(Vol.63 No.4 1999)
Time-dependent Changes of Amino Acids in the Serum, Liver, Brain
and Urine of Rats Administered with Theanine
Takehiko TERASHIMA, Jun TAKIDO, and Hidehiko YOKOGOSHI＊
Comparative Composition of Brown Rice Lipids (Lipid Fractions) of Indica and Japonica Rices
Yasuo MANO・, Kumi KAWAMINAMI, Michiyuki KOJIMA, Masao OHNISHI,
and Seisuke ITO
Complementary Effects of Bifidogenic Growth Stimulators and Ammonium
Sulfate in Natural Rubber Serum Powder on Bifidobacterium bifidum
Shin-ichi ETOH, Kenji SONOMOTO, and Ayaaki ISHIZAKI＊
Two Cytosolic Cyclophilin Genes of Arabidopsis thaliana Differently Regulated
in Temporal- and Organ-Specific Expression
Takeshi SAITO,1 Kei TADAKUMA,1 Naoko TAKAHASHI,1 Hiroyuki ASHIDA,1
Katsunori TANAKA,2 Makoto KAWAMUKAI,2 Hideyuki MATSUDA,2
and Tsuyoshi NAKAGAWA1,＊
Effective Moisture Diffusivity of Fresh Japanese Noodle (udon) as a Function of Temperature
Tadao INAZU・ and Ken-ichi IWASAKI
Complete Growth Inhibition of Bacillus subtilis by Nisin-Producing Lactococci
in Fermented Soybeans
Takeo KATO,・ Kumiko MAEDA,＊ Haruko KASUYA,＊＊ and Tsukasa MATSUDA＊＊＊
Preparation and Application of Anti-idiotypic Antibody against
Anti-gibberellin A4 Antibody
Yoshihito SUZUKI,・ Naoki SHIMADA, Rimpei NIWA, Michinori YOKOI, Masatoshi NAKAJIMA,
Noboru MUROFUSHI, and Isomaro YAMAGUCHI
Effects of DFA IV in Rats: Calcium Absorption and Metabolism
of DFA IV by Intestinal Microorganisms
Katsuichi SAITO, Toru HIRA,＊ Takuya SUZUKI,＊ Hiroshi HARA,＊
Atsushi YOKOTA, and Fusao TOMITA・
Kinetic Analysis of the Autoxidation of Ethyl Eicosapentaenoate at Different Oxygen Levels
Hidefumi YOSHII,・ Takeshi FURUTA, Takatoki ITOH, Kentaro YOSHIMURA,
Yoshihisa MISAWA,＊ Noriaki HATA,＊ Hideaki KOBAYASHI,＊＊ Yu-Yen LINKO,＊＊＊
and Pekka LINKO＊＊＊
Expression of Elongation Factor 1β′ in Escherichia coli and Its Interaction
with Elongation Factor 1α from Silk Gland
Katsuyoshi KAMIIE,1,・ Hideharu TAIRA,2 Kohmei KOBAYASHI,1 Tetsuro YAMASHITA,2
Shin-ichiro KIDOU,3 and Shin-ichiro EJIRI3
Construction of an L-Isoleucine Overproducing Strain of Escherichia coli K-12
Ken-ichi HASHIGUCHI,＊ Hiroko TAKESADA,＊＊ Eiichiro SUZUKI,＊＊ and Hiroshi MATSUI＊＊
Recovery of Caco-2 Cell Monolayers to Normal from the Transport-enhanced
State Induced by Capric Acid Sodium Salt and its Monoacylglycerol
Motohiro SHIMA,・ Yukitaka KIMURA, Shuji ADACHI and Ryuichi MATSUNO
A New Carboxylesterase from Brevibacterium linens IFO 12171 Responsible
for the Conversion of 1,4-Butanediol Diacrylate to 4-Hydroxybutyl Acrylate:
Purification, Characterization, Gene Cloning, and Gene Expression in Escherichia coli
Yasuyoshi SAKAI, Junko ISHIKAWA, Shunji FUKASAKA, Hiroya YURIMOTO, Ryoji MITSUI,
Hideshi YANASE,＊ and Nobuo KATO・
Elevated Body Fat in Rats by the Dietary Nitric Oxide Synthase Inhibitor,
L-Nω Nitroarginine
Abdelkrim KHEDARA, Tsuyoshi GOTO, Masako MORISHIMA, Jun KAYASHITA,＊
and Norihisa KATO・
Production of Recombinant Der fI (a major mite allergen) by Aspergillus oryzae
Hiroshi SHOJI,・ Hiroyuki HORIUCHI,＊ and Masamichi TAKAGI＊
High-multiplicity of Chitinase genes in Streptomyces coelicolor A3(2)
Akihiro SAITO,1,2 Takeshi FUJII,1 Tadakatsu YONEYAMA,2 Matthias REDENBACH,3
Tsuyoshi OHNO,4 Takeshi WATANABE,4 and Kiyotaka MIYASHITA1＊
Phloretin-induced Apoptosis in B16 Melanoma 4A5 Cells and HL60 Human
Leukemia Cells
Masuko KOBORI,・ Keiko IWASHITA,＊ Hiroshi SHINMOTO, and Tojiro TSUSHIDA
An Isoelectric Separation of Soybean β-Amylase Isoforms and Their Enzymic
Characteristics
Yuhuan WANG, Masaomi ARAHIRA, and Chikafusa FUKAZAWA＊
(2E,6R)-8-Hydroxy-2,6-dimethyl-2-octenoic Acid,
a Novel Anti-osteoporotic Monoterpene, Isolated from Cistanche salsa
Kohji YAMAGUCHI, Chikara SHINOHARA, Sachiko KOJIMA, Mikiko SODEOKA,
and Tomoko TSUJI・
Synthesis of 4′-C-Ethynyl-β-D-ribo-pentofuranosyl Pyrimidines
Ritsuko YAMAGUCHI, Tatsuma IMANISHI, Satoru KOHGO,
Hiroko HORIE, and Hiroshi OHRUI＊
Potentiation of GABAA Receptors Expressed in Xenopus Oocytes
by Perfume and Phytoncid
Hitoshi AOSHIMA・ and Koutaro HAMAMOTO
Note
Effect of Media Constituents on the Formation by Halophilic Yeast
of the 2 (or 5)-Ethyl-5 (or 2)-methyl-4-hydroxy-3 (2H)-furanone
Aroma Component Specific to Miso
Etsuko SUGAWARA＊,・ and Yonekichi SAKURAI＊＊
Note
Improved Suppression by Dietary Taurine of the Fecal Excretion of Bile Acids
from Hypothyroid Rats
Hideki MOCHIZUKI, Jun TAKIDO, and Hidehiko YOKOGOSHI・
Note
Isolation and Structural Determination of a Novel Bicyclic Taxane Diterpene
from Needles of the Chinese Yew, Taxus mairei
Qing-Wen SHI, Takayuki ORITANI,＊ Takeyoshi SUGIYAMA, and Teiko YAMADA
Note
Insecticidal Activity of α-Methylene-γ-butyrolactone against Several Insect Pests
Probal Kanti DATTA, Chul-Sa KIM,・ Tetsuro HARA, Eiji ITOH,
and Michio HORIIKE
Note
Differential Accumulation of Transcripts Encoding Sulfur Assimilation Enzymes
upon Sulfur and/or Nitrogen Deprivation in Arabidopsis thaliana
