(Vol.60. No.12 1996)
Molecular Asymmetry
and Pheromone Science
Kenji Mori 1925
Vol. 60. No. 12(1996)
Strategy and Design of Novel Reagents for the Fluorometric Analysis of Biomolecules
Hiroshi Meguro and Hiroshi Ohrui
Department of Applied Biological Chemistry, Faculty of Agriculture, Tohoku University, Tsutsumidohri-Amamiyamachi 1-1, Sendai 981, Japan
This article covers molecular designs to develop several new fluorometric reagents and their applications to increase the sensitivities up to the picomole level using HPLC for the measurement of biomolecules. The methods were designed to demonstrate the physiological activities, for example (1) N-(9-acridinyl)maleimide (NAM) for the measurement of SH, -S-S-, and sulfite such as cysteine, (2) diphenyl-1-pyrenylphosphine (DPPP) for the hydroperoxides in lipids, serum, tissues, and foodstuffs, (3) 9-bromomethylacridine (9-BrMA), (4) 2-(anthracene-2,3-dicarboxylimide)ethyltrifluoromethane sulfonate (AE-OTf ) for carboxylic acids, and (5) The chiral fluorometric labelling reagent (S )-(+)-2-tert-butyl-2-methyl-1,3-benzodioxole-4-carboxylic acid (TBMB) to identify the chiralities of amino acids, sugars, and mono- and diacylglycerols. Key words: fluorometric reagent; d,l-analysis; amino acids; acylglycerols; sugars
Molecular Asymmetry and Pheromone Science
Kenji Mori
Department of Chemistry, Faculty of Science, Science University of Tokyo, 1-3 Kagurazaka, Shinjuku-ku, Tokyo 162, Japan
In pheromone science, molecular asymmetry is important both chemically and biologically. Techniques for the determination of the absolute configuration of pheromones are discussed first. Enantioselective synthesis of pheromones is the central part of the research on molecular asymmetry in pheromone science. Synthesis of sordidin, the banana weevil pheromone, is given as an example in that area. Molecular asymmetry governs the biodiversity of pheromone perception as illustrated by the detailed discussion on the relationships between absolute configuration and pheromone activity. Key words: pheromone, absolute configuration of ; pheromone, synthesis of ; stereochemistry-bioactivity relationships
Comparison of the Response of Serum Ceruloplasmin and Cholesterol, and of Tissue Ascorbic Acid, Metallothionein, and Nonprotein Sulfhydryl in Rats to the Dietary Level of Cystine and Cysteine
Ben-Shan Yang, Michikazu Yamazaki, Qin Wan, and Norihisa Kato
Department of Applied Biochemistry, Faculty of Applied Biological Science, Hiroshima Univerisity, Higashi-Hiroshima 739, Japan
Received December 18, 1995
The effects were compared of the addition of graded levels of L-cystine and of L-cysteine (0.3, 3, or 5%) to a 10% casein diet on several metabolic parameters in rats. The growth-promoting effect of cystine was equivalent to that of cysteine. Supplementation of these two amino acids elevated serum cholesterol, liver ascorbic acid, liver nonprotein sulfhydryl (SH) and kidney metallothionein, and reduced the activity of serum ceruloplasmin. The responses of serum cholesterol, liver nonprotein SH, and serum ceruloplasmin to cystine were greater than of those to cysteine. When the basal diet was supplemented with 0.3% of these amino acids, the elevation of liver ascorbic acid by cystine supplementation was less than that by cysteine supplementation. However, when supplemented with 5% of these amino acids, the elevation of liver ascorbic acid by cystine was greater than that by cysteine. There was no difference in the influence of cystine and cysteine on kidney metallothionein. This study demonstrates that dietary cystine and cysteine had the same influence on growth, but had a differential influence on such metabolic parameters as liver nonprotein SH, serum ceruloplasmin, serum cholesterol, and tissue ascorbic acid. Key words: l-cystine; l-cysteine; metabolic response; nonprotein sulfhydryl; rat
Characterization of the Bovine -Casein Gene Promoter
Takahiro Adachi, Jung-Youb Ahn, Kazue Yamamoto, Naohito Aoki, Ryo Nakamura, and Tsukasa Matsuda
Department of Applied Biological Sciences, School of Agricultural Sciences, Nagoya University, Chikusa-ku, Nagoya 464-01, Japan
Received February 22, 1996
-Casein gene promoter was localized within a 570-bp fragment (-552/+18) of a 5'-flanking region by the gene transfection assay. Deletion mutation analysis in mammary epithelial cell line, HC11, suggested that there are regulatory element in a region from -439 through -125. Some nuclear proteins from lactating rate mammary gland bind to this region specifically. One of them expressed preferentially during pregnancy bound to a 132-bp fragment (-439/-308) and another expressed preferentially during lactation bound to a 183-bp fragment (-307/-125). Key words: casein; milk protein; gene expression; promoter
Purification and Characterization of Serine Hydroxymethyltransferase from Mitochondria of Euglena gracilis z
Masako Sakamoto,*,*** Tsutomu Masuda,* Yukio Yanagimoto,* Yoshihisa Nakano,** Shozaburo Kitaoka,** and Yoshinori Tanigawa***
* Shitenno-ji International Buddhist University, Habikino, Osaka 583, Japan ** Department of Agriculture, University of Osaka Prefecture, Sakai, Osaka 593, Japan *** Department of Biochemistry, Shimane Medical University, Izumo, Shimane 693, Japan
Received February 28, 1996
Mitochondrial serine hydroxymethyltransferase, L-serine: tetrahydrofolate 5,10-hydroxymethyltransferase (EC 2.1.2.1), (m-SHMT) was extracted and highly purified from Euglena gracilis z. The specific activity increased from the crude extract with 10% yield up to 580-fold through the following steps: ammonium sulfate fractionation, DEAE-cellulose column chromatography and rechromatography, and affinity chromatography with L-lysine-Sepharose 4B. The molecular weight of the purified m-SHMT was 88,000 by gel filtration through Sephadex G-200, and 44,000 by SDS-PAGE. One mol of the purified enzyme contained two mol of pyridoxal 5'-phosphate (PLP), indicating that the enzyme is a dimer. Characteristics of the enzyme were examined and compared with SHMTs of other origins. The m-SHMT of Euglena gracilis z had L-threonine aldolase activity as did s-SHMT of the same origin in addition to the usual SHMT activity. Key word: serine hydroxymethyltransferase
Effect of Solute Adsorption Properties on Its Separation from Supercritical Carbon Dioxide with a Thin Porous Silica Membrane
Tomoyuki Fujii, Yoshihiro Tokunaga, and Kozo Nakamura
Department of Applied Biological Chemistry, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113, Japan
Received March 15, 1996
