(Vol.68 No.7 2004)
Purification and Characterization of a 24 kDa Protein from Tartary
Buckwheat Seeds
Zhuanhua Wang,1,† Zheng Zhang,1 Zhuohui Zhao,1 Gunilla Wieslander,2 Dan Norbäck,2
and Ivan Kreft3 p.1409
Cloning and Characterization of the Catabolite Control Protein
A Gene from Bacillus stearothermophilus No. 236
Il-Dong Choi,1 Gyong-Sik Ha,1 Kyung-Nam Kim,2 and Yong-Jin Choi1,† p.1414
Mass Spectrometric Assignment of Smith Degradation Glycopeptides
Derived from Ribonuclease B
Suthasinee Pitchayawasin and Minoru Isobe† p.1424
Cloning and Characterization of the Genes Encoding Enzymes for
the Protocatechuate Meta-degradation Pathway of Pseudomonas ochraceae NGJ1*
Kiyofumi Maruyama,1,† Tomohiko Shibayama,1 Aki Ichikawa,1 Yukari Sakou,1 Satsue
Yamada,1 and Hiroyuki Sugisaki2 p.1434
Superior Molasses Assimilation, Stress Tolerance, and Trehalose
Accumulation of Baker’s Yeast Isolated from Dried Sweet Potatoes (hoshi-imo)
Osamu Nishida,*,** Seigo Kuwazaki,** Chise Suzuki, and Jun Shima† p.1442
Characterization of the L-Lysine Biosynthetic Pathway in the Obligate
Methylotroph Methylophilus methylotrophus
Yoshiya Gunji, Nobuharu Tsujimoto,* Megumi Shimaoka, Yuri Ogawa-miyata, Shin-ichi
Sugimoto, and Hisashi Yasueda† p.1449
Preparation and Biological Activity of Molecular Probes to Identify
and Analyze Jasmonic Acid-binding Proteins
Yusuke Jikumaru,1 Tadao Asami,2 Hideharu Seto,2 Shigeo Yoshida,2 Tadashi Yokoyama,3
Naomi Obara,4 Morifumi Hasegawa,4 Osamu Kodama,4 Makoto Nishiyama,1 Kazunori
Okada,1 Hideaki Nojiri,1 and Hisakazu Yamane1,† p.1461
Divergent Structures of Carbazole Degradative car Operons Isolated
from Gram-negative Bacteria
Kengo Inoue,1 Jaka Widada,1 Seiichiro Nakai,1 Takayuki Endoh,1 Masaaki Urata,1
Yuji Ashikawa,1 Masaki Shintani,1 Yuko Saiki,1,2 Takako Yoshida,1 Hiroshi Habe,1
Toshio Omori,1,* and Hideaki Nojiri1,† p.1467
Cloning and Overexpression of the Exiguobacterium sp. F42 Gene
Encoding a New Short Chain Dehydrogenase, Which Catalyzes the Stereoselective
Reduction of Ethyl 3-Oxo-3-(2-thienyl)propanoate to Ethyl (S)-3-Hydroxy-3-(2-thienyl)propanoate
Masaru Wada,1,† Ayumi Yoshizumi,1 Yuki Furukawa,1 Hiroshi Kawabata,2 Makoto
Ueda,2 Hiroshi Takagi,1 and Shigeru Nakamori1 p.1481
Genetic Analysis of chs1+ and chs2+ Encoding Chitin Synthases
from Schizosaccharomyces pombe
Yasuhiro Matsuo,1 Katsunori Tanaka,1 Tsuyoshi Nakagawa,2 Hideyuki Matsuda,1
and Makoto Kawamukai1,† p.1489
Absorption of Acylated Anthocyanins in Rats and Humans after Ingesting
an Extract of Ipomoea batatas Purple Sweet Potato Tuber
Katsuhisa Harada,1,† Mitsuyoshi Kano,1 Tomomi Takayanagi,1 Osamu Yamakawa,2
and Fumiyasu Ishikawa1 p.1500
Molecular Cloning of a Genomic DNA Encoding Yam Class IV Chitinase
Takuji Mitsunaga,1 Minoru Iwase,1 Wimal Ubhayasekera,2 Sherry L. Mowbray,2 and
Daizo Koga1,† p.1508
Intracellular Localization of a Class IV Chitinase from Yam
Takuji Mitsunaga, Minoru Iwase, Dai Yuki, and Daizo Koga† p.1518
Metabolism of Benzoquinone by Yeast Cells and Oxidative Characteristics
of Corresponding Hydroquinone: Application to Highly Sensitive Measurement of
Yeast Cell Density by Using Benzoquinone and a Chemiluminescent Probe
Tadayuki Tsukatani,1,† Seiji Ide,1 Hiroyuki Ukeda,2 and Kiyoshi Matsumoto3
p.1525
Gene Cloning and Characterization of a Bacillus vietnamensis Metalloprotease
Mihwan Kim, Yoshitaka Nishiyama, Kiyoshi Mura,† Chiyoko Tokue, and Soichi Arai
p.1533
apg15-1, a UGA Mutant Allele in the Saccharomyces cerevisiae APG16
Gene, and Its Suppression by a Cytoplasmic Factor
Hiroshi Okazaki,1 Bun-ichiro Ono,3 Yoshinori Ohsumi,4 and Mariko Ohsumi1,2,†
p.1541
Glucosyldiacylglycerol Enhances Reciprocal Activation of Prourokinase
and Plasminogen
Wenhui Wu, Ritsuko Narasaki, Fumihiko Maeda, and Keiji Hasumi† p.1549
Cloning of the Ruminococcus albus cel5D and cel9A Genes Encoding
Dockerin Module-containing Endoglucanases and Expression of cel5D in Escherichia
coli
Hidenori Taguchi, Daisuke Hagiwara, Tsutomu Genma, Shuichi Karita,† Tetsuya
Kimura, Kazuo Sakka, and Kunio Ohmiya p.1557
Note
Expression of a Single-chain Antibody against Indole-3-acetic Acid in Escherichia
coli
Nobuhito Mitani1,2,† and Ryoji Matsumoto1 p.1565
Note
Effects of Protein Transport Inhibitors on the Distribution and Secretion of
the Fusion Protein RntA-EGFP in Aspergillus oryzae
Kumiko Masai, Jun-ichi Maruyama, Harushi Nakajima, and Katsuhiko Kitamoto†
p.1569
Note
Identification of (+)-Phyllocladene, (-)-Sandaracopimaradiene, and (+)-Kaurene
as New Fungal Metabolites from Fusicoccin-producing Phomopsis amygdali F6
Hiromichi Kenmoku,1,* Mio Tanaka,2 Koichi Ogiyama,3 Nobuo Kato,4 and Takeshi
Sassa1,2,† p.1574
Note
Running Inhibits Osteoporosis Induced by Protein-deficient (PD) Food Intake
Hajime Tamaki,1 Liman Sun,1 Yutaka Ohta,2 Naofumi Katsuyama,2 Tetsuji Ishimaru,3
and Isao Chinen1,† p.1578
Note
Purification and Characterization of O-Acetylserine Sulfhydrylase of Corynebacterium
glutamicum
Masaru Wada,† Naoki Awano, Hiroyuki Yamazawa, Hiroshi Takagi, and Shigeru Nakamori
p.1581
Note
New 2(3→ 20)Abeotaxane and 3,11-Cyclotaxane from Needles of Taxus cuspidata
Qing-Wen Shi,1,† Zuo-Ping Li,1 Ding Zhao,1 Ji-Shun Gu,2 Takayuki Oritani,3 and
Hiromasa Kiyota3,† p.1584
Note
8-Hydroxydaidzein, an Aldose Reductase Inhibitor from Okara Fermented with Aspergillus
sp. HK-388
Tomoyuki Fujita,† Tomoyoshi Funako, and Hideo Hayashi p.1588
