Contents and Abstracts of BBB

(Vol.68 No.12 2004)

Review
Suppressive Effect of a Hot Water Extract of Adzuki Beans (Vigna angularis) on Hyperglycemia after Sucrose Loading in Mice and Diabetic Rats
Tomohiro Itoh1, Nobuyuki Kita1, Yuko Kurokawa2, Misato Kobayashi3, Fumihiko Horio3, and Yukio Furuichi2 p.2421

Isolation of Lactobacillus sakei Strain KJ-2008 and Its Removal of Characteristic Malodorous Gases under Anaerobic Culture Conditions
Jeong-Dong Kim1 and Kook-Hee Kang2 p.2427

Synthesis of Chondroitin Sulfate E Hexasaccharide in the Repeating Region by an Effective Elongation Strategy toward Longer Chondroitin Oligosaccharide
Jun-ichi Tamura and Mihoko Tokuyoshi p.2436

Isolation and Structural Elucidation of 4-(²-D-Glucopyranosyldisulfanyl)butyl Glucosinolate from Leaves of Rocket Salad (Eruca sativa L.) and Its Antioxidative Activity
Sun-Ju Kim, Shigeki Jin, and Gensho Ishii p.2444

Analysis of Bacterial Glucose Dehydrogenase Homologs from Thermoacidophilic Archaeon Thermoplasma acidophilum: Finding and Characterization of Aldohexose Dehydrogenase
Yoshiaki Nishiya1, Noriko Tamura2, and Tomohiro Tamura2,3 p.2451

Genes Involved in Aniline Degradation by Delftia acidovorans Strain 7N and Its Distribution in the Natural Environment
Masaaki Urata1, Eiji Uchida1, Hideaki Nojiri1, Toshio Omori1, Rie Obo2, Noriko Miyaura2, and Naoki Ouchiyama2 p.2457

Role of the Carbohydrate Chain and Two Phosphate Moieties in the Heat-Induced Aggregation of Hen Ovalbumin
Fumito Tani1, Nobuaki Shirai2, Yukiko Nakanishi1, Kyoden Yasumoto3, and Naofumi Kitabatake1 p.2466

Phenolic Composition and Radical Scavenging Activity of Sweetpotato-Derived Shochu Distillery By-Products Treated with Koji
Makoto Yoshimoto1, Rie Kurata-azuma1, Makoto Fujii2, De-Xing Hou2, Kohji Ikeda3, Tomohisa Yoshidome3, and Miho Osako3 p.2477

Decreased mRNA Expression of the PTH/PTHrP Receptor and Type II Sodium-Dependent Phosphate Transporter in the Kidney of Rats Fed a High Phosphorus Diet Accompanied with a Decrease in Serum Calcium Concentration
Shin-ichi Katsumata, Ritsuko Masuyama, Mariko Uehara, and Kazuharu Suzuki p.2484

Recognition of Native and/or Thermally Induced Denatured Forms of the Major Food Allergen, Ovomucoid, by Human IgE and Mouse Monoclonal IgG Antibodies
Junko Hirose1, Naofumi Kitabatake1, Akihiro Kimura2, and Hiroshi Narita3 p.2490

A Nonconserved Carboxy-Terminal Segment of GroEL Contributes to Reaction Temperature
Takamichi Nakamura1, Mitsuko Tanaka1, Akihiko Maruyama2, Yowsuke Higashi2, and Yasurou Kurusu1 p.2498

Efficacy of Wogonin in the Production of Immunoglobulins and Cytokines by Mesenteric Lymph Node Lymphocytes in Mouse Colitis Induced with Dextran Sulfate Sodium
Beong Ou Lim p.2505

Binding Energy of Tea Catechin/Caffeine Complexes in Water Evaluated by Titration Experiments with 1H-NMR
Nobuyuki Hayashi, Tomomi Ujihara, and Katsunori Kohata p.2512

Involvement of Sulfide:Quinone Oxidoreductase in Sulfur Oxidation of an Acidophilic Iron-Oxidizing Bacterium, Acidithiobacillus ferrooxidans NASF-1
Satoshi Wakai1, Mei Kikumoto1, Tadayoshi Kanao2, and Kazuo Kamimura2 p.2519

Cyclic Tetrasaccharide-Synthesizing Enzymes from Arthrobacter globiformis A19
Kazuhisa Mukai, Kazuhiko Maruta, Kazuhiro Satouchi, Michio Kubota, Shigeharu Fukuda, Masashi Kurimoto, and Yoshio Tsujisaka p.2529

Efflux of Sphingoid Bases by P-Glycoprotein in Human Intestinal Caco-2 Cells
Tatsuya Sugawara1,2, Mikio Kinoshita3, Masao Ohnishi3, Tsuyoshi Tsuzuki4, Teruo Miyazawa4, Junichi Nagata1, Takashi Hirata5, and Morio Saito1 p.2541

Purification and Characterization of a Co(II)-Sensitive +--Mannosidase from Ginkgo biloba Seeds
Kwan Kit Woo1, Mitsuhiro Miyazaki2, Shintaro Hara1, Mariko Kimura3, and Yoshinobu Kimura1,2 p.2547

Roles of the HSP70-Subunit in a Eukaryotic Multi-Site-Specific Endonuclease, Endo.SceI: Autophosphorylation and Heat Stability
Katsumi Kawasaki1,2, Takehiko Shibata2, and Fumiaki Ito1 p.2557

N-Linked Glycan Structures of Human Lactoferrin Produced by Transgenic Rice
Kazuhito Fujiyama1, Yohei Sakai1, Ryo Misaki1, Itaru Yanagihara2, Takeshi Honda2, Hiroyuki Anzai3, and Tatsuji Seki1 p.2565

Biosynthesis of Abscisic Acid by the Direct Pathway via Ionylideneethane in a Fungus, Cercospora cruenta
Masahiro Inomata1, Nobuhiro Hirai2, Ryuji Yoshida3, and Hajime Ohigashi1 p.2571

Identification of Three Clones Which Commonly Interact with the Kinase Domains of Highly Homologous Two Receptor-Like Kinases, RLK902 and RKL1
Yoshiaki Tarutani1, Akiko Sasaki1, Michiko Yasuda2, Hideo Nakashita2, Shigeo Yoshida2, Isomaro Yamaguchi1, and Yoshihito Suzuki1 p.2581

A recQ Gene Homolog from the Basidiomycetous Mushroom Lentinula edodes: Sequence Analysis and Expression
Shiho Katsukawa, Takashi Yamazaki, Susumu Kajiwara, and Kazuo Shishido p.2588

Molecular Characterization of the Cytoplasmic Interacting Protein of the Receptor Kinase IRK Expressed in the Inflorescence and Root Apices of Arabidopsis
Junichiro Hattan, Hirosuke Kanamoto, Miho Takemura, Akiho Yokota, and Takayuki Kohchi p.2598

Analysis of Secreted Proteins during Conidial Germination of Aspergillus oryzae RIB40
Li Ying Zhu, Cong Ha Nguyen, Tsutomu Sato, and Michio Takeuchi p.2607

Note
Inhibition of Osteoporosis Due to Restricted Food Intake by the Fish Oils DHA and EPA and Perilla Oil in the Rat
Liman Sun1, Hajime Tamaki1, Tetsuji Ishimaru2, Tomokazu Teruya1, Yutaka Ohta3, Naofumi Katsuyama3, and Isao Chinen1 p.2613

Note
Anti-Oxidative Compounds in Barley Tea
Hideo Etoh1, Kazushi Murakami1, Tokiyasu Yogoh1, Hajime Ishikawa2, Yoshiyasu Fukuyama3, and Hitoshi Tanaka4 p.2616

