Bioscience, Biotechnology, and Biochemistry
Online ISSN : 1347-6947
Print ISSN : 0916-8451
Volume 77 , Issue 8
Showing 1-35 articles out of 35 articles from the selected issue
Award Reviews
  • Takumi MISAKA
    2013 Volume 77 Issue 8 Pages 1613-1616
    Published: August 23, 2013
    Released: August 23, 2013
    [Advance publication] Released: August 07, 2013
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    Objective quantification of taste intensity would be extremely useful for product development and quality control in the food industry. Progress has been made toward using the responses of cultured cells expressing human taste receptors as an indicator in evaluating gustatory intensity objectively. Effective utilization of such evaluation systems should not only provide information regarding taste intensities as perceived by humans, but also make possible the characterization of taste modulators to be used for commercial food products. Moreover, such an evaluation system should be also useful in advancing our understanding of the ways tastants are recognized by taste receptors.
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  • Hiromichi MINAMI
    2013 Volume 77 Issue 8 Pages 1617-1622
    Published: August 23, 2013
    Released: August 23, 2013
    [Advance publication] Released: August 07, 2013
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    Higher plants produce diverse chemicals, including alkaloids, terpenoids, and phenolic compounds (phenylpropanoids and flavonoids) as secondary metabolites. These chemicals are widely used for human health and nutrition. Alkaloids, for example, are valued in medicine due to their high biological activities, but most of these metabolites accumulate at low levels in plant cells, resulting in poor extraction yields. Increasingly, attention is devoted to the production of plant metabolites by reconstructing plant biosynthetic pathways in microorganisms. This technology has been aided by advances in synthetic biology and metabolic engineering. Here, the review a fermentation platform for low-cost production of numerous alkaloids using bioengineered Escherichia coli and/or Saccharomyces cerevisiae.
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Organic Chemistry Regular Paper
  • Toshiya MASUDA, Yukari MIURA, Miyuki INAI, Akiko MASUDA
    2013 Volume 77 Issue 8 Pages 1753-1758
    Published: August 23, 2013
    Released: August 23, 2013
    [Advance publication] Released: August 07, 2013
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    The enhancing effect of a cysteinyl thiol N-benzoylcysteine methyl ester on the antioxidant activity of several flavonoids was investigated in a lipid oxidation system. Obvious enhancement was apparent for catechin, myricetin, quercetin, and taxifolin, the activity for myricetin being the most potent among them. An HPLC analysis of the products from the antioxidation reaction of myricetin in the presence of the thiol was carried out and the structures of the products were determined to clarify the enhancing effect chemically. The obtained data indicated that two thiol adducts on the B ring, and probably C-ring adducts, which were produced in the antioxidation process, exerted an enhancing effect on the antioxidant activity of myricetin.
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Organic Chemistry Notes
Biochemistry & Molecular Biology Regular Papers
  • Akio SAITO
    2013 Volume 77 Issue 8 Pages 1628-1632
    Published: August 23, 2013
    Released: August 23, 2013
    [Advance publication] Released: August 07, 2013
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    Pigment epithelium-derived factor (PEDF) is a member of the serine protease inhibitor family. It is present in a variety of tissues and organs, including plasma. Here, PEDF was purified from human plasma by use of a dermatan sulfate affinity column, and then hydroxyapatite, gel filtration and ion exchange columns. It did not form a complex with various proteases, including chymotrypsin, elastase, kallikrein, thrombin, plasmin, cathepsins G, activated protein C, and urokinase, but collagen type I facilitated the degradation of PEDF by chymotrypsin more than 10-fold. Dermatan sulfate, heparan sulfate, and heparin showed similar effects, but other glycosaminoglycans, such as hyaluronic acid, chondroitin sulfate A, C, D, E, and keratan sulfate, had no effect on PEDF degradation.
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  • Yuichi SHIGEOKA, Tetsuro FUJISAWA, Satoshi TESHIBA, Hisayoshi FUKUMORI ...
