Contents and Abstracts of Latest Issue of BBB

(Vol.65 No.7 2001)

Review
New Aspects of Physiological and Pathophysiological Functions
of Adenosine A2A Receptor in Basal Ganglia
Hiroshi KASE・ p.1447

Effects of α-D-Glucosylglycerol on the in Vitro Digestion of Disaccharides
by Rat Intestinal Enzymes・
Fumihito TAKENAKA・・ and Hirofumi UCHIYAMA p.1458

Establishment of a New Cross of the Rice Blast Fungus Derived from Japanese
Differential Strain Ina168 and Hermaphroditic Rice Pathogen Guy11
Satoru FUKIYA, Megumi KODAMA, Hideki KITO, Teruo SONE, and Fusao TOMITA・ p.1464

Isolation and Characterization of Desulfitobacterium sp. strain Y51 Capable
of Efficient Dehalogenation of Tetrachloroethene and Polychloroethanes
Akiko SUYAMA,1,2 Ryo IWAKIRI,1 Keiichirou KAI,1 Takashi TOKUNAGA,3 Nobuyuki SERA,3
and Kensuke FURUKAWA1,・ p.1474

Synthesis of Optically Active N-(2-Pyridyloxiran-2-ylmethyl)
benzenesulfonamide Derivatives and Their Herbicidal Activity＊
Akemi HOSOKAWA,・ Manabu KATSURADA, Osamu IKEDA, Noriko MINAMI,
and Tetsuo JIKIHARA p.1482

Accelerative Effect of Olive Oil on Liver Glycogen Synthesis in Rats Subjected
to Water-immersion Restraint Stress
Hisanao TAKEUCHI,・ Norio SUZUKI, Masakazu TADA, and Puming HE p.1489

Characterization of the Replication Region of the Lactobacillus reuteri Plasmid pTC82 Potentially Used in the Construction of Cloning Vector
Chuen-Fu LIN, Jin-Li HO, and Tung-Ching CHUNG・ p.1495

Visualization of Nuclei in Aspergillus oryzae with EGFP and Analysis
of the Number of Nuclei in Each Conidium by FACS
Jun-ichi MARUYAMA, Harushi NAKAJIMA, and Katsuhiko KITAMOTO＊ p.1504

1-Aminocyclopropane-1-carboxylate Synthase of Penicillium citrinum: Primary
Structure and Expression in Escherichia coli and Saccharomyces cerevisiae
Yukiko KAKUTA, Toshinori IGARASHI, Toyotaka MURAKAMI, Hiroyuki ITO,
Hirokazu MATSUI, and Mamoru HONMA p.1511

In Vitro Biosynthesis of Homogalacturonan by a Membrane-bound
Galacturonosyltransferase from Epicotyls of Azuki Bean
Yoshimi TAKEUCHI and Yoichi TSUMURAYA・ p.1519

In Vitro Model using Mouse Hepatocytes for Study of Alcohol Stress
Jae-Won YANG, Jun-Seop SHIN, Jae-Jeong LEE, Hyo-Ihl CHANG and Chan-Wha KIM
p.1528

Insecticidal and Neural Activities of Candidate Photoaffinity Probes
for Neonicotinoid Binding Sites
Kazuhiko MATSUDA,1,・ Makoto IHARA,1 Keiichiro NISHIMURA,2
David B. SATTELLE,3 and Koichiro KOMAI1 p.1534

Identification of Amino Acid Residues Essential for the Substrate Specificity
of Flavobacterium sp. Endo-β-N-acetylglucosaminidase
Kiyotaka FUJITA, Ryo-ich NAKATAKE, Kayo YAMABE, Akira WATANABE,
Yasuhiko ASADA, and Kaoru TAKEGAWA・ p.1542

Purification and Characterization of Aminopeptidase B
from Escherichia coli K-12
Hideyuki SUZUKI,・ Sachiko KAMATANI, and Hidehiko KUMAGAI p.1549

First Stereoselective Synthesis of (+)-Magnostellin C, a Tetrahydrofuran
Type of Lignan Bearing a Chiral Secondary Benzyl Alcohol
Satoshi YAMAUCHI・ and Yoshiro KINOSHITA p.1559

In Vivo and in Vitro Analyses of the AmyR Binding Site of the Aspergillus
nidulans agdA Promoter; Requirement of the CGG Direct Repeat
for Induction and High Affinity Binding of AmyR
Shuji TANI, Tomoyuki ITOH, Masashi KATO, Tetsuo KOBAYASHI, and Norihiro TSUKAGOSHI
p.1568

Molecular Cloning, Sequencing, and Expression of cDNA Encoding Serine
Protease with Fibrinolytic Activity from Earthworm＊
Manabu SUGIMOTO1 and Nobuyoshi NAKAJIMA2,・ p.1575

Structural Change and Catalytic Activity of Horseradish Peroxidase
in Oxidative Polymerization of Phenol
Masaru AKITA, Daisuke TSUTSUMI, Masami KOBAYASHI, and Hideo KISE・ p.1581

A Novel Screening for Inhibitors of a Pleiotropic Drug Resistant Pump,
Pdr5, in Saccharomyces cerevisiae
Kazumi HIRAGA, Anoja WANIGASEKERA, Hisanori SUGI, Nobuyuki HAMANAKA,1
and Kohei ODA・ p.1589

Characteristics of Wine Produced by Mushroom Fermentation
Tokumitsu OKAMURA,・ Tomoko OGATA, Norie MINAMIMOTO, Tomomi TAKENO,
Hiroko NODA, Shoko FUKUDA, and Masahiro OHSUGI p.1596

Effects of Double Mutation at Two Distant IgE-binding Sites
in the Three-dimensional Structure of the Major House Dust Mite
Allergen Der f 2 on IgE-binding and Histamine-releasing Activity
Toshiro TAKAI,1,・ Hideki HATANAKA,2 Saori ICHIKAWA,2,3 Toyokazu YOKOTA,1
Fuyuhiko INAGAKI,2,4 and Yasushi OKUMURA1 p.1601

Purification and Substrate Specificity of Honeybee,
Apis mellifera L., α-Glucosidase III
Mamoru NISHIMOTO, Masaki KUBOTA, Masahisa TSUJI, Haruhide MORI,
Atsuo KIMURA, Hirokazu MATSUI, and Seiya CHIBA・ p.1610

Azurin Involved in Alcohol Oxidation System in Pseudomonas putida HK5:
Expression Analysis and Gene Cloning
Hirohide TOYAMA,・ Nahoko AOKI, Kazunobu MATSUSHITA, and Osao ADACHI p.1617

Cloning of a Gene Cluster Encoding Enzymes Responsible for the Mevalonate
Pathway from a Terpenoid-antibiotic-producing Streptomyces Strain
Yoshimitsu HAMANO,1 Tohru DAIRI,1,・ Masahiro YAMAMOTO,1 Takashi KAWASAKI,1
Kazuhide KANEDA,2 Tomohisa KUZUYAMA,2 Nobuya ITOH,1 and Haruo SETO2,・ p.1627

Is Your Ribozyme Design Really Correct?:
A proposal of simple single turnover competition assay to evaluate ribozymes
Terumichi TANAKA,1,・ Osamu INUI,1 Natsuki DOHI,2 Noriko OKADA,2
Hidechika OKADA,2 and Yo KIKUCHI1 p.1636

Note
Inhibition by Agaricus blazei Murill Fractions of Cytopathic Effect Induced
by Western Equine Encephalitis (WEE) Virus on VERO Cells in Vitro
Kenji SORIMACHI,1 Yukari IKEHARA,2 Genzo MAEZATO,2 Akira OKUBO,3,・
Sunao YAMAZAKI,3 Kazumi AKIMOTO,4 and Akira NIWA1 p.1645

Note
Purification of Saponin Compounds in Bupleurum falcatum
by Solvent Partitioning and Preparative LC
Namsoo KIM・ and In-Seon PARK p.1648

Note
Antimutagenicity of Deacylated Anthocyanins in Purple-fleshed Sweetpotato
Makoto YOSHIMOTO,・ Shigenori OKUNO, Masaatu YAMAGUCHI,＊ and Osamu YAMAKAWA
p.1652

Note
Acetyl-CoA Carboxylase Inhibitors from Avocado
(Persea americana Mill) Fruits
Hironori HASHIMURA, Chihoko UEDA, Jun KAWABATA,・ and Takanori KASAI p.1656

