(Vol.69 No.01 2005)
Review
Coumarin and Flavone Derivatives from Estragon and Thyme as Inhibitors of Chemical Mediator Release from RBL-2H3 Cells
Jun Watanabe1,2, Hiroshi Shinmoto1, and Tojiro Tsushida1 p.1
Interaction between Trehalose and Alkaline-Earth Metal Ions
Kazuyuki Oku1, Mayumi Kurose1, Michio Kubota1, Shigeharu Fukuda1, Masashi Kurimoto1, Yoshio Tujisaka1, and Minoru Sakurai2 p.7
Comparative Effects of Non-Gelatinized Corn and Rice Starches on
the Life-Span of ICR Mice
Kohji Yamaki1, Takashi Ide2, Yuko Takano-ishikawa2, and Kazuki Shinohara2 p.13
Immunological Effects of Partially Hydrolyzed Arabinoxylan from
Corn Husk in Mice
Koichi Ogawa1, Masayasu Takeuchi1, and Nobuyuki Nakamura2 p.19
Characterization of a Metal-Chelating Substance in Coffee
Makiko Takenaka, Naoko Sato, Hiromi Asakawa, Xu Wen, Masatsune Murata, and Seiichi Homma p.26
Effects of Dietary Protein of Korean Foxtail Millet on Plasma Adiponectin,
HDL-Cholesterol, and Insulin Levels in Genetically Type 2 Diabetic Mice
You-Young Choi1, Kyoichi Osada2, Yoshiaki Ito3, Takashi Nagasawa3, Myeong-Rak Choi4, and Naoyuki Nishizawa3 p.31
Purification and Characterization of Serine Proteinase from a Halophilic
Bacterium, Filobacillus sp. RF2-5
Kazumi Hiraga1, Yasushi Nishikata1, Sirilak Namwong2, Somboon Tanasupawat2, Katsumi Takada3, and Kohei Oda1 p.38
Mode of Action of Cellulases on Dyed Cotton with a Reactive Dye
Minoru Yamada1,2, Yoshihiko Amano2, Eisuke Horikawa2, Kouichi Nozaki2, and Takahisa Kanda2 p.45
Enhanced Expression of Apoptin by the Myc–Max Binding Motif and
SV40 Enhancer for SCLC Gene Therapy
Joon-Seok Song p.51
Adenovirus-Mediated Suicide SCLC Gene Therapy Using the Increased
Activity of the hTERT Promoter by the MMRE and SV40 Enhancer
Joon-Seok Song p.56
Transient RNA Silencing of Scoulerine 9-O-Methyltransferase Expression
by Double Stranded RNA in Coptis japonica Protoplasts
Joseph Gogo Dubouzet1, Takashi Morishige1, Nanae Fujii1, Chung-Il An2, Ei-ichiro Fukusaki2, Kentaro Ifuku1, and Fumihiko Sato1 p.63
Isolation and Characterization of a cDNA Encoding Phytochrome
A in the Non-Photosynthetic Parasitic Plant, Orobanche minor Sm.
Chitra Trakulnaleamsai1, Atsushi Okazawa1, Chung-Il An1, Shin’ichiro Kajiyama1, Ei’ichiro Fukusaki1, Koichi Yoneyama2, Yasutomo Takeuchi2, and Akio Kobayashi1 p.71
Purification and Characterization of a Yeast Carbonyl Reductase
for Synthesis of Optically Active (R)-Styrene Oxide Derivatives
Noriyuki Kizaki1, Ikuo Sawa1, Miho Yano1, Yoshihiko Yasohara1, and Junzo Hasegawa2 p.79
The Class V Chitin Synthase Gene csmA Is Crucial for the Growth
of the chsA chsC Double Mutant in Aspergillus nidulans
Emi Yamada, Masayuki Ichinomiya, Akinori Ohta, and Hiroyuki Horiuchi p.87
Confirmation and Quantification of Strigolactones, Germination
Stimulants for Root Parasitic Plants Striga and Orobanche, Produced by Cotton
Daisuke Sato, Ayman A. Awad, Yasutomo Takeuchi, and Koichi Yoneyama p.98
Enzymatic Properties of Rhea Lysozyme
Jureerut Pooart, Takao Torikata, and Tomohiro Araki p.103
Synthesis and Antioxidant Activity of Olivil-Type Lignans
Satoshi Yamauchi1, Yoshimasa Hayashi1, Takuya Kirikihira2, and Toshiya Masuda2 p.113
Stimulation of Mycelia Growth in Several Mushroom Species by Rice
Husks
Hidetoshi Hanai1, Sayuri Ishida1, Chiaki Saito1, Tetsuya Maita1, Miyako Kusano2, Shigeru Tamogami1, and Masana Noma1 p.123
Preparation and Properties of Gelatin-Immobilized β-Glucosidase
from Pyrococcus furiosus
Hidetaka Nagatomo, Yoh-ichi Matsushita, Kazuhiro Sugamoto, and Takanao Matsui p.136
Occurrence of Lewis a Epitope in N-Glycans of a Glycoallergen,
Jun a 1, from Mountain Cedar (Juniperus ashei) Pollen
Yoshinobu Kimura1,2, Maiko Kamamoto1, Megumi Maeda2, Mitsuhiro Okano3, Minehiko Yokoyama4, and Kosuke Kino4 p.137
Short-Step and Scalable Synthesis of (±)-Cytoxazone
Masayoshi Asano, Chiaki Nagasawa, Masumi Suzuki, Shigeru Nishiyama, and Takeshi Sugai p.145
Electron Transfer Ability from NADH to Menaquinone and from NADPH
to Oxygen of Type II NADH Dehydrogenase of Corynebacterium glutamicum
Nawarat Nantapong, Asuka Otofuji, Catharina T. migita, Osao Adachi, Hirohide Toyama, and Kazunobu Matsushita p.149
Functions of Family-22 Carbohydrate-Binding Module in Clostridium
thermocellum Xyn10C
Ehsan Ali, Guangshan Zhao, Makiko Sakka, Tetsuya Kimura, Kunio Ohmiya, and Kazuo Sakka p.160
Design and Facile Synthesis of Neoglycolipids as Lactosylceramide
Mimetics and Their Transformation into Glycoliposomes
Yoichiro Harada1, Takeomi Murata1,2, Kazuhide Totani1, Tetsuya Kajimoto3, Shah Md. Masum4, Yukihiro Tamba4, Masahito Yamazaki4, and Taichi Usui1,2 p.166
Modulating Effect of Sesamin, a Functional Lignan in Sesame Seeds,
on the Transcription Levels of Lipid- and Alcohol-Metabolizing Enzymes in Rat
Liver: A DNA Microarray Study
Nobuo Tsuruoka1, Ayako Kidokoro1, Ichiro Matsumoto2, Keiko Abe2, and Yoshinobu Kiso1 p.179
Purification, Characterization, and Antifungal Activity of Chitinases
from Pineapple (Ananas comosus) Leaf
Toki Taira, Noriko Toma, and Masanobu Ishihara p.189
Note
Screening for Tyrosinase Inhibitors among Extracts of Seashore Plants and Identification
of Potent Inhibitors from Garcinia subelliptica
Toshiya Masuda1, Daiki Yamashita1, Yoshio Takeda1, and Shigetomo Yonemori2 p.197
Note
Stimulation of IL-8 Production by Aralia cordate Lectin in Human Colon Carcinoma
Caco-2 Cells
Yu Koyama, Takuji Suzuki, Akane Kajiya, and Mamoru Isemura p.202
Note
Improvement of the Aspergillus oryzae Enolase Promoter (P-enoA) by the Introduction
of cis-Element Repeats
Hirokazu Tsuboi1, Akio Koda1, Tomomi Toda2, Toshitaka Minetoki1, Masato Hirotsune1, and Masayuki Machida2 p.206
Note
Y-700, a Novel Inhibitor of Xanthine Oxidase, Suppresses the Development of
Colon Aberrant Crypt Foci and Cell Proliferation in 1,2-Dimethylhydrazine-Treated
Mice
Takao Hashimoto1, Atsushi Fukunari1,2, Ichimaro Yamada1, Noriyuki Yanaka1, Dong Chen1, and Norihisa Kato1 p.209
Note
Ergosterol Peroxide, an Apoptosis-Inducing Component Isolated from Sarcodon
aspratus (Berk.) S. Ito
Toshiyuki Takei1, Mitsuru Yoshida2, Mayumi Ohnishi-kameyama2, and Masuko Kobori2 p.212
Note
Identification of Insertion Sequence from a γ-Hexachlorocyclohexane Degrading
Bacterium, Sphingomonas paucimobilis UT26
Keisuke Miyauchi1,2, Masao Fukuda1, Masataka Tsuda3, Masamichi Takagi2,4, and Yuji Nagata2,3 p.216
Note
Contribution of Conserved Asn Residues to the Inhibitory Activities of Kunitz-Type
