Bioscience, Biotechnology, and Biochemistry
Online ISSN : 1347-6947
Print ISSN : 0916-8451
Volume 65 , Issue 7
Showing 1-38 articles out of 38 articles from the selected issue
Review
  • Hiroshi KASE
    2001 Volume 65 Issue 7 Pages 1447-1457
    Published: 2001
    Released: July 31, 2002
    JOURNALS FREE ACCESS
    There is now growing interest in the functional role of adenosine A2A receptors. Their distribution within the brain is restricted in the basal ganglia, particularly abundant in the striatum, which are thought to play a crucial role in the control of motor behavior. Indeed, newly developed A2A receptor selective antagonists have a profound influence on motor functions, with anti-Parkinsonian activities in several animal models. Striatal spiny neurons serve as a major anatomical locus for the relay of cortical information flow through the basal ganglia. The GABA releasing projection neurons represent the A2A receptor-mediated main target of adenosine. The GABAergic synaptic neurotransmission is regulated by adenosine via A2A receptors on the presynaptic terminals. Blockade of this modulatory function by A2A antagonists could repair striatopallidal abnormal neuronal activities provoked by striatal dopamine depletion in the Parkinsonian state. A2A receptor antagonists provide a novel therapeutic potential for the treatment of Parkinson’s disease.
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Organic Chemistry
Biochemistry & Molecular Biology
  • Yukiko KAKUTA, Toshinori IGARASHI, Toyotaka MURAKAMI, Hiroyuki ITO, Hi ...
    2001 Volume 65 Issue 7 Pages 1511-1518
    Published: 2001
    Released: July 31, 2002
    JOURNALS FREE ACCESS
    A plant hormone, ethylene, is formed through 1-aminocyclopropane-1-carboxylic acid (ACC). A fungus, Penicillium citrium, was found to synthesize ACC and to degrade ACC into 2-oxobutyrate and ammonia. ACC synthase, responsible for ACC synthesis in P. citrinum, was characterized on the molecular level by sequencing of N terminal and proteolytic peptides of the enzyme, and cloning and sequencing of its cDNA. The ACC synthase from P. citrinum had 430 amino acid residues and a shorter C terminal than the plant enzyme. The enzyme purified from Escherichia coli transformed with ACC-synthase-encoding DNA showed similar properties to those of the purified enzyme from P. citrinum. Saccharomyces cerevisiae with ACC synthase accumulated ACC in the medium with increasing time of incubation. The sequence of ACC synthase from P. citrinum was compared with that of the plant enzyme with discussion about important residues for catalysis.
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  • Yoshimi TAKEUCHI, Yoichi TSUMURAYA
    2001 Volume 65 Issue 7 Pages 1519-1527
    Published: 2001
    Released: July 31, 2002
    JOURNALS FREE ACCESS
    A membrane preparation of 7-d-old seedlings from azuki bean (Vigna angularis) contained galacturonosyltransferase (GalAT) capable of transferring galacturonic acid (GalA) from UDP-GalA into polygalacturonic acid (PGA) as an exogenous acceptor. The enzyme was maximally active at pH 6.8-7.8 and 25-35°C in the presence of 5 mM Mn2+ and 0.5% (w/v) Triton X-100. Acid-soluble low-Mr (average Mr 10,000) PGA was a more efficient acceptor substrate than acid-insoluble polymer (Mr 70,000). The apparent Michaelis constants for UDP-GalA and low-Mr PGA were 0.14 mM and 0.02 mg/ml, respectively. Various pectins with different degrees of methyl-esterification (DE) were poor acceptors, and the enzyme activity tended to decrease with decreasing DE of the pectins. The transfer products from incubation of the enzyme with UDP-14C-GalA and the low-Mr PGA yielded 14C-GalA2 as the major product upon digestion with an endopolygalacturonase (EPGase), confirming the incorporation of GalA into PGA through contiguous α-1,4-linkages.
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  • Jae-Won YANG, Jun-Seop SHIN, Jae-Jeong LEE, Hyo-Ihl CHANG, Chan-Wha KI ...
    2001 Volume 65 Issue 7 Pages 1528-1533
    Published: 2001
    Released: July 31, 2002
    JOURNALS FREE ACCESS
    In this study, the effects of ethanol and allyl alcohol on primary mouse hepatocytes were investigated. No cytotoxicity was observed by ethanol treatments, but more toxicity to cells was found in the response to allyl alcohol treatment. The expression of cytochrome P450 2E1 (CYP2E1), phase I enzyme was examined in response to ethanol and allyl alcohol. Both xenobiotics induced CYP2E1 up to 1.5∼5 fold at the protein level. The effects of insulin on CYP2E1 expression were also measured. Insulin, which has been regarded as an essential hormone for primary hepatocytes, was shown to decrease the level of CYP2E1 protein, and did not affect cell viability. These results on CYP2E1 induction demonstrate that primary mouse hepatocytes, when using ethanol and allyl alcohol as substrates and in insulin-free medium, provide a suitable system for the studies of the role of CYP2E1 in xenobiotic metabolism and toxicity.
