Bioscience, Biotechnology, and Biochemistry
Online ISSN : 1347-6947
Print ISSN : 0916-8451
Volume 71 , Issue 11
Showing 1-35 articles out of 35 articles from the selected issue
Award Review
  • Yukiko YOSHIDA
    2007 Volume 71 Issue 11 Pages 2623-2631
    Published: November 23, 2007
    Released: November 23, 2007
    [Advance publication] Released: November 07, 2007
    JOURNALS FREE ACCESS
    F-box proteins are the substrate-recognition subunits of the SCF (Skp1-Cul1-F-box protein) complex, which is the largest known class of E3 ubiquitin ligases. They play important roles in ubiquitin-dependent proteolysis in eukaryotes. The human genome contains about 70 genes for F-box proteins, and at least five homologous F-box proteins containing a conserved motif in their C-termini are thought to recognize sugar chain of N-linked glycoproteins. Among theses, Fbs1 and Fbs2 are perhaps involved in the endoplasmic reticulum-associated degradation pathway. In this review, I focus on the in vivo function of Fbs1 and homologous proteins, novel intracellular oligosaccharide recognition molecules involved in the quality control system.
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  • Hiroshi SHIBA, Seiji TAKAYAMA
    2007 Volume 71 Issue 11 Pages 2632-2646
    Published: November 23, 2007
    Released: November 23, 2007
    [Advance publication] Released: November 07, 2007
    JOURNALS FREE ACCESS
    In many organisms, allelic diversity generates phenotypic variations and contributes to many events, such as development, adaptation to changing environment, and genome evolution. Allelic diversity is generally defined by the difference in nucleotide sequences that code for a gene. However, a heritable epigenetic modification, in which the modification is attributable to the degree of methylation of a gene and not to the change in its sequence, sometimes occurs and can affect the level of gene expression by reducing its transcriptional level. Some examples of epigenetic phenomena mediated by allele-specific DNA methylation in plants found to date include genomic imprinting, nucleolar dominance, and paramutation. Unlike the case in mammals, epigenetic modifications of plant genes are thought to be mitotically and meiotically stable, but recent studies of allele-specific demethylation at the FWA and MEDEA loci and recessive allele-specific methylation of Brassica self-incompatibility alleles indicate that DNA methylation patterns in plants can vary temporally and spatially in each generation. In this review, we describe various epigenetic phenomena regulated by allele-specific DNA methylation and their possible underlying mechanisms.
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Organic Chemistry Regular Papers
Organic Chemistry Notes
  • Jian-Bin LI, Fumio HASHIMOTO, Keiichi SHIMIZU, Yusuke SAKATA
    2007 Volume 71 Issue 11 Pages 2833-2836
    Published: November 23, 2007
    Released: November 23, 2007
    JOURNALS FREE ACCESS
    Ten anthocyanins, cyanidin 3-sambubioside, 3-glucoside and their acylated derivatives, cyanidin 3-lathyroside and cyanidin 3-galactoside, were isolated from red flowers of Camellia reticulata. Their structures were determined on the basis of spectroscopic analyses, and the chemotaxonomic distribution of the accumulated anthocyanins in the petals of wild Camellia reticulata and C. pitardii var. yunnanica is discussed.
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  • Keiji MORIMURA, Chihiro YAMAZAKI, Yasunao HATTORI, Hidefumi MAKABE, Ts ...
    2007 Volume 71 Issue 11 Pages 2837-2840
    Published: November 23, 2007
    Released: November 23, 2007
    [Advance publication] Released: November 07, 2007
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    A chromene-type compound, daedalin A (1), was isolated from mycelial culture broth of Daedalea dickinsii. Based on spectroscopic data, the structure of 1 was found to be (2R)-6-hydroxy-2-hydroxymethyl-2-methyl-2H-chromene. Daedalin A (1) strongly inhibited the activity of tyrosinase (IC50: 194 μM). In addition, 1 also showed 1,1-diphenyl-2-picrylhydrazyl scavenging activity (IC50: 16.9 μM) and superoxide anion scavenging activity (IC50: 28.5 μM).
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Organic Chemistry Communication
  • Masanobu YAMAMOTO, Tomoya KAMATA, Nguyen Duc DO, Yasushi ADACHI, Masak ...