A thin porous silica membrane (average pore size of 3.3 mm) was prepared by the sol-gel method and used to separate the solute from supercritical carbon dioxide. The characteristics of solute permeation were investigated in respect of the adsorption properties of the solute, the desorption rate of the solute from the membrane being measured and the potential energy of solute near the silica surface being calculated by the molecular modeling technique. It was found that caffeine was strongly adsorbed to the surface and then slowly desorbed to form an adsorption layer, making the pores narrower and causing a molecular-sieving effect. Therefore, the rejection value was positive. On the other hand, the rejection value of n-octanoic acid, which was well adsorbed and rapidly desorbed, was negative. It is presumed that the molecules filled the pores due to their potential energy and were then forced to flow through the pores by the transmembrane pressure. Key words: supercritical carbon dioxide; membrane separation; silica membrane; adsorption; molecular modeling
Xyloglucan Endotransglycosylation in Suspension-cultured Poplar Cells
Takumi Takeda, Yasushi Mitsuishi,* Fukumi Sakai, and Takahisa Hayashi
Wood Research Institute, Kyoto University, Gokasho, Uji, Kyoto 611, Japan * National Institute of Bioscience and Human Technology, AIST, 1-1 Higashi, Tsukuba, Ibaraki 305, Japan
Received April 8, 1996
Xyloglucan endotransglycosylase activity was identified and defined by transfer of a part of xyloglucan to reduced xyloglucan heptasaccharide ([3H]XXXGol) in an enzyme preparations from suspension-cultured poplar cells. Although the activity was distributed in buffer-soluble and buffer-insoluble fractions associated with cells and in the extracellular fraction, it was mostly recovered in the buffer-insoluble fraction, suggesting that the enzyme was bound to the cell wall. The affinity for acceptor XXXGol was increased at a higher concentration of donor xyloglucan with a constant Vmax. The Vmax for donor xyloglucan was increased at a higher concentration of the oligosaccharide without any change in affinity. These kinetic data suggest that the acceptor acts by combining with the enzyme independently of the donor. The velocity of the reaction decreased gradually as the heptasaccharide units was increased from two to four, suggesting that the xyloglucan endotransglycosylase reaction caused donor xyloglucan substantially to decrease in molecular size. The activity in buffer-soluble fraction was increased by ABA in auxin-starved cells, when cultured in MS medium containing various plant hormones. Nevertheless, the activity increased markedly at the exponential growth and decreased immediately at the stationary phase of cells in the presence of 2,4-D. The activity of xyloglucan endotransglycosylase is developmentally regulated during growth but is not directly induced by plant hormones. Key words: Poplus alba L.; xyloglucan endotransglycosylase; xyloglucan oligosaccharide
Three Extracellular Chitinases in Suspension-cultured Rice Cells Elicited by N-Acetylchitooligosaccharides
Hiroshi Inui, Yasuhiro Yamaguchi, Yoshitsugu Ishigami, Satoru Kawaguchi, Taku Yamada, Hideshi Ihara,* and Shigehiro Hirano
Department of Agricultural Biochemistry and Biotechnology, Tottori University, Tottori 680, Japan * College of Integrated Arts and Sciences, Osaka Prefecture University, Sakai, Osaka 593, Japan
Received April 8, 1996
In rice suspension culture, a large part (about 90% of total activity in the culture) of the chitinase activity was found in the medium. Two extracellular chitinases (which we named RCH-A and -B) were separated from the cell suspension by DEAE-cellulofine column chromatography. When cells were treated with N-acetylchitooligosaccharides (chitin oligosaccharides) for 3 days, extracellular chitinase activity increased about 3-fold over the control culture. After the treatment, another extracellular chitinase (named RCH-C) appeared in addition to increases in the levels of RCH-A and -B. Partial amino acid sequences of these enzymes indicated that RCH-A (33.5 kDa) and -B (34 kDa) were class Ib chitinases but RCH-C (27 kDa) was a class III chitinase. RCH-A and -B were capable of actively degrading water-insoluble chitin with high affinities, while RCH-C had less affinity for the substrate. However, when a water-soluble chitin derivative, 6-O-hydroxyethylchitin (glycolchitin) was used, RCH-C as well as RCH-A and -B degraded actively with a high affinity. A synergistic effect was observed when these three chitinases acted simultaneously in the hydrolysis of chitin. Key words: chitinase; plant defense reaction; N-acetylchitooligosaccharide; elicitor; rice (Oryza sativa L. var. japonica)
Ginsenosides Increase Secretion of Lipoprotein Lipase by 3T3-L1 Adipocytes
Hiroshi Masuno, Takashi Kitao, and Hiromichi Okuda*
Department of Medical Laboratory Technology, Ehime College of Health Science, Takooda, Tobe-cho, Iyo-gun, Ehime 791-21, Japan * Department of Medical Biochemistry, School of Medicine, Ehime University, Shigenobu, Onsen-gun, Ehime 791-02, Japan
Received April 11, 1996
Treatment of 3T3-L1 adipocytes with either an oleanolic acid glycoside or a 20(S)-protopanaxatriol glycoside increased the secretion of lipoprotein lipase activity into the medium dose-dependently. At a concentration of 100 g/ml, ginsenosides Ro, Re, Rg1, and Rh1 increased the secretion of lipase activity into the medium by 119, 107, 56, and 32%, respectively. The ratio of lipase activity in the medium to cellular lipase activity was 4.7% in control cells and 8.6% in ginsenoside Ro-treated cells, 8.3% in ginsenoside Re-treated cells, 7.0% in ginsenoside Rg1-treated cells, and 6.3% in ginsenoside Rh1-treated cells. Ginsenoside Rb2, which is a 20(S )-protopanaxadiol glycoside, increased the secretion of lipase activity by 16% at 25 g/ml, and the ratio of lipase activity in the medium to cellular lipase activity was higher in ginsenoside Rb2-treated cells than in control cells. However, at 100 and 200 g/ml, ginsenoside Rb2 decreased the secretion of lipase activity in parallel with cellular lipase activity. Ginsenoside Rd also decreased the secretion of lipase activity in the same dose-dependent manner. Thus, the effective dose for the secretion of lipoprotein lipase activity with ginsenosides varies with their aglycone structure. Key words: 3T3-L1 adipocytes; ginsenoside; lipoprotein lipase; secretion
Participation of the Superoxide Radical in the Beneficial Effect of Ascorbic Acid on Heat-induced Fish Meat Gel (Kamaboko)
Kimio Nishimura, Masahiro Goto,* and Junichi Mano**
Department of Food Science & Nutrition, Faculty of Human Life & Science, Doshisha Women's College of Liberal Art, Kamigyo-ku, Kyoto 602, Japan * Department of Food & Nutrition, Faculty of Home Economics, Kochi Women's University, Eikokuji-cho, Kochi 780, Japan ** Research Institute for Food Science, Kyoto University, Uji 611, Japan
Received April 22, 1996
