Contents and Abstracts of BBB

(Vol.67 No.7 2003)

New Potent Antioxidative Hydroxyflavanones Produced with Aspergillus saitoi from Flavanone Glycoside in Citrus Fruit
Yoshiaki MIYAKE,1, Kenichiro MINATO,2 Syuichi FUKUMOTO,1 Kanefumi YAMAMOTO,1 Tomoko OYA-ITO,3 Syunro KAWAKISHI,3 and Toshihiko OSAWA4　p.1443

Anti-obesity Effect of Dioscorea nipponica Makino with Lipase-inhibitory Activity in Rodents
Chong-Suk KWON,2 Ho Yong SOHN,2 Sung Hee KIM,1 Ji Hyun KIM,2 Kun Ho SON,2 Jeong Soon LEE,1 Jin Kyu LIM,1 and Jong-Sang KIM1,† p.1451

High-cholesterol Diets Induce Changes in Lipid Composition of Rat Erythrocyte Membrane Including Decrease in Cholesterol, Increase in α-Tocopherol and Changes in Fatty Acids of Phospholipids
Shiro MAWATARI,1,† Yasushi OHNISHI,2 Yoshikazu KAJI,2 Tohru MARUYAMA,3 Kaori MURAKAMI,1 Koichiro TSUTSUI,3 and Takehiko FUJINO3 p.1457

Production of Galactinol from Sucrose by Plant Enzymes
Nariaki WAKIUCHI,1,2, Ryohei SHIOMI,1 and Hajime TAMAKI3 p.1465

Either Soluble or Plastidic Expression of Recombinant Protoporphyrinogen Oxidase Modulates Tetrapyrrole Biosynthesis and Photosynthetic Efficiency in Transgenic Rice
Sunyo JUNG,1 Jung Sung CHUNG,2 Sun Mi JANG,3 Ja Ock GUH,2 Hee Jae LEE,4 Sang-Uk CHON,5 Kyung-Moon KIM,1 Suk Bong HA,3 and Kyoungwhan BACK3,† p.1472

Effects of High Concentrations of Inorganic Salts on Swarming Ability in Fluorescent Pseudomonas Strains
Masao SAKAI, Hiroyuki FUTAMATA, and Shinjiro KANAZAWA p.1479

Purification and Characterization of Thermostable α-Galactosidase from Ganoderma lucidum
Thida SRIPUAN,1,2,† Kazuhiro AOKI,3 Kenji YAMAMOTO,3 Dararat TONGKAO,1 and Hidehiko KUMAGAI3 p.1485

Active Stress during Compression Testing of Various Foods Measured Using a Multiple-point Sheet Sensor
Kaoru KOHYAMA,1, Tomoko SASAKI,1 and Haruka DAN1,2 p.1492

Stimulation of Glucose Uptake in Muscle Cells by Prolonged Treatment with Scriptide, a Histone Deacetylase Inhibitor
Hisako TAKIGAWA-IMAMURA, Takumi SEKINE, Mitsuo MURATA, Kiyoshi TAKAYAMA, Kiyoshi NAKAZAWA, and Junichi NAKAGAWA p.1499

Amino Acid Residue Substitution at T-Cell Determinant-flanking Sites in β-Lactoglobulin Modulates Antigen Presentation to T Cells through Subtle Conformational Change
Akio AMETANI,1, Toshio SAKURAI,1,2 Yoshinori KATAKURA,1,3, Satoru KUHARA,3 Hideki HIRAKAWA,3 Tomohiro HOSOI,1, Shun-ichi DOSAKO,2 and Shuichi KAMINOGAWA1,† p.1507

Genomic Structure and 5′-Flanking Sequences of Rat N-Acetylglucosaminyltransferase I Gene and Regulatory Role of Its Transcriptional Diversity
Takashi FUKADA,1,2, Masanobu ONO,2 Shohei SAKATA,2 Noriyuki KIOKA,2 Hiroshi SAKAI,2,3 and Tohru KOMANO2,4 p.1515

Interaction between Elongation Factors 1β and 1γ from Bombyx mori Silk Gland
Katsuyoshi KAMIIE,1,† Tetsuro YAMASHITA,2 Hideharu TAIRA,2 Shin-ichiro KIDOU,3 and Shin-ichiro EJIRI3 p.1522

Synthesis of Glyceroyl β-N-Acetyllactosaminide and Its Derivatives through a Condensation Reaction by Cellulase
Nozomu YASUTAKE,1 Kazuhide TOTANI,2 Yoichiro HARADA,2 Shinobu HARAGUCHI,2 Takeomi MURATA,2 and Taichi USUI2,† p.1530

Volatilization of Mercury by an Iron Oxidation Enzyme System in a Highly Mercury-resistant Acidithiobacillus ferrooxidans Strain MON-1
Tsuyoshi SUGIO,1,† Mitsuko FUJII,1 Fumiaki TAKEUCHI,2 Atsunori NEGISHI,3 Terunobu MAEDA,4 and Kazuo KAMIMURA1 p.1537

Fourth 3D Structure of the Chitosan Molecule: Conformation of Chitosan in Its Salts with Medical Organic Acids Having a Phenyl Group
Makoto KAWAHARA,1 Toshifumi YUI,2 Kunio OKA,1 Peter ZUGENMAIER,3 Shiho SUZUKI,4 Shinichi KITAMURA,4 Kenji OKUYAMA,5 and Kozo OGAWA1,† p.1545

Deactivation of Gibberellin by 2-Oxidation during Germination of Photoblastic Lettuce Seeds
Kentaro NAKAMINAMI,1 Yoshiaki SAWADA,2 Miwako SUZUKI,2 Hiromichi KENMOKU,1 Hiroshi KAWAIDE,3 Wataru MITSUHASHI,1,2 Takeshi SASSA,1,2 Yasunori INOUE,4 Yuji KAMIYA,5 and Tomonobu TOYOMASU1,2,† p.1551

Synthesis of the Four Components of the Female Sex Pheromone of the Painted Apple Moth, Teia anartoides
Shin-etsu MUTO and Kenji MORI p.1559

Structural Studies and Antifungal Activity of Unique Polyene Amides, Clathrynamide A and Three New Derivatives, from a Marine Sponge, Psammoclemma Sp.
Makoto OJIKA, Yu ITOU, and Youji SAKAGAMI p.1568

Note
Development of an Effective Sample Preparation Method for the Proteome Analysis of Body Fluids Using 2-D Gel Electrophoresis
Won-A JOO, Do-Youn LEE, and Chan-Wha KIM p.1574

Note
Quinone Hemiacetal Formation from Protocatechuic Acid during the DPPH Radical Scavenging Reaction
Shizuka SAITO, Yasuko OKAMOTO, Jun KAWABATA, and Takanori KASAI p.1578

Note
A Novel Isoindoline, Porritoxin Sulfonic Acid, from Alternaria porri and the Structure-phytotoxicity Correlation of Its Related Compounds
Masayuki HORIUCHI,1,† Keiichiro OHNISHI,2,† Noriyasu IWASE,2 Yoshikazu NAKAJIMA,2 Kenji TOUNAI,2 Masakazu YAMASHITA,2 and Yasumasa YAMADA3 p.1580

Note
Conjugated Linoleic Acid (CLA) Inhibits Fatty Acid Synthetase Activity in Vitro
Hirosuke OKU,1,† Sawitree WONGTANGTINTHARN,2 Hironori IWASAKI,1 and Takayoshi TODA3 p.1584

