashi 400-8511, Japan
2Laboratory of Microbiology, The Institute of Physical and Chemical Research (RIKEN), Hirosawa,
Wako, Saitama 351-0198, Japan

Received December 25, 2000; Accepted April 4, 2001
The L-2,3-butanediol dehydrogenase produced in E. coli JM109/pLBD2-CTC was purified by 5 steps. The molecular mass of this enzyme was estimated at 110 kDa and the subunit was measured to be 30 kDa. The L-BDH had some differences from the BDHs from other sources in substrate specificity, pI value, pH stability, effects of divalent cations, and organic acids.
Key words: 2,3-butanediol dehydrogenase; 2,3-butanediol; acetoin; Brevibacterium saccharolyticum C-1012; short-chain dehydrogenase/reductase family

-29-
Note
Synthesis of the L-Enantiomer of 4′-C-Ethynyl-2′-deoxycytidine

Satoru KOHGO,1 Hiroaki MITSUYA,2,3 and Hiroshi OHRUI1,†

1Division of Life Science, Graduate School of Agricultural Science, Tohoku University,
1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan
2Department of Immunopathophysiology and Internal Medicine II, Kumamoto University School of Medicine,
Honjo, Kumamoto 860-8556, Japan
3Experimental Retrovirology Section, Medicine Branch, Division of Clinical Sciences, National Cancer Institute,
Bethesda, Maryland 20892, USA

Received January 9, 2001; Accepted March 2, 2001
The L-enantiomer of 4′-C-ethynyl-2′-deoxycytidine (2) was synthesized, but did not show any activity against HIV-1 up to 100 μM.
Key words: 4′-C-ethynyl-2′-deoxycytidine; L-configuration nucleosides; anti-HIV activity

-30-
Note
Chitinase in Cucumber Xylem Sap

Susumu MASUDA,1,† Hiroshi KAMADA,2 and Shinobu SATOH2

1Noda Institute for Scientific Research, Noda 399, Noda, Chiba 278-0037, Japan
2University of Tsukuba, Institute of Biological Sciences, Tsukuba, Ibaraki 305-8572, Japan

Received January 11, 2001; Accepted March 16, 2001
A chitinase activity was detected in fractions of xylem sap collected from the cut surface of cucumber stems. A 28-kDa acidic protein was purified from the active fractions and its N-terminal amino acid sequence was found to be identical to that of a chitinase gene. Cucumber roots produce and secrete an acidic chitinase, one of the PR proteins, into xylem sap and deliver it to aboveground organs.
Key words: cucumber; chitinase; root; xylem sap

-31-
Note
Specific Antimicrobial Synergism of Synthetic Hydroxy Isothiocyanates
with Aminoglycoside Antibiotics

Hirokuni TAJIMA, Hisashi KIMOTO,1 and Akira TAKETO2

Fukui Research Laboratory, Rengo Co., Ltd., 10-8-1, Jiyugaoka, Kanazu-cho, Sakai-gun, Fukui 919-0604,
Japan
1Department of Biochemistry I, Fukui Medical School, Matsuoka, Fukui 910-1103, Japan
2Department of Applied Physics and Chemistry, Fukui University of Technology, Fukui 910-8505, Japan

Received January 18, 2001; Accepted March 29, 2001
Hydroxy isothiocyanates, especially 2-(4-hydroxyphenyl)ethyl isothiocyanate (hITC), were examined for antimicrobial synergism with several antibiotics against Escherichia coli and Staphylococcus aureus, using a multiwell plate system. hITC had antibacterial synergism, specifically with aminoglycoside antibiotics. The synergism was observed in synthetic medium (M9 minimal medium) or soybean casein digest broth, but not in nutrient broth. Synergism was seen in the presence of certain sugars such as glucose, fructose, and maltose in the medium.
Key words: hydroxy isothiocyanate; 2-(4-hydroxyphenyl)ethyl isothiocyanate; synergism; aminoglycoside; streptomycin

-32-
Note
Increased Transglycosylation Activity of Rhodotorula glutinis
Endo-β-Glucanase in Media Containing Organic Solvent