Yube YAMAGUCHI, Tatsuo NAKAMURA, Emiko HARADA, Nozomu KOIZUMI, and Hiroshi SANO・
Note
Sequence of a cDNA Encoding Mouse F1F0-ATP Synthase g Subunit
Deahyun JEON, Hyungha KIM, and Myunggi BAIK・
Note
Detection and Analysis of Translation Elongation Factor 3 Genes
from Various Yeasts
Masahiro URITANI,・ Yasuhito SHOUMURA, and Shinpei YAMADA＊
Note
Simultaneous Determination of Different Classes of Lipid Hydroperoxides
by High-Performance Liquid Chromatography with Post Column Detection
by a Ferrous/Xylenol Orange Reagent
Keiji SUGINO・
Note
Construction and Characterization of Escherichia coli Disruptants Defective
in the yaeM Gene
Tomohisa KUZUYAMA, Shunji TAKAHASHI＊, and Haruo SETO
Prelimimary Communication
Expression of a Functional Single-chain Antibody against GA24/19
in Transgenic Tobacco
Naoki SHIMADA, Yoshihito SUZUKI, Masatoshi NAKAJIMA, Udo CONRAD,＊
Noboru MUROFUSHI, and Isomaro YAMAGUCHI
-1-
Time-dependent Changes of Amino Acids in the Serum, Liver, Brain
and Urine of Rats Administered with Theanine
Takehiko TERASHIMA, Jun TAKIDO, and Hidehiko YOKOGOSHI＊
School of Food and Nutritional Sciences, The University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan
Received July 16, 1998; Accepted November 26, 1998
Time-dependent changes of theanine (γ-glutamylethylamide) and other amino acids in various tissues of rats were investigated during the 24 hrs after theanine administration. When theanine (4 g/kg of body weight) was intragastrically administered to rats, the concentrations of theanine in the serum, liver and brain were significantly increased 1 hr after its administration, and thereafter gradually decreased, but reached the maximum level in the brain after 5 hrs. Theanine in these tissues had completely disappeared 24 hrs after its administration. In contrast, the administration of theanine resulted in the concentrations of theanine, urea, ethylamine and glutamic acid in the urine being significantly enhanced. These results suggest that theanine might be degraded via glutamic acid.
theanine; glutamic acid; ethylamine; amino acids; rat
-2-
Comparative Composition of Brown Rice Lipids (Lipid Fractions) of Indica
and Japonica Rices
Yasuo MANO・, Kumi KAWAMINAMI, Michiyuki KOJIMA, Masao OHNISHI,
and Seisuke ITO・Obihiro Otani Junior College, 3-Kib￣・ ogaoka, Otofuke-cho, Hokkaido 080-0335, Japan
Department of Bioresource Chemistry, Obihiro University of Agriculture and Veterinary Medicine,
Inada, Obihiro, Hokkaido 080-0835, JapanReceived August 25, 1998; Accepted October 8, 1998
The brown rice lipids were analyzed from three japonica and two indica rices. They had substantially no difference in the ratio of NL, GL, and PL, the C16/C18 (16:0/the sum of 18:0, 18:1, 18:2, and 18:3) ratio in PL classes, and in the order of unsaturated index in each glycerolipid class among the five varieties. However, the chemical compositions of glyceroglycolipids and cerebroside were different in indica and japonica rices. The contents of 18:3 of MGDG and DGDG were higher in japonica than indica rices, but those of 16:0 were reversed. The ratio of C14--20/C21--26 of the hydroxy fatty acids in cerebroside was 1.18 in japonica and 0.62 in indica rices. Furthermore, the ratio of the trihydroxy sphingoid bases to dihydroxy ones showed a great difference between the subspecies.
Unsaturated fatty acid contents in the TG and PL classes were largely different from each other among japonica rices harvested in separate districts.
japonica; indica; unsaturated index; hydroxy fatty acid; sphingoid base
-3-
Complementary Effects of Bifidogenic Growth Stimulators and Ammonium
Sulfate in Natural Rubber Serum Powder on Bifidobacterium bifidum
Shin-ichi ETOH, Kenji SONOMOTO, and Ayaaki ISHIZAKI＊
Laboratory of Microbial Technology, Department of Food Science and Technology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan
Recieved August 31, 1998; Accepted December 9, 1998
Natural rubber serum powder, rich in crude protein and carbohydrates, had a strong growth-stimulating activity for Bifidobacterium bifidum JCM 1254, which was unable to grow in a fully synthetic medium, B12 assay medium. Natural rubber serum powder was fractionated by ultrafiltration (molecular weight cutoff 1000). The active ultrafiltrate was further concentrated and desalted with an adsorptive microconcentrator, which adsorbs virtually all amino acids and peptides. Through this purification step, it was found that the adsorbed fraction obtained did not stimulate growth independently but acted complementarily with a small amount of ammonium sulfate. The adsorbed fraction was subsequently analyzed on reversed-phase high pressure liquid chromatography, and the activities of the eluates were measured on B12 assay medium with ammonium sulfate. Consequently, it was proved that several peptidic ingredients in the adsorbed fraction increased the growth of B. bifidum.
