Bioscience, Biotechnology, and Biochemistry
Online ISSN : 1347-6947
Print ISSN : 0916-8451
Volume 66 , Issue 7
Showing 1-31 articles out of 31 articles from the selected issue
Analytical Chemistry Note
Organic Chemistry Regular Papers
  • Keun-Hong PARK, You Han BAE
    2002 Volume 66 Issue 7 Pages 1473-1478
    Published: 2002
    Released: June 10, 2003
    JOURNALS FREE ACCESS
      The spheroid of specific cells is often regarded as the better form in artificial organs and mammalian cell bioreactors for improved cell-specific functions. In this study, freshly harvested primary rat hepatocytes, which had been cultivated as spheroids and entrapped in a synthetic thermo-reversible extracellular matrix, were examined for differentiated morphology and enhanced liver-specific functions as compared to a control set (hepatocytes in single-cell form). A copolymer of N-isopropylacrylamide (98 mole % in the feed) and acrylic acid (poly(NiPAAm-co-AAc)), and the adhesion molecule, an Arg-Gly-Asp (RGD)-incorporated thermo-reversible matrix, were used to entrap hepatocytes in the form of either spheroids or single cells. In a 28-day culture period, the spheroids in the RGD-incorporated gel maintained higher viability and produced albumin and urea at constant rates, while there was lower cell viability and less albumin secretion by the spheroids in p(NiPAAm-co-AAc). Hepatocytes cultured as spheroids in the RGD-incorporated gel would constitute a potentially useful three-dimensional cell system for application in a bio-artificial liver device.
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  • Satoshi YAMAUCHI, Satoshi BANDO, Yoshiro KINOSHITA
    2002 Volume 66 Issue 7 Pages 1495-1499
    Published: 2002
    Released: June 10, 2003
    JOURNALS FREE ACCESS
      (1S,2S,5R,6S )-6-(3,4-Methylenedioxyphenyl)-3,7-dioxabicyclo[3.3.0]octan-1,2-diol ((+)-1-hydroxysamin 1) was synthesized, starting from olefin 8. Stereoselective α-hydroxylation was achieved after converting 8 to aldehyde 13. Resulting unstable α-hydroxy aldehyde 14 was then transformed to (+)-1-hydroxysamin (1). This is a new efficient synthetic route to 1,2-oxygenated 6-arylfurofuran lignans.
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  • Jun HIRATAKE, Takayuki IRIE, Nobuya TOKUTAKE, Jun'ichi ODA
    2002 Volume 66 Issue 7 Pages 1500-1514
    Published: 2002
    Released: June 10, 2003
    JOURNALS FREE ACCESS
      A series of sulfoximine-based transition-state analogue inhibitors with a varying alkyl side chain was synthesized to probe the recognition of a Cys substrate by E. coli γ-glutamylcysteine synthetase (γ-GCS). The sulfoximines with a small alkyl group (H, methyl, ethyl, propyl, butyl and CH2OH) each served as a slow-binding inhibitor, the sulfoximine with an ethyl being by far the most potent inhibitor to cause facile and irreversible enzyme inhibition. As the size of the side chain changed from an ethyl, the inhibition potency markedly decreased to reduce the overall affinity with concomitant loss in the inactivation rate and with facile enzyme reactivation by dilution. The sulfoximine without a side chain inhibited the enzyme with almost the same potency as that of L-buthionine-(SR)-sulfoximine (BSO). The free energy difference calculated from the inhibition constants indicates that the side chain of Cys was recognized by its size through hydrophobic interaction and contributed almost equally or even more than the carboxy group to the overall binding of Cys in the transition state.
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  • Yui MASUDA, Masao YOSHIDA, Kenji MORI
    2002 Volume 66 Issue 7 Pages 1531-1537
    Published: 2002
    Released: June 10, 2003
    JOURNALS FREE ACCESS
      The ceramide sex pheromone [(2S,2′R,3S,4R)-2-(2′-hydroxy-21′-methyldocosanoylamino)-1,3,4-pentadecanetriol (1)] of the female hair crab (Erimacrus isenbeckii) was synthesized by starting from (S)-serine and 12-bromo-1-dodecanol.
