(Vol.64 No.9 2000)
Erythropoietin: Multiple Physiological Functions and Regulation
of Biosynthesis
Ryuzo SASAKI,・・・ Seiji MASUDA, and Masaya NAGAO p.1775
Effects of Germinated Barley Foodstuff on Microflora and Short
Chain Fatty
Acid Production in Dextran Sulfate Sodium-induced Colitis in Rats.
Yoshio ARAKI,1 Akira ANDOH,2 Shigeki KOYAMA,2 Yoshihide FUJIYAMA,2
Osamu KANAUCHI,3 and Tadao BAMBA2 p.1794
Ascorbic Acid Stimulation of Production of a Highly Branched
β-1,3-Glucan by
Aureobasidium pullulans K-1---Oxalic Acid, a Metabolite of Ascorbic Acid
as the Stimulating Substance---
Nobutake HAMADA,・・・ Kohji DEGUCHI,＊ Takashi OHMOTO, Kiyofumi SAKAI,
Tatsuhiko OHE, and Hajime YOSHIZUMI＊ p.1801
Production of Betacyanins by a Cell Suspension Culture of Table
Beet
(Beta vulgaris L.)
Toru AKITA,・・・ Yasuhiko HINA, and Toyoyuki NISHI p.1807
Flavonoids Inhibit Cell Growth and Induce Apoptosis
in B16 Melanoma 4A5 Cells
Keiko IWASHITA,・・・ Masuko KOBORI,＊ Kohji YAMAKI,＊ and Tojiro TSUSHIDA＊
p.1813
Synthesis of α-D-Glucosylglycerol by α-Glucosidase and Some
of Its Characteristics・・・
Fumihito TAKENAKA・・・・・・ and Hirofumi UCHIYAMA p.1821
Interaction of Gum Arabic, Maltodextrin and Pullulan
with Lipids in Emulsions
Yasuki MATSUMURA,・・・ Chikako SATAKE, Masaaki EGAMI, and Tomohiko MORI p.1827
Preparative 2?-Reduction of ATP Catalyzed by Ribonucleotide
Reductase Purified by Liquid-Liquid Extraction
Andr"e BRUNELLA,1 Miguel ABRANTES,2 and Oreste GHISALBA3 p.1836
Absolute Configuration of a Ceramide with a Novel Branched-chain
Fatty
Acid Isolated from the Epiphytic Dinoflagellate, Coolia monotis
Kazuaki AKASAKA,1 Seiya SHICHIJYUKARI,1 Shigeru MATSUOKA,2 Michio MURATA,2
Hiroshi MEGURO,3 and Hiroshi OHRUI1＊ p.1842
Structural Analysis of Free N-Glycans Occurring in Soybean
Seedlings
and Dry Seeds＊
Yoshinobu KIMURA・・・ and Erika KITAHARA p.1847
Epitope Analysis and Primary Structures of Variable Regions
of Anti-human FcεRI Monoclonal Antibodies, and Expression
of the Chimeric Antibodies Fused with Human Constant Regions
Toshiro TAKAI,1,2,・・・ Toshifumi YUUKI,2 Namiko IWAMOTO-YASUE,2
Ko OKUMURA,1 and Chisei RA1,3 p.1856
Preventive Effect of Lactobacillus delbrueckii subsp. bulgaricus
on the Oxidation of LDL
Masaki TERAHARA,・・・ Sachiko NISHIDE, and Tsutomu KANEKO p.1868
Characteristics of Serine Acetyltransferase from Escherichia
coli Deleting
Different Lengths of Amino Acid Residues from the C-Terminus
Koshiki MINO, Kenji HIRAOKA, Koreyoshi IMAMURA, Takaharu SAKIYAMA,
Naoki EISAKI,＊ Asahi MATSUYAMA,＊ and Kazuhiro NAKANISHI・・・ p.1874
Safety Assessment of Rice Genetically Modified with Soybean
Glycinin
by Feeding Studies on Rats
Keiko MOMMA,・・・ Wataru HASHIMOTO, Hye-Jin YOON, Sachiko OZAWA, Yasuki FUKUDA,＊
Shigeyuki KAWAI, Fumio TAKAIWA,＊＊ Shigeru UTSUMI, and Kousaku MURATA p.1881
Meat Allergy: Investigation of Potential Allergenic Proteins
in Beef
Gi Dong HAN,a Masatomo MATSUNO,b Giichi ITO,c Yoshihide IKEUCHI,d
and Atsushi SUZUKId,・・・ p.1887
Purification and Characterization of Chitosanase and Exo-β-D-Glucosaminidase
from a Koji Mold, Aspergillus oryzae IAM2660
Xiao-Yong ZHANG, An-Lan DAI, Xue-Kun ZHANG, Kouji KUROIWA,
Ritsuko KODAIRA, Makoto SHIMOSAKA,・・・ and Mitsuo OKAZAKI＊ p.1896
Cloning and Expression of a Novel Murine Anti-human Fas Antibody
Hiroko YOSHIDA-KATO,1 Kimihisa ICHIKAWA,1 Junko YAMAGUCHI,1 Kenji WATANABE,1
Jun OHSUMI,1 Shin YONEHARA,2 and Nobufusa SERIZAWA1,・・・ p.1903
Kurosu, a Traditional Vinegar Produced from Unpolished Rice,
Suppresses Lipid Peroxidation in Vitro and in Mouse Skin
Shoko NISHIDAI, Yoshimasa NAKAMURA,＊ Koji TORIKAI,＊ Mikako YAMAMOTO,
Nobuhiro ISHIHARA, Hirotaka MORI, and Hajime OHIGASHI＊,・・・ p.1909
Synthesis of 2-C-Methyl-D-erythritol and 2-C-Methyl-L-threitol;
Determination
of the Absolute Configuration of 2-C-Methyl-1,2,3,4-butanetetrol Isolated
from Phlox sublata L
Isao SAKAMOTO,1 Kazuo ICHIMURA,2 and Hiroshi OHRUI1,・・・ p.1915
Thermally Induced Disintegration of the Bacillus stearothermophilus
Dihydrolipoamide Dehydrogenase・・・
Yasuaki HIROMASA, Yoichi ASO,＊,・・・・・・ Shoji YAMASHITA,＊＊ and Kohji MENO＊ p.1923
Gene Regulation in Response to Overexpression of Cytochrome
P450
and Proliferation of the Endoplasmic Reticulum
in Saccharomyces cerevisiae
Thomas ZIMMER,1,2 Atsushi OGURA,1 Tsuyoshi TAKEWAKA,1 Rose-Marie ZIMMER,1,2
Akinori OHTA,1,・・・ and Masamichi TAKAGI1 p.1930
Defect in Cell Wall Integrity of the Yeast Saccharomyces cerevisiae
Caused
by a Mutation of the GDP-mannose Pyrophosphorylase Gene VIG9
Koji YODA,・・・ Tsuyoshi KAWADA, Chiaki KAIBARA, Akihiko FUJIE, Masato ABE, Hitoshi
HASHIMOTO, Jiro SHIMIZU, Nario TOMISHIGE, Yoichi NODA, and Makari YAMASAKI・・・・・・
p.1937
A Positive Screening for Drugs that Specifically Inhibit the
Ca2+-Signaling
Activity on the Basis of the Growth Promoting Effect on a Yeast Mutant
with a Peculiar Phenotype
Atsunori SHITAMUKAI,1 Masaki MIZUNUMA,1 Dai HIRATA,1 Hidetoshi TAKAHASHI,2
and Tokichi MIYAKAWA1,・・・ p.1942
Molecular Cloning and Characterization of a cDNA and a Gene
for Subtilisin-like Serine Proteases from Rice (Oryza sativa L.)
and Arabidopsis thaliana＊
Hiroshi YAMAGATA,・・・ Maki UESUGI, Kazunori SAKA, Teruo IWASAKI,＊＊ and Yasuo
AIZONO
p.1947
Note
Degradation of Bisphenol A by the Lignin-Degrading Enzyme,
Manganese Peroxidase, Produced by the White-rot Basidiomycete,
Pleurotus ostreatus
Taeko HIRANO, Yoichi HONDA, Takashi WATANABE, and Masaaki KUWAHARA p.1958
Note
Enzymatic Assay of Histamine by Amperometric Detection of H2O2
with a Peroxidase-based Sensor
Takao HIBI・・・ and Mitsugi SENDA p.1963
Note
Tyrosinase Inhibitory Activity of Bangladeshi Indigenous Medicinal Plants
Firoza KHANOM, Hiroshi KAYAHARA,1,・・・ and Koji TADASA1 p.1967
Note
A dihydroxy-γ-lactone as an Oviposition Stimulant
for the Swallowtail Butterfly, Papilio bianor,
from the Rutaceous Plant, Orixa japonica
Hajime ONO, Ritsuo NISHIDA,・・・ and Yasumasa KUWAHARA p.1970
Note
Practical Synthesis of the Disaccharide Epitope, D-Galactopyranosyl-α-1,3-D-
galactopyranose, by using 1,2;5,6-Di-O-cyclohexylidene-α-D-galactofuranose
as the Glycosyl Acceptor
Isao SAKAMOTO and Hiroshi OHRUI・・・ p.1974
Note
Purification and Characterization of Biliverdin-binding Protein from Larval
Hemolymph of the Swallowtail Butterfly, Papilio xuthus L.
