(Vol.64 No.10 2000)
Expression of Human Cytochromes P450 1A1 and P450 1A2 as Fused
Enzymes
with Yeast NADPH-cytochrome P450 Oxidoreductase
in Transgenic Tobacco Plants
Noriaki SHIOTA,・・・ Susumu KODAMA, Hideyuki INUI, and Hideo OHKAWA・・・ p.2025
Contribution of Cyanide-Insensitive Respiratory Pathway, Catalyzed
by the Alternative Oxidase, to Citric Acid Production in Aspergillus niger
Kohtaro KIRIMURA,・・・ Masashi YODA, Hideki SHIMIZU, Shuji SUGANO,
Makoto MIZUNO, Kuniki KINO, and Shoji USAMI p.2034
Regulation by Organic Acids of Polysaccharide-mediated
Microbe-plant Interactions
Kuniho NAKATA,・・・,＊ Takayoshi KOBAYASHI,＊＊ Yasuyuki TAKIGUCHI,＊＊
and Tatsuaki YAMAGUCHI＊＊ p.2040
Inhibition of Tumor Cell Growth by A Specific 6-Phosphofructo-2-kinase
Inhibitor, N-Bromoacetylethanolamine Phosphate, and Its Analogues
Takafumi HIRATA,＊ Mitsuaki WATANABE,・・・ Shinji MIURA,・・・ Katsushi IJICHI,・・・
Masashi FUKASAWA,・ and Ryuzo SAKAKIBARA§,1 p.2047
Effect of Cobalamin on the Allergic Response in Mice
Ulala FUNADA,＊ Masahiro WADA,＊＊,・・・ Tetsunori KAWATA,＊＊＊ Nobuo TANAKA,＊＊＊＊
Tadahiro TADOKORO,＊ and Akio MAEKAWA＊ p.2053
Isolation and Some Properties of a Mesophilic and Mixotrophic
Iron-oxidizing Bacterium, OKM-9
Takao INOUE,1 Kazuo KAMIMURA,2 and Tsuyoshi SUGIO1・・・ p.2059
Enzymatic Properties of Phenylalanine101 Mutant Enzyme
of Ribonuclease Rh from Rhizopus niveus
Kazuko OHGI,・・・ Sanae KUDO, Mitsuaki TAKEUCHI, Masanori IWAMA, and Masachika
IRIE
p.2068
Early Biosynthetic Pathway to Abscisic Acid in Cercospora cruenta
Hirotaka YAMAMOTO,・・・ Masahiro INOMATA, Shinobu TSUCHIYA, Makoto NAKAMURA,
Takeo UCHIYAMA, and Takayuki ORITANI＊ p.2075
Purification and Identification of a Growth-Stimulating Peptide
for Bifidobacterium bifidum from Natural Rubber Serum Powder
Shin-ichi ETOH, Kayoko ASAMURA, Azumi OBU, Kenji SONOMOTO, and Ayaaki ISHIZAKI＊
p.2083
Changes in the Content and Biosynthesis of Phytoalexins in
Banana Fruit
Tsunashi KAMO,a,b Nobuhiro HIRAI,a,b,・・・ Mitsuya TSUDA,c Daie FUJIOKA,d
and Hajime OHIGASHIa p.2089
Stereocontrolled Reduction of α- and β-Keto Esters
with Micro Green Algae, Chlorella Strains
Kohji ISHIHARA,1,・・・ Hitomi YAMAGUCHI,2 Noriko ADACHI,1
Hiroki HAMADA,3 and Nobuyoshi NAKAJIMA4 p.2099
Substrate Specificity of Cucumisin on Synthetic Peptides
Hiroo YONEZAWA,・・・ Hideki KAIZUKA, Tetsuya UCHIKOBA, Kazunari ARIMA,＊
and Makoto KANEDA p.2104
Structural Features of N-Glycans Linked to Royal Jelly Glycoproteins:
Structures of high-mannose type, hybrid type, and biantennary type glycans
Yoshinobu KIMURA,1,＊＊ Chiyoko MIYAGI,1 Mariko KIMURA,1 Teruhiko NITODA,1
Nobuyuki KAWAI,2 and Hiroyuki SUGIMOTO2 p.2109
Role of the Non-essential Region Encompassing the N-Terminal
Two Transmembrane Stretches of Escherichia coli SecE
Ken-ichi NISHIYAMA, Hirofumi SUZUKI, and Hajime TOKUDA・・・ p.2121
Characterization of Human Recombinant Transglutaminase 1 Purified
from Baculovirus-infected Insect Cells
Kiyotaka HITOMI,・・・ Yoshihisa YAMAGIWA, Koji IKURA,＊ Kiyofumi YAMANISHI,＊＊
and Masatoshi MAKI p.2128
Production of Humanized Fab Fragment against Human High Affinity
IgE Receptor in Pichia pastoris
Kyoko TAKAHASHI,1 Toshifumi YUUKI,1 Toshiro TAKAI,1 Chisei RA,2,3
Ko OKUMURA,2 Toyokazu YOKOTA,1 and Yasushi OKUMURA1 p.2138
Regulation of Sex Pheromone Biosynthesis in Three Plusiinae
Moths:
Macdunnoughia confusa, Anadevidia peponis, and Chrysodeixis eriosoma
Mitsuaki KOMODA, Shin-ichi INOMATA, Akihiko ONO, Hayaki WATANABE,
and Tetsu ANDO・・・p.2145
Genetic Analysis of Growth Inhibition of Yeast Cells Caused
by Expression
of Aspergillus oryzae RNase T1
Gen NONAKA, Tomoki ISHIKAWA, Tin-tin LIU, Harushi NAKAJIMA,
and Katsuhiko KITAMOTO・・・ p.2152
Dietary Conjugated Linoleic Acid Increases Immunoglobulin Productivity
of Sprague-Dawley Rat Spleen Lymphocytes
Masao YAMASAKI, Koji KISHIHARA, Keiko MANSHO, Yuuya OGINO, Masaaki KASAI,＊
Michihiro SUGANO,＊＊ Hirofumi TACHIBANA, and Koji YAMADA p.2159
Improved Hypolipidemic Effects of Xanthan Gum-Galactomannan
Mixtures in Rats
Yukiko YAMAMOTO,・・・ Ikuyo SOGAWA, Akiko NISHINA, Shigeru SAEKI,
Naoki ICHIKAWA, and Shiho IIBATA p.2165
Solution Properties of Antitumor Sulfated Derivative of α-(1→3)-D-Glucan
from Ganoderma lucidum
Lina ZHANG,・・・ Mei ZHANG, Qi ZHOU, Jinghua CHEN, and Fanbo ZENG＊ p.2172
Purification and Characterization of an α-L-Rhamnosidase
from Pichia angusta X349
Takaaki YANAI and Michikatsu SATO・・・ p.2179
Purification and Characterization of a Basic Amino Acid-Specific
Peptidase
from Seeds of Jack Bean (Canavalia ensiformis)
Kiyotaka OSHIKAWA, Ken-ichi AOKI, Yuuki YOSHINO, and Shigeyuki TERADA・・・ p.2186
Some Molecular and Inhibitory Specifications of a Dipeptidyl
Carboxypeptidase from the Polychaete Neanthes virens
Resembling Angiotensin I Converting Enzyme
Toshiya KAWAMURA,・・・ Kiyofumi KIKUNO,＊ Tatsuya ODA,＊ and Tsuyoshi MURAMATSU＊
p.2193
Primary Amino Acid and DNA Sequences of Gassericin T, a Lactacin
F-Family
Bacteriocin Produced by Lactobacillus gasseri SBT2055
Yasushi KAWAI,・・・ Boku SAITOH, Osamu TAKAHASHI, Haruki KITAZAWA,
Tadao SAITO, Hadjime NAKAJIMA,＊ and Takatoshi ITOH p.2201
Improved Stereoselective Synthesis of Optically Active Methylene
Lactone,
Key Intermediate for the Synthesis of 1,2-Oxidized Furofuran Lignan,
by Direct α-Methylenation to Butanolide
Satoshi YAMAUCHI,・・・ Nobuyuki YAMAMOTO, and Yoshiro KINOSHITA p.2209
Note
An Antibiotic from Penicillium sp. Covering the Cocoon
of the Leaf-rolling Moth, Dactylioglypha tonica
Nobutaka IMAMURA,・・・ Takahiro ISHIKAWA, Tomoko OHTSUKA, Kazumi YAMAMOTO,
Masato DEKURA, Hiroshi FUKAMI, and Ritsuo NISHIDA＊ p.2216
Note
Antioxidative Activity of (--)-Epigallocatechin-3-(3!-O-methyl)gallate Isolated
from Fresh Tea Leaf and Preliminary Results on Its Biological Activity
Masaya KAWASE,＊,＃ Rong WANG,＊＊ Teruaki SHIOMI,＊＊＊ Ryoyasu SAIJO,・・・,＊＊
and Kiyohito YAGI＊＊＊ p.2218
Note
A cDNA Encoding Ribosomal Protein RPL15 from the Desiccation-tolerant
Bryophyte Tortula ruralis: mRNA Transcripts are Stably Maintained
in Desiccated and Rehydrated Gametophytes
Qin ZENG and Andrew J. WOOD・・・ p.2221
Note
Induction of Osteopontin Gene Expression during Mammary Gland Involution
and Effects of Glucocorticoid on its Expression in Mammary Epithelial Cells
Mijoung LEE,＊ Sanghyun NA,＊ Deahyun JEON,＊ Hyungha KIM,＊ Yunjaie CHOI,＊＊
and Myunggi BAIK・・・,＊ p.2225
Note
Synthesis and Lateral Root-Inducing Activity of Novel
N-Substituted-2-Piperidones with a 1,4-Benzodioxan Ring
Hidetaka TSUKADA, Naotaka YAMADA, Eiji TANIGUCHI, and Eiichi KUWANO・・・ p.2229
Note
Sites of Expression of DnaJ Homologs and Hsp70 in Male and Female Flowers
of the Japanese Willow Salix gilgiana Seemen
Norihiro FUTAMURA,1 Hiroshi KOUCHI,2 and Kenji SHINOHARA1 p.2232
Note
Effects of pH and Light on the Storage Stability of the Purple Pigment,
Hordeumin, from Uncooked Barley Bran Fermented Broth
Tomoaki DEGUCHI, Shuji SHOHARA, Riichiro OHBA, and Seinosuke UEDA p.2236
Note
Differential Response of Obese Gene Expression from Fasting
in Bovine Adipose Tissues
Hyungha KIM,＊ Yountae CHI,＊ Kichul CHUNG,＊ Kwanghyun KIM,＊＊
Yunjaie CHOI,・・・ and Myunggi BAIK＊,・・・ p.2240
Note
Molecular Cloning and Expression of the Nucleoside Deoxyribosyltransferase-II
Gene from Lactobacillus helveticus
Kiyoshi OKUYAMA and Toshitada NOGUCHI p.2243
Note
Naringin Glycosides α-Glucosylated on Ring B Found in the Natural Food
Additive, Enzymatically Modified Naringin
Takumi AKIYAMA, Mitsuo YAMADA, Takashi YAMADA, and Tamio MAITANI p.2246
Note
Decrease in Rice Allergenic Proteins of Polished Rice Grains by Incubating
with a Miso Solution
Hidehiko IZUMI,1 Shiho KONDO,1 Hajime KASHIO,1 Tsukasa MATSUDA,2,・・・ and Ryo
NAKAMURA3 p.2250
Note
Cloning and Expression of the ccpA Gene Encoding Catabolite Control Protein
from Thermoactinomyces sp. E79＊
Min-Woo MOON, Sun-Yang PARK, Hyung-Kwoun KIM, Do-Young YUM,§
Hahn-Shik KWAK,1 Tae-Kwang OH, and Jung-Kee LEE・・・ p.2254
Note
Phosphorylation of Guanosine Using Guanosine-inosine Kinase
from Exiguobacterium acetylicum Coupled with ATP Regeneration
Hisashi KAWASAKI, Yoshihiro USUDA, Megumi SHIMAOKA, and Takashi UTAGAWA p.2259
Note
Glycosidic Flavonoids as Rat-Liver Injury Preventing Compounds
from Green Tea
Shingo WADA, Puming HE, Ikue HASHIMOTO, Naoharu WATANABE,・・・
and Kimio SUGIYAMA p.2262
Note
Trypsin Inhibitory Activity of Bovine Fetuin De-O-glycosylated