The influence of ascorbic acid (AsA) on the proteins in raw meat paste (surimi) of walleye pollack during the formation of a gel by incubating at 40C, or 40C and 90C was examined by SDS-polyacrylamide gel electrophoresis. AsA significantly enhanced the decrease in the amount of myosin heavy chain (MHC), by promoting the polymerization of MHC via disulfide bridging. Diethylenetriaminepentaacetic acid, a chelator, and tiron, a scavenger of the superoxide radical, both inhibited the promoting effect of AsA on MHC polymerization in surimi. Therefore, the superoxide radical generated via the metal-catalyzed autoxidation of AsA participated in the polymerization of MHC. The production of the cysteine (Cys) thiyl radical due to superoxide in a 10 mM Cys solution was confirmed by electron spin resonance spectroscopy, in which superoxide was generated by photo-activation of riboflavin. These results suggest that the polymerization of MHC by AsA proceeds through a radical-radical reaction of the thiyl radicals of Cys residues that are generated by superoxide radicals. Key words: ascorbic acid; fish gel; superoxide radical; cysteine thiyl radical; disulfide bridging
Solid-state Spin Trapping of the Hydroxyl Radical Formed by Gamma-irradiation in Ice and the Scavenging Effect of Sodium L-Ascorbate
Hisashi Yoshioka, Hiroe Yoshioka,* and Kunihiko Hasegawa*
Graduate School of Nutritional and Environmental Sciences, University of Shizuoka, 52-1 Yada, Shizuoka-shi 422, Japan * Radiochemical Research Laboratory, Faculty of Science, Shizuoka University, 836 Ohya, Shizuoka-shi 422, Japan
Received April 24, 1996
Spin trapping of the hydroxyl (OH) radical formed by -irradiation in ice was performed by using 5,5-dimethyl pyrroline N-oxide (DMPO) as a trapping agent. A frozen aqueous solution of DMPO was -irradiated at -70C for three days, and ESR spectra were then measured at room temperature as a function of time after melting. A strong four-line absorption characteristic of the spin adduct (DMPO-OH) was observed and the intensity decreased with time, while no signal was observed when -irradiation was carried out at room temperature. The intensity of DMPO-OH increased linearly with radiation dose, but it was not changed by the standing time at -70C after -irradiation, suggesting that the OH radical formed by -irradiation in ice was trapped by DMPO in the frozen state, DMPO-OH being stable for more than three days in contrast to the case at room temperature. The quantity of DMPO-OH was decreased in the presence of sodium L-ascorbate. The analysis of the intensity change shows that this solid-state spin trapping technique is useful to measure separately the OH radical scavenging and DMPO-OH scavenging abilities of antioxidants. Key words: spin trapping; hydroxyl radical; gamma irradiation; 5,5-dimethyl pyrroline N-oxide; sodium l-ascorbate
Effects of Ganglioside GD1a on the Fluidity and Phase Transition of Phosphatidylcholine Model Membrane Immobilized on Porous Cellulose Acetate Filter
Minoru Inagaki, Yoko Matsunaga, Noriko Konagaya, Shiro Nishikawa, and Naoki Kashimura
Laboratory of Biofunctional Chemistry, Department of Bioscience, Faculty of Bioresources, Mie University, Kamihama-cho 1515, Tsu, Mie 514, Japan
Received April 26, 1996
The thermotropic effect of ganglioside GD1a on the phosphatidylcholine membrane immobilized on the porous cellulose acetate filter was investigated by ESR spin-labeling and DSC. The spin-labeled GD1a having 12-DOXYL-stearic acid instead of the long acyl chain of the ceramide portion (GD1a*) was incorporated into the model membrane. An ESR examination of the membrane showed that GD1a* undergoes an anisotropic rotation in the wide range of temperature -30-70C. By monitoring the overall splitting (2T//) of spin-labeled phosphatidylcholine (PC*), the model membranes were found to show decreased fluidity in accordance with the GD1a content. The phase transition temperature (Tm) of distealoylphosphatidylcholine (DSPC) model membranes could be estimated by the measurement of ESR and DSC. The effects of GD1a were found to be more significant in broadening the phase transition rather than in elevating the Tm of DSPC model membranes. Key words: ganglioside GD1a; phosphatidylcholine membrane; phase transition; ESR spin-labeling; DSC
Base Specificity and Primary Structure of Poly U-preferential Ribonuclease from Chicken Liver
Takashi Uchida, Kumi Hayano, Masanori Iwama, Hideaki Watanabe, Akihiro Sanda,* Kazuko Ohgi, and Masachika Irie
Department of Microbiology, Hoshi College of Pharmacy, Ebara 2-4-41, Shinagawa-ku, Tokyo 142, Japan * Faculty of Public Health, Azabu University, Fuchinobe, Sagamihara, Kanagawa 229, Japan
Received May 7, 1996
The primary structure and base specificity of chicken liver RNase CL1 which has been reported by Miura et al. [Chem. Pharm. Bull., 32, 4053-4060 (1984)] as poly U-preferential RNase, were extensively studied. The sequence study of this enzyme and comparison of the amino acid sequence of the enzyme with homologous RNases from oyster and Drosophila melanogaster suggested that RNase CL1 consists of three peptides with 17, 19, and 163 amino acid residues. The amino acid sequence of these three peptides were identified. The two small peptides are joined to the large peptide by disulfide bridges. The amino acid sequence of RNase CL1 had 62 (31.2%) and 63 residues (31.6%) identical with oyster RNase and D. melanogaster RNase, respectively, and belongs to the RNase T2 family RNase. Reassessment of the base specificity of RNase CL1 found that it is guanylic acid, then uridylic acid-preferential, and not poly U preferential. Key words: chicken liver; poly U; ribonuclease; primary structure; base specificity
Cytotoxicity of 7-Ketocholesterol toward Cultured Rat Hepatocytes and the Effect of Vitamin E
Kimiko Ohtani, Kaoru Miyabara, Emi Okamoto, Masaharu Kamei,* and Isao Matsui-Yuasa
Faculty of Human Life Science, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558, Japan * Osaka City Institute of Public Health and Environmental Science, 8-34 Higashiuemachi, Tennoji-ku, Osaka 543, Japan
Received May 9, 1996
The effects of 7-ketocholesterol on rat hepatocytes prepared by collagenase perfusion were examined. The viability of cells incubated with 100 M 7-ketocholesterol was significantly lower than those with cholesterol, although the LDH activity in the cultured medium remained unchanged during the incubation. Hepatocytes treated with 7-ketocholesterol produced large amounts of ENO and O-(/)2 in the early stage of incubation. Treatment of the hepatocytes with Carboxy-PTIO, which selectively scavenged ENO, or with L-NMMA, an inhibitor of ENO synthase, increased the cell viability. The addition of 7-ketocholesterol to the culture medium tended to increase the ratio of total sterol to phospholipid of the hepatocytes in a time-dependent manner without changing the content of phospholipid. No lipid peroxidation or oxidation of the cellular SH groups, protein SH and glutathione, was apparent. Vitamin E added 1 h before the addition of 7-ketocholesterol prevented the hepatocytes from cell death by suppressing the incorporation of 7-ketocholesterol into the hepatocytes and by scavenging O-2. Key words: cholesterol oxide; 7-ketocholesterol; cytotoxicity; hepatocyte; vitamin E
