776 research outputs found

    Mutational analysis of feedback inhibition and catalytic sites of prephenate dehydratase from Corynebacterium glutamicum

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    Prephenate dehydratase is a key regulatory enzyme in the phenylalanine-specific pathway of Corynebacterium glutamicum. PCR-based random mutagenesis and functional complementation were used to screen for m-fluorophenylalanine (mFP)-resistant mutants. Comparison of the amino acid sequence of the mutant prephenate dehydratases indicated that Ser-99 plays a role in the feedback regulation of the enzyme. When Ser-99 of the wild-type enzyme was replaced by Met, the specific activity of the mutant enzyme was 30% lower than that of the wild-type. The Ser99Met mutant was active in the presence of 50 muM phenylalanine, whereas the wild-type enzyme was not. The functional roles of the eight conserved residues of prephenate dehydratase were investigated by site-directed mutagenesis. Glu64Asp substitution reduced enzyme activity by 15%, with a 4.5- and 1.7-fold increase in K-m and k(cat) values, respectively. Replacement of Thr-183 by either Ala or Tyr resulted in a complete loss of enzyme activity. Substitution of Arg-184 with Leu resulted in a 50% decrease of enzyme activity. The specific activity for Phe185Tyr was more than 96% lower than that of the wild-type, and the K-m value was 26-fold higher. Alterations in the conserved Asp-76, Glu-89, His-115, and Arg-236 residues did not cause a significant change in the K-m and k(cat) values. These results indicated that Glu-64, Thr-183, Arg-184, and Phe-185 residues might be involved in substrate binding and/or catalytic activity

    Roles of insulin-like growth factor II in cardiomyoblast apoptosis and in hypertensive rat heart with abdominal aorta ligation

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    Although IGF-II activating the IGF-II receptor signaling pathway has been found to stimulate cardiomyocyte hypertrophy, the role of IGF-II in cardiac cell apoptosis remains unclear. This study aimed to identify the roles of IGF-II and/or IGF-II receptors (IGF-II/IIR) in cardiomyoblast apoptosis and in hypertensive rat hearts with abdominal aorta ligation. Cultured rat heart-derived H9c2 cardiomyoblasts and excised hearts from Sprague-Dawley rats with 0- to 20-day complete abdominal aorta ligation, a model of ANG II elevation and hypertension, were used. IGF-II/IIR expression, caspase activity, DNA fragmentation, and apoptotic cells were measured by RT-PCR, Western blot, agarose gel electrophoresis, and TUNEL assay following various combinations of ANG II, IGF-II/IIR antibody, CsA (calcineurin inhibitor), SP-600125 (JNK inhibitor), SB-203580 (p38 inhibitor), U-0126 (MEK inhibitor), or Staurosporine (PKC inhibitor) in H9c2 cells. ANG II-induced DNA fragmentation and TUNEL-positive cells were blocked by IGF-II/IIR antibodies and antisense IGF-II, but not by IGF-II sense. IGF-II-induced apoptosis was blocked by IGF-IIR antibody and CsA. The increased gene expressions of IGF-II and -IIR induced by ANG II were reversed by U-0126 and Sp600125, respectively. Caspase 8 activities induced by ANG II were attenuated by U-0126, SP-600125, and CsA. DNA fragmentation induced by ANG II was totally blocked by SP-600125, and CsA and was attenuated by U-0126. In rats with 0- to 20-day complete abdominal aorta ligation, the increases in IGF-II/IIR levels in the left ventricle were accompanied by hypertension as well as increases in caspase 9 activities and TUNEL-positive cardiac myocytes. ANG II-induced apoptosis was reversed by IGF-II/IIR blockade and coexisted with increased transactivation of IGF-II and -IIR, which are mediated by ERK and JNK pathways, respectively, both of which further contributed to cardiomyoblast apoptosis via calcineurin signaling. The increased cardiac IGF-II, IGF-IIR, caspase 9, and cellular apoptosis were also found in hypertensive rats with abdominal aorta ligation

    Role of calcineurin in Porphyromonas gingivalis-induced myocardial cell hypertrophy and apoptosis

