9 research outputs found

    Mapping of the PKAN-linked Pank2 Y227C mutation onto the Pank3 structure und partial alignment of human Pank proteins.

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    <p>A) Pank3 dimer structure (PDB ID 2i7P) is shown in blue (chain A) and yellow (chain B), respectively. B) The magnification reveals a polar interaction (dashed line) in the WT Pank3 between Y27 (magenta, corresponding to Y227 in Pank2) and R49 (green, corresponding to R249 in Pank2). Side chains of F14, L25, L45 and I85 (cyan) are in close proximity to Y27 (less than 5 Ångström) constituting a hydrophobic environment around it. C) A partial multiple alignment of human Pank proteins, Pank2 (GI: 85838513), Pank1 (GI:23510400) and Pank3 (GI:119581908) is shown. Elements of secondary structure, helices (α) and β-strands (-) are indicated above and numbered accordingly. Known sites of PKAN missense mutations are boxed in yellow, the Pank2 Y227 site described here is boxed in magenta, the Pank2 R249 site which is in polar contact with Pank2 Y227 and is itself a known PKAN-linked Pank2 mutation is boxed in green. D) The magnification shows the effect of a Pank3 Y27C mutation (magenta) corresponding to the PKAN-linked Pank2 Y227C mutation disrupting the tyrosine-specific polar contact and local hydrophobic packing. PANK3 structure views and mutation Y27C were edited and modeled by PyMOL (<a href="http://www.pymol.org/" target="_blank">http://www.pymol.org/</a>). The contact map of Y27 was calculated using the Protein Interactions Calculator (PIC) server [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125861#pone.0125861.ref024" target="_blank">24</a>].</p

    Degree of acanthocytosis in patients and control donors.

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    <p>Acanthocyte counts of donors that are heterozygous (n = 36), homozygous (n = 23) and wild-type (22) with respect to the c.680 A>G mutation in the PANK2 gene were microscopically assessed as described in the Materials and Methods section. The samples were grouped in four classes of acanthocytosis (normal (acanthocyte count <6% of total cells), mild (6–10%), elevated (10–20%) and high (>20%)) and the number of samples of each class is shown as relative percent of the total number in each subset of donors.</p

    Distribution of acanthocytosis in the patient and control samples.

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    <p>The distribution of the amount of acanthocytes (in % of total cells) in the samples of donors that are heterozygous (n = 36), homozygous (n = 23) and wild-type (n = 22) with respect to the c.680 A>G mutation in the PANK2 gene is shown in a Box-Whiskers blot. The circles are <i>outliers</i>, and the asterisk is a <i>far outlier</i>.</p

    Impaired drug-induced endovesiculation in erythrocytes of patients with acanthocytosis.

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    <p>Erythrocytes from patients (ChAc, PKAN+, PKAN-) and control donors were subjected to drug-induced endovesiculation using 3 mM primaquine. The amount of FITC-dextran positive cells (in %) was assessed by flow cytometry as described. Respective pairs of patient and control donors are connected by lines. The data of the MPAN patient within the NBIA/PKAN- cohort is shown as a dashed line.</p

    Microscopic comparison of patient’s and control erythrocytes in drug-induced endovesiculation.

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    <p>Erythrocytes of a PKAN+ patient (B and D) and a control donor (A and C) were treated with 3 mM primaquine (A and B) or 0.8 mM chlorpromazine (C and D) in the presence of FITC-dextran to monitor the formation of endovesicles by confocal microscopy. Representative phase contrast (left panels), fluorescence (middle panels) and overlay (right panels) images are shown.</p

    Correlations between drug-induced endovesiculation, LPA-induced PS exposure and calcium uptake.

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    <p>The data of ChAc patients (circles) and respective control samples (triangles) for % of cells with endovesicles upon incubation with primaquine, for % annexin V-positive cells and % Fluo-3-positive cells upon LPA treatment are blotted against each other as indicated (data derived from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076715#pone-0076715-g004" target="_blank">Figures 4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076715#pone-0076715-g007" target="_blank">7</a>). Linear regression lines are shown with R2 (shown as inserts). In each combination a positive correlation is observed with Pearson’s r and the 2-tailed significance (given as inserts).</p

    Dose-dependent uptake of fluid phase FITC-dextran by erythrocytes treated with amphiphilic drugs.

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    <p>Erythrocytes were suspended in FITC-labeled dextran and incubated with the indicated amphiphilic drugs to induce endovesiculation. Upon washing, the uptake of fluorescent label was quantified by flow cytometry. Representative histograms are shown. The concentrations were 1.5, 0.4 and 0.375 mM (blue) and 3.0, 0.6 and 0.75 mM (green) for primaquine, chlorpromazine and imipramine, respectively (red is a control incubation without drug).</p

    Altered LPA-induced PS exposure and calcium uptake in erythrocytes of patients with acanthocytes.

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    <p>Erythrocytes from patients and control donors were treated with LPA as described in Materials and Methods and either PS exposure (A) or calcium uptake (B) was monitored by flow cytometry. The percentage of FITC-annexin V-positive cells (A) and of Fluo-3-positive cells (B) of patient and control samples are shown and respective pairs are connected by lines. The data of the MPAN patient within the NBIA/PKAN- cohort is shown as a dashed line.</p

    Differences in LPA-induced PS exposure and calcium uptake in erythrocytes of neuroacanthocytosis patients.

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    <p>Erythrocytes from ChAc, PKAN+ and PKAN- patients and control donors were treated with LPA as described in Materials and Methods and stained for PS exposure with FITC-annexin V (upper panels) or calcium uptake with Fluo-3 (lower panels) and analysed by flow cytometry. Overlays of representative histograms of patients (red) and controls (blue) show reduced PS exposure and calcium uptake in both ChAc and PKAN+ samples. The PKAN- sample does not differ from the respective control.</p
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