Tadao OIKAWA,1,2 Yasuyuki TSUKAGAWA,1 Masashi CHINO,1 and Kenji SODA1,2,†

1Department of Biotechnology, Faculty of Engineering, Kansai University; 2Kansai University
High Technology Research Center, Suita-shi, Osaka 564-8680, Japan

Received January 22, 2001; Accepted March 21, 2001
The transglycosylation of p-nitrophenyl-β-D-cellotrioside to cellotetraose catalyzed by endo-1,4-β-glucanase (cellulase, EC 3.2.1.4) from a psychrotrophic yeast, Rhodotorula glutinis KUJ 2731, was increased by addition of a miscible organic solvent in the reaction mixture. Among various organic solvents tested, acetone was most effective. The transglycosylation activity increased with an increase in acetone concentrations, while hydrolysis activity was suppressed. The transglycosylation preferably occurred at acidic pH with the optimum pH at 2 in 10 mM Gly-HCl buffer. The optimum temperature of transglycosylation was found to be 50°C in the presence of 40％ acetone.
Key words: transglycosylation; endo-1,4-β-glucanase; Rhodotorula glutinis; psychrotrophic yeast

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Effect of Germanium-132 on Low-density Lipoprotein Oxidation
and Atherosclerosis in Kurosawa and Kusanagi Hypercholesterolemic Rabbits

Yasuhisa WAKABAYASHI†

Department of Medicine, University of Kitasato Medical School, 1-15-1 Kitasato, Sagamihara 228-8555, Japan

Received January 29, 2001; Accepted March 16, 2001
Germanium-132 (Ge-132) was given at 200 mg/kg of body weight to 8-week-old Kurosawa and Kusanagi hypercholesterolemic (KHC) rabbits. Thirty-six weeks later, the susceptibility of plasma low-density lipoprotein to oxidation and the morphology of atherosclerosis in the aorta and coronary artery were investigated. Treatment with Ge-132 resulted in decreases in the oxidation rate and in the formation rate of thiobarbituric acid-reactive substances following copper-induced oxidation of LDL. Ge-132 is suggested to possess antioxidative properties, but this did not lead to any attenuation of atherosclerotic progression in the KHC rabbits.
Key words: germanium-132 (Ge-132); antioxidant; low-density lipoprotein; atherosclerosis; Kurosawa and Kusanagi hypercholesterolemic (KHC) rabbits

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Enzymatic Glycosylation of Lincomycin

Lenka WEIGNEROV<0193>A, Jaroslav SP<0193>I<0189>ZEK, Lucie NAJMANOV<0193>A, and Vladim<0225><0131>r K<0189>REN†

Institute of Microbiology, Academy of Sciences of the Czech Republic, V<0225><0131>de<0190>nsk<0193>a 1083,
CZ 142 20 Prague 4, Czech Republic

Received January 29, 2001; Accepted April 3, 2001
Lincomycin (1), a glycosidic antibiotic, active against Gram-positive bacteria, was modified enzymatically with the aim of improving its physico-chemical and biological properties. Compound 1 was glycosylated using jack bean α-mannosidase to produce 7-O-α-D-mannopyranosyl-lincomycin (2).
Key words: lincomycin; enzymatic glycosylation; mannosylation

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Influences of Nitrogen Sources on Usnic Acid Production in a Cultured
Mycobiont of the Lichen Usnea hirta (L.) Wigg.

Yasuhiro KINOSHITA,1 Yoshikazu YAMAMOTO,2,† Teiko KUROKAWA,3 and Isao YOSHIMURA3

1Basic Research Department, Nippon Paint Co., Ltd., 4-1-15, Minamishinagawa, Shinagawa,
Tokyo 140-8675, Japan
2Department of Biological Production, Faculty of Bioresource Sciences, Akita Prefectural University,
Akita 010-0195, Japan
3Kochi Gakuen College, 292 Asahitenjincho, Kochi 780-0955, Japan

Received February 1, 2001; Accepted March 15, 2001
Effects of the nitrogen sources in the medium for the production of secondary metabolites in lichens were examined. The usnic acid production by a mycobiont of the lichen Usnea hirta was higher in the liquid medium containing ammonium and nitrate ions than in those containing amino acids.
Key words: Usnea hirta; lichen mycobiont culture; usnic acid production; nitrogen source