Note
Mitogenic Properties of Pokeweed Lectin-D Isoforms on Human Peripheral Blood
Lymphocytes: Non-mitogen PL-D1 and Mitogen PL-D2
Kenichi Yamaguchi,1,2,† Mizuki Uechi,1 Yoshinori Katakura,3 Tatsuya Oda,2 and
Masatsune Ishiguro1,* p.1591
Note
Growth and Carotenoid Production of Thraustochytrium sp. CHN-1 Cultured under
Superbright Red and Blue Light-emitting Diodes
Yukiho Yamaoka,1,† Marvelisa L. Carmona,2 and Shinji Oota3 p.1594
Note
Enhanced Calcium Absorption in the Small Intestine by a Phytate-removed Deamidated
Soybean Globulin Preparation
Hitomi Kumagai,1,† Atsushi Koizumi,1 Akihiro Suda,1 Noriko Sato,1 Hidetoshi
Sakurai,1 and Hitoshi Kumagai2 p.1598
Note
Antimicrobial Activities of Diterpene Dialdehydes, Constituents from Myoga (Zingiber
mioga Roscoe), and Their Quantitative Analysis
Masako Abe,1 Yoshio Ozawa,1,† Yasushi Uda,2 Fuminori Yamada,3 Yasujiro Morimitsu,4
Yoshimasa Nakamura,5 and Toshihiko Osawa5 p.1601
Note
Classification of Brassinosteroid-regulated Genes Based on Expression Profiles
in bri1 and in Response to a Protein Kinase Inhibitor, Staurosporin
Hideki Goda, Yukihisa Shimada,† Shozo Fujioka, and Shigeo Yoshida p.1605
Note
Fusicocca-3(16),10(14)-diene, and β - and δ -Araneosenes, New Fusicoccin Biosynthesis-related
Diterpene Hydrocarbons from Phomopsis amygdali
Takeshi Sassa,1,2,† Hiromichi Kenmoku,2 Kayoko Nakayama,1 and Nobuo Kato3
p.1608
Communication
Production of Lupin Acid Phosphatase in Transgenic Rice for Use as a Phytate-hydrolyzing
Enzyme in Animal Feed
Akira Hamada,1 Ken-ichi Yamaguchi,1,* Michiko Harada,1 Seiya Nikumaru,1 Jun
Wasaki,2 Takuro Shinano,2 Hirokazu Matsui,3 Mitsuru Osaki,3 and Hideo Honda1,†
p.1611
Communication
Dihydrocoronatine, Promising Candidate for a Chemical Probe to Study Coronatine-,
Jasmonoid- and Octadecanoid-binding Protein
Munenori Suzuki,1 Morifumi Hasegawa,1,2 Osamu Kodama,1,2 and Hiroaki Toshima1,2,†
p.1617
Communication
Interaction between a Negative Regulator (Msa2/Nrd1) and a Positive Regulator
(Cpc2) of Sexual Differentiation in Schizosaccharomyces pombe
Hee Tae Jeong, Yasuo Oowatari, Michiyo Abe, Katsunori Tanaka, Hideyuki Matsuda,
and Makoto Kawamukai† p.1621
-1-
Purification and Characterization of a 24 kDa Protein from Tartary Buckwheat
Seeds
Zhuanhua Wang,1,† Zheng Zhang,1 Zhuohui Zhao,1 Gunilla Wieslander,2 Dan Norbäck,2 and Ivan Kreft3
1College of Life Science and Biotechnology, Shanxi University, Taiyuan 030006, China
2Department of Medical Sciences/Occupational and Environmental Medicine, Uppsala University, University Hospital, S-751 85 Uppsala, Sweden
3Biotechnical Faculty, University of Ljubljana, P.O. Box 2995, SI-1001 Ljubljana, Slovenia
A 24 kDa protein was isolated from tartary buckwheat seeds by using chromatography of Superdex 75 gel filtration and Resource Q ion-exchange column. SDS-PAGE and Sephacryl S-200 gel filtration were used to provide information about the molecular mass of the protein purified from tartary buckwheat. The protein was composed of 215 amino acid residues and showed strong IgE binding activity in an ELISA test to the sera colleted from two patients allergic to buckwheat. These results suggested that the purified 24 kDa protein from tartary buckwheat seeds was an important functional protein and was relatively specific for buckwheat-allergic patients. It should be a very useful tool in the diagnosis of buckwheat allergy in the future.
-2-
Cloning and Characterization of the Catabolite Control Protein A Gene from Bacillus
stearothermophilus No. 236
Il-Dong Choi,1 Gyong-Sik Ha,1 Kyung-Nam Kim,2 and Yong-Jin Choi1,†
1Graduate School of Life Science and Biotechnology, Korea University, Seoul 136-701, Korea
2Molecular Biology, Sejong University, Seoul 143-747, Korea
The gene encoding the catabolite control protein A (CcpA) of Bacillus stearothermophilus No. 236, a strong xylanolytic bacterium, was cloned, sequenced, and expressed in Escherichia coli. The nucleotide sequence of the ccpA gene corresponded to an open reading frame of 1,005 bp that encodes a polypeptide of 334 amino acid residues with a calculated molecular mass of 36,902 kDa. The CcpA protein belonging to the LacI/GalR family of transcriptional regulators was produced by a recombinant E. coli strain expressing the B. stearothermophilus No. 236 ccpA gene and purified to apparent homogeneity. The transcription start site was mapped at a position 63 nucleotides upstream of the translation initiation codon, and a presumed promoter sequence was also identified. The deduced amino acid sequence of the ccpA gene product contained the helix-turn-helix motif found in many DNA-binding proteins, and showed the highest identity (62%) with CcpA from B. subtilis. The B. stearothermophilus No. 236 ccpA gene was demonstrated to be able to complement a B. subtilis ccpA mutant that exhibited two distinct mutant phenotypes: a growth defect and a release of carbon catabolite repression (CCR). These results indicate that the ccpA gene product of B. stearothermophilus No. 236 is functionally active also in B. subtilis. Electrophoretic mobility shift assay with the purified CcpA revealed that the CcpA of B. stearothermophilus No. 236 bound specifically to the xynA creB (catabolite responsive element B) sequence. Taken together, these results strongly suggest that the CcpA protein participates in CCR of B. stearothermophilus No. 236 xynA gene.
