Contents and Abstracts of Latest Issue of BBB

(Vol.67 No.2 2003)

Review
Genetics of Polycyclic Aromatic Hydrocarbon Metabolism in Diverse Aerobic Bacteria
Hiroshi HABE and Toshio OMORI p.225

Effects of Corticosterone on Ca2＋ Uptake and Myofibrillar Disassembly in Primary Muscle Cell Culture
Kazue MACHIDA,1 Ryouji ISHIBASHI,2 Tomoko HARA,2 Akira OHTSUKA,1 and Kunioki HAYASHI1, p.244

Role of Endo-1,4-β-glucanases from Neisseria sicca SB in Synergistic Degradation of Cellulose Acetate
Kunihiko MORIYOSHI, Takashi OHMOTO, Tatsuhiko OHE, and Kiyofumi SAKAI p.250

Enzymatically Depolymerized Alginate Oligomers That Cause Cytotoxic Cytokine Production in Human Mononuclear Cells
Yoshiko IWAMOTO, Xu XU, Tadashi TAMURA, Tatsuya ODA, and Tsuyoshi MURAMATSU p.258

Substrate Specificities of Deuterolysin from Aspergillus oryzae and Electron Paramagnetic Resonance Measurement of Cobalt-substituted Deuterolysin
Yuko DOI,1 Byung Rho LEE,2 Masamichi IKEGUCHI,1,2 Yasunori OHOBA,3 Tadaaki IKOMA,3 Shozo TERO-KUBOTA,3 Seigo YAMAUCHI,3 Koji TAKAHASHI,4 and Eiji ICHISHIMA1,2, p.264

Wound-responsive cis-Element in the 5′-Upstream Region of Cucumber Ascorbate Oxidase Gene
Hiroshi ASAO,1,2 Kazuya YOSHIDA,2, Yukio NISHI,3 and Atsuhiko SHINMYO2 p.271

Reduction of Paraquat-induced Oxidative Stress in Rats by Dietary Soy Peptide
Asako TAKENAKA,1, Hideyuki ANNAKA,1 Yuki KIMURA,1 Hisa AOKI,2 and Kiharu IGARASHI1
p.278

Fermentation Conditions and Properties of a Chitosanase from Acinetobacter sp. C-17
Xu-Fen ZHU, Xiao-Yun WU, and Yun DAI p.284

Structure of Folding Intermediates at pH 4.0 and Native State of Microbial Transglutaminase
Kei-ichi YOKOYAMA,2 Daisuke EJIMA,2 Yoshiko KITA,1 John S. PHILO,1 and Tsutomu ARAKAWA1, p.291

Reduction Mechanism of Tetrazolium Salt XTT by a Glucosamine Derivative
Tomoko SHIMAMURA, Atsuko TAKAMORI, Hiroyuki UKEDA, and Masayoshi SAWAMURA p.295

Expression, Purification, and Characterization of 2′-Aminobiphenyl-2,3-diol 1,2-dioxygenase from Carbazole-degrader Pseudomonas resinovorans Strain CA10
Kenichi IWATA,1 Hideaki NOJIRI,1 Kentaro SHIMIZU,2 Takako YOSHIDA,1 Hiroshi HABE,1 and Toshio OMORI1, p.300

(E)-2-(2-Hydroxyethylidene)-6-methyl-5-heptenal (α-Acariolal) and (E)-2-(2-Hydroxyethyl)-6-methyl-2,5-heptadienal (β-Acariolal), Two New Types of Isomeric Monoterpenes from Caloglyphus polyphyllae (Acari: Acaridae)
Nobuhiro SHIMIZU, Hirokazu TARUI, Naoki MORI, Ritsuo NISHIDA, and Yasumasa KUWAHARA
p.308

Insulin-Dependent Adipogenesis in Stromal ST2 Cells Derived from Murine Bone Marrow
Jin DING, Kazuo NAGAI, and Je-Tae WOO p.314

N-Cyanomethyl-2-chloroisonicotinamide Induces Systemic Acquired Resistance in Arabidopsis without Salicylic Acid Accumulation
Michiko YASUDA,2,3,4 Hideo NAKASHITA,1,2, Satoru HASEGAWA,2,6 Masanori NISHIOKA,2,5 Yuko ARAI,2,4, Masakazu URAMOTO,6 Isamu YAMAGUCHI,2,4, and Shigeo YOSHIDA1,3,4
p.322

Determination of the Absolute Configuration of (＋)-Xestoaminol C [(2S, 3R)-2-Amino-3-tetradecanol], a Metabolite of Fiji Sponge, Xestospongia sp., by the Synthesis of Its N,O-Diacetyl Derivative
Miwako ICHIHASHI1 and Kenji MORI2, p.329

Isolation of Paenibacillus illinoisensis That Produces Cyclodextrin Glucanotransferase Resistant to Organic Solvents
Noriyuki DOUKYU, Hirokazu KUWAHARA, and Rikizo AONO p.334

Mutational Analysis of Amino Acid Residues Involved in Catalytic Activity of a Family 18 Chitinase from Tulip Bulbs
Keisuke SUZUKAWA,1 Takeshi YAMAGAMI,1 Takayuki OHNUMA,1 Hideki HIRAKAWA,2 Satoru KUHARA,2 Yoichi ASO,1 and Masatsune ISHIGURO1, p.341

Novel Gene Encoding a Ca2＋-Binding Protein and under Hexokinase-dependent Sugar Regulation
Shigeo OTSUKI,1 Akira IKEDA,1 Tomomi SUNAKO,1 Shoshi MUTO,2 Junshi YAZAKI,3 Keiko NAKAMURA,3 Fumiko FUJII,3 Kanako SHIMBO,3 Yoshimi OTSUKA,3 Kimiko YAMAMOTO,3 Katsumi SAKATA,4 Takuji SASAKI,4 Naoki KISHIMOTO,4 Shoshi KIKUCHI,4 and Junji YAMAGUCHI1,
p.347

Novel Chitosanase from Streptomyces griseus HUT 6037 with Transglycosylation Activity
Toshiaki TANABE,1 Kazuko MORINAGA,1 Tamo FUKAMIZO,2 and Masaru MITSUTOMI1, p.354

Note
Effects of Degree of Hydrolysis and pH on the Solubility of Heme-iron Enriched Peptide in Hemoglobin Hydrolysate
Man-Jin IN,2 Dong Chung KIM,3 Hee Jeong CHAE,4 and Nam-Soon OH1, p.365

Note
Periplasmic Secretion of Native Ovalbumin without Signal Cleavage in Escherichia coli
Yasuhiro ARII, Nobuyuki TAKAHASHI, and Masaaki HIROSE p.368

Note
Effect of a Selected Amino Acid Mixture on the Recovery from Muscle Fatigue during and after Eccentric Contraction Exercise Training
Masaaki SUGITA,1 Masaru OHTANI,2, Naokata ISHII,2 Kimiaki MARUYAMA,3 and Kando KOBAYASHI2 p.372

Note
New Phenolic Compounds from Kokuto, Non-centrifuged Cane Sugar
Kensaku TAKARA,1, Daigo MATSUI,1 Koji WADA,1 Toshio ICHIBA,2 Isao CHINEN,1 and Yoko NAKASONE1 p.376

Note
Prolyl Endopeptidase Inhibitory Peptides in Wine
Takaaki YANAI, Yumiko SUZUKI, and Michikatsu SATO p.380

Note
Response of the Induction of Rat Liver Serine Dehydratase to Changes in the Dietary Protein Requirement
Saeko IMAI,1 Ryuhei KANAMOTO,1, Iyo YAGI,1 Makoto KOTARU,2 Tohru SAEKI,1 and Kimikazu IWAMI1 p.383

Note
Presence of Free D-Amino Acids in Microalgae
Takehiko YOKOYAMA,1 Nobuhiro KAN-NO,1 Takehiko OGATA,1 Yuichi KOTAKI,1 Minoru SATO,2 and Eizoh NAGAHISA1, p.388

Note
High IgM Production by Human-human Hybridoma HB4C5 Cells Cultured in Microtubes
Takuya SUGAHARA, Satomi YANO, and Takeshi SASAKI p.393

Note
Evaluation of the Anti-oxidative Effect (in vitro) of Tea Polyphenols
Fumio HASHIMOTO,1, Masateru ONO,2 Chikako MASUOKA,2 Yasuyuki ITO,2 Yusuke SAKATA,1 Keiichi SHIMIZU,1 Gen-ichiro NONAKA,3 Itsuo NISHIOKA,3 and Toshihiro NOHARA4 p.396

Note
Effect of α-Tocopherol on the Oxidative Modification of Apolipoprotein E in Human Very-low-density Lipoprotein
Hirofumi ARAI,1 Koji UCHIDA,2 Kenji FUKUNAGA,3 Yuji NAGASAKA,4 Satoshi MOHRI,5 Hiroko FURUMOTO,1 Yasuhiro KURAMITSU,1 and Kazuyuki NAKAMURA1, p.402