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    Background and objective: Periodontal pathogen Porphyromonas gingivalis (P. gingivalis) increased cardiomyocyte hypertrophy and apoptosis whereas Actinobaeillus actinomycetemcomitans and Prevotella intermedia had no effects. The purpose of this study is to clarify the role of calcineurin signaling pathway in P. gingivalis-induced H9c2 myocardial cell hypertrophy and apoptosis. Methods: DNA fragmentation, nuclear condensation, cellular morphology, calcineurin protein, Bcl2- associated death promoter (Bad) and nuclear factor of activated T cell (NFAT)-3 protein products in cultured H9c2 myocardial cell were measured by agarose gel electrophoresis, DAPI, immunofluorescence, and Western blotting following P. gingivalis and/or pre-administration of CsA (calcineurin inhibitors cyclosporin A). Results: P. gingivalis not only increased calcineurin protein, NFAT-3 protein products and cellular hypertrophy, but also increased DNA fragmentation, nuclear condensation and Bad protein products in H9c2 cells. The increased cellular sizes, DNA fragmentation, nuclear condensation, and Bad of H9c2 cells treated with P. gingivalis were all significantly reduced after pre-administration of CsA. Conclusion: Our findings suggest that the activity of calcineurin signal pathway may be initiated by P. gingivalis and further lead to cell hypertrophy and death in culture H9c2 myocardial cells

    Competing Orders in Coupled Luttinger Liquids

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    We consider the problem of two coupled Luttinger liquids both at half filling and at low doping levels, to investigate the problem of competing orders in quasi-one-dimensional strongly correlated systems. We use bosonization and renormalization group equations to investigate the phase diagrams, to determine the allowed phases and to establish approximate boundaries among them. Because of the chiral translation and reflection symmetry in the charge mode away from half filling, orders of charge density wave (CDW) and spin-Peierls (SP) diagonal current (DC) and dd-density wave (DDW) form two doublets and thus can be at most quasi-long range ordered. At half-filling, umklapp terms break this symmetry down to a discrete group and thus Ising-type ordered phases appear as a result of spontaneous breaking of the residual symmetries. Quantum disordered Haldane phases are also found, with finite amplitudes of pairing orders and triplet counterparts of CDW, SP, DC and DDW. Relations with recent numerical results and implications to similar problems in two dimensions are discussed.Comment: 16 pages, 5 figures, 4 tables. Revised manuscript; a misprint in Eq. B3 has been corrected. The paper is already in print in PR

    The profile of cardiac cytochrome c oxidase (COX) expression in an accelerated cardiac-hypertrophy model

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    The contribution of the mitochondrial components, the main source of energy for the cardiac hypertrophic growth induced by pressure overload, is not well understood. In the present study, complete coarctation of abdominal aorta was used to induce the rapid development of cardiac hypertrophy in rats. One to two days after surgery, we observed significantly higher blood pressure and cardiac hypertrophy, which remained constantly high afterwards. We found an early increased level of cytochrome c oxidase ( COX) mRNA determined by in-situ hybridization and dot blotting assays in the hypertrophied hearts, and a drop to the baseline 20 days after surgery. Similarly, mitochondrial COX protein level and enzyme activity increased and, however, dropped even lower than baseline 20 days following surgery. In addition, in natural hypertension- induced hypertrophic hearts in genetically hypertensive rats, the COX protein was significantly lower than in normotensive rats. Taken together, the lower efficiency of mitochondrial activity in the enlarged hearts of long-term complete coarcted rats or genetically hypertensive rats could be, at least partially, the cause of hypertensive cardiac disease. Additionally, the rapid complete coarctation-induced cardiac hypertrophy was accompanied by a disproportionate COX activity increase, which was suggested to maintain the cardiac energy-producing capacity in overloaded hearts

    Time-Dependent Fatigue Crack Propagation Behavior of Two Solid-Solution-Strengthened Ni-Based Superalloys—INCONEL 617 and HAYNES 230