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Organic Chemistry Note
Organic Chemistry Preliminary Communication
Biochemistry & Molecular Biology Regular Papers
  • Fernando SORIA, Guillermo ELLENRIEDER
    2002 Volume 66 Issue 7 Pages 1442-1449
    Published: 2002
    Released: June 10, 2003
    JOURNALS FREE ACCESS
      The kinetics of thermal inactivation of A. terreus α-rhamnosidase was studied using the substrate p-nitrophenyl α-L-rhamnoside between 50°C and 70°C. Up to 60°C the inactivation of the purified enzyme was completely reversible, but samples of crude or partially purified enzyme showed partial reversibility. The presence of the product rhamnose, the substrate naringin, and other additives reduced the reversible inactivation, maintaining in some cases full enzyme activity at 60°C. A mechanism for the inactivation process, which permitted the reproduction of experimental results, was proposed. The products rhamnose (inhibition constant, 2.1 mM) and prunin (2.6 mM) competitively inhibited the enzyme reaction. The maximum hydrolysis of supersaturated naringin solution, without enzyme inactivation, was observed at 60°C. Hydrolysis of naringin reached 99% with 1% naringin solution, although the hydrolysis degree of naringin was only 40% due to products inhibition when the initial concentration of flavonoid was 10%. The experimental results fitted an equation based on the integrated Michaelis-Menten's, including competitive inhibition by products satisfactorily.
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  • Toshiya IIDA, Yuki MUKOUZAKA, Kaoru NAKAMURA, Isamu YAMAGUCHI, Toshiak ...
    2002 Volume 66 Issue 7 Pages 1462-1472
    Published: 2002
    Released: June 10, 2003
    JOURNALS FREE ACCESS
      Sixteen actinomycetes capable of utilizing dibenzofuran as a sole source of carbon and energy were isolated, including Rhodococcus, Microbacterium, and Terrabacter genera. Heretofore, no dibenzofuran-utilizing strain belonging to the genus Microbacterium has been reported. Five extradiol dioxygenase genes (dfdB, and edi1 to 4) of the strain Rhodococcus sp. YK2 were cloned and analyzed. The nucleotide sequence of dfdB gene was almost identical to the bphC1 gene of Terrabacter sp. DPO360, which was involved in dibenzofuran metabolism in this strain. Southern and Northern hybridization analyses using these extradiol dioxygenase genes as probes suggest that the dfdB gene in YK2 was conserved in diverse dibenzofuran-utilizing actinomycetes; also, the dfdB gene was the only expressed gene among five extradiol dioxygenase genes in the medium containing DF as a sole carbon source. These results suggest that the dfdB gene is important for dibenzofuran metabolism not only in the strain YK2, but also in diverse dibenzofuran-degrading actinomycetes.
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  • Dal-Ho HAN, Michael S. DENISON, Hirofumi TACHIBANA, Koji YAMADA
    2002 Volume 66 Issue 7 Pages 1479-1487
    Published: 2002
    Released: June 10, 2003
    JOURNALS FREE ACCESS
      In this study, we investigated the estrogenic activity of environmental estrogens by a competition binding assay using a human recombinant estrogens receptor (hERβ) and by a proliferation assay using MCF-7 cells and a sulforhodamine-B assay. In the binding assay, pharmaceuticals had a stronger binding activity to hERβ than that of some phytoestrogens (coumestrol, daidzein, genistein, luteolin, chrysin, flavone, and naringenin) or industrial chemicals, but phytoestrogens such as coumestrol had a binding activity as strong as pharmaceuticals such as 17α-ethynylestradiol (EE), tamoxifen (Tam), and mestranol. In the proliferation assay, pharmaceuticals such as diethylstilbestrol, EE, Tam, and clomiphene, and industrial chemicals such as 4-nonylphenol, bisphenol A, and 4-dihydroxybiphenyl had a proliferation-stimulating activity as strong as 17β-estradiol (ES). In addition, we found that phytoestrogens such as coumestrol, daidzein, luteolin, and quercetin exerted a proliferation stimulating activity as strong as ES. Furthermore, we examined the suppression of proliferation-stimulating activity, induced by environmental estrogen, by flavonoids, such as daidzein, genistein, quercetin, and luteolin, and found that these flavonoids suppressed the induction of the proliferation-stimulating activity of environmental estrogens. The suppressive effect of flavonoids suggests that these compounds have anti-estrogenic and anti-cancer activities.