    2007 Volume 71 Issue 11 Pages 2860-2863
    Published: November 23, 2007
    Released: November 23, 2007
    [Advance publication] Released: November 07, 2007
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    Female moths of Lyclene dharma dharma (Arctiidae, Lithosiinae) produced three pheromone components (IIII), which strongly stimulated male antennae. Using GC–MS analysis and chemical derivatizations, the following structures were estimated: 6-methyl-2-octadecanone (I), 14-methyl-2-octadecanone (II), and 6,14-dimethyl-2-octadecanone (III). While the stereochemistry of the chiral centers could not be determined because it was difficult to collect a sufficient amount of the natural pheromone, the plain structures of I and II were confirmed by synthesis of the racemic mixtures starting from diols. These methyl-branched ketones have not been identified as a natural product, indicating that they constitute a new chemical group of lepidopteran female sex pheromones.
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Biochemistry & Molecular Biology Regular Papers
  • Jiaqi CHEN, Gang BAI, Yu CAO, Zhihui GAO, Qi ZHANG, Yuanyuan ZHU, Wenb ...
    2007 Volume 71 Issue 11 Pages 2655-2662
    Published: November 23, 2007
    Released: November 23, 2007
    [Advance publication] Released: November 07, 2007
    JOURNALS FREE ACCESS
    Glucagon-like peptide-1 (GLP-1) has great therapeutic potential to treat diabetes type 2, mainly due to its unique glucose-dependent stimulation of insulin secretion profiles, but its clinical application is limited by its short half-life in vivo, which resultes from degradation by dipeptidyl peptidase IV and/or renal clearance. Developing long-acting GLP-1 analogs is therefore an important step toward using them therapeutically. In this study, the GLP-1/human serum albumin (HSA) fusion protein gene was cloned into the secretor type expression vector pPIC9K and subsequently expressed in Pichia pastoris. The expression quantity reached 58.5 mg/l in small-scale incubation. After optimization and characterization, the GLP-1/HSA fusion protein was successfully purified from the supernatant of the broth using immunomagnetic cellulose microspheres. HPLC showed that the purified GLP-1/HSA had an overall purity of 93.9%, and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) confirmed the fusion protein exhibited the expected molecular mass of 70 kDa. Furthermore, that analysis of in vivo activity indicated that GLP-1/HSA reduced the blood glucose level after intraperitoneal administration to Chinese Kunming mice in a dose-dependent manner, and the effects held significantly 4 h after administration. Overall, this study illustrates the development of a long-acting GLP-1/HSA fusion protein expressed in Pichia pastoris.
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  • Hidekatsu MAEDA, Shinya WADA, Masamichi IKEGUCHI, Norihiko MINOURA, Sh ...
    2007 Volume 71 Issue 11 Pages 2670-2679
    Published: November 23, 2007
    Released: November 23, 2007
    [Advance publication] Released: November 07, 2007
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    DNA is known to be aggregated by metal ions including Mn2+ ions, but analysis of the aggregation process from a chemical viewpoint, which means identification of the product yielded during the process, has not been performed yet. On examination of the kinds of degraded materials that were in the supernatant obtained on centrifugation of a DNA mixture aggregated under conditions of 10 mM Mn2+ ions ([Mn]/[P] = 46.3) at 70 °C for 1 h, the degradation products were found to be dAMP, dCMP, dGMP, and TMP. These dNMPs were purified by HPLC on TSKgel ODS-80Ts and identified by LC-TOF/MS. The degradation activity was lost on pretreatment of the DNA with a phenol–chloroform mixture, and the activity was recovered by pretreatment with a mixture of DMSO and a buffer containing surfactants. Mn2+, Co2+, Ni2+, Cu2+, Zn2+, and Cd2+, as transition element metal ions, were effective as to the degradation into dNMP. Mg2+, Ca2+, Sr2+, and Ba2+, as alkali earth element metal ions, were not effective as to the degradation. Monovalent anions such as Cl, CH3OO, and NO3 were found to increase the degradation rate. Sixty μg of the 120 μg of the starting DNA in 450 μl was degraded into dNMP on reaction for 1 h in the presence of 100 mM NaCl and 10 mM Mn2+ ions. In this process, aggregation did not occur, and thus was not considered to be necessary for degradation. The degradation was found not to occur at pH 7.0, and to be very sensitive to pH. The OH ion should have a critical role in cleavage of the phosphodiester linkages in this case. The dNMP obtained in the degradation process was found to be only 5′-NMP, based on the H1NMR spectra. This prosess should prove to be a new process for the production of 5′-dNMP in addtion to the exonuclease.