by Endo-α-N-acetylgalactosaminidase
Hisashi ASHIDA,・・・ Kenji YAMAMOTO, and Hidehiko KUMAGAI p.2266
Note
cis-N-(p-Coumaroyl)serotonin from Konnyaku,
Amorphophallus konjac K. Koch
Tatsuya NIWA, Hideo ETOH,＊,・・・ Atsushi SHIMIZU,＊＊ and Yasuo SHIMIZU＊＊
p.2269
Note
Complete Amino Acid Sequences of Three Proteinase Inhibitors
from White Sword Bean (Canavalia gladiata)
Sung-Soo PARK, Toshihisa SUMI, Hideki OHBA,・・・ Osamu NAKAMURA,
and Makoto KIMURA＊ p.2272
Preliminary Communication
New Scheme of the Biosynthesis of Formononetin Involving
2,7,4'-Trihydroxyisoflavanone but Not Daidzein as the Methyl Acceptor
Tomoyoshi AKASHI, Yuji SAWADA, Toshio AOKI, and Shin-ichi AYABE・・・ p.2276
-1-
Expression of Human Cytochromes P450 1A1 and P450 1A2 as Fused Enzymes
with Yeast NADPH-cytochrome P450 Oxidoreductase
in Transgenic Tobacco Plants
Noriaki SHIOTA,・・・ Susumu KODAMA, Hideyuki INUI, and Hideo OHKAWA・・・
Department of Biological and Environmental Science, Faculty of Agriculture, Kobe University,
Rokkodai-cho 1-1, Nada, Kobe 657-8501, Japan
Received August 16, 1999; Accepted June 15, 2000
Among 11 isoforms of the human cytochrome P450 enzymes metabolizing xenobiotics, CYP 1A1 and CYP 1A2 were major P450 species in the metabolism of the herbicides chlortoluron and atrazine in a yeast expression system. CYP1A2 was more active in the metabolism of both herbicides than CYP1A1. The fused enzymes of CYP1A1 and CYP1A2 with yeast NADPH-cytochrome P450 oxidoreductase were functionally active in the microsomal fraction of the yeast Saccharomyces cerevisiae and showed increased specific activity towards 7-ethoxyresorufin as compared to CYP1A1 and CYP1A2 alone. Then, both fused enzymes were each expressed in the microsomes of tobacco (Nicotiana tabacum cv. Samsun NN) plants. The transgenic plants expressing the CYP1A2 fusion enzyme had higher resistance to the herbicide chlortoluron than the plants expressing the CYP1A1 fusion enzyme did. The transgenic plants expressing the CYP1A2 fused enzyme metabolized chlortoluron to a larger extent to its non-phytotoxic metabolites through N-demethylation and ring-methyl hydroxylation as compared to the plants expressing the CYP1A1 fused enzyme. Thus, the possibility of increasing the herbicide resistance in the transgenic plants by the selection of P450 species and the fusion with P450 reductase is discussed.
Key words: human cytochrome P450; P450 oxidoreductase; transgenic tobacco plants; herbicide resistance
-2-
Contribution of Cyanide-Insensitive Respiratory Pathway, Catalyzed
by the Alternative Oxidase, to Citric Acid Production in Aspergillus niger
Kohtaro KIRIMURA,・・・ Masashi YODA, Hideki SHIMIZU, Shuji SUGANO,
Makoto MIZUNO, Kuniki KINO, and Shoji USAMI
Department of Applied Chemistry, School of Science and Engineering, Waseda University,
Ohkubo 3-4-1, Shinjuku-ku, Tokyo 169-8555, Japan
Received February 9, 2000; Accepted June 8, 2000
In Aspergillus niger, a cyanide (CN)- and antimycin A-insensitive and salicylhydroxamic acid (SHAM)-sensitive respiratory pathway exists besides the cytochrome pathway and is catalyzed by the alternative oxidase (AOX). In this study, A. niger WU-2223L, a citric acid-producing strain, was cultivated in a medium containing 120 g/l of glucose, which is the concentration usually needed for citric acid production, and the effects of 2％ (v/v) methanol, an inducer of citric acid, 2 μM antimycin A, and 1 mM SHAM on AOX activities and citric acid production were investigated. The AOX activity, measured as duroquinol oxidase, was localized in the purified mitochondria regardless of the presence of any additives. When WU-2223L was cultivated with antimycin A or methanol, both citric acid production and citric acid productivity, shown as the ratio of production per mycelial dry weight, increased with the increase of both the activity of AOX and the rate of CN-insensitive and SHAM--sensitive respiration. On the other hand, when WU-2223L was cultivated with SHAM, an inhibitor of AOX, the CN-insensitive and SHAM-sensitive respiration was not detected and the citric acid production and the productivity drastically decreased, although mycelial growth was not affected. These results clearly indicated that the CN-insensitive and SHAM-sensitive respiration catalyzed by AOX, localized in the mitochondria, contributed to citric acid production by A. niger.
Key words: alternative oxidase; Aspergillus niger; citric acid; cyanide-insensitive respiration; mitochondria
-3-
Regulation by Organic Acids of Polysaccharide-mediated
Microbe-plant Interactions
Kuniho NAKATA,・・・,＊ Takayoshi KOBAYASHI,＊＊ Yasuyuki TAKIGUCHI,＊＊
and Tatsuaki YAMAGUCHI＊＊
＊Central Research Laboratories, Mercian Corporation, 4-9-1, Johnan, Fujisawa 251-0057, Japan
＊＊Chemistry of Organic Resources Laboratory, Department of Industrial Chemistry, Faculty
of Technology, Chiba Institute of Technology, 2-17-1, Tsudanuma, Narashino 275-0016, Japan
Received February 23, 2000; Accepted June 5, 2000
A polysaccharide flocculant of Klebsiella pneumoniae H12 has been suggested to mediate microbe-plant interactions with the aid of Ca2+ [K. Nakata et al., Biosci. Biotechnol. Biochem., 64, 459--465, 2000]. Here, two-way regulation of polysaccharide-mediated interactions between K. pneumoniae and Raphanus sativus was studied using organic acids. Namely, 10 mM equivalents of organic acids promoted production of the polysaccharide by the bacterium, but inhibited flocculation of bacterial cells by the polysaccharide. These phenomena were counterbalanced by equi-molar equivalents of Ca2+, suggesting competition for Ca2+ between the carboxylic residues of the polysaccharide and those of the aliphatic acids. By electron microscopy observations, bacterial cell aggregates were sparsely distributed over the main roots and root hairs, had various sizes, and seemed to tightly adhere to root tissues. Their shapes seemed to be distorted and abundant in cavities. In brief, these microscopical observations may be explained by a two-way regulation system of bacterial adhesion to a plant by organic acids.
Key words: flocculant; Klebsiella; organic acid; polysaccharide; symbiosis
-4-
Inhibition of Tumor Cell Growth by A Specific 6-Phosphofructo-2-kinase
Inhibitor, N-Bromoacetylethanolamine Phosphate, and Its Analogues
Takafumi HIRATA,＊ Mitsuaki WATANABE,・・・ Shinji MIURA,・・・ Katsushi IJICHI,・・・
Masashi FUKASAWA,・ and Ryuzo SAKAKIBARA§,1
＊Department of Clinical Pharmaceutics, Graduate School of Pharmaceutical Sciences, and ・・・Center for
Instrumental Analysis, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan
・・・Biology Laboratory, Biochemicals Division, Yamasa Corporation, 10-1, Araoi-Cho 2-Chome, Choshi,
Chiba 288-0056, Japan
・Department of Microbiology, National Defense Medical College, Namiki 3-2, Tokorozawa,
Sitama 359-8513, Japan
§Department of Home Economics, Nutrition and Food Science Course, Kyushu Women′s University,
1-1 Jiyugaoka, Yahatanishi-Ku, Fukuoka 807-8586, Japan
Recieved February 28, 2000; Accepted June 15, 2000
The high rate of glycolysis despite the presence of oxygen and mitochondria in tumor cells implies an important role for this process in cell division. The rate of glycolysis is assumed to be dependent on the cellular concentration of fructose 2,6-bisphosphate, the concentration of which in turn depends on a bifunctional enzyme and the ratio of this enzyme′s 6-phosphofructo-2-kinase versus its fructose 2,6-bisphosphatase activities. To prove the hypothesis that inhibition of glycolysis in tumor cells by 6-phosphofructo-2-kinase inhibitors would cause inhibition of tumor cell proliferation, ten N-bromoacetylethanolamine phosphate analogues were designed, synthesized, and tested. They were screened for their activities against various human tumor cell lines to study the effects of inhibition of glycolysis on cell proliferation. The relationship between the structure of these compounds and their inhibitory activity on cell proliferation was also discussed. It was found that the activity of N-(2-methoxyethyl)-bromoacetamide, N-(2-ethoxyethyl)-bromoacetamide, and N-(3-methoxypropyl)-bromoacetamide was comparable to that of the positive control AraC. These three inhibitors showed in vivo anticancer effects in P388 transplant BDF1 mice.