Note
Isothermal Titration Calorimetric Studies on the Binding of a Family 6 Carbohydrate-binding Module of Clostridium thermocellum XynA with Xlylooligosaccharides
Kazuo SAKKA, Mari NAKANISHI, Mari SOGABE, Takamitsu ARAI, Hiroki OHARA, Akiyoshi TANAKA, Tetsuya KIMURA, and Kunio OHMIYA p.406

Note
Antioxidative Compounds from Crotalaria sessiliflora
Abdul MUN′IM, Osamu NEGISHI, and Tetsuo OZAWA p.410

Note
(−)-Olivil and (＋)-1-Acetoxypinoresinol from the Olive Tree (Olea europaea LINNE; Oleaceae) as Feeding Stimulants of the Olive Weevil (Dyscerus perforatus)
Emiko KADOWAKI,1 Yasuhiro YOSHIDA,3 Teruhiko NITODA,1 Naomichi BABA,2 and Shuhei NAKAJIMA2, p.415

Note
Effect of an Oral Administration of Lactobacillus casei Strain Shirota on the Natural Killer Activity of Blood Mononuclear Cells in Aged Mice
Tetsuji HORI, Junko KIYOSHIMA, and Hisako YASUI p.420

Note
Inhibition of Osteoporosis in Rats Fed with Sugar Cane Wax
Hajime TAMAKI,1 Sun Li MAN,1 Yutaka OHTA,2 Naofumi KATSUYAMA,2 and Isao CHINEN1,
p.423

Note
Release Characteristics of Flavor from Spray-dried Powder in Boiling Water and during Rice Cooking
Hirokazu SHIGA,1 Hidefumi YOSHII,1, Rumiko TAGUCHI,1 Taiji NISHIYAMA,1 Takeshi FURUTA,1 and Pekka LINKO2　p.426

Note
Effect of Dietary Fiber on the Lipid Metabolism and Immune Function of Aged Sprague-Dawley Rats
Koji YAMADA,1, Yoko TOKUNAGA,1 Atsushi IKEDA,1 Ken-ichi OHKURA,1 Shihoko KAKU-OHKURA,1 Soichi MAMIYA,2 Beong Ou LIM,3 and Hirofumi TACHIBANA1 p.429

Note
Synthesis of Hydroxymethylglutathione from Glutathione and L-Serine Catalyzed by Carboxypeptidase Y
Ryosuke OKUMURA, Yukio KOIZUMI, and Jiro SEKIYA p.434

Note
Cloning and Overexpression of the 3-Hydroxyisobutyrate Dehydrogenase Gene from Pseudomonas putida E23
Emran Kabir CHOWDHURY, Yuka AKAISHI, Shinji NAGATA, and Haruo MISONO p.438

Note
Hydroxysulochrin, a Tea Pollen Growth Inhibitor from the Fungus Aureobasidium sp.
Atsumi SHIMADA,1 Chisako SHIOKAWA,2 Miyako KUSANO,2 Shozo FUJIOKA,3 and Yasuo KIMURA2, p.442

Note
6-Hydroxyflavonoids as α-Glucosidase Inhibitors from Marjoram (Origanum majorana) Leaves
Jun KAWABATA, Kenji MIZUHATA, Eri SATO, Tetsuo NISHIOKA, Yoritaka AOYAMA, and Takanori KASAI p.445

Note
Cooperation of Sly1/SM-Family Protein and Sec18/NSF of Saccharomyces cerevisiae in Disassembly of cis-SNARE Membrane-Protein Complexes
Yoichi KOSODO, Yoichi NODA, Hiroyuki ADACHI, and Koji YODA p.448

Note
Evidence of Isozymes for Δ6 Fatty Acid Desaturase in Rat Hepatocytes
Katsuya INAGAKI, Tsunehiro AKI, Taketoshi SHIOTA, Seiji KAWAMOTO, Seiko SHIGETA, Osamu SUZUKI, and Kazuhisa ONO p.451

Preliminary Communication
In vivo Visualization of the Distribution of a Secretory Protein in Aspergillus oryzae Hyphae Using the RntA-EGFP Fusion Protein
Kumiko MASAI,1 Jun-ichi MARUYAMA,1,2 Harushi NAKAJIMA,1 and Katsuhiko KITAMOTO1,
p.455

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Review
Genetics of Polycyclic Aromatic Hydrocarbon Metabolism in Diverse Aerobic Bacteria

Hiroshi HABE and Toshio OMORI

Biotechnology Research Center, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

Polycyclic aromatic hydrocarbons (PAHs), which consist of two or more fused aromatic rings, are widespread in the environment and persist over long periods of time. The decontamination of a PAH-polluted environment is of importance because some PAHs are toxic, mutagenic, and carcinogenic and therefore are health hazards. As part of the efforts to establish remediation processes, the use of aerobic bacteria has been extensively studied, and both enzymologic and genetic studies are underway for the purpose of effective biodegradation. In the last two decades, one highly conserved group of PAH-catabolic genes from Pseudomonas species, called the nah-like genes, has been well investigated, and much has been found, including the structure-function relationships and the evolutionary trails of the catabolic enzymes. However, recently, PAH-catabolic genes, which are evolutionarily different from the nah-like genes, have been characterized from both Gram-negative bacteria other than Pseudomonas species and Gram-positive bacteria, and the information about these genes is expanding. This review is an outline of genetic knowledge about bacterial PAH catabolism.
Key words: fluorene degradation; naphthalene degradation; phenanthrene degradation; polycyclic aromatic hydrocarbons; pyrene degradation

-2-
Effects of Corticosterone on Ca2＋ Uptake and Myofibrillar Disassembly in Primary Muscle Cell Culture

Kazue MACHIDA,1 Ryouji ISHIBASHI,2 Tomoko HARA,2 Akira OHTSUKA,1 and Kunioki HAYASHI1,

1United Graduate School of Agricultural Sciences, Kagoshima University, Korimoto 1-21-24, Kagoshima 890-0065, Japan 2Faculty of Agriculture, Kagoshima University, Korimoto 1-21-24, Kagoshima 890-0065, Japan

Received December 6, 2001; Accepted September 24, 2002
This study was done to examine the effects of corticosterone, a glucocorticoid, on Ca2＋ uptake, proteolysis, and Ca2＋ channels in primary cultures of chick muscle cells, to clarify the mechanism of glucocorticoid action on muscle proteolysis. Chick muscle cells were incubated for 24 h in a medium containing corticosterone (30 ng/ml) when the cells were confluent (6 days). To examine the contribution of Ca2＋ channels, nifedipine, a Ca2＋ channels antagonist, was used. Ca2＋ uptake measured with 45CaCl2 was increased three-fold by corticosterone, with a peak at 12 h after the treatment started. The growth of the cells estimated from the protein content and creatine kinase activity was not affected by corticosterone. Proteolysis, evaluated with [3H]tyrosine as a label of the protein and Nτ-methylhistidine release, was unchanged by corticosterone. However, the amount of easily releasable myofilament as a measure of myofibrillar disassembly in the muscle cells was increased by corticosterone, and prevented by nifedipine. These results show that corticosterone increases Ca2＋ uptake and starts myofibrillar protein breakdown.
Key words: corticosterone; calcium uptake; proteolysis; muscle cell

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Role of Endo-1,4-β-glucanases from Neisseria sicca SB in Synergistic Degradation of Cellulose Acetate

Kunihiko MORIYOSHI, Takashi OHMOTO, Tatsuhiko OHE, and Kiyofumi SAKAI

Department of Biochemistry, Osaka Municipal Technical Research Institute, 1-6-50 Morinomiya, Joto-ku, Osaka 536-8553, Japan

Received March 15, 2002; Accepted October 7, 2002
An enzyme hydrolyzing β-1,4 bonds in cellulose acetate was purified 10.5-fold to electrophoretic homogeneity from a culture supernatant of Neisseria sicca SB, which assimilate cellulose acetate as the sole carbon and energy source. The enzyme was an endo-1,4-β-glucanase, to judge from the substrate specificity and hydrolysis products of cellooligosaccharides, we named it endo-1,4-β-glucanase I (EG I). Its molecular mass was 50 kDa, 9 kDa larger than EG II from this strain, and its isoelectric point was 5.0. Results of N-terminal and inner-peptide sequences of both enzymes, and a similarity search, suggested that EG I contained a carbohydrate-binding module at the N-terminus and that EG II lacked this module. The pH and temperature optima of EG I were 5.0--6.0 and 45°C. It hydrolyzed water-soluble cellulose acetate (degree of substitution, 0.88) and carboxymethyl cellulose. The Km and Vmax for these compounds were 0.296％ and 1.29 μmol min−1 mg−1, and 0.448％ and 13.6 μmol min−1 mg−1, respectively. Both glucanases and cellulose acetate esterase from this strain degraded water-insoluble cellulose acetate synergistically.
Key words: cellulose acetate; biodegradation; Neisseria sicca; endo-1,4-β-glucanase

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Enzymatically Depolymerized Alginate Oligomers That Cause Cytotoxic Cytokine Production in Human Mononuclear Cells

Yoshiko IWAMOTO, Xu XU, Tadashi TAMURA, Tatsuya ODA, and Tsuyoshi MURAMATSU

Division of Biochemistry, Faculty of Fisheries, Nagasaki University, Nagasaki 852-8521, Japan