Purification and Characterization of Six Kiwifruit Proteases Isolated with Two Ion-exchange Resins, Toyopearl-SuperQ and Bakerbond WP-PEI
Sumi Sugiyama, Kozo Ohtsuki, Kenji Sato, and Makoto Kawabata*
Department of Food Science and Nutrition, Kyoto Prefectural University, Nakaragi-cho, Shimogamo, Sakyo-ku, Kyoto 606, Japan * Department of Food Science and Nutrition, Koka Woman's Junior College, Kadono-cho, Nishikyogoku, Ukyo-ku, Kyoto 615, Japan
Received May 9, 1996
Kiwifruit (Actinidia chinensis) contains a cysteine protease, actinidin, and it was suggested to contain two components, A1 and A2. However, the separation of two components was not shown, and the comparison of the two components has not been thoroughly done. We have now shown that actinidin can be separated into six proteases, named KP1, KP2, KP3, KP4, KP5, and KP6, by improved polyacrylamide gel electrophoresis at pH 4.0. Each kiwifruit protease was purified with two ion-exchange resins, Toyopearl-SuperQ and Bakerbond WP-PEI. Before the purification of kiwifruit proteases, excess p-chloromercuribenzoate was added to crude kiwifruit protease to prevent the autodigestion. Each kiwifruit protease had a molecular mass of 23,500 and the same amino terminal sequences from the first to the thirteenth. They had different pI 's. These six kiwifruit proteases were divided into two groups by the effects of DTT and Zn2+ on the activity. These results indicated that the six components must be A1, A2, and four previously unknown proteases. Thus we have separated the kiwifruit proteases which were thought to be two, into six components. Key words: kiwifruit protease; cysteine protease; actinidin; Toyopearl-SuperQ; Bakerbond WP-PEI
Oxidative Depolymerization of Chitosan by Hydroxyl Radical
Shinichiro Tanioka, Yoshihisa Matsui,* Takao Irie,* Takahiko Tanigawa, Yoshinori Tanaka, Hitoshi Shibata,* Yoshihiro Sawa,* and Yasuhisa Kono*,
Department of Bacteriology, Faculty of Medicine, Tottori University, Yonago, Tottori 683, Japan * Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, Matsue, Shimane 690, Japan
Received May 10, 1996
Oxidative depolymerization of chitosan induced by oxygen radical-generating systems was studied. Chitosan, but not chitin, was susceptible to oxidative depolymerization by hydroxyl radical generated through Cu(II)-ascorbate and ultraviolet-H2O2 systems in time- and concentration-dependent manners. Superoxide, H2O2, and singlet oxygen did not cause depolymerization. Metal ion chelators inhibited depolymerization by Cu(II)-ascorbate system, suggesting that the formation of chitosan-copper ion complex is important in the oxidative depolymerization. The molecular weight of the initial product during depolymerization was similar to that of glucosamine. The results suggest that copper ion could tend to coordinate to the NH2-groups at the terminal of chitosan and hydroxyl radical generated at its binding site cut off chitosan at the near position. Key words: chitosan; oxidative depolymerization; hydroxyl radical; site-specific Fenton reaction
Oxidative Depolymerization of Chitosan by Hydroxyl Radical
Shinichiro Tanioka, Yoshihisa Matsui,* Takao Irie,* Takahiko Tanigawa, Yoshinori Tanaka, Hitoshi Shibata,* Yoshihiro Sawa,* and Yasuhisa Kono*,
Department of Bacteriology, Faculty of Medicine, Tottori University, Yonago, Tottori 683, Japan * Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, Matsue, Shimane 690, Japan
Received May 10, 1996
Oxidative depolymerization of chitosan induced by oxygen radical-generating systems was studied. Chitosan, but not chitin, was susceptible to oxidative depolymerization by hydroxyl radical generated through Cu(II)-ascorbate and ultraviolet-H2O2 systems in time- and concentration-dependent manners. Superoxide, H2O2, and singlet oxygen did not cause depolymerization. Metal ion chelators inhibited depolymerization by Cu(II)-ascorbate system, suggesting that the formation of chitosan-copper ion complex is important in the oxidative depolymerization. The molecular weight of the initial product during depolymerization was similar to that of glucosamine. The results suggest that copper ion could tend to coordinate to the NH2-groups at the terminal of chitosan and hydroxyl radical generated at its binding site cut off chitosan at the near position. Key words: chitosan; oxidative depolymerization; hydroxyl radical; site-specific
Cloning and Nucleotide Sequence of the -D-Glucosidase Gene from Bifidobacterium breve clb, and Expression of -D-Glucosidase Activity in Escherichia coli
Naoki Nunoura, Kohji Ohdan, Keiko Tanaka, Hisanori Tamaki, Toshihiro Yano,* Masayuki Inui,** Hideaki Yukawa,** Kenji Yamamoto, and Hidehiko Kumagai
Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-01, Japan * Department of Food Science, Ishikawa Agricultural College, 1-308 Suematsu, Nonoichi-machi, Ishikawa 921, Japan ** Research Institute of Innovative Technology for the Earth (RITE), 9-2 Kizugawadai, Kizu-cho, Soraku-gun, Kyoto 619-02, Japan
Received May 27, 1996
Genomic DNA encoding a -D-glucosidase (EC 3.2.1.21), which has -D-fucosidase activity, was cloned from Bifidobacterium breve clb. We sequenced a 1.9-kbp cloned DNA fragment that contained a single open reading frame encoding 460 amino acids with a calculated molecular mass of 51,513 Da. A putative ribosome binding site was found 5 bp upstream of the initiation codon. The amino acid sequence of this -D-glucosidase from Bifidobacterium breve clb had 46% identity with that of -glucosidase from Microbispore bispore. The enzyme of Bifidobacterium breve clb was expressed in Escherichia coli. A cell-free extract prepared from the recombinant strain showed 80 to 90-fold more -D-glucosidase activity than that from Bifidobacterium breve clb. The recombinant enzyme was purified to homogeneity from cell-free extracts of the recombinant strain using 4 column chromatographies. The recovery of enzyme from the recombinant strain was about 138-fold-higher than that of Bifidobacterium breve clb. The enzymatic properties were similar to those of Bifidobacterium breve clb. For application of this recombinant enzyme, we attempted to synthesize a disaccharide that seemed to be specifically assimilated by Bifidobacteria using the condensation activity of the enzyme. Key words: Bifidobacterium breve; Escherichia coli; -d-glucosidase; -d-fucosidase; Bifidus factor
In Vitro Survey of -Glucosidase Inhibitory Food Components
Toshiro Matsui, Chiho Yoshimoto, Katsuhiro Osajima,* Tomoyuki Oki, and Yutaka Osajima
Department of Food Science and Technology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812, Japan * Research Laboratories, Senmi Ekisu Co., Hirano, Ozu 795, Japan
Received June 3, 1996