Note
Presence of Higher Alcohols as Ferulates in Potato Pulp and Its Radical-Scavenging Activity
Keita Yunoki1,2, Renaguli Musa1,2, Mikio Kinoshita1, Hiroyuki Tazaki1,2, Yuji Oda3, and Masao Ohnishi1,2 p.2619

Note
Fatty Acid Compositions of Commercial Red Wines
Keita Yunoki1,2, Mikio Tanji1, Yukie Murakami1, Yoshihiro Yasui3, Shuji Hirose3, and Masao Ohnishi1,2 p.2623

Note
Whole-Mount Silver Staining of Arabidopsis and Maize: A New Method for Staining Secondary Wall Thickenings in Tracheary Elements
Sakihito Kitajima, Yoko Hasegawa, and Yukio Sugimura p.2627

Note
Mutational Analysis of the Length of the J3/4 Domain of Escherichia coli Ribonuclease P Ribozyme
Shinnosuke Haga, Terumichi Tanaka, and Yo Kikuchi p.2630

Note
Isolation and Structures of Acremolactones B and C, Novel Plant-Growth Inhibitory ³-Lactones from Acremonium roseum I4267
Takeshi Sassa, Takahiro Ooi, and Hiroshi Kinoshita p.2633

Note
Convenient and Sensitive Evaluation of a Superoxide Anion-Generating Reagent Methyl Viologen by Escherichia coli Harboring a soxS::gfp Reporter Plasmid
Akihito Shibuya, Norihiko Tsukagoshi, Iwao Ohtsu, Noriyuki Dokyu, and Rikizo Aono p.2637

Note
Anti-Fungal Sesquiterpenoid from the Root Exudate of Solanum abutiloides
Toshiyuki Yokose, Kozue Katamoto, Sun Park, Hideyuki Matsuura, and Teruhiko Yoshihara p.2640

Note
The 9cis,11trans,13cis Isomer of Conjugated Linolenic Acid Reduces Apolipoprotein B100 Secretion and Triacylglycerol Synthesis in HepG2 Cells
Keisuke Arao1, Hiroaki Yotsumoto2, Seo-Young Han1, Koji Nagao1, and Teruyoshi Yanagita1 p.2643

Note
Production of Dihydroxy C50-Carotenoid by Aureobacterium sp. FERM P-18698
Satoshi Fukuoka1, Yusuke Ajiki2, Takahiko Ohga3, Yasuhiro Kawanami3, and Ken Izumori3 p.2646

Note
Inhibitory Effects of Alcohols on the Activity of Human Matrix Metalloproteinase 7 (Matrilysin)
Yuko Muta, Hiroshi Oneda, and Kuniyo Inouye p.2649

Communication
Neurite Outgrowth-Stimulating Peptide Derived from Bovine ⁰-Casein
Minoru Sakaguchi, Chieko Ishikawa, Tomoaki Nishimura, Emi Sugihara, and Eiko Matsumura p.2653

Communication
Formation Mechanism of 2,6-Dimethyl-2,6-Octadienes from Thermal Decomposition of Linalyl ²-D-Glucopyranoside
Shoji Hattori1, Chiami Kawaharada2, Hiroyuki Tazaki2, Takane Fujimori1, Koji Kimura2, Masao Ohnishi2, and Kensuke Nabeta2 p.2656

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Review
Suppressive Effect of a Hot Water Extract of Adzuki Beans (Vigna angularis) on Hyperglycemia after Sucrose Loading in Mice and Diabetic Rats

Tomohiro Itoh1, Nobuyuki Kita1, Yuko Kurokawa2, Misato Kobayashi3, Fumihiko Horio3, and Yukio Furuichi2

1Imuraya Confectionary Co., Ltd., Takachaya 7-1-1, Tsu, Mie 514-8530, Japan
2Faculty of Bioresources, Mie University, Kamihama 1515, Tsu, Mie 514-8507, Japan
3Nagoya University, Graduate School of Bioagricultural Sciences, Chikusa, Nagoya 464-8601, Japan

A hot water extract obtained by boiling adzuki beans (Vigna angularis) to produce bean paste for Japanese cake showed inhibitory activity against alpha-glucosidase, alpha-amylase, maltase, sucrase, and isomaltase after HP-20 column chromatography. The IC50 values for each hydrolylase were 0.78 mg/ml (+--amylase), 2.45 mg/ml (maltase), 5.37 mg/ml (sucrase), and 1.75 mg/ml (isomaltase). The active fraction showed potential hypoglycemic activity in both normal mice and streptozotocin (STZ)-induced diabetic rats after an oral administration of sucrose, but did not show any effect on the blood glucose concentration after glucose administration, suggesting that the active fraction suppressed the postprandial blood glucose level by inhibiting +--glucosidase and +--amylase, irrespective of the endogenous blood insulin level.

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Isolation of Lactobacillus sakei Strain KJ-2008 and Its Removal of Characteristic Malodorous Gases under Anaerobic Culture Conditions

Jeong-Dong Kim1 and Kook-Hee Kang2

1Department of Life Science, Hanyang University, Seoul 133-791, Korea
2Department of Food and Biotechnology, Sungkyunkwan University, Suwon 440-746, Korea

A number of different sources, such as composts, leachates, and pig feces samples were collected from different pig farms in Korea. Several microorganisms were screened for their ability to deodorize the malodorous gases. As a result, a novel malodorous gas-deodorizing bacterial strain KJ-2008 was isolated due to the most abundant of nitrate-supplemented minimal media under anaerobic conditions. Crimp-sealed serum bottles containing nitrate-supplemented minimal medium (MM-NO3-) in airtight conditions were inoculated with KJ-2008. Nitrate concentration decreased rapidly after 20 h incubation and nitrite production reached almost zero during the time the experimental was carried out. Taxonomic identification including 16S rDNA base sequencing and phylogenetic analysis indicated that the isolate KJ-2008 had a 99.8% homology in its 16S rDNA base sequence with Lactobacillus sakei. Among the volatile fatty acids, acetic acid contained in large amounts in fresh piggery slurry decreased about 40% after 50 h incubation of the strain KJ-2008. n-Butyric acid, n-valeric acid, and iso-valeric acid gradually decreased, and iso-butyric acid and capronic acid dramatically eliminated at initial time with the treatment. Moreover, NH3 removal efficiency reached a maximum of 98.5% after 50 h of incubation. The concentration of H2S did not change.

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Synthesis of Chondroitin Sulfate E Hexasaccharide in the Repeating Region by an Effective Elongation Strategy toward Longer Chondroitin Oligosaccharide

Jun-ichi Tamura and Mihoko Tokuyoshi

Department of Regional Environment, Faculty of Regional Sciences, Tottori University, Koyamacho-minami 4-101, Tottori 680-8551, Japan

We synthesized chondroitin and its sulfate E hexasaccharides (1 and 2) composed of the trimer of the repeating disaccharide, ²-D-GalNAc(+-4,6-di-O-SO3Na)-(1'4)-²-D-GlcA, by employing an efficient synthetic strategy for longer chondroitin oligosaccharide. Successful elongation with the ²-D-GalNAc-(1'4)-²-D-GlcA unit instead of the corresponding disaccharide possessing an azide group avoided problematic reduction of the multiple azide groups on the hexasaccharide.

