(Vol.64 No.8 2000)
Review
Growth Regulation by Insulin-like Growth Factor-I in Fish
Shunsuke MORIYAMA,1 Felix G. AYSON,2 and Hiroshi KAWAUCHI1 p.1553
Stereoselective Model Synthesis of the Optically Active Olivil
Type of Lignan from D-Xylose
Satoshi YAMAUCHI＊ and Yoshiro KINOSHITA p.1563
Hydroxycinnamic Acids and Their Dimers Involved in the Cessation
of Cell Elongation in Mentha Suspension Culture
Jian-Gang YANG and Takeo UCHIYAMA＊,・・・ p.1572
Cloning and Heterologous Expression of Gene Encoding
A Polygalacturonase from Aspergillus awamori
Masaru NAGAI, Akira OZAWA,＊ Tohoru KATSURAGI,＊＊ Haruhiko KAWASAKI,＊
and Takuo SAKAI・・・ p.1580
Quinochalcones and Flavonoids from Fresh Florets in Different
Cultivars
of Carthamus tinctorius L.
Kohei KAZUMA, Takashi TAKAHASHI, Katsura SATO, Hisatomo TAKEUCHI,
Takeshi MATSUMOTO,＊ and Toshikatsu OKUNO・・・ p.1588
Preventive Effects of Dietary Cabbage Acylated Anthocyanins
on Paraquat-induced Oxidative Stress in Rats
Kiharu IGARASHI,・・・ Yuriko KIMURA, and Asako TAKENAKA p.1600
Cyclodextrin Encapsulation to Prevent the Loss of l-Menthol
and its Retention during Drying
Xiang-Dong LIU, Takeshi FURUTA,・・・ Hidefumi YOSHII,
Pekka LINKO,＊ and W. Jan COUMANS＊＊ p.1608
Antifungal Compound, Feruloylagmatine, Induced in Winter
Wheat Exposed to a Low Temperature
Shigeki JIN・・・ and Midori YOSHIDA p.1614
Scaling Analysis of the Concentration Dependence on Elasticity
of Agarose Gel
Tomoyuki FUJII,・・・ Toshimasa YANO, Hitoshi KUMAGAI, and Osato MIYAWAKI p.1618
Time-Resolved Fluorescence Studies on the Internal Motion of
Chlorophyll a
of Light-Harvesting Chlorophyll a/b-Protein Complex in Lipid Membranes
Makio FURUICHI,a Etsuko NISHIMOTO,b Toshiaki KOGA,c and Shoji YAMASHITAd,・・・
p.1623
Effects of Bienzyme Complex Formation of Cysteine Synthetase
from Escherichia coli on Some Properties and Kinetics
Koshiki MINO, Tsuyoshi YAMANOUE, Takaharu SAKIYAMA, Naoki EISAKI,＊
Asahi MATSUYAMA,＊ and Kazuhiro NAKANISHI・・・ p.1628
C19 Odd-chain Polyunsaturated Fatty Acids (PUFAs) Are Metabolized
to C21-PUFAs in a Rat Liver Cell Line, and Curcumin, Gallic Acid,
and Their Related Compounds Inhibit Their Desaturation
Nobuhiro NAKANO, Norifumi SHIRASAKA, Hiroko KOYAMA, Miki HINO,
Tetsuo MURAKAMI, Sakayu SHIMIZU,＊ and Hajime YOSHIZUMI・・・ p.1641
Chemical characteristics of dehydro-L-ascorbic acid
Tadao KURATA and Yoko NISHIKAWA・・・ p.1651
Cryoprotective Activities of Group 3 Late Embryogenesis Abundant
Proteins from Chlorella vulgaris C-27
Ken-ichi HONJOH,＊,・・・ Hiroko MATSUMOTO,＊ Hideyuki SHIMIZU,＊ Kanae OOYAMA,＊
Kageyuki TANAKA,＊ Yuichi ODA,＊ Ryoji TAKATA,＊ Toshio JOH,＊＊
Koushirou SUGA,＊,＊＊＊ Takahisa MIYAMOTO,＊ Masayoshi IIO,＊
and Shoji HATANO＊＊＊＊ p.1656
Sulfated Fibroin, a Novel Sulfated Peptide Derived from Silk,
Inhibits Human Immunodeficiency Virus Replication in Vitro
Kazuyo GOTOH,1 Hiroyuki IZUMI,1,2 Taisei KANAMOTO,1 Yasushi TAMADA,3
and Hideki NAKASHIMA1,・・・ p.1664
Synthesis of (±)-Methyl Bishomononactate, a Monomeric Component
of Polynactin Antibiotics
Tadaatsu HANADATE, Hiromasa KIYOTA,・・・ and Takayuki ORITANI p.1671
Isolation and Characterization of a Novel Polysaccharide
As a Possible Allergen Occurring in Wheat Flour
Soichi TANABE,1,＊ Jun WATANABE,2 Kazunobu OYAMA,2 Eri FUKUSHI,2
Jun KAWABATA,2 Soichi ARAI,3 Tasuku NAKAJIMA,4 and Michiko WATANABE1 p.1675
Characterization and Affinity Purification of Juvenile Hormone
Esterase
from Bombyx mori
Takahiro SHIOTSUKI,＊,・・・ Bryony C. BONNING,＊＊ Makoto HIRAI,＊ Kyoko KIKUCHI,＊
and Bruce D. HAMMOCK＊＊＊ p.1681
Addition of Signal Leader Sequences to the N-Termini of Olfactory
Receptor Proteins Enhances Their Expression in Xenopus Oocyte・・・
Akihito YASUOKA,1,・・・・・・ Yasufumi EMORI,2 and Keiko ABE3 P.1688
CYP78A1 Preferentially Expressed in Developing Inflorescences
of Zea mays
Encoded a Cytochrome P450-Dependent Lauric Acid 12-Monooxygenase
Hiromasa IMAISHI,1 Satoshi MATSUO,2 Eri SWAI,2 and Hideo OHKAWA2,・・・ p.1696
Hydrolysis of β-Galactosyl Ester Linkage by β-Galactosidases
Taro KISO,1 Hirofumi NAKANO,1 Hirofumi NAKAJIMA,2 Tadamasa TERAI,2
Katsuyuki OKAMOTO,3 and Sumio KITAHATA1 p.1702
Triterpene Phytoalexins from Strawberry Fruit
Nobuhiro HIRAI,1,2,・・・ Megumi SUGIE,1 Mitsuo WADA,1 El Hassan LAHLOU,2
Tsunashi KAMO,1,2 Ryuji YOSHIDA,3 Mitsuya TSUDA,4 and Hajime OHIGASHI1 p.1707
Note
New Syntheses of the Rice Moth and Stink Bug Pheromones by Employing
(2R, 6S)-7-Acetoxy-2,6-dimethyl-1-heptanol as a Building Block・・・
Yoshihide NAKAMURA・・・・・・ and Kenji MORI・・・・・・・・・ p.1713
Note
Purification and Characterization of Subtilisin DJ-4 Secreted
by Bacillus sp. Strain DJ-4 Screened from Doen-Jang
Seung-Ho KIM＊ and Nack-Shick CHOI p.1722
Note
Synthesis of (3E, 5Z)-3,5-Dodecadienylacetate, the Sex Pheromone
of Phtheochroa cranaodes (Lepidoptera: Tortricidae)
Jhillu Singh YADAV,＊ and Etukala Jagan REDDY p.1726
Note
Purification and Characterization of an Endo-Polygalacturonase
from Aspergillus awamori
Masaru NAGAI, Tohoru KATSURAGI,＊ Takao TERASHITA,
Kentaro YOSHIKAWA, and Takuo SAKAI・・・ p.1729
Note
Molecular Structure of rDNA Repeat Unit in Magnaporthe grisea
Teruo SONE, Satoru FUKIYA, Megumi KODAMA, and Fusao TOMITA＊ p.1726
Note
Phylogenetic Analysis of Methanogens in Sheep Rumen Ecosystem
and Detection of Methanomicrobium mobile
by Fluorescence In Situ Hybridization
Kazuhiro YANAGITA,1,2 Yoichi KAMAGATA,1＊ Mamoru KAWAHARASAKI,1
Toshihiko SUZUKI,1 Yutaka NAKAMURA,2 and Hajime MINATO2 p.1737
Note
Efficient Synthesis of a Sialyl T-antigen-linked Glycopeptide
by the Chemoenzymatic Method
Katsumi AJISAKA＊ and Mariko MIYASATO p.1743
Note
Molecular Cloning of Genomic DNA for Fructose-1,6-bisphosphatase
from Aspergillus oryzae
Motoaki SANO, Keiichi NAKAJIMA,＊ Kumiko TAKASE, Midori YAMAMOTO,
and Masayuki MACHIDA p.1747
Note
Oxidation and Reduction of Nitrite Ion in the TiO2 Photo-induced
Catalytic Reaction
Hitoshi SHIBATA,＊・・・ Namiko NODA,＊ Yasushi OGURA,＊ Kunihisa SOGABE,＊＊
and Yoshihiro SAWA＊ p.1751
Note
Mutational Analysis of the Multicopy hao Gene Coding for Hydroxylamine