Note
Inhibitory Effects of Resveratrol Derivatives from Dipterocarpaceae Plants on Tyrosinase Activity
Kenji OHGUCHI,1, Toshiyuki TANAKA,2 Tetsuro ITO,2 Munekazu IINUMA,3 Kenji MATSUMOTO,1 Yukihiro AKAO,1 and Yoshinori NOZAWA1 p.1587

Note
Degradation of Car Engine Base Oil by Rhodococcus sp. NDKK48 and Gordonia sp. NDKY76A
Daisuke KOMA,1 Yuichi SAKASHITA,1 Kenzo KUBOTA,1 Yoshihide FUJII,1 Fumihiko HASUMI,2 Seon-Yong CHUNG,3 and Motoki KUBO1,† p.1590,

Note
Isolation and Identification of Sodium 2-Propenyl Thiosulfate from Boiled Garlic (Allium sativum) That Oxidizes Canine Erythrocytes
Osamu YAMATO,1,† Yuko SUGIYAMA,1, Hideyuki MATSUURA,2 Keun-Woo LEE,3 Koichi GOTO,1, Mohammad Alamgir HOSSAIN,1 Yoshimitsu MAEDE,1 and Teruhiko YOSHIHARA2 p.1594

Note
New Plant Growth Promoters, Repraesentins A, B and C, from Lactarius repraesentaneus
Mitsuru HIROTA,1, Yuji SHIMIZU,1 Tsunashi KAMO,1 Hidefumi MAKABE,2 and Hisao SHIBATA1
p.1597

Note
Conversion of the Cleavage Specificity of Subtilisin YaB on Oxidized Insulin Chains to an Elastase-like Specificity by Replacement of Gly124 with Ala
Hui-Ching MEI,1,2 Ywan-Feng LI,1 Chi-Cheng HSU,1 Ying-Chieh TSAI,1, and Hiroshi TAKAGI3, p.1601

Note
Transfer of Pro-R Hydrogen from NADH to Dihydroxyacetonephosphate by sn-Glycerol-1-phosphate Dehydrogenase from the Archaeon Methanothermobacter thermautotrophicus
Yosuke KOGA,1,† Nobuhito SONE,2 Shunsuke NOGUCHI,2 and Hiroyuki MORII1 p.1605

Note
Purification and Characterization of Purple Acid Phosphatase PAP1 from Dry Powder of Sweet Potato
Tatsuya KUSUDO, Toshiyuki SAKAKI, and Kuniyo INOUYE p.1609

Note
Identification and Classification of Two-component Systems That Affect rpoS Expression in Escherichia coli
Masahito SUGIURA, Hirofumi AIBA, and Takeshi MIZUNO p.1612

Communication
Identification of the Lantibiotic Nisin Q, a New Natural Nisin Variant Produced by Lactococcus lactis 61-14 Isolated from a River in Japan
Takeshi ZENDO,1 Masanori FUKAO,1 Kyoko UEDA,2 Tomoko HIGUCHI,2 Jiro NAKAYAMA,1 and Kenji SONOMOTO1,† p.1616

Communication
Enterostatin (VPDPR) and Its Peptide Fragment DPR Reduce Serum Cholesterol Levels after Oral Administration in Mice
Yasuyuki TAKENAKA, Futoshi NAKAMURA, Taichi YAMAMOTO, and Masaaki YOSHIKAWA p.1620

Communication
Anti-angiogenic Activity of Tocotrienol
Hitoshi INOKUCHI, Hisako HIROKANE, Tsuyoshi TSUZUKI, Kiyotaka NAKAGAWA, Miki IGARASHI, and Teruo MIYAZAWA p.1623

-1-
New Potent Antioxidative Hydroxyflavanones Produced with Aspergillus saitoi from Flavanone Glycoside in Citrus Fruit

Yoshiaki MIYAKE,1, Kenichiro MINATO,2 Syuichi FUKUMOTO,1 Kanefumi YAMAMOTO,1 Tomoko OYA-ITO,3 Syunro KAWAKISHI,3 and Toshihiko OSAWA4

1Fundamental Technical Research Department of Pokka Corporation Ltd., 45-2 Kumanosyo, Shikatsu-cho, Nishikasugai-gun, Aichi 481-8515, Japan 2Departmnet of Horticulture, Miyagi Agricultural College, Taihaku, Sendai 982-0215, Japan 3Department of Food and Nutrition, Sugiyama Jogakuen University, Nagoya 464-8662, Japan 4Laboratory of Food and Biodynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan

Received Octover 4, 2001; Accepted February 21, 2003
Potent antioxidative hydroxyflavanones were produced with Aspergillus saitoi from hesperidin or naringin, which are flavanone glycosides in citrus fruit with weak antioxidative activity. The hydroxyflavanone produced from hesperidin was identified as 8-hydroxyhesperetin (8-HHE), a novel substance, and those from naringin were identified as carthamidin (6-hydroxynaringenin) and isocarthamidin (8-hydroxynaringenin) by FAB-MS, 1H-NMR and 13C-NMR analyses. The antioxidative activity of these hydroxyflavanones was examined by using the free radical-scavenging system of 1,1-diphenyl-2-picrylhydrazyl (DPPH) and the methyl linoleate oxidation system. The hydroxyflavanones (8-HHE, carthamidin, and isocarthamidin) exhibited stronger activity than the flavanone glycosides (hesperidin or naringin) and their aglycones (hesperetin or naringenin). The activity of 8-HHE and isocarthamidin was comparable to that of α-tocopherol, and that of carthamidin was weaker than that of isocarthamidin. The hydroxyflavanones, which were hydroxylated on A ring of flavanone by Aspergillus saitoi, were obtained as potent antioxidants.
Key words: antioxidant; Aspergillus saitoi; carthamidin; 8-hydroxyhesperetin; isocarthamidin

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Anti-obesity Effect of Dioscorea nipponica Makino with Lipase-inhibitory Activity in Rodents

Chong-Suk KWON,2 Ho Yong SOHN,2 Sung Hee KIM,1 Ji Hyun KIM,2 Kun Ho SON,2 Jeong Soon LEE,1 Jin Kyu LIM,1 and Jong-Sang KIM1,†

1Department of Animal Science and Biotechnology, Kyungpook National University, Taegu 702-701, S. Korea 2Department of Food Science and Nutrition, Andong National University, Andong 760-749, S. Korea

Received September 27, 2002; Accepted Febrary 28, 2003
In the process of screening for pancreatic lipase inhibitors, which could be used as an anti-obesity measure, the methanol extract of Dioscorea nipponica Makino powder (DP) appeared to have potent inhibitory activity against porcine pancreatic lipase with an IC50 value of 5--10 μg/ml, where the enzyme activity was assayed by using 4-methylumbelliferyl oleate as a substrate. Further purification of active components present in the herb generated dioscin that belongs to the saponin family. Dioscin and its aglycone, diosgenin, both suppressed the time-dependent increase of blood triacylglycerol level when orally injected with corn oil to mice, suggesting their inhibitory potential against fat absorption. Sprague-Dawley rats fed on a high-fat diet containing 5％ Dioscorea nipponica Makino and 40％ beef tallow gained significantly less body weight and adipose tissue than control animals fed on a high-fat diet alone during an 8-week experimental period (P＜0.05).
Key words: obesity; Dioscorea nipponica; lipase inhibition; dioscin; diosgenin