Received June 21, 2002; Accepted October 21, 2002
Enzymatically depolymerized guluronate and mannuronate oligomers were prepared from polyuronates with an alginate lyase from a Pseudoalteromonas sp., and their effects on mononuclear cells from human peripheral blood were examined. Conditioned medium prepared by the incubation of cells with an untreated polyuronate had little effect on growth of human leukemic U937 cells, but a medium prepared with depolymerized uronate oligomers inhibited their growth. Inhibition was greater in a medium prepared with guluronate oligomer than one prepared with mannuronate oligomer. The cytotoxic activity of the medium was heat-labile and nondialyzable. Apoptotic nuclear morphological changes and increased caspase-3-like activity were found in U937 cells treated with a medium prepared with depolymerized uronates. The medium prepared with purified tetra-guluronate and tetra-mannuronate also was cytotoxic; these effects were inhibited by antibodies to tumor necrosis factor-α. Our results suggested that enzymatically depolymerized guluronate and mannuronate oligomers induced the production of cytotoxic cytokines in human mononuclear cells, although the uronate polymers before depolymerization had no such activity.
Key words: alginate; oligoguluronate; oligomannuronate; human mononuclear cells; immunomodulation

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Substrate Specificities of Deuterolysin from Aspergillus oryzae and Electron Paramagnetic Resonance Measurement of Cobalt-substituted Deuterolysin

Yuko DOI,1 Byung Rho LEE,2 Masamichi IKEGUCHI,1,2 Yasunori OHOBA,3 Tadaaki IKOMA,3 Shozo TERO-KUBOTA,3 Seigo YAMAUCHI,3 Koji TAKAHASHI,4 and Eiji ICHISHIMA1,2,

1Department of Bioengineering, Graduate School of Engineering, Soka University, Hachioji, Tokyo 192-8577, Japan 2Department of Bioengineering, Faculty of Engineering, Soka University, Hachioji, Tokyo 192-8577, Japan 3Institute of Multidisciplinary Research for Advanced Materials (IMRAM), Tohoku University, Katahira 2-1-1, Sendai 980-8577, Japan 4Department of Applied Biological Science, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-0054, Japan

Received June 25, 2002; Accepted October 9, 2002
The substrate specificities of deuterolysin, a 19-kDa zinc-protease (EC 3.4.24.39) from Aspergillus oryzae, were investigated at pH 9.0 with various fluorogenic
acyl-peptide-4-methylcoumaryl-7-amides (peptide-
MCAs). N-Butoxycarbonyl-Arg-Val-Arg-Arg-MCA
was the best substrate for deuterolysin. We therefore measured its kinetic parameters. Deuterolysin had high activity toward the peptide bonds next to pairs of basic residues in calf thymus histone H4. The specificity of cobalt-substituted deuterolysin (Co-deuterolysin) for peptide-MCAs was similar to that of native deuterolysin. The CD spectrum of Co-deuterolysin was similar to that of the native deuterolysin. The metal coordination sphere of Co-deuterolysin was analyzed by Q-band (33.9570 GHz) electron paramagnetic resonance (EPR) spectroscopy. Using computer simulation of EPR, we found the g principal values to be gxx＝5.20, gyy＝4.75, and gzz＝2.24; the metal center was a divalent cobalt ion in a high spin state.
Key words: cobalt-substituted; deuterolysin; electron paramagnetic resonance; substrate specificity; zinc-protease

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Wound-responsive cis-Element in the 5′-Upstream Region of Cucumber Ascorbate Oxidase Gene

Hiroshi ASAO,1,2 Kazuya YOSHIDA,2, Yukio NISHI,3 and Atsuhiko SHINMYO2

1Nara Agricultural Experiment Station, Shijo-cho, Kashihara, Nara 634-0813, Japan 2Graduate School of Biological Sciences, Nara Institute of Science and Technology, Takayama-cho, Ikoma, Nara 630-0101, Japan 3Department of Biotechnology, Faculty of Engineering, Osaka University, Yamadaoka, Suita, Osaka 565-0871, Japan

Received June 27, 2002; Accepted October 9, 2002
The cucumber (Cucumis sativas) AAO1 gene (former name, Aso1) encodes an ascorbate oxidase that catalyzes the oxidation by molecular oxygen of ascorbic acid to dehydroascorbate. CsAAO1 mRNA concentrations rose rapidly after mechanical wounding of cucumbers. To study the wound-responsive expression of CsAAO1 in detail, we examined transgenic tobacco plants harboring a CsAAO1 promoter-β-glucuronidase fusion gene. CsAAO1 promoter activity in leaves of the tobacco was induced by wounding. Analysis of the regulatory properties of 5′-deleted promoter fragments showed that a putative wound-responsive cis-element (WRE) was located −736 to −707 bp from the translation initiation site. DNA binding factors that bound specifically to the putative WRE sequence were identified in tobacco nuclear extracts by gel retardation assays.
Key words: ascorbate oxidase; cucumber; promoter; wound; cis-element

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Reduction of Paraquat-induced Oxidative Stress in Rats by Dietary Soy Peptide

Asako TAKENAKA,1, Hideyuki ANNAKA,1 Yuki KIMURA,1 Hisa AOKI,2 and Kiharu IGARASHI1

1Department of Bioresource Engineering, Faculty of Agriculture, Yamagata University, Tsuruoka, Yamagata 997-8555, Japan 2Course of the Science of Bioresources, The United Graduate School of Agricultural Sciences, Iwate University, Morioka, Iwate 020-8550, Japan

Received July 8, 2002; Accepted October 23, 2002
The effect of a dietary soy protein isolate (SPI), soy peptide (PEP) and the amino acids in soy protein on paraquat (PQ)-induced oxidative stress was investigated in rats. In the first experiment, male Wistar rats were fed on experimental diets containing casein (CAS), SPI and PEP as nitrogen sources with or without 0.025％ PQ. The reduced food intake and body weight gain of the rats fed with PQ was mitigated by either the SPI or PEP intake. Both SPI and PEP prevented the elevation of the serum TBARS concentration and tended to prevent the elevation of lung weight induced by PQ. In the second experiment, the rats were fed on diets containing an amino acid mixture resembling casein (CASAA) or soy protein (SPIAA) with or without PQ. The SPIAA intake did not affect the reduction of food intake and body weight gain, nor the elevation of lung weight and TBARS in the serum and liver induced by PQ. These results demonstrate that the intake of either dietary SPI or PEP, but not an amino acid mixture resembling soy protein, had the effect of reducing PQ-induced oxidative stress in rats.
Key words: rat; soy peptide; paraquat; oxidative stress

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Fermentation Conditions and Properties of a Chitosanase from Acinetobacter sp. C-17

Xu-Fen ZHU, Xiao-Yun WU, and Yun DAI

College of Life Science, Zhejiang University, Hangzhou, Zhejiang 310012, People′s Republic of China

Received July 8, 2002; Accepted September 22, 2002
A species of bacterium with high chitosanase activity was isolated from soil samples in Haiyan City, China, and identified as an Acinetobacter species. This strain, named Acinetobacter sp. strain C-17, produced a chitosanase that was inducible and secreted into the medium. The optimal conditions for enzyme production were cells used to inoculate a medium containing 1％ chitosan (pH 7.0) followed by culture at 30°C. The chitosanase activity reached 1.7 U/ml when strain C-17 was incubated in a 250-ml flask under the optimal conditions for 24 h, and reached 2.8 U/ml when cells were incubated in a 3-l fermentor. The optimal pH and temperature for hydrolysis of chitosanase were 7.0 and 36°C, respectively. The chitosanase activity was stable in the pH range of 5--8 and temperature range of 30--40°C. The chitosanase of the strain was extracted by zinc acetate and ammonium sulfate precipitation. The molecular mass was estimated to be 35.4 kDa by SDS-PAGE.
Key words: Acinetobacter sp. strain C-17; chitosanase; chitosan; fermentation

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Structure of Folding Intermediates at pH 4.0 and Native State of Microbial Transglutaminase

Kei-ichi YOKOYAMA,2 Daisuke EJIMA,2 Yoshiko KITA,1 John S. PHILO,1 and Tsutomu ARAKAWA1,

1Alliance Protein Laboratories, Thousand Oaks, CA 91360, USA 2Central Research Laboratories, Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-shi, Kawasaki 210-8681, Japan

Received July 10, 2002; Accepted Octover 28, 2002
Recombinant microbial transglutaminase has been expressed in Escherichia coli as insoluble inclusion bodies. After we searched for refolding conditions, refolding of the protein could be done by first dilution of the unfolded enzyme in a buffer at pH 4.0, and then by titration of the pH from 4.0 to 6.0. CD analysis showed that a burst of secondary structure formation occurred within the dead time of the experiment and accounted for 75％ of the signal change in the far UV CD, with little tertiary structure being formed. This burst was followed by slow rearrangement of the secondary structure accompanied by formation of tertiary structure. The secondary and tertiary structures of the final sample at pH 4.0, corresponding to the folding intermediate, were different from these structures at pH 6.0. Once the native structure was obtained, acidification of the native protein to pH 4.0 did not lead to a structure like that of the folding intermediate. Sedimentation velocity analysis showed that the folding intermediate had an expanded structure and contained no other structure species including large aggregates.
Key words: transglutaminase; sedimentation; refolding; circular dichroism; secondary structure

