Bioscience, Biotechnology, and Biochemistry
Online ISSN : 1347-6947
Print ISSN : 0916-8451
Volume 73 , Issue 4
Showing 1-36 articles out of 36 articles from the selected issue
Award Review
  • Hiroya YURIMOTO
    2009 Volume 73 Issue 4 Pages 793-800
    Published: April 23, 2009
    Released: April 23, 2009
    [Advance publication] Released: April 07, 2009
    JOURNALS FREE ACCESS
    Methanol is a promising feedstock for biotechnological and chemical processes as well as a primary source of energy to replace coal and petroleum. Methylotrophic yeasts, that can utilize methanol as the sole source of carbon and energy, have been studied intensively in terms of both physiological activities and potential applications. During growth on methanol, the enzymes involved in methanol metabolism are massively produced in these yeasts, indicating that the gene promoters of these enzymes are strong methanol-inducible promoters. Using these promoters, high-level heterologous gene expression systems have been developed in several methylotrophic yeast strains, such as Pichia pastoris, Hansenula polymorpha, and Candida boidinii. To achieve efficient industrial use of methanol and efficient protein production by methylotrophic yeasts, it is important to elucidate the molecular basis of methanol-inducible gene expression in these yeasts. This review describes recent advances in understanding of the regulation of methanol-inducible gene expression and the molecular mechanism of transcriptional activation in the methylotrophic yeast C. boidinii. Application of this gained knowledge led to successful production of useful enzymes in this yeast, which is also reviewed.
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Biochemistry & Molecular Biology Regular Papers
  • Xiujie XIE, Wei HUANG, Chengzhe XUE, Qun WEI
    2009 Volume 73 Issue 4 Pages 801-804
    Published: April 23, 2009
    Released: April 23, 2009
    [Advance publication] Released: April 07, 2009
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    Protein phosphatase-1 (PP1) is one of the most important Ser/Thr phosphatases in eukaryotic cells. G274, E275, and F276 are located at the tip of the β12-β13 loop of protein phosphatase-1. Without Mn2+, the basal activities and intrinsic fluorescence spectra of all single and double deletion mutants of G274, E275, and F276 were similar to those of PP1, but deletion mutants ΔE275 and ΔE275F276 showed hyperactivity and corresponding changes in intrinsic fluorescence spectra when Mn2+ was present. This suggests that the conformation transition resulting from the combined effect of mutation and Mn2+ accounts for the hyperactivity of mutants. These observations imply that E275 and F276 play a role in resisting enzyme activation by Mn2+ in PP1.
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  • Seo Young LEE, Geun Tae KIM, Si Hun ROH, Jin-Su SONG, Hie-Joon KIM, So ...
    2009 Volume 73 Issue 4 Pages 811-816
    Published: April 23, 2009
    Released: April 23, 2009
    [Advance publication] Released: April 07, 2009
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    Ginseng is a well known herbal medicine in Asia, and ginsenoside Rg3 has anti-cancer and various pharmacological effects. In particular, 20S-ginsenoside Rg3 may increase the anti-proliferative effects of chemotherapy. The authors investigated the mechanism of the anti-proliferative effect of 20S-Rg3 at the protein level in HT29 colon cancer cells. MTT, caspase-3 assays, and flow cytometry analysis were performed to determine cytotoxicity and apoptosis, and proteomic analysis was performed by two-dimensional gel electrophoresis and MALDI-TOF/TOF MS, and a database was used to identify protein changes in 20S-Rg3 treated HT29 cells. The proteins identified included down-regulated Rho GDP dissociation inhibitor, up-regulated tropomyosin1, and annexin5 and glutathione s-transferase p1, which are apoptosis associated proteins. The anti-proliferative mechanism of 20S-Rg3 was found to be involved in mitotic inhibition, DNA replication, and repair and growth factor signaling. The findings of this study suggest that the cytotoxicity of 20S-Rg3 in colon cancer is dependent on several mechanisms, including apoptosis.
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  • Toshiya IIDA, Yasuhiro MOTEKI, Kaoru NAKAMURA, Katsuhiko TAGUCHI, Masa ...
