(Vol.68 No.10 2004)
Review
Protein Traffic for Secretion and Related Machinery of Bacillus subtilis
Kunio Yamane, Keigo Bunai, and Hiroshi Kakeshita p.2007
Effect of MMP/ADAM Inhibitors on Goblet Cell Hyperplasia in Cultured
Human Bronchial Epithelial Cells
Hajime Yoshisue and Kazuhide Hasegawa p.2024
Glucosylation of Phenolic Compounds by Pharbitis nil Hairy Roots:
I. Glucosylation of Coumarin and Flavone Derivatives
Hideki Kanho1, Sayaka Yaoya1, Tomio Itani1, Takahisa Nakane2, Nobuo Kawahara3,
Yoichi Takase4, Kazuo Masuda4, and Masanori Kuroyanagi1 p.2032
Cloning and Enhanced Expression of the Cytochrome P450nor Gene
(nicA; CYP55A5) Encoding Nitric Oxide Reductase from Aspergillus oryzae
Masahiko Kaya1, Kengo Matsumura1, Katsuya Higashida1, Yoji Hata1, Akitsugu Kawato1,
Yasuhisa Abe1, Osamu Akita2, Naoki Takaya3, and Hirofumi Shoun4 p.2040
Hazelnut Oil Administration Reduces Aortic Cholesterol Accumulation
and Lipid Peroxides in the Plasma, Liver, and Aorta of Rabbits Fed a High-cholesterol
Diet
Aydan Hatipoglu1, Oznur Kanba li1, Jale Balkan1, Mutlu Kucuk2, U ur Cevikba
3, Gulcin Aykac-toker1, Hakan Berkkan4, and Mujdat Uysal1 p.2050
The Pro-peptide of Streptomyces mobaraensis Transglutaminase Functions
in cis and in trans to Mediate Efficient Secretion of Active Enzyme from Methylotrophic
Yeasts
Hiroya Yurimoto1, Maiko Yamane1, Yoshimi Kikuchi2, Hiroshi Matsui2, Nobuo Kato1,
and Yasuyoshi Sakai1 p.2058
Substrate Specificities of Several Prenyl Chain Elongating Enzymes
with Respect to 4-Methyl-4-pentenyl Diphosphate
Masahiko Nagaki1, Yohei Miki1, Minori Nakada1, Jun Kawakami1, Haruo Kitahara2,
Yuji Maki3, Yoshinori Gotoh3, Tokuzo Nishino4, and Tanetoshi Koyama5 p.2070
A Practical Method for Measuring Deoxynivalenol, Nivalenol, and
T-2 + HT-2 Toxin in Foods by an Enzyme-linked Immunosorbent Assay Using Monoclonal
Antibodies
Takumi Yoshizawa1, Hiroaki Kohno2, Kazuyuki Ikeda2, Tatsuya Shinoda2, Hiroaki
Yokohama2, Kazuki Morita2, Osamu Kusada3, and Yoshinori Kobayashi4 p.2076
Regioselectivity in β-Galactosidase-catalyzed Transglycosylation
for the Enzymatic Assembly of D-Galactosyl-D-mannose
Mariko Miyasato1 and Katsumi Ajisaka2 p.2086
Effect of β-Casein (1-28) on Proliferative Responses and Secretory
Functions of Human Immunocompetent Cell Lines
Takeshi Kawahara, Daisuke Katayama, and Hajime Otani p.2091
GroEL Chaperone Binding to Beetle Luciferases and the Implications
for Refolding When Co-expressed
Balan Venkatesh1, Mohammad Arifuzzaman2, Hirotada Mori2, Takahisa Taguchi1,
and Yoshihiro Ohmiya1 p.2096
Effects of Cross-sectional Area on Human Bite Studied with Raw
Carrot and Surimi Gel
Kaoru Kohyama1, Tomoko Sasaki1, Fumiyo Hayakawa1, and Eiko Hatakeyama2 p.2104
Cloning and Expression of a Novel Trichoderma viride Laminarinase
AI Gene (lamAI)
Rika Nobe1, Yoichi Sakakibara1, Kihachiro Ogawa2, and Masahito Suiko1 p.2111
A Novel Zinc-containing α-Keto Ester Reductase from Actinomycete:
An Approach Based on Protein Chemistry and Bioinformatics
Kohji Ishihara1, Hitomi Yamaguchi2, Taketo Omori3, Tadashi Uemura3, Nobuyoshi
Nakajima4, and Nobuyoshi Esaki3 p.2120
Cleavage of Various Peptides with Pitrilysin from Escherichia
coli: Kinetic Analyses Using β-Endorphin and Its Derivatives
Joel Cornista1, Satoshi Ikeuchi1, Mitsuru Haruki1, Atsuko Kohara2, Kazufumi
Takano1,3, Masaaki Morikawa1, and Shigenori Kanaya1 p.2128
Gene Cloning and Biochemical Characterizations of Thermostable
Ribonuclease HIII from Bacillus stearothermophilus
Hyongi Chon1, Rikita Nakano1, Naoto Ohtani1, Mitsuru Haruki1, Kazufumi Takano1,2,
Masaaki Morikawa1, and Shigenori Kanaya1 p.2138
Utility of Dry Gel from Two-dimensional Electrophoresis for Peptide
Mass Fingerprinting Analysis of Silkworm Proteins
Pingbo Zhang1, Kohji Yamamoto1, Yongqiang Wang1, Yutaka Banno1, Hiroshi Fujii1,
Fumio Miake2, Nobuhiro Kashige2, and Yoichi Aso3 p.2148
Dietary Resistant Starch Alters the Characteristics of Colonic
Mucosa and Exerts a Protective Effect on Trinitrobenzene Sulfonic Acid-induced
Colitis in Rats
Tatsuya Morita1, Hiroki Tanabe1, Kimio Sugiyama1, Seiichi Kasaoka2, and Shuhachi
Kiriyama3 p.2155
Spectrophotometric and Kinetic Studies on the Binding of the Bioflavonoid
Quercetin to Bovine Serum Albumin
Trevor M. Kitson p.2165
Biotransformation of Various Alkanes Using the Escherichia coli
Expressing an Alkane Hydroxylase System from Gordonia sp. TF6
Tadashi Fujii1, Tatsuya Narikawa1, Koji Takeda1, and Junichi Kato2 p.2171
Structure-activity Relationship for FR901464: A Versatile Method for the Conversion
and Preparation of Biologically Active Biotinylated Probes
Hajime Motoyoshi1, Masato Horigome1, Ken Ishigami1, Tatsuhiko Yoshida2, Sueharu