||natural rubber serum powder; Bifidobacteri||um; bifidogenic growth stimulator; ammonium sulfate
-4-
Two Cytosolic Cyclophilin Genes of Arabidopsis thaliana Differently Regulated
in Temporal- and Organ-Specific Expression
Takeshi SAITO,1 Kei TADAKUMA,1 Naoko TAKAHASHI,1 Hiroyuki ASHIDA,1
Katsunori TANAKA,2 Makoto KAWAMUKAI,2 Hideyuki MATSUDA,2
and Tsuyoshi NAKAGAWA1,＊1Research Institute of Molecular Genetics, Shimane University, 1060 Nishikawatsu, Matsue 690-8504, Japan
2Department of Applied Bioscience and Biotechnology, Fuculty of Life and Environmental Science,
Shimane University, 1060 Nishikawatsu, Matsue 690-8504, JapanReceived September 1, 1998; Accepted December 4, 1998
We have previously isolated two closely related genes (ATCYP1 and ATCYP2) each encoding a cytosolic cyclophilin of Arabidopsis thaliana. Here we tested expression patterns of these two genes by Northern analysis and by histochemical analysis with transgenic plants carrying the promoter: β-glucuronidase (GUS) fusion. The results showed that ATCYP1 is predominantly transcribed in vascular tissue and flowers, but ATCYP2 is at higher levels in younger leaves. The different expression patterns seemed to be conferred by the quite different promoter structures carrying various cis elements. Our finding suggests that the two cyclophilins have different roles in Arabidopsis thaliana cells.
Arabidopsis thaliana; β-glucuronidase; cyclophilin; vascular tissue
-5-
Effective Moisture Diffusivity of Fresh Japanese Noodle (udon) as a Function
of Temperature
Tadao INAZU・ and Ken-ichi IWASAKI
Food Research Institute, Kagawa Prefectural Government 587-1 Goto, Takamatsu, Kagawa 761-8031, Japan
Received September 4, 1998; Accepted December 28, 1998
The effect of moisture content and temperature on the effective moisture diffusivity was investigated to identify the optimal drying condition for Japanese noodle (udon) by using a diffusion model. The drying of fresh udon with different moisture contents was carried out under constant conditions of relative humidity and airflow at 20°C, 30°C and 40°C. The effective moisture diffusivity calculated from diffusion model was found to be constant at each temperature, and not to be influenced by the initial moisture content of the fresh udon. The moisture content calculated from the effective moisture diffusivity (2.1--3.7×10－7 cm2 s－1) agrees well with the experimental data. The effect of temperature on the effective moisture diffusivity was adequately modeled by the Arrhenius relationship. The activation energy was 21.3 kJ mol－1.
Japanese noodle (udon); effective moisture diffusivity; diffusion model; drying; temperature dependence
-6-
Complete Growth Inhibition of Bacillus subtilis by Nisin-Producing Lactococci
in Fermented Soybeans
Takeo KATO,・ Kumiko MAEDA,＊ Haruko KASUYA,＊＊ and Tsukasa MATSUDA＊＊＊
・Food Research Institute, Aichi Prefectural Government, 2-1-1-Shinpukuji-cho, Nishi-ku, Nagoya 451-0083, Japan
＊Marusan-Ai Co. Ltd, 1-Niki-cho, Okazaki 444-2193, Japan
＊＊Faculty of Agriculture, Meijo University, Shiogamaguchi, Tenpaku-ku, Nagoya 468-0073, Japan
＊＊＊Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku,
Nagoya 464-8601, JapanReceived September 11, 1998; Accepted December 1, 1998
The growth inhibition by nisin-producing lactococci against Bacillus subtilis and its application to soybean miso fermentation were investigated. Lactococcus lactis subsp. lactis IFO12007 (nisin-producing, salt-intolerant) rapidly proliferated to more than 109 cells/g in cooked soybeans without any excessive pH decrease. In spite of the mild decrease in pH, the growth of B. subtilis was completely inhibited; no living cells were detected in a soybean sample inoculated with 106 cells/g and incubated for 24 to 72 h. This Lc. lactis was applied to soybean miso fermentation as a starter culture. It produced high nisin activity (1.28×105 AU/g) in cooked soybean, resulting in the complete growth inhibition of B. subtilis, which had been inoculated at the beginning of the koji fermentation, throughout the process of miso production. Over-acidification, which is undesirable for miso quality, was successfully prevented simply by adding salt which killed the salt-intolerant Lc. lactis. Furthermore, the nisin activity in miso disappeared with aging.
bacteriocinogenic lactic acid bacteria; nisin; Bacillus subtilis inhibition; soybean miso fermentation
-7-
Preparation and Application of Anti-idiotypic Antibody against
Anti-gibberellin A4 Antibody
Yoshihito SUZUKI,・ Naoki SHIMADA, Rimpei NIWA, Michinori YOKOI, Masatoshi NAKAJIMA,
Noboru MUROFUSHI, and Isomaro YAMAGUCHIDepartment of Applied Biological Chemistry, Division of Agriculture and Agricultural Life Sciences,
The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, JapanReceived September 11, 1998; Accepted December 2, 1998
A monoclonal anti-idiotypic antibody was raised against anti-gibberellin A4 (GA4) antibody, which recognizes biologically active gibberellins such as GA1 and GA4 specifically. Amino acid sequences of variable regions of both anti-GA4 and anti-idiotypic antibodies were analyzed. By using the property of the anti-idiotypic antibody to compete with GA1/4 in binding to the anti-GA4 antibody, we successfully applied the anti-idiotypic antibody to ELISA as a tracer for measuring GA1/4. The single-chain Fv (scFv) gene of the anti-idiotypic antibody was constructed, and scFv expressed in E. coli showed binding activity to anti-GA4 antibody. These results suggest the possible application of anti-idiotypic antibody as a handy and stable source of an enzymatic tracer for ELISA by production of fusion protein of the scFv and an appropriate enzyme.
anti-idiotypic antibody; ELISA; gibberellin; scFv
-8-
Effects of DFA IV in Rats: Calcium Absorption and Metabolism
of DFA IV by Intestinal Microorganisms
Katsuichi SAITO, Toru HIRA,＊ Takuya SUZUKI,＊ Hiroshi HARA,＊
Atsushi YOKOTA, and Fusao TOMITA・Laboratory of Applied Microbiology, Department of Bioscience and Chemistry, Faculty of Agriculture,
Hokkaido University, Sapporo 060-8589, Japan
＊Laboratory of Nutritional Biochemistry, Department of Bioscience and Chemistry, Faculty of Agriculture,
Hokkaido University, Sapporo 060-8589, JapanReceived September 24, 1998; Accepted November 27, 1998
Di-D-fructose-2,6′:6,2′-dianhydride (DFA IV) is a disaccharide consisting of two fructose residues that can be prepared from levan by levan fructotransferase from Arthrobacter nicotinovorans GS-9, and it can be expected to have novel physiological functions from its unique structure. In this study, the effects of DFA IV on calcium absorption and the metabolism of DFA IV by intestinal microorganisms were studied in rats to examine the physiological functions of DFA IV.
