Bioscience, Biotechnology, and Biochemistry
Online ISSN : 1347-6947
Print ISSN : 0916-8451
Volume 64 , Issue 8
Showing 1-37 articles out of 37 articles from the selected issue
Review
  • Shunsuke MORIYAMA, Felix G. AYSON, Hiroshi KAWAUCHI
    2000 Volume 64 Issue 8 Pages 1553-1562
    Published: 2000
    Released: February 08, 2005
    JOURNALS FREE ACCESS
      Insulin-like growth factor-I (IGF-I) is a mitogenic polypeptide that plays an essential role in the regulation of development and somatic growth of vertebrates, mainly by mediating growth hormone actions. It has clearly been established that the structure of IGF-I and its biological function has been highly conserved among vertebrates. In this paper, we review the recent developments in the molecular, biochemical, and physiological properties of IGF-I in fish.
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Analytical Chemistry Note
Organic Chemistry Regular Papers
Organic Chemistry Notes
Biochemistry & Molecular Biology Regular Papers
  • Jian-Gang YANG, Takeo UCHIYAMA
    2000 Volume 64 Issue 8 Pages 1572-1579
    Published: 2000
    Released: February 08, 2005
    JOURNALS FREE ACCESS
      The contents of wall-bound hydroxycinnamic acids and their dimers were compared between elongated and non-elongated cells of suspension-cultured Mentha. Wall-bound peroxidase activity was also investigated. The main hydroxycinnamic acids esterified to these two kinds of cell walls were ferulic and caffeic acids. Eleven dehydrodicaffeic acid isomers and six dehydrodiferulic acid isomers formed through C-C and C-O-C coupling processes, were detected by GC-MS from the extracts released from the walls of non-elongated cells. On the other hand, only four dehydrodicaffeic acid isomers and three dehydrodiferulic acid isomers were found in the walls of elongated cells. Amounts of monomers of ferulic and caffeic acids and their 5,5′-dehydrodimers in non-elongated cell walls were about ten and twenty times higher, respectively, than those in the elongated cell walls. There was a close correlation between the amount of 5,5′-dehydrodimers and activity of wall-bound peroxidase in non-elongated and elongated cells. The level of 5,5′-dehydrodimers accumulated at a higher rate than monomers in non-elongated cell walls. These results suggest that the dimerization of ester-linked ferulic and caffeic acids by peroxidase and the increase in amounts of their 5,5′-dehydrodimers are important factors in the cessation of cell elongation in Mentha suspension culture.
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  • Makio FURUICHI, Etsuko NISHIMOTO, Toshiaki KOGA, Shoji YAMASHITA
    2000 Volume 64 Issue 8 Pages 1623-1627
    Published: 2000
    Released: February 08, 2005
    JOURNALS FREE ACCESS
      By analyzing the steady state and time-resolved fluorescence anisotropy, the internal motions of chlorophyll a of light-harvesting chlorophyll a/b-protein complex (LHCII) were characterized in a dimyristoylphosphatidylcholine (DMPC) liposome. Corresponding to the thermotropic phase of the membrane, chlorophyll a showed an unique internal motion in LHCII. At the gel phase, two motional components, one fast and the other slow, were observed, which would originate in the heterogeneity of the mutual orientation and the binding site of the chlorophyll a in LHCII. Interestingly, the faster motion was suppressed and only the slower segmental rotation with the larger motional amplitude was allowed on the phase transition to a liquid crystalline phase.
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  • Koshiki MINO, Tsuyoshi YAMANOUE, Takaharu SAKIYAMA, Naoki EISAKI, Asah ...