    2013 Volume 77 Issue 8 Pages 1637-1644
    Published: August 23, 2013
    Released: August 23, 2013
    [Advance publication] Released: August 07, 2013
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    The Bacillus stearothermophilus lipoate acetyltransferase (E2), composed of sixty identical, subunits is the core component of the pyruvate dehydrogenase complex (PDC). E2 polypeptide is composed of LD, PSBD, and CD domains. Most studies had focused on a truncated E2 that is deficient in LD and PSBD, because CD mainly contributes to maintaining the multimeric structure. We examined salt-induced changes in E2 without truncation and constructed reaction models. We speculate that in the presence of KCl, E2 is dissociated into a monomer and then assembled into an aggregative complex (CA) and a quasi-stable complex (CQ). CA was larger than CQ, but smaller than intact E2. CA and CQ were dominant complexes at about neutral pH and at basic pH respectively. PDC, in which PSBD is occupied by other components, and a truncated E2 undergo dissociation only. LD-PSBD region besides CD might then contribute to the partial association of dissociated E2.
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  • Taku OZAKI, Sei-ichi ISHIGURO, Hideaki ITOH, Kazuhisa FURUHAMA, Mitsur ...
    2013 Volume 77 Issue 8 Pages 1645-1649
    Published: August 23, 2013
    Released: August 23, 2013
    [Advance publication] Released: August 07, 2013
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    Cisplatin is a widely used chemotherapeutic agent, but its use is limited by nephrotoxicity associated with mitochondrial dysfunction. Because its mechanisms are poorly understood, we aimed to identify the mitochondrial proteins targeted by cisplatin. We isolated renal mitochondrial proteins from Sprague-Dawley (SD) rats and performed cisplatin-affinity column chromatography. The proteins eluted were detected on SDS–PAGE and subjected to in-gel tryptic digestion and LC-MS/MS analysis. We identified glutamate oxaloacetate transaminase (GOT) and mitochondrial malate dehydrogenase (MDH). Next, we administered cisplatin intraperitoneally to SD rats to induce nephrotoxicity and assayed the activities of the enzymes. The results indicated that cisplatin caused a severe decrease in mitochondrial GOT activity on day 1 after cisplatin administration. Three d later, we also identified a decrease in mitochondrial MDH activity. Our results indicate that cisplatin binds to mitochondrial GOT and inhibits its activity, causing mitochondrial dysfunction and subsequent nephrotoxicity.
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  • Tomoaki FUKUNAGA, Mikiko NAKAMURA, Takao KITAGAWA, Ramida WATANAPOKASI ...
    2013 Volume 77 Issue 8 Pages 1669-1676
    Published: August 23, 2013
    Released: August 23, 2013
    [Advance publication] Released: August 07, 2013
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    Drugs affecting cellular morphological changes leading to tumor cell migration and invasion are desirable for cancer therapy. In the present study, we screened for small-molecule compounds that affect the cellular morphology of both unicellular yeast and mammalian HEK293 cells to identify drug candidates. The yeast formin protein Bni1 and Src homology 3 (SH3)-pleckstrin homology (PH) domain protein Boi1, which are required for proper morphogenesis, cause growth defects when overexpressed in yeast. Using this system, we screened a chemical library consisting of ~8000 compounds to identify drug candidates that suppress these growth defects. None of the screened compounds induced morphological changes in vegetatively growing yeast cells, but several compounds had inhibitory effects on pheromone-induced projection formation and actin localization, suggesting that these compounds affected a specific stage of morphogenesis. Five of the compounds also induced morphological changes in mammalian HEK293 cells. Among the identified compounds, BTB03156, 2-[(4-chlorophenyl)sulfonyl]-1-methyl-3,5-dinitrobenzene, and BTB02467, 1-[(4-chlorophenyl)sulfonyl]-2-nitro-4-(trifluoromethyl)benzene, although they have similar structures, displayed differing effects on the yeast growth defects caused by latrunculin A, an actin polymerization inhibitor. The chemical library compounds identified using this in vivo screening approach are simple, cell-permeable molecules, and therefore may be useful in the development of therapeutic drugs for cancer metastasis and other actin-related diseases.