    2009 Volume 73 Issue 4 Pages 822-827
    Published: April 23, 2009
    Released: April 23, 2009
    [Advance publication] Released: April 07, 2009
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    The activity of Rieske non-heme iron oxygenases (aromatic hydrocarbon dioxygenases, AhDOs) is important for the bacterial degradation of aromatic pollutants such as polycyclic aromatic hydrocarbons and dioxins. During our analysis of the role of AhDOs in dioxin bioremediation, some enzymes derived from high G + C Gram-positive actinomycetes were difficult to produce in active form in the Escherichia coli protein expression system. In this study, we constructed a heterologous expression system for AhDOs in Rhodococcus species using a constitutive expression promoter, PdfdB, and a shuttle vector, pRK401, and analyzed the ability of these enzymes to degrade dibenzofuran and deplete several chlorinated dioxins. Three active AhDOs expressed in Rhodococcus strains that were difficult to obtain by the E. coli system showed different regiospecificities for dibenzofuran bioconversion as well as different substrate depletion specificities for chlorinated dioxins. Moreover, AhDO derived from R. erythropolis TA421 showed relatively diverse depletion-substrate specificity for chlorinated dioxins.
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  • Yuji IWATA, Mari YONEDA, Yuki YANAGAWA, Nozomu KOIZUMI
    2009 Volume 73 Issue 4 Pages 865-869
    Published: April 23, 2009
    Released: April 23, 2009
    [Advance publication] Released: April 07, 2009
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    Accumulation of unfolded proteins in the endoplasmic reticulum (ER) of eukaryotic cells triggers the transcriptional induction of ER-resident molecular chaperones to maintain cellular homeostasis, termed the ER stress response. Previously we isolated AtbZIP60, a membrane-bound transcription factor involved in the Arabidopsis ER stress response whose activity is controlled by proteolytic cleavage. In this study we characterized the active form of AtbZIP60 localized in the nucleus during the ER stress response. Transient assay using Arabidopsis protoplasts revealed that activation of BiP promoters by AtbZIP60 is dependent on the cis-elements plant-unfolded protein response element (P-UPRE) and ER stress response element (ERSE). Transcriptional activation activity of AtbZIP60 was mainly located in the region for amino acids 41–80 of AtbZIP60. Size exclusion chromatography analysis showed that the nuclear form of AtbZIP60 exists as a protein complex of approximately 260 kDa. On the basis of the present study combined with observations described in the literature, possible mechanisms of AtbZIP60’s action in the nucleus are discussed.
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  • Junji ITOU, Yoko EGUCHI, Ryutaro UTSUMI
    2009 Volume 73 Issue 4 Pages 870-878
    Published: April 23, 2009
    Released: April 23, 2009
    [Advance publication] Released: April 07, 2009
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    Using an EvgS-active mutant (evgS1) in combination with gene deletions, we clarified the molecular mechanism of the transcriptional cascade of acid resistance and multidrug resistance genes initiated by the EvgS/EvgA two-component system in Escherichia coli, followed by sequential induction of the transcriptional regulators, YdeO and GadE. Overexpression of EvgA, the response regulator of the EvgS/EvgA system, is known to induce the expression of a number of acid resistance and multidrug resistance genes, in which the EvgA-YdeO-GadE circuit is involved, but the role of the sensor EvgS in this circuit has remained unsolved. Our results suggest that the transcriptional cascade initiated by the EvgS/EvgA system in fact functions for acid and drug resistance in E. coli.
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  • Hokuto OHTSUKA, Yuki OGAWA, Hiroyuki MIZUNO, Satoka MITA, Hirofumi AIB ...
    2009 Volume 73 Issue 4 Pages 885-889
    Published: April 23, 2009
    Released: April 23, 2009
    [Advance publication] Released: April 07, 2009
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    In fission yeast, we identified two genes, named ecl2+ and ecl3+, that are paralogous to ecl1+, which extends the chronological lifespan. Both ecl2+ and ecl3+ extend the chronological lifespan when overexpressed as ecl1+. ecl2+ and ecl3+ encode 84- and 89-amino acid polypeptides respectively that are not annotated in the current database. The Ecl2 protein is localized mainly in the nucleus, as Ecl1. These results suggest that ecl1+, ecl2+, and ecl3+ have overlapping functions in the regulation of chronological lifespan.
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  • Ayako NISHIZAWA-YOKOI, Eriko YOSHIDA, Yukinori YABUTA, Shigeru SHIGEOK ...