Note
Purification and Properties of Two Malate Dehydrogenases
from Candida sp. N-16 Grown on Methanol
Jun YOSHIKAWA, Katsura SEKI, Hirofumi SHINOYAMA, and Takaaki FUJII・ p.1659

Note
A Basic Class I Chitinase Expression in Winged Bean is Up-regulated
by Osmotic Stress
Yoshiko TATEISHI, Yoshimi UMEMURA, and Muneharu ESAKA p.1663

Note
Synthesis of cis-Lactone Lignan, cis-(2S,3R)-Parabenzlactone,
from L-Arabinose
Satoshi YAMAUCHI・ and Yoshiro KINOSHITA p.1669

Note
Anti-inflammatory and Anti-allergic Actions by Oral Administration
of a Perilla Leaf Extract in Mice
Hiroshi UEDA and Masatoshi YAMAZAKI p.1673

Note
Stereoselective Reduction of Ethyl 4-chloro-3-oxobutanoate by Fungi
Yuri SARATANI,1 Eiji UHEDA,1 Hiroaki YAMAMOTO,2 Atsuo NISHIMURA,1
and Fumiki YOSHIZAKO1,・ p.1676

Note
Purification and Characterization of an Esterase from Micrococcus sp.
YGJ1 Hydrolyzing Phthalate Esters
Keiichi AKITA, Chikako NAITOU, and Kiyofumi MARUYAMA・ p.1680

Note
Cloning and Nucleotide Sequence of the Mycodextranase Gene
from Streptomyces sp. J-13-3
Katsuichiro OKAZAKI,1,・ Takashi AMANO,1 Tetsuhiro MORIMOTO,1 Takahiro IEMOTO,1
Toshiyuki KAWABATA,1 Shigeru HAYAKAWA,2 and Kazuya AKIMITSU1 p.1684

Preliminary Communication
Introduction of Bacterial Metabolism into Higher Plants
by Polycistronic Transgene Expression
Hideo NAKASHITA,1・ Yuko ARAI,1,2 Toshiharu SHIKANAI,3 Yoshiharu DOI,1
and Isamu YAMAGUCHI1 p.1688

Preliminary Communication
The Expression of a Barley HvNAS1 Nicotianamine Synthase Gene
Promoter-gus Fusion Gene in Transgenic Tobacco is Induced
by Fe-deficiency in Roots
Kyoko HIGUCHI,1,2,・ Masaharu TANI,1 Hiromi NAKANISHI,1 Toshihiro YOSHIWARA,3
Fumiyuki GOTO,3 Naoko K. NISHIZAWA,4 and Satoshi MORI1,2 p.1692

-1-
Review
New Aspects of Physiological and Pathophysiological Functions
of Adenosine A2A Receptor in Basal Ganglia

Hiroshi KASE・

Pharmaceutical Research ＆ Development Division, Kyowa Hakko Kogyo Co. Ltd., 1-6-1 Ohtemachi,
Chiyoda-ku, Tokyo 100-8185, Japan

There is now growing interest in the functional role of adenosine A_{2A} receptors. Their distribution within the brain is restricted in the basal ganglia, particularly abundant in the striatum, which are thought to play a crucial role in the control of motor behavior. Indeed, newly developed A_{2A} receptor selective antagonists have a profound influence on motor functions, with anti-Parkinsonian activities in several animal models. Striatal spiny neurons serve as a major anatomical locus for the relay of cortical information flow through the basal ganglia. The GABA releasing projection neurons represent the A_{2A} receptor-mediated main target of adenosine. The GABAergic synaptic neurotransmission is regulated by adenosine via A_{2A} receptors on the presynaptic terminals. Blockade of this modulatory function by A_{2A} antagonists could repair striatopallidal abnormal neuronal activities provoked by striatal dopamine depletion in the Parkinsonian state. A_{2A} receptor antagonists provide a novel therapeutic potential for the treatment of Parkinson′s disease.
Key words: adenosine; receptor; movement; basal ganglia; neuromodulation

-2-
Effects of α-D-Glucosylglycerol on the in Vitro Digestion of Disaccharides
by Rat Intestinal Enzymes・

Fumihito TAKENAKA・・ and Hirofumi UCHIYAMA

Tatsuuma-honke Brewing Co. Ltd., 2-6 Tateishi-cho, Nishinomiya, Hyogo 662-0943, Japan

Recived October 30, 2000; Accepted March 2, 2001
α-D-Glucosylglycerol (GG) is a mixture of 2--O--α-D-glucosylglycerol (GG-II), (2R)-1--O--α-D-glucosylglycerol (R-GG-I) and (2S)-1--O--α-D-glucosylglycerol (S-GG-I). GG has been found to be slightly hydrolyzed in vitro only by rat intestinal enzymes, but hardly at all by other digestive juices. GG suppressed the hydrolysis of maltose, sucrose and isomaltose by rat intestinal enzymes because the amount of glucose in the digestion of a mixture of GG and disaccharide was less than the sum of that in each individual digestion. The consumption of GG was suppressed by isomaltose, but promoted by maltose, with the hydrolysis of GG being suppressed. Sucrose appeared to suppress only the consumption of S-GG-I, suggesting that S-GG-I was hydrolyzed by the active site of sucrase in a sucrase-isomaltase complex. Transglucosylation seems to have occurred more frequently in the individual digestion of maltose and isomaltose than in that of GG and sucrose. GG seemed to promote transglucosylation in the presence of maltose, to suppress it with sucrose, and to delay it with isomaltose.
Key words: α-D-glucosylglycerol; intestinal enzyme; disaccharidase; membrane digestion; digestive suppressor

-3-
Establishment of a New Cross of the Rice Blast Fungus Derived from Japanese
Differential Strain Ina168 and Hermaphroditic Rice Pathogen Guy11

Satoru FUKIYA, Megumi KODAMA, Hideki KITO, Teruo SONE, and Fusao TOMITA・

Laboratory of Applied Microbiology, Department of Molecular Bioscience
Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido
University, Sapporo 060-8589, Japan

Received November 1, 2000; Accepted March 19, 2001
Mating experiments between Magnaporthe grisea Japanese rice pathogens and Guy11, a hermaphroditic fertile rice pathogen, were done aimed at identification of avirulence genes. A cross named cross 2107 with thirty-six random progenies was obtained. Segregation analyses of genetic markers found that the cross was less suitable for genetic analysis. Backcrosses with cross 2107 progenies and Guy11 were done and another cross named cross 5307 with sixty-five progenies was obtained. A locus controlling kasugamycin resistance named Ksg1R was identified and used for a model case of genetic mapping. Bulked segregant analysis was done to find adjacent RAPD markers for mapping of the gene. Three adjacent markers to Ksg1R were obtained and a genetic map around the Ksg1R was made, but these markers were not located on a single chromosome. These results suggest that genetic analysis to identify a gene locus is available in cross 5307. Infection assay of parental strains of cross 5307 to Japanese differential rice cultivars suggested the possibility of genetic analysis of cultivar specificity toward four rice cultivars: Aichi-asahi, Kusabue, Tsuyuake, and K59.
Key words: Magnaporthe grisea; cultivar specificity; genetic cross; genetic analysis; random amplified polymorphic DNAs (RAPD)

-4-
Isolation and Characterization of Desulfitobacterium sp. strain Y51 Capable
of Efficient Dehalogenation of Tetrachloroethene and Polychloroethanes

Akiko SUYAMA,1,2 Ryo IWAKIRI,1 Keiichirou KAI,1 Takashi TOKUNAGA,3 Nobuyuki SERA,3
and Kensuke FURUKAWA1,・

1Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Hakozaki 6-10-1,
Higashi-ku, Fukuoka 812-8581, Japan
2Towa Kagaku Co. Ltd., Hunairimachi 6-5, Naka-ku, Hiroshima 730-0841, Japan
3Fukuoka Institute of Health and Environmental Sciences, Aza-Mukaida 39, Mukaizano, Dazaifu,
Fukuoka 818-0135, Japan

