(Vol.65 No.11 2001)
Effects of Polysaccharides from Rhizomes of Curcuma zedoaria
on Macrophage Functions
Kyung Im KIM, Kwang Soon SHIN,1 Woo Jin JUN, Bum Shik HONG,† Dong Hoon SHIN,
Hong Yon CHO, Hyo Il CHANG, Seong Mo YOO,2 and Han Chul YANG p.2369
Antimicrobial Characteristics of Chitosans against Food Spoilage
Microorganisms in Liquid Media and Mayonnaise
Hoon Il OH,† Yong Jin KIM, Eun Jung CHANG, and Jee Young KIM p.2378
Structural Characterization and Immunomodulating Activity
of a Complex Glucan from Spores of Ganoderma lucidum
Xingfeng BAO, Jinian FANG,† and Xiaoyu LI p.2384
Adsorption Properties and Activities of Lipase on a Gold Substrate
Modified
by Self-assembled Monolayers
Atsushi KOBAYASHI, Yukari SATO,† and Fumio MIZUTANI p.2392
Purification and Characterization of a Monohalomethane-producing
Enzyme
S-adenosyl-L-methionine: Halide Ion Methyltransferase
from a Marine Microalga, Pavlova pinguis
Noboru OHSAWA,1 Mika TSUJITA,2 Satoru MORIKAWA,2 and Nobuya ITOH1,† p.2397
Immunochemical and Mutational Analyses of P-type ATPase Spf1p
Involved
in the Yeast Secretory Pathway
Chise SUZUKI p.2405
Hydrolysis of a Chitosan-induced Milk Aggregate by Pepsin, Trypsin
and Pancreatic Lipase
Salvador F. AUSAR, Carlos A. LANDA, Ismael D. BIANCO, Leonardo F. CASTAGNA,
and Dante M. BELTRAMO† p.2412
Binding Properties of Bacillus thuringiensis Cry4A Toxin to
the Apical
Microvilli of Larval Midgut of Culex pipiens
Masashi YAMAGIWA,＊ Shinya KAMAUCHI,＊ Takayuki OKEGAWA,＊ Motoyuki ESAKI,＊＊
Kanao OTAKE,＊＊ Teruo AMACHI,＊＊ Tohru KOMANO,＊＊＊ and Hiroshi SAKAI＊
p.2419
Role of Conserved Residues of the WRKY Domain in the DNA-binding
of Tobacco WRKY Family Proteins
Kenichiro MAEO,† Shingo HAYASHI, Hisae KOJIMA-SUZUKI, Atsushi MORIKAMI,1
and Kenzo NAKAMURA p.2428
Purification and Characterization of Two Lectins from a Toxic
Moray,
Gymnothrax javanicus
Hirokazu KAWAGISHI,1† Masaaki YASUI,1 Akinori UNO,1 Takeomi MURATA,1
Taichi USUI,1 and Syoei FURUKAWA2 p.2437
Capsaicin-, Resiniferatoxin-, and Olvanil-induced Adrenaline
Secretions
in Rats via the Vanilloid Receptor
Tatsuo WATANABE, Nobuo SAKURADA, and Kenji KOBATA p.2443
Intestinal Absorbability of Wheat Allergens, Subunits of a
Wheat α-Amylase
Inhibitor, Expressed by Bacteria
Mioko KUSABA-NAKAYAMA, Masami KI, Emiko KAWADA, Masao SATO, Ikuo IKEDA,
Toshihiro MOCHIZUKI,＊ and Katsumi IMAIZUMI† p.2448
Enzymatic Synthesis of Sulfated Disaccharides using β-D-Galactosidase-
catalyzed Transglycosylation
Takeomi MURATA,1,† Masaki KOSUGI,1 Tadashi NAKAMURA,2 Tadasu URASHIMA,2
and Taichi USUI1 p.2456
Substrate Recognition Mechanism of Carboxypeptidase Y
Hiroshi NAKASE, Shoichi MURATA, Hiroshi UENO, and Rikimaru HAYASHI† p.2465
Oxygenation Reactions of Various Tricyclic Fused Aromatic Compounds
Using Escherichia coli and Streptomyces lividans Transformants
Carrying Several Arene Dioxygenase Genes
Kazutoshi SHINDO,1 Yasuo OHNISHI,2 Hyo-Kon CHUN,2 Haruko TAKAHASHI,3 Miki HAYASHI,4
Atsushi SAITO,5 Kazuo IGUCHI,3 Kensuke FURUKAWA,6 Shigeaki HARAYAMA,5
Sueharu HORINOUCHI,2 and Norihiko MISAWA4† p.2472
Glycoproteins Secreted from Suspension-cultured Tobacco BY2
Cells have
Distinct Glycan Structures from Intracellular Glycoproteins
Ryo MISAKI, Yoshinobu KIMURA,＊ Kazuhito FUJIYAMA,† and Tatsuji SEKI p.2482
Effects of Bovine β-Casein (1--28) and Its Chemically Synthesized
Partial
Fragments on Proliferative Responses and Immunoglobulin Production
in Mouse Spleen Cell Cultures
Hajime OTANI†, Takehito WATANABE, and Yasuhito TASHIRO＊ p.2489
Lecithin:Cholesterol Acyltransferase Reduces the Adverse Effects
of Oxidized
Low-Density Lipoprotein while Incurring Damage Itself
Zakir H. HOWLADER, Shin KAMIYAMA, Yohei MURAKAMI, Michiko ITO,
Michio KOMAI, and Yuji FURUKAWA† p.2496
Regulation of Hydroxyindole-O-methyltransferase Gene Expression
in Japanese Quail (Coturnix coturnix japonica)
Zhengwei FU,1 Hisanori KATO,1,† Naoko KOTERA,2 Tadashi NOGUCHI,1
Kunio SUGAHARA,2 and Tatsuo KUBO2 p.2404
Characterization and Overproduction of EcoO109I Methyltransferase
Keiko KITA, Junko TSUDA, and Ryu-ichi NISHIGAKI p.2512
A Comparative Study on Structural Elucidation of Sialyl Oligosaccharides
by Mass Spectrometry with Fast Atom Bombardment, Electrospray Ionization,
and Matrix Assisted Laser Desorption/Ionization
Masahiko OKAMOTO p.2519
Radicicol Binding to Swo1/Hsp90 and Inhibition of Growth of
Specific
Temperature-sensitive Cell Cycle Mutants of Fission Yeast
Se Won KI,1,＊ Koji KASAHARA,1,<Oh><0167><Wa> Ho Jeong KWON,1,‡
Ken ISHIGAMI,2 Takeshi KITAHARA,2
Teruhiko BEPPU,1, Minoru YOSHIDA,1,3,† and Sueharu HORINOUCHI1 p.2528
Dietary Manipulations of Body Fat-reducing Potential
of Conjugated Linoleic Acid in Rats
Michihiro SUGANO,1 Asuka AKAHOSHI,1 Kazunori KOBA,2 Kazunari TANAKA,2
Tomoka OKUMURA,1 Hiroko MATSUYAMA,1 Yuki GOTO,1 Tomoko MIYAZAKI,1
Kayoko MURAO,1 Masao YAMASAKI,3 Michiko NONAKA,3 and Koji YAMADA3 p.2535
Crystalline Behavior of Chitosan Organic Acid Salts
Jumpei KAWADA,† Toshifumi YUI,1 Kenji OKUYAMA,2 and Kozo OGAWA†† p.2542
Note
Regulation of Cigarette Smoke-Induced Cytochrome P4501A1 Gene
Expression in Osteogenic Disorder Shionogi Rat Liver and in Lung
by Large Ascorbic Acid Dose
Etsuko UETA,1 Emiko SUZUKI,2 Eiji NANBA,3 Yuko TADOKORO,1
Yuzuru OTSUKA,4,† and Tadao KURATA1 p.2548
Note
Extremely Low Frequency Magnetic Fields Suppress the Reduction
of Germination Rate of Arabidopsis thaliana Seeds Kept
in Saturated Humidity
Koichi TAKIMOTO,1,† Hiroko YAGUCHI,2,3,†† and Junji MIYAKOSHI3 p.2552
Note
Protective Effect of Ajoene on Acetaminophen-induced Hepatic Injury in Mice
Atsuhiko HATTORI, Norihiko YAMADA, Tomoaki NISHIKAWA,
Hiroyuki FUKUDA, and Tsuchiyoshi FUJINO† p.2555
Note
A New Type of Allantoinase in Amphibian Liver
Wataru MASUDA, Satoko FUJIWARA and Tomoo NOGUCHI p.2558
Note
Induction and Secretion of RNA-degrading Enzymes
by Phosphate Deficiency in Pholiota nameko
Toshio JOH,1 Yuji TASAKI,2 Takashi HARA,1 and Toshiro HAYAKAWA1 p.2561
Note
Changes of Pyridine Nucleotide Levels during Adipocyte Differentiation
of Mouse 3T3-L1 Cells
Tsutomu FUKUWATARI,1,† Miki DOI,1 Etsuro SUGIMOTO,1 Teruo KAWADA,2
and Katsumi SHIBATA1 p.2565
Note
Synthesis of (R)-(+)-Hippospongic Acid A, a Triterpene Isolated