    2009 Volume 73 Issue 4 Pages 890-895
    Published: April 23, 2009
    Released: April 23, 2009
    [Advance publication] Released: April 07, 2009
    JOURNALS FREE ACCESS
    Supplementary material
    We have isolated a high-light and heat-shock inducible gene, Arabidopsis heat shock transcription factor (HsfA2), which induces expression of various types of target gene such as heat shock protein 18.2-CI (Hsp18.1-CI), galactinol synthase 1 (GolS1), and Bcl-2-associated athanogene 6 (Bag6). Here we investigated the regulatory system of target genes operating via HsfA2. A transient reporter assay using a luciferase reporter construct with different fragments of the Hsp18.1-CI, the GolS1, or the Bag6 promoter showed that two modules of a TATA-proximal heat shock element (HSE) are essential for transcriptional activation by HsfA2. Electrophoretic mobility shift assay demonstrated that the increase in protein complex formation onto the HSE was markedly suppressed during high-light stress and recovery from the stress in knockout HsfA2 plants. HsfA2 appears to function not only in the triggering of response to environmental stress, but also in the amplification of the signal in the response.
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  • Eriko IKEDA, Satomi ANDOU, Ubu IWAMA, Chiaki KATO, Koki HORIKOSHI, Hid ...
    2009 Volume 73 Issue 4 Pages 896-900
    Published: April 23, 2009
    Released: April 23, 2009
    [Advance publication] Released: April 07, 2009
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    The deep-sea denitrifier Pseudomonas sp. strain MT-1 has two distinct gene clusters encoding dissimilatory nitrate reductases, periplasmic nitrate reductase (Nap) and membrane-bound nitrate reductase (Nar). In order to investigate the physiological roles of these enzymes, we determined the nitrate reductase activity of the soluble and membrane fractions from MT-1 and the type strain of Pseudomonas stutzeri (closely related with MT-1) grown under various conditions. In MT-1, the activities of both fractions were highest when the cells were grown anaerobically in the presence of nitrate under atmospheric pressure. However, the activity of the soluble fraction decreased when the cells were grown under high pressure, whereas that of membrane fraction remained constant. Further, the activity of the soluble fraction decreased when the enzyme reaction was performed at low temperature, although that of membrane fraction was not similarly affected. Additionally, the results of RT-PCR showed that expression of the nar genes was strongly induced under high pressure. In contrast, P. stutzeriT showed no such response following a shift in growth pressure. These results suggest that MT-1 possesses a special mechanism for adaptation to the low-temperature and high-pressure environments of the deep sea, and that Nar is the main dissimilatory nitrate reductase in MT-1 in such environments.
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  • Kuniki KINO, Yoichi KOTANAKA, Toshinobu ARAI, Makoto YAGASAKI
    2009 Volume 73 Issue 4 Pages 901-907
    Published: April 23, 2009
    Released: April 23, 2009
    [Advance publication] Released: April 07, 2009
    JOURNALS FREE ACCESS
    Supplementary material
    L-Amino acid ligase catalyzes the formation of an α-peptide bond from unprotected L-amino acids in an ATP-dependent manner, and this enzyme is very useful in efficient peptide production. We performed enzyme purification to obtain a novel L-amino acid ligase from Bacillus subtilis NBRC3134, a microorganism producing peptide-antibiotic rhizocticin. Rhizocticins are dipeptide or tripeptide antibiotics and commonly possess L-arginyl-L-2-amino-5-phosphono-3-cis-pentenoic acid. The purification was carried out by detecting L-arginine hydroxamate synthesis activity, and a target enzyme was finally purified 1,280-fold with 0.8% yield. The corresponding gene was then cloned and designated rizA. rizA was 1,242 bp and coded for 413 amino acid residues. Recombinant RizA was prepared, and it was found that the recombinant RizA synthesized dipeptides whose N-terminus was L-arginine in an ATP-dependent manner. RizA had strict substrate specificity toward L-arginine as the N-terminal substrate; on the other hand, the substrate specificity at the C-terminus was relaxed.
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  • Rui TADA, Asuka TANIOKA, Ken-ichi ISHIBASHI, Yoshiyuki ADACHI, Kazufum ...