Horinouchi2, Minoru Yoshida2,3,4, Hidenori Watanabe1, and Takeshi Kitahara1
p.2178
Note
Synthesis and Antioxidative Activity of 3 ,4 ,6,7-Tetrahydroxyaurone, a Metabolite
of Bidens frondosa
Somepalli Venkateswarlu, Gopala K. Panchagnula, and Gottumukkala V. Subbaraju
p.2183
Note
Effect of Moisture Content on the Expansion Volume of Popped Amaranth Seeds
by Hot Air and Superheated Steam Using a Fluidized Bed System
Yotaro Konishi1, Hiroyuki Iyota2, Kaori Yoshida1, Junko Moritani1, Tamotsu Inoue2,
Nobuya Nishimura2, and Tomohiro Nomura2 p.2186
Note
Detection of Antigen-antibody Reaction Using a Fluorescent Probe and Its Application
to Homogeneous Competitive-type Immunoassay for Insulin
Hiroshi Oneda and Kuniyo Inouye p.2190
Note
Purification and Antifungal Activity of Recombinant Chitinase from Escherichia
coli Carrying the Family 19 Chitinase Gene of Streptomyces sp. J-13-3
Yousuke Yamashita and Katsuichiro Okazaki p.2193
Note
A Synthetic Polymer, Poly(2-methacryloyloxyethyl phosphorylcholine-co-n-stearyl
methacrylate), Stimulates Insulin Release from RINm5F Insulinoma Cells
Susumu Maruyama, Jianen Hu, Akiko Yamanaka, and Toshiaki Ichimura p.2197
Note
Absolute Stereochemistry of Acremolactone A, a Novel Herbicidal Epoxydihydropyranyl γ-Lactone from Acremonium roseum I4267
Takeshi Sassa1, Takahiro Ooi1, Hiroshi Kinoshita1, and Katsuhide Okada2 p.2201
Note
Content and Chemical Compositions of Cerebrosides in Lactose-assimilating Yeasts
Mikio Tanji1,2, Kanae Namimatsu1, Mikio Kinoshita1,2, Hidemasa Motoshima3, Yuji
Oda4, and Masao Ohnishi1,2 p.2205
Communication
Transcriptional Coactivator p300/CBP-Associated Factor and p300/CBP-Associated
Factor Type B Are Required for Normal Estrogen Response of the Mouse Uterus
Erina Inoue1,2, Miho Hanai1,3, Kazuhiko Yamada1, Takatoshi Esashi1,3, and Jun
Yamauchi1,2 p.2209
Communication
Biosynthesis of Fukinolic Acid Isolated from Petasites japonicus
Yasuhiro Hasa1 and Hiroyuki Tazaki1,2 p.2212
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Review
Protein Traffic for Secretion and Related Machinery of Bacillus subtilis
Kunio Yamane, Keigo Bunai, and Hiroshi Kakeshita
Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan
Gram-positive sporulating Bacillus subtilis secretes high levels of protein. Its complete genome sequence, published in 1997, encodes 4,106 proteins. Bioinformatic searches have predicted that about half of all B. subtilis proteins are related to the cell membrane through export to the extracellular medium, insertion, and attachment. Key features of the B. subtilis protein secretion machinery are the absence of an Escherichia coli SecB homolog and the presence of an SRP (signal recognition particle) that is structurally rather similar to human SRP. In addition, B. subtilis contains five type I signal peptidases (SipS, T, U, V, and W). Our in vitro assay system indicated that co-operation between the SRP-protein targeting system to the cell membrane and the Sec protein translocation machinery across the cytoplasmic membrane constitutes the major protein secretion pathway in B. subtilis. Furthermore, the function of the SRP-Sec pathway in protein localization to the cell membrane and spore was analyzed.
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Effect of MMP/ADAM Inhibitors on Goblet Cell Hyperplasia in Cultured Human Bronchial
Epithelial Cells
Hajime Yoshisue and Kazuhide Hasegawa
Pharmaceutical Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 1188 Shimotogari, Nagaizumi, Sunto, Shizuoka 411-8731, Japan
While epidermal growth factor receptor (EGFR) plays a pivotal role in the repair process of epithelial cells, it is also involved in the overproduction of mucus and goblet cell hyperplasia (GCH), which occurs in chronic airway diseases such as asthma. Among the EGFR ligands, transforming growth factor (TGF)-α is thought to be the most important in the synthesis of mucus. Pro-TGF-α is cleaved to give an active form by members of the matrix metalloproteinases (MMP)/a disintegrin and metalloproteinases (ADAM) family. Thus MMP/ADAM inhibitors might prevent GCH by inhibiting transactivation of EGFR. Upon stimulation of differentiating normal human bronchial epithelial (NHBE) cells by IL-13, GCH was induced. The mucin genes MUC5AC, MUC5B, and MUC2 were upregulated whereas the expression of ciliated cell markers was greatly repressed. GM6001, a broad-spectrum inhibitor for MMP/ADAM, inhibited IL-13-induced mucin gene expression and mucus production as measured by periodic acid-Schiff staining. This was accompanied by an inhibition of TGF-α release. These results suggest that MMP/ADAMs play a pivotal role in the development of GCH in lung epithelial cells.