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Enantioselective Synthesis of Ancepsenolide and its Analogs

Katsuki TAKAI and Ryozo IRIYE†

Department of Bioscience and Biotechnology, Faculty of Agriculture, Shinshu University (United Graduate
School of Gifu University), 8304 Minamiminowa, Kamiina, Nagano 399-4598, Japan

Received February 2, 2001; Accepted March 12, 2001
Ancepsenolide (1a-s) and the enantiomer (1a-r) were respectively synthesized from (S)- and (R)-2-[(R)-O-MEM-mandeloyloxy]propanal (3a-s and 3a-r) and diisopropyl hexadecanedioate (5). The analogs (1b, 2a and 2b) were synthesized by a similar method.
Key words: ancepsenolide; aldol reaction; optical resolution; butenolide

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Multiplicity of Aromatic Ring Hydroxylation Dioxygenase Genes
in a Strong PCB Degrader, Rhodococcus sp. Strain RHA1 Demonstrated
by Denaturing Gradient Gel Electrophoresis

Wataru KITAGAWA,1 Asaka SUZUKI,2 Toshihiro HOAKI,2 Eiji MASAI,1 and Masao FUKUDA1

1Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata 940-2188, Japan
2TAISEI Research Institute, Yokohama, Kanagawa 245-0051, Japan

Received February 9, 2001; Accepted March 30, 2001
To address the multiplicity of aromatic ring hydroxylation dioxygenases, we used PCR amplification and denaturing gradient gel electrophoresis (DGGE). The amplified DNA fragments separated into five bands, A to E. Southern hybridization analysis of RHA1 total DNA using the probes for each band showed that band C originated from a couple of homologous genes. The nucleotide sequences of the bands showed that bands A, C, and E would be parts of new dioxygenase genes in RHA1. That of band B agreed with the bphA1 gene, which was characterized previously. That of band D did not correspond to any known gene sequences. The regions including the entire open reading frames (ORFs) were cloned and sequenced. The nucleotide sequences of ORFs suggested that the genes of bands A, C, and E may respectively encode benzoate, biphenyl, and poly-hydrocarbon dioxygenases. Northern hybridization indicated the induction of the gene of band A by benzoate and biphenyl, and that of the gene of band C by biphenyl and ethylbenzene, supporting the above notions. The gene of band E was not induced by any of these substrates. Thus the combination of DGGE and Southern hybridization enable us to address the multiplicity of the ring hydroxylation dioxygenase genes and to isolate some of them.
Key words: DGGE; Rhodococcus; PCB; aromatic ring dioxygenase; diversity

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Mutational Analyses of Dictyostelium IQGAP-Related Protein GAPA:
Possible Interaction with Small GTPases in Cytokinesis

Masao SAKURAI, Hiroyuki ADACHI,＃,† and Kazuo SUTOH

Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, 3-8-1 Komaba,
Meguro-ku, Tokyo 153-8902, Japan

Received February 13, 2001; Accepted March 13, 2001
GAPA is an IQGAP-related protein and is involved in Dictyostelium cytokinesis. Since mammalian IQGAPs are effectors for Rac/Cdc42, GAPA is also predicted to bind to small GTPases, which are to be identified. In this study, mutant GAPAs were examined for functions in cytokinesis by genetic complementation of gapA- cells. Positively charged side chains of Arg442 and Lys474 of GAPA, predicted to be present on the surface of interaction with small GTPases, were found to be essential, suggesting an interaction between GAPA and putative small GTPase in cytokinesis. Also, results from truncated GAPAs indicated that almost the entire region of GAPA homologous to IQGAP is required for cytokinesis in Dictyostelium.
Key words: cytokinesis; Dictyostelium; GAPA; IQGAP; Rho family small GTPases

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A New Double Coupling System: Synthesis of Citronellyl Acetate via
Transacetylation to Citronellol from Acetyl Coenzyme A Produced
from Glucose and Free Fatty Acids