Akira YAMANAKA,・・・ Takamasa ITO, Daizo KOGA,＊ Toshiyuki SATO,＊＊
Masanori OCHIAI,＊＊＊ and Katsuhiko ENDO p.1978
Note
On the Sweetness of N-(Trifluoroacetyl)aspartame
Michael FRANK and David J. AITKEN・・・ p.1982
Note
Staurosporine Promotion of Formation of Continuous Monolayers
of Primary Rat Hepatocytes by Improving Attachment and Spreading
Sumio MAEDA,・・・ Kong-Hua LIN, Hidetoshi INAGAKI, and Takao SAITO p.1985
Note
Syntheses of Stereochemically Restricted Lactone-type Analogues
of Jasmonic Acids
Hiroaki TOSHIMA,・・・ Hisateru ARAMAKI, and Akitami ICHIHARA p.1988
Note
Recognition of Receptor Lipopolysaccharides by Spike G Protein
of Bacteriophage fX174
Tomoko KAWAURA, Minoru INAGAKI,・・・ Shuichi KARITA,＊ Muneharu KATO,
Shiro NISHIKAWA, and Naoki KASHIMURA p.1993
Note
Fungicidal and Herbicidal Activities of Berberine Related Alkaloids
Kinuko IWASA,＊,・・・ Masataka MORIYASU,＊ and Bassam NADER＊＊ p.1998
Note
Suppression of D-Galactosamine-induced Liver Injury by Mushrooms
in Rats
Eun Woo LEE, Puming HE, Hirokazu KAWAGISHI, and Kimio SUGIYAMA・・・ p.2001
Note
Producing a Low Ovomucoid Egg White Preparation by Precipitation
with Aqueous Ethanol
Soichi TANABE,・・・ Shoko TESAKI, and Michiko WATANABE p.2005
Note
Application of a Metal Switch to Aqualysin I, a Subtilisin-type
Bacterial
Serine Protease, to the S3 Site Residues, Ser102 and Gly131
Terumichi TANAKA,1,・・・ Yo KIKUCHI,1 Hiroshi MATSUZAWA,2 and Takahisa OHTA3
p.2008
Note
High Expression of the Second Lysine Decarboxylase Gene, ldc,
in Escherichia coli WC196 Due to the Recognition of the Stop Codon (TAG),
at a Position Which Corresponds to the 33th Amino Acid Residue of σ38,
as a Serine Residue by the Amber Suppressor, supD
Takahiro NAGANO,1 Yoshimi KIKUCHI,2 and Yoshiyuki KAMIO1,・・・ p.2012
Note
The aman6 Gene Encoding a Yeast Mannan Backbone Degrading
1,6-α-D-Mannanase in Bacillus circulans: Cloning, Sequence Analysis,
and Expression・・・・・・
Yutaka MARUYAMA and Tasuku NAKAJIMA・・・ p.2018
Preliminary Communication
Detection of Biomarkers for Apoptosis in Rat Liver after Perfusion
with 3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1)
Hitoshi ASHIDA,・・・ Kaori KIHARA, Bunsyo SHIOTANI, Takashi HASHIMOTO,
Kazuki KANAZAWA, and Gen-ichi DANNO p.2021
-1-
Erythropoietin: Multiple Physiological Functions and Regulation of Biosynthesis
Ryuzo SASAKI,・・・ Seiji MASUDA, and Masaya NAGAO
Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan
Erythropoietin (Epo), which is produced by the kidney in the adult and by the liver in the fetus, increases red blood cells by supporting the survival of erythroid progenitor cells and stimulating their differentiation and proliferation via binding to Epo receptor (EpoR). The main signal in the control of Epo production is oxygen; hypoxia stimulates Epo production through activation of Epo gene transcription. Tremendous progress in our understanding of molecular mechanisms of Epo action on erythroid cells and regulation of the Epo production has been made by manipulation of cDNAs and genes of Epo and EpoR. Studies on hypoxic induction of Epo gene transcription led to the identification of hypoxia-inducible factor (HIF-1), a transcriptional factor, that functions as a global regulator of hypoxic gene expression. Paracrine Epo/EpoR systems that are independent of the endocrine erythropoietic system (kidney/bone marrow) have been found in the central nervous system and uterus. Novel functions of Epo at these local sites and tissue-specific regulation of Epo production including a newly found potent regulator (estrogen) have been proposed. The tissue-specific regulation rationalizes the specific functions of Epo produced by individual tissues.
Key words: erythropoietin; kidney; brain; uterus; hypoxia
-2-
Effects of Germinated Barley Foodstuff on Microflora and Short Chain Fatty
Acid Production in Dextran Sulfate Sodium-induced Colitis in Rats.
Yoshio ARAKI,1 Akira ANDOH,2 Shigeki KOYAMA,2 Yoshihide FUJIYAMA,2
Osamu KANAUCHI,3 and Tadao BAMBA2
1Department of Internal Medicine, Nagahama Red Cross Hospital, 14-7 Miyamae-chou,
Nagahama, Shiga 526-8585, Japan
2Department of Internal Medicine, Shiga University of Medical Science, Seta Tukinowa, Otsu,
Shiga 520-2100, Japan
3Applied Bioresearch Center, Corporate Research and Development Division, Kirin Brewery Co. Ltd.,
3 Miyaharacho, Takasaki, Gunma 370-12, Japan
Received September 28, 1999; Accepted May 17, 2000
Germinated barley foodstuff (GBF) administration has been previously reported to suppress dextran sulfate sodium (DSS)-induced experimental colitis. In this study, we investigated the roles of the intestinal microflora and short chain fatty acids (SCFAs) following administration of GBF in DSS-induced rat colitis. Sprague-Dawley rats were fed 3％ (w/w of diet) DSS in GBF-diets for 5 days. The control rats were fed 3％ DSS in cellulose-diets for 5 days. The administration of GBF effectively prevented bloody diarrhea and mucosal damage as compared to control rats. GBF significantly elevated fecal acetic acid and n-butyric acid levels. GBF tended to increase the number of eubacteria and that of bifidobacteria as compared to control rats. In addition, the number of enterobacteriaceae, the total number of aerobes and bacteroidaseae, were significantly lower in rats fed GBF than in the control group. It is suggested that the therapeutic effects of GBF for DSS-induced colitis depend mainly on increased SCFAs, which are accompanied by changes of composition of intestinal bacteria.
Key words: germinated barley foodstuff; microflora; dextran sulfate sodium-induced colitis; short chain fatty acids; eubacteria
-3-
Ascorbic Acid Stimulation of Production of a Highly Branched β-1,3-Glucan by
Aureobasidium pullulans K-1---Oxalic Acid, a Metabolite of Ascorbic Acid
as the Stimulating Substance---
Nobutake HAMADA,・・・ Kohji DEGUCHI,＊ Takashi OHMOTO, Kiyofumi SAKAI,
Tatsuhiko OHE, and Hajime YOSHIZUMI＊
Osaka Municipal Technical Research Institute, 6-50, Morinomiya 1-chome, Joto-ku, Osaka 536-8553, Japan
＊Agriculture Faculty of Kinki University, 3327-204, Nakamachi, Nara 631-0052, Japan
Received January 7, 2000; Accepted May 9, 2000
The production of a highly branched β-1,3-glucan by Aureobasidium pullulans K-1 in Czapek′s medium has been found to be stimulated by ascorbic acid. When the culture supernatant, after removal of polysaccharide from the culture filtrate by ethanol precipitation, was concentrated, then added to a new medium and this strain was cultured in the medium, the polysaccharide production was stimulated the same as when L-ascorbic acid was added to the medium. The stimulating substance was partially purified from the supernatant, and was found to be oxalic acid; 0.03％ oxalic acid was the most effective concentration for the stimulation of polysaccharide production. The stimulating substance, oxalic acid, was proved to be derived from ascorbic acid added to a medium in an experiment using L-[1-14C]ascorbic acid. We suggest that oxalic acid generated from the metabolism of ascorbic acid in cells of Aureobasidium pullulans K-1 participated in the stimulation of the polysaccharide production by ascorbic acid.
Key words: Aureobasidium pullulans; β-1,3-glucan; polysaccharide production stimulating substance; ascorbic acid; oxalic acid
-4-
Production of Betacyanins by a Cell Suspension Culture of Table Beet
(Beta vulgaris L.)