Received November 1, 2000; Accepted February 28, 2001
A strict anaerobic bacterium, strain Y51, was isolated from soil contaminated with tetrachloroethene (PCE). Strain Y51 is capable of very efficiently dehalogenating PCE via trichloroethene (TCE) to cis-1,2-dichloroethene (cis-1,2-DCE) at concentrations as high as 960 μM and as low as 0.6 μM. Strain Y51 was Gram-negative, motile with some lateral flagella, and curved rod-shaped. On the basis of the 16S rDNA sequence, the organism was identified to be a species within the genus Desulfitobacterium. Strain Y51 also had dehalogenation activities toward polychloroethanes such as hexa-, penta-, and tetrachloroethanes, from which dichloroethenes were produced as the final products. The cell extracts mediated the dehalogenation of PCE with reduced methyl viologen as an electron carrier at the specific rate of 5.0 nmol・min−1・mg cell protein−1 (pH 7.2, 37°C). Dehalogenation was highly susceptible to air oxidation, and to potential alternative electron acceptors such as nitrite or sulfite.
Key words: Desulfitobacterium sp.; tetrachloroethene (PCE); polychloroethanes; reductive dehalogenation

-5-
Synthesis of Optically Active N-(2-Pyridyloxiran-2-ylmethyl)
benzenesulfonamide Derivatives and Their Herbicidal Activity＊

Akemi HOSOKAWA,・ Manabu KATSURADA, Osamu IKEDA, Noriko MINAMI,
and Tetsuo JIKIHARA

Agricultural Chemicals Laboratory, Yokohama Research Center, Mitsubishi Chemical Co. Ltd.,
Kamoshida-cho 1000, Aoba-ku, Yokohama 227-8502, Japan

Received November 7, 2000; Accepted February 7, 2001
Novel herbicidally active sulfonamide compounds having a 2-arylsubstituted oxiranylmethyl structure are racemates due to a chiral carbon in the oxirane moiety. To clarify the stereochemical structure-activity relationship, we synthesized each enantiomer of 4-chloro-N-[2-(6-chloropyridin-2-yl)-2-oxiran-2-ylmethyl]-3,N-dimethylbenzenesulfonamide and N-[2-(6-chloropyridin-2-yl)-2-oxiran-2-ylmethyl]-N-methyl-5,6,7,8-tetrahydronaphthalene-2-sulfonamide by chemical methods including Sharpless asymmetric chlorohydroxylation. The results of herbicidal tests indicated that the (S)-isomers were the active forms.
Key words: herbicide; sulfonamide derivatives; oxirane derivatives; Echinochloa oryzicola; asymmetric synthesis

-6-
Accelerative Effect of Olive Oil on Liver Glycogen Synthesis in Rats Subjected
to Water-immersion Restraint Stress

Hisanao TAKEUCHI,・ Norio SUZUKI, Masakazu TADA, and Puming HE

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

Received November 10, 2000; Accepted January 30, 2001
The effects of dietary oils on stress-induced changes in the liver glycogen metabolism of male Wistar rats at 6 weeks of age were investigated. The rats were subjected to repetitive water-immersion restraint and fed with a 20％ saturated fatty acid mixture (PSC), olive oil (OLI), safflower oil (SAF), or linseed oil (LIS) diet. Stress loading decresed the body weight gain, although the food intake was hardly changed, and the weights of the liver and spleen generally declined regardless of the elapsed time after stress loading and the type of dietary oil. The adrenal weight was generally enhanced by stress in all deitary groups, and particularly tended to be greater in the OLI and PSC groups than in the other two. The plasma corticosterone concentration increased immediately after stressing (Stress-1), but approached the level of the rats with no stress (No stress) 2 h after releasing the stress load (Stress-2) in all groups. The enhancement of corticosterone level in the Stress-1 animals was large in the PSC and OLI groups, and the decline of this level in the Stress-2 animals was small in the OLI group when compared with the other groups. Although the concentrations of total cholesterol (T-CHOL) and triacylglycerol (TG) in the plasma were decreased by stress loading in all groups, these concentrations in the PSC and OLI groups were nearly always higher than in the other groups. The liver serine dehydratase (SDH) activity enhanced by stress was high in the OLI group and tended to be high in the PSC group when compared with the other groups. The contents of liver glycogen were reduced in the Stress-1 animals and extremely elevated in the Stress-2 animals of all groups, and particularly in the OLI group, the reduction in the Stress-1 animals was smaller and the enhancement in the Stress-2 animals was greater than in the other groups. These results suggest that feeding oleic acid to rats exposed to water-immersion restraint further accelerated liver glycogen synthesis through the rise in liver SDH activity due to inceased corticosterone secretion when compared with the effect from linoleic and α-linolenic acids.
Key words: olive oil; adrenal corticosterone; water-immersion restraint; glycogen synthesis; serine dehydratase

-7-
Characterization of the Replication Region of the Lactobacillus reuteri Plasmid pTC82 Potentially Used in the Construction of Cloning Vector

Chuen-Fu LIN, Jin-Li HO, and Tung-Ching CHUNG・

Department of Veterinary Medicine, National Chung-Hsing University, Taichung, Taiwan, ROC

Received December 4, 2000; Accepted March 16, 2001
A 3.2 kb DNA fragment containing the replication region (RR) from pTC82 was cloned, sequenced, and found to contain elements typical of plasmids that replicate via a rolling-circle mechanism of replication (RCR), including double-strand origin (DSO), replication protein gene (rep), and single-strand origin (SSO). The DSO of pTC82 contains two domains showing 55.5％ and 84.6％ similarities in nucleotide (nt) sequence to the conserved functional elements bind and nic, respectively, which are required for the initiation of the leading strand typical of the pC194-RCR family. Although the predicted rep gene product of pTC82 (Rep82) shares little identity (less than 24％) with other known Reps, a region containing three motifs, characteristic of the pC194-family Reps, was identified, indicating the Rep82 as a novel Rep protein of this family. Downstream of the rep82 gene, strong similarity to the typical palT type-SSO could be detected. This is the first palT type-SSO to be identified from Lactobacillus. Through a series of deletion studies, the minimal replicon of the cloned RR was found to be 2.66 kb in size including the DSO region and rep gene. This RR was further identified as being highly stable in L. reuteri and also bearing a very narrow host-range property, suggesting it to be a good replicon potentially useful in vector construction for developing L. reuteri as a vaccine carrier.
Key words: replication region; Lactobacillus reuteri; indigenous plasmid; Rep protein; pC194 family

-8-
Visualization of Nuclei in Aspergillus oryzae with EGFP and Analysis
of the Number of Nuclei in Each Conidium by FACS

Jun-ichi MARUYAMA, Harushi NAKAJIMA, and Katsuhiko KITAMOTO＊

Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

Received December 12, 2000; Accepted February 15, 2001
Aspergillus oryzae has been reported to form conidia with multinuclei. In order to analyze nuclei in living cells, we developed an expression system of the A. nidulans histone H2B protein tagged by EGFP (H2B::EGFP). In both A. oryzae niaD300 and A. nidulans FGSC89 transformants expressing H2B::EGFP, fluorescence was detected in nuclear regions of hyphae and conidia. While a conidium contained only one fluorescent spot in the A. nidulans transformant, approximately 66％ of conidia had two, 24％ had one, and 10％ had three or more in the A. oryzae transformant. The conidia expressing H2B::EGFP were put through FACS (fluorescence-activated cell sorting) analysis and two sharp peaks, corresponding to one and two nuclei in each conidium, were noted in the A. oryzae transformant. In addition, the A. oryzae uninucleate conidia that were successfully isolated by FACS reproduced conidia with almost the same number distribution of nuclei as that of the original. Conidia of five A. oryzae strains used in sake brewing were scored for the number of nuclei, showing that a varied number of nuclei existed in each conidium and some strains had a small number of uninucleate conidia.
Key words: Aspergillus oryzae; nuclei; conidia; EGFP; FACS

-9-
1-Aminocyclopropane-1-carboxylate Synthase of Penicillium citrinum: Primary
Structure and Expression in Escherichia coli and Saccharomyces cerevisiae

Yukiko KAKUTA, Toshinori IGARASHI, Toyotaka MURAKAMI, Hiroyuki ITO,
Hirokazu MATSUI, and Mamoru HONMA