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Glucosylation of Phenolic Compounds by Pharbitis nil Hairy Roots: I. Glucosylation
of Coumarin and Flavone Derivatives
Hideki Kanho1, Sayaka Yaoya1, Tomio Itani1, Takahisa Nakane2, Nobuo Kawahara3, Yoichi Takase4, Kazuo Masuda4, and Masanori Kuroyanagi1
1School of Bioresources, Hiroshima Prefectural University, 562 Nanatsuka, Shobara, Hiroshima 727-0023, Japan
2Tsukuba Medicinal Plant Research Station, National Institute of Health Sciences, 1 Hachimandai, Tsukuba, Ibaraki 305-0843, Japan
3National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya, Tokyo 158-8501, Japan
4Showa Pharmaceutical University, 3-3165 Higashi-Tamagawagakuen, Machida, Tokyo 194-8543, JapanHairy roots of medicinal morning glory (Pharbitis nil) showed potent glucosylation activity against umbelliferone and aesculetin, so the glucosylation activity against several phenolic compounds was tested. Some coumarin derivatives and flavone derivatives having phenolic hydroxyl groups were incubated with the hairy roots. The coumarin derivatives and flavone derivatives almost disappeared from the culture medium in half a day. In the case of the coumarin derivatives, a 7-hydroxyl group was easily glucosylated. A methyl group at C-8 somewhat decreased the glucosylation to a hydroxyl group at C-7 of the coumarin skeleton. The 4-hydroxy coumarin derivatives were changed to acetophenone-type glucosides by incubation with the hairy roots through decarboxylation.
Several flavonol derivatives were tested for glucosylation by the hairy roots. 3-Hydroxy flavone, 3.6-dihydroxyflavone and 3,7-dihydroxyflavone were glucosylated to give 3-glucosylated derivatives. Of these, 3,6-dihydroxyflavone was highly glucosylated, but not 3-hydroxyflavone or 3,7-dihydroxyflavone to the same degree. In the case of the flavones, a 3-hydroxy group could be predominantly glucosylated, and hydroxyl groups on the A and B ring of the flavones affected glucosylation by the hairy roots.
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Cloning and Enhanced Expression of the Cytochrome P450nor Gene (nicA; CYP55A5)
Encoding Nitric Oxide Reductase from Aspergillus oryzae
Masahiko Kaya1, Kengo Matsumura1, Katsuya Higashida1, Yoji Hata1, Akitsugu Kawato1, Yasuhisa Abe1, Osamu Akita2, Naoki Takaya3, and Hirofumi Shoun4
1Research Institute, Gekkeikan Sake Co., Ltd., 300 Katahara-cho, Fushimi-ku, Kyoto 612-8361, Japan
2National Research Institute of Brewing (NRIB), 3-7-1 Kagamiyama, Higashi-hiroshima, Hiroshima 739-0046, Japan
3Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan
4Department of Biotechnology, Graduate School of Agricultural and Life Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113-8657, JapanWe cloned and characterized the gene and cDNA of Aspergillus oryzae cytochrome P450nor (Anor). The Anor gene (nicA; CYP55A5) has a different gene structure from other P450nor genes in that it has an extra intron. There were not only two kinds of mRNA but also two sets of TATA-box and CCAAT-box, and it appears that this gene has two expression patterns, like CYP55A1 of Fusarium oxysporum. A reporter analysis using the uidA gene indicated that gene expression of CYP55A5 was induced under anaerobic conditions, like CYP55A1. When the CYP55A5 gene was overexpressed in A. oryzae, a large amount of active Anor were accumulated as intracellular protein. Anor employed both NADH and NADPH as electron donors for reducing nitric oxide to nitrous oxide. Anor measured the amount of NO generated from 3-(2-Hydroxy-1-(1-methylethyl)-2-nitrosohydrazino)-1-propanamine (NOC5) with a spectrophotometer. The sensitivity was 10 nmol/ml.
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Hazelnut Oil Administration Reduces Aortic Cholesterol Accumulation and Lipid
Peroxides in the Plasma, Liver, and Aorta of Rabbits Fed a High-cholesterol
Diet
Aydan Hatipoglu1, Oznur Kanba li1, Jale Balkan1, Mutlu Kucuk2, U ur Cevikba 3, Gulcin Aykac-toker1, Hakan Berkkan4, and Mujdat Uysal1
1Department of Biochemistry, Istanbul Faculty of Medicine, University of Istanbul, 34093 Capa-Istanbul, Turkey
2The Experimental and Medical Research Center, University of Istanbul, Istanbul, Turkey
3Department of Pathology, Istanbul Faculty of Medicine, University of Istanbul, Istanbul, Turkey
4Department of Biochemistry, Pharmacy Faculty, University of Istanbul, Istanbul, TurkeyHazelnut oil (HO) is rich in monounsaturated fatty acids and antioxidants. We wanted to investigate the effect of HO on lipid levels and prooxidant-antioxidant status in rabbits fed a high-cholesterol (HC) diet. An HC diet caused significant increases in lipids and lipid peroxide levels in the plasma, liver, and aorta together with histopathological atherosclerotic changes in the aorta. Glutathione levels, glutathione peroxidase, and glutathione transferase activities decreased significantly, but superoxide dismutase activity and vitamin E and C levels remained unchanged in the livers of rabbits following HC diet. HO supplementation reduced plasma, liver, and aorta lipid peroxide levels and aorta cholesterol levels together with amelioration in atherosclerotic lesions in the aortas of rabbits fed an HC diet, without any decreasing effect on cholesterol levels in the plasma or liver. HO did not alter the antioxidant system in the liver in the HC group. Our findings indicate that HO reduced oxidative stress and cholesterol accumulation in the aortas of rabbits fed an HC diet.
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The Pro-peptide of Streptomyces mobaraensis Transglutaminase Functions in cis
and in trans to Mediate Efficient Secretion of Active Enzyme from Methylotrophic
Yeasts
Hiroya Yurimoto1, Maiko Yamane1, Yoshimi Kikuchi2, Hiroshi Matsui2, Nobuo Kato1, and Yasuyoshi Sakai1
1Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto 606-8502, Japan
2Ajinomoto Institute of Life Sciences, 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki 210-8681, JapanTransglutaminase (TGase) from the actinomycete Streptomyces mobaraensis is a useful enzyme in the food industry, and development of an efficient production system for it would be desirable. Herein we report secretion of TGase in an enzymatically active form by methylotrophic yeasts as expression hosts. Secretory production of active TGase required a pro-peptide from TGase. When an artificial Kex2-endopeptidase recognition site was placed between the pro-peptide and mature TGase, secretion and in vitro maturation of TGase depended on Kex2-dependent cleavage. Unexpectedly, coexpression of unlinked pro-peptide with mature TGase yielded efficient secretion of the active enzyme. These results indicate that the pro-peptide from TGase functions not only in an intramolecular but also in an intermolecular manner. Site-directed mutagenesis of putative N-glycosylation sites increased the productivity of the active TGase further. A recombinant Candida boidinii strain was found to secrete active TGase up to 1.83 U/ml (about 90 mg/l) after 119 h of cultivation.