The apparent calcium absorption in rats fed with DFA IV was significantly higher than that in the control rats, and it seems that calcium absorption had almost been completed at the end of the small intestine. DFA IV also increased the calcium absorption in in vitro experiments, using everted jejunal and ileal sacs, and this result supports the finding obtained in the in vivo experiments. These results indicate that DFA IV may have a function for increasing the calcium absorption in the small intestine of rats. However, the effect in the large intestine could not be clearly observed because of the lack of calcium that reached there.
The results of analyses of organic acids in the cecal and colonic contents and of DFA IV in the fecal, cecal, and colonic contents showed that the metabolism of DFA IV by microorganisms in the large intestine progressed gradually, and that DFA IV was converted mainly to acetate, butyrate, and lactate.
DFA IV; calcium absorption; rats
-9-
Kinetic Analysis of the Autoxidation of Ethyl Eicosapentaenoate
at Different Oxygen Levels
Hidefumi YOSHII,・ Takeshi FURUTA, Takatoki ITOH, Kentaro YOSHIMURA,
Yoshihisa MISAWA,＊ Noriaki HATA,＊ Hideaki KOBAYASHI,＊＊ Yu-Yen LINKO,＊＊＊
and Pekka LINKO＊＊＊Department of Biotechnology, Tottori University, Tottori 680-0945, Japan
＊Harima Chemicals Inc., Tsukuba 300-2635, Japan
＊＊R＆D Div., Q.P. Corporation, Tokyo 183-0034, Japan
＊＊＊Department of Chemical Technology, Helsinki University of Technology, Espoo, FIN-02015 HUT, FinlandReceived October 6, 1998; Accepted December 7, 1998
The autoxidation of such n-3 polyunsaturated fatty acid (PUFA) ethyl esters as ethyl eicosapentaenoate and ethyl docosahexaenoate was investigated at various temperatures. Extensive studies of the oxidative reaction for ethyl eicosapentaenoate were carried out at different oxygen levels. At the same oxygen level and temperature, the autocatalytic reaction rate, by which oxidation progressed in the first half period, was about 1.5--2.5 times larger than the first-order reaction rate which governed the oxidation in the second half period. The reaction rate constants for ethyl eicosapentaenoate at different oxygen levels correlated well with the Langmuir-type equation of the oxygen concentration, in which the Langmuir parameter was independent of temperature.
autoxidation; ethyl eicosapentaenoate; ethyl docosahexaenoate; polyunsaturated fatty acid; kinetic energy
-10-
Expression of Elongation Factor 1β′ in Escherichia coli and Its Interaction
with Elongation Factor 1α from Silk Gland
Katsuyoshi KAMIIE,1,・ Hideharu TAIRA,2 Kohmei KOBAYASHI,1 Tetsuro YAMASHITA,2
Shin-ichiro KIDOU,3 and Shin-ichiro EJIRI31Department of Bioscience and Biotechnology, Faculty of Engineering, Aomori University,
Aomori 030-0943, Japan
2Department of Bioscience and Technology and 3Cryobiosystem Research Center, Faculty of Agriculture,
Iwate University, Morioka 020-8550, JapanReceived October 8, 1998; Accepted December 8, 1998
Silk gland elongation factor 1 (EF-1) consists of four subunits: α, β, β′, and γ. EF-1ββ′γ catalyzes the exchange of GDP for GTP on EF-1α and stimulates the binding of EF-1α-dependent aminoacyl-tRNA to ribosomes. The carboxy-terminal regions of the EF-1β subunits from various species are highly conserved. We examined the region of EF-1β′ that binds to EF-1α by in vitro binding assays, and examined the GDP/GTP exchange activity using deletion mutants of a GST-EF1β′ fusion protein. We thereby suggested a pivotal amino acid region, residues 189-222, of EF-1β′ for binding to EF-1α.
elongation factor 1β′; elongation factor 1α; subunit interaction; glutathione S-transferase (GST) fusion protein; Bombyx mori
-11-
Construction of an L-Isoleucine Overproducing Strain of Escherichia coli K-12
Ken-ichi HASHIGUCHI,＊ Hiroko TAKESADA,＊＊ Eiichiro SUZUKI,＊＊ and Hiroshi MATSUI＊＊
＊Fermentation ＆ Biotechnology Laboratories, and
＊＊Central Research Laboratories, Ajinomoto Co. Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki 210-8681, JapanReceived October 13, 1998; Accepted December 9, 1998
The genes for a threonine deaminase that is resistant to feedback inhibition by L-isoleucine and for an active acetohydroxyacid synthase II were introduced by a plasmid into a L-threonine-producing recombinant strain of Escherichia coli K-12. Analysis of culture broth of the strain using 13C nuclear magnetic resonance suggested that α, β-dihydroxy-β-methylvalerate (DHMV) and α-keto-β-methylvalerate (KMV), the third and the fourth intermediates in the L-isoleucine biosynthetic pathway from L-threonine, respectively, accumulated in the medium in amounts comparable to that of L-isoleucine. The ratio of accumulated L-isoleucine:DHMV:KMV were approximately 2:1:1. The concentration of accumulated L-isoleucine increased by twofold after the additional introduction of the genes for dihyroxyacid dehydratase (DH) and transaminase-B (TA-B), and the intermediates no longer accumulated. The resultant strain TVD5 accumulated 10 g/l of L-isoleucine from 40 g/l of glucose.
