(Vol.67 No.3 2003)
Type II Antifreeze Protein from a Mid-latitude Freshwater Fish,
Japanese Smelt (Hypomesus nipponensis)
Yasuhiro YAMASHITA,1, Rikako MIURA,1 Yukari TAKEMOTO,1 Sakae TSUDA,2 Hidehisa
KAWAHARA,1 and Hitoshi OBATA1 p.461
Elimination of Cell-cycle Regulators during Caspase-3-dependent
Apoptosis Caused by an Immunosuppressant, FTY720
Yun-Sik LEE,1 Hiroo NAKAJIMA,2 Mie TSURUGA,3 and Junji MAGAE3, p.467
(＋)-Menthol and Its Hydroxy Derivatives, Novel Fungal Monoterpenols
from the Fusicoccin-producing Fungi, Phomopsis amygdali F6a and Niigata 2
Takeshi SASSA,1,2, Hiromichi KENMOKU,1 Mitsuyoshi SATO,2 Tetsuya MURAYAMA,1,2
and Nobuo KATO3 p.475
High-throughput PCR Screening of Genes for Three-component Regulatory
System Putatively Involved in Quorum Sensing from Low-G＋C Gram-positive Bacteria
Jiro NAKAYAMA,1, Antoon D. L. AKKERMANS,2 and Willem M. DE VOS2 p.480
Characterization of Aspartate Aminotransferase from the Cyanobacterium
Phormidium lapideum
Hyeung KIM, Koji IKEGAMI, Masaki NAKAOKA, Mayumi YAGI, Hitoshi SHIBATA, and
Yoshihiro SAWA p.490
Regioselective Glucosylation of Pyridoxine by Microorganisms
Yasuhisa ASANO and Koichi WADA p.499
Improvement in 5′-Position-selective Glucosylation of Pyridoxine
by Verticillium dahliae TPU 4900
Koichi WADA and Yasuhisa ASANO p.508
Characterization of a Saccharomyces cerevisiae Mutant with Pseudohyphae
and Cloning of a Gene Complementing the Mutation
Jaruwan MANEESRI, Masayuki AZUMA, Satoshi TORII, Koichi IGARASHI, and Hiroshi
OOSHIMA
p.517
Maltosyl-erythritol, a Major Transglycosylation Product of Erythritol
by Bacillus stearothermophilus Maltogenic Amylase
Jong-Won YOON,1, Eun-Joo JEON,1 Il-Hun JUNG,1 Mee-Jung MIN,1 Hye-Young LEE,1
Myo-Jeong KIM,2 Jin-Sook BAEK,1 Hee-Seob LEE,1 Cheon-Seok PARK,3 Sangsuk OH,4
Kwan-Hwa PARK,1 and Tae-Wha MOON1, p.525
Acarviosine-simmondsin, a Novel Compound Obtained from Acarviosine-glucose
and Simmondsin by Thermus Maltogenic Amylase and Its in vivo Effect on Food
Intake and Hyperglycemia
Jin-Sook BAEK,1 Hye-Young KIM,2 Thomas P. ABBOTT,3 Tae-Wha MOON,1 Soo-Bok LEE,4
Cheon-Seok PARK,5, and Kwan-Hwa PARK1, p.532
Histidine-114 at Subsites E and F Can Explain the Characteristic
Enzymatic Activity of Guinea Hen Egg-white Lysozyme
Gen TOSHIMA, Shunsuke KAWAMURA, Tomohiro ARAKI, and Takao TORIKATA p.540
Characterization of the Prr1 Response Regulator with Special
Reference to Sexual Development in Schizosaccharomyces pombe
Norihito NAKAMICHI, Hisami YANADA, Hirofumi AIBA, Keisuke AOYAMA, Ryusuke OHMIYA,
and Takeshi MIZUNO p.547
Characterization of Rice Functional Monosaccharide Transporter,
OsMST5
Budsaraporn NGAMPANYA,1,2 Anna SOBOLEWSKA,3 Taito TAKEDA,3 Kyoko TOYOFUKU,3
Jarunya NARANGAJAVANA,2 Akira IKEDA,1,4 and Junji YAMAGUCHI1, p.556
The N-Terminal Sequence of Paratropomyosin Binding Fragments
from β-Connectin
Minoru YAMANOUE,1, Shuji UEDA,2, Akiko OHASHI,1 Yumi YOSHIMURA,3 and Shigemi
NORIOKA3
p.563
Enzymatic Properties of Glutamine 32 Mutants of RNase Rh from
Rhizopus niveus, a Trial to Alter the Most Preferential Inter-nucleotidic Linkages
of RNase Rh
Kazuko OHGI,1, Masanori IWAMA,2 Norio INOKUCHI,3 and Masachika IRIE1 p.570
Effects of Bovine α-Lactalbumin on Gastric Defense Mechanisms
in Naive Rats
Yoshihiko USHIDA, Yukiko SHIMOKAWA, Hiroshi MATSUMOTO, Tomohiro TOIDA, and Hirotoshi
HAYASAWA p.577
Cloning of the Xylitol Dehydrogenase Gene from Gluconobacter
oxydans and Improved Production of Xylitol from D-Arabitol
Masakazu SUGIYAMA, Shun-ichi SUZUKI, Naoto TONOUCHI, and Kenzo YOKOZEKI p.584
Functional Analysis of Individual Oligosaccharide Chains of Sendai
Virus Hemagglutinin-neuraminidase Protein
Hiroaki SEGAWA,1,2 Akira INAKAWA,1 Tetsuro YAMASHITA,1 and Hideharu TAIRA1,
p.592
Isotope Ratio Analysis by HRGC-MS of Monoterpene Hydrocarbons
from Citrus Essential Oils
Atsushi SATAKE,1,2, Akitoshi UNE,1 Takao UENO,1 Hiroyuki UKEDA,1 and Masayoshi
SAWAMURA1 p.599
Functional Analysis of a β-Ketoacyl-CoA Synthase Gene, MpFAE2,
by Gene Silencing in the Liverwort Marchantia polymorpha L.
Masataka KAJIKAWA,1 Shohei YAMAOKA,1 Katsuyuki T. YAMATO,1 Hiroyuki KANAMARU,2
Eiji SAKURADANI,2 Sakayu SHIMIZU,2 Hideya FUKUZAWA,1 and Kanji OHYAMA1, p.605
Note
Cloning of Structural Gene of Deinococcus radiodurans UV-Endonuclease β
Shigeru KITAYAMA,1,2, Issay NARUMI,2 Tomoo FUNAYAMA,2 and Hiroshi WATANABE2
p.613
Note
Effects of Adding Some Dietary Fibers to a Cystine Diet on the Activities of
Liver Antioxidant Enzymes and Serum Enzymes in Rats
Guochun HE and Yoritaka AOYAMA p.617
Note
Genomic Organization of the S-Locus Region of Brassica
Hiroshi SHIBA,1, Masayuki KENMOCHI,1 Minoru SUGIHARA,1 Megumi IWANO,1 Shinji
KAWASAKI,2 Go SUZUKI,3 Masao WATANABE,4 Akira ISOGAI,1 and Seiji TAKAYAMA1 p.622
Note
Purification and Characterization of Extracellular β-Galactosidase Secreted
by Supension Cultured Rice (Oryza sativa L.) Cells
Satoshi KANEKO and Hideyuki KOBAYASHI p.627
Note
Mushroom Tyrosinase Inhibitory Activity of Esculetin Isolated from Seeds of
Euphorbia lathyris L.