-3-
Mass Spectrometric Assignment of Smith Degradation Glycopeptides Derived from
Ribonuclease B
Suthasinee Pitchayawasin and Minoru Isobe†
Laboratory of Organic Chemistry, School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan
We established a method to determine the glycosyl linkage structure by a combination of Smith degradation and liquid chromatography-electrospray ionization-quadrupole-time of flight-mass spectrometry (LC-ESI-Q-TOF-MS) and tandem MS (MS/MS). To assign the sugar linkage of N-glycoprotein, we employed a typical ribonuclease B containing oligosaccharides (Man5–9GlcNAc2). Tryptic digestion of ribonuclease B provided a mixture of high-mannose glycopeptides consisting of the four amino acids, Asn34-Leu-Thr-Lys37 (NLTK, T6). The mixture of glycopeptides was separated by high-performance liquid chromatography (HPLC) in a reversed phase column and was characterized by ESI-Q-TOF-MS and MS/MS. Comparison of the data with and without Smith degradation allowed us to make reasonable assignments to support such linkage patterns as (1→ 2), (1→ 3), (1→ 6) and their multiples. These assignments were limited to six mannoses or lower due to the unstable nature of the higher derivatives. This method should be applicable to determine the linkage pattern of an unknown glycoprotein in about a 6-microgram amount.
-4-
Cloning and Characterization of the Genes Encoding Enzymes for the Protocatechuate
Meta-degradation Pathway of Pseudomonas ochraceae NGJ1*
Kiyofumi Maruyama,1,† Tomohiko Shibayama,1 Aki Ichikawa,1 Yukari Sakou,1 Satsue Yamada,1 and Hiroyuki Sugisaki2
1Department of Biomolecular Science, Faculty of Engineering, Gifu University, Yanagido 1-1, Gifu 501-1193, Japan
2Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan
The 2-pyrone-4,6-dicarboxylate lactonase gene (proL), the protocatechuate 4,5-dioxygenase α and β subunits genes (proOa and proOb), and the 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase gene (proD) were cloned from the chromosomal DNA of Pseudomonas ochraceae NGJ1. These genes were in the order proLOaObD on the DNA, and a possible transcription terminator sequence followed. The proL and proD genes were over-expressed in Escherichia coli, and their gene products were purified for identification, while the expression of proOaOb was at a lower level. The protocatechuate meta-degradation operon was reconstituted with the recombinant plasmids and expressed successfully in E. coli.
-5-
Superior Molasses Assimilation, Stress Tolerance, and Trehalose Accumulation
of Baker’s Yeast Isolated from Dried Sweet Potatoes (hoshi-imo)
Osamu Nishida,*,** Seigo Kuwazaki,** Chise Suzuki, and Jun Shima†
National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan
Yeast strains were isolated from dried sweet potatoes (hoshi-imo), a traditional preserved food in Japan. Dough fermentation ability, freeze tolerance, and growth rates in molasses, which are important characteristics of commercial baker’s yeast, were compared between these yeast strains and a commercial yeast derivative that had typical characteristics of commercial strains. Classification tests including pulse-field gel electrophoresis and fermentation/assimilation ability of sugars showed that almost the stains isolated belonged to Saccharomyces cerevisiae. One strain, ONY1, accumulated intracellular trehalose at a higher level than commercial strain T128. Correlated with intracellular trehalose contents, the fermentation ability of high-sugar dough containing ONY1 was higher. ONY1 also showed higher freeze tolerance in both low-sugar and high-sugar doughs. The growth rate of ONY1 was significantly higher under batch and fed-batch cultivation conditions using either molasses or synthetic medium than that of strain T128. These results suggest that ONY1 has potential commercial use as baker’s yeast for frozen dough and high-sugar dough.
-6-
Characterization of the L-Lysine Biosynthetic Pathway in the Obligate Methylotroph
Methylophilus methylotrophus
Yoshiya Gunji, Nobuharu Tsujimoto,* Megumi Shimaoka, Yuri Ogawa-miyata, Shin-ichi Sugimoto, and Hisashi Yasueda†
Fermentation and Biotechnology Laboratories, Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki 210-8681, Japan
The L-lysine biosynthetic pathway of the gram-negative obligate methylotroph Methylophilus methylotrophus AS1 was examined through characterization of the enzymes aspartokinase (AK), aspartsemialdehyde dehydrogenase, dihydrodipicolinate synthase (DDPS), dihydrodipicolinate reductase, and diaminopimelate decarboxylase. The AK was inhibited by L-threonine and by a combination of L-threonine and L-lysine, but not by L-lysine alone, and the activity of DDPS was moderately reduced by L-lysine. In an L-lysine producing mutant (G49), isolated as an S-(2-aminoethyl)-L-cysteine (lysine analog) resistant strain, both AK and DDPS were partially resistant to feedback inhibition. The ask and dapA genes encoding AK and DDPS respectively were isolated from the parental strain, AS1, and its G49 derivative. Comparison of the sequences revealed a point mutation in each of these genes in G49. The mutation in the ask gene altered aspartic acid in a key region involved in the allosteric regulation common to AKs, while a novel mutation in the dapA gene altered tyrosine-106, which was assumed to be involved in the binding of L-lysine to DDPS.
-7-
Preparation and Biological Activity of Molecular Probes to Identify and Analyze
Jasmonic Acid-binding Proteins
Yusuke Jikumaru,1 Tadao Asami,2 Hideharu Seto,2 Shigeo Yoshida,2 Tadashi Yokoyama,3 Naomi Obara,4 Morifumi Hasegawa,4 Osamu Kodama,4 Makoto Nishiyama,1 Kazunori Okada,1 Hideaki Nojiri,1 and Hisakazu Yamane1,†
1Biotechnology Research Center, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan
2The Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
3Tokyo University of Agriculture and Technology, 3-5-8 Saiwaicho, Fuchu, Tokyo 183-8509, Japan
4College of Agriculture, Ibaraki University, 3-21-1 Chuo, Ami, Ibaraki 300-0393, Japan
Several types of jasomonic acid (JA) derivatives, including JA–amino acid conjugates, a JA–biotin conjugate, a JA–dexamethasone heterodimer, and a JA–fluoresceine conjugate, were prepared as candidates for molecular probes to identify JA-binding proteins. These JA derivatives, excepting the JA–fluoresceine conjugate, exhibited significant biological activities in a rice seedling assay, a rice phytoalexin-inducing assay, and/or a soybean phenylalanine ammonia-lyase-inducing assay. These JA derivatives could therefore be useful probes for identifying JA-binding proteins. The activity spectra of the prepared compounds were different from each other, suggesting that different types of JA receptors were involved in the perception of JA derivatives in the respective bioassays.