Protease Inhibitors from Plants
Shiroh Iwanaga1, Nobuyuki Yamasaki1, Makoto Kimura1, and Yoshiaki Kouzuma2 p.220
Note
Characterization of Apoptosis Induced by Fucoxanthin in Human Promyelocytic
Leukemia Cells
Eiichi Kotake-nara, Masaru Terasaki, and Akihiko Nagao p.224
Note
Optimum Culture Conditions for the Production of N-Substituted Formamide Deformylase
by Arthrobacter pascens F164
Hiroshi Fukatsu, Masahiko Goda, Yoshiteru Hashimoto, Hiroki Higashibata, and Michihiko Kobayashi p.228
Note
Enantioselective Synthesis of Four Isomers of 3-Hydroxy-4-Methyltetradecanoic
Acid, the Constituent of Antifungal Cyclodepsipeptides W493 A and B
Ken-ichi Nihei1, Kimiko Hashimoto2, Kazuo Miyairi1, and Toshikatsu Okuno1 p.231
Note
Transformation of Escherichia coli Mediated by Natural Phospholipids
Yuko Sato1,3, Noriyuki Kumazawa2, Kenichi Yoshikawa3, and Yasurou Kurusu1 p.235
Note
Pradimicin Resistance of Yeast Is Caused by a Mutation of the Putative N-Glycosylation
Sites of Osmosensor Protein Sln1
Fumitaka Hiramoto, Nobuhiko Nomura, Tamotsu Furumai, Yasuhiro Igarashi, and Toshikazu Oki p.238
Note
Sugar Responsible and Tissue Specific Expression of a Gene Encoding AtCIPK14,
an Arabidopsis CBL-Interacting Protein Kinase
Eun-Jeong Lee, Hiromichi Iai, Hiroshi Sano, and Nozomu Koizumi p.242
Note
Interaction of Modified Cyclodextrins with Cytochrome P-450
Minoru Ishikawa, Hidefumi Yoshii, and Takeshi Furuta p.246
Note
Oral Administration of Freeze-Dried Kefir Reduces Intestinal Permeation of and
Oral Sensitization to Ovalbumin in Mice
Chisato Umeda1, Kei Sonoyama1, Natsu Yamaguchi1, Ryo Saito1, Keiko Akashi2, Hidemasa Motoshima2, and Jun Kawabata1 p.249
Note
Regeneration of Bacteriorhodopsin from Thermally Unfolded Bacterio-Opsin and
All-trans Retinal at High Temperatures
Ganga D. Ghimire1, Hiroyuki Sugiyama2, Masashi Sonoyama2, and Shigeki Mitaku1,2 p.252
Note
Acid Hydrolysis of Protein in a Microcapillary Tube for the Recovery of Tryptophan
Abayomi Peter Adebiyi, Dong-Hao Jin, Tomohisa Ogawa, and Koji Muramoto p.255
Communication
Occurrence of Fructosyl-Amino Acid Oxidase-Reactive Compounds in Fungal Cells
Nobuyuki Yoshida, Kenji Takatsuka, Tohoru Katsuragi, and Yoshiki Tani p.258
-1-
Review
Coumarin and Flavone Derivatives from Estragon and Thyme as Inhibitors of Chemical Mediator Release from RBL-2H3 Cells
Jun Watanabe1,2, Hiroshi Shinmoto1, and Tojiro Tsushida1
1National Food Research Institute, Kannondai, Tsukuba 305-8642, Japan
2Japan Society for the Promotion of Science, Ichibancho, Chiyoda-ku, Tokyo 102-8471, JapanEstragon and thyme extracts showed potent inhibitory activities against chemical mediator release from rat basophilic leukemia RBL-2H3 cells. 7-Methoxycoumarin was isolated from estragon, and 5,4’-dihydroxy-6,7,3’-trimethoxyflavone, 5,4’-dihydroxy-6,7,8,3’-tetramethoxyflavone, 5-hydroxy-6,7,8,3’,4’-pentamethoxyflavone and luteolin were isolated from thyme as active components. Structure-activity relationship studies among the active isolates and their related compounds indicated that the oxygen-containing functional group at the 7-position of the coumarin structure was advantageous for the inhibitory activity and that methylation of the hydroxyl group at the 4’-position of the flavone structure was disadvantageous. It was also found that coumarin derivatives inhibited an earlier step than intracellular calcium release and proteinkinase C activation, while flavones inhibited a later step or both earlier and later steps.
-2-
Interaction between Trehalose and Alkaline-Earth Metal Ions
Kazuyuki Oku1, Mayumi Kurose1, Michio Kubota1, Shigeharu Fukuda1, Masashi Kurimoto1, Yoshio Tujisaka1, and Minoru Sakurai2
1Amase Institute, Hayashibara Biochemical Laboratories, Inc., 7-7 Amaseminami-machi, Okayama 700-0834, Japan
2Center for Biological Resources and Informatics, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, JapanWe investigated the interaction between trehalose and alkaline-earth metal ions. The nuclear relaxation times of carbon atoms of trehalose were shortened by addition of the alkaline-earth chloride salts, MgCl2, CaCl2, and SrCl2, indicating that trehalose formed metal-complexes with the alkaline-earth metal chlorides. From the data of the 1H–1H coupling constants of trehalose in the presence of the alkaline-earth chlorides, it appeared that trehalose formed complexes with MgCl2, and CaCl2 at the various complexing sites: Mg2+ was coordinated to O-4 and O-4’ of trehalose, and Ca2+ to O-2 and O-3. We succeeded in the preparation of two types of crystals of the trehalose/CaCl2. One was a crystal consisting of trehalose, CaCl2, and water in a ratio of 1:1:1. The other was an anhydrous crystal containing trehalose and CaCl2 in a ratio of 1:2. Several applications of the complexing between trehalose and the metal ions for food processing are proposed.
-3-
Comparative Effects of Non-Gelatinized Corn and Rice Starches on the Life-Span of ICR Mice
Kohji Yamaki1, Takashi Ide2, Yuko Takano-ishikawa2, and Kazuki Shinohara2
1National Agricultural Research Center for Tohoku Region, National Agricultural Research Organization, Arai, Fukushima 960-2156, Japan
2National Food Research Institute, Kannondai 2-1-12, Tsukuba, Ibaraki 305-8642, JapanWe compared the effects of non-gelatinized rice and corn starches on the life-span of ICR mice. Six groups of male ICR mice consisting of 30 animals each were maintained on purified experimental diets containing either corn or rice starch and different amounts of soybean oil (6, 12 or 24%) throughout their life-time. Plots of the survival rates of the mice indicate that rice compared to corn starch conferred a longer life-span to ICR mice, although a significant difference due to the starch type was only observed in the mice fed on the 24% fat diet ( p=0.012). A divergent effect of rice and corn starches on the survival rate was apparent when observations were combined with respect to the starch type regardless of the dietary fat level ( p=0.005). In addition, two-way ANOVA data indicate that the mean survival time was longer for the mice given rice starch (593–645 days) than for those fed corn starch (538–580 days) ( p=0.011). However, no significant difference in these parameters due to dietary fat levels was observed. The results of our study indicate that starch type is one of the determinants of longevity in mice.