Yukimitsu MASAMOTO,1 Hideya ANDO,2 Yoshiyuki MURATA,3 Yasuaki SHIMOISHI,3 Mikiro
TADA,1 and Kyoya TAKAHATA3, p.631
Note
Inhibitory Activity of Analogs of AM-Toxin, a Host-specific Phytotoxin from
the Alternaria alternata Apple Pathotype, on Photosynthetic O2 Evolution in
Apple Leaves
Masahiro MIYASHITA,1, Tomoko NAKAMORI,1 Hisashi MIYAGAWA,1 Miki AKAMATSU,2 and
Tamio UENO1 p.635
Note
Continuous Cell-free Protein Synthesis Directed by Messenger DNA and Catalyzed
by Extract of Thermus thermophilus HB27
Taketoshi UZAWA,, Akihiko YAMAGISHI, and Tairo OSHIMA p.639
Note
Formation of 3′-O-β-Galactosyl Compounds of 5-Bromouridine by Sporobolomyces
singularis
Kei UCHIDA and Yukio SUZUKI p.643
Note
Cloning and Overexpression of β-N-Acetylglucosaminidase Encoding Gene nagA from
Aspergillus oryzae and Enzyme-catalyzed Synthesis of Human Milk Oligosaccharide
Ichiro MATSUO,2, Sunhwa KIM,1 Yuichi YAMAMOTO,2, Katsumi AJISAKA,2 Jun-ich MARUYAMA,1
Harushi NAKAJIMA,1 and Katsuhiko KITAMOTO1, p.646
Note
Targeted Gene Disruption of the Neuronal Calcium Sensor 1 Homologue in Rice
Blast Fungus, Magnaporthe grisea
Ken-ichiro SAITOH,1 Tsutomu ARIE,1 Tohru TERAOKA,1 Isamu YAMAGUCHI,2, and Takashi
KAMAKURA2, p.651
Note
Molecular Cloning of Rat Transcription Factor YY1
Chiharu NISHIYAMA,1,2, Toyokazu YOKOTA,2 Makoto NISHIYAMA,3 Chisei RA,1 Ko OKUMURA,1
and Hideoki OGAWA1 p.654
Preliminary Communication
Total Synthesis of 0231B, an Inhibitor of 3α-Hydroxysteroid Dehydrogenase Produced
by Streptomyces sp. HKI0231
Taichi KOMODA, Yoshihiko SHINODA, and Shin-ichi NAKATSUKA p.659
Preliminary Communication
Gnetol as a Potent Tyrosinase Inhibitor from Genus Gnetum
Kenji OHGUCHI,1, Toshiyuki TANAKA,2 Ibrahim ILIYA,2 Tetsuro ITO,2 Munekazu IINUMA,3
Kenji MATSUMOTO,1 Yukihiro AKAO,1 and Yoshinori NOZAWA1 p.663
-1-
Type II Antifreeze Protein from a Mid-latitude Freshwater Fish, Japanese Smelt
(Hypomesus nipponensis)
Yasuhiro YAMASHITA,1, Rikako MIURA,1 Yukari TAKEMOTO,1 Sakae TSUDA,2 Hidehisa KAWAHARA,1 and Hitoshi OBATA1
1Department of Biotechnology, Faculty of Engineering, Kansai University, 3-3-35 Yamate-cho, Suita, Osaka 564-8680, Japan 2Research Institute of Biological Resources, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, Toyohira, Sapporo 062-8517, Japan
Received April 23, 2002; Accepted October 10, 2002
A lot of reports of antifreeze protein (AFP) from fish have been published, but no report has mentioned of commercialized mid-latitude fresh water fish which producing AFP in its body fluid. We found that the AFP in the body fluid of Japanese smelt (Hypomesus nipponensis) from mid-latitude fresh water was purified and characterized. The N-terminal amino acid sequence of the Japanese smelt AFP was 75.0％ identical to Type II AFP from herring. Results of EDTA treatment and ruthenium red staining suggested that the Japanese smelt AFP had at least one Ca＾{2＋}-binding domain. Interestingly, the antifreeze activity of the Japanese smelt AFP did not completely disappear when Ca＾{2＋} ions were removed. The molecular mass of the Japanese smelt AFP was calculated to be 16,756.8 by the TOF-mass analysis. The Open reading flame of the gene coding for the Japanese smelt AFP was 444 bp long and was 85.0％ identical with the entire herring AFP gene. The cDNA and amino acid sequence of the Japanese smelt AFP were the same length as those of herring AFP.
Key words: antifreeze protein; Type II; Japanese smelt
-2-
Elimination of Cell-cycle Regulators during Caspase-3-dependent Apoptosis Caused
by an Immunosuppressant, FTY720
Yun-Sik LEE,1 Hiroo NAKAJIMA,2 Mie TSURUGA,3 and Junji MAGAE3,
1National Institute of Bioscience and Human Technology, 1-1 Higashi, Tsukuba 305-8566, Japan 2Laboratory of Breast Surgery, Department of Endocrine, Kyoto Prefectural University of Medicine, Kawaramachi, Hirokoji, Kamikyo-ku, Kyoto 602-0841, Japan 3Department of Biotechnology, Institute of Research and Innovation, 1201 Takada, Kashiwa 277-0861, Japan
Received July 11, 2002; Accepted November 15, 2002
The immunosuppressant, FTY720 causes apoptosis of lymphocytes, reduces numbers of lymphocytes in peripheral blood, and prevents infiltration of lymphocytes into allografts, which may be one of the mechanisms involved in its effects. Here we compared caspase activation and expression of cell-cycle regulators during apoptosis caused by FTY720, and Fas-stimulation in a mouse lymphoma transfected with human Fas antigen. FTY720 activated caspases-3, -8, and -9 as rapidly as did Fas-mediated apoptosis. The activation was blocked by a peptide inhibitor for caspase-3, DEVD-CHO. Fas-induced activation of caspases-8 and -9 was unaffected by the inhibitor. FTY720 eliminated proliferating cell nuclear antigen, retinoblastoma family members, differentiation regulated transcription factor polypetide-1, and cyclin H. These cell-cycle regulators were not eliminated when the peptide inhibitor was used. Dysfunction of cell-cycle regulators may play a critical role in the signal transduction pathway for activation of FTY720-mediated apoptosis.
Key words: apoptosis; FTY720; caspase; cell-cycle; pRb
-3-
(＋)-Menthol and Its Hydroxy Derivatives, Novel Fungal Monoterpenols from the
Fusicoccin-producing Fungi, Phomopsis amygdali F6a and Niigata 2
Takeshi SASSA,1,2, Hiromichi KENMOKU,1 Mitsuyoshi SATO,2 Tetsuya MURAYAMA,1,2 and Nobuo KATO3
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 3Institute of Advanced Material Study, Kyushu University, Kasuga 816-8580, Japan
Received July 15, 2002; Accepted November 1, 2002
In our search for new fusicoccins of unique diterpene glucosides from Phomopsis amygdali, we found that a fragrant substance was formed in the early stage of fusicoccin fermentation. This fragrant constituent was isolated and identified as (＋)-menthol, which is a novel fungal metabolite as the enantiomer of well-known peppermint (－)-menthol. (＋)-7-Hydroxymenthol and new (＋)-(6S)-hydroxymenthol were also isolated and identified as fungal metabolites. In addition, p-menthanetriol, which has been reported as the first fungal monoterpene from the fungus, was also isolated. The possible biosynthetic relationship of these metabolites is discussed.
