Contents and Abstracts of Latest Issue of BBB

(Vol.67 No.5 2003)

Effect of Methanol Extract of Zanthoxylum piperitum Leaves and of Its Compound, Protocatechuic Acid, on Hepatic Drug Metabolizing Enzymes and Lipid Peroxidation in Rats
Jong Moon HUR,1 Ju Gwon PARK,1 Ki Ho YANG,1 Jong Cheol PARK,1,† Jeong Ro PARK,2 Soon Sil CHUN,2 Jae Sue CHOI,3 and Jong Won CHOI4 p.945

Decrease in Ovalbumin Specific IgE of Mice Serum after Oral Uptake of Lactic Acid Bacteria
Yuu ISHIDA, Izuki BANDOU, Hiroki KANZATO, and Naoyuki YAMAMOTO p.951

Purification and Characterization of Two NAD-Dependent Alcohol Dehydrogenases (ADHs) Induced in the Quinoprotein ADH-Deficient Mutant of Acetobacter pasteurianus SKU1108
Piyawan CHINNAWIROTPISAN,1 Kazunobu MATSUSHITA,2,† Hirohide TOYAMA,2 Osao ADACHI,2 Savitree LIMTONG,1 and Gunjana THEERAGOOL1 p.958

A Root-specific O-Methyltransferase Gene Expressed in Salt-tolerant Barley
Manabu SUGIMOTO,1,† Yoshihiro OKADA,2 Kazuhiro SATO,1 Kazutoshi ITO,2 and Kazuyoshi TAKEDA1 p.966

cGMP-Phosphodiesterase Activity Is Up-regulated in Response to Pressure Overload of Rat Ventricles
Noriyuki YANAKA,1, Yukie KUROSAWA,2 Kouichi MINAMI,2 Eri KAWAI,1 and Kenji OMORI2,† p.973

Asymmetric Chloronicotinyl Insecticide, 1-[1-(6-Chloro-3-pyridyl)ethyl]- 2-nitroiminoimidazolidine: Preparation, Resolution and Biological Activities toward Insects and Their Nerve Preparations
Shinzo KAGABU,1,† Kazuhisa KIRIYAMA,2 Hisashi NISHIWAKI,3 Yuko KUMAMOTO,1 Toshiji TADA,2 and Keiichiro NISHIMURA2 p.980

Enzymatic Synthesis of 2′-Deoxyguanosine with Nucleoside Deoxyribosyltransferase-II
Kiyoshi OKUYAMA, Susumu SHIBUYA, Tomoki HAMAMOTO, and Toshitada NOGUCHI p.989

Eritadenine-induced Alterations of Plasma Lipoprotein Lipid Concentrations and Phosphatidylcholine Molecular Species Profile in Rats Fed Cholesterol-free and Cholesterol-enriched Diets
Yasuhiko SHIMADA, Tatsuya MORITA, and Kimio SUGIYAMA p.996

Induction Mechanism of 3-Hydroxy-3-methylglutaryl-CoA Reductase in Potato Tuber and Sweet Potato Root Tissues
Katsuyoshi KONDO, Ikuzo URITANI, and Kazuko OBA p.1007

The Production of a New Tempeh-like Fermented Soybean Containing a High Level of γ-Aminobutyric Acid by Anaerobic Incubation with Rhizopus
Hideyuki AOKI,1,† Ichiyo UDA,1 Keiko TAGAMI,1 Yuji FURUYA,1 Yasushi ENDO,2 and Kenshiro FUJIMOTO2 p.1018

Glycosidase-catalyzed Deoxy Oligosaccharide Synthesis. Practical Synthesis of Monodeoxy Analogs of Ethyl β-Thioisomaltoside Using Aspergillus niger α-Glucosidase
Toshiyuki NISHIO,1, Chika KANAI,1 Wataru HAKAMATA,2 Masahiro OGAWA,1 Kousuke NAKAJIMA,1 Shigeki HOSHINO,1 Akari MATSUISHI,1 Ryu KAWACHI,1 and Tadatake OKU1
p.1024

Analysis of Molecular Interactions in Heat-induced Aggregation of a Non-inhibitory Serpin Ovalbumin Using a Molecular Chaperone
Fumito TANI,1,† Nobuaki SHIRAI,2 Yukiko NAKANISHI,1, and Naofumi KITABATAKE1
p.1030

Respiratory Isozyme, Two Types of Rusticyanin of Acidithiobacillus ferrooxidans
Kazuhiro SASAKI,1 Chigusa IDA,2 Akikazu ANDO,2 Norio MATSUMOTO,1 Hiroshi SAIKI,1 and Naoya OHMURA1, † p.1039

Isolation and Characterization of Agar-degrading Paenibacillus spp. Associated with the Rhizosphere of Spinach
Akifumi HOSODA,1 Masao SAKAI,2,† and Shinjiro KANAZAWA2 p.1048

Combinatorial Effects of Nonsteroidal Anti-inflammatory Drugs and Food Constituents on Production of Prostaglandin E2 and Tumor Necrosis Factor-α in RAW264.7 Murine Macrophages
Akira MURAKAMI,1, Daisuke TAKAHASHI,2 Kazuma HAGIHARA,2 Koichi KOSHIMIZU,2 and Hajime OHIGASHI1 p.1056

Structure, Heterologous Expression, and Properties of Rice (Oryza sativa L.) Family 19 Chitinases
Nam-Hai TRUONG,1, Seung-Moon PARK,1, Yoko NISHIZAWA,2 Takeshi WATANABE,3 Takuji SASAKI,2 and Yoshifumi ITOH1,† p.1063

Antiviral Activity of a Hot Water Extract of Black Soybean against a Human Respiratory Illness Virus
Masafumi YAMAI,1,2,† Kazunori TSUMURA,3 Mariko KIMURA,4 Seiji FUKUDA,2 Tsukasa MURAKAMI,5 and Yoshinobu KIMURA1,3, † p.1071

Purification, Characterization, and Sequence Analysis of Two α-Amylase Isoforms from Azuki Bean, Vigna angularis, Showing Different Affinity towards β-Cyclodextrin Sepharose
San San MAR, Haruhide MORI, Jin-Ha LEE, Kenji FUKUDA, Wataru SABURI, Arinobu FUKUHARA, Masayuki OKUYAMA, Seiya CHIBA, and Atsuo KIMURA p.1080

Transglycosylation of Glycosyl Residues to Cyclic Tetrasaccharide by Bacillus stearothermophilus Cyclomaltodextrin Glucanotransferase Using Cyclomaltodextrin as the Glycosyl Donor
Takashi SHIBUYA, Hajime AGA, Hikaru WATANABE, Tomohiko SONODA, Michio KUBOTA, Shigeharu FUKUDA, Masashi KURIMOTO, and Yoshio TSUJISAKA p.1094

Chromosomal Circularization in Streptomyces griseus by Nonhomologous Recombination of Deletion Ends
Shoichi INOUE, Koji HIGASHIYAMA, Tetsuya UCHIDA, Keiichiro HIRATSU, and Haruyasu KINASHI p.1101

A Possible Role of NADPH-Dependent Cytochrome P450nor Isozyme in Glycolysis under Denitrifying Conditions
Tomo-o WATSUJI,1 Naoki TAKAYA,1 Akira NAKAMURA,1 and Hirofumi SHOUN2,† p.1109

Denitrification of Nitrate by the Fungus Cylindrocarpon tonkinense
Tomo-o WATSUJI,1 Naoki TAKAYA,1 Akira NAKAMURA,1 and Hirofumi SHOUN2, † p.1115

Note
Degradation of Chlorinated Biphenyl, Dibenzofuran, and Dibenzo-p-dioxin by Marine Bacteria That Degrade Biphenyl, Carbazole, or Dibenzofuran
Hiroyuki FUSE, Osamu TAKIMURA, Katsuji MURAKAMI, Hiroyuki INOUE, and Yukiho YAMAOKA
p.1121

Note
Effect of the Flavonoid Components Obtained from Scutellaria Radix on the Histamine, Immunoglobulin E and Lipid Peroxidation of Spleen Lymphocytes of Sprague-dawley Rats
Beong Ou LIM,1,2,† Ryo Won CHOUE,1,2 Hyeon Yong LEE,3 Nak Sul SEONG,4 and Jong Dai KIM3 p.1126

Note
Change in the Concentration of Vitamins C and E in Rat Tissues by Paraquat Administration
Kazumi IKEDA, Yumi KUMAGAI, Yuka NAGANO, Naoko MATSUZAWA, and Shosuke KOJO p.1130

Note
Induction of Differentiation in Human Promyelocytic Leukemia HL-60 Cell Line by Niacin-related Compounds
Keiko IWATA, Shin OGATA, Katsuzumi OKUMURA, and Hiroshi TAGUCHI p.1132

Note
Inhibition of Myeloperoxidase-catalyzed Tyrosylation by Phenolic Antioxidants in vitro
Yoji KATO,1,† Akihiko NAGAO,2 Junji TERAO,3 and Toshihiko OSAWA4 p.1136

Note
Procyanidin B1 Is Detected in Human Serum after Intake of Proanthocyanidin-rich Grape Seed Extract
Atsushi SANO,1, Jun YAMAKOSHI,1 Shoichi TOKUTAKE,1 Koichiro TOBE,1 Yoshiro KUBOTA,2 and Mamoru KIKUCHI1 p.1140

Note
Rhizoremediation of Dioxin-like Compounds by a Recombinant Rhizobium tropici Strain Expressing Carbazole 1,9a-Dioxygenase Constitutively
Yuko SAIKI,1 Hiroshi HABE,2 Toshifumi YUUKI,1 Mitsuo IKEDA,1 Takako YOSHIDA,2 Hideaki NOJIRI,2 and Toshio OMORI2,† p.1144