-8-
Divergent Structures of Carbazole Degradative car Operons Isolated from Gram-negative
Bacteria
Kengo Inoue,1 Jaka Widada,1 Seiichiro Nakai,1 Takayuki Endoh,1 Masaaki Urata,1 Yuji Ashikawa,1 Masaki Shintani,1 Yuko Saiki,1,2 Takako Yoshida,1 Hiroshi Habe,1 Toshio Omori,1,* and Hideaki Nojiri1,†
1Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
2Fundamental Research Laboratory and Asahi Breweries Ltd., 1-21 Midori 1-Chome, Moriya-shi, Ibaraki 302-0106, Japan
Southern hybridization analysis of the genomes from the newly-isolated 10 carbazole (CAR)-utilizing bacteria revealed that 8 of the isolates carried gene clusters homologous to the CAR-catabolic car operon of Pseudomonas resinovorans strain CA10. Sequencing analysis showed that two car operons and the neighboring regions of Pseudomonas sp. strain K23 are nearly identical to that of strain CA10. In contrast to strains CA10 and K23, carEF genes did not exist downstream of the car gene cluster of Janthinobacterium sp. strain J3. In the car gene clusters, strains CA10, K23 and J3 have Rieske-type ferredoxin as a component of carbazole dioxygenase, although Sphingomonas sp. strain KA1 possesses a putidaredoxin-type ferredoxin. We confirmed that this putidaredoxin-type ferredoxin CarAc can function as an electron mediator to CarAa of strain KA1. In the upstream regions of the carJ3 and carKA1 gene clusters, ORFs whose deduced amino acid sequences showed homology to GntR-family transcriptional regulators were identified.
-9-
Cloning and Overexpression of the Exiguobacterium sp. F42 Gene Encoding a New
Short Chain Dehydrogenase, Which Catalyzes the Stereoselective Reduction of
Ethyl 3-Oxo-3-(2-thienyl)propanoate to Ethyl (S)-3-Hydroxy-3-(2-thienyl)propanoate
Masaru Wada,1,† Ayumi Yoshizumi,1 Yuki Furukawa,1 Hiroshi Kawabata,2 Makoto Ueda,2 Hiroshi Takagi,1 and Shigeru Nakamori1
1Department of Bioscience, Fukui Prefectural University, 4-1-1 Kenjyojima, Matsuoka-cho, Fukui 910-1195, Japan
2Life Science Laboratory, Mitsubishi Chemical Group Science and Technology Research Center, Inc., 1000 Kamoshida-cho, Aoba-ku, Yokohama 227-8502, Japan
Exiguobacterium sp. F42 was screened as a producer of an enzyme catalyzing the NADPH-dependent stereoselective reduction of ethyl 3-oxo-3-(2-thienyl)propanoate (KEES) to ethyl (S)-3-hydroxy-3-(2-thienyl)propanoate ((S)-HEES). (S)-HEES is a key intermediate for the synthesis of (S)-duloxetine, a potent inhibitor of the serotonin and norepinephrine uptake carriers. The responsible enzyme (KEES reductase) was partially purified, and the gene encoding KEES reductase was cloned and sequenced via an inverse PCR approach. Sequence analysis of the gene for KEES reductase revealed that the enzyme was a member of the short chain dehydrogenase/reductase family. The probable NADPH-interacting site and 3 catalytic residues (Ser-Tyr-Lys) were fully conserved. The gene was highly expressed in Escherichia coli, and the gene product was purified to homogeneity from the recombinant E. coli by simpler procedures than from the original host. The molecular mass of the purified enzyme was 27,500 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and 55,000 as determined by gel filtration chromatography. Our results show that this enzyme can be used for the practical production of (S)-HEES.
-10-
Genetic Analysis of chs1+ and chs2+ Encoding Chitin Synthases from Schizosaccharomyces
pombe
Yasuhiro Matsuo,1 Katsunori Tanaka,1 Tsuyoshi Nakagawa,2 Hideyuki Matsuda,1 and Makoto Kawamukai1,†
1Department of Applied Bioscience and Biotechnology, Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue 690-8504, Japan
2Center for Integrated Research in Science, Shimane University, 1060 Nishikawatsu, Matsue 690-8504, Japan
To explore the function of chitin in Schizosaccharomyces pombe, we have cloned chs1+ and chs2+, encoding putative chitin synthases, based on sequences in the Sanger Centre database. The synthetic lethal phenotype of the S. cerevisiae chs1 chs2 chs3 mutant was complemented by expression of S. pombe chs1+ or chs2+, indicating that both chs1+ and chs2+ in fact encode chitin synthase. The homothallic Δchs1 strain formed abnormal asci that contained 1, 2, or 3 spores, while the Δchs2 strain had no noticeable phenotype. The chs1 chs2 double disruptant looked similar phenotypically to the Δchs1 strain. The Chs2-GFP fusion protein predominantly localized at the septum after the septum was formed during vegetative growth. The level of chs2+ mRNA increased just before the septum was formed. Levels of Chs2-13Myc synthesis also changed during the cell cycle. Thus, chs1+ is required for proper spore formation, and chs2+ is perhaps involved in septum formation.
-11-
Absorption of Acylated Anthocyanins in Rats and Humans after Ingesting an Extract
of Ipomoea batatas Purple Sweet Potato Tuber
Katsuhisa Harada,1,† Mitsuyoshi Kano,1 Tomomi Takayanagi,1 Osamu Yamakawa,2 and Fumiyasu Ishikawa1
1Yakult Central Institute for Microbiological Research, 1796 Yaho, Kunitachi-shi, Tokyo 186-8650, Japan
2National Agricultural Research Center for Kyushu Okinawa Region, Suya 2421, Nishigoshi, Kikuchi, Kumamoto 861-1102, Japan
We evaluated the absorbability of anthocyanins in humans and rats administered with a beverage prepared from an extract of the tuber of purple sweet potato (Ipomoea batatas Cultivar Ayamurasaki), or with an anthocyanin concentrate. Two major anthocyanin components, cyanidin 3-O-(2-O-(6-O-(E)-caffeoyl-β-D-glucopyranosyl)-β-D-glucopyranoside)-5-O-β-D-glucopyranoside) and peonidin 3-O-(2-O-(6-O-(E)-caffeoyl-β-D-glucopyranosyl)-β-D-glucopyranoside)-5-O-β-D-glucopyranoside), were detected in the plasma and urine of both rats and humans by HPLC or liquid chromatography/mass spectrometry (LC/MS). The plasma concentration of anthocyanins in humans reached a maximum 90 minutes after ingestion, and the recovery of anthocyanins in the urine was estimated as 0.01–0.03%. These results indicate that acylated anthocyanins could be selectively absorbed after ingesting food.
-12-
Molecular Cloning of a Genomic DNA Encoding Yam Class IV Chitinase
Takuji Mitsunaga,1 Minoru Iwase,1 Wimal Ubhayasekera,2 Sherry L. Mowbray,2 and Daizo Koga1,†
1Laboratory of Biochemistry, Department of Biological Science, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan
2Department of Molecular Biology, Swedish University of Agricultural Sciences, S-751 24 Uppsala, Sweden
Genomic DNA for a class IV chitinase was cloned from yam (Dioscorea opposita Thunb) leaves and sequenced. The deduced amino acid sequence shows 50 to 59% identity to class IV chitinases from other plants. The yam chitinase, however, has an additional sequence of 8 amino acids (a C-terminal extension) following the cysteine that was reported as the last amino acid for other class IV chitinases; this extension is perhaps involved in subcellular localization. A homology model based on the structure of a class II chitinase from barley was used as an aid to interpreting the available data. The analysis suggests that the class IV enzyme recognizes an even shorter segment of the substrate than class I or II enzymes. This observation might help to explain why class IV enzymes are better suited to attack against pathogen cell walls.