-4-
Immunological Effects of Partially Hydrolyzed Arabinoxylan from Corn Husk in Mice
Koichi Ogawa1, Masayasu Takeuchi1, and Nobuyuki Nakamura2
1Research Institute, Nihon Shokuhin Kako Co., Ltd., 30 Tajima, Fuji-shi, Shizuoka 417-8530, Japan
2Fine Chemicals Department, Nihon Shokuhin Kako Co., Ltd., 33-8 Sendagaya, Shibuya-ku, Tokyo 151-0051, JapanThe effect of oral administration of partially hydrolyzed water-soluble corn husk arabinoxylan (CHAX), the average molecular weight of which is about 53 kDa, on immunopotentiating activity was investigated in mice. Oral administration of CHAX to healthy mice significantly augmented the production of interleukin (IL)-2 and interferon (IFN)-γ with a slight increase in IL-4 in mitogen-induced proliferation of spleen cells. Natural killer (NK) cell activity in spleen cells from mice, which were transplanted tumor and administrated CHAX, was augmented about 2-fold. In model mice of atopic dermatitis, the average ear thickness of mice administrated CHAX was induced by dinitrophenyl-fluorobenzene (DNFB) after injection of an anti-dinitrophenyl (DNP)-IgE monoclonal antibody was much smaller than that in control animals. These results suggest that CHAX has the ability to increase the level of immunopotentiating activity without causing over response of immunological reaction even if it is administrated orally to mice.
-5-
Characterization of a Metal-Chelating Substance in Coffee
Makiko Takenaka, Naoko Sato, Hiromi Asakawa, Xu Wen, Masatsune Murata, and Seiichi Homma
Department of Nutrition and Food Science, Ochanomizu University, 2-1-1 Otsuka, Bunkyo-ku, Tokyo 112-8610, Japan
A metal-chelating substance in brewed coffee was separated and characterized by its chemical structure. This substance was a brown polymer. The contents of sugars, amino acids and phenolics in the substance were evaluated. This polymer contained small amounts of sugars and amino acids in its partial structure. After being decomposed by alkaline fusion, the decomposition products were identified by HPLC and GC–MS. Several phenolics were detected in the decomposed products. To characterize this substance, various types of model compounds were prepared by roasting chlorogenic acid, sucrose, and (or) protein with cellulose powder. Among these model compounds, the polymer-forming ability was highest in the model prepared from all four of materials, but the metal-chelating ability was the highest in the model prepared from chlorogenic acid and cellulose. These results suggest that this metal-chelating substance was a melanoidin-like polymer formed by the decomposition and polymerization of sugars, amino acids and phenolics.
-6-
Effects of Dietary Protein of Korean Foxtail Millet on Plasma Adiponectin, HDL-Cholesterol, and Insulin Levels in Genetically Type 2 Diabetic Mice
You-Young Choi1, Kyoichi Osada2, Yoshiaki Ito3, Takashi Nagasawa3, Myeong-Rak Choi4, and Naoyuki Nishizawa3
1United Graduate School of Agricultural Sciences, Iwate University, Morioka, Iwate 020-8550, Japan
2Department of Biochemistry and Biotechnology, Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki, Aomori 036-8561, Japan
3Department of Agro-Bioscience, Faculty of Agriculture, Iwate University, Morioka, Iwate 020-8550, Japan
4Division of Biotechnology and Chemical Engineering, Yosu National University, Yeosu 550-749, The Republic of KoreaWe examined the effects of intake of Korean foxtail millet protein (FMP) on plasma levels of lipid, glucose, insulin, and adiponectin in genetically type 2 diabetic KK-Ay mice. When mice were fed a normal FMP diet or a high-fat-high-sucrose diet containing FMP for 3 weeks, in both experiments plasma concentrations of high-density lipoprotein cholesterol (HDL-cholesterol) and adiponectin increased remarkably in comparison with a casein diet group, whereas concentrations of insulin decreased greatly and that of plasma glucose was comparable to that in the casein diet group. Considering the role of adiponectin, insulin, and HDL-cholesterol in diabetes, atherosclerosis, and obesity, it appears likely that FMP may improve insulin sensitivity and cholesterol metabolism through an increase in adiponectin concentration. Therefore, FMP would serve as another beneficial food component in obesity-related diseases such as type 2 diabetes and cardiovascular diseases.
-7-
Purification and Characterization of Serine Proteinase from a Halophilic Bacterium, Filobacillus sp. RF2-5
Kazumi Hiraga1, Yasushi Nishikata1, Sirilak Namwong2, Somboon Tanasupawat2, Katsumi Takada3, and Kohei Oda1
1Department of Applied Biology, Faculty of Textile Science, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan
2Department of Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand
3Peptide Institute Inc., Minoh, Osaka 562-8686, JapanIn order to find a unique proteinase, proteinase-producing bacteria were screened from fish sauce in Thailand. An isolated moderately halophilic bacterium was classified and named Filobacillus sp. RF2-5. The molecular weight of the purified enzyme was estimated to be 49 kDa. The enzyme showed the highest activity at 60 °C and pH 10–11 under 10% NaCl, and was highly stable in the presence of about 25% NaCl. The activity was strongly inhibited by phenylmethane sulfonyl fluoride (PMSF), chymostatin, and α-microbial alkaline proteinase inhibitor (MAPI). Proteinase activity was activated about 2-fold and 2.5-fold by the addition of 5% and 15–25% NaCl respectively using Suc–Ala–Ala–Phe–pNA as a substrate. The N-terminal 15 amino acid sequence of the purified enzyme showed about 67% identity to that of serine proteinase from Bacillus subtilis 168 and Bacillus subtilis (natto). The proteinase was found to prefer Phe, Met, and Thr at the P1 position, and Ile at the P2 position of peptide substrates, respectively. This is the first serine proteinase with a moderately thermophilic, NaCl-stable, and NaCl-activatable, and that has a unique substrate specificity at the P2 position of substrates from moderately halophilic bacteria, Filobacillus sp.
-8-
Mode of Action of Cellulases on Dyed Cotton with a Reactive Dye
Minoru Yamada1,2, Yoshihiko Amano2, Eisuke Horikawa2, Kouichi Nozaki2, and Takahisa Kanda2
1Techno Center, Tokai Senko K.K., 1-2 Neshinden, Nishibiwajima-cho, Nishikasugai-gun, Aichi 452-0068, Japan
2Department of Chemistry and Material Engineering, Faculty of Engineering, Shinshu University, 4-17-1 Wakasato, Nagano 380-8553, JapanCotton woven fabrics which were previously dyed with a reactive dye were treated with a commercial cellulase preparation. Dyeing with a reactive dye for cotton apparently inhibited the weight loss activity and saccharification activity of cellulase. In addition, dyed cotton was treated with highly purified cellulases which were exo-type cellulases (Cellobiohydrolase I (CBH I) and Cellobiohydrolase II (CBH II)) and endo-type cellulase (Endoglucanase II (EG II)). Exo-type cellulases were inhibited more than endo-type cellulase by dyeing in the case of saccharification activity. CBH I was severely inhibited by dyeing as compared with CBH II or EG II from the viewpoint of morphological changes in the fiber surface. Dyes on the cellulose substrates severely influenced CBH I in spite of the rare modification, because CBH I hydrolyzed cellulose with true-processive action. The change in the activity of each cellulase component on dyed cotton can affect the synergistic action of cellulases.
-9-
Enhanced Expression of Apoptin by the Myc–Max Binding Motif and SV40 Enhancer for SCLC Gene Therapy
Joon-Seok Song
Institute of Biotechnology, Korea University, Seoul 136-701, Korea
Apoptin is derived from chicken anemia virus (CAV) and known to induce tumor specific apoptosis but not normal cells. The aim of this study was to use increased expression of apoptin by the Myc–Max response element (MMRE) and SV40 enhancer in small-cell lung cancer (SCLC) gene therapy. To investigate the possibility of the utilization of the MMRE, apoptin, and SV40 promoter/enhancer in targeted cancer gene therapy, adenovirus vector expressing apoptin controlled by the MMRE, and SV40 promoter/enhancer was constructed. Ad-MMRE-apoptin-enh infected SCLC cells were significantly suppressed and induced apoptosis more than those of Ad-apoptin or Ad-apoptin-enh. Infection with Ad-MMRE-apoptin-enh of normal cells did not increase apoptosis. About 85% of SCLC tumors show overexpression of the myc family, so the increased expression of apoptin by MMRE and SV40 enhancer can be used in targeted SCLC gene therapy. These results indicate that apoptin expression was increased by the MMRE and SV40 promoter/enhancer, and that this strategy can be used in SCLC targeted cancer gene therapy.