Note
Construction of an Efficient Expression System for Aspergillus Isopullulanase in Pichia pastoris, and a Simple Purification Method
Hiromi AKEBOSHI,1 Yutaka KASHIWAGI,2 Hiroyoshi AOKI,1, Takashi TONOZUKA,1 Atsushi NISHIKAWA,1 and Yoshiyuki SAKANO1, † p.1149

Note
Identification of β and γ Subunits of Laminins Localized in the Basement Membrane of Rat Circumvallate Papillae
Mikiya KISHI,1,2 Kaori SANO,1 Misaki ASANO-MIYOSHI,1,3 Yoshinori TSUKAMOTO,2 Yasufumi EMORI,3 and Keiko ABE1,† p.1154

Note
Authentic and Recombinant Bilirubin Oxidases Are in Different Resting Forms
Takeshi SAKURAI,1, Lei ZHAN,1 Takahiro FUJITA,1 Kunishige KATAOKA,1 Atsushi SHIMIZU,2 Tatsuya SAMEJIMA,2 and Shotaro YAMAGUCHI3 p.1157

Note
Transglucosylation Activities of Multiple Forms of α-Glucosidase from Spinach
Manabu SUGIMOTO,1,† Satoshi FURUI,1, Kenji SASAKI,2 and Yukio SUZUKI1 p.1160

Note
Microbial Synthesis of trans Isomer of Eicosapentaenoic Acid (EPA) from the Chemically Synthesized Trans Isomer of Linolenic Acid by a Δ12 Desaturase-defective Mutant of Mortierella alpina 1S-4
Norifumi SHIRASAKA,1,† Shigenori MIYAMOTO,1 Tetsuo MURAKAMI,1 Hajime YOSHIZUMI,1 and Sakayu SHIMIZU2 p.1164

Note
Identification of Volicitin-related Compounds from the Regurgitant of Lepidopteran Caterpillars
Naoki MORI,1, Naoko YOSHINAGA,1 Yoshitsugu SAWADA,1 Masao FUKUI,2 Masami SHIMODA,3 Kenji FUJISAKI,2 Ritsuo NISHIDA,1 and Yasumasa KUWAHARA1 p.1168

Note
Escherichia coli tRNAs Are Resistant to the Hyperprocessing Reaction of Homologous E. coli Ribonuclease P Ribozyme
Terumichi TANAKA, Tomoaki ANDO, Etsuko SAKAI, Taka-aki HAHISBA, Yoshiaki HORI, and Yo KIKUCHI p.1172

Note
Preparation of Antiserum against Rat Δ6-Desaturase and Its Use to Evaluate the Desaturase Protein Levels in Rats Treated with Gemfibrozil, a Ligand for Peroxisome Proliferator-activated Receptor α
Yoko SHOJI, Ayako SANEKATA, Masao SATO, and Katsumi IMAIZUMI p.1177

Note
Escherichia coli Can Be Transformed by a Liposome-mediated Lipofection Method
Yoshikazu KAWATA, Shin-ichi YANO, and Hiroyuki KOJIMA p.1179

Note
Synthesis of 3-O-β-N-Acetylglucosaminyl Cyclic Tetrasaccharide through a Lysozyme-catalyzed Transfer Reaction
Hikaru WATANABE, Hajime AGA, Tomohiko SONODA, Michio KUBOTA, Shigeharu FUKUDA, Masashi KURIMOTO, and Yoshio TSUJISAKA p.1182

Communication
Novel Oxidative Dimer from Caffeic Acid
Hiroyuki TAZAKI,1,† Jun KAWABATA,2 and Takashi FUJITA3 p.1185

Communication
Effects of Physiological Changes in Potato Tubers (Solanum tuberosum L.) after Low Temperature Storage on the Level of Acrylamide Formed in Potato Chips
Yoshihiro CHUDA,1 Hiroshi ONO,2,† Hiroshi YADA,2 Akiko OHARA-TAKADA,3 Chie MATSUURA-ENDO,3 and Motoyuki MORI3 p.1188

Communication
Inportance of Phosphatidylinositol 3-Phosphate in Sporulation of Schizosaccharomyces pombe
Masayuki ONISHI,1 Yoko NAKAMURA,1 Takako KOGA,1, Aiko HIRATA,2 and Yasuhisa FUKUI1,† p.1191

Communication
Uzu Mutation in Barley (Hordeum vulgare L.) Reduces the Leaf Unrolling Response to Brassinolide
Ichiro HONDA,1,† Haruko ZENIYA,1,2 Koichi YONEYAMA,3 Makiko CHONO,1 Shigenobu KANEKO,1 and Yoshiaki WATANABE1 p.1194

-1-
Effect of Methanol Extract of Zanthoxylum piperitum Leaves and of Its Compound, Protocatechuic Acid, on Hepatic Drug Metabolizing Enzymes and Lipid Peroxidation in Rats

Jong Moon HUR,1 Ju Gwon PARK,1 Ki Ho YANG,1 Jong Cheol PARK,1,† Jeong Ro PARK,2 Soon Sil CHUN,2 Jae Sue CHOI,3 and Jong Won CHOI4

1Department of Oriental Medicine Resources and Research Institute of Korean Oriental Medicine, 2Department of Food and Nutrition, Sunchon National University, Suncheon, Jeonnam 540-742, Korea 3Department of Food and Life Science, Pukyung National University, Busan 608-737, Korea 4Department of Pharmacy, Kyungsung University, Busan 608-736, Korea

Received May 8, 2002; Accepted December 26, 2002
The effect of methanol extract and protocatechuic acid from the leaves of Zanthoxylum piperitum on lipid peroxidation and drug metabolizing enzymes were investigated in the liver of bromobenzene-treated rats. The methanol extract and protocatechuic acid reduced the level of lipid peroxide induced by bromobenzene. The methanol extract and protocatechuic acid reduced the activity of aniline hydroxylase that had been increased by bromobenzene, while did not affect the activities of aminopyrine N-demethylase and glutathione S-transferase. The methanol extract and compound effectively restored the activity of epoxide hydrolase which had been decreased by bromobenzene. These results may suggest that the methanol extract of Z. piperitum and protocatechuic acid prevented lipid peroxidation by reducing the activity of aniline hydroxylase, an epoxide-producing enzyme, and by enhancing the activity of epoxide hydrolase, an epoxide-removing enzyme, in rats that had been intoxicated with bromobenzene.
Key words: bromobenzene; Zanthoxylum piperitum; protocatechuic acid; drug-metabolizing enzyme; lipid peroxidation

-2-
Decrease in Ovalbumin Specific IgE of Mice Serum after Oral Uptake of Lactic Acid Bacteria

Yuu ISHIDA, Izuki BANDOU, Hiroki KANZATO, and Naoyuki YAMAMOTO

R＆D Center, Calpis Co., Ltd., 11-10, 5-Chome, Fuchinobe, Sagamihara, Kanagawa 229-0006, Japan

Received September 25, 2002; Accepted January 27, 2003
Different kinds of lactobacilli and Bifidobacteria fermented milk were fed to ovalbumin-specific IgE-elevated mice for 3 days, and after the final administration, changes in the ovalbumin-specific IgE values for each sample were compared to the value for non-fermented milk. Seven of the Lactobacillus-fermented milks caused a significant decrease in the serum ovalbumin-specific IgE levels. Above all, Lactobacillus acidophilus L92, Lactobacillus acidophilus CP1613, and Lactobacillus fermentum CP34 fermented milk had the most significant effects of decreasing the serum ovalbumin-specific IgE levels compared to a control group. The L. acidophilus L92 and L. fermentum CP34 cells also showed significant ovalbumin-specific IgE lowering activities. From these results, an active component seems to exist in the cells of L. acidophilus L92 and L. fermentum CP34 strains. Recovery of the radiolabeled L. acidophilus L92 and L. fermentum CP34 cells from the small intestine and the large intestine of the mouse 13 h after oral administration were higher than the recovery of any other strain.
Key words: antigen-specific IgE; Lactobacillus acidophilus; Lactobacillus fermentum; gastrointestinal tract; re-isolation

-3-
Purification and Characterization of Two NAD-Dependent Alcohol Dehydrogenases (ADHs) Induced in the Quinoprotein ADH-Deficient Mutant of Acetobacter pasteurianus SKU1108

Piyawan CHINNAWIROTPISAN,1 Kazunobu MATSUSHITA,2,† Hirohide TOYAMA,2 Osao ADACHI,2 Savitree LIMTONG,1 and Gunjana THEERAGOOL1

1Department of Microbiology, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand 2Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan

Received September 27, 2002; Accepted December 21, 2002
High NAD-dependent alcohol dehydrogenase (ADH) activity was found in the cytoplasm when a membrane-bound, quinoprotein, ADH-deficient mutant strain of Acetobacter pasteurianus SKU1108 was grown on ethanol. Two NAD-dependent ADHs were separated and purified from the supernatant fraction of the cells. One (ADH I) is a trimer, consisting of an identical subunit of 42 kDa, while the other (ADH II) is a homodimer, having a subunit of 31 kDa. One of the two ADHs, ADH II, easily lost the activity during the column chromatographies, which could be stabilized by the addition of DTT and MgCl2 in the column buffer. ADH I but not ADH II contained approximately one zinc atom per subunit. The N-terminal amino acid analysis indicated that ADH I and ADH II have homology to the long-chain and short-chain ADH families, respectively. ADH I showed a preference for primary alcohols, while ADH II had a preference for secondary alcohols. The two ADHs showed clear difference in their kinetics on ethanol, acetaldehyde, NAD, and NADH. The physiological function of both ADH I and ADH II are also discussed.
Key words: acetic acid bacteria; Acetobacter pasteurianus; quinoprotein alcohol dehydrogenase; NAD-dependent alcohol dehydrogenase