A survey of food components with -glucosidase (AGH) inhibitory activity was conducted to identify a prophylactic effect for diabetes in food. Sardine muscle hydrolyzed by alkaline protease showed potent activity (IC50=48.7 mg/ml) as well as green and oolong teas (IC50=11.1 and 11.3 mg/ml, respectively). Furthermore, hydrolyzates prepared by various proteases gave differing AGH inhibitory activity. DEAE-Sephadex chromatography of the alkaline protease hydrolyzate eluted potent AGH inhibitors (IC50= 15.6 mg/ml) with a 50 mM phosphate buffer (pH 7.0) containing 0.3 M NaCl, and their subsequent separation by HPLC in an ODS column showed that there were some inhibitors possessing primary amino groups. This indicates that they would have been high anionic and peptidic compounds. Key words: -glucosidase inhibition; diabetes; physiologically functional food
Elucidation of the Partial Structure of Polymeric Thearubigins from Black Tea by Chemical Degradation
Tetsuo Ozawa, Mari Kataoka, Keiko Morikawa, and Osamu Negishi
Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305, Japan
Received June 4, 1996
Butanol-soluble neutral and acidic thearubigins were prepared by a combination of solvent extraction, fractional precipitation, and Toyopearl column chromatography. These pigments showed similar properties to those of Roberts' thearubigins on the paper chromatogram, and a convex broad band with several peaks on reversed-phase HPLC. The thearubigins were methylated, degallated, and chemically degraded with KMnO4 under alkaline conditions. From the degradation products, methyl esters of 4-methoxy benzoic acid, 3,4-dimethoxy benzoic acid, 3,4,5-trimethoxy benzoic acid, 3,4-dimethoxy-1,2-benzenedicarboxylic acid, 3,4-dimethoxy-1,5-benzenedicarboxylic acid, 3,4-dimethoxy-1,6-benzenedicarboxylic acid, 3,4,5-trimethoxy-1,2-benzenedicarboxylic acid, 4,5,6-trimethoxy-1,2,3-benzenetricarboxylic acid, 4,5,4',5'-tetramethoxy-2,2'-diphenic acid, and 3,4,5,3',4',5'-hexamethoxy-2,2'-diphenic acid were detected by using GC-MS and GC-SIM. The butanol-soluble neutral thearubigins, which had been purified by Toyopearl column chromatography, were hydrolyzed by treating with HCl-BuOH. Cyanidin and delphinidin were identified in the reaction mixture. These results suggest that thearubigins are heterogeneous polymers of flavan-3-ols and flavan-3-ol gallates which have a bond at C4, C6, C8, C2', C5', and C6' in the flavan-3-ol units. In addition to the C4-C8 or C6 interflavanoid linkage, a C6'-C6' linkage was also found, and a possible partial structure for thearubigin is proposed. Key words: black tea; thearubigin
Effects of Hexametaphosphate on Soybean Pectic Polysaccharide Extraction
Fumihide Yamaguchi, Hiroshi Kojima, Manabu Muramoto, Yasuhide Ota,* and Chitoshi Hatanaka*
Food R & D Center, Japan Tobacco Inc., 6-2 Umegaoka, Aoba-ku, Yokohama, Kanagawa 227, Japan * Department of Applied Biochemistry, Faculty of Applied Biological Science, Hiroshima University, Higashi-Hiroshima, Hiroshima 724, Japan
Received June 6, 1996
The effects of sodium hexametaphosphate concentration, incubation temperature, and pH on the extraction of polysaccharide from soybean okara (soybean curd waste) were examined. The results suggest that the soybean polysaccharides were almost completely extracted with 50 times their volume of a 2% hexametaphosphate solution at 100C for 2 h, avoiding protein contamination. The viscosity, molecular weight, and sugar composition of the polysaccharides were compared with those of the commercially available samples. No differences were observed in the molecular weight or neutral sugar composition. However, it was found that the galacturonate distribution in the molecules of the two polysaccharides was different. The viscosity of the polysaccharide solution was changed by adding small amount of salt. Key words: soybean pectin; pectic polysaccharide; extraction; hexametaphosphate
Dipeptidyl Aminopeptidase IV from Pseudomonas sp. WO24
Wataru Ogasawara, Yoshiyuki Ogawa, Keiichi Yano,* Hirofumi Okada, and Yasushi Morikawa
Department of Bioengineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata 940-21, Japan * Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 3-6-6 Asahimachi, Machida, Tokyo 194, Japan
Received June 10, 1996
Dipeptidyl aminopeptidase IV from Pseudomonas sp. WO24 was purified as two molecular forms of 84 and 82-kDa by SDS-PAGE. Peptide mapping and N-terminal sequence analyses indicated that both proteins might be derived from the same protein, and that the 82-kDa molecule might be a truncated form from the 84-kDa molecule at least at the N-terminus. The DAP IV gene of Pseudomonas sp. WO24 was cloned and expressed in E. coli. The enzyme expressed in E. coli JM109 harboring a hybrid plasmid, pYO-6A, with about a 3-kbp fragment containing the DAP IV gene, was purified with an activity recovery of 24%. The recombinant enzyme also had the same two molecular forms, though the ratio of the two forms (about 1 : 1) was different from that of the native ones (about 1 : 4). The native and recombinant enzyme preparations had similar specific activities, suggesting that the 84 and 82-kDa molecules are in an active form and have almost the same specific activity. The molecular mass, the subunit number, the substrate specificity, and the effects of various inhibitors of the native enzyme indicated that this enzyme was a typical DAP IV and had properties similar to those of Flavobacterium meningosepticum rather than others. Key words: dipeptidyl aminopeptidase IV; Pseudomonas; purification; cloning; expression
Hydrolytic Activity of -Mannosidase against Deoxy Derivatives of p-Nitrophenyl -D-Mannopyranoside
Toshiyuki Nishio, Yayoi Miyake, Hitomi Tsujii, Wataru Hakamata, Kazunari Kadokura, and Tadatake Oku
Laboratory of Bio-organic Chemistry, College of Bioresource Sciences, Nihon University, 3-34-1 Shimouma, Setagaya-ku, Tokyo 154, Japan
Received June 10, 1996
Deoxy derivatives of p-nitrophenyl (PNP) -D-mannopyranoside, PNP 2-deoxy--D-arabino-hexopyranoside, 3-deoxy--D-arabino-hexopyranoside, 4-deoxy--D-lyxo-hexopyranoside, and -D-rhamnopyranoside, were synthesized and hydrolytic activities of jack bean and almond -mannosidases against them were investigated. These -mannosidases scarcely acted on the 2-, 3-, and 4-deoxy derivatives, while the 6-deoxy one was hydrolyzed by the enzymes as fast as PNP -D-mannopyranoside, which is a common substrate for -mannosidase. These results indicate that the hydroxyl groups at C-2, 3, and 4 of the mannopyranoside are necessary to be recognized as a substrate by these enzymes, while that at C-6 does not have so a crucial role in substrate discrimination. Values of Km and Vmax of the enzymes on the hydrolysis of PNP -D-rhamnopyranoside were obtained from kinetic studies. Key words: -mannosidase; substrate specificity; glycon hydroxyl groups; PNP deoxy--d-mannopyranosides; synthesis