Key words: 6-Phosphofructo-2-kinase; tumor cell growth; enzyme inhibitor; glycolysis; N-bromoacetylethanolamine phosphate
-5-
Effect of Cobalamin on the Allergic Response in Mice
Ulala FUNADA,＊ Masahiro WADA,＊＊,・・・ Tetsunori KAWATA,＊＊＊ Nobuo TANAKA,＊＊＊＊
Tadahiro TADOKORO,＊ and Akio MAEKAWA＊
＊Department of Applied Biology and Chemistry, Faculty of Applied Bioscience, Tokyo University
of Agriculture, Setagaya-ku, Tokyo 156-8502, Japan
＊＊Department of Nutritional Science, Faculty of Applied Bioscience, Tokyo University of Agriculture,
Setagaya-ku, Tokyo 156-8502, Japan
＊＊＊Faculty of Education, Okayama University, Tsushimanaka, Okayama 700-0082, Japan
＊＊＊＊Seifuso Hospital, Niiza, Saitama 352-0023, Japan
Received March 3, 2000; Accepted May 16, 2000
Cyanocobalamin, deoxyadenosylcobalamin and methylcobalamin were separately administered to mice sensitized with the ovalbumin antigen to investigate the allergic response. The serum IgE and pulmonary histamine concentrations were significantly lower in all cobalamin (Cbl)-administered groups. The production of interleukin (IL)-2 and IL-4 in splenocytes was also lower in all Cbl-administered groups. These results show that Cbls were effective in mitigating allergic reactions and IL-2 production. CD3+CD28+ (CD28 is an accessory molecule related to IL-2 production) and CD4+CD28+ in splenocytes were higher in all the Cbl-
administered groups. However, CD3+CD28--, CD4+CD28-- and CD5+CD25-- (CD25: IL-2 Rα/p55) were lower in the Cbl-administered groups. In addition, Cbl specifically inhibited the cellular phosphorylation of tyrosine induced by ovalbumin sensitization. These results indicate that the signal in a cell by CD 28 was restrained by Cbl. We infer that Cbl administration significantly reduced the IL-2 concentration, and secondarily the IL-4, IgE and histamine concentrations.
Key words: cobalamin; allergy; IgE; IL-2; IL-4
-6-
Isolation and Some Properties of a Mesophilic and Mixotrophic
Iron-oxidizing Bacterium, OKM-9
Takao INOUE,1 Kazuo KAMIMURA,2 and Tsuyoshi SUGIO1・・・
1Division of Science and Technology for Energy Conversion, Graduate School of Natural Science
and Technology, Okayama University, Tsushima Naka, Okayama 700-8530, Japan
2Department of Biological Function, Faculty of Agriculture, Okayama University, Tsushima Naka,
Okayama, 700-8530, Japan
Received March 17, 2000; Accepted May 30, 2000
An iron-oxidizing bacterium strain, OKM-9, isolated from mud obtained from the bottom of a pond, Minamikata Ohike, in Okayama prefecture, Japan, grew well in an FeSO_{4}・7H_{2}O (3％)-medium (pH 2.5) with 0.03％ yeast extract. However, the strain could not grow either in an FeSO_{4}・7H_{2}O (3％)-medium without yeast extract or in a yeast extract (0.03％)-medium (pH 2.5) without Fe＾{2+}. The strain did not use elemental sulfur as an energy source and did not have the activity to fix carbon dioxide. Strain OKM-9 could grow in an FeSO_{4}・7H_{2}O (3％)-medium with twenty different L-amino acids instead of yeast extract. Incorporation of [U-＾{14}C] glutamic acid into the cells was dependent on the energy produced by the oxidation of Fe＾{2+}. Strain OKM-9 did not grow heterotrophically using amino acids and hexoses as a sole energy and carbon source. The results that strain OKM-9 absolutely required ferrous iron (Fe＾{2+}) as a sole energy source and yeast extract or L-amino acids as a carbon source for growth strongly suggest that the strain is a mixotrophic iron-oxidizing bacterium. Strain OKM-9 was a Gram-negative and rod-shaped bacterium (0.4--0.6×1.6--2.2 μm) and the mean G+C content of the DNA of the bacterium was 59.6 mol％. The optimum temperature and pH for growth were 30°C and 2.1, respectively. However, the strain could not grow at temperatures above 45°C. Iron-oxidizing activities of strain OKM-9 measured with intact cells and the plasma membrane were 14.3 and 5.7 μl O_{2} uptake/mg protein/min, respectively. The pyridine ferrohemochromes prepared from the plasma membrane of this strain showed absorption peaks characteristic of α-bands of heme a and b, but not heme c, at 587 and 557 nm, respectively. The results suggest that the cytochromes composing an iron-oxidation system of strain OKM-9 are different from those of the well-known mesophilic iron-oxidizing bacteria Thiobacillus ferrooxidans and Leptospirillum ferrooxidans.
iron-oxidizing bacterium; mesophile; mixotroph; Thiobacillus ferrooxidans; Leptospirillum ferrooxidans
-7-
Enzymatic Properties of Phenylalanine101 Mutant Enzyme
of Ribonuclease Rh from Rhizopus niveus
Kazuko OHGI,・・・ Sanae KUDO, Mitsuaki TAKEUCHI, Masanori IWAMA, and Masachika IRIE
Department of Microbiology, Hoshi College of Pharmacy, Ebara 2-4-41, Shinagawa-ku,
Tokyo 142-8501, Japan
Received March 17, 2000; Accepted June 15, 2000
To investigate the role of Phe101, a component of a base recognition site (B2 site) of a base-nonspecific RNase Rh from Rhizopus niveus, we prepared several enzymes mutated at this position, F101W, F101L, F101I, F101A, F101Q, F101R, and F101K, and their enzymatic activities towards RNA, 16 dinucleoside phosphates, and 2?, 3?-cyclic pyrimidine nucleotides were measured. Enzymatic activity toward RNA of F101W, F101L, and F101I were about 7, 20, and 3.8％ of the native enzyme, respectively, and those of the other mutants were less than 1％ of the RNase Rh. Similar results were also obtained with GpG as substrate. Thus, it was concluded that Phe101 is a very important residue as a component of the B2 site of RNase Rh, and its role could be replaced by Leu, then Trp and Ile, though in less effectively. The results suggested that some kind of interaction between B2 base and the side chain of amino acid residue at the 101th position, such as π/π or CH/π interaction is very important for the enzymatic activity of RNase Rh. The mutation of Phe101 markedly affected the enzymatic activity toward dinucleoside phosphates and polymer substrates, but only moderately the rate of hydrolysis of cyclic nucleotides, indicating the presence of secondary effect of the mutation on B1 site.
Key words: base non-specific RNase; B2-subsite; ribonuclease Rh; Rhizopus niveus; site-directed-mutagenesis
-8-
Early Biosynthetic Pathway to Abscisic Acid in Cercospora cruenta
Hirotaka YAMAMOTO,・・・ Masahiro INOMATA, Shinobu TSUCHIYA, Makoto NAKAMURA,
Takeo UCHIYAMA, and Takayuki ORITANI＊
Faculty of Agriculture, Niigata University, Ikarashi, Niigata 950-2181, Japan
＊Graduate School of Agricultural Science, Tohoku University, Aoba-ku, Sendai 981-8555, Japan
Received March 21, 2000; Accepted May 13, 2000
A new biosynthetic intermediate of ABA, (2Z,4E)-γ-ionylideneacetaldehyde, was isolated from young mycelia of Cercospora cruenta. Under an 18O2 atmosphere, an oxygen atom of this endogenous aldehyde was exclusively labeled. Similarly, three 18O atoms were incorporated into the ABA molecule recovered after prolonged incubation; selectively labeled were one of the carboxyl oxygen atoms and the two on the ring portion of ABA. A feeding experiment with [1-13C]glucose proved the exclusive operation of the mevalonate pathway for the formation of both ABA and β-carotene. These results suggest that (2Z,4E)-γ-ionylideneacetaldehyde can be a key ABA biosynthetic intermediate formed by the oxidative cleavage of a carotenoid precursor.
Key words: abscisic acid; (2Z,4E)-γ-ionylideneacetaldehyde; carotenoid pathway; mevalonate pathway; Cercospora cruenta
-9-
Purification and Identification of a Growth-Stimulating Peptide
for Bifidobacterium bifidum from Natural Rubber Serum Powder
Shin-ichi ETOH, Kayoko ASAMURA, Azumi OBU, Kenji SONOMOTO, and Ayaaki ISHIZAKI＊
Laboratory of Microbial Science and Technology, Division of Bioscience and Biotechnology,
Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University,
6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan
Received March 23, 2000; Accepted May 25, 2000
Natural rubber serum powder, which is a by-product obtained in the production of latex rubber, has a strong growth-stimulating activity for Bifidobacterium bifidum JCM 1254. The retained fraction obtained by ultrafiltration (molecular weight cutoff 1000) showed a growth- stimulating activity in a dose-dependent manner on B12 assay medium with ammonium sulfate. One of the growth stimulators was purified from the retained fraction by acetone precipitation, solid-phase extraction with a hydrophobic pretreatment column, and multi-stage reversed-phase HPLC. An increase of 53-fold in the specific activity, and a recovery of 1.3％ were obtained. The amino acid composition and N-terminal sequence analysis of this growth stimulator provided the structure of Ala-Thr-Pro-Glu-Lys-Glu-Glu-Pro-Thr-
Ala. The molecular mass was 1075 by MALDI-TOF MS analysis. These results showed that this growth stimulator was a decapeptide with the sequence shown above. This is the first report that clarified the structure of an active peptide for the growth of Bifidobacterium.
