Contents and Abstracts of BBB

(Vol.67 No.12 2003)

Amino Acid Composition of Soybean Protein Increased Postprandial Carbohydrate Oxidation in Diabetic Mice
Kengo ISHIHARA,1,† Yoshiko FUKUCHI,1 Wataru MIZUNOYA,2 Yukiko MITA,1 Yoko FUKUYA,1 Tohru FUSHIKI,2 and Kyoden YASUMOTO1 p.2505

Induction of Anti-inflammatory Responses by Dietary Momordica charantia L. (Bitter Gourd)
Mariko MANABE, Ryo TAKENAKA, Teruko NAKASA, and Osamu OKINAKA p.2512

Effects of Voluntary Resistance Exercise and High-protein Snack on Bone Mass, Composition, and Strength in Rats Given Glucocorticoid Injections
Tatsuhiro MATSUO,1,† Tsutomu NOZAKI,1 Koji OKAMURA,2 Keitaro MATSUMOTO,3 Tatsuya DOI,3 Shoich GOHTANI,1 and Masashige SUZUKI4 p.2518

TransaldolaseGlucose-6-phosphate Isomerase Bifunctional Enzyme and Ribulokinase as Factors to Increase Xylitol Production from D-Arabitol in Gluconobacter oxydans
Masakazu SUGIYAMA, Shun-ichi SUZUKI, Naoto TONOUCHI, and Kenzo YOKOZEKI p.2524

Variable Interactions between Sucrose Non-fermented 1-Related Protein Kinases and Regulatory Proteins in Higher Plants
Akira NOZAWA, Yasutaka SAWADA, Tsuyoshi AKIYAMA, Nozomu KOIZUMI, and Hiroshi SANO
p.2533

Neurite Outgrowth-stimulating Activities of β-Casomorphins in Neuro-2a Mouse Neuroblastoma Cells
Minoru SAKAGUCHI,1,† Kouichi MURAYAMA,1 Yunden JINSMAA,2 Masaaki YOSHIKAWA,2 and Eiko MATSUMURA1 p.2541

Enhancing Effect of Lipids and Emulsifiers on the Accumulation of Quercetin Metabolites in Blood Plasma after the Short-term Ingestion of Onion by Rats
Keiko AZUMA,1,† Katsunari IPPOUSHI,1 Hidekazu ITO,1 Hideki HORIE,1 and Junji TERAO2
p.2548

Synthesis and Activity of Pyrimidinylpropenamide Antibiotics: The Alkyl Analogues of Sparsomycin
Noriyuki NAKAJIMA, Takeshi ENOMOTO, Takehiro WATANABE, Nobuyasu MATSUURA, and Makoto UBUKATA p.2556

Anti-cariogenic Properties of a Water-soluble Extract from Cacao
Kyoko ITO,1,† Yuko NAKAMURA,1 Takahisa TOKUNAGA,1 Daisuke IIJIMA,2 and Kazuo FUKUSHIMA2 p.2567

Genomic Cloning of Ribonucleases in Nicotiana glutinosa Leaves, as Induced in Response to Wounding or to TMV-Infection, and Characterization of Their Promoters
Takeshi HAYASHI, Daiki KOBAYASHI, Tohru KARIU, Maino TAHARA, Kazumasa HADA, Yoshiaki KOUZUMA, and Makoto KIMURA p.2574

Synthetic Racemate and Enantiomers of Cytosporone E, a Metabolite of an Endophytic Fungus, Show Indistinguishably Weak Antimicrobial Activity
Tomoya OHZEKI1 and Kenji MORI2,† p.2584

SS33410, an Inhibitor of V-ATPase, Blocks Intracellular Protein Transport of the VSV-G Protein in the Golgi Compartment
Dae-Hyun SEOG p.2591

Purification, Characterization, and Molecular Cloning of a Pyranose Oxidase from the Fruit Body of the Basidiomycete, Tricholoma matsutake
Yoshimitsu TAKAKURA†, and Shigeru KUWATA p.2598

Identification and Expression Analysis of cDNA Encoding a Chloroplast Recombination Protein REC1, the Chloroplast RecA Homologue in Chlamydomonas reinhardtii
Emi NAKAZATO,1 Hideya FUKUZAWA,2 Satoshi TABATA,3 Hideo TAKAHASHI,1 and Kan TANAKA1,† p.2608

Pyrazolecarboxylic Acid Derivative Induces Systemic Acquired Resistance in Tobacco
Michiko YASUDA,2,5, Masanori NISHIOKA,1,4, Hideo NAKASHITA,3,4,† Isamu YAMAGUCHI,4, and Shigeo YOSHIDA2,3,5 p.2614

The Amino Acid Sequence of Satyr Tragopan Lysozyme and Its Activity
Tomohiro ARAKI, Gen TOSHIMA, Tomomi KUSAO, Yuki CHIJIIWA, Shunsuke KAWAMURA, and Takao TORIKATA p.2621

Synthesis of the Sex Pheromone of the Citrus Mealybug, Pseudococcus cryptus
Takashi NAKAHATA,1 Noriaki ITAGAKI,1 Tomonori ARAI,2 Hajime SUGIE,3 and Shigefumi KUWAHARA1,† p.2627

Production of Hydrogen Peroxide by Polyphenols and Polyphenol-rich Beverages under Quasi-physiological Conditions
Mitsugu AKAGAWA, Tomoko SHIGEMITSU, and Kyozo SUYAMA p.2632

Note
Suppression by Hydrangeae Dulcis Folium of D-Galactosamine-induced Liver Injury in Vitro and in Vivo
Ryusuke NAKAGIRI, Erika HASHIZUME, Shun KAYAHASHI, Yasushi SAKAI, and Toshikazu KAMIYA p.2641

Note
Oxygenated Lycopene and Dehydrated Lutein in Tomato Puree
Tadashi YOKOTA,1,† Hideo ETOH,1 Shunji OSHIMA,2 Kirou HAYAKAWA,2 and Yukio ISHIGURO2 p.2644

Note
Coenzyme Specificity of Enzymes in the Oxidative Pentose Phosphate Pathway of Gluconobacter oxydans
Naoto TONOUCHI, Masakazu SUGIYAMA, and Kenzo YOKOZEKI p.2648

