34 research outputs found

    SNP genotyping in HS mice.

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    <p>Genomic location is given based on mouse genome assembly NCBI build 37. Details of the <i>Sod1</i> and <i>Il1-r1</i> SNPs are available from the Sanger Centre (<a href="http://www.sanger.ac.uk/cgi-bin/modelorgs/mousegenomes/snps.pl" target="_blank">www.sanger.ac.uk/cgi-bin/modelorgs/mousegenomes/snps.pl</a>). All polymorphisms were analysed by allele discrimination using a 7500 Fast real time PCR system (Applied Biosystems). For probe details see Table S1. For all genotypes, the statistical test used was the Kruskal-Wallis non-parametric ANOVA. The allelic test used was the Mann-Whitney test.</p

    Kaplan-Meier survival curves.

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    <p>Data are shown as % of animals (female) surviving (y-axis) plotted against the number of days post inoculation (x-axis). (A) Transmission of Chandler/RML prion strain to <i>Sod1<sup>−/−</sup></i> and <i>Sod1<sup>+/+</sup></i> (WT) litter mate controls (B) Transmission of ME7 prion strain to <i>Sod1<sup>−/−</sup></i> and <i>Sod1<sup>+/+</sup></i> (WT). (C) Transmission of MRC2 mouse adapted BSE prion strain to <i>Sod1<sup>−/−</sup></i> and <i>Sod1<sup>+/+</sup></i> (WT). A reduction in mean incubation time of 20%, 13%, and 24% was seen in A-C respectively. This reduction in survival was statistically significant for each transmission (P<0.001, Kaplan-Meier log-rank survival test).</p

    <i>Sod1</i> expression in mouse brain.

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    <p>Panel A and B. Quantification of <i>Sod1</i> mRNA expression in whole brain by real-time RT-PCR. N = 6 for all groups and samples were run in triplicate. All samples were duplexed for <i>Sod1</i> (Fam-label) and an endogenous control <i>GAPDH, β-actin</i> or <i>Thy-1</i> (Vic-label). Expression level is expressed in arbitrary units as normalised by the geometric mean of the quantity of the endogenous controls (<i>y</i>-axis). Error bars represent the standard deviation. (A) <i>Sod1</i> mRNA expression level for parental strain of the HS mice (except LP). (B) <i>Sod1</i> mRNA expression level grouped by allele (A/G) (A = A, BALB, C3H, C57; G = AKR, CBA, DBA). No significant difference was observed between the groups. Panels (C) and (D). Quantification of Sod1 protein in whole brain (10% homogenate, weight/volume) from n = 3 A allele mice (C57Bl/6) and G allele mice (FVB/N). FVB/N mice were used to represent a G allele strain rather than an HS parental strain due to availability of tissue. Samples were immunoblotted with rabbit polyclonal anti-human SOD1(Abcam) and detected with IRDye800CW (green) conjugated goat anti-rabbit IgG (Li-Cor). Anti-β-actin mouse monoclonal antibody (Sigma) was also included as a loading control and was detected using IRDye680 (red) conjugated goat anti-mouse IgG (Li-Cor). Fluorescence was visualised using an Odyssey infra-red imager (Li-Cor). (C) Uninoculated mice, (D) Terminally sick Chandler/RML prion inoculated mice.</p

    Western blots of PrP<sup>Sc</sup> from infected mouse brains.

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    <p>10% w/v brain homogenates (n = 3 per group) were digested with proteinase K and immunoblotted with anti-PrP monoclonal antibody ICSM35 (D-Gen Ltd, UK). (A) Transmission of Chandler/RML prions to <i>Sod1<sup>−/−</sup></i> and <i>Sod1<sup>+/+</sup></i> (WT) litter mate controls. (B) Transmission of ME7 prion strain to <i>Sod1<sup>−/−</sup></i> and <i>Sod1<sup>+/+</sup></i> (WT) litter mate controls. (C) Transmission of MRC2 mouse adapted BSE prion strain to <i>Sod1<sup>−/−</sup></i> and <i>Sod1<sup>+/+</sup></i> (WT) litter mate controls. No differences were seen between the two groups regardless of prion strain.</p

    RML histology.