Toru AKITA,・・・ Yasuhiko HINA, and Toyoyuki NISHI
The Nippon Shinyaku Institute for Botanical Research, 39 Sakanotsuji-cho, Ohyake, Yamashina-ku,
Kyoto 607-8182, Japan
Received January 14, 2000; Accepted May 2, 2000
A cell suspension culture of table beet (Beta vulgaris L.) was established for efficient betacyanin production from violet callus induced from the hypocotyls of aseptic seedlings. This suspension culture produced large amounts of betacyanins. The betacyanin content increased with increasing cell growth during the log phase. Reducing the total nitrogen concentration (30 mM) and modifying the ratio of ammonium to nitrate (1:14) resulted in an increased betacyanin content. Supplementation of Fe2+ to the LS medium also promoted betacyanin production. The maximal betacyanin yield was achieved with a 2 mM Fe2+ concentration. Combining these conditions, we established a revised LS medium to improve betacyanin productivity (250 mg/l for a 14-day culture).
Key words: Beta vulgaris L.; betacyanin; cell suspension culture
-5-
Flavonoids Inhibit Cell Growth and Induce Apoptosis
in B16 Melanoma 4A5 Cells
Keiko IWASHITA,・・・ Masuko KOBORI,＊ Kohji YAMAKI,＊ and Tojiro TSUSHIDA＊
Institute of Applied Biochemistry, University of Tsukuba, 1-1-1 Tennodai, Tsukuba,
Ibaraki 305-0006, Japan
＊National Food Research Institute, Ministry of Agriculture, Forestry and Fisheries,
2-1-2 Kannondai, Tsukuba, Ibaraki 305-8642, Japan
Received January 28, 2000; Accepted April 24, 2000
We investigated the growth inhibitory activity of several flavonoids, including apigenin, luteolin, kaempherol, quercetin, butein, isoliquiritigenin, naringenin, genistein, and daizein against B16 mouse melanoma 4A5 cells. Isoliquiritigenin and butein, belonging to the chalcone group, markedly suppressed the growth of B16 melanoma cells and induced cell death. The other flavonoids tested showed little growth inhibitory activity and scarcely caused cell death. In cells treated with isoliquiritigenin or butein, condensation of nuclei and fragmentation of nuclear DNA, which are typical phenomena of apoptosis, were observed by Hoechst 33258 staining and by agarose gel electrophoresis of DNA. Flowcytometric analysis showed that isoliquiritigenin and butein increased the proportion of hypodiploid cells in the population of B16 melanoma cells. These results demonstrate that isoliquiritigenin and butein inhibit cell proliferation and induce apoptosis in B16 melanoma cells. Extracellular glucose decreased the proportion of hypodiploid cells that appeared as a result of isoliquiritigenin treatment. p53 was not detected in cells treated with either of these chalcones, however, protein of the Bcl-2 family were detected. The level of expression of Bax in cells treated with either of these chalcones was markedly elevated and the level of Bcl-XL decreased slightly.
Isoliquiritigenin did not affect Bcl-2 expression, but butein down-regulated Bcl-2 expression. From these results, it seems that the pathway by which the chalcones induce apoptosis may be independent of p53 and dependent on proteins of the Bcl-2 family. It was supposed that isoliquiritigenin induces apoptosis in B16 cells by a mechanism involving inhibition of glucose transmembrane transport and promotion of Bax expression. On the other hand, it was suggested that butein induces apoptosis via down-regulation of Bcl-2 expression and promotion of Bax expression. This mechanism differs from the isoliquiritigenin induction pathway.
・
Key words: flavonoid; antiproliferation; apoptosis; melanoma cells; Bcl-2 family
-6-
Synthesis of α-D-Glucosylglycerol by α-Glucosidase and Some
of Its Characteristics・・・
Fumihito TAKENAKA・・・・・・ and Hirofumi UCHIYAMA
Tatsuuma-honke Brewing Co., Ltd., 2-6 Tateishi-cho, Nishinomiya, Hyogo 662-0943, Japan
Received January 31, 2000; Accepted April 13, 2000
It has been found that α-D-glucosylglycerol (GG) is contained in such traditional Japanese foods brewed by using koji as sake, miso and mirin, and that GG is formed by transglucosylation to glycerol that is produced by yeast with α-glucosidase (EC 3.2.1.20) from koji in the sake mash. GG has also been found to consist of three components, 2-O-α-D-glucosylglycerol (GG-II), (2R)-1-O-α-D-glucosylglycerol (R-GG-I) and (2S)-1-O-α-D-glucosylglycerol (S-GG-I). GG was synthesized from a mixture of maltose and glycerol by the batch method, using α-glucosidase (transglucosidase L-AMANO). α-Glucosidase seemed to be so stable that the amount of GG increased about 5-fold compared with that in the first reaction by the daily addition of maltose for 10 d. Syrupy GG obtained was found to have the following characteristics: about 0.55-fold sweetness compared with sucrose, high thermo-stability, low heat-colorability, low Maillard reactivity, low hygroscopicity, high water-holding capacity, non-cariogenicity and low digestibility.
Key words: α-D-glucosylglycerol; enzymatic synthesis; characteristics; α-glucosidase; batch method
-7-
Interaction of Gum Arabic, Maltodextrin and Pullulan
with Lipids in Emulsions
Yasuki MATSUMURA,・・・ Chikako SATAKE, Masaaki EGAMI, and Tomohiko MORI
Research Institute for Food Science, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan
Received February 4, 2000; Accepted April 18, 2000
The interaction of gum arabic, maltodextrin and pullulan with lipids in emulsion systems was investigated. Interfacial tension and interfacial viscosity measurements revealed that only gum arabic could adsorb and form a viscoelastic film at the oil-water interface. Good emulsifying activity was demonstrated for gum arabic, whereas fine emulsions could not be produced from the other polysaccharide solutions and oil. Frequency-dependent increases in the storage and loss moduli were observed for all the polysaccharide solutions. Such rheological behavior did not substantially change when maltodextrin and pullulan were mixed with oil to form emulsions. However, the frequency-dependence of the dynamic moduli disappeared in the gum arabic-stabilized emulsion, suggesting the formation of a network structure in which oil droplets could form junctions with gum arabic chains. The results on the inhibition of lipid oxidation by polysaccharides suggest that gum arabic protected lipids from the attack of lipoxygenase and free radicals by adsorbing at the oil droplet surface.
Key words: emulsion; gum arabic; maltodextrin; pullulan; polysaccharide and lipid interaction
-8-
Preparative 2?-Reduction of ATP Catalyzed by Ribonucleotide
Reductase Purified by Liquid-Liquid Extraction
Andr"e BRUNELLA,1 Miguel ABRANTES,2 and Oreste GHISALBA3
1Outcomes International, Malzgasse 9, CH-4052 Basel, Switzerland
2Logoplaste SA, P-1000 Lisboa, Portugal
3Novartis Pharma AG, WSJ-508.202A, CH-4002 Basel, Switzerland
Received February 7, 2000; Accepted April 27, 2000
Recombinant Lactobacillus leichmannii ribonucleosidetriphosphate reductase (RTPR, E.C.1.17.4.2) constitutively expressed by E. coli HB101 pSQUIRE has been purified from sonicated cell material in a one-step procedure by PEG 4000 (16％ (w/w))/phosphate (7％ (w/w)) liquid-liquid extraction. A high yield of 75.1％ RTPR in the top phase and a partitioning of 8.5:1 between total RTPR activity in top and bottom phase were obtained in this preparative system. The RTPR-containing top phase was used to reduce ATP in the 2?-position on a gram scale with high final conversion and yield proving the ribonucleotide reductase approach feasible for the preparative synthesis of 2?-deoxyribonucleotides. High concentrations of sodium acetate in the reaction served to substitute for allosteric effectors of RTPR. 1,4-Dithio-DL-threitol was used as an artificial reducing agent for RTPR.
Key words: liquid-liquid extraction; enzymatic synthesis; ribonucleotide reductase; ATP; 2?-deoxyadenosine-5?-triphosphate
-9-
Absolute Configuration of a Ceramide with a Novel Branched-chain Fatty
Acid Isolated from the Epiphytic Dinoflagellate, Coolia monotis
Kazuaki AKASAKA,1 Seiya SHICHIJYUKARI,1 Shigeru MATSUOKA,2 Michio MURATA,2
Hiroshi MEGURO,3 and Hiroshi OHRUI1＊
1Graduate School of Agricultural Science, Tohoku University, Tsutsumidori-Amamiyamachi 1-1,
Aoba-ku, Sendai 981-8555, Japan
2Graduate School of Science, Osaka University, Machikaneyama 1-1, Toyonaka-city,
Osaka 560-0043, Japan
3Tohoku Fukushi University, Kunimi, Aoba-ku, Sendai 981-0943, Japan
Recived February 14, 2000; Accepted April 13, 2000
The absolute configuration of the chiral center at the C15 position of a novel branched-chain fatty acid derived from a new ceramide isolated from the epiphytic dinoflagellate Coolia monotis was determined to be of R from by reversed-phase HPLC after cleavage to 12-methylpentadecanoic acid and subsequent conversion with the chiral fluorescent reagent, (1R,2R)-2-(2,3-anthracenedicarboximido)cyclohexanol.