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  • Sotaro FUJII, Misa MASANARI, Hiroki INOUE, Masaru YAMANAKA, Satoshi WA ...
    2013 Volume 77 Issue 8 Pages 1677-1681
    Published: August 23, 2013
    Released: August 23, 2013
    [Advance publication] Released: August 07, 2013
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    Sequence analysis indicated that thermophilic Hydrogenophilus thermoluteolus cytochrome c′ (PHCP) and its mesophilic homolog, Allochromatium vinosum cytochrome c′ (AVCP), closely resemble each other in a phylogenetic tree of the cytochrome c′ family, with 55% sequence identity. The denaturation temperature of PHCP was 87 °C, 35 °C higher than that of AVCP. Furthermore, PHCP exhibited a larger enthalpy change value during its thermal denaturation than AVCP. While AVCP was dimeric, as observed previously, PHCP was trimeric, and this was the first observation as a cytochrome c′. Dissociation of trimeric PHCP and its protein denaturation reversibly occurred at the same time in a two-state transition manner. Therefore, PHCP is enthalpically more stable than AVCP, perhaps due to its unique trimeric form, in addition to the lower number of Gly residues in its putative α-helical regions.
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  • Taiji IMOTO
    2013 Volume 77 Issue 8 Pages 1703-1708
    Published: August 23, 2013
    Released: August 23, 2013
    [Advance publication] Released: August 07, 2013
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    It is a common practice to employ kcat[E]0/Km as a first-order rate constant for the analysis of an enzymatic reaction, where [E]0 is the total enzyme concentration. I describe in this report a serious shortcoming in analyzing enzymatic reactions when kcat[E]0/Km is employed and show that kcat[E]0/Km can only be applied under very limited conditions. I consequently propose the use of a more universal first-order rate constant, kcat[ES]K/[S]0, where [ES]K is the initial equilibrium concentration of the ES-complex derived from [E]0, [S]0 and Km. Employing kcat[ES]K/[S]0 as the first-order rate constant enables all enzymatic reactions to be reasonably simulated under a wide range of conditions, and the catalytic and binding contributions to the rate constant of any enzyme can be determined under any and all conditions.
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  • Hirokazu SUZUKI, Ayano MURAKAMI, Ken-ichi YOSHIDA
    2013 Volume 77 Issue 8 Pages 1709-1714
    Published: August 23, 2013
    Released: August 23, 2013
    [Advance publication] Released: August 07, 2013
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    Supplementary material
    Members of glycoside hydrolase family 1 (GH1) cleave glycosidic linkages with a variety of physiological roles. Here we report a unique GH1 member encoded in the genome of Bifidobacterium adolescentis ATCC 15703. This enzyme, BAD0156, was identified from over 2,000 GH1 sequences accumulated in a database by a genome mining approach based on a motif sequence. A recombinant BAD0156 protein was characterized to confirm that this enzyme alone specifically hydrolyzes p-nitrophenyl-α-L-arabinofuranoside among the 24 p-nitrophenyl-glycosides examined. Among natural glycosides, α-1,5-linked arabino-oligosaccharides served as substrates, but arabinan, debranched arabinan, arabinoxylan, and arabinogalactan did not. A time course analysis of arabino-oligosaccharide hydrolysis indicated that BAD0156 is an exo-acting enzyme. These results suggest that BAD0156 is an α-L-arabinofuranosidase. This is the first report of a GH1 enzyme that acts specifically on arabinosides, providing information on GH1 substrate specificity.