    2000 Volume 64 Issue 8 Pages 1628-1640
    Published: 2000
    Released: February 08, 2005
    JOURNALS FREE ACCESS
      Some properties and kinetics of the free and bound serine acetyltransferases (SATs) and O-acetylserine sulfhydrylase-As (OASS-As) from Escherichia coli were investigated. In some cases, SATΔC20, deleting 20 amino acid residues from the C-terminus of the wild-type SAT (Biosci. Biotechnol. Biochem., 63, 168-179 (1999)) was tested for comparison. The optimum pH and stability against some reagents for the free and bound wild-type SATs were similar except for the resistance to cold inactivation. The kinetics for the wild-type SAT and SATΔC20 followed a Ping-Pong Bi Bi mechanism with a mixed-type inhibition by L-cysteine. The kinetics and kinetic constants for the wild-type SAT were not changed by the complex formation with OASS-A. The optimum pH for OASS-A was shifted towards an alkaline pH by the complex formation. Thermal stability and stability against some reagents for the free and bound OASS-As were almost the same. On the other hand, the maximum velocity for OASS-A was lowered and dissociation constants for the substrates and products were increased by forming the complex with the wild-type SAT, although the kinetics for the free and bound enzymes followed the same Ping-Pong Bi Bi mechanism. From comparisons of computed courses of L-cysteine formation from L-serine using SAT (wild-type SAT and SATΔC20) and OASS-A with the experimental results and changes in the stability of the wild-type SAT by the complex formation, we discuss the role and significance of a complex formation for the cysteine synthetase.
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  • Ken-ichi HONJOH, Hiroko MATSUMOTO, Hideyuki SHIMIZU, Kanae OOYAMA, Kag ...
    2000 Volume 64 Issue 8 Pages 1656-1663
    Published: 2000
    Released: February 08, 2005
    JOURNALS FREE ACCESS
      The nucleotide sequence of hiC12, isolated as a cDNA clone of hardening-induced Chlorella (hiC) genes, was identified. The clone encodes a late embryogenesis abundant (LEA) protein having six repeats of a 11-mer amino acid motif, although in a slightly imperfect form. To overexpress the hiC61) and hiC12 genes, their coding regions were PCR amplified and subcloned into a pGEX-1λT vector. The HIC6 and HIC12 proteins were expressed as GST fusion proteins in E. coli, then purified. The two HIC proteins were found to be effective in protecting a freeze-labile enzyme, LDH, against freeze-inactivation. On a molar concentration basis, they were about 3.1×106 times more effective in protecting LDH than sucrose and as effective as BSA. Cryoprotection tests with five kinds of chain-shortened polypeptides, synthesized based on the 11-mer amino acid motif of the HIC6 protein showed that the cryoprotective activity decreased with a decrease in the repeating units of the 11-mer motif. In fact, cryoprotective activities of three kinds of single 11-mer amino acids were very low even at high concentrations. All the results suggested that the sufficiently repeated 11-mer motif is required for the cryoprotective activities of Chlorella LEA proteins.
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  • Takahiro SHIOTSUKI, Bryony C. BONNING, Makoto HIRAI, Kyoko KIKUCHI, Br ...
    2000 Volume 64 Issue 8 Pages 1681-1687
    Published: 2000
    Released: February 08, 2005
    JOURNALS FREE ACCESS
      Juvenile hormone esterase (JHE) from hemolymph of the silkworm moth Bombyx mori was characterized for substrate specificity and inhibitor sensitivity. B. mori JHE hydrolyzed the juvenile hormone surrogate substrate methyl n-heptylthioacetothioate (HEPTAT) more efficiently than p-nitrophenyl acetate and 1-naphthyl acetate substrates widely used to assay total carboxylesterase activity. B. mori JHE was sensitive to 3-octylthio-1,1,1-trifluoro-2-propanone (OTFP), which was developed as a selective inhibitor for lepidopteran JHE, and relatively insensitive to diisopropyl fluorophosphate (DFP), an inhibitor of serine esterases but not of all JHEs. Affinity purification with a trifluoromethyl ketone ligand was more efficient for purification of B. mori JHE than DEAE ion exchange chromatography.
