Bioscience, Biotechnology, and Biochemistry
Online ISSN : 1347-6947
Print ISSN : 0916-8451
Volume 72 , Issue 6
Showing 1-38 articles out of 38 articles from the selected issue
Analytical Chemistry Regular Paper
  • KiBeom LEE
    2008 Volume 72 Issue 6 Pages 1464-1474
    Published: June 23, 2008
    Released: June 23, 2008
    [Advance publication] Released: June 07, 2008
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    One method of improving the protein profiling of complex mammalian proteomes is the use of prefractionation followed by application of narrow pH range two dimensional (2-D) gels. The success of this strategy relies on sample solubilization; poor solubilization has been associated with missing protein fractions and diffuse, streaked, and/or trailing protein spots. In this study, I sought to optimize the solubilization of prefractionated human cancer cell samples using isoelectric focusing (IEF) rehydration buffers containing a variety of commercially available reducing agents, detergents, chaotropes, and carrier ampholytes. The solubilized proteins were resolved on 2-D gels and compared. Among five tested IEF rehydration buffers, those containing 3-[(3-cholamidopropyl)dimethylamino]-1-propane sulfonate (CHAPS) and dithiothreitol (DTT) provided superior resolution, while that containing Nonidet P-40 (NP-40) did not significantly affect protein resolution, and the tributyl phosphine (TBP)-containing buffer yielded consistently poor results. In addition, I found that buffers containing typically high urea and ampholyte levels generated sharper 2-D gels. Using these optimized conditions, I was able to apply 2-D gel analysis successfully to fractionated proteins from human breast cancer tissue MCF-7, across a pH range of 4–6.7.
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Organic Chemistry Notes
Biochemistry & Molecular Biology Regular Papers
  • Harumi OKADA, Toshihiro MAEDA, Makoto HATTORI, Tadashi YOSHIDA, Keiji ...
    2008 Volume 72 Issue 6 Pages 1438-1447
    Published: June 23, 2008
    Released: June 23, 2008
    [Advance publication] Released: June 07, 2008
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    Pepsin-solubilized collagen (PSC) was conjugated with carboxymethyl dextran (CMD) using cyanogen bromide to obtain a PSC-CMD film having improved physical properties, physiological properties, and cell affinity. The conjugation was confirmed by the loss of the α- and β-subunit chains and the polymerized band on SDS–PAGE, and by a decrease in the isoelectric point to 3.2. PSC-CMD had a large polymerized structure with the 6 PSC and 228 CMD molecules. PSC-CMD was readily soluble in water, reconstructed a matrix with a less-ordered structure and a characteristic morphological shape, and lost platelet aggregation-inducing ability. The PSC-CMD film, cross-linked by ultraviolet irradiation, exhibited reduced solubility, moderate water vapor permeability, and increased flexibility. PSC-CMD coatings exhibited good cell attachment and growth for fibroblasts and vein endothrical cells.
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  • Kittapong TANG, Rutchadaporn Sriprang KOBAYASHI, Verawat CHAMPREDA, Li ...
    2008 Volume 72 Issue 6 Pages 1448-1456
    Published: June 23, 2008
    Released: June 23, 2008
    [Advance publication] Released: June 07, 2008
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    A gene encoding a thermostable pullulan-hydrolyzing enzyme was isolated from environmental genomic DNA extracted from soil sediments of Bor Khleung hot spring in Thailand. Sequence comparison with related enzymes suggested that the isolated enzyme, designated Env Npu193A, was most likely a neopullulanase-like enzyme. Env Npu193A was expressed in Pichia pastoris as a monomeric recombinant protein. The purified Env Npu193A exhibited pH stability ranging from 3 to 9. More than 60% of enzyme activity was retained after incubation at 60 °C for 1 h. Env Npu193A was found to hydrolyze various substrates, including pullulan, starch, and γ-cyclodextrin. The optimal working condition for Env Npu193A was at pH 7 at 75 °C with Km and Vmax toward pullulan of 1.22±0.3% and 23.24±1.7 U/mg respectively. Env Npu193A exhibited distinct biochemical characteristics as compared with the previously isolated enzyme from the same source. Thus, a culture-independent approach with sequence-basing was found to be an effective way to discover novel enzymes displaying unique substrate specificity and high thermostability from natural bioresources.