-4-
A Root-specific O-Methyltransferase Gene Expressed in Salt-tolerant Barley

Manabu SUGIMOTO,1,† Yoshihiro OKADA,2 Kazuhiro SATO,1 Kazutoshi ITO,2 and Kazuyoshi TAKEDA1

1Laboratory of Biochemistry, Research Institute for Bioresources, Okayama University, Kurashiki, Okayama 710--0046, Japan 2Plant Bioengineering Research Laboratories, Sapporo Breweries Ltd., Nitta, Gunma 370--0393, Japan

Received October 15, 2002; Accepted February 3, 2003
A cDNA encoding an O-methyltransferase (OMT) was isolated from salt-tolerant barley roots by subtraction hybridization with cDNAs of salt-tolerant barley roots as a tester cDNA and cDNAs of the salt-sensitive barley roots as a driver cDNA. The deduced amino acid sequence showed significant identity with plant caffeic acid/5-hydroxyferulic acid OMTs. Southern blot analysis showed that the OMT gene was a single copy in both salt-tolerant and -sensitive barley. The cloned gene was expressed in a wheat germ cell-free system to produce the OMT, which had methylating activity for caffeic acid. Northern blot analysis showed that the OMT gene was expressed constitutively in the salt-tolerant barley roots and the expression level was increased 1.5 times by salt stress, but the salt-sensitive barley showed no expression of the gene in roots and leaves.
Key words: Hordeum vulgare L.; O-methyltransferase; subtraction hybridization; salt stress; root

-5-
cGMP-Phosphodiesterase Activity Is Up-regulated in Response to Pressure Overload of Rat Ventricles

Noriyuki YANAKA,1, Yukie KUROSAWA,2 Kouichi MINAMI,2 Eri KAWAI,1 and Kenji OMORI2,†

1Discovery Research Laboratory, Tanabe Seiyaku Co., Ltd., Yodogawa-ku, Osaka 532-8505, Japan 2Discovery Research Laboratory, Tanabe Seiyaku Co., Ltd., Toda, Saitama 335-8305, Japan

Revised October 17, 2002; Accepted January 30, 2003
Although expression of natriuretic peptides in cardiac tissues is up-regulated in response to pressure overload, no significant change in cGMP level in hypertrophied ventricles was observed. Activities of two cyclic nucleotide phosphodiesterase (PDE) isoforms, Ca2＋/calmodulin-stimulated PDE (PDE1) and cGMP-stimulated PDE (PDE2), were significantly higher in rat left ventricles 14 days after aortic banding. The absence of significant changes in PDE1A and PDE2A mRNA levels indicated that the two PDE activities were post-transcriptionally up-regulated. These results suggested that the increased cGMP-PDE activity in response to pressure overload plays an important role in neutralizing cGMP action in cardiac tissue.
Key words: cGMP; natriuretic peptide; phosphodiesterase; pressure overload

-6-
Asymmetric Chloronicotinyl Insecticide, 1-[1-(6-Chloro-3-pyridyl)ethyl]- 2-nitroiminoimidazolidine: Preparation, Resolution and Biological Activities toward Insects and Their Nerve Preparations

Shinzo KAGABU,1,† Kazuhisa KIRIYAMA,2 Hisashi NISHIWAKI,3 Yuko KUMAMOTO,1 Toshiji TADA,2 and Keiichiro NISHIMURA2

1Department of Chemistry, Faculty of Education, Gifu University, Gifu 501-1193, Japan 2Research Institute for Advanced Science and Technology, Osaka Prefecture University, Gakuen-cho, Sakai, Osaka 599-8570, Japan 3Laboratory of Pesticide Chemistry, Department of Agricultural Chemistry, Faculty of Agriculture, Kinki University, Nakamachi, Nara 631-8505, Japan

Received October 23, 2002; Accepted December 27, 2002
The asymmetric chloronicotinyl insecticide, 1-[1-(6-chloro-3-pyridyl)ethyl]-2-nitroiminoimidazolidine, was prepared, and the absolute configurations of the enantiomers were determined by an X-ray analysis. The insecticidal activity against the housefly measured with metabolic inhibitors showed the (S) enantiomer to be slightly more active than the (R) isomer. Electrophysiological measurements on the American cockroach central nerve cord showed the compounds to elicite the impulses and subsequently blocked them. The neuroblocking potency of the (S) isomer was 5.9 μM, while that of the (R) isomer was as high as 73 μM. The molar concentrations required for 50％ inhibition of the specific binding of [3H]imidacloprid to the housefly head membrane preparation were respectively 0.19 μM and 0.95 μM for the (S) and (R) isomers. This enatioselectivity ratio was smaller than 35 for nicotine isomers but greater than 2 for epibatidine isomers.
Key words: imidacloprid; asymmetric chloronicotinyl insecticide; insecticidal activity; neuroblocking potency; binding affinity

-7-
Enzymatic Synthesis of 2′-Deoxyguanosine with Nucleoside Deoxyribosyltransferase-II

Kiyoshi OKUYAMA, Susumu SHIBUYA, Tomoki HAMAMOTO, and Toshitada NOGUCHI

Biochemicals Division, Yamasa Corporation, Choshi, Chiba 288-0056, Japan

Recived October 24, 2002; Accepted January 10, 2003
Nucleoside deoxyribosyltransferase-II (NdRT-II) of Lactobacillus helveticus, which catalyzes the transfer of a glycosyl residue from a donor deoxyribonucleoside to an acceptor base, has a broad specificity for the acceptor bases. Six-substituted purines were found to be substrates as acceptor bases for NdRT-II. Using this property of the enzyme, we established a practical procedure for enzymatic synthesis of 2′-deoxyguanosine (dGuo), consisting of the transglycosylation from thymidine to 6-substituted purine (2-amino-6-chloropurine; ACP) instead of natural guanine and the conversion of 2-amino-6-chloropurine-2′-deoxyriboside (ACPdR) to dGuo with bacterial adenosine deaminase. Through the successive reactions, dGuo was synthesized in high yield.
Key words: nucleoside deoxyribosyltransferase; 2′-deoxyribonucleoside; 2′-deoxyguanosine; 2-amino-6-chloropurine; adenosine deaminase

-8-
Eritadenine-induced Alterations of Plasma Lipoprotein Lipid Concentrations and Phosphatidylcholine Molecular Species Profile in Rats Fed Cholesterol-free and Cholesterol-enriched Diets

Yasuhiko SHIMADA, Tatsuya MORITA, and Kimio SUGIYAMA

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

Received October 30, 2002; Accepted February 12, 2003
The effects of dietary eritadenine on the concentration of plasma lipoprotein lipids and the molecular species profile of plasma lipoprotein phosphatidylcholine (PC) were investigated in rats fed cholesterol-free and cholesterol-enriched diets to obtain insights into the relationship between the changes in PC molecular species profile and the hypocholesterolemic action of eritadenine. The effect of eritadenine on the secretion rate of very low density lipoprotein (VLDL) from the liver was also estimated. Rats were fed the control or eritadenine-supplemented (50 mg/kg) diets with or without exogenous cholesterol for 14 d. Eritadenine supplementation significantly decreased the cholesterol of major plasma lipoproteins, high density lipoprotein and VLDL, in rats fed cholesterol-free and cholesterol-enriched diets, respectively. The ratio of PC to phosphatidylethanolamine, Δ6-desaturase activity, and the ratio of arachidonic acid to linoleic acid in liver microsomes were markedly decreased by eritadenine irrespective of the presence or absence of exogenous cholesterol. Dietary eritadenine increased the proportion of 16:0--18:2 molecular species with a decrease in 18:0--20:4 in plasma lipoprotein PC in both rats fed cholesterol-free and cholesterol-enriched diets. Eritadenine did not depress the secretion rate of VLDL in rats fed a cholesterol-free diet containing a high level of choline. The results indicate that dietary eritadenine elicits its hypocholesterolemic action with modulations of the fatty acid and molecular species profiles of PC irrespective of the presence or absence of exogenous cholesterol. The eritadenine-induced alteration of PC molcular species profile is discussed in relation to the hypocholesterolemic action of eritadenine.
Key words: eritadenine; cholesterol; phosphatidylcholine; linoleic acid metabolism; Δ6-desaturase

-9-
Induction Mechanism of 3-Hydroxy-3-methylglutaryl-CoA Reductase in Potato Tuber and Sweet Potato Root Tissues

Katsuyoshi KONDO, Ikuzo URITANI, and Kazuko OBA

Laboratory of Food Biochemistry, Nagoya Women′s University, 3-40 Shioji, Mizuho, Nagoya 467-8610, Japan

Received October 30, 2002; Accepted January 15, 2003
3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR, EC1.1.1.34), the key enzyme in isoprenoid biosynthesis, was purified from microsomes of potato tuber tissue, and a polyclonal antibody and two monoclonal antibodies against the purified enzyme were prepared. HMGR protein content was measured by immunotitration and radioimmunoassay using these antibodies. HMGR activity was very low in the fresh tissues of both potato tuber and sweet potato root. The activity in potato tuber was increased by cutting and further by additional fungal infection of the cut tissues. In sweet potato root tissue, the activity was scarcely increased after cutting alone, but was markedly increased by additional fungal infection or chemical treatment. The HMGR protein contents in both fresh potato tuber and sweet potato root tissues were also very low, and increased markedly in response to cutting and fungal infection. From these results, we proposed a hypothesis on the induction mechanism of HMGR after cutting and fungal infection in potato tuber and sweet potato root tissues.
Key words: 3-hydroxy-3-methylglutaryl coenzyme A reductase; sesqueterpenes; sweet potato root; potato tuber; fungal infection

-10-
The Production of a New Tempeh-like Fermented Soybean Containing a High Level of γ-Aminobutyric Acid by Anaerobic Incubation with Rhizopus

Hideyuki AOKI,1,† Ichiyo UDA,1 Keiko TAGAMI,1 Yuji FURUYA,1 Yasushi ENDO,2 and Kenshiro FUJIMOTO2