-3-
High-cholesterol Diets Induce Changes in Lipid Composition of Rat Erythrocyte Membrane Including Decrease in Cholesterol, Increase in α-Tocopherol and Changes in Fatty Acids of Phospholipids

Shiro MAWATARI,1,† Yasushi OHNISHI,2 Yoshikazu KAJI,2 Tohru MARUYAMA,3 Kaori MURAKAMI,1 Koichiro TSUTSUI,3 and Takehiko FUJINO3

1Department of Nutrition and Health Science, Faculty of Human Environmental Science, Fukuoka Women′s University, Fukuoka 813-8529, Japan 2First Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka 812-8582, Japan 3The Institute of Rheological Functions of Food Co., Ltd., Fukuoka 811-2501, Japan

Received November 7, 2002; Accepted March 26, 2003
Effects of high dietary cholesterol on erythrocyte membrane lipids were studied. Feeding rats with a diet containing 0.5％ cholesterol and 0.15％ sodium cholate for two weeks induced changes in erythrocyte membrane lipids including a decrease in cholesterol, an increase in α-tocopherol (α-Toc) and changes in the fatty acid composition of phospholipids. Oleic acid and linoleic acid increased, while arachidonic acid decreased in phosphatidylcholine. Saturated fatty acids decreased and unsaturated fatty acids increased in phosphatidylethanolamine. Almost the same changes in membrane lipids were also noted after six weeks of feeding rats with the diet. A diet containing 0.5％ cholesterol but without sodium cholate caused a decrease in erythrocyte cholesterol and an increase in erythrocyte α-Toc after two weeks of feeding, as compared to the basal diet, indicating that high dietary cholesterol, but not sodium cholate, was responsible for these changes in the erythrocyte membrane.
Key words: high cholesterol diets; rat erythrocyte; cholesterol; fatty acids of phospholipids; tocopherol

-4-
Production of Galactinol from Sucrose by Plant Enzymes

Nariaki WAKIUCHI,1,2, Ryohei SHIOMI,1 and Hajime TAMAKI3

1The Graduate School of Science and Technology, Kobe University, 1-1 Rokkodaicho, Nada, Kobe 657-8501, Japan 2Department of Biofunctional Chemistry, Faculty of Agriculture, Kobe University, 1-1 Rokkodaicho, Nada, Kobe 657-8501, Japan 3Laboratories of Applied Biochemistry, Faculty of Agriculture, University of the Ryukyus, 1 Senbaru, Nishihara-cho, Okinawa 903-0213, Japan

Received December 2, 2002; Accepted March 20, 2003
Galactinol, 1-O-(α-D-galactopyranosyl)-myo-inositol, was produced from sucrose as a starting material. UDP-Glc was prepared with sucrose and UDP using sucrose synthase partially purified from sweet potato roots. Then, the UDP-Glc was converted to UDP-Gal using yeast UDP-Gal 4-epimerase from a commercial source. Finally, galactinol was produced from the UDP-Gal and myo-inositol using galactinol synthase partially purified from cucumber leaves. The product was identified as galactinol by the retention times of HPLC, α-galactosidase digestion, and NMR spectrometry.
Key words: galactinol; galactinol synthase; cucumber leaves; UDP-Galactose 4-epimerase; sucrose synthase

-5-
Either Soluble or Plastidic Expression of Recombinant Protoporphyrinogen Oxidase Modulates Tetrapyrrole Biosynthesis and Photosynthetic Efficiency in Transgenic Rice

Sunyo JUNG,1 Jung Sung CHUNG,2 Sun Mi JANG,3 Ja Ock GUH,2 Hee Jae LEE,4 Sang-Uk CHON,5 Kyung-Moon KIM,1 Suk Bong HA,3 and Kyoungwhan BACK3,†

1Scigen Harvest Research Center, Suwon 441-744, Korea 2Institute of Agricultural Science and Technology, Chonnam National University, Gwangju 500-757, Korea 3Department of Biotechnology, Biotechnology Research Institute, Chonnam National University, Gwangju 500-757, Korea 4School of Plant Science, Seoul National University, Suwon 441--744, Korea 5Biotechnology Industrialization Center, Dongshin University, Naju 520-714, Korea

Received December 2, 2002; Accepted March 18, 2003
Protoporphyrinogen oxidase (Protox) is the last shared enzyme of the porphyrin pathway. As a continuation of our previous work in which the transgenic rice plants expressing the Bacillus subtilis Protox in the cytoplasm or the plastid showed resistance to diphenyl ether herbicide, this study was undertaken to identify the effects of tertapyrrole biosynthesis in these transgenic rice plants. The transgenic plants either targeted into plastids or expressed in cytoplasm showed higher Protox activity than wild-type plants did. Photosynthetic activity, measured as a quantum yield of photosystem II, was slightly higher in transgenic plants than in wild-type plants, but chlorophyll contents were not significantly different between transgenic and wild-type plants. As for porphyrin biosynthesis, both cytoplasm-expressed and plastid-targeted transgenic plants showed increased synthesis of aminolevulinic acid, Mg-Proto IX, and protoheme in comparison to wild-type plants whereas synthesis of protoporphyrin IX was similar for wild-type and transgenic plants. These results indicate that either cytoplasm or plastid expression of B. subtilis Protox in rice can upregulate the porphyrin pathway leading to increase in photosynthetic efficiency in plants.
Key words: protoporphyrinogen oxidase; Bacillus subtilis; transgenic rice; protoporphyrin IX; photosynthetic activity

-6-
Effects of High Concentrations of Inorganic Salts on Swarming Ability in Fluorescent Pseudomonas Strains

Masao SAKAI, Hiroyuki FUTAMATA, and Shinjiro KANAZAWA

Laboratory of Soil Microbiology, Department of Plant Resources, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan

Received January 7, 2003; Accepted March 26, 2003
We did tests using swarm plates, to examine the effects of various salts and their concentrations on the chemotaxis of fluorescent Pseudomonas strains. As a result, we found that the swarming ability of the Pseudomonas strains was inhibited by high concentrations of Ca2＋. The growth of the strains was not affected at the high concentration of Ca2＋, but the cells grown in swarm agar under the condition were extended in the filaments. Most of the cells had reached 10 μm to 40 μm in length. Such cell elongation was not observed with salts other than calcium salts. A significant correlation between the cell elongation and the decrease of swarming ability by the high concentration of Ca2＋ was observed.
Key words: fluorescent Pseudomonas; chemotaxis; inorganic salts; calcium; root colonization

-7-
Purification and Characterization of Thermostable α-Galactosidase from Ganoderma lucidum

Thida SRIPUAN,1,2,† Kazuhiro AOKI,3 Kenji YAMAMOTO,3 Dararat TONGKAO,1 and Hidehiko KUMAGAI3

1Department of Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand 2Institute for Science and Technology Research and Development, Chiang Mai University, Chiang Mai 50200, Thailand 3Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kyoto, Japan

Received January 9, 2003; Accepted March 17, 2003
α-Galactosidase was purified from a fresh fruiting body of Ganoderma lucidum by precipitation with ammonium sulfate and column chromatographies with DEAE-Sephadex and Con A-Sepharose. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis. Its N-terminal amino acid sequence was similar to that of Mortierella vinacea α-galactosidase. The molecular mass of the enzyme was about 56 kDa by SDS-polyacrylamide gel electrophoresis, and about 249 kDa by gel filtration column chromatography. The optimum pH and temperature were 6.0 and 70°C, respectively. The enzyme was fully stable to heating at 70°C for 30 min. It hydrolyzed p-nitrophenyl-α-D-galactopyranoside (Km＝0.4 mM) but hydrolyzed little o-nitrophenyl-α-D-galactopyranoside. It also hydrolyzed melibiose, raffinose, and stachyose. The enzyme catalyzed the transgalactosylation reaction which synthesized melibiose. The product was confirmed by various analyses.
Key words: α-galactosidase; Ganoderma lucidum; thermostable enzyme; transglycosylation; galactooligosaccharide