L-isoleucine; overproduction; Escherichia coli; intermediates, NMR
-12-
Recovery of Caco-2 Cell Monolayers to Normal from the Transport-enhanced
State Induced by Capric Acid Sodium Salt and its Monoacylglycerol
Motohiro SHIMA,・ Yukitaka KIMURA, Shuji ADACHI and Ryuichi MATSUNO
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University,
Sakyo-ku, Kyoto 606-8502, JapanReceived October 14, 1998; Accepted December 14, 1998
Caco-2 cell monolayers were used as a model of the intestinal epithelium to investigate the recovery profile from the transport-enhanced state induced by the transport enhancers, capric acid sodium salt (C10FANa) and capric acid monoacylglycerol (C10MG). The transepithelial electrical resistance, MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide) assay, and lactate dehydrogenase (LDH) release rate were investigated. The cell monolayer recovered depending on the concentration of the enhancer and on the exposure time. The MTT assay revealed that the cells recovered their mitochondrial dehydrogenase activity without proliferation. The cell monolayer exposed to C10FANa released LDH to both the apical and basolateral sides, but to C10MG, only to the apical side. The results were compared with those for SDS and taurocholic acid sodium salt, and the effect of C10FANa was found to be different. These results suggest that the damage by MCFA compounds is recoverable and that the recovery can be assessed by an MTT assay, but that the LDH-release behavior is different among the enhancers.
Caco-2; cell damage; MTT assay; LDH; medium-chain fatty acid
-13-
A New Carboxylesterase from Brevibacterium linens IFO 12171 Responsible
for the Conversion of 1,4-Butanediol Diacrylate to 4-Hydroxybutyl Acrylate:
Purification, Characterization, Gene Cloning, and Gene Expression
in Escherichia coli
Yasuyoshi SAKAI, Junko ISHIKAWA, Shunji FUKASAKA, Hiroya YURIMOTO, Ryoji MITSUI,
Hideshi YANASE,＊ and Nobuo KATO・Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University,
Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan
＊Department of Biotechnology, Tottori University, Koyama-cho Minami, Tottori 680-0945, JapanReceived October 15, 1998; Accepted December 9, 1998
A carboxylesterase that is responsible for conversion of 1,4-butanediol diacrylate (BDA) to 4-hydroxybutyl acrylate (4HBA) was found in Brevibacterium lines IFO 12171, and purified to homogeneity. The purified enzyme was active toward a variety of diesters of ethylene glycol, 1,4-butanediol, and 1,6-hexanediol. The Km and kcat of the enzyme for BDA were 3.04 mM and 203,000 s－1, respectively. The reaction with the purified enzyme gave 98 mM 4HBA from 100 mM BDA for 60 min.
The enzyme gene was cloned from the chromosomal DNA of the bacterium. The open reading frame encoding the enzyme was 1176 bp long, corresponding to a protein of 393 amino acid residues (molecular mass＝42,569 Da). The deduced amino acid sequence contained the tetra peptide motif sequence, STTK, and the serine residue was confirmed to be the catalytic center of BDA esterase by site-directed mutagenesis for several amino acid residues. The gene was expressed in Escherichia coli under the control of the lac promoter, and the gene product (a fusion protein with 6 amino acid residues from β-galactosidase) showed the same catalytic properties as the enzyme from the parent strain.
Brevibacterium linens IFO 12171; 1,4-butanediol diacrylate esterase; carboxylesterase; 4-hydroxybutyl acrylate; porcine liver esterase
-14-
Elevated Body Fat in Rats by the Dietary Nitric Oxide Synthase Inhibitor,
L-Nω Nitroarginine
Abdelkrim KHEDARA, Tsuyoshi GOTO, Masako MORISHIMA, Jun KAYASHITA,＊
and Norihisa KATO・Department of Applied Biochemistry, Faculty of Applied Biological Science, Hiroshima University,
Higashi-Hiroshima 739-8528, Japan
＊Development, Health Care, Kissei Pharmaceutical Company, Matsumoto 399-8710, JapanReceived October 19, 1998; Accepted December 12, 1998
The influence of the dietary nitric oxide (NO) synthase inhibitor, L-Nω nitroarginine (L-NNA) on body fat was examined in rats. In experiment 1, all rats were fed with the same amount of diet with or without 0.02％ L-NNA for 8 wk. L-NNA intake caused elevations in serum triglyceride and body fat, and reduction in serum nitrate (a metabolite of nitric oxide). The activity of hepatic carnitine palmitoyltransferase was reduced by L-NNA. In experiment 2, rats were fed for 8 wk with the same amount of diets with or without 0.02％ L-NNA supplemented or not with 4％ L-arginine. The elevation in body fat, and the reductions in serum nitrate and in the activity of hepatic carnitine palmitoyltransfererase by L-NNA were all suppressed by supplemental L-arginine. The results suggest that lower NO generation elevated not only serum triglyceride, but also body fat by reduced fatty acid oxidation.
nitric oxide; serum triglyceride; body fat; fatty acid oxidation; rat
-15-
Production of Recombinant Der fI (a major mite allergen) by Aspergillus oryzae
Hiroshi SHOJI,・ Hiroyuki HORIUCHI,＊ and Masamichi TAKAGI＊
Institute for Production Research and Development, The Nikka Whisky Distilling Co., Ltd.,
967, Matsuyama, Masuo, Kashiwa, Chiba 277-0033, Japan
＊Laboratory of Cellular Genetics, Department of Biotechnology, Division of Agriculture and Agricultural
Life Science, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, JapanReceived October 20, 1998; Accepted December 11, 1998
Der fI is a major mite allergen. To produce Der fI by Aspergillus oryzae, we placed a DNA fragment encoding precursor-type recombinant Der fI E(-1)K (reDer fI E(-1)K), which had the C-terminal amino acid of the pro-sequence (Glu) changed to Lys, downstream of the glaA gene||promoter and introduced it into Aspergillus oryzae. In liq||uid culture, most of the reDer fI E(-1)K produced by the transformants was degraded when culture was shaken vigorously. However, the degradation of reDer fI E(-1)K was suppressed when it was shaken gently. The processed reDer fI E(-1)K could be obtained after lysylendopeptidase and endoglycosidase Hf (Endo Hf) treatment. The yield of processed reDer fI E(-1)K was 8 mg/l. When the transformant was grown on a wheat bran culture, the yield of processed reDer fI E(-1)K reached 48 mg/kg. Because processed reDer fI E(-1)Ks obtained from both cultures had almost the same IgE-binding activity and elicited the same skin reaction as native Der fI, they could be very useful for diagnostic purposes or immunotherapy.
mite; allergen; fungi; glycosidase
-16-
High-multiplicity of Chitinase genes in Streptomyces coelicolor A3(2)
Akihiro SAITO,1,2 Takeshi FUJII,1 Tadakatsu YONEYAMA,2 Matthias REDENBACH,3
Tsuyoshi OHNO,4 Takeshi WATANABE,4 and Kiyotaka MIYASHITA1＊1National Institute of Agro-Environmental Sciences, 3-1-1 Kan-nondai, Tsukuba, Ibaraki 305-8604, Japan
2Institute of Applied Biochemistry, University of Tsukuba, 1-1-1 Ten-nodai, Tsukuba, Ibaraki 305-8572, Japan
3Department of Genetics, Genome Research Unit, Kaiserslautern University, 67663 Kaiserslautern, Germany
4Department of Applied Biological Chemistry, Faculty of Agriculture, Niigata University, Niigata 950-2102, JapanReceived October 20, 1998; Accepted December 2, 1998
Six different genes for chitinase from ordered cosmids of the chromosome of Streptomyces coelicolor A3(2) were identified by hybridization, using the chitinase genes from other Streptomyces spp. as probes, and cloned. The genes were sequenced and analyzed. The genes, together with an additional chitinase gene obtained from the data bank, can be classified into either family 18 or family 19 of the glycosyl hydrolase classification. The five chitinases that fall into family 18 show diversity in their multiple domain structures as well as in the amino acid sequences of their catalytic domains. The remaining two chitinases are members of family 19 chitinases, since their C-terminus shares more than 70％ identity with the catalytic domain of ChiC||of Streptomyces griseus, the sole gene for family 19||chitinase so far found in an organism other than higher plants.
Streptomyces coelicolor; chitinase genes; family 19 chitinase
-17-
Phloretin-induced Apoptosis in B16 Melanoma 4A5 Cells and HL60 Human
Leukemia Cells
Masuko KOBORI,・ Keiko IWASHITA,＊ Hiroshi SHINMOTO, and Tojiro TSUSHIDA
National Food Research Institute, Ministry of Agriculture, Forestry and Fisheries,
2-1-2 Kannondai, Tsukuba, Ibaraki 305-8642, Japan
＊Institute of Applied Biochemistry, university of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-0006, JapanReceived October 22, 1998; Accepted December 8, 1998
The dihydrochalcone phloretin induced apoptosis in B16 mouse melanoma 4A5 cells and HL60 human leukemia cells. Phloretin was suggested to induce apoptosis in B16 cells mainly through the inhibition of glucose transmembrane transport. The phloretin-induced apoptosis in B16 cells was inhibited by actinomycin D, Ac-YVAD-CHO caspase-1-like inhibitor, and Ac-DEVD-CHO caspase-3-like inhibitor. During the induction of apoptosis by phloretin, the expression of Bax protein in B16 cells increased and the levels of p53, Bcl-2, and Bcl-XL proteins did not change. Our results suggested that phloretin induced apoptosis through the promotion of Bax protein expression and caspases activation. On the other hand, phloretin may induce apoptosis in HL60 cells through the inhibition of protein kinase C activity because phloretin inhibited protein kinase C activity in HL60 cells more than that in B16 cells. The phloretin induced-apoptosis in HL60 cells was not inhibited by actinomycin D and the caspase-1-like inhibitor, but slightly inhibited by the caspase-3-like inhibitor. Phloretin reduced the level of caspase 3 protein in HL60 cells, but not the level of the Bcl-2 protein. Phloretin did not increase the level of Bax protein. Phloretin was suggested to induce apoptosis in HL60 cells through the inhibition of protein kinase C activity, followed by the pathway, which is different from that in B16 cells.
phloretin; apoptosis; melanoma cells; leukemia cells; glucose transport
-18-
An Isoelectric Separation of Soybean β-Amylase Isoforms and Their Enzymic
Characteristics
Yuhuan WANG, Masaomi ARAHIRA, and Chikafusa FUKAZAWA＊
Genetic Engineering Laboratory, National Food Research Institute, Ministry of Agriculture,
Forestry and Fisheries, Tsukuba, Ibaraki 305-8642, JapanReceived October 28, 1998; Accepted December 2, 1998
Authentic soybean β-amylase preparation, purified to homogeneity as judged by SDS-PAGE by using an affinity purification step, was composed of four pI-differing isoforms. By chromatofocusing, these isoforms were separated into three fractions, designated as fractions 1--3 in the order of elution. Fraction 1 contained two isoforms having the same molecular mass (55,989 Da), as measured by mass spectrometric analysis, with different pIs, 5.32 (Isoform I) and 5.22 (Isoform II). Fraction 2 showed a single isoform having a molecular mass of 55,994 Da and having a pI of 5.09. This component, named Isoform III, existed rather in excess in a mixture of the authentic enzyme isoforms. The remainder (fraction 3) also contained a single component (Isoform IV) which has a molecular mass of 56,310 Da with a pI of 4.97. Chemical analyses indicated that the N-termini and the C-terminal tripeptides of four pI-separated isoforms mentioned are similar to one another, and are blocked and are NH2-Val-Asp-Gly-COOH, respectively. Moreover, enzymic properties involving specific activity||and the value of kcat/Km for the above three fractions are||almost the same, and also agreed completely with those of an unfractionated authentic β-amylase preparation.
soybean β-amylase; pI-heterogeneity; kinetic analyses; chromatofocusing; mass spectrometric analyses
-19-
(2E,6R)-8-Hydroxy-2,6-dimethyl-2-octenoic Acid,
a Novel Anti-osteoporotic Monoterpene, Isolated from Cistanche salsa
Kohji YAMAGUCHI, Chikara SHINOHARA, Sachiko KOJIMA, Mikiko SODEOKA,
and Tomoko TSUJI・Sagami Chemical Research Center, 4-4-1 Nishi-ohnuma, Sagamihara, Kanagawa 229-0012, Japan
Received November 4, 1998; Accepted December 21, 1998
(2E,6R)-8-Hydroxy-2,6-dimethyl-2-octenoic acid [(R)-HDOA], a novel monoterpene from Cistanche salsa, a Chinese herb, was found to be an anti-osteoporotic compound. The extract of Cistanche salsa significantly suppressed the bone weight loss in ovariectomized mice, a postmenopausal osteoporosis model. The active substance was then purified by using this osteoporotic model and the chemical structure was determined. The active compound from Cistanche salsa, (R)-HDOA, suppressed the decrease of bone weight and the mechanical strength in the ovariectomized mice. Furthermore, (R)- and (S)-HDOA were synthesized and the activity of each was evaluated. (R)-HDOA suppressed the bone weight loss, although (S)-HDOA did not showed any activity.