Note
Cellulose as Extracellular Polysaccharide of Hot Spring Sulfur-turf Bacterial Mat
Kazutoshi OGAWA1 and Yonosuke MAKI2,† p.2652

Note
Specific Incorporation of L-Glutamine into Volicitin in the Regurgitant of Spodoptera litura
Naoko YOSHINAGA, Yoshitsugu SAWADA, Ritsuo NISHIDA, Yasumasa KUWAHARA, and Naoki MORI
p.2655

Note
Efficient Synthesis of Akolactone A via Pd-Catalyzed Carbonylation
Hidefumi MAKABE,1,† Motonori OKAJIMA,1 Hiroyuki KONNO,2 Tsunashi KAMO,3 and Mitsuru HIROTA3 p.2658

Note
Transformation of Aspergillus aculeatus Using the Drug Resistance Gene of Aspergillus oryzae and the pyrG Gene of Aspergillus nidulans
Shin KANAMASA, Kana YAMAOKA, Takashi KAWAGUCHI, Jun-ichi SUMITANI, and Motoo ARAI
p.2661

Note
Stereochemistry of 2-Phenylethylamine Oxidation Catalyzed by Bacterial Copper Amine Oxidase
Mayumi UCHIDA,1 Akifumi OHTANI,1 Naoki KOHYAMA,1 Toshihide OKAJIMA,2 Katsuyuki TANIZAWA,2 and Yukio YAMAMOTO1,† p.2664

Communication
Protein Profile of Symbiotic Bacteria Mesorhizobium loti MAFF303099 in Mid-growth Phase
Hideyuki KAJIWARA,1,† Takayo KANEKO,1 Masami ISHIZAKA,2 Shigeyuki TAJIMA,3 and Hiroshi KOUCHI4 p.2668

Communication
A Transient RNA Interference Assay System Using Arabidopsis Protoplasts
Chung-Il AN, Aki SAWADA, Ei-ichiro FUKUSAKI, and Akio KOBAYASHI p.2674

Communication
Cloning and Characterization of a Caenorhabditis elegans cDNA Encoding a New InsulinIGF-Like Peptide
Tsuyoshi KAWANO,1,† Kyoko TAKUWA,2 Masaji ISHIGURO,2 Terumi NAKAJIMA,2 and Yasuo KIMURA1 p.2678

Communication
Tissue Distribution and Intracellular Localization of Catechins in Tea Leaves
Takao SUZUKI,1 Naoki YAMAZAKI,1 Yasutoshi SADA,2 Itaro OGUNI,1 and Yuji MORIYASU1,† p.2683

-1-
Amino Acid Composition of Soybean Protein Increased Postprandial Carbohydrate Oxidation in Diabetic Mice

Kengo ISHIHARA,1,† Yoshiko FUKUCHI,1 Wataru MIZUNOYA,2 Yukiko MITA,1 Yoko FUKUYA,1 Tohru FUSHIKI,2 and Kyoden YASUMOTO1

1Department of Food and Nutrition, School of Life Studies, Sugiyama Jogakuen University, Nagoya 464-8662, Japan 2Laboratory of Nutrition Chemistry, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan

Received March 27, 2003; Accepted September 11, 2003
The effects of an amino acid mixture simulating dietary soybean protein on the postprandial energy metabolism was investigated using type II diabetic mice. KK-Ay strain mice were fed restrictive isoenergetic and isonitrogenous diets (35％ of energy as protein and 5％ as fat) based on either casein, soybean protein isolate hydrolysate (SPI-H), SPI-HET (ethanol unsoluble fraction of SPI-H), SPI-AA and casein-AA (amino acid mixtures simulating SPI-H and casein). To measure dietary carbohydrate oxidation, the animals were fed a diet containing 13C-glucose. Postprandial respiratory quotient and expired 13CO2 were higher in the SPI-AA than in the casein-AA group, as the differences were similarly observed in mice fed SPI-H and casein diet. No significant differences were observed in the postprandial respiratory quotient and expired 13CO2 between the SPI-H and SPI-HET group. In conclusion, this study on food-restricted mice indicates that the amino acid mixtures simulating SPI-H or casein could affect postprandial energy metabolism in diabetic mice, as observed in those fed SPI-H or casein in the form of peptide or protein.
Key words: KK-Ay; energy restriction; diabetes; 13C; respiratory quotient

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Induction of Anti-inflammatory Responses by Dietary Momordica charantia L. (Bitter Gourd)

Mariko MANABE, Ryo TAKENAKA, Teruko NAKASA, and Osamu OKINAKA

Department of Food Science and Nutrition, Faculty of Human Life and Science, Doshisha Women′s College of Liberal Arts, Kamigyo-ku, Kyoto 602-0893, Japan

Received May 14, 2003; Accepted August 26, 2003
We assessed the immunomodulatory activity of Momordica charantia L. (bitter gourd), a vegetable that has been reported to possess various bioactivities. We examined the effect of bitter gourd on intestinal immunity by monitoring the TGF-β and IL-7 secretion from Caco-2 cells and the IL-10 and IL-12 secretion from THP-1 cells that are used as in vitro models of the intestinal epithelium and monocytemacrophages, respectively. We also determined the in vivo immunological responses of rats fed on bitter gourd for 3 weeks. We found that bitter gourd induced a decrease in the intestinal secretion of IL-7 and an increase in the secretions of TGF-β and IL-10, these effects reflecting the bitter gourd-induced changes in systemic immunity, i.e., a decrease in the number of lymphocytes, increases in the populations of Th cells and NK cells, and increase in the Ig production of lymphocytes. Dietary bitter gourd may therefore induce both intestinal and also systemic anti-inflammatory responses.
Key words: Momordica charantia L. (bitter gourd); immunomodulatory activity; IL-7;

-3-
Effects of Voluntary Resistance Exercise and High-protein Snack on Bone Mass, Composition, and Strength in Rats Given Glucocorticoid Injections

Tatsuhiro MATSUO,1,† Tsutomu NOZAKI,1 Koji OKAMURA,2 Keitaro MATSUMOTO,3 Tatsuya DOI,3 Shoich GOHTANI,1 and Masashige SUZUKI4

1Faculty of Agriculture, Kagawa University, Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0795, Japan 2Osaka University of Health and Sport Sciences, Osaka 590-0496, Japan 3Saga Nutraceuticals Research Institute, Otsuka Pharmaceutical Co., Saga 841-0195, Japan 4School of Sport Sciences, Waseda University, Tokorozawa, Saitama 359-1192, Japan