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Substrate Specificities of Several Prenyl Chain Elongating Enzymes with Respect
to 4-Methyl-4-pentenyl Diphosphate
Masahiko Nagaki1, Yohei Miki1, Minori Nakada1, Jun Kawakami1, Haruo Kitahara2, Yuji Maki3, Yoshinori Gotoh3, Tokuzo Nishino4, and Tanetoshi Koyama5
1Department of Materials Science and Technology, Faculty of Science and Technology, Hirosaki University, 3 Bunkyo-cho, Hirosaki, Aomori 036-8561, Japan
2Department of Natural Science, Faculty of Education, Hirosaki University, 1 Bunkyo-cho, Hirosaki, Aomori 036-8560, Japan
3Department of Biological and Material Chemistry, Faculty of Science, Yamagata University, Kojirakawa-machi, Yamagata 990-8560, Japan
4Department of Biochemistry and Engineering, Faculty of Engineering, Tohoku University, Aoba-ku, Sendai, Miyagi 980-8579, Japan
5Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Aoba-ku, Sendai, Miyagi 980-8577, JapanIn order to develop synthetic methods for biologically active homoallylic terpene sulfates, we examined the applicability and substrate specificities of several prenyl chain elongating enzymes with respect to 4-methyl-4-pentenyl diphosphate (homoIPP).
The reaction of dimethylallyl diphosphate with homoIPP by use of Bacillus stearothermophilus (all-trans)-farnesyl diphosphate synthase resulted in efficient yields of cis-(yield: 45.9%) and trans-4,8-dimethylnona-3,7-dien-1-ol (homoGOH, 25.5%), which has a carbon skeleton of 4,8-dimethylnona-3-en-1-sulfate, an antiproliferative compound from a marine organism (Aiello, A. et al., Tetrahedron, 53, 11489-11492 (1997)). The homoIPP was found to be also active as a homoallylic substrate in place of isopentenyl diphosphate for Sulfolobus acidocaldarius geranylgeranyl diphosphate synthase to give diphosphate of cis- and trans-4,8,12-trimethyltrideca-3,7,11-trien-1-ol, for Micrococcus luteus B-P 26 hexaprenyl diphosphate synthase to give cis- and trans-4,8,12,16-tetramethylheptadeca-3,7,11,15-tetraen-1-ol (homoGGOH), and for Micrococcus luteus B-P 26 undecaprenyl diphosphate synthase to give cis-homoGGOH exclusively.
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A Practical Method for Measuring Deoxynivalenol, Nivalenol, and T-2 + HT-2 Toxin
in Foods by an Enzyme-linked Immunosorbent Assay Using Monoclonal Antibodies
Takumi Yoshizawa1, Hiroaki Kohno2, Kazuyuki Ikeda2, Tatsuya Shinoda2, Hiroaki Yokohama2, Kazuki Morita2, Osamu Kusada3, and Yoshinori Kobayashi4
1Department of Biochemistry and Food Science, Faculty of Agriculture, Kagawa University, Miki, Kagawa 761-0795, Japan
2Fuji Research Laboratories, Kyowa Medex Co., Ltd., Nagaizumi-cho, Shizuoka 411-0932, Japan
3KM Assay Center, Kyowa Medex Co., Ltd., Nagaizumi-cho, Shizuoka 411-0932, Japan
4Tsukuba Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., Tsukuba, Ibaragi 305-0841, JapanWe have developed and tested an enzyme-linked immunosorbent assay system for individual measurement of deoxynivalenol, nivalenol, and T-2 + HT-2 toxin using monoclonal antibodies for 3,4,15-triacetyl-nivalenol, for both 3,4,15-triacetyl-nivalenol and 3,15-diacetyl-deoxynivalenol, and for acetyl-T-2 toxin. The assay system comprised three kits (desinated the DON + NIV kit, the NIV kit, and the T-2 + HT-2 kit). The practical performance of the enzyme-linked immunosorbent assay system was assessed by assaying trichothecene mycotoxins in wheat kernels. The enzyme-linked immunosorbent assay system meets all the requirements for use in a routine assay in terms of sensitivity (detection limit: deoxynivalenol 80 ng/g, nivalenol 80 ng/g, T-2 toxin 30 ng/g), reproducibility (total coefficient of variation: 1.9-6.2%), accuracy (recovery: 93.8-112.0%), simplicity and rapidity (time required: <2 h), mass handling (>42 samples/assay), and a good correlation with gas chromatography-mass spectrometry (r=0.9146-0.9991). Components derived from the wheat extract did not interfere with the assay kits. The enzyme-linked immunosorbent assay system is a useful alternative method to gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, or liquid chromatography-ultraviolet absorption for screening cereals and foods for trichothecene mycotoxin contamination.
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Regioselectivity in β-Galactosidase-catalyzed Transglycosylation for the Enzymatic
Assembly of D-Galactosyl-D-mannose
Mariko Miyasato1 and Katsumi Ajisaka2
1Food Science Institute, Meiji Dairies Co., 540 Naruda, Odawara, Kanagawa 250-0862, Japan
2Niigata University of Pharmacy and Applied Life Sciences, 265-1 Higashijima, Niitsu, Niigata 956-8603, JapanThe regioselectivity of β-galactosidase derived from Bacillus circulans ATCC 31382 (β-1,3-galactosidase) in transgalactosylation reactions using D-mannose as an acceptor was investigated. This D-mannose associated regioselectivity was found to be different from reactions using either GlcNAc or GalNAc as acceptors, not only for β-1,3-galactosidase but also for β-galactosidases of different origins. The relative hydrolysis rate of Galβ-pNP and D-galactosyl-D-mannoses, of various linkages, was also measured in the presence of β-1,3-galactosidase and was found to correlate well with the ratio of disaccharides formed by transglycosylation. The unexpected regioselectivity using D-mannose can therefore be explained by an anomalous specificity in the hydrolysis reaction. By utilizing the identified characteristics of both regioselectivity and hydrolysis specificity using D-mannose, an efficient method for enzymatic synthesis of β-1,3-, β-1,4- and β-1,6-linked D-galactosyl-D-mannose was subsequently established.