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  • Kiyotaka FUJITA, Ryo-ich NAKATAKE, Kayo YAMABE, Akira WATANABE, Yasuhi ...
    2001 Volume 65 Issue 7 Pages 1542-1548
    Published: 2001
    Released: July 31, 2002
    JOURNALS FREE ACCESS
    The gene encoding the endo-β-N-acetylglucosaminidase from Flavobacterium sp. (Endo-Fsp) was sequneced. The Endo-Fsp gene was overexpressed in Escherichia coli cells, and was purified from inclusion bodies after denaturation by 8 M urea. The renatured Endo-Fsp had the same optimum pH and substrate specificity as the native enzyme. Endo-Fsp had 60% sequence identity with the endo-β-N-acetylglucosaminidase from Streptomyces plicatus (Endo-H), and the putative catalytic residues were conserved. Site-directed mutagenesis was done at conserved residues based on the three-dimensional structure and mutagenesis of Endo-H. The mutant of Glu-128, corresponding to Glu-132 in Endo-H and identified as an active site residue, was inactivated. Mutagenesis around the predicted active site of Endo-Fsp reduced the enzymatic activity. Moreover, the hydrolytic activity toward hybrid-type oligosaccharides was decreased compared to that toward high-mannose type oligosaccharides by mutagenesis of Asp-126 and Asp-127. Therefore, sitedirected mutagenesis of some of these conserved residues indicates that the predicted active sites are essential to the enzymatic activity of Endo-Fsp, and may have similar roles in catalysis as their counterparts in Endo-H.
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  • Shuji TANI, Tomoyuki ITOH, Masashi KATO, Tetsuo KOBAYASHI, Norihiro TS ...
    2001 Volume 65 Issue 7 Pages 1568-1574
    Published: 2001
    Released: July 31, 2002
    JOURNALS FREE ACCESS
    The α-glucosidase gene (agdA) of Aspergillus nidulans has a single CGGN8CGG type AmyR binding site in its promoter region. The binding site is functional in vivo as a cis-element responsible for induction by starch, and mutational studies indicated that both the CGG triplets are required for high-level induction. A part of AmyR (residues 1-411; AmyR1-411), which was produced as a MalE fusion protein in E. coli, bound to the CGGN8CGG site of the agdA promoter. DNA binding profiles to the mutant binding sites that lacked both or either one of the CGG triplets suggested that AmyR1-411 can bind to a single CGG triplet site with low affinity and that two AmyR molecules cooperatively bind to the CGG direct repeat.
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  • Manabu SUGIMOTO, Nobuyoshi NAKAJIMA
    2001 Volume 65 Issue 7 Pages 1575-1580
    Published: 2001
    Released: July 31, 2002
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    An earthworm, Lumbricus rubellus, produces alkaline trypsin-like proteases that are greater than trypsins in their stability and strong tolerance to organic solvents. cDNAs encoding strong fibrinolytic proteases (F-III-2 and F-III-1) in the six isozymes were cloned and sequenced to study their stability-structure relationship. The cDNAs of F-III-2 and F-III-1 comprised 1011 and 973 bp and included open reading frames that encode polypeptides of 245 and 246 amino acid residues, respectively. The deduced amino acid sequences of F-III-2 and F-III-1 have 7 and 8 activation peptides in the N-termini respectively, indicating that they are translated as proenzymes and modified to active forms by post-translational processing. They showed similarity to mammalian serine proteases and conserved the catalytic amino acid residues, however, neither arginine nor lysine residues were present in the autolysis region. The gene encoding the native form of F-III-2 was expressed in Pichia pastoris to produce and secrete the earthworm protease in the culture medium, which dissolves an artificial fibrin plate.
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  • Masaru AKITA, Daisuke TSUTSUMI, Masami KOBAYASHI, Hideo KISE
    2001 Volume 65 Issue 7 Pages 1581-1588
    Published: 2001
    Released: July 31, 2002
    JOURNALS FREE ACCESS
    The effects of solvent and reaction conditions on the catalytic activity of horseradish peroxidase (HRP) were investigated for oxidative polymerization of phenol in water/organic mixtures using hydrogen peroxide as an oxidant. Also, the structural changes of HRP were investigated by CD and absorption spectroscopy in these solvents. The results suggest that the yield of phenol polymer (the conversion of phenol to polymer) is strongly affected by the reaction conditions due to the structural changes of HRP, that is, the changes in higher structure of the apo-protein and dissociation or decomposition of the prosthetic heme. Optimum solvent compositions for phenol polymerization depend on the nature of the organic solvents owing to different effects of the solvents on HRP structure. In addition to initial rapid changes, slower changes of HRP structure occur in water/organic solvents especially at high concentrations of organic solvents. In parallel with these structural changes, catalytic activity of HRP decreases with time in these solvents. At higher reaction temperatures, the yield of the polymer decreases, which is also ascribed to modification of HRP structure. It is known that hydrogen peroxide is an inhibitor of HRP, and the yield of phenol polymer is strongly dependent on the manner of addition of hydrogen peroxide to the reaction solutions. The polymer yield decreases significantly when hydrogen peroxide was added to the reaction solution in a large amount at once. This is probably due to inactivation of HRP by excess hydrogen peroxide. From the CD and absorption spectra, it is suggested that excess hydrogen peroxide causes not only decomposition of the prosthetic heme but also modification of the higher structure of HRP.