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  • Zhi-Hua CHEN, Yoshiro SAITO, Yasukazu YOSHIDA, Etsuo NIKI
    2008 Volume 72 Issue 6 Pages 1491-1497
    Published: June 23, 2008
    Released: June 23, 2008
    [Advance publication] Released: June 07, 2008
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    Free radicals induce oxidative stress in vivo, leading to various disorders and diseases. In the present study, the effect of oxygen pressure on the cytotoxicity induced by free radicals was studied. It was found that alkyl radicals markedly aggravated Jurkat cell apoptosis under low oxygen pressure and this was ascribed to a hypoxic condition caused by the consumption of oxygen by alkyl radicals giving peroxyl radicals and subsequent lipid peroxidation by a chain mechanism. The intracellular lipid hydroperoxides significantly increased at an early time point even under hypoxia. Cytochrome c was released from the mitochondria, and caspase-9 as well as caspase-3 was activated during apoptosis, indicating that cell death followed by the intrinsic, mitochondrial apoptosis pathway. Pretreatment with VAD-FMK, a caspase inhibitor, attenuated the apoptosis induced by alkyl radicals under hypoxia. Moreover, pretreatment with various antioxidants also significantly rescued the cells from apoptosis. Taken together, the results indicate that free radicals induced hypoxic conditions, which accelerated mitochondria-dependent cell apoptosis.
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  • Daisuke TAKAHASHI, Shuzo MATSUMOTO, Etsuko NISHIMOTO, Takuhiro OTOSU, ...
    2008 Volume 72 Issue 6 Pages 1498-1505
    Published: June 23, 2008
    Released: June 23, 2008
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    The conformation and dynamics of a protein are essential in characterizing the protein folding/unfolding intermediate state. They are closely involved in the packing and site-specific interactions of peptide elements to build and stabilize the tertiary structure of the protein. In this study, it was confirmed that trypsin inhibitor obtained from seeds of bitter gourd (BGTI) adopted a peculiar but plausible conformation and dynamics in the unfolding intermediate state. The fluorescence spectrum of one of two tryptophan residues of BGTI, Trp9, shifted to the blue side in the presence of 2–3 M guanidine hydrochloride, although the other, Trp54, did not show this spectral shift. At the same time, the motional freedom of Trp9 revealed by a time-resolved fluorescence study decreased, suggesting that the segmental motion of this residue was more restricted. These results indicate that BGTI takes such a conformation state that the hydrophobic core and loop domains arranging Trp9 and Trp54 respectively are heterogeneously packed in the unfolding intermediate state.
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  • Techawaree UEATRONGCHIT, Ai KAYO, Hidenobu KOMEDA, Yasuhisa ASANO, Ara ...
    2008 Volume 72 Issue 6 Pages 1513-1522
    Published: June 23, 2008
    Released: June 23, 2008
    [Advance publication] Released: June 07, 2008
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    A hydroxynitrile lyase was isolated and purified to homogeneity from seeds of Eriobotrya japonica (loquat). The final yield, of 36% with 49-fold purification, was obtained by 30–80% (NH4)2SO4 fractionation and column chromatography on DEAE-Toyopearl and Concanavalin A Sepharose 4B, which suggested the presence of a carbohydrate side chain. The purified enzyme was a monomer with a molecular mass of 72 kDa as determined by gel filtration, and 62.3 kDa as determined by SDS-gel electrophoresis. The N-terminal sequence is reported. The enzyme was a flavoprotein containing FAD as a prosthetic group, and it exhibited a Km of 161 μM and a kcatKm of 348 s−1 mM−1 for mandelonitrile. The optimum pH and temperature were pH 5.5 and 40 °C respectively. The enzyme showed excellent stability with regard to pH and temperature. Metal ions were not required for its activity, while activity was significantly inhibited by CuSO4, HgCl2, AgNO3, FeCl3, β-mercaptoethanol, iodoacetic acid, phenylmethylsulfonylfluoride, and diethylpyrocarbonate. The specificity constant (kcatKm) of the enzyme was investigated for the first time using various aldehydes as substrates. The enzyme was active toward aromatic and aliphatic aldehydes, and showed a preference for smaller substrates over bulky one.