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Isolation and Structural Elucidation of 4-(²-D-Glucopyranosyldisulfanyl)butyl Glucosinolate from Leaves of Rocket Salad (Eruca sativa L.) and Its Antioxidative Activity

Sun-Ju Kim, Shigeki Jin, and Gensho Ishii

National Agricultural Research Center for Hokkaido Region, Toyohira-ku, Sapporo 062-8555, Japan

A structurally unique glucosinolate (GSL) was identified to be 4-(²-D-glucopyranosyldisulfanyl)butyl GSL in rocket leaves. The positive-ion electrospray ionization mass spectrometry (ESI-MS) data indicated that the new GSL had a molecular weight of 521 (m/z 522, [M+H]+, as desulfo-GSL). The molecular formula of the substance was determined to be C17H32O11NS3 (m/z 522.1143, [M+H]+) based on its positive-ion high-resolution fast atom bombardment mass spectrometry (HR-FAB-MS) data. For the further confirmation, desulfated GSL of 4-(²-D-glucopyranosyldisulfanyl)butyl GSL was prepared by commercial 1-thio-²-D-glucose and dimeric 4-mercaptobutyl desulfo-GSL, which was also isolated from rocket leaves, and its chemical structure was then confirmed by MS data and nuclear magnetic resonance (NMR) spectroscopy. In addition, the antioxidative activity of 4-(²-D-glucopyranosyldisulfanyl)butyl desulfo-GSL was measured by means of chemiluminescence (CL) for evaluating the functional properties. The antioxidative activity (2.089 unit/g) was relatively higher than that of dimeric 4-mercaptobutyl desulfo-GSL (1.227).

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Analysis of Bacterial Glucose Dehydrogenase Homologs from Thermoacidophilic Archaeon Thermoplasma acidophilum: Finding and Characterization of Aldohexose Dehydrogenase

Yoshiaki Nishiya1, Noriko Tamura2, and Tomohiro Tamura2,3

1Department of Biochemistry, Toyobo Co., Ltd., 17-9 Koami-cho, Nihonbashi, Chuo-ku, Tokyo 103-8530, Japan
2Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology, 2-17-2-1 Tsukisamu-higashi, Toyohira-ku, Sapporo 062-8517, Japan
3Laboratory of Molecular Environmental Microbiology, Graduate School of Agriculture, Hokkaido University, Kita-9, Nishi-9, Kita-ku, Sapporo 060-8589, Japan

The NADP+-preferring glucose dehydrogenase from thermoacidophilic archaeon Thermoplasma acidophilum has been characterized, and its crystal structure has been determined (Structure, 2:385-393, 1994). Its sequence and structure are not homologous to bacterial NAD(P)+-dependent glucose dehydrogenases, and its molecular weight is also quite defferent. On the other hand, three functionally unknown genes with homologies to bacterial NAD(P)+-dependent glucose dehydrogenases have been sequenced as part of the T. acidophilum genome project (gene names: Ta0191, Ta0747, and Ta0754 respectively). We expressed two genes of three, Ta0191 and Ta0754, in Escherichia coli, and purified the gene products to homogeneity. Dehydrogenase activities were thereby detected from the purified proteins. The Ta0754 gene product exhibited aldohexose dehydrogenase activity, and the Ta0191 gene product exhibited weak 2-deoxyglucose dehydrogenase activity. No aldohexose dehydrogenase gene has been isolated, while the enzyme was reported in 1968. This is the first report of the gene and primary structure. The purified Ta0754 gene product, designated AldT, was characterized. The enzyme AldT effectively catalyzed the oxidation of various aldohexoses, especially D-mannose. Lower activities on D-2-deoxyglucose, D-xylose, D-glucose, and D-fucose were detected although no activities were shown on other aldohexoses or additional sugars. As a cofactor, NAD+ was much more suitable for the activity than NADP+. The NAD+-preferring dehydrogenase most effectively reacting to D-mannose is for the first time. AldT was most active at pH 10 and above 70 ℃, and completely stable up to 60℃ after incubation for 15 min. Other enzymatic properties were also investigated.

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Genes Involved in Aniline Degradation by Delftia acidovorans Strain 7N and Its Distribution in the Natural Environment

Masaaki Urata1, Eiji Uchida1, Hideaki Nojiri1, Toshio Omori1, Rie Obo2, Noriko Miyaura2, and Naoki Ouchiyama2

1Biotechnology Research Center, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
2Kurume Laboratory, Chemicals Evaluation and Research Institute (CERI), 19-14 Chuo-machi, Kurume-shi, Fukuoka 830-0023, Japan

Aniline-degraders were isolated from activated sludge and environmental samples and classified into eight phylogenetic groups. Seven groups were classified into Gram-negative bacteria, such as Acidovorax sp., Acinetobacter sp., Delftia sp., Comamonas sp., and Pseudomonas sp., suggesting the possible dominance of Gram-negative aniline-degraders in the environment. Aniline degradative genes were cloned from D. acidovorans strain 7N, and the nucleotide sequence of the 8,039-bp fragment containing eight open reading frames was determined. Their deduced amino acid sequences showed homologies to glutamine synthetase (GS)-like protein, glutamine amidotransferase (GA)-like protein, large and small subunits of aniline dioxygenase, reductase, LysR-type regulator, small ferredoxin-like protein, and catechol 2,3-dioxygenase, suggesting a high similarity of this gene cluster to those in P. putida strain UCC22 and Acinetobacter sp. strain YAA. Polymerase chain reaction (PCR) and sequencing analyses of GS-like protein gene segments of other Gram-negative bacteria suggested that Gram-negative bacteria have aniline degradative gene that can be divided into two distinctive groups.

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Role of the Carbohydrate Chain and Two Phosphate Moieties in the Heat-Induced Aggregation of Hen Ovalbumin

Fumito Tani1, Nobuaki Shirai2, Yukiko Nakanishi1, Kyoden Yasumoto3, and Naofumi Kitabatake1

1Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Goka-sho, Uji, Kyoto 611-0011, Japan
2Industrial Research Center of Shiga Prefecture, 232 Kamitoyama, Ritto, Shiga 520-3004, Japan
3Department of Food and Nutrition, Sugiyama Jogakuen University, 17-3 Hoshigaoka-motomachi, Chikusa-ku, Nagoya, Aichi 464-8662, Japan

We investigated the effect of the carbohydrate chain and two phosphate moieties on heat-induced aggregation of hen ovalbumin. The dephosphorylated form of ovalbumin was obtained by treating the original protein with acid phosphatase. The single carbohydrate chain was removed by digestion of heat-denatured ovalbumin with glycopeptidase F, and the resulting polypeptide without this carbohydrate chain was correctly refolded to acquire protease-resistance. Thermal unfolding can be approximated by a mechanism involving a two-state transition between the folded and unfolded states with a midpoint temperature of 76 ℃ for the original form, of 74 ℃ for the dephosphorylated form, and of 71 ℃ for the carbohydrate-free form. The conformational stability of the original form was higher than that of the carbohydrate-free form. When the three forms of ovalbumin were heated to 80 ℃ and then cooled rapidly in an ice bath, the polypeptide chains were compactly collapsed to metastable intermediates with secondary structures whose properties were indistinguishable. Upon incubation at 60℃, renaturation was possible for a large portion of the intermediates of the original form, but for only a small portion of those of the carbohydrate-free form. Light scattering experiments showed that in the presence of sulfate anions, the intermediates of the carbohydrate-free form aggregated to a greater extent than did those of the original form. The intermediates of the carbohydrate-free form bound to the chaperonin GroEL with about 10-fold higher affinity than those of the original form. It follows that the carbohydrate chain and the two phosphate moieties do not affect hydrophobic collapse in the kinetic refolding of hen ovalbumin but play an important role in the slow rearrangement. They block the off-pathway reaction that competes with correct refolding by effectively decreasing surface hydrophobicity.