Received May 15, 2003; Accepted September 16, 2003
We examined the effects of a voluntary resistance exercise (climbing) together with high-protein snacks (60％ protein) on bone mass and strength in rats given glucocorticoid-injections (2 mgkgday) as a model of age-related osteopenia. Fifty-two male Sprague-Dawley rats, 8 weeks age, were assigned to exercise or sedentary groups. These groups were further divided into groups that received no snack, snack during activity or a snack during rest. All groups were meal-fed 7:30--8:30 h and 19:30--20:30 h and the snack was fed 23:30--0:30 h (active) or 11:30--12:30 h (resting). Energy and protein intake were approximately equal in all groups. The exercise groups were allowed to climb a wire-mesh tower cage (；20 cm×200 cm) to drink water from a bottle set at the top. Weight gain during the 8-week experimental period was inhibited by a glucocorticoid-injection. Bone mass and strength were increased by climbing exercise with a high-protein snack, while no effect of snack nor any effect of snack timing was observed. Bone weight, calcium content and protein content were positively correlated to maximum load or structural stiffness. These results suggest that resistance exercise and high-protein supplementation may be a preventive therapy for osteoporosis associated with aging.
Key words: resistance exercise; high-protein snack; bone mass; bone strength; glucocorticoid

-4-
TransaldolaseGlucose-6-phosphate Isomerase Bifunctional Enzyme and Ribulokinase as Factors to Increase Xylitol Production from D-Arabitol in Gluconobacter oxydans

Masakazu SUGIYAMA, Shun-ichi SUZUKI, Naoto TONOUCHI, and Kenzo YOKOZEKI

AminoScience Laboratories, Ajinomoto Co., Inc., Suzuki-cho, Kawasaki-ku, Kawasaki-shi 210-8681, Japan

Received May 20, 2003; Accepted September 8, 2003
Xylitol production from D-arabitol by the membrane and soluble fractions of Gluconobacter oxydans was investigated. Two proteins in the soluble fraction were found to have the ability to increase xylitol production. Both of these xylitol-increasing factors were purified, and on the basis of their NH2-terminal amino acid sequences the genes encoding both of the factors were cloned. Expression of the cloned genes in Escherichia coli showed that one of the xylitol-increasing factors is the bifunctional enzyme transaldolaseglucose-6-phosphate isomerase, and the other is ribulokinase. Using membrane and soluble fractions of G. oxydans, 3.8 gl of xylitol were produced from 10 gl D-arabitol after incubation for 40 h, and addition of purified recombinant transaldolaseglucose-6-phosphate isomerase or ribulokinase increased xylitol to 5.4 gl respectively, confirming the identity of the xylitol-increasing factors.
Key words: xylitol; Gluconobacter; pentose phosphate pathway; NADH regeneration

-5-
Variable Interactions between Sucrose Non-fermented 1-Related Protein Kinases and Regulatory Proteins in Higher Plants

Akira NOZAWA, Yasutaka SAWADA, Tsuyoshi AKIYAMA, Nozomu KOIZUMI, and Hiroshi SANO

Research and Education Center for Genetic Information, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan

Received May 21, 2003; Accepted August 26, 2003
WPK4 is a sucrose non-fermented 1 (SNF1)-related wheat protein kinase, and was previously reported to interact with 14-3-3 proteins. We identified four Arabidopsis thaliana WPK4-like genes, and designated them AtWL1 through AtWL4. Yeast two-hybrid analysis, however, indicated that none of the AtWLs interacted with any of A. thaliana 14-3-3 (At14-3-3) proteins, although WPK4 itself interacted with six of them. Structurally, AtWLs were classified into a subfamiliy of AtCIPK, which generally interacts with calucineurin B-like proteins (CBL). This was also the case for AtWL1 and AtWL2, showing an efficient interaction with AtCBL2. In contrast, WPK4 interacted with none of the CBLs. In addition, to ascertain the possible interaction in vivo, expression of those genes was examined with a promoter-GUS assay. These results suggested that the interacting partner of SNF1-related protein kinases varies among plant species, and that, in the case of A. thaliana, it was CBLs, some of which were predicted to broadly regulate multiple CIPKs.
Key words: Arabidopsis thaliana; calcineurin B-like protein; protein kinase

-6-
Neurite Outgrowth-stimulating Activities of β-Casomorphins in Neuro-2a Mouse Neuroblastoma Cells

Minoru SAKAGUCHI,1,† Kouichi MURAYAMA,1 Yunden JINSMAA,2 Masaaki YOSHIKAWA,2 and Eiko MATSUMURA1

1Laboratory of Cell Biology, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan 2Division of Food Bioscience and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan

Received May 22, 2003; Accepted September 11, 2003
Endogenous opioid peptides and opiate drugs are known to affect the development of the nervous system. β-Casomorphins (β-CMs) belong to a family of exogenous opioid peptides derived from the milk protein β-casein by proteolytic fragmentation. We investigated the effects of various fragments and analogues of β-CM on neurite outgrowth in Neuro-2a mouse neuroblastoma cells. The fragments β-CM-5 to -9 and β-CM-5 amide stimulated neurite outgrowth. Fragments shorter than β-CM-5 (β-CM-3, -4, and β-CM-4 amide) and longer than β-CM-9 (β-CM-13 and -21) had no effects. The activity of β-CMs to promote neurite outgrowth does not correlate with their opioid activity in guinea-pig ileum. The effect of the most potent fragment, β-CM-5, was prevented by the μ-opioid receptor-selective
antagonist D-Phe-Cys2-Tyr3-D-Trp-Orn5-Thr6-Pen7-
Thr8-NH2 (CTOP), or by pretreatment with pertussis toxin. These results suggest that the stimulatory effects of β-CMs on neurite outgrowth were mediated through G protein-coupled μ-opioid receptors.
Key words: β-casomorphin; neuroblastoma; neurite outgrowth; μ-opioid receptor; G-protein

-7-
Enhancing Effect of Lipids and Emulsifiers on the Accumulation of Quercetin Metabolites in Blood Plasma after the Short-term Ingestion of Onion by Rats