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Reduction Mechanism of Tetrazolium Salt XTT by a Glucosamine Derivative

Tomoko SHIMAMURA, Atsuko TAKAMORI, Hiroyuki UKEDA, and Masayoshi SAWAMURA

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

Received July 11, 2002; Accepted October 1, 2002
XTT (3′-｛1-[(phenylamino)-carbonyl]-3,4-tetrazolium｝-bis(methoxy-6-nitro)benzenesulfonic acid hydrate) was reduced by incubated glucosamine hydrochloride. The XTT reducibility by incubated glucosamine was linearly related with the DNA-breaking activity. In order to elucidate the reaction mechanism, the glucosamine derivatives formed during the incubation process were separated by HPLC, and the compound responsible for the reduction was analyzed. Among the incubated products, fructosazine and deoxyfructosazine were identified by LC-MS, FAB-MS, and 1H- and 13C-NMR. These products showed no XTT reducibility, but an unstable intermediate with a molecular weight of 322 displayed reducibility. Since the intermediate gave fructosazine by oxidation with XTT and was a precursor of deoxyfructosazine, we conclude that the intermediate could have been dihydrofructosazine. Therefore, the XTT reducibility by incubated glucosamine was based on dihydrofructosazine formed by the condensation of two molecules of glucosamine.
Key words: glucosamine; DNA-breaking activity; fructosazine; XTT

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Expression, Purification, and Characterization of 2′-Aminobiphenyl-2,3-diol 1,2-dioxygenase from Carbazole-degrader Pseudomonas resinovorans Strain CA10

Kenichi IWATA,1 Hideaki NOJIRI,1 Kentaro SHIMIZU,2 Takako YOSHIDA,1 Hiroshi HABE,1 and Toshio OMORI1,

1Biotechnology Research Center, and 2Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

Received July 17, 2002; Accepted October 17, 2002
The two-subunit meta-cleavage enzyme, 2′-aminobiphenyl-2,3-diol 1,2-dioxygenase (CarBaBb), from the carbazole degrader Pseudomonas resinovorans strain CA10 was purified to homogeneity from an Escherichia coli strain carrying the expression vector pUCA503, in which two copies of the carBaBb genes are tandemly linked. SDS-PAGE and gel filtration showed that CarB was a α2β2-heterotetrameric enzyme with subunit molecular masses of approximately 10,000 for CarBa and 29,000 for CarBb. The optimum pH for activity was 8.5 and that of temperature was 35°C. The CarB enzyme had a Km of 14 μM and a kcat/Km of 0.25 μM−1 s−1 for 2′-aminobiphenyl-2,3-diol, and the catalytic activities for biphenyl-type catecholic substrates were higher than those for monoaromatic catechol derivatives. The enzyme was originally isolated as a meta-cleavage enzyme for 2′-aminobiphenyl-2,3-diol involved in carbazole degradation, but the enzyme was highly specific for 2,3-dihydroxybiphenyl.
Key words: carbazole; biodegradation; meta-cleavage enzyme; purification; Pseudomonas resinovorans

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(E)-2-(2-Hydroxyethylidene)-6-methyl-5-heptenal (α-Acariolal) and (E)-2-(2-Hydroxyethyl)-6-methyl-2,5-heptadienal (β-Acariolal), Two New Types of Isomeric Monoterpenes from Caloglyphus polyphyllae (Acari: Acaridae)

Nobuhiro SHIMIZU, Hirokazu TARUI, Naoki MORI, Ritsuo NISHIDA, and Yasumasa KUWAHARZ

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

Received July 19, 2002; Accepted September 26, 2002
The opisthonotal gland secretion of the acarid mite, Caloglyphus polyphyllae, contained two new monoterpenes, (E)-2-(2-hydroxyethylidene)-6-methyl-5-heptenal (1) and (E)-2-(2-hydroxyethyl)-6-methyl-2,5-heptadienal (2), to which we have given the trivial names α- and β-acariolal in relation to α- and β-acaridial (3 and 4), respectively. Elucidation of the structure of 1 was established mainly from 1H-NMR and GC/MS spectral data after partial purification, together with the fact that 1 was recovered in the more-polar fraction from a silica gel column than α- and β-acaridial (3 and 4) present in the secretion. Compound 2 was obtained in the same fraction as a mixture with 1. Based on the facts that 2 had the same molecular weight by GC/MS and the same polarity as that of 1, compound 2 was assumed to be a structural analog of 1. The structures of compounds 1 and 2 were confirmed by their synthesis in nine and ten respective steps starting from α-bromo-γ-butyrolactone.
Key words: Acaridae; Caloglyphus polyphyllae; monoterpene; α-acariolal; β-acariolal

-13-
Insulin-Dependent Adipogenesis in Stromal ST2 Cells Derived from Murine Bone Marrow

Jin DING, Kazuo NAGAI, and Je-Tae WOO

Department of Bioscience and Biochemistry, College of Bioscience and Biotechnology, Chubu University, 1200 Matsumotomachi, Kasugai-shi, Aichi-ken 487-8501, Japan

Received July 26, 2002; Accepted September 27, 2002
ST2 cells, a cloned stromal-cell line from mouse bone marrow have the phenotypes of osteoblasts and hematopoietic supporting cells, but little is known about the adipogenesis in this cell line. We found that treatment of ST2 cells with a cocktail, a combination of insulin, dexamethasone (DEX) and 3-isobutyl-1-methylxanthine (IBMX), induced adipocyte differentiation. The cells were adipocytic, as seen by the accumulation of triglycerides and with lipid droplets stained with Oil Red O. Expressions of the adipogenic transcriptional factors peroxisome-proliferator-activated receptor γ and CCAAT-enhancer binding protein α were stimulated, triggering the expression of the late adipocyte-specific marker α-glycerophosphate-3-dehydrogenase. Unlike another bone marrow stromal cell line PA6, ST2 cells responded to the adipogenic effects of insulin, as do the extramedullary preadipocytes 3T3-L1. Like PA6 and 3T3-L1 cells, adipogenesis in ST2 cells was inhibited by 1,25-dihydroxyvitamin D3, retinoic acid, tumour necrosis factor α, and transforming growth factor β. Therefore, ST2 cells can differentiate into osteoblasts, adipocytes, and hematopoietic supporting cells, in which the process of adipogenesis differs from that of bone marrow preadipocytes PA6, and resembles that of the extramedullary preadipocytes 3T3-L1.
Key words: adipocyte; adipogenesis; bone; mouse ST2 stromal cell line; growth factor

-14-
N-Cyanomethyl-2-chloroisonicotinamide Induces Systemic Acquired Resistance in Arabidopsis without Salicylic Acid Accumulation

Michiko YASUDA,2,3,4 Hideo NAKASHITA,1,2, Satoru HASEGAWA,2,6 Masanori NISHIOKA,2,5 Yuko ARAI,2,4, Masakazu URAMOTO,6 Isamu YAMAGUCHI,2,4, and Shigeo YOSHIDA1,3,4

1Plant Functions Laboratory and 2Microbial Toxicology Laboratory, RIKEN Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan 3Laboratory for Growth Regulation, Plant Science Center, RIKEN Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan 4Graduate School of Science and Engineering, Saitama University, 255 Shimookubo, Saitama-shi, Saitama 338-8570, Japan 5Biological Research Laboratories, Nissan Chemical Industries Ltd., 1470 Shiraoka, Minamisaitama, Saitama 349-0294, Japan 6Department of Applied Biological Chemistry, Tamagawa University, Machida, Tokyo 194-8610, Japan

Received July 29, 2002; Accepted October 17, 2002
Systemic acquired resistance (SAR) is a potent innate immunity system in plants that is induced through the salicylic acid-mediated pathway. N-cyanomethyl-2-chloroisonicotinamide (NCI) is able to induce a broad range of disease resistance in tobacco and rice and induces SAR marker gene expression without SA accumulation in tobacco. To clarify the detailed mode of action of NCI, we analyzed its ability to induce defense gene expression and resistance in Arabidopsis mutants that are defective in various defense signaling pathways. Wild-type Arabidopsis treated with NCI exhibited increased expression of several pathogenesis-related genes and enhanced resistance to the bacterial pathogen, Pseudomonas syringae pv. tomato DC3000. NCI induced disease resistance and PR gene expression in NahG transgenic plants, but not in the npr1 mutant. NCI could induce PR gene expression in the etr1-1, ein2-1 and jar1-1 mutants. Thus, NCI activates SAR, independently from ethylene and jasmonic acid, by stimulating the site between SA and NPR1.
Key words: systemic acquired resistance; Arabidopsis; salycilic acid; PR protein; Pseudomonas syringae

-15-
Determination of the Absolute Configuration of (＋)-Xestoaminol C [(2S, 3R)-2-Amino-3-tetradecanol], a Metabolite of Fiji Sponge, Xestospongia sp., by the Synthesis of Its N,O-Diacetyl Derivative

Miwako ICHIHASHI1 and Kenji MORI2,

1Department of Chemistry, Graduate School of Science, Science University of Tokyo, Kagurazaka 1-3, Shinjuku-ku, Tokyo 162-8601, Japan 2Insect Pheromone and Traps Division, Fuji Flavor Co., Ltd., Midorigaoka 3-5-8, Hamura-City, Tokyo 205-8503, Japan

Received July 29, 2002; Accepted September 23, 2002
The N,O-diacetyl derivative of (＋)-xestoaminol C (2-amino-3-tetradecanol, 1), an inhibitor of reverse transcriptase isolated from Xestospongia sp., was synthesized from (S)-alanine, and its absolute configuration was determined as 2S, 3R.
Key words: amino alcohol; configuration determination; marine sponge; xestoaminol C; Xestospongia sp.