Key words: (＋)-menthol; (＋)-7-hydroxymenthol; (＋)-(6S)-hydroxymenthol; fungal monoterpene; Phomopsis amygdali
-4-
High-throughput PCR Screening of Genes for Three-component Regulatory System
Putatively Involved in Quorum Sensing from Low-G＋C Gram-positive Bacteria
Jiro NAKAYAMA,1, Antoon D. L. AKKERMANS,2 and Willem M. DE VOS2
1Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan 2Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, Hesselink van Suchtelenweg 4, 6703 CT Wageningen, The Netherlands
Received July 24, 2002; Accepted October 19, 2002
Quorum sensing of Gram-positive bacteria is often regulated by three-component regulatory system composed of autoinducing peptide, sensor kinase and response regulator. We used PCR to study a gene cassette encoding this three-component regulatory system. Degenerate primers were designed from consensus amino acid sequences in the HPK10 subfamily, mostly involved in quorum sensing. Products amplified from genomic DNA of Lactobacillus, Enterococcus, and Clostridium species were cloned and sequenced; their deduced amino acid sequences were similar to those of members of the HPK10 subfamily. Complete genes for the putative gene cassette were cloned by inverse PCR from L. paracasei E93490 and L. plantarum WCFS6. Phylogenetic analysis grouped the cloned putative HPKs into the HPK10 subfamily. These results indicated the usefulness of this high-throughput gene screening and suggested that the three-component regulatory gene cassette are widely present.
Key words: PCR; three component regulatory system; histidine protein kinase; autoinducing peptide; quorum sensing
-5-
Characterization of Aspartate Aminotransferase from the Cyanobacterium Phormidium
lapideum
Hyeung KIM, Koji IKEGAMI, Masaki NAKAOKA, Mayumi YAGI, Hitoshi SHIBATA, and Yoshihiro SAWA
Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue, Shimane 690-8504, Japan
Received July 30, 2002; Accepted September 21, 2002
Aspartate aminotransferase (AspAT) was purified to homogeneity from cell extracts of the non-N2-fixing cyanobacterium Phormidium lapideum. The NH2-terminal sequence of 25 amino acid residues was different from the sequences of the subfamily Iα of AspATs from eukaryotes and Escherichia coli, but it was similar to sequences of the subfamily Iγ of AspATs from archaebacteria and eubacteria. The enzyme was most active at 80°C and was stable at up to 75°C. Thermal inactivation (60--85°C) of the enzyme followed first-order kinetics, with 2-oxoglutarate causing a shift of the thermal inactivation curves to higher temperatures. However, at 25°C the kcat of P. lapideum AspAT was nearly equal to the values of AspATs from mesophilic organisms. The enzyme used L-aspartate and L-cysteine sulfinate as amino donors and 2-oxoglutarate as an amino acceptor. The Km values were 5.0 mM for L-aspartate, 5.7 mM for L-glutamate, 0.2 mM for 2-oxoglutarate, and 0.032 mM for oxaloacetate.
Key words: aspartate aminotransferase; cyanobacterium; Phormidium lapideum; thermotolerant enzyme
-6-
Regioselective Glucosylation of Pyridoxine by Microorganisms
Yasuhisa ASANO and Koichi WADA
Biotechnology Research Center, Toyama Prefectural University, 5180 Kurokawa, Kosugi, Toyama 939-0398, Japan
Received August 1, 2002; Accepted November 13, 2002
Microorganisms from culture collections and isolates from nature were screened for the ability to catalyze the regioselective glucosylation of pyridoxine (PN) to produce pyridoxine 5′-α-D-glucoside (PN-5′-α-G) or pyridoxine 4′-α-D-glucoside (PN-4′-α-G). Transglucosylation activity specific to 5′-position of PN was found in fungi belonging to genera such as Coriolus and Verticillium, and activity at the 4′-position of PN was found in bacteria belonging to genera such as Bacillus and Serratia. From 100 mM PN, intact cells of Verticillium dahliae TPU 4900 produced 42 mM (13.9 mg/mL) PN-5′-α-G after 70 h of reaction. Intact cells of Bacillus cereus TPU 5504 produced 33 mM (10.9 mg/mL) PN-4′-α-G after 19 h of reaction. The selectivities for 5′- and 4′-positions were 80％ and 90％, respectively.
Key words: Verticillium; Coriolus; Bacillus; pyridoxine 5′-α-D-glucoside; pyridoxine 4′-α-D-glucoside
-7-
Improvement in 5′-Position-selective Glucosylation of Pyridoxine by Verticillium
dahliae TPU 4900
Koichi WADA and Yasuhisa ASANO
Biotechnology Research Center, Toyama Prefectural University, 5180 Kurokawa, Kosugi, Toyama 939-0398, Japan
Received August 1, 2002; Accepted November 13, 2002
Optimization of culture and reaction conditions for 5′-position-selective transglucosylation to pyridoxine by Verticillium dahliae TPU 4900 was investigated. V. dahliae TPU 4900 had high transglucosylation activity when grown with soluble starch as a carbon source and organic nitrogens such as Esusan meat as a nitrogen source at 15--20°C. Both the yield of pyridoxine 5′-α-D-glucoside (PN-5′-α-G) and the 5′-position-selectivity reached a maximum when an intact-cell reaction was done at 50--60°C and pH 7 with additions of dextrin. The transglucosylation activity in culture broth was 71 times with the optimization of culture conditions that under the conditions used for screening. The productivity of PN-5′-α-G synthesis was 6.9 times that under the initial conditions when the reaction conditions of intact cells were optimized. From 1000 mM (206 g/L) pyridoxine hydrochloride, PN-5′-α-G was synthesized to the concentration of 300 mM (98.4 g/L as PN-5′-α-G) with 5′-selectivity of 85％ in 53 h by intact cells of V. dahliae TPU 4900.
Key words: Verticillium dahliae; transglucosylation; optimization; pyridoxine; pyridoxine 5′-α-D-glucoside
-8-
Characterization of a Saccharomyces cerevisiae Mutant with Pseudohyphae and
Cloning of a Gene Complementing the Mutation
Jaruwan MANEESRI, Masayuki AZUMA, Satoshi TORII, Koichi IGARASHI, and Hiroshi OOSHIMA
Department of Applied and Bioapplied Chemistry, Graduate School of Engineering, Osaka City University, Sugimoto 3-3-138, Sumiyoshi-ku, Osaka 558-8585, Japan
Received August 2, 2002; Accepted September 25, 2002
Screening for morphological mutants of a haploid strain of Saccharomyces cerevisiae was done on the basis of their cell-shape on a solid medium containing isoamyl alcohol, which causes cell elongation, to obtain information on the morphogenesis. Mutant J19, which had pseudohyphae in liquid medium even in the absence of isoamyl alcohol, had many elongated cells. Few reports exist of haploid cells growing as pseudohyphae in liquid culture. Cell-wall analysis showed that J19 had ordinary amounts of alkali-insoluble glucan and chitin, but that isoamyl alcohol in the medium caused structural changes in the cell wall. Addition of a DNA fragment that included the wild-type SCL1 gene to J19 complemented its morphological phenotype. Sequencing of J19 SCL1 showed that the glycine at position 226 in the Scl1 protein had been replaced by asparatic acid, suggesting that this mutation in the protein, a subunit of proteasomes, may be involved in the morphological change.