-10-
Adenovirus-Mediated Suicide SCLC Gene Therapy Using the Increased Activity of the hTERT Promoter by the MMRE and SV40 Enhancer
Joon-Seok Song
Institute of Biotechnology, Korea University, Seoul 136-701, Korea
Telomerase is a ribonucleoprotein complex of which the function is to add telomeric repeats to chromosomal ends. Telomerase consists of two essential components, the telomerase RNA template (hTR) and the catalytic subunit (hTERT). hTERT is expressed only in cells and tissues positive for telomerase activity, i.e., tumor or stem cells. The aim of this study was to use increased telomerase promoter activity in small-cell lung cancer (SCLC) gene therapy. The hTERT promoter and Myc–Max response elements (MMRE) in pGL3-Control vector containing SV40 enhancer resulted in strong expression of the luciferase gene only in telomerase positive and myc overexpressing SCLC cell line but not in normal human cell line. To investigate the possibility of the utilization of the MMRE, hTERT promoter, and SV40 enhancer in targeted SCLC gene therapy, adenovirus vector expressing HSV-TK controlled by the MMRE, hTERT promoter, and SV40 enhancer for the induction of telomerase positive and myc-overexpressing cancer specific cell death was constructed. SCLC cells infected with Ad-MMRE-hT-TK-enh were significantly suppressed and induced apoptosis more than those of Ad-hT-TK or Ad-hT-TK-enh infected cells. Telomerase and c-myc are activated in 60∼80% of SCLC, so the increased activity of telomerase promoter can be used for targeted SCLC gene therapy. These results show that the MMRE, hTERT promoter, and SV40 enhancer can be used in SCLC targeted cancer gene therapy.
-11-
Transient RNA Silencing of Scoulerine 9-O-Methyltransferase Expression by Double Stranded RNA in Coptis japonica Protoplasts
Joseph Gogo Dubouzet1, Takashi Morishige1, Nanae Fujii1, Chung-Il An2, Ei-ichiro Fukusaki2, Kentaro Ifuku1, and Fumihiko Sato1
1Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan
2Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871, JapanRNAi (RNA interference, RNA silencing) is a powerful tool for functional genomics, but the construction of an RNAi vector(s) and the establishment of stable transformants are time-consuming and laborious. Here we report the transient RNAi of endogenous biosynthetic genes involved in isoquinoline alkaloid biosynthesis in Coptis japonica protoplasts. Double stranded (ds) RNA fragments of various lengths prepared from several different positions of the coding sequence of scoulerine 9-O-methyltransferase (SMT) were introduced into C. japonica protoplasts by polyethylene glycol-mediated transformation, and their effects were monitored by reverse transcription-polymerase chain reaction. Substantial silencing of SMT gene expression was obtained by the introduction of these SMT dsRNAs. A significant reduction in SMT protein levels was also observed. The potentials of this transient RNAi system to evaluate the functions of biosynthetic genes in Coptis alkaloid research are discussed.
-12-
Isolation and Characterization of a cDNA Encoding Phytochrome A in the Non-Photosynthetic Parasitic Plant, Orobanche minor Sm.
Chitra Trakulnaleamsai1, Atsushi Okazawa1, Chung-Il An1, Shin’ichiro Kajiyama1, Ei’ichiro Fukusaki1, Koichi Yoneyama2, Yasutomo Takeuchi2, and Akio Kobayashi1
1Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan
2Center for Research on Wild Plants, Utsunomiya University, 350 Mine-machi, Utsunomiya 321-8505, JapanIn this study, the isolation and characterization of a phytochrome A (PHYA) homologous cDNA (OmPHYA) in the non-photosynthetic holoparasitic plant Orobanche minor are described. The present findings provide the first report of the presence of a PHYA homolog in the holoparasite. This study found that OmPHYA is of similar size to the other PHYAs of green plants and shows 72, 77, and 77% amino acid sequence identity with PHYA in Arabidopsis, potato, and tobacco respectively. The OmPHYA contains a conserved chromophore attachment cysteine at position 323. Although OmPHYA shows high sequence identity with other PHYAs in green plants, 13 amino acid substitutions located in both the N and C-terminal domains are observed (a total of 26 amino acids). OmPHYA is encoded by a single gene within the O. minor genome. The abundance of the OmPHYA transcript as well as nuclear translocation of OmphyA occurs in a light-dependent manner.
-13-
Purification and Characterization of a Yeast Carbonyl Reductase for Synthesis of Optically Active (R)-Styrene Oxide Derivatives
Noriyuki Kizaki1, Ikuo Sawa1, Miho Yano1, Yoshihiko Yasohara1, and Junzo Hasegawa2
1Fine Chemicals Research Laboratories, Kaneka Corporation, 1-8 Miyamae, Takasago, Hyogo 676-8688, Japan
2Life Science RD Center, Kaneka Corporation, 1-8 Miyamae, Takasago, Hyogo 676-8688, JapanOptically active styrene oxide derivatives are versatile chiral building blocks. Stereoselective reduction of phenacyl halide to chiral 2-halo-1-phenylethanol is the key reaction of the most economical synthetic route. Rhodotorula glutinis var. dairenensis IFO415 was discovered on screening as a potent microorganism reducing a phenacyl halide to the (R)-form of the corresponding alcohol. An NADPH-dependent carbonyl reductase was purified to homogeneity through four steps from this strain. The relative molecular mass of the enzyme was estimated to be 40,000 on gel filtration and 30,000 on SDS-polyacrylamide gel electrophoresis. This enzyme reduced a broad range of carbonyl compounds in addition to phenacyl halides. Some properties of the enzyme and preparation of a chiral styrene oxide using the crude enzyme are reported herein.
-14-
The Class V Chitin Synthase Gene csmA Is Crucial for the Growth of the chsA chsC Double Mutant in Aspergillus nidulans
Emi Yamada, Masayuki Ichinomiya, Akinori Ohta, and Hiroyuki Horiuchi
Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
chsA and chsC are genes encoding class II and I chitin synthases of Aspergillus nidulans respectively. In a previous study, chsA chsC double mutants showed various growth defects, suggesting that their cell wall architecture was disorganized and their cell wall integrity diminished. Here, we constructed chsA chsC chsD triple mutants and chsA chsC csmA triple mutants to investigate the role of the class IV and class V chitin synthases, ChsD and CsmA respectively, in maintaining the cell wall structure of the chsA chsC double mutant. The former triple mutant grew a little slower than the chsA chsC double mutant, but the two showed similar phenotypes. In contrast, the latter triple mutant exhibited severe growth defects, particularly under low osmotic conditions. The levels of the csmA transcript of the wild-type strain and chsA or chsC single mutants were markedly elevated under low osmotic conditions, while that of the chsA chsC double mutants was high even under such conditions. These and other results suggest that the function of csmA is important for the maintenance of cell wall integrity and the polarized growth of the chsA chsC double mutant.
-15-
Confirmation and Quantification of Strigolactones, Germination Stimulants for Root Parasitic Plants Striga and Orobanche, Produced by Cotton
Daisuke Sato, Ayman A. Awad, Yasutomo Takeuchi, and Koichi Yoneyama
Center for Research on Wild Plants, Utsunomiya University, Utsunomiya 321-8505, Japan
The germination stimulants for root parasitic plants Striga and Orobanche produced by cotton (Gossypium hirsutum L.) were examined in detail. Seeds of cotton were germinated and grown on glass wool wetted with sterile distilled water in sterile filter units. The root exudate was collected daily and extracted with ethyl acetate. Each of these ethyl acetate extracts was analyzed directly by high-performance liquid chromatography linked with tandem mass spectrometry (LC/MS/MS). The results demonstrate that cotton roots exuded strigol and strigyl acetate, but no other known strigolactones such as orobanchol and alectrol. The production of strigol was detected even in the root exudate collected during the first 24 h of incubation and reached a maximum 5–7 days later. The average exudation of strigol and strigyl acetate during the incubation period was ca. 15 and 2 pg/plant/day, respectively, indicating that strigol mainly contributed to germination stimulation by the cotton root exudate.