1Research Laboratory, Ikeda Tohka Industries Co., Ltd., 95-7 Minooki-Cho, Fukuyama, Hiroshima 721-0956, Japan 2Department of Science of Biological Function, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Sendai 981-8555, Japan

Received November 1, 2002; Accepted February 14, 2003
A cultivation procedure for the preparation of a new tempeh-like fermented soybean containing a high level of γ-aminobutyric acid was developed. Steamed soybeans were incubated aerobically with Rhizopus microsporus var. oligosporus IFO 8631 for 20 h, and then anaerobically incubated for 5 h by replacement of the atmosphere with nitrogen. The GABA content in the aerobically fermented soybeans was about 30 mg per 100 g dry fermented soybeans, while the anaerobically cultivation was about 370 mg/100 g dry fermented soybeans. The incubation with several strains of Rhizopus species showed that all of R. microsporus var. oligosporus and R. oryzae examined accumulated GABA in the anaerobically fermented soybeans. In particular, R. microsporus var. oligosporus IFO 32002 and IFO 32003 showed the highest content of GABA (1,740 mg/100 g dry fermented soybeans and 1,500 mg/100 g dry fermented soybeans, respectively). Moreover, the free protein amino acids increased greatly in the fermented soybeans during the anaerobic cultivation.
Key words: γ-aminobutyric acid; fermented soybean; GABA; Rhizopus; tempeh

-11-
Glycosidase-catalyzed Deoxy Oligosaccharide Synthesis. Practical Synthesis of Monodeoxy Analogs of Ethyl β-Thioisomaltoside Using Aspergillus niger α-Glucosidase

Toshiyuki NISHIO,1, Chika KANAI,1 Wataru HAKAMATA,2 Masahiro OGAWA,1 Kousuke NAKAJIMA,1 Shigeki HOSHINO,1 Akari MATSUISHI,1 Ryu KAWACHI,1 and Tadatake OKU1

1Laboratory of Bio-organic Chemistry, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa-shi, Kanagawa 252-8510, Japan 2Division of Organic Chemistry, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan

Received November 7, 2002; Accepted January 15, 2003
Enzymatic transglycosylation using four possible monodeoxy analogs of p-nitrophenyl α-D-glucopyranoside (Glcα-O-pNP), modified at the C-2, C-3, C-4, and C-6 positions (2D-, 3D-, 4D-, and 6D-Glcα-O-pNP, respectively), as glycosyl donors and six equivalents of ethyl β-D-thioglucopyranoside (Glcβ-S-Et) as a glycosyl acceptor, to yield the monodeoxy derivatives of glucooligosaccharides were done. The reaction was catalyzed using purified Aspergillus niger α-glucosidase in a mixture of 50 mM sodium acetate buffer (pH 4.0)/CH3CN (1: 1 v/v) at 37°C. High activity of the enzyme was observed in the reaction between 2D-Glcα-O-pNP and Glcβ-S-Et to afford the monodeoxy analogs of ethyl β-thiomaltoside and ethyl β-thioisomaltoside that contain a 2-deoxy α-D-glucopyranose moiety at their glycon portions, namely ethyl 2-deoxy-α-D-arabino-hexopyranosyl-(1,4)-β-D-thioglucopyranoside and ethyl
2-deoxy-α-D-arabino-hexopyranosyl-(1,6)-β-D-thioglucopyranoside, in 6.72％ and 46.6％ isolated yields (based on 2D-Glcα-O-pNP), respectively. Moreover, from 3D-Glcα-O-pNP and Glcβ-S-Et, the enzyme also catalyzed the synthesis of the 3-deoxy analog of ethyl β-thioisomaltoside that was modified at the glycon α-D-glucopyranose moiety, namely ethyl 3-deoxy-α-D-ribo-hexopyranosyl-(1,6)-β-D-thioglucopyranoside, in 23.0％ isolated yield (based on 3D-Glcα-O-pNP). Products were not obtained from the enzymatic reactions between 4D- or 6D-Glcα-O-pNP and Glcβ-S-Et.
Key words: transglycosylation; α-glucosidase; Aspergillus niger; 2-deoxy isomaltoside; 3-deoxy isomaltoside

-12-
Analysis of Molecular Interactions in Heat-induced Aggregation of a Non-inhibitory Serpin Ovalbumin Using a Molecular Chaperone

Fumito TANI,1,† Nobuaki SHIRAI,2 Yukiko NAKANISHI,1, and Naofumi KITABATAKE1

1Laboratory of Food and Environmental Sciences, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Goka-sho, Uji, Kyoto 611-0011, Japan 2Industrial Research Center of Shiga Prefecture, 232 Kamitoyama, Ritto, Shiga 520--3004, Japan

Received November 7, 2002; Accepted December 27, 2002
Aggregation occurs through hydrophobic interactions when a polypeptide chain refolds in non-native states or when genetic variants of biologically active proteins assume inappropriate conformations, as observed in the case of dysfunctional serpins. Here, using the molecular chaperone BiP from bovine liver microsomes, we characterized the hydrophobic nature of the peptide segment which is considered to be a site required for aggregation among a non-inhibitory serpin ovalbumin in a heat-denatured state. Screening of the peptide scan for binding of BiP showed that BiP-binding sites are mostly buried in the folded ovalbumin. When ovalbumin was heat-denatured, the denatured protein was recognized by the antibody that reacts with the hydrophobic surface of the amino-terminal segment of ovalbumin. This antibody significantly suppressed the binding of BiP to denatured ovalbumin. BiP also bound the immobilized peptide in an ATP-dependent manner and the peptide stimulated the ATPase activity of BiP with a Km of 165 μM and a Vmax of 0.4 nmol/min per milligram. Measurement of surface plasmon resonance showed that the peptide had a Kd of 0.52 μM by BiP, lower than that for RCMLA (Kd＝1.1 μM) and even lower than that of the peptide P10K, PLSRTLSVAAKK, (Kd＝21 μM). These results demonstrate that the aggregation-prone site on heat-denatured ovalbumin has almost the same hydrophobic nature of interacting with the molecular chaperone BiP as the conventionally known peptides that bind to the Escherichia coli chaperone DnaK.
Key words: aggregation; hydrophobic interaction; molecular chaperone; ovalbumin; serpin

-13-
Respiratory Isozyme, Two Types of Rusticyanin of Acidithiobacillus ferrooxidans

Kazuhiro SASAKI,1 Chigusa IDA,2 Akikazu ANDO,2 Norio MATSUMOTO,1 Hiroshi SAIKI,1 and Naoya OHMURA1,

1Central Research Institute of Electric Power Industry, Department of Bio-science, 1646 Abiko, Abiko City, Chiba 270-1194, Japan 2Chiba University, Department of Biotechnology, Graduate School of Science and Technology, 648 Matsudo, Matsudo City, Chiba 271-8510, Japan

Received November 8, 2002; Accepted January 10, 2003
Among the members of the copper protein superfamily, the type I enzyme rusticyanin, which is found as an electron carrier in the oxidative respiratory chain of Acidithiobacillus ferrooxidans, is the only one to have both a high redox potential and acid stability. Here we report that two forms of the rusticyanin gene (rus) are present in the genomes of some strains of A. ferrooxidans. The more common form of rus (type-A) was found to be present in all six strains studied, including those harboring only a single copy of the gene. In addition a less common form (type-B) occurred in strains harboring multiple copies of the gene. The two genes were expressed as rusticyanin isozymes with differing surface charges due to differences in their amino acid composition. Still, the copper coordination sites were completely conserved, thereby maintaining the high redox potential necessary for an electron carrier.
Key words: Acidithiobacillus ferrooxidans; rusticyanin; isozyme; respiration; iron

-14-
Isolation and Characterization of Agar-degrading Paenibacillus spp. Associated with the Rhizosphere of Spinach

Akifumi HOSODA,1 Masao SAKAI,2,† and Shinjiro KANAZAWA2

1Laboratory of Soil Microbiology, Department of Plant Resources, Graduate School of Bioresource and Bioenvironmental Sciences, and 2Laboratory of Soil Microbiology, Department of Plant Resources, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan

Received November 8, 2002; Accepted January 11, 2003
Agar-degrading bacteria in spinach plant roots cultivated in five soils were screened, and four strains of Paenibacillus sp. were isolated from roots cultivated in three soils. The agar-degrading bacteria accounted for 1.3％ to 2.5％ of the total bacteria on the roots. In contrast, no agar-degrading colony was detected in any soil (non-rhizosphere soil samples) by the plate dilution method, and thus these agar-degrading bacteria may specifically inhabit plant roots. All isolates produced extracellular agarase, and could grow using agar in the culture medium as the sole carbon source. Zymogram analyses of agarase showed that all four isolates extracellularly secreted multiple agarases (75--160 kDa). In addition, the isolates degraded not only agar but also various plant polysaccharides, i.e., cellulose, pectin, starch, and xylan.
Key words: rhizosphere; agarase; Paenibacillus; zymogram

-15-
Combinatorial Effects of Nonsteroidal Anti-inflammatory Drugs and Food Constituents on Production of Prostaglandin E2 and Tumor Necrosis Factor-α in RAW264.7 Murine Macrophages

Akira MURAKAMI,1, Daisuke TAKAHASHI,2 Kazuma HAGIHARA,2 Koichi KOSHIMIZU,2 and Hajime OHIGASHI1

1Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan 2Department of Biotechnological Science, Faculty of Biology-Oriented Science and Technology, Kinki University, Wakayama 649-6493, Japan