-13-
Intracellular Localization of a Class IV Chitinase from Yam
Takuji Mitsunaga, Minoru Iwase, Dai Yuki, and Daizo Koga†
Laboratory of Biochemistry, Department of Biological Science, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan
Genomic DNA encoding a class IV chitinase was cloned from yam (Dioscorea opposita Thunb) leaves in previous research (Biosci. Biotechnol. Biochem., 68, 1508–1517 (2004)). But this chitinase had an additional sequence composed of eight amino acids (a C-terminal extension) at the C-terminal, compared with class IV chitianses from other plants. In order to clarify the role of this C-terminal extension in cellular localization, plants and suspension-cultured cells of Nicotiana tabacum were transformed with either the cloned yam class IV chitinase gene carrying the C-terminal extension or its truncated gene by the Agrobacterium-mediated method, and then their localization was investigated. The results suggest that the C-terminal extension of yam class IV chitinase plays a role as a targeting signal for plant vacuoles. This is the first report presenting the existence of vacuolar type class IV chitinase.
-14-
Metabolism of Benzoquinone by Yeast Cells and Oxidative Characteristics of Corresponding
Hydroquinone: Application to Highly Sensitive Measurement of Yeast Cell Density
by Using Benzoquinone and a Chemiluminescent Probe
Tadayuki Tsukatani,1,† Seiji Ide,1 Hiroyuki Ukeda,2 and Kiyoshi Matsumoto3
1Biotechnology and Food Research Institute, Fukuoka Industrial Technology Center, Kurume 839-0861, Japan
2Department of Bioresources Science, Faculty of Agriculture, Kochi University, Nankoku 783-8502, Japan
3Institute of Food Biotechnology, Department of Bioscience and Biotechnology, Division of Bioresource and Bioenvironmental Sciences, Graduate School Kyushu University, Fukuoka 812-8581, Japan
The metabolic efficiency of seven derivatives of 1,4-benzoquinone (BQ) by yeast cells and the oxidative characteristics of the corresponding hydroquinones (HQs) were studied by electrochemical, spectrophotometric and chemiluminescent methods. The spectrophotometric method was based on the reduction of a tetrazolium salt to formazan dye during the autoxidation of HQs generated by yeast cells under alkaline conditions. The amounts of HQs detected directly by the electrochemical method did not agree with those calculated from the formazan dye obtained by the spectrophotometric method. A tetrazolium salt was reduced to a formazan dye by both the superoxide anion radical (O2-·) generated during the autoxidation of 2,3,5,6-tetramethyl-1,4-HQ and by HQ itself. Little formazan dye was formed, and hydrogen peroxide (H2O2) was then finally produced during the autoxidation of 1,4-HQ or 2-methyl-1,4-HQ. Formazan dye and H2O2 were generated at a certain ratio during the autoxidation of derivatives of dimethyl-1,4-HQ or 2,3,5-trimethyl-1,4-HQ. The analytical method based on chemiluminescence with lucigenin and 2,3,5,6-tetramethyl-1,4-BQ was applied to highly sensitive measurement of the yeast cell density. A linear relationship between the chemiluminescence intensity and viable cell density was obtained in the range of 1.2× 103–4.8× 104 cells/ml. The detection limit was 4.8× 102 cells/ml.
-15-
Gene Cloning and Characterization of a Bacillus vietnamensis Metalloprotease
Mihwan Kim, Yoshitaka Nishiyama, Kiyoshi Mura,† Chiyoko Tokue, and Soichi Arai
Department of Nutritional Science, Faculty of Applied Bioscience, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan
A Bacillus vietnamensis metalloprotease (BVMP) with high affinity toward collagen was isolated and purified from the culture supernatant of Bacillus vietnamensis 11-4 occurring in Vietnamese fish sauces. The BVMP gene was cloned and its nucleotide and coded amino acid sequences determined. BVMP consists of 547 amino acid residues, with the zinc-binding sites conserved in common metalloproteases. It shares 57% amino acid identity with thermolysin originating from Bacillus thermoproteolyticus. The three-dimensional structure of BVMP was deduced by computer-aided modeling with the use of the known three-dimensional thermolysin structure as a template. Like thermolysin, BVMP cleaved the oxidized insulin B-chain at the peptide bonds involving the N-terminal sides of hydrophobic and aromatic amino acids. BVMP also showed high hydrolytic activity toward gelatin, collagen, casein, and elastin, especially toward the skeletal proteins at increased NaCl concentration. The high activity was found to be due to enhanced affinity to the substrates. Kinetical data on BVMP indicated that the Km values for the hydrolysis of Cbz-GPGGPA as a collagen model decreased as the concentration of added NaCl increased. Some contribution of this enzyme during the aging of fish sauces at high salt concentrations can thus be expected.
-16-
apg15-1, a UGA Mutant Allele in the Saccharomyces cerevisiae APG16 Gene, and
Its Suppression by a Cytoplasmic Factor
Hiroshi Okazaki,1 Bun-ichiro Ono,3 Yoshinori Ohsumi,4 and Mariko Ohsumi1,2,†
1Department of Bioscience, Teikyo University of Science and Technology, Uenohara, Kitatsuru-gun, Yamanashi 409-0193, Japan
2Biotechnology Research Center, Teikyo University of Science and Technology, Uenohara, Kitatsuru-gun, Yamanashi 409-0193, Japan
3Department of Biotechnology, Faculty of Science and Engineering, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan
4Department of Cell Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan
Autophagy is a complex cellular process by which starving cells utilize cytoplasmic macromolecules as nutritional resources. In Saccharomyces cerevisiae, more than 15 genes are involved in this process and most of them have been cloned and characterized by now. But there remains a complementation group represented by a single mutation, apg15-1, unclear as to its molecular nature. We obtained DNA fragments that functionally complemented apg15-1 and found that the responsible ORF, YMR159C, was already assigned as APG16. It was further found that apg15-1 was a UGA allele in which the 243rd base of the 450 bp coding region of APG16 was converted from C to T, and that the previously observed complementation between apg15-1 and apg16D was attributable to the action of a cytoplasmic omnipotent suppressor. This suppressor was readily cured by guanidine-HCl and also by overexpression or disruption of HSP104, indicating its close similarity to the PSI prion-like factor. Since apg15-1 is a mutation highly sensitive to termination suppression, it can be used as a tool to detect weak termination suppressors.