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  • Kazumi HIRAGA, Anoja WANIGASEKERA, Hisanori SUGI, Nobuyuki HAMANAKA, K ...
    2001 Volume 65 Issue 7 Pages 1589-1595
    Published: 2001
    Released: July 31, 2002
    JOURNALS FREE ACCESS
    Yeast is an excellent model system of eukaryotes for the study of molecular mechanisms of ATP-binding cassette transporters. Pdr5 protein is a yeast Saccharomyces cerevisiae ATP-binding cassette transporter conferring resistance to several unrelated drugs. Here, we described a novel drug screening system designated to detect compounds that inhibit the function of Pdr5. An indicator strain with increased drug sensitivity was constructed with an ergosterol-deficient background (Δsyr1/erg3 null mutation). The sensitivity of the indicator strain (Δsyr1/erg3Δpdr5Δsnq2) to the Pdr5 substrates, cycloheximide and cerulenin, was increased 16-fold and 4-fold against wild type, respectively. The screening system is mainly based on the growth inhibition of the PDR5-overexpressed indicator strain with the combination of a sample and cycloheximide or cerulenin. The effect of an mdr inhibitor, FK506 on the screening system was clearly detected even at a low concentration (∼0.5 μg/ml). In addition, accumulation of rhodamine 6G in the cells was detected as a result of Pdr5 inhibition by FK506. These results indicated that the screening system is useful for a sensitive screening of Pdr5-specific inhibitors with low toxicity.
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  • Toshiro TAKAI, Hideki HATANAKA, Saori ICHIKAWA, Toyokazu YOKOTA, Fuyuh ...
    2001 Volume 65 Issue 7 Pages 1601-1609
    Published: 2001
    Released: July 31, 2002
    JOURNALS FREE ACCESS
    Recently, we reported that introduction of mutations that induced conformational changes of the major mite allergen Der f 2 was an efficient strategy to reduce the allergenicity for safer allergen-specific immunotherapy. In this study, we evaluated another strategy, disruption of two independent IgE epitopes without inducing conformational change. We analyzed allergenicities of the wild-type Der f 2, two single mutants with a mutation at either of the two IgE-binding sites (K15A and K77A), and a double mutant with mutations at both of the sites (K15/77A). Purified recombinant forms of Der f 2 expressed in Escherichia coli had correct disulfide bonds, equivalent apparent molecular masses of approximately 15 kDa, and similar secondary structures. The mutants of Der f 2 had less IgE reactivities than the wild-type Der f 2 and reduced inhibitory activities for IgE-binding to the wild-type Der f 2. However, the mutations did not significantly reduce histamine-releasing activity.
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  • Mamoru NISHIMOTO, Masaki KUBOTA, Masahisa TSUJI, Haruhide MORI, Atsuo ...
    2001 Volume 65 Issue 7 Pages 1610-1616
    Published: 2001
    Released: July 31, 2002
    JOURNALS FREE ACCESS
    α-Glucosidase III, which was different in substrate specificity from honeybee α-glucosidases I and II, was purified as an electrophoretically homogeneous protein from honeybees, by salting-out chromatography, DEAE-cellulose, DEAE-Sepharose CL-6B, Bio-Gel P-150, and CM-Toyopearl 650M column chromatographies. The enzyme preparation was confirmed to be a monomeric protein and a glycoprotein containing about 7.4% of carbohydrate. The molecular weight was estimated to approximately 68,000, and the optimum pH was 5.5. The substrate specificity of α-glucosidase III was kinetically investigated. The enzyme did not show unusual kinetics, such as the allosteric behaviors observed in α-glucosidases I and II, which are monomeric proteins. The enzyme was characterized by the ability to rapidly hydrolyze sucrose, phenyl α-glucoside, maltose, and maltotriose, and by extremely high Km for substrates, compared with those of α-glucosidases I and II. Especially, maltotriose was hydrolyzed over 3 times as rapidly as maltose. However, maltooligosaccharides of four or more in the degree of polymerization were slowly degraded. The relative rates of the k0 values for maltose, sucrose, p-nitrophenyl α-glucoside and maltotriose were estimated to be 100, 527, 281 and 364, and the Km values for these substrates, 11, 30, 13, and 10 mM, respectively. The subsite affinities (Ai’s) in the active site were tentatively evaluated from the rate parameters for maltooligosaccharides. In this enzyme, it was peculiar that the Ai value at subsite 3 was larger than that of subsite 1.