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  • Yajing ZHOU, Qingli XIAO, Zhifang ZHANG, Jialu HE, Yuanxing ZHANG
    2002 Volume 66 Issue 7 Pages 1488-1494
    Published: 2002
    Released: June 10, 2003
    JOURNALS FREE ACCESS
       Via a transient expression assay system, an experimental study was undertaken to characterize the effects of insect ecdysone and juvenile hormone analogue on the transient expression of the luciferase gene under the control of the immediate-early gene (ie-1) promoter of Bombyx mori nuclear polyhedrosis virus. The results demonstrated that the transcriptional activity of the ie-1 promoter was increased to a certain extent by different insect hormone treatments in uninfected insect cells or fifth instar silkworm larvae transfected with a plasmid containing a luciferase gene driven by the ie-1 promoter. By ecdysone treatment alone, an increase of 5-7 fold was reached in Bm-N, or Bm-5 cells, or in the early developmental stage of fifth instar larvae. By treatment with juvenile hormone analogue alone, about 2-fold, in Bm-N, Bm-5, and Sf-21 cells, or about 5-fold increase in the middle developmental stage of larvae was given, respectively. By co-treatment with ecdysone and juvenile hormone analogue, the incease was given between that of ecdysone and juvenile hormone analogue treatment alone. In addition, the synergistic effects of foreign/endogenous hormones on the activity of ie-1 promoter are discussed.
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  • Chizuko FUJITA, Akiko NISHIMURA, Ryoko IWAMOTO, Kenji IKEHARA
    2002 Volume 66 Issue 7 Pages 1515-1523
    Published: 2002
    Released: June 10, 2003
    JOURNALS FREE ACCESS
      Escherichia coli SpoT protein, with 702 amino acid residues, is a bifunctional enzyme catalyzing both guanosine 5′-diphosphate 3′-diphosphate (ppGpp) degradation and its synthesis. First, we investigated how many domains are included in SpoT protein, by limited hydrolysis of the protein with serine proteases, α-chymotrypsin, and elastase. Based on the results, we deduced that SpoT protein is composed of two major domains, an N-terminal half domain from Met1 to Phe373 and a C-terminal half domain from Glu374 to Asn702 (C-terminal end). In addition, by a further α-chymotrypsin digestion, two cleaved sites were found at Arg196 in the N-terminal half domain (D12) and at Lys475 in the C-terminal half domain (D34), to produce four minor domains, D1, D2, D3, and D4. Next, plasmids expressing the two major domains (D12 and D34) and four minor domains (D1, D2, D3, and D4) were constructed. Consequently, the deduced SpoT minor domains as well as the major domains were expressed as stable protein units, except for D4. D4 may also be folded into a stable protein in E. coli cells, since high expression of D4 from a plasmid results in host cell lethality. E. coli relA, spoT double null strains expressing D1, D2, and D12 recovered cell growth in M9 minimal medium, but the transformants of D3, D4, and D34 did not grow in the minimal medium. This indicates that ppGpp synthetic activities could be restricted in the N-terminal half domain (D12, D1, and D2).