    2009 Volume 73 Issue 4 Pages 908-911
    Published: April 23, 2009
    Released: April 23, 2009
    [Advance publication] Released: April 07, 2009
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    We recently determined the structure of a unique type of 1,3-β-D-glucan obtained from Aureobasidium pullulans (AP-FBG) and found that it reacted with the antibodies in human sera. The reactivity of AP-FBG to the antibodies was stronger than that of 1,3-β-D-glucan obtained Grifola frondosa (GRN) but weaker than that of 1,3-β-D-glucan from Candida albicans (CSBG). Here, we demonstrated that AP-FBG reacted to IgG antibodies, especially those of the subclasses IgG2, IgG1, and IgG3, in human sera. Moreover, the results of competitive enzyme-linked immunosorbent assays (ELISAs) using various glucan competitors showed that these IgGs recognized branched chains at position 6. This is the first study to report that the branched chains at position 6 of β-D-glucan strongly contribute to its recognition by antibodies in human sera. This high reactivity of AP-FBG to human IgG could be advantageous for the use of this glucan in medicine, e.g., as an immunostimulatory agent.
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  • Satomi OKUYAMA, Sachiko NAKAMURA-TSURUTA, Hiroaki TATENO, Jun HIRABAYA ...
    2009 Volume 73 Issue 4 Pages 912-920
    Published: April 23, 2009
    Released: April 23, 2009
    [Advance publication] Released: April 07, 2009
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    Small-sized isolectins (9 KDa) from Hypnea japonica belong to a new lectin family. Here, we describe the carbohydrate-binding properties of the three isolectins (hypninA1, A2, and A3) and the amino acid sequence of hypninA3 (P85888). In frontal affinity chromatography with about 100 pyridylaminated oligosaccharides, the isolectins, which had no affinity for monosaccharides, commonly bound only core (α1-6) fucosylated N-glycans, and did not the other oligosaccharides examined, including (α1-2), (α1-3), and (α1-4) fucosylated glycans. The specific binding of hypninA3 with the fucosylated N-glycans (Ka; 0.52–7.58×106 M−1) was confirmed by surface plasmon resonance analyses on an immobilized glycoprotein with and without core (α1-6) fucose. Such specificity of hypninA is clearly distinct from those of other known fucose-binding lectins, making it a valuable tool for cancer diagnosis and quality control of medicinal antibodies. HypninA3 is a polypeptide composed of 90 amino acids containing four half-cystines.
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Biochemistry & Molecular Biology Notes
  • Yoshihiro NOMURA, Yasuhiro ISHII, Koji TAKAHASHI
    2009 Volume 73 Issue 4 Pages 926-929
    Published: April 23, 2009
    Released: April 23, 2009
    [Advance publication] Released: April 07, 2009
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    The effect of adding a polyanionic saccharide (alginic acid, pectic acid, agarose, κ-carragenan or λ-carragenan) to a collagen solution on the reconstruction rate of collagen molecular assembly was evaluated. The rate of collagen molecular assembly was reduced by the addition of alginic acid and of pectic acid that was containing carboxy groups. Dextran, agarose, and soluble starch did not affect the rate, while dextran sulfate and λ-carragenan accelerated the rate of collagen molecular assembly. The denaturation curves of collagen reconstructed with λ-carragenan showed two peaks at about 45 °C and 55 °C. The use of a polyanionic saccharide made it possible to endow the reconstructed collagen fibrils with flexibility to enhance the application of collagen as a scaffold material for tissue engineering.
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  • Aftab AHMAD, Izumi KAJI, Yukiko MURAKAMI, Nana FUNATO, Takeshi OGAWA, ...
    2009 Volume 73 Issue 4 Pages 936-938
    Published: April 23, 2009
    Released: April 23, 2009
    [Advance publication] Released: April 07, 2009
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    Most genetically modified plants are made with antibiotic-resistant genes and promoters derived from plant pathogens. To promote public acceptance, we have adapted a selectable marker and promoters all of which were derived from Arabidopsis thaliana to transformation. We have employed the gene for acetolactate synthase with its own promoter as a selectable marker, and the actin 2 gene promoter to strongly express an objective gene.
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  • Yoshiaki YAMANO, Atsushi ASANO, Masanori OHTA, Shuji HIRATA, Tomoko SH ...