3-Hydroxyisobutyrate Dehydrogenase from Pseudomonas putida E23: Purification and Characterization Emran Kabir Chowdhury,
Shinji Nagata, and Haruo Misono
Laboratory of Applied Microbiology, Department of Bioresources Science, Kochi University, Nankoku, Kochi 783, Japan
Received June 11, 1996
The NAD+-dependent 3-hydroxyisobutyrate dehydrogenase [EC 1.1.1.31] was purified to homogeneity from Pseudomonas putida E23. The enzyme was a tetramer (molecular mass, 120 kDa) consisted of identical subunits (molecular mass, 30 kDa). The enzyme was specific for NAD+ (Km, 0.44 mM). The maximal activity was obtained at about pH 10. The enzyme was specific for the L-isomer of 3-hydroxyisobutyrate. In addition to L-3-hydroxyisobutyrate, L-serine, 2-methyl-DL-serine, and 3-hydroxypropionate were substrates. The Km for L-3-hydroxyisobutyrate, L-serine, 2-methyl-DL-serine, and 3-hydroxypropionate were 0.12, 18, 44, and 83 mM, respectively. The enzyme was inhibited by p-chloromercuribenzoate, HgCl2, and AgNO2, but not by EDTA, ,'-dipyridyl, and o-phenanthroline. The N-terminal 26 amino acid sequence was compared with the sequences deduced from the enzyme genes of rat liver and Pseudomonas aeruginosa. Key words: 3-hydroxyisobutyrate dehydrogenase; Pseudomonas putida; oxidation of l-serine
Purification and Characterization of a Lectin from Amaranthus hypochondriacus var. Mexico Seeds
Munetaka Ozeki, Kazuo Kamemura, Kikuko Moriyama, Yayoi Itoh, Yukio Furuichi, Hayato Umekawa, and Takao Takahashi
Department of Agricultural Chemistry, Faculty of Bioresources, Mie University, Tsu, Mie 514, Japan
Received June 13, 1996
A lectin from Amaranthus hypochondriacus var. Mexico (AHML) was purified by affinity chromatography using asialofetuin-Sepharose 4B. AHML is specific for N-acetyl-D-galactosamine as are the other Amaranthus lectins. AHML has no carbohydrate moiety and requires no metal ion for the hemagglutination activity. The pI of AHML is 6.8. AHML has a native molecular mass of 45.0 kDa and is composed of homo-subunits having molecular masses of 36.8 kDa. Key words: Amaranthus; lectin; hemagglutinin; purification
Stable Isotope Labeled Cytochrome c2 from Desulfovibrio vulgaris on a Defined Medium as Sole Nitrogen Source
Tomoaki Ohmura, Hideo Akutsu,* and Takeshi Nakamura
Biotechnology Laboratory, Advanced Technology Research Center, Mitsubishi Heavy Industries, Ltd., Kanazawa-ku, Yokohama, Kanagawa 236, Japan * Department of Bioengineering, Faculty of Engineering, Yokohama National University, Hodogaya-ku, Yokohama, Kanagawa 240, Japan
Received June 26, 1996
The conditions of cultivation and the composition of medium for Desulfovibrio vulgaris Miyazaki F (DvMF) were examined to obtain cytochrome c2 labeled with a stable isotope. The growth of DvMF was steady and reproducible under purging with N2 and under pH control. DvMF was able to grow on a defined medium without natural products. The composition of medium containing a small amount of NH4Cl as sole nitrogen source was established. Then, [U-15N]cytochrome c2 was obtained during the culture of DvMF in a defined medium with 15NH4Cl; it was confirmed by 1H-15N HMQC. This is the first report of [U-15N]cytochrome c2. Key words: sulfate-reducing bacteria; Desulfovibrio vulgaris; cytochrome c2; stable isotope
Steady-State Kinetic and Calorimetric Studies on the Binding of Aspergillus niger Glucoamylase with Gluconolactone, 1-Deoxynojirimycin, and -Cyclodextrin
Akiyoshi Tanaka
Department of Chemistry, Faculty of Education, Mie University, 1515 Kamihama, Tsu, Mie 514, Japan
Received July 15, 1996
Binding equilibria of Aspergillus niger glucoamylase with its several ligands were observed to analyze the binding modes of the ligands. Steady-state kinetic studies using p-nitrophenyl -glucoside as a substrate showed that 1-deoxynojirimycin, which is a mixed type inhibitor for Rhizopus glucoamylase, was a competitive type inhibitor bound at the active site of the enzyme, but gluconolactone, which is also a mixed type inhibitor for Rhizopus glucoamylase, was a non-competitive type inhibitor forming a nonproductive ternary complex with the enzyme and the substrate. -Cyclodextrin, which binds to the starch-binding domain of the enzyme, did not inhibit the enzyme activity, showing that there was no interaction between the catalytic domain and the starch-binding domain for the binding of the substrate and -cyclodextrin. Isothermal titration calorimetry showed that one 1-deoxynojirimycin molecule and two -cyclodextrin molecules bind to the catalytic domain and the starch-binding domain of the enzyme, respectively, and there is no significant interaction between the binding of these ligands. Key words: domain structure; domain interaction; glucoamylase; steady-state kinetics; titration calorimetry
Efficient Lipase-catalyzed Preparation of Long-chain Fatty Acid Esters of Bile Acids: Biological Activity and Synthetic Application of the Products
Takeshi Sugai, Masahiro Takizawa, Mikio Bakke, Yoshikazu Ohtsuka, and Hiromichi Ohta
Department of Chemistry, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223, Japan
Received July 22, 1996
A highly regioselective (3-position) and efficient (quantitative yield) acylation of bile acids catalyzed by immobilized Candida antarctica lipase was established. Methyl cholate derivatives acylated with long-chain fatty acids (C12-C16) showed an inhibitory effect on the growth of some strains of Gram-positive and -negative bacteria (27-400 g/ml). The anti-bacterial activity was slightly weaker than has been observed for methyl cholate, while the increased lipophilicity and lower melting points of the present derivatives are well suited for a potential germicide which would be safe and be topically applied. This enzyme-catalyzed transesterification is also demonstrated as an expeditious route to ursodeoxycholic acid, in respect of the regioselective introduction of acyl protecting groups on the hydroxyl groups of the intermediates. 7-Ketolithocholic acid, a known direct precursor of ursodeoxycholic acid, was obtained from cholic acid via chenodeoxycholic acid in a 46% yield and 9 steps. Key words: Candida antarctica lipase; transesterification; bile acid; antibacterial activity; ursodeoxycholic acid
Dextran Raffinose Derivative Assay for Affinity Purified Corn Coleoptile Lectin
Adriana Merida, Leonardo Vasquez, Margarito Martinez, Teresa Aquino, Eduardo Perez-Campos, and Felix Cordoba*,
Biochemistry and Immunology Unit of Technological Institute of Oaxaca, and * Departments of Biochemistry and Experimental Medicine, National University of Mexico School of Medicine, P.O. Box 872, Oaxaca 68000, Oaxaca, Mexico
Received September 21, 1995