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    <p>Histological features of Chandler/RML prion transmission to <i>Sod1<sup>−/−</sup></i> (A–C) and wild type control (D–F) mice. Panels A and D show distribution of disease-associated PrP by immunohistochemistry using anti-PrP monoclonal antibody ICSM35. Panels B, C, E and F show detail from the hippocampus and are stained with haematoxylin and eosin (H&E) to visualise spongiform change and neuronal loss. There is almost no neuronal loss in the <i>Sod1<sup>−/−</sup></i> mice but mild neuronal loss is seen in the wild type animals. Overall, the pattern of spongiosis, gliosis and PrP distribution are similar between the two groups, however, the distribution of disease-associated PrP is patchier in the knockouts especially in the cortex. Scale bar corresponds to 3 mm (A, D), 660 µm (B, E) or 160 µm (C, F).</p

    Quantification of Sod and PrP<sup>C</sup>

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    <p>. 10% weight/volume brain homogenates immunoblotted with rabbit polyclonal anti-human SOD1 (Abcam) and detected with IRDye800CW (green) conjugated goat anti-rabbit IgG (Li-Cor). Anti-β-actin mouse monoclonal antibody (Sigma) was also included as a loading control and was detected using IRDye680 (red) conjugated goat anti-mouse IgG (Li-Cor). Fluorescence was visualised using an Odyssey infra-red imager (Li-Cor). (A) Brains from <i>Sod1<sup>+/+</sup></i> wild type mice inoculated with PBS compared with end stage Sod1<sup>+/+</sup> wild type mice inoculated with RML, ME7 and MRC2 prion strains. No differences were seen between the groups. (B) Uninfected mice. (C) Total SOD enzymatic activity in 10% (w/v) <i>Sod1<sup>+/+</sup></i> (WT) brains. Brains from terminally sick mice infected with RML, ME7 and MRC2 were compared with uninfected mice. Samples were run in triplicate with n = 6 for each group. Data are shown normalised by total protein content (µg/ml) as determined by a Bradford protein assay (mean ± standard deviation). No significant difference was seen between the groups. (D) PrP<sup>c</sup> levels in <i>Sod1<sup>−/−</sup></i> and <i>Sod1<sup>+/+</sup></i> (WT) litter mate control mice by ELISA. PrP<sup>c</sup> levels (µg/ml) were determined in triplicate using 10% (weight/volume) brain homogenate for <i>Sod1<sup>−/−</sup></i> (n = 3) and <i>Sod1<sup>+/+</sup></i> (n = 3) in a PrP specific ELISA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054454#pone.0054454-Wadsworth2" target="_blank">[32]</a>. Data are shown normalised by total protein content (µg/ml and x 1000) as determined by a BCA assay (mean ± standard deviation). No significant difference was seen between the two groups.</p

    Major strain distribution patterns genotyped for HS mice.

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    <p>The number of SNPs is taken from genomic sequence generated by the Sanger Institute (<a href="http://www.sanger.ac.uk/cgi-bin/modelorgs/mousegenomes/snps.pl" target="_blank">www.sanger.ac.uk/cgi-bin/modelorgs/mousegenomes/snps.pl</a>) and spans 5 kb upstream of the 5′UTR start site to the end of the 3′UTR (NCBI Build 37). Ambiguous SNPs have been excluded. The strain distribution pattern (BALB, CBA); (A, AKR, C3H, C57, DBA, LP) was also seen for <i>App</i> (239/978) but this was not genotyped in the HS mice. Other individual strain distribution patterns were seen ≤5 times.</p

    Panel Showing the Protein-Based Phylogenies of the Cytoplasmic Dynein Subunit Families

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    <div><p>Species names are shown with NCBI/GenBank gene/protein names. NCBI/GenBank protein-sequence accession numbers are given in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020001#pgen-0020001-st001" target="_blank">Table S1</a>. Orthologous human, mouse, and rat gene names use the revised systematized consensus nomenclature (e.g. DYNC1H1 in humans, mouse, and rat). Relationships amongst dynein sequences of different species do <i>not</i> necessarily reflect the evolutionary relationships amongst species; see [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020001#pgen-0020001-b208" target="_blank">208</a>] and [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020001#pgen-0020001-b209" target="_blank">209</a>] for further details. Named clades are indicated in the right margins. Bayesian and maximum-likelihood bootstrap values are shown as percentages (top and bottom, respectively), adjