80 research outputs found
THE DEVELOPMENT AND INVESTIGATION OF MEASURING CHAINS OF THE CAPACITANGE AND INDUCTANCE TRANSMITTERS
Offered have been the methods of conversion and improvement of accuracy of the capacitance and inductive transmitter measuring chains, pritected by the Authorized Certificates. The measuring chains of the capacitance and inductive transmitters, possessing the wide functional abilities and high metrological characteristics have been developed, they are protected by the Autorized Certificates. The measuring chains of the instruments and transmitters have been introduced. The introduction of the obtained results will allow to widen the functional abilities and metrological characteristics of measuring chains and the transmittersAvailable from VNTIC / VNTIC - Scientific & Technical Information Centre of RussiaSIGLERURussian Federatio
Measurement of <i>In Vivo</i> Protein Binding Affinities in a Signaling Network with Mass Spectrometry
Protein interaction networks play
a key role in signal processing.
Despite the progress in identifying the interactions, the quantification
of their strengths lags behind. Here we present an approach to quantify
the <i>in vivo</i> binding of proteins to their binding
partners in signaling-transcriptional networks, by the pairwise genetic
isolation of each interaction and by varying the concentration of
the interacting components over time. The absolute quantification
of the protein concentrations was performed with targeted mass spectrometry.
The strengths of the interactions, as defined by the apparent dissociation
constants, ranged from subnanomolar to micromolar values in the yeast
galactose signaling network. The weak homodimerization of the Gal4
activator amplifies the signal elicited by glucose. Furthermore, combining
the binding constants in a feedback loop correctly predicted cellular
memory, a characteristic network behavior. Thus, this genetic-proteomic
binding assay can be used to faithfully quantify how strongly proteins
interact with proteins, DNA and metabolites
Additional file 3: of Hapl-o-Mat: open-source software for HLA haplotype frequency estimation from ambiguous and heterogeneous data
Methods. (PDF 609 kb
Additional file 2: of Hapl-o-Mat: open-source software for HLA haplotype frequency estimation from ambiguous and heterogeneous data
Translation between Typing Resolutions. (PDF 303 kb
Geochronology, geochemistry, and tectonic setting of the Oligocene magmatic rocks (Marmaros Magmatic Assemblage) in Gökçeada Island, northwest Turkey
<p>Through the İzmir–Ankara–Erzincan and the Vardar oceans suture zones, convergence between the Eurasian and African plates played a key role in controlling Palaeogene magmatism in northwestern Anatolia, northern Aegean, and eastern Balkans. LA-ICP-MS dating of U and Pb isotopes on zircon separates from the tuffs of the Harmankaya Volcanic Rocks, which are inter-fingered with the lower-middle Eocene deposits of the Gaziköy Formation to the north of the Ganos Fault and the Karaağaç Formation in the Gelibolu Peninsula, yielded a late Ypresian (51 Ma) age. The chemical characteristics suggest that the lavas and tuffs of the Harmankaya Volcanic Rocks are products of syn- or post-collision magmas. These volcanic rocks show also close affinities to the subduction-related magmas. In addition to the already known andesitic volcanic rocks, our field observations in Gökçeada Island indicate also the existence of granitic and rhyolitic rocks (Marmaros Magmatic Assemblage). Our U–Pb zircon age data has shown that the newly discovered Marmaros granitic plutons intruded during late Oligocene (26 Ma) into the deposits of the Karaağaç Formation in Gökçeada Island. LA-ICP-MS dating of U and Pb isotopes on zircon separates from the Marmaros rhyolitic rocks yielded a late Oligocene (26 Ma) crystallization age. Geochemical characteristics indicate that the more-evolved Oligocene granitic and rhyolitic rock of the Marmaros Magmatic Assemblage possibly assimilated a greater amount of crustal material than the lower Eocene Harmankaya Volcanic Rocks. Geochemical features and age relationships suggest increasing amounts of crustal contamination and a decreasing subduction signature during the evolution of magmas in NW Turkey from the early Eocene to the Oligocene. The magmatic activity developed following the northward subduction of the İzmir–Ankara–Erzincan oceanic lithosphere and the earliest Palaeocene final continental collision between the Sakarya and Anatolide–Tauride zones.</p
A Proteomic View at the Biochemistry of Syntrophic Butyrate Oxidation in <em>Syntrophomonas wolfei</em>