from the Marine Sponge, Hippospongia sp.†
Miwako ICHIHASHI, Hirosato TAKIKAWA,†† and Kenji MORI††† p.2569
Note
Measurement of Free Choline in Plant Leaves by Capillary Electrophoresis
Jinghua ZHANG,† Akira OKUBO,†† and Sunao YAMAZAKI＊ p.2573
Note
A Novel Protein, Mpm1, of the Mitochondria of the Yeast
Saccharomyces cerevisiae
Hironori INADOME, Yoichi NODA, Hiroyuki ADACHI, and Koji YODA† p.2557
Note
Induction of Phagocyte Oxidase Components during Human Myeloid
Differentiation: Independent Protein Expression and Discrepancy
with the Function
Yoko INOUE, Masako YAGISAWA, Kumiko SAEKI, Shinobu IMAJOH-OHMI,＊
Shiro KANEGASAKI,＊ and Akira YOU p.2581
Preliminary Communication
Structure Elucidation of Ostreocin D, a Palytoxin Analog Isolated
from the Dinoflagellate Ostreopsis siamensis
Takanori UKENA,1 Masayuki SATAKE,1,† Masaya USAMI,1 Yasukatsu OSHIMA,1
Hideo NAOKI, 2 Tsuyoshi FUJITA,2 Yukiko KAN,2 and Takeshi YASUMOTO1 p.2585
-1-
Effects of Polysaccharides from Rhizomes of Curcuma zedoaria
on Macrophage Functions
Kyung Im KIM, Kwang Soon SHIN,1 Woo Jin JUN, Bum Shik HONG,† Dong Hoon SHIN,
Hong Yon CHO, Hyo Il CHANG, Seong Mo YOO,2 and Han Chul YANGGraduate School of Biotechnology, Korea University, Seoul 136-701, Korea
1Department of Food Science and Biotechnology, Kyonggi University, Suwon, Kyonggi-do 442-760, Korea
2Department of Informational Statistics, Korea University, Seochang, Chungnam 339-700, KoreaReceived January 19, 2001; Accepted July 5, 2001
The effects of Curcuma zedoaria, which is used as a condiment, in perfumery, and as a medicine, on immune response were investigated by measuring macrophage-stimulating activity in macrophages and RAW 264.7 cells. In this study, CZ-1 and CZ-1-III, the fractions partially purified from C. zedoaria, had a strong, dose-dependent lysosomal enzyme activity. It was suggested that active portions of CZ-1-III were polysaccharides rather than proteins. Phagocytic activity increased as a similar pattern in both the Gram-negative and Gram-positive bacteria, time-dependently. It was demonstrated that CZ-1-III can augment the oxygen burst response but had an even higher activity in vivo than in vitro. Also a significant increase of H2O2, NO, and TNF-α production was observed. However, the production of TNF-α at the concentration of 1,000 μg/ml decreased. These data suggested that C. zedoaria had macrophage-stimulating activity and the possibility of being used as a biological response modifier.
Key words: macrophages; Curcuma zedoaria; phagocytosis; reactive oxygen species; TNF-α
-2-
Antimicrobial Characteristics of Chitosans against Food Spoilage
Microorganisms in Liquid Media and Mayonnaise
Hoon Il OH,† Yong Jin KIM, Eun Jung CHANG, and Jee Young KIM
Department of Food Science and Technology, Sejong University, Seoul 143-747, Korea
Received February 28, 2001; Accepted July 2, 2001
Four different kinds of chitosans were prepared by treating crude chitin with various NaOH concentrations. The antimicrobial activities of the chitosans were tested against four species of food spoilage microorganisms (Lactobacillus plantarum, Lactobacillus fructivorans, Serratia liquefaciens, and Zygosaccharomyces bailii). The initial effect of the chitosans was biocidal, and counts of viable cells were significantly reduced. After an extended lag phase, some strains recovered and resumed growth. The activities of chitosan against these microorganisms increased with the concentration. Chitosan-50 was most effective against L. fructivorans, but inhibition of L. plantarum was greatest with chitosan-55. There was no significant difference among the chitosans in their antimicrobial activity against S. liquefaciens and Z. bailii. The addition of chitosan to mayonnaise significantly decreased the viable cell counts of L. fructivorans and Z. bailii during storage at 25°C. These results suggest that chitosan can be used as a food preservative to inhibit the growth of spoilage microorganisms in mayonnaise.
Key words: chitosan; molecular weight; antimicrobial activity; mayonnaise
-3-
Structural Characterization and Immunomodulating Activity
of a Complex Glucan from Spores of Ganoderma lucidum
Xingfeng BAO, Jinian FANG,† and Xiaoyu LI
Shanghai Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy
of Sciences, Shanghai 200031, People′s Republic of ChinaReceived March 16, 2001; Accepted July 9, 2001
A polysaccharide with a molecular weight of 1.26×105, obtained from the sporoderm-broken spores of Ganoderma lucidum, was purified by anion-exchange and gel-permeation chromatography. This polysaccharide had a strong effect on suppressing the antibody production and the Con A or LPS induced lymphocyte proliferation in mice. Chemically, the structure of the polysaccharide was identified by methylation analysis, 1 D, 2 D NMR and ESI-MS spectroscopic studies of the native one and of the oligosaccharide fragments generated by partial acid hydrolysis, Smith degradation, and acetolysis. It was concluded that the intact polysaccharide was a complex β-D-glucan consisting of a (1→6)-linked backbone chain, in which every other glucosyl residue was substituted at C-3 or C-4 with mono-, di- and trisaccharide branches.
Key words: Ganoderma lucidum; spores; glucan; antibody production; lymphocyte proliferation
-4-
Adsorption Properties and Activities of Lipase on a Gold Substrate Modified
by Self-assembled Monolayers
Atsushi KOBAYASHI, Yukari SATO,† and Fumio MIZUTANI
National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba,
Ibaraki 305-8566, JapanReceived March 30, 2001; Accepted July 12, 2001
The adsorption properties, amount and specific activity of lipase D from Rhizopus delemar were investigated by employing a gold substrate modified with seven kinds of thiol monolayer. Quartz crystal microbalance measurements revealed that the amount of the enzyme adsorbed to the hydrophobic monolayers (e.g. benzenethiol) was much higher than that to the hydrophilic monolayers (e.g. 3-mercaptopropanoic acid). In contrast, lipase D adsorbed to the hydrophilic, 2-amino-1-ethanethiol monolayer showed the highest specific activity, the value being 300-fold higher than for the same enzyme dissolved in an aqueous medium.
