Purification and characterization of membrane-bound hydrogenase from Hydrogenobacter thermophilus strain TK-6, an obligately autotrophic, thermophilic, hydrogen-oxidizing bacterium.
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A membrane-bound hydrogenase was purified to electrophoretic homogeneity from the cells of Hydrogenobacter thermophilus strain TK-6, an obligately autotrophic, thermophilic, hydrogen-oxidizing bacterium. Solubilization and purification were done aerobically in the presence of Triton X-100. Three chromatography steps were done for purification; Butyl-Sepharose, Mono-Q, and Superose 6, in this order. Purification was completed with 6.73% yield of total activity and with 21.4-fold increase of specific activity when compared with the values for the membrane fraction. The purified hydrogenase was shown to be a tetramer with alpha2beta2 structure, with a molecular mass of 60,000 Da for the large subunit and 38,000 Da for the small subunit. The purified hydrogenase directly reduced methionaquinone with an apparent Km of around 300 microM and with a turnover number around 2900 (min(-1)). Metal analysis and EPR properties of the hydrogenase have shown that the enzyme is one of the [NiFe]-hydrogenases. Also, optimum pH and temperature for reaction, thermal stability, and electron acceptor specificity were reported. Finally, a model is presented for energy and central metabolism of H. thermophilus strain TK-6. (+info)
Nuclear export of heat shock and non-heat-shock mRNA occurs via similar pathways.
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Several studies of the yeast Saccharomyces cerevisiae support differential regulation of heat shock mRNA (hs mRNA) and non-hs mRNA nuclear export during stress. These include the finding that hs mRNA export at 42 degrees C is inhibited in the absence of the nucleoporinlike protein Rip1p (also called Nup42p) (C. A. Saavedra, C. M. Hammell, C. V. Heath, and C. N. Cole, Genes Dev. 11:2845-2856, 1997; F. Stutz, J. Kantor, D. Zhang, T. McCarthy, M. Neville, and M. Rosbash, Genes Dev. 11:2857-2868, 1997). However, the results reported in this paper provide little evidence for selective non-hs mRNA retention or selective hs mRNA export under heat shock conditions. First, we do not detect a block to non-hs mRNA export at 42 degrees C in a wild-type strain. Second, hs mRNA export appears to be mediated by the Ran system and several other factors previously reported to be important for general mRNA export. Third, the export of non-hs mRNA as well as hs mRNA is inhibited in the absence of Rip1p at 42 degrees C. As a corollary, we find no evidence for cis-acting hs mRNA sequences that promote transport during heat shock. Taken together, our data suggest that a shift to 42 degrees C in the absence of Rip1p impacts a late stage of transport affecting most if not all mRNA. (+info)
Mapping of genes for cooking and eating qualities in Thai jasmine rice (KDML105).
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Thai jasmine rice, KDML 105, is known as the best quality rice. It is known not only for its aroma but also for its good cooking and eating qualities. Amylose content (AC), gel consistency (GC) and gelatinization temperature (GT) are important traits determining rice quality. A population of recombinant inbred lines (RIL) derived from KDML105 x CT9993 cross was used to study the genetic control of AC, GC and GT traits. A total of 191 markers were used in the linkage map construction. The 1605.3 cM linkage map covering nearly the whole rice genome was used for QTL (define QTL) analysis. Four QTLs for AC were detected on chromosomes 3, 4, 6 and 7. These QTLs accounted for 80% of phenotypic variation explained (PVE) in AC. The presence of one major gene as well as several modifiers was responsible for the expression of the trait. Two QTLs on chromosome 6 and one on chromosome 7 were detected for GC, which accounts for 57% of PVE. A single gene of major effect along with modifier genes controls GC from this cross. The QTLs in the vicinity of waxy locus were major contributors in the expression of AC and GC. The finding that the position of QTLs for AC and GC were near each other may reflect tight linkage or pleiotropy. Three QTLs were detected, one on chromosome 2 and two on chromosome 6, which accounted for 67% of PVE in GT. Just like AC and GC, one major gene and modifier genes governed the variation in GT resulting from the KDML105 x CT9993 cross. Breeding for cooking and eating qualities will largely rely on the preferences of the end users. (+info)
The reduced folate carrier in L1210 murine leukemia cells is a 58-kDa protein.
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The reduced folate carrier (RFC1) is a major transporter for both natural reduced folates and antifolate chemotherapeutics. Using polyclonal antibodies targeted to epitopes at the loop between the sixth and seventh predicted transmembrane domains or the distal C-terminus, we were able to demonstrate by Western blot analysis that the molecular size of RFC1 expressed in murine leukemia L1210 cells is 58 kDa as predicted by the open reading frame of its cDNA. 46- and 38-kDa proteins detected only in plasma membrane preparations were proteolytic degradation products that appeared during membrane preparation or treatment with the conventional SDS-PAGE loading buffer. These data resolve discrepancies reported previously for the molecular size of RFC1. (+info)
Some properties of a macromolecular conjugate of lysozyme prepared by modification with a monomethoxypolyethylene glycol derivative.