Oxidoreductase in Nitrosomonas sp. Strain ENI-11
Akira YAMAGATA, Ryuichi HIROTA, Junichi KATO, Akio KURODA,
Tsukasa IKEDA, Noboru TAKIGUCHI, and Hisao OHTAKE・・・ p.1754
Note
Synthesis of the Spirosuccinimide Moiety of Asperparaline A
Shinji TANIMORI,・・・ Kouji FUKUBAYASHI, and Mitsunori KIRIHATA p.1758
Note
Absolute Configuration at C45 in 45-Hydroxyyessotoxin,
a Marine Polyether Toxin Isolated from Shellfish
Akio MOROHASHI, Masayuki SATAKE, Yasukatsu OSHIMA, and Takeshi YASUMOTO・・・ p.1761
Note
Native and Acid-denatured L-Lactate Dehydrogenase Are Different
in the Lysosomal Proteinases Involving in Their Overall Degradation
Takeyuki OHSHITA p.1764
Note
Concanavalin A-Induced Discharge of Glycocalyx of Raphidophycean
Flagellates, Chattonella marina and Heterosigma akashiwo
Tarou OKAMOTO,1 Daekyung KIM,1 Tatsuya ODA,1,・・・ Kazumi MATSUOKA,2
Atsushi ISHIMATSU,3 and Tsuyoshi MURAMATSU1 p.1767
Preliminary Communication
mRNA Expression of MT1-MMP, MMP-9, Cathepsin K, and TRAP
in Highly Enriched Osteoclasts Cultured on Several Matrix Proteins
and Ivory Surfaces
Toshimasa UEMURA,1,2 Yin-kun LIU,3 and Yoshinori KUBOKI4 p.1771
-1-
Review
Growth Regulation by Insulin-like Growth Factor-I in Fish
Shunsuke MORIYAMA,1 Felix G. AYSON,2 and Hiroshi KAWAUCHI1
1Laboratory of Molecular Endocrinology, School of Fisheries Sciences, Kitasato University,
Sanriku, Iwate 022-0101, Japan
2Aquaculture Department, Southeast Asian Fisheries Development Center (SEAFDEC AQD),
Iloilo, Philippines
Insulin-like growth factor-I (IGF-I) is a mitogenic polypeptide that plays an essential role in the regulation of development and somatic growth of vertebrates, mainly by mediating growth hormone actions. It has clearly been established that the structure of IGF-I and its biological function has been highly conserved among vertebrates. In this paper, we review the recent developments in the molecular, biochemical, and physiological properties of IGF-I in fish.
Keywords: fish; growth; insulin-like growth factor; structure; function
-2-
Stereoselective Model Synthesis of the Optically Active Olivil
Type of Lignan from D-Xylose
Satoshi YAMAUCHI＊ and Yoshiro KINOSHITA
College of Agriculture, Ehime University, Tarumi 3-5-7, Matsuyama, Ehime 790-8566, Japan
Received November 19, 1999; Accepted March 7, 2000
The olivil type of lignan, (2S,3R,4R)-4-benzyl-4-
hydroxy - 3 - hydroxymethyl - 2 - (3,4 - methylenedioxyphenyl)tetrahydrofuran, was stereoselectively synthesized from D-xylose.
Key words: lignan; tetrahydrofuran lignan; olivil
-3-
Hydroxycinnamic Acids and Their Dimers Involved in the Cessation
of Cell Elongation in Mentha Suspension Culture
Jian-Gang YANG and Takeo UCHIYAMA＊,・・・
Graduate School of Science and Technology, Niigata University, Ikarashi,
Niigata 950-2102, Japan
＊Faculty of Agriculture, Niigata University, Ikarashi, Niigata 950-2181, Japan
Received December 1, 1999; Accepted February 23, 2000 The contents of wall-bound hydroxycinnamic acids and their dimers were compared between elongated and non-elongated cells of suspension-cultured Mentha. Wall-bound peroxidase activity was also investigated. The main hydroxycinnamic acids esterified to these two kinds of cell walls were ferulic and caffeic acids. Eleven dehydrodicaffeic acid isomers and six dehydrodiferulic acid isomers formed through C-C and C-O-C coupling processes, were detected by GC-MS from the extracts released from the walls of non-elongated cells. On the other hand, only four dehydrodicaffeic acid isomers and three dehydrodiferulic acid isomers were found in the walls of elongated cells. Amounts of monomers of ferulic and caffeic acids and their 5,5?-dehydrodimers in non-elongated cell walls were about ten and twenty times higher, respectively, than those in the elongated cell walls. There was a close correlation between the amount of 5,5?-dehydrodimers and activity of wall-bound peroxidase in non-elongated and elongated cells. The level of 5,5?-dehydrodimers accumulated at a higher rate than monomers in non-elongated cell walls. These results suggest that the dimerization of ester-linked ferulic and caffeic acids by peroxidase and the increase in amounts of their 5,5?-dehydrodimers are important factors in the cessation of cell elongation in Mentha suspension culture.
Key words: cell elongation; cell wall; dehydrodicaffeic acid; dehydrodiferulic acid; wall-bound peroxidase
-4-
Cloning and Heterologous Expression of Gene Encoding
A Polygalacturonase from Aspergillus awamori
Masaru NAGAI, Akira OZAWA,＊ Tohoru KATSURAGI,＊＊ Haruhiko KAWASAKI,＊
and Takuo SAKAI・・・
Department of Food Science and Nutrition, Faculty of Agriculture, Kinki University,
3327-204, Nakamachi, Nara 631-8505, Japan
＊Department of Applied Biochemistry, College of Agriculture, Osaka Prefecture University,
Gakuen-cho 1-1, Sakai, Osaka 593-8231, Japan
＊＊Graduate School of Biological Sciences, Nara Institute of Science and Technology,
Takayama Science Town, Ikoma, Nara 630-0101, Japan
Received December 1, 1999; Accepted April 12, 2000
A polygalacturonase gene of Aspergillus awamori IFO 4033 was cloned by genomic Southern hybridization with a probe of a DNA fragment synthesized by PCR. This was done using primers constructed based on the N-terminal amino acid sequence of a polygalacturonase, protopectinase-AS, produced by the strain and the consensus internal amino acid sequence of fungal polygalacturonases. The cloned polygalacturonase gene, containing an ORF, encodes 362 amino acids, including a 52-bp intron. It contains the consensus nucleotide sequence of PacC binding sites, and its expression was appeared to be regulated by ambient pH. After the intron was excised, the cloned gene was inserted into an expression plasmid for yeast, pMA91, and introduced into Saccharomyces cerevisiae to be expressed. The expressed gene product was purified to a homogeneous preparation, and this confirmed that the polygalacturonase produced was the product of the cloned gene.