<div><p>In syntrophic conversion of butyrate to methane and CO<sub>2</sub>, butyrate is oxidized to acetate by secondary fermenting bacteria such as <i>Syntrophomonas wolfei</i> in close cooperation with methanogenic partner organisms, e.g., <i>Methanospirillum hungatei</i>. This process involves an energetically unfavourable shift of electrons from the level of butyryl-CoA oxidation to the substantially lower redox potential of proton and/or CO<sub>2</sub> reduction, in order to transfer these electrons to the methanogenic partner <i>via</i> hydrogen and/or formate.</p> <p>In the present study, all prominent membrane-bound and soluble proteins expressed in <i>S. wolfei</i> specifically during syntrophic growth with butyrate, in comparison to pure-culture growth with crotonate, were examined by one- and two-dimensional gel electrophoresis, and identified by peptide fingerprinting-mass spectrometry. A membrane-bound, externally oriented, quinone-linked formate dehydrogenase complex was expressed at high level specifically during syntrophic butyrate oxidation, comprising a selenocystein-linked catalytic subunit with a membrane-translocation pathway signal (TAT), a membrane-bound iron-sulfur subunit, and a membrane-bound cytochrome. Soluble hydrogenases were expressed at high levels specifically during growth with crotonate. The results were confirmed by native protein gel electrophoresis, by formate dehydrogenase and hydrogenase-activity staining, and by analysis of formate dehydrogenase and hydrogenase activities in intact cells and cell extracts. Furthermore, constitutive expression of a membrane-bound, internally oriented iron-sulfur oxidoreductase (DUF224) was confirmed, together with expression of soluble electron-transfer flavoproteins (EtfAB) and two previously identified butyryl-CoA dehydrogenases.</p> <p>Our findings allow to depict an electron flow scheme for syntrophic butyrate oxidation in <i>S. wolfei</i>. Electrons derived from butyryl-CoA are transferred through a membrane-bound EtfAB:quinone oxidoreductase (DUF224) to a menaquinone cycle and further <i>via</i> a <i>b</i>-type cytochrome to an externally oriented formate dehydrogenase. Hence, an ATP hydrolysis-driven proton-motive force across the cytoplasmatic membrane would provide the energy input for the electron potential shift necessary for formate formation.</p> </div
Changes in the expression of Alix do not alter HPIV1 replication or release.
<p><b>A.</b> Over-expression of Alix did not affect HPIV1 replication. 293 T cells were transfected with plasmid expressing Alix and then infected with WT or P(C-) HPIV1 at a MOI of 5 (the same MOI that was used in previous experiments). 0.5 mL was aliquoted from the overlying tissue culture medium and replaced with 0.5 mL fresh medium at the indicated time points. The aliquots were assayed for infectious virus by limiting dilution. <b>B.</b> siRNA-mediated knock-down of Alix expression did not affect HPIV1 replication. 293 T cells were transfected twice with control non-targeting (siCON) or Alix-specific (siALIX) siRNA and infected with a low MOI (0.01) of WT or P(C-) HPIV1. HPIV1 replication was assayed as in part A. <b>C.</b> Analysis of viral proteins released into the overlying tissue culture medium or present intracellularly during Alix knock-down. On day 5 of the experiment described in part B (with the addition of 293 T cells transfected with siGAPDH as an additional control), total protein from the tissue culture supernatant and from the cell lysate was collected and analyzed by SDS-PAGE and Western blotting. This confirmed the presence of N protein in virions released into the overlying culture medium and in cell lysates for both viruses, the absence of intracellular C protein for the P(C-) virus, and the reduced accumulation of C protein from WT HPIV1 during Alix knock-down. <b>D.</b> Confirmation of siRNA-mediated knock-down of Alix expression. From the experiment in part C, the knock-down of Alix and GAPDH expression was confirmed by measuring intracellular mRNA by RT-qPCR.</p
Alix Serves as an Adaptor That Allows Human Parainfluenza Virus Type 1 to Interact with the Host Cell ESCRT System
<div><p>The cellular ESCRT (endosomal sorting complex required for transport) system functions in cargo-sorting, in the formation of intraluminal vesicles that comprise multivesicular bodies (MVB), and in cytokinesis, and this system can be hijacked by a number of enveloped viruses to promote budding. The respiratory pathogen human parainfluenza virus type I (HPIV1) encodes a nested set of accessory C proteins that play important roles in down-regulating viral transcription and replication, in suppressing the type I interferon (IFN) response, and in suppressing apoptosis. Deletion or mutation of the C proteins attenuates HPIV1 <i>in vivo</i>, and such mutants are being evaluated preclinically and clinically as vaccines. We show here that the C proteins interact and co-localize with the cellular protein Alix, which is a member of the class E vacuolar protein sorting (Vps) proteins that assemble at endosomal membranes into ESCRT complexes. The HPIV1 C proteins interact with the Bro1 domain of Alix at a site that is also required for the interaction between Alix and Chmp4b, a subunit of ESCRT-III. The C proteins are ubiquitinated and subjected to proteasome-mediated degradation, but the interaction with Alix<sub>Bro1</sub> protects the C proteins from degradation. Neither over-expression nor knock-down of Alix expression had an effect on HPIV1 replication, although this might be due to the large redundancy of Alix-like proteins. In contrast, knocking down the expression of Chmp4 led to an approximately 100-fold reduction in viral titer during infection with wild-type (WT) HPIV1. This level of reduction was similar to that observed for the viral mutant, P(C-) HPIV1, in which expression of the C proteins were knocked out. Chmp4 is capable of out-competing the HPIV1 C proteins for binding Alix. Together, this suggests a possible model in which Chmp4, through Alix, recruits the C proteins to a common site on intracellular membranes and facilitates budding.</p> </div
The C protein isoform is subject to proteasome-mediated degradation.