-8-
Active Stress during Compression Testing of Various Foods Measured Using a Multiple-point Sheet Sensor

Kaoru KOHYAMA,1, Tomoko SASAKI,1 and Haruka DAN1,2

1National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan 2RETS Division, Nitta Corporation, 8-2-1 Ginza, Chuo-ku, Tokyo 104-0061, Japan

Received January 14, 2003; Accepted March 18, 2003
Using a multiple-point sheet sensor (MSS), load and contact area were directly measured for compression of four different foods. The MSS provided temporal and spatial changes in stress applied on the sample surface during the testing. The sum of load value detected by the MSS corresponded to the load measured by a universal testing machine during the compression. The contact area between a flat probe and food surface varied with the variety of foods even though under a small strain, and increased as compression strain increased. The active stress, that is, the load divided by the contact area, was different from conventional stress, that is, the load divided by the initial cross-sectional area. The value of active stress provided a better explanation of textural characteristics of food, because texture is often sensed under a large deformation and mixed assessment of mechanical and geometrical properties.
Key words: texture; large deformation; compression testing; multiple-point sheet sensor; stress distribution

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Stimulation of Glucose Uptake in Muscle Cells by Prolonged Treatment with Scriptide, a Histone Deacetylase Inhibitor

Hisako TAKIGAWA-IMAMURA, Takumi SEKINE, Mitsuo MURATA, Kiyoshi TAKAYAMA, Kiyoshi NAKAZAWA, and Junichi NAKAGAWA

Medicinal Research Laboratories, Taisho Pharmaceutical Co., Ltd., 1-403 Yoshino-cho, Kita-ku, Saitama-shi, Saitama 331-9530, Japan

Received January 21, 2003; Accepted April 10, 2003
Glucose incorporation is regulated mainly by GLUT4 in skeletal muscles. Here we report that treatment of L6 myotubes with scriptide, a hydroxamic acid-based histone deacetylase (HDAC) inhibitor, stimulated 2-deoxyglucose uptake. The effect appeared only after 24 hr, resulting in 2.4-fold glucose uptake at treatment day 6. Scriptide acted synergistically with insulin, indicating it stimulated a distinct pathway from the insulin signaling pathway. It was not observed in undifferentiated myoblasts or 3T3-L1 adipocytes, suggesting a muscle-specific effect of scriptide. A five-carbon chain and hydroxamic acid, essential for histone deacetylase inhibition, were indispensable for this effect, and trichostatin A stimulated glucose uptake as well. Scriptide increased the cellular content of GLUT4, and induced GLUT4 translocation, but GLUT4 mRNA level did not change, indicating scriptide functions posttranslationally. Our results indicated a novel function for HDAC inhibitors of increasing GLUT4 content and its translocation in muscle cells, resulting in stimulation of glucose uptake.
Key words: glucose transport; diabetes mellitus; muscle; histone deacetylase; scriptide

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Amino Acid Residue Substitution at T-Cell Determinant-flanking Sites in β-Lactoglobulin Modulates Antigen Presentation to T Cells through Subtle Conformational Change

Akio AMETANI,1, Toshio SAKURAI,1,2 Yoshinori KATAKURA,1,3, Satoru KUHARA,3 Hideki HIRAKAWA,3 Tomohiro HOSOI,1, Shun-ichi DOSAKO,2 and Shuichi KAMINOGAWA1,†

1Department of Applied Biological Chemistry, The University of Tokyo, Tokyo 113-8657, Japan 2Technology and Research Institute, Snow Brand Co., Ltd., Kawagoe-city, Saitama 350-1165, Japan 3Graduate School of Genetic Resources Technology, Kyushu University, Fukuoka 812-8581, Japan

Received January 27, 2003; Accepted April 11, 2003
We compared T-cell responses to regions in residues 21--40 of A and B variants of bovine milk β-lactoglobulin (β-LG) that vary by two different amino acid residues at 64 and 118. Results showed that T cells from C57/BL6 and C3H/HeN mice immunized with peptide 21--40 or BALB/c mice immunized with peptide 21--32 or 25--40 responded more vigorously to β-LG B than to β-LG A. This difference in response to 25--40 in BALB/c mice was not observed when β-LGs B and A were denatured, suggesting that the conformation difference affects display of the determinant 25--40. Reactivity of anti-β-LG monoclonal antibodies and molecular modeling using molecular dynamics calculations revealed subtle differences in the three-dimensional structure of these two variants. Furthermore, substitution of two amino acid residues at sites distant from the T-cell determinant induced differential determinant display on antigen-presenting cells, possibly due to subtle conformational changes in β-LG.
Key words: amino acid substitution; antigen presentation; conformational change; T-cell response

-11-
Genomic Structure and 5′-Flanking Sequences of Rat N-Acetylglucosaminyltransferase I Gene and Regulatory Role of Its Transcriptional Diversity

Takashi FUKADA,1,2, Masanobu ONO,2 Shohei SAKATA,2 Noriyuki KIOKA,2 Hiroshi SAKAI,2,3 and Tohru KOMANO2,4

1Laboratory of Applied Molecular Biology, Division of Applied Biochemistry, Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University, Gakuen-cho 1-1, Sakai, Osaka 599-8531, Japan 2Laboratory of Cellular Biochemistry, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwake-cho, Sakyou-ku, Kyoto 606-8502, Japan 3Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, Tsushima-naka 3-1-1, Okayama 700-8530, Japan 4Department of Genetic Engineering, Faculty of Biology-Oriented Science and Technology, Kinki University, Wakayama 649-6493, Japan

Received January 27, 2003; Accepted March 27, 2003
It has been known that mouse, rat, and human N-acetylglucosaminyltransferase I (GnT-I) genes produce at least two transcripts, which differ in their 5′-untranslated region (5′-UTR) length, and the longer transcript is preferentially expressed in brains. However, the physiological meaning of this brain-specific expression pattern was unknown. We cloned the rat GnT-I gene and analyzed its structure. It consisted of five exons, and four of them coded only 5′-UTRs. A putative NF-κB binding site was found in the 5′-flanking sequence for the transcript that was previously shown to be induced by inflammation. The unusually long 5′-UTR of the major GnT-I transcript in rat brain was shown to inhibit protein production from the following coding sequence in COS7 cells. Comparison of the GnT-I protein/mRNA ratio in rat brain and liver showed that GnT-I mRNA in the brain was translated 3.8-times less efficiently than in the liver. Implications are discussed of these results in regulation of GnT-I expression in rat brain.
Key words: N-acetylglucosaminyltransferase I; untranslated region; NF-κB; gene regulation; tissue-specific expression

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Interaction between Elongation Factors 1β and 1γ from Bombyx mori Silk Gland

Katsuyoshi KAMIIE,1,† Tetsuro YAMASHITA,2 Hideharu TAIRA,2 Shin-ichiro KIDOU,3 and Shin-ichiro EJIRI3