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  • Akihito HARADA, Hiroyuki AZAKAMI, Akio KATO
    2008 Volume 72 Issue 6 Pages 1523-1530
    Published: June 23, 2008
    Released: June 23, 2008
    [Advance publication] Released: June 07, 2008
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    Stable and unstable mutant lysozymes in long helices B and C were constructed to evaluate the effect of the helices on amyloid fibril formation at pH 2. Stable mutant N27D and unstable mutant K33D in the B-helix did not change in amyloid fibril formation. In contrast, stable mutant N93D and unstable mutant K97D in the C-helix showed big differences in behavior as to amyloid fibril formation. Stable mutant N93D showed a longer lag phase of aggregation and suppressed the amyloid fibril formation, whereas unstable mutant K97D showed a shorter lag phase of aggregation and accelerated amyloid fibril formation. These results suggest that the long C-helix is involved mainly in the α-helix to β-sheet transition during amyloid formation of lysozyme.
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  • Koei OKAZAKI, Osami NIWA
    2008 Volume 72 Issue 6 Pages 1531-1538
    Published: June 23, 2008
    Released: June 23, 2008
    [Advance publication] Released: June 07, 2008
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    Supplementary material
    Dikaryons, cells with two haploid nuclei contributed by the members of a mating pair, are part of the life cycle of many filamentous fungi, but the molecular mechanisms underlying the division of dikaryons are largely unknown. We found that the fission yeast Schizosaccharomyces pombe has a latent ability to divide as a dikaryon. Cells capable of restarting the mitotic cycle with two nuclei were prepared by transient inactivation of the septation initiation network. Close pairing of the two nuclei before mitosis was dependent on minus-end-directed kinesin Klp2p and was essential for propagation as a dikaryon. The two spindles extended in opposite directions, keeping their old spindle pole bodies at the prospective site of cell division until the mid-anaphase. The spindles then overlapped, exchanging the inner nuclei. Finally, twin mitosis was followed by a single cytokinesis, producing two daughter dikaryons carrying copies of the original pair of nuclei.
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  • Takeshi KUMAGAI, Shogo ITO, Norihito NAKAMICHI, Yusuke NIWA, Masaya MU ...
    2008 Volume 72 Issue 6 Pages 1539-1549
    Published: June 23, 2008
    Released: June 23, 2008
    [Advance publication] Released: June 07, 2008
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    Over 1,600 genes encoding putative transcription factors have been identified in the Arabidopsis genome sequence, however, their physiological functions are not yet fully understood. In this study, a small subfamily of double B-box zinc finger (DBB, DOUBLE B-BOX) genes, encoding eight putative transcription factors, were characterized with reference to the circadian rhythm and the early photomorphogenic regulation of hypocotyl elongation in response to light signals. Among these, it was found that the transcriptions of five DBB genes were under the control of circadian rhythm. To gain insight into the physiological roles of these putative transcription factors, forward and reverse genetic studies were carried out. The results suggested that they are commonly implicated in light signal transduction during early photomorphogenesis, however, their functions are not totally redundant, as judged by the fact that their circadian-expression profiles (or phases) were distinctive from each other, and by the fact that some DBBs (named DBB1a, DBB1b, STO, and STH) were apparently implicated in light signal transduction in a negative manner, whereas another (named DBB3) was implicated in a positive manner with regard to light-induced inhibition of elongation of hypocotyls. We also found that homologous B-box zinc finger genes are widely conserved in higher plants (e.g., Oryza sativa). Taking this altogether, it is probable that in addition to previously characterized bZIP-type transcription factors (e.g., HY5 and HYH) and bHLH-type transcription factors (e.g., PIF4 and PIF5/PIL6), a set of B-box zinc finger transcription factors should also be taken into consideration for a better understanding of the complex molecular mechanisms underlying the early photomorphogenic development of Arabidopsis thaliana.