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  • Taisuke WATANABE, Ichiro MATSUO, Jun-ichi MARUYAMA, Katsuhiko KITAMOTO ...
    2007 Volume 71 Issue 11 Pages 2688-2696
    Published: November 23, 2007
    Released: November 23, 2007
    [Advance publication] Released: November 07, 2007
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    Recently, asparagine-linked oligosaccharides (N-glycans) have been found to play a pivotal role in glycoprotein quality control in the endoplasmic reticulum (ER). In order to screen proteins interacting with N-glycans, we developed affinity chromatography by conjugating synthetic N-glycans on sepharose beads. Using the affinity beads with the dodecasaccharide Glc1Man9GlcNAc2, one structure of the N-glycans, a 75-kDa protein, was isolated from the membranous fraction including the ER in Aspergillus oryzae. By LC-MS/MS analysis using the A. oryzae genome database, the protein was identified as one (AO090009000313) sharing similarities with calnexin. Further affinity chromatographic experiments suggested that the protein specifically bound to Glc1Man9GlcNAc2, similarly to mammalian calnexins. We designated the gene AoclxA and expressed it as a fusion gene with egfp, revealing the ER localization of the AoClxA protein. Our results suggest that our affinity chromatography with synthetic N-glycans might help in biological analysis of glycoprotein quality control in the ER.
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  • Noriko KAJITANI, Hirotsugu KOBUCHI, Hirofumi FUJITA, Hiromi YANO, Taku ...
    2007 Volume 71 Issue 11 Pages 2701-2711
    Published: November 23, 2007
    Released: November 23, 2007
    [Advance publication] Released: November 07, 2007
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    Calcium ions (Ca2+) are involved in a number of physiological cellular functions including apoptosis. An elevation in intracellular levels of Ca2+ in A23187-treated HL-60 cells was associated with the generation of both intracellular and extracellular reactive oxygen species (ROS) and induction of apoptotic cell death. A23187-induced apoptosis was prevented by cyclosporin A, a potent inhibitor of mitochondrial permeability transition (MPT). The generation of extracellular ROS was suppressed by the NADPH oxidase inhibitor diphenylene iodonium, and by superoxide dismutase, but these agents had no effect on A23187-induced apoptosis. In contrast, the blocking of intracellular ROS by a cell-permeant antioxidant diminished completely the induction of MPT and apoptosis. In isolated mitochondria, the addition of Ca2+ induced a typical MPT concomitant with the generation of ROS, which leads to augmentation of intracellular ROS levels. These results indicate that intracellular not extracellular ROS generated by A23187 is associated with the opening of MPT pores that leads to apoptotic cell death.
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  • Hideki SEZUTSU, Hideyuki KAJIWARA, Katsura KOJIMA, Kazuei MITA, Toshik ...
    2007 Volume 71 Issue 11 Pages 2725-2734
    Published: November 23, 2007
    Released: November 23, 2007
    [Advance publication] Released: November 07, 2007
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    Hornet silk, a fibrous protein in the cocoon produced by the larva of the vespa, is composed of four major proteins. In this study, we constructed silk-gland cDNA libraries from larvae of the hornet Vespa simillima xanthoptera Cameron and deduced the full amino acid sequences of the four hornet silk proteins, which were named Vssilk 1–4 in increasing order of molecular size. Portions of the amino acid sequences of the four proteins were confirmed by Matrix-assisted laser desorption/ionization-time of flight/mass spectrometry (MALDI-TOF/MS) and N-terminal protein sequencing. The primary sequences of the four Vssilk proteins (1–4) were highly divergent, but the four proteins had some common properties: (i) the amino acid compositions of all four proteins were similar to each other in that the well-defined and characteristic repetitive patterns present in most of the known silk proteins were absent; and (ii) the characteristics of the amino acid sequences of the four proteins were also similar in that Ser-rich structures such as sericin were localized at both ends of the chains and Ala-rich structures such as fibroin were found in the center. These characteristic primary structures might be responsible for the coexisting α-helix and β-sheet conformations that make up the unique secondary structure of hornet silk proteins in the native state. Because heptad repeat sequences of hydrophobic residue are present in the Ala-rich region, we believe that the Ala-rich region of hornet silk predominantly forms a coiled coil with an α-helix conformation.
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  • Satoshi WAKAI, Mizuho TSUJITA, Mei KIKUMOTO, Mohammed A. MANCHUR, Tada ...
