Prevalence of H63D, S65C and C282Y hereditary hemochromatosis gene mutations in Slovenian population by an improved high-throughput genotyping assay-2

<p><b>Copyright information:</b></p><p>Taken from "Prevalence of H63D, S65C and C282Y hereditary hemochromatosis gene mutations in Slovenian population by an improved high-throughput genotyping assay"</p><p>http://www.biomedcentral.com/1471-2350/8/69</p><p>BMC Medical Genetics 2007;8():69-69.</p><p>Published online 23 Nov 2007</p><p>PMCID:PMC2253505.</p><p></p>es for all genotypes are presented. Curves in the plots correspond to the indicated fluorophores or water (see the legend); ROX, internal reference dye; mse, mean squared error; NTC, no-template control. (B) End-point fluorescence detection is shown by the scattered diagram. Clustering of genotypes is based on the relative fluorescence from each well on 96-well plate. The expected area for 193 mutant homozygote samples clustering is indicated by red circle

Prevalence of H63D, S65C and C282Y hereditary hemochromatosis gene mutations in Slovenian population by an improved high-throughput genotyping assay-1

<p><b>Copyright information:</b></p><p>Taken from "Prevalence of H63D, S65C and C282Y hereditary hemochromatosis gene mutations in Slovenian population by an improved high-throughput genotyping assay"</p><p>http://www.biomedcentral.com/1471-2350/8/69</p><p>BMC Medical Genetics 2007;8():69-69.</p><p>Published online 23 Nov 2007</p><p>PMCID:PMC2253505.</p><p></p>requency for Slovenia is presented in the last column (total). In the region 9 H63D allele frequency is significant higher comparing to other regions (p = 0.005). (B) Geographic regions of Slovenia

Temperature- and time-dependent degradation of PrP^Sc by the R30 fraction, in post-nuclear homogenates from CJD-infected brain.

<p>A post-nuclear homogenate from CJD-infected brain was incubated as indicated, without (lane 1) or with (lanes 2–6) the R30 fraction from the Superdex 200 preparative grade gel filtration column, under the following conditions: at room temperature for 60 min (lane 2; RT); or at 92°C for 10 min (lane 3), 20 min (lane 4), 40 min (lane 5), and 60 min (lanes 1, 6). The proteins were separated by SDS-PAGE and transferred to nitrocellulose membrane. The immunoreactive species were detected as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039548#s2" target="_blank">Materials and Methods</a>, with the 6H4 anti-PrP monoclonal antibody used as the primary antibody.</p

Detection of phosphotungstate-precipitable PrP.

<p>Post-nuclear homogenates from CJD-infected brain were incubated as indicated, without (lane 1) or with (lane 2) 0.75 µg proteinase K (PK), for 20 min at 37°C, or without (lane 3) or with (lane 4) the R30 fraction from the Superdex 200 preparative grade gel filtration column, for 20 min at 92°C. Following NaPTA precipitation as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039548#s2" target="_blank">Materials and Methods</a>, the proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. The immunoreactive species were detected as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039548#s2" target="_blank">Materials and Methods</a>, with the 6H4 anti-PrP monoclonal antibody.</p

Digestion of PrP^C and PrP^Sc of different species by the R30 fraction.

<p>Post-nuclear homogenates from human (A), bovine (B) and mouse (C) uninfected (lanes 1–4), TSE infected (lanes 5 to 8) and Alzheimer’s disease (lanes 9 to 12) brains were incubated as indicated, without (lanes 1, 5, 9) or with (lanes 2, 6, 10) 0.75 µg proteinase K (PK), for 20 min at 37°C, or without (lanes 3, 7, 11) or with (lanes 4, 8, 12) the R30 fraction from the Superdex 200 preparative grade gel filtration column, for 20 min at 92°C. The proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. The immunoreactive species were detected as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039548#s2" target="_blank">Materials and Methods</a>, with the 6H4 anti-PrP monoclonal antibody used as the primary antibody.</p

Residual protease activity of the purified pernisine in the absence and presence of CaCl₂ and protease inhibitors.

<p>Residual protease activity of the purified pernisine in the absence and presence of CaCl₂ and protease inhibitors.</p

Azocasein assays of the purified pernisine: effects of CaCl₂ and NaCl.

<p>Relative activities of the purified pernisine according to increasing CaCl₂ concentrations (A), and according to increasing NaCl concentrations in the presence (gray, dot-dash line) and absence (black line) of 1 mM CaCl₂ (B). Assays were carried out in 50 mM Tris/HCl, pH 8.0, with 0.1% (w/v) azocasein, for 20 min at 92°C. Data are means ± SD from three independent experiments. The lines shown represent the best fit of the data according to OriginPro 8.0 program.</p

Detection of smaller sized peptides as a result of PrP^Sc digestion.

<p><b>A.</b> Post-nuclear homogenates from uninfected (lane 1) and CJD-infected (3, 6, 9 and 12 µL; lanes 2–5, respectively) brains were incubated as indicated, without (lane 1) and with the R30 fraction from the Superdex 200 preparative grade gel filtration column, for 20 min at 92°C. The proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. The immunoreactive species were detected as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039548#s2" target="_blank">Materials and Methods</a>, with the EB8 anti-PrP (N-terminal) monoclonal antibody used as the primary antibody. <b>B.</b> Diluted (20x) post-nuclear homogenates from CJD-infected brain were incubated as indicated, without (lane 1) or with (lane 2) the R30 fraction from the Superdex 200 preparative grade gel filtration column, for 20 min at 92°C. The proteins were dot blotted onto nitrocellulose membranes. The immunoreactive species were detected as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039548#s2" target="_blank">Materials and Methods</a>, with the C7/5 anti-PrP (C-terminal) and the 6H4 anti-PrP monoclonal antibody used as the primary antibodies, as indicated.</p

Azocasein assays of the purified pernisine: effects of temperature and pH.

<p>Relative activities of the purified pernisine in the presence (gray dashed lines) and absence (black lines) of 1 mM CaCl₂ according to temperature (<b>A</b>), in 50 mM Tris/HCl, pH 8.0, and according to pH (<b>B</b>), in 50 mM glycine/HCl, pH 2 to 5, 50 mM HEPES, pH 6 to 8, or 50 mM glycine/NaOH, pH 9 to 11, at 92°C. Assays were carried out for 20 min. Data are means ± SD from three independent experiments. The lines shown represent the best fit of the data according to OriginPro 8.0 program.</p

Thermostability of pernisine.

<p>Residual activity of pernisine incubated at different temperatures (70°C and 90°C) in the presence and absence of Ca²⁺ ions at pH 8.0 for prolonged incubation times (0 min, 20 min, 40 min, 120 min) as marked.</p