Key words: natural rubber serum powder; Bifidobacterium; growth-stimulating peptide; purification
-10-
Changes in the Content and Biosynthesis of Phytoalexins in Banana Fruit
Tsunashi KAMO,a,b Nobuhiro HIRAI,a,b,・・・ Mitsuya TSUDA,c Daie FUJIOKA,d
and Hajime OHIGASHIa
aDivision of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
bCREST, Japan Science and Technology Corporation (JST)
cDivision of Environmental Science and Technology, Graduate School of Agriculture, Kyoto University,
Kyoto 606-8502, Japan
dKansai Consumer′s Cooperative Union, 3-3-3 Tenjinbashi, Kita-ku, Osaka 530-0041, Japan
Received March 24, 2000; Accepted June 5, 2000
Changes in the phytoalexin content in unripe fruit of banana, Musa acuminata, were analyzed after various treatments. The results show that level of hydroxyanigorufone started to increase 1--2 day after either wounding or inoculation with conidia of Colletotrichum musae. Inoculation followed by wounding induced the formation of many other phenylphenalenones. The accumulation of hydroxyanigorufone decreased, after its transient maximum, on ripening by exposure of the wounded fruit to ethylene. The level of production of hydroxyanigorufone in ripe fruit treated by wounding and/or by inoculation was much lower than that in unripe fruit. 2-Aminooxyacetic acid, an inhibitor of phenylalanine ammonia-lyase (PAL), inhibited the accumulation of hydroxyanigorufone in wounded fruit, and the PAL activity increased after wounding and ethylene treatment, respectively. Feeding experiments with [1-＾{13}C] and [2-＾{13}C]cinnamic acids, and [2-＾{13}C]malonate show that two molecules of cinnamic acid and one of malonate were incorporated into each molecule of hydroxyanigorufone. The phytoalexins isolated from fruit to which deuterated hydroxyanigorufone and irenolone had been administered revealed that 2-(4?-hydroxyphenyl)-1,8-naphthalic anhydride was biosynthesized from hydroxyanigorufone rather than from irenolone.
Key words: Musa acuminata; banana; phytoalexin; phenylphenalenone; phenylalanine ammonia-lyase (PAL)
-11-
Stereocontrolled Reduction of α- and β-Keto Esters
with Micro Green Algae, Chlorella Strains
Kohji ISHIHARA,1,・・・ Hitomi YAMAGUCHI,2 Noriko ADACHI,1
Hiroki HAMADA,3 and Nobuyoshi NAKAJIMA4
1Department of Chemistry, Kyoto University of Education, Fushimi-ku, Kyoto 612-8522, Japan
2Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan
3Department of Applied Science, Okayama University of Science, Ridai-cho, Okayama 700-0005, Japan
4Department of Nutritional Science, Okayama Prefectural University, Soja, Okayama 719-1197, Japan
Received March 27, 2000; Accepted May 29, 2000
The stereocontrolled reduction of α- and β-keto esters using micro green algae was accomplished by a combination of the cultivation method and the introduction of an additive. The reduction of ethyl pyruvate and ethyl benzoylformate by the photoautotrophically cultivated Chlorella sorokiniana gave the corresponding alcohol in high e.e. (>99％ e.e. (S) and >99％ e.e. (R), respectively). In the presence of glucose as an additive, the reduction of ethyl 3-methyl-2-oxobutanoate by the heterotrophically cultivated C. sorokiniana afforded the corresponding (R)-alcohol. On the other hand, the reduction in the presence of ethyl propionate gave the (S)-alcohol. Ethyl 2-methyl-3-oxobutanoate was reduced in the presence of glycerol by the photoautotrophically cultivated C. sorokiniana or the heterotrophically cultivated C. sorokiniana to the corresponding syn-(2R,3S)-hydroxy ester with high diastereo- and enantiomeric excess (e.e.). Some additives altered the stereochemical course in the reduction of α- and β-keto esters.
Key words: Chlorella; asymmetric reduction; keto ester; heterotrophic cultivation; photoautotrophic cultivation
-12-
Substrate Specificity of Cucumisin on Synthetic Peptides
Hiroo YONEZAWA,・・・ Hideki KAIZUKA, Tetsuya UCHIKOBA, Kazunari ARIMA,＊
and Makoto KANEDA
Department of Chemistry, Faculty of Science, Kagoshima University, 1-Korimoto, Kagoshima 890-0065, Japan
＊Plant Gene Expression Center, Department of Plant Biology, University of California,
800 Buchanan st. Albany, CA 94710, U.S.A.
Received March 28, 2000; Accepted May 30, 2000
The substrate specificity of cucumisin [EC 3.4.21.25] was identified by the use of the synthetic peptide substrates Leum-Pro-Glu-Ala-Leun (m＝0--4, n＝0--3). Neither Pro-Glu-Ala-Leu (m＝0) nor Leu-Pro-Glu-Ala (n＝0) was cleaved by cucumisin, however other analogus peptides were cleaved between Glu-Ala. The hydrolysis rates of Leum-Pro-Glu-Ala-Leu increased with the increase of m＝1 to 2 and 3, but was however, essentially same with the increase of m＝3 to 4. Similarly, the hydrolysis rates of Leu-Leu-Pro-Glu-Ala-Leun increased with the increase of n＝0 to 1 and 2, but was essentially same with the increase of n＝2 to 3. Then, it was concluded that cucumisin has a S5-S3? subsite length. In order to identify the substrate specificity at P1 position, Leu-Leu-Pro-X-Ala-Leu (X; Gly, Ala, Val, Leu,
Ile, Pro, Asp, Glu, Lys, Arg, Asn, Gln, Phe, Tyr, Ser,
Thr, Met, Trp, His) were synthesized and digested by cucumisin. Cucumisin showed broad specificity at the P1 position. However, cucumisin did not cleave the C-terminal side of Gly, Ile, Pro, and preferred Leu, Asn, Gln, Thr, and Met, especially Met. Moreover, the substrates, Leu-Leu-Pro-Glu-Y-Leu (Y; Gly, Ala, Ser, Leu, Val, Glu, Lys, Phe) were synthesized and digested by cucumisin. Cucumisin did not cleave the N-terminal side of Val but preferred Gly, Ser, Ala, and Lys especially Ser. The specificity of cucumisin for naturally occurring peptides does not agree strictly with the specificity obtained by synthetic peptides at the P1 or P1? position alone, but it becomes clear that the most of the cleavage sites on naturally occurring peptides by cucumisin contain suitable amino acid residues at P1 and (or) P1? positions. Moreover, cucumisin prefers Pro than Leu at P2 position, indicating that the specificity at P2 position differs from that of papain.
Key words: cucumisin; plant protease; substrate specificity
-13-
Structural Features of N-Glycans Linked to Royal Jelly Glycoproteins:
Structures of high-mannose type, hybrid type, and biantennary type glycans
Yoshinobu KIMURA,1,＊＊ Chiyoko MIYAGI,1 Mariko KIMURA,1 Teruhiko NITODA,1
Nobuyuki KAWAI,2 and Hiroyuki SUGIMOTO2
1Department of Bioresources Chemistry, Faculty of Agriculture, Okayama University, Tsushima-Naka 1-1-1,
Okayama 700-8530, Japan
2Yamada Bee Farm, Ichiba 194, Kagamino-Cho, Tomada-Gun, Okayama 708-0393, Japan
Received April 5, 2000; Accepted June 22, 2000
The structures of N-glycans of total glycoproteins in royal jelly have been explored to clarify whether antigenic N-glycans occur in the famous health food. The structural feature of N-glycans linked to glycoproteins in royal jelly was first characterized by immunoblotting with an antiserum against plant complex type N-glycan and lectin-blotting with Con A and WGA. For the detail structural analysis of such N-glycans, the pyridylaminated (PA-) N-glycans were prepared from hydrazinolysates of total glycoproteins in royal jelly and each PA-sugar chain was purified by reverse-phase HPLC and size-fractionation HPLC. Each structure of the PA-sugar chains purified was identified by the combination of two-dimensional PA-sugar chain mapping, ESI-MS and MS/MS analyses, sequential exoglycosidase digestions, and 500 MHz 1H-NMR spectrometry.
The immunoblotting and lectinblotting analyses preliminarily suggested the absence of antigenic N-glycan bearing β1-2 xylosyl and/or α1-3 fucosyl residue(s) and occurrence of β1-4GlcNAc residue in the insect glycoproteins.
The detailed structural analysis of N-glycans of total royal jelly glycoproteins revealed that the antigenic N-glycans do not occur but the typical high mannose-type structure (Man9~4GlcNAc2) occupies 71.6％ of total N-glycan, biantennary-type structures (GlcNAc2Man3GlcNAc2) 8.4％, and hybrid type structure (GlcNAc1Man4GlcNAc2) 3.0％. Although the complete structures of the remaining 17％ N-glycans; C4, (HexNAc3Hex3HexNAc2: 3.0％), D2 (HexNAc2Hex5HexNAc2: 4.5％), and D3 (HexNAc3Hex4HexNAc2: 9.5％) are still obscure so far, ESI-MS analysis, exoglycosidase digestions by two kinds of β-N-acetylglucosaminidase, and WGA blotting suggested that these N-glycans might bear a β1-4 linkage N-acetylglucosaminyl residue.
Key words: royal jelly; insect glycoprotein; N-glycan glycoform; antigenic N-glycan
-14-
Role of the Non-essential Region Encompassing the N-Terminal
Two Transmembrane Stretches of Escherichia coli SecE
Ken-ichi NISHIYAMA, Hirofumi SUZUKI, and Hajime TOKUDA・・・
Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku,
Tokyo 113-0032, Japan
Received April 5, 2000; Accepted May 20, 2000
SecE is an essential component of the protein translocation machinery of Escherichia coli and has three transmembrane stretches. An N-terminal region (SecE-N) encompassing the first two transmembrane stretches is dispensable for protein translocation but a SecE derivative (SecE-C) lacking this region is very unstable. We show here that FtsH, the AAA (ATPases associated with diverse cellular activities) family protease, causes the instability of SecE-C. SecE-C became stable when SecE-N was co-expressed. Deletion of the N-terminal region of SecE also rendered the SecE-SecY-SecG complex unstable. In spite of these alterations, the N-terminal region of SecE had little stimulatory effect on protein translocation in vivo or in vitro.