    2007 Volume 71 Issue 11 Pages 2735-2742
    Published: November 23, 2007
    Released: November 23, 2007
    [Advance publication] Released: November 07, 2007
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    Sulfide:quinone oxidoreductase (SQR) was purified from membrane of acidophilic chemolithotrophic bacterium Acidithiobacillus ferrooxidans NASF-1 cells grown on sulfur medium. It was composed of a single polypeptide with an apparent molecular mass of 47 kDa. The apparent Km values for sulfide and ubiquinone were 42 and 14 μM respectively. The apparent optimum pH for the SQR activity was about 7.0. A gene encoding a putative SQR of A. ferrooxidans NASF-1 was cloned and sequenced. The gene was expressed in Escherichia coli as a thioredoxin-fusion protein in inclusion bodies in an inactive form. A polyclonal antibody prepared against the recombinant protein reacted immunologically with the purified SQR. Western blotting analysis using the antibody revealed an increased level of SQR synthesis in sulfur-grown A. ferrooxidans NASF-1 cells, implying the involvement of SQR in elemental sulfur oxidation in sulfur-grown A. ferrooxidans NASF-1 cells.
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  • Jun XIE, Xue-Zhi OUYANG, Kuai-Fei XIA, Yu-Feng HUANG, Wen-Bi PAN, Ying ...
    2007 Volume 71 Issue 11 Pages 2759-2765
    Published: November 23, 2007
    Released: November 23, 2007
    [Advance publication] Released: November 07, 2007
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    SaPIN2a, a plant proteinase inhibitor from nightshade (Solanum americanum), was located to the enucleate sieve elements (SEs) of phloem. The expressed SaPIN2a in transgenic lettuce showed inhibition of plant endogenous trypsin- and chymotrypsin-like activities, suggesting that SaPIN2a can regulate proteolysis in plant cells. To further investigate the physiological role of SaPIN2a, we produced transgenic nightshade and lettuce plants overexpressing SaPIN2a from the cauliflower mosaic virus (CaMV) 35S promoter using Agrobacterium-mediated transformation. Overexpression of SaPIN2a in transgenic plants was demonstrated by northern blot and western blot analysis. SaPIN2a-overexpressing transgenic nightshade plants showed significantly lower height than wild-type plants. Transmission electron microscopy analysis showed that chloroplast-like organelles with thylakoids, which are not present in enucleate SEs of wild-type plants, were present in the enucleate SEs of SaPIN2a-overexpressing transgenic plants. This finding is discussed in terms of the possible role played by SaPIN2a in the regulation of proteolysis in SEs.
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  • Masayuki ICHINOMIYA, Hirotaka UCHIDA, Yukako KOSHI, Akinori OHTA, Hiro ...
    2007 Volume 71 Issue 11 Pages 2787-2799
    Published: November 23, 2007
    Released: November 23, 2007
    [Advance publication] Released: November 07, 2007
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    A protein kinase C (PKC)-encoding gene (pkcA) was isolated from the filamentous fungus Aspergillus nidulans. Although we attempted to isolate pkcA deletion mutants, we obtained only heterokaryons that had both ΔpkcA and pkcA+ nuclei. Conidia produced by the heterokaryon germinated. The germ tubes, however, lysed frequently and no colony formation was observed, indicating that the pkcA gene is essential to the viability of A. nidulans. We constructed conditional mutants (alcA(p)-pkcA mutants) that expressed pkcA under the control of the alcA promoter (alcA(p)). Under alcA(p)-repressing conditions, their colonies were smaller than those of the wild-type strains and their hyphae lysed frequently. These phenotypes were not remedied under moderate- or high-osmolarity conditions; the growth defect deteriorated further under the latter. Under alcA(p)-inducing conditions, the alcA(p)-pkcA mutants also showed growth-sensitivity to cell wall destabilizing agents. These results indicate that pkcA plays an important role in the maintenance of cell integrity.
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  • Naoki YAMANAKA, Naoko HONDA, Noe OSATO, Ryusuke NIWA, Akira MIZOGUCHI, ...