1Department of Bioscience and Biotechnology, Faculty of Engineering, Aomori University, Aomori 030-0943, Japan 2Department of Agro-science, and 3Cryobiosystem Research Center, Faculty of Agriculture, Iwate University, Morioka 020-8550, Japan

Received January 28, 2003; Accepted March 26, 2003
Elongation factor 1 (EF-1) from the silk gland of Bombyx mori consists of four subunits: α (51 kDa), β (26 kDa), γ (49 kDa), and δ (33 kDa). The EF-1α subunit catalyzes the binding of aminoacyl-tRNA to the ribosome concomitant with the hydrolysis of GTP. The EF-1α-bound GDP is then exchanged for GTP by the EF-1βgd complex. To facilitate analysis of the roles of the individual EF-1β, γ, and δ subunits in GDP/GTP exchange on EF-1α, we cloned the cDNAs for these subunits and expressed them in Escherichia coli. EF-1β, EF-1γ, and the carboxyl-terminal half of EF-1δ were expressed, purified, and examined for protein:protein interactions by gel filtration chromatography and by a quartz-crystal microbalance method. An 80-kDa species containing EF-1β and γ subunits in a 1:1 molar ratio was detected by gel filtration. A higher molecular weight species containing an excess of EF-1γ relative to EF-1β was also detected. The amino-terminal region of EF-1β (amino acid residues 1--129) was sufficient for binding to EF-1γ. The carboxyl-terminal half of EF-1δ did not appear to form a complex with EF-1γ.
Key words: elongation factor 1β (EF-1β); elongation factor 1γ (EF-1γ); subunit interaction; quartz-crystal microbalance (QCM); Bombyx mori

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Synthesis of Glyceroyl β-N-Acetyllactosaminide and Its Derivatives through a Condensation Reaction by Cellulase

Nozomu YASUTAKE,1 Kazuhide TOTANI,2 Yoichiro HARADA,2 Shinobu HARAGUCHI,2 Takeomi MURATA,2 and Taichi USUI2,†

1Science of Biological Resource, The United Graduate School of Agricultural Science, Gifu University, Yanagido 1-1, Gifu 501-1193, Japan 2Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, Ohya 836, Shizuoka 422-8529, Japan

Received January 31, 2003; Accepted March 27, 2003
A condensation reaction between N-acetyllactosamine and glycerol was directly catalyzed by using a commercially available cellulase preparation from Trichoderma reesei. 1-O-β-N-Acetyllactosaminyl-(R, S)-glycerols (1) were readily synthesized in a 5％ yield based on the N-acetyllactosamine added and conveniently isolated by two-step column chromatographies. The use of a partially purified enzyme increased 2.3-fold the yield of 1, compared to that of the crude enzyme containing β-D-galactosidase activity. When various alkanols (n:2--4) were used in the condensation reaction, the corresponding alkyl β-N-acetyllactosaminides were obtained in yields of 0.3--1.1％ of the desired compounds.
Key words: condensation; N-acetyllactosaminide; Trichoderma reesei; cellulase; glycerol

-14-
Volatilization of Mercury by an Iron Oxidation Enzyme System in a Highly Mercury-resistant Acidithiobacillus ferrooxidans Strain MON-1

Tsuyoshi SUGIO,1,† Mitsuko FUJII,1 Fumiaki TAKEUCHI,2 Atsunori NEGISHI,3 Terunobu MAEDA,4 and Kazuo KAMIMURA1

1Graduate School of Natural Science and Technology, Science and Technology for Energy Conversion, Okayama University, Tsushima Naka 1-1-1, Okayama 700-8530, Japan 2Administration Center for Environmental Science and Technology, Okayama University, Tsushima Naka 1-1-1, Okayama 700-8530, Japan 3Technical Research Institute, Hazama Corporation, 515-1 Nishimukai, Karima, Tsukuba 305-0822, Japan 4Civil Chemical Engineering Corporation, 3411 Sanuki-machi, Ryugasaki 301-0033, Japan

Received February 5, 2003; Accepted April 7, 2003
A highly mercury-resistant strain Acidithiobacillus ferrooxidans MON-1, was isolated from a culture of a moderately mercury-resistant strain, A. ferrooxidans SUG 2-2 (previously described as Thiobacillus ferrooxidans SUG 2-2), by successive cultivation and isolation of the latter strain in a Fe2＋ medium with increased amounts of Hg2＋ from 6 μM to 20 μM. The original stain SUG 2-2 grew in a Fe2＋ medium containing 6 μM Hg2＋ with a lag time of 22 days, but could not grow in a Fe2＋ medium containing 10 μM Hg2＋. In contrast, strain MON-1 could grow in a Fe2＋ medium containing 20 μM Hg2＋ with a lag time of 2 days and the ability of strain MON-1 to grow rapidly in a Fe2＋ medium containing 20 μM Hg2＋ was maintained stably after the strain was cultured many times in a Fe2＋ medium without Hg2＋. A similar level of NADPH-dependent mercury reductase activity was observed in cell extracts from strains SUG 2-2 and MON-1. By contrast, the amounts of mercury volatilized for 3 h from the reaction mixture containing 7 μM Hg2＋ using a Fe2＋-dependent mercury volatilization enzyme system were 5.6 nmol for SUG 2-2 and 67.5 nmol for MON-1, respectively, indicating that a marked increase of Fe2＋-dependent mercury volatilization activity conferred on strain MON-1 the ability to grow rapidly in a Fe2＋ medium containing 20 μM Hg2＋. Iron oxidizing activities, 2,3,5,6-tetramethyl-p-phenylenediamine (TMPD) oxidizing activities and cytochrome c oxidase activities of strains SUG 2-2 and MON-1 were 26.3 and 41.9 μl O2 uptake/mg/min, 15.6 and 25.0 μl O2 uptake/mg/min, and 2.1 and 6.1 mU/mg, respectively. These results indicate that among components of the iron oxidation enzyme system, especially cytochrome c oxidase activity, increased by the acquisition of further mercury resistance in strain MON-1. Mercury volatilized by the Fe2＋-dependent mercury volatilization enzyme system of strain MON-1 was strongly inhibited by 1.0 mM sodium cyanide, but was not by 50 nM rotenone, 5 μM 2-n-heptyl-4-hydroxy-quinoline-N-oxide (HQNO), 0.5 μM antimycin A, or 0.5 μM myxothiazol, indicating that cytochrome c oxidase plays a crucial role in mercury volatilization of strain MON-1 in the presence of Fe2＋.
Key words: iron-oxidizing bacterium; Acidithiobacillus ferrooxidans; mercury resistance; mercury reduction; cytochrome c oxidase

-15-
Fourth 3D Structure of the Chitosan Molecule: Conformation of Chitosan in Its Salts with Medical Organic Acids Having a Phenyl Group

Makoto KAWAHARA,1 Toshifumi YUI,2 Kunio OKA,1 Peter ZUGENMAIER,3 Shiho SUZUKI,4 Shinichi KITAMURA,4 Kenji OKUYAMA,5 and Kozo OGAWA1,†

1Research Institute for Advanced Science and Technology (RIAST), Osaka Prefecture University, Sakai, Osaka 599-8570, Japan 2Faculty of Engineering, Miyazaki University, Miyazaki 889-2192, Japan 3Technische Universit<Oh><0228><Wa>at Clausthal, Institut f<Oh><0228><Wa>ur Physikalische Chemie, Arnold-Sommerfeld-Str. 4, D-38678 Clausthal-Zellerfeld, Germany 4Graduate School of Agriculture and Biological Science, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan 5Faculty of Technology, Tokyo University of Agriculture and Technology, Tokyo 184-8588, Japan