Corn coleoptile lectin was isolated using lactose/agarose affinity chromatograpy. The purified lectin, displaying a single band in polyacrylamide gel electrophoresis, was studied by a technique using dextran-raffinose biotinylated derivatives. Raffinose, lactose, lactulose, N-acetyl-D-galactosamine, glucose, and L-fucose were used to measure the inhibition and binding activity of the lectin. The inhibition pattern obtained agreed with previous hemagglutination inhibition data. Key words: derivative; raffinose; corn; coleoptile; lectin
Analysis of (-)-Epigallocatechin Gallate in Human Serum Obtained after Ingesting Green Tea
Tomonori Unno, Kazuo Kondo,* Hiroshige Itakura,* and Tadakazu Takeo
Central Research Institute, Itoen Ltd., 21 Mekami, Sagara-cho, Haibara-gun, Shizuoka 421-05, Japan * Division of Clinical Nutrition, The National Institute of Health and Nutrition, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162, Japan
Received November 29, 1995
A method for analyzing the EGCg concentration in human serum was developed by using high-performance liquid chromatography with electrochemical detection. EGCg was detected in human serum after the ingestion of 5 g of green tea powder (matsu-cha) dissolved in 200 ml of hot water. The concentration of EGCg in the serum reached the highest level about 2 h after ingesting the green tea, and then decreased. Key words: HPLC analysis; EGCg; human serum; HPLC-ECD; absorption
Isolation and Characterization of a Plasmid from Lactobacillus helveticus CP53
Naoyuki Yamamoto and Toshiaki Takano
R & D Center, The Calpis Food Industry Co., Ltd., 11-10 Fuchinobe 5-chome, Sagamihara, Kanagawa 229, Japan
Received April 26, 1996
A plasmid, pCP53 (11.5 kilo base pairs long), was isolated from Lactobacillus helveticus CP53 and its restriction map was constructed. A chimeric plasmid, pCP53D (4.7 kb), which had a 2.0-kb Hin dIII fragment from pCP53 and a tetracycline resistance (Tc) gene from Streptococcus faecalis replicated and expressed tetracycline resistance in Lactobacillus casei CP680. Fifteen strains of L. helveticus were transformed with pCP53D by electroporation and selected for Tc resistance. Transformants were obtained from two out of 15 host strains, L. helveticus CP611 and CP791, with 5~103 and 50 transformants per g of DNA, respectively. Key words: plasmid; Lactobacillus helveticus; tetracycline resistance; replication origin; plasmid
Effect of Sesamin on the Composition of Eicosapentaenoic Acid in Liver and on Its Lymphatic Absorption in Rats
Rumi Umeda-Sawada, Megumi Ogawa, Yuko Okada, and Osamu Igarashi
Institute of Environmental Science for Human Life, Ochanomizu University, 2-1-1 Otsuka, Bunkyo-ku, Tokyo 112, Japan
Received May 30, 1996
Changes in the hepatic concentration of n-3 fatty acids, e.g., eicosapentaenoic acid and linolenic acid, were significantly reduced by their simultaneous administration with sesamin, whereas there was no such effect of sesamin for n-6 and n-9 fatty acids. However, there was no significant difference in lymphatic absorption between eicosapentaenoic acid (n-3) and arachidonic acid (n-6), irrespective of the presence or absence of sesamin. Key words: sesamin; EPA; lymphatic absorption; n-3 fatty acid; n-6 fatty acid
Characterization of Endogenous Gibberellins in Dwarf Rice Mutants
Ichiro Honda, Kazuhisa Sudo, Shinya Iwasaki, Tadashi Yanagisawa, Isomaro Yamaguchi,* Hiroshi Kato,** Ryoichi Ikeda,** Hideo Hirasawa,** and Nobutaka Takahashi ***
Department of Applied Biochemistry, Utsunomiya University, Utsunomiya 321, Japan * Department of Applied Biological Chemistry, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan ** National Agriculture Research Center, Ministry of Agriculture, Forestry, and Fisheries, Tsukuba 305, Japan *** Frontier Research Program, The Institute of Physical and Chemical Research (RIKEN), Wako 351-01, Japan
Received May 30, 1996
The elongation response of 17 near-isogenic dwarf lines of the Shiokari rice cultivar to gibberellins (GAs) was examined. Two strains, ID-18h and ID-19, were selected for further analysis of their endogenous GA levels. It is suggested that ID-18h dwarfism is caused by the deficiency of GA 3-hydroxylation activity, and ID-19 dwarfism by the insensitivity of the elongation response to physiologically active GAs. Key words: dwarf; Oryza sativa; gibberellin; Shiokari; GC-MS
Transduction of a Cosmid with the R4 Phage cos Sequence by Heterogeneous Actinophages, SPA10 and SPA38
Tomio Morino and Hideo Takahashi *
Research and Development Division, Pharmaceutical Group, Nippon Kayaku Co., Ltd., 3-31-12 Shimo, Kita-ku, Tokyo 115, Japan * Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113, Japan
Received May 31, 1996
A cosmid, pR4C1, composed of the actinophage R4 cos sequence and Streptomyces plasmid pIJ365, was encapsidated in R4 phage particles in vivo. [T. Morino et al., Mol. Gen. Genet., 198, 228-233 (1985)] In this report a cosmid derivative, pR4C4, is shown to be also encapsidated by heterogeneous actinophages, SPA10 and SPA38, and transferred to Streptomyces lividans. Use of this transduction and conservation of the DNA packaging mechanism are discussed. Key words: cosmid transduction; R4 phage; SPA phage(s); cos sequence; in vivo packaging
Purification and Partial Characterization of an Endo-(13,14)--glucanase from Rice, Oryza sativa L.
Takashi Akiyama, Hanae Kaku,* and Naoto Shibuya*
National Hokkaido Agricultural Experiment Station, 1 Hitsujigaoka, Toyohira, Sapporo 062, Japan * Department of Cell Biology, National Institute of Agrobiological Resources, Tsukuba, Ibaraki 305, Japan
Received June 5, 1996
(13,14)--Glucanase (EC 3.2.1.73), with a molecular weight of 34,000 and an isoelectric point of 4.9, was purified to homogeneity from extracts of fresh rice bran. The enzyme specifically hydrolyzed (13,14)--glucans such as barley -glucan and lichenans, but laminarins and CM-cellulose were not substrates. Endproduct analysis using barley -glucan as the substrate suggested that the enzyme is an endo-type (13,14)--glucanase. Key words: (13,14)--glucanase; rice grain; Oryza sativa
Fungerin, a New Antifungal Alkaloid from Fusarium sp. Yumiko Kato, Hiroyuki Koshino,*,
Jun Uzawa,* and Kentaro Anzai
Department of Chemistry, Faculty of Science, Science University of Tokyo, Kagurazaka, Shinjuku-ku, Tokyo 162, Japan * The Institute of Physical and Chemical Research (RIKEN), Wako, Saitama 351-01, Japan
Received June 6, 1996
A new antifungal alkaloid named fungerin was isolated as a fungal metabolite of Fusarium sp. The structure of fungerin was elucidated principally by NMR spectroscopy involving 15N-NMR studies using the pulsed field gradient (PFG)-HMBC technique at natural abundance. Key words: fungerin; antifungal substance; Fusarium; 15N-NMR; 1H-15N HMBC
Inhibitory Effect of Dietary Wheat Bran on Formation of Aberrant Crypt Foci in Rat Colon Induced by a Single Injection of 1,2-Dimethylhydrazine
Satoshi Ishizuka and Takanori Kasai
Department of Bioscience and Chemistry, Faculty of Agriculture, Hokkaido University, Kita-9, Nishi-9, Kita-ku, Sapporo 060, Japan
Received June 13, 1996