    2007 Volume 71 Issue 11 Pages 2808-2814
    Published: November 23, 2007
    Released: November 23, 2007
    [Advance publication] Released: November 07, 2007
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    The regulatory mechanisms of ecdysteroidogenic P450 gene expression were investigated in the silkworm, Bombyx mori. Bommo-FMRFamide (BRFa), a neural suppressor of prothoracic gland (PG) activity, was found to suppress the expression of several P450 genes induced by prothoracicotropic hormone (PTTH) in the PG. A transcription inhibitor suppressed PTTH-induced expression of the P450 genes and the opposing effects of BRFa, while their short-term effects on ecdysteroidogenesis remained unchanged. This result suggests that the effects of these factors on the P450 gene transcripts become obvious on a longer time scale. Moreover, spontaneous expression of a P450 gene was observed in long-term PG culture, and was repressed by juvenile hormone. These results explain well the developmental fluctuation patterns of the P450 gene transcripts in the PG, indicating that multiple factors coordinate to regulate basal PG activity during insect development.
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Biochemistry & Molecular Biology Note
Biochemistry & Molecular Biology Communication
  • Yusuke KAZAMA, Hiroyuki SAITO, Makoto FUJIWARA, Tomoki MATSUYAMA, Yori ...
    2007 Volume 71 Issue 11 Pages 2864-2869
    Published: November 23, 2007
    Released: November 23, 2007
    [Advance publication] Released: November 07, 2007
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    We have developed an efficient system to detect and analyze DNA mutations induced by heavy-ion beams in Arabiopsis thaliana. In this system, a stable transgenic Arabidopsis line that constitutively expresses a yellow fluorescent protein (YFP) by a single-copy gene at a genomic locus was constructed and irradiated with heavy-ion beams. The YFP gene is a target of mutagenesis, and its loss of function or expression can easily be detected by the disappearance of YFP signals in planta under microscopy. With this system, a 12C6+-induced mutant with single deletion and multiple base changes was isolated.
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Food & Nutrition Science Regular Papers
  • Naoki TSURUOKA, Rumiko YAMATO, Yasuo SAKAI, Yoshino YOSHITAKE, Hideto ...
    2007 Volume 71 Issue 11 Pages 2680-2687
    Published: November 23, 2007
    Released: November 23, 2007
    [Advance publication] Released: November 07, 2007
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    Peptides produced by the enzymatic degradation of collagens are reported to have various activities of biological and medical interest. The mechanisms underlying their actions are, however, poorly understood. We have produced, by collagenase digestion of type I collagen, a highly purified, non-antigenic, and low allergenic tripeptide fraction (collagen tripeptide, Ctp). We report here the effects of Ctp on the in vivo bone fracture healing and in vitro calcification of osteoblastic cells. An oral administration of Ctp to rats with a femur fracture accelerated the fracture healing. Ctp apparently stimulated the calcification of human osteoblastic cells in culture. This osteotrophic effect was accompanied by a significant increase in type I collagen protein production and its mRNA levels. DNA microarray and quantitative RT-PCR analyses demonstrated that Ctp upregulated the bone-specific transcription factor, Osterix, suggesting that the induction of type I collagen gene expression by Ctp was mediated by upregulation of this factor.
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  • Yoo-Hyun LEE, Soon Won HONG, Woojin JUN, Hong Yon CHO, Han-Cheon KIM, ...
    2007 Volume 71 Issue 11 Pages 2712-2719
    Published: November 23, 2007
    Released: November 23, 2007
    [Advance publication] Released: November 07, 2007
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    Histone acetylation depends on the activity of two enzyme families, histone acetyltransferase (HAT) and deacetylase (HDAC). In this study, we screened various plant extracts to find potent HAT inhibitors. Hot water extracts of allspice inhibited HAT activity, especially p300 and CBP (40% at 100 μg/ml). The mRNA levels of two androgen receptor (AR) regulated genes, PSA and TSC22, decreased with allspice treatment (100 μg/ml). Importantly, in IP western analysis, AR acetylation was dramatically decreased by allspice treatment.
    Furthermore, chromatin immunoprecipitation indicated that the acetylation of histone H3 in the PSA and B2M promoter regions was also repressed. Finally, allspice treatment reduced the growth of human prostate cancer cells, LNCaP (50% growth inhibition at 200 μg/ml). Taken together, our data indicate that the potent HAT inhibitory activity of allspice reduced AR and histone acetylation and led to decreased transcription of AR target genes, resulting in inhibition of prostate cancer cell growth.