Key words: absolute configuration; chiral discrimination; branched-chain fatty acid; chiral conversion reagent
-11-
Structural Analysis of Free N-Glycans Occurring in Soybean Seedlings
and Dry Seeds＊
Yoshinobu KIMURA・・・ and Erika KITAHARA
Department of Bioresources Chemistry, Faculty of Agriculture, Okayama University,
Okayama 700-8530, Japan
Received February 16, 2000; Accepted April 27, 2000
The structures of unconjugated or free N-glycans in stems of soybean seedlings and dry seeds have been identified. The free N-glycans were extracted from the stems of seedlings or defatted dry seeds. After desalting by two kinds of ion-exchange chromatography and a gel filtration, the free N-glycans were coupled with 2-aminopyridine. The resulting fluorescence-labeled (PA-) N-glycans were purified by gel filtration, Con A affinity chromatography, reverse-phase HPLC, and size-fractionation HPLC. The structures of the PA-sugar chains purified were analyzed by the combination of two-dimensional sugar chain mapping, jack bean α-mannosidase digestion, α-1,2-mannosidase digestions, partial acetolysis, and ESI-MS/MS. The free N-glycan structures found showed that two categories of free N-glycans occur in the stems of soybean seedlings. One is a high-mannose type structure having one GlcNAc residue at the reducing end (Man9~5GlcNAc1, 93％), that would be derived by endo-GM (Kimura, Y. et al., Biochim. Biophys. Acta, 1381, 27--36 (1998)). The other small component is a xylose-containing type one having two GlcNAc residues at the reducing end (Man3Xyl1GlcNAc2, 7％), which would be derived by PNGase-GM (Kimura, Y. and Ohno, A., Biosci. Biotechnol. Biochem., 62, 412--418 (1998)). The detailed structural analysis of free glycans showed that high-mannose type free N-glycans (Man9~5GlcNAc1) in the soybean seedlings have a common core structural unit; Manα1--
6(Man1--3)Manα1--6(Manα1--3)Manβ1--4GlcNAc.
Comparing the amount of free N-glycans in the seedling stems and dry seeds, the amount in the stems of seedlings was much higher than that in the dry seeds; approximately 700 pmol per one stem, 8 pmol in one dry seed. This fact suggested that free N-glycans in soybean seedlings could be produced by two kinds of N-glycan releasing enzymes during germination or seedling-development.
Key words: free N-glycan; N-glycan metabolism; end-β-N-acetylglucosaminidase; pepetid:N-glycanase; Glycine max
-12-
Epitope Analysis and Primary Structures of Variable Regions
of Anti-human FcεRI Monoclonal Antibodies, and Expression
of the Chimeric Antibodies Fused with Human Constant Regions
Toshiro TAKAI,1,2,・・・ Toshifumi YUUKI,2 Namiko IWAMOTO-YASUE,2
Ko OKUMURA,1 and Chisei RA1,3
1Department of Immunology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku,
Tokyo 113-8421, Japan
2Bioscience Research ＆ Development Laboratory, Asahi Breweries, Ltd., 1-21 Midori 1-chome,
Moriya-machi, Kitasoma-gun, Ibaraki 302-0106, Japan
3Division of Molecular Biology, Allergy Research Center, Juntendo University School of Medicine,
2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan
Received February 17, 2000; Accepted April 24, 2000
The structural analysis of monoclonal antibodies (mAbs) against the α subunit of the high affinity IgE receptor (FcεRIα) is an alternative approach to obtaining information for the design of inhibitors that will block complementary interaction between IgE and FcεRIα and to analyzing the various biological effects induced by anti-FcεRIα autoantibodies in chronic urticaria. In this study, epitopes for mouse anti-human FcεRIα mAbs and primary structures of variable regions of the mAbs were analyzed. Three mAbs inhibitory for IgE-binding reacted to the deletion mutants of FcεRIα containing the whole second immunoglobulin-like domain as well as IgE did. On the other hand, two uninhibitory mAbs reacted to those containing the whole first immunoglobulin-like domain. The cDNAs for variable regions of the five mAbs were cloned and sequenced. Two inhibitory mouse/human chimeric antibodies were expressed in COS7 cells and bound to Chinese hamster ovary transfectant cells expressing FcεRI (CHO/αβγ), and these inhibited the binding of IgE to CHO/αβγ cells.
Key words: high-affinity IgE receptor; monoclonal antibody; epitope; variable region; chimeric antibody
-13-
Preventive Effect of Lactobacillus delbrueckii subsp. bulgaricus
on the Oxidation of LDL
Masaki TERAHARA,・・・ Sachiko NISHIDE, and Tsutomu KANEKO
Food Functionality Research Institute, Meiji Milk Products Co., Ltd., 1-21-3 Sakae-Cho,
Higashimurayama, Tokyo 189-8530, Japan
Received February 18, 2000; Accepted April 23, 2000
Lactobacillus delbrueckii subsp. bulgaricus 2038 was examined for its activity to prevent the oxidation of the erythrocyte membrane in vitro, and the oxidation of LDL in vivo.
Strain 2038 produced radical scavengers that reacted with 1,1-diphenyl-2-picrylhydrazl (DPPH) during cultivation. Moreover, the ethereal extract from the supernatant of the culture prevented the oxidation of the erythrocyte membrane in vitro.
As an in vivo study, male F344 rats were fed on diets containing 20％ fresh soybean oil (or 13％ oxidized oil and 7％ fresh oil) with 10％ freeze-dried powder of the 2038 culture (or with skim milk powder) for 4 weeks. The level of thiobarbituric acid-reactive substances was lower in the low-density lipoproteins (per milligram of cholesterol) from rats fed on the oxidized oil with freeze-dried powder of the 2038 culture than without it. The level of vitamin E in the plasma was higher in the rats fed on the oxidized oil with the freeze-dried powder than without it.
Key words: 1,1-diphenyl-2-picrylhydrazl (DPPH); erythrocyte membrane; thiobarbituric acid-reactive substances (TBARS); malondialdehyde (MDA); vitamin E
-14-
Characteristics of Serine Acetyltransferase from Escherichia coli Deleting
Different Lengths of Amino Acid Residues from the C-Terminus
Koshiki MINO, Kenji HIRAOKA, Koreyoshi IMAMURA, Takaharu SAKIYAMA,
Naoki EISAKI,＊ Asahi MATSUYAMA,＊ and Kazuhiro NAKANISHI・・・
Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University,
3-1-1 Tsushima-naka, Okayama 700-8530, Japan
＊Research and Development Division, Kikkoman Corporation, 399 Noda, Chiba 278-0037,
Japan/Research Institute of Innovative Technology for the Earth (RITE), 2-8-11 Nishi-shinbashi,
Minato-ku, Tokyo 105-0003, Japan
Received March 1, 2000; Accepted May 16, 2000
Some properties of serine acetyltransferases (SATs) from Escherichia coli, deleting 10--25 amino acid residues from the C-terminus (SATΔC10--ΔC25) were investigated. The specific activity depended only slightly on the length of the C-terminal region deleted. Although the sensitivity of SATΔC10 to inhibition by L-cysteine was similar to that for the wild-type SAT, it became less with further increases in the length of the amino acid residues deleted. SATΔC10 was inactivated on cooling to 0°C and dissociated into dimers or trimers in the same manner as the wild-type SAT, but Met-256-Ile mutant SAT as well as SATΔC14, SATΔC20, and SATΔC25 were stable. Since SATΔC10, SATΔC14, and SATΔC25 did not form a complex with O-acetylserine sulfhydrylase-A (OASS-A) in a way similar to SATΔC20, it was indicated that 10 amino acid residues or fewer from the C-terminus of the wild-type SAT are responsible for the complex formation with OASS-A. The C-terminal peptide of the 10 amino acid residues interacted competitively with OASS-A with respect to OAS although its affinity was much lower than that for the wild-type SAT.
Key words: serine acetyltransferase; O-acetylserine sulfhydrylase; cysteine synthetase; cold inactivation; C-terminal region
-15-
Safety Assessment of Rice Genetically Modified with Soybean Glycinin
by Feeding Studies on Rats
Keiko MOMMA,・・・ Wataru HASHIMOTO, Hye-Jin YOON, Sachiko OZAWA, Yasuki FUKUDA,＊
Shigeyuki KAWAI, Fumio TAKAIWA,＊＊ Shigeru UTSUMI, and Kousaku MURATA
Research Institute for Food Science, Kyoto University, Uji, Kyoto 611-0011, Japan
＊Chukyo Community College, Mizunami, Gifu 509-6192, Japan
＊＊National Institute of Agrobiological Resources, Tsukuba, Ibaraki 305-8056, Japan
Received March 8, 2000; Accepted April 26, 2000
Feeding studies on rice genetically modified with soybean glycinin were performed on rats for four weeks. The rats were divided into three groups, each being fed on (I) only a commercial diet, (II) this diet plus control rice and (III) this diet plus rice genetically modified with glycinin. The rats were fed with 10 g/kg-weight of rice every day by oral administration. During the test period, the rats in every group grew well without marked differences in appearance, food intake, body weight, or cumulative body weight gain. There were also no significant differences in the blood count, blood composition or internal organ weights among the rats. Necropsy at the end of the experiment indicated neither pathological symptoms nor histopathological abnormalities in the liver and kidney. Judging from these results, the rice genetically modified with glycinin is considered to have been essentially the same in nutritional and biochemical characteristics as the control rice.