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  • Akihito YASUOKA, Yasufumi EMORI, Keiko ABE
    2000 Volume 64 Issue 8 Pages 1688-1695
    Published: 2000
    Released: February 08, 2005
    JOURNALS FREE ACCESS
      Several recent papers have reported the difficulties in expressing olfactory receptor proteins (ORs) in heterologous systems, and proposed that some sequences in ORs have negative effects on their efficient expression. To obtain an efficient expression system of ORs, we modified N-terminal sequences of ORs through the addition of exogenous sequences. Three kinds of sequences, designated as 5HT, V, and VL, were used. 5HT and V corresponded to the signal leader (SL) sequences of 5HT3R and VIPR, respectively. VL corresponded to the first extracellular region of VIPR containing the SL sequence and three potential asparagine- (Asn-) linked glycosylation sites. The myc epitope was also added to the C-termini of the sequences. Several ORs including I7 of rat, GUST43 of rat, Y1 of medaka, FOR1-3 of pufferfish, 47E of carp, and ODR-10 of nematode were subjected to the modifications, and the RNAs encoding modified ORs were injected into Xenopus oocytes. The membrane fraction of the oocytes were analyszed by Western blotting to examine the expression of the proteins. In the cases of ORs modified with 5HT and V, only ODR-10 and 47E, both of which have more than two Asn-linked glycosylation sites in their extracellular regions, were detected as the bands of predicted molecular weights. On the other hand, most of the ORs modified with VL showed the bands of predicted molecular weights. These results suggest that SL sequences together with potential Asn-linked glycosylation sites have positive effects on the expression of ORs in heterologous systems.
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  • Hiromasa IMAISHI, Satoshi MATSUO, Eri SWAI, Hideo OHKAWA
    2000 Volume 64 Issue 8 Pages 1696-1701
    Published: 2000
    Released: February 08, 2005
    JOURNALS FREE ACCESS
      Cytochrome P450 (P450 or CYP) monooxygenases play an important role in the oxidation of a number of lipophilic substrates including secondary metabolites in higher plants. Larkin reported that CYP78A1 was preferentially expressed in developing inflorescences of Zea mays (Larkin, Plant Mol. Biol. 25: 343-353, 1994). However, the enzymatic function of CYP78A1 hasn’t been clarified yet. To characterized the enzymatic activity of CYP78A1, in this study, CYP78A1 cDNA and tobacco or yeast NADPH-cytochrome P450 oxidoreductase (P450 reductase) was expressed in the yeast Saccharomyces cerevisiae AH22 cells under the control of alcohol dehydrogenase promoter I and terminator. The reduced CO-difference spectrum of a microsomal fraction prepared from the transformed yeast cells expressing CYP78A1 and yeast P450 reductase showed a peak at 449 nm. Based on the spectrum, the content of a P450 molecule was estimated to be 45 pmol P450 equivalent/mg of protein in the microsomal fraction. The recombinant yeast microsomes containing CYP78A1 and yeast P450 reductase were found to catalyze 12-monooxygenation of lauric acid. Based on these results, CYP78A1 preferentially expressed in developing inflorescences of Zea mays appeared to have participated in the monooxygenation of fatty acids.
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  • Taro KISO, Hirofumi NAKANO, Hirofumi NAKAJIMA, Tadamasa TERAI, Katsuyu ...
    2000 Volume 64 Issue 8 Pages 1702-1706
    Published: 2000
    Released: February 08, 2005
    JOURNALS FREE ACCESS
      p-Hydroxybenzoyl β-galactose (pHB-Gal) was synthesized chemically to examine the hydrolytic activity of β-galactosyl ester linkage by β-galactosidases. The enzyme from Penicillium multicolor hydrolyzed the substrate as fast as p-nitrophenyl β-galactoside (pNP-Gal), a usual substrate with a β-galactosidic linkage. The enzymes from Escherichia coli and Aspergillus oryzae hydrolyzed pHB-Gal with almost the same rates as pNP-Gal. The enzymes from Bacillus circulans, Saccharomyces fragilis, and bovine liver showed much lower activities. pH-activity profiles, inhibition analysis, and kinetic properties of the enzymic reaction on pHB-Gal suggested that β-galactosidase had only one active site for hydrolysis of both galactosyl ester and galactoside. The Penicillium enzyme hydrolyzed pHB-Gal in the presence of H218O to liberate galactose containing 18O. This result suggests the degradation occurs between the anomeric carbon and an adjacent O atom in the ester linkage of pHB-Gal.