    2009 Volume 73 Issue 4 Pages 946-949
    Published: April 23, 2009
    Released: April 23, 2009
    [Advance publication] Released: April 07, 2009
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    We analyzed the gene expression of sperm flagellum-movement associated proteins, namely, adenylate kinase domain containing protein (RGD1303144) and outer dense fiber protein 1. These gene expressions were up-regulated in a maturing rat testis, and RGD1303144 gene was expressed dominantly in spermatocytes. Although 2,3,7,8-tetrachlorodibenzo-p-dioxin administration reduced sperm motility, these gene expressions were not affected.
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  • Seiichirou NAKAYASU, Ryo SOEGIMA, Kenichi YAMAGUCHI, Tatsuya ODA
    2009 Volume 73 Issue 4 Pages 961-964
    Published: April 23, 2009
    Released: April 23, 2009
    [Advance publication] Released: April 07, 2009
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    A fucose-containing, sulfated polysaccharide ascophyllan was isolated from the brown alga Ascophyllum nodosum. Composition analysis demonstrated that ascophyllan mainly contains uronic acid, xylose, fucose, and sulfate half ester in approximately equimolecular proportions, which are evidently distinct from those of alginate and fucoidan. Ascophyllan inhibited the proliferation of U937 cells in a concentration-dependent manner, and DNA-fragmentation and typical apoptotic nuclear morphological changes were observed in the ascophyllan-treated cells. Furthermore, ascophyllan induced the secretion of tumor necrosis factor-α (TNF-α) and granulocyte colony-stimulating factor (G-CSF) from mouse macrophage cell line RAW264.7.
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Biochemistry & Molecular Biology Communication
Food & Nutrition Science Regular Papers
  • Shigeki MURAKAMI, Shigekazu KURIHARA, Natsue KOIKAWA, Akira NAKAMURA, ...
    2009 Volume 73 Issue 4 Pages 817-821
    Published: April 23, 2009
    Released: April 23, 2009
    [Advance publication] Released: April 07, 2009
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    Athletes become increasingly susceptible to infection with intense training that results in immune suppression. The immune state was investigated after administering cystine/theanine (CT), which has been reported to have an immune reinforcement effect, to athletes before training involving a prolonged period of intense exercise. Fifteen long-distance runners were each allocated to the CT or placebo group, and the test food was ingested for 10 d prior to the start of training. Clinical examinations were performed before and after the training. The results indicate a significant increase in the high-sensitivity C-reactive protein (hs-CRP) and neutrophil count in the blood, as well as a decreasing tendency for lymphocytes in the placebo group, but not the CT group. These observations suggest that the ingestion of CT contributed to suppressing the change in inflammatory response, prevented a decrease in the immune function, and prevented infection and reduced symptoms when infected associated with continuous intense exercise.
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  • Mi-Young PARK, Hoon-Jeong KWON, Mi-Kyung SUNG
    2009 Volume 73 Issue 4 Pages 828-832
    Published: April 23, 2009
    Released: April 23, 2009
    [Advance publication] Released: April 07, 2009
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    The aloe ingredients responsible for physiological effects and the concentrations required to exert their biological activities are not fully understood. This study compares the anti-inflammatory effects of aloin and aloe-emodin with other polyphenols. Our results demonstrated that aloe-emodin dose-dependently inhibited inducible nitric oxide synthase (iNOS) mRNA expression and nitric oxide (NO) production at 5–40 μM. In addition, the levels of cyclooxygenase-2 (COX-2) mRNA and prostaglandin E2 (PGE2) production were suppressed by 40 μM aloe-emodin. Aloin also suppressed the production of NO at 5–40 μM, although it did not suppress PGE2 production. The present results indicate that aloin and aloe-emodin possibly suppress the inflammatory responses by blocking iNOS and COX-2 mRNA expression. The anti-inflammatory effect of aloe-emodin was comparable to that of kaempferol and quercetin, indicating aloe-emodin as a possible key constituent responsible for the anti-inflammatory activity of aloe.
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  • Ayumi KUNIMOTO, Takako SISINO, Kentaro SAKAI, Tomoaki MATSUMOTO, Kyoko ...