Received February 12, 2003; Accepted March 29, 2003
Chitosan salts with two medical organic acids having phenyl groups (salicylic and gentisic acids) exhibited fiber diffraction patterns of a new type of crystal which does not compare with known types I and II. The crystals, called type III salts, showed a fiber repeat of 2.550 nm and a meridional reflection at the 5th layer line. These results coupled with a conformational analysis indicate the chain conformation of chitosan with the salts to be a 5/3 helix, this helix differing from those of type I (an extended two-fold helix) and type II (a relaxed two-fold helix or a 4/1 helix). The fiber patterns of all the type III salts were similar. This observation has also been found with type II salts and is an indication that the acid ions are not arranged in regular positions in the crystals. A comparison of solid-state 13C-NMR spectra of the gentisic acid salt and the aspirin salt, which could not be crystallized, suggests that, in the latter salt, the chitosan molecules also formed a 5/3 helix.
Key words: chitosan-salicylic acid salt; chitosan-gentisic acid salt; chitosan-aspirin salt; type III salt of chitosan; 5/3 conformation of chitosan

-16-
Deactivation of Gibberellin by 2-Oxidation during Germination of Photoblastic Lettuce Seeds

Kentaro NAKAMINAMI,1 Yoshiaki SAWADA,2 Miwako SUZUKI,2 Hiromichi KENMOKU,1 Hiroshi KAWAIDE,3 Wataru MITSUHASHI,1,2 Takeshi SASSA,1,2 Yasunori INOUE,4 Yuji KAMIYA,5 and Tomonobu TOYOMASU1,2,†

1Course of the Science of Bioresources, The United Graduate School of Agricultural Science, Iwate University, Morioka, Iwate 020-8550, Japan 2Department of Bioresource Engineering, Yamagata University, Tsuruoka, Yamagata 997-8555, Japan 3Department of Applied Biological Science, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan 4Department of Applied Biological Science, Tokyo University of Science, Noda, Chiba 278-8510, Japan 5RIKEN Plant Science Center, Wako, Saitama 351-0198, Japan

Received February 14, 2003; Accepted Feburuary 21, 2003
Gibberellin (GA) plays an important role in the induction of germination of photoblastic lettuce (Lactuca sativa L. cv. Grand Rapids) seeds. We have previously shown that gene expression of a GA 3-oxidase (Ls3h1) increased after a red light treatment, resulting in an increase in the endogenous content of GA1, bioactive GA. Since the metabolism of GAs is also important for determining the endogenous levels of bioactive GAs, cDNAs encoding GA 2-oxidases (LsGA2ox1 and LsGA2ox2, for L. sativa GA 2-oxidase), which catalyze the deactivation of GAs, were isolated from lettuce seeds to investigate the regulation of these genes by light. An expression analysis shows that the mRNA levels of both enzymes was not markedly altered under different light conditions during germination. However, the amount of LsGA2ox2 transcripts was decreased to approximately half the level by red light. This reduction might play a role in the increase in GA1 level by red light in the lettuce seeds.
Key words: lettuce; gibberellin; deactivation; germination

-17-
Synthesis of the Four Components of the Female Sex Pheromone of the Painted Apple Moth, Teia anartoides

Shin-etsu MUTO and Kenji MORI

Insect Pheromone and Traps Division, Fuji Flavor Co., Ltd., Midorigaoka 3-5-8, Hamura-City, Tokyo 205-8503, Japan

Received February 24, 2003; Accepted March 28, 2003
Four pheromone components of the female painted apple moth (Teia anartoides), an Australian insect pest, were synthesized. These were (Z)-6-henicosen-11-one (1), (6Z, 8E)-6,8-henicosadien-11-one (2), (Z)-cis-9,10-epoxy-6-henicosene (3), and (Z)-cis-9,10-epoxy-6-icosene (4). 2-Dodecanone was converted to 1 and 2, and both the enantiomers of 3 and 4 were synthesized from the enantiomers of 4-tert-butyldimethylsilyloxy-cis-2,3-epoxy-1-butanol.
Key words: chiral epoxide; ketone; painted apple moth; pheromone; Teia anartoides

-18-
Structural Studies and Antifungal Activity of Unique Polyene Amides, Clathrynamide A and Three New Derivatives, from a Marine Sponge, Psammoclemma Sp.

Makoto OJIKA, Yu ITOU, and Youji SAKAGAMI

Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan

Received February 27, 2003; Accepted March 27, 2003
Known polyene amide, clathrynamide A, and three novel related metabolites, debromoclathrynamide A, (4E,6E)-debromoclathrynamide A and (6E)-clathrynamide A, were isolated from an Okinawan marine sponge, Psammoclemma sp. The absolute stereochemistry of clathrynamide A was determined to be 3R,12R by the modified Mosher method. The structures of the three new compounds were elucidated by spectroscopic analyses as debromo derivatives and geometrical isomers. Their antifungal activities were also evaluated with a phytopathogenic fungus, indicating that both the bromine atom and double-bond geometry affected the activity.
Key words: clathrynamide; polyene; Psammoclemma; marine sponge; antifungal activity

-19-
Note
Development of an Effective Sample Preparation Method for the Proteome Analysis of Body Fluids Using 2-D Gel Electrophoresis

Won-A JOO, Do-Youn LEE, and Chan-Wha KIM

Graduate School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Korea

Received November 18, 2002; Accepted March 25, 2003
A sample preparation is still the most critical step in two-dimensional electrophoresis (2-DE) and should be optimized for each type of sample. In this study, a protein extraction method from body fluids was developed using a combined centrifugal filter device and a sample treating buffer. When plasma, amniotic fluid, urine, and tear were tested with this method, the recovery of protein reached almost 90％ and high-quality separation of 2-DE gel was obtained.
Key words: body fluids; sample preparation of 2-DE; centrifugal filter device, detergent; IEF

-20-
Note
Quinone Hemiacetal Formation from Protocatechuic Acid during the DPPH Radical Scavenging Reaction

Shizuka SAITO, Yasuko OKAMOTO, Jun KAWABATA, and Takanori KASAI

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

Received January 6, 2003; Accepted February 21, 2003
Protocatechuic acid was rapidly converted to protocatechuquinone 3-methyl hemiacetal and protocatechuquinone during the reaction with DPPH radical in methanol. The structure of the acetal was determined by comparing the NMR data with those of an authentic compound prepared by (diacetoxy)iodobenzene oxidation of protocatechuic acid.
Key words: protocatechuic acid; radical scavenging reaction; DPPH radical; quinone hemiacetal

-21-
Note
A Novel Isoindoline, Porritoxin Sulfonic Acid, from Alternaria porri and the Structure-phytotoxicity Correlation of Its Related Compounds

Masayuki HORIUCHI,1,† Keiichiro OHNISHI,2,† Noriyasu IWASE,2 Yoshikazu NAKAJIMA,2 Kenji TOUNAI,2 Masakazu YAMASHITA,2 and Yasumasa YAMADA3

1Research Institute for Production Development, 15 Shimogamo Morimoto-cho, Sakyo-ku, Kyoto 606-0805, Japan 2Department of Molecular Science and Technology, Faculty of Engineering, Doshisha University, 1-3 Tatara Miyakotani, Kyotanabe-shi, Kyoto 610-0312, Japan 3Department of Food Science and Nutrition, Doshisha Women′s College of Liberal Arts, Teramachi-Imadegawa, Kamigyo-ku, Kyoto 602-0893, Japan