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  • Daiki MIURA, Yutaka MIURA, Kazumi YAGASAKI
    2007 Volume 71 Issue 11 Pages 2743-2750
    Published: November 23, 2007
    Released: November 23, 2007
    [Advance publication] Released: November 07, 2007
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    The effect of apple polyphenol extract (APE) on the proliferation and invasion of a rat ascites hepatoma cell line of AH109A was examined in vitro. APE suppressed both the hepatoma proliferation and invasion in a dose-dependent manner up to 200 μg/ml. Serum obtained from rats orally given APE also inhibited hepatoma proliferation and invasion when added to the culture medium. Subsequently, the effect of dietary APE on growth and the metastasis of AH109A hepatomas were investigated in vivo. APE reduced the growth and metastasis of solid hepatomas and significantly suppressed the serum lipid peroxide level in rats transplanted with AH109A. APE also suppressed the serum very-low-density lipoprotein + low-density lipoprotein (VLDL + LDL)-cholesterol level. These in vitro and in vivo findings suggest that APE has anti-hepatoma activities.
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  • Yoshifumi TANIGUCHI, Akiko MIZOTE, Keizo KOHNO, Kanso IWAKI, Kazuyuki ...
    2007 Volume 71 Issue 11 Pages 2766-2773
    Published: November 23, 2007
    Released: November 23, 2007
    [Advance publication] Released: November 07, 2007
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    In this study, we examined the effects of dietary lactosucrose (LS, a non-digestible oligosaccharide) on the IgE response in mice immunized with ovalbumin (OVA)/alum. In addition to IgG1 and IgG2a responses, the anti-OVA IgE response in mice fed LS diets was dose-dependently suppressed, as compared with the control mice, while the serum total IgG levels were comparable. Moreover, dietary LS feeding inhibited antigen-specific IgE and IgG1 productions even after a second immunization. Regarding with cytokine production, when stimulated in vitro with OVA, splenocytes obtained from LS-fed mice produced a similar level of IFN-γ, and lower levels of IL-4 and IL-5, as compared with the control mice. But IL-10 production by OVA-stimulated splenocytes was augmented in LS-fed mice, suggesting that IL-10 producing cells are responsible for the immunoregulatory effect of LS. Our findings indicate the further possibility that dietary LS supplementation can be used to prevent IgE-mediated allergic diseases.
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  • Kyoko TOMITA, Taizo NAGURA, Yasuhide OKUHARA, Haruyo NAKAJIMA-ADACHI, ...
    2007 Volume 71 Issue 11 Pages 2774-2780
    Published: November 23, 2007
    Released: November 23, 2007
    [Advance publication] Released: November 07, 2007
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    We examined how dietary melibiose affected the T-helper (Th) cell responses induced by an orally fed antigen in ovalbumin (OVA)-specific T cell receptor transgenic mice (OVA 23-3). Dietary melibiose markedly decreased the Th2 type responses as shown by a significant decrease in the interleukin (IL)-4 production and T cell proliferative response induced by sensitization from the 7-d oral administration of OVA. It was additionally observed that the Th1 type responses tended to decrease. We therefore examined the effect of melibiose feeding on the induction of immunological tolerance induced by the oral administration of an antigen (oral tolerance). The Th cell responses induced in BALB/c mice by subcutaneous immunization with OVA were suppressed by the prior oral administration of OVA. Such responses in the OVA-fed and immunized mice were further diminished by dietary melibiose. These results suggest that dietary melibiose strongly affected the Th cell responses to an ingested antigen, and further demonstrate the potential of melibiose to enhance the induction of oral tolerance.
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Food & Nutrition Science Note
Microbiology & Fermentation Technology Regular Papers
  • Miho KOBAYASHI, Masaru NOMURA, Hiromi KIMOTO
    2007 Volume 71 Issue 11 Pages 2647-2654
    Published: November 23, 2007
    Released: November 23, 2007
    [Advance publication] Released: November 07, 2007
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    This study was designed selectively to eliminate a θ-plasmid from Lactococcus lactis strains by transforming synthetic competitors. A shuttle vector for Escherichia coli and L. lactis, pDB1, was constructed by ligating a partial replicon of pDR1-1B, which is a 7.3 kb θ-plasmid in L. lactis DRC1, with an erythromycin resistance gene into pBluescript II KS+. This versatile vector was used to construct competitors to common lactococcal θ-plasmids. pDB1 contains the 5′ half of the replication origin and the 3′ region of repB of pDR1-1B, but lacks the 1.1-kb region normally found between these two segments. A set of primers, Pv3 and Pv4, was designed to amplify the 1.1-kb middle parts of the general θ-replicons of lactococcal plasmids. When the PCR products were cloned into the Nru I and Xho I sites of pDB1, synthetic replicons were constructed and replication activity was restored. A number of θ-plasmids in L. lactis ssp. lactis and cremoris were eliminated selectively by transforming the synthetic competitors. These competitors were easily eliminated by subculture for a short time in the absence of selection. The resulting variants contained no exogenous DNA and are suitable for food products, since part of the phenotype was altered without altering other plasmids indispensable for fermentation.