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  • Mayumi OKUMURA, Angela M. KATSUYAMA, Hideki SHIBATA, Masatoshi MAKI
    2013 Volume 77 Issue 8 Pages 1715-1721
    Published: August 23, 2013
    Released: August 23, 2013
    [Advance publication] Released: August 07, 2013
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    Supplementary material
    The endosomal sorting complex required for transport (ESCRT) system comprises a series of protein complexes that play essential roles in multivesicular body (MVB) sorting of ubiquitylated membrane proteins, enveloped RNA virus budding, and cytokinesis in mammalian cells. The complex, named ESCRT-I, consists of four subunits (TSG101, VPS28, VPS37, and MVB12). There are four VPS37 isoforms. We have reported that ALIX (an ALG-2-interacting protein and accessory protein in the ESCRT system) is physically linked with TSG101 by ALG-2 in a Ca2+-dependent manner, but the role of ALG-2 as an adaptor protein for the ESCRT-I complex remains unknown. To characterize this adaptor function, initially we investigated the binding of ALG-2 to ESCRT-I complexes containing each one of the four different VPS37 isoforms by two approaches: first, Far-Western blot analysis with biotin-labeled ALG-2 probe, and second, a pulldown assay to determine the binding of the four recombinant ESCRT-I complexes to Strep-tagged ALG-2 after co-expression in HEK293T cells. VPS37B and VPS37C appeared to interact with ALG-2 in a stronger manner than TSG101 does. The results of in vitro binding assays using purified recombinant proteins indicated that ALG-2 functions as a Ca2+-dependent adaptor protein that bridges ALIX and ESCRT-I to form a ternary complex, ESCRT-I/ALIX/ALG-2.
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  • Zhifeng CUI, Yongming ZHANG, Hironori INOUE, Syun YOGO, Eiji HIRASAWA
    2013 Volume 77 Issue 8 Pages 1728-1733
    Published: August 23, 2013
    Released: August 23, 2013
    [Advance publication] Released: August 07, 2013
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    Semicarbazide-sensitive amine oxidase activity was detected in Narcissus tazetta. The enzyme was purified to homogeneity by the criterion of native polyacrylamide gel electrophoresis (PAGE) with DEAE-Sephacel, hydroxyapatite, and phenyl-Sepharose columns. The molecular mass of the enzyme, determined using a GS-520 HQ column, was estimated to be 135 kDa. SDS–PAGE yielded two bands of, 75 kDa and 65 kDa. The enzyme, which had catalytic activity for some aliphatic and aromatic monoamines, belongs to a class of monoamine oxidases (MAOs). The Km value for n-propylamine was 5.9 × 10−5 M. A substrate analog, 2-bromoethylamine, inhibited enzyme activity. Redox-cycling staining detected a quinone in the MAO protein. By inductively coupled plasma mass analysis, it was determined that there were 2.44 moles of copper atoms per mole of the enzyme. Protein sequence analysis revealed that there was no identity between two N-terminal residues of the 75 kDa and 65 kDa proteins of narcissus MAO.
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  • Ryuto MARUYAMA, Yuki KAMOSHIDA, Makoto SHIMIZU, Jun INOUE, Ryuichiro S ...
    2013 Volume 77 Issue 8 Pages 1734-1738
    Published: August 23, 2013
    Released: August 23, 2013
    [Advance publication] Released: August 07, 2013
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    The activating transcription factor 6α (ATF6α) is a sensor of the endoplasmic reticulum stress response that regulates the expression of genes involved in the unfolded protein response. Here we found that forced expression of a constitutively active form of ATF6α, ATF6(N), stimulated the expression of cholesterogenic genes, including 3-hydroxy-3-methyl-glutaryl (HMG)-CoA reductase, HMG-CoA synthase, and squalene synthase, and de novo cholesterol synthesis in hepatoma Huh-7 cells. An ATF6α mutant lacking the DNA-binding domain ATF6(N)ΔbZip failed to show these effects. Luciferase assays indicated that ATF6(N) overexpression stimulated the promoter activities of HMG-CoA reductase, HMG-CoA synthase, and squalene synthase. Chromatin immunoprecipitation assays revealed that ATF6(N) interacted with the promoter region of the HMG-CoA synthase gene. Collectively, these results indicate that ATF6α can regulate de novo cholesterol synthesis through stimulation of cholesterogenic gene expression.