Key words: polygalacturonase gene; A. awamori; gene cloning; PacC binding site
-5-
Quinochalcones and Flavonoids from Fresh Florets in Different Cultivars
of Carthamus tinctorius L.
Kohei KAZUMA, Takashi TAKAHASHI, Katsura SATO, Hisatomo TAKEUCHI,
Takeshi MATSUMOTO,＊ and Toshikatsu OKUNO・・・
Faculty of Agriculture and Life science, Hirosaki University, Bunkyo-cho, Hirosaki 036-8561, Japan
＊School of Engineering, Hokkaido Tokai University, Minami-ku, Sapporo 005-8601, Japan
Received December 24, 1999; Accepted March 23, 2000
The flavonoid constituents in fresh florets of the three distinctive cultivars of Carthamus tinctorius L. were purified and identified to investigate flavonoid biosynthesis in the petals. From the orange flower of cv. Ken-ba (K.), four new compounds, anhydrosafflor yellow B (1), two kaempferols, 9 and 13, and a quercetin, 17, were isolated, as well as the twelve known compounds, and their structures were determined by spectral data, chemical reactions, and molecular mechanics calculations. From the yellow flower of cv. Ogon-hanagasa (O.), two flavonols and two quinochalcones, and from the white flower of cv. Shiro-bana (S.), three flavonols were isolated. These compounds were the same as those contained in cv. K. To compare the flavonoid constituents among the three cultivars, crude extracts were analyzed by a LC/PDA/MS system. In cv. K., six quinochalcones and eleven flavonols were identified. In cv. O., three quinochalcones and nine flavonols were identified, but the red pigment, carthamin (4), and its precursor, precarthamin (3), were not detected. In cv. S., four flavonols without a 6-hydroxyl group were identified. On the basis of a comparative study on the constituents among these three cultivars, a possible biosynthetic pathway to form quinochalcones via the intermediate, pentahydroxychalcone (19), is proposed.
Key words: Carthamus tinctorius L.; flower pigment; quinochalcone; flavonol; biosynthesis
-6-
Preventive Effects of Dietary Cabbage Acylated Anthocyanins
on Paraquat-induced Oxidative Stress in Rats
Kiharu IGARASHI,・・・ Yuriko KIMURA, and Asako TAKENAKA
Department of Bioresource Engineering, Faculty of Agriculture, Yamagata University,
1-23 Wakaba-machi, Tsuruoka-shi, Yamagata 997-0037, Japan
Received January 11, 2000; Accepted March 30, 2000
The preventive effects of acylated anthocyanins from red cabbage on paraquat-induced oxidative stress were determined in rats. Decreased food intake and body weight gain, and increased lung weight and atherogenic index by feeding the rats on a diet containing paraquat were clearly suppressed by supplementing acylated anthocynins to the paraquat diet. Paraquat feeding increased the concentration of thiobarbituric acid-reactive substances (TBARS) in liver lipids, and decreased the liver triacylglycerol level. These effects tended to be suppressed by supplementing acylated anthocynins to the paraquat diet. In addition, the catalase activity in the liver mitochondrial fraction was markedly decreased by feeding on the paraquat diet, this decrease being partially suppressed by supplementing the paraquat diet with acylated anthocyanins. An increase in the NADPH-cytochrome-P450-reductase activity in the liver microsome fraction by paraquat was supressed by supplementing the paraquat diet with acylated anthocyanins. These results suggest that acylated anthocyanins from red cabbage acted preventively against the oxidative stress in vivo that may have been due to active oxygen species formed through the action of paraquat.
Key words: red cabbage; acylated anthocyanins; oxidative stress; paraquat
-7-
Cyclodextrin Encapsulation to Prevent the Loss of l-Menthol
and its Retention during Drying
Xiang-Dong LIU, Takeshi FURUTA,・・・ Hidefumi YOSHII,
Pekka LINKO,＊ and W. Jan COUMANS＊＊
Department of Biotechnology, Tottori University, Tottori 680-8552, Japan
＊Department of Chemical Technology, Helsinki University of Technology,
FIN-02150 Espoo, Finland
＊＊Faculty of Chemical Engineering, Eindhoven University of Technology,
P.O. Box 513, 5600 MB Eindhoven, The Netherlands
Received January 12, 2000; Accepted March 8, 2000
The taste and flavor of spray-dried powdered products are the most important quality factors. In the present study, molecular encapsulation in cyclodextrin was applied to prevent the loss of a hydrophobic flavor compound (l-menthol) during the drying of a droplet. β-Cyclodextrin appeared to be a better encapsulant for menthol than α- and γ-cyclodextrin. The retention of menthol increased with increasing concentration of both cyclodextrin and maltodextrin. A simple mathematical model is proposed for estimating the flavor retention. The theoretical results by this model estimated well the final retention of menthol encapsulated in a blend of β-cyclodextrin and maltodextrin.
Key words: flavor retention; cyclodextrin; spray drying; diffusion; l-menthol
-8-
Antifungal Compound, Feruloylagmatine, Induced in Winter
Wheat Exposed to a Low Temperature
Shigeki JIN・・・ and Midori YOSHIDA
Hokkaido National Agricultural Experiment Station, Toyohira-ku, Sapporo 062-8555, Japan
Received January 13, 2000; Accepted March 13, 2000
An antifungal compound, feruloylagmatine [1-(trans - 4? - hydroxy - 3? - methoxycinnamoylamino) - 4 - guanidinobutane], was isolated from crowns of winter wheat (Triticum aestivum L. cv Chihokukomugi). Its structure was identified by NMR, MS and UV spectral analyses. It was also confirmed by an HPLC analysis that the compound was induced in wheat by a low temperature.
Key words: antifungal; Triticum aestivum; wheat; low temperature; feruloylagmatine
-9-
Scaling Analysis of the Concentration Dependence on Elasticity
of Agarose Gel
Tomoyuki FUJII,・・・ Toshimasa YANO, Hitoshi KUMAGAI, and Osato MIYAWAKI
Department of Applied Biological Chemistry, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku,
Tokyo 113-8657, Japan
Received January 14, 2000; Accepted March 27, 2000
Since the critical exponent of the elastic modulus is related to the spatial dimension and the critical exponent of the correlation length, depending on the characteristics of elasticity, we experimentally evaluated both the elastic modulus of a sol-gel transition system and also the correlation length. We could determine the correlation length of agarose gel by the dynamic light scattering method; it was well described by the power law as a function of the deviation from the sol-gel transition point. Three scaling laws between the critical exponent of the correlation length (ν) and that of the elastic shear modulus (t) were compared, and the critical exponent of the elastic modulus was described by the equation of de Gennes expression (t＝1+ν(d--2), where d is the spatial dimension). This result suggests that agarose fibers are stiff enough to show scalar elasticity.
Key words: agarose gel; percolation; correlation length; dynamic shear modulus; scaling law
-10-
Time-Resolved Fluorescence Studies on the Internal Motion of Chlorophyll a
of Light-Harvesting Chlorophyll a/b-Protein Complex in Lipid Membranes
Makio FURUICHI,a Etsuko NISHIMOTO,b Toshiaki KOGA,c and Shoji YAMASHITAd,・・・
aStructural Biophysics Laboratory, RIKEN Harima Institute, Hyogo 679-5148, Japan
bDepartment of Medical Biochemistry, Kurume University School of Medicine,
Kurume, Fukuoka 830-0011, Japan
cDepartment of Material Chemistry, Kyushu National Industrial Research Institute,
Tosu, Saga 841-0052, Japan
dInstitute of Biophysics, Faculty of Agriculture, Kyushu University, Hakozaki, Higashi-ku,
Fukuoka 812-8581, Japan
Received January 20, 2000; Accepted April 18, 2000
By analyzing the steady state and time-resolved fluorescence anisotropy, the internal motions of chlorophyll a of light-harvesting chlorophyll a/b-protein complex (LHCII) were characterized in a dimyristoylphosphatidylcholine (DMPC) liposome. Corresponding to the thermotropic phase of the membrane, chlorophyll a showed an unique internal motion in LHCII. At the gel phase, two motional components, one fast and the other slow, were observed, which would originate in the heterogeneity of the mutual orientation and the binding site of the chlorophyll a in LHCII. Interestingly, the faster motion was suppressed and only the slower segmental rotation with the larger motional amplitude was allowed on the phase transition to a liquid crystalline phase.