Keiko AZUMA,1,† Katsunari IPPOUSHI,1 Hidekazu ITO,1 Hideki HORIE,1 and Junji TERAO2

1National Institute of Vegetable and Tea Science, National Agricultural Research Organization, Ano, Mie 514-2392, Japan 2Department of Nutrition, School of Medicine, The University of Tokushima, Kuramoto-cho 3, Tokushima 770-8503, Japan

Received May 23, 2003; Accepted August 8, 2003
The effects of co-ingested lipids and emulsifiers on the accumulation of quercetin metabolites in blood plasma after the short-term ingestion of onion by rats were investigated. Plasma extracts of rats that had been fed onion-containing diets for one and two weeks were analyzed by HPLC with electrochemical detection after a treatment with sulfataseβ-glucuronidase. Almost all of the quercetin metabolites in the plasma were sulfateglucuronide conjugates of quercetin and isorhamnetin. More than 4.6％ (ww) of soybean oil in the diets significantly enhanced the accumulation of quercetin metabolites in the plasma. Fish oil and beef tallow increased this to an extent similar to that with soybean oil, and lecithin was more effective than the other three lipids. Two emulsifiers, sodium caseinate and sucrose fatty acid ester, also showed an enhancing effect on the accumulation of quercetin metabolites. These results indicate that co-ingested lipids and emulsifiers could enhance the bioavailability of quercetin glucosides in onion.
Key words: onion; quercetin metabolite; plasma; lipid; emulsifier

-8-
Synthesis and Activity of Pyrimidinylpropenamide Antibiotics: The Alkyl Analogues of Sparsomycin

Noriyuki NAKAJIMA, Takeshi ENOMOTO, Takehiro WATANABE, Nobuyasu MATSUURA, and Makoto UBUKATA

Biotechnology Research Center, Toyama Prefectural University, Kosugi, Toyama 939-0398, Japan

Received June 2, 2003; Accepted July 31, 2003
Facile syntheses of sparsomycin (3) and its four analogues (4--7) based on diastereoselective oxidation of sulfide, sulfenylation, and coupling of 6-methyluracylacryllic acid with monooxodithioacetal amine, are described. Studies on the biological activity of morphological reversion on srcts-NRK cells were also carried out.
Key words: total synthesis; src＾{ts}-NRK cell; sparoxomycin A1; pyridinylpropenamide antibiotic; structure-activation relationship

-9-
Anti-cariogenic Properties of a Water-soluble Extract from Cacao

Kyoko ITO,1,† Yuko NAKAMURA,1 Takahisa TOKUNAGA,1 Daisuke IIJIMA,2 and Kazuo FUKUSHIMA2

1Health ＆ Bioscience Laboratories, Meiji Seika Kaisha, Ltd., 5--3-1 Chiyoda, Sakado, Saitama 350-0289, Japan 2Department of Microbiology, Nihon University School of Dentistry at Matsudo, 2-870-1 Sakae-cho Nishi, Matsudo 271-8587, Japan

Received June 5, 2003; Accepted August 28, 2003
The addition of a water-soluble extract from cacao-extracted powder (CEPWS) to a cariogenic model food, a white chocolate-like diet that contains 35％ sucrose, significantly reduced caries scores in SPF rats infected with Streptococcus sobrinus 6715, compared to control rats fed a white chocolate-like diet. CEPWS markedly inhibited water-insoluble glucan (WIG) synthesis through crude glucosyltransferases (GTFs) from Streptococcus sobrinus B13N in vitro. GTF-inhibitor(s) in CEPWS was prepared through three-step fractionation, and was termed CEPWS-BT, which is a high molecular weight (＞10 kDa) heat-stable matrix of sugar, protein, and polyphenol. When the inhibitory effect of CEPWS-BT on glucan synthesis was examined using the purified GTF-I, GTF-T, and GTF-U enzymes from S. sobrinus B13N, significant reduction in GTF-I and GTF-T activity as a result of adding CEPWS-BT at low concentrations was observed. These results suggest that the addition of CEPWS to cariogenic food could be useful in controlling dental caries.
Key words: dental caries; glucosyltransferase; inhibitor; Streptococcus sobrinus; cacao

-10-
Genomic Cloning of Ribonucleases in Nicotiana glutinosa Leaves, as Induced in Response to Wounding or to TMV-Infection, and Characterization of Their Promoters

Takeshi HAYASHI, Daiki KOBAYASHI, Tohru KARIU, Maino TAHARA, Kazumasa HADA, Yoshiaki KOUZUMA, and Makoto KIMURA

Laboratory of Biochemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Hakozaki 6-10-1, Higashi-ku, Fukuoka 812-8581, Japan

Received June 18, 2003; Accepted September 3, 2003
We previously cloned two distinct cDNA clones, NGR1 and NGR3, encoding S-like ribonucleases (RNases) induced by wounding and tobacco mosaic virus (TMV) infection, respectively, in Nicotiana glutinosa leaves. To gain insight into the regulatory mechanism of the RNase genes, we analyzed nucleotide sequences of the genes ngr1 (4.1 kbp) and ngr3 (5.3 kbp), containing their structural genes as well as 5′-flanking regions. The ngr1 gene is organized in three exons with two intervening introns, and ngr3 has four exons interrupted by three introns. Primer extension analyses localized single transcription initiation sites at −32 and −99 upstream of the translation initiation codons ATG in the genes ngr1 and ngr3, respectively. The β-glucuronidase (GUS) reporter gene analysis with serial 5′-deletion mutants as well as a gel shift assay defined the wound-responsive region at residues −509 to −288 in gene ngr1 and a TMV-responsive region at the residues −401 to −174 in ngr3, respectively. Sequence search using PLACE and PlantCARE data bases showed that a wound-responsive element: the WUN-motif, occurs within the wound-responsive region in ngr1, while ngr3 contains several potential cis-regulating elements, such as the elicitor responsiveness element: the W-box, a TMV responsive element: GT1, and the WUN-motif at positions between −401 and −174. These findings suggested that some of these cis-elements may be involved in inducible expressions of ngr1 and ngr3. Furthermore, the gel shift assay suggested that the dissociation of protein factor(s) upon TMV-infection from the regulatory region may cause an inducible expression of ngr3.
Key words: Nicotiana glutinosa; promoter; ribonuclease; tobacco mosaic virus (TMV)-infection; wound-inducible