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Effect of β-Casein (1-28) on Proliferative Responses and Secretory Functions
of Human Immunocompetent Cell Lines
Takeshi Kawahara, Daisuke Katayama, and Hajime Otani
Integrated Department of Sciences of Functional Foods, Graduate School of Agriculture, Shinshu University, 8304 Minamiminowa, Kamiina, Nagano 399-4598, Japan
The effect of bovine β-casein (1-28) purified from commercial casein phosphopeptide preparations on human T, B, and monocyte cell lines was evaluated. Beta-casein (1-28) enhanced the proliferation of the following: T cell lines HUT-78, Jurkat Clone E6-1, and MOLT-4; B cell lines BALL, KHM-1B, and U266B1; and monocyte cell lines U937 and HL-60. Moreover, β-casein (1-28) stimulated IgA production by KHM-1B over 96 h of culture. Semiquantitative reverse transcriptional-polymerase chain reaction analysis indicated that β-casein (1-28) enhanced mRNA expression of interleukin (IL)-6 in U266B1 and KHM-1B. These results suggest that β-casein (1-28) exerts a mitogenic effect on human T, B, and monocyte cells, and an IgA-enhancing effect on B cells.
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GroEL Chaperone Binding to Beetle Luciferases and the Implications for Refolding
When Co-expressed
Balan Venkatesh1, Mohammad Arifuzzaman2, Hirotada Mori2, Takahisa Taguchi1, and Yoshihiro Ohmiya1
1Cell Dynamics Research Group, National Institute of Advanced Industrial Science and Technology, Midorigaoka, Ikeda, Osaka 563-8577, Japan
2Research and Education Center for Genetic Information, Nara Institute of Science and Technology, Ikoma 630-0101, JapanThe folding of many proteins including luciferase in vivo requires the assistance of molecular chaperone proteins. To understand how a chaperone targets luciferase, we took three luciferases that give different bioluminescence with the same luciferin substrate and with differences in homology. The three luciferase genes, firefly luciferase (FF-Luc) (from Pyrocoelia miyako), and red (RE-Luc) and green (GR-Luc) bioluminescence-emitting luciferases (from Phrixothrix railroad-worms), were expressed in Escherichia coli to produce fusion proteins with predicted molecular masses. Subsequently, we observed that DnaK and GroEL were co-purified along with recombinant luciferase. Although the amount of co-purified DnaK was almost the same compared to FF-Luc, GroEL was 25 and 32 times higher in GR-Luc and RE-Luc respectively. Furthermore, co-expression of GroEL/GroES along with luciferase substantially refolded RE-Luc and GR-Luc compared to FF-Luc.
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Effects of Cross-sectional Area on Human Bite Studied with Raw Carrot and Surimi
Gel
Kaoru Kohyama1, Tomoko Sasaki1, Fumiyo Hayakawa1, and Eiko Hatakeyama2
1National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan
2Kansei Fukushi Research Center, Tohoku Fukushi University, 6-149-1 Kunimigaoka, Aoba-ku, Sendai 989-3201, JapanThe effects of the cross-sectional area of food samples on bite force with molar teeth were investigated using raw carrots and surimi gels. We evaluated human bite force for food samples with different sizes between the upper and lower molars using a multiple-point sheet sensor and electromyography (EMG). The bite force curve and EMG clearly showed textural characteristics of the carrot and gel. In particular, the first peak in the bite curves corresponded to breaking point in the compression test. With increasing cross-sectional area of both foodstuffs, the bite force and contact area increased and the average stress to which the specimen was subjected (mean stress) tended to decrease, while the stress produced between the teeth and the specimen (active stress) did not change. Chewing rhythm and EMG activities were not greatly influenced by sample size. These findings suggest that higher bite force might cause difficulty in biting food with a larger cross-sectional area.
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Cloning and Expression of a Novel Trichoderma viride Laminarinase AI Gene (lamAI)
Rika Nobe1, Yoichi Sakakibara1, Kihachiro Ogawa2, and Masahito Suiko1
1Department of Biochemistry and Applied Biosciences, Faculty of Agriculture, University of Miyazaki, 1-1 Gakuen Kibanadai Nishi, Miyazaki 889-2192, Japan
2Department of Food Science for Health, Faculty of Health and Nutrition, Minami Kyushu University, 5-1-2 Kirishima, Miyazaki 880-0032, JapanThe gene lamAI, which encodes a novel laminarinase AI of Trichoderma viride U-1, was cloned using RT-PCR in conjunction with the rapid amplification of cDNA ends (RACE) technique. The open reading frame consisted of 2,277 bp encoding a protein of 759 amino acid residues, including a 32-residue signal prepropeptide. The protein showed 91% sequence similarity to the putative Trichoderma virens β-1,3-glucanase BGN1, but no significant similarity to fungal β-1,6-glucanases or β-1,3-glucanases from other organisms. On 40 h incubation with a solo carbon source, northern analysis revealed that the gene was induced by 0.5% laminaran from Eisenia bicyclis but was not by the same concentration of glucose. The lamAI cDNA was functionally expressed in the methylotrophic yeast Pichia pastoris, resulting in a recombinant enzyme with as high activity against laminaran as native LAMAI. Based on these data, the probable existence of endo-β-1,3:1,6-glucan hydrolases as a subclass of endo-β-1,3-glucanases in some mycoparasitic fungi is suggested.
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A Novel Zinc-containing α-Keto Ester Reductase from Actinomycete: An Approach
Based on Protein Chemistry and Bioinformatics
Kohji Ishihara1, Hitomi Yamaguchi2, Taketo Omori3, Tadashi Uemura3, Nobuyoshi Nakajima4, and Nobuyoshi Esaki3
1Department of Life Science, Okayama University of Science, 1-1 Ridai-cho, Okayama 700-0005, Japan
2Research and Development Center, Nagase & Co., Ltd., Nishi-ku, Kobe 651-2241, Japan
3Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan
4Graduate School of Health and Welfare Science, Okayama Prefectural University, Soja, Okayama 719-1197, JapanWe have achieved the purification of an α-keto ester reductase (SCKER) from S. coelicolor A3(2) whole cells. SCKER proved to be a homotetramer of 132 kDa containing one equivalent of zinc ion per subunit. The enzyme differed from other α-keto ester reductases from microorganisms with regard to subunit structure and metal ion dependency. From a computer search using the protein data banks, the N-terminal amino acid sequence of SCKER was consistent with that of a possible zinc containing alcohol dehydrogenase in S. coelicolor A3(2). None of three hypothetical proteins of S. coelocor A3(2) having a high homology sequence with those of already purified α-keto ester reductases from S. thermocyaneoviolaceus [Yamaguchi, H., et al., Biosci. Biotechnol. Biochem., 66, 588-597 (2002)] was identical with that of SCKER.