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  • Zhangzhi WANG, Mu YANG, Xinyan WANG, Hong ZHOU
    2008 Volume 72 Issue 6 Pages 1550-1557
    Published: June 23, 2008
    Released: June 23, 2008
    [Advance publication] Released: June 07, 2008
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    Pituitary adenylate cyclase-activating polypeptide-38 (PACAP38) is a potent secretagog for growth hormone and gonadotropin in fish species. To obtain recombinant grass carp PACAP38, its open reading frame was subcloned in pET32a(+) vector to express thioredoxin (Trx)-PACAP fusion protein in Escherichia coli BL21 (DE3). The resulting expression level of the thioredoxin-PACAP reached 36% of the total proteins, and more than 85% of fusion protein existed as soluble form. Using Ni2+-chelating affinity chromatography, 102 mg of Trx-PACAP38 with a purity of 97% was obtained from 342 mg of crude proteins from a 1-liter culture of Escherichia coli. The purified Trx-PACAP specifically inhibited T98G human glioblastoma cell proliferation, but the fusion partner had no effect in this regard. Moreover, this inhibition was totally abolished by PACAP-specific antibody.
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  • Keiko KAWAUCHI, Kei TOBIUME, Kimi IWASHITA, Hiroko INAGAKI, Tetsunori ...
    2008 Volume 72 Issue 6 Pages 1564-1570
    Published: June 23, 2008
    Released: June 23, 2008
    [Advance publication] Released: June 07, 2008
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    Cycloprodigiosin hydrochloride (cPrG-HCl), a member of the prodigiosin family of compounds, has been reported to act as an H+/Cl symporter. This compound induces apoptosis in several cancer cells and acts as an antitumor drug in animal models. In this study, we found a novel function of cPrG-HCl; to suppress cell death in PC12 cells, which is caused by protein synthesis inhibitors cycloheximide and actinomycin D. cPrG-HCl activated Akt and suppressed apoptosis, and this was accompanied by inhibition of caspase-3 activity and DNA fragmentation independently of its H+/Cl symporter activity. Wortmannin, a phosphatidylinositol 3-kinase (PI3K) inhibitor, and dominant-negative Ras attenuated the anti-apoptotic activity of cPrG-HCl, which indicates that cPrG-HCl activated the Ras–PI3K–Akt pathway suppressing apoptosis. On the other hand, serum-deprivation-induced apoptosis was not suppressed by cPrG-HCl.
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  • Chien-Ho CHEN, Der-Zen LIU, Hsu-Wei FANG, Hong-Jen LIANG, Tzu-Sheng YA ...
    2008 Volume 72 Issue 6 Pages 1586-1594
    Published: June 23, 2008
    Released: June 23, 2008
    [Advance publication] Released: June 07, 2008
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    We studied the effects of multi- and single-target liposomal drugs on human gastric cancer cell AGS both in vitro and in vivo. The cytotoxic effect of dihydrotanshinone I was significantly enhanced by treatment with octreotide-polyethylene glycol(PEG)-liposome, Arg-Gly-Asp(RGD)-PEG-liposome, and RGD/octreotide-PEG-liposome encapsulated with 0.5 μg/ml of dihydrotanshinone I to AGS cell for 24 h, compared to control. Furthermore, the AGS cell survival rate for multi-target versus single target liposomal drugs was significantly suppressed. Microsocpic examination revealed that significant cell death occurred in the multi- and single-target liposomal encapsulated drug groups. Significant suppression of tumor growth in AGS cell xenograft nude mice given octreotide-PEG-liposome, RGD/octreotide-PEG-liposome encapsulated drug, versus those given a free drug was noted after 13 d of experimentation with the multi-targeted liposome: up to 60.75% and 41.2% reduction of tumor volume as compared to dimethylsulfoxide (DMSO) control and the free drug groups respectively. The treated animals showed no gross signs of toxicity. The results have potential clinical application.
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Biochemistry & Molecular Biology Notes
Biochemistry & Molecular Biology Communications
Food & Nutrition Science Regular Papers
  • Yuji NONAKA, Hiroko ISHIBASHI, Masaaki NAKAI, Hiroshi SHIBATA, Yoshino ...
    2008 Volume 72 Issue 6 Pages 1399-1408
    Published: June 23, 2008
    Released: June 23, 2008
    [Advance publication] Released: June 07, 2008
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    We examined the alleviation of cyclophosphamide-induced immunodepression by the antlered form of Ganoderma lucidum (G. lucidum AF) and also evaluated the anti-tumor and anti-metastatic effects of G. lucidum AF in cyclophosphamide-treated mice. G. lucidum AF alleviated cyclophosphamide-induced decrease in body weight, natural killer (NK) activity, interferon (IFN)-γ production, and cytotoxic T lymphocyte (CTL) activity, and inhibited the abnormal increase and decrease in interleukine (IL)-4 level due to cyclophosphamide administration. Post-treatment with cyclophosphamide and G. lucidum AF significantly inhibited tumor growth in MM 46-bearing mice. When Lewis lung carcinoma cells were injected into mice after a cyclophosphamide administration, metastasis of these cells to the lung was increased, but G. lucidum AF suppressed it. The anti-tumor and anti-metastatic effects of the combination of G. lucidum AF and cyclophosphamide might influence the modulatory effects of G. lucidum AF on both cellular and humoral immunity. These findings suggest that G. lucidum AF would be beneficial in alleviating the reduction of immune response by chemotherapeutic anti-cancer drugs.
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  • Kenji OISHI, Tadashi SATO, Wakae YOKOI, Yasuto YOSHIDA, Masahiko ITO, ...
    2008 Volume 72 Issue 6 Pages 1409-1415
    Published: June 23, 2008
    Released: June 23, 2008
    [Advance publication] Released: June 07, 2008
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    Bisphenol A (BPA), a putative endocrine disruptor, may be taken up by humans via the diet and have adverse effects on human health. In this study, we evaluated whether the probiotics, Bifidobacterium breve strain Yakult (BbY) and Lactobacillus casei strain Shirota (LcS), could exert a protective effect against dietary exposure to BPA. A group of rats fed on a diet containing 5% BbY or 5% LcS showed three advantageous effects compared to the control group; (i) the area under the blood concentration-time curve of BPA after its oral administration was significantly decreased, (ii) the amount of BPA excreted in the feces was significantly greater (2.4 times), and (iii) the percentage of BPA bound to the sediment fraction of the feces was significantly higher. These results suggest that BbY and LcS reduced the intestinal absorption by facilitating the excretion of BPA, and that these probiotics may suppress the adverse effects of BPA on human health.
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  • Bei-Wei ZHU, Lu-Lu ZHAO, Li-Ming SUN, Dong-Mei LI, Yoshiyuki MURATA, L ...