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Phenolic Composition and Radical Scavenging Activity of Sweetpotato-Derived Shochu Distillery By-Products Treated with Koji

Makoto Yoshimoto1, Rie Kurata-azuma1, Makoto Fujii2, De-Xing Hou2, Kohji Ikeda3, Tomohisa Yoshidome3, and Miho Osako3

1Department of Upland Farming Research, National Agricultural Research Center for Kyushu Okinawa Region, Yokoichi 6651-2, Miyakonojo, Miyazaki 885-0091, Japan
2Department of Biological Science and Technology, Faculty of Agriculture, Kagoshima University, Korimoto 1-21-24, Kagoshima 890-0065, Japan
3Den-en Shuzo Co., Ltd., Hiwaki Tounohara 11356-1, Satsumasendai, Kagoshima 895-1295, Japan

Phenolic composition and radical scavenging activity in the shochu distillery by-products of sweetpotato (Ipomoea batatas L.) treated with koji (Aspergillus awamori mut.) and cellulase (Cellulosin T2) were investigated to develop new uses. Koji and Cellulosin T2 treatment of shochu distillery by-products from sweetpotatoes, rice, and barley increased phenolic content. Caffeic acid was identified as a dominant phenolic component in the shochu distillery by-products of the sweetpotato. Adding koji and/or Cellulosin T2 to the shochu distillery by-product indicated that koji was involved in caffeic acid production. Caffeic acid was not detected in raw or steamed roots of Koganesengan, the material of sweetpotato for shochu production, suggesting that it is produced during shochu fermentation. The phenolic content and radical scavenging activity the shochu distillery by-product treated with koji and Cellulosin T2 were superior to those of commercial vinegar. These results suggest that koji treatment of sweetpotato-derived shochu distillery by-products has potential for food materials with physiological functions. Further koji treatment of sweetpotato shochu-distillery by-products may be applicable to mass production of caffeic acid.

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Decreased mRNA Expression of the PTH/PTHrP Receptor and Type II Sodium-Dependent Phosphate Transporter in the Kidney of Rats Fed a High Phosphorus Diet Accompanied with a Decrease in Serum Calcium Concentration

Shin-ichi Katsumata, Ritsuko Masuyama, Mariko Uehara, and Kazuharu Suzuki

Department of Nutritional Science, Faculty of Applied Bioscience, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan

This study investigates the phosphorus (P) homeostasis in the process of an altered parathyroid hormone (PTH) action in the kidney of rats fed a high P diet. Four-week-old male Wistar strain rats were fed diets containing five different P levels (0.3, 0.6, 0.9, 1.2 and 1.5%) for 21 days. The serum PTH concentration and urinary excretion of P were elevated with increasing dietary P level. Compared to rats fed the 0.3% P diet, the serum calcium (Ca) concentration remained unchanged, while the serum 1,25(OH)2D3 concentration and urinary excretion of cAMP were elevated with increasing dietary P level in rats fed the high P diets containing 0.6-0.9% P. On the other hand, a lower serum Ca concentration was observed in rats fed the high P diets containing 1.2% or greater P. The serum 1,25(OH)2D3 concentration remained unchanged in rats fed the high P diets containing 1.2% or greater P, comparison with rats fed the 0.3% P diet. The urinary excretion of cAMP and PTH/PTH-related peptide (PTHrP) receptor and type II sodium-dependent phosphate transporter (NaPi-2) mRNA in the kidney were both decreased in rats fed the high P diets containing 1.2% or greater P. In conclusion, a high P diet with subsequent decrease in serum Ca concentration suppressed the PTH action in the kidney due to PTH/PTHrP receptor mRNA down-regulation. Furthermore, an increase in the urinary excretion of P might have been caused by decreased NaPi-2 mRNA expression without the effects of PTH and 1,25(OH)2D3.

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Recognition of Native and/or Thermally Induced Denatured Forms of the Major Food Allergen, Ovomucoid, by Human IgE and Mouse Monoclonal IgG Antibodies

Junko Hirose1, Naofumi Kitabatake1, Akihiro Kimura2, and Hiroshi Narita3

1Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji 611-0011, Japan
2Itayado Clinic, Shoyama 1-9-12, Nagata, Kobe, Hyogo 653-0853, Japan
3Department of Food and Nutrition, Kyoto Women's University, Kyoto 605-8501, Japan

Human sera obtained from children with egg allergy reacted well with both native and heated ovomucoid (OM). Ovalbumin is present in egg white in a 5 times greater quantity than OM; however, it easily aggregates and becomes difficult to extract by heating. For accurate food allergen labeling of processed food, therefore, OM should be evaluated with the determination of egg white protein in consideration of heat denaturation. Three kinds of monoclonal antibodies and sandwich ELISA tests were established which are able to recognize the native and/or heat-denatured forms of OM. The usefulness of these characteristic mAbs and ELISA tests are discussed in relation to allergen labeling, monitoring food processing, and movement or change of dietary protein in vivo.

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A Nonconserved Carboxy-Terminal Segment of GroEL Contributes to Reaction Temperature

Takamichi Nakamura1, Mitsuko Tanaka1, Akihiko Maruyama2, Yowsuke Higashi2, and Yasurou Kurusu1

1Laboratory of Molecular Microbiology, College of Agriculture, Ibaraki University, Ami 3-21-1, Inashiki, Ibaraki 300-0393, Japan
2National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566, Japan

The role of the C-terminal segment of the GroEL equatorial domain was analyzed. To understand the molecular basis for the different active temperatures of GroEL from three bacteria, we constructed a series of chimeric GroELs combining the C-terminal segment of the equatorial domain from one species with the remainder of GroEL from another. In each case, the foreign C-terminal segment substantially altered the active temperature range of the chimera. Substitution of L524 of Escherichia coli GroEL with the corresponding residue (isoleucine) from psychrophilic GroEL resulted in a GroE with approximately wild-type activity at 25 ℃, but also at 10 ℃, a temperature at which wild-type E. coli GroE is inactive. In a detailed look at the temperature dependence of the GroELs, normal E. coli GroEL and the L524I mutant became highly active above 14 ℃ and 12 ℃ respectively. Similar temperature dependences were observed in a surface plasmon resonance assay of GroES binding. These results suggested that the C-terminal segment of the GroEL equatorial domain has an important role in the temperature dependence of GroEL. Moreover, E. coli acquired the ability to grow at low temperature through the introduction of cold-adapted chimeric or L524I mutant groEL genes.

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Efficacy of Wogonin in the Production of Immunoglobulins and Cytokines by Mesenteric Lymph Node Lymphocytes in Mouse Colitis Induced with Dextran Sulfate Sodium

Beong Ou Lim

Department of Applied Biochemistry, Konkuk University, Chungju, Chungbuk 380-701, Korea

We previously examined wogonin, isolated from Scutellaria baicalensis, chemical mediators, and IgE by mesenteric lymph node (MLN) lymphocytes in rats. The present study explores the effect of wogonin on the MLN lymphocyte function of mice given orally at 20 mg/kg for 2 weeks with dextran sulfate sodium (DS)-induced colitis. The results indicate that IgA levels in MLN lymphocytes were high, while IgE was low, in mice given wogonin compared to those given water. Also, fecal IgA concentration of DS in the wogonin group mice was significantly higher than in the DS group. Concentrations of interferon-³ and interleukin (IL)-2 of T cells by concanavalin A treatment was significantly higher in the wogonin fed group than in the normal group. Activation-induced IL-4, IL-5 and IL-10 secretion was lower in wogonin fed mice compared control mice after DS-induced colitis. For these reasons, we conclude that wogonin can alleviate the inflammation in DS-induced colitis brought about by an abnormal Th2 response.