Key words: self-assembled monolayer; lipase; interfacial activation; quartz crystal microbalance
-5-
Purification and Characterization of a Monohalomethane-producing Enzyme
S-adenosyl-L-methionine: Halide Ion Methyltransferase
from a Marine Microalga, Pavlova pinguis
Noboru OHSAWA,1 Mika TSUJITA,2 Satoru MORIKAWA,2 and Nobuya ITOH1,†
1Biotechnology Research Center, Toyama Prefectural University, 5180 Kurokawa Kosugi,
Toyama 939-0398, Japan
2Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Fukui University,
Bunkyo 3-9-1, Fukui 910-8507, JapanReceived April 26, 2001; Accepted July 17, 2001
A monohalomethane-producing enzyme, S-adenosyl-L-methionine-dependent halide ion methyltransferase (EC 2.1.1.-) was purified from the marine microalga Pavlova pinguis by two anion exchange, hydroxyapatite and gel filtration chromatographies. The methyltransferase was a monomeric molecule having a molecular weight of 29,000. The enzyme had an isoelectric point at 5.3, and was optimally active at pH 8.0. The Km for iodide and SAM were 12 mM and 12 μM, respectively, which were measured using a partially purified enzyme. Various metal ions had no significant effect on methyl iodide production, suggesting that the enzyme does not require metal ions. The enzyme reaction strictly depended on SAM as a methyl donor, and the enzyme catalyzed methylation of the I－, Br－, and Cl－ to corresponding monohalomethanes and of bisulfide to methyl mercaptan.
Key words: S-adenosyl-L-methionine; halide ion methyltransferase; marine microalga; monohalomethane; Pavlova pinguis
-6-
Immunochemical and Mutational Analyses of P-type ATPase Spf1p Involved
in the Yeast Secretory Pathway
Chise SUZUKI
National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan
Received May 7, 2001; Accepted July 16, 2001
The yeast SPF1 gene encodes a novel P-type ATPase, the substrate of which specificity has not been identified. It is required for sensitivity to SMKT, a killer toxin produced by the halotolerant yeast Pichia farinosa. To investigate the function of Spf1p, Asp487, the putative phosphorylation site of Spf1p, was replaced by Asn. Expression of the altered SPF1, with Asp487 replaced by Asn, did not suppress the SMKT-resistant phenotype of spf1 mutants, suggesting that the catalytic activity of this ATPase is required for acquisition of sensitivity to SMKT. Subcellular fractionation experiments indicated that the fractionation pattern of Spf1p was similar to that of an early Golgi protein, Och1p. Cells lacking Spf1p had an abnormal fractionation pattern of Sec12p. The spf1 disruptant also showed increased expression of Kar2p and sensitivity to tunicamycin. The glycosylation-defective phenotype and possible role of Spf1p in the secretory pathway are discussed.
Key words: SMKT; Pichia farinosa; killer toxin; Spf1p; Kar2p
-7-
Hydrolysis of a Chitosan-induced Milk Aggregate by Pepsin, Trypsin
and Pancreatic Lipase
Salvador F. AUSAR, Carlos A. LANDA, Ismael D. BIANCO, Leonardo F. CASTAGNA,
and Dante M. BELTRAMO†Center of Excellence in Products and Processes of Cordoba (CEPROCOR), Cordoba Science Agency,
Pabellon CEPROCOR, CP 5164, Santa Maria de Punilla, Cordoba, ArgentineReceived May 10, 2001; Accepted July 10, 2001
The addition of chitosan to whole milk results in dose dependent destabilization and coagulation of the casein micelles and milk fat. The present study evaluates how the presence of chitosan could affect the hydrolysis of this chitosan-induced aggregate by different gastrointestinal proteases (pepsin and trypsin) and by pancreatic lipase. The chitosan-milk aggregate was hydrolyzed by pepsin and trypsin, as evaluated by the UV absorbance of TCA-soluble peptides and by urea-PAGE. The kinetics and extent of hydrolysis were independent of the casein being soluble or aggregated. The release of soluble peptides from the aggregate was independent of the presence of chitosan.
A progressive inhibition of pancreatic lipase was observed in proportion to the increase in molecular weight of the chitosan employed to induce the formation of the aggregate. Interestingly, the presence of chitosan not only affected the initial velocity of the reaction, but also reduced its extent.
The results indicate that a milk aggregate induced by chitosan was very well digested by gastric and intestinal proteases independently of the molecular weight of the chitosan used, and that the aggregate could retain the lipid-lowering effect of chitosan.
Key words: chitosan; casein; milk fat; protease; lipase
-8-
Binding Properties of Bacillus thuringiensis Cry4A Toxin to the Apical
Microvilli of Larval Midgut of Culex pipiens
Masashi YAMAGIWA,＊ Shinya KAMAUCHI,＊ Takayuki OKEGAWA,＊ Motoyuki ESAKI,＊＊
Kanao OTAKE,＊＊ Teruo AMACHI,＊＊ Tohru KOMANO,＊＊＊ and Hiroshi SAKAI＊＊Department of Bioscience and Biotechnology, Okayama University, Okayama 700-8530, Japan
＊＊Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University,
Kyoto 606-8502, Japan
＊＊＊Department of Genetic Engineering, Faculty of Biology-oriented Science and Technology,
Kinki University, Wakayama 649-6493, JapanReceived May 17, 2001; Accepted July 23, 2001
Cry4A is a dipteran-specific δ-endotoxin produced by Bacillus thuringiensis, and toxic to Culex pipiens (mosquito) larvae. The immunohistochemical staining of the midgut sections of C. pipiens larvae revealed that Cry4A bound in vitro and in vivo to the microvilli of the epithelial cells of posterior midgut and gastric caecae. The binding of digoxigenin-labeled Cry4A (DIG-Cry4A) to the apical microvilli was almost abolished in the presence of excess unlabeled Cry4A, suggesting that the binding of Cry4A to the microvilli was specific. Several Cry4A-specific binding proteins were detected using the ligand blotting technique with DIG-Cry4A. Moreover, an insertion assay was done, where the binding of DIG-Cry4A to the BBMVs was completely irreversible and did not compete with excess unlabeled Cry4A. On the basis of these results, we propose a schematic interpretation for the binding process of Cry4A.
Key words: Bacillus thuringiensis; Cry4A; Culex pipiens; midgut; membrane binding
-9-
Role of Conserved Residues of the WRKY Domain in the DNA-binding
of Tobacco WRKY Family Proteins
Kenichiro MAEO,† Shingo HAYASHI, Hisae KOJIMA-SUZUKI, Atsushi MORIKAMI,1
and Kenzo NAKAMURALaboratory of Biochemistry, Department of Cellular Mechanisms and Functions, Graduate School
of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, JapanReceived May 21, 2001; Accepted July 2, 2001
Four cDNA clones of tobacco that could code for polypeptides with two WRKY domains were isolated. Among four NtWRKYs and other WRKY family proteins, sequence similarity was basically limited to the two WRKY domains. Glutathione S-transferase fusion proteins with the C-terminal WRKY domain of four NtWRKYs bound specifically to the W-box (TTGACC), and the N-terminal WRKY domain showed weaker binding activity with the W-box compared to the C-terminal domain. The DNA-binding activity of the WRKY domain was abolished by o-phenanthroline and this inhibition was recovered specifically by Zn2＋. Substitution of the conserved cysteine and histidine residues of the plant-specific C2H2-type zinc finger-like motif in the WRKY domain abolished the DNA binding. In addition, mutations in the invariable WRKYGQK sequence at the N-terminal side of the zinc finger-like motif also significantly reduced the DNA-binding activity, suggesting that these residues are required for proper folding of the DNA-binding zinc finger.