-16-
Isolation of Paenibacillus illinoisensis That Produces Cyclodextrin Glucanotransferase Resistant to Organic Solvents

Noriyuki DOUKYU, Hirokazu KUWAHARA, and Rikizo AONO

Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta 4259, Midori-ku, Yokohama 226-8501, Japan

Received August 13, 2002; Accepted September 26, 2002
A bacterium that secreted cyclodextrin glucanotransferase (CGTase) in a medium overlaid with n-hexane was isolated and identified as Paenibacillus illinoisensis strain ST-12 K. The CGTase of the strain was purified from the culture supernatant. The molecular mass was 70 kDa. The enzyme was stable at pH 6 to 10 and active at pH 5.0 to 8.0. The optimum temperature at pH 7.0 was 65°C in the presence of 5 mM CaCl2. The enzyme produced mainly β-cyclodextrin. The total yield of α-, β-, and γ- cyclodextrins was increased 1.4-fold by the addition of ethanol. In particular, the yield of β-cyclodextrins in the presence of 10％ (vol/vol) ethanol was 1.6-fold that without ethanol. The CGTase was stable and active in the presence of large amounts of various organic solvents.
Key words: cyclodextrin glucanotransferase; cyclodextrin; organic solvent; organic-solvent-tolerant; Paenibacillus illinoisensis

-17-
Mutational Analysis of Amino Acid Residues Involved in Catalytic Activity of a Family 18 Chitinase from Tulip Bulbs

Keisuke SUZUKAWA,1 Takeshi YAMAGAMI,1 Takayuki OHNUMA,1 Hideki HIRAKAWA,2 Satoru KUHARA,2 Yoichi ASO,1 and Masatsune ISHIGURO1,

1Laboratories of Protein Chemistry and Engineering and 2Genetic Regulation, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan

Received August 16, 2002; Accepted October 6, 2002
We expressed chitinase-1 (TBC-1) from tulip bulbs (Tulipa bakeri) in E. coli cells and used site-directed mutagenesis to identify amino acid residues essential for catalytic activity. Mutations at Glu-125 and Trp-251 completely abolished enzyme activity, and activity decreased with mutations at Asp-123 and Trp-172 when glycolchitin was the substrate. Activity changed with the mutations of Trp-251 to one of several amino acids with side-chains of little hydrophobicity, suggesting that hydrophobic interaction of Trp-251 is important for the activity. Molecular dynamics (MD) simulation analysis with hevamine as the model compound showed that the distance between Asp-123 and Glu-125 was extended by mutation of Trp-251. Kinetic studies of Trp-251-mutated chitinases confirmed these various phenomena. The results suggested that Glu-125 and Trp-251 are essential for enzyme activity and that Trp-251 had a direct role in ligand binding.
Key words: chitinase; tulip bulb; Tulipa bakeri; site-directed mutagenesis

-18-
Novel Gene Encoding a Ca2＋-Binding Protein and under Hexokinase-dependent Sugar Regulation

Shigeo OTSUKI,1 Akira IKEDA,1 Tomomi SUNAKO,1 Shoshi MUTO,2 Junshi YAZAKI,3 Keiko NAKAMURA,3 Fumiko FUJII,3 Kanako SHIMBO,3 Yoshimi OTSUKA,3 Kimiko YAMAMOTO,3 Katsumi SAKATA,4 Takuji SASAKI,4 Naoki KISHIMOTO,4 Shoshi KIKUCHI,4 and Junji YAMAGUCHI1,

1Division of Biological Sciences, Graduate School of Science, Hokkaido University, Kita-ku N10-W8, Sapporo 060-0810, Japan 2Bioscience Center, Nagoya University, Chikusa, Nagoya 464-8601, Japan 3STAFF Institute, 446-1 Ippaizuka, Kamiyokoba, Tsukuba, Ibaraki 305-0854, Japan 4NIAS, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan

Received September 5, 2002; Accepted October 7, 2002
A cDNA encoding a predicted 15-kDa protein was earlier isolated from sugar-induced genes in rice embryos (Oryza sativa L.) by cDNA microarray analysis. Here we report that this cDNA encodes a novel Ca2＋-binding protein, named OsSUR1 (for Oryza sativa sugar-up-regulated-1). The recombinant OsSUR1 protein expressed in Escherichia coli had 45Ca2＋-binding activity. Northern analysis showed that the OsSUR1 gene was expressed mainly in the internodes of mature plants and in embryos at an early stage of germination. Expression of the OsSUR1 gene was induced by sugars that could serve as substrates of hexokinase, but expression was not repressed by Ca2＋ signaling inhibitors, calmodulin antagonists and inhibitors of protein kinase or protein phosphatase. These results suggested that OsSUR1 gene expression was stimulated by a hexokinase-dependent pathway not mediated by Ca2＋.
Key words: EF-hand motif; hexokinase; Oryza sativa L.; signal transduction; sugar sensing

-19-
Novel Chitosanase from Streptomyces griseus HUT 6037 with Transglycosylation Activity

Toshiaki TANABE,1 Kazuko MORINAGA,1 Tamo FUKAMIZO,2 and Masaru MITSUTOMI1,

1Department of Applied Biological Sciences, Faculty of Agriculture, Saga University, Saga 840-8502, Japan 2Laboratory of Biophysical Chemistry, Faculty of Agriculture, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan

Received September 20, 2002; Accepted October 26, 2002
Streptomyces griseus HUT 6037 inducibly produced two chitosanases when grown on chitosan. To elucidate the mechanism of degradation of chitinous compound by this strain, chitosanases I and II of S. griseus HUT 6037 were purified and characterized. The purified enzymes had a molecular mass of 34 kDa. Their optimum pH was 5.7, and their optimum temperature was 60°C. They hydrolyzed not only partially deacetylated chitosan, but also carboxymethylcellulose. Time-dependent 1H-NMR spectra showing hydrolysis of (GlcN)6 by the chitosanases were obtained for identification of the anomeric form of the reaction products. Both chitosanases produced the β-form specifically, indicating that they were retaining enzymes. These enzymes catalyzed a glycosyltransfer reaction in the hydrolysis of chitooligosaccharides. The N-terminal and internal amino acid sequences of chitosanase II were identified. A PCR fragment corresponding to these amino acid sequences was used to screen a genomic library for the entire gene encoding chitosanase II. Sequencing of the choII gene showed an open reading frame encoding a protein with 359 amino acid residues. The deduced primary structure was similar to endoglucanase E-5 of Thermomonospora fusca, which enzyme belongs to family 5 of the glycosyl hydrolases. This is the first report of a family 5 chitosanase with transglycosylation activity.
Key words: chitosanase; Streptomyces griseus; family 5; transglycosylation

-20-
Note
Effects of Degree of Hydrolysis and pH on the Solubility of Heme-iron Enriched Peptide in Hemoglobin Hydrolysate

Man-Jin IN,2 Dong Chung KIM,3 Hee Jeong CHAE,4 and Nam-Soon OH1,

1Department of Food Science and Technology, Kongju National University, Yesan 340-802, Korea 2Department of Human Nutrition and Food Science, Chungwoon University, Hongsung 350-701, Korea 3Department of Food Science, Sunchon First College, Sunchon 540-744, Korea 4Department of Food Science and Technology and Department of Innovative Industrial Technology, Hoseo Graduate School of Venture, Hoseo University, Asan 336-795, Korea

Received January 21, 2002; Accepted October 8, 2002
Hemoglobin was hydrolyzed with Esperase and Flavourzyme as the endopeptidase and exopeptidase, respectively. The solubility of the heme-iron enriched peptide fraction decreased as the degree of hydrolysis of the hydrolysate increased. When the pH of a hydrolysate was adjusted to 5.0 after simultaneous hydrolysis with the two enzymes, the solubility of heme-iron enriched peptide was nearly zero, and 98％ of the heme-iron enriched peptide fraction was recovered as a precipitate. These results indicated that an effective separation method for the production of heme-iron enriched peptide could be established by pH adjustment of the hemoglobin hydrolysate with high degree of hydrolysis.
Key words: heme-iron enriched peptide; hemoglobin; pH adjustment; degree of hydrolysis; solubility

-21-
Note
Periplasmic Secretion of Native Ovalbumin without Signal Cleavage in Escherichia coli