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Binding Energy of Tea Catechin/Caffeine Complexes in Water Evaluated by Titration Experiments with 1H-NMR

Nobuyuki Hayashi, Tomomi Ujihara, and Katsunori Kohata

National Institute of Vegetable and Tea Science, 2769 Kanaya, Shizuoka 428-8501, Japan

Evaluating the binding energy of a catechin/caffeine complex in water is important in order to elucidate the ability for molecular recognition of tea catechins. The results of this study revealed that the stoichiometric ratio of the complexation between tea chatechins (EGCg, ECg, EGC, and EC) and caffeine was 1:1 at least up to a concentration of 5.0 mM. The free energy (-"G) values for binding in water at 301 K were evaluated to be 2.7, 2.6, 2.2, and 2.0 kcal/mol for EGCg, ECg, EGC, and EC, respectively, by the titration method with 1H-NMR. An investigation of the 1H-NMR chemical shift change and NOESY spectra in the catechin/caffeine solutions showed the participation of the A-rings of the catechins in complexation, as well as that of the galloyl groups or B-rings.

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Involvement of Sulfide:Quinone Oxidoreductase in Sulfur Oxidation of an Acidophilic Iron-Oxidizing Bacterium, Acidithiobacillus ferrooxidans NASF-1

Satoshi Wakai1, Mei Kikumoto1, Tadayoshi Kanao2, and Kazuo Kamimura2

1Division of Science and Technology for Energy Conversion, Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima-Naka, Okayama 700-8530, Japan
2Faculty of Agriculture, Okayama University, 1-1-1 Tsushima-Naka, Okayama 700-8530, Japan

The effects of cyanide, azide, and 2-n-Heptyl-4-hydroxy-quinoline-N-oxide (HQNO) on the oxidation of ferrous ion or elemental sulfur with Acidithiobacillus ferrooxidans NASF-1 cells grown in iron- or sulfur-medium were examined. The iron oxidation of both iron- and sulfur-grown cells was strongly inhibited by cyanide and azide, but not by HQNO. Sulfur oxidation was relatively resistant to cyanide and azide, and inhibited by HQNO. Higher sulfide oxidation, ubiquinol dehydrogenase activity, and sulfide:quinone oxidoreductase (SQR) activity were observed in sulfur-grown cells more than in iron-grown cells. Sulfide oxidation in the presence of ubiquinone with the membrane fraction was inhibited by HQNO, but not by cyanide, azide, antimycin A, and myxothiazol. The transcription of three genes, encoding an aa3-type cytochrome c oxidase (coxB), a bd-type ubiquinol oxidase (cydA), and an sqr, were measured by real-time reverse transcription polymerase chain reaction. The transcriptional levels of coxB and cydA genes were similar in sulfur- and iron-grown cells, but that of sqr was 3-fold higher in sulfur-grown cells than in iron-grown cells. A model is proposed for the oxidation of reduced inorganic sulfur compounds in A. ferrooxidans NASF-1 cells.

-15-
Cyclic Tetrasaccharide-Synthesizing Enzymes from Arthrobacter globiformis A19

Kazuhisa Mukai, Kazuhiko Maruta, Kazuhiro Satouchi, Michio Kubota, Shigeharu Fukuda, Masashi Kurimoto, and Yoshio Tsujisaka

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

A bacterial strain Arthrobacter globiformis A19 producing cyclic tetrasaccharide (CTS) was isolated from soil. The enzymes, 6-+--glucosyltransferase (6GT) and 3-+--isomaltosyltransferase (IMT), involved in the synthesis of CTS were purified to homogeneity. The molecular and enzymatic properties of IMT from A. globiformis were similar to those of enzymes from Bacillus globisporus C11 and N75. Arthrobacter 6GT had a smaller molecular mass of 108 kDa and a higher optimum pH of 8.4 than the enzymes from strains of B. globisporus. The genes for IMT (ctsY) and 6GT (ctsZ) were cloned from the genome of A. globiformis A19. The two genes linked together in tandem and formed a gene cluster, ctsYZ. Both of the gene products showed similarities to +--glucosidases belonging to glycoside hydrolase family 31, and conserved two aspartic acids corresponding to the putative catalytic residues of the family enzymes. The enzymatic system for the production of CTS consisting of 6GT and IMT might be widespread among bacteria.

-16-
Efflux of Sphingoid Bases by P-Glycoprotein in Human Intestinal Caco-2 Cells

Tatsuya Sugawara1,2, Mikio Kinoshita3, Masao Ohnishi3, Tsuyoshi Tsuzuki4, Teruo Miyazawa4, Junichi Nagata1, Takashi Hirata5, and Morio Saito1

1Division of Food Science, Incorporated Administrative Agency, National Institute of Health and Nutrition, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8636, Japan
2Japan Society for the Promotion of Science, Tokyo 102-8471, Japan
3Department of Agricultural and Life Science, Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080-8555, Japan
4Food and Biodynamic Chemistry laboratory, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan
5Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan

The aim of this study was to determine whether sphingoid bases that originated from various dietary sources, such as mammals, plants, and fungi, are substrates for P-glycoprotein in differentiated Caco-2 cells, which are used as a model of intestinal epithelial cells. In Caco-2 cells, the uptake of sphingosine, the most common sphingoid base found in mammals, was significantly higher at physiological temperatures than those of cis/trans-8-sphingenine, trans-4, cis/trans-8-sphingadienine, 9-methyl-trans-4, trans-8-sphingadienine, or sphinganine. Verapamil, a potent P-glycoprotein inhibitor, increased the cellular accumulation of sphingoid bases, except for sphingosine, in a dose-dependent manner. Incubation with 1 1/4M digoxin for 48 h caused up-regulation of murtidrug-resistance (MDR)1 mRNA and decreased the accumulation of sphingoid bases in Caco-2 cells, except for sphingosine. Thus P-glycoprotein probably contributes to the selective absorption of sphingosine from dietary sphingolipids in the digestive tract.

-17-
Purification and Characterization of a Co(II)-Sensitive +--Mannosidase from Ginkgo biloba Seeds

Kwan Kit Woo1, Mitsuhiro Miyazaki2, Shintaro Hara1, Mariko Kimura3, and Yoshinobu Kimura1,2

1Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan
2Department of Bioresources Chemistry, Faculty of Agriculture, Okayama University, Okayama 700-8530, Japan
3Faculty of Food Culture, Department of Food Systems, Kurashiki Sakuyo University, Nagao-Tamashima, Kurashiki 710-0292, Japan

An +--mannosidase was purified from developing Ginkgo biloba seeds to apparently homogeneity. The molecular weight of the purified +--mannosidase was estimated to be 120 kDa by SDS-PAGE in the presence of 2-mercaptoethanol, and 340 kDa by gel filtration, indicating that Ginkgo +--mannosidase may function in oligomeric structures in the plant cell. The N-terminal amino acid sequence of the purified enzyme was Ala-Phe-Met-Lys-Tyr-X-Thr-Thr-Gly-Gly-Pro-Val-Ala-Gly-Lys-Ile-Asn-Val-His-Leu-. The +--mannosidase activity for Man5GlcNAc1 was enhanced by the addition of Co2+, but the addition of Zn2+, Ca2+, or EDTA did not show any significant effect. In the presence of cobalt ions, the hydrolysis rate for pyridylaminated Man6GlcNAc1 was significantly faster than that for pyridylaminated Man6GlcNAc2, suggesting the possibility that this enzyme is involved in the degradation of free N-glycans occurring in developing plant cells (Kimura, Y., and Matsuo, S., J. Biochem., 127, 1013-1019 (2000)). To our knowledge, this is the first report showing that plant cells contain an +--mannosidase, which is activated by Co2+ and prefers the oligomannose type free N-glycans bearing only one GlcNAc residue as substrate.