Received January 8, 2003; Accepted February 26, 2003
Novel zinniol-related compound 3, named porritoxin sulfonic acid, with an isoindoline skeleton was isolated from the culture liquid of Alternaria porri. The structure was determined to be 2-(2″-sulfoethyl)-4-methoxy-5-methyl-6-(3′-methyl-2′-butenyloxy)-2,3-dihydro-1H-isoindol-1-one. The phytotoxic activities of three isoindolines (1--3) were evaluated in a seedling-growth assay against stone leek and lettuce.
Key words: Alternaria porri; porritoxin sulfonic acid; zinniol; phytotoxin; seedling-growth assay

-22-
Note
Conjugated Linoleic Acid (CLA) Inhibits Fatty Acid Synthetase Activity in Vitro

Hirosuke OKU,1,† Sawitree WONGTANGTINTHARN,2 Hironori IWASAKI,1 and Takayoshi TODA3

1Division of Molecular Biotechnology, Center of Molecular Bioscience, University of the Ryukyus, Nishihara, Okinawa 903-0213, Japan 2Department of Bioscience and Biotechnology, Faculty of Agriculture, University of the Ryukyus, Nishihara, Okinawa 903-0213, Japan 3Department of Clinical Laboratory Medicine, University of the Ryukyus Hospital, School of Medicine, Uehara 207, Nishihara, Okinawa 903-0125, Japan

Received January 20, 2003; Accepted April 11, 2003
This paper describes the in vitro effect of conjugated linoleic acid (CLA) on fatty acid biosynthesis. Among the rat liver enzymes involved in fatty acid biosynthesis, fatty acid synthetase (FAS) showed the largest activity fluctuation with the types of fatty acids. Of the fatty acids, CLA was the most potent inhibitor of FAS, and the 9c, 11t-rather than the 10t, 12c-isomer showed greater inhibition. CLA also significantly lowered the incorporation of [14C]-acetate into phospholipid in breast cancer cells, supporting the view that CLA inhibits fatty acid biosynthesis through the interaction with FAS.
Key words: conjugated linoleic acid; fatty acid biosynthesis; fatty acid synthetase; inhibition; cytotoxicity

-23-
Note
Inhibitory Effects of Resveratrol Derivatives from Dipterocarpaceae Plants on Tyrosinase Activity

Kenji OHGUCHI,1, Toshiyuki TANAKA,2 Tetsuro ITO,2 Munekazu IINUMA,3 Kenji MATSUMOTO,1 Yukihiro AKAO,1 and Yoshinori NOZAWA1

1Gifu International Institute of Biotechnology, 1-1 Naka-fudogaoka, Kakamigahara, Gifu 504-0838, Japan 2Gifu Prefectural Institute of Health and Environmental Sciences, Kakamigahara, Gifu 504-0838, Japan 3Gifu Pharmaceutical University, 5-6-1 Mitahora-higashi, Gifu 502-5858, Japan

Received January 27, 2003; Accepted March 27, 2003
Stilbene derivatives, which are resveratrol (3,4′,5-trihydroxy-trans-stilbene) oligomers ranging from monomer to tetramer, isolated from Dipterocarpaceae plants were tested for their inhibitory effects against murine tyrosinase activity. The structure-activity relationships obtained in this study suggest that the double bond in the stilbene skeleton is critical for the inhibition, and also that molecular size is important for inhibitory potency.
Key words: tyrosinase; resveratrol; stilbene; Dipterocarpaceae

-24-
Note
Degradation of Car Engine Base Oil by Rhodococcus sp. NDKK48 and Gordonia sp. NDKY76A

Daisuke KOMA,1 Yuichi SAKASHITA,1 Kenzo KUBOTA,1 Yoshihide FUJII,1 Fumihiko HASUMI,2 Seon-Yong CHUNG,3 and Motoki KUBO1,†

1Department of Bioscience ＆ Technology, Faculty of Science ＆ Engineering, Ritsumeikan University, Nojihigashi 1-1-1, Kusatsu, Shiga 525-8577, Japan 2Department of Biochemistry, Numazu College of Technology, Ooka 3600, Numazu, Shizuoka 410-8501, Japan 3Department of Environmental Engineering, College of Engineering, Chonnam National University, Kwangju 500757, Korea

Received January 28, 2003; Accepted April 5, 2003
Two microorganisms (NDKK48 and NDKY76A) that degrade long-chain cyclic alkanes (c-alkanes) were isolated from soil samples. Strains NDKK48 and NDKY76A were identified as Rhodococcus sp. and Gordonia sp., respectively. Both strains used not only normal alkane (n-alkane) but also c-alkane as a sole carbon and energy source, and the strains degraded more than 27％ of car engine base oil (1％ addition).
Key words: c-alkane; car engine oil; Rhodococcus sp.; Gordonia sp.; biodegradation

-25-
Note
Isolation and Identification of Sodium 2-Propenyl Thiosulfate from Boiled Garlic (Allium sativum) That Oxidizes Canine Erythrocytes

Osamu YAMATO,1,† Yuko SUGIYAMA,1, Hideyuki MATSUURA,2 Keun-Woo LEE,3 Koichi GOTO,1, Mohammad Alamgir HOSSAIN,1 Yoshimitsu MAEDE,1 and Teruhiko YOSHIHARA2

1Laboratory of Internal Medicine, Department of Veterinary Clinical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan 2Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan 3Department of Veterinary Internal Medicine, College of Veterinary Medicine, Kyungpook National University, Taegu 702-701, Korea

Received January 31, 2003; Accepted March 26, 2003
Sodium 2-propenyl thiosulfate was identified in boiled garlic (Allium sativum). When canine erythrocytes were incubated with sodium 2-propenyl thiosulfate, the methemoglobin concentration and Heinz body percentage in erythrocytes were both increased, indicating that the compound induced oxidative damage in canine erythrocytes. It seems that this compound is one of the causative agents of garlic-induced hemolysis in dogs.
Key words: sodium 2-propenyl thiosulfate; garlic; canine erythrocyte; oxidant

-26-
Note
New Plant Growth Promoters, Repraesentins A, B and C, from Lactarius repraesentaneus

Mitsuru HIROTA,1, Yuji SHIMIZU,1 Tsunashi KAMO,1 Hidefumi MAKABE,2 and Hisao SHIBATA1

1Department of Bioscience and Biotechnology, Faculty of Agriculture, Shinshu University, 8304 Minami-minowa, Kami-ina, Nagano 399-4598, Japan 2Sciences of Functional Foods, Graduate School of Agriculture, Shinshu University, 8304 Minami-minowa, Kami-ina, Nagano 399-4598, Japan

Received February 14, 2003; Accepted March 20, 2003
Three new plant growth regulatory sesquiterpenoids were isolated from the Lactarius repraesentaneus fungus. Their structures were elucidated to be a protoilludene sesquiterpene, namely repraesentin A (1), and two related sesquiterpenes, namely repraesentins B (2) and C (3). Compounds 1--3 showed promotion activities toward the radicle elongation of lettuce seedlings by 136％, 118％ and 184％ at 67 ppm, respectively.
Key words: Lactarius repraesentaneus; 6-protoilludene; sesquiterpene; plant growth regulator

-27-
Note
Conversion of the Cleavage Specificity of Subtilisin YaB on Oxidized Insulin Chains to an Elastase-like Specificity by Replacement of Gly124 with Ala