Key words: protein translocation; proteoliposome; FtsH; SecE; Escherichia coli
-15-
Characterization of Human Recombinant Transglutaminase 1 Purified
from Baculovirus-infected Insect Cells
Kiyotaka HITOMI,・・・ Yoshihisa YAMAGIWA, Koji IKURA,＊ Kiyofumi YAMANISHI,＊＊
and Masatoshi MAKI
Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences,
Nagoya University, Chikusa, Nagoya, 464-8601, Japan
＊Department of Applied Biology, Kyoto Institute of Technology, Kyoto, 606-8585, Japan
＊＊Department of Dermatology, Kyoto Prefectural University of Medicine, Kamigyou-ku,
Kyoto, 602-8566, Japan
Received April 5, 2000; Accepted June 1, 2000
Transglutaminase 1 (TGase 1) is required for the formation of a cornified envelope in stratified squamous epithelia. Recombinant human TGase 1 expressed in baculovirus-infected cells was purified in a soluble form at the molecular mass of 92 kDa. Recombinant TGase 1 was susceptible to limited proteolysis by both μ- and m-calpains, the calcium-dependent intracellular cysteine proteases. Although the proteolysis did not induce the elevation of the specific enzyme activity of TGase 1, the requirement of calcium ion in the enzymatic reaction was reduced. Furthermore, the effects of GTP, nitric oxide, and sphingosylphosphocholine, known as regulatory factors for tissue-type isozyme (TGase 2), on the enzymatic activity of TGase 1 were investigated.
Key words: transglutaminase; calpain; proteolysis; calcium; baculovirus
-16-
Production of Humanized Fab Fragment against Human High Affinity
IgE Receptor in Pichia pastoris
Kyoko TAKAHASHI,1 Toshifumi YUUKI,1 Toshiro TAKAI,1 Chisei RA,2,3
Ko OKUMURA,2 Toyokazu YOKOTA,1 and Yasushi OKUMURA1
1Foods ＆ Pharmaceuticals Research ＆ Development Laboratory, Asahi Breweries Ltd., 1-1-21, Midori,
Moriya-machi, Kitasoma-gun, Ibaraki 302-0106, Japan
2Department of Immunology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku,
Tokyo 113-8421, Japan
3Allergy Research Center, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo
113-8421, Japan
Received April 6, 2000; Accepted June 6, 2000
Binding of allergen-IgE complexes to the high affinity IgE receptor (FcεRI) on mast cells and basophils leads to the release of various mediaters such as histamine. Fab fragments prepared by the papain digestion of humanized antibody against human FcεRI inhibited the release of histamine from human basophils. Here we established an expression system to directly produce Fab fragments of the humanized anti-human FcεRI antibody in methylotropic yeast, P. pastoris. Fab fragments were efficiently secreted into the medium at a concentration of 10--40 mg/L using a signal sequence from the P. pastoris phosphatase gene. They were consisted of disulfide-linked light and heavy chains correctly starting from the first amino acid residues by proper cleavage of the signal peptides. The obtained Fab fragments inhibited the binding between IgE and FcεRI as efficiently as the counterpart prepared by papain digestion of the whole antibody.
Key words: Fab; humanize; FcεRI α chain; secretion; Pichia pastoris
-17-
Regulation of Sex Pheromone Biosynthesis in Three Plusiinae Moths:
Macdunnoughia confusa, Anadevidia peponis, and Chrysodeixis eriosoma
Mitsuaki KOMODA, Shin-ichi INOMATA, Akihiko ONO, Hayaki WATANABE,
and Tetsu ANDO・・・
Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture
and Technology, Koganei, Tokyo 184-8588, Japan
Received April 11, 2000; Accepted June 19, 2000
Virgin females of M. confusa, A. peponis, and C. eriosoma secrete (Z)-7-dodecenyl acetate as a common main pheromone component. Their pheromone titers decreased after decapitation, and increased in the decapitated females after injection of a synthetic hormone, pheromone biosynthetic activating neuropeptide (PBAN) of Bombyx mori. In addition, an extract of brain-subesophageal ganglion complexes of each Plusiinae species activated pheromone biosynthesis in decapitated females of not only the corresponding species, but also that of Mamestra brassicae. These results indicate that pheromone biosynthesis of the three Plusiinae species is also controlled by a PBAN-like substance. However, the Plusiinae females exceptionally contained remarkable amounts of the pheromone even 1 day after decapitation. Since it has been reported that pheromones completely disappear at least 1 day after decapitation in females of many other lepdidoptran species including B. mori and M. brassicae, a different mechanism is likely regarding the regulation of the studied Plusiinae pheromone biosynthesis. Furthermore, an incorporation experiment with a labeled pheromone precursor, D9-(Z)-7-dodecenoic acid, showed that moderate biosynthesis still proceeded in the pheromone glands of M. confusa females 1 day after decapitation, providing an evidence why complete disappearance of the pheromone was not observed in the females which otherwise lacked a source of the pheromonotropic neuropeptide.
Key words: sex pheromones; biosynthesis regulation; PBAN; neuropeptide hormone; deuterium incorporation
-18-
Genetic Analysis of Growth Inhibition of Yeast Cells Caused by Expression
of Aspergillus oryzae RNase T1
Gen NONAKA, Tomoki ISHIKAWA, Tin-tin LIU, Harushi NAKAJIMA,
and Katsuhiko KITAMOTO・・・
Department of Biotechnology, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan
Received April 12, 2000; Accepted June 2, 2000
Even though most fungal hydrolytic enzymes have been successfully secreted in S. cerevisiae cells by expression of corresponding cDNA, overexpression of A. oryzae RNase T1 causes severe growth inhibition in yeast. We observed that yeast strains carrying RNase T1 cDNA under control of the GAL1 promoter with a single-copy vector were able to grow on galactose medium while those with a multi-copy vector were not. It was found that overexpression of three mutated versions of RNase T1 with low enzymatic activity did not affect the growth. We also observed that expression of RNase T1 without a signal sequence severely inhibited growth of the transformant even on the single-copy plasmid. Subcellular fractionation showed that overexpressed myc-tagged RNase T1 was localized in the membrane fraction. In the yeast secretory pathway, while the mutants defective in translocation into the ER, ER-Golgi trafficking and vacuole formation had severe growth inhibition during expression of RNase T1 from the single-copy plasmid. These results suggest that a mislocalization of active RNase T1 in cytosol by overflow from the secretory apparatus has toxic effects on the host cells.
Key words: Aspergillus oryzae; RNase T1; Saccharomyces cerevisiae; secretory pathway
-19-
Dietary Conjugated Linoleic Acid Increases Immunoglobulin Productivity
of Sprague-Dawley Rat Spleen Lymphocytes
Masao YAMASAKI, Koji KISHIHARA, Keiko MANSHO, Yuuya OGINO, Masaaki KASAI,＊
Michihiro SUGANO,＊＊ Hirofumi TACHIBANA, and Koji YAMADA
Laboratory of Food Chemistry, Department of Bioscience and Biotechnology, Division of Bioresource
and Bioenvironmental Sciences, Graduate School, Kyushu University, Fukuoka 812-8581, Japan
＊Nagoya Factory, Rinol Oil Mills, Co., Ltd., 37-15 Shiomi-cho, Minato-ku, Nagoya 455-0028, Japan
＊＊Faculty of Environmental and Symbiotic Sciences, Prefectural University of Kumamoto,
Kumamoto 862-8502, Japan
Received April 14, 2000; Accepted May 26, 2000
The dietary effects of conjugated linoleic acid (CLA) on Ig production of Sprague-Dawley rats were examined at various doses such as 0 (control), 0.05, 0.10, 0.25, and 0.50％. CLA increased IgG and IgM production of spleen lymphocytes in a dose-dependent manner, and these levels reached a plateau at 0.25％. IgA production was not detected in the control group, while it was detected in all CLA-fed groups and IgA productivity of spleen lymphocytes increased in a dose-dependent manner at the doses from 0.05 to 0.50％. Dietary CLA did not affect serum Ig levels. The major fatty acid composition of spleen lymphocytes was not affected by dietary CLA, which itself was hardly incorporated into the cells. In an in vitro assay, the effects of CLA and its oxidative derivatives, furan type fatty acids, on Ig productivity were also examined. As a result, 100 μM CLA suppressed Ig production of spleen lymphocytes and the degree was as follows IgA>IgG>IgM. Each CLA isomer and the furan type fatty acids also suppressed Ig production but the degree was weaker than the mixture of CLA isomers. In this result, dietary CLA increased Ig productivity of spleen lymphocytes in vivo.
Key words: CLA; immunoglobulin; spleen lymphocytes; furan type fatty acid
-20-
Improved Hypolipidemic Effects of Xanthan Gum-Galactomannan
Mixtures in Rats
Yukiko YAMAMOTO,・・・ Ikuyo SOGAWA, Akiko NISHINA, Shigeru SAEKI,
Naoki ICHIKAWA, and Shiho IIBATA
Department of Food and Nutrition, Faculty of Human Life Science, Osaka City University, Sugimoto 3-3-138,
Sumiyoshi-ku, Osaka 558-8585, Japan
Received April 14, 2000; Accepted June 12, 2000
This study describes the effects of mixtures of xanthan gum and galactomannan, guar gum, or locust bean gum, on the lipids in plasma and liver in non-diabetic and diabetic rats. Non-diabetic rats were fed cholesterol-free diets with 3％ guar gum, locust bean gum, or xanthan gum (3G, 3L, and 3X), or a mixture of xanthan gum and guar gum or locust bean gum (1:2, w/w) (2G1X, 2L1X) for 2 weeks. Rats fed diets not containing these polysaccharides were used as controls. The total cholesterol in plasma and the triacylglycerol in liver were significantly lowered in rats fed the 2G1X diet. The 3G, 3X, 3L, and 2L1X diets showed no significant effect on the total cholesterol and triacylglycerol in plasma and liver. In the streptozotocin-induced (STZ) diabetic rats, the total cholesterol in plasma was lowered in rats fed the 3G, 3X or 2G1X diet for 4 weeks, and the 2G1X diet was more effective than the 3G and 3X diets. The triacylglycerol in plasma in STZ diabetic rats was also significantly lowered by the 2G1X diet. These results showed that a mixture of xanthan gum and guar gum has an improved hypolipidemic effect on non-diabetic and STZ diabetic rats. The effects of the 2G1X diet on the diabetic symptoms in STZ diabetic rats, suppression of food and water intakes, decrease in glucose in urine, and lowering of plasma glucose, were also observed.