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Biochemistry & Molecular Biology Notes
  • Motoaki SANO, Keiichi NAKAJIMA, Kumiko TAKASE, Midori YAMAMOTO, Masayu ...
    2000 Volume 64 Issue 8 Pages 1747-1750
    Published: 2000
    Released: February 08, 2005
    JOURNALS FREE ACCESS
      We have cloned and sequenced an Aspergillus oryzae genomic DNA fragment that encodes a fructose-1,6-bisphosphatase gene (fbpA) with the aim of studying transcriptional regulation mechanisms involved in basic metabolism. Expression of fbpA was repressed in the presence of glucose, but not in the presence of pyruvate or sodium acetate in the medium. The CreA and FacB element found in the fbpA 5′-flanking region may be important in fbpA regulation.
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  • Akira YAMAGATA, Ryuichi HIROTA, Junichi KATO, Akio KURODA, Tsukasa IKE ...
    2000 Volume 64 Issue 8 Pages 1754-1757
    Published: 2000
    Released: February 08, 2005
    JOURNALS FREE ACCESS
      The ammonia-oxidizing bacterium Nitrosomonas sp. strain ENI-11 contains three copies of the hao gene (hao1, hao2, and hao3) coding for hydroxylamine oxidoreductase (HAO). Three single mutants (hao1::kan, hao2::kan, or hao3::kan) had 68 to 75% of the wild-type growth rate and 58 to 89% of the wild-type HAO activity when grown under the same conditions. A double mutant (hao1::kan and hao3::amp) also had 68% of the wild-type growth and 37% of the wild-type HAO activity.
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  • Takeyuki OHSHITA
    2000 Volume 64 Issue 8 Pages 1764-1766
    Published: 2000
    Released: February 08, 2005
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      When native and acid-denatured lactate dehydrogenase (LDH) were incubated with total lysosomal enzymes in vitro, amino acids from their degradation were produced at various acidic pH. The pH profile in the overall degradation of native LDH was markedly different from that of acid-denatured LDH. Disappearance of the 35-kDa subunit of native LDH was markedly suppressed by a low level of cystatin α as well as by a general cysteine proteinase inhibitor, N-(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucine-3-methylbutylamide (E-64-c). On the other hand, the degradation of acid-denatured LDH was only slightly suppressed by these inhibitors. It was concluded that at least a part of the proteinases involved in the overall degradation of native LDH is different from the proteinases involved in the degradation of acid-denatured form and a role of a cystatin α-sensitive cysteine proteinase is critical in the lysosomal degradation of native LDH, but not in that of acid-denatured form.
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  • Tarou OKAMOTO, Daekyung KIM, Tatsuya ODA, Kazumi MATSUOKA, Atsushi ISH ...
    2000 Volume 64 Issue 8 Pages 1767-1770
    Published: 2000
    Released: February 08, 2005
    JOURNALS FREE ACCESS
      Chattonella marina and Heterosigma akashiwo, known as red tide phytoplankton, are naturally wall-less and have quite fragile cell structures. In this study, we found that an equilibrium dialysis technique allowed the study of lectin binding to these flagellates. The results suggested that concanavalin A (Con A) binds to these flagellate cells through the specific carbohydrate moieties on the cell surface. Interestingly, the binding of an excess of Con A on the cell surface caused morphological changes concomitant with discharge of glycocalyx, a polysaccharide-containing common structure on the external cell surface of these flagellates. Fluorescent microscopic observation using FITC-labeled Con A (F-Con A) confirmed that F-Con A molecules are localized on the discharged glycocalyx.