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  • Tsuyoshi SUGIO, Taher M. TAHA, Tadayoshi KANAO, Fumiaki TAKEUCHI
    2007 Volume 71 Issue 11 Pages 2663-2669
    Published: November 23, 2007
    Released: November 23, 2007
    [Advance publication] Released: November 07, 2007
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    When Acidithiobacillus ferrooxidans ATCC23270 cells, grown for many generations on sulfur were grown in sulfur medium with and without Fe3+, the bacterium markedly increased not only in iron oxidase activity but also in Fe2+-producing sulfide:ferric ion oxidoreductase (SFORase) activity during the early log phase, and retained part of these activities during the late log phase. The activity of SFORase, which catalyzes the production of Fe2+ from Fe3+ and sulfur, of sulfur-grown cells was approximately 10–20 fold higher than that of iron-grown cells. aa3 type cytochrome c oxidase, an important component of iron oxidase in A. ferrooxidans, was partially purified from sulfur-grown cells. A. ferrooxidans ATCC23270 cells grown for many generations on sulfur had the ability to grow on iron as rapidly as that did iron-grown cells. These results suggest that both iron oxidase and Fe2+-producing SFORase have a role in the energy generation of A. ferrooxidans ATCC23270 from sulfur.
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  • Shingo IZAWA, Takeomi KITA, Kayo IKEDA, Takeo MIKI, Yoshiharu INOUE
    2007 Volume 71 Issue 11 Pages 2800-2807
    Published: November 23, 2007
    Released: November 23, 2007
    [Advance publication] Released: November 07, 2007
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    Recent studies have revealed that cytoplasmic processing bodies (P-bodies) play important roles in the control of eukaryotic gene expression in response to stress. Since the formation of P-bodies is in dynamic competition with translation, the status of translation is reflected in the assembly and disassembly of P-bodies in eukaryotic cells. During the brewing of Japanese sake and the making of wine, yeast cells are exposed to stress caused by increases in the concentration of ethanol. Here we found that ethanol stress enhances the formation of P-bodies in yeast cells in SD medium. In the wine-making process, P-body formation was also enhanced as alcoholic fermentation proceeded, but the formation of P-bodies was not simply affected by the ethanol concentration in the sake mash. These findings suggest differences in the rate of translation and the cytoplasmic mRNA flux during the sake brewing and wine making processes.
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  • Takashi OHSHIRO, Ryo OHKITA, Takeshi TAKIKAWA, Masanori MANABE, Woo Ch ...
    2007 Volume 71 Issue 11 Pages 2815-2821
    Published: November 23, 2007
    Released: November 23, 2007
    [Advance publication] Released: November 07, 2007
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    In the microbial dibenzothiophene desulfurization pathway, 2′-hydroxybiphenyl-2-sulfinate is converted to 2-hydroxybiphenyl and sulfinate by desulfinase (DszB) at the last step, and this reaction is rate-limiting for the whole pathway. The catalytic activity and thermostability of DszB were enhanced by the two amino acid substitutions. Based on information on the 3-D structure of DszB and a comparison of amino acid sequences between DszB and reported thermophilic and thermostable homologs (TdsB and BdsB), two amino acid residues, Tyr63 and Gln65, were selected as targets to mutate and improve DszB. These two residues were replaced by several amino acids, and the promising mutant enzymes were purified and their properties were examined. Among the wild-type and mutant enzymes, Y63F had higher catalytic activity but similar thermostability, and Q65H showed higher thermostability but less catalytic activity and affinity for the substrate. To compensate for these drawbacks, the double mutant enzyme Y63F-Q65H was purified and its properties were investigated. This mutant enzyme showed higher thermostability without loss of catalytic activity or affinity for the substrate. These superior properties of the mutant enzyme have also been confirmed with resting cells harboring the mutant gene.
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Microbiology & Fermentation Technology Notes
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