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  • Hirohide TOYAMA, Nahoko AOKI, Kazunobu MATSUSHITA, Osao ADACHI
    2001 Volume 65 Issue 7 Pages 1617-1626
    Published: 2001
    Released: July 31, 2002
    JOURNALS FREE ACCESS
    Expression of azurin in Pseudomonas putida HK5 was examined by immunoblot analysis. Similar amounts of azurin were found in the cells grown into the stationary phase on any carbon sources, including LB medium without alcohol, where no quinoprotein alcohol dehydrogenases appeared. In the early exponential phase, the highest amount of azurin was found in the cells grown on 1-butanol, but here was none in the case of LB medium, suggesting that expression of azurin is cooperative with that of the alcohol oxidase system, especially the system including quinohemoprotein alcohol dehydrogenase IIB. The azurin gene (azu) was cloned and sequenced. azu is monocistronic, and in its promoter region, FNR-binding consensus sequence was found. However, its relative position suggests different transcriptional regulation from that in azu of P. aeruginosa. The molecular weight of the mature protein without copper ion calculated from the amino acid sequence was consistent with the value of the purified azurin measured by mass spectrometry.
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  • Terumichi TANAKA, Osamu INUI, Natsuki DOHI, Noriko OKADA, Hidechika OK ...
    2001 Volume 65 Issue 7 Pages 1636-1644
    Published: 2001
    Released: July 31, 2002
    JOURNALS FREE ACCESS
    Today, many nucleic acid enzymes are used in gene therapy and gene regulations. However, no simple assay methods to evaluate enzymatic activities, with which we judge the enzyme design, have been reported. Here, we propose a new simple competition assay for nucleic acid enzymes of different types to evaluate the cleaving efficiency of a target RNA molecule, of which the recognition sites are different but overlapped. Two nucleic acid enzymes were added to one tube to make a competition of these two enzymes for one substrate. The assay was used on two ribozymes, hammerhead ribozyme and hairpin ribozyme, and a DNA-enzyme. We found that this assay method is capable of application to those enzymes, as a powerful tool for the selection and designing of RNA-cleaving enzymes.
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  • Kenji SORIMACHI, Yukari IKEHARA, Genzo MAEZATO, Akira OKUBO, Sunao YAM ...
    2001 Volume 65 Issue 7 Pages 1645-1647
    Published: 2001
    Released: July 31, 2002
    JOURNALS FREE ACCESS
    Anti-viral activities of Agaricus blazei Murill were investigated. The water extracts of the cultured mycelia and fruiting bodies were fractionated with different concentrations of ethanol. To several viruses which have cytopathic effects (CPE) on VERO cells, inhibition of these effects by the ethanol fractions was tested. Strong inhibition of CPE induced by western equine encephalitis (WEE) virus was observed in the mycelial fractions but not those of fruiting bodies.
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  • Yoshiko TATEISHI, Yoshimi UMEMURA, Muneharu ESAKA
    2001 Volume 65 Issue 7 Pages 1663-1668
    Published: 2001
    Released: July 31, 2002
    JOURNALS FREE ACCESS
    We isolated a cDNA for basic class I chitinase (ChitiWb1). ChitiWb1 cDNA encodes a protein that consists of 315 amino acid residues and has a signal peptide. Northern blot analysis indicated that the class I chitinase mRNA in leaves and cultured cells of winged bean was increased by treatments with NaCl, KCl, CaCl2, mannitol or saccharose, but not with abscisic acid. Thus, class I chitinase expression was shown to be up-regulated by osmotic stress.
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  • Keiichi AKITA, Chikako NAITOU, Kiyofumi MARUYAMA
    2001 Volume 65 Issue 7 Pages 1680-1683
    Published: 2001
    Released: July 31, 2002
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    An esterase hydrolyzing phthalate esters has been purified from Micrococcus sp. YGJ1. The enzyme, a monomeric protein (Mr=56 kDa) with a pI of 4.0, hydrolyzes various aliphatic and aromatic carboxylesters. The medium chain (C3-C4) esters are the most preferred substrates. The enzyme is inhibited by HgCl2 and p-chloromercuribenzoate but not by phenylmethyl-sulfonyl fluoride.
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  • Katsuichiro OKAZAKI, Takashi AMANO, Tetsuhiro MORIMOTO, Takahiro IEMOT ...
    2001 Volume 65 Issue 7 Pages 1684-1687
    Published: 2001
    Released: July 31, 2002
    JOURNALS FREE ACCESS
    Mycodextranase (EC 3.2.1.61) is an α-glucanase that cleaves α-1,4-bonds of alternating α-1,3- and α-1,4-linked D-glucan (nigeran). The gene encoding mycodextranase from Streptomyces sp. J-13-3 was cloned by hybridization with a degenerate oligonucleotide probe from the amino-terminal amino acid sequence of the enzyme and its nucleotide structure was analyzed. The open reading frame consisted of 1,803 base pairs encoding a signal peptide of 60 amino acids and a mature protein of 540 amino acids with a calculated molecular weight of 56,078. The deduced amino acid sequence showed weak similality to a chitinase homolog from Streptomyces lividans and a chitinase from Xanthomonas sp.