Key words: transcription factor; DNA-binding domain; WRKY domain; tobacco; plant-specific zinc finger
-10-
Purification and Characterization of Two Lectins from a Toxic Moray,
Gymnothrax javanicus
Hirokazu KAWAGISHI,1† Masaaki YASUI,1 Akinori UNO,1 Takeomi MURATA,1
Taichi USUI,1 and Syoei FURUKAWA21Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya,
Shizuoka 422-8529, Japan
2Department of Molecular Biology, Gifu Pharmaceutical University, 5-6-1 Mitahora-Higashi,
Gifu 502-8585, JapanReceived May 21, 2001; Accepted July 3, 2001
Two lectins, Gymnothrax javanicus lectin-I (GJL-I) and Gymnothrax javanicus lectin-II (GJL-II) were isolated from the stomach and intestine, and the liver, respectively, of a toxic moray eel, Gymnothrax javanicus,. GJL-I is a polymer of two heterogeneous subunits of 67 and 51 kDa. In a hemagglutination inhibition assay, it had sugar-binding specificity toward lactose and lactulose among the mono- or oligo-saccharides and bovine submaxillary mucin (BSM) among the glycoproteins tested. The lectin stimulated nerve growth factor (NGF) synthesis by astroglial cells. GJL-II was a polymer of subunit of 41 kDa. This lectin had N-acetyllactosamine binding specificity.
Key words: lectin; Gymnothrax javanicus; lactose-specific; N-acetyllactosamine-specific; nerve growth factor-synthesis
-11-
Capsaicin-, Resiniferatoxin-, and Olvanil-induced Adrenaline Secretions
in Rats via the Vanilloid Receptor
Tatsuo WATANABE, Nobuo SAKURADA, and Kenji KOBATA
School of Food and Nutritional Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan
Received May 21, 2001; Accepted July 24, 2001
The effects of capsaicin analogs on adrenaline secretion were investigated in rats. Capsaicin (20--100 μg/kg, iv) caused biphasic adrenaline secretion. Capsazepine (20 mg/kg, iv), a specific competitive antagonist of the vanilloid (capsaicin) receptor, strongly inhibited both phases of adrenaline secretion by capsaicin (50 μg/kg). Next, the effects of two capsaicin analogs on the adrenal catecholamine secretion were examined. Resiniferatoxin (20--200 ng/kg, iv), a naturally occurring phorbol-
ester-like compound, provoked slow onset adrenaline secretion in a dose-dependent manner. Olvanil (2.46--246 μg/kg, iv), a synthesized non pungent capsaicin analog, also stimulated delayed catecholamine secretion dose-dependently. Capsazepine (20 mg/kg, iv) pretreatment prevented the resiniferatoxin (50 ng/kg)- and olvanil (24.6 μg/kg)-induced catecholamine secretion. These results suggest that some vanilloids (capsaicin, resiniferatoxin, olvanil) excite adrenaline secretion and such excitation is via the vanilloid receptor.
Key words: capsaicin; resiniferatoxin; olvanil; adrenaline secretion; vanilloid receptor
-12-
Intestinal Absorbability of Wheat Allergens, Subunits of a Wheat α-Amylase
Inhibitor, Expressed by Bacteria
Mioko KUSABA-NAKAYAMA, Masami KI, Emiko KAWADA, Masao SATO, Ikuo IKEDA,
Toshihiro MOCHIZUKI,＊ and Katsumi IMAIZUMI†Laboratory of Nutrition Chemistry, Division of Bioresource and Bioenvironmental Sciences, Graduate School
and ＊University Farm, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, JapanReceived May 22, 2001; Accepted July 13, 2001
Wheat CM2, CM3 and CM16 proteins are known as subunits of the tetrameric α-amylase inhibitor as well as major allergens to baker′s asthma. The purpose of this study is to produce these CM proteins by bacteria in a quantity adequate for studying the penetration characteristics of the CM proteins through intestinal mucosa in rats and Caco-2 cells. cDNAs encoding the mature proteins were expressed in Escherichia coli and purified by an Ni2+-chelating column. The recombinant proteins were radioiodinated and admministered orally to rats or applied to the apical site of the Caco-2 cell monolayer. The radioactivity in the trichloroacetic acid-insoluble fraction, which was mainly composed of peptides with molecular mass less than that of the intact CM proteins, in the serum and the basolateral medium was highest in recombinant CM3. Accordingly, the intestinal absorption of these three proteins in the form present in wheat should be evaluated.
Key words: allergen absorption; baker′s asthma; Caco-2 cell; recombinant protein; wheat α-amylase inhibitor
-13-
Enzymatic Synthesis of Sulfated Disaccharides using β-D-Galactosidase-
catalyzed Transglycosylation
Takeomi MURATA,1,† Masaki KOSUGI,1 Tadashi NAKAMURA,2 Tadasu URASHIMA,2
and Taichi USUI11Department of Applied Biological Chemistry, Shizuoka University, 836, Ohya, Shizuoka 422-8529, Japan
2Department of Bioresource Science, Obihiro University of Agriculture and Veterinary Medicine,
Inada-cho, Obihiro, Hokkaido 080-8555, JapanReceived May 24, 2001; Accepted July 13, 2001
We have established a unique enzymatic approach for obtaining sulfated disaccharides using Bacillus circulans β-D-galactosidase-catalyzed 6-sulfo galactosylation. When 4-methyl umbelliferyl 6-sulfo β-D-galactopyranoside (S6Galβ-4MU) was used as a donor, the enzyme induced transfer of 6-sulfo galactosyl residue to GlcNAc acceptor. As a result, the desired compound 6′-sulfo N-acetyllactosamine (S6Galβ1--4GlcNAc) and its positional isomer 6′-sulfo N-acetylisolactosamine (S6Galβ1--6GlcNAc) were observed by HPAEC-PAD, in 49％ total yield based on the donor added, and in a molar ratio of 1:3.5. With a glucose acceptor, the regioselectivity was substantially changed and S6Galβ1--2Glc was mainly produced along with β-(1--1)α, β-(1--3), β-(1--6) isomers in 74％ total yield. When methyl α-D-glucopyranoside (Glcα-OMe) was an acceptor, the enzyme also formed mainly S6Galβ1--2Glcα-OMe with its β-(1--6)-linked isomer in 41％ total yield based on the donor added. In both cases, it led to the predominant formation of β-(1--2)-linked disaccharides. In contrast, with the corresponding methyl β-D-glucopyranoside (Glcβ-OMe) acceptor, S6Galβ1--3Glcβ-OMe and S6Galβ1--6Glcβ-OMe were formed in a low total yield of 12％. These results indicate that the regioselectivity and efficiency on the β-D-galactosidase-mediated transfer reaction significantly depend on the anomeric configuration in the glucosyl acceptors.
Key words: Bacillus circulans; β-D-galactosidase; sulfated disaccharide; transglycosylation; regioselectivity
-14-
Substrate Recognition Mechanism of Carboxypeptidase Y
Hiroshi NAKASE, Shoichi MURATA, Hiroshi UENO, and Rikimaru HAYASHI†
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo,
Kyoto 606-8502, JapanReceived June 1, 2001; Accepted July 10, 2001
To clarify the substrate-recognition mechanism of carboxypeptidase Y, Fmoc-(Glu)nAla-OH (n＝1 to 6), Fmoc-(Glu)nAla-NH2 (1 to 5), and Fmoc-Lys(Glu)3Ala-NH2 were synthesized, and kinetic parameters for these substrates were measured. Km for Fmoc-peptides significantly decreased as peptide length increased from n＝1 to n＝5 with only slight changes in kcat. Km for Fmoc-(Glu)5,6Ala-OH were almost the same as one for protein substrates described previously (Nakase et al., Bull. Chem. Soc. Jpn., 73, 2587--2590). These results show that the enzyme has six subsites (S1′ and S1-S5). Each subsite affinity calculated from the Km revealed subsite properties, and from the differences of subsite affinity between pH 6.5 and 5.0, the residues in each subsite were predicted. For Fmoc-peptide amide substrates, the priorities of amidase and carboxamide peptidase activities were dependent on the substrate. It is likely that the interactions between side chains of peptide and subsites compensate for the lack of P1′-S1′ interaction, so the amidase activity prevailed for Fmoc-(Glu)3,5Ala-NH2. These results suggest that these subsites contribute extensively to substrate recognition rather than a hydrogen bond network.