-18-
Roles of the HSP70-Subunit in a Eukaryotic Multi-Site-Specific Endonuclease, Endo.SceI: Autophosphorylation and Heat Stability

Katsumi Kawasaki1,2, Takehiko Shibata2, and Fumiaki Ito1

1Department of Biochemistry, Faculty of Pharmaceutical Sciences, Setsunan University, 45-1 Nagaotoge-cho, Hirakata, Osaka 573-0101, Japan
2Laboratory of Cellular and Molecular Biology, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan

The 70 kDa heat shock proteins (HSP70) are a family of molecular chaperones that bind transiently to unfolded proteins in an ATP/ADP dependent manner. Endo.SceI comprises a unique example for mitochondrial HSP70, which exists in a stable complex with a nucleolytic subunit as a multi-site specific DNase. The HSP70-subunit in Endo.SceI was autophosphorylated by ATP in vitro. The autophosphorylation was higher in the Endo.SceI complex form than in the free form. Although the autophosphorylation had no significant effect on the endonucleolytic activity of Endo.SceI, the factors favoring autophosphorylation protected the endonucleolytic activity of Endo.SceI against heat inactivation. ATP, adenosine 5-O-(3-thiotriphosphate) (ATP-³-S), and ADP not only protected the endonucleolytic activity against heat inactivation in the presence of Ca2+ ions, but also reduced the labeling of the HSP70-subunit by [³-32P]ATP in Endo.SceI. These findings suggest that the HSP70-subunit shields Endo.SceI from heat inactivation through ATP/ADP binding.

-19-
N-Linked Glycan Structures of Human Lactoferrin Produced by Transgenic Rice

Kazuhito Fujiyama1, Yohei Sakai1, Ryo Misaki1, Itaru Yanagihara2, Takeshi Honda2, Hiroyuki Anzai3, and Tatsuji Seki1

1International Center for Biotechnology, Osaka University, 2-1 Yamada-oka, Suita, Osaka 565-0871, Japan
2Department of Bacterial Infections, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan
3Gene Research Center, Ibaraki University, Ami, Ibaraki 300-0393, Japan

Human lactoferrin was produced in genetically engineered rice. N-linked glycan structures of recombinant human lactoferrin were determined. The oligosaccharides liberated by hydrazinolysis were labeled with 2-aminopyridine (PA). The PA-labeled glycans were purified by reverse-phase and size-fractionation HPLCs. The structures of these glycans were identified by HPLC, exoglycosidase digestion, and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The glycan structures determined were ManFucXylGlcNAc2 (3.4%), Man2FucGlcNAc2 (2.1%), Man3FucGlcNAc2 (2.5%), Man3FucXylGlcNAc2 (42.5%), two isomers of Man2FucXylGlcNAc2 (39.1%), Man3XylGlcNAc2 (6.5%), and Man2XylGlcNAc2 (3.9%).

-20-
Biosynthesis of Abscisic Acid by the Direct Pathway via Ionylideneethane in a Fungus, Cercospora cruenta

Masahiro Inomata1, Nobuhiro Hirai2, Ryuji Yoshida3, and Hajime Ohigashi1

1Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
2International Innovation Center, Kyoto University, Kyoto 606-8501, Japan
3Department of Agriculture Technology, Toyama Prefectural University, Toyama 939-0311, Japan

We examined the biosynthetic pathway of abscisic acid (ABA) after isopentenyl diphosphate in a fungus, Cercospora cruenta. All oxygen atoms at C-1, -1, -1, and -4 of ABA produced by this fungus were labeled with 18O from 18O2. The fungus did not produce the 9Z-carotenoid possessing ³-ring that is likely a precursor for the carotenoid pathway, but produced new sesquiterpenoids, 2E,4E-³-ionylideneethane and 2Z,4E-³-ionylideneethane, along with 2E,4E,6E-allofarnesene. The fungus converted these sesquiterpenoids labeled with 13C to ABA, and the incorporation ratio of 2Z,4E-³-ionylideneethane was higher than that of 2E,4E-³-ionylideneethane. From these results, we concluded that C. cruenta biosynthesized ABA by the direct pathway via oxidation of ionylideneethane with molecular oxygen following cyclization of allofarnesene. This direct pathway via ionylideneethane in the fungus is consistent with that in Botrytis cinerea, except for the positions of double bonds in the rings of biosynthetic intermediates, suggesting that the pathway is common among ABA-producing fungi.

-21-
Identification of Three Clones Which Commonly Interact with the Kinase Domains of Highly Homologous Two Receptor-Like Kinases, RLK902 and RKL1

Yoshiaki Tarutani1, Akiko Sasaki1, Michiko Yasuda2, Hideo Nakashita2, Shigeo Yoshida2, Isomaro Yamaguchi1, and Yoshihito Suzuki1

1Department of Applied Biological Chemistry, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
2Plant Functions Laboratory, RIKEN Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan

We have previously reported the characterization of highly homologous two leucine-rich repeat (LRR)-receptor-like kinase (RLK) genes, RLK902 and RKL1, which showed 75% identity at the amino acid sequence level. To investigate the RLK902 and RKL1 mediated signal transduction pathways, we performed yeast two-hybrid screening using the kinase domains of RLK902 and RKL1 as baits. Three clones, Y-1, 2 and 3, were found to interact commonly with the kinase domain of RLK902 and RKL1 and not to interact with the kinase domain of BRI1, a member of LRR-RLKs. This result suggests that RLK902 and RKL1 may have common biochemical functions, especially in their downstream signal transduction. Furthermore, the detail analysis of their responsiveness to various conditions suggests their involvement in such stress conditions as mechanical wounding, treatment with salicylic acid, and pathogen infection.

-22-
A recQ Gene Homolog from the Basidiomycetous Mushroom Lentinula edodes: Sequence Analysis and Expression

Shiho Katsukawa, Takashi Yamazaki, Susumu Kajiwara, and Kazuo Shishido

Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama 226-8501, Japan

We cloned and sequenced a recQ gene homolog from the basidiomycetous mushroom Lentinula edodes (Le.). This gene, named Le.recQ, was found to have a coding capacity of 945 amino acids (aa) and be interrupted by 11 small introns. The deduced Le.RECQ protein was clearly smaller than other fungal RecQ proteins such as Neurospora crassa QDE3 (1955 aa), Schizosaccharomyces pombe Rqh1 (1328 aa), and Saccharomyces cerevisiae SGS1 (1447 aa). It exhibited the highest homology to the Arabidopsis thaliana RecQl4A protein (1182 aa) in its size and aa sequence. Northern-blot analysis showed that the Le.recQ gene is transcribed at similar levels during mycelial development in L. edodes fruiting-body formation on a sawdust-corn bran medium. The L. edodes dikaryotic mycelial cells, which had been vegetatively grown in SMY liquid medium, were found to contain a clearly larger amount of Le.recQ transcript than the L. edodes two compatible monokaryotic mycelial cells. Expression of Le.recQ cDNA in S. cerevisiae might partially complement defects associated with the loss of its homolog S. cerevisiae SGS1 gene.

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Molecular Characterization of the Cytoplasmic Interacting Protein of the Receptor Kinase IRK Expressed in the Inflorescence and Root Apices of Arabidopsis

Junichiro Hattan, Hirosuke Kanamoto, Miho Takemura, Akiho Yokota, and Takayuki Kohchi

Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan

Meristem maintenance and differentiation is regulated by intercellular communication through receptor-like kinases (RLKs) in plants, but the underlying molecular mechanisms of RLK signaling remain largely unknown. A cytoplasmic interactor for inflorescence and root apices receptor-like kinase (IRK), which is a typical meristematic RLK with leucine-rich repeats in Arabidopsis, was identified using a yeast two-hybrid assay and named IRK-interacting protein (IRKI). IRKI is a novel but highly conserved protein found in higher plants. The interaction between IRK and IRKI was confirmed by an in vitro pull-down assay and supported by their simultaneous expression in actively dividing cells in meristems. In the root tip, IRKI expression and localization visualized by green fluorescence protein (GFP) were observed in the quiescent center, initial cells, and immature stele cells. IRKI expression was expanded by exogenous auxin treatment and repressed by inhibitor treatment of polar auxin transport.