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Glucosyldiacylglycerol Enhances Reciprocal Activation of Prourokinase and Plasminogen
Wenhui Wu, Ritsuko Narasaki, Fumihiko Maeda, and Keiji Hasumi†
Department of Applied Biological Science, Tokyo Noko University, Fuchu, Tokyo 183-8509, Japan
Reciprocal activation of prourokinase (pro-u-PA) and plasminogen is an important mechanism in the initiation and propagation of local fibrinolytic activity. We found that glucosyldiacylglycerol (GDG) enhanced the reciprocal activation by 1.5- to 2-fold at 0.7–16 μM, accompanying increased conversions of both zymogens to active two-chain forms. The reciprocal activation system consists of (i) plasminogen activation by pro-u-PA to form plasmin, (ii) pro-u-PA activation by the resulting plasmin to form two-chain u-PA (tcu-PA), and (iii) plasminogen activation by the resulting tcu-PA. Whereas GDG minimally affected steps (ii) and (iii) in isolated systems, it markedly enhanced step (i) in the absence of the conversion of pro-u-PA to tcu-PA. GDG significantly increased the intrinsic fluorescence of pro-u-PA (6.7%), but not that of tcu-PA or plasminogen. The large change in intrinsic fluorescence suggests that GDG selectively affects pro-u-PA to alter its conformation, and this mechanism may account for enhancement of its intrinsic plasminogen activator activity.
-18-
Cloning of the Ruminococcus albus cel5D and cel9A Genes Encoding Dockerin Module-containing
Endoglucanases and Expression of cel5D in Escherichia coli
Hidenori Taguchi, Daisuke Hagiwara, Tsutomu Genma, Shuichi Karita,† Tetsuya Kimura, Kazuo Sakka, and Kunio Ohmiya
Faculty of Bioresources, Mie University, 1515 Kamihama, Tsu 514-8507, Japan
An EcoRI chromosomal DNA fragment of Ruminococcus albus F-40 that conferred endoglucanase activity on Escherichia coli was cloned. An open reading frame (ORF1) and another incomplete reading frame (ORF2) were found in the EcoRI fragment. The ORF2 was completed using inverse PCR genome walking technique. ORF1 and ORF2, which confront each other, encoded cellulases belonging to families 5 and 9 of the glycoside hydrolases and were designated cel5D and cel9A respectively. The cel5D gene encodes 753 amino acids with a deduced molecular weight of 83,409. Cel5D consists of a signal peptide of 24 amino acids, a family-5 catalytic module, a dockerin module, and two family-4 carbohydrate-binding modules (CBMs). The cel9A gene encodes 936 amino acids with a deduced molecular weight of 104,174, consisting of a signal peptide, a family-9 catalytic module, a family-3 CBM, and a dockerin module. The catalytic module polypeptide (rCel5DCat) derived from Cel5D was constructed, expressed, and purified from a recombinant E. coli. The truncated enzyme hydrolyzed cellohexaose, cellopentaose, and cellotetraose to yield mainly cellotriose and cellobiose with glucose as a minor product, but the enzyme was less active toward cellotriose and not active toward cellobiose, suggesting that this enzyme is a typical endoglucanase. rCel5DCat had a Km of 3.9 mg/ml and a Vmax of 37.2 μmol/min/mg for carboxymethycellulose.
-19-
Note
Expression of a Single-chain Antibody against Indole-3-acetic Acid in Escherichia
coli
Nobuhito Mitani1,2,† and Ryoji Matsumoto1
1Department of Citrus Research, National Institute of Fruit Tree Science, Kuchinotsu, Nagasaki 859-2501, Japan
2Department of Grape and Persimmon Research, National Institute of Fruit Tree Science, Akitsu, Hiroshima 729-2494, Japan
A hybridoma cell line that produces a monoclonal antibody specific for indole-3-acetic acid (IAA) was prepared. The DNA fragments coding the variable regions of the light and the heavy chains of the antibody were prepared by PCR using the cDNA of the antibody as a template. A chimera DNA for a single chain variable fragment (scFv) was constructed, and expressed in Escherichia coli. The scFv antibody expressed in E. coli as well as the original monoclonal antibody showed a specific binding to IAA.
-20-
Note
Effects of Protein Transport Inhibitors on the Distribution and Secretion of
the Fusion Protein RntA-EGFP in Aspergillus oryzae
Kumiko Masai, Jun-ichi Maruyama, Harushi Nakajima, and Katsuhiko Kitamoto†
Department of Biotechnology, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
The distribution of the secreted protein ribonuclease T1 (RntA) fused with the enhanced green fluorescent protein (EGFP), RntA-EGFP, was visualized in hyphae of Aspergillus oryzae in the presence of a protein transport inhibitor, brefeldin A, cytochalasin A, or nocodazole. During treatment with the protein transport inhibitors, the distribution of RntA-EGFP changed and distinct patterns of fluorescence accumulation were observed. The addition of brefeldin A caused RntA-EGFP fluorescence to appear in reticular networks, and the disruption of the polymerization of actin filaments by cytochalasin A caused an increase in RntA-EGFP fluorescence intensity in the hyphae without accumulation in a specific cellular component. In contrast, RntA-EGFP fluorescence was distributed in different parts of a hypha during treatment with nocodazole, a compound that depolymerizes microtubules. In addition, quantitative analysis was performed using the RntA-EGFP visualization system to analyze the relative amount of RntA-EGFP secreted into the culture medium during treatment with the protein transport inhibitors.
-21-
Note
Identification of (+)-Phyllocladene, (-)-Sandaracopimaradiene, and (+)-Kaurene
as New Fungal Metabolites from Fusicoccin-producing Phomopsis amygdali F6
Hiromichi Kenmoku,1,* Mio Tanaka,2 Koichi Ogiyama,3 Nobuo Kato,4 and Takeshi Sassa1,2,†
1Course of the Science of Bioresources, The United Graduate School of Agricultural Sciences, Iwate University (Yamagata University), Wakaba-cho, Tsuruoka 997-8555, Japan
2Department of Bioresource Engineering, Faculty of Agriculture, Yamagata University, Wakaba-cho, Tsuruoka 997-8555, Japan
3Department of Bioenvironment, Faculty of Agriculture, Yamagata University, Wakaba-cho, Tsuruoka 997-8555, Japan
4Institute of Scientific and Industrial Research, Osaka University, Mihogaoka-cho, Ibaraki 567-0047, Japan
A chemical analysis of the diterpene hydrocarbons produced by fusicoccin-producing fungus Phomopsis amygdali F6 identified five phyllocladene-related tri- and tetracyclic diterpene hydrocarbons. The presence of (+)-phyllocladene, (-)-sandaracopimaradiene, (+)-isopimara-8,15-diene, and (+)-pimara-8(14),15-diene in the fungus was demonstrated by GC-MS, 1H-NMR, and [α ]D measurements. (+)-Kaurene was also identified by GC-MS and chiral capillary GC. The possible biosynthetic relationship of these metabolites is discussed.
-22-
Note
Running Inhibits Osteoporosis Induced by Protein-deficient (PD) Food Intake
Hajime Tamaki,1 Liman Sun,1 Yutaka Ohta,2 Naofumi Katsuyama,2 Tetsuji Ishimaru,3 and Isao Chinen1,†
1Laboratories of Applied Biochemistry, Faculty of Agriculture, University of the Ryukyus, Senbaru 1, Nishihara-cho, Okinawa 903-0123, Japan
2Faculty of Medicine, University of the Ryukyus, Nishihara-cho, Okinawa 903-0123, Japan
3Musashi Junior High School, Nariyoshi 810, Musashi-machi Higashikunisaki-gun, Oita 873-0404, Japan
Running at 0.7 km/h for 10 min every day inhibited development of osteoporosis caused by protein deficient (PD) food intake. Urine alkaline phosphatase (ALP), a marker of bone formation osteoporosis, was not elevated in rats fed PD, when the osteoporosis was inhibited by running. Estrogen supplementation increased bone-breaking energy (BBE), but did not increase bone mineral density (BMD), and did not decrease urinary ALP levels.