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Cleavage of Various Peptides with Pitrilysin from Escherichia coli: Kinetic
Analyses Using β-Endorphin and Its Derivatives
Joel Cornista1, Satoshi Ikeuchi1, Mitsuru Haruki1, Atsuko Kohara2, Kazufumi Takano1,3, Masaaki Morikawa1, and Shigenori Kanaya1
1Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan
2Protein Engineering Research Institute, 6-2-3 Furuedai, Suita, Osaka 565-0874, Japan
3PRESTO, JST, 2-1 Yamadaoka, Suita, Osaka 565-0871, JapanPitrilysin from Escherichia coli was overproduced, purified, and analyzed for enzymatic activity using 14 peptides as a substrate. Pitrilysin cleaved all the peptides, except for two of the smallest, at a limited number of sites, but showed little amino acid specificity. It cleaved β-endorphin (β-EP) most effectively, with a Km value of 0.36 μM and a kcat value of 750 min-1. β-EP consists of 31 residues and was predominantly cleaved by the enzyme at Lys19-Asn20. Kinetic analyses using a series of β-EP derivatives with N and/or C-terminal truncations and with amino acid substitutions revealed that three hydrophobic residues (Leu14, Val15, and Leu17) and the region 22-26 in β-EP are responsible for high-affinity recognition by the enzyme. These two regions are located on the N- and C-terminal sides of the cleavage site in β-EP, suggesting that the substrate binding pocket of pitrilysin spans its catalytic site.
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Gene Cloning and Biochemical Characterizations of Thermostable Ribonuclease
HIII from Bacillus stearothermophilus
Hyongi Chon1, Rikita Nakano1, Naoto Ohtani1, Mitsuru Haruki1, Kazufumi Takano1,2, Masaaki Morikawa1, and Shigenori Kanaya1
1Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan
2PRESTO, JST, 2-1 Yamadaoka, Suita, Osaka 565-0871, JapanThe gene encoding RNase HIII from the thermophilic bacterium Bacillus stearothermophilus was cloned and overexpressed in Escherichia coli, and the recombinant protein (Bst-RNase HIII) was purified and biochemically characterized. Bst-RNase HIII is a monomeric protein with 310 amino acid residues, and shows an amino acid sequence identity of 47.1% with B. subtilis RNase HIII (Bsu-RNase HIII). The enzymatic properties of Bst-RNase HIII, such as pH optimum, metal ion requirement, and cleavage mode of the substrates, were similar to those of Bsu-RNase HIII. However, Bst-RNase HIII was more stable than Bsu-RNase HIII, and the temperature (T1/2) at which the enzyme loses half of its activity upon incubation for 10 min was 55 -C for Bst-RNase HIII and 35 -C for Bsu-RNase HIII. The optimum temperature for Bst-RNase HIII activity was also shifted upward by roughly 20 -C as compared to that of Bsu-RNase HIII. The availability of such a thermostable enzyme will facilitate structural studies of RNase HIII.
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Utility of Dry Gel from Two-dimensional Electrophoresis for Peptide Mass Fingerprinting
Analysis of Silkworm Proteins
Pingbo Zhang1, Kohji Yamamoto1, Yongqiang Wang1, Yutaka Banno1, Hiroshi Fujii1, Fumio Miake2, Nobuhiro Kashige2, and Yoichi Aso3
1Laboratory of Insect Genetic Resources, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan
2Microbiology Laboratory, Faculty of Pharmaceutical Sciences, Fukuoka University, Fukuoka 814-0181, Japan
3Laboratory of Genetic and Protein Engineering, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, JapanWe compared the use of wet and dry two-dimensional electrophoresis (2-DE) gels for in-gel tryptic digestion and subsequent analysis by mass spectrometry, first using bovine serum albumin (BSA) as a model protein and then using unknown proteins from an extract of the silkworm midgut. The gel was either dried at 80 -C or left wet. Upon analysis of BSA, there was little difference in peptide recovery from 2-DE or in mass spectrum between the dry and the wet gels. The midgut extract was resolved into more than 1,100 protein spots by 2-DE, and 40 of these spots were sampled for further analysis. For all of the 40 proteins, the results obtained from dry and wet gels were quite similar in mass spectra and protein identification, although the relative amounts of peptides from tryptic digestion ranged from 45 to 146%. Based on these results, we confirmed the utility of dry electrophoretic gels for proteomics of insect extracts.
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Dietary Resistant Starch Alters the Characteristics of Colonic Mucosa and Exerts
a Protective Effect on Trinitrobenzene Sulfonic Acid-induced Colitis in Rats
Tatsuya Morita1, Hiroki Tanabe1, Kimio Sugiyama1, Seiichi Kasaoka2, and Shuhachi Kiriyama3
1Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka 422-8529, Japan
2Bunkyo University Women s College, 1100 Namegaya, Chigasaki, Kanagawa 253-8550, Japan
3Faculty of Nutritional Sciences, University of Shizuoka, Yada 52-1, Shizuoka 422-8526, JapanThe protective effect of a dietary high-amylose cornstarch (HAS) against trinitrobenzene sulfonic acid (TNBS)-induced colitis was examined in rats. Rats were fed a HAS-free basal diet or, a 15% or 30% HAS supplemented diet for 10 d, and then received intracolonic TNBS to induce colitis and fed the respective diets for a further 8 d. HAS ingestion significantly protected colonic injuries as evidenced by lower colonic myeloperoxidase activity. Rats fed the HAS diet showed greater cecal short-chain fatty acid (SCFA) production than those fed the basal diet. Further, just before TNBS administration, HAS ingestion dose-dependently increased fecal and cecal mucin contents, and protein and nucleic acid contents in the colonic mucosa. HAS ingestion also reduced colonic permeability. The protective effect of HAS ingestion on TNBS-induced colitis is perhaps exerted through alterations in colonic mucosa, possibly due to cecal SCFA production.