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  • Ken I HOHMURA, Hua SHI, Kazunori HIRAYOSHI
    2013 Volume 77 Issue 8 Pages 1739-1746
    Published: August 23, 2013
    Released: August 23, 2013
    [Advance publication] Released: August 07, 2013
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    Control of interactions among proteins is critical in the treatment of diseases, but the specificity required is not easily incorporated into small molecules. Macromolecules could be more suitable as antagonists in this situation, and RNA aptamers have become particularly promising. Here we describe a novel selection procedure for RNA aptamers against a protein that constitutes a single structural domain, the Drosophila TATA-binding protein (TBP). In addition to the conventional filter partitioning method with free TBP as target, we performed another experiment, in which the TATA-bound form of TBP was targeted. Aptamers generated by both selections were able to bind specifically to TBP, but the two groups showed characteristics which were clearly different in terms of their capability to compete with TATA-DNA, their effects on the TATA-bound form of TBP, and their effects on in vitro transcription. The method used to generate these two groups of aptamers can be used with other targets to direct aptamer specificity to discrete sites on the surface of a protein.
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  • Kyung-Mo SONG, Masayuki OKUYAMA, Mariko NISHIMURA, Takayoshi TAGAMI, H ...
    2013 Volume 77 Issue 8 Pages 1759-1765
    Published: August 23, 2013
    Released: August 23, 2013
    [Advance publication] Released: August 07, 2013
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    The specificity for the α-1,4- and α-1,6-glucosidic linkages varies among glycoside hydrolase family 31 α-glucosidases. This difference in substrate specificity has been considered to be due to the difference in an aromatic residue on β→α loop 1 in the catalytic domain with a (β/α)8 barrel fold; i.e., the enzymes having Tyr and Trp on β→α loop 1 were respectively described as α-1,4-specific and α-1,6-specific α-glucosidases. Schwanniomyces occidentalis α-glucosidase, however, prefers the α-1,4-glucosidic linkage, although the enzyme possesses Trp324 at the corresponding position. The mutation of Trp324 to Tyr decreased the ability for hydrolysis of the α-1,6-glucosidic linkage and formation of the α-1,6-glucosidic linkage in transglycosylation, indicating Trp324 to be closely associated with α-1,6 specificity, even if the enzyme preferred the α-1,4-glucosidic linkage. The mutant enzyme was found to catalyze the production of the branched oligosaccharide, 2,4-di-O-(α-D-glucopyranosyl)-D-glucopyranose, more efficiently than the wild-type enzyme.
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Biochemistry & Molecular Biology Notes
Biochemistry & Molecular Biology Communication
Food & Nutrition Science Regular Papers
  • Takahiko MITANI, Asako HORINISHI, Kunihiro KISHIDA, Tomoaki KAWABATA, ...
    2013 Volume 77 Issue 8 Pages 1623-1627
    Published: August 23, 2013
    Released: August 23, 2013
    [Advance publication] Released: August 07, 2013
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    The fruit of mume, Japanese apricot (Prunus mume Sieb. et Zucc.), was evaluated for its phenolics content, high performance liquid chromatography (HPLC) profile and antioxidative activities. The phenolics content of mume fruit was relatively high, the flesh of fully matured fruit containing up to 1% of phenolics on a dry weight basis. Reflecting such a high content of phenolics, the ORAC (oxygen radical absorbance capacity) value for mume fruit flesh showed high values, ranging from 150 to 320 µmol/g Trolox equivalent, depending upon the stage of maturation. 5-O-Caffeoylqunic acid (chlorogenic acid), 3-O-caffeoylquinic acid and tetra-O-acylated sucrose-related compounds were isolated from the flesh of mume fruit, although many unknown peaks were also apparent in the HPLC chromatogram. An alkali hydrolysate comprised four main phenolic acids, caffeic acid, cis/trans-p-coumaric acid and ferulic acid. No flavonoids were observed in the analysis. These results suggest that the majority of phenolics in mume fruit were hydroxycinnamic acid derivatives.