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  • Minoru TAKEDA, Shogo NOMOTO, Jun-ichi KOIZUMI
    2002 Volume 66 Issue 7 Pages 1546-1551
    Published: 2002
    Released: June 10, 2003
    JOURNALS FREE ACCESS
      An extracellular polysaccharide (EPS) was recovered and purified from the culture fluid of a sheathed bacterium, Sphaerotilus natans. Glucose, rhamnose, and aldobiouronic acid were detected in the acid hydrolysate of EPS by thin-layer chromatography (TLC). The aldobiouronic acid was found to be composed of glucuronic acid and rhamnose by TLC and gas-liquid chromatography analyses of the corresponding neutral disaccharide. The structure of EPS was identified by methylation linkage analysis and nuclear magnetic resonance. Additionally, partial acid hydrolysates of EPS were prepared and put through fast atom bombardment-mass spectrometry to determine the sugar sequence of EPS. The resulting data showed that EPS produced by S. natans is a new gellan-like polysaccharide constructed from a tetrasaccharide repeating unit, as shown below.
    →4)-α-D-Glcp-(1→2)-β-D-GlcAp-(1→2)-α-L-Rhap- (1→3)-β-L-Rhap-(1→
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Biochemistry & Molecular Biology Notes
Food & Nutrition Science Regular Papers
  • Tsutomu FUKUWATARI, Etsuro SUGIMOTO, Katsumi SHIBATA
    2002 Volume 66 Issue 7 Pages 1435-1441
    Published: 2002
    Released: June 10, 2003
    JOURNALS FREE ACCESS
      We have recently reported that the antituberculosis drug, pyrazinamide (PZA), caused a significant increase in the conversion ratio of tryptophan to niacin in rats. In the present work, we investigated whether or not pyrazinoic acid (POA), a putative metabolite of PZA, increased the conversion ratio of tryptophan to niacin. Weaning rats were fed with a niacin-free and tryptophan-limited diet (negative control diet), or with the negative control diet supplemented with 0.003% nicotinic acid (positive control diet) or 1% POA (test diet) for 27 days. The growth rate was almost same between the groups fed on the positive control diet and the test diet. Dietary POA significantly increased the conversion ratio of tryptophan to niacin. Although POA did not directly inhibit the activity of α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD), the rate-limiting enzyme in the tryptophan-niacin pathway, liver ACMSD activity was only not detected in the test diet group. These results suggest that a derivative of POA metabolized by rats inhibited the ACMSD activity.
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  • Hiroki MATSUOKA, Asaka TAKAHASHI, Yoshio OZAWA, Yoichi YAMADA, Yasushi ...
    2002 Volume 66 Issue 7 Pages 1450-1454
    Published: 2002
    Released: June 10, 2003
    JOURNALS FREE ACCESS
      The structure of the yellow pigment found in salted radish roots was studied. It was found that 1-(2-thioxopyrrolidin-3-yl)-1,2,3,4-tetrahydro-β-carboline-3- carboxylic acid (TPCC) was unstable under neutral pH, and was easily converted into the yellow pigment. The yellow pigment was isolated and identified as 2-[3-(2-thioxopyrrolidin-3-ylidene)methyl]-tryptophan (TPMT) by IR, MS, 1H-, and 13C-NMR spectroscopy. In addition, we proved that this compound was the main yellow pigment in salted radish roots. This compound induced no mutagenicity in Salmonella typhimurium TA98 and TA100, either with or without prior activation.
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  • Hitomi KUMAGAI, Hirotaka SETO, Yuko NORIMATSU, Kenji ISHII, Hitoshi KU ...
    2002 Volume 66 Issue 7 Pages 1455-1461
    Published: 2002
    Released: June 10, 2003
    JOURNALS FREE ACCESS
      The changes in the interaction between food proteins and water and in their surface functional property during enzymatic hydrolysis were investigated. Ovalbumin, a soy protein isolate (SPI), and casein were hydrolyzed with trypsin, and the degree of hydrolysis, water activity aw, and foaming capacity of each hydrolysate were measured. Ovalbumin showed the minimum value for aw, and the values for SPI and casein progressively decreased during hydrolysis. Therefore, the activity coefficient of water, γw (=aw/xw, where xw is the mole fraction of water) was obtained to remove the influence of mole change and to examine the interaction of protein hydrolysates with water. In order to calculate xw in a sample during protein hydrolysis, a method for roughly estimating the number of moles of the protein hydrolysate in a solution was developed. The strategy was to modify the TNBS (2, 4, 6-trinitrobenzenesulfonic acid) method and to combine this method with the modified Ellman method and the determination of lysine by an amino acid analyzer. During enzymatic hydrolysis, each protein sample showed a minimum γw value and maximum foaming capacity.