-16-
Enzymatic Properties of Rhea Lysozyme
Jureerut Pooart, Takao Torikata, and Tomohiro Araki
Department of Bioscience, School of Agriculture, Kyushu Tokai University, Aso, Kumamoto 869-1404, Japan
Rhea lysozyme was analyzed for its enzymatic properties both lytic and oligomer activities to reveal the structural and functional relationships of goose type lysozyme. Rhea lysozyme had the highest lytic activity at pH 6, followed by ostrich and goose at pH 5.5–6, whereas the optimum of cassowary was at pH 5. pH profile was correlated to the net charge of each molecule surface. On the other hand, the pH optimum for oligomer substrate was found to be pH 4, indicating the mechanism of rhea catalysis as a general acid. The time-course of the reaction was studied using β-1,4-linked oligosaccharide of N-acetylglucosamine (GlcNAc) with a polymerization degree of n ((GlcNAc)n) (n=4, 5, and 6) as the substrate. This enzyme hydrolyzed (GlcNAc)6 in an endo-splitting manner, which produced (GlcNAc)3 + (GlcNAc)3 predominating over that to (GlcNAc)2+ (GlcNAc)4. This indicates that the lysozyme hydrolyzed preferentially the third glycosidic linkage from the nonreducing end. Theoretical analysis has shown the highest rate constant value at 1.5 s-1 with (GlcNAc)6. This confirmed six substrate binding subsites as goose lysozyme (Honda, Y., and Fukamizo, T., Biochim. Biophys. Acta, 1388, 53–65 (1998)). The different binding free energy values for subsites B, C, F, and G from goose lysozyme might responsible for the amino acid substitutions, Asn122Ser and Phe123Met, located at the subsite B.
-17-
Synthesis and Antioxidant Activity of Olivil-Type Lignans
Satoshi Yamauchi1, Yoshimasa Hayashi1, Takuya Kirikihira2, and Toshiya Masuda2
1Faculty of Agriculture, Ehime University, Tarumi 3-5-7, Matsuyama, Ehime 790-8566, Japan
2Faculty of Integrated Arts and Sciences, University of Tokushima, Tokushima 770-8502, JapanOlivil-type lignans, an enantiomeric type of natural olivil, were synthesized for the first time to evaluate the relationship between the structure of olivil and its antioxidant activity. A comparison of the antioxidant activity with that of other synthesized tetrahydrofuran lignans indicated reduced activity with the tertiary hydroxy group. A different effect of the two phenolic groups of olivil on the antioxidant activity was also observed.
-18-
Stimulation of Mycelia Growth in Several Mushroom Species by Rice Husks
Hidetoshi Hanai1, Sayuri Ishida1, Chiaki Saito1, Tetsuya Maita1, Miyako Kusano2, Shigeru Tamogami1, and Masana Noma1
1Faculty of Bioresource Sciences, Akita Prefectural University, Akita 010-0195, Japan
2Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 263-8522, JapanWhen supplemented to the culture medium of mushroom Coprinus cinereus, rice husks soaked beforehand in methanol stimulated mycelia growth up to a concentration of 80 mg/ml dose-dependently, whereas the non-treated stimulated mycelia growth up to 20 mg/ml. This result suggests the existence of both stimulatory and inhibitory substances in rice husks. Since momilactone A (MLA) is recognized as one of the phytoalexins in rice husks, its biological activity against mycelia growth was tested. Momilactone A inhibited mycelia growth at 5 μg/disc, whereas the methanol extract of husks did so at 1 mg/disc, wherein 0.2 μg of MLA was estimated by LC/MS/MS. Thus the phytoalexins including MLA should inhibit mycelia growth. Rice husks stimulated mycelia growth in some edible mushroom species such as Grifola frondosa (maitake), Lentinus edodes (shiitake), Pleurotus eryngii (eringi), and P. ostreatus (hiratake). Our findings might lead to the development of new profitable cultivation methods for mushrooms using rice husks.
-19-
Preparation and Properties of Gelatin-Immobilized β-Glucosidase from Pyrococcus furiosus
Hidetaka Nagatomo, Yoh-ichi Matsushita, Kazuhiro Sugamoto, and Takanao Matsui
Faculty of Engineering, University of Miyazaki, Gakuen-Kibanadai-Nishi, Miyazaki 889-2192, Japan
Hyperthermostable β-glucosidase from Pyrococcus furiosus was enclosed in gelatin gel by cross-linking with transglutaminase. Gelatin-immobilized β-glucosidase was considerably more thermostable than the native enzyme. Lyophilized immobilisate was stored at 90 °C for 1 month without loss of activity. The immobilized β-glucosidase catalyzed transglucosylation of 5-phenylpentanol with 10.0 equivalent of cellobiose at pH 5.0 and 70 °C for 12 h to afford 5-phenylpentyl β-D-glucopyranoside in 41% yield. The immobilized enzyme was more effective than the native one in transglucosylation. The gelatin-immobilized Pfu-β-glucosidase recovered from the first run of the reaction was reusable on successive runs.
-20-
Occurrence of Lewis a Epitope in N-Glycans of a Glycoallergen, Jun a 1, from Mountain Cedar (Juniperus ashei) Pollen
Yoshinobu Kimura1,2, Maiko Kamamoto1, Megumi Maeda2, Mitsuhiro Okano3, Minehiko Yokoyama4, and Kosuke Kino4
1Department of Bioresources Chemistry, Faculty of Agriculture, Okayama University, Okayama 700-8530, Japan
2Division of Biomolecular Science, Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan
3Department of Otolaryngology-Head and Neck Surgery, Okayama University Graduate School of Medicine and Dentistry, Okayama 700-8558, Japan
4Meiji Co., Odawara 250-0862, JapanWe have determined the structures of N-glycans linked to major allergens in the mountain cedar (Juniperus ashei) pollen, Jun a 1. First, two kinds of the pollen glycoallergen (Jun a 1-A and Jun a 1-B) were purified from partially purified Jun a 1 by cation exchange chromatography. The N-glycans were liberated by hydrazinolysis from the two glycoallergens and the resulting sugar chains were N-acetylated and then coupled with 2-aminopyridine. Three pyridylaminated sugar chains were purified by reversed-phase HPLC and size-fractionation HPLC from Jun a 1-A and Jun a 1-B respectively. The structures were determined by a combination of exo- and endo-glycosidase digestions, two dimensional sugar chain mapping, and electrospray ionization mass spectrometry (ESI-MS) analysis. Structural analysis indicated that Lewis a epitope (Galβ1–3(Fucα1–4)GlcNAcβ1–) occurs in the N-glycans of the pollen allergens.
-21-
Short-Step and Scalable Synthesis of (±)-Cytoxazone
Masayoshi Asano, Chiaki Nagasawa, Masumi Suzuki, Shigeru Nishiyama, and Takeshi Sugai
Department of Chemistry, Keio University, 3-14-1 Hiyoshi, Yokohama 223-8522, Japan
A five-step and scalable synthesis of racemic cytoxazone, a novel cytokine modulator, was accomplished in a total yield of 51% from p-methoxycinnamyl alcohol without any protective groups. The keystep was the new one-pot azidohydroxylation procedure by the combined use of NaN3–H2O2–CH3CN. The epoxidation of an olefin by means of an in situ-formed iminohydroperoxide worked well, accompanied by the concomitant regioselective ring opening reaction of the resulting highly reactive epoxide with an azide ion.
-22-
Electron Transfer Ability from NADH to Menaquinone and from NADPH to Oxygen of Type II NADH Dehydrogenase of Corynebacterium glutamicum
Nawarat Nantapong, Asuka Otofuji, Catharina T. migita, Osao Adachi, Hirohide Toyama, and Kazunobu Matsushita
Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi, Yamaguchi 753-8515, Japan
Type II NADH dehydrogenase of Corynebacterium glutamicum (NDH-2) was purified from an ndh overexpressing strain. Purification conferred 6-fold higher specific activity of NADH:ubiquinone-1 oxidoreductase with a 3.5-fold higher recovery than that previously reported (K. Matsushita et al., 2000). UV–visible and fluorescence analyses of the purified enzyme showed that NDH-2 of C. glutamicum contained non-covalently bound FAD but not covalently bound FMN. This enzyme had an ability to catalyze electron transfer from NADH and NADPH to oxygen as well as various artificial quinone analogs at neutral and acidic pHs respectively. The reduction of native quinone of C. glutamicum, menaquinone-2, with this enzyme was observed only with NADH, whereas electron transfer to oxygen was observed more intensively with NADPH. This study provides evidence that C. glutamicum NDH-2 is a source of the reactive oxygen species, superoxide and hydrogen peroxide, concomitant with NADH and NADPH oxidation, but especially with NADPH oxidation. Together with this unique character of NADPH oxidation, phylogenetic analysis of NDH-2 from various organisms suggests that NDH-2 of C. glutamicum is more closely related to yeast or fungal enzymes than to other prokaryotic enzymes.