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

Received December 13, 2000; Accepted February 21, 2001
A plant hormone, ethylene, is formed through 1-aminocyclopropane-1-carboxylic acid (ACC). A fungus, Penicillium citrinum, was found to synthesize ACC and to degrade ACC into 2-oxobutyrate and ammonia. ACC synthase, responsible for ACC synthesis in P. citrinum, was characterized on the molecular level by sequencing of N terminal and proteolytic peptides of the enzyme, and cloning and sequencing of its cDNA. The ACC synthase from P. citrinum had 430 amino acid residues and a shorter C terminal than the plant enzyme. The enzyme purified from Escherichia coli transformed with ACC-synthase-encoding DNA showed similar properties to those of the purified enzyme from P. citrinum. Saccharomyces cerevisiae with ACC synthase accumulated ACC in the medium with increasing time of incubation. The sequence of ACC synthase from P. citrinum was compared with that of the plant enzyme with discussion about important residues for catalysis.
Key words: 1-aminocyclopropane-1-carboxylic acid (ACC); ACC synthase; Penicillium citrinum

-10-
In Vitro Biosynthesis of Homogalacturonan by a Membrane-bound
Galacturonosyltransferase from Epicotyls of Azuki Bean

Yoshimi TAKEUCHI and Yoichi TSUMURAYA・

Department of Biochemistry and Molecular Biology, Faculty of Science, Saitama University,
255 Shimo-okubo, Saitama 338-8570, Japan

Received December 15, 2000; Accepted March 19, 2001
A membrane preparation of 7-d-old seedlings from azuki bean (Vigna angularis) contained galacturonosyltransferase (GalAT) capable of transferring galacturonic acid (GalA) from UDP-GalA into polygalacturonic acid (PGA) as an exogenous acceptor. The enzyme was maximally active at pH 6.8--7.8 and 25--35°C in the presence of 5 mM Mn2+ and 0.5％ (w/v) Triton X-100. Acid-soluble low-Mr (average Mr 10,000) PGA was a more efficient acceptor substrate than acid-insoluble polymer (Mr 70,000). The apparent Michaelis constants for UDP-GalA and low-Mr PGA were 0.14 mM and 0.02 mg/ml, respectively. Various pectins with different degrees of methyl-esterification (DE) were poor acceptors, and the enzyme activity tended to decrease with decreasing DE of the pectins. The transfer products from incubation of the enzyme with UDP-14C-GalA and the low-Mr PGA yielded 14C-GalA2 as the major product upon digestion with an endopolygalacturonase (EPGase), confirming the incorporation of GalA into PGA through contiguous α-1,4-linkages.
Key words: cell wall (pectin); galacturonan; galacturonosyltransferase; pectin; Vigna (epicotyl)

-11-
In Vitro Model using Mouse Hepatocytes for Study of Alcohol Stress

Jae-Won YANG, Jun-Seop SHIN, Jae-Jeong LEE, Hyo-Ihl CHANG and Chan-Wha KIM

Graduate School of Biotechnology, Korea University, Seoul 136-701, Korea

Received December 15, 2000; Accepted March 19, 2001
In this study, the effects of ethanol and allyl alcohol on primary mouse hepatocytes were investigated. No cytotoxicity was observed by ethanol treatments, but more toxicity to cells was found in the response to allyl alcohol treatment. The expression of cytochrome P450 2E1 (CYP2E1), phase I enzyme was examined in response to ethanol and allyl alcohol. Both xenobiotics induced CYP2E1 up to 1.5~5 fold at the protein level. The effects of insulin on CYP2E1 expression were also measured. Insulin, which has been regarded as an essential hormone for primary hepatocytes, was shown to decrease the level of CYP2E1 protein, and did not affect cell viability. These results on CYP2E1 induction demonstrate that primary mouse hepatocytes, when using ethanol and allyl alcohol as substrates and in insulin-free medium, provide a suitable system for the studies of the role of CYP2E1 in xenobiotic metabolism and toxicity.
Key words: mouse hepatocyte; ethanol; allyl alcohol; CYP2E1; hepatotoxicity

-12-
Insecticidal and Neural Activities of Candidate Photoaffinity Probes
for Neonicotinoid Binding Sites

Kazuhiko MATSUDA,1,・ Makoto IHARA,1 Keiichiro NISHIMURA,2
David B. SATTELLE,3 and Koichiro KOMAI1

1Department of Agricultural Chemistry, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan
2Research Institute for Advanced Science and Technology, Osaka Prefecture University, 1-2 Gakuencho,
Sakai 599-8570, Osaka, Japan
3MRC Functional Genetics Unit, Department of Human Anatomy and Genetics, University of Oxford,
South Parks Road, Oxford OX1 3QX, United Kingdom

Received December 20, 2000; Accepted February 16, 2001
Photoreactive derivatives of imidacloprid and its nitromethylene analogue were synthesized as candidate photoaffinity probes for identifying the amino acid residues of nicotinic acetylcholine receptors (nAChRs) that interact with the neonicotinoid insecticides. When the candidate probes were injected into American cockroaches, the nerve cord neural activity initially increased, then ceased and death of the insect followed. Both the nerve cord and toxicity were enhanced by changing the photoreactive substituent from the para position to the meta position on the spacer benzyl moiety. When tested on a Drosophila SAD/chicken β2 hybrid, recombinant nAChR expressed in Xenopus oocytes, the nitromethylene candidate probes showed agonist activity similar to that previously observed for imidacloprid.
Key words: nicotinic acetylcholine receptor; neonicotinoid; photoaffinity probe; imidacloprid; trifluoromethyldiazirine

-13-
Identification of Amino Acid Residues Essential for the Substrate Specificity
of Flavobacterium sp. Endo-β-N-acetylglucosaminidase

Kiyotaka FUJITA, Ryo-ich NAKATAKE, Kayo YAMABE, Akira WATANABE,
Yasuhiko ASADA, and Kaoru TAKEGAWA・

Department of Life Sciences, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa 761-0795, Japan

Received January 12, 2001; Accepted February 16, 2001
The gene encoding the endo-β-N-acetylglucosaminidase from Flavobacterium sp. (Endo-Fsp) was sequenced. The Endo-Fsp gene was overexpressed in Escherichia coli cells, and was purified from inclusion bodies after denaturation by 8 M urea. The renatured Endo-Fsp had the same optimum pH and substrate specificity as the native enzyme. Endo-Fsp had 60％ sequence identity with the endo-β-N-acetylglucosaminidase from Streptomyces plicatus (Endo-H), and the putative catalytic residues were conserved. Site-directed mutagenesis was done at conserved residues based on the three-dimensional structure and mutagenesis of Endo-H. The mutant of Glu-128, corresponding to Glu-132 in Endo-H and identified as an active site residue, was inactivated. Mutagenesis around the predicted active site of Endo-Fsp reduced the enzymatic activity. Moreover, the hydrolytic activity toward hybrid-type oligosaccharides was decreased compared to that toward high-mannose type oligosaccharides by mutagenesis of Asp-126 and Asp-127. Therefore, site-directed mutagenesis of some of these conserved residues indicates that the predicted active sites are essential to the enzymatic activity of Endo-Fsp, and may have similar roles in catalysis as their counterparts in Endo-H.
Key words: endo-β-N-acetylglucosaminidase; Flavobacterium sp.; site-directed mutagenesis; substrate specificity

-14-
Purification and Characterization of Aminopeptidase B
from Escherichia coli K-12

Hideyuki SUZUKI,・ Sachiko KAMATANI, and Hidehiko KUMAGAI

Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University,
Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan

Received January 15, 2001; Accepted March 13, 2001
Aminopeptidase B, which is one of the four cysteinylglycinases of Escherichia coli K-12, was purified to electrophoretic homogeneity and its enzymatic characteristics were observed. Aminopeptidase B was activated by various divalent cations such as Ni2+, Mn2+, Co2+, and Cd2+, and lost its activity completely on dialysis against EDTA. This indicates that aminopeptidsase B is a metallopeptidase. It was stabilized against heat in the presence of Mn2+ or Co2+. The activity of aminopeptidase B, which was saturated with one of above divalent cations, was enhanced on the addition of a very small amount of a second divalent cation. α-Glutamyl p-nitroanilide, leucine p-nitroanilide, and methionine p-nitroanilide were good substrates for aminopeptidase B, while native peptides, cysteinylglycine and leucylglycine, were far better substrates. The kcat/Km for cysteinylglycine was much bigger than those for leucylglycine or leucine p-nitroanilide.
Key words: aminopeptidase; cysteinylglycinase; Escherichia coli K-12; metallopeptidase; glutathione metabolism