Key words: hypolipidemic effect; guar gum; xanthan gum; viscosity; diabetes
-21-
Solution Properties of Antitumor Sulfated Derivative of α-(1→3)-D-Glucan
from Ganoderma lucidum
Lina ZHANG,・・・ Mei ZHANG, Qi ZHOU, Jinghua CHEN, and Fanbo ZENG＊
Department of Chemistry, Wuhan University, Wuhan 430072, China
＊Pharmacy College, Tongji Medical University, Wuhan 430030, China
Received April 17, 2000; Accepted June 8, 2000
Four fractions of a water-insoluble α-(1→3)-D-glucan GL extracted from fruiting bodies of Ganoderma lucidum were dissolved in 0.25 M LiCl/DMSO, and then reacted with sulfur trioxide-pyridine complex at 80°C to synthesize a series of water-soluble sulfated derivatives S-GL. The degree of substitution of DS was measured by using IR infrared spectra, elemental analysis, and 13C NMR to be 1.2--1.6 in the non-selective sulfation. Weight-average molecular weight Mw and intrinsic viscosity [η] of the sulfated derivatives S-GL were measured by multi-angle laser light scattering and viscometry. The Mw value (2.4×104) of sulfated glucan S-GL-1 was much lower than that (44.5×104) of original α-(1→3)-D-glucan GL-1. The Mark-Houwink equation and average value of characteristic ratio C∞ for the S-GL in 0.2 M NaCl aqueous solution at 25°C were found to be: [η]＝1.32×10--3M1.06w (cm3 g--1) and 16, respectively, in the Mw range from 1.1×104 to 2.4×104. It indicated that the sulfated derivatives of the α-(1→3)-D-glucan in the aqueous solution behave as an expanded chain, owing to intramolecular hydrogen bonding or interaction between charge groups. Interestingly, two sulfated derivatives synthesized from the α-(1→3)-D-glucan and curdlan, a β-(1→3)-D-glucan, all had significant higher antitumor activity against Ehrlich ascites carcinoma (EAC) than the originals. The effect of expanded chains of the sulfated glucan in the aqueous solution on the improvement of the antitumor activity could not be negligible.
Key words: Ganoderma lucidum; polysaccharide; sulfated modification; molecular weight; antitumor activity
-22-
Purification and Characterization of an α-L-Rhamnosidase
from Pichia angusta X349
Takaaki YANAI and Michikatsu SATO・・・
Mercian Corporation, Wine ＆ Spirits Research Institute, 9-1, Johnan 4-chome, Fujisawa 251-0057, Japan
Received April 18, 2000; Accepted May 28, 2000
An intracellular α-L-rhamnosidase from Pichia angusta X349 was purified to homogeneity through four chromatographic steps. The α-L-rhamnosidase appeared to be a monomeric protein with a molecular mass of 90 kDa. The enzyme had an isoelectric point at 4.9, and was optimally active at pH 6.0 and at around 40°C. The Ki for L-rhamnose inhibition was 25 mM. The enzyme was inhibited by Cu2+, Hg2+, and p-chloromercuribenzoate. The α-L-rhamnosidase was highly specific for α-L-rhamnopyranoside and liberated rhamnose from naringin, rutin, hesperidin, and 3-quercitrin. The α-L-rhamnosidase was active at the ethanol concentrations of wine. It efficiently released monoterpenols, such as linalool and geraniol, from an aroma precursor extracted from Muscat grape juice.
Key words: α-L-rhamnosidase; Pichia angusta; aroma improvement; monoterpenol
-23-
Purification and Characterization of a Basic Amino Acid-Specific Peptidase
from Seeds of Jack Bean (Canavalia ensiformis)
Kiyotaka OSHIKAWA, Ken-ichi AOKI, Yuuki YOSHINO, and Shigeyuki TERADA・・・
Department of Chemistry, Faculty of Science, Fukuoka University, 8-19-1 Nanakuma, Jonan-ku,
Fukuoka 814-0180, Japan
Received April 19, 2000; Accepted June 12, 2000
A peptidase was purified from seeds of Canavalia ensiformis by extraction with water, ammonium sulfate precipitation, and successive chromatographies on DEAE-Toyopearl 650M, butyl-Toyopearl 650M, and G-3000 SW columns. The enzyme has an apparent molecular weight of 41,000. Activity is maximal at pH 9 and 60°C. The enzyme hydrolyzed synthetic substrates at Arg-X and Lys-X bonds more rapidly than bovine trypsin did, and did not cleave protein or ester substrates. The enzyme was inhibited by alkylamines and several serine protease inhibitors such as diisopropylfluorophosphate, chymostatin, leupeptin, and benzamidine. Cysteine protease-, metalloprotease-, and proteinous trypsin inhibitors were ineffective. Inhibition by alkylamines was dependent on length of the alkyl chains. From the substrate specificity and susceptibility to chemicals, the enzyme is a unique peptidase with trypsin-like specificity.
Key words: Canavalia ensiformis; Jack bean seeds; peptidase; purification; trypsin-like specificity
-24-
Some Molecular and Inhibitory Specifications of a Dipeptidyl
Carboxypeptidase from the Polychaete Neanthes virens
Resembling Angiotensin I Converting Enzyme
Toshiya KAWAMURA,・・・ Kiyofumi KIKUNO,＊ Tatsuya ODA,＊ and Tsuyoshi MURAMATSU＊
Industrial Technology Center of Nagasaki, 2-1303-8 Ikeda, Omura, Nagasaki 856-0026, Japan
＊Division of Biochemistry, Faculty of Fisheries, Nagasaki University, 1-14 Bunkyo, Nagasaki 852-8521, Japan
Received April 28, 2000; Accepted June 2, 2000
Dipeptidyl carboxypeptidase (DCP) from the polychaete Neanthes virens, resembling mammalian angiotensin I converting enzyme (ACE), was studied to discover some of its molecular and inhibitory properties, as the first evidence of these in a marine invertebrate. Amino acid and carbohydrate contents were analyzed. The N-terminal amino acid sequence of N. virens DCP was (NH2)D-E-E-A-G-R-Q-W-L-A-E-Y-D-L-R-N-Q-T-V-L-. Peptide maps of N. virens DCP from lysyl endopeptidase digestion were different from rabbit p-ACE. The far-ultraviolet circular dichroic spectra of N. virens DCP indicated that the secondary structure of this enzyme seemed to be an α-helical structure and was similar to that of rabbit p-ACE, but the near-ultraviolet circular dichroic spectra of N. virens DCP indicated that the aromatic amino acid residue circumambience of this enzyme was different from rabbit p-ACE. The effects of several reagents for chemical modification of amino acids on the activity of N. virens DCP were tested. Arg, Tyr, Glu, and/or Asp, His, Trp, and Met caused loss of the activity. In addition, the IC50 and Ki values for a well-known ACE inhibitor, Val-Tyr, which was a competitive inhibitor of N. virens DCP, were 263 and 20 μM, respectively. These results suggested that N. virens DCP is different from mammalian ACE in the molecular and inhibitory properties, although the same substrate specificity was demonstrated in a previous paper.
Key words: angiotensin I converting enzyme; dipeptidyl carboxypeptidase; inhibitory property; molecular property; Neanthes virens
-25-
Primary Amino Acid and DNA Sequences of Gassericin T, a Lactacin F-Family
Bacteriocin Produced by Lactobacillus gasseri SBT2055
Yasushi KAWAI,・・・ Boku SAITOH, Osamu TAKAHASHI, Haruki KITAZAWA,
Tadao SAITO, Hadjime NAKAJIMA,＊ and Takatoshi ITOH
Laboratory of Animal Products Chemistry, Biological Resource Science, Graduate School of Agricultural
Science, Tohoku University, Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan
＊Technology and Research Institute, Snow Brand Milk Products Co. Ltd., Minamidai 1-1-2,
Kawagoe 350-1165, Japan
Received May 1, 2000; Accepted June 6, 2000
A broad-spectral bacteriocin, named gassericin T, produced by Lactobacillus gasseri SBT 2055 (from human feces) was isolated to homogeneity from the culture supernatant by hydrophobic chromatography. By SDS-PAGE and in situ activity assay, the purified gassericin T migrated as a single band with bacteriocin activity and molecular size of 5,400. A 2.9-kbp HindIII--HindIII fragment of chromosome DNA was hybridized with the oligonucleotide probe designed from the partial N-terminal amino acid sequence of gassericin T and was cloned. Six ORFs including the structural gene of gassericin T were deduced by computer analysis and the data bases. The structural gene of gassericin T (gatA) was identified as the fourth ORF, which encoded a protein composed of 75 amino acids that included the GG motif of the cleavage site. Chemical sequencing analysis of the complete amino acid sequence showed that gassericin T (57 amino acids) had a disulfide bond in the molecule and no modified amino acid residues, making it a class II bacteriocin. The gassericin T had 60％ sequence similarity to mature LafA (57 amino acids, lactacin F, bacteriocins produced by L. johnsonii VPI11088), and the sequences around the processing site and C-terminal area were well conserved. The fifth ORF was designated as gatX, encoded as a peptide composed of 65 amino acids containing the GG motif of the putative cleavage site, however mature GatX and its antibacterial activity were not detected in the culture supernatant. GatX has higher similarity with LafX than with lactobin A (50 amino acids) belonging to the first lactacin F-family. These results indicated that gassericin T belongs to the hydrophobic class II bacteriocins and the most vicinal lactacin F-family.
Key words: bacteriocin; Lactobacillus gasseri; gassericin T; lactacin F; lactobin A
-26-
Improved Stereoselective Synthesis of Optically Active Methylene Lactone,
Key Intermediate for the Synthesis of 1,2-Oxidized Furofuran Lignan,
by Direct α-Methylenation to Butanolide
Satoshi YAMAUCHI,・・・ Nobuyuki YAMAMOTO, and Yoshiro KINOSHITA
College of Agriculture, Ehime University, Tarumi 3-5-7, Matsuyama, Ehime 790-8566, Japan
Received May 11, 2000; Accepted June 6, 2000
(3R) - 3 - [(1R) - 1 - (tert - Butyldimethylsilyl)oxy - 1 - (2 -methoxy - 4,5 - methylenedioxyphenyl) methyl] - 2 - methylene-4-butanolide, which is a key intermediate for the synthesis of 1,2-oxidized furofuran lignan, was stereoselectively synthesized from L-glutamic acid by applying direct methylenation to butanolide.
Key words: lignan; furofuran lignan; stereoselective synthesis
-27-
Note
An Antibiotic from Penicillium sp. Covering the Cocoon
of the Leaf-rolling Moth, Dactylioglypha tonica
Nobutaka IMAMURA,・・・ Takahiro ISHIKAWA, Tomoko OHTSUKA, Kazumi YAMAMOTO,
Masato DEKURA, Hiroshi FUKAMI, and Ritsuo NISHIDA＊
Department of Bioscience and Biotechnology, Faculty of Science and Engineering, Ritsumeikan University,
Noji-higashi, Kusatsu 525-8577, Japan
＊Division of Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502,
Japan
Received December 16, 1999; Accepted May 19, 2000
An antibiotic-producing Penicillium sp. strain was isolated from cocoons of the leaf-rolling moth, Dactylioglypha tonica. An antibacterial compound was isolated from the cultured broth, and the chemical structure of the principle was determined by spectroscopic data to be a derivative of isocoumarincarboxylate.