Hui-Ching MEI,1,2 Ywan-Feng LI,1 Chi-Cheng HSU,1 Ying-Chieh TSAI,1, and Hiroshi TAKAGI3,

1Institute of Biochemistry, National Yang-Ming University, Taipei, Taiwan 11221, R.O.C. 2Department of Medical Technology, China Medical College, Taichung, Taiwan 40443, R.O.C. 3Department of Bioscience, Fukui Prefectural University, Fukui 910-1195, Japan

Received February 19, 2003; Accepted April 7, 2003
Replacement of Gly124 on the S1 pocket of subtilisin YaB with Ala changed the cleavage pattern on oxidized insulin B-chain from the subtilisin type to the elastase type. The initial cleavage site in the B-chain shifted from L15-Y16 for wild-type YaB to A14-L15 for the G124A mutant. Upon complete hydrolysis with the G124A mutant, four of the six major cleavage sites on the B-chain were identical to porcine pancreatic elastase cleavage sites.
Key words: subtilisin YaB; substrate specificity; oxidized insulin chains

-28-
Note
Transfer of Pro-R Hydrogen from NADH to Dihydroxyacetonephosphate by sn-Glycerol-1-phosphate Dehydrogenase from the Archaeon Methanothermobacter thermautotrophicus

Yosuke KOGA,1,† Nobuhito SONE,2 Shunsuke NOGUCHI,2 and Hiroyuki MORII1

1Department of Chemistry, University of Occupational and Environmental Health, Kitakyushu 807-8555, Japan 2Department of Biochemical Engineering and Science, Faculty of Computer Science and System Engineering, Kyushu Institute of Technology, Iizuka, Fukuoka 820-8502, Japan

Received February 24, 2003; Accepted April 7, 2003
sn-Glycerol-1-phosphate dehydrogenase is responsible for the formation of the sn-glycerol-1-phosphate backbone of archaeal lipids. [4-3H]NADH that had 3H at the R side was produced from [4-3H]NAD and glucose with glucose dehydrogenase (a pro-S type enzyme). The 3H of this [4-3H]NADH was transferred to dihydroxyacetonephosphate during the sn-glycerol-1-phosphate dehydrogenase reaction. On the contrary, in a similar reaction using alcohol dehydrogenase (a pro-R type enzyme), 3H was not incorporated into glycerophosphate. These results confirmed a prediction of the tertiary structure of sn-glycerol-1-phosphate dehydrogenase by homology modeling.
Key words: sn-glycerol-1-phosphate dehydrogenase; Archaea; phospholipid backbone; stereospecificity; NAD

-29-
Note
Purification and Characterization of Purple Acid Phosphatase PAP1 from Dry Powder of Sweet Potato

Tatsuya KUSUDO, Toshiyuki SAKAKI, and Kuniyo INOUYE

Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kitashirakawa, Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan

Received March 12, 2003; Accepted April 8, 2003
Purple acid phosphatase (PAP) was purified from sweet potato dry powder, which is used as a food additive. Spectrometric and enzymatic analyses, and analysis of the amino-terminal sequence indicated that the purified purple acid phosphatase was PAP1. High activity in neutral and acidic conditions, broad substrate specificity, and good thermal stability of PAP1 suggest the possibility of practical applications of PAP1.
Key words: purple acid phosphatase (PAP); sweet potato PAP1; dry powder of sweet potato; binuclear Fe--Mn center

-30-
Note
Identification and Classification of Two-component Systems That Affect rpoS Expression in Escherichia coli

Masahito SUGIURA, Hirofumi AIBA, and Takeshi MIZUNO

Laboratory of Molecular Microbiology, School of Agriculture, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan

Received March 18, 2003; Accepted April 11, 2003
The rpoS-encoded σS subunit of RNA polymerase regulates the expression of stationary phase and stress response genes in Escherichia coli. Recent study of our DNA microarray analysis suggested that the rpoS expression is affected by multiple two-component systems. In this study, we identified two-component-system mutants in which the rpoS expression increased. The regulatory manner of the systems on rpoS expression is suggested.
Key words: RpoS; Escherichia coli; two-component system; His-Asp phosphorelay; sigma factor

-31-
Communication
Identification of the Lantibiotic Nisin Q, a New Natural Nisin Variant Produced by Lactococcus lactis 61-14 Isolated from a River in Japan

Takeshi ZENDO,1 Masanori FUKAO,1 Kyoko UEDA,2 Tomoko HIGUCHI,2 Jiro NAKAYAMA,1 and Kenji SONOMOTO1,†

1Laboratory of Microbial Technology, Division of Microbial Science and Technology, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan 2Biotechnology ＆ Food Research Institute, Fukuoka Industrial Technology Center, 1465-1 Aikawacho, Kurume, Fukuoka 839-0861, Japan

Received January 17, 2003; Accepted April 25, 2003
Lactococcus lactis 61-14 isolated from river water produced a bacteriocin active against a wide range of Gram-positive bacteria. N-terminal amino acid sequencing, mass spectral analysis of the purified bacteriocin, and genetic analysis using nisin-specific primers showed that the bacteriocin was a new natural nisin variant, termed nisin Q. Nisin Q and nisin A differ in four amino acids in the mature peptide and two in the leader sequence.
Key words: nisin; bacteriocin; lantibiotic; lactic acid bacteria; Lactococcus lactis

-32-
Communication
Enterostatin (VPDPR) and Its Peptide Fragment DPR Reduce Serum Cholesterol Levels after Oral Administration in Mice

Yasuyuki TAKENAKA, Futoshi NAKAMURA, Taichi YAMAMOTO, and Masaaki YOSHIKAWA

Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan

Received March 24, 2003; Accepted May 6, 2003
We found that enterostatin (VPDPR), an anorexigenic peptide for a high-fat diet, significantly reduces serum cholesterol levels after oral administration of 100 mg/kg for 3 days in mice fed a high cholesterol-cholic acid diet. DPR, a peptide fragment of VPDPR, also had hypocholesterolemic activity at a dose of 50 mg/kg. Food intake was not suppressed under these dietary conditions. Fecal excretion of cholesterol and bile acids was increased significantly by both VPDPR and DPR. Interestingly, DPR induced hypocholesterolemic effects just two hours after a single oral administration at a dose of 100 mg/kg.
Key words: enterostatin; hypocholesterolemic effect; bile acids

-33-
Communication
Anti-angiogenic Activity of Tocotrienol

Hitoshi INOKUCHI, Hisako HIROKANE, Tsuyoshi TSUZUKI, Kiyotaka NAKAGAWA, Miki IGARASHI, and Teruo MIYAZAWA

Food ＆ Biodynamic Chemistry Laboratory, Graduate School of Life Science and Agriculture, Tohoku University, Sendai 981-8555, Japan

Received April 7, 2003; Accepted May 19, 2003
The anti-angiogenic property of vitamin E compounds, with particular emphasis on tocotrienol, has been investigated in vitro. Tocotrienol, but not tocopherol, inhibited both the proliferation and tube formation of bovine aortic endothelial cells, with δ-tocotrienol appearing the highest activity. Also, δ-tocotrienol reduced the vascular endothelial growth factor-stimulated tube formation by human umbilical vein endothelial cells. Our findings suggest that tocotrienol has potential use as a therapeutic dietary supplement for minimizing tumor angiogenesis.
Key words: angiogenesis inhibitor; cancer; tocopherol; tocotrienol; vitamin E