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    The fatigue crack propagation (FCP) as well as the sustained loading crack growth (SLCG) behavior of two solid-solution-strengthened Ni-based superalloys, INCONEL 617 (Special Metals Corporation Family of Companies) and HAYNES 230 (Haynes International, Inc., Kokomo, IN), were studied at increased temperatures in laboratory air under a constant stress-intensity- factor (K) condition. The crack propagation tests were conducted using a baseline cyclic triangular waveform with a frequency of 1 3 Hz. Various hold times were imposed at the maximum load of a fatigue cycle to study the hold time effect. The results show that a linear elastic fracture mechanics (LEFM) parameter, stress intensity factor (K), is sufficient to describe the FCP and SLCG behavior at the testing temperatures ranging from 873 K to 1073 K (600 C to 800 C). As observed in the precipitation-strengthened superalloys, both INCONEL 617 and HAYNES 230 exhibited the time-dependent FCP, steady SLCG behavior, and existence of a damage zone ahead of crack tip. A thermodynamic equation was adapted to correlate the SLCG rates to determine thermal activation energy. The fracture modes associated with crack propagation behavior were discussed, and the mechanism of time-dependent FCP as well as SLCG was identified. Compared with INCONEL 617, the lower crack propagation rates of HAYNES 230 under the time-dependent condition were ascribed to the different fracture mode and the presence of numerous W-rich M6C-type and Cr-rich M23C6-type carbides. Toward the end, a phenomenological model was employed to correlate the FCP rates at cycle/time-dependent FCP domain. All the results suggest that an environmental factor, the stress assisted grain boundary oxygen embrittlement (SAGBOE) mechanism, is mainly responsible for the accelerated time dependent FCP rates of INCONEL 617 and HAYNES 230

    Improving seed germination of the eggplant rootstock Solanum torvum by testing multiple factors using an orthogonal array design

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    [EN] Solanum torvum is a highly vigorous relative of eggplant that is resistant to a number of harmful soil-borne diseases and is compatible for grafting with eggplant. Being a potential rootstock, this plant frequently presents poor and erratic germination, which makes its practical use difficult. We used an L8 (2(7)) orthogonal array design to evaluate the primary effects of seven factors (soaking of seeds, scarification with sodium hypochlorite (NaClO), application of gibberellic acid (GA(3)), use of potassium mitrate (KNO3) as a moistening agent, cold stratification, application of a heatshock, and light irradiation during germination) at two levels (L0 and L1) using four germination parameters (early and final germination, germination rate and vigour index) in fresh S. torvum seeds. S. torvum seeds had a strong dormancy with no germination in the untreated seeds and high early and final germination (approximately 100%) in certain treatments. An evaluation of the main effects revealed highly positive effects on germination from seed soaking, and the use of GA(3), KNO3, and light irradiation, whereas NaClO scarification had a negative effect. The application of cold stratification and heat shock treatments also had a positive effect on seed germination but to a lesser extent than the other treatments. An improved proposed protocol that consisted of subjecting seeds to soaking, the application of GA(3) and KNO3, cold stratification, heat shock, and light irradiation was validated and demonstrated to be highly effective, with seed germination success greater than 60% being observed at 3 days and final germination reaching a plateau at 6 days. A second validation experiment using a commercial growing substrate also showed a high emergence (approximately 50%) at 7 days and a final germination of approximately 80% was recorded with application of the improved protocol. The seed germination protocol that we have developed will facilitate the use of S. torvum as a rootstock for eggplant and its use in breeding programmes. Our results also reveal that orthogonal array designs are a powerful tool for establishing improved protocols for seed germination. (C) 2015 Elsevier B.V. All rights reserved.This work was completed as part of the initiative "Adapting Agriculture to Climate Change: Collecting, Protecting and Preparing Crop Wild Relatives", which is supported by the Government of Norway. The project is managed by the Global Crop Diversity Trust with the Millennium Seed Bank of the Royal Botanic Gardens, Kew and is implemented in partnership with national and international gene banks and plant breeding institutes. For further information see the project website: http://www.cwrdiversity.org/. Isabel Andujar and Pietro Gramazio are grateful to Universitat Politecnica de Valencia for their post-doctoral (PAID-10-14) and pre-doctoral (Programa FPI de la UPV-Subprograma 1) contracts, respectively.Ranil, RH.; Niran, HML.; Plazas Ávila, MDLO.; Fonseka, R.; Hemal Fonseka, H.; Vilanova Navarro, S.; Andújar Pérez, I.... (2015). Improving seed germination of the eggplant rootstock Solanum torvum by testing multiple factors using an orthogonal array design. Scientia Horticulturae. 193:174-181. https://doi.org/10.1016/j.scienta.2015.07.030S17418119
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