    2009 Volume 73 Issue 4 Pages 840-848
    Published: April 23, 2009
    Released: April 23, 2009
    [Advance publication] Released: April 07, 2009
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    Tropomyosins have been identified as a common allergen in crustaceans, but their allergenicity is not well understood. In the present study, we isolated an allergen, Pen j 1, a tropomyosin from kuruma prawn Penaeus japonicus, and determined its N-terminal amino acid sequence. The cDNA encoding the allergen was cloned by 5′- and 3′-rapid amplification of cDNA ends (RACE), and was found to code for a protein which consists of 284 amino acid residues. Sequencing analyses indicated for the first time that mature tropomyosin is formed by the elimination of a leader peptide of nine amino acid residues. To elucidate the binding sites of IgE antibodies in the sera of shrimp-sensitive patients, various recombinant peptides were expressed in Escherichia coli as fusion proteins with glutathione S-transferase (GST), and the examined with regard to reactivity with IgE antibodies. The IgE-binding epitopes were found to locate over the whole sequence of the allergen, and the IgE antibodies in the sera were found to recognize strongly its C-terminal region.
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  • Jishu QUAN, Long PIAO, Huixian XU, Tian LI, Xuezhe YIN
    2009 Volume 73 Issue 4 Pages 849-854
    Published: April 23, 2009
    Released: April 23, 2009
    [Advance publication] Released: April 07, 2009
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    The protective effect of iridoid glucosides from Boschniakia rossica (BRI) against carbon tetrachloride (CCl4)-induced liver injury was examined. CCl4 at a dose of 0.5 ml/kg of body weight was given intraperitoneally to rats to induce liver damage. The rats were sacrificed 16 h after the CCl4 injection. The CCl4 challenge caused a marked increase in the levels of serum animotransferases, tumor necrosis factor-α (TNF-α) and of hepatic inducible nitric oxide synthase (iNOS) protein, depleted reduced glutathione (GSH), and propagated lipid peroxidation. The liver antioxidative defense system, including superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione reductase (GR), as well as the cytochrome P450 2E1 (CYP2E1) expression were suppressed, however. Preadministration of BRI reversed the significant changes of all liver function parameters induced by CCl4 and restored the liver CYP2E1 content and function. These results demonstrate that BRI produced a protective action on CCl4-induced acute hepatic injury via reduced oxidative stress, suppressed inflammatory response and improved CYP2E1 function in the liver.
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  • Yu-Ting HUANG, Jun-ichi ONOSE, Naoki ABE, Kunie YOSHIKAWA
    2009 Volume 73 Issue 4 Pages 855-860
    Published: April 23, 2009
    Released: April 23, 2009
    [Advance publication] Released: April 07, 2009
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    Increasing attention has been focused on food-drug interactions. We have investigated the inhibitory effect of Chinese edible mushrooms, Boletus calopus and Suillus bovinus, on cytochrome P450 (CYP) 1A2, 2C9, 2D6, and 3A4, the main drug-metabolizing enzymes. Three pulvinic acid derivatives, atromentic acid (1), variegatic acid (2), and xerocomic acid (3), isolated from Boletus calopus and Suillus bovinus, revealed nonspecific inhibitory effects on all four CYPs. Using these compounds, the maximum IC50 values obtained with CYP3A4 in vitro were atromentic acid (1), 65.1±3.9 μM; variegatic acid (2), 2.2±0.1 μM; and xerocomic acid (3), 2.4±0.1 μM. Variegatic acid (2) and xerocomic acid (3) were effective inhibitors, comparable to cimetidine, dicoumarol, erythromycin, safrole, and uniconazole. Variegatic acid (2) and xerocomic acid (3) efficiently reduced ferryl myoglobin in CYPs. Reduction of ferryl heme to ferric heme is likely the mechanism of the nonspecific inhibitory effects of these compounds on CYPs.
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Food & Nutrition Science Notes
Microbiology & Fermentation Technology Regular Papers
  • Tadashi FUJII, Yoshikazu FUJII, Kazuhiro MACHIDA, Atsushi OCHIAI, Masa ...