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Food & Nutrition Science Notes
Food & Nutrition Science Preliminary Communication
Microbiology & Fermentation Technology Regular Papers
  • Takayoshi KOBAYASHI, Yasuyuki TAKIGUCHI, Yuuki YAZAWA, Kuniho NAKATA, ...
    2002 Volume 66 Issue 7 Pages 1524-1530
    Published: 2002
    Released: June 10, 2003
    JOURNALS FREE ACCESS
      The glycoside composition and sequence of an extracellular polysaccharide flocculant of Klebsiella pneumoniae H12 was analyzed. GC and HPLC analysis of the acid-hydrolysate identified its constituent monosaccharides as D-Glc, D-Man, D-Gal, and D-GlcA in an approximate molar ratio of 3.9:1.0:2.3:3.6. To analyze the glycoside sequence, the polysaccharide was partially hydrolyzed by acid and enzyme treatment. GC, HPLC, TLC, MALDI-TOF/MS, and 1H- and 13C- NMR spectroscopy characterized the obtained oligosaccharides.
      The results clarified the partial structure of H12 polysaccharide as a linear polymer of a unit of pentasaccharide with a side chain of one D-GlcA to D-Glc moiety (see below). Although the existence of other sequences or other constituent glycosides could not be fully excluded, H12 polysaccharide must be a novel types as such a complicated unit for a polymer has not so far been reported. The partial structure of a H12 polysaccharide flocculant is also discussed in this report.
    →4)- α-D-Glcp-(1→ 2)-α-D-Manp-(1→3)-4,6-Pyr-β-D-
      3 Galp-(1→4)-β-D-Galp-(1→
     
    1
     
      β-D-GlcpA
     
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  • Hideyuki TAMEGAI, Eriko NANGO, Ayumi KOIKE-TAKESHITA, Fumitaka KUDO, K ...
    2002 Volume 66 Issue 7 Pages 1538-1545
    Published: 2002
    Released: June 10, 2003
    JOURNALS FREE ACCESS
      A gene (btrC2) encoding the 20-kDa subunit of 2-deoxy-scyllo-inosose (DOI) synthase, a key enzyme in the biosynthesis of 2-deoxystreptamine, was identified from the butirosin-producer Bacillus circulans by reverse genetics. The deduced amino acid sequence of BtrC2 closely resembled that of YaaE of B. subtilis, but the function of the latter has not been known to date. Instead, BtrC2 appeared to show sequence similarity to a certain extent with HisH of B. subtilis, an amidotransferase subunit of imidazole glycerol phosphate synthase. Disruption of btrC2 reduced the growth rate compared with the wild type, and simultaneously antibiotic producing activity was lost. Addition of NH4Cl to the medium complemented only the growth rate of the disruptant, and both the growth rate and antibiotic production were restored by addition of yeast extract. In addition, a heterologous co-expression system of btrC2 with btrC was constructed in Escherichia coli. The simultaneously over-expressed BtrC2 and BtrC constituted a heterodimer, the biochemical features of which resembled those of DOI synthase from B. circulans more than those of the recombinant homodimeric BtrC. Despite the similarity of BtrC2 to HisH the heterodimer showed neither aminotransfer nor amidotransfer activity for 2-deoxy-scyllo-inosose as a substrate. All the observations suggest that BtrC2 is involved not only in the secondary metabolism, but also in the primary metabolism in B. circulans. The function of BtrC2 in the butirosin biosynthesis appears to be indirect, and may be involved in stabilization of DOI synthase and in regulation of its enzyme activity.
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Microbiology & Fermentation Technology Notes
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