Key words: pseudohyphal growth; cell extension; cell wall; SCL1; isoamyl alcohol
-9-
Maltosyl-erythritol, a Major Transglycosylation Product of Erythritol by Bacillus
stearothermophilus Maltogenic Amylase
Jong-Won YOON,1, Eun-Joo JEON,1 Il-Hun JUNG,1 Mee-Jung MIN,1 Hye-Young LEE,1 Myo-Jeong KIM,2 Jin-Sook BAEK,1 Hee-Seob LEE,1 Cheon-Seok PARK,3 Sangsuk OH,4 Kwan-Hwa PARK,1 and Tae-Wha MOON1,
1Center for Agricultural Biomaterials, National Laboratory for Functional Food Carbohydrates, and Department of Food Science and Technology, School of Agricultural Biotechnology, Seoul National University, Suwon 441-744, Korea 2Food Science and Nutrition Research Center and School of Food and Life Sciences, Inje University, Kimhae 621-749, Korea 3Department of Food Science and Technology, Kyunghee University, Suwon 449-701, Korea 4Department of Food and Nutrition, Ewha Womans University, Seoul 120-750, Korea
Received August 5, 2002; Accepted October 12, 2002
This study was done to modify erythritol to change its physicochemical and sensory properties. Erythritol, a four-carbon sugar alcohol, was transglycosylated by Bacillus stearothermophilus maltogenic amylase with maltotriose as a donor molecule. The presence of various transglycosylation products of erythritol was confirmed by TLC and high performance ion exchange chromatography (HPIC). The major transfer product was purified by gel filtration chromatography on Bio-Gel P-2. Examination by LC-MS, matrix-assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF-MS), and 13C NMR showed that the major transfer product was maltosyl-erythritol. Results of 13C NMR of maltosyl-erythritol suggested that linkage was formed between the C1 carbon of glucose unit in maltose and either one of the two carbon atoms of the terminal hydroxyl groups of erythritol, so that a mixture of 1-O- and 4-O-α-maltosyl-erythritol was produced. The sweetness of maltosyl-erythritol was about 40％ that of sucrose, and its negative sensory properties were less than those of erythritol.
Key words: maltosyl-erythritol; Bacillus stearothermophilus maltogenic amylase; transglycosylation; sweetness
-10-
Acarviosine-simmondsin, a Novel Compound Obtained from Acarviosine-glucose and
Simmondsin by Thermus Maltogenic Amylase and Its in vivo Effect on Food Intake
and Hyperglycemia
Jin-Sook BAEK,1 Hye-Young KIM,2 Thomas P. ABBOTT,3 Tae-Wha MOON,1 Soo-Bok LEE,4 Cheon-Seok PARK,5, and Kwan-Hwa PARK1,
1National Laboratory for Functional Food Carbohydrates, and Department of Food Science and Technology, School of Agricultural Biotechnology, Seoul National University, Suwon 441-744, Korea 2Food Chemistry and Biotechnology Division, Korea Food Research Institute, Sungnam 463-420, Korea 3National Center for Agricultural Utilization Research, USDA, IL 61604, USA 4Department of Food and Nutrition, Yonsei University, Seoul 120-749, Korea 5Department of Food Science and Technology, Kyunghee University, Youngin 449-701, Korea
Received August 22, 2002; Accepted October 31, 2002
Simmondsin was modified with acarviosine-glucose using the transglycosylation activity of Thermus maltogenic amylase to synthesize a novel compound with both antiobesity and hypoglycemic activity. The LC/MS and 13C NMR analyses confirmed that the structure of the major transglycosylation product was acarviosine-simmondsin (Acv-simmondsin), in which acarviosine was attached to the glucose moiety of simmondsin by an α-(1,6)-glycosidic linkage. It was found that Acv-simmondsin was a potent competitive inhibitor of α-glucosidase with the Ki value of 0.69 μM and a mixed type inhibitor of α-amylase with the Ki and KI of 20.78 μM and 26.31 μM, respectively. The administration of Acv-simmondsin (0.1 g/100 g diet/day) to mice for 5 days significantly reduced food intake by 35％, compared to 25％ with simmondsin in control obese mice. Acv-simmondsin (50 mg/kg BW) suppressed the postprandial blood glucose response to sucrose (1 g/kg BW) by 74％, compared to 71％ with acarbose, in normal rats.
Key words: Thermus maltogenic amylase; transglycosylation; acarviosine-simmondsin; acarbose; food intake
-11-
Histidine-114 at Subsites E and F Can Explain the Characteristic Enzymatic Activity
of Guinea Hen Egg-white Lysozyme
Gen TOSHIMA, Shunsuke KAWAMURA, Tomohiro ARAKI, and Takao TORIKATA
Department of Bioscience, School of Agriculture, Kyushu Tokai University, Aso, Kumamoto 869-1404, Japan
Received August 26, 2002; Accepted November 21, 2002
The courses of the reaction catalyzed by guinea hen egg-white lysozyme (GHL), in which Asn113 and Arg114 at subsites E and F in hen egg-white lysozyme (HEL) are replaced by Lys and His, respectively, was studied with the substrate N-acetylglucosamine pentamer, (GlcNAc)5. Although GHL was found to retain the main-chain folding similar to HEL as judged from CD spectroscopy, the courses of GHL showed increased production of (GlcNAc)4 and reduced production of (GlcNAc)2 when compared with HEL. To identify critical residue(s) involved in the alteration in the courses of GHL, two mutant enzymes as to subsites E and F in HEL, N113K and R114H, were prepared by site-directed mutagenesis. Kinetic analysis of these mutants revealed that the mutation of Asn113 to Lys had little effect on the courses of HEL, while the Arg114 to His mutation completely reproduced the courses of GHL, demonstrating that His114 in GHL is the key residue responsible for the characteristic courses of GHL. Computer simulation of the reaction courses of the R114H mutant revealed that this substitution decreased not only the binding free energies for subsites E and F, but also the rate constant of transglycosylation. The Arg residue at position 114 may play an important role in the transglycosylation activity of HEL.
Key words: lysozyme; lysozyme-catalyzed reactions; site-directed mutagenesis; subsite; transglycosylation
-12-
Characterization of the Prr1 Response Regulator with Special Reference to Sexual
Development in Schizosaccharomyces pombe
Norihito NAKAMICHI, Hisami YANADA, Hirofumi AIBA, Keisuke AOYAMA, Ryusuke OHMIYA, and Takeshi MIZUNO
Laboratory of Molecular Microbiology, School of Agriculture, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan
Received August 28, 2002; Accepted November 8, 2002
The histidine (His)-to-Aspartate (Asp) phosphorelay is a paradigm of intracellular signaling systems through protein phosphorylation in both prokaryotes and eukaryotes. The fission yeast Schizosaccharomyces pombe has three histidine kinases (Phk1/Mak2, Phk2/Mak3, and Phk3/Mak1), together with two response regulators (Mcs4 and Prr1). The results of recent extensive studies suggested that these His-to-Asp phosphorelay components are involved in oxidative stress responses through the transcriptional regulation of several scavenger genes for toxic free radicals. It was also suggested that they were somehow implicated in control of both the mitotic and meiotic cell proliferations. Among these S. pombe His-to-Asp phosphorelay components, however, the function of Prr1 is less clear. We here characterized a mutant, named prr1-D418N, specifying an altered Prr1 protein that presumably acts as a gain-of-function (or constitutive-active) mutant, with special reference to sexual development. The mutant cells showed a striking phenotype in that they underwent mating even in a nitrogen-sufficient medium, under which conditions the wild-type cells hardly did so. Furthermore, the mutant cells underwent mating very rapidly in a nitrogen-deficient medium. Under anaerobic (or micro-aerobic) growth conditions, the wild-type cells were not capable of undergoing sexual development even in a nitrogen-deficient medium. The prr1-D418N cells underwent mating efficiently under such anaerobic growth conditions. Taken these together, it was suggested that the function of Prr1 is closely linked to the well-characterized signaling pathways for induction of the sexual development, in a way that this response regulator regulates a critical step of the initiation of meiosis through activating the transcription of ste11＋, mam2＋, and mei2＋, in S. pombe.