Key words: animal feeding study; food safety; genetically modified rice; soybean glycinin
-16-
Meat Allergy: Investigation of Potential Allergenic Proteins in Beef
Gi Dong HAN,a Masatomo MATSUNO,b Giichi ITO,c Yoshihide IKEUCHI,d
and Atsushi SUZUKId,・・・
aDoctor′s Program in Functional Biology, Graduate School of Science and Technology,
University of Niigata, Niigata 950-2181, Japan
bDepartment of Pediatrics, Yoshida Hospital, Yoshida 959-9213, Japan
cHealth Administration Center, University of Niigata, Niigata 950-2181, Japan
dDepartment of Applied Biological Chemistry, Faculty of Agriculture, University of Niigata,
Niigata 950-2181, Japan
Received March 9, 2000; Accepted April 15, 2000
The potential allergenic proteins in beef were investigated. The sera of ten beef-allergic patients suffering from atopic dermatitis and having a positive RAST score to beef, aged 3--18 years, were obtained from Yoshida Hospital in Japan, and five non-allergic individuals were subjected to this study. The sera of the ten patients reacted strongly to a beef extract, but not to pork and chicken extracts by both ELISA and immunoblotting. The sera of the five control subjects did not react to any of these meat extracts. Three bands having molecular masses of ~200 kDa, ~67 kDa and ~60 kDa were observed by immunoblotting after SDS-PAGE. Two fractions of the beef extract from a Sephadex-gel (G-200) filtration column strongly reacted with the sera of the beef-allergic patients by ELISA and immunoblotting: one fraction had the ~67 kDa component and the other had the ~200 kDa and ~60 kDa components. One of them (~67 kDa) was confirmed to be bovine serum albumin (BSA) by an analysis of the N-terminal amino acid sequence. We could not identify the others by sequencing, but the ~200 kDa and ~60 kDa components were presumed to be glycoproteins. Bovine gamma globulin (BGG: M.W. ~160 kDa) is a glycoprotein and has several subunits. The beef-allergic patients showed strong reactivity to the ~200 kDa and ~60 kDa components of pure BGG by immunoblotting. Inhibition-ELISA showed that pure BGG preparations strongly inhibited the binding of sera from the beef-allergic patients to the beef extract. These results suggest that the ~200 kDa, ~67 kDa and ~60 kDa components in the beef extract had strong allergenicity: ~67 kDa was BSA, and ~200 kDa and ~60 kDa were presumably aggregated BGG and it′s heavy chain, respectively.
Key words: meat allergy; beef allergy; beef extract; BSA; BGG
-17-
Purification and Characterization of Chitosanase and Exo-β-D-Glucosaminidase
from a Koji Mold, Aspergillus oryzae IAM2660
Xiao-Yong ZHANG, An-Lan DAI, Xue-Kun ZHANG, Kouji KUROIWA,
Ritsuko KODAIRA, Makoto SHIMOSAKA,・・・ and Mitsuo OKAZAKI＊
Department of Applied Biology, Faculty of Textile Science and Technology, and ＊Gene Research Center,
Shinshu University, 3-15-1 Tokida, Ueda, Nagano 386-8567, JapanReceived March 13, 2000; Accepted May 22, 2000
Chitosan-degrading activity was detected in the culture fluid of Aspergillus oryzae, A. sojae, and A. flavus among various fungal strains belonging to the genus Aspergillus. One of the strong producers, A. oryzae IAM2660 had a higher level of chitosanolytic activity when N-acetylglucosamine (GlcNAc) was used as a carbon source. Two chitosanolytic enzymes, 40 kDa and 135 kDa in molecular masses, were purified from the culture fluid of A. oryzae IAM2660. Viscosimetric assay and an analysis of reaction products by thin-layer chromatography clearly indicated the endo- and exo-type cleavage manner for the 40-kDa and 135-kDa enzymes, respectively. The 40-kDa enzyme, designated chitosanase, catalyzed a hydrolysis of glucosamine (GlcN) oligomers larger than pentamer, glycol chitosan, and chitosan with a low degree of acetylation (0--30％). The 135-kDa enzyme, named exo-β-D-glucosaminidase, released a single GlcN residue from the GlcN oligomers and chitosan, but did not release GlcNAc residues from either GlcNAc oligomer or colloidal chitin.
Key words: chitosan; chitosanase; exo-β-D-glucosaminidase; Aspergillus
-18-
Cloning and Expression of a Novel Murine Anti-human Fas Antibody
Hiroko YOSHIDA-KATO,1 Kimihisa ICHIKAWA,1 Junko YAMAGUCHI,1 Kenji WATANABE,1
Jun OHSUMI,1 Shin YONEHARA,2 and Nobufusa SERIZAWA1,・・・
1Biomedical Research Laboratories, Sankyo Co., Ltd., Tokyo 140-8710, Japan
2Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan
Received March 13, 2000; Accepted May 1, 2000
Agonistic anti-human Fas antibodies that can induce apoptosis are thought to have therapeutic effects for various diseases resulting from an abnormality of the Fas/FasL system. However, some anti-Fas antibodies show toxicity, and it is difficult to investigate their therapeutic and toxicological effect using animals because of their species specificity. We previously obtained a murine anti-human Fas mAb, HFE7A. HFE7A reacted with both human and murine Fas, and mitigated lymphadenopathy without any sign of hepatotoxicity in MRLgld/gld mice. It is suggested that humanized HFE7A would be a therapeutic treatment for various diseases resulting from an abnormality of the Fas/FasL system. Here we isolated the cDNAs that code for the heavy and light chains of HFE7A and identified the corresponding nucleotide sequences. The recombinant HFE7A was indistinguishable in binding and apoptosis-inducing activity to that from a hybridoma cell line. These data provide essential information for the humanization and clinical application of the humanized HFE7A.
Key words: Fas; apoptosis; antibody; cloning
-19-
Kurosu, a Traditional Vinegar Produced from Unpolished Rice,
Suppresses Lipid Peroxidation in Vitro and in Mouse Skin
Shoko NISHIDAI, Yoshimasa NAKAMURA,＊ Koji TORIKAI,＊ Mikako YAMAMOTO,
Nobuhiro ISHIHARA, Hirotaka MORI, and Hajime OHIGASHI＊,・・・
Research Center, Tamanoi Vinegar Co. Ltd., 100 Nishimachi, Yamatokoriyama,
Nara 639-1038, Japan
＊Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University,
Kyoto 606-8502, Japan
Received March 15, 2000; Accepted April 28, 2000
The in vitro antioxidative activities of various kinds of vinegar were investigated by using a linoleic acid autoxidation model detected by the thiobarbituric acid (TBA) method and the 1,1-diphenyl-2-picrylhydrazyl radical system. An ethyl acetate extract of Kurosu (EK), a vinegar made from unpolished rice, exhibited the highest antioxidative activity in both systems. EK (5 mg) inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced edema formation (14％) and myeloperoxidase activity (52％, P<0.01) in female ICR mouse skin. Furthermore, EK significantly suppressed double TPA application-induced H2O2 generation (53％, P<0.01) and lipid peroxidation determined by the TBA-reacting substance level (95％, P<0.01). In a two-stage carcinogenesis experiment with dimethylbenz[a]anthracene/TPA, EK significantly reduced the number of tumors per mouse by 36％ (P<0.05) at 15 weeks after promotion. These results suggest that the antitumor-promoting effect may be partially due to the antioxidative properties of EK such as the decomposition of free radicals and interference with free radical-generating leukocytes.
Key words: antioxidative activity; Kurosu; vinegar; phorbol ester; tumor promotion
-20-
Synthesis of 2-C-Methyl-D-erythritol and 2-C-Methyl-L-threitol; Determination
of the Absolute Configuration of 2-C-Methyl-1,2,3,4-butanetetrol Isolated
from Phlox sublata L
Isao SAKAMOTO,1 Kazuo ICHIMURA,2 and Hiroshi OHRUI1,・・・
1Division of Life Science, Graduate School of Agricultural Science, Tohoku University,
1-1 Tsutsumidori Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan
2National Research Institute of Vegetables, Ornamental Plants and Tea, Ano, Mie 514-2392, Japan
Received March 16, 2000; Accepted April 26, 2000
2-C-Methyl-D-erythritol (A) and 2-C-methyl-L-threitol (B) were respectively synthesized from D-glucose and D-galactose. The 2-C-methyl-1,2,3,4-butanetetrol compound (C) recently isolated from Phlox sublata L was confirmed to be A by comparing the CD and 1H-NMR spoectra of its tri-O-benzoate with those of A and B.
Key words: 2-C-methyl-D-erythritol; non-mevalonate isoprenoid biosynthesis; Phlox sublata L; absolute configuration
-21-
Thermally Induced Disintegration of the Bacillus stearothermophilus
Dihydrolipoamide Dehydrogenase・・・
Yasuaki HIROMASA, Yoichi ASO,＊,・・・・・・ Shoji YAMASHITA,＊＊ and Kohji MENO＊
RIKEN, Institute of Physical and Chemical Research, Sayo-gun, Hyogo 679-5143, Japan
＊Laboratory of Protein Chemistry and Engineering, and ＊＊ Laboratory of Biophysics,
Kyushu University, Fukuoka 812-8581, Japan
Received March 17, 2000; Accepted May 9, 2000
Upon heat treatment of the pyruvate dehydrogenase complex from Bacillus stearothermophilus, the most thermostable component is a dihydrolipoamide dehydrogenase (E3c). To understand this stability, the thermal disintegration of E3 dissociated from the complex (E3d) was examined, comparing with that of E3c. Judging from residual activity and inactivation rate, E3d was less thermostable than E3c; E3d and E3c lost half of their original activities upon incubations for 30 min at 79°C and 90°C, respectively. Heat treatment of E3d raised the fluorescence intensities of Trp residue, intrinsic FAD, and extrinsic 8-anilinonaphthalene-1-sulfonate. E3d lost FAD, and inactive E3d polypeptides were aggregated. The sulfonate bound to the aggregate became notably fluorescent. The thermal disintegration of E3d was speculated to be a consecutive reaction that was different from the concurrent disintegration reaction of the complex. Some interactions with other component polypeptides was suggested to improve the thermostability of E3c.
Key words: Bacillus stearothermophilus; dihydrolipoamide dehydrogenase; pyruvate dehydrogenase complex
-22-
Gene Regulation in Response to Overexpression of Cytochrome P450
and Proliferation of the Endoplasmic Reticulum
in Saccharomyces cerevisiae
Thomas ZIMMER,1,2 Atsushi OGURA,1 Tsuyoshi TAKEWAKA,1 Rose-Marie ZIMMER,1,2
Akinori OHTA,1,・・・ and Masamichi TAKAGI1
1Department of Biotechnology, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan
2Friedrich Schiller University Jena, Institute of Physiology and Institute of Pathophysiology, 07740 Jena,
Germany
Received March 17, 2000; Accepted May 10, 2000
In this study, we addressed the question of which genes are transcriptionally co-regulated upon proliferation of the endoplasmic reticulum, as induced by an experimental overexpression of cytochrome P450Cm2 (CYP52A4) in Saccharomyces cerevisiae. Using the mRNA differential display technique, six genes were found to be up-regulated: ASN2, MDJ1, YLR194c, YNL208w, YER175, and YGL121c. Genes coding for Dur1.2p, Dal2p, and Sps19p were down-regulated. Two strongly induced genes, which were found to accommodate the peroxisome box (YLR194c) and a 10-bp consensus sequence of genes involved in lipid metabolism (YNL208w) in their promoter regions, were further analyzed with respect to the course of induction, the necessity of the P450 membrane anchor for induction, and the effects of gene disruption on P450Cm2 overexpression. We found that both genes are not essential to overproduce P450Cm2, but their induction was dependent on P450Cm2 membrane integration.
Key words: cytochrome P450; ER proliferation; mRNA differential display; yeast
-23-
Defect in Cell Wall Integrity of the Yeast Saccharomyces cerevisiae Caused
by a Mutation of the GDP-mannose Pyrophosphorylase Gene VIG9
Koji YODA,・・・ Tsuyoshi KAWADA, Chiaki KAIBARA, Akihiko FUJIE, Masato ABE, Hitoshi HASHIMOTO, Jiro SHIMIZU, Nario TOMISHIGE, Yoichi NODA, and Makari YAMASAKI・・・・・・
Department of Biotechnology, The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
Received March 27, 2000; Accepted May 18, 2000
The Saccharomyces cerevisiae VIG9 gene encodes GDP-mannose pyrophosphorylase, which synthesizes GDP-mannose from GTP and mannose-1-phosphate. Although the null mutant was lethal, the vig9 mutants so far obtained showed no growth defect but immature protein glycosylation and drug hypersensitivity. During our search for cell-wall mutants, we found a novel temperature-sensitive mutant, JS30, which required an osmotic stabilizer for viability. JS30 excreted cell surface proteins in the medium without any indication of cell lysis. Although conventional genetic analysis using mating was impossible, by detailed characterization of JS30 including an in vitro enzyme assay and nucleotide sequencing, we found the defect of JS30 was due to a mutation in the VIG9 gene. These results indicated a critical role of GDP-mannose in maintenance of cell-wall integrity.
Key words: GDP-mannose pyrophosphorylase; protein glycosylation; VIG9; cell wall; Saccharomyces cerevisiae
-24-
A Positive Screening for Drugs that Specifically Inhibit the Ca2+-Signaling
Activity on the Basis of the Growth Promoting Effect on a Yeast Mutant
with a Peculiar Phenotype
Atsunori SHITAMUKAI,1 Masaki MIZUNUMA,1 Dai HIRATA,1 Hidetoshi TAKAHASHI,2
and Tokichi MIYAKAWA1,・・・
1Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter,
Hiroshima University, Higashi-Hiroshima 739-8526 Japan
2Research Institute of Life Science, Snow Brand Milk Products Co., Ltd., Ishibashi-machi,
Shimotsuga-gun, Tochigi, 329-0512, Japan
Received March 27, 2000; Accepted May 1, 2000
An inappropriate activation of a signaling pathway in yeast often has a deleterious physiological effect and causes various defects, including growth defects. In a certain genetic background (Δzds1) of Saccharomyces cerevisiae, the cell-cycle progression in G2 is specifically blocked in the medium with CaCl2 by the hyperactivation of the Ca2+-signaling pathways. Here, we developed a novel drug screening procedure designed to detect the active compounds that specifically attenuate the Ca2+-signaling activity on the basis of the ability to abrogate the growth defect of the cells suffering from the hyperactivated Ca2+ signal. Using known calcineurin inhibitors as model compounds, we have established the screening conditions for the drugs that suppress the Ca2+-induced growth inhibition. An indicator strain with an increased drug sensitivity was constructed with a syr1/erg3 null mutation.
Key words: Ca2+ signal; calcineurin; drug screening; immunosuppressant; MAP kinase
-25-
Molecular Cloning and Characterization of a cDNA and a Gene
for Subtilisin-like Serine Proteases from Rice (Oryza sativa L.)
and Arabidopsis thaliana＊
Hiroshi YAMAGATA,・・・ Maki UESUGI, Kazunori SAKA, Teruo IWASAKI,＊＊ and Yasuo AIZONO
Laboratory of Biological Chemistry, Faculty of Agriculture, Kobe University, Nada, Kobe 657-8501, Japan
Received March 27, 2000; Accepted May 9, 2000
The complete nucleotide sequences of a cDNA (RSP1) that encodes a subtilisin-like serine protease (subtilase) of rice (Oryza sativa L.) and a gene (ASP48) for Arabidopsis subtilase were analyzed. The RSP1 cDNA and ASP48 DNA encoded 736- and 757-residue pre-pro-polypeptides including a signal peptide with molecular masses of 78,668 Da and 79,414 Da, respectively. RSP1 is the first known serine protease in rice, and ASP48 is a gene for ara12 cDNA. Sequence comparison and phylogenetic analysis showed that RSP1 is distantly related to all other plant subtilases and ASP48 is closely related to a tomato subtilase, SBT1. The ASP48 gene was found to lack introns. The Arabidopsis subtilase gene appears to consist of a small gene family. The RSP1 was found to be expressed in seed and shoots of seedlings while ASP48 transcripts was found to be accumulated in immature silique and flowers, indicating that both RSP1 and ASP48 are organ-specific and may be involved in the specific proteolytic events that occur during organ development.
Key words: serine protease; subtilisin; plant subtilase; rice; Arabidopsis thaliana
-26-
Note
Degradation of Bisphenol A by the Lignin-Degrading Enzyme,
Manganese Peroxidase, Produced by the White-rot Basidiomycete,
Pleurotus ostreatus
Taeko HIRANO, Yoichi HONDA, Takashi WATANABE, and Masaaki KUWAHARA
Wood Research Institute, Kyoto University, Uji, Kyoto 611-0011, Japan
Received August 12, 1999; Accepted April 27,2000
Degradation of 2,2-Bis(4-hydroxyphenyl)propane (bisphenol A, BPA), an endocrine-disturbing chemical, by the growing mycelia of the white-rot basidiomycete, Pleurotus ostreatus, was examined. About 80％ of BPA initially present decreased in 12 days of culture with this fungus. By in vitro experiments using the lignin-degrading enzyme manganese peroxidase (MnP), BPA was metabolized to phenol, 4-isopropenylphenol, 4-isopropylphenol, and hexestrol. The degradation products of BPA were assumed to be formed by the one-electron oxidation of the substrate.