    2008 Volume 72 Issue 6 Pages 1430-1437
    Published: June 23, 2008
    Released: June 23, 2008
    [Advance publication] Released: June 07, 2008
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    Cathepsin L-like enzyme was purified from the body wall of the sea cucumber Stichopus japonicus by an integral method involving ammonium sulfate precipitation and a series of column chromatographies on DEAE Sepharose CL-6B, Sephadex G-75, and TSK-GEL. The molecular mass of the purified enzyme was estimated to be 63 kDa by SDS–PAGE. The enzyme cleaved N-carbobenzoxy-phenylalanine-arginine
    7-amido-4-methylcoumarin with Km (69.92 μM) and kcat (12.80/S) hardly hydrolyzed N-carbobenzoxy-arginine-arginine 7-amido-4-methylcoumarin and L-arginine 7-amido-4-methylcoumarin. The optimum pH and temperature for the purified enzyme were found to be 5.0 and 50 °C. It showed thermal stability below 40 °C. The activity was inhibited by sulfhydryl reagents and activated by reducing agents. These results suggest that the purified enzyme was a cathepsin L-like enzyme and that it existed in the form of its enzyme-inhibitor complex or precursor.
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  • Younghoon KIM, Jin Young WHANG, Kwang Youn WHANG, Sejong OH, Sae Hun K ...
    2008 Volume 72 Issue 6 Pages 1483-1490
    Published: June 23, 2008
    Released: June 23, 2008
    [Advance publication] Released: June 07, 2008
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    This study characterizes the factors responsible for the cholesterol reduction by Lactobacillus acidophilus ATCC 43121. In addition, two-dimensional gel electrophoresis (2-DE) and protein profiling was also used to study the response of ATCC 43121 at the proteome level in the presence of cholesterol. The results show that the cell-free supernatant (CFS) produced by ATCC 43121 in the presence of bile salts could also reduce the cholesterol in the broth, whereas all previous reports have suggested a mechanism by live cells. The active fraction was partially purified by 60% ammonium sulfate precipitation, and subsequent Sephacryl S-100 column chromatography. The molecular weight of the component with cholesterol-reducing activity was estimated to be approximately 12 kDa by SDS–PAGE. These results suggest that the novel protein isolated from CFS may be an important factor in the mechanism for cholesterol reduction by ATCC 43121. In addition, the proteins expressed by ATCC 43121 in the presence of cholesterol micelles were detected by 2-DE, five protein spots with at least a 2.5-fold increase in amounts being identified. The responsible proteins may be involved in the stress-response, translation, and metabolic processes. These results may suggest a new possibility for the mechanism underlying cholesterol reduction by lactic acid bacteria (LAB), differing from the conclusions of previous reports.
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  • Ryosuke MATSUOKA, Mamoru KIMURA, Ayano MUTO, Yasunobu MASUDA, Masao SA ...
    2008 Volume 72 Issue 6 Pages 1506-1512
    Published: June 23, 2008
    Released: June 23, 2008
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    Eggs are a popular source of dietary cholesterol, but their consumption does not necessarily result in an increased serum cholesterol concentration. We investigated the cholesterol-lowering activity of egg white protein (EWP) and its potential mechanism in rats. The consumption of EWP resulted in a decreased concentration of cholesterol in the serum, liver and intestinal mucosa. The excretion of fecal neutral sterols and bile acids was greater by rats fed with EWP than by those fed with casein. The ratio of cholesterol and bile acids in the micellar phase to those in the solid phase was lower in the intestinal contents from rats fed with EWP than from those fed with casein.
    These results suggest that the cholesterol-lowering activity of EWP can be attributed to lowering the cholesterol absorption by intervening in the micellar formation in the intestines.
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  • Jun TAKEBAYASHI, Yasuyuki YAGI, Rie ISHII, Shigeki ABE, Kazuhiko YAMAD ...
    2008 Volume 72 Issue 6 Pages 1558-1563
    Published: June 23, 2008
    Released: June 23, 2008
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    The antioxidant activity of a provitamin C agent, 2-O-β-D-glucopyranosyl-L-ascorbic acid (AA-2βG), was compared to that of 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G) and ascorbic acid (AA) using four in vitro methods, 1,1-diphenyl-picrylhydrazyl (DPPH) radical-scavenging assay, 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS•+)-scavenging assay, oxygen radical absorbance capacity (ORAC) assay, and 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced erythrocyte hemolysis inhibition assay. AA-2βG slowly and continuously scavenged DPPH radicals and ABTS•+ in roughly the same reaction profiles as AA-2G, whereas AA quenched these radicals immediately. In the ORAC assay and the hemolysis inhibition assay, AA-2βG showed similar overall activities to AA-2G and to AA, although the reactivity of AA-2βG against the peroxyl radical generated in both assays was lower than that of AA-2G and AA. These data indicate that AA-2βG had roughly the same radical-scavenging properties as AA-2G, and a comprehensive in vitro antioxidant activity of AA-2βG appeared to be comparable not only to that of AA-2G but also to that of AA.