Key words: light-harvesting chlorophyll a/b-protein complex (LHCII); fluorescence anisotropy; phase transition; chlorophyll a
-11-
Effects of Bienzyme Complex Formation of Cysteine Synthetase
from Escherichia coli on Some Properties and Kinetics
Koshiki MINO, Tsuyoshi YAMANOUE, 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 January 24, 2000; Accepted March 31, 2000
Some properties and kinetics of the free and bound serine acetyltransferases (SATs) and O-acetylserine sulfhydrylase-As (OASS-As) from Escherichia coli were investigated. In some cases, SATΔC20, deleting 20 amino acid residues from the C-terminus of the wild-type SAT (Biosci. Biotechnol. Biochem., 63, 168--179 (1999)) was tested for comparison. The optimum pH and stability against some reagents for the free and bound wild-type SATs were similar except for the resistance to cold inactivation. The kinetics for the wild-type SAT and SATΔC20 followed a Ping-Pong Bi Bi mechanism with a mixed-type inhibition by L-cysteine. The kinetics and kinetic constants for the wild-type SAT were not changed by the complex formation with OASS-A. The optimum pH for OASS-A was shifted towards an alkaline pH by the complex formation. Thermal stability and stability against some reagents for the free and bound OASS-As were almost the same. On the other hand, the maximum velocity for OASS-A was lowered and dissociation constants for the substrates and products were increased by forming the complex with the wild-type SAT, although the kinetics for the free and bound enzymes followed the same Ping-Pong Bi Bi mechanism. From comparisons of computed courses of L-cysteine formation from L-serine using SAT (wild-type SAT and SATΔC20) and OASS-A with the experimental results and changes in the stability of the wild-type SAT by the complex formation, we discuss the role and significance of a complex formation for the cysteine synthetase.
Key words: serine acetyltransferase; O-acetylserine sulfhydrylase; cysteine synthetase; enzyme complex; truncated serine acetyltransferase
-12-
C19 Odd-chain Polyunsaturated Fatty Acids (PUFAs) Are Metabolized
to C21-PUFAs in a Rat Liver Cell Line, and Curcumin, Gallic Acid,
and Their Related Compounds Inhibit Their Desaturation
Nobuhiro NAKANO, Norifumi SHIRASAKA, Hiroko KOYAMA, Miki HINO,
Tetsuo MURAKAMI, Sakayu SHIMIZU,＊ and Hajime YOSHIZUMI・・・
Department of Food Science and Nutrition, Faculty of Agriculture, Kinki University,
3327-204 Naka-machi, Nara 631-8505, Japan
＊Division of Applied Life Science, Graduate School of Agriculture, Kyoto University,
Sakyo-ku, Kyoto 606-8502, Japan
Received January 24, 2000; Accepted March 26, 2000
It was demonstrated that the rat liver cell line BRL-3A converted exogenous C19 odd chain-polyunsaturated fatty acids (PUFAs) into the corresponding C21- and C23-PUFAs as follows: 21:3n-8, 21:4n-8, 23:3n-8, and 23:4n-8 (from 19:3n-8); 21:4n-5, 21:5n-5, 23:4n-5, and 23:5n-5 (from 19:4n-5); 21:5n-2, 21:6n-2, 23:5n-2, and 23:6n-2 (from 19:5n-2). It presumed that these C19 PUFAs were converted through the mimic route to docosahexaenoic acid (22:6n-3) from eicosapentaenoic acid (20:5n-3). In addition, the characterization of the change of fatty acid composition of cellular lipids in rat liver cells were examined, using 19:4n-5 and several fatty acid desaturation inhibitors. Curcumin related compounds, curcumin, capsaicin, isoeugenol, 4-(4-hydroxy-3-methoxyphenyl)-3-buten-2-one, and gallic acid esters with near five carbon numbered alcohol had great changes of fatty acid composition of cellular lipids based on inhibition of the Δ6 desaturation of C24-PUFAs in rat liver cells.
Key words: C19-polyunsaturated fatty acids; desaturation; curcumin; gallic acid; rat liver cell line
-13-
Chemical characteristics of dehydro-L-ascorbic acid
Tadao KURATA and Yoko NISHIKAWA・・・
Institute of Environmental Science for Human Life, Ochanomizu University, 2-1-1 Otsuka,
Bunkyo-ku, Tokyo 112-8610, Japan
Received January 27, 2000; Accepted March 23, 2000
Dehydro-L-ascorbic acid (DAA) exists mainly in its C2 hydrated bicyclic form (5) in an aqueous solution, and monocyclic DAA (3), which is the expected reaction product immediately after the oxidation of AA, has not been observed by NMR spectroscopy. The formation mechanism for 5 from 3 and the stability of 5 were examined by the semi-empirical molecular orbital method (MOPAC). It was indicated that the protonation reaction was the key step in the formation of 5, therefore, the formation of 5 is thought to be more difficult under physiological conditions which mostly involve in the neutral or slightly alkaline state. However, by NMR, it was confirmed that, even in a neutral or slightly alkaline state very close to physiological conditions, the predominant form of DAA existing in an aqueous solution immediately after the enzymatic oxidation of AA was confirmed to be 5, although the possible existence of other forms of DAA at very low concentrations could not be completely excluded.
Key words: dehydro-L-ascorbic acid; L-ascorbic acid; NMR; hydrophobic reaction
-14-
Cryoprotective Activities of Group 3 Late Embryogenesis Abundant
Proteins from Chlorella vulgaris C-27
Ken-ichi HONJOH,＊,・・・ Hiroko MATSUMOTO,＊ Hideyuki SHIMIZU,＊ Kanae OOYAMA,＊
Kageyuki TANAKA,＊ Yuichi ODA,＊ Ryoji TAKATA,＊ Toshio JOH,＊＊
Koushirou SUGA,＊,＊＊＊ Takahisa MIYAMOTO,＊ Masayoshi IIO,＊
and Shoji HATANO＊＊＊＊
＊Department of Bioscience and Biotechnology, Division of Bioresource and Bioenvironmental Sciences,
Graduate School, Kyushu University, Fukuoka 812-8581, Japan
＊＊Department of Applied Biological Chemistry, Faculty of Agriculture, Niigata University, 2-Ikarashi,
Niigata 950-2181, Japan
＊＊＊Chlorella Industries Co. Ltd., 1343 Hisatomi, Chikugo, Fukuoka 833-0056, Japan
＊＊＊＊Graduate School of Health and Social Welfare Science, Nishikyushu University, 4490-9 Ooazaozaki,
Kanzaki-machi, Kanzaki-gun, Saga 842-8585, Japan
Received January 28, 2000; Accepted April 4, 2000
The nucleotide sequence of hiC12, isolated as a cDNA clone of hardening-induced Chlorella (hiC) genes, was identified. The clone encodes a late embryogenesis abundant (LEA) protein having six repeats of a 11-mer amino acid motif, although in a slightly imperfect form. To overexpress the hiC61) and hiC12 genes, their coding regions were PCR amplified and subcloned into a pGEX-1λT vector. The HIC6 and HIC12 proteins were expressed as GST fusion proteins in E. coli, then purified. The two HIC proteins were found to be effective in protecting a freeze-labile enzyme, LDH, against freeze-inactivation. On a molar concentration basis, they were about 3.1×106 times more effective in protecting LDH than sucrose and as effective as BSA. Cryoprotection tests with five kinds of chain-shortened polypeptides, synthesized based on the 11-mer amino acid motif of the HIC6 protein showed that the cryoprotective activity decreased with a decrease in the repeating units of the 11-mer motif. In fact, cryoprotective activities of three kinds of single 11-mer amino acids were very low even at high concentrations. All the results suggested that the sufficiently repeated 11-mer motif is required for the cryoprotective activities of Chlorella LEA proteins.