Key words: carboxypeptidase Y; Fmoc-peptide; Fmoc-peptide amide; subsite affinity; substrate recognition mechanism
-15-
Oxygenation Reactions of Various Tricyclic Fused Aromatic Compounds
Using Escherichia coli and Streptomyces lividans Transformants
Carrying Several Arene Dioxygenase Genes
Kazutoshi SHINDO,1 Yasuo OHNISHI,2 Hyo-Kon CHUN,2 Haruko TAKAHASHI,3 Miki HAYASHI,4
Atsushi SAITO,5 Kazuo IGUCHI,3 Kensuke FURUKAWA,6 Shigeaki HARAYAMA,5
Sueharu HORINOUCHI,2 and Norihiko MISAWA4†1Pharmaceutical Research Laboratories, Kirin Brewery Co., Ltd., 3, Miyahara-cho, Takasaki 370-1295, Japan
2Department of Biotechnology, Graduate School of Agriculture and Life Sciences, The University of Tokyo,
Bunkyo-ku, Tokyo 113-8657, Japan
3Tokyo University of Pharmacy and Life Science, 1432-1, Horinouchi, Hachioji, Tokyo 192-0392, Japan
4Central Laboratories for Key Technology, Kirin Brewery Co. Ltd., 1-13-5, Fukuura, Kanazawa-ku,
Yokohama 236-0004, Japan
5Marine Biotechnology Institute, Heita, Kamaishi 026-0001, Japan
6Division of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka 812-8581, JapanReceived June 6, 2001; Accepted July 19, 2001
Bioconversion (biotransformation) experiments on arenes (aromatic compounds), including various tricyclic fused aromatic compounds such as fluorene, dibenzofuran, dibenzothiophene, carbazole, acridene, and phenanthridine, were done using the cells of Escherichia coli transformants expressing several arene dioxygenase genes. E. coli carrying the phenanthrene dioxygenase (phdABCD) genes derived from the marine bacterium Nocardioides sp. strain KP7 converted all of these tricyclic aromatic compounds, while E. coli carrying the Pseudomonas putida F1 toluene dioxygenase (todC1C2BA) genes or the P. pseudoalcaligenes KF707 biphenyl dioxygenase (bphA1A2A3A4) genes was not able to convert these substrates. Surprisingly, E. coli carrying hybrid dioxygenase (todC1::bphA2A3A4) genes with a subunit substitution between the toluene and biphenyl dioxygenases was able to convert fluorene, dibenzofuran, and dibenzothiophene. The cells of a Streptomyces lividans transformant carrying the phenanthrene dioxygenase genes were also evaluated for bioconversion of various tricyclic fused aromatic compounds. The ability of this actinomycete in their conversion was similar to that of E. coli carrying the corresponding genes. Products converted from the aromatic compounds with these recombinant bacterial cells were purified by column chromatography on silica gel, and identified by their MS and 1H and 13C NMR analyses. Several products, e.g., 4-hydroxyfluorene converted from fluorene, and cis-1,2-dihydroxy-1,2-
dihydrophenanthridine, cis-9,10-dihydroxy-9,10-dihydrophenanthridine, and 10-hydroxyphenanthridine, which were converted from phenanthridine, were novel compounds.
Key words: tricyclic aromatic compounds; phenanthridine; arene dioxygenase; Escherichia coli; Streptomyces lividans
-16-
Glycoproteins Secreted from Suspension-cultured Tobacco BY2 Cells have
Distinct Glycan Structures from Intracellular Glycoproteins
Ryo MISAKI, Yoshinobu KIMURA,＊ Kazuhito FUJIYAMA,† and Tatsuji SEKI
The International Center for Biotechnology, Osaka University, Yamada-oka 2-1, Suita-shi, Osaka 565-0871,
Japan
＊Department of Bioresources Chemistry, Faculty of Agriculture, Okayama University, Tsushima-naka 1-1-1,
Okayama 700-8530, JapanReceived June 6, 2001; Accepted July 13, 2001
Glycan structures of glycoproteins secreted in the spent medium of tobacco BY2 suspension-cultured cells were analyzed. The N-glycans were liberated by hydrazinolysis and the resulting oligosaccharides were labeled with 2-aminopyridine. The pyridylaminated (PA) glycans were purified by reversed-phase and size-fractionation HPLC. The structures of the PA sugar chains were identified by a combination of the two-dimensional PA sugar chain mapping, MS analysis, and exoglycosidase digestion. The ratio (40:60) of the amount of glycans with high-mannose-type structure to that with plant-complex-type structure of extracellular glycoproteins is significantly different from that (ratio 10:90) previously found in intracellular glycoproteins [Palacpac et al., Biosci. Biotechnol. Biochem. 63 (1999) 35--39]. Extracellular glycoproteins have six distinct N-glycans (marked by ＊) from intracellular glycoproteins, and the high-mannose-type structures account for nearly 40％ (Man5GlcNAc2, 28.8％; Man6GlcNAc2＊, 6.4％; and Man7GlcNAc2＊, 3.8％), while the plant-complex-type structures account for nearly 60％ (GlcNAc2Man3Xyl1GlcNAc2＊, 32.1％; GlcNAc1Man3Xyl1GlcNAc2 (containing two isomers)＊, 6.2％; GlcNAc2Man3GlcNAc2＊, 4.9％; Man3Xyl1Fuc1GlcNAc2, 8.3％; and Man3Xyl1GlcNAc2, 3.7％).
Key words: N-glycan; tobacco BY2 cells; spent medium; extracellular glycoprotein
-17-
Effects of Bovine β-Casein (1--28) and Its Chemically Synthesized Partial
Fragments on Proliferative Responses and Immunoglobulin Production
in Mouse Spleen Cell Cultures
Hajime OTANI†, Takehito WATANABE, and Yasuhito TASHIRO＊
Integrated Department of Sciences of Functional Foods, Graduate School of Agriculture, Shinshu University,
8304 Minamiminowa-mura, Kamiina-gun, Nagano-ken 399-4598, Japan
＊New Materials Division, Meiji Seika Kaisha, Ltd., 580 Horikawa-cho, Saiwai-ku, Kawasaki-shi,
Kanagawa-ken 210-0913, JapanReceived June 11, 2001; Accepted July 26, 2001
Effects of bovine β-casein (1--28) having a phosphoserine-rich region (Glu14-SerP-Leu-SerP-SerP-SerP-Glu-Glu21) and its chemically synthesized partial fragments on proliferation of lymphocytes and immunoglobulin production were investigated in mouse spleen cell cultures. The parent fragment 1--28 and all fragments containing SerP-Leu-SerP and/or SerP-SerP-SerP had a significant mitogenic effect, stimulated proliferation of lymphocytes induced by lipopolysaccharide, phytohemagglutinin, or concanavalin A, and increased immunoglobulin (IgG＋IgM＋IgA) or IgA levels in the cell cultures. In contrast, dephosphorylated β-casein (14--21) and SerP-SerP amide had hardly any immunoregulatory activity. On the other hand, SerP-Leu-SerP amide reacted little with antibodies specific to bovine β-casein (1--28), but β-casein (14--21), and SerP-SerP-SerP amide obviously reacted with the antibody. These results confirm that the immunoregulatory activity of casein phosphopeptides is attributable to SerP-X-SerP, which may well be available as a non-allergic food ingredient having an adjuvant activity for mucosal IgA responses.