-15-
First Stereoselective Synthesis of (+)-Magnostellin C, a Tetrahydrofuran
Type of Lignan Bearing a Chiral Secondary Benzyl Alcohol

Satoshi YAMAUCHI・ and Yoshiro KINOSHITA

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

Received January 15, 2001; Accepted February 23, 2001
(+)-Magnostellin C, which is a tetrahydrofuran type of lignan bearing a chiral secondary benzylic hydroxy group, was stereoselectively synthesized from L-arabinose by using threo selective aldol condensation.
Key words: lignan; tetrahydrofuran lignan; Magnostellin

-16-
In Vivo and in Vitro Analyses of the AmyR Binding Site of the Aspergillus
nidulans agdA Promoter; Requirement of the CGG Direct Repeat
for Induction and High Affinity Binding of AmyR

Shuji TANI, Tomoyuki ITOH, Masashi KATO, Tetsuo KOBAYASHI, and Norihiro TSUKAGOSHI

Department of Biological Mechanisms and Functions, Graduate School of Bioagricultural Sciences,
Nagoya University, Chikusa-ku, Nagoya-shi, Aichi 464-8601, Japan

Received January 16, 2001; Accepted March 7, 2001
The α-glucosidase gene (agdA) of Aspergillus nidulans has a single CGGN8CGG type AmyR binding site in its promoter region. The binding site is functional in vivo as a cis-element responsible for induction by starch, and mutational studies indicated that both the CGG triplets are required for high-level induction.
A part of AmyR (residues 1-411; AmyR1-411), which was produced as a MalE fusion protein in E. coli, bound to the CGGN8CGG site of the agdA promoter. DNA binding profiles to the mutant binding sites that lacked both or either one of the CGG triplets suggested that AmyR1-411 can bind to a single CGG triplet site with low affinity and that two AmyR molecules cooperatively bind to the CGG direct repeat.
Key words: AmyR; Aspergillus nidulans; amylase induction; DNA binding; transcriptional activator

-17-
Molecular Cloning, Sequencing, and Expression of cDNA Encoding Serine
Protease with Fibrinolytic Activity from Earthworm＊

Manabu SUGIMOTO1 and Nobuyoshi NAKAJIMA2,・

1Research Institute for Bioresources, Okayama University, Kurashiki, Okayama 710-0046, Japan
2Graduate School of Health and Welfare Science, Okayama Prefectural University, Soja,
Okayama 719-1197, Japan

Received January 17, 2001; Accepted March 1, 2001
An earthworm, Lumbricus rubellus, produces alkaline trypsin-like proteases that are greater than trypsins in their stability and strong tolerance to organic solvents. cDNAs encoding strong fibrinolytic proteases (F-III-2 and F-III-1) in the six isozymes were cloned and sequenced to study their stability-structure relationship. The cDNAs of F-III-2 and F-III-1 comprised 1011 and 973 bp and included open reading frames that encode polypeptides of 245 and 246 amino acid residues, respectively. The deduced amino acid sequences of F-III-2 and F-III-1 have 7 and 8 activation peptides in the N-termini respectively, indicating that they are translated as proenzymes and modified to active forms by post-translational processing. They showed similarity to mammalian serine proteases and conserved the catalytic amino acid residues, however, neither arginine nor lysine residues were present in the autolysis region. The gene encoding the native form of F-III-2 was expressed in Pichia pastoris to produce and secrete the earthworm protease in the culture medium, which dissolves an artificial fibrin plate.
Key words: earthworm; serine protease; fibrinolytic enzyme; gene expression; nucleotide sequence

-18-
Structural Change and Catalytic Activity of Horseradish Peroxidase
in Oxidative Polymerization of Phenol

Masaru AKITA, Daisuke TSUTSUMI, Masami KOBAYASHI, and Hideo KISE・

Institute of Materials Science, University of Tsukuba, Tennoudai, Tsukuba, Ibaraki 305-8573, Japan

Received January 17, 2001; Accepted March 9, 2001
The effects of solvent and reaction conditions on the catalytic activity of horseradish peroxidase (HRP) were investigated for oxidative polymerization of phenol in water/organic mixtures using hydrogen peroxide as an oxidant. Also, the structural changes of HRP were investigated by CD and absorption spectroscopy in these solvents. The results suggest that the yield of phenol polymer (the conversion of phenol to polymer) is strongly affected by the reaction conditions due to the structural changes of HRP, that is, the changes in higher structure of the apo-protein and dissociation or decomposition of the prosthetic heme. Optimum solvent compositions for phenol polymerization depend on the nature of the organic solvents owing to different effects of the solvents on HRP structure. In addition to initial rapid changes, slower changes of HRP structure occur in water/organic solvents especially at high concentrations of organic solvents. In parallel with these structural changes, catalytic activity of HRP decreases with time in these solvents. At higher reaction temperatures, the yield of the polymer decreases, which is also ascribed to modification of HRP structure. It is known that hydrogen peroxide is an inhibitor of HRP, and the yield of phenol polymer is strongly dependent on the manner of addition of hydrogen peroxide to the reaction solutions. The polymer yield decreases significantly when hydrogen peroxide was added to the reaction solution in a large amount at once. This is probably due to inactivation of HRP by excess hydrogen peroxide. From the CD and absorption spectra, it is suggested that excess hydrogen peroxide causes not only decomposition of the prosthetic heme but also modification of the higher structure of HRP.
Key words: horseradish peroxidase; phenol; oxidative polymerization; enzyme structure; circular dichroism

-19-
A Novel Screening for Inhibitors of a Pleiotropic Drug Resistant Pump,
Pdr5, in Saccharomyces cerevisiae

Kazumi HIRAGA, Anoja WANIGASEKERA, Hisanori SUGI, Nobuyuki HAMANAKA,1
and Kohei ODA・

Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Matsugasaki,
Sakyo-ku, Kyoto 606-8585, Japan
1Minase Research Institute, Ono Pharmaceutical Ltd., Shimamoto, Mishima, Osaka 618-8585, Japan

Received January 18, 2001; Accepted February 28, 2001
Yeast is an excellent model system of eukaryotes for the study of molecular mechanisms of ATP-binding cassette transporters. Pdr5 protein is a yeast Saccharomyces cerevisiae ATP-binding cassette transporter conferring resistance to several unrelated drugs. Here, we described a novel drug screening system designated to detect compounds that inhibit the function of Pdr5. An indicator strain with increased drug sensitivity was constructed with an ergosterol-deficient background (Δsyr1/erg3 null mutation). The sensitivity of the indicator strain (Δsyr1/erg3Δpdr5Δsnq2) to the Pdr5 substrates, cycloheximide and cerulenin, was increased 16-fold and 4-fold against wild type, respectively. The screening system is mainly based on the growth inhibition of the PDR5-overexpressed indicator strain with the combination of a sample and cycloheximide or cerulenin. The effect of an mdr inhibitor, FK506 on the screening system was clearly detected even at a low concentration (~0.5 μg/ml). In addition, accumulation of rhodamine 6G in the cells was detected as a result of Pdr5 inhibition by FK506. These results indicated that the screening system is useful for a sensitive screening of Pdr5-specific inhibitors with low toxicity.
Key words: pleiotropic drug resistance; PDR5; Saccharomyces cerevisiae; ergosterol; immunosuppressant

-20-
Characteristics of Wine Produced by Mushroom Fermentation

Tokumitsu OKAMURA,・ Tomoko OGATA, Norie MINAMIMOTO, Tomomi TAKENO,
Hiroko NODA, Shoko FUKUDA, and Masahiro OHSUGI

Department of Food Science and Nutrition, School of Human Environmental Sciences,
Mukogawa Women′s University, 6-46 Ikebiraki-cho, Nishinomiya, Hyogo 663-8137, Japan