-24-
Analysis of Secreted Proteins during Conidial Germination of Aspergillus oryzae RIB40

Li Ying Zhu, Cong Ha Nguyen, Tsutomu Sato, and Michio Takeuchi

Department of Agriscience and Bioscience, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan

To broaden our understanding of extracellular proteins of Aspergillus oryzae at the conidial germination stage, analyses of the secreted proteins during germination were carried out. Taka-amylase A (TAA), glucoamylase (GLAA), and aspergillopepsin A (PEPA) were identified as the main products by peptide mass fingerprinting. TAA and PEPA were detected simultaneously with the formation of germ tubes. With the development of germination, the pH of the medium fell from 5.5 to 3.5. The secreted PEPA had a pro-sequence and likely shifted from 42 kDa to 41 kDa below pH 4.6, indicating that the precursor of PEPA was secreted and underwent pH-dependent processing. Furthermore, the 41 kDa protein was trapped by the addition of pepstatin A, the specific inhibitor of PEPA, suggesting that the maturation of pro-PEPA was a stepwise autoprocessing upon acidification of the medium and itself was an intermediate of the processing. It was implied that PEPA plays an important role at the early germination stage.

-25-
Note
Inhibition of Osteoporosis Due to Restricted Food Intake by the Fish Oils DHA and EPA and Perilla Oil in the Rat

Liman Sun1, Hajime Tamaki1, Tetsuji Ishimaru2, Tomokazu Teruya1, Yutaka Ohta3, Naofumi Katsuyama3, and Isao Chinen1

1Laboratories of Applied Biochemistry, Faculty of Agriculture, University of the Ryukyus, Senbaru 1, Nishihara-cho, Okinawa 903-0213, Japan
2Musashi Junior High School, 810 Nariyoshi, Musashi-mati, Higashikunisaki-gun, Oita 873-0404, Japan
3Faculty of Medicine, University of the Ryukyus, Senbaru 1, Nishihara-cho, Okinawa 903-0213, Japan

Seven-week old female rats fed restricted foods including the fish oils Docosahesaenoic Acid (DHA) and Eicosapentaenoic Acid (EPA) and perilla oil with food intake decreased by 50%, had increases of fracture force and bone mineral density (BMD) and decreases in levels of Deoxypiridinoline (Dpd) and Calcium (Ca) in the urine, compared with those of rats with osteoporosis due to restricted soy bean oil food intake. Therefore, the fish oils DHA and EPA and perilla oil depressed excretion of urinary Ca and inhibited osteoporosis due to restricted food intake.

-26-
Note
Anti-Oxidative Compounds in Barley Tea

Hideo Etoh1, Kazushi Murakami1, Tokiyasu Yogoh1, Hajime Ishikawa2, Yoshiyasu Fukuyama3, and Hitoshi Tanaka4

1Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka 422-8529, Japan
2Living Environmental Division, Aichi Environmental Research Center, 7-6 Nagare, Tsuji-machi, Kita, Nagoya 462-0032, Japan
3Faculty of Pharmaceutical Sciences, Tokushima Bunri University, 180 Yamashiro-cho, Tokushima 770-8514, Japan
4Faculty of Pharmacy, Meijo University, Yagotoyama, Tempaku, Nagoya 468-8503, Japan

Five phenolic compounds, p-hydroxyacetophenone, 5,7-dihydroxychromone, naringenin, quercetin, and iso-americanol A, were found first time in the barley tea, together with the known compounds, p-hydroxybenzaldehyde, 3,4-dihydroxybenzaldehyde, p-hydroxybenzoic acid, vanillic acid, and p-coumaric acid. The anti-oxidative properties were evaluated by measuring their peroxynitrite-scavenging activities. Among these compounds, 3,4-dihydroxybenzaldehyde, p-coumaric acid, quercetin, and isoamericanol A showed stronger activities than that of BHT (butylated hydroxytoluene) at 400 1/4M.

-27-
Note
Presence of Higher Alcohols as Ferulates in Potato Pulp and Its Radical-Scavenging Activity

Keita Yunoki1,2, Renaguli Musa1,2, Mikio Kinoshita1, Hiroyuki Tazaki1,2, Yuji Oda3, and Masao Ohnishi1,2

1Department of Bioresource Science, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan
2The United Graduate School of Agricultural Science, Iwate University, Morioka, Iwate 020-8550, Japan
3Department of Upland Agriculture, National Agricultural Research Center for Hokkaido Region, Memuro, Kasai, Hokkaido 082-0071, Japan

Higher alcohols with a carbon length ranging from 16 to 30 found in the lipophilic fraction from potato pulp were shown to be present as ferulate and in a free form, but not as wax. Thin-layer chromatography of the neutral lipids in potato pulp indicated a few spots with scavenging activity toward the 1,1-diphenyl-2-picrylhydrazyl (DPPH) stable radical, the major active component being characterized as alkyl ferulate which showed almost the same level of activity as ³-oryzanol.

-28-
Note
Fatty Acid Compositions of Commercial Red Wines

Keita Yunoki1,2, Mikio Tanji1, Yukie Murakami1, Yoshihiro Yasui3, Shuji Hirose3, and Masao Ohnishi1,2

1Department of Bioresource Science, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan
2The United Graduate School of Agricultural Science, Iwate University, Morioka, Iwate 020-8550, Japan
3Tokachi-Ikeda Research Institute for Viticulture and Enology, Ikeda, Hokkaido 083-0002, Japan

A determination was made of fatty acid compositions in twelve commercial red wines made from grapes differing in kind and vintage. Twelve fatty acids were identified, palmitic, myristic, and lauric acids being found predominant. Total acyls (32<81 nmol/100 ml) differed considerably. Changes in fatty acid constituents in must from grape berries and wines according to the process of manufacture were also examined.

-29-
Note
Whole-Mount Silver Staining of Arabidopsis and Maize: A New Method for Staining Secondary Wall Thickenings in Tracheary Elements

Sakihito Kitajima, Yoko Hasegawa, and Yukio Sugimura

Graduate School of Science and Technology, Kyoto Institute of Technology, Kyoto 606-8585, Japan

Secondary wall thickenings in tracheary elements were specifically stained by incubation of Arabidopsis and maize in Silver Stain Plus (Bio-Rad) staining solution, after pretreatment with SDS and ethanol solution. Scanning electron microscopic analysis of sections of celery revealed that silver particles were deposited on the secondary wall thickenings, indicating that the staining was due to the deposition of silver through the interaction of the stain with lignin. This method is more sensitive than the acidified phloroglucinol method.

-30-
Note
Mutational Analysis of the Length of the J3/4 Domain of Escherichia coli Ribonuclease P Ribozyme

Shinnosuke Haga, Terumichi Tanaka, and Yo Kikuchi

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

We prepared a series of length variants of the J3/4 domain of Escherichia coli ribonuclease P (RNase P) ribozyme: the four-base long J3/4 domain (A62G63G64A65) was replaced with GGA (denoted "A), GA ("AG), A ("AGG), AAGGA (£A), AAAGGA (£AA), and AAAAGGA (£AAA). The results indicated that truncating and inserting operations of the J3/4 domain drastically reduced ribozyme activity (WT\gg£AA>£A>£AAA\gg"AG>"A, "AGG), but did not affect the cleavage site selection of a substrate by the ribozyme. The reduced ribozyme activity of each mutant was rescued to some extent by the addition of a high concentration of magnesium ions. Our data indicate that the conserved AGGA sequence was important for efficient ribozyme reactions, and suggested that the length mutations affected ribozyme activity through metal ion binding steps.