Key words: casein phosphopeptide; SerP-X-SerP; mitogenic activity; lymphocytes; non-allergic IgA stimulator
-18-
Lecithin:Cholesterol Acyltransferase Reduces the Adverse Effects of Oxidized
Low-Density Lipoprotein while Incurring Damage Itself
Zakir H. HOWLADER, Shin KAMIYAMA, Yohei MURAKAMI, Michiko ITO,
Michio KOMAI, and Yuji FURUKAWA†Laboratory of Nutrition, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori,
Amamiya Machi, Aoba-Ku, Sendai 981-8555, JapanReceived June 15, 2001; Accepted July 30, 2001
In this study, we tried to determine (a) whether there is any permanent effect of oxLDL on the LCAT molecule and its activator lipoprotein apoA-I, and (b) the fate of oxLDL after its exposure to LCAT and apoA-I. Purified LCAT and LDL/oxLDL was incubated at 37°C for 1h, and separated by gel-permeation chromatography. Its activity was significantly less (30％ less) after separating from oxLDL compared to that of LDL. Cofactor activity of isolated apoA-I (incubated with LDL/oxLDL) was found affected by oxLDL. These results indicate modification of the LCAT and apoA-I molecule. But LCAT was found more affected compared to apoA-I in terms of LCAT activity. We are also reporting a novel function of LCAT. It was found to reduce the adverse effects of oxLDL, for example, ability to affect the LCAT activity and TBARS value. This ability of LCAT to repair oxLDL was dose-dependent. But there was no change in its REM values or fluorescence intensity.
Key words: apolipoprotein A-I; irreversible modification; lecithin:cholesterol acyltransferase; oxidized LDL; adverse effects of oxLDL
-19-
Regulation of Hydroxyindole-O-methyltransferase Gene Expression
in Japanese Quail (Coturnix coturnix japonica)
Zhengwei FU,1 Hisanori KATO,1,† Naoko KOTERA,2 Tadashi NOGUCHI,1
Kunio SUGAHARA,2 and Tatsuo KUBO21Laboratory of Nutritional Biochemistry, Department of Applied Biological Chemistry, Graduate School
of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan
2Faculty of Agriculture, Utsunomiya University, Mine-machi, Utsunomiya 321-8505, JapanReceived June 15, 2001; Accepted August 6, 2001
Hydroxyindole-O-methyltrasferase (HIOMT) plays an important role as the final enzyme in the synthesis of melatonin. In this study, the expression of the HIOMT gene in Japanese quail was investigated with respect to tissue distribution and the effects of light and vitamin A deficiency. HIOMT mRNA in the pineal gland and eye had a clear daily rhythm with peak values in daytime. The testis also contained a detectable amount of HIOMT mRNA, which did not display a rhythmic change over a 24-h period. When birds were rendered vitamin A deficient through feeding with a vitamin A-free diet, the daily rhythm of the HIOMT gene almost disappeared in both the pineal gland and eye due to increases in the nighttime values. Our previous observations and these results suggest that vitamin A and a photo-signal are required to maintain the rhythmic expression of the HIOMT gene as well as the arylalkylamine N-acetyltransferase gene.
Key words: hydroxyindole-O-methyltrasferase; arylalkylamine N-acetyltransferase; melatonin; vitamin A; pineal gland
-20-
Characterization and Overproduction of EcoO109I Methyltransferase
Keiko KITA, Junko TSUDA, and Ryu-ichi NISHIGAKI
Department of Biotechnology, Tottori University, 4-101 Koyamaminami, Tottori 680-8552, Japan
Received June 15, 2001; Accepted July 24, 2001
In order to characterize EcoO109I methyltransferase, a recombinant Escherichia coli clone that overproduces the enzyme was constructed. The coding region of M.EcoO109I was joined to the lac promoter of an expression vector, pUC118, and the resulting plasmid was introduced into E. coli HB101. M.EcoO109I was purified homogeneously from IPTG-induced cells, and was found to consist of a monomer subunit. M.EcoO109I uniquely methylates the inner deoxycytidylate residue in the sequence 5′-(A/G)GGNCC(C/T)-3′ to produce 5-methylcytosine. The enzyme was most active at pH 8.0--8.5 and 50°C. The enzyme activity was not affected by the addition of Mg2＋ or EDTA.
Key words: restriction-modification system; 5-methylcytosine; methyltransferase; overproduction
-21-
A Comparative Study on Structural Elucidation of Sialyl Oligosaccharides
by Mass Spectrometry with Fast Atom Bombardment, Electrospray Ionization,
and Matrix Assisted Laser Desorption/Ionization
Masahiko OKAMOTO
Environmental Health Science Laboratory, Sumitomo Chemical Co. Ltd., 1-98, 3-chome, Kasugade-naka,
Konohana-ku, Osaka 554-0022, JapanReceived June 20, 2001; Accepted August 4, 2001
To establish a new protocol for sensitive detection and structural characterization of sialyl oligosaccharides, their sensitivities and structural information from mass spectrometry and tandem mass spectrometry with FAB-, ESI-, and MALDI were evaluated in detail. Among these ionization methods, FAB-MS and FAB-MS/MS gave reproducible and predictable spectra carrying information on sequence and branching of sialyl oligosaccharides after derivatization with 2-aminopyridine (PA). With both positive and negative ion modes, their structural elucidation promises to be straightforward, MS/MS spectra being measurable at as low as 200 pmol. Thus, this method constitutes a powerful tool for sensitive detection and structural characterization of limited quantities of sialyl oligosaccharides by FAB-MS and FAB-MS/MS.
Key words: sialyl oligosacchareide; sialyl oligosaccharides sequence; mass spectrometry; inner fragment ion
-22-
Radicicol Binding to Swo1/Hsp90 and Inhibition of Growth of Specific
Temperature-sensitive Cell Cycle Mutants of Fission Yeast
Se Won KI,1,＊ Koji KASAHARA,1,<Oh><0167><Wa> Ho Jeong KWON,1,‡ Ken ISHIGAMI,2 Takeshi KITAHARA,2
Teruhiko BEPPU,1, Minoru YOSHIDA,1,3,† and Sueharu HORINOUCHI11Department of Biotechnology, 2Department of Applied Biological Chemistry, Graduate School of Agriculture
and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, and 3CREST Research Project,
Japan Science and Technology Corporation, JapanReceived June 28, 2001; Accepted July 30, 2001
A panel screening using cdc mutants of Schizosaccharomyces pombe identified radicicol as a potent growth inhibitor of certain mutants at the permissive temperature. The strains sensitive to radicicol were cdc7, cdc11, and cdc14, all of which are defective in early septum formation. Cytokinesis but not nuclear division of these mutants was inhibited by radicicol, but that of cells with the wild-type background was not. A biologically active derivative of radicicol with a biotin moiety at the C-11 position bound Swo1, an Hsp90 homologue in S. pombe. Increased Swo1 expression partially suppressed radicicol sensitivity of cdc14 and almost completely rescued morphological abnormalities in cdc14 and cdc7 cells induced by radicicol at the permissive temperature. On the other hand, the increased Swo1 expression did not restore septum formation at the nonpermissive temperature. These results suggest that Swo1, as a molecular chaperone, plays a role in stabilizing these temperature-sensitive proteins at the permissive temperature or in activating the cytokinesis signaling cascade.