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Synthetic Racemate and Enantiomers of Cytosporone E, a Metabolite of an Endophytic Fungus, Show Indistinguishably Weak Antimicrobial Activity

Tomoya OHZEKI1 and Kenji MORI2,†

1Department of Chemistry, Graduate School of Science, Science University of Tokyo, Kagurazaka 1-3, Shinjuku-ku, Tokyo 162-8601, Japan 2Glycosphingolipid Synthesis Group, Laboratory for Immune Regulation, RIKEN Research Center for Allergy and Immunology, co Seikagaku Corporation, Tateno 3-1253, Higashiyamato City, Tokyo 207-0021, Japan

Received June 19, 2003; Accepted July 28, 2003
The racemate and the enantiomers of cytosporone E [3-heptyl-4,5,6-trihydroxyphthalide (1)], a metabolite of the endophytic fungus, CR200 (Cytospora sp.), were synthesized. The key steps were (i) Sharpless asymmetric dihydroxylation of an alkene (8) and (ii) HPLC separation of the enantiomers of tert-butyldimethylsilyl ether (12) on a chiral stationary phase. The racemate and enantiomers of cytosporone E showed only weak antimicrobial activity with no difference among them.
Key words: antimicrobial compound; asymmetric dihydroxylation; enantiomeric separation; endophytic fungus; phthalide

-12-
SS33410, an Inhibitor of V-ATPase, Blocks Intracellular Protein Transport of the VSV-G Protein in the Golgi Compartment

Dae-Hyun SEOG

Department of Microbiology, College of Medicine, Inje University, Busan, 614-735, Korea

Received June 25, 2003; Accepted September 3, 2003
An earlier report suggested that SS33410, structurally related to folimycin and bafilomycin A1, blocked secretion of the glycoprotein (G protein) of vesicular stomatitis virus (VSV) into the medium and, instead, G protein was accumulated intracellulary. To identify the inhibition site of SS33410 in intracellular protein transport, I have analyzed the oligosaccharide chain structure of the intracellularly accumulated G protein. In SS33410-treated VSV-infected cells, G protein oligosaccharide was suggested to have a composition of GlcNAc-Man5-GlcNAc2 as analyzed by Bio-Gel P-4 column chromatography following digestion with α-mannosidase, β-N-acetylhexosaminidase, and then with α-mannosidase. SS33410 specifically inhibited vacuolar-type ATPase (V-ATPase). These studies thus suggest that SS33410 blocks the intracellular protein transport before the step of trimming by mannosidase II, which is confined to the medial Golgi compartment.
Key words: protein transport; VSV-G protein; vacuolar type ATPase; Golgi apparatus; glycoprotein

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Purification, Characterization, and Molecular Cloning of a Pyranose Oxidase from the Fruit Body of the Basidiomycete, Tricholoma matsutake

Yoshimitsu TAKAKURA†, and Shigeru KUWATA

Plant Breeding and Genetics Research Laboratory, Japan Tobacco, Inc., 700 Higashibara Toyoda, Iwata, Shizuoka 438-0802, Japan

Received June 26, 2003; Accepted August 27, 2003
A new H2O2-generating pyranose oxidase was purified as a strong antifungal protein from an arbuscular mycorrhizal fungus, Tricholoma matsutake. The protein showed a molecular mass of 250 kDa in gel filtration, and probably consisted of four identical 62 kDa subunits. The protein contained flavin moiety and it oxidized D-glucose at position C-2. H2O2 and D-glucosone produced by the pyranose oxidase reaction showed antifungal activity, suggesting these compounds were the molecular basis of the antifungal property. The Vmax, Km, and kcat for D-glucose were calculated to be 26.6 Umg protein, 1.28 mM, and 111s, respectively. The enzyme was optimally active at pH 7.5 to 8.0 and at 50°C. The preferred substrate was D-glucose, but 1,5-anhydro-D-glucitol, L-sorbose, and D-xylose were also oxidized at a moderate level. The cDNA encodes a protein consisting of 564 amino acids, showing 35.1％ identity to Coriolus versicolor pyranose oxidase. The recombinant protein was used for raising the antibody.
Key words: pyranose oxidase; Tricholoma matsutake; H2O2; cDNA; antifungal activity

-14-
Identification and Expression Analysis of cDNA Encoding a Chloroplast Recombination Protein REC1, the Chloroplast RecA Homologue in Chlamydomonas reinhardtii

Emi NAKAZATO,1 Hideya FUKUZAWA,2 Satoshi TABATA,3 Hideo TAKAHASHI,1 and Kan TANAKA1,†

1Laboratory of Molecular Genetics, Department of Molecular Biology, Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan 2Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan 3Kazusa DNA Research Institute, 2-6-7 Kazusa-Kamatari, Kisarazu, Chiba 292-0818, Japan

Received June 30, 2003; Accepted September 5, 2003
Chloroplasts of plant cells have their own genome, and a basic recombination protein homologous to the eubacterial RecA was suggested to be involved in the perpetuation of chloroplast DNA. A candidate cDNA sequence encoding the chloroplast RecA protein was identified from the Kazusa EST database for the unicellular green alga, Chlamydomonas reinhardtii (http://www.kazusa.or.jpenplantchlamyEST). Analysis of the cDNA sequence identified an open reading frame (ORF) of 414 amino acids encoding a eubacteria-type RecA protein. Thus the corresponding gene was named REC1. The predicted protein contains an N-terminal extension that does not show any similarity with other RecA proteins. Transient expression of a REC1-sGFP (green fluorescent protein) fusion construct in tobacco cells has indicated that this N-terminal sequence functions as a transit peptide for import into chloroplasts. Since DNA-damaging reagents induced the REC1 mRNA, REC1 was suggested to have roles in DNA recombination and repair of the chloroplast DNA in C. reinhardtii.
Key words: Chlamydomonas reinhardtii; recombination; DNA repair; chloroplast; DNA damage

-15-
Pyrazolecarboxylic Acid Derivative Induces Systemic Acquired Resistance in Tobacco

Michiko YASUDA,2,5, Masanori NISHIOKA,1,4, Hideo NAKASHITA,3,4,† Isamu YAMAGUCHI,4, and Shigeo YOSHIDA2,3,5