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Spectrophotometric and Kinetic Studies on the Binding of the Bioflavonoid Quercetin
to Bovine Serum Albumin
Trevor M. Kitson
Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand
This work investigates the binding of the bioflavonoid, quercetin, to bovine serum albumin (BSA) by spectrophotometric techniques involving both the conventional and stopped-flow methods. Both the neutral and negatively-charged forms of quercetin bound to BSA with a red shift in the maximal absorption. At high pH values, quercetin was rapidly degraded in an oxygen-dependent process, but this decomposition was substantially slower when the flavonoid was bound to BSA. At pH 7.4, the difference spectrum of quercetin with and without BSA was maximal at 425 nm; this wavelength can be conveniently used to monitor the extent and speed of binding. Spectrophotometric studies with a range of equimolar mixtures of quercetin and BSA at pH 7.4 suggest the binding was maximal when the concentration was 10 μM. It is postulated that the binding site of BSA for quercetin was less available at higher protein concentrations, perhaps because of conformational change or self-association. The rate of spectrophotometric change when quercetin bound to BSA was fairly slow; the process was not quite complete within 45 seconds and was biphasic. When a pre-mixed equimolar mixture of BSA and quercetin was diluted with an equal volume of the buffer, there was a surprising further increase in absorbance at 425 nm (rather than the fall anticipated if the binary complex were to dissociate). It is concluded that, upon dilution, the effective concentration of BSA s binding site increased, providing more scope for quercetin to bind.
-20-
Biotransformation of Various Alkanes Using the Escherichia coli Expressing an
Alkane Hydroxylase System from Gordonia sp. TF6
Tadashi Fujii1, Tatsuya Narikawa1, Koji Takeda1, and Junichi Kato2
1Bioresource Laboratories, Mercian Corporation, 1808 Nakaizumi, Iwata, Shizuoka 438-0078, Japan
2Department of Molecular Biotechnology Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8530, JapanBiotransformation using alkane-oxidizing bacteria or their alkane hydroxylase (AH) systems have been little studied at the molecular level. We have cloned and sequenced genes from Gordonia sp. TF6 encoding an AH system, alkB2 (alkane 1-monooxygenase), rubA3 (rubredoxin), rubA4 (rubredoxin), and rubB (rubredoxin reductase). When expressed in Escherichia coli, these genes allowed the construction of biotransformation systems for various alkanes. Normal alkanes with 5 to 13 carbons were good substrates for this biotransformation, and oxidized to their corresponding 1-alkanols. Surprisingly, cycloalkanes with 5 to 8 carbons were oxidized to their corresponding cycloalkanols as well. This is the first study to achieve biotransformation of alkanes using the E. coli expressing the minimum component genes of the AH system. Our biotransformation system has facilitated assays and analysis leading to improvement of AH systems, and has indicated a cycloalkane oxidation pathway in microorganisms for the first time.
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Structure-activity Relationship for FR901464: A Versatile Method for the Conversion
and Preparation of Biologically Active Biotinylated Probes
Hajime Motoyoshi1, Masato Horigome1, Ken Ishigami1, Tatsuhiko Yoshida2, Sueharu Horinouchi2, Minoru Yoshida2,3,4, Hidenori Watanabe1, and Takeshi Kitahara1
1Department 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
2Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
3Chemical Genetics Laboratory, RIKEN, Wako, Saitama 351-0198, Japan
4CREST Research Project, Japan Science and Technology Agency, Saitama 332-0012, JapanThe structure-activity relationship for FR901464, a potent cell-cycle inhibitor, was examined by synthesizing its analogs. A versatile method for converting FR901464 was devised. This method made it possible to synthesize biologically active FR901464-biotin conjugates which could be used to isolate the binding proteins.
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Note
Synthesis and Antioxidative Activity of 3 ,4 ,6,7-Tetrahydroxyaurone, a Metabolite
of Bidens frondosa
Somepalli Venkateswarlu, Gopala K. Panchagnula, and Gottumukkala V. Subbaraju
Laila Impex R & D Centre, Unit I, Phase III, Jawahar Autonagar, Vijayawada 520 007, India
3 ,4 ,6,7-Tetrahydroxyaurone (1a), an aurone isolated from Bidens frondosa, and five analogues (1b-1f) were synthesized from pyrogallol in three steps. The antioxidative activity of 1a-1f was determined by the superoxide free radical and 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging methods.
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Note
Effect of Moisture Content on the Expansion Volume of Popped Amaranth Seeds
by Hot Air and Superheated Steam Using a Fluidized Bed System
Yotaro Konishi1, Hiroyuki Iyota2, Kaori Yoshida1, Junko Moritani1, Tamotsu Inoue2, Nobuya Nishimura2, and Tomohiro Nomura2
1Department of Food Science and Nutrition, Graduate School of Human Life Science, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-8585, Japan
2Department of Mechanical and Physical Engineering, Graduate School of Engineering, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-8585, JapanThe effect of the initial moisture content (X0) of amaranth seeds on expansion volume after popping was examined in hot air and superheated steam (SHS), using a fluidized bed system (FBS). The moisturized seeds were prepared under various vapor pressures due to various saturated salt solutions. In hot air, the maximum expansion volume was shown by seeds having X0 of 0.16 at 260 -C for 15 sec, reaching 8.7-fold of the pre-popped seeds. Heating by SHS decreased the volume slightly. Thus, popping of amaranth seeds is influenced not only by the moisture content of the seeds, but also by moisture in the heat media.
-24-
Note
Detection of Antigen-antibody Reaction Using a Fluorescent Probe and Its Application
to Homogeneous Competitive-type Immunoassay for Insulin
Hiroshi Oneda and Kuniyo Inouye
Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
The interaction of bovine insulin with anti-human insulin antibody (mAb) was examined using a fluorescent probe. The fluorescence intensity of fluoresceinthiocarbamyl (FTC)-insulin was increased by adding mAb, and the increase was saturated at 53% at a molar ratio of FTC-insulin to mAb of 2.0. Based on the change in fluorescence intensity, a standard curve of the homogeneous competitive-type immunoassay was constructed, and the detection range of insulin was found to be 50-400 nM.