-23-
Note
Purification and Characterization of O-Acetylserine Sulfhydrylase of Corynebacterium
glutamicum
Masaru Wada,† Naoki Awano, Hiroyuki Yamazawa, Hiroshi Takagi, and Shigeru Nakamori
Department of Bioscience, Fukui Prefectural University, 4-1-1 Kenjyojima, Matsuoka-cho, Fukui 910-1195, Japan
We highly purified O-acetylserine sulfhydrylase from the glutamate-producing bacterium Corynebacterium glutamicum. The molecular mass of the purified enzyme was 34,500 as determined by SDS-polyacrylamide gel electrophoresis, and 70,800 as determined by gel filtration chromatography. It had an apparent Km of 7.0 mM for O-acetylserine and a Vmax of 435 μ mol min-1 (mg · protein)-1. This is the first report of the cysteine biosynthetic enzyme of C. glutamicum in purified form.
-24-
Note
New 2(3→ 20)Abeotaxane and 3,11-Cyclotaxane from Needles of Taxus cuspidata
Qing-Wen Shi,1,† Zuo-Ping Li,1 Ding Zhao,1 Ji-Shun Gu,2 Takayuki Oritani,3 and Hiromasa Kiyota3,†
1Department of Natural Product Chemistry, School of Pharmaceutical Sciences, Hebei Medicinal University, 336 Zhongshan East Road, 050017, Shijiazhuang, Hebei Province, People’s Republic of China
2Department of Pharmacy, Central Hospital of Cangzhou, 061028, Cangzhou, Hebei Province, People’s Republic of China
3Laboratory of Applied Bioorganic Chemistry, Division of Bioscience & Biotechnology for Future Bioindustries, Graduate School of Agricultural Sciences, Tohoku University, 1-1 Tsutsumidori-Amamiya, Aoba-ku, Sendai 981-8555, Japan
Two new taxoid metabolites, 2 α ,7 β ,10 β -triacetoxy-5 α,13 α -dihydroxy-2(3 → 20)abeotaxa-4(20),11-dien-9-one (1) and 2 α -acetoxy-5 α -cinnamoyloxy-9 α,10 β -dihydroxy-3,11-cyclotax-4(20)-en-13-one (2), were isolated from the methanol extract of needles of the Japanese yew, Taxus cuspidata.
-25-
Note
8-Hydroxydaidzein, an Aldose Reductase Inhibitor from Okara Fermented with Aspergillus
sp. HK-388
Tomoyuki Fujita,† Tomoyoshi Funako, and Hideo Hayashi
Division of Applied Biological Chemistry, Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai, Osaka 599-8531, Japan
The aldose reductase (AR) inhibitor, 8-hydroxydaidzein, was isolated and identified from a methanolic extract of okara (soybean pulp) fermented with the fungal strain, Aspergillus sp. HK-388. 8-Hydroxydaidzein showed non-competitive inhibition of human recombinant AR with respect to DL-glyceraldehyde, its Ki value being evaluated as 7.0 μ M.
-26-
Note
Mitogenic Properties of Pokeweed Lectin-D Isoforms on Human Peripheral Blood
Lymphocytes: Non-mitogen PL-D1 and Mitogen PL-D2
Kenichi Yamaguchi,1,2,† Mizuki Uechi,1 Yoshinori Katakura,3 Tatsuya Oda,2 and Masatsune Ishiguro1,*
1Laboratory of Protein Chemistry & Engineering, Graduate School of Genetic Resources Technology, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan
2Division of Biochemistry, Faculty of Fisheries, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan
3Cellular Regulation Technology, Graduate School of Genetic Resources Technology, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan
We investigated native structures and mitogenic properties of pokeweed lectin-D isoforms (PL-D1 and -D2) on human peripheral blood lymphocytes along with other isolectins (PL-A to -C). Both native PL-D isoforms appeared to behave as monomers. PL-D2 proliferated the lymphocytes like PL-C, whereas PL-D1 had no mitogenicity. PL-D1 acquired mitogenic activity after trimming of the C-terminal dipeptide.
-27-
Note
Growth and Carotenoid Production of Thraustochytrium sp. CHN-1 Cultured under
Superbright Red and Blue Light-emitting Diodes
Yukiho Yamaoka,1,† Marvelisa L. Carmona,2 and Shinji Oota3
1National Institute of Advanced Industrial Science and Technology, 2-2-2 Hirosuehiro, Kure, Hiroshima 737-0197, Japan
2School of Biosphere Sciences, Hiroshima University, 1-4-4 Kagamiyama, Higashi-Hiroshima 737-8528, Japan
3Instrument Center for Chemical Analysis, Hiroshima University, 1-4-4 Kagamiyama, Higashi-Hiroshima 737-8528, Japan
Isomers of astaxanthin produced by Thraustochytrium sp. CHN-1 are identified as (3S,3S’)-trans-astaxanthin, (3R,3R’)-trans-astaxanthin and (3S,3S’)-cis-astaxanthin by chirality column HPLC, and 1H and 13C NMR. We studied the effects of light generated by superbright blue, red and near-red LEDs on the growth and carotenoid production of Thraustochytrium sp. CHN-1. Thraustochytrium sp. CHN-1 responded to blue LEDs light: It produced carotenoid pigments (astaxanthin)
-28-
Note
Enhanced Calcium Absorption in the Small Intestine by a Phytate-removed Deamidated
Soybean Globulin Preparation
Hitomi Kumagai,1,† Atsushi Koizumi,1 Akihiro Suda,1 Noriko Sato,1 Hidetoshi Sakurai,1 and Hitoshi Kumagai2
1Department of Agricultural and Biological Chemistry, Nihon University, 1866 Kameino, Fujisawa-shi, Kanagawa 252-8510, Japan
2Department of Food Science and Nutrition, Kyoritsu Women’s University, 1-710 Motohachioji, Hachioji-shi, Tokyo 193-8501, Japan
Soybean globulins were deamidated after removing phytate using ion-exchange resins, and then hydrolyzed by digestive enzymes. The phytate-removed deamidated soybean globulins (PrDS) retained high calcium-binding ability even after the hydrolysis by digestive enzymes. PrDS and its hydrolysates enhanced calcium absorption from the small intestine when injected into the small intestine together with a calcium solution.