-23-
Functions of Family-22 Carbohydrate-Binding Module in Clostridium thermocellum Xyn10C
Ehsan Ali, Guangshan Zhao, Makiko Sakka, Tetsuya Kimura, Kunio Ohmiya, and Kazuo Sakka
Faculty of Bioresources, Mie University, Tsu 514-8507, Japan
Clostridium thermocellum xylanase Xyn10C (formerly XynC) is a modular enzyme, comprising a family-22 carbohydrate-binding module (CBM), a family-10 catalytic module of the glycoside hydrolases, and a dockerin module responsible for cellulosome assembly consecutively from the N-terminus. To study the functions of the CBM, truncated derivatives of Xyn10C were constructed: a recombinant catalytic module polypeptide (rCM), a family-22 CBM polypeptide (rCBM), and a polypeptide composed of the family-22 CBM and CM (rCBM–CM). The recombinant proteins were characterized by enzyme and binding assays. Although the catalytic activity of rCBM–CM toward insoluble xylan was four times higher than that of rCM toward the same substrate, removal of the CBM did not severely affect catalytic activity toward soluble xylan or β-1,3-1,4-glucan. rCBM showed an affinity for amorphous celluloses and insoluble and soluble xylan in qualitative binding assays. The optimum temperature of rCBM–CM was 80 °C and that of rCM was 60 °C. These results indicate that the family-22 CBM of C. thermocellum Xyn10C not only was responsible for the binding of the enzyme to the substrates, but also contributes to the stability of the CM in the presence of the substrate at high temperatures.
-24-
Design and Facile Synthesis of Neoglycolipids as Lactosylceramide Mimetics and Their Transformation into Glycoliposomes
Yoichiro Harada1, Takeomi Murata1,2, Kazuhide Totani1, Tetsuya Kajimoto3, Shah Md. Masum4, Yukihiro Tamba4, Masahito Yamazaki4, and Taichi Usui1,2
1Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka 422-8529, Japan
2CREST, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan
3Kyoto Pharmaceutical University, 5 Misasagi-Nakauchicho, Yamashinaku, Kyoto 607-8414, Japan
4Department of Physics, Faculty of Science, Shizuoka University, 836 Ohya, Shizuoka 422-8529, Japan
Neoglycolipids composed of disaccharide glycoside and phospholipid were designed and prepared as mimetics of lactosylceramide. The lactosyl- and N-acetyllactosaminyl-phospholipids (Lac-DPPA and LacNAc-DPPA) were enzymatically synthesized from lactose and LacNAc respectively by cellulase-mediated condensation with 1,6-hexanediol, followed by conjugation of the resulting glycosides and dipalmitoylphosphatidyl choline (DPPC) mediated by Streptomyces phospholipase D. Alternatively, allyl β-lactoside was ozonolyzed to give an aldehyde, which was condensed with dipalmytoyl phosphatidyl ethanolamine to afford a second type of glycolipid (Lac-DPPE). NMR spectroscopy indicated that the neoglycolipids behave differently in different solvent systems. X-ray diffraction clearly showed that multilamellar vesicles (MLVs) of Lac-DPPE and Lac-DPPA-MLV are in the bilayer gel phase at 20 °C, whereas those of Lac-DPPE-MLV were in the lamellar liquid-crystalline phase at 50 °C. Differential scanning calorimetry showed that Lac-DPPE-MLV had complex thermotropic behavior depending on the incubation conditions. After a long incubation at 10 °C, endothermic transitions are observed at 39.6, 42.3 °C, and 42.9 °C. These neoglycolipids have the ability to trap calcein, a chelating derivative of fluorescein, in MLVs and showed specific binding to lectin in plate assays using fluorescently labeled compounds.
-25-
Modulating Effect of Sesamin, a Functional Lignan in Sesame Seeds, on the Transcription
Levels of Lipid- and Alcohol-Metabolizing Enzymes in Rat Liver: A DNA Microarray
Study
Nobuo Tsuruoka1, Ayako Kidokoro1, Ichiro Matsumoto2, Keiko Abe2, and Yoshinobu Kiso1
1Suntory Institute for Health Care Science, 1-1-1 Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 618-8503, Japan
2Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, JapanSesamin, a major lignan in sesame seeds, has multiple functions such as cholesterol-lowering and anti-hypertensive activities. To investigate the effect of sesamin on gene expression in the liver, a DNA microarray analysis was carried out. The ingestion of sesamin dissolved in olive oil up-regulated the expression of 38 genes, 16 of which encode proteins possessing a lipid-metabolizing function, and 16 of which encode proteins possessing a xenobiotic/endogenous substance metabolizing function. In particular, sesamin significantly increased the expression of β-oxidation-associated enzymes in peroxisomes and auxiliary enzymes required for degradation, via the β-oxidation pathway, of unsaturated fatty acids in mitochondria. The ingestion of sesamin also resulted in an increase in the gene expression of acyl-CoA thioesterase involved in acyl-CoA hydrolase and very-long-chain acyl-CoA thioesterase. Interestingly, it induced the expression of the gene for aldehyde dehydrogenase, an alcohol-metabolizing enzyme. These results suggest that sesamin regulates the metabolism of lipids, xenobiotics, and alcohol at the mRNA level.
-26-
Purification, Characterization, and Antifungal Activity of Chitinases from Pineapple
(Ananas comosus) Leaf
Toki Taira, Noriko Toma, and Masanobu Ishihara
epartment of Bioscience and Biotechnology, Faculty of Agriculture, Ryukyu University, Okinawa 903-0213, Japan
Three chitinases, designated pineapple leaf chitinase (PL Chi)-A, -B, and -C were purified from the leaves of pineapple (Ananas comosus) using chitin affinity column chromatography followed by several column chromatographies. PL Chi-A is a class III chitinase having a molecular mass of 25 kDa and an isoelectric point of 4.4. PL Chi-B and -C are class I chitinases having molecular masses of 33 kDa and 39 kDa and isoelectric points of 7.9 and 4.6 respectively. PL Chi-C is a glycoprotein and the others are simple proteins. The optimum pHs of PL Chi-A, -B, and -C toward glycolchitin are pH 3, 4, and 9 respectively. The chitin-binding ability of PL Chi-C is higher than that of PL Chi-B, and PL Chi-A has lower chitin-binding ability than the others. At low ionic strength, PL Chi-B exhibits strong antifungal activity toward Trichoderma viride but the others do not. At high ionic strength, PL Chi-B and -C exhibit strong and weak antifungal activity respectively. PL Chi-A does not have antifungal activity.
-27-
Note
Screening for Tyrosinase Inhibitors among Extracts of Seashore Plants and Identification of Potent Inhibitors from Garcinia subelliptica
Toshiya Masuda1, Daiki Yamashita1, Yoshio Takeda1, and Shigetomo Yonemori2
1Faculty of Integrated Arts and Sciences, University of Tokushima, Tokushima 770-8502, Japan
2Tropical Biosphere Research Center, University of Ryukyus, Taketomi, Okinawa 907-1541, JapanThe tyrosinase inhibitory activity of methanol extracts of the leaves of 39 plant species growing on the seashore of Iriomote island (Okinawa, Japan) was investigated. The extracts of Hibiscus tiliaceus, Carex pumila, and Garcinia subelliptica showed potent activity among them. The inhibitors in the extract of Garcinia subelliptica were purified by assay-guided fractionation to give two biflavonoids. These were known compounds (2R,3S-5,7,4’,5”,7”,3”’,4”’-heptahydroxy flavanone[3-8”] flavone and 5,7,4’,5”,7”,3”’,4”’-heptahydroxy[3-8”] biflavanone), although their strong inhibitory activity toward tyrosinase is revealed for the first time in this work. One of these biflavonoids (2R,3S-5,7,4’,5”,7”,3”’,4”’-heptahydroxy flavanone[3-8”] flavone) showed much stronger activity (IC50 2.5 μM) than that of kojic acid (IC50 9.1 μM) when L-tyrosine was used as the substrate.