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  • Yoshiaki MIYAKE, Mika MOCHIZUKI, Chihiro ITO, Masataka ITOIGAWA, Toshi ...
    2008 Volume 72 Issue 6 Pages 1580-1585
    Published: June 23, 2008
    Released: June 23, 2008
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    Antioxidants having a 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging capacity in rice mold starters, which are used for the preparation of various Japanese fermented foods, and their effectiveness against the expression of blood adhesion molecules were examined. An antioxidant was isolated from the rice mold starters used for shochu and identified as pyranonigrin-S (PG-S) by 1H-NMR, 13C-NMR, and FAB-MS analyses. It was a derivative of pyranonigrin-A (PG-A), which has been isolated as an antioxidant from the rice mold starters. Pyranonigrins PG-A and PG-S were found to exist in spores on rice mold starters which had been prepared by Aspergillus awamori, A. kawachii, and A. saitoi. PG-S exhibited a higher level of DPPH radical scavenging activity than PG-A. PG-A was found to have a significant suppressive effect on the expression of vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVECs) induced by tumor necrosis factor-α (TNF-α) (P<0.05).
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  • Saki TAKEDA, Noriko BANDO, Rintaro YAMANISHI
    2008 Volume 72 Issue 6 Pages 1595-1600
    Published: June 23, 2008
    Released: June 23, 2008
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    To elucidate health benefits of β-carotene, especially on immunity, we measured redox-related indices in spleen cells from BALB/c mice supplemented with various amounts of β-carotene. In mice supplemented with β-carotene in their diet, glutathione, an intracellular anti-oxidation agent, increased in their splenocytes. This change was highly correlated with the accumulation of β-carotene, but not with that of retinol. The increase in glutathione was accompanied by an increase in mRNA for γ-glutamylcysteine synthetase, a rate-limiting enzyme for glutathione synthesis. The higher the glutathione content was in the spleen cells, the higher the activity of cysteine cathepsin became in crude antigen-presenting cells contained in the spleen. These data suggest that accumulated β-carotene in splenocytes, without being metabolized, caused an increase in the intracellular glutathione level, thereby anti-oxidatively supporting the activity of redox-sensitive lysosomal protease, which is involved in antigen-presentation.
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Food & Nutrition Science Notes
Microbiology & Fermentation Technology Regular Papers
  • Muhammad Saiful ISLAM, Hiroko KAWASAKI, Yuki MURAMATSU, Yasuyoshi NAKA ...
    2008 Volume 72 Issue 6 Pages 1416-1429
    Published: June 23, 2008
    Released: June 23, 2008
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    Supplementary material
    A polyphasic study was performed to determine the taxonomic position of strain EK05T isolated from a root-outgrowth of Entada koshunensis, a legume available in Okinawa, Japan. Phylogenetic analysis of the 16S rRNA gene showed that the strain belongs to the genus Bradyrhizobium. Subsequent multilocus sequence analysis with ITS, glnII, recA, gyrB, and atpD sequences revealed that the isolate represents a distinct evolutionary lineage within the genus Bradyrhizobium. DNA-DNA hybridization indicated that strain EK05T shares <61% DNA relatedness with the type strains of all six recognized species of Bradyrhizobium, confirming that this strain is a novel species within the genus. Phylogenetic trees based on symbiotic loci, nifH and nodC, also placed strain EK05T clearly in a novel branch. On the basis of its phylogenetic distinctiveness, we propose Bradyrhizobium iriomotense sp. nov. for strain EK05T. The type strain is EK05T (= NBRC 102520T = LMG 24129T).