Key words: fission yeast; phosphorelay; response regulator; sexual development
-13-
Characterization of Rice Functional Monosaccharide Transporter, OsMST5
Budsaraporn NGAMPANYA,1,2 Anna SOBOLEWSKA,3 Taito TAKEDA,3 Kyoko TOYOFUKU,3 Jarunya NARANGAJAVANA,2 Akira IKEDA,1,4 and Junji YAMAGUCHI1,
1Graduate School of Science, Hokkaido University, Kita-ku N10-W8, Sapporo 060-0810, Japan 2Department of Biotechnology, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand 3Bioscience Center, Nagoya University, Chikusa-ku, Nagoya 464-0860, Japan 4CREST, JST (Japan Science and Technology Corporation)
Received August 29, 2002; Accepted October 15, 2002
cDNA of a monosaccharide transporter in rice, OsMST5 (Oryza sativa monosaccharide transporter 5) was cloned and its sugar transport activity was characterized by heterologous expression analysis. The amino acid sequence and topology were similar to the sequences and topology of other plant monosaccharide transporters. Yeast cells co-expressed with OsMST5 cDNA transported some monosaccharide substrates. The transport rate increased when ethanol as an electron donor was added, so the transporter was an energy-dependent active one. Most of the OsMST5 was expressed in panicles before pollination, indicating that it is associated with pollen development in rice.
Key words: energy-dependent active transport; heterologous yeast expression; Oryza sativa L.; pollen development; sink cell
-14-
The N-Terminal Sequence of Paratropomyosin Binding Fragments from β-Connectin
Minoru YAMANOUE,1, Shuji UEDA,2, Akiko OHASHI,1 Yumi YOSHIMURA,3 and Shigemi NORIOKA3
1Faculty of Agriculture, and 2Graduate School of Science and Technology, Kobe University, Kobe, Hyogo 657-8501, Japan 3Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan
Received September 5, 2002; Accepted November 1, 2002
In order to clarify the position where paratropomyosin binds to connectin in the A-I junction region of a sarcomere, chicken β-connectin was digested by Staphylococcus aureus V8 protease under denaturing conditions and the digested peptides were electrophoretically separated. Five peptides, 150-kDa, 100-kDa, 70-kDa, and 43-kDa fragments, were simultaneously detected by biotinylated paratropomyosin and an anti-connectin monoclonal antibody. The N-terminal sequence of the 43-kDa fragment was found to be YQFRVYAVNK, similar to the sequence of 7556--7565 amino acids in the I51 fibronectin type 3 domain that was located at the A-I junction region of human cardiac titin/connectin. Therefore, we propose that paratropomyosin binds to the 43-kDa fragment from β-connectin at the A-I junction region in both living muscle and in muscle immediately postmortem, and the N-terminus of the 43-kDa fragment is localized in the I51 domain.
Key words: paratropomyosin; β-connectin; A-I junction; myofibrils; postmortem ageing
-15-
Enzymatic Properties of Glutamine 32 Mutants of RNase Rh from Rhizopus niveus,
a Trial to Alter the Most Preferential Inter-nucleotidic Linkages of RNase Rh
Kazuko OHGI,1, Masanori IWAMA,2 Norio INOKUCHI,3 and Masachika IRIE1
1Department of Applied Microbiology, Hoshi University, School of Pharmacy and Pharmaceutical Sciences, 2-4-41 Ebara, Shinagawa-ku, Tokyo 142-8501, Japan 2Department of Materials Engineering, Nagaoka National College of Technology, 888 Nishikatagaichou, Nagaoka, Niigata 940-8532, Japan 3Department of Microbiology, College of Pharmacy, Nihon University, 7-7-1 Narashinodai, Funabashi, Chiba 274-8555, Japan
Received September 9, 2002; Accepted November 16, 2002
In order to investigate the effects of mutation of Gln32, a component of a base recognition site (B2 site) of a base-nonspecific RNase from Rhizopus niveus, we prepared several enzymes mutant at this position, Q32F, Q32L, Q32V, Q32T, Q32D, Q32N, and Q32E, and their enymatic activities toward RNA and 16 dinucleoside phosphates were measured. Enzymatic activities of the mutant enzymes towards RNA were between 10--125％ of the native enzyme. From the rates of hydrolysis of 16 dinucleoside phosphates by mutant enzymes, we estimated the base specificity of both B1 and B2 sites. The results indicated that mutation of Gln32 to Asp, Asn, and Glu caused the B2 site to prefer cytosine more and to a less extent, to prefer uracil (Q32N), and that Q32F made the enzyme more guanine-base preferential. The results suggested that we are able to construct an enzyme that preferentially cleaves internucleotidic linkages, at the 5′-side of cytosine residues (Q32D, Q32N, and Q32E) and guanine residues (Q32F and Q32T), thus, cleaves purine-C(Q32D, Q32N, Q32E) and GpG and ApG (Q32F, and Q32T) most easily. The results seemed to suggest converting a base-non-specific RNase to a base-specific one.
Key words: base non-specific RNase; B2 subsite; ribonuclease Rh; Rhizopus niveus; site-directed mutagenesis
-16-
Effects of Bovine α-Lactalbumin on Gastric Defense Mechanisms in Naive Rats
Yoshihiko USHIDA, Yukiko SHIMOKAWA, Hiroshi MATSUMOTO, Tomohiro TOIDA, and Hirotoshi HAYASAWA
Biochemical Research Laboratory, Morinaga Milk Industry Co., Ltd., 5-1-83 Higashihara, Zama, Kanagawa 228-8583, Japan
Received September 17, 2002; Accepted November 8, 2002
We recently investigated the effects of the major proteins in cow′s milk on gastric mucosal injuries in rat ulcer models. We found that α-lactalbumin (α-LA) has marked preventive effects against gastric mucosal injuries and that prostaglandin (PG) synthesis may contribute to these effects [Matsumoto et al., Biosci. Biotechnol. Biochem., 65, 1104--1111, 2001]. In this study, we investigated the effects of α-LA on several defense mechanisms of gastric mucosa by evaluating gastric PGE2 content, gastric mucin content, gastric luminal pH, gastric fluid volume, and gastric emptying in naive rats. Oral administration of α-LA (200, 500, and 1000 mg/kg) elevated endogenous PGE2 levels in gastric tissue and increased the gastric mucin contents of both the gastric fluid and the adherent mucus gel layer. In addition to these PG-related responses, α-LA also caused PG-independent responses such as elevation of gastric luminal pH, increase in gastric fluid volume, and delay in gastric emptying. These responses were observed to be dose-dependent (200--1000 mg/kg of α-LA). Thus, we demonstrated that α-LA enhances both PG-dependent and PG-independent gastric defense mechanisms in naive rats. Both of these mechanisms are probably involved in its gastroprotective action.