Received November 27, 2002; Accepted February 15, 2003
Combinatorial chemopreventive strategies, in contrast to those with individual agents, show potential in terms of potentially lower toxicity and higher efficacy. In this study, we combined several agents and examined their suppressive effects on the combined lipopolysaccharide (LPS)- and interferon(IFN)-γ-induced formation of proinflammatory mediators, including prostaglandin (PG) E2 and tumor necrosis factor (TNF)-α, in RAW264.7 murine macrophages. The combinatorial effects of indomethacin/genistein (GEN) and aspirin/GEN were found to be synergistic for PGE2 suppression, while the nimesulide/GEN combination was antagonistic. Further, while (−)-epigallocatechin gallate (EGCG) alone increased LPS/IFM-γ-induced production of PGE2 and TNF-α as well as cyclooxygenase-2 expression, the EGCG/GEN combination markedly suppressed these parameters. Our results suggest that certain chemopreventive agents act complexly and that, when used in combination, they affect the intracellular signaling pathways of the paired agents to exert additive, synergistic, or antagonistic effects.
Key words: epigallocatechin gallate; genistein; cyclooxygenase; RAW264.7 cells; inflammation

-16-
Structure, Heterologous Expression, and Properties of Rice (Oryza sativa L.) Family 19 Chitinases

Nam-Hai TRUONG,1, Seung-Moon PARK,1, Yoko NISHIZAWA,2 Takeshi WATANABE,3 Takuji SASAKI,2 and Yoshifumi ITOH1,†

1National Food Research Institute, Kannondai 2-1-12, Tsukuba, Ibaraki 305-8642, Japan 2National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan 3Department of Biosystem Science, Graduate School of Science and Technology, Niigata University, 8050 Ikarashi-2, Niigata 950-2102, Japan

Received December 3, 2002; Accepted January 31, 2003
We identified four new family 19 chitinases in Oryza sativa L. cv. Nipponbare: one class I (OsChia1d), two class II (OsChia2a and OsChia2b), and one class IV (OsChia4a). OsChia2a resembled (about 60％ identity) the catalytic domains of class I chitinases, but OsChia2b was almost identical (95％ identity) to that of the class IV enzyme. OsChia1c, OsChia1cΔCBD (a deletion of OsChia1c lacking a chitin-binding domain, CBD), and OsChia2b were separately expressed and purified in Pichia pastoris. OsChia1c inhibited fungal growth significantly more than OsChia1cΔCBD or OsChia2b. The activities of these enzymes on chitin polymers were similar, but they acted differently on N-acetylchitooligosaccharides, (GlcNAC)n. OsChia1c slowly hydrolyzed (GlcNAC)6 and very poorly hydrolyzed (GlcNAC)4 and (GlcNAC)5. In contrast, OsChia2b efficiently hydrolyzed these oligosaccharides. The high antifungal activity and low hydrolytic activity of the class I enzyme towards (GlcNAC)n imply that it participates in the generation of N-acetylchitooligosaccharide elicitors from the cell walls of infecting fungi.
Key words: rice (Oryza sativa L); chitinase; N-acetylchitooligosaccharides; antifungal activity; chitin-binding domain

-17-
Antiviral Activity of a Hot Water Extract of Black Soybean against a Human Respiratory Illness Virus

Masafumi YAMAI,1,2,† Kazunori TSUMURA,3 Mariko KIMURA,4 Seiji FUKUDA,2 Tsukasa MURAKAMI,5 and Yoshinobu KIMURA1,3,

1Division of Biomolecular Science, The Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tushima-Naka, Okayama 700-8530, Japan 2R＆D Laboratory, Taiyo Corporation, 1-6-27 Higashiawaji, Higashiyodogawa-ku, Osaka 533-0023, Japan 3Department of Bioresources Chemistry, Faculty of Agriculture, Okayama University, 1-1-1 Tushima-Naka, Okayama 700-8530, Japan 4Department of Food Systems, Faculty of Food Culture, Kurashiki Sakuyo University, 3515 Nagao-Tamashima, Kurashiki 710-0292, Japan 5Department of Microbiology, Osaka City Institute of Public Health and Environmental Sciences, 8-34 Tojo-cho, Tennoji-ku, Osaka 543-0026, Japan

Received December 4, 2002; Accepted December 30, 2002
Significant antiviral activity against respiratory illness viruses has been found in a hot-water extract of black soybean. This black soybean extract showed significant antiviral activity against human adenovirus type 1 and coxsackievirus B1 in a dose-dependent manner, while the hot-water extract from common yellowish soybean showed only weak activity. The antiviral activity could not be extracted from the black soybean by 70％ aqueous ethanol, suggesting that saponin in the seed did not contribute to this activity. The antiviral activity was only recovered from cotyledons and not from seed coats with the hot water, showing that the activity was distributed in the cotyledons and that antocyanins in the black soybean seed coats did not contribute to the antiviral activity. The antiviral compound(s) in the black soybean was partially purified by up to 166 times by a combination of gel filtration, reversed phase HPLC, and ion-exchange HPLC. The partially purified antiviral compound showed hydrophilic and anionic properties, and a maximum absorption at 260 nm, suggesting that this antiviral fraction may contain a phenyl group(s). On the other hand, an amino acid analysis with the acid hydrolyzate and a neutral sugar analysis showed that the antiviral compound from black soybean might not be a polypeptide or glycoconjugate bearing neutral sugar(s).
Key words: black soybean; hot-water extract; antiviral activity; respiratory illness virus; Glycine max

-18-
Purification, Characterization, and Sequence Analysis of Two α-Amylase Isoforms from Azuki Bean, Vigna angularis, Showing Different Affinity towards β-Cyclodextrin Sepharose

San San MAR, Haruhide MORI, Jin-Ha LEE, Kenji FUKUDA, Wataru SABURI, Arinobu FUKUHARA, Masayuki OKUYAMA, Seiya CHIBA, and Atsuo KIMURA

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

Received December 18, 2002; Accepted January 23, 2003
Two α-amylase isoforms designated VAAmy1 and VAAmy2 were purified from cotyledons of germinating seedlings of azuki bean (Vigna angularis). VAAmy1 apparently had lower affinity towards a β-cyclodextrin Sepharose column than VAAmy2. Molecular weights of VAAmy1 and VAAmy2 were estimated to be 47,000 and 44,000, respectively. However, no considerable difference was found between them in effects of pH, temperature, CaCl2, and EDTA, as well as the kinetic parameters for amylose (average degree of polymerization 17): kcat, 71.8 and 55.5 s−1, Km, 0.113 and 0.097 mg/ml; for blocked 4-nitrophenyl α-D-maltoheptaoside: kcat, 62.4 and 85.3 s−1, Km, 0.22 and 0.37 mM, respectively. Primary structures of the two enzymes were analyzed by N-terminal sequencing, cDNA cloning, and MALDI-TOF mass spectrometry, implying that the two enzymes have the same peptide. The results indicated that the low affinity of VAAmy1 towards β-cyclodextrin Sepharose was due to some modification on/near carbohydrate binding site in the limited sequence regions, resulting in higher molecular weight.
Key words: azuki bean α-amylase; isoforms; possible posttranslational modification; carbohydrate binding site; alternative splicing

-19-
Transglycosylation of Glycosyl Residues to Cyclic Tetrasaccharide by Bacillus stearothermophilus Cyclomaltodextrin Glucanotransferase Using Cyclomaltodextrin as the Glycosyl Donor

Takashi SHIBUYA, Hajime AGA, Hikaru WATANABE, Tomohiko SONODA, Michio KUBOTA, Shigeharu FUKUDA, Masashi KURIMOTO, and Yoshio TSUJISAKA

Hayashibara Biochemical Laboratories, Inc., 7-7 Amase-minami machi, Okayama 700-0834, Japan

Received December 18, 2002; Accepted February 13, 2003
Cyclomaltodextrin glucanotransferase (EC 2.4.1.19, abbreviated as CGTase) derived from Bacillus stearothermophilus produced a series of transfer products from a mixture of cyclomaltohexaose and cyclic tetrasaccharide (cyclo｛→6)-α-D-Glcp-(1→3)-α-D-Glcp-(1→6)-α-D-Glcp-(1→3)-α-D-Glcp-(1→｝, CTS). Of the transfer products, only two components, saccharides A and D, remained and accumulated after digestion with glucoamylase. The total combined yield of the saccharides reached 63.4％ of total sugars, and enzymatic and instrumental analyses revealed the structures of both saccharides. Saccharide A was identified as
4-mono-O-α-glucosyl-CTS, ｛→6)-[α-D-Glcp-(1→4)]-α-D-Glcp-(1→3)-α-D-Glcp-(1→6)-α-D-Glcp-(1→3)-α-D-Glcp-(1→｝, and sachharide D was 4,4′-di-O-α-glucosyl-CTS, ｛→6)-[α-D-Glcp-(1→4)]-α-D-Glcp-(1→3)-α-D-Glcp-(1→6)-[α-D-Glcp-(1→4)]-α-D-Glcp-(1→3)-α-D-Glcp-(1→｝. These structures led us to conclude that the
glycosyltransfer catalyzed by CGTase was specific to the C4-OH of the 6-linked glucopyranosyl residues in CTS.
Key words: cyclic tetrasaccharide; cyclomaltodextrin glucanotransferase; cyclomaltodextrin; transglycosylation

-20-
Chromosomal Circularization in Streptomyces griseus by Nonhomologous Recombination of Deletion Ends

Shoichi INOUE, Koji HIGASHIYAMA, Tetsuya UCHIDA, Keiichiro HIRATSU, and Haruyasu KINASHI

Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8530, Japan

Received December 24, 2002; Accepted January 29, 2003
Streptomyces linear chromosomes frequently cause deletions at both ends spontaneously or by various mutagenic treatments, and concomitantly display dynamic structural changes such as circularization and arm replacement. We have cloned and sequenced the fusion junctions of circularized chromosomes in two deletion mutants of Streptomyces griseus. No homology and a 1-bp overlap were found between the deletion ends of the mutant chromosomes. Taking this together with previous results, we concluded that chromosomal circularization in Streptomyces occurs by nonhomologous recombination between deletion ends.
Key words: Streptomyces; chromosomal circularization; chromosomal deletion; genome fluidity; nonhomologous recombination