Shinobu ODA1,† and Hiromichi OHTA2

1Technical Research Laboratory, Kansai Paint Co., Ltd., 4-17-1 Higashi-Yawata, Hiratsuka,
Kanagawa 254-8562, Japan
2Department of Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi,
Kohoku-ku, Yokohama, Kanagawa 223-8522, Japan

Received February 14, 2001; Accepted March 19, 2001
A double coupling system, which couples metabolism of glucose and transacetylation, is a unique procedure for the production of acetic esters. In the novel coupling system described in this article, acetyl coenzyme A (acetyl-CoA) was supplied via metabolism of both glucose and exogenous saturated fatty acids. While short and middle chain fatty acids having C4--8 were very biotoxic, myristic acid (C14) was effectively used as a source of acetyl-CoA.
Key words: interface bioreactor; coupling system; transacetylation; β-oxidation; acetic ester

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NMR Determination of the Absolute Configuration of a Macrophomate
Synthase Inhibitor by Using an Axial Chiral Reagent

Hiroki FUKUI,1 Yukiharu FUKUSHI,1,2,† Satoshi OHASHI,1 Hideaki OIKAWA,1
and Satoshi TAHARA1,2

1Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Kita-ku,
Sapporo 060-8589, Hokkaido, Japan
2CREST, Japan Science and Technology Corporation, 4-1-8 Honmachi, Kawaguchi 332-0012, Saitama, Japan

Received February 21, 2001; Accept April 7, 2001
Macrophomate synthase catalyzes an extraordinary four-step transformation from oxalacetate and 2-pyrone to macrophomic acid by an intermolecular Diels-Alder reaction. The absolute configuration of the most potent macrophomate synthase inhibitor; (-)-2-carboxylmethyl-1-methoxybicyclo[2.2.2]oct-5-ene-2-carboxylic acid, was determined to be (1S, 2R, 4R) by using an axial chiral reagent.
Key words: macrophomate synthase; absolute configuration; axially chiral reagent; MBCC; NOE

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Suppressive Effect of Coffee on Lipopolysaccharide-induced Hepatitis
in D-Galactosamine-sensitized Rats

Puming HE, Yasuhiro NODA, and Kimio SUGIYAMA†

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

Received February 23, 2001; Accepted April 12, 2001
A coffee extract significantly suppressed lipopolysaccharide (LPS)-induced hepatitis in D-galactosamine-sensitized rats, as assessed by the plasma alanine and aspartate aminotransferase activities, when it was added to the diet (30 g/kg) and fed to rats for 14 days. Its effect was as strong as that of a green tea extract. The coffee extract suppressed LPS-induced hepatitis when singly force-fed (1.2 g/kg) 1.5 h prior to the injection of the drugs, whereas a decaffeinated coffee extract had no significant effect. The hepatoprotective effect of caffeine was stronger than that of theobromine. These results indicate that coffee can protect animals from LPS-induced hepatitis, and that the effect of coffee might be mainly due to caffeine.
Key words: coffee; caffeine; theobromine; lipopolysaccharide; hepatitis

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A Transformation System for an Ectomycorrhizal Basidiomycete,
Lyophyllum shimeji

Takeshi SAITO,1,2 Norio TANAKA,2 and Takao SHINOZAWA1,†

1Depertment of Biological and Chemical Engineering, Faculty of Engineering, Gunma University,
Kiryu, Gunma 376-8515, Japan
2The Mushroom Research Institute of Japan, Kiryu, Gunma 376-0051, Japan

Received March 16, 2001; Accepted May 7, 2001
A transformation vector, pLS-hph, was constructed with the promoter and terminator of the glyceraldehyde-3-phosphate dehydrogenase (GPD) gene derived from an ectomycorrhizal basidiomycete, Lyophyllum shimeji, and with the hygromycin B (HmB) phosphotransferase (hph) gene from Escherichia coli. This vector was introduced into protoplasts of L. shimeji and 3.4 transformants per μg plasmid DNA were obtained. In most of the transformants, multiple copies of the vector were integrated into the genomic DNA. The results indicate that pLS-hph is a useful vector for L. shimeji.
Key words: Lyophyllum shimeji; transformation; ectomycorrhizal basidiomycete; hygromycin B