Key words: α-lactalbumin; gastroprotection; ulcer; defense mechanism; prostaglandin
-17-
Cloning of the Xylitol Dehydrogenase Gene from Gluconobacter oxydans and Improved
Production of Xylitol from D-Arabitol
Masakazu SUGIYAMA, Shun-ichi SUZUKI, Naoto TONOUCHI, and Kenzo YOKOZEKI
AminoScience Laboratories, Ajinomoto Co., Inc., Suzuki-cho, Kawasaki-ku, Kawasaki-shi 210-8681, Japan
Received September 20, 2002; Accepted November 11, 2002
Xylitol dehydrogenase (XDH) was purified from the cytoplasmic fraction of Gluconobacter oxydans ATCC 621. The purified enzyme reduced D-xylulose to xylitol in the presence of NADH with an optimum pH of around 5.0. Based on the determined NH2-terminal amino acid sequence, the gene encoding xdh was cloned, and its identity was confirmed by expression in Escherichia coli. The xdh gene encodes a polypeptide composed of 262 amino acid residues, with an estimated molecular mass of 27.8 kDa. The deduced amino acid sequence suggested that the enzyme belongs to the short-chain dehydrogenase/reductase family. Expression plasmids for the xdh gene were constructed and used to produce recombinant strains of G. oxydans that had up to 11-fold greater XDH activity than the wild-type strain. When used in the production of xylitol from D-arabitol under controlled aeration and pH conditions, the strain harboring the xdh expression plasmids produced 57 g/l xylitol from 225 g/l D-arabitol, whereas the control strain produced 27 g/l xylitol. These results demonstrated that increasing XDH activity in G. oxydans improved xylitol productivity.
Key words: xylitol; xylitol dehydrogenase; Gluconobacter; short-chain dehydrogenase/reductase family
-18-
Functional Analysis of Individual Oligosaccharide Chains of Sendai Virus Hemagglutinin-neuraminidase
Protein
Hiroaki SEGAWA,1,2 Akira INAKAWA,1 Tetsuro YAMASHITA,1 and Hideharu TAIRA1,
1Department of Agro-bioscience, Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka City 020-8550, Japan 2Department of Biochemistry, University of Kentucky Medical Center, College of Medicine, 800 Rose Street, University of Kentucky Medical Center, Lexington, KY 40536, U.S.A.
Received September 25, 2002; Accepted November 7, 2002
The roles of N-linked glycosylation in the intracellular transport and biological activities of the Sendai virus hemagglutinin-neuraminidase (HN) protein were studied. The protein contains four potential N-glycosylation sites: N77, N448, N499, and N511. By site-directed mutagenesis of these positions, the mature protein contained three N-linked oligosaccharides attached to N77, N499, and N511. The role of each added oligosaccharide in the structure and functions of the protein was identified by characterization of surface expression, hemadsorption, and neuraminidase activities of the corresponding mutant proteins. Elimination of the sites of N499 and N511 had the most detrimental effect, decreasing surface expression and hemadsorption. Elimination of the sites of N77 and N448 had similar but weaker effects. Mutants missing the sites of N499 and N511 were not able to induce syncytia formation in cells expressing mutant HN proteins and the fusion protein. Therefore, the N-linked oligosaccharides attached to N499 and N511 were important for intracellular transport and for the fusion promotion.
Key words: hemagglutinin-neuraminidase; intracellular transport; N-linked oligosaccharide chain; Sendai virus
-19-
Isotope Ratio Analysis by HRGC-MS of Monoterpene Hydrocarbons from Citrus Essential
Oils
Atsushi SATAKE,1,2, Akitoshi UNE,1 Takao UENO,1 Hiroyuki UKEDA,1 and Masayoshi SAWAMURA1
1Department of Bioresources Science, Faculty of Agriculture, Kochi University, Nankoku, Kochi 783-8502, Japan 2Nagaoka Perfumery Co., Ltd., 3-30, 1-chome, Itsukaichi, Ibaraki-City, Osaka 567-0005, Japan
Received October 18, 2002; Accepted November 12, 2002
The isotope ratio of monoterpene hydrocarbons in citrus essential oils of different origins was measured by ordinary high-resolution gas chromatography-mass spectrometry (HRGC-MS). The isotope ratio (Ir) was determined by the ratio of the isotope peak intensity (m/z 137) to the molecular mass peak intensity (m/z 136) of the monoterpene hydrocarbons. The accuracy of Ir was examined by measuring monoterpene hydrocarbon standards and 13C-labeled compounds. The isotope fingerprints based on the values of monoterpene hydrocarbons from lemon, lime and yuzu essential oils were determined. These citrus essential oils were also discriminated by a principal component analysis of their Ir data. The characteristic vectors showed that α-terpinene, β-pinene and β-phellandrene were important components for distinguishing between the citrus species. It is suggested that this technique will be applicable to evaluate the quality, genuineness and origin of citrus fruits and their products.
Key words: citrus essential oil; isotope effect; isotope ratio; HRGC/MS; monoterpene hydrocarbon
-20-
Functional Analysis of a β-Ketoacyl-CoA Synthase Gene, MpFAE2, by Gene Silencing
in the Liverwort Marchantia polymorpha L.
Masataka KAJIKAWA,1 Shohei YAMAOKA,1 Katsuyuki T. YAMATO,1 Hiroyuki KANAMARU,2 Eiji SAKURADANI,2 Sakayu SHIMIZU,2 Hideya FUKUZAWA,1 and Kanji OHYAMA1,
1Laboratory of Plant Molecular Biology, Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan 2Laboratory of Fermentation Physiology and Applied Microbiology, Department of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
Received November 5, 2002; Accepted November 25, 2002
We have isolated a β-ketoacyl CoA synthase (KCS) gene, MpFAE2, from a liverwort, Marchantia polymorpha, and identified its substrate specificity using the technique of dsRNA-mediated gene silencing and overexpression. KCS catalyzes an essential reaction in the fatty acid elongation process, i.e., condensation of malonyl-CoA with acyl-CoA. By introducing a construct with a hairpin structure containing a partial MpFAE2 gene, the level of the MpFAE2 gene expression was suppressed constitutively. The transgenic plants showed a specific accumulation of fatty acid 18:0. In contrast, in transgenic M. polymorpha plants overexpressing the MpFAE2 gene, fatty acid 22:0 is accumulated. These results indicate that the MpFAE2 gene product catalyzes the elongation steps of 18:0 to 20:0 and possibly also of 20:0 to 22:0.
Key words: β-ketoacyl-CoA synthase gene; fatty acid chain elongation; dsRNA-mediated gene silencing; overexpression; Marchantia polymorpha
-21-
Note
Cloning of Structural Gene of Deinococcus radiodurans UV-Endonuclease β
Shigeru KITAYAMA,1,2, Issay NARUMI,2 Tomoo FUNAYAMA,2 and Hiroshi WATANABE2
1Cell Physiology Laboratory, The Institute of Physical ＆ Chemical Research, Wako, Saitama 350-0198, Japan 2Biotechnology Laboratory, Japan Atomic Energy Research Institute, Takasaki, Gunma 370-1292, Japan
Received July 26, 2002; Accepted November 8, 2002
To characterize its enzymic property we cloned and sequenced the gene of Deinococcus radiodurans encoding UV-endonuclease β, an alternative enzyme to UvrABC repairing damaged DNA. Amino acid substitutions were found in UV-sensitive mutants. The putative amino acid sequence had some similarity with those of eukaryotic UV-endonucleases and with a sequence found in a protein data base of Bacillus subtilis.