Yasuhiro ARII, Nobuyuki TAKAHASHI, and Masaaki HIROSE

Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan

Received May 22, 2002; Accepted October 16,2002
In Escherichia coli cells carrying wild-type ovalbumin cDNA, some of the recombinant protein was secreted into the periplasmic space. In contrast, a signal-region mutant form of ovalbumin (deletion, Gly1 to Ala39) was not detected in the periplasm despite being synthesized at the same level as the wild-type protein. Chemical and spectroscopic analyses showed that periplasmic ovalbumin assumes a conformation indistinguishable from that of native egg white ovalbumin. We concluded that a process resembling the secretion of ovalbumin process in the oviduct occurs also in bacteria.
Key words: ovalbumin; recombinant ovalbumin; internal signal sequence; serpin superfamily

-22-
Note
Effect of a Selected Amino Acid Mixture on the Recovery from Muscle Fatigue during and after Eccentric Contraction Exercise Training

Masaaki SUGITA,1 Masaru OHTANI,2, Naokata ISHII,2 Kimiaki MARUYAMA,3 and Kando KOBAYASHI2

1Department of Health ＆ Physical Education, Mie University, Tsu City 514-8507, Japan 2Department of Life Science (Sports Sciences), The University of Tokyo, Tokyo 153-8902, Japan 3Department of Life Science, Meiji University, Kawasaki 214-8571, Japan

Received June 3, 2002; Accepted October 21, 2002
The effect of an amino acid mixture on the recovery from muscle fatigue after eccentric exercise (ECEX) training was examined in twenty-two male college students. The administration of 5.6 g of the amino acid mixture twice daily resulted faster recovery of muscle strength than that with a placebo. The oral administration of the amino acid mixture was proved to effective for muscle strength recovery after the eccentric exercise.
Key words: amino acid; eccentric exercise training; resistance training; muscle strength recovery

-23-
Note
New Phenolic Compounds from Kokuto, Non-centrifuged Cane Sugar

Kensaku TAKARA,1, Daigo MATSUI,1 Koji WADA,1 Toshio ICHIBA,2 Isao CHINEN,1 and Yoko NAKASONE1

1Laboratory of Applied Biochemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, University of the Ryukyus, Senbaru 1, Nishihara-cho, Okinawa 903-0213, Japan 2Okinawa Industrial Technology Center, Suzaki 12-2, Gushikawa-shi, Okinawa 904-2234, Japan

Received June 21, 2002; Accepted October 2, 2002
Five new phenolic compounds, 4-(β-D-glucopyranosyloxy)-3,5-dimethoxyphenyl-propanone (8), 3-[5-[(threo) 2,3-dihydro-2-(4-hydroxy-3-methoxyphenyl)-3-hydroxymethyl-7-methoxybenzofurany]]-propanoic acid (12), 2-[4-(3-hydroxy-1-propenyl)-2,6-dimethoxyphenoxy]-3-hydroxy-3-(4-hydroxy-3,5-dimethoxyphenyl)propyl-β-D-glucopyranoside (13), 4-[(erythro) 2,3-dihydro-3(hydroxymethyl)-5-(3-hydropropyl)-7-methoxy-2-benzofuranyl]-2,6-dimethoxyphenyl-β-D-
glucopyranoside (14), 9-O-β-D-xylopyranoside of icariol A2 (15), and known phenolic compounds were isolated from Kokuto, non-centrifuged cane sugar (Saccharum officinarum L.). Their structures were determined by a spectral investigation.
Key words: cane sugar; Saccharum officinarum L.; Kokuto; phenolic compounds

-24-
Note
Prolyl Endopeptidase Inhibitory Peptides in Wine

Takaaki YANAI, Yumiko SUZUKI, and Michikatsu SATO

Mercian Corporation, Wine ＆ Spirits Research Institute, 9-1 Johnan 4-chome, Fujisawa 251-0057, Japan

Received June 25, 2002; Accepted October 1, 2002
Two peptides that inhibit prolyl endopeptidase were isolated from a red wine made from Cabernet Sauvignon grapes. Their amino acid sequences and IC50 values were Val-Glu-Ile-Pro-Glu (17.0 μM) and Tyr-Pro-Ile-Pro-Phe (87.8 μM). The peptides also suppressed the degradation levels of the bioactive peptides vasopressin,substance P, and neurotensin fragments 8--13, which are involved in memory and neural communication.
Key words: prolyl endopeptidase; inhibitor; peptide; wine

-25-
Note
Response of the Induction of Rat Liver Serine Dehydratase to Changes in the Dietary Protein Requirement

Saeko IMAI,1 Ryuhei KANAMOTO,1, Iyo YAGI,1 Makoto KOTARU,2 Tohru SAEKI,1 and Kimikazu IWAMI1

1Department of Biological Resource Chemistry, Kyoto Prefectural University, Shimogamo, Sakyo-ku, Kyoto 606-8522, Japan 2Department of Food Science, College of Human Ecology, Koka Women′s University, Nishikyogoku, Ukyo-ku, Kyoto 615-0882, Japan

Received June 26, 2002; Accepted October 16, 2002
Growing and mature rats were examined for the effect of a change in dietary protein requirements on the induction of liver serine dehydratase (SDH). The rats were fed on diets varying in casein content, and the weight change and nitrogen balance was determined. SDH activity and its gene expression were induced in both growing and mature rats when their protein intake exceeded their nutritional requirements.
Key words: protein requirement; serine dehydratase; nitrogen balance; growing rat; mature rat

-26-
Note
Presence of Free D-Amino Acids in Microalgae

Takehiko YOKOYAMA,1 Nobuhiro KAN-NO,1 Takehiko OGATA,1 Yuichi KOTAKI,1 Minoru SATO,2 and Eizoh NAGAHISA1,

1School of Fisheries Sciences, Kitasato University, Ofunato, Iwate 022-0101, Japan 2Graduate School of Agriculture, Tohoku University, Sendai 981-8555, Japan

Received July 3, 2002; Accepted September 24, 2002
Several species of microalgae (phytoplankton), 4 species of freshwater algae and 4 species of marine diatoms, were cultured germ-free in the laboratory. The presence of free D-amino acids was verified in these species by a reversed-phase HPLC analysis. D-Aspartate was detected in all the microalgae examined, but D-alanine was only present in the marine diatoms. The D-amino acid content in Asterionella sp. of the marine diatoms increased from the exponential phase to the stationary phase and then decreased to the phase of decline.
Key words: D-aspartate; D-alanine; phytoplankton; diatom; microalga

-27-
Note
High IgM Production by Human-human Hybridoma HB4C5 Cells Cultured in Microtubes

Takuya SUGAHARA, Satomi YANO, and Takeshi SASAKI

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

Received July 5, 2002; Accepted October 16, 2002
HB4C5 cells, a human-human hybridoma, were cultured in microtubes. After 24 h of cultivation, growth of the cells cultured in microtubes was 1.2- to 1.5-fold that in culture dishes or 96-well culture plates. The production of IgM was 2.6- to 3.3-fold that in the 96-well culture plates.
Key words: cell culture vessel; human-human hybridoma; immunoglobulin production; physical stimulation

-28-
Note
Evaluation of the Anti-oxidative Effect (in vitro) of Tea Polyphenols

Fumio HASHIMOTO,1, Masateru ONO,2 Chikako MASUOKA,2 Yasuyuki ITO,2 Yusuke SAKATA,1 Keiichi SHIMIZU,1 Gen-ichiro NONAKA,3 Itsuo NISHIOKA,3 and Toshihiro NOHARA4

1Faculty of Agriculture, Kagoshima University, Korimoto 1-21-24, Kagoshima 890-0065, Japan 2Faculty of Agriculture, Kyushu Tokai University, Choyo 5435, Aso, Kumamoto 869-1404, Japan 3Faculty of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-0082, Japan 4Faculty of Pharmaceutical Sciences, Kumamoto University, Oe-honmachi 5-1, Kumamoto 862-0973, Japan

Received July 17, 2002; Accepted September 25, 2002
Forty-three polyphenols from tea leaves were evaluated for their anti-oxidative effect against lipid peroxidation by the ferric thiocyanate method in vitro. Among these, 1,4,6-tri-O-galloyl-β-D-glucose (hydrolyzable tannin) showed the highest anti-oxidative activity against lipid peroxidation, even stronger than that of 3-tert.-butyl-4-hydroxyanisole (BHA). The assay demonstrates that tea polyphenols, except for desgalloylated dimeric proanthocyanidins that possess a catechin structure in the upper unit and desgalloylated flavan-3-ols, and excepting theaflavin 3,3′-di-O-gallate, had more anti-oxidative activity than that of α-tocopherol. The chemical structure-activity relationship shows that the anti-oxidative action advanced with the condensation of two molecules of flavan-3-ols as well as with 3-O-acylation in the flavan skeleton such as that by galloyl, (3′-O-methyl)-galloyl, and p-coumaroyl groups.
Key words: antioxidant; Camellia sinensis; galloyl glucose; polyphenol; tea

-29-
Note
Effect of α-Tocopherol on the Oxidative Modification of Apolipoprotein E in Human Very-low-density Lipoprotein