-31-
Note
Isolation and Structures of Acremolactones B and C, Novel Plant-Growth Inhibitory ³-Lactones from Acremonium roseum I4267

Takeshi Sassa, Takahiro Ooi, and Hiroshi Kinoshita

Department of Bioresource Engineering, Faculty of Agriculture, Yamagata University, Wakaba-cho, Tsuruoka 997-8555, Japan

Novel acremolactones B and C were isolated from the acremolactone A-producing Acremonium roseum I4267. The structure of acremolactone B having a phenylpyridyl ³-lactone was elucidated by spectroscopic methods. It showed plant growth inhibitory activity toward Chinese cabbage seedlings. The congener of acremolactone C having a phenylcyclopentenone ³-lactone showed weak activity.

-32-
Note
Convenient and Sensitive Evaluation of a Superoxide Anion-Generating Reagent Methyl Viologen by Escherichia coli Harboring a soxS::gfp Reporter Plasmid

Akihito Shibuya, Norihiko Tsukagoshi, Iwao Ohtsu, Noriyuki Dokyu, and Rikizo Aono

Department of Biological Information, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan

We constructed a reporter system to detect a superoxide-generating methyl viologen using SoxRS of Escherichia coli and GFP of Aequorea victoria. E. coli carrying this plasmid exhibited strong fluorescence when grown in the presence of a superoxide-generating reagent methyl viologen. The fluorescence intensity observed in the stationary phase culture of the transformant increased in response to the methyl viologen concentration in a range of 0.01 1/4M to 10 1/4M.

-33-
Note
Anti-Fungal Sesquiterpenoid from the Root Exudate of Solanum abutiloides

Toshiyuki Yokose, Kozue Katamoto, Sun Park, Hideyuki Matsuura, and Teruhiko Yoshihara

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

The Solanum abutiloides plant is highly resistant to soil-borne pathogens such as Fusarium oxysporum f. sp. melongenae, Verticillium dahliae, and Ralstonia solanacearum. This species is utilized as a mating source of resistant cultivars and is also used as a rootstock. The root exudate of Solanum abutiloides was extracted from a soil system composed of charcoal and vermiculite. Anti-fungal activity was found in the extract, and an active ingredient was isolated. The chemical structure of the active compound was determined to be 3-²-acetoxysolavetivone, a new sesquiterpenoid. The anti-fungal activity of 3-²-acetoxysolavetivone examined by the inhibition of spore germination of Fusarium oxysporum was close to that of lubimin, and higher than that of solavetivone.

-34-
Note
The 9cis,11trans,13cis Isomer of Conjugated Linolenic Acid Reduces Apolipoprotein B100 Secretion and Triacylglycerol Synthesis in HepG2 Cells

Keisuke Arao1, Hiroaki Yotsumoto2, Seo-Young Han1, Koji Nagao1, and Teruyoshi Yanagita1

1Department of Applied Biological Sciences, Saga University, Honjyo-1, Saga 840-8502, Japan
2Department of Food and Nutritional Science, Saga Junior College, Kamizono 3-18-15, Saga 840-0806, Japan

The physiological effects of 9cis,11trans,13cis-conjugated linolenic acid (9c,11t,13c-CLNA), one of the CLNA isomers, were studied in human hepatoma HepG2 cells. 9c,11t,13c-CLNA significantly decreased apolipoprotein B100 secretion compared with +--linolenic acid (+--LNA). The uptake of 14C-oleate into newly synthesized cellular triacylglycerol was also decreased by 9c,11t,13c-CLNA more than by +--LNA treatment. This is the first study to show the hypolipidemic effect of 9c,11t,13c-CLNA.

-35-
Note
Production of Dihydroxy C50-Carotenoid by Aureobacterium sp. FERM P-18698

Satoshi Fukuoka1, Yusuke Ajiki2, Takahiko Ohga3, Yasuhiro Kawanami3, and Ken Izumori3

1National Institute of Advanced Industrial Science and Technology, AIST Shikoku, 2217-14 Hayashi-cho, Takamatsu 761-0395, Japan
2Yamagata Research Institute of Technology, Shonai Testing Facility, Mikawa, Yamagata 997-1321, Japan
3Kagawa University, Faculty of Agriculture, Miki-cho, Kagawa 761-0795, Japan

The dihydroxy C50-carotenoid, decaprenoxanthin, was produced by Aureobacterium sp. collected from sea water. The addition of D-psicose to the culture medium improved the growth of cells and the yield of the carotenoid. The 13C-NMR spectrum of decaprenoxanthin, which has not previously been reported, was successfully measured.

-36-
Note
Inhibitory Effects of Alcohols on the Activity of Human Matrix Metalloproteinase 7 (Matrilysin)

Yuko Muta, Hiroshi Oneda, and Kuniyo Inouye

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

Aliphatic alcohols inhibited the activity of human matrix metalloproteinase 7 (matrilysin) competitively with Ki of 6.1-19.4% (v/v) or 0.66-4.80 M. From the relationship between the structures of alcohols and their Ki values, alcohols are considered to bind the hydrophobic S1 subsite most plausibly, and the size of the pocket was estimated to be large enough to accommodate the length of 1-butanol (4-carbon chain) and the bulk of tertiary alcohols. Alcohols might be suitable probes for exploring the active-site geometry of enzymes.

-37-
Communication
Neurite Outgrowth-Stimulating Peptide Derived from Bovine ⁰-Casein

Minoru Sakaguchi, Chieko Ishikawa, Tomoaki Nishimura, Emi Sugihara, and Eiko Matsumura

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

We found a novel peptide that stimulates neurite outgrowth in Neuro-2a mouse neuroblastoma cells, from the pepsin-pancreatin digest of bovine ⁰-casein. The amino acid sequence of this peptide is Phe-Leu-Pro-Tyr-Pro-Tyr (FLPYPY), corresponding to peptidic sequence 76-81 of bovine ⁰-casein. The neurite outgrowth-stimulating activity of FLPYPY was seen over 10-9 M. On the other hand, FLPYP and FLPYPYY, which corresponded to sequences 76-80 and 76-82 of bovine ⁰-casein respectively, were ineffective.

-38-
Communication
Formation Mechanism of 2,6-Dimethyl-2,6-Octadienes from Thermal Decomposition of Linalyl ²-D-Glucopyranoside

Shoji Hattori1, Chiami Kawaharada2, Hiroyuki Tazaki2, Takane Fujimori1, Koji Kimura2, Masao Ohnishi2, and Kensuke Nabeta2

1Soda Aromatic Co., Ltd., Analysis Research Department, 1573-4 Funakata, Noda-shi, Chiba 270-0233, Japan
2Department of Bioresource Science, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro 080-8555, Japan

Thermally decomposed products of (+-)-linalyl ²-D-glucoside were analyzed by GC and GC/MS. 2,6-Dimethyl-2,6-octadienes produced by mild pyrolysis of linalyl ²-D-glucopyranoside under a vacuum were detected and characterized by MS and NMR spectroscopy. This suggests that 2,6-dimethyl-2,6-octadienes are produced during thermal decomposition of the glucoside via proton transfer from the anomeric position to C-6 in the aglycon moiety. A stable isotope labeling experiment directly indicated the new reaction mechanism.