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  • Satoko SOGA, Noriyasu OTA, Akira SHIMOTOYODOME
    2013 Volume 77 Issue 8 Pages 1633-1636
    Published: August 23, 2013
    Released: August 23, 2013
    [Advance publication] Released: August 07, 2013
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    Long-term ingestion of coffee polyphenols (chlorogenic acids, CGAs) reduces body fat in humans and rodents. While CGA supplementation has been shown to increase fat utilization in rodents, evidence in humans is still limited. The present study clarifies the effect of daily CGA consumption on energy metabolism in humans. Eighteen healthy male subjects (36.1 ± 7.4 y of age) participated in a placebo-controlled, double-blind, crossover, intervention study with two different test beverages. The subjects consumed 185 mL of a test beverage with or without CGAs (329 mg) daily for 4 wk. The energy metabolism was evaluated by using indirect calorimetry before and after the test period during fasting and up to 180 min postprandially. Indirect calorimetry showed that a 4-wk ingestion of the CGA beverage led to a significantly higher postprandial energy expenditure than that of the control beverage. The subjects ingesting the CGA beverage exhibited higher postprandial fat utilization than those consuming the control beverage. The daily CGA consumption therefore increased postprandial fat utilization in healthy humans.
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  • Chun-Shan LIU, Tae-Gyu NAM, Min-Woo HAN, Soo-mi AHN, Han Seok CHOI, Ta ...
    2013 Volume 77 Issue 8 Pages 1682-1688
    Published: August 23, 2013
    Released: August 23, 2013
    [Advance publication] Released: August 07, 2013
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    Oxidative stress due to the over-production of reactive oxygen species (ROS) is associated with human skin aging. This study was designed to identify the bioactive phenolics in detoxified Rhus verniciflua Stokes (DRVS) that may protect human skin against oxidative stress. Under oxidative stress caused by H2O2, the 40% (v/v) aqueous methanol extract of DRVS protected human keratinocytes in a dose-dependent manner. The expression of matrix metalloproteinase-1 (MMP-1) was also inhibited by the DRVS extract in human dermal fibroblasts-neonatal cells exposed to ultraviolet A. The major bioactive phenolics of DRVS were tentatively identified by LC/Q-TOF-ESI-MS/MS, and included gallic acid, 2-(ethoxymethoxy)-3-hydroxyphenol, fustin, a fustin isomer, tetragalloyl glucose, pentagalloyl glucose, fisetin, sulfuretin, a sulfuretin isomer, and butein. The results suggest that a DRVS extract may be effective in slowing skin aging through its antioxidative properties and by down-regulating MMP-1 expression. Further studies are needed to examine whether this effect would be mediated by the phenolics identified in this study.
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  • Kumiko TAKEMORI, Aki TAHARA, Tetsuo MURAKAMI, Takashi KOMETANI
    2013 Volume 77 Issue 8 Pages 1689-1693
    Published: August 23, 2013
    Released: August 23, 2013
    [Advance publication] Released: August 07, 2013
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    Oxidative stress was induced in 12-week-old offspring of protein-restricted (9% protein) and control (20% protein) protein-restricted stroke-prone spontaneously hypertensive rats (SHRSP) by administering phorbol 12-myristate 13-acetate (PMA) for 4 weeks to determine the effects of oxidative stress on the vascular function of the SHRSP offspring. There was no significant difference in the blood pressure of offspring of the protein-restricted dams and control dams. The plasma diacron-reactive oxygen metabolite (dROM) level at 16 weeks of age was significantly higher in offspring of the protein-restricted dams, whereas the anti-oxidative enzyme activity was similar in both groups. Acetylcholine (Ach)-induced relaxation was significantly reduced in offspring of the protein-restricted dams. The expression of endothelial nitric oxide synthase (eNOS) was lower and the expression of soluble guanylic acid cyclase (sGC) was higher in offspring of the protein-restricted dams. These results indicate that SHRSP offspring of the protein-restricted dams were sensitive to oxidative stress, and displayed the vascular dysfunction.