Received January 19, 2001; Accepted March 1, 2001
Saccharomyces cerevisiae is the main microorganism used in wine brewing, because this microbe has potent ability to produce alcohol dehydrogenase. We have recently discovered that some genera of mushroom produced alcohol dehydrogenase, and made wine by using a mushroom in place of S. cerevisiae. The highest alcohol concentration in this wine was achieved with Pleurotus ostreatus (2.6 M, 12.2％). In the case of Agaricus blazei, the same alcohol concentration (1.7 M, 8％) was produced under both aerobic and anaerobic conditions. This wine produced by A. blazei contained about 0.68％ β-D-glucan, which is known to have a preventive effects against cancer. The wine made by using Flammulina velutipes showed thrombosis-preventing activity, giving a prolonged thrombin clotting time 2.2-fold that of the control. Thus, the wine made by using mushroom seems to be a functional food which can be expected to have preventive effects against cancer and thrombosis.
Key words: wine; mushroom; alcohol fermentation; β-D-glucan; anti-thrombin substance

-21-
Effects of Double Mutation at Two Distant IgE-binding Sites
in the Three-dimensional Structure of the Major House Dust Mite
Allergen Der f 2 on IgE-binding and Histamine-releasing Activity

Toshiro TAKAI,1,・ Hideki HATANAKA,2 Saori ICHIKAWA,2,3 Toyokazu YOKOTA,1
Fuyuhiko INAGAKI,2,4 and Yasushi OKUMURA1

1Bioscience Research and Development Laboratory, Asahi Breweries, Ltd., 1-21, Midori 1-chome,
Moriya-machi, Kitasoma-gun, Ibaraki 302-0106, Japan
2Department of Molecular Physiology, Tokyo Metropolitan Institute of Medical Science,
3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613
3Department of Material and Biological Science, Faculty of Science, Japan Women′s University,
2-8-1 Mejirodai, Bunkyo-ku Tokyo 112-8681, Japan
4Division of Structural Biology, Graduate School of Pharmaceutical Sciences, Hokkaido University,
N-12, W-6, Kita-ku, Sapporo 060-0812, Japan

Received January 19, 2001; Accepted February 21, 2001
Recently, we reported that introduction of mutations that induced conformational changes of the major mite allergen Der f 2 was an efficient strategy to reduce the allergenicity for safer allergen-specific immunotherapy. In this study, we evaluated another strategy, disruption of two independent IgE epitopes without inducing conformational change. We analyzed allergenicities of the wild-type Der f 2, two single mutants with a mutation at either of the two IgE-binding sites (K15A and K77A), and a double mutant with mutations at both of the sites (K15/77A). Purified recombinant forms of Der f 2 expressed in Escherichia coli had correct disulfide bonds, equivalent apparent molecular masses of approximately 15 kDa, and similar secondary structures. The mutants of Der f 2 had less IgE reactivities than the wild-type Der f 2 and reduced inhibitory activities for IgE-binding to the wild-type Der f 2. However, the mutations did not significantly reduce histamine-releasing activity.
Key words: mite group 2 allergens; IgE epitopes; tertiary structure; allergen engineering; recombinant allergen

-22-
Purification and Substrate Specificity of Honeybee,
Apis mellifera L., α-Glucosidase III

Mamoru NISHIMOTO, Masaki KUBOTA, Masahisa TSUJI, Haruhide MORI,
Atsuo KIMURA, Hirokazu MATSUI, and Seiya CHIBA・

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

Received January 22, 2001; Accepted February 27, 2001
α-Glucosidase III, which was different in substrate specificity from honeybee α-glucosidases I and II, was purified as an electrophoretically homogeneous protein from honeybees, by salting-out chromatography, DEAE-cellulose, DEAE-Sepharose CL-6B, Bio-Gel P-150, and CM-Toyopearl 650M column chromatographies. The enzyme preparation was confirmed to be a monomeric protein and a glycoprotein containing about 7.4％ of carbohydrate. The molecular weight was estimated to approximately 68,000, and the optimum pH was 5.5. The substrate specificity of α-glucosidase III was kinetically investigated. The enzyme did not show unusual kinetics, such as the allosteric behaviors observed in α-glucosidases I and II, which are monomeric proteins. The enzyme was characterized by the ability to rapidly hydrolyze sucrose, phenyl α-glucoside, maltose, and maltotriose, and by extremely high Km for substrates, compared with those of α-glucosidases I and II. Especially, maltotriose was hydrolyzed over 3 times as rapidly as maltose. However, maltooligosaccharides of four or more in the degree of polymerization were slowly degraded. The relative rates of the k0 values for maltose, sucrose, p-nitrophenyl α-glucoside and maltotriose were estimated to be 100, 527, 281 and 364, and the Km values for these substrates, 11, 30, 13, and 10 mM, respectively. The subsite affinities (Ai′s) in the active site were tentatively evaluated from the rate parameters for maltooligosaccharides. In this enzyme, it was peculiar that the Ai value at subsite 3 was larger than that of subsite 1.
Key words: honeybee α-glucosidase; substrate specificity; allosteric behavior; subsite affinity

-23-
Azurin Involved in Alcohol Oxidation System in Pseudomonas putida HK5:
Expression Analysis and Gene Cloning

Hirohide TOYAMA,・ Nahoko AOKI, Kazunobu MATSUSHITA, and Osao ADACHI

Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University,
Yamaguchi 753-8515, Japan

Received January 23, 2001; Accepted March 2, 2001
Expression of azurin in Pseudomonas putida HK5 was examined by immunoblot analysis. Similar amounts of azurin were found in the cells grown into the stationary phase on any carbon sources, including LB medium without alcohol, where no quinoprotein alcohol dehydrogenases appeared. In the early exponential phase, the highest amount of azurin was found in the cells grown on 1-butanol, but here was none in the case of LB medium, suggesting that expression of azurin is cooperative with that of the alcohol oxidase system, especially the system including quinohemoprotein alcohol dehydrogenase IIB.
The azurin gene (azu) was cloned and sequenced. azu is monocistronic, and in its promoter region, FNR-binding consensus sequence was found. However, its relative position suggests different transcriptional regulation from that in azu of P. aeruginosa. The molecular weight of the mature protein without copper ion calculated from the amino acid sequence was consistent with the value of the purified azurin measured by mass spectrometry.
Key words: azurin; quinoprotein; pyrroloquinoline quinone; PQQ; alcohol dehydrogenase

-24-
Cloning of a Gene Cluster Encoding Enzymes Responsible for the Mevalonate
Pathway from a Terpenoid-antibiotic-producing Streptomyces Strain

Yoshimitsu HAMANO,1 Tohru DAIRI,1,・ Masahiro YAMAMOTO,1 Takashi KAWASAKI,1
Kazuhide KANEDA,2 Tomohisa KUZUYAMA,2 Nobuya ITOH,1 and Haruo SETO2,・

1Biotechnology Research Center, Toyama Prefectural University, Toyama 939-0398, Japan
2Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku 113-0032, Japan

Received January 24, 2001; Accepted February 28, 2001
A gene cluster encoding enzymes responsible for the mevalonate pathway was isolated from Streptomyces griseolosporeus strain MF730-N6, a terpenoid-antibiotic terpentecin producer, by searching a flanking region of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene, which had been previously isolated by complementation. By DNA sequencing of an 8.9-kb BamHI fragment, 7 genes encoding geranylgeranyl diphosphate synthase (GGDPS), mevalonate kinase (MK), mevalonate diphosphate decarboxylase (MDPD), phosphomevalonate kinase (PMK), isopentenyl diphosphate (IPP) isomerase, HMG-CoA reductase, and HMG-CoA synthase were suggested to exist in that order. Heterologous expression of these genes in E. coli and Streptomyces lividans, both of which have only the nonmevalonate pathways, suggested that the genes for the mevalonate pathway were included in the cloned DNA fragment. The GGDPS, MK, MDPD, PMK, IPP isomerase, and HMG-CoA synthase were expressed in E. coli. Among them, the recombinant GGDPS, MK, and IPP isomerase were confirmed to have the expected activities. This is the first report, to the best of our knowledge, about eubacterial MK with direct evidence.
Key words: mevalonate pathway; gene cloning; Streptomyces; terpenoid; terpentecin

-25-
Is Your Ribozyme Design Really Correct?:
A proposal of simple single turnover competition assay to evaluate ribozymes

Terumichi TANAKA,1,・ Osamu INUI,1 Natsuki DOHI,2 Noriko OKADA,2
Hidechika OKADA,2 and Yo KIKUCHI1

1Division of Bioscience and Biotechnology, Department of Ecological Engineering, Toyohashi University
of Technology, Tempaku-cho, Toyohashi, Aichi 441-8580, Japan
2Department of Molecular Biology, Nagoya City University School of Medicine, Mizuho,
Nagoya 467-8601, Japan