Hirofumi ARAI,1 Koji UCHIDA,2 Kenji FUKUNAGA,3 Yuji NAGASAKA,4 Satoshi MOHRI,5 Hiroko FURUMOTO,1 Yasuhiro KURAMITSU,1 and Kazuyuki NAKAMURA1,

1Department of Biochemistry and Biomolecular Recognition, Yamaguchi University School of Medicine, Ube 755-8505, Japan 2Laboratory of Food and Biodynamics, Nagoya University School of Agricultural Science, Nagoya 464-8601, Japan 3Department of Public Health, Kansai Medical University, Moriguchi 570-8506, Japan 4Department of Nutrition, Yamaguchi Prefectural University, Yamaguchi 753-8502, Japan 5Industrial Technology Institute, Miyagi Prefectural Government, Sendai 981-3206, Japan

Received July 26, 2002; Accepted October 1, 2002
The oxidative modification of apolipoprotein (apo) E and lipid peroxidation in human very-low-density lipoprotein (VLDL) induced by peroxynitrite and cupric ions in vitro were strongly suppressed by enrichment with α-tocopherol (α-Toc; 170 μM). α-Toc also suppressed the decrease in the heparin-binding activity of apoE in the VLDL oxidation. These results suggest that α-Toc protected apoE in VLDL from oxidative stress.
Key words: apolipoprotein E; oxidation; α-tocopherol; very-low-density lipoprotein

-30-
Note
Isothermal Titration Calorimetric Studies on the Binding of a Family 6 Carbohydrate-binding Module of Clostridium thermocellum XynA with Xlylooligosaccharides

Kazuo SAKKA, Mari NAKANISHI, Mari SOGABE, Takamitsu ARAI, Hiroki OHARA, Akiyoshi TANAKA, Tetsuya KIMURA, and Kunio OHMIYA

Faculty of Bioresources, Mie University, 1515 Kamihamacho, Tsu 514-8507, Japan

Received July 29, 2002; Accepted October 17, 2002
The family 6 carbohydrate-binding module (CBM) of Clostridium thermocellum XynA was expressed, and the binding equilibria of the CBM with xylooligosaccharides (degree of polymerization DP＝2--8) were observed by isothermal titration calorimetry (ITC) at pH 8. The association constant, Ka, increased with increasing DP from 5×103 M−1 (DP＝2) to approximately 5×105 M−1 (DP＝5--8) at 20°C. The Ka values at 60°C were about 1/10 of those at 20°C. The binding was found to be an enthalpy-driven reaction. The DP dependence of the thermodynamic parameters of the binding reaction suggested the size of the ligand-binding site to be 5 xylose units long.
Key words: Clostridium thermocellum; xylanase; carbohydrate-binding module; isothermal titration calorimetry

-31-
Note
Antioxidative Compounds from Crotalaria sessiliflora

Abdul MUN′IM, Osamu NEGISHI, and Tetsuo OZAWA

Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan

Received August 2, 2002; Accepted October 17, 2002
Seven antioxidative compounds were isolated from the EtOAc extract of the aerial part of C. sessiliflora (Japanese name, tanukimame) by activity-guided fractionation with 2,2-diphenyl-1-picrylhydrazyl (DPPH). Among the isolated compounds, hydroxyeucomic acid showed the strongest free radical-scavenging activity, which was almost identical to that of epigallocatechin gallate, against DPPH. Orientin and isoorientin showed strong anti-peroxidative activities toward linoleic acid and protective effects against the bactericidal action of the tert-butyl peroxyl radical. Their activities were nearly equal to that of epigallocatechin gallate.
Key words: antioxidative compound; Crotalaria sessiliflora; free radical; tert-butyl peroxyl radical

-32-
Note
(−)-Olivil and (＋)-1-Acetoxypinoresinol from the Olive Tree (Olea europaea LINNE; Oleaceae) as Feeding Stimulants of the Olive Weevil (Dyscerus perforatus)

Emiko KADOWAKI,1 Yasuhiro YOSHIDA,3 Teruhiko NITODA,1 Naomichi BABA,2 and Shuhei NAKAJIMA2,

1Graduate School of Natural Science and Technology, Okayama University, Tsushimanaka, Okayama 700-8530, Japan 2Department of Bioresources Chemistry, Faculty of Agriculture, Okayama University, Tsushimanaka, Okayama 700-8530, Japan 3Nippon Olive Co., Ltd., 3911-10 Ushimado, Ushimado-cho, Oku-gun, Okayama 701-4394, Japan

Received August 9, 2002; Accepted September 25, 2002
Guided by a feeding stimulant activity test on the olive weevil (Dyscerus perforatus), two compounds that showed potent feeding stimulant activity were isolated from the olive tree (Olea europaea). Based on their spectral data and a literature survey, they were identified as (−)-olivil (1) and (＋)-1-acetoxypinoresinol (2). The activities of these minor lignans were significantly higher for the female than for the male weevil.
Key words: olive weevil; olive; feeding stimulant; (−)-olivil; (＋)-1-acetoxypinoresinol

-33-
Note
Effect of an Oral Administration of Lactobacillus casei Strain Shirota on the Natural Killer Activity of Blood Mononuclear Cells in Aged Mice

Tetsuji HORI, Junko KIYOSHIMA, and Hisako YASUI

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

Received August 9, 2002; Accepted October 3, 2002
The natural killer (NK) activity of blood mononuclear cells and splenocytes in aged mice fed on a diet containing Lactobacillus casei strain Shirota (LcS group) was significantly (P＜0.01) higher than that in mice fed on a diet without LcS. In the LcS group, there was a significant positive correlation (r＝0.63, P＜0.01) between the NK activity of blood mononuclear cells and the NK activity of splenocytes.
Key words: Lactobacillus casei strain Shirota; natural killer activity; splenocytes; blood mononuclear cells; aged mice

-34-
Note
Inhibition of Osteoporosis in Rats Fed with Sugar Cane Wax

Hajime TAMAKI,1 Sun Li MAN,1 Yutaka OHTA,2 Naofumi KATSUYAMA,2 and Isao CHINEN1,

1Laboratories of Applied Biochemistry, Faculty of Agriculture, University of the Ryukyus, Senbaru 1, Nishihara-cho, Okinawa 903-0213, Japan 2Faculty of Medicine, University of the Ryukyus, Senbaru 1, Nishiharas-cho, Okinawa 903-0213, Japan

Received August 13, 2002; Accepted September 27, 2002
Rats fed on a restricted, semi-purified diet containing a 50％-reduced level of carbohydrate and oil, but normal levels of protein, minerals and vitamins, exhibited osteoporosis. However, rats fed on this restricted diet, but containing sugar cane wax, did not exhibit this bone disease. Sugar cane wax, containing a long-chain carbohydrate with an OH radical, prevented the development of osteoporosis via a non-estrogenic mechanism.
Key words: osteoporosis; sugar cane wax; bone fracture energy; bone mineral density; estrogen

-35-
Note
Release Characteristics of Flavor from Spray-dried Powder in Boiling Water and during Rice Cooking

Hirokazu SHIGA,1 Hidefumi YOSHII,1, Rumiko TAGUCHI,1 Taiji NISHIYAMA,1 Takeshi FURUTA,1 and Pekka LINKO2

1Department of Biotechnology, Tottori University, Tottori 680-8552, Japan 2Department of Chemical Technology, Helsinki University of Technology, P.O. Box 6100, FIN-02015 HUT (Espoo), Finland

Received August 22, 2002; Accepted October 28, 2002
The release characteristics of flavor in boiling water and the flavor retention in the rice after cooking were investigated by using spray dried powder in encapsulated in or emulsified with d-limonene or ethyl n-hexanoate in cyclodextrin and maltodextrin, or in gum arabic and maltodextrin. The behavior of flavor release into the boiling water was well simulated by Avrami′s equation. The retention of d-limonene and ethyl n-hexanoate in cooked rice was correlated in each case with the flavor amount of spray-dried powder added.
Key words: β-cyclodextrin; ethyl n-hexanoate; d-limonene; release; retention

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Note
Effect of Dietary Fiber on the Lipid Metabolism and Immune Function of Aged Sprague-Dawley Rats

Koji YAMADA,1, Yoko TOKUNAGA,1 Atsushi IKEDA,1 Ken-ichi OHKURA,1 Shihoko KAKU-OHKURA,1 Soichi MAMIYA,2 Beong Ou LIM,3 and Hirofumi TACHIBANA1

1Division of Applied Biological Chemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan 2Department of Research and Development, Nutritional Foods Division, Taiyo Kagaku Co., Ltd., Yokkaichi 510-0844, Japan 3Department of Medical Nutrition, Graduate School of East-West Medical Science, Kyung Hee University, 1 Hoegi-Dong, Dongdaemoon-ku, Seoul 130-701, Korea