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  • Hideo NAKASHITA, Yuko ARAI, Toshiharu SHIKANAI, Yoshiharu DOI, Isamu Y ...
    2001 Volume 65 Issue 7 Pages 1688-1691
    Published: 2001
    Released: July 31, 2002
    JOURNALS FREE ACCESS
    Multiple-gene transformation is required to improve or change plant metabolisms effectively; but this many-step procedure is time-consuming and costing. We succeeded in the metabolic engineering of tobacco plants by introducing multiple genes as a bacteria-type operon into a plastid genome. The tobacco plastid was transformed with a polycistron consisting of three bacterial genes for the biosynthesis of a biodegradable polyester, polyhydroxybutyrate (PHB). Accumulation of PHB in the leaves of the transgenic tobacco indicated that the introduced genes were polycistronically expressed. This “phyto-fermentation” system can be used in plant production of various chemical commodities and pharmaceuticals.
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  • Kyoko HIGUCHI, Masaharu TANI, Hiromi NAKANISHI, Toshihiro YOSHIWARA, F ...
    2001 Volume 65 Issue 7 Pages 1692-1696
    Published: 2001
    Released: July 31, 2002
    JOURNALS FREE ACCESS
    Nicotianamine (NA) is a precursor for mugineic acid-family phytosiderophores, which are a critical component of the Fe aquisition process in graminaceous plants. In addition, nicotianamine synthase (NAS) is strongly induced in these plants by Fe deficiency. NA is essential for Fe metabolism also in dicots, but NAS is not induced by Fe deficiency. We introduced a barley HνNAS1 promoter-gus fusion gene into tobacco. GUS activity was induced in the roots of these plants by Fe deficiency, and was constitutively expressed at a low level in their leaves.
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Food & Nutrition Science
  • Fumihito TAKENAKA, Hirofumi UCHIYAMA
    2001 Volume 65 Issue 7 Pages 1458-1463
    Published: 2001
    Released: July 31, 2002
    JOURNALS FREE ACCESS
    α-D-Glucosylglycerol (GG) is a mixture of 2-O-α-D-glucosylglycerol (GG-II), (2R)-1-O-α-D-glucosyl-glycerol (R-GG-I) and (2S)-1-O-α-D-glucosylglycerol (S-GG-I). GG has been found to be slightly hydrolyzed in vitro only by rat intestinal enzymes, but hardly at all by other digestive juices. GG suppressed the hydrolysis of maltose, sucrose and isomaltose by rat intestinal enzymes because the amount of glucose in the digestion of a mixture of GG and disaccharide was less than the sum of that in each individual digestion. The consumption of GG was suppressed by isomaltose, but promoted by maltose, with the hydrolysis of GG being suppressed. Sucrose appeared to suppress only the consumption of S-GG-I, suggesting that S-GG-I was hydrolyzed by the active site of sucrase in a sucrase-isomaltase complex. Transglucosylation seems to have occurred more frequently in the individual digestion of maltose and isomaltose than in that of GG and sucrose. GG seemed to promote transglucosylation in the presence of maltose, to suppress it with sucrose, and to delay it with isomaltose.
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  • Hisanao TAKEUCHI, Norio SUZUKI, Masakazu TADA, Puming HE
    2001 Volume 65 Issue 7 Pages 1489-1494
    Published: 2001
    Released: July 31, 2002
    JOURNALS FREE ACCESS
    The effects of dietary oils on stress-induced changes in the liver glycogen metabolism of male Wistar rats at 6 weeks of age were investigated. The rats were subjected to repetitive water-immersion restraint and fed with a 20% saturated fatty acid mixture (PSC), olive oil (OLI), safflower oil (SAF), or linseed oil (LIS) diet. Stress loading decresed the body weight gain, although the food intake was hardly changed, and the weights of the liver and spleen generally declined regardless of the elapsed time after stress loading and the type of dietary oil. The adrenal weight was generally enhanced by stress in all deitary groups, and particularly tended to be greater in the OLI and PSC groups than in the other two. The plasma corticosterone concentration increased immediately after stressing (Stress-1), but approached the level of the rats with no stress (No stress) 2 h after releasing the stress load (Stress-2) in all groups. The enhancement of corticosterone level in the Stress-1 animals was large in the PSC and OLI groups, and the decline of this level in the Stress-2 animals was small in the OLI group when compared with the other groups. Although the concentrations of total cholesterol (T-CHOL) and triacylglycerol (TG) in the plasma were decreased by stress loading in all groups, these concentrations in the PSC and OLI groups were nearly always higher than in the other groups. The liver serine dehydratase (SDH) activity enhanced by stress was high in the OLI group and tended to be high in the PSC group when compared with the other groups. The contents of liver glycogen were reduced in the Stress-1 animals and extremely elevated in the Stress-2 animals of all groups, and particularly in the OLI group, the reduction in the Stress-1 animals was smaller and the enhancement in the Stress-2 animals was greater than in the other groups. These results suggest that feeding oleic acid to rats exposed to water-immersion restraint further accelerated liver glycogen synthesis through the rise in liver SDH activity due to inceased corticosterone secretion when compared with the effect from linoleic and α-linolenic acids.