Key words: Deinococcus radiodurans; DNA repair gene; endonuclease; flap endonuclease 1; UV-endonuclease β
-22-
Note
Effects of Adding Some Dietary Fibers to a Cystine Diet on the Activities of
Liver Antioxidant Enzymes and Serum Enzymes in Rats
Guochun HE and Yoritaka AOYAMA
Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Nishi-9, Kita-9, Kita-ku, Sapporo 060-8589, Japan
Receivesd July 30, 2002; Accepted November 6, 2002
This study investigates whether some dietary fibers can the toxicity due to cystine added to the diet. Wistar rats were investigated for the effects of adding pectin, sugar beet fiber or konjac mannan to a cystine diet on the growth rate and on the activities of liver antioxidant enzymes and serum enzymes. The addition of pectin, sugar beet fiber or konjac mannan to the cystine diet resulted in a significant increase in both the food intake and body weight gain. Feeding the cystine diet caused lower activities of total and Cu, Zn-superoxide dismutase, and of catalase in the liver. The addition of pectin to the cystine diet counteracted the activities of the total and Cu,Zn-superoxide dismutase, and of catalase in liver. Of the dietary fibers tested, konjac mannan prevented the elevation of the two enzyme activities in the serum induced by feeding the cystine diet, indicating that this fiber might have the ability to alleviate hepatic damage due to dietary cystine.
Key words: cystine; dietary fiber; superoxide dismutase; serum ornithine carbamoyltransferase; serum alanine aminotransferase
-23-
Note
Genomic Organization of the S-Locus Region of Brassica
Hiroshi SHIBA,1, Masayuki KENMOCHI,1 Minoru SUGIHARA,1 Megumi IWANO,1 Shinji KAWASAKI,2 Go SUZUKI,3 Masao WATANABE,4 Akira ISOGAI,1 and Seiji TAKAYAMA1
1Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma 630-0101, Japan 2National Institute of Agrobiological Resources, Tsukuba 305-0856, Japan 3Division of Natural Science, Osaka Kyoiku University, Kashiwara 582-8582, Japan 4Faculty of Agriculture, Iwate University, Morioka 020-8550, Japan
Received August 20, 2002; Accepted November 1, 2002
To gain some insights into the structure of the S-locus and the mechanisms that have kept its diversity, a 75-kb genomic fragment containing the self-incompatibility (S) locus region was isolated from the S12-haplotype of Brassica rapa and compared with those of other S-haplotypes. The region around the S determinant genes was highly polymorphic and filled with S-haplotype-specific intergenic sequences. The diverse genomic structure must contribute to the suppression of recombination at the S-locus.
Key words: self-incompatibility; genomic organization; intergenic region; repetitive sequence; S-locus
-24-
Note
Purification and Characterization of Extracellular β-Galactosidase Secreted
by Supension Cultured Rice (Oryza sativa L.) Cells
Satoshi KANEKO and Hideyuki KOBAYASHI
National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan
Received August 30, 2002; Accepted November 11, 2002
A β-galactosidase was purified 1300-fold by lactosyl-Sepharose 4B and Sephacryl S-200 column chromatographies from the cultured medium of a rice-cell suspension. The purified enzyme appeared as 47 kD and 40 kD polypeptides on SDS-PAGE and had a specific activity of 65.1 units/mg. Optimum activity was observed at pH 3.5 and 60°C. The enzyme released galactose from galactoxyloglucan and pectic galactans.
Key words: β-galactosidase; purification of β-galactosidase; Oryza sativa L.; rice
-25-
Note
Mushroom Tyrosinase Inhibitory Activity of Esculetin Isolated from Seeds of
Euphorbia lathyris L.
Yukimitsu MASAMOTO,1 Hideya ANDO,2 Yoshiyuki MURATA,3 Yasuaki SHIMOISHI,3 Mikiro TADA,1 and Kyoya TAKAHATA3,
1Graduate School of Natural Science and Technology, Okayama University, 1-1 Naka 3-chome, Tsushima, Okayama 700-8530, Japan 2R＆D Center, Sunstar Inc., 3-1 Asahi-Machi, Takatsuki, Osaka 569-1195, Japan 3Faculty of Agriculture, Okayama University, 1-1 Naka 1-chome, Tsushima, Okayama 700-8530, Japan
Received September 6, 2002; Accepted November 11, 2002
A tyrosinase inhibitor was isolated from the seeds of Euphorbia lathyris L. by bioassay-guided fractionation and purification, using silica gel column chromatography. It was identified as esculetin by comparing its physical properties and spectral data with those of an authentic sample. The IC50 value of esculetin in the mushroom tyrosinase activity test was 43 μM. The kinetic study indicates that esculetin exhibited competitive inhibition against the oxidation of 3-(3,4-dihydroxyphenyl)-alanine by mushroom tyrosinase. The structure-activity relationships among five esculetin analogs suggest that hydroxyl groups at the C6 and C7 positions of the coumarin skeleton played an important role in the expression of tyrosinase inhibitory activity.
Key words: esculetin; mushroom tyrosinase inhibitory activity; coumarin; Euphorbia lathyris L.
-26-
Note
Inhibitory Activity of Analogs of AM-Toxin, a Host-specific Phytotoxin from
the Alternaria alternata Apple Pathotype, on Photosynthetic O2 Evolution in
Apple Leaves
Masahiro MIYASHITA,1, Tomoko NAKAMORI,1 Hisashi MIYAGAWA,1 Miki AKAMATSU,2 and Tamio UENO1
1Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan 2Division of Environmental Science and Technology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
Received September 9, 2002; Accepted October 17, 2002
The effect of the host-specific phytotoxins, AM-toxins, on the photosynthetic activity of leaves from susceptible apple cultivars was investigated by using an oxygen electrode. The photosynthetic O2 evolution was inhibited by AM-toxin I in a host-specific manner. The inhibitory activity of several AM-toxin analogs against photosynthesis was also evaluated and the findings were correlated with their necrosis-inducing activity.
Key words: AM-toxin; photosynthesis; host-specific toxin; oxygen electrode; necrosis
-27-
Note
Continuous Cell-free Protein Synthesis Directed by Messenger DNA and Catalyzed
by Extract of Thermus thermophilus HB27
Taketoshi UZAWA,, Akihiko YAMAGISHI, and Tairo OSHIMA
Department of Life Science, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama 226-8501, Japan
Received September 11, 2002; Accepted October 17, 2002
Polypeptide synthesis directed by DNA as the messenger in a cell-free system of Thermus thermophilus was investigated. Polypeptides were synthesized with the addition of neomycin in the presence of DNA catalyzed by the cell extract. The stability of messenger DNA was greater than that of messenger RNA. Continuous cell-free translation with messenger DNA produced polypeptides at the rate of more than 8 μg/h in the presence of spermine.