-21-
A Possible Role of NADPH-Dependent Cytochrome P450nor Isozyme in Glycolysis under Denitrifying Conditions

Tomo-o WATSUJI,1 Naoki TAKAYA,1 Akira NAKAMURA,1 and Hirofumi SHOUN2,†

1Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan 2Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-Ku, Tokyo 113-8657, Japan

Received December 27, 2002; Accepted January 30, 2003
The denitrifying fungus Cylindrocarpon tonkinense contains two isozymes of cytochrome P450nor. One isozyme, P450nor1, uses NADH specifically as its electron donor whereas the other isozyme P450nor2 prefers NADPH to NADH. Here we show that P450nor1 is localized in both cytosol and mitochondria, like P450nor of Fusarium oxysporum, while P450nor2 is exclusively in cytosol. We also found that the addition of glucose as a carbon source to the culture media leads to the production of much more P450nor2 in the fungal cells than a non-fermentable substrate (glycerol or acetate) does. These results suggest that the NADP-dependent pentose phosphate cycle acts predominantly in C. tonkinense as the glycolysis pathway under the denitrifying conditions, which was confirmed by the observation that glucose induced enzyme activities involved in the cycle. These results showed that P450nor2 should act as the electron sink under anaerobic, denitrifying conditions to regenerate NADP＋ for the pentose phosphate cycle.
Key words: denitrification; cytochrome P450nor; nitric oxide reductase; mitochondria; Cylindrocarpon tonkinense

-22-
Denitrification of Nitrate by the Fungus Cylindrocarpon tonkinense

Tomo-o WATSUJI,1 Naoki TAKAYA,1 Akira NAKAMURA,1 and Hirofumi SHOUN2,

1Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan 2Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-Ku, Tokyo 113-8657, Japan

Received January 14, 2003; Accepted February 4, 2003
The denitrifying fungus Cylindrocarpon tonkinense was thought to be able to denitrify only nitrite (NO−2) but not nitrate (NO−3) to form nitrous oxide (N2O). Here we found, however, that C. tonkinense can denitrify NO−3 under certain conditions. Presence of ammonium (NH＋3) in addition to NO−3 and the use of a fermentable sugar as an electron donor were key conditions for inducing the denitrifying activity. Such induction accompanied a remarkable increase in the intracellular level of the enzyme activities related to NO−3 metabolism. These activities contained assimilatory type NADPH (or NADH)-dependent NO−3 reductase (aNar), dissimilatory nitrite reductase (dNir), and nitric oxide reductase (P450nor), but did not contain ubiquinol-dependent, dissimilatory NO−3 reductase (dNar). The denitrification was inhibited by tungstate, an inhibitor of Nar. These results demonstrated occurrence of a novel type of denitrification in C. tonkinense, in which assimilatory type Nar is possibly involved.
Key words: denitrification; nitrate respiration; nitrate assimilation; nitrate reductase; Cylindrocarpon tonkinense

-23-
Note
Degradation of Chlorinated Biphenyl, Dibenzofuran, and Dibenzo-p-dioxin by Marine Bacteria That Degrade Biphenyl, Carbazole, or Dibenzofuran

Hiroyuki FUSE, Osamu TAKIMURA, Katsuji MURAKAMI, Hiroyuki INOUE, and Yukiho YAMAOKA

Institute for Marine Resources and Environment, National Institute of Advanced Industrial Science and Technology, 2-2-2 Hirosuehiro, Kure, Hiroshima 737-0197, Japan

Received August 19, 2002; Accepted December 20, 2002
Marine bacterial strains (BP-PH, CAR-SF, and DBF-MAK) were isolated using biphenyl, carbazole (CAR), or dibenzofuran (DF) respectively as substrates for growth. Their 16S ribosomal DNA sequences showed that the species closest to strain BP-PH, strain CAR-SF, and strain DBF-MAK are Alteromonas macleodii (96.3％ identity), Neptunomonas naphthovorans (93.1％ identity), and Cycloclasticus pugetii (97.3％ identity), respectively. The metabolites produced suggested that strain CAR-SF degrades CAR via dioxygenation in the angular position and by the meta-cleavage pathway, and that strain DBF-MAK degrades DF via both lateral and angular dioxygenation. Polychlorinated biphenyl (KC-300) and 2,3-dichlorodibenzo-p-dioxin were partially degraded by strain BP-PH and strain DBF-MAK, while 2,7-dichlorodibenzo-p-dioxin and 2,4,8-trichlorodibenzofuran remained virtually unchanged.
Key words: carbazole; dibenzofuran; biphenyl; dibenzo-p-dioxin; marine bacteria

-24-
Note
Effect of the Flavonoid Components Obtained from Scutellaria Radix on the Histamine, Immunoglobulin E and Lipid Peroxidation of Spleen Lymphocytes of Sprague-dawley Rats

Beong Ou LIM,1,2,† Ryo Won CHOUE,1,2 Hyeon Yong LEE,3 Nak Sul SEONG,4 and Jong Dai KIM3

1Graduate School of East-West Medical Science, Department of Medical Nutrition, Kyung Hee University, 1 Hoeki-Dong, Dongdaemoon-Ku, Seoul 130-701, Korea 2Research Institute of Clinical Nutrition, Kyung Hee University, 1 Hoeki-Dong, Dongdaemoon-Ku, Seoul 130-701, Korea 3School of Biotechnology and Bioengineering, Kangwon National University, 200-701, Korea 4Crop Experiment Station, RDA, Suwon, 441-100, Korea

The effect on the IgE content induced by concanavalin A in spleen lymphocytes of the presence wogonin, ganhuangenin, wogonoside and 3,5,7,2′,6′-pentahydroxyl flavanone was investigated. These flavonoid components markedly inhibited the histamine released from cells stimulated with the calcium ionophore, A23187. However, the magnitude of the inhibitory effect on the degree of lipid peroxidation by ConA of these components was in order of PHF＞GHG＞WG＞WGS. Interestingly, WG, GHG and WGS, with a methoxyl group in the A and B rings, strongly inhibited histamine and IgE production, whereas PHF without a methoxyl group was much stronger than that for lipid peroxidation. We suggest that WG, GHG and WGS might block the pathway for the release of histamine, and that the IgE level in spleen lymphocytes is responsible for the lipid peroxidation.
Key words: Scutellaria Radix; histamine; spleen; immmunoglobulin E; lipid peroxidation

-25-
Note
Change in the Concentration of Vitamins C and E in Rat Tissues by Paraquat Administration

Kazumi IKEDA, Yumi KUMAGAI, Yuka NAGANO, Naoko MATSUZAWA, and Shosuke KOJO

Department of Food Science and Nutrition, Nara Women′s University, Nara 630-8506, Japan

Received September 10, 2002; Accepted December 26, 2002
Paraquat causes lung injury by oxidative stress. After 48 h of intraperitoneal administration of paraquat (50 mg/kg of body weight) to rats, the vitamin C concentration in the lungs was significantly decreased, while lung vitamin E content was increased after 12 h. These results indicate that vitamin C directly reflected the oxidative stress in the lungs.
Key words: oxidative stress; paraquat; radical; vitamin C; vitamin E

-26-
Note
Induction of Differentiation in Human Promyelocytic Leukemia HL-60 Cell Line by Niacin-related Compounds

Keiko IWATA, Shin OGATA, Katsuzumi OKUMURA, and Hiroshi TAGUCHI

Laboratory of Molecular and Cellular Biology, Department of Life Science, Faculty of Bioresources, Mie University, Tsu, Mie 514-8507, Japan

Received September 17, 2002; Accepted January 15, 2003
Water-soluble vitamin, niacin, and its related compounds were examined for their differentiation-inducing activity in human promyelocytic leukemia cells (HL-60). Among the compounds, which inhibited cell proliferation measured by MTT assay, isonicotinic acid, nicotinamide N-oxide, and nicotinamide induced NBT reducing activity. HL-60 cells were differentiated into granulocyte-like cells by these compounds, judging from morphological changes and loss of nonspecific esterase activity. The differentiation-inducing activity of water-soluble vitamin and its related compounds suggest that these compounds may be applicable for medical use.
Key words: niacin; isonicotinic acid; nicotinamide N-oxide; differentiation; HL-60

-27-
Note
Inhibition of Myeloperoxidase-catalyzed Tyrosylation by Phenolic Antioxidants in vitro

Yoji KATO,1,† Akihiko NAGAO,2 Junji TERAO,3 and Toshihiko OSAWA4

1School of Humanities for Environmental Policy and Technology, Himeji Institute of Technology, 1-1-12 Shinzaike-honcho, Himeji, Hyogo 670-0092, Japan 2National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan 3Department of Nutrition School of Medicine, the University of Tokushima, Kuramoto-cho, Tokushima 770-0042, Japan 4Nagoya University Graduate School of Bioagricultural Sciences, Nagoya 464-8601, Japan

Received October 7, 2002; Accepted January 27, 2003
We have developed an in vitro assay system for the evaluation of the inhibitory effects of phenolic antioxidants on myeloperoxidase (MPO) activity. The formation of dityrosine from the MPO/H2O2/L-tyrosine system was used as an indicator of the MPO activity. Because the buffer system used does not include chloride ion, this assay has the advantage of exclusion of direct reaction between an antioxidant and HOCl. In this assay, ferulic acid, gallic acid, and quercetin strongly inhibited the dityrosine formation, and curcumin and caffeic acid were also effective.
Key words: myeloperoxidase; dityrosine; phenolic antioxidants; polyphenol

-28-
Note
Procyanidin B1 Is Detected in Human Serum after Intake of Proanthocyanidin-rich Grape Seed Extract