1Biological Research Laboratories, Nissan Chemical Industries, LTD., 1470 Shiraoka, Minamisaitama, Saitama 349-0294, Japan 2Laboratory for Growth Regulation, Plant Science Center, RIKEN, 1-7-22 Suehiro, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan 3Plant Functions Laboratory, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan 4Microbial Toxicology Laboratory, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan 5Graduate School of Science and Engineering, Saitama University, 255 Shimookubo, Saitama-shi, Saitama 338-8570, Japan

Received July 8, 2003; Accepted September 8, 2003
Systemic acquired resistance (SAR) is a potent innate immunity system in plants that is induced through a
salicylic acid (SA)-mediated pathway. Here, we characterized 3-chloro-1-methyl-1H-pyrazole-5-carboxylic
acid (CMPA) as an effective SAR inducer in tobacco. Soil drench application of CMPA induced PR gene expression and a broad range of disease resistance without antibacterial activity in tobacco. Both analysis of CMPA′s effects on NahG transgenic tobacco plants and SA measurement in wild-type plants indicated that CMPA-induced resistance enhancement does not require SA. Therefore, it is suggested that CMPA induces SAR by triggering the signaling at the same level as or downstream of SA accumulation as do both benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester and N-cyanomethyl-2-chloroisonicotinamide.
Key words: Nicotiana tabacum; salicylic acid; systemic acquired resistance; pathogenesis-related gene; Pseudomonas syringae pv. tabaci

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The Amino Acid Sequence of Satyr Tragopan Lysozyme and Its Activity

Tomohiro ARAKI, Gen TOSHIMA, Tomomi KUSAO, Yuki CHIJIIWA, Shunsuke KAWAMURA, and Takao TORIKATA

Department of Bioscience, School of Agriculture, Kyushu Tokai University, Aso, Kumamoto 869-1404, Japan

Received July 14, 2003; Accepted September 12, 2003
The amino acid sequence of satyr tragopan lysozyme and its activity was analyzed. Carboxymethylated lysozyme was digested with trypsin and the resulting peptides were sequenced. The established amino acid sequence had three amino acid substitutions at positions 103 (Asn to Ser), 106 (Ser to Asn), and 121 (His to Gln) comparing with Temminck′s tragopan lysozyme and five amino acid substitutions at positions 3 (Phe to Tyr), 15 (His to Leu), 41 (Gln to His), 101 (Asp to Gly) and 103 (Asn to Ser) with chicken lysozyme. The time course analysis using N-acetylglucosamine pentamer as a substrate showed a decrease of binding free energy change, 1.1 kcalmol at subsite A and 0.2 kcalmol at subsite B, between satyr tragopan and chicken lysozymes. This was assumed to be responsible for the amino acid substitutions at subsite A-B at position 101 (Asp to Gly), however another substitution at position 103 (Asn to Ser) considered not to affect the change of the substrate binding affinity by the observation of identical time course of satyr tragopan lysozyme with turkey and Temminck′s tragopan lysozymes that carried the identical amino acids with chicken lysozyme at this position. These results indicate that the observed decrease of binding free energy change at subsites A-B of satyr tragopan lysozyme was responsible for the amino acid substitution at position 101 (Asp to Gly).
Key words: lysozyme; amino acid sequence; egg white

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Synthesis of the Sex Pheromone of the Citrus Mealybug, Pseudococcus cryptus

Takashi NAKAHATA,1 Noriaki ITAGAKI,1 Tomonori ARAI,2 Hajime SUGIE,3 and Shigefumi KUWAHARA1,†

1Laboratory of Applied Bioorganic Chemistry, Division of Life Science, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan 2Department of Citrus Research, National Institute of Fruit Tree Science, Kuchinotsu, Nagasaki 859-2501, Japan 3National Institute of Agro-Environmental Sciences, 3-1-3 Kannondai, Tsukuba, Ibaraki 305-8604, Japan

Received July 16, 2003; Accepted August 13, 2003
The sex pheromone of the citrus mealybug (Pseudococcus cryptus), [(1R,3R)-3-isopropenyl-2,2-dimethylcyclobutyl]methyl 3-methyl-3-butenoate, was synthesized from (＋)-α-pinene in five operational steps in a 43％ overall yield. The synthetic pheromone was identical with the natural pheromone in 1H-NMR and mass spectroscopic properties, and showed almost the same pheromonal activity as the natural pheromone.
Key words: Pseudococcus cryptus; pheromone; terpenoid

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Production of Hydrogen Peroxide by Polyphenols and Polyphenol-rich Beverages under Quasi-physiological Conditions

Mitsugu AKAGAWA, Tomoko SHIGEMITSU, and Kyozo SUYAMA

Department of Applied Bioorganic Chemistry, Division of Life Science, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan

Received August 28, 2003; Accepted September 9, 2003
To investigate the ability of the production of H2O2 by polyphenols, we incubated various phenolic compounds and natural polyphenols under a quasi-physiological pH and temperature (pH 7.4, 37°C), and then measured the formation of H2O2 by the ferrous ion oxidation-xylenol orange assay. Pyrocatechol, hydroquinone, pyrogallol, 1,2,4-benzenetriol, and polyphenols such as catechins yielded a significant amount of H2O2. We also examined the effects of a metal chelator, pH, and O2 on the H2O2-generating property, and the generation of H2O2 by the polyphenol-rich beverages, green tea, black tea, and coffee, was determined. The features of the H2O2-generating property of green tea, black tea, and coffee were in good agreement with that of phenolic compounds, suggesting that polyphenols are responsible for the generation of H2O2 in beverages. From the results, the possible significances of the H2O2-generating property of polyphenols for biological systems are discussed.
Key words: hydrogen peroxide; polyphenol; catechin; tea; coffee

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Note
Suppression by Hydrangeae Dulcis Folium of D-Galactosamine-induced Liver Injury in Vitro and in Vivo

Ryusuke NAKAGIRI, Erika HASHIZUME, Shun KAYAHASHI, Yasushi SAKAI, and Toshikazu KAMIYA

Kyowa Hakko Kogyo Co., Ltd., Tsukuba Research Laboratories, 2 Miyukigaoka, Tsukuba-shi, Ibaraki 305-0841, Japan