The frequency of appearance of aberrant crypt foci (ACF) in the distal colon was significantly lower in rats fed a high fiber (20% wheat bran) diet than in those fed a fiber-free one at 4 weeks after a single injection of 1,2-dimethylhydrazine (DMH, 20 mg/kg), although crypt/ACF was high in the former relative to the latter. This result suggests that dietary wheat bran effectively serves as a regulator of ACF frequency at early stages after DMH injection. Key words: aberrant crypt foci; 1,2-dimethylhydrazine; wheat bran; rat
Antimutagenic Effects of Ajoene, an Organosulfur Compound Derived from Garlic
Keiko Ishikawa, Rie Naganawa, Hisae Yoshida, Nami Iwata, Hiroyuki Fukuda, Tsuchiyoshi Fujino, and Atsushi Suzuki
Bio Development Division, Central Institute, Nagoya Seiraku Co., Ltd., 310 Nakasuna-cho, Tenpaku-ku, Nagoya 468, Japan
Received June 17, 1996
The antimutagenic effects of ajoene, which is an organosulfur compound derived from garlic, were investigated by the Ames test. Ajoene inhibited mutagenesis induced by both benzo[a]pyrene (B[a]P) and 4-nitro-1,2-phenylenediamine (NPD) in a dose-dependent manner. In particular, NPD-induced mutagenesis was more effectievly suppressed by ajoene than the B[a]P-induced type. Furthermore, the inhibition of mutagenesis by ajoene was more effective for transition-type mutations than for the frame shift type. HPLC analysis of B[a]P metabolism in the presence of the rat liver microsomal fraction (S-9) showed that ajoene dose-dependently inhibited the metabolic activation of B[a]P. This suggests that ajoene affected the metabolic enzymes in the S-9 fraction. Key words: antimutagenic effect; ajoene; garlic; Ames test
Bacterial Production and Purification of Phosphorylatable Phosphoenolpyruvate Carboxylase from Tobacco
Nozomu Koizumi,*, Fumihiko Sato, and Yasuyuki Yamada
Department of Agricultural Chemistry, Faculty of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-01, Japan
Received June 17, 1996
Tobacco phosphoenolpyruvate carboxylase (PEPC) [EC 4.1.1.31] cDNA was efficiently expressed in E. coli under the control of the lacZ promoter. The enzyme, purified to homogeneity, had the same catalytic activity, and was phosphorylated in vitro by maize PEPC kinase. Key words: bacterial expression; cDNA; phosphoenolpyruvate carboxylase; phosphorylation; tobacco
Preparation and Some Properties of Type I Collagen from Fish Scales
Yoshihiro Nomura, Hiromitu Sakai,* Yasuhiro Ishii, and Kunio Shirai
Applied Protein Chemistry, Scleroprotein and Leather Research Institute, Faculty of Agriculture, Tokyo University of Agriculture and Technology, Fuchu-shi, Tokyo 183, Japan
Received June 24, 1996
Soluble collagen from fish (sardine) scales was yielded at about 5% with 0.5 M acetic acid after demineralization with EDTA, while a great portion of the collagen remained insoluble. The solubility of this insoluble collagen was about 20% at 45C (denaturation temperature of soluble collagen) for 24 h. The remaining 80% of the insoluble collagen was denatured in the form of insoluble gelatin, and that may be an interesting food material. Key words: collagen; scale; denaturation temperature; DSC; optical rotation
Synthesis of Tropolone Glucopyranosides
Zong-Wen Li, Zhi-Hong Li, Zhong-Tian Jin, and Kimiaki Imafuku*,
Department of Chemistry, Yanbian University, Yanji, Jilin Province 133002, People's Republic of China * Department of Chemistry, Faculty of Science, Kumamoto University, Kurokami, Kumamoto 860, Japan
Received June 26, 1996
Reactions of tropolone (1a) and 3-isopropenyl-, 3-acetyl-, 3-acetamido-, and 5-bromotropolones 1b-e with 2,3,4,6-tetra-O-acetyl--D-glucopyranosyl bromide were carried out in the presence of silver carbonate at 80C. Unsubstituted, 3'-/7'-isopropenyl-, 7'-acetyl-, 7'-acetamido-, and 5'-bromo-substituted 2'-troponyl 2,3,4,6-tetra-O-acetyl--D-glucopyranosides were respectively obtained. Key words: tropolone; glucopyranosides; Koenigs-Knorr method
Microbial Transformation of Indene by the Pyricularia zingiberi Nishikado Fungus
Manabu Nukina, Shin Ito, and Takayuki Kurebayashi
Department of Bioproduction, Faculty of Agriculture, Yamagata University, Wakaba-machi, Tsuruoka-shi, Yamagata 997, Japan
Received June 26, 1996
Indene was found to be microbially transformed to (-)-(1R,2S )-cis-1,2-indandiol (7.3% yield, 15% e.e.), (+)-(1R,2R)-trans-1,2-indandiol (3% yield, 83% e.e.), (-)-(R)-indenol (0.1% yield, 89% e.e.), (+)-(S )-2-hydroxy-1-indanone (0.1% yield, 31% e.e.), 1-indanone (0.2% yield), and (+)-(S )-1-indanol (trace, 99% e.e.) by the Pyricularia zingiberi fungus. Key words: indene metabolism; microbial transformation; Pyricularia zingiberi
N-Linked Sugar Chain of 55-kDa Royal Jelly Glycoprotein
Yoshinobu Kimura,* Shin-ichiro Kajiyama,*, Jun Kanaeda,** Tomomi Izukawa,*** and Masami Yonekura***,
* Department of Bioresources Chemistry, Faculty of Agriculture, Okayama University, Tsushima-Naka, Okayama 700, Japan ** Research Institute, API Company, Limited, Kanoh, Gifu 500, Japan *** Department of Applied Biological Resource Sciences, School of Agriculture, Ibaraki University, Ami-machi, Ibaraki 300-03, Japan
Received July 22, 1996
An N-linked sugar chain from 55-kDa royal jelly glycoportein (RJGP), which maintains the high viability of rat liver primary cultured cell and is a different molecular species from 350-kDa RJGP [Kimura et al., Biosci. Biotech. Biochem., 59, 507-509 (1995)], has been identified. The sugar chains were released by hydrazinolysis followed by N-acetylation and pyridylamination. The structural analysis of the pyridylaminated sugar chain was done by a combination of sequential exo-mannosidase digestions, MALDI-TOF MS, and 500 MHz 1H-NMR. For the carbohydrate moiety of 55-kDa RJGP, only one N-linked sugar chain has been detected. The structure has been found to be Man12Man16(Man1 2Man13)Man16(Man12Man12Man13)Man1 4GlcNAc14GlcNAc, which is a non-processed high mannose type structure. Key words: royal jelly; insect glycoprotein; N-glycan; PA-sugar chain; MALDI-TOF MS
Isolation and Structure of Ciguatoxin-4A, a New Ciguatoxin Precursor, from Cultures of Dinoflagellate Gambierdiscus toxicus and Parrotfish Scarus gibbus
Masayuki Satake, Yoshihiko Ishibashi, Anne-Marie Legrand,* and Takeshi Yasumoto
Faculty of Agriculture, Tohoku University, Tsutsumidori-Amamiya, Aoba-ku, Sendai 981, Japan * Institut Territorial de Recherches Medicales Louis Malarde, BP 30, Papeete, Tahiti, French Polynesia
Received August 16, 1996
A new ciguatoxin congener, ciguatoxin-4A (CTX4A), was isolated from cultures of marine dinoflagellate Gambierdiscus toxicus, and its structure was elucidated to be 52-epiciguatoxin-4B on the basis of spectroscopic data. Chromatographic and spectral comparisons indicated that CTX4A was identical with a structurally unelucidated congener known as scaritoxin or SG1. Key words: ciguatera; ciguatoxin-4A (CTX4A); dinoflagellate; Gambierdiscus toxicus; scaritoxin