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  • Hisashi SEMBA, Yukio DOBASHI, Toru MATSUI
    2008 Volume 72 Issue 6 Pages 1457-1463
    Published: June 23, 2008
    Released: June 23, 2008
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    Hydroxynitrile lyase from cassava, Manihot esculenta (MeHNL), catalyzes the formation of (S)-cyanohydrins from HCN and aldehydes or ketones. (S)-Mandelonitrile was produced on a bench scale with immobilized MeHNL, after optimizing the enzyme expression system using recombinant technology. MeHNL was cloned from a cDNA library prepared from a leaf of Manihot esculenta, and then expressed in a multi-auxotrophic mutant of Saccharomyces cerevisiae cells. The maximum yield of active MeHNL was obtained by integrating transformation 4 times with a tandemly repeated expression cassette. Silica gel was the most suitable support for immobilization of the prepared enzyme from the recombinant yeast. Using this immobilized enzyme, 22 batches of (S)-mandelonitrile synthesis were performed in a 20 liters bioreactor (1 M benzaldehyde and 1.5 M HCN). During this operation, about 29 kg of (S)-mandelonitrile was produced from 23.3 kg of benzaldehyde, giving 98 mol % yield and a mean enantio excess of 98.9% ee.
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  • Osao ADACHI, Yoshitaka ANO, Hirohide TOYAMA, Kazunobu MATSUSHITA
    2008 Volume 72 Issue 6 Pages 1475-1482
    Published: June 23, 2008
    Released: June 23, 2008
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    In addition to the cytoplasmic soluble form of 3-dehydroquinate dehydratase (sDQD) (EC 4.1.2.10), a novel form of DQD occurring in the periplasmic space was found in Gluconobacter oxydans IFO 3244. The novel DQD, tentatively designated as pDQD, appeared to have a practical function involved in oxidative fermentation extracellularly coupling with membrane-bound quinoprotein quinate dehydrogenase (QDH) yielding 3-dehydroshikimate from quinate via 3-dehydroquinate. pDQD was not detached from the membrane by mechanical disruption or extraction with high salt, but was solubilized only with detergent. pDQD and sDQD were purified to homogeneity and compared as to their enzymatic properties. They showed the same apparent molecular weights and same catalytic properties, but they were distinct each other in subunit molecular mass, 16 kDa for pDQD and 47 kDa for sDQD.
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  • Yi-Xin DING, Xiang OU-YANG, Chang-Hua SHANG, Ang REN, Liang SHI, Yu-Xi ...
    2008 Volume 72 Issue 6 Pages 1571-1579
    Published: June 23, 2008
    Released: June 23, 2008
    [Advance publication] Released: June 07, 2008
    JOURNALS FREE ACCESS
    A farnesyl-diphosphate synthase gene, designated GlFPS, was isolated from a triterpene-producing basidiomycetous fungus, Ganoderma lucidum. The GlFPS cDNA was found to contain an open reading frame of 1,083 bp, encoding a protein of 360 amino acids with a calculated molecular mass of 41.27 kDa. The deduced amino acid sequence of the GlFPS cDNA exhibited a high homology with other fungal FPS genes, and contained four conserved domains. Phylogenetic analysis showed that GlFPS belonged to the basidiomycete FPS group. Competitive PCR revealed that GlFPS was constitutively expressed in the mycelium growth stage, whereas the transcripts of GlFPS accumulated to high levels rapidly during the process of mushroom primordia. Treatment of mycelia with exogenous methyl jasmonate also caused a large accumulation of GlFPS mRNA. Subsequently, promoter analysis indicated that the 5′ upstream region of GlFPS possessed various potential regulatory elements associated with physiological and environmental factors. Functional complementation of GlFPS in an ERG20-disrupted yeast strain indicated that the cloned cDNA encoded a farnesyl-diphosphate synthase.
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