Received January 30, 2001; Accepted March 12, 2001
Today, many nucleic acid enzymes are used in gene therapy and gene regulations. However, no simple assay methods to evaluate enzymatic activities, with which we judge the enzyme design, have been reported. Here, we propose a new simple competition assay for nucleic acid enzymes of different types to evaluate the cleaving efficiency of a target RNA molecule, of which the recognition sites are different but overlapped. Two nucleic acid enzymes were added to one tube to make a competition of these two enzymes for one substrate. The assay was used on two ribozymes, hammerhead ribozyme and hairpin ribozyme, and a DNA-enzyme. We found that this assay method is capable of application to those enzymes, as a powerful tool for the selection and designing of RNA-cleaving enzymes.
Key words: hammerhead ribozyme; hairpin ribozyme; DNA-enzyme; 5I2 antigen mRNA; competition assay

-26-
Note
Inhibition by Agaricus blazei Murill Fractions of Cytopathic Effect Induced
by Western Equine Encephalitis (WEE) Virus on VERO Cells in Vitro

Kenji SORIMACHI,1 Yukari IKEHARA,2 Genzo MAEZATO,2 Akira OKUBO,3,・
Sunao YAMAZAKI,3 Kazumi AKIMOTO,4 and Akira NIWA1

1Department of Microbiology and 4 Institute of Medical Research, Dokkyo University School of Medicine,
Mibu, Tochigi 321-0293, Japan
2Okinawa Fermentative Chemicals Co. Ltd., Nishizakicho, Itoman, Okinawa 901-0305, Japan
3Department of Applied Biological Chemistry, The University of Tokyo, Bunkyo, Tokyo 113-8657, Japan

Received August 29, 2000; Accepted March 8, 2001
Anti-viral activities of Agaricus blazei Murill were investigated. The water extracts of the cultured mycelia and fruiting bodies were fractionated with different concentrations of ethanol. To several viruses which have cytopathic effects (CPE) on VERO cells, inhibition of these effects by the ethanol fractions was tested. Strong inhibition of CPE induced by western equine encephalitis (WEE) virus was observed in the mycelial fractions but not those of fruiting bodies.
Key words: Agaricus blazei Murill; cytopathic effect; western equine encephalitis (WEE) virus; VERO cells

-27-
Note
Purification of Saponin Compounds in Bupleurum falcatum
by Solvent Partitioning and Preparative LC

Namsoo KIM・ and In-Seon PARK

Korea Food Research Institute, San 46-1, Backhyun-dong, Bundang-ku, Songnam-si,
Kyonggi-do 463-420, Republic of Korea

Received August 30, 2000; Accepted March 14, 2001
Saponin compounds (saikosaponin c, a, and d) in Bupleurum falcatum were partially purified by solvent partitioning of the herbal extract using diethyl ether, distilled water, n-butanol, and acetone. After separation of the saponins by preparative LC, the purity of each saikosaponin was more than 94％. The identities of purified individual saikosaponins were confirmed by TLC, analytical LC, and fast-atom bombardment mass spectrometry.
Key words: purification; saikosaponins; solvent partitioning; preparative LC

-28-
Note
Antimutagenicity of Deacylated Anthocyanins in Purple-fleshed Sweetpotato

Makoto YOSHIMOTO,・ Shigenori OKUNO, Masaatu YAMAGUCHI,＊ and Osamu YAMAKAWA

Department of Upland Farming, Kyushu National Agricultural Experiment Station, Miyakonojo,
Miyazaki 885-0091, Japan
＊College of Horticulture, Minami-Kyushu University, Takanabe, Miyazaki 884-0001, Japan

Received October 19, 2000; Accepted February 9, 2001
The antimutagenicity of the 3-sophoroside-5-glucoside of cyanidin and 3-sophoroside-5-glucoside of peonidin, the anthocyanin derivatives deacylated from the 3-(6,6?-caffeylferulylsophoroside)-5-glucoside of cyanidin (YGM-3) and 3-(6,6?-caffeylferulylsophoroside)-5-glucoside of peonidin (YGM-6) which had been purified from the sweetpotato with purple-colored flesh, was investigated by using Salmonella typhimurium TA 98. A comparison of the antimutagenicity between YGM-3 and YGM-6 and the deacylated derivatives showed that the activity of cyanidin was stronger than that of peonidin. Deacylation of the peonidin-type pigment markedly decreased this antimutagenicity. Caffeic acid showed the strongest antimutagenicity of the constituent organic acids of the anthocyanin pigments, caffeic acid, ferulic acid, and p-hydroxybenzoic acid. These results suggest that the cathecol structure plays an important role in the strong antimutagenicity of anthocyanin pigments.
Key words: antimutagenicity; sweetpotato; anthocyanin; deacylated derivative

-29-
Note
Acetyl-CoA Carboxylase Inhibitors from Avocado
(Persea americana Mill) Fruits

Hironori HASHIMURA, Chihoko UEDA, Jun KAWABATA,・ and Takanori KASAI

Laboratory of Food Biochemistry, Division of Applied Bioscience, Graduate School of Agriculture,
Hokkaido University, Kita-ku, Sapporo 060-8589, Japan

Received November 17, 2000; Accepted March 19, 2001
A methanol extract of avocado fruits showed potent inhibitory activity against acetyl-CoA carboxylase, a key enzyme in fatty acid biosynthesis. The active principles were isolated and identified as (5E,12Z,15Z)-2 - hydroxy - 4 - oxoheneicosa - 5, 12, 15 - trienyl (1), (2R,12Z,15Z)-2-hydroxy-4-oxoheneicosa-12,15-dienyl(2), (2R＊,4R＊)-2,4-dihydroxyheptadec-16-enyl (3) and (2R＊,4R＊)-2,4-dihydroxyheptadec-16-ynyl (4) acetates by instrumental analyses. The IC50 of the compounds were 4.0×10−6, 4.9×10−6, 9.4×10−6, and 5.1×10−6M, respectively.
Key words: acetyl-CoA carboxylase inhibitor; avocado; long-chain fatty alcohol

-30-
Note
Purification and Properties of Two Malate Dehydrogenases
from Candida sp. N-16 Grown on Methanol

Jun YOSHIKAWA, Katsura SEKI, Hirofumi SHINOYAMA, and Takaaki FUJII・

Department of Bioresources Science, Graduate School of Science and Technology, Chiba University,
648, Matsudo, Matsudo-shi, Chiba 271-8510, Japan

Received December 5, 2000; Accepted March 12, 2001
Two malate dehydrogenases (MDH-M1 and MDH-M2) were found in a methanol-using yeast, Candida sp. N-16. MDH-M2 was induced with methanol. These enzymes were purified as electrophoretically and isoelectrophoretically homogeneous proteins. The molecular weights of MDH-M1 and MDH-M2 were estimated to be about 78,000 (homodimer) and 160,000 (homotetramer). Several kinetic properties were significantly different between the two enzymes. The value (2.07) of V_{max(oxaloacetate)}/V_{max(malate)} and K_{cat}s (555 s＾{--1} for oxaloacetate, 481 s＾{--1} for NADH) of MDH-M2 were higher than the ratio (1.37) of V_{max} and K_{cat}s (241 s＾{--1} for oxaloacetate, 271 s＾{--1} for NADH) of MDH-M1, respectively. The activity of MDH-M2 was inhibited by a high concentration of NAD＾{+} and the activity of MDH-M1 by oxaloacetate.
Key words: malate dehydrogenase; methanol-using yeast; Candida; malate; metabolism of methanol

-31-
Note
A Basic Class I Chitinase Expression in Winged Bean is Up-regulated
by Osmotic Stress

Yoshiko TATEISHI, Yoshimi UMEMURA, and Muneharu ESAKA

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

Received December 22, 2000; Accepted March 1, 2001
We isolated a cDNA for basic class I chitinase (ChitiWb1). ChitiWb1 cDNA encodes a protein that consists of 315 amino acid residues and has a signal peptide. Northern blot analysis indicated that the class I chitinase mRNA in leaves and cultured cells of winged bean was increased by treatments with NaCl, KCl, CaCl_{2}, mannitol or saccharose, but not with abscisic acid. Thus, class I chitinase expression was shown to be up-regulated by osmotic stress.
Key words: cDNA cloning; chitinase (EC 3.2.1.14); osmotic stress; winged bean (Psophocarpus tetragonolobus)