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  • Eun-Hei LEE, Woo-Chan SON, Seong-Eun LEE, Bae-Hwan KIM
    2013 Volume 77 Issue 8 Pages 1694-1702
    Published: August 23, 2013
    Released: August 23, 2013
    [Advance publication] Released: August 07, 2013
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    This study investigated the effects of administering poly-γ-glutamic acid (γ-PGA), isoflavones, and γ-PGA with isoflavones on the lipid, fatty liver, and gene expression levels associated with fatty acid oxidation and adipose synthesis in high-fat diet (HFD)-induced C57BL/6 mice. The results demonstrate a significant decrease in the body weight gain, food intake, food efficiency, liver weight, and epididymal adipose tissue of the experimental groups in comparison with the HFD-induced control group. The serum biochemistry indices for hepatic damage, hyperlipidemia, hyperglycemia, and lipid deposits in the liver and adipose tissue were also lower in the experimental groups than in the control group. The anti-oxidative index, and cytokine and enzyme levels associated with obesity (e.g., leptin, adiponectin, AMPK, CPT-1, PPARα, GLUT-4, and UCP-2) were enhanced in the experimental groups in comparison with the control group. These results demonstrate that γ-PGA and isoflavones improved the blood lipid level, insulin resistance, and hyperglycemia. Increased fatty acid oxidation inhibited the synthesis and accumulation of adipose tissue. The results suggest that γ-PGA and isoflavones could be used as new functional foods for preventing obesity.
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Food & Nutrition Science Notes
Microbiology & Fermentation Technology Regular Papers
  • Makoto HIBI, Nobuyuki HORINOUCHI, Weihao TU, Chee-Leong SOONG, Masashi ...
    2013 Volume 77 Issue 8 Pages 1650-1654
    Published: August 23, 2013
    Released: August 23, 2013
    [Advance publication] Released: August 07, 2013
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    A succinimide-assimilating bacterium, Pseudomonas putida s52, was found to be a potent producer of pyruvate from fumarate. Using washed cells from P. putida s52 as catalyst, 400 mM pyruvate was produced from 500 mM fumarate in a 36-h reaction. Bromopyruvate, a malic enzyme inhibitor, was used for the selection of mutants with higher pyruvate productivity. A bromopyruvate-resistant mutant, P. putida 15160, was found to be an effective catalyst for pyruvate production. Moreover, under batch bioreactor conditions, 767 mM of pyruvate was successfully produced from 1,000 mM fumarate in a 72-h reaction with washed cells from P. putida 15160 as catalyst.
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  • Fanglong ZHAO, Chaoyou XUE, Meiling WANG, Xiaoyang WANG, Wenyu LU
    2013 Volume 77 Issue 8 Pages 1661-1668
    Published: August 23, 2013
    Released: August 23, 2013
    [Advance publication] Released: August 07, 2013
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    Metabolomics analysis of three Saccharopolyspora spinosa strains (wild type strain WT, ultraviolet mutant strain WH124, and metabolic engineering strain LU104) with different spinosad producing levels was performed by liquid chromatograph coupled to mass spectrometry (LC-MS). The metabolite profiles were subjected to hierarchal clustering analysis (HCA) and principal component analysis (PCA). The results of HCA on a heat map revealed that the large numbers of primary metabolism detected were more abundant in WH124 and less abundant in LU104 during the early fermentation stage as compared to the WT strain. PCA separated the three strains clearly and suggested nine metabolites that contributed predominantly to the separation. These biomarkers were associated with central carbon metabolism (succinic acid, α-ketoglutarate, acetyl-CoA, and ATP), amino acid metabolism (glutamate, glutamine, and valine), and secondary metabolism (pseudoaglycone), etc. These findings provide insight into the metabolomic characteristics of the two high-yield strains and for further regulation of spinosad production.