-28-
Note
Stimulation of IL-8 Production by Aralia cordate Lectin in Human Colon Carcinoma Caco-2 Cells
Yu Koyama, Takuji Suzuki, Akane Kajiya, and Mamoru Isemura
School of Food and Nutritional Sciences and Center of Excellence for the 21st Century, University of Shizuoka, Yada, Shizuoka 422-8526, Japan
Some lectins are known to stimulate interleukin-8 (IL-8) productions in human colon carcinoma Caco-2 cells. Since IL-8 may cause deleterious effects, we examined this stimulatory activity of Aralia cordate lectin (ACL) and Wasabia japonica lectin (WJL), both of which we isolated recently. The results indicate that ACL exhibited strong stimulatory activity for IL-8 protein production, while WJL showed marginal activity. The activity of ACL was associated with high enhancement of IL-8 gene expression. The effect of ACL was abolished almost completely in the presence of brefeldin A, indicating that internalization into cells is necessary for ACL to exert activity. The findings suggest that ingestion of a large amount of raw vegetable Aralia cordate might cause unfavorable effects on the colon.
-29-
Note
Improvement of the Aspergillus oryzae Enolase Promoter (P-enoA) by the Introduction of cis-Element Repeats
Hirokazu Tsuboi1, Akio Koda1, Tomomi Toda2, Toshitaka Minetoki1, Masato Hirotsune1, and Masayuki Machida2
1General Research Laboratory, Ozeki Corporation, 4-9 Imazu Dezaike-cho, Nishinomiya-shi, Hyogo 663-8227, Japan
2National Institute of Advanced Industrial Science and Technology (AIST), Central 6, 1-1 Higashi, Tsukuba, Ibaraki 305-8566, JapanWe constructed a protein expression vector with an improved enoA promoter that harbored 12 tandem repeats of the cis-acting element (region III) of Aspergillus oryzae. The improved promoter yielded reporter β-glucuronidase (GUS) activity approximately 30-fold of the original promoter. Northern blot analysis confirmed that GUS expression was increased at the transcriptional level. The transformant harboring seven copies of the novel vector showed more than 100,000 U/mg GUS protein, which was approximately 30% of all the cell-free soluble proteins.
-30-
Note
Y-700, a Novel Inhibitor of Xanthine Oxidase, Suppresses the Development of Colon Aberrant Crypt Foci and Cell Proliferation in 1,2-Dimethylhydrazine-Treated Mice
Takao Hashimoto1, Atsushi Fukunari1,2, Ichimaro Yamada1, Noriyuki Yanaka1, Dong Chen1, and Norihisa Kato1
1Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima 739-8528, Japan
2Pharmaceutical Research Unit, Mitsubishi Pharma Corporation, Yokohama 227-0033, JapanY-700, 1-[3-cyano-4-(2,2-dimethylpropoxy)phenyl]-1H-pyrazole-4-carboxylic acid, is a newly synthesized inhibitor of xanthine oxidase. This study found that feeding of Y-700 suppressed the development of colonic aberrant crypt foci, precursor lesions of colon cancer, and cell proliferation in 1,2-dimethylhydrazine-treated mice, accompanied by reduced serum urate. These results suggest that Y-700 is a useful agent for the prevention of colon tumorigenesis and that xanthine oxidase plays an important role in the development of colon cancer.
-31-
Note
Ergosterol Peroxide, an Apoptosis-Inducing Component Isolated from Sarcodon aspratus (Berk.) S. Ito
Toshiyuki Takei1, Mitsuru Yoshida2, Mayumi Ohnishi-kameyama2, and Masuko Kobori2
1Fukushima Prefectural Forestry Research Centre, 1 Nishishimasaka, Narita, Asaka-machi, Koriyama, Fukushima 963-0112, Japan
2National Food Research Institute, Tsukuba, Ibaraki 305-8642, JapanAmong the lipophilic extracts of seven traditional edible mushrooms, the acetone extract of Sarcodon aspratus markedly inhibited the growth of HL60 human leukemia cells and induced apoptosis after 24 h incubation. The major active component was identified as ergosterol peroxide by NMR and ESI-MS analysis. Ergosterol peroxide completely inhibited growth and induced apoptosis of HL60 cells at a concentration of 25 μM.
-32-
Note
Identification of Insertion Sequence from a γ-Hexachlorocyclohexane Degrading Bacterium, Sphingomonas paucimobilis UT26
Keisuke Miyauchi1,2, Masao Fukuda1, Masataka Tsuda3, Masamichi Takagi2,4, and Yuji Nagata2,3
1Department of Bioengineering, Nagaoka University of Technology, Nagaoka, Niigata 940-2188, Japan
2Department of Biotechnology, University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
3Graduate School of Life Sciences, Tohoku University, Katahira, Sendai 980-8577, Japan
4Department of Applied Life Sciences, Niigata University of Pharmacy and Applied Life Sciences, Niitsu, Niigata 956-8603, JapanTn5-derived mutants of the γ-hexachlorocyclohexane-degrading bacterium Sphingomonas paucimobilis UT26 were genetically characterized, and an endogenous insertion sequence (IS) which belongs to the IS1380 family was identified. The IS, named ISsp1, existed as multi copies in UT26, and its transposition appeared to be activated during the process of Tn5-mutagenesis. It was found that transposon mutagenesis can cause endogenous mutations.
-33-
Note
Contribution of Conserved Asn Residues to the Inhibitory Activities of Kunitz-Type Protease Inhibitors from Plants
Shiroh Iwanaga1, Nobuyuki Yamasaki1, Makoto Kimura1, and Yoshiaki Kouzuma2
1Laboratory of Biochemistry, Faculty of Agriculture, Graduate School, Kyushu University, Hakozaki 6-10-1, Higashi-ku, Fukuoka 812-8581, Japan
2Laboratory of Food Molecular Functionality, College of Agriculture, Ibaraki University, 3-21-1 Chuo, Ami-machi, Inashiki-gun, Ibaraki 300-0393, JapanPlant Kunitz-type protease inhibitors contain a conserved Asn residue in the N-terminal region. To investigate the role of Asn residue in protease inhibitory activities, Erythrina variegata trypsin inhibitor a (ETIa), E. variegata chymotrypsin inhibitor (ECI), and their mutants, ETIa-N12A and ECI-N13A, were used. Both mutants exhibit weaker inhibitory activities toward their cognate proteases than the wild-type proteins and were readily cleaved at reactive sites. Furthermore, kinetic analysis of the interactions of the mutated proteins with their cognate proteases by surface plasmon resonance (SPR) measurement indicated that replacements of the Asn residue mainly affected dissociation rate constants. The conserved Asn residues of Kunitz-type inhibitors play an important role in exhibiting effective inhibitory activity by stabilizing the structures of the primary binding loop and protease-inhibitor complex.
-34-
Note
Characterization of Apoptosis Induced by Fucoxanthin in Human Promyelocytic Leukemia Cells
Eiichi Kotake-nara, Masaru Terasaki, and Akihiko Nagao
National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan
Apoptosis induced by fucoxanthin in HL-60 cells was associated with a loss of mitochondrial membrane potential at an early stage, but not with an increase in reactive oxygen species. Fucoxanthin treatment caused cleavages of procaspase-3 and poly (ADP-ribose) polymerase without any effect on the protein level of Bcl-2, Bcl-XL, or Bax. Apoptosis induction by fucoxanthin may be mediated via mitochondrial membrane permeabilization and caspase-3 activation.
-35-
Note
Optimum Culture Conditions for the Production of N-Substituted Formamide Deformylase by Arthrobacter pascens F164
Hiroshi Fukatsu, Masahiko Goda, Yoshiteru Hashimoto, Hiroki Higashibata, and Michihiko Kobayashi
Institute of Applied Biochemistry, and Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan
We investigated the optimum culture conditions for the production of a novel enzyme, N-substituted formamide deformylase, which acts mainly on N-benzylformamide, in Arthrobacter pascens F164. The highest enzyme activity was obtained when this strain F164 was cultivated in a synthetic medium with N-benzylformamide as sole nitrogen source. This deformylase was found to be an inducible enzyme depending on N-benzylformamide.