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  • Namsoo KIM, In-Seon PARK
    2001 Volume 65 Issue 7 Pages 1648-1651
    Published: 2001
    Released: July 31, 2002
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    Saponin compounds (saikosaponin c, a, and d) in Bupleurum falcatum were partially purified by solvent partitioning of the herbal extract using diethyl ether, distilled water, n-butanol, and acetone. After separation of the saponins by preparative LC, the purity of each saikosaponin was more than 94%. The identities of purified individual saikosaponins were confirmed by TLC, analytical LC, and fast-atom bombardment mass spectrometry.
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  • Makoto YOSHIMOTO, Shigenori OKUNO, Masaatu YAMAGUCHI, Osamu YAMAKAWA
    2001 Volume 65 Issue 7 Pages 1652-1655
    Published: 2001
    Released: July 31, 2002
    JOURNALS FREE ACCESS
    The antimutagenicity of the 3-sophoroside-5-glucoside of cyanidin and 3-sophoroside-5-glucoside of peonidin, the anthocyanin derivatives deacylated from the 3-(6,6'-caffeylferulylsophoroside)-5-glucoside of cyanidin (YGM-3) and 3-(6,6'-caffeylferulyl-sophoroside)-5-glucoside of peonidin (YGM-6) which had been purified from the sweetpotato with purple-colored flesh, was investigated by using Salmonella typhimurium TA 98. A comparison of the antimutagenicity between YGM-3 and YGM-6 and the deacylated derivatives showed that the activity of cyanidin was stronger than that of peonidin. Deacylation of the peonidin-type pigment markedly decreased this antimutagenicity. Caffeic acid showed the strongest antimutagenicity of the constituent organic acids of the anthocyanin pigments, caffeic acid, ferulic acid, and p-hydroxybenzoic acid. These results suggest that the cathecol structure plays an important role in the strong antimutagenicity of anthocyanin pigments.
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  • Hironori HASHIMURA, Chihoko UEDA, Jun KAWABATA, Takanori KASAI
    2001 Volume 65 Issue 7 Pages 1656-1658
    Published: 2001
    Released: July 31, 2002
    JOURNALS FREE ACCESS
    A methanol extract of avocado fruits showed potent inhibitory activity against acetyl-CoA carboxylase, a key enzyme in fatty acid biosynthesis. The active principles were isolated and identified as (5E,12Z,15Z)-2-hydroxy-4-oxoheneicosa-5,12,15-trienyl (1), (2R,12Z,15Z)-2-hydroxy-4-oxoheneicosa-12,15-dienyl (2), (2R*,4R*)-2,4-dihydroxyheptadec-16-enyl (3) and (2R*,4R*)-2,4-dihydroxyheptadec-16-ynyl (4) acetates by instrumental analyses. The IC50 of the compounds were 4.0×10-6, 4.9×10-6, 9.4×10-6, and 5.1×10-6M, respectively.
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  • Hiroshi UEDA, Masatoshi YAMAZAKI
    2001 Volume 65 Issue 7 Pages 1673-1675
    Published: 2001
    Released: July 31, 2002
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    The anti-inflammatory and anti-allergic activity of perilla leaf extract was investigated. The oral administration of perilla leaf extract to mice inhibited two types of acute inflammatory models, arachidonic acid-induced ear edema and 12-o-tetradecanoylphorbol-13-acetate-induced ear edema. Oral administration of perilla leaf extract also inhibited the contact dermatitis model, oxazolone-induced ear edema, by affecting sensitization.
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Microbiology & Fermentation Technology
  • Satoru FUKIYA, Megumi KODAMA, Hideki KITO, Teruo SONE, Fusao TOMITA
    2001 Volume 65 Issue 7 Pages 1464-1473
    Published: 2001
    Released: July 31, 2002
    JOURNALS FREE ACCESS
    Mating experiments between Magnaporthe grisea Japanese rice pathogens and Guy11, a hermaphroditic fertile rice pathogen, were done aimed at identification of avirulence genes. A cross named cross 2107 with thirty-six random progenies was obtained. Segregation analyses of genetic markers found that the cross was less suitable for genetic analysis. Backcrosses with cross 2107 progenies and Guy11 were done and another cross named cross 5307 with sixty-five progenies was obtained. A locus controlling kasugamycin resistance named Ksg1R was identified and used for a model case of genetic mapping. Bulked segregant analysis was done to find adjacent RAPD markers for mapping of the gene. Three adjacent markers to Ksg1R were obtained and a genetic map around the Ksg1R was made, but these markers were not located on a single chromosome. These results suggest that genetic analysis to identify a gene locus is available in cross 5307. Infection assay of parental strains of cross 5307 to Japanese differential rice cultivars suggested the possibility of genetic analysis of cultivar specificity toward four rice cultivars: Aichiasahi, Kusabue, Tsuyuake, and K59.