-29-
Note
Antimicrobial Activities of Diterpene Dialdehydes, Constituents from Myoga (Zingiber
mioga Roscoe), and Their Quantitative Analysis
Masako Abe,1 Yoshio Ozawa,1,† Yasushi Uda,2 Fuminori Yamada,3 Yasujiro Morimitsu,4 Yoshimasa Nakamura,5 and Toshihiko Osawa5
1Department of Health and Nutrition, Takasaki University of Health and Welfare, Takasaki 370-0033, Japan
2Deparment of Bioproductive Sciences, Utsunomiya University, Utsunomiya 321-8505, Japan
3Department of Horticultural Breeding, Gunma Agricultural Technology Center, 493 Azuma-mura, Sawagun 379-2224, Japan
4Laboratory of Food Chemistry, Ochanomizu University, 211 Ohtsuka, Bunkyo-ku, Tokyo 112-8610, Japan
5Laboratory of Food and Biodynamics, Nagoya University Graduate School of Bioagricultural Sciences, Nagoya 464-8601, Japan
The antimicrobial activities of the three diterpene dialdehydes, miogadial, galanal A and galanal B, isolated from flower buds of the myoga (Zingiber mioga Roscoe) plant were investigated with some strains of bacteria, yeasts and molds. Among the three compounds, miogadial exhibited relatively greater antimicrobial activity than the others against Gram-positive bacteria and yeasts. Galanals A and B also behaved as antimicrobial agents against Gram-positive bacteria and yeasts. The content of miogadial in the flower buds was much higher than that in the leaves, whereas galanals A and B were contained at high levels in the leaves and rhizomes.
-30-
Note
Classification of Brassinosteroid-regulated Genes Based on Expression Profiles
in bri1 and in Response to a Protein Kinase Inhibitor, Staurosporin
Hideki Goda, Yukihisa Shimada,† Shozo Fujioka, and Shigeo Yoshida
Plant Science Center, RIKEN, Suehiro 1-7-22, Tsurumi, Yokohama, Kanagawa 230-0045, Japan
To gain insight into the brassinosteroid (BR) signaling pathway, the expression of BR-regulated genes was analysed in the BR-signaling mutant br-insensitive 1 (bri1), and in the presence of a protein kinase inhibitor, staurosporin. BR-regulated genes were classified based on the results. This classification will perhaps prove useful in BR-signalling studies using BR-regulated genes as molecular markers.
-31-
Note
Fusicocca-3(16),10(14)-diene, and β - and δ -Araneosenes, New Fusicoccin Biosynthesis-related
Diterpene Hydrocarbons from Phomopsis amygdali
Takeshi Sassa,1,2,† Hiromichi Kenmoku,2 Kayoko Nakayama,1 and Nobuo Kato3
1Department of Bioresource Engineering, Faculty of Agriculture, Yamagata University, Wakaba-cho, Tsuruoka 997-8555, Japan
2Course of the Science of Bioresources, The United Graduate School of Agricultural Sciences, Iwate University (Yamagata University), Wakaba-cho, Tsuruoka 997-8555, Japan
3The Institute of Scientific and Industrial Research, Osaka University, Mihogaoka, Ibaraki 567-0047, Japan
Further isolation and examination of fusicoccane hydrocarbons biosynthetically related to fusicoccin from Phomopsis amygdali allowed us to identify new fungal diterpene hydrocarbons of fusicoccadiene and araneosene. These were assigned as (+)-fusicocca-3(16),10(14)-diene, and (+)- β - and (+)- δ -araneosenes. These findings led to the experimental clarification of the structures of the biosynthetic hydrocarbon intermediates presumed earlier.
-32-
Communication
Production of Lupin Acid Phosphatase in Transgenic Rice for Use as a Phytate-hydrolyzing
Enzyme in Animal Feed
Akira Hamada,1 Ken-ichi Yamaguchi,1,* Michiko Harada,1 Seiya Nikumaru,1 Jun Wasaki,2 Takuro Shinano,2 Hirokazu Matsui,3 Mitsuru Osaki,3 and Hideo Honda1,†
1Functional Chemicals Laboratory, Mitsui Chemicals, Inc., Togo 1144, Mobara 297-0017, Japan
2Creative Research Initiative ‘Sousei’ (CRIS), Hokkaido University, N20W10 Kita-ku, Sapporo 001-0020, Japan
3Graduate School of Agriculture, Hokkaido University, N9W9 Kita-ku, Sapporo 060-8589, Japan
The acid phosphatase gene from lupin was expressed in transgenic rice plants under the control of the maize ubiquitin promoter or rice chlorophyll a/b binding protein (Cab) promoter. Transgenic rice leaves exhibited up to an 18-fold increase in phytate-hydrolyzing activity. Based on the phytate-hydrolyzing activity at pH 5.5, more than 85% this activity was retained after heat-treatment at 80 °C for 15 min, and the heterologous enzyme in leaf sections and leaf extracts was relatively stable during storage. A distinct increase in released phosphate was observed when the heterologous enzyme was mixed with the feed extract. These results suggest that the heterologous enzyme in rice plants may maintain its desired characteristics as a phytate-hydrolyzing enzyme when added to animal feed.
-33-
Communication
Dihydrocoronatine, Promising Candidate for a Chemical Probe to Study Coronatine-,
Jasmonoid- and Octadecanoid-binding Protein
Munenori Suzuki,1 Morifumi Hasegawa,1,2 Osamu Kodama,1,2 and Hiroaki Toshima1,2,†
1United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu-city, Tokyo 183-8509, Japan
2Department of Bioresource Science, College of Agriculture, Ibaraki University, 3-21-1 Chuo, Ami, Ibaraki 300-0393, Japan
Coronatine (1), its synthetic analogs (6-13) and jasmonic acid induced various volatiles in rice leaves. In the range of 0.01–0.1 mM, dihydrocoronatine (7) exhibited 4-687 times higher activity for linalool emission than that of 1. The radioactive derivative of 7, [4,5-3H]-7, was employed to identify the putative coronatine-binding protein in rice leaves. 7 would be a promising candidate for a chemical probe to study cornatine-binding protein related to the jasmonoid and octadecanoid signaling pathway in higher plants. A detailed study of coronatine-binding protein in rice leaves and cell culture with [4,5-3H]-7 is now in progress.
-34-
Communication
Interaction between a Negative Regulator (Msa2/Nrd1) and a Positive Regulator
(Cpc2) of Sexual Differentiation in Schizosaccharomyces pombe
Hee Tae Jeong, Yasuo Oowatari, Michiyo Abe, Katsunori Tanaka, Hideyuki Matsuda, and Makoto Kawamukai†
Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue 690-8504, Japan
The sexual differentiation of Schizosaccharomyces pombe is controlled by many cellular components which have not been fully characterized. We isolated a gene called msa2 as a multi-copy suppressor of a sporulation abnormal mutant (sam1). Msa2p is identical with Nrd1p which has been characterized as a factor that blocks the onset of sexual differentiation. The yeast two-hybrid system was used to identify Cpc2p, a fission yeast homolog of the RACK1 protein, that interacted with Msa2p/Nrd1p. We confirmed that Msa2p/Nrd1p interacted with Cpc2p in S. pombe cells. An epistatic analysis of msa2/nrd1 and cpc2 suggests that Msa2p/Nrd1p was an upstream regulator for Cpc2p. A localization analysis of Cpc2p and Msa2p/Nrd1p indicates that both proteins were predominantly localized in the cytoplasm. The interaction of negative regulator Msa2p/Nrd1p with positive regulator Cpc2p suggests a new regulatory circuit in the sexual differentiation of S. pombe.