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Hen egg-white lysozyme was modified with a succinyl ester derivative of monomethoxypolyethylene glycol (mPEG-COONSu), and some properties of the resulting conjugate (mPEG-lysozyme) were studied. The conjugate was prepared by modification of lysozyme with mPEG-COONSu and purified with use of columns of CM-Toyopearl 650M and Sephadex G-75. Analytical data indicated that in the conjugate, 1.05 moles of mPEG with an average molecular weight of 5,000 were covalently attached to the lysozyme molecule. Tryptic peptide analysis of the conjugate showed that Lys 33 in lysozyme is the residue mainly modified with mPEG-COONSu. Covalent attachment of the mPEG-derivative to amino groups greatly increased the thermostability of lysozyme without any conformational change of the protein molecule. mPEG-lysozyme retained full enzyme activity for glycol chitin, but lytic activity for Micrococcus luteus cells in neutral media was 75% of that of native lysozyme and its optimal pH was at pH 5.0. It was also found that the reactivity of lysozyme with anti-lysozyme antibody from BALB/c mice or human lymphocytes was decreased by modification with mPEG-COONSu. From these findings, it was suggested that mPEG-COONSu can be advantageously used for protein tailoring of lysozyme. (+info)
Efficient selection for thermostable protease in Thermus thermophilus.
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An efficient procedure was established to select for thermostable proteases in an extreme thermophile, Thermus thermophilus. A non-protease-secreting mutant derived from T. thermophilus TH125 was used as host and the expression plasmid for aqualysin I from T. aquaticus YT-1 was constructed as a source of thermostable protease. T. thermophilus cells harboring the recombinant plasmid produced active aqualysin I into the medium and were able to grow on a minimal medium containing milk casein as the sole source of carbon and nitrogen. (+info)
Enhanced binding of antibodies to neutralization epitopes following thermal and chemical inactivation of human immunodeficiency virus type 1.
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Inactivation of viral particles is the basis for several vaccines currently in use. Initial attempts to use simian immunodeficiency virus to model a killed human immunodeficiency virus type 1 (HIV-1) vaccine were unsuccessful, and limited subsequent effort has been directed toward a systematic study of the requirements for a protective killed HIV-1 vaccine. Recent insights into HIV-1 virion and glycoprotein structure and neutralization epitopes led us to revisit whether inactivated HIV-1 particles could serve as the basis for an HIV-1 vaccine. Our results indicate that relatively simple processes involving thermal and chemical inactivation can inactivate HIV-1 by at least 7 logs. For some HIV-1 strains, significant amounts of envelope glycoproteins are retained in high-molecular-weight fractions. Importantly, we demonstrate retention of each of three conformation-dependent neutralization epitopes. Moreover, reactivity of monoclonal antibodies directed toward these epitopes is increased following treatment, suggesting greater exposure of the epitopes. In contrast, treatment of free envelope under the same conditions leads only to decreased antibody recognition. These inactivated virions can also be presented by human dendritic cells to direct a cell-mediated immune response in vitro. These data indicate that a systematic study of HIV-1 inactivation, gp120 retention, and epitope reactivity with conformation-specific neutralizing antibodies can provide important insights for the development of an effective killed HIV-1 vaccine. (+info)
Reduced levels of chloroplast FtsH protein in tobacco mosaic virus-infected tobacco leaves accelerate the hypersensitive reaction.
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In tobacco cultivars resistant to tobacco mosaic virus (TMV), infection results in the death of the infected cells accompanying the formation of necrotic lesions. To identify the genes involved in this hypersensitive reaction, we isolated the cDNA of tobacco DS9, the transcript of which decreases before the appearance of necrotic lesions. The DS9 gene encodes a chloroplastic homolog of bacterial FtsH protein, which serves to maintain quality control of some cytoplasmic and membrane proteins. A large quantity of DS9 protein was found in healthy leaves, whereas the quantity of DS9 protein in infected leaves decreased before the lesions appeared. In transgenic tobacco plants containing less and more DS9 protein than wild-type plants, the necrotic lesions induced by TMV were smaller and larger, respectively, than those on wild-type plants. These results suggest that a decrease in the level of DS9 protein in TMV-infected cells, resulting in a subsequent loss of function of the chloroplasts, accelerates the hypersensitive reaction. (+info)