Cistanche salsa; (2E,6R)-8-hydroxy-2,6-dimethyl-2-octenoic acid; osteoporosis; ovariectomized mice
-20-
Synthesis of 4′-C-Ethynyl-β-D-ribo-pentofuranosyl Pyrimidines
Ritsuko YAMAGUCHI, Tatsuma IMANISHI, Satoru KOHGO,
Hiroko HORIE, and Hiroshi OHRUI＊Department of Applied Biological Chemistry, Faculty of Agriculture, Tohoku University,
1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, JapanReceived November 9, 1998; Accepted December 21, 1998
The 4′-C-ethynyl-β-D-ribo-pentofuranosylpyrimidines were prepared from D-glucose through properly protected 4′-C-formyl-D-ribo-ribofuranose as the key intermediate, and preliminary biological tests against some viruses and tumor cells showed that the compounds were not active.
4′-C-substituted nucleosides; ethynyl group
-21-
Potentiation of GABAA Receptors Expressed in Xenopus Oocytes
by Perfume and Phytoncid
Hitoshi AOSHIMA・ and Koutaro HAMAMOTO
Department of Physics, Biology and Informatics, Faculty of Science, Yamaguchi University,
1677-1 Yoshida, Yamaguchi 753-8512, JapanReceived November 11, 1998; Accepted January 8, 1999
To study the effects of perfume and phytoncid on GABAA receptors, ionotropic GABAA receptors were expressed in Xenopus oocytes by injecting mRNAs that had been prepared from rat whole brain. Essential oil, perfume and such phytoncid as leaf alcohol, hinokitiol, pinene, eugenol, citronellol and citronellal potentiated the response in the presence of GABA at low concentrations (10 and 30 μM), possibly because they bound to the potentiation-site in GABAA receptors and increased the affinity of GABA to the receptors. Since it is known that the potentiation of GABAA receptors by benzodiazepine, barbiturate, steroids and anesthetics induces the anxiolytic, anticonvulsant and sedative activity or anesthetic effect, these results suggest the possibility that the intake of perfume or phytoncid through the lungs, the skin or the intestines modulates the neural transmission in the brain through ionotropic GABAA receptors and changes the frame of the human mind, as alcohol or tobacco does.
aromatherapy; essential oil; perfume; phytoncid; GABAA receptor
-22-
Note
Effect of Media Constituents on the Formation by Halophilic Yeast
of the 2 (or 5)-Ethyl-5 (or 2)-methyl-4-hydroxy-3 (2H)-furanone
Aroma Component Specific to Miso
Etsuko SUGAWARA＊,・ and Yonekichi SAKURAI＊＊
＊Faculty of Education, Iwate University, 3-18-33 Ueda, Morioka 020-8550, Japan
＊＊Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka 020-8550, JapanReceived September 7, 1998; Accepted November 26, 1998
The formation of HEMF [2 (or 5)-ethyl-5 (or 2)-methyl-4-hydroxy-3 (2H)-furanone] by yeast was examined in an attempt to investigate its mechanism and involved factors. HEMF formation was promoted by yeast cultivation in a heat-sterilized medium which included glucose, ribose, and a nitrogenous compound such as an extract of shoyu koji, poly-peptone, casamino acid, or an amino acid (glutamic acid, threonine, serine, or alanine).
2 (or 5)-ethyl-5 (or 2)-methyl-4-hydroxy-3 (2H)-furanone; aroma component; miso; halophilic yeast
-23-
Note
Improved Suppression by Dietary Taurine of the Fecal Excretion of Bile Acids
from Hypothyroid Rats
Hideki MOCHIZUKI, Jun TAKIDO, and Hidehiko YOKOGOSHI・
School of Food and Nutritional Sciences, The University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan
Received September 8, 1998; Accepted December 15, 1998
The effect of dietary taurine, 2-aminoethanesulfonic acid, on hypercholesterolemia caused by thiouracil-induced hypothyroidism was investigated in hypothyroid rats. Serum total- and HDL-cholesterol were significantly increased, and the excretion of fecal bile acids was significantly decreased. Taurine did not change the hypercholesterolemia, but significantly recovered the excretion of bile acids.
taurine; cholesterol; bile acids; hypothyroid rat
-24-
Note
Isolation and Structural Determination of a Novel Bicyclic Taxane Diterpene
from Needles of the Chinese Yew, Taxus mairei
Qing-Wen SHI, Takayuki ORITANI,＊ Takeyoshi SUGIYAMA, and Teiko YAMADA
Laboratory of Applied Bioorganic Chemistry, Division of Life Science, Graduate School of Agricultural Science,
Tohoku University, 1-1 Tsutsumidori-Amamiya machi, Aoba-ku, Sendai 981-8555, JapanReceived Septemer 14, 1998; Accepted December 2, 1998
A novel bicyclic taxane diterpene with a rare 12-membered ring was isolated from needles of the Chinese yew, Taxus mairei, and its structure was established as (3E, 8E)-2α, 7β, 9, 10β, 13α, 20-hexaacetoxy-5(2′-acetoxy-cinnamoyloxy)-3,8-secotaxa-3,8,11-triene (1) with the help of 1D and 2D NMR data. The relative stereochemistry was deduced from a NOESY experiment.
Taxus mairei; Taxaceae; taxoids; bicyclic taxanes; needles
-25-
Note
Insecticidal Activity of α-Methylene-γ-butyrolactone against Several Insect Pests
Probal Kanti DATTA, Chul-Sa KIM,・ Tetsuro HARA, Eiji ITOH,
and Michio HORIIKEDepartment of Bioresources Science, Faculty of Agriculture, Kochi University, B200 Monobe,
Nankoku 783-8502, JapanReceived September 24, 1998; Accepted November 27, 1998
α-Methylene-γ-butyrolactone (tulipalin A), which has been found to possess effective insecticidal activity against Thrips palmi (Thysanoptera; Thripinae), was examined on several insect pests. This compound caused high mortality against thrips species such as Frankliniella occidentallis and Frankliniella intonsa. In addition, some mortality was observed against other agricultural pest species. It is considered that α-methylene-γ-butyrolactone has a wide spectrum of insecticidal activity.
insecticidal activity; α-methylene-γ-butyrolactone (tulipalin A); Thrips palmi; Frankliniella intonsa; Frankliniella occidentallis
-26-
Note
Differential Accumulation of Transcripts Encoding Sulfur Assimilation Enzymes
upon Sulfur and/or Nitrogen Deprivation in Arabidopsis thaliana
Yube YAMAGUCHI, Tatsuo NAKAMURA, Emiko HARADA, Nozomu KOIZUMI, and Hiroshi SANO・
Research and Education Center for Genetic Information, Nara Institute of Science and Technology,
8916-5 Takayama-cho, Ikoma, Nara 630-0101, JapanReceived September 24, 1998; Accepted November 27, 1998
Expression of nine genes encoding enzymes involved in the sulfur assimilation pathway was examined by RNA blot hybridization. Significantly increased levels of transcripts encoding ATP sulfurylase and APS reductase were apparent under sulfur deprivation. However, in the absence of nitrogen, their responsiveness to sulfur deprivation was markedly reduced. Results suggest that the sulfur assimilation pathway is regulated at the transcriptional level by both nitrogen and sulfur sources.