Key words: bisphenol A; bioremediation; manganese peroxidase; Pleurotus ostreatus; lignin-degrading enzyme
-27-
Note
Enzymatic Assay of Histamine by Amperometric Detection of H2O2
with a Peroxidase-based Sensor
Takao HIBI・・・ and Mitsugi SENDA
Department of Bioscience, Fukui Prefectural University, 4-1-1 Kenjyojima, Matsuoka-cho,
Yoshida-gun, Fukui 910-1195, Japan
Received October 21, 1999; Accepted April 17, 2000
A method for an enzymatic assay of histamine by using histamine oxidase from Arthrobacter globiformis in combination with amperometric determination of H2O2 is described. Histamine could be quantified at a level as low as 10--7 M. The assay is adaptable to determine histamine in food samples including tuna fish with good sensitivity and selectivity.
Key words: histamine; food analysis; histamine oxidase; biosensor; hydrogen peroxide
-28-
Note
Tyrosinase Inhibitory Activity of Bangladeshi Indigenous Medicinal Plants
Firoza KHANOM, Hiroshi KAYAHARA,1,・・・ and Koji TADASA1
The United Graduate School of Agricultural Science, Gifu University, 1-1 Yanagido, Gifushi,
Gifu 501-1193, Japan
1Division of Bio-organic Chemistry, Department of Bioscience and Biotechnology, Faculty of Agriculture,
Shinshu University, 8304 Minamiminowa-mura, Kamiina-gun, Nagano-ken 399-4598, Japan
Received January 24, 2000; Accepted April 13, 2000
The tyrosinase-inhibitory activity of 15 kinds of Bangladeshi medicinal plants was evaluated. Methanol extracts were prepared for screening tests, and other kinds of extracts were also studied for those with high activity. Swertia chirata, Piper nigrum, Glycyrrhiza glabra, Piper longam and Ocimum americanum were screened as highly inhibiting samples. Methanol was found to be the most efficient solvent for extracting the active compounds. The 50％ tyrosinase-inhibitory concentration of the Glycyrrhiza glabra methanol extract was 21.2 μg/ml.
Key words: Bangladeshi; medicinal plant; tyrosinase; inhibitory activity
-29-
Note
A dihydroxy-γ-lactone as an Oviposition Stimulant
for the Swallowtail Butterfly, Papilio bianor,
from the Rutaceous Plant, Orixa japonica
Hajime ONO, Ritsuo NISHIDA,・・・ and Yasumasa KUWAHARA
Laboratory of Chemical Ecology, Division of Applied Life Sciences, Graduate School of Agriculture,
Kyoto University, Kyoto 606-8502, Japan
Received February 10, 2000; Accepted April 15, 2000
The oviposition response of the Rutaceae-feeding swallowtail butterfly, Papilio bianor, was induced by a methanolic extract from leaves of its major host, Orixa japonica. Several components were responsible for this oviposition response. One of the stimulants was isolated and identified as (--)-2-C-methyl-D-erythrono-1,4-lactone. The compound was inactive alone, but elicited oviposition behavior when mixed with other fractions.
Key words: Papilio bianor; Orixa japonica; oviposition stimulant; (--)-2-C-methyl-D-erythrono-1,4-lactone; Papilionidae
-30-
Note
Practical Synthesis of the Disaccharide Epitope, D-Galactopyranosyl-α-1,3-D-
galactopyranose, by using 1,2;5,6-Di-O-cyclohexylidene-α-D-galactofuranose
as the Glycosyl Acceptor
Isao SAKAMOTO and Hiroshi OHRUI・・・
Division of Life Science, Graduate School of Agricultural Science, Tohoku University,
1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan
Received February 17, 2000; Accepted April 23, 2000
D-Galactosyl-α-1,3-D-galactopyranose (1) was chemically prepared in a good yield by coupling phenyl 2,3,4,6-tetra-O-benzyl-1-thio-β-D-galactopyranoside (5) or 2,3,4,6-tetra-O-benzyl-α-D-galactopyranosyl bromide (8) with 1,2:5,6-di-O-cyclohexylidene-α-D-galactofuranose (3) with subsequent de-O-benzylation and de-O-cyclohexylidenation of the resulting protected α-1,3-disaccharide.
Key words: 1,2:5,6 - di - O - cyclohexylidene - α - D - galactofuranose; D-galactopyranosyl-α-1,3-D-galactopyranose; xenotransplantation; α-glycosydation
-31-
Note
Purification and Characterization of Biliverdin-binding Protein from Larval
Hemolymph of the Swallowtail Butterfly, Papilio xuthus L.
Akira YAMANAKA,・・・ Takamasa ITO, Daizo KOGA,＊ Toshiyuki SATO,＊＊
Masanori OCHIAI,＊＊＊ and Katsuhiko ENDO
Department of Physics, Biology and Informatics, Faculty of Science, Yamaguchi University,
Yamaguchi 753-8512, Japan
＊Department of Environmental and Biological Science, Faculty of Agriculture, Yamaguchi University,
Yamaguchi 753-0841, Japan
＊＊Department of Biology, Faculty of Science, Shinshu University, Matsumoto 390-0802, Japan
＊＊＊Biochemical Laboratory, The Institute of Low Temperature Science, Hokkaido University,
Sapporo 060-0819, Japan
Receivd Februrary 17, 2000; Accepted May 12, 2000
The biliverdin-binding protein from the larval hemolymph of the swallowtail butterfly, Papilio xuthus L., was purified and characterized. The crude biliverdin-binding protein, obtained by ammonium sulfate fractionation, was purified in two steps, the first one by gel filtration chromatography and the second one by ion-exchange chromatography. The molecular mass of the purified protein was analyzed by SDS-polyacrylamide gel electrophoresis and estimated to be 21 kDa. The N-amino terminal sequence of P. xuthus biliverdin-binding protein analyzed up to the 19th residue showed that 42％ of the amino acid sequence are sequence similarity to the bilin-binding protein from Pieris brassicae. These results suggest that the P. xuthus biliverdin-binding protein belongs to the insecticyanin-type.
Key words: biliverdin-binding protein; Papilio xuthus; butterfly; insecticyanin
-32-
Note
On the Sweetness of N-(Trifluoroacetyl)aspartame
Michael FRANK and David J. AITKEN・・・
Laboratoire SEESIB-CNRS--UMR 6504, D"epartement de Chimie, Universit"e Blaise
Pascal--Clermont-Ferrand II, 24 avenue des Landais, 63177 Aubifere cedex, France
Received February 28, 2000; Accepted May 19, 2000
A panel of tasters has found that the N-trifluoroacetyl derivative of aspartame is five times less sweet than the parent compound, contrary to the tenet in the literature, but consistent with sweet receptor models which require this nitrogen to exist in protonated form.
Key words: artificial sweetener; aspartame derivative; sweetness test
-33-
Note
Staurosporine Promotion of Formation of Continuous Monolayers
of Primary Rat Hepatocytes by Improving Attachment and Spreading
Sumio MAEDA,・・・ Kong-Hua LIN, Hidetoshi INAGAKI, and Takao SAITO
Department of Chemistry, National Industrial Research Institute of Nagoya, 1-1 Hirate-cho,
Kita-ku, Nagoya 462-8510, Japan
Received March 1, 2000; Accepted May 8, 2000
Primary rat hepatocytes form discontinuous monolayers even at their maximum density. Here, we show that staurosporine promotes attachment and spreading of hepatocytes onto culture substrates, so that hepatocytes form a close, continuous monolayer. This treatment did not attenuate major hepatic functions. Therefore, this technique is promising for making seamless cell-sheet structures, which will be applicable for cell-polarity experiments or artificial liver construction.
Key words: staurosporine; primary hepatocyte; monolayer
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Note
Syntheses of Stereochemically Restricted Lactone-type Analogues
of Jasmonic Acids
Hiroaki TOSHIMA,・・・ Hisateru ARAMAKI, and Akitami ICHIHARA
Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University,
Sapporo 060-8589, Japan
Received March 2, 2000; Accepted April 10, 2000
5-Oxa-7-epi-jasmonic acid and 5-oxa-jasmonic acid, which are stereochemically restricted lactone-type analogues of jasmonic acids, were synthesized via three-component coupling of 2(5H)-furanone, tert-butyl acetate and 1-bromo-2-pentyne. After acidic deprotection of the tert-butyl esters, the (Z)-olefin was introduced by catalytic partial reduction with the Lindlar catalyst to give the desired analogues.
Key words: 7-epi-jasmonic acid; jasmonic acid; phytohormone; jasmonic acid analogue
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Note
Recognition of Receptor Lipopolysaccharides by Spike G Protein
of Bacteriophage fX174
Tomoko KAWAURA, Minoru INAGAKI,・・・ Shuichi KARITA,＊ Muneharu KATO,
Shiro NISHIKAWA, and Naoki KASHIMURA
Department of Bioscience, and ＊Center for Molecular Biology and Genetics, Mie University,
1515 Kamihama, Tsu, Mie 514-8507, Japan
Received March 3, 2000; Accepted April 23, 2000
The spike G protein of bacteriophage fX174 was prepared as a hexa histidine-tagged G protein (HisG). In the enzyme-linked plate assay, HisG bound specifically to lipopolysaccharides (LPSs) of the fX174-sensitive strains, and did not bind to LPSs of the fX174-insensitive strains. The truncated G protein obtained after trypsin digestion of HisG had the similar affinity to the LPSs to HisG, indicating that eight amino acid residues from the N-terminus are not essential to the binding with the LPSs.