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Biochemistry & Molecular Biology Preliminary Communication
Food & Nutrition Science Regular Papers
  • Kiharu IGARASHI, Yuriko KIMURA, Asako TAKENAKA
    2000 Volume 64 Issue 8 Pages 1600-1607
    Published: 2000
    Released: February 08, 2005
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      The preventive effects of acylated anthocyanins from red cabbage on paraquat-induced oxidative stress were determined in rats. Decreased food intake and body weight gain, and increased lung weight and atherogenic index by feeding the rats on a diet containing paraquat were clearly suppressed by supplementing acylated anthocynins to the paraquat diet. Paraquat feeding increased the concentration of thiobarbituric acid-reactive substances (TBARS) in liver lipids, and decreased the liver triacylglycerol level. These effects tended to be suppressed by supplementing acylated anthocynins to the paraquat diet. In addition, the catalase activity in the liver mitochondrial fraction was markedly decreased by feeding on the paraquat diet, this decrease being partially suppressed by supplementing the paraquat diet with acylated anthocyanins. An increase in the NADPH-cytochrome-P450-reductase activity in the liver microsome fraction by paraquat was supressed by supplementing the paraquat diet with acylated anthocyanins. These results suggest that acylated anthocyanins from red cabbage acted preventively against the oxidative stress in vivo that may have been due to active oxygen species formed through the action of paraquat.
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  • Xiang-Dong LIU, Takeshi FURUTA, Hidefumi YOSHII, Pekka LINKO, W. Jan C ...
    2000 Volume 64 Issue 8 Pages 1608-1613
    Published: 2000
    Released: February 08, 2005
    JOURNALS FREE ACCESS
      The taste and flavor of spray-dried powdered products are the most important quality factors. In the present study, molecular encapsulation in cyclodextrin was applied to prevent the loss of a hydrophobic flavor compound (l-menthol) during the drying of a droplet. β-Cyclodextrin appeared to be a better encapsulant for menthol than α- and γ-cyclodextrin. The retention of menthol increased with increasing concentration of both cyclodextrin and maltodextrin. A simple mathematical model is proposed for estimating the flavor retention. The theoretical results by this model estimated well the final retention of menthol encapsulated in a blend of β-cyclodextrin and maltodextrin.
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  • Tomoyuki FUJII, Toshimasa YANO, Hitoshi KUMAGAI, Osato MIYAWAKI
    2000 Volume 64 Issue 8 Pages 1618-1622
    Published: 2000
    Released: February 08, 2005
    JOURNALS FREE ACCESS
      Since the critical exponent of the elastic modulus is related to the spatial dimension and the critical exponent of the correlation length, depending on the characteristics of elasticity, we experimentally evaluated both the elastic modulus of a sol-gel transition system and also the correlation length. We could determine the correlation length of agarose gel by the dynamic light scattering method; it was well described by the power law as a function of the deviation from the sol-gel transition point. Three scaling laws between the critical exponent of the correlation length (ν) and that of the elastic shear modulus (t) were compared, and the critical exponent of the elastic modulus was described by the equation of de Gennes expression (t=1+ν(d-2), where d is the spatial dimension). This result suggests that agarose fibers are stiff enough to show scalar elasticity.
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  • Nobuhiro NAKANO, Norifumi SHIRASAKA, Hiroko KOYAMA, Miki HINO, Tetsuo ...
    2000 Volume 64 Issue 8 Pages 1641-1650
    Published: 2000
    Released: February 08, 2005
    JOURNALS FREE ACCESS
      It was demonstrated that the rat liver cell line BRL-3A converted exogenous C19 odd chain-polyunsaturated fatty acids (PUFAs) into the corresponding C21- and C23-PUFAs as follows: 21:3n-8, 21:4n-8, 23:3n-8, and 23:4n-8 (from 19:3n-8); 21:4n-5, 21:5n-5, 23:4n-5, and 23:5n-5 (from 19:4n-5); 21:5n-2, 21:6n-2, 23:5n-2, and 23:6n-2 (from 19:5n-2). It presumed that these C19 PUFAs were converted through the mimic route to docosahexaenoic acid (22:6n-3) from eicosapentaenoic acid (20:5n-3). In addition, the characterization of the change of fatty acid composition of cellular lipids in rat liver cells were examined, using 19:4n-5 and several fatty acid desaturation inhibitors. Curcumin related compounds, curcumin, capsaicin, isoeugenol, 4-(4-hydroxy-3-methoxyphenyl)-3-buten-2-one, and gallic acid esters with near five carbon numbered alcohol had great changes of fatty acid composition of cellular lipids based on inhibition of the Δ6 desaturation of C24-PUFAs in rat liver cells.