APS reductase; Arabidopsis thaliana; ATP sulfurylase; nitrogen source; sulfur assimilation
-27-
Note
Sequence of a cDNA Encoding Mouse F1F0-ATP Synthase g Subunit
Deahyun JEON, Hyungha KIM, and Myunggi BAIK・
Department of Genetic Engineering, Institute of Biotechnology, College of Agriculture,
Chonnam National University, Kwangju 500-757, KoreaReceived October 20, 1998; Accepted December 1, 1998
A pregnant-induced clone was identified by differential screening from a cDNA library of mouse mammary gland. The clone was identified as a full-length cDNA encoding the F1F0-ATP synthase g subunit. Comparison of the deduced amino acid sequences of mouse ATP synthase g subunit with those of bovine species showed 86％ identity. The high levels of ATP synthase g subunit mRNA were detected in heart and uterine tissues.
mouse F1F0-ATP synthase g subunit; mammary gland
-28-
Note
Detection and Analysis of Translation Elongation Factor 3 Genes
from Various Yeasts
Masahiro URITANI,・ Yasuhito SHOUMURA, and Shinpei YAMADA＊
Department of Chemistry, and ＊Department of Biology and Geoscience, Faculty of Science,
Shizuoka University, 836 Oya, Shizuoka-shi, Shizuoka 422-8529, JapanReceived Nomvember 2, 1998; Accepted December 7, 1998
Yeast translation requires a unique elongation factor, EF-3. However, information about EF-3 genes has been limited to only a few yeast species. Here, we developed a PCR-based system to detect the EF-3 genes specifically, and identified EF-3 gene fragments from various yeast species in which EF-3 genes have not yet been found.
EF-3; translational elongation; yeast; ABC protein
-29-
Note
Simultaneous Determination of Different Classes of Lipid Hydroperoxides
by High-Performance Liquid Chromatography with Post Column Detection
by a Ferrous/Xylenol Orange Reagent
Keiji SUGINO・
Nissin Central Research Institute, Nissin Food Products Company, Kusatsu, Shiga 552-0055, Japan
Received November 20, 1998; Accepted January 8, 1999
A novel high-performance liquid chromatographic (HPLC) method with post-column detection was developed to simultaneously analyze the lipid hydroperoxides of polar and nonpolar lipids. The HPLC method uses a reversed-phase column (octyl-bonded silica) and a mobile phase of water/acetonitrile/methanol. Hydroperoxides were detected at 592 nm by using a ferrous (II)/xylenol orange (FeXO) reagent. This method enabled the separation of fatty acid hydroperoxides, phosphatidylcholine hydroperoxides, and hydroperoxides of neutral lipids, including triacylglycerol hydroperoxides and cholesterol ester hydroperoxides, by chromatography. These hydroperoxides could be quantified in a range between 40 pmol and 2 nmol. This new method was applied to estimate the lipid hydroperoxides formed during the photosensitized oxidation of rat plasma.
hydroperoxide; high-performance liquid chromatography; xylenol orange; plasma
-30-
Note
Construction and Characterization of Escherichia coli Disruptants Defective
in the yaeM Gene
Tomohisa KUZUYAMA, Shunji TAKAHASHI＊, and Haruo SETO
Institute of Molecular and Cellular Biosciences, University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan
Received December 9, 1998; Accepted January 4, 1999
Escherichia coli disruptants defective in the yaeM gene, which is located at 4.2 min on the chromosome map, were constructed and characterized. The disruptants showed auxotrophy for 2-C-methylerythritol, a free alcohol of 2-C-methyl-D-erythritol 4-phosphate that is a biosynthetic precursor in the nonmevalonate pathway. This result clearly shows that the yaeM gene is indeed involved in this pathway in E. coli.
biosynthesis; terpenoid; nonmevalonate pathway; 2-C-methyl-D-erythritol 4-phosphate; Escherichia coli
-31-
Preliminary Communication
Expression of a Functional Single-chain Antibody against GA24/19
in Transgenic Tobacco
Naoki SHIMADA, Yoshihito SUZUKI, Masatoshi NAKAJIMA, Udo CONRAD,＊
Noboru MUROFUSHI, and Isomaro YAMAGUCHIDepartment of Applied Biological Chemistry, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan
＊Institute of Plant Genetics and Crop Plant Research, Corrensstra・e 3, D-06466 Gatersleben, GermanyReceived January 11, 1999; Accepted February 8, 1999
An anti-gibberellin A24/19 single-chain Fv gene was constructed from γ and κ genes cloned from a hybridoma cell line producing monoclonal antibody against gibberellin A24/19, biosynthetic precursors of gibberellin A4/1 which are biologically active per se. The single-chain Fv gene was introduced into tobacco plants after the binding activity of the single-chain Fv expressed in Escherichia coli was confirmed. When the single-chain Fv expression is targeted to endoplasmic reticulum, the plants could accumulate the single-chain Fv protein with the antigen binding activity up to 3.6％ of the total soluble protein. On the other hand, when the expression is targeted to cytosol, accumulation of the single-chain Fv protein was not detected at all. The dwarf phenotype of the transgenic plants expressing the single-chain Fv protein, together with the preliminary analytical data indicating a decreased level of gibberellin A1 in the dwarf transgenics, suggested that the single-chain Fv decreased the concentration of bioactive gibberellins by trapping and inhibiting the metabolism of gibberellin A24 and/or A19 to gibberellin A4 and/or A1.
single-chain Fv; gibberellin; GA24; GA19