Key words: Dactylioglypha tonica; cocoon; antibiotic; Penicillium; isocoumarincarboxylate
-28-
Note
Antioxidative Activity of (--)-Epigallocatechin-3-(3!-O-methyl)gallate Isolated
from Fresh Tea Leaf and Preliminary Results on Its Biological Activity
Masaya KAWASE,＊,＃ Rong WANG,＊＊ Teruaki SHIOMI,＊＊＊ Ryoyasu SAIJO,・・・,＊＊
and Kiyohito YAGI＊＊＊
＊Faculty of Agriculture, and ＊＊Faculty of Education, Kagawa University, 1-1 Saiwai-cho,
Takamatsu 760-8522, Japan
＊＊＊Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita 565-0871, Japan
Received January 20, 2000; Accepted June 14, 2000
Antioxidative activity of (--)-epigallocatechin-3-(3!-O-methyl)gallate (catechin e) was examined. Catechin e showed a strong antioxidative activity. A preliminary test using rat cancer cells suggests that catechin e also has a strong cytotoxic activity. Among tested catechins, only catechin e has strong activity for both.
Key words: antioxidative activity; catechins; (--)-epigallocatechin-3-(3!-O-methyl)gallate
-29-
Note
A cDNA Encoding Ribosomal Protein RPL15 from the Desiccation-tolerant
Bryophyte Tortula ruralis: mRNA Transcripts are Stably Maintained
in Desiccated and Rehydrated Gametophytes
Qin ZENG and Andrew J. WOOD・・・
Department of Plant Biology, Southern Illinois University-Carbondale, Carbondale, IL 62901-6509, U.S.A.
Received February 10, 2000; Accepted June 12, 2000
A Tortula ruralis cDNA, Rpl15, encoding a predicted polypeptide with significant similarity to the large-subunit ribosomal protein L15 (RPL15) was isolated from a desiccated gametophyte cDNA library, and is the first L15 homologue identified from a bryophyte. The deduced 203 amino acid polypeptide is approximately 24.1 kDa, with a predicted pI of 11.1, and shares extensive identity with rat RPL15 deduced polypeptide (>57％). RNA blot hybridization using total RNA demonstrated that Rpl15 is constitutively expressed in moss gametophytes during a desiccation-rehydration cycle and that Rpl15 mRNA transcripts are maintained in desiccated gametophytes as conserved mRNAs.
Key words: bryophyte; desiccation; expressed sequence tag; Tortula ruralis
-30-
Note
Induction of Osteopontin Gene Expression during Mammary Gland Involution
and Effects of Glucocorticoid on its Expression in Mammary Epithelial Cells
Mijoung LEE,＊ Sanghyun NA,＊ Deahyun JEON,＊ Hyungha KIM,＊ Yunjaie CHOI,＊＊
and Myunggi BAIK・・・,＊
＊Department of Genetic Engineering, Biotechnology Research Institute, College of Agriculture,
Chonnam National University, Kwangju 500-757, Korea
＊＊School of Agricultural Biotechnology, Seoul National University, Suweon 441-744, Korea
Received February 21, 2000; Accepted June 7, 2000
To understand the molecular mechanisms of mammary gland involution, an involution-induced clone was identified from a cDNA library of mouse mammary gland by differential screening. Characterization of a clone by sequencing and northern analysis showed that expression of the osteopontin gene was induced during involution of mouse mammary gland. But induction of the osteopontin gene was not observed in apoptotic HC11 mammary epithelial cells under serum starvation. In HCll cells, dexamethasone treatment from the seeding stage showed five-fold induction of osteopontin gene expression, but the expression was not changed when dexamethasone was added to confluent cells.
Key words: osteopontin gene; involution; mouse mammary gland; dexamethasone
-31-
Note
Synthesis and Lateral Root-Inducing Activity of Novel
N-Substituted-2-Piperidones with a 1,4-Benzodioxan Ring
Hidetaka TSUKADA, Naotaka YAMADA, Eiji TANIGUCHI, and Eiichi KUWANO・・・
Laboratory of Pesticide Chemistry, Division of Bioresource and Bioenvironmental Sciences, Graduate School,
Kyushu University, Fukuoka 812-8581, Japan
Received March 6, 2000; Accepted June 14, 2000
Novel N-substituted-2-piperidones with a 1,4-benzodioxan ring were prepared and evaluated for their activity to induce lateral roots in lettuce seedlings. Compounds were obtained by aldol condensation of the lithium enolate of N-substituted-2-piperidones with 1,4-benzodioxan-6-carbaldehyde. Of the series compounds tested, N-cinnamyl-3-[1-(1,4-benzodioxan-6-yl)-1-hydroxymethyl]-2-piperidone (2e) had the highest activity. In seedlings treated with 10 ppm of 2e, all of the primary roots formed lateral roots. Only erythro-2e showed lateral root-inducing activity, while threo-2e was inactive.
Key words: 2-piperidones; 1,4-benzodioxan; lateral root; lettuce seedling
-32-
Note
Sites of Expression of DnaJ Homologs and Hsp70 in Male and Female Flowers
of the Japanese Willow Salix gilgiana Seemen
Norihiro FUTAMURA,1 Hiroshi KOUCHI,2 and Kenji SHINOHARA1
1Molecular and Cell Biology Section, Bio-Resources Technology Division, Forestry and Forest Products
Research Institute (FFPRI), Inashiki, Ibaraki 305-8687, Japan
2National Institute of Agrobiological Resources, Tsukuba, Ibaraki 305-8602, Japan
Received March 16, 2000; Accepted June 2, 2000
We did in situ hybridization to identify the sites at which DnaJ homologs (SGJ1 and SGJ3) and Hsp70 were expressed in male and female flowers of the Japanese willow, Salix gilgiana Seemen. Transcripts of SGJ1, SGJ3, and Hsp70 were detected in microspores and in the tapetal layers of immature anthers, in the inner integument of ovules, and in the funicular epidermal layers of ovaries. Transcripts of SGJ3 and Hsp70 were also detected in the nectaries of male and female flowers, in which transcripts of SGJ1 were undetectable.
Key words: DnaJ-like proteins; flower development; heat shock proteins; willow (Salix gilgiana)
-33-
Note
Effects of pH and Light on the Storage Stability of the Purple Pigment,
Hordeumin, from Uncooked Barley Bran Fermented Broth
Tomoaki DEGUCHI, Shuji SHOHARA, Riichiro OHBA, and Seinosuke UEDA
Department of Applied Microbial Technology, Faculty of Engineering, Kumamoto Institute of Technology,
4-22-1 Ikeda, Kumamoto 860-0082, Japan
Received March 21, 2000; Accepted June 7, 2000
The pigment retention rate of hordeumin was higher than that of two standard anthocyanidins, cyanidin and delphinidin, when hordeumin and anthocyanidins were dissolved in Walpole buffer (pH 1.0) and stored. Moreover, when pigment solutions were stored at 15°C under light irradiation, the pigment retention rate of the hordeumin solution became higher than those of standard anthocyanidins (2 to 10 times) as the storage period increased. Comparing various pH buffers (MacIlvaine buffer, pH 2.2 to 7.0), the pigment retention rate of hordeumin at pH 5.0 was highest. Furthermore, the half-life of hordeumin at pH 5.0 was increased from 9 days to 17.5 days when nitrogen gas was bubbled into the hordeumin solution. We considered that the storage stability of hordeumin is higher than standard anthocyanidins because hordeumin is a complex with anthocyanin, tannin, and protein.
Key words: hordeumin; barley bran; anthocyanidin; storage stability; pH stability
-34-
Note
Differential Response of Obese Gene Expression from Fasting
in Bovine Adipose Tissues
Hyungha KIM,＊ Yountae CHI,＊ Kichul CHUNG,＊ Kwanghyun KIM,＊＊
Yunjaie CHOI,・・・ and Myunggi BAIK＊,・・・
＊Department of Genetic Engineering and ＊＊Department of Animal Resources, Biotechnology Research
Institute, College of Agriculture, Chonnam National University, Kwangju 500-757, Korea
・・・School of Agricultural Biotechnology, Seoul National University, Suweon 441-744, Korea
Received March 21, 2000; Accepted June 7, 2000
To understand the molecular mechanism for intramuscular fat deposition, the expression of the obese gene was examined in response to fasting. Food deprivation for 48 h induced a decrease in the level of obese mRNA in pooled adipose tissues (abdominal, perirenal, subcutaneous, intermuscular and intramuscular). The expression of obese mRNA was examined for individual adipose tissue from several fat depots. It was highly expressed in perirenal adipose tissue, but fasting did not affect its expression level in this tissue. Moderate levels were detected in subcutaneous and intermuscular adipose tissues, and a fasting-induced decrease in obese mRNA was apparent in these tissues. The expression level of the obese gene in intramuscular adipose tissue was very low and did not respond to fasting.
Key words: obese gene; bovine adipose tissues; fasting
-35-
Note
Molecular Cloning and Expression of the Nucleoside Deoxyribosyltransferase-II
Gene from Lactobacillus helveticus
Kiyoshi OKUYAMA and Toshitada NOGUCHI
Biochemicals Division, Yamasa Corporation, Choshi, Chiba 288-0056, Japan
Received March 23, 2000; Accepted May 25, 2000
Nucleoside deoxyribosyltransferase-II, which catalyzes transfer of glycosyl residues from a donor deoxynucleoside to an acceptor base, was purified from Lactobacillus helveticus and its gene was cloned. Analysis of the nucleotide sequence showed the presence of a 474-nucleotide open reading frame encoding a protein of 158 amino acids with a molecular weight of 18,317. The active enzyme can be produced in large quantities in E. coli cells using the cloned gene.
Key words: Lactobacillus helveticus; nucleoside deoxyribosyltransferase-II; gene cloning
-36-
Note
Naringin Glycosides α-Glucosylated on Ring B Found in the Natural Food
Additive, Enzymatically Modified Naringin
Takumi AKIYAMA, Mitsuo YAMADA, Takashi YAMADA, and Tamio MAITANI
National Institute of Health Sciences, Kamiyoga 1-18-1, Setagaya, Tokyo 158-8501, Japan
Received March 28, 2000; Accepted June 5, 2000
The constituents of the natural food additive, enzymatically modified naringin, were structurally analyzed. Four constituents were isolated from the glucoamylase-treated sample. An NMR analysis revealed that two of them were novel compounds having 4?-O-α-glucosyl moieties on ring B of the naringenin aglycone. Both the aglycone and the glucose moiety in naringin are shown to be simultaneously glucosylated.