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  • Tadao KURATA, Yoko NISHIKAWA
    2000 Volume 64 Issue 8 Pages 1651-1655
    Published: 2000
    Released: February 08, 2005
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      Dehydro-L-ascorbic acid (DAA) exists mainly in its C2 hydrated bicyclic form (5) in an aqueous solution, and monocyclic DAA (3), which is the expected reaction product immediately after the oxidation of AA, has not been observed by NMR spectroscopy. The formation mechanism for 5 from 3 and the stability of 5 were examined by the semi-empirical molecular orbital method (MOPAC). It was indicated that the protonation reaction was the key step in the formation of 5, therefore, the formation of 5 is thought to be more difficult under physiological conditions which mostly involve in the neutral or slightly alkaline state. However, by NMR, it was confirmed that, even in a neutral or slightly alkaline state very close to physiological conditions, the predominant form of DAA existing in an aqueous solution immediately after the enzymatic oxidation of AA was confirmed to be 5, although the possible existence of other forms of DAA at very low concentrations could not be completely excluded.
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  • Soichi TANABE, Jun WATANABE, Kazunobu OYAMA, Eri FUKUSHI, Jun KAWABATA ...
    2000 Volume 64 Issue 8 Pages 1675-1680
    Published: 2000
    Released: February 08, 2005
    JOURNALS FREE ACCESS
      A new polysaccharide with a molecular weight of 5.0×104 was isolated as a possible wheat allergen from a water-soluble fraction of flour by affinity chromatography and gel filtration. The isolated polysaccharide was found to be a possible wheat allergen, as it bound specifically to IgE antibodies in the sera of patients allergic to the water-soluble fraction of flour. Chemically, the sugar moiety of the polysaccharide consisted of D-glucose and D-mannose with β-1,4-linkages in a molar ratio of 4.4:1. Since this mannoglucan is thought to be stable in our body, it would act as a remaining allergen to cause a long-lasting allergic reaction to wheat flour.
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Microbiology & Fermentation Technology Regular Papers
  • Masaru NAGAI, Akira OZAWA, Tohoru KATSURAGI, Haruhiko KAWASAKI, Takuo ...
    2000 Volume 64 Issue 8 Pages 1580-1587
    Published: 2000
    Released: February 08, 2005
    JOURNALS FREE ACCESS
      A polygalacturonase gene of Aspergillus awamori IFO 4033 was cloned by genomic Southern hybridization with a probe of a DNA fragment synthesized by PCR. This was done using primers constructed based on the N-terminal amino acid sequence of a polygalacturonase, protopectinase-AS, produced by the strain and the consensus internal amino acid sequence of fungal polygalacturonases. The cloned polygalacturonase gene, containing an ORF, encodes 362 amino acids, including a 52-bp intron. It contains the consensus nucleotide sequence of PacC binding sites, and its expression was appeared to be regulated by ambient pH. After the intron was excised, the cloned gene was inserted into an expression plasmid for yeast, pMA91, and introduced into Saccharomyces cerevisiae to be expressed. The expressed gene product was purified to a homogeneous preparation, and this confirmed that the polygalacturonase produced was the product of the cloned gene.
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  • Kazuyo GOTOH, Hiroyuki IZUMI, Taisei KANAMOTO, Yasushi TAMADA, Hideki ...
    2000 Volume 64 Issue 8 Pages 1664-1670
    Published: 2000
    Released: February 08, 2005
    JOURNALS FREE ACCESS
      We prepared two kinds of sulfated silk fibroins, SclFib30 and SclFib31, which contain different amounts of sulfate. These sulfated silk fibroins have anti-HIV-1 activity in vitro, apparently due to interference with the adsorption of virus particles to CD4+ cells, and completely blocked virus binding to the cells at a concentration of 100 μg/ml. Sulfated fibroins also abolished cell-to-cell infection-induced syncytium formation upon cocultivation of MOLT-4 and MOLT-4/HIV-1IIIB cells, suggesting that they would interfere with gp120 and prevent the formation of gp120/CD4 complex. Silk is used in biomaterials such as surgical sutures and is believed to be a safe material for humans. In accordance with low anticoagulant activity and high anti-HIV-1 activity against both X4 HIV-1 and R5 HIV-1 strains, sulfated silk fibroins have potential as antiviral material such for a vaginal anti-HIV formulation.