Atsushi SANO,1, Jun YAMAKOSHI,1 Shoichi TOKUTAKE,1 Koichiro TOBE,1 Yoshiro KUBOTA,2 and Mamoru KIKUCHI1

1Research and Development Division, Kikkoman Corporation, 399 Noda, Noda, Chiba 278-0037, Japan 2Kikkoman General Hospital, 100 Miyazaki, Noda, Chiba 278-0005, Japan

Received October 22, 2002; Accepted January 27, 2003
To confirm the absorption of proanthocyanidin (PA) into the human body, four healthy adults were administered 2.0 g of PA-rich grape seed extract (GSE). Blood were drawn before intake and 2 h after intake. Through the enzymatic treatment of sulfatase and β-glucuronidase, blood samples were analyzed by HPLC coupled with mass-spectrometry (LC/MS). Procyanidin B1 [epicatechin-(4β→8)-catechin] was detected in human serum 2 h after intake. Its concentration was 10.6±2.5 nmol/l.
Key words: proanthocyanidin; procyanidin B1; absorption; grape seed extract; human

-29-
Note
Rhizoremediation of Dioxin-like Compounds by a Recombinant Rhizobium tropici Strain Expressing Carbazole 1,9a-Dioxygenase Constitutively

Yuko SAIKI,1 Hiroshi HABE,2 Toshifumi YUUKI,1 Mitsuo IKEDA,1 Takako YOSHIDA,2 Hideaki NOJIRI,2 and Toshio OMORI2,†

1Fundamental Research Laboratory, Asahi Breweries Ltd., 1-21 Midori 1-chome, Moriya-shi, Ibaraki 302-0106, Japan 2Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

Received October 28, 2002; Accepted January 28, 2003
A recombinant Rhizobium strain, PBK3-IS, that constitutively expressed the oxygenase component of carbazole 1,9a-dioxygenase from Sphingomonas sp. strain KA1, was constructed. In the water-cultured siratro rhizospheres inoculated with strain PBK3-IS, 48％ of the dibenzofuran was removed within 3 days (initial substrate, 25 μg). Similar results were obtained in soil-cultured siratro rhizospheres using sterile vermiculite. When non-sterile field soils were used instead of sterile vermiculite, the inoculated recombinant strain could grow on the siratro root in all soils tested, except for wet paddy field.
Key words: carbazole 1,9a-dioxygenase; dioxin-like compounds; rhizoremediation; Rhizobium; Sphingomonas

-30-
Note
Construction of an Efficient Expression System for Aspergillus Isopullulanase in Pichia pastoris, and a Simple Purification Method

Hiromi AKEBOSHI,1 Yutaka KASHIWAGI,2 Hiroyoshi AOKI,1, Takashi TONOZUKA,1 Atsushi NISHIKAWA,1 and Yoshiyuki SAKANO1,

1Department of Applied Biological Science, Faculty of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan 2National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan

Received November 11, 2002; Accepted January 20, 2003
Aspergillus niger ATCC 9642 isopullulanase (IPU) was heterologously expressed by Pichia pastoris GS115 under three different signal sequences of Saccharomyces cerevisiae acid phosphatase, S. cerevisiae α-factor prepro peptide, and A. niger isopullulanase. One-step purification using lectin Con A affinity chromatography yielded recombinant IPU (IPU-PP) with high purity. IPU-PP had a higher carbohydrate content than native IPU and IPU-AO expressed in A. oryzae M-2--3. IPU-PP hydrolyzed various substrates containing the structure of panose, which indicated a strict subsite recognition of the panose motif.
Key words: Aspergillus niger; heterologous expression; isopullulanase; Pichia pastoris

-31-
Note
Identification of β and γ Subunits of Laminins Localized in the Basement Membrane of Rat Circumvallate Papillae

Mikiya KISHI,1,2 Kaori SANO,1 Misaki ASANO-MIYOSHI,1,3 Yoshinori TSUKAMOTO,2 Yasufumi EMORI,3 and Keiko ABE1,†

1Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan 2Mitsukan Group Corporation, Central Research Institute, 2-6 Nakamura-cho, Handa, Aichi 475-8585, Japan 3Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Tokyo 113-0033, Japan

Received November 12, 2002; Accepted January 27, 2003
Taste bud cells have elongated shape and are anchored to the basement membrane. Here we analyzed the subunits of laminin, a major component of the basement membrane, in circumvallate papillae of rat tongue. Two β subunits, β1 and β2, were detected by RT-PCR, of which the β2 subunit was immunohistochemically identified as a major component of the basement membrane. The γ1 subunit was also immunohistochemically identified as a major component. Profiles of these two laminin subunits were nearly the same as that obtained by an anti-laminin antibody, indicating that laminins of the basement membrane in the papillae contain β2 and γ1 as major β and γ subunits, respectively. As potential receptors for these laminin ligands, integrin subunits were analyzed by RT-PCR. Three integrin β subunit species, β1, β4, and β5, were shown to be expressed in the epithelium of the circumvallate papillae by RT-PCR among the five species examined.
Key words: laminins; basement membrane; circumvallate papillae; taste buds

-32-
Note
Authentic and Recombinant Bilirubin Oxidases Are in Different Resting Forms

Takeshi SAKURAI,1, Lei ZHAN,1 Takahiro FUJITA,1 Kunishige KATAOKA,1 Atsushi SHIMIZU,2 Tatsuya SAMEJIMA,2 and Shotaro YAMAGUCHI3

1Department of Chemistry, Faculty of Science, Kanazawa University, Kakuma, Kanazawa 920-1192, Japan 2Department of Chemistry, College of Science and Engineering, Aoyamagakuin University, Chitosedai, Setagaya-ku, Tokyo 157-8572, Japan 3Development Division, Amano Enzyme Co., Ltd., Nishiharu, Nishi-kasugai, Aichi 481-0041, Japan

Received November 13, 2002; Accepted January 15, 2003
Myrothecium verrucaria bilirubin oxidase expressed in Aspergillus oryzae is in a resting form different from that of the authentic bilirubin oxidase, but reaches the resting form of the authentic enzyme after one cycle of reduction and reoxidation with dioxygen as shown by the absorption and electron paramagnetic resonance spectra.
Key words: bilirubin oxidase; multicopper oxidase; trinuclear Cu center

-33-
Note
Transglucosylation Activities of Multiple Forms of α-Glucosidase from Spinach

Manabu SUGIMOTO,1,† Satoshi FURUI,1, Kenji SASAKI,2 and Yukio SUZUKI1

1Research Institute for Bioresources, Okayama University, Kurashiki, Okayama 710-0046, Japan 2Faculty of Pharmaceutical Sciences, Okayama University, Okayama, Okayama 700-8530, Japan

Received November 13, 2002; Accepted February 6, 2003
Transglucosylation activities of spinach α-glucosidase I and IV, which have different substrate specificity for hydrolyzing activity, were investigated. In a maltose mixture, α-glucosidase I, which has high activity toward not only maltooligosaccharides but also soluble starch and can hydrolyze isomaltose, produced maltotriose, isomaltose, and panose, and α-glucosidase IV, which has high activity toward maltooligosaccharides but faint activity toward soluble starch and isomaltose, produced maltotriose, kojibiose, and 2,4-di-α-D-glucosyl-glucose. Transglucosylation to sucrose by α-glucosidase I and IV resulted in the production of theanderose and erlose, respectively, showing that spinach α-glucosidase I and IV are useful to synthesize the α-1,6-glucosylated and α-1,2- and 1,4-glucosylated products, respectively.
Key words: α-glucosidase; spinach; transglucosylation

-34-
Note
Microbial Synthesis of trans Isomer of Eicosapentaenoic Acid (EPA) from the Chemically Synthesized Trans Isomer of Linolenic Acid by a Δ12 Desaturase-defective Mutant of Mortierella alpina 1S-4

Norifumi SHIRASAKA,1,† Shigenori MIYAMOTO,1 Tetsuo MURAKAMI,1 Hajime YOSHIZUMI,1 and Sakayu SHIMIZU2

1Department of Food and Nutrition, Faculty of Agriculture, Kinki University, Nakamachi, Nara, Nara 631-8505, Japan 2Division of Applied Life Science, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan

Received November 21, 2002; Accepted January 21, 2003
The mono trans geometrical isomer of eicosapentaenoic acid, 5c,8c,11c,14c,17t-eicosapenntaenoic acid (20:5Δ5c,8c,11c,14c,17t), was synthesized by fatty acid microbial conversion using a Δ12-desaturase defective mutant of an arachidonic acid (AA)-producing fungus, Mortierella alpina 1S-4. The substrate for the bioconversion, a geometrical isomer of linolenic acid, was prepared by isomerization of linseed oil methyl ester by the nitrous acid method, followed by purification on a AgNO3-silica gel column. The structure and double bond geometry were identified after hydrazine reduction followed by permanganate oxidation to 20:5Δ5c,
8c,11c,14c,17t. The biosynthetic route from 18:3Δ6c,
9c,12t to 20:5Δ5c,8c,11c,14c,17t was presumed to mimic the route from linoleic acid to arachidonic acid.
Key words: Mortierella mutant; microbial conversion; trans isomer; linolenic acid; eicosapentaenoic acid

-35-
Note
Identification of Volicitin-related Compounds from the Regurgitant of Lepidopteran Caterpillars

Naoki MORI,1, Naoko YOSHINAGA,1 Yoshitsugu SAWADA,1 Masao FUKUI,2 Masami SHIMODA,3 Kenji FUJISAKI,2 Ritsuo NISHIDA,1 and Yasumasa KUWAHARA1

1Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo, Kyoto 606-8502, Japan 2Division of Biological Sciences, Graduate School of Agriculture, Kyoto University, Sakyo, Kyoto 606-8502, Japan 3National Institute of Agrobiological Sciences, 1-2 Ohwashi, Tsukuba 305-8634, Japan