Received February 27, 2003; Accepted August 4, 2003
Hydrangeae Dulcis Folium, the fermented and dried leaves of Hydrangea macrophylla SER. var. thunbergii MAKINO, suppressed D-galactosamine-induced liver injury by 85.2％ when added to the diet at 1％ and fed to rats for fifteen days. The hepatoprotective effect is more potent than that of a milk thistle extract and turmeric powder. Some fractionated extracts showed hepatoprotective activity in the D-galactosamine-induced in vitro liver injury model.
Key words: hepatocyte; hepatoprotective; liver injury; Hydrangeae Dulcis Folium; D-galactosamine

-20-
Note
Oxygenated Lycopene and Dehydrated Lutein in Tomato Puree

Tadashi YOKOTA,1,† Hideo ETOH,1 Shunji OSHIMA,2 Kirou HAYAKAWA,2 and Yukio ISHIGURO2

1Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka 422-8529, Japan 2Kagome Co., Ltd., 17 Nishitomiyama, Nishinasuno-cho, Nasu-gun, Tochigi 329-2762, Japan

Received May 19, 2003; Accepted August 20, 2003
Oxygenated lycopenes, (2S,5S,6R)-2,6-cyclolycopene-1-methoxy-5-ol, (2S,5S,6R)-1,16-didehydro-2,6-cyclolycopene-5-ol and (3R,6′R)-3-hydroxy-3′,4′-didehydro-β,γ-carotene (anhydrolutein I) were isolated from tomato puree. The structures of these compounds were elucidated on the basis of spectroscopic evidence.
Key words: 1,16 - didehydro - 2,6 - cyclolycopene - 5-ol; 2,6-cyclolycopene-1-methoxy-5-ol; (3R,6′R)-3-hydroxy-3′,4′-didehydro-β,γ-carotene (anhydrolutein I); tomato puree

-21-
Note
Coenzyme Specificity of Enzymes in the Oxidative Pentose Phosphate Pathway of Gluconobacter oxydans

Naoto TONOUCHI, Masakazu SUGIYAMA, and Kenzo YOKOZEKI

AminoScience Laboratories, Ajinomoto Co., Inc., Suzuki-cho, Kawasaki-ku, Kawasaki-shi 210-8681, Japan

Received May 20, 2003; Accepted September 8, 2003
The coenzyme specificity of enzymes in the oxidative pentose phosphate pathway of Gluconobacter oxydans was investigated. By investigation of the activities of glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) in the soluble fraction of G. oxydans, and cloning and expression of genes in Escherichia coli, it was found that both G6PDH and 6PGDH have NADNADP dual coenzyme specificities. It was suggested that the pentose phosphate pathway is responsible for NADH regeneration in G. oxydans.
Key words: glucose-6-phosphate dehydrogenase; 6-phosphogluconate dehydrogenase; pentose phosphate pathway; Gluconobacter; NADH regeneration

-22-
Note
Cellulose as Extracellular Polysaccharide of Hot Spring Sulfur-turf Bacterial Mat

Kazutoshi OGAWA1 and Yonosuke MAKI2,†

1Department of Environmental Science, College of Science and Engineering, Iwaki Meisei University, Iwaki 970-8551, Japan 2Unit of Environmental Science, Faculty of Humanities and Social Sciences, Iwate University, Morioka 020-8550, Japan

Received June 2, 2003; Accepted August 28, 2003
The carbohydrate fraction of a hot spring sulfur-turf bacterial mat was shown to contain cellulose by the examination of neutral sugar composition, methylation analysis, and the identification of free oligosacchrides obtained from an acetolyzate of the desulfurized sulfur-turf mat. This suggested that the sulfur-oxidizing bacteria composing the sulfur-turf were producers of cellulose.
Key words: cellulose; extracellular polysaccharide; sulfur-turf; sulfur-oxidizing bacteria

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Note
Specific Incorporation of L-Glutamine into Volicitin in the Regurgitant of Spodoptera litura

Naoko YOSHINAGA, Yoshitsugu SAWADA, Ritsuo NISHIDA, Yasumasa KUWAHARA, and Naoki MORI

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

Received June 24, 2003; Accepted August 5, 2003
Volicitin, [N-(17-hydroxylinolenoyl)-L-glutamine], was identified as an elicitor of plant volatiles from a Spodoptera exigua regurgitant. It has been proposed that gut microbes synthesize volicitin from glutamine, a predominant amino acid component in the insect gut. However, we found that glutamine was not a major component in the regurgitant of Spodoptera litura, although L-glutamine was exclusively incorporated into volicitin by S. litura fed on diets enriched with various amino acids. This selectivity of glutamine as a substrate was not due to a dominant occurrence in the insect gut.
Key words: insect-produced elicitor; volicitin; biosynthesis; Spodoptera litura

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Note
Efficient Synthesis of Akolactone A via Pd-Catalyzed Carbonylation

Hidefumi MAKABE,1,† Motonori OKAJIMA,1 Hiroyuki KONNO,2 Tsunashi KAMO,3 and Mitsuru HIROTA3

1Sciences of Functional Foods, Graduate School of Agriculture, Shinshu University, 8304 Minami-minowa, Kamiina, Nagano 399-4598, Japan 2Department of Biological Science and Technology, Faculty of Engineering, University of Tokushima, 2-1 Minamijosanjimacho, Tokushima 770-8506, Japan 3Department of Bioscience and Biotechnology, Faculty of Agriculture, Shinshu University, 8304 Minami-minowa, Kamiina, Nagano 399-4598, Japan

Received July 3, 2003; Accepted August 26, 2003
The first synthesis of (＋)- and (−)-akolactone A is described by using Pd-catalyzed carbonylation. A comparison of the optical rotation of both enantiomers of akolactone A and the natural compound suggests that the absolute configuration at the 4-position of akolactone A is R.
Key words: Pd-catalyzed carbonylation; α,β-unsaturated-γ-lactone

-25-
Note
Transformation of Aspergillus aculeatus Using the Drug Resistance Gene of Aspergillus oryzae and the pyrG Gene of Aspergillus nidulans

Shin KANAMASA, Kana YAMAOKA, Takashi KAWAGUCHI, Jun-ichi SUMITANI, and Motoo ARAI

Graduate School of Agriculture and Biological Sciences, and Research Institute for Advanced Science and Technology, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai, Osaka 599-8531, Japan