Key words: Chlorella vulgaris C-27; cryoprotection; freezing tolerance; LEA protein
-15-
Sulfated Fibroin, a Novel Sulfated Peptide Derived from Silk,
Inhibits Human Immunodeficiency Virus Replication in Vitro
Kazuyo GOTOH,1 Hiroyuki IZUMI,1,2 Taisei KANAMOTO,1 Yasushi TAMADA,3
and Hideki NAKASHIMA1,・・・
1Department of Microbiology and Immunology, Kagoshima University Dental School,
8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan
2Shin Nippon Biomedical Laboratories, 2438 Miyanoura, Yoshida-cho, Kagoshima 891-1394, Japan
3Biomimetics Laboratory, National Institute of Sericultural and Entomological Science,
1-2 Owashi, Tukuba, Ibaraki 305-8634, Japan
Received February 1, 2000; Accepted April 13, 2000
We prepared two kinds of sulfated silk fibroins, SclFib30 and SclFib31, which contain different amounts of sulfate. These sulfated silk fibroins have anti-HIV-1 activity in vitro, apparently due to interference with the adsorption of virus particles to CD4+ cells, and completely blocked virus binding to the cells at a concentration of 100 μg/ml. Sulfated fibroins also abolished cell-to-cell infection-induced syncytium formation upon cocultivation of MOLT-4 and MOLT-4/HIV-1IIIB cells, suggesting that they would interfere with gp120 and prevent the formation of gp120/CD4 complex. Silk is used in biomaterials such as surgical sutures and is believed to be a safe material for humans. In accordance with low anticoagulant activity and high anti-HIV-1 activity against both X4 HIV-1 and R5 HIV-1 strains, sulfated silk fibroins have potential as antiviral material such for a vaginal anti-HIV formulation.
Keywords: AIDS; silk fibroin; sulfated peptides; binding inhibitor; anti-HIV formulation
-16-
Synthesis of (±)-Methyl Bishomononactate, a Monomeric Component
of Polynactin Antibiotics
Tadaatsu HANADATE, Hiromasa KIYOTA,・・・ and Takayuki ORITANI
Laboratory of Applied Bioorganic Chemistry, Division of Life Science, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiya, Aoba-ku, Sendai 981-8555, Japan
Received February 9, 2000; Accepted March 9, 2000
The synthesis of (±)-methyl bishomononactate, one of the monomeric components of polynactin antibiotics (macrotetrolides), was achieved via cis-selective iodoetherification as the key step.
Keywords: total synthesis; iodoetherification; macrotetrolides; polynactin; antibiotics
-17-
Isolation and Characterization of a Novel Polysaccharide
As a Possible Allergen Occurring in Wheat Flour
Soichi TANABE,1,＊ Jun WATANABE,2 Kazunobu OYAMA,2 Eri FUKUSHI,2
Jun KAWABATA,2 Soichi ARAI,3 Tasuku NAKAJIMA,4 and Michiko WATANABE1
1Food Science Laboratory, Faculty of Education, Tokyo Gakugei University, Tokyo 184-8501, Japan
2Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan
3Department of Nutritional Science, Tokyo University of Agriculture, Tokyo 156-0054, Japan
4Division of Life Science, Graduate School of Agricultural Science, Tohoku University,
Sendai 981-8555, Japan
Received February10, 2000; Accepted March 28, 2000
A new polysaccharide with a molecular weight of 5.0×104 was isolated as a possible wheat allergen from a water-soluble fraction of flour by affinity chromatography and gel filtration. The isolated polysaccharide was found to be a possible wheat allergen, as it bound specifically to IgE antibodies in the sera of patients allergic to the water-soluble fraction of flour. Chemically, the sugar moiety of the polysaccharide consisted of D-glucose and D-mannose with β-1,4-linkages in a molar ratio of 4.4:1. Since this mannoglucan is thought to be stable in our body, it would act as a remaining allergen to cause a long-lasting allergic reaction to wheat flour.
Keywords: wheat allergy; allergen; polysaccharide; mannoglucan
-18-
Characterization and Affinity Purification of Juvenile Hormone Esterase
from Bombyx mori
Takahiro SHIOTSUKI,＊,・・・ Bryony C. BONNING,＊＊ Makoto HIRAI,＊ Kyoko KIKUCHI,＊
and Bruce D. HAMMOCK＊＊＊
＊Department of Insect Physiology and Behavior, National Institute of Sericultural
and Entomological Science, Tsukuba, Ibaraki 305-8634, Japan
＊＊Department of Entomology, Iowa State University, Ames, IA 50011, U.S.A.
＊＊＊Department of Entomology, University of California, Davis, CA 95616, U.S.A.
Received February 16, 2000; Accepted April 7, 2000
Juvenile hormone esterase (JHE) from hemolymph of the silkworm moth Bombyx mori was characterized for substrate specificity and inhibitor sensitivity. B. mori JHE hydrolyzed the juvenile hormone surrogate substrate methyl n-heptylthioacetothioate (HEPTAT) more efficiently than p-nitrophenyl acetate and 1-naphthyl acetate substrates widely used to assay total carboxylesterase activity. B. mori JHE was sensitive to 3-octylthio-1,1,1-trifluoro-2-propanone (OTFP), which was developed as a selective inhibitor for lepidopteran JHE, and relatively insensitive to diisopropyl fluorophosphate (DFP), an inhibitor of serine esterases but not of all JHEs. Affinity purification with a trifluoromethyl ketone ligand was more efficient for purification of B. mori JHE than DEAE ion exchange chromatography.
Keywords: affinity purification; Bombyx mori; juvenile hormone esterase; transition-state analog; trifluoromethyl ketone
-19-
Addition of Signal Leader Sequences to the N-Termini of Olfactory
Receptor Proteins Enhances Their Expression in Xenopus Oocyte・・・
Akihito YASUOKA,1,・・・・・・ Yasufumi EMORI,2 and Keiko ABE3
1Bio-oriented Technology Research Advancement Institution, 1-40-2, Nisshin-cho, Oomiya,
Saitama 331-8537, Japan
2Department of Biophysics and Biochemistry, Faculty of Science, The University of Tokyo, 7-3-1,
Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
3Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences,
The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
Recieved February 16, 2000; Accepted March 27, 2000
Several recent papers have reported the difficulties in expressing olfactory receptor proteins (ORs) in heterologous systems, and proposed that some sequences in ORs have negative effects on their efficient expression. To obtain an efficient expression system of ORs, we modified N-terminal sequences of ORs through the addition of exogenous sequences. Three kinds of sequences, designated as 5HT, V, and VL, were used. 5HT and V corresponded to the signal leader (SL) sequences of 5HT3R and VIPR, respectively. VL corresponded to the first extracellular region of VIPR containing the SL sequence and three potential asparagine- (Asn-) linked glycosylation sites. The myc epitope was also added to the C-termini of the sequences. Several ORs including I7 of rat, GUST43 of rat, Y1 of medaka, FOR1-3 of pufferfish, 47E of carp, and ODR-10 of nematode were subjected to the modifications, and the RNAs encoding modified ORs were injected into Xenopus oocytes. The membrane fraction of the oocytes were analyszed by Western blotting to examine the expression of the proteins. In the cases of ORs modified with 5HT and V, only ODR-10 and 47E, both of which have more than two Asn-linked glycosylation sites in their extracellular regions, were detected as the bands of predicted molecular weights. On the other hand, most of the ORs modified with VL showed the bands of predicted molecular weights. These results suggest that SL sequences together with potential Asn-linked glycosylation sites have positive effects on the expression of ORs in heterologous systems.