Received August 28, 2002; Accepted September 24, 2002
Eight-month-old Sprague-Dawley rats were fed on diets containing dietary fiber at the 5％ level for 3 weeks to examine the effect on the lipid metabolism and immune function. Among cellulose, guar gum, partially hydrolyzed guar gum (PHGG), glucomannan and highly methoxylated pectin, guar gum induced a significant decrease in the food intake and weight gain, as well as a significant increase in the liver weight. In addition, the epidydimal adipose tissue weight of the rats fed on PHGG was significantly higher than that of the rats fed on cellulose. There was no significant effect on the serum lipid levels, but the serum IgG level of the rats fed on guar gum was significantly lower than that of the rats fed on cellulose. The IgA and IgG productivity in mesenteric lymph node (MLN) lymphocytes was significantly higher in the rats fed on guar gum, glucomannan and pectin than in those fed on cellulose, while the effect on Ig productivity in spleen lymphocytes was not as marked. In addition, only guar gum induced a significant increase of IgM productivity in MLN lymphocytes when compared to the cellulose group. These results suggest that enhancement of the immune function by dietary fiber is mainly expressed in the gut immune system.
Key words: dietary fiber; guar gum; IgA; IgG; mesenteric lymph node

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Note
Synthesis of Hydroxymethylglutathione from Glutathione and L-Serine Catalyzed by Carboxypeptidase Y

Ryosuke OKUMURA, Yukio KOIZUMI, and Jiro SEKIYA

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

Receivd August 29, 2002; Accepted October 28, 2002
Hydroxymethylglutathione (γ-L-glutamyl-L-cysteinyl-L-serine; hmGSH) occurs in many species belonging to the family Gramineae, but the biosynthetic pathway for hmGSH has not been identified. We found that carboxypeptidase Y (CPY), but not carboxypeptidase A, catalyzed hmGSH synthesis from glutathione and L-serine in vitro at acidic pH. CPY also catalyzed methylglutathione synthesis from glutathione and L-alanine. These findings suggested that a carboxypeptidase-like enzyme may be involved in hmGSH synthesis in vivo.
Key words: aminolysis; carboxypeptidase Y; glutathione; hydroxymethylglutathione; methylglutathione

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Note
Cloning and Overexpression of the 3-Hydroxyisobutyrate Dehydrogenase Gene from Pseudomonas putida E23

Emran Kabir CHOWDHURY, Yuka AKAISHI, Shinji NAGATA, and Haruo MISONO

Department of Bioresources Science, Kochi University, Nankoku, Kochi 783-8502, Japan

Received September 6, 2002; Accepted October 17, 2002
The structural gene for NAD＋-dependent 3-hydroxyisobutyrate dehydrogenase (EC 1.1.1.31) from Pseudomonas putida E23 was cloned in Escherichia coli cells to obtain a large amount of the enzyme and its nucleotides were sequenced to study its structural relationship with other proteins. The gene encoded a polypeptide containing 295 amino acid residues and was in a cluster with the gene for methylmalonate semialdehyde dehydrogenase. Transformed E. coli cells overproduced 3-hydroxyisobutyrate dehydrogenase, and the recombinant enzyme was purified to homogeneity with a high yield. Lysine and asparagine residues, which are important in catalysis of the 3-hydroxyacid dehydrogenase family, are conserved in this enzyme.
Key words: 3-Hydroxyisobutyrate dehydrogenase; Pseudomonas putida; valine metabolism; nucleotide sequence; amino acid sequence

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Note
Hydroxysulochrin, a Tea Pollen Growth Inhibitor from the Fungus Aureobasidium sp.

Atsumi SHIMADA,1 Chisako SHIOKAWA,2 Miyako KUSANO,2 Shozo FUJIOKA,3 and Yasuo KIMURA2,

1Department of Environmental Chemistry, Faculty of Engineering, Kyushu Kyoritsu University, 1-8 Jiyugaoka, Yahatanishi, Kitakyushu 807-8585, Japan 2Department of Biological and Environmental Chemistry, Faculty of Agriculture, Tottori University, Koyama, Tottori 680-8553, Japan 3The Institute of Physical and Chemical Research (RIKEN), Hirosawa, Wako-shi 351-0198, Japan

Received September 6, 2002; Accepted October 28, 2002
A new plant growth regulator, hydroxysulochrin (1), together with sulochrin (2) was isolated from the culture filtrate of Aureobasidium sp. grown on a malt extract medium. The structures of 1 and 2 were established by spectroscopic methods. 1 and 2 inhibited tea pollen tube growth by 41％ and 36％ of the control value at a concentration of 100 mg/l, respectively. However, 1 and 2 showed no inhibitory effect on the growth of lettuce seedlings from 0.1 mg/l to 100 mg/l.
Key words: hydroxysulochrin; Aureobasidium; tea pollen

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Note
6-Hydroxyflavonoids as α-Glucosidase Inhibitors from Marjoram (Origanum majorana) Leaves

Jun KAWABATA, Kenji MIZUHATA, Eri SATO, Tetsuo NISHIOKA, Yoritaka AOYAMA, and Takanori KASAI

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

Received September 6, 2002; Accepted October 16, 2002
A methanol extract of marjoram leaves strongly inhibited rat intestinal α-glucosidase. Five 6-hydroxyflavonoids, 6-hydroxyapigenin (scutellarein; IC50 for sucrose hydrolysis by rat intestinal α-glucosidase, 12 μM), 6-hydroxyapigenin-7-O-β-D-glucopyranoside (＞500 μM), 6-hydroxyluteolin-7-O-β-D-glucopyranoside (300 μM), 6-hydroxyapigenin-7-O-(6-O-feruloyl)-β-D-glucopyranoside (＞500 μM), and 6-hydroxyluteolin-7-O-(6-O-feruloyl)-β-D-glucopyranoside (＞500 μM), were isolated as active principles and related compounds. The two feruloylglucosides are novel compounds.
Key words: 6-hydroxyflavone; Origanum majorana; marjoram; α-glucosidase inhibitor

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Note
Cooperation of Sly1/SM-Family Protein and Sec18/NSF of Saccharomyces cerevisiae in Disassembly of cis-SNARE Membrane-Protein Complexes

Yoichi KOSODO, Yoichi NODA, Hiroyuki ADACHI, and Koji YODA

Department of Biotechnology, University of Tokyo, Yayoi, Bunkyo-Ku, Tokyo 113-8657, Japan

Received September 9, 2002; Accepted October 31, 2002
Assembly and disassembly of the SNARE membrane-protein complexes plays a key role in vesicular trafficking. The SM-family Sly1 protein binds to the tSNARE Sed5 protein and stimulates its assembly into a trans-SNARE complex. Disassembly of the resulting cis-SNARE complex containing Sed5 was retarded in a temperature-sensitive yeast mutant of Sly1 protein with a defect in binding to Sed5. A temperature-sensitive mutation (sec18-1) of Sec18/NSF disassembly ATPase showed synthetic lethality with the sly1ts mutation. These results suggest that Sly1 and Sec18 proteins work cooperatively and that the binding of Sly1 to Sed5 stimulates the disassembly of the cis-SNARE complex by Sec18 ATPase.
Key words: Sly1/SM-protein; Sec18/NSF; SNARE complex; Saccharomyces cerevisiae; vesicle transport

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Note
Evidence of Isozymes for Δ6 Fatty Acid Desaturase in Rat Hepatocytes

Katsuya INAGAKI, Tsunehiro AKI, Taketoshi SHIOTA, Seiji KAWAMOTO, Seiko SHIGETA, Osamu SUZUKI, and Kazuhisa ONO

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

Received September 11, 2002; Accepted October 28, 2002
The expression of Δ6 fatty acid desaturase, previously identified, was suppressed almost completely by hyper expression of the corresponding antisense gene in a transformant of the rat hepatic cell line BRL-3A. Conversion rates of [1-14C] linoleic acid, α-linolenic acid, and tetracosapentaenoic acid into the respective Δ6 fatty acids were equivalent to those in control cells. This finding suggested that all of these reactions were catalyzed by at least two Δ6 desaturase isozymes in rat hepatocytes.
Key words: antisense; Δ6 desaturase; isozyme; polyunsaturated fatty acids; rat hepatocyte

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Preliminary Communication
In vivo Visualization of the Distribution of a Secretory Protein in Aspergillus oryzae Hyphae Using the RntA-EGFP Fusion Protein

Kumiko MASAI,1 Jun-ichi MARUYAMA,1,2 Harushi NAKAJIMA,1 and Katsuhiko KITAMOTO1,

1Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan 2Bio-oriented Technology Research Advancement Institution (BRAIN), 1-40-2 Nisshin, Saitama-shi, Saitama 331-8537, Japan

Received October 4, 2002; Accepted November 20, 2002
A fusion gene encoding ribonuclease T1-EGFP (rntA-egfp) was constructed and expressed to use it as a tool for studies on the secretory pathway in Aspergillus oryzae. The successful secretion of the intact RntA-EGFP fusion protein was detected by fluorescence measurement and Western analysis. With use of the RntA-EGFP system, we were able to see high fluorescence at hyphal tips and observe concentrated fluorescence at septa in basal cells during growth at optimal conditions. Cold or heat shock during growth caused the accumulation of EGFP fluorescence in vacuoles.
Key words: Aspergillus oryzae; secretory pathway; fusion protein; EGFP