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  • Akiko SUYAMA, Ryo IWAKIRI, Keiichirou KAI, Takashi TOKUNAGA, Nobuyuki ...
    2001 Volume 65 Issue 7 Pages 1474-1481
    Published: 2001
    Released: July 31, 2002
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    A strict anaerobic bacterium, strain Y51, was isolated from soil contaminated with tetrachloroethene (PCE). Strain Y51 is capable of very efficiently dehalogenating PCE via trichloroethene (TCE) to cis-1,2-dichloroethene (cis-1,2-DCE) at concentrations as high as 960 μM and as low as 0.6 μM. Strain Y51 was Gram-negative, motile with some lateral flagella, and curved rodshaped. On the basis of the 16S rDNA sequence, the organism was identified to be a species within the genus Desulfitobacterium. Strain Y51 also had dehalogenation activities toward polychloroethanes such as hexa-, penta-, and tetrachloroethanes, from which dichloroethenes were produced as the final products. The cell extracts mediated the dehalogenation of PCE with reduced methyl viologen as an electron carrier at the specific rate of 5.0 nmol·min-1·mg cell protein-1 (pH 7.2, 37°C). Dehalogenation was highly susceptible to air oxidation, and to potential alternative electron acceptors such as nitrite or sulfite.
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  • Chuen-Fu LIN, Jin-Li HO, Tung-Ching CHUNG
    2001 Volume 65 Issue 7 Pages 1495-1503
    Published: 2001
    Released: July 31, 2002
    JOURNALS FREE ACCESS
    A 3.2 kb DNA fragment containing the replication region (RR) from pTC82 was cloned, sequenced, and found to contain elements typical of plasmids that replicate via a rolling-circle mechanism of replication (RCR), including double-strand origin (DSO), replication protein gene (rep), and single-strand origin (SSO). The DSO of pTC82 contains two domains showing 55.5% and 84.6% similarities in nucleotide (nt) sequence to the conserved functional elements bind and nic, respectively, which are required for the initiation of the leading strand typical of the pC194-RCR family. Although the predicted rep gene product of pTC82 (Rep82) shares little identity (less than 24%) with other known Reps, a region containing three motifs, characteristic of the pC194-family Reps, was identified, indicating the Rep82 as a novel Rep protein of this family. Downstream of the rep82 gene, strong similarity to the typical palT type-SSO could be detected. This is the first palT type-SSO to be identified from Lactobacillus. Through a series of deletion studies, the minimal replicon of the cloned RR was found to be 2.66 kb in size including the DSO region and rep gene. This RR was further identified as being highly stable in L. reuteri and also bearing a very narrow host-range property, suggesting it to be a good replicon potentially useful in vector construction for developing L. reuteri as a vaccine carrier.
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  • Jun-ichi MARUYAMA, Harushi NAKAJIMA, Katsuhiko KITAMOTO
    2001 Volume 65 Issue 7 Pages 1504-1510
    Published: 2001
    Released: July 31, 2002
    JOURNALS FREE ACCESS
    Aspergillus oryzae has been reported to form conidia with multinuclei. In order to analyze nuclei in living cells, we developed an expression system of the A. nidulans histone H2B protein tagged by EGFP (H2B::EGFP). In both A. oryzae niaD300 and A. nidulans FGSC89 transformants expressing H2B::EGFP, fluorescence was detected in nuclear regions of hyphae and conidia. While a conidium contained only one fluorescent spot in the A. nidulans transformant, approximately 66% of conidia had two, 24% had one, and 10% had three or more in the A. oryzae transformant. The conidia expressing H2B::EGFP were put through FACS(fluorescence-activated cell sorting) analysis and two sharp peaks, corresponding to one and two nuclei in each conidium, were noted in the A. oryzae transformant. In addition, the A. oryzae uninucleate conidia that were successfully isolated by FACS reproduced conidia with almost the same number distribution of nuclei as that of the original. Conidia of five A. oryzae strains used in sake brewing were scored for the number of nuclei, showing that a varied number of nuclei existed in each conidium and some strains had a small number of uninucleate conidia.
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  • Hideyuki SUZUKI, Sachiko KAMATANI, Hidehiko KUMAGAI
    2001 Volume 65 Issue 7 Pages 1549-1558
    Published: 2001
    Released: July 31, 2002
    JOURNALS FREE ACCESS
    Aminopeptidase B, which is one of the four cysteinyl-glycinases of Escherichia coli K-12, was purified to electrophoretic homogeneity and its enzymatic characteristics were observed. Aminopeptidase B was activated by various divalent cations such as Ni2+, Mn2+, Co2+, and Cd2+, and lost its activity completely on dialysis against EDTA. This indicates that aminopeptidase B is a metallopeptidase. It was stabilized against heat in the presence of Mn2+ or Co2+. The activity of aminopeptidase B, which was saturated with one of above divalent cations, was enhanced on the addition of a very small amount of a second divalent cation. α-Glutamyl p-nitroanilide, leucine p-nitroanilide, and methionine p-nitroanilide were good substrates for aminopeptidase B, while native peptides, cysteinylglycine and leucylglycine, were far better substrates. The kcat/Km for cysteinylglycine was much bigger than those for leucylglycine or leucine p-nitroanilide.