Keywords: olfactory receptor; signal leader sequence; heterologous expression; Xenopus oocyte
-20-
CYP78A1 Preferentially Expressed in Developing Inflorescences of Zea mays
Encoded a Cytochrome P450-Dependent Lauric Acid 12-Monooxygenase
Hiromasa IMAISHI,1 Satoshi MATSUO,2 Eri SWAI,2 and Hideo OHKAWA2,・・・
・1Center for Cooperative Research and Development, and 2Department of Biological and Environmental Science,・
Faculty of Agriculture, Kobe University, Rokkodaid-cho 1-1, Nada-ku, Kobe 657-8501, Japan
Received February 17, 2000; Accepted April 11, 2000
Cytochrome P450 (P450 or CYP) monooxygenases play an important role in the oxidation of a number of lipophilic substrates including secondary metabolites in higher plants. Larkin reported that CYP78A1 was preferentially expressed in developing inflorescences of Zea mays (Larkin, Plant Mol. Biol. 25: 343--353, 1994). However, the enzymatic function of CYP78A1 hasn′t been clarified yet. To characterized the enzymatic activity of CYP78A1, in this study, CYP78A1 cDNA and tobacco or yeast NADPH-cytochrome P450 oxidoreductase (P450 reductase) was expressed in the yeast Saccharomyces cerevisiae AH22 cells under the control of alcohol dehydrogenase promoter I and terminator. The reduced CO-difference spectrum of a microsomal fraction prepared from the transformed yeast cells expressing CYP78A1 and yeast P450 reductase showed a peak at 449 nm. Based on the spectrum, the content of a P450 molecule was estimated to be 45 pmol P450 equivalent/mg of protein in the microsomal fraction. The recombinant yeast microsomes containing CYP78A1 and yeast P450 reductase were found to catalyze 12-monooxygenation of lauric acid. Based on these results, CYP78A1 preferentially expressed in developing inflorescences of Zea mays appeared to have participated in the monooxygenation of fatty acids.
Keywords: cytochrome P450; Zea mays; lauric acid; Saccharomyces cerevisiae
-21-
Hydrolysis of β-Galactosyl Ester Linkage by β-Galactosidases
Taro KISO,1 Hirofumi NAKANO,1 Hirofumi NAKAJIMA,2 Tadamasa TERAI,2
Katsuyuki OKAMOTO,3 and Sumio KITAHATA1
1Osaka Municipal Technical Research Institute, 1-6-50 Morinomiya, Joto-ku, Osaka 536-8553, Japan
2Osaka Institute of Technology, 5-16-1, Ohmiya, Asahi-ku, Osaka 535-8585, Japan
3Showa sangyo Co., Ltd., 1-16, Sakura, Tsukubashi, Ibaraki 350-0003, Japan
Received February 17, 2000; Accepted March 28, 2000
p-Hydroxybenzoyl β-galactose (pHB-Gal) was synthesized chemically to examine the hydrolytic activity of β-galactosyl ester linkage by β-galactosidases. The enzyme from Penicillium multicolor hydrolyzed the substrate as fast as p-nitrophenyl β-galactoside (pNP-Gal), a usual substrate with a β-galactosidic linkage. The enzymes from Escherichia coli and Aspergillus oryzae hydrolyzed pHB-Gal with almost the same rates as pNP-Gal. The enzymes from Bacillus circulans, Saccharomyces fragilis, and bovine liver showed much lower activities. pH-activity profiles, inhibition analysis, and kinetic properties of the enzymic reaction on pHB-Gal suggested that β-galactosidase had only one active site for hydrolysis of both galactosyl ester and galactoside. The Penicillium enzyme hydrolyzed pHB-Gal in the presence of H218O to liberate galactose containing 18O. This result suggests the degradation occurs between the anomeric carbon and an adjacent O atom in the ester linkage of pHB-Gal.
Keywords: β-galactosidase; galactosyl ester linkage; active site
-22-
Triterpene Phytoalexins from Strawberry Fruit
Nobuhiro HIRAI,1,2,・・・ Megumi SUGIE,1 Mitsuo WADA,1 El Hassan LAHLOU,2
Tsunashi KAMO,1,2 Ryuji YOSHIDA,3 Mitsuya TSUDA,4 and Hajime OHIGASHI1
1Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
2CREST, Japan Science and Technology Corporation, Hon-cho 4-1-8, Kawaguchi 332-0012, Japan
3College of Agricultural Technology, Toyama Prefectural University, Toyama 939-0311, Japan
4Division of Environmental Science and Technology, Graduate School of Agriculture,
Kyoto University, Kyoto 606-8502, Japan
Received February 18, 2000; Accepted March 18, 2000
Strawberry cv. Houkouwase is resistant to infection by Colletotrichum fragariae. The formation of antifungal compounds was observed in unripe fruit which had been wounded and inoculated with conidia of C. musae. Three antifungal compounds were isolated and identified as euscaphic acid, tormentic acid and myrianthic acid. Myrianthic acid inhibited the growth of C. musae at 3 μg, and euscaphic and tormentic acids showed inhibitory effects at 100 μg. A quantitative analysis of their contents showed that the triterpenes increased in wounded fruit, and in wounded and inoculated fruit, but not in non-treated fruit. These findings indicate that unripe fruit of Houkouwase produced the triterpenes as phytoalexins. The triterpene phytoalexins seem to be involved in the resistance of strawberry to the fungus.
Keywords: antifungal compound; Fragaria ananassa; phytoalexin; strawberry; triterpene
-23-
Note
New Syntheses of the Rice Moth and Stink Bug Pheromones by Employing
(2R, 6S)-7-Acetoxy-2,6-dimethyl-1-heptanol as a Building Block・・・
Yoshihide NAKAMURA・・・・・・ and Kenji MORI・・・・・・・・・
Department of Chemistry, Faculty of Science, Science University of Tokyo, Kagurazaka 1-3,
Shinjuku-ku, Tokyo 162-8601, Japan
Received February 21, 2000; Accepted April 4, 2000
(2R, 6R, 10R) - 6, 10, 14 - Trimethyl - 2 - pentadecanol, the female pheromone of the rice moth (Corcyra cephalonica), and methyl (2R, 6R, 10R)-2,6,10-trimethyltridecanoate, the male pheromone of the stink bug (Euschistus heros) were synthesized by employing (2R, 6S)-7-acetoxy-2,6-dimethyl-1-heptanol as the common chiral building block.
Keywords: Corcyra cephalonica; Euschistus heros; pheromone; rice moth; stink bug
-24-
Note
Purification and Characterization of Subtilisin DJ-4 Secreted
by Bacillus sp. Strain DJ-4 Screened from Doen-Jang
Seung-Ho KIM＊ and Nack-Shick CHOI
Protein Engineering Group, Korea Research Institute of Bioscience and Biotechnology,
Yusong, Taejon 305-600, Korea
Received September 17, 1999; Accepted April 9, 2000
Bacillus sp. strain DJ-4, which produces extracellular proteases, was screened from Doen-Jang, a traditional Korean fermented food. A fibrinolytic enzyme (subtilisin DJ-4) was purified using commercial chromatographic techniques. The relative molecular mass of the isolated protein was 29 kDa by SDS-PAGE and fibrin zymography assay. The enzyme was characterized as a serine protease by an inhibitor assay on the fibrin zymography gel and by an amidolytic assay using a chromogenic substrate. The enzyme was inhibited by PMSF, but not by EDTA or leupeptin. The first 14 amino acids of the N-terminal sequence were identical to that of subtilisin BPN?, but the activity of subtilisin DJ-4 was 2.2 and 4.3 times higher than those of subtilisin BPN? and subtilisin Carlsberg, respectively.