Key words: spike G protein; φX174; lipopolysaccharide; enzyme-linked assay; phage receptor
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Note
Fungicidal and Herbicidal Activities of Berberine Related Alkaloids
Kinuko IWASA,＊,・・・ Masataka MORIYASU,＊ and Bassam NADER＊＊
＊Kobe Pharmaceutical University, 4-19-1 Motoyamakita, Higashinada, Kobe 658-8558, Japan
＊＊Dow Argo Sciences LLC, 9330 Zionsville Road, Indianapolis, Indiana 46268, USA
Received March 6, 2000; Accepted May 1, 2000
The 23 quaternary and tertiary protoberberines related to berberine were tested for in vitro and/or in vivo fungicidal and herbicidal activities. Among the compounds tested, there was some activity observed with some of only the protoberberinium salts, but not sufficiently strong or broad spectrum for agrochemical use. From the structure-activity point of view, some features can be pointed out.
Key words: berberine analog; fungicidal and herbicidal activity; structure-activity relationship
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Note
Suppression of D-Galactosamine-induced Liver Injury by Mushrooms in Rats
Eun Woo LEE, Puming HE, Hirokazu KAWAGISHI, and Kimio SUGIYAMA・・・
Department of Applied Biochemistry, Faculty of Agriculture, Shizuoka University, Shizuoka 422-8529, Japan
Received March 6, 2000; Accepted May 1, 2000
Six species of edible mushroom were found to suppress D-galactosamine-induced enhancement of plasma alanine and aspartate aminotransferase activities when powdered mushrooms were added to the diet (5％) and fed to rats for 2 wk. Grifola frondosa exhibited the most potent effect in a dose-dependent manner. A significant effect was observed only from the water-soluble low-molecular-weight fraction of G. frondosa. The results indicate that several mushrooms possess a protective effect against liver injury induced by D-galactosamine.
Key words: mushroom; Grifola frondosa; liver injury; D-galactosamine; rat
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Note
Producing a Low Ovomucoid Egg White Preparation by Precipitation
with Aqueous Ethanol
Soichi TANABE,・・・ Shoko TESAKI, and Michiko WATANABE
Food Science Laboratory, Faculty of Education, Tokyo Gakugei University, Tokyo 184-8501, Japan
Received March 29, 2000; Accepted April 28, 2000
A novel method for producing a low ovomucoid egg white preparation is proposed. Egg white powder (0.5 g) was dissolved in a 10-fold weight of distilled water and adjusted to pH 5, and ethanol was added to the solution at a final concentration of 20％ (v/v). The mixture was vigorously stirred and centrifuged. The precipitate was washed three times with 20％ ethanol (6.25 ml each), with about 65％ of egg white proteins occurring in the precipitate. The use of ELISA demonstrated that 70％ of ovomucoid was recovered from the supernatant fraction. However, functionally important proteins such as ovalbumin, ovotransferrin, and lysozyme still remained in the precipitate. These results may be due primarily to the much higher solubility of ovomucoid in this aqueous ethanol. Food quality evaluation showed that high whippability and foam stability were retained in the low ovomucoid preparation as in its material egg white. This product would thus be applicable as a new processed food for ovomucoid-sensitive allergic patients.
Key words: allergy; egg white; ovomucoid; food processing
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Note
Application of a Metal Switch to Aqualysin I, a Subtilisin-type Bacterial
Serine Protease, to the S3 Site Residues, Ser102 and Gly131
Terumichi TANAKA,1,・・・ Yo KIKUCHI,1 Hiroshi MATSUZAWA,2 and Takahisa OHTA3
1Division of Bioscience and Biotechnology, Department of Ecological Engineering, Toyohashi University
of Technology, Tempaku-cho, Toyohashi, Aichi 441-8580, Japan
2Aomori University, 2-3-1, Sachihata, Aomori-shi, Aomori 030-0943, Japan
3Kogakuin University, 1-24-2, Nishi-shinjuku, Shinjuku-ku, Tokyo 163-8677, Japan
Received April 5, 2000; Accepted May 19, 2000
We applied `metal switch′ experiments to the S3 site residues, Ser102 and Gly131, of aqualysin I, a subtilisin-type serine protease. We showed that two histidines introduced at these positions did take part in histidine-metal-histidine bridge formation, and metal ions inhibited the protease activities. These results indicate that two histidines are near each other, and both side chains are metal-accessible. This is the first report on application of the metal-switch technique to a subtilisin-related enzyme.
Key words: aqualysin I; subtilisin; Thermus aquaticus YT-1; metal-switch; S3 site
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Note
High Expression of the Second Lysine Decarboxylase Gene, ldc,
in Escherichia coli WC196 Due to the Recognition of the Stop Codon (TAG),
at a Position Which Corresponds to the 33th Amino Acid Residue of σ38,
as a Serine Residue by the Amber Suppressor, supD
Takahiro NAGANO,1 Yoshimi KIKUCHI,2 and Yoshiyuki KAMIO1,・・・
1Laboratory of Applied Microbiology, Department of Molecular and Cell Biology, Graduate School
of Agricultural Science, Tohoku University, 1-1 Tsutsumi-dori Amamiya-machi, Aoba-ku,
Sendai 981-8555 Japan
2Central Research Laboratories, Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki 210-0801, Japan
Received April 6, 2000; Accepted May 1, 2000
Escherichia coli WC196, which was obtained from the strain W3110 by nitrosoguanidine mutagenesis as an overproducer of lysine, produced approximately twenty times more cadaverine than did W3110, and had a twenty fold higher level of rpoS gene product, σ38, than in W3110. Both WC196 and W3110 had a stop codon (TAG) in rpoS at position which corresponds to the 33th residue of σ38 protein. In addition, WC196 but not W3110 had a mutation in the gene encoding Ser-tRNA (SerU), called, supD. Analysis of the amino acid sequence of a σ38 preparation from WC196 showed that the 33th residue of σ38 is a serine residue. The ΔrpoS ΔcadA mutant of E. coli W3110 harboring the plasmid containing rpoS, in which the TAG codon was converted to a TCG codon for serine-33 residue of σ38, expressed a significant amount of Ldc and accumulated a large amount of σ38. However, the ΔrpoS ΔcadA mutant of W3110 with the plasmid containing the intact rpoS from W3110 could synthesize neither σ38 nor Ldc significantly.
Key words: second lysine decarboxylase Ldc; Escherichia coli W3110; rpoS<+U=11.25CallMacro=80>rpoS gene product σ38; amber suppressor supD
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Note
The aman6 Gene Encoding a Yeast Mannan Backbone Degrading
1,6-α-D-Mannanase in Bacillus circulans: Cloning, Sequence Analysis,
and Expression・・・・・・
Yutaka MARUYAMA and Tasuku NAKAJIMA・・・
Laboratory of Enzymology, Division of Life Science, Graduate School of Agricultural Science,
Tohoku University, Sendai, 981-8555, Japan
Received April 7, 2000; Accepted May 23, 2000
A gene (aman6) encoding endo-1,6-α-D-mannanase, a yeast mannan backbone degrading enzyme from Bacillus circulans was cloned. The putative aman6 was 1767 base pairs long and encoded a mature 1,6-α-D-mannanase protein of 589 amino acids and a signal peptide of 36 amino acids. The purified mature 1,6-α-D-mannanase from the Escherichia coli transformant showed 61-kDa protein, and N-terminal amino acid sequence and other general properties of the recombinant enzyme were identical to those of 1,6-α-D-mannanase from Bacillus circulans TN-31.
Key words: yeast mannoprotein; 1,6-α-D-mannanase; recombinant enzyme; aman6 gene; Bacillus circulans
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Preliminary Communication
Detection of Biomarkers for Apoptosis in Rat Liver after Perfusion
with 3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1)
Hitoshi ASHIDA,・・・ Kaori KIHARA, Bunsyo SHIOTANI, Takashi HASHIMOTO,
Kazuki KANAZAWA, and Gen-ichi DANNO
Department of Biofunctional Chemistry, Faculty of Agriculture, Kobe University, Rokkodai-cho 1,
Nada-ku, Kobe 657-8501, Japan
Received March 16, 2000; Accepted June 13, 2000
We previously demonstrated that 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) induced apoptosis in primary cultured rat hepatocytes. In this study, we investigated apoptotic biomarkers in rat liver after perfusion with 30 μM Trp-P-1 as preliminary experiments for in vivo study. Induction of c-Myc and p53 protein and the activities of caspase-3, -6, and -8 were detected in Trp-P-1-perfused liver. In addition, Trp-P-1 modulated the DNA binding activity of the apoptosis-related transcription factors, NF-κB and AP-1. These results imply a possibility that Trp-P-1 would induce apoptosis in vivo.
Key words: heterocyclic amine; Trp-P-1; apoptosis; NF-κB; hepatocytes