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  • Tokumitsu OKAMURA, Tomoko OGATA, Norie MINAMOTO, Tomomi TAKENO, Hiroko ...
    2001 Volume 65 Issue 7 Pages 1596-1600
    Published: 2001
    Released: July 31, 2002
    JOURNALS FREE ACCESS
    Saccharomyces cerevisiae is the main microorganism used in wine brewing, because this microbe has potent ability to produce alcohol dehydrogenase. We have recently discovered that some genera of mushroom produced alcohol dehydrogenase, and made wine by using a mushroom in place of S. cerevisiae. The highest alcohol concentration in this wine was achieved with Pleurotus ostreatus (2.6 M, 12.2%). In the case of Agaricus blazei, the same alcohol concentration (1.7 M, 8%) was produced under both aerobic and anaerobic conditions. This wine produced by A. blazei contained about 0.68% β-D-glucan, which is known to have a preventive effects against cancer. The wine made by using Flammulina velutipes showed thrombosis-preventing activity, giving a prolonged thrombin clotting time 2.2-fold that of the control. Thus, the wine made by using mushroom seems to be a functional food which can be expected to have preventive effects against cancer and thrombosis
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  • Yoshimitsu HAMANO, Tohru DAIRI, Masahiro YAMAMOTO, Takashi KAWASAKI, K ...
    2001 Volume 65 Issue 7 Pages 1627-1635
    Published: 2001
    Released: July 31, 2002
    JOURNALS FREE ACCESS
    A gene cluster encoding enzymes responsible for the mevalonate pathway was isolated from Streptomyces griseolosporeus strain MF730-N6, a terpenoid-antibiotic terpentecin producer, by searching a flanking region of th 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene, which had been previously isolated by complementation. By DNA sequencing of an 8.9-kb BamHI fragment, 7 genes encoding geranylgeranyl diphosphate synthase (GGDPS), mevalonate kinase (MK), mevalonate diphosphate decarboxylase (MDPD), phosphomevalonate kinase (PMK), isopentenyl diphosphate (IPP) isomerase, HMG-CoA reductase, and HMG-CoA synthase were suggested to exist in that order. Heterologous expression of these genes in E. coli and Streptomyces lividans, both of which have only the nonmevalonate pathways, suggested that the genes for the mevalonate pathway were included in the cloned DNA fragment. The GGDPS, MK, MDPD, PMK, IPP isomerase, and HMG-CoA synthase were expressed in E. coli. Among them, the recombinant GGDPS, MK, and IPP isomerase were confirmed to have the expected activities. This is the first report, to the best of our knowledge, about eubacterial MK with direct evidence.
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  • Jun YOSHIKAWA, Katsura SEKI, Hirofumi SHINOYAMA, Takaaki FUJII
    2001 Volume 65 Issue 7 Pages 1659-1662
    Published: 2001
    Released: July 31, 2002
    JOURNALS FREE ACCESS
    Two malate dehydrogenases (MDH-M1 and MDH-M2) were found in a methanol-using yeast, Candida sp. N-16. MDH-M2 was induced with methanol. These enzymes were purified as electrophoretically and isoelectrophoretically homogeneous proteins. The molecular weights of MDH-M1 and MDH-M2 were estimated to be about 78,000 (homodimer) and 160,000 (homotetramer). Several kinetic properties were significantly different between the two enzymes. The value (2.07) of Vmax(oxaloacetate)/Vmax(malate) and Kcats (555 s-1 for oxaloacetate, 481 s-1 for NADH) of MDH-M2 were higher than the ratio (1.37) of Vmax and Kcats(241 s-1 for oxaloacetate, 271 s-1 for NADH) of MDH-M1, respectively. The activity of MDH-M2 was inhibited by a high concentration of NAD+ and the activity of MDH-M1 by oxaloacetate.
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  • Yuri SARATANI, Eiji UHEDA, Hiroaki YAMAMOTO, Atsuo NISHIMURA, Fumiki Y ...
    2001 Volume 65 Issue 7 Pages 1676-1679
    Published: 2001
    Released: July 31, 2002
    JOURNALS FREE ACCESS
    The enantioselectivity of ECAA to ECHB by eight fungi of four genus was evaluated. All strains showed (S)-selectivity, and Cylindrocarpon sclerotigenum IFO 31855 gave the highest yield and good optical purity (e.e.; >99%). Cell-free extract and acetone-dried cells of C. sclerotigenum IFO 31855 reduced ECAA to (S)-ECHB in the presence of NADPH (e.e.; >99%) and the e.e. was not decreased by heat treatment of the cell-free extract or the acetone-dried cells. The active fractions shown by two peaks on a DEAE-Toyopearl 650 M column gave preferentially (S)-ECHB (e.e.; >99%).
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