Key words: cdc mutant; cell cycle; heat-shock protein; radicicol; septum formation
-23-
Dietary Manipulations of Body Fat-reducing Potential
of Conjugated Linoleic Acid in Rats
Michihiro SUGANO,1 Asuka AKAHOSHI,1 Kazunori KOBA,2 Kazunari TANAKA,2
Tomoka OKUMURA,1 Hiroko MATSUYAMA,1 Yuki GOTO,1 Tomoko MIYAZAKI,1
Kayoko MURAO,1 Masao YAMASAKI,3 Michiko NONAKA,3 and Koji YAMADA31Faculty of Environmental and Symbiotic Sciences, Prefectural University of Kumamoto, 3-1-100 Tsukide,
Kumamoto 862-8502, Japan
2Faculty of Nursing and Nutrition, Siebold University of Nagasaki, Nagayo, Nagasaki 851-2195, Japan
3Division of Bioresource and Bioenvironmental Sciences, Graduate School, Kyushu University, Higashi-ku,
Fukuoka 812-8581, JapanReceived June 28, 2001; Accepted July 31, 2001
To study whether the body fat-reducing potential of conjugated linoleic acid (CLA) could be increased through dietary manipulations, the effects of the combination of CLA with different proteins, fats, and sesamin were examined in rats. Male rats were fed diets containing 1％ CLA or linoleic acid (LA) in combination with different proteins (20％ of casein or soybean protein), fats (7％ perilla oil or soybean oil) and 0.2％ sesamin (SES) for 3 or 4 weeks. When the dietary fat source was soybean oil, CLA, as compared with LA, significantly reduced weights of epididymal and perirenal adipose tissues, irrespective of the dietary protein sources. However, the highest reducing effect was shown when soybean protein was given as a protein source. SES stimulated the reduction of epididymal and perirenal adipose tissue weights in both protein diets. In contrast, CLA increased the weight of brown adipose tissue, and SES further increased it in combination with soybean oil but not with perilla oil. No effect of dietary manipulation was observed on serum leptin and TNF-α levels. Thus, the body fat-reducing potential of CLA can be increased by an appropriate combination with food factors that may stimulate fatty acid β-oxidation.
Key words: conjugated linoleic acid; adipose tissue; dietary protein and fat; sesamin; leptin
-24-
Crystalline Behavior of Chitosan Organic Acid Salts
Jumpei KAWADA,† Toshifumi YUI,1 Kenji OKUYAMA,2 and Kozo OGAWA††
Research Institute for Advanced Science and Technology (RIAST), Osaka Prefecture University, Sakai,
Osaka 599-8570, Japan
1Faculty of Engineering, Miyazaki University, Miyazaki 889-2192, Japan
2Faculty of Technology, Tokyo University of Agriculture and Technology, Tokyo 184-8588, JapanReceived July 12, 2001; Accepted August 4, 2001
The crystal structures of chitosan acid salts were studied by X-ray diffraction measurements on a fiber diagram and a new procedure to obtain an anhydrous polymorph of chitosan was found. The salts prepared by immersing a chitosan into a mixture of acid solution and isopropanol were classified into two types (Types I and II) depending on their conformation. Molecular conformation of the Type I salt retains the extended 2-fold helical structure of the original chitosan, but that of Type II salt is a twisted 2-fold helix. All the Type II salts changed to the anhydrous ''Annealed'' polymorph of chitosan when soaking in 75％ aqueous isopropanol, but when the Type I salts were immersed in the solution, they returned to the hydrated ''Tendon'' polymorph which is that of the original chitosan. The strange transformation observed in Type II salt may be related to the stability of the molecular conformation of chitosan in the salt.
Key words: crystalline transformation; chitosan organic acid salt; hydrated crystal of chitosan; anhydrous crystal of chitosan
-25-
Note
Regulation of Cigarette Smoke-Induced Cytochrome P4501A1 Gene
Expression in Osteogenic Disorder Shionogi Rat Liver and in Lung
by Large Ascorbic Acid Dose
Etsuko UETA,1 Emiko SUZUKI,2 Eiji NANBA,3 Yuko TADOKORO,1
Yuzuru OTSUKA,4,† and Tadao KURATA11Institute of Environmental Science for Human Life, Ochanomizu University, Tokyo 112-8610, Japan
2Faculty of Life Science, Ochanomizu University, Tokyo 112-8610, Japan
3Gene Research Center, Tottori University, Yonago 683-0826, Japan
4Faculty of Education and Regional Sciences, Tottori University, Tottori 680-8551, JapanReceived September 14, 2000; Accepted June 27, 2001
The effects of a large ascorbic acid dose on cytochrome P4501A1 gene expression induced by cigarette smoke exposure was studied in Osteogenic Disorder Shionogi rats, which lack ascorbic acid biosynthesis. The rats were divided into four groups and were administered either a minimal amount (4 mg/day, 4S and 4C) or a large amount (40 mg/day, 40S and 40C) of ascorbic acid. The 4S group and 40S group were daily exposed to cigarette smoke for 2 hours, while the 4C group and 40C group were not. At the end of the 25-day experiment, the rats were killed. The cytochrome P4501A1 mRNA level both in the liver and lung was measured by a competitive reverse transcription---
polymerase chain reaction method. When a minimal amount of ascorbic acid was administered, the cytochrome P4501A1 mRNA increased in the liver of the cigarette smoke-exposed group (4S) compared with the control group (4C). On the other hand, when a large amount of ascorbic acid was administered, this increase was not observed in the cigarette smoke-induced group (40S) in liver. On the other hand, in lung, an increased mRNA level in 4S group was not decreased by large ascorbic acid administration (40S). This is the first direct mRNA-level evidence of the effects of a large ascorbic acid dose on the gene expression stimulated by cigarette smoke.
Key words: large ascorbic acid dose; gene expression; cigarette smoke; cytochrome P4501A1; competitive RT-PCR
-26-
Note
Extremely Low Frequency Magnetic Fields Suppress the Reduction
of Germination Rate of Arabidopsis thaliana Seeds Kept
in Saturated Humidity
Koichi TAKIMOTO,1,† Hiroko YAGUCHI,2,3,†† and Junji MIYAKOSHI3
1Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, 1677-1 Yoshida
Yamaguchi 753-8515, Japan
2Graduate School of Human and Environmental Studies, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku,
Kyoto 606-8501, Japan
3Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho,
Sakyo-ku, Kyoto 606-8501, JapanReceived April 2, 2001; Accepted July 2, 2001
The effects of an extremely low frequency magnetic field (ELFMF) on the germination of plant seeds were examined. The decrease in the germination activity of the seeds of Arabidopsis thaliana WS kept in saturated humidity and high temperature (37°C) was suppressed by the exposure to a 400 mT ELFMF.
Key words: magnetic field; saturated humidity; plant seed; Arabidopsis; germination
-27-
Note
Protective Effect of Ajoene on Acetaminophen-induced Hepatic Injury in Mice
Atsuhiko HATTORI, Norihiko YAMADA, Tomoaki NISHIKAWA,
Hiroyuki FUKUDA, and Tsuchiyoshi FUJINO†Biodevelopment Division, Central Institute, Nagoya Seiraku Co. Ltd., 310 Nakasuna-cho,
Tempaku-ku, Nagoya, Aichi 468-0065, JapanReceived May 14, 2001; Accepted July 11, 2001
Ajoene, a garlic-derived sulfur-containing compound, exhibited a hepatoprotective effect against acetaminophen-induced liver injury in mice. A pretreatment with ajoene suppressed the rise in serum glutamic-pyruvic transaminase activity and the reduction in the hepatic reduced glutathione level. These effects of ajoene were observed dose-dependently (20--100 mg/kg). The pretreatment by ajoene also suppressed the decrease in hepatic protein thiol content resulting from acetaminophen administration.