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  • Dongyeop KIM, Atiqur RAHMAN, Irnayuli R. SITEPU, Yasuyuki HASHIDOKO
    2013 Volume 77 Issue 8 Pages 1722-1727
    Published: August 23, 2013
    Released: August 23, 2013
    [Advance publication] Released: August 07, 2013
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    Supplementary material
    In modified Winogradsky's (MW) medium supplemented with excessive indole (1), Burkholderia unamae strain CK43B isolated from polyphenol-rich Shorea rhizosphere showed almost no cell growth, but it showed drastic cell growth given further supplementation of gallic acid, a simple plant polyphenol. This active cell growth of B. unamae CK43B was due to the stimulating effect of gallic acid on 1-degradation of bacterial cells, which acquired a nitrogen source in 1. Under aerobic culture conditions with appropriate concentrations (0.5–2.0 mM) of gallic acid, B. unamae CK43B started to decompose exogenous 1 in a dose-dependent manner, and finally accumulated catechol (5) via anthranilic acid (4). Pyrogallol also showed a cometabolic effect on decarboxylation-coupled oxidative deamination of B. unamae CK43B, producing 5 from 4, as gallic acid did. These results suggest that pyrogallol-type plant polyphenols act as stimulators on B. unamae CK43B, causing it to degrade an N-heterocyclic aromatic compound (NHAC) including nitrogen-containing humic substances.
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  • Hao FANG, Masanori TOYOFUKU, Tatsunori KIYOKAWA, Akihiro ICHIHASHI, Ka ...
    2013 Volume 77 Issue 8 Pages 1747-1752
    Published: August 23, 2013
    Released: August 23, 2013
    [Advance publication] Released: August 07, 2013
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    Bacteria participate in social behaviors by communicating with each other and forming surface-associated biofilms. In Pseudomonas aeruginosa, such social behaviors are affected greatly by the environment. Although P. aeruginosa survive under anaerobic conditions, previous studies indicate that quorum sensing is attenuated under such conditions, and that this leads to decreased activity of extracellular virulence factors as compared to aerobic conditions. Hence it has come into question whether P. aeruginosa are virulent under anaerobic conditions. Here, we compared various phenotypes between PAO1 and clinical isolates under anaerobic conditions. Our data revealed that when grown anaerobically, growth and cell morphology greatly differed among the strains. One of the clinical isolates produced comparable amounts of quorum-sensing signaling molecules and extracellular virulence factors under aerobic and anaerobic conditions, while the other strains showed low production under anaerobic conditions. Biofilm formation also exhibited strain-dependent variations, suggesting that there are several mechanisms that lead to biofilm formation under anaerobic conditions. Taken together, these results indicate that the impact of anaerobiosis on the social interactions of P. aeruginosa is strain dependent, and suggest that multiple regulatory mechanisms are involved in the regulation of quorum sensing and biofilm formation under anaerobic conditions.
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Environmental Science Regular Paper
  • Ginga SHIMAKAWA, Tatsuya IWAMOTO, Tomohito MABUCHI, Ryota SAITO, Hiros ...
    2013 Volume 77 Issue 8 Pages 1655-1660
    Published: August 23, 2013
    Released: August 23, 2013
    [Advance publication] Released: August 07, 2013
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    Supplementary material
    In this study, we sought to determine whether and how an α,β-unsaturated carbonyl, acrolein, can inhibit the growth of the cyanobacterium Synechocystis sp. PCC6803 (S. 6803). Treatment of S. 6803 with 200 µM acrolein for 3 d significantly and irreversibly inhibited its growth. To elucidate the inhibitory mechanism, we examined the effects of acrolein on photosynthesis. In contrast to dark conditions, the addition of acrolein to S. 6803 under conditions of illumination lowered the CO2-dependent O2 evolution rate (photosynthetic activity). Furthermore, treatment with acrolein lowered the activity reducing dimethyl benzoquinone in photosystem II (PSII). Acrolein also suppressed the reduction rate for the oxidized form of the reaction center chlorophyll of photosystem I (PSI), P700. These results indicate that acrolein inhibited PSII activity in thylakoid membranes. The addition of 200 µM acrolein to the illuminated S. 6803 cells gradually increased the steady-state level (Fs) of Chl fluorescence and decreased the quantum yield of PSII. These results suggested that acrolein damaged the acceptor side of PSII. On the other hand, acrolein did not inhibit respiration. From the above results, we gained insight into the metabolism of acrolein and its physiological effects in S. 6803.
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Environmental Science Note
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