20 research outputs found

    Ability of membTF mutants to support activation of FX and FIX, and clotting of plasma.

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    a<p>Data are mean ± standard deviation of normalized rates of FX and FIX activation, and clotting activities, using membTF mutants in 5% PS/95% PC vesicles. Eight mutants selected for study are boldfaced.</p>b<p>Not determined.</p>c<p>Kirchhofer et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088675#pone.0088675-Kirchhofer1" target="_blank">[3]</a>, using sTF with either SW-13 cell membranes or 500 µM 70% PS/30% PC liposomes.</p>d<p>Ruf et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088675#pone.0088675-Ruf1" target="_blank">[24]</a>, using detergent lysates of cells transfected to express membrane-anchored TF.</p>e<p>Rehemtulla et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088675#pone.0088675-Rehemtulla1" target="_blank">[32]</a>, using detergent lysates of cells transfected to express membrane-anchored TF.</p>f<p>Roy et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088675#pone.0088675-Roy1" target="_blank">[30]</a>, using detergent lysates of cells transfected to express membrane-anchored TF, diluted in a buffer with 100% PC vesicles.</p>g<p>Ruf et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088675#pone.0088675-Ruf2" target="_blank">[31]</a>, using detergent lysates of cells transfected to express membrane-anchored TF.</p

    Ability of membTF mutants to support FVIIa amidolytic activity and FX activation in solution (with 0.1% Triton X-100).

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    <p>Ability of membTF mutants to support FVIIa amidolytic activity and FX activation in solution (with 0.1% Triton X-100).</p

    Summary of kinetic constants for activation of FX.

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    <p>*Liposomes contained the indicated % PS (balance = PC).</p

    PS dependence of the effects of membTF mutations on FX and FIX activation.

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    <p>Initial rates were quantified for activation of FX (A,B) or FIX (C,D) by FVIIa bound to membTF mutants in liposomes of the indicated mol% PS (balance = PC). Data in panels A, C and D are normalized to those obtained with wild-type membTF at the same PS content. <i>Solid lines</i> were plotted if they fit the data with an r<sup>2</sup>≥0.95. <i>Dashed lines</i> connect data points if an attempt to fit a line yielded an r<sup>2</sup><0.95. For clarity, the <i>solid</i> and <i>dashed lines</i> for FIX activation are plotted in separate panels (C and D). In panel B, the absolute initial rates were plotted for FX activation by FVIIa bound to wild-type membTF (W.T.) and two mutants (S162A and S163A). Data in all panels are mean ± SD, <i>n = </i>3.</p

    Influence of PE on FX activation by membTF mutants.

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    <p>Initial rates were quantified for activation of FX by FVIIa bound to membTF mutants S162A (inverted triangles) and S163A (squares) in liposomes of the indicated mol% PS in the absence (filled symbols) and presence (open symbols) of 30 mol% PE (balance = PC). Data are normalized to wild-type membTF at the same phospholipid content. Data are mean ± SD, <i>n = </i>3.</p

    Procoagulant activities of membTF mutants.

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    <p>Clotting times of normal plasma, with no added FVIIa (A) or with 10 nM added FVIIa (B), were quantified in assays triggered by relipidated wild-type or mutant membTF. The membTF-liposomes contained 5% PS (<i>solid bars</i>), 20% PS (<i>hatched bars</i>) or 30% PS (<i>gray bars</i>) (balance = PC). Specific activities of mutants are plotted as percent of wild-type activity at the same phospholipid composition. Data are mean ± SD, <i>n = </i>2 or 3.</p

    TF mutants selected by yeast surface display for enhanced FVIIa binding.

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    <p>After random mutagenesis, sTF mutants were expressed on the surface of yeast cells and selected for increased FVIIa binding as described in Methods. 37 clones were sequenced and the frequencies of the resulting mutations are plotted by residue number. The actual numbers of observed amino acid substitutions by amino acid type are indicated on the graph for residues K165, K166 and M210.</p

    TF residues that putatively interact with PS.

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    <p>The crystal structure of sTF (Protein Data Bank file 1BOY <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088675#pone.0088675-Harlos1" target="_blank">[33]</a>) is arranged so that the area proposed to interact with membrane is facing the viewer. In (A) and (B), residues are colored by percentage of time they directly interacted with PS in simulations <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088675#pone.0088675-Ohkubo1" target="_blank">[10]</a>: <i>green</i>, <30% of the time; <i>blue</i>, 30–70% of the time; and <i>red</i>, >70% of the time. A) is from simulations of sTF alone on the membrane, and B) from simulations of sTF/FVIIa on the membrane. C) TF residues are colored according to their effects, when mutated to Ala in this study, on normalized FX activation rates using membTF-liposomes with 5% PS/95% PC: <i>red</i>, <30%; <i>blue</i>, 30–70%; and <i>green</i>, >70%.</p

    Assaying RNA Localization <i>in Situ</i> with Spatially Restricted Nucleobase Oxidation

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    We report herein a novel chemical-genetic method for assaying RNA localization within living cells. RNA localization is critical for normal physiology as well as the onset of cancer and neurodegenerative disorders. Despite its importance, there is a real lack of chemical methods to directly assay RNA localization with high resolution in living cells. Our novel approach relies on <i>in situ</i> nucleobase oxidation by singlet oxygen generated from spatially confined fluorophores. We demonstrate that our novel method can identify RNA molecules localized within specific cellular compartments. We anticipate that this platform will provide the community with a much-needed methodology for tracking RNA localization within living cells, and set the stage for systematic large scale analysis of RNA localization in living systems
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