Keywords: Bacillus sp. DJ-4; subtilisin DJ-4; fibrin zymography; fermented food
-25-
Note
Synthesis of (3E, 5Z)-3,5-Dodecadienylacetate, the Sex Pheromone
of Phtheochroa cranaodes (Lepidoptera: Tortricidae)
Jhillu Singh YADAV,＊ and Etukala Jagan REDDY
Pheromone Laboratory, Organic Division-I, Indian Institute of Chemical Technology,
Hyderabad 500 007, India
Received September 29, 1999; Accepted March 21, 2000
(3E, 5Z)-3,5-Dodecadienyl acetate, the female sex pheromone of Phtheochroa cranaodes, was regio and stereo-selectively synthesized from 1-octyne and (E)-4-bromo-3-buten-1-ol by using Pd(PPh3)4, CuI and piperidine to afford the enyne (5). Further elaboration afforded the target pheromone. The synthetic pheromone was identified with the natural product by its MS and IR, data GLC retention time and biological activity.
Keywords: sex pheromone; Phtheochroa cranaode; 1-octyne; (E)-4-bromo-3-buten-1-ol, (E,Z)- dodecadienyl acetate; stereo-selective reaction
-26-
Note
Purification and Characterization of an Endo-Polygalacturonase
from Aspergillus awamori
Masaru NAGAI, Tohoru KATSURAGI,＊ Takao TERASHITA,
Kentaro YOSHIKAWA, and Takuo SAKAI・・・
Department of Food Science and Nutrition, Faculty of Agriculture, Kinki University, 3327-204,
Nakamachi, Nara 631-8505, Japan
＊Graduate School of Biological Sciences, Nara Institute of Science and Technology,
Takayama Science Town, Ikoma, Nara 630-0101, Japan
Received December 1, 1999; Accepted April 12, 2000
An extracellular endo-polygalacturonase (PGase) produced by Aspergillus awamori IFO 4033 was isolated from the culture filtrate. The enzyme was purified to a homogeneous preparation with cation-exchange and size-exclusion chromatographies. Its properties were investigated, comparing them with that of recombinant pgx2 gene product, a PGase having protopectinase activity. This enzyme was a monomeric protein of 41 kDa, with an isoelectric point of pH 6.1. The characteristics of this PGase substantially coincide, with that of recombinant pgx2 gene product, and the PGase is assumed to be native pgx2 gene product. The production of PGase-X2 was confirmed to be regulated by ambient pHs.
Keywords: polygalacturonase; Aspergillus awamori; pH regulation
-27-
Note
Molecular Structure of rDNA Repeat Unit in Magnaporthe grisea
Teruo SONE, Satoru FUKIYA, Megumi KODAMA, and Fusao TOMITA＊
Laboratory of Applied Microbiology, Graduate School of Agriculture, Hokkaido University,
Kita-9, Nishi-9, Kita-ku, Sapporo 060-8589, Japan
Received December 6, 1999; Accepted March 29, 2000
The 8-kb repeat unit of M. grisea rRNA-encoding DNA (rDNA) was cloned as three subclones, pM50, pM21, and pM86. Nucleotide sequencing of these subclones uncovered the structure of an rDNA repeat unit similar to those of other ascomycetes. The intergenic spacer (IGS) of the rDNA cistron contained a repetitive (R) region, which was rich in two kinds of short tandemly repeated elements.
Keywords: Pyricularia; rice blast; rDNA; intergenic spacer; tandem repeat
-28-
Note
Phylogenetic Analysis of Methanogens in Sheep Rumen Ecosystem
and Detection of Methanomicrobium mobile
by Fluorescence In Situ Hybridization
Kazuhiro YANAGITA,1,2 Yoichi KAMAGATA,1＊ Mamoru KAWAHARASAKI,1
Toshihiko SUZUKI,1 Yutaka NAKAMURA,2 and Hajime MINATO2
1National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, Tsukuba, Ibaraki 305-8566, Japan,
2Department of Animal Sciences, Ibaraki University, Ami, Ibaraki 300-0331, Japan
Received December 13, 1999; Accepted March 29, 2000
The population of methanogens in the sheep rumen microbial ecosystem was studied by using 16S rDNA cloning analysis, epifluorescence microscopy (which detects autofluorescence of a specific cofactor F_{420} in methanogens) and the 16S rRNA-targeted in situ hybridization technique. The 16S rDNA clone libraries were constructed by PCR amplification with an Archaea-specific primer set and partial sequencing of the clonal 16S rDNAs was done. Phylogenetic analysis indicated that the clones were affiliated with Methanomicrobium mobile, Methanobrevibacter ruminantium and Methanobrevibacter smithii. Epifluorescence microscopy (F_{420} autofluorescence) and in situ hybridization by using a newly designed M. mobile-specific 16S rRNA-targeted oligonucleotide probe found that methanogens accounted for approximately 3.6％ of total ruminal microorganisms and approximately 54％ of the total methanogens were M. mobile.
Keywords: rumen; methanogens; Methanomicrobium; Methanobrevibacter; in situ hybridization
-29-
Note
Efficient Synthesis of a Sialyl T-antigen-linked Glycopeptide
by the Chemoenzymatic Method
Katsumi AJISAKA＊ and Mariko MIYASATO
Meiji Institute of Health Science, Meiji Milk Products Co. Ltd., 540 Naruda, Odawara 250-0862, Japan
Received Jannuary 17, 2000; Accepted March 8, 2000
A sialyl T-antigen-linked tetrapeptide was prepared by the combined method of chemical synthesis and enzymatic synthesis. The GalNAc-linked peptide was first obtained by using a commercial peptide synthesizer, and then a galactose residue was attached with β-(1→3)-linkage by transglycosylating with a recombinant β-galactosidase from Bacillus circulans. The sialic acid residue was then combined by α-(2→3)-linkage with sialytransferase from rat liver.
Keywords: peptide synthesis; glycopeptide; mucin-type; β-galactosidase; sialyltransferase
-30-
Note
Molecular Cloning of Genomic DNA for Fructose-1,6-bisphosphatase
from Aspergillus oryzae
Motoaki SANO, Keiichi NAKAJIMA,＊ Kumiko TAKASE, Midori YAMAMOTO,
and Masayuki MACHIDA
Department of Molecular Biology, National Institute of Bioscience and Humam-Techology, Higashi 1-1, Ibaraki 305-8566, Japan
＊United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology,
Fuchu, Tokyo 183-8509, Japan
Received February 3, 2000; Accepted April 15, 2000
We have cloned and sequenced an Aspergillus oryzae genomic DNA fragment that encodes a fructose-1,6-bisphosphatase gene (fbpA) with the aim of studying transcriptional regulation mechanisms involved in basic metabolism. Expression of fbpA was repressed in the presence of glucose, but not in the presence of pyruvate or sodium acetate in the medium. The CreA and FacB element found in the fbpA 5?-flanking region may be important in fbpA regulation.
Keywords: fructose-1,6-bisphosphatase; Aspergillus oryzae; genomic DNA; transcriptional regulation
-31-
Note
Oxidation and Reduction of Nitrite Ion in the TiO2 Photo-induced
Catalytic Reaction
Hitoshi SHIBATA,＊・・・ Namiko NODA,＊ Yasushi OGURA,＊ Kunihisa SOGABE,＊＊
and Yoshihiro SAWA＊
＊Department of Life Science and Biotechnology, Faculty of Life and Environmental Science,
Shimane University, Matsue, Shimane 690-8504, Japan
＊＊Faculty of Education, Shimane University, Matsue, Shimane 690-8504, Japan
Received February 7, 2000; Accepted April 4, 2000
During a photo-induced catalytic reaction under near UV irradiation to an aqueous suspension of TiO_{2}, about 95％ of NO_{2}＾{--} was oxidized to NO_{3}＾{--}, but NH_{4}＾{+} was not detected. The oxidation was inhibited by the addition of mannitol or under anaerobic conditions. The nitration of HPA was observed in the presence of t-buthanol, suggesting the formation of ONOO＾{--}. An ESR spectrum gave a triplet signal at g＝2.041, in the presence of NO_{2}＾{--}, mannitol, FeSO_{4}, and MGD, indicating the reduction of NO_{2}＾{--} to NO.