Key words: natural food additive; naringin; enzymatically modified naringin; cyclodextrin glucanotransferase; nuclear magnetic resonance
-37-
Note
Decrease in Rice Allergenic Proteins of Polished Rice Grains by Incubating
with a Miso Solution
Hidehiko IZUMI,1 Shiho KONDO,1 Hajime KASHIO,1 Tsukasa MATSUDA,2,・・・ and Ryo NAKAMURA3
1Department of Food and Nutrition, Nagoya College of Nutrition, Naka, Nagoya 460-0007, Japan
2Department of Applied Molecular Biosciences, School of Bioagricultural Sciences, Nagoya University,
Chikusa, Nagoya 464-8601, Japan
3Department of Food Science and Technology, Nihon University, Fujisawa 252-8510, Japan
Received April 5, 2000; Accepted May 11, 2000
The effect of miso on allergenic proteins in rice seeds was investigated. When polished rice grains were incubated at 37°C for 30--120 min with a 10％ miso solution, but not with heat-treated miso or 1％ NaCl, the amount of soluble proteins extracted from the rice grains with 1 M NaCl markedly decreased. SDS-PAGE, immunoblotting and densitometric analyses of these soluble proteins and insoluble proteins indicate that 26 kDa globulin and 14--16 kDa allergens in the grains were decreased to 15--60％ during incubation with the miso solution, especially soybean-koji miso, without any large change in the content of major insoluble proteins.
Key words: rice; allergen; food allergy; miso
-38-
Note
Cloning and Expression of the ccpA Gene Encoding Catabolite Control Protein
from Thermoactinomyces sp. E79＊
Min-Woo MOON, Sun-Yang PARK, Hyung-Kwoun KIM, Do-Young YUM,§
Hahn-Shik KWAK,1 Tae-Kwang OH, and Jung-Kee LEE・・・
Environmental Bioresources Laboratory, Korea Research Institute of Bioscience and Biotechnology,
P.O. Box 115, Yusong, Taejon 305-600, Korea
1Department of Genetic Engineering, Paichai University, Taejon 302-735, Korea
Received April 5, 2000; Accepted June 3, 2000
The gene ccpA encoding the catabolite control protein A (CcpA) of Thermoactinomyces sp. E79 has been cloned and characterized. Nucleotide sequence analysis of the CcpA clone showed that the cloned fragment contained the full structural gene for a protein of 346 amino acids. The predicted amino acid sequence shows similarity to the transcriptional regulators of the LacI-GalR family; a highly conserved helix-turn-helix motif, which might bind to DNA, was identified through comparison with regulator proteins in this family. The highest sequence identity was obtained when it was compared with the CcpA of Bacillus subtilis (60％) or Bacillus megaterium (60％). The expression of ccpA in Thermoactinomyces sp. E79 was dependent on glucose, which is contrast to the cases of B. subtilis, B. megaterium and S. xylosus. The complementation experiment with the B. megaterium ccpA mutant indicated that the cloned gene was a ccpA.
Key words: catabolite control protein A; catabolite repression; Thermoactinomyces; alkaline protease
-39-
Note
Phosphorylation of Guanosine Using Guanosine-inosine Kinase
from Exiguobacterium acetylicum Coupled with ATP Regeneration
Hisashi KAWASAKI, Yoshihiro USUDA, Megumi SHIMAOKA, and Takashi UTAGAWA
Applied Microbiology Laboratory, Fermentation and Biotechnology Laboratories, Ajinomoto Co. Inc.,
1-1 Suzuki-cho, Kawasaki-ku, Kawasaki-shi 210-8681, Japan
Received April 5, 2000; Accepted May 29, 2000
Guanosine 5?-monophosphate (5?-GMP) and inosine 5?-monophosphate (5?-IMP) are widely used as flavor enhancers. Recently, a novel process for 5?-IMP production by phosphorylation of inosine using guanosine-inosine kinase coupled with ATP regeneration was reported.1) In this study, we demonstrated the practical possibility of producing 5?-GMP by phosphorylation of guanosine using a guanosine-inosine kinase from Exiguobacterium acetylicum coupled with ATP regeneration.
Key words: guanosine 5?-monophosphate; guanosine-inosine kinase; ATP regeneration
-40-
Note
Glycosidic Flavonoids as Rat-Liver Injury Preventing Compounds
from Green Tea
Shingo WADA, Puming HE, Ikue HASHIMOTO, Naoharu WATANABE,・・・
and Kimio SUGIYAMA
Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University,
Shizuoka 422-8529, Japan
Received April 12, 2000; Accepted June 3, 2000
A glycosidic flavonoids-rich fraction from green tea leaves was purified to isolate five glycosidic flavonoids, guided by the detection of a preventive effect on D-galactosamine-induced liver injury in rats. These were identified as a flavone C-glycoside (1) and trisaccharide flavonols (2--5) based on the spectroscopic analyses. These compounds suppressed the D-galactosamine-induced increase of plasma alanine aminotransferase and asparatate aminotransferase activities in rats.
Key words: green tea; flavonoids; liver injury; D-galactosamine
-41-
Note
Trypsin Inhibitory Activity of Bovine Fetuin De-O-glycosylated
by Endo-α-N-acetylgalactosaminidase
Hisashi ASHIDA,・・・ Kenji YAMAMOTO, and Hidehiko KUMAGAI
Laboratory of Molecular Biology of Bioresponce, Division of Integrated Life Science,
Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan
Received April 17, 2000; Accepted June 20, 2000
The effects of bovine fetuin O-glycans on its trypsin inhibitory activity were examined. De-sialylated (asialo-) and de-O-glycosylated fetuin were prepared from native fetuin using Arthrobacter neuraminidase and the mixture of it and Bacillus endo-α-N-acetylgalactosaminidase, respectively. De-sialylation and de-O-glycosylation from fetuin were confirmed with SDS-PAGE followed by western blotting using anti-human Thomsen-Friedenreich antigen (T antigen) antibody which recognizes O-linked galactosyl β1,3 N-acetylgalactosamine (Galβ1→3GalNAc). Native fetuin completely inhibited the trypsin activity at about a 1:1 molar ratio. In contrast, the trypsin inhibitory activity of asialo- and de-O-glycosylated fetuin decreased about a half and one-third of that of native fetuin, respectively.
Key words: fetuin; trypsin inhibitor; endo-α-N-acetylgalactosaminidase; O-glycan
-42-
Note
cis-N-(p-Coumaroyl)serotonin from Konnyaku,
Amorphophallus konjac K. Koch
Tatsuya NIWA, Hideo ETOH,＊,・・・ Atsushi SHIMIZU,＊＊ and Yasuo SHIMIZU＊＊
The United Graduate School of Agricultural Sciences, Gifu University (Shizuoka University),
Shizuoka 422-8529, Japan
＊Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka 422-8529, Japan
＊＊Toa Co. Ltd., 2-8-14 Toyota, Shizuoka 422-8027, Japan
Received April 18, 2000; Accepted June 15, 2000
Tobiko is produced as a by-product in the manufacture of konnyaku and comes to about 45％ of dried konnyaku-imo. For a possible use of this powder, we studied its components. Serotonin, trans-N-(p-coumaroyl) serotonin, and a new compound, cis-N-(p-coumaroyl)serotonin, were characterized from this powder.
Key words: konnyaku; tobiko; serotonin; cis-N-
(p-coumaroyl)serotonin; trans-N-(p-coumaroyl)serotonin
-43-
Note
Complete Amino Acid Sequences of Three Proteinase Inhibitors
from White Sword Bean (Canavalia gladiata)
Sung-Soo PARK, Toshihisa SUMI, Hideki OHBA,・・・ Osamu NAKAMURA,
and Makoto KIMURA＊
Materials Chemistry Department, Kyushu National Industrial Research Institute, Shuku-machi,
Tosu, Saga 841-0052, Japan
＊Laboratory of Biochemistry, Faculty of Agriculture, Kyushu University, Fukuoka 812-0053, Japan
Received May 8, 2000; Accepted June 6, 2000
Three major serine proteinase inhibitors (SBI-1, -2, and -3) were purified from the seeds of white sword bean (Canavalia gladiata) by FPLC and reversed-phase HPLC. The sequences of these inhibitors were established by automatic Edman degradation and TOF-mass spectrometry. SBI-1, -2, and -3 consisted of 72, 73, and 75 amino acid residues, with molecular masses of 7806.5, 7919.8, and 8163.4, respectively. The sequences of SBI-1 and -2 coincided with those of CLT I and II [Terada et al. (1994) Biosci. Biotech. Biochem., 58, 376--379] except only N- or C-terminal amino acid residues. Analysis of the amino acid sequences showed that the active sites of the inhibitors contained a Lys21-Ser22 against trypsin and Leu48-Ser49 against chymotrypsin, respectively. Further, it became apparent that about seven disulfide bonds were present. These results suggest that sword bean inhibitors are members of the Bowman-Birk proteinase inhibitor family.
Key words: amino acid sequence; Canavalia gladiata; sword bean proteinase inhibitor; Bowman-Birk family
-44-
Preliminary Communication
New Scheme of the Biosynthesis of Formononetin Involving
2,7,4'-Trihydroxyisoflavanone but Not Daidzein as the Methyl Acceptor
Tomoyoshi AKASHI, Yuji SAWADA, Toshio AOKI, and Shin-ichi AYABE・・・
Department of Applied Biological Sciences, Nihon University, Fujisawa, Kanagawa 252-8510, Japan
Received May 19, 2000; Accepted June 26, 2000
Glycyrrhiza echinata cell-free extract produced isoformononetin by the 7-O-transmethylation of daidzein from S-adenosyl-L-methionine (SAM). When the yeast microsome expressing 2-hydroxyisoflavanone synthase was mixed with the cell-free extract and incubated with liquiritigenin and SAM, formononetin emerged. Furthermore, the cell-free extract yielded formononetin on incubation with 2,7,4?-trihydroxyisoflavanone and SAM. We propose a novel pathway of formononetin biosynthesis involving 2,7,4?-trihydroxyisoflavanone as the methyl acceptor.
Key words: Glycyrrhiza echinata; Fabaceae; cytochrome P450; flavonoid; O-methyltransferase