Received July 4, 2003; Accepted September 3, 2003
Transformation systems for Aspergillus aculeatus has been developed, based on the use of the pyrithiamine resistance gene of Aspergillus oryzae and the orotidine-5′-monophosphate decarboxylase gene (pyrG) of Aspergillus nidulans. An A. aculeatus mutant which can be transformed effectively by the A. nidulans pyrG gene was isolated as a transformation host. This is the first report of transformation of A. aculeatus.
Key words: Aspergillus aculeatus; transformation; pyrG; pyrithiamine; drug resistance gene

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Note
Stereochemistry of 2-Phenylethylamine Oxidation Catalyzed by Bacterial Copper Amine Oxidase

Mayumi UCHIDA,1 Akifumi OHTANI,1 Naoki KOHYAMA,1 Toshihide OKAJIMA,2 Katsuyuki TANIZAWA,2 and Yukio YAMAMOTO1,†

1Graduate School of Human and Environmental Studies, Kyoto University, Kyoto, Kyoto 606-8501, Japan 2Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0047, Japan

Received July 28, 2003; Accepted September 10, 2003
The stereochemical course of the reaction catalyzed by a copper amine oxidase from Arthrobacter globiformis has been investigated using 2-phenylethylamine stereospecifically deuterium-labeled at the C1 position. Measurements of deuterium content in the product, phenylacetaldehyde, by gas chromatography-mass spectrometry revealed stereospecific abstraction of the pro-S hydrogen during the enzymatic oxidation, as predicted from the structure modeling for the enzyme-bound substrate.
Key words: 2-phenylethylamine; copper-containing amine oxidase; topa quinone; stereospecifity; deuterium-labeled substrate

-27-
Communication
Protein Profile of Symbiotic Bacteria Mesorhizobium loti MAFF303099 in Mid-growth Phase

Hideyuki KAJIWARA,1,† Takayo KANEKO,1 Masami ISHIZAKA,2 Shigeyuki TAJIMA,3 and Hiroshi KOUCHI4

1Department of Biochemistry, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan 2Chemical Analysis Research Center, National Institute for Agro-Environmental Sciences, Tsukuba, Ibaraki 305-8602, Japan 3Department of Life Science, Faculty of Agriculture, Kagawa University, Miki, Kagawa 761-0795, Japan 4Department of Plant Physiology, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki 305-8602, Japan

Received June 20, 2003; Accepted October 11, 2003
Expressed proteins in cultured symbiotic bacteria (Mesorhizobium loti MAFF303099) in the mid-growth phase were proteomically analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and capillary high-performance liquid chromatography coupled with an ion-trap mass spectrometry (MS). The genome sequence data of M. loti were used to identify the analyzed proteins. We identified 114 of the 127 proteins analyzed on 2D-PAGE gel with some microheterogenities which were caused by post-translational modifications.
Key words: mass spectrometry; Mesorhizobium loti; proteome; two-dimensional polyacrylamide gel electrophoresis

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Communication
A Transient RNA Interference Assay System Using Arabidopsis Protoplasts

Chung-Il AN, Aki SAWADA, Ei-ichiro FUKUSAKI, and Akio KOBAYASHI

Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan

Received July 25, 2003; Accepted October 15, 2003
Double-stranded RNA (dsRNA) induces sequence-specific gene silencing in eukaryotes through a process known as RNA interference (RNAi). RNAi is now used as a powerful tool for functional genomics in many eukaryotes, including plants. We herein report a dsRNA-mediated transient RNAi assay system using protoplasts from Arabidopsis mesophyll cells and suspension-cultured cells (cell line T87). Introduction of dsRNA into protoplasts led to marked silencing of target transgenes. Our assay system would provide a convenient and efficient way to induce RNAi in protoplasts of the model plant Arabidopsis thaliana.
Key words: RNA interference; double-stranded RNA; Arabidopsis thaliana; protoplasts

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Communication
Cloning and Characterization of a Caenorhabditis elegans cDNA Encoding a New InsulinIGF-Like Peptide

Tsuyoshi KAWANO,1,† Kyoko TAKUWA,2 Masaji ISHIGURO,2 Terumi NAKAJIMA,2 and Yasuo KIMURA1

1Department of Biological and Environmental Chemistry, Faculty of Agriculture, Tottori University, Koyama-cho, Tottori City, Tottori 680-8553, Japan 2Suntory Institute for Bioorganic Research, Shimamoto-cho, Mishima-gun, Osaka 618-8503, Japan

Received August 18, 2003; Accepted September 24, 2003
A Caenorhabditis elegans cDNA encoding a new insulinIGF-like peptide was cloned and examined. The predicted peptide shows significant sequence similarity with the peptide Ceinsulin-1 reported previously and contains a characteristic insertion consisting of three residues in the putative B domain as with the Ceinsulin-1. The gene expression pattern during development is almost identical to that of Ceinsulin-1. The predicted tertiary structure of the peptide is quite similar to that of Ceinsulin-1, and their predicted receptor-recognition surfaces also closely match. These facts suggest that both peptides could recognize the same receptor.
Key words: cDNA cloning; Caenorhabditis elegans; insulin-like peptide; receptor-recognition; tertiary structure

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Communication
Tissue Distribution and Intracellular Localization of Catechins in Tea Leaves

Takao SUZUKI,1 Naoki YAMAZAKI,1 Yasutoshi SADA,2 Itaro OGUNI,1 and Yuji MORIYASU1,†

1School of Food and Nutritional Sciences, University of Shizuoka, 52-1 Yada Shizuoka-shi, Shizuoka 422-8526, Japan 2Shizuoka Tea Experiment Station, 1706-11 Kurasawa, Kikugawa, Shizuoka 439-0002, Japan

Received August 25, 2003; Accepted October 16, 2003
We investigated the leaf tissue and cellular morphology of tea (Camellia sinensis). Osmiophilic material, presumably catechins, was present in mesophyll cells, but not in epidermal cells. Electron microscopy showed that catechins were localized to restricted regions within the central vacuoles. In addition, two kinds of small vacuoles of 0.5--3 μm were present in mesophyll cells. One vacuole had catechins within its whole lumen, while the other had an electron-lucent lumen. We found fusion profiles between a large central vacuole and these small vacuoles. We propose that after catechins are synthesized, they are incorporated into small vacuoles and transported to the large central vacuoles.
Key words: Camellia; catechin; tannin; tea; vacuole