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Microbiology & Fermentation Technology Notes
  • Seung-Ho KIM, Nack-Shick CHOI
    2000 Volume 64 Issue 8 Pages 1722-1725
    Published: 2000
    Released: February 08, 2005
    JOURNALS FREE ACCESS
      Bacillus sp. strain DJ-4, which produces extracellular proteases, was screened from Doen-Jang, a traditional Korean fermented food. A fibrinolytic enzyme (subtilisin DJ-4) was purified using commercial chromatographic techniques. The relative molecular mass of the isolated protein was 29 kDa by SDS-PAGE and fibrin zymography assay. The enzyme was characterized as a serine protease by an inhibitor assay on the fibrin zymography gel and by an amidolytic assay using a chromogenic substrate. The enzyme was inhibited by PMSF, but not by EDTA or leupeptin. The first 14 amino acids of the N-terminal sequence were identical to that of subtilisin BPN′, but the activity of subtilisin DJ-4 was 2.2 and 4.3 times higher than those of subtilisin BPN′ and subtilisin Carlsberg, respectively.
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  • Masaru NAGAI, Tohoru KATSURAGI, Takao TERASHITA, Kentaro YOSHIKAWA, Ta ...
    2000 Volume 64 Issue 8 Pages 1729-1732
    Published: 2000
    Released: February 08, 2005
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      An extracellular endo-polygalacturonase (PGase) produced by Aspergillus awamori IFO 4033 was isolated from the culture filtrate. The enzyme was purified to a homogeneous preparation with cation-exchange and size-exclusion chromatographies. Its properties were investigated, comparing them with that of recombinant pgx2 gene product, a PGase having protopectinase activity. This enzyme was a monomeric protein of 41 kDa, with an isoelectric point of pH 6.1. The characteristics of this PGase substantially coincide, with that of recombinant pgx2 gene product, and the PGase is assumed to be native pgx2 gene product. The production of PGase-X2 was confirmed to be regulated by ambient pHs.
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  • Teruo SONE, Satoru FUKIYA, Megumi KODAMA, Fusao TOMITA
    2000 Volume 64 Issue 8 Pages 1733-1736
    Published: 2000
    Released: February 08, 2005
    JOURNALS FREE ACCESS
      The 8-kb repeat unit of M. grisea rRNA-encoding DNA (rDNA) was cloned as three subclones, pM50, pM21, and pM86. Nucleotide sequencing of these subclones uncovered the structure of an rDNA repeat unit similar to those of other ascomycetes. The intergenic spacer (IGS) of the rDNA cistron contained a repetitive (R) region, which was rich in two kinds of short tandemly repeated elements.
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  • Kazuhiro YANAGITA, Yoichi KAMAGATA, Mamoru KAWAHARASAKI, Toshihiko SUZ ...
    2000 Volume 64 Issue 8 Pages 1737-1742
    Published: 2000
    Released: February 08, 2005
    JOURNALS FREE ACCESS
      The population of methanogens in the sheep rumen microbial ecosystem was studied by using 16S rDNA cloning analysis, epifluorescence microscopy (which detects autofluorescence of a specific cofactor F420 in methanogens) and the 16S rRNA-targeted in situ hybridization technique. The 16S rDNA clone libraries were constructed by PCR amplification with an Archaea-specific primer set and partial sequencing of the clonal 16S rDNAs was done. Phylogenetic analysis indicated that the clones were affiliated with Methanomicrobium mobile, Methanobrevibacter ruminantium and Methanobrevibacter smithii. Epifluorescence microscopy (F420 autofluorescence) and in situ hybridization by using a newly designed M. mobile-specific 16S rRNA-targeted oligonucleotide probe found that methanogens accounted for approximately 3.6% of total ruminal microorganisms and approximately 54% of the total methanogens were M. mobile.
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