-32-
Note
Synthesis of cis-Lactone Lignan, cis-(2S,3R)-Parabenzlactone,
from L-Arabinose

Satoshi YAMAUCHI・ and Yoshiro KINOSHITA

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

Received January 9, 2001; Accepted February 10, 2001
As a model synthesis on cis-2,3-dibenzyl-4-butanolide lignan, cis-(2S,3R)-parabenzlactone bearing a chiral benzyl alcohol moiety was stereoselectively synthesized from L-arabinose.
Key words: lignan; cis-2,3-dibenzyl-4-butanolide

-33-
Note
Anti-inflammatory and Anti-allergic Actions by Oral Administration
of a Perilla Leaf Extract in Mice

Hiroshi UEDA and Masatoshi YAMAZAKI

Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, Teikyo University,
Sagamiko-machi, Kanagawa 199-0195, Japan

Received January 11, 2001; Accepted March 19, 2001
The anti-inflammatory and anti-allergic activity of perilla leaf extract was investigated. The oral administration of perilla leaf extract to mice inhibited two types of acute inflammatory models, arachidonic acid-induced ear edema and 12-o-tetradecanoylphorbol-13-acetate-induced ear edema. Oral administration of perilla leaf extract also inhibited the contact dermatitis model, oxazolone-induced ear edema, by affecting sensitization.
Key words: Perilla frutescens; anti-inflammation; anti-allergy

-34-
Note
Stereoselective Reduction of Ethyl 4-chloro-3-oxobutanoate by Fungi

Yuri SARATANI,1 Eiji UHEDA,1 Hiroaki YAMAMOTO,2 Atsuo NISHIMURA,1
and Fumiki YOSHIZAKO1,・

1Research Institute for Advanced Science and Technology, Osaka Prefecture University, 1-2 Gakuen-cho,
Sakai, Osaka 599-8570, Japan
2Tsukuba Research Center, Daicel Chemical Industries Ltd., 27 Miyukigaoka, Tsukuba-City,
Ibaraki 305-0841, Japan

Received January 11, 2001; Accepted February 24, 2001
The enantioselectivity of ECAA to ECHB by eight fungi of four genus was evaluated. All strains showed (S)-selectivity, and Cylindrocarpon sclerotigenum IFO 31855 gave the highest yield and good optical purity (e.e.; >99％). Cell-free extract and acetone-dried cells of C. sclerotigenum IFO 31855 reduced ECAA to (S)-ECHB in the presence of NADPH (e.e.;>99％) and the e.e. was not decreased by heat treatment of the cell-free extract or the acetone-dried cells. The active fractions shown by two peaks on a DEAE-Toyopearl 650 M column gave preferentially (S)-ECHB (e.e.;>99％).
Key words: stereoselective reduction; ethyl 4-chloro-3-oxobutanoate; (S)-ethyl 4-chloro-3-hydroxybutanoate; Cylindrocarpon sclerotigenum IFO 31855

-35-
Note
Purification and Characterization of an Esterase from Micrococcus sp.
YGJ1 Hydrolyzing Phthalate Esters

Keiichi AKITA, Chikako NAITOU, and Kiyofumi MARUYAMA・

Department of Biomolecular Science, Faculty of Engineering, Gifu University, Yanagido 1-1,
Gifu 501-1193, Japan

Received January 31, 2001; Accepted March 8, 2001
An esterase hydrolyzing phthalate esters has been purified from Micrococcus sp. YGJ1. The enzyme, a monomeric protein (Mr=56 kDa) with a pI of 4.0, hydrolyzes various aliphatic and aromatic carboxylesters. The medium chain (C3-C4) esters are the most preferred substrates. The enzyme is inhibited by HgCl2 and p-chloromercuribenzoate but not by phenylmethylsulfonyl fluoride.
Key words: esterase; phthalate esters; Micrococcus

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Note
Cloning and Nucleotide Sequence of the Mycodextranase Gene
from Streptomyces sp. J-13-3

Katsuichiro OKAZAKI,1,・ Takashi AMANO,1 Tetsuhiro MORIMOTO,1 Takahiro IEMOTO,1
Toshiyuki KAWABATA,1 Shigeru HAYAKAWA,2 and Kazuya AKIMITSU1

1Department of Life Sciences and 2Department of Biochemistry and Food Science, Faculty of Agriculture,
Kagawa University, Miki, Kagawa 761-0795, Japan

Received February 9, 2001; Accepted March 13, 2001
Mycodextranase (EC 3.2.1.61) is an α-glucanase that cleaves α-1,4-bonds of alternating α-1,3- and α-1,4-linked D-glucan (nigeran). The gene encoding mycodextranase from Streptomyces sp. J-13-3 was cloned by hybridization with a degenerate oligonucleotide probe from the amino-terminal amino acid sequence of the enzyme and its nucleotide structure was analyzed. The open reading frame consisted of 1,803 base pairs encoding a signal peptide of 60 amino acids and a mature protein of 540 amino acids with a calculated molecular weight of 56,078. The deduced amino acid sequence showed weak similality to a chitinase homolog from Streptomyces lividans and a chitinase from Xanthomonas sp.
Key words: Streptomyces sp.; mycodextranase; gene cloning; nucleotide sequencing

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Preliminary Communication
Introduction of Bacterial Metabolism into Higher Plants
by Polycistronic Transgene Expression

Hideo NAKASHITA,1・ Yuko ARAI,1,2 Toshiharu SHIKANAI,3 Yoshiharu DOI,1
and Isamu YAMAGUCHI1

1Microbial Toxicology Laboratory, RIKEN Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
2Graduate School of Science and Engineering, Saitama University, 255 Shimo-Okubo, Urawa,
Saitama 338-8570, Japan
3Graduate School of Biological Sciences, Nara Institute of Technology, 8916-5 Takayama-cho, Ikoma,
Nara 630-0101, Japan

Received February 5, 2001; Accepted April 24, 2001
Multiple-gene transformation is required to improve or change plant metabolisms effectively; but this many-step procedure is time-consuming and costing. We succeeded in the metabolic engineering of tobacco plants by introducing multiple genes as a bacteria-type operon into a plastid genome. The tobacco plastid was transformed with a polycistron consisting of three bacterial genes for the biosynthesis of a biodegradable polyester, polyhydroxybutyrate (PHB). Accumulation of PHB in the leaves of the transgenic tobacco indicated that the introduced genes were polycistronically expressed. This ``phyto-fermentation′′ system can be used in plant production of various chemical commodities and pharmaceuticals.
Key words: plastid transformation; operon; metabolic engineering; phyto-fermentation; polyhydroxybutyrate

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Preliminary Communication
The Expression of a Barley HvNAS1 Nicotianamine Synthase Gene
Promoter-gus Fusion Gene in Transgenic Tobacco is Induced
by Fe-deficiency in Roots

Kyoko HIGUCHI,1,2,・ Masaharu TANI,1 Hiromi NAKANISHI,1 Toshihiro YOSHIWARA,3
Fumiyuki GOTO,3 Naoko K. NISHIZAWA,4 and Satoshi MORI1,2

1Laboratory of Plant Molecular Physiology, Department of Applied Biological Chemistry,
The University of Tokyo, 1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
2Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology
Corporation (JST), 4-1-8 Honcho, Kawaguchi-shi, Saitama 332-0012, Japan
3Department of Bio-Science, Central Research Institute of Electric Power Industry, 1646 Abiko,
Chiba 270-1194, Japan
4Laboratory of Plant Biotechnology, Department of Global Agricultural Sciences, The University of Tokyo,
1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

Received March 12, 2001; Accepted April 13, 2001
Nicotianamine (NA) is a precursor for mugineic acid-family phytosiderophores, which are a critical component of the Fe aquisition process in graminaceous plants. In addition, nicotianamine synthase (NAS) is strongly induced in these plants by Fe deficiency. NA is essential for Fe metabolism also in dicots, but NAS is not induced by Fe deficiency. We introduced a barley HvNAS1 promoter-gus fusion gene into tobacco. GUS activity was induced in the roots of these plants by Fe deficiency, and was constitutively expressed at a low level in their leaves.
Key words: barley; Fe-deficiency; heterologous transformation; nicotianamine synthase gene; tobacco