Keywords: nitric oxide; nitrite; peroxynitrite; photo-induced catalysis; titanium dioxide
-32-
Note
Mutational Analysis of the Multicopy hao Gene Coding for Hydroxylamine
Oxidoreductase in Nitrosomonas sp. Strain ENI-11
Akira YAMAGATA, Ryuichi HIROTA, Junichi KATO, Akio KURODA,
Tsukasa IKEDA, Noboru TAKIGUCHI, and Hisao OHTAKE・・・
Department of Fermentation Technology, Hiroshima University, Higashi-Hiroshima,
Hiroshima 739-8527, Japan
Received February 7, 2000; Accepted April 7, 2000
The ammonia-oxidizing bacterium Nitrosomonas sp. strain ENI-11 contains three copies of the hao gene (hao1, hao2, and hao3) coding for hydroxylamine oxidoreductase (HAO). Three single mutants (hao1::kan, hao2::kan, or hao3::kan) had 68 to 75％ of the wild-type growth rate and 58 to 89％ of the wild-type HAO activity when grown under the same conditions. A double mutant (hao1::kan and hao3::amp) also had 68％ of the wild-type growth and 37％ of the wild-type HAO activity.
Keywords: ammonia-oxidizing bacterium; gene disruption; hydroxylamine oxidoreductase; multicopy gene; Nitrosomonas
-33-
Note
Synthesis of the Spirosuccinimide Moiety of Asperparaline A
Shinji TANIMORI,・・・ Kouji FUKUBAYASHI, and Mitsunori KIRIHATA
Department of Applied Biochemistry, College of Agriculture, Osaka Prefecture University,
1-1 Gakuen-cho, Sakai, Osaka 599-8531, Japan
Received February 16, 2000; Accepted March 21, 2000
2,6,6-Trimethyl-2-azaspiro[4.4]nonane-1,3-dione (9), a spirosuccinimide moiety of asperparaline A (1), was synthesized by starting from 2,2-dimethylcyclopentanone (4) via trinitrile 6 in five steps in a moderate yield. This conversion establishes a model study for synthesis of the spirosuccinimide moiety of asperparaline A (1).
Keywords: asperparaline; indolizine alkaloid; spirosuccinimide
-34-
Note
Absolute Configuration at C45 in 45-Hydroxyyessotoxin,
a Marine Polyether Toxin Isolated from Shellfish
Akio MOROHASHI, Masayuki SATAKE, Yasukatsu OSHIMA, and Takeshi YASUMOTO・・・
Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiya,
Aoba-ku, Sendai 981-8555, Japan
Received February 22, 2000; Accepted March 24, 2000
The marine polyether toxin, 45-hydroxyyessotoxin, was isolated together with yessotoxin from the scallop, Patinopecten yessoensis. The 45-hydroxy group in the side chain was esterified with (S)- and (R)-α-methoxy-α-trifluoromethylphenylacetic acids (MTPA). A detailed analysis of the 2D NMR spectra of the two esters established the R configuration at C45.
Keywords: 45-hydroxyyessotoxin; modified Mosher method; MTPA ester; shellfish metabolite
-35-
Note
Native and Acid-denatured L-Lactate Dehydrogenase Are Different
in the Lysosomal Proteinases Involving in Their Overall Degradation
Takeyuki OHSHITA
School of Food and Nutrition, Shokei Junior College, Kuhonji-2, Kumamoto 862-0976, Japan
Received February 24, 2000; Accepted April 4, 2000
When native and acid-denatured lactate dehydrogenase (LDH) were incubated with total lysosomal enzymes in vitro, amino acids from their degradation were produced at various acidic pH. The pH profile in the overall degradation of native LDH was markedly different from that of acid-denatured LDH. Disappearance of the 35-kDa subunit of native LDH was markedly suppressed by a low level of cystatin α as well as by a general cysteine proteinase inhibitor, N-(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucine-3-methylbutylamide (E-64-c). On the other hand, the degradation of acid-denatured LDH was only slightly suppressed by these inhibitors. It was concluded that at least a part of the proteinases involved in the overall degradation of native LDH is different from the proteinases involved in the degradation of acid-denatured form and a role of a cystatin α-sensitive cysteine proteinase is critical in the lysosomal degradation of native LDH, but not in that of acid-denatured form.
Keywords: lysosomes; cysteine proteinases; cathepsins; cystatin; L-lactate dehydrogenase
-36-
Note
Concanavalin A-Induced Discharge of Glycocalyx of Raphidophycean
Flagellates, Chattonella marina and Heterosigma akashiwo
Tarou OKAMOTO,1 Daekyung KIM,1 Tatsuya ODA,1,・・・ Kazumi MATSUOKA,2
Atsushi ISHIMATSU,3 and Tsuyoshi MURAMATSU1
1Division of Biochemistry, Faculty of Fisheries, Nagasaki University, Bunkyo-machi, Nagasaki 852-8521, Japan
2Laboratory of Coastal Environmental Sciences, Faculty of Fisheries, Nagasaki University, Bunkyo-machi,
Nagasaki 852-8521, Japan
3Marine Research Institute, Nagasaki University, Taira-cho, Nagasaki 851-2213, Japan
Received March 8, 2000; Accepted April 10, 2000
Chattonella marina and Heterosigma akashiwo, known as red tide phytoplankton, are naturally wall-less and have quite fragile cell structures. In this study, we found that an equilibrium dialysis technique allowed the study of lectin binding to these flagellates. The results suggested that concanavalin A (Con A) binds to these flagellate cells through the specific carbohydrate moieties on the cell surface. Interestingly, the binding of an excess of Con A on the cell surface caused morphological changes concomitant with discharge of glycocalyx, a polysaccharide-containing common structure on the external cell surface of these flagellates. Fluorescent microscopic observation using FITC-labeled Con A (F-Con A) confirmed that F-Con A molecules are localized on the discharged glycocalyx.
Keywords: red tide; concanavalin A; glycocalyx; phytoplankton
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Preliminary Communication
mRNA Expression of MT1-MMP, MMP-9, Cathepsin K, and TRAP
in Highly Enriched Osteoclasts Cultured on Several Matrix Proteins
and Ivory Surfaces
Toshimasa UEMURA,1,2 Yin-kun LIU,3 and Yoshinori KUBOKI4
1National Institute for Advanced Interdisciplinary Research (NAIR), Tsukuba Research Center,
Tsukuba, Ibaraki 305-8562, Japan
2CREST, JST (Japan Science and Technology)
3Liver Cancer Institute, Shanghai Medical University, Shanghai 200032, P.R. China
4Department of Biochemistry, School of Dentistry, Hokkaido University, Nishi 7, Kita 13, Kitaku,
Sapporo, Hokkaido 060-8586, Japan
Received February 8, 2000; Accepted May 1, 2000
We demonstrated that the transcriptional expression of MT1-MMP, MMP-9, cathepsin K, and TRAP in highly enriched osteoclasts were regulated by different matrix proteins that bind to integrin on osteoclast, such as collagen type I (CoI), fibronectin (FN), vitronectin (VN), osteopontin (OPN), and ivory. Results suggested that the OPN-integrin αVβ3 binding plays a more important role than CoI-α2β1 binding in the regulation of osteoclast activity.
Keywords: osteoclast; osteopontin; matrix protein