Received November 25, 2002; Accepted January 7, 2003
Volicitin-related compounds were found in the oral secretion of the three noctuid species, Helicoverpa armigera, Mythimna separata and Spodoptera litura, and one sphingid species, Agrius convolvuli. Volicitin
[N-(17-hydroxylinolenoyl)-L-glutamine],N-(17-hydroxy-
linoleoyl)-glutamine, N-linolenoylglutamine and N-linoleoylglutamine were identified in the secretion from the noctuid larvae. In secretions from the sphingid larvae, N-linolenoylglutamine and N-linoleoylglutamine were the main components. Furthermore, there were significant differences in the amounts of the N-acylamino acid conjugates in the secretions from the three noctuid species. These results suggest that the proportion of volicitin-related compounds in the regurgitant was species-specific.
Key words: insect-produced elicitors; Helicoverpa armigera; Mythimna separata; Spodoptera litura; Agrius convolvuli

-36-
Note
Escherichia coli tRNAs Are Resistant to the Hyperprocessing Reaction of Homologous E. coli Ribonuclease P Ribozyme

Terumichi TANAKA, Tomoaki ANDO, Etsuko SAKAI, Taka-aki HAHISBA, Yoshiaki HORI, and Yo KIKUCHI

Division of Bioscience and Biotechnology, Department of Ecological Engineering, Toyohashi University of Technology, Tempaku-cho, Toyohashi, Aichi 441-8580, Japan

Received November 27, 2002; Accepted January 17, 2003
Bacterial ribonuclease P RNA ribozyme can do the hyperprocessing reaction, the internal cleavage reaction of some floppy eukaryotic tRNAs. The hyperprocessing reaction can be used as a detection tool to examine the stability of the cloverleaf shape of tRNA. Until now, the hyperprocessing reaction has been observed in the heterologous combination of eukaryotic tRNAs and bacterial RNase P enzymes. In this paper, we examined the hyperprocessing reaction of Escherichia coli tRNAs by homologous E. coli RNase P, to find that these homologous tRNAs were resistant to the toxic hyperprocessing reaction. Our results display the evidence for molecular co-evolution between homologous tRNAs and RNase P in the bacterium E. coli.
Key words: tRNA; hyperprocessing; ribonuclease P; evolution; Escherichia coli

-37-
Note
Preparation of Antiserum against Rat Δ6-Desaturase and Its Use to Evaluate the Desaturase Protein Levels in Rats Treated with Gemfibrozil, a Ligand for Peroxisome Proliferator-activated Receptor α

Yoko SHOJI, Ayako SANEKATA, Masao SATO, and Katsumi IMAIZUMI

Laboratory of Nutrition Chemistry, Faculty of Bioresources and Bioenvironmental Sciences, Kyushu University, Fukuoka 812-8581, Japan

Received December 2, 2002; Accepted February 17, 2003
Anti-rat Δ6-desaturase serum was produced by immunizing rabbits with the 14 N-terminal amino acids (2--15) of rat Δ6-desaturase. The antiserum prevented the enzymatic activity of Δ6-desaturase in microsomes. Subsequently, the antiserum was used to demonstrate that gemfibrozil, a ligand for peroxisome proliferator-activated receptor α, is involved in activating Δ6-desaturase gene expression, thereby elevating the protein level and the activity.
Key words: Δ6-desaturase; gemfibrozil; rat

-38-
Note
Escherichia coli Can Be Transformed by a Liposome-mediated Lipofection Method

Yoshikazu KAWATA, Shin-ichi YANO, and Hiroyuki KOJIMA

Special Division for Green Life Technology, National Institute of Advanced Industrial Science and Technology (AIST), 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan

Received December 2, 2002; Accepted January 28, 2003
Transformation of Escherichia coli is a basic technique for genetic engineering. We used a liposome-mediated lipofection method to transform electrocompetent E. coli cells which has little natural competence of foreign DNA without electroporation treatment, and got transformants with simple and quick treatment by a plasmid or a transposon and transposase complex.
Key words: lipofection; Escherichia coli; transformation; cation liposome

-39-
Note
Synthesis of 3-O-β-N-Acetylglucosaminyl Cyclic Tetrasaccharide through a Lysozyme-catalyzed Transfer Reaction

Hikaru WATANABE, Hajime AGA, Tomohiko SONODA, Michio KUBOTA, Shigeharu FUKUDA, Masashi KURIMOTO, and Yoshio TSUJISAKA

Amase Institute, Hayashibara Biochemical Laboratories, Inc., 7-7 Amase-minami machi, Okayama 700-0834, Japan

Received December 6, 2002; Accepted January 17, 2003
Egg white lysozyme was found to catalyze the transfer of N-acetylglucosamine to cyclo{→6)-α-D-Glcp-(1→3)-α-D-Glcp-(1→6)-α-D-Glcp-(1→3)-α-D-Glcp-(1→} (CTS). Structural analysis showed that the transfer product was
3-O-β-N-acetylglucosaminyl CTS, cyclo{→6)-α-D-Glcp-(1→3)-α-D-Glcp-(1→6)-[β-GlcNAc-(1→3)]-α-D-Glcp-(1→3)-α-D-Glcp-(1→}. This branched saccharide is anticipated to be a model compound of the sugar chains of
glycoproteins.
Key words: cyclic tetrasaccharide; N-acetylglucosamine; lysozyme; transglycosylation

-40-
Communication
Novel Oxidative Dimer from Caffeic Acid

Hiroyuki TAZAKI,1,† Jun KAWABATA,2 and Takashi FUJITA3

1Department of Bioresource Science, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro 080-8555, Japan 2Laboratory of Food Biochemistry, Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Kita-9 Nishi-9, Kita-ku, Sapporo 060-8589, Japan 3Technology and Research Institute, Snow Brand Milk Products Co., LTD., 1-2 Minamidai 1-chome, Kawagoe, Saitama 350-1165, Japan

Received January 9, 2003; Accepted February 10, 2003
The novel Diels-Alder adduct, dicaffeoyl quinone as its hydrate, was formed from the oxidation of 3,4-dihydroxycinnamic acid (caffeic acid) with NaIO4. The structure of this hydrate was determined by spectroscopic methods.
Key words: caffeic acid; ortho-quinone; Diels-Alder reaction

-41-
Communication
Effects of Physiological Changes in Potato Tubers (Solanum tuberosum L.) after Low Temperature Storage on the Level of Acrylamide Formed in Potato Chips

Yoshihiro CHUDA,1 Hiroshi ONO,2,† Hiroshi YADA,2 Akiko OHARA-TAKADA,3 Chie MATSUURA-ENDO,3 and Motoyuki MORI3

1Center for Food Quality, Labeling and Consumer Services, 2-1 Shintoshin, Chuo-ku, Saitama 330-9731, Japan 2National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan 3National Agricultural Research Center for Hokkaido Region, National Agricultural Research Organization, Memuro-cho, Hokkaido 082-0071, Japan

Received January 23, 2003; Accepted March 5, 2003
Acrylamide in potato chips made from tubers stored at 2 or 20°C for two weeks after harvest was analyzed by GC-MS. The acrylamide level in the former chips was higher than ten times of that in the latter, which was highly correlated with both glucose and fructose levels in the tubers.
Key words: acrylamide; potato chips; potato tuber; storage; reducing sugar

-42-
Communication
Inportance of Phosphatidylinositol 3-Phosphate in Sporulation of Schizosaccharomyces pombe

Masayuki ONISHI,1 Yoko NAKAMURA,1 Takako KOGA,1, Aiko HIRATA,2 and Yasuhisa FUKUI1,†

1Laboratory of Biological Chemistry, Graduate School of Agricultural and Life Science, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan 2Department of Integrated Biosciences, Graduate School of Frontier Science, University of Tokyo, Chiba 277-8562, Japan

Received January 24, 2003; Accepted February 24, 2003
In Schizosaccharomyces pombe, Pik3p phosphorylates phosphatidylinositol (PI) to produce PI 3-P, which is further phosphorylated by Ste12p to yield PI 3,5-P2. Pik3p is required for both conjugation and sporulation. To test which of PI 3-P and PI 3,5-P2 is required for sporulation, diploid cells defective in production of PI 3,5-P2 were used. They underwent sporulation almost normally provided that the osmotic pressure of the medium was controlled, suggesting that not PI 3,5-P2 but PI 3-P was important. Electron microscopic analysis confirmed normal sporulation in the absence of PI 3,5-P2 although the forespore membrane was found to be less dense in these cells.
Key words: Shizosaccharomyces pombe; conjugation; phosphatidylinositol 3-phosphate 5-kinase; vacuolar protein sorting; suppressor mutation

-43-
Communication
Uzu Mutation in Barley (Hordeum vulgare L.) Reduces the Leaf Unrolling Response to Brassinolide

Ichiro HONDA,1,† Haruko ZENIYA,1,2 Koichi YONEYAMA,3 Makiko CHONO,1 Shigenobu KANEKO,1 and Yoshiaki WATANABE1

1Department of Wheat and Barley, National Institute of Crop Science, National Agricultural Research Organization, 2-1-18 Kannondai, Tsukuba 305-8518, Japan 2Department of Applied Biochemistry, 350 Mine-machi, Utsunomiya University, Utsunomiya 321-8505, Japan 3Center for Research on Wild Plants, Utsunomiya University, 350 Mine-machi, Utsunomiya 321-8505, Japan

Received February 5, 2003; Accepted March 17, 2003
A sensitive method to examine the brassinolide (BL) response of barley (Hordeum vulgare L.) using dark-grown leaf segments was established based on the known method for wheat. BL responses of 53 dwarf isogenic lines of barley were examined, and two lines were found having a uzu gene that doesn′t respond significantly. These results indicate that uzu dwarfism may be caused by the non-responding character to BL.
Key words: dwarf; barley; brassinolide; brassinosteroids; uzu