Key words: Thermus thermophilus; messenger DNA; cell-free protein synthesis; extreme thermophile; polyamine
-28-
Note
Formation of 3′-O-β-Galactosyl Compounds of 5-Bromouridine by Sporobolomyces
singularis
Kei UCHIDA and Yukio SUZUKI
Research Institute for Bioresources, Okayama University, Kurashiki, Okayama-ken 710-0046, Japan
Received September 11, 2002; Accepted November 19, 2002
Two new compounds of 5-bromouridine, 3′-O-(β-D-
galactopyranosyl)-5-bromouridine and 3′-O-[β-D-galactopyranosyl-(1→4)-β-D-galactopyranosyl]-5-bromouridine, were found to be selectively formed in a high yield in a culture filtrate of Sporobolomyces singularis, when
grown on a medium containing lactose and 5-bromouridine.
Key words: Sporobolomyces singularis; transglycosylation; 3′-O-β-galactosyl-5-bromouridine; 3′-O-β-galactobiosyl-5-bromouridine
-29-
Note
Cloning and Overexpression of β-N-Acetylglucosaminidase Encoding Gene nagA from
Aspergillus oryzae and Enzyme-catalyzed Synthesis of Human Milk Oligosaccharide
Ichiro MATSUO,2, Sunhwa KIM,1 Yuichi YAMAMOTO,2, Katsumi AJISAKA,2 Jun-ich MARUYAMA,1 Harushi NAKAJIMA,1 and Katsuhiko KITAMOTO1,
1Department of Biotechnology, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan 2Meiji Institute of Health Science, Naruda 540, Odawara 250-0862, Japan
Received September 18, 2002; Accepted November 20, 2002
We isolated a β-N-acetylglucosaminidase encoding gene from the filamentous fungus Aspergillus oryzae, and designated it nagA. The nagA gene encoded a polypeptide of 600 amino acids with significant similarity to glucosaminidases and hexosaminidases of various eukaryotes. A. oryzae strain carrying the nagA gene under the control of the improved glaA promoter produced large amounts of β-N-acetylglucosaminidase in a wheat bran solid culture. The β-N-acetylglucosaminidase was purified from crude extracts of the solid culture by column chromatographies on Q-Sepharose and Sephacryl S-200. This enzyme was used for synthesis of lacto-N-triose II, which is contained in human milk. By reverse hydrolysis reaction, lacto-N-triose II and its positional isomer were synthesized from lactose and D-N-acetylglucosamine in 0.21％ and 0.15％ yield, respectively.
Key words: β-N-acetylglucosaminidase; nagA; Aspergillus oryzae; enzymatic oligosaccharide synthesis
-30-
Note
Targeted Gene Disruption of the Neuronal Calcium Sensor 1 Homologue in Rice
Blast Fungus, Magnaporthe grisea
Ken-ichiro SAITOH,1 Tsutomu ARIE,1 Tohru TERAOKA,1 Isamu YAMAGUCHI,2, and Takashi KAMAKURA2,
1Faculty of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan 2Microbial Toxicology Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
Received September 27, 2002; Accepted November 19, 2002
We isolated a neuronal calcium sensor 1/frequenin-like gene, Mg-NCS-1, from Magnaporthe grisea and evaluated the phenotypes of null-mutants of the gene. The putative Mg-NCS-1 protein showed high similarity to the other NCS-1 proteins. The null-mutants had normal growth and pathogenicity similar to the parental strain, but their growth was suppressed in high concentrations of Ca2＋ or acidic conditions.
Key words: neuronal calcium sensor 1; frequenin; calcium binding protein; Magnaporthe grisea; rice blast fungus
-31-
Note
Molecular Cloning of Rat Transcription Factor YY1
Chiharu NISHIYAMA,1,2, Toyokazu YOKOTA,2 Makoto NISHIYAMA,3 Chisei RA,1 Ko OKUMURA,1 and Hideoki OGAWA1
1Atopy (Allergy) Research Center, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan 2Foods ＆ Pharmaceuticals Research ＆ Development Laboratory, Asahi Breweries, Ltd., 1-1-21 Midori, Moriya-shi, Ibaraki 302-0106, Japan 3Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
Received October 29, 2002; Accepted November 7, 2002
YY1 is a ubiquitously expressed multifunctional transcription factor that is involved in both positive and negative regulation of gene expression as well as initiation of transcription. Here, we isolated cDNA encoding a full-length open reading frame (ORF) of rat YY1. Rat YY1 is composed of 411 amino acid residues and its amino acid sequence is 97.6％ identical to that of mouse YY1 and 97.8％ identical to that of human YY1. The transactivating abilities of wild-type rat YY1 and four truncated mutant forms of YY1 were examined by transient reporter assays. When residues 114--193, which sequence includes a portion of the activation region and most of the Gly/Lys-rich region, were lacking, transactivation activity decreased somewhat, but the further deletion in the activation region (of residues 56--113) did not cause further decrease of the activity. On the other hand, N-terminus of the activation region (1--78/100--106) did not have transactivation activity by itself as well as synergistic activity with an erythroid specific transcription factor GATA-1.
Key words: YY1; transcription factor; cDNA; GATA-1
-32-
Preliminary Communication
Total Synthesis of 0231B, an Inhibitor of 3α-Hydroxysteroid Dehydrogenase Produced
by Streptomyces sp. HKI0231
Taichi KOMODA, Yoshihiko SHINODA, and Shin-ichi NAKATSUKA
The United Graduate School of Agricultural Science, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
Received November 7, 2002; Accepted December 17, 2002
The new inhibitors of 3α-hydroxysteroid dehydrogenase, 0231A 1 and 0231B 2, have a unique benz[c,d]indol-3(1H)-one structure in their molecules. In our advanced studies on indole chemistry, we have developed an efficient synthetic method for benz[c,d]indol-3(1H)-one derivatives. We report here its application to the synthesis of 0231B in 10 steps (8.1％ overall yield) from 6-methylindole 8 by introducing an acyl group into the 3-position of the indole nucleus, cyclization of the side chain at the 3-position to the 4-position and subsequent elimination of the phenyl group, and conjugate addition of the substituted phenyl group.
Key words: inhibitor; 3α-hydroxysteroid dehydrogenase; anti-inflammatory agent; indole
-33-
Preliminary Communication
Gnetol as a Potent Tyrosinase Inhibitor from Genus Gnetum
Kenji OHGUCHI,1, Toshiyuki TANAKA,2 Ibrahim ILIYA,2 Tetsuro ITO,2 Munekazu IINUMA,3 Kenji MATSUMOTO,1 Yukihiro AKAO,1 and Yoshinori NOZAWA1
1Gifu International Institute of Biotechnology, Mitake, Kani-gun, Gifu 505-0116, Japan 2Gifu Prefectural Institute of Health and Environmental Sciences, Kakamigahara, Gifu 504-0838, Japan 3Gifu Pharmaceutical University, 5-6-1 Mitahora-higashi, Gifu 502-5858, Japan
Received November 13, 2002; Accepted December 9, 2002
Gnetol (2,3′,5′,6-tetrahydroxy-trans-stilbene), a naturally occurring compound particularly found in the genus Gnetum, had a strong inhibitory effect on murine tyrosinase activity. Gnetol (IC50, 4.5 μM) was stronger than kojic acid (IC50, 139 μM) as a standard inhibitor for murine tyrosinase activity. Moreover, gnetol significantly suppressed melanin biosynthesis in murine B16 melanoma cells.
Key words: tyrosinase; melanin; gnetaceae; gnetol