-25-
Note
Purification and Antifungal Activity of Recombinant Chitinase from Escherichia
coli Carrying the Family 19 Chitinase Gene of Streptomyces sp. J-13-3
Yousuke Yamashita and Katsuichiro Okazaki
Department of Life Sciences, Faculty of Agriculture, Kagawa University, Miki, Kagawa 761-0795, Japan
A recombinant chitinase was purified from the cell extract of Escherichia coli JM109 transformed by plasmid pUC19 carrying the gene encoding family 19 chitinase of Streptomyces sp. J-13-3 by column chromatography on DEAE-Sepharose, CM-Sepharose, and Bio-Gel P-100. The final preparation was homogenous in polyacrylamide gel electrophoresis. The molecular weight of the purified enzyme was estimated to be 32,000. The recombinant chitinase hydrolyzed the trimer to hexamer of N-acetylglucosamine and had the identical N-terminal amino acid sequence of the mature protein, indicating removal of the signal sequence by E. coli signal peptidase. The fungal growth in well (200 μl of medium) of microplate by measurement of absorbance at 595 nm indicated that the chitinase (10 μg) completely and half inhibited growth of Trichoderma reesei and Aspergillus niger respectively.
-26-
Note
A Synthetic Polymer, Poly(2-methacryloyloxyethyl phosphorylcholine-co-n-stearyl
methacrylate), Stimulates Insulin Release from RINm5F Insulinoma Cells
Susumu Maruyama, Jianen Hu, Akiko Yamanaka, and Toshiaki Ichimura
Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology, AIST Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan
A water-soluble phospholipid-like polymer, poly(2-methacryloyloxyethyl phosphorylcholine-co-n-stearyl methacrylate) (PMC18, average molecular weight = 4.3ﾗ104), at a concentration (0.5-5 mg/ml) showing no inhibition of cell proliferation, stimulated insulin release from RINm5F rat insulinoma cells in a concentration- and time-related manner. But poly(2-methacryloyloxyethyl phosphorylcholine) and other synthetic phospholipid-like polymers failed to stimulate insulin release.
-27-
Note
Absolute Stereochemistry of Acremolactone A, a Novel Herbicidal Epoxydihydropyranyl γ-Lactone from Acremonium roseum I4267
Takeshi Sassa1, Takahiro Ooi1, Hiroshi Kinoshita1, and Katsuhide Okada2
1Department of Bioresource Engineering, Faculty of Agriculture, Yamagata University, Wakaba-cho, Tsuruoka 997-8555, Japan
2Faculty of Education, Yamagata University, Koshirakawa-cho, Yamagata 990-8560, JapanAcremolactone A was chemically degraded to the bicyclic hemiacetal γ-lactone and an epoxycyclohexenol, and their stereochemistry was determined by spectroscopic methods. These observations and data from NOE experiments on acremolactone A led to the configurational assignment of all asymmetric carbons in acremolactone A, enabling its stereostructure to be established.
-28-
Note
Content and Chemical Compositions of Cerebrosides in Lactose-assimilating Yeasts
Mikio Tanji1,2, Kanae Namimatsu1, Mikio Kinoshita1,2, Hidemasa Motoshima3, Yuji Oda4, and Masao Ohnishi1,2
1Department of Bioresource Science, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan
2The United Graduate School of Agricultural Science, Iwate University, Morioka, Iwate 020-8550, Japan
3Research Center, Yotsuba Milk Products Co., Ltd., Kitahiroshima, Hokkaido 061-1264, Japan
4Department of Upland Agriculture, National Agricultural Research Center for Hokkaido Region, Memuro, Hokkaido 082-0071, JapanCerebrosides were found in ten lactose-assimilating yeasts. Representative component ceramide moieties of cerebrosides from nine of these yeasts contained 9-methyl-4-trans, 8-trans-sphingadienine, and 2-hydroxy fatty acid with carbon chain lengths of 16 or 18. The major ceramide moieties in Brettanomyces anomalus, however, differed from those in other yeasts, and were predominately moieties containing 2-hydroxymyristic acid. Thus we found that various cerebroside molecular species are present in yeasts.
-29-
Communication
Transcriptional Coactivator p300/CBP-Associated Factor and p300/CBP-Associated
Factor Type B Are Required for Normal Estrogen Response of the Mouse Uterus
Erina Inoue1,2, Miho Hanai1,3, Kazuhiko Yamada1, Takatoshi Esashi1,3, and Jun Yamauchi1,2
1Division of Applied Food Research, National Institute of Health and Nutrition, 1-23-1 Toyama, Shinjyuku, Tokyo 162-8636, Japan
2Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, 4-1-8 Honchyo, Kawaguchi, Saitama 332-0012, Japan
3Department of Human Life and Culture, Faculty of Humanities, Seitoku University, 550 Iwase, Matsudo, Chiba 271-8555, JapanMice with targeted gene disruption of one of the estrogen receptor coactivators, p300/CBP-associated factor (PCAF), and its counterpart, PCAF-B, were used to investigate the possible involvement of PCAF and PCAF-B in estrogen receptor-mediated actions in vivo. Among ovariectomized mice that were treated with estrogen, PCAF and PCAF/PCAF-B knockouts showed abnormal growth of the uterus compared with the wild type. The level of c-fos gene expression in the uterus was not induced by estrogen in the knockouts. These observations suggest that PCAF and PCAF-B are required for estrogen-dependent normal growth of the uterus via estrogen receptor-mediated transcriptional regulations.
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Communication
Biosynthesis of Fukinolic Acid Isolated from Petasites japonicus
Yasuhiro Hasa1 and Hiroyuki Tazaki1,2
1The United Graduate School of Agricultural Science, Iwate University, Morioka 020-8550, Japan
2Department of Bioresource Science, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro 080-8555, JapanThe biosynthesis of fukinolic acid, which had been isolated from the Japanese fuki vegetable, Petasites japonicus, was investigated by feeding selected 13C-labeled compounds to axenic cultures of P. japonicus. [1,2-13C2] sodium acetate and [1-13C] L-tyrosine were incorporated into the fukiic acid sub group, while [3-13C] L-phenylalanine was incorporated into the caffeic acid moiety.