Key words: acetaminophen; hepatoprotective effect; ajoene; garlic; Allium sativum
-28-
Note
A New Type of Allantoinase in Amphibian Liver
Wataru MASUDA, Satoko FUJIWARA and Tomoo NOGUCHI
Department of Biochemistry, Kyushu Dental College, Kokura, Kitakyushu 803-8580, Japan
Received May 18, 2001; Accepted August 1, 2001
Allantoinase and allantoicase are known to form a complex in amphibian liver. In this study, a new type of allantoinase that did not form a complex with allantoicase was found in the amphibian liver. Purified enzyme had a molecular mass of about 44 kDa both in SDS-PAGE and gel-filtrations. The enzyme cross-reacted with anti-sardine allantoinase polyclonal antibody, and it weakly cross-reacted with anti-bullfrog allantoinase polyclonal antibody.
Key words: allantoinase; allantoicase; amphibian liver; purine degradation
-29-
Note
Induction and Secretion of RNA-degrading Enzymes
by Phosphate Deficiency in Pholiota nameko
Toshio JOH,1 Yuji TASAKI,2 Takashi HARA,1 and Toshiro HAYAKAWA1
1Department of Applied Biological Chemistry, Faculty of Agriculture, and 2Department of Biosystem Science,
Graduate School of Science and Technology, Niigata University, Ikarashi 2-8050, Niigata 950-2181, JapanReceived May 18, 2001; Accepted July 28, 2001
Changes in activity of RNA-degrading enzyme and amounts of phosphorus in mycelia and culture filtrate during Pi-sufficient and -deficient cultures of Pholiota nameko were investigated. The results showed that the intracellular and extracellular activities are controlled by the Pi concentration in the medium. Moreover, the induction and secretion of RNA-degrading enzymes under the Pi-deficient condition were analyzed by activity staining.
Key words: Pholiota nameko; phosphate deficiency; RNA-degrading enzyme; activity staining
-30-
Note
Changes of Pyridine Nucleotide Levels during Adipocyte Differentiation
of Mouse 3T3-L1 Cells
Tsutomu FUKUWATARI,1,† Miki DOI,1 Etsuro SUGIMOTO,1 Teruo KAWADA,2
and Katsumi SHIBATA11Course of Food Science and Nutrition, Department of Life Style Studies, School of Human Cultures,
The University of Shiga Prefecture, Hikone, Shiga 522-8533, Japan
2Laboratory of Nutrition Chemistry, Division of Food Science and Biotechnology, Graduate School
of Agriculture, Kyoto University, Kyoto 606-8502, JapanReceived May 24, 2001; Accepted July 10, 2001
The levels of NAD and NADP were measured in 3T3-L1 cells during a differentiation from preadipocytes to adipocytes. The cells were grown in the ordinary medium and differentiated in the medium by adding dexamethasone, 1-methyl-3-isobutylxanthine, and insulin for 2 days, and then they were grown in the medium by adding only insulin for another 8 days to accumulate fat. The levels of cellular NAD and NADP increased abruptly with days after differentiation, and the levels of NAD and NADP reached maximum at day 7, and at day 10 the values were decreased compared with the maximum values. These results suggest that expression of the pyridine nucleotide biosynthesis genes is induced in the differentiation process.
Key words: adipocyte; NAD; NADP; differentiation; measurement
-31-
Note
Synthesis of (R)-(+)-Hippospongic Acid A, a Triterpene Isolated
from the Marine Sponge, Hippospongia sp.†
Miwako ICHIHASHI, Hirosato TAKIKAWA,†† and Kenji MORI†††
Department of Chemistry, Science University of Tokyo, Kagurazaka 1-3, Shinjuku-ku, Tokyo 162-8601, Japan
Received May 31, 2001; Accepted July 5, 2001
Hippospongic acid A (1) is a triterpene metabolite of the marine sponge, Hippospongia sp., with inhibitory activity against the gastrulation of starfish embryos. (R)-(+)-1 was synthesized by employing enzymatic kinetic resolution as the key step.
Key words: marine natural product; enantioselective synthesis; triterpenoid; enzymatic resolution
-32-
Note
Measurement of Free Choline in Plant Leaves by Capillary Electrophoresis
Jinghua ZHANG,† Akira OKUBO,†† and Sunao YAMAZAKI＊
Department of Applied Biological Chemistry, The University of Tokyo, 1-1-1 Yayoi, Bunkyo,
Tokyo 113-8657, Japan
＊Faculty of Environmental Studies, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan
Received June 19, 2001; Accepted August 1, 2001
A low pH capillary electrophoresis (CE) was used for the measurement of free choline in plant leaves. Choline in the leaf extract was first converted to the benzoyl ester and put into CE. A well-resolved peak in the electropherogram was easily obtained. Involvement of enzymes in a two-step oxidation of choline to glycine betaine was evaluated in different plant species with the same method developed for glycine betaine and betaine aldehyde.
Key words: betaine aldehyde; capillary electrophoresis; choline; glycine betaine; salt-stress
-33-
Note
A Novel Protein, Mpm1, of the Mitochondria of the Yeast
Saccharomyces cerevisiae
Hironori INADOME, Yoichi NODA, Hiroyuki ADACHI, and Koji YODA†
Department of Biotechnology, the University of Tokyo, Yayoi, Bunkyo-Ku, Tokyo 113-8657, Japan
Received June 19, 2001; Accepted August 6, 2001
A previously uncharacterized yeast protein, YJL066c, was discovered in the membrane fraction although it has no hydrophobic stretch. The protein was partly solubilized by Triton X-100 in an oligomeric form, while it was insoluble in alkali or salt. By immunofluorescent microscopy, its localization coincided with the mitochondria. We therefore propose it should be named Mpm1 (mitochondrial peculiar membrane protein 1).
Key words: mitochondria; MPM1; YJL066c; yeast; Saccharomyces cerevisiae
-34-
Note
Induction of Phagocyte Oxidase Components during Human Myeloid
Differentiation: Independent Protein Expression and Discrepancy
with the Function
Yoko INOUE, Masako YAGISAWA, Kumiko SAEKI, Shinobu IMAJOH-OHMI,＊
Shiro KANEGASAKI,＊ and Akira YUODepartment of Hematology, Research Institute, International Medical Center of Japan, Tokyo 162-8655, Japan
＊The Institute of Medical Science, University of Tokyo, Tokyo 108-8639, JapanReceived July 23, 2001; Accepted August 3, 2001
We investigated the content of four components of the O－2-producing enzyme (p47, p67, p22, and gp91) and the O－2-producing capacity in human myeloid cell lines. The content of the four components of the phagocyte oxidase was minimal before differentiation induction. During differentiation, expression of p22 and gp91 was at consistently low levels, even when the O－2-producing capacity was equivalent to that of normal neutrophils. On the other hand, p47 was consistently and rapidly induced to the level comparable to normal neutrophils. The results indicate that low expression of p22 and gp91 is sufficient to obtain normal O－2 production, and that p47 might play an important regulatory role in the functional differentiation.
Key words: phagocyte oxidase; superoxide production; differentiation
-35-
Preliminary Communication
Structure Elucidation of Ostreocin D, a Palytoxin Analog Isolated
from the Dinoflagellate Ostreopsis siamensis
Takanori UKENA,1 Masayuki SATAKE,1,† Masaya USAMI,1 Yasukatsu OSHIMA,1
Hideo NAOKI, 2 Tsuyoshi FUJITA,2 Yukiko KAN,2 and Takeshi YASUMOTO11Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiya, Aoba-ku,
Sendai 981-8555, Japan
2Suntory Institute for Bioorganic Research, Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka, JapanReceived June 6, 2001; Accepted July 31, 2001
The structure of ostreocin D, a palytoxin analog isolated from the marine dinoflagellate Ostreopsis siamensis, was found to be 42-hydroxy-3,26-didemethyl-19,44-dideoxypalytoxin by detailed 2D NMR analyses of intact ostreocin D and its ozonolysis products. Partial stereochemical assignments were done. This result indicates that the dinoflagellate O. siamensis is one of the biogenetic origins of palytoxin.
Key words: palytoxin; ostreocin; biogenetic origin; dinoflagellate; ozonolysis