    2009 Volume 73 Issue 4 Pages 805-810
    Published: April 23, 2009
    Released: April 23, 2009
    [Advance publication] Released: April 07, 2009
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    We report here some efficient biotransformations using Escherichia coli strains with disruptions for the AcrAB-TolC efflux pump system. Biotransformations of compactin into pravastatin (6α-hydroxy-iso-compactin) were performed using E. coli strains with tolC and/or acrAB mutations expressing a cytochrome P450 (P450) gene. The production levels of pravastatin using strains with acrAB, tolC, and tolC acrAB mutations increased by 3.7-, 7.0-, and 7.1-fold, respectively. Likewise, the production levels of 25-hydroxy vitamin D3 and 25-hydroxy 4-cholesten 3-one using tolC acrAB mutant strains expressing an individual P450 gene increased by 2.2- and 16-fold, respectively. The enhancement of this biotransformation efficiency could be explained by increases in the intracellular amounts of substrates and the concentrations of active P450s. These results demonstrate that we have achieved versatile methods for efficient biotransformations using E. coli strains with tolC acrAB mutations expressing P450 genes.
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  • Pattraporn JOMMUENGBOUT, Surapong PINITGLANG, Khin Lay KYU, Khanok RAT ...
    2009 Volume 73 Issue 4 Pages 833-839
    Published: April 23, 2009
    Released: April 23, 2009
    [Advance publication] Released: April 07, 2009
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    The three-dimensional structure (3D structure) of Xyn11A, a family 11 xylanase from Bacillus firmus K-1, was obtained through homology modeling. To study the substrate-binding site of Xyn11A, six xylooligosaccharides, xylobiose to xyloheptaose (X2–X7), were docked into the active site of Xyn11A by molecular docking. Based on the docked energy and estimated free energy of binding combined with modeled enzyme-substrate complexes, the substrate-binding site of Xyn11A probably contained six subsites, defined as −3, −2, −1, +1, +2, and +3. Focus on possible stacking interaction presented seven aromatic residues, that played an important role in six subsites of Xyn11A such as Tyr165 (−3), Trp9 and Tyr69 (−2), Tyr80 (−1), Tyr65 (+1), Tyr88 (+2) and Tyr173 (+3). The bond-cleavage positions showed that X2 and X3 did not bind at the cleft (subsites −1 and +1) of Xyn11A. Related to the experiment, the end products of larchwood xylan hydrolysis by purified Xyn11A were X2 and X3. X2 and X3 acted as the end product inhibitors of Xyn11A.
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  • Hideki KITO, Ayumi ABE, I-Nengah SUJAYA, Yuji ODA, Kozo ASANO, Teruo S ...
    2009 Volume 73 Issue 4 Pages 861-864
    Published: April 23, 2009
    Released: April 23, 2009
    [Advance publication] Released: April 07, 2009
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    Twenty-one strains of Amylomyces rouxii isolated from starters of Asian fermented foods were divided into two groups, lactic acid (LA) and fumaric and malic acid (FMA) producers, by organic acid productivity in liquid culture. Phylogenetic analysis based on the ldhB gene, ribosomal RNA encoding DNA (rDNA) internal transcribed spacer (ITS) sequence, and genome-wide amplified fragment length polymorphism (AFLP) revealed that A. rouxii was grouped into two major clusters as to organic acid accumulation, corresponding to Rhizopus oryzae and Rhizopus delemar. These observations suggest that the species A. rouxii is composed of two distinct types, derived from R. oryzae or R. delemar via domestication in the starters.
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  • Shigehito IKUSHIMA, Toshio FUJII, Osamu KOBAYASHI
    2009 Volume 73 Issue 4 Pages 879-884
    Published: April 23, 2009
    Released: April 23, 2009
    [Advance publication] Released: April 07, 2009
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    In order to take full advantage of the industrially important yeast Candida utilis, we developed a practical recombinant DNA tool for multiple gene disruption in C. utilis based on the Cre-loxP system, which makes possible the reuse of selection markers. For this purpose, two plasmids were constructed: one harbored a heterologous loxP-flanked selection marker cassette carrying the gene responsible for hygromycin B-resistance, and the other had an autonomous replication sequence (ARS) and a Cre-recombinase expression module. Multiple disruption of C. utilis NBRC0988 URA3 genes (CuURA3), encoding orotidine-5′-phosphate decarboxylase, validated the efficiency of the system. The fourth round of deletion yielded a null mutant, i.e., a uracil auxotroph, giving some support to the possibility that C. utilis NBRC0988 is a tetraploid. This agreed very well with the outcomes of FACS analysis, which showed that various strains of this yeast contained 3–5 times more DNA than a Saccharomyces cerevisiae haploid.
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Microbiology & Fermentation Technology Notes
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