<p><b>A and B.</b> Analysis of the effect of various proteolytic inhibitors on C protein accumulation. A549 cells were infected with WT HPIV1 and then treated with the proteasome inhibitor MG132 (<b>A and B)</b>, the pan-caspase inhibitor zVAD-FMK (<b>A</b>) or the lysosomal protease inhibitor leupeptin (<b>B)</b>. Analysis of cell extracts by SDS-PAGE and Western blotting showed that only the proteasome inhibitor MG132 was associated with increased accumulation of the C isoform, whereas the C’ isoform was unaffected. <b>C and D.</b> Evidence for ubiquitination of the HPIV1 C protein. <b>C.</b> 293 T cells were transfected with plasmids expressing Myc-tagged C protein and HA-tagged ubiquitin (Ub). Two serial immunoprecipitations were performed, the first using anti-Myc antibodies to isolate the C protein, and the second using immobilized anti-HA antibodies to isolate ubiquitinated proteins. The proteins were separated by SDS-PAGE and analyzed by Western blotting to visualize the ubiquitinated proteins. Bands 1–8 were excised from a duplicate gel that had been run in parallel and stained with Coomassie blue, and the gel slices were analyzed using mass spectrometry. Bands 1–4 served as a negative control. <b>D.</b> Amino acid sequences of the C protein (top) and ubiquitin (bottom) showing peptide sequences identified by mass spectrometry (bold). In addition, the Lys-48 residue of ubiquitin (underlined), but not Lys-63, was found to have evidence of a Gly-Gly modification corresponding to the presence of a poly-ubiquitin linkage. A Gly-Gly modification (underlined) corresponding to a poly-ubiquitin linkage was found at Lys-48 and not at Lys-63. <b>E.</b> Confirmation that the C protein is ubiquitinated. 293 T cells were transfected with Myc-tagged C’<sup>WT</sup><sub>GTG</sub> and HA-tagged ubiquitin expression constructs and incubated in the presence or absence of the proteasome inhibitor lactacystin. The Myc-tagged C proteins were isolated by immunoprecipitation using anti-Myc antibodies, separated by SDS-PAGE, and then analyzed by Western blotting. <b>F.</b> Confirmation that the poly-ubiquitin linkage involves Lys-48. Plasmids expressing HA-tagged ubiquitin with every lysine mutated into an arginine except for Lys-63 (Ub<sub>K63</sub>) or Lys-48 (Ub<sub>K48</sub>) were transfected into 293 T cells together with plasmid expressing Myc-tagged C’<sup>WT</sup><sub>GTG</sub>. Cell lysates were subjected to immunoprecipitation with anti-Myc antibodies to isolate the C protein and analyzed by SDS-PAGE and Western blotting to detect the presence of poly-ubiquitin in the high molecular weight bands. <b>G.</b> The accumulation of C protein isoforms was increased by the over-expression of ubiquitin to a level comparable to that seen after treatment with a proteasome inhibitor. 293 T cells were transfected with HA-tagged ubiquitin and Myc-tagged C and subsequently treated with the proteasome inhibitor lactacystin. C protein expression was examined by Western blotting. <b>H.</b> The accumulation of C protein was also increased by the over-expression of Alix to a level comparable to that seen after treatment with a proteasome inhibitor. 293 T cells were transfected with HA-tagged Alix and Myc-tagged C and subsequently treated with the proteasome inhibitor lactacystin. C protein expression was examined by Western blotting as in part G. <b>I.</b> The F170S mutation-containing C proteins also undergo ubiquitination. 293 T cells were transfected with plasmid expressing HA-tagged ubiquitin with plasmids expressing Myc-tagged C’<sup>WT</sup><sub>GTG</sub> or C’<sup>F170S</sup><sub>GTG</sub>. As in panel <b>F</b>, cell lysates were subjected to immunoprecipitation with anti-Myc antibodies to isolate the C protein and analyzed by SDS-PAGE and Western blotting to detect the presence of poly-ubiquitin in the high molecular weight bands. <b>J.</b> Alix over-expression increases the abundance of F170S mutation-containing C proteins but to a lesser extent compared to the WT C proteins. 293 T cells were transfected with plasmid expressing Alix and then infected with WT of F170S HPIV1. C protein expression was then examined by Western blotting. The * represents a non-specific band.</p
Representative two-dimensional IEF/SDS-PAGE gels of soluble proteins of butyrate- or crotonate-grown <i>S. wolfei</i> cells.
<p>All major protein spots on the gel from butyrate-grown cells (A), and the proteins spots that appeared to be differentially expressed in crotonate-grown cells (B), were excised and identified by PF-MS (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056905#pone-0056905-t002" target="_blank">Table 2</a>).</p
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