-36-
Note
Enantioselective Synthesis of Four Isomers of 3-Hydroxy-4-Methyltetradecanoic Acid, the Constituent of Antifungal Cyclodepsipeptides W493 A and B
Ken-ichi Nihei1, Kimiko Hashimoto2, Kazuo Miyairi1, and Toshikatsu Okuno1
1Department of Biochemistry and Biotechnology, Faculty of Agriculture and Life Science, Hirosaki University, 3 Bunkyo-cho, Hirosaki 036-8561, Japan
2Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522, JapanFour possible stereoisomers of 3-hydroxy-4-methyltetradecanoic acid were enantioselectively synthesized by using Sharpless epoxidation and a subsequent epoxide-ring opening reaction with trimethylaluminum as the key steps. The absolute configuration of the β-oxyacid component of antifungal cyclodepsipeptides W493 A and B was consequently determined as 3S,4R.
-37-
Note
Transformation of Escherichia coli Mediated by Natural Phospholipids
Yuko Sato1,3, Noriyuki Kumazawa2, Kenichi Yoshikawa3, and Yasurou Kurusu1
1Laboratory of Molecular Microbiology, College of Agriculture, Ibaraki University, Ami 3-21-1, Inashiki, Ibaraki 300-0393, Japan
2Faculty of Engineering, Ibaraki University, Bunkyo 2-1-1, Mito, Ibaraki 310-8512, Japan
3Department of Physics, Graduate School of Science, Kyoto University, and CREST, Kyoto 606-8502, JapanTransformation system for Escherichia coli based upon introduction of plasmid DNA by natural phospholipids has been developed. Transformants are easily obtained by treatment with natural phospholipids such as phosphatidylethanolamine, phosphatidylcoline, and phosphatidylserine, where the presence of MgCl2 or CaCl2 is essential. This method of transformation is applicable not only for small plasmid pHSG399 (2.3 Kb) but also for giant plasmid R6K (100 Kb).
-38-
Note
Pradimicin Resistance of Yeast Is Caused by a Mutation of the Putative N-Glycosylation Sites of Osmosensor Protein Sln1
Fumitaka Hiramoto, Nobuhiko Nomura, Tamotsu Furumai, Yasuhiro Igarashi, and Toshikazu Oki
Biotechnology Research Center, Toyama Prefectural University, 5180 Kurokawa, Kosugi, Toyama 939-0398, Japan
Pradimicin, a mannose-binding antifungal antibiotic, induces apoptosis-like cell death in Saccharomyces cerevisiae. Previously we found that the substitution of the 74th amino acid from glycine to cysteine in Ypd1 yields a mutant resistant to pradimicin. In this study, the involvement of a membrane-spanning osomosensor, Sln1, which is located upstream of Ypd1, was investigated. A mutant, sln1 ΔNG, that lacks the putative N-glycosylation sites in the extracellular domain became resistant to pradimicin. On the other hand, the null mutants of Ssk1, Pbs2, and Hog1, which are located downstream of the Sln1 cascade, were sensitive to pradimicin as well as the wild-type strain. In conclusion, pradimicin exerts its fungicidal action with the involvement of Sln1, but the downstream branch, Ssk1 and the HOG pathway, is not involved.
-39-
Note
Sugar Responsible and Tissue Specific Expression of a Gene Encoding AtCIPK14, an Arabidopsis CBL-Interacting Protein Kinase
Eun-Jeong Lee, Hiromichi Iai, Hiroshi Sano, and Nozomu Koizumi
Research and Education Center for Genetic Information, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan
xpression of AtCIPK14 (an Arabidopsis CBL-interacting protein kinase 14) was induced by metabolic sugars. Two A/T-rich sequences similar to elements involved in sugar-inducible expression of other genes were found within the -183 bp 5’ region of the AtCIPK14 promoter that was responsible for the sugar induction. Histochemical analysis using a reporter gene indicated vascular-specific expression of AtCIPK14.
-40-
Note
Interaction of Modified Cyclodextrins with Cytochrome P-450
Minoru Ishikawa, Hidefumi Yoshii, and Takeshi Furuta
Department of Biotechnology, Faculty of Engineering, Tottori University, 4-101 Koyama-Minami, Tottori 680-8552, Japan
The effects of modified cyclodextrins (CDs) hydroxypropyl-β-CD and methyl-β-CD were studied in vitro on cDNA-expressed human cytochrome P-450 (CYP) activities (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4). The modified CDs inhibited the activities of CYP2C19 and CYP3A4 while enhancing CYP2C9 activity by 140 to 176% relative to the control values at lower concentrations. In addition, methyl-β-CD inhibited CYP1A2 and CYP2D6 at higher concentrations.
-41-
Note
Oral Administration of Freeze-Dried Kefir Reduces Intestinal Permeation of and Oral Sensitization to Ovalbumin in Mice
Chisato Umeda1, Kei Sonoyama1, Natsu Yamaguchi1, Ryo Saito1, Keiko Akashi2, Hidemasa Motoshima2, and Jun Kawabata1
1Laboratory of Food Biochemistry, Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan
2Research Center, Yotsuba Milk Products Co., Ltd., Kitahiroshima 061-1264, JapanAn increase in plasma ovalbumin concentrations after intragastric administration of ovalbumin was suppressed by concomitant freeze-dried kefir in BALB/c mice. Serum levels of ovalbumin-specific immunoglobulin G and proliferation of splenic mononuclear cells in mice immunized orally with ovalbumin were suppressed by feeding freeze-dried kefir. We propose that kefir reduces intestinal permeation of food antigen, which contributes to suppression of oral sensitization.
-42-
Note
Regeneration of Bacteriorhodopsin from Thermally Unfolded Bacterio-Opsin and All-trans Retinal at High Temperatures
Ganga D. Ghimire1, Hiroyuki Sugiyama2, Masashi Sonoyama2, and Shigeki Mitaku1,2
1Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16 Nakacho, Koganei, Tokyo 184-8588, Japan
2Department of Applied Physics, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, JapanThe temperature dependence of regeneration of bacteriorhodopsin (bR) from its apoprotein, bacterio-opsin (bO), and all-trans retinal was investigated using two different procedures to probe the structural properties of bO at high temperatures. Regeneration experiments performed at 25 °C after incubation of bO within the temperature range of 35–75 °C indicate that irreversible thermal unfolding begins at 50 °C. When bO is incubated for one hour and mixed with retinal at the same elevated temperatures, however, a greater extent of regeneration to bR occurs, even at temperatures ranging from 50 to 65 °C. These experimental results indicate that regeneration of bR occurs from thermally unfolded bO and suggest dynamic structural fluctuation of bO in the unfolded state.
-43-
Note
Acid Hydrolysis of Protein in a Microcapillary Tube for the Recovery of Tryptophan
Abayomi Peter Adebiyi, Dong-Hao Jin, Tomohisa Ogawa, and Koji Muramoto
Department of Biomolecular Science, Graduate School of Life Sciences, Tohoku University, Sendai 981-8555, Japan
The acid hydrolysis of proteins was miniaturized and simplified by employing microcapillary tubes (100 μl in volume) with 6 M HCl containing 1% 2-mercaptoethanol and 3% phenol for an amino acid compositional analysis. The method not only eliminated the laborious evacuation step for the hydrolysis tube but also decreased the destruction of tryptophan during hydrolysis. The recovery of tryptophan was 79% by acid hydrolysis at 145 °C for 4 h. Since the acid mixture could be removed under vacuum, the hydrolysate was subjected to an amino acid analysis without neutralization or dilution.
-44-
Communication
Occurrence of Fructosyl-Amino Acid Oxidase-Reactive Compounds in Fungal Cells
Nobuyuki Yoshida, Kenji Takatsuka, Tohoru Katsuragi, and Yoshiki Tani
Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma 630-0192, Japan
Fructosyl-amino acid oxidase (FAOD)-reactive fraction (FRY) was found in commercial yeast extract. FRY showed very hydrophilic property and was adsorbed to phenylboronate silica gel, indicating that it contained the Amadori compound. TLC and amino acid analyses revealed that glucosone, lysine, and arginine were produced from FRY after incubation with FAOD. TOF-MS analysis confirmed that FRY is a mixture of fructosyl lysine and fructosyl arginine. These compounds were also detected in mycelial extract of an FAOD-producer, Aspergillus terreus GP1, grown on the minimum medium, suggesting that a glycation reaction occurs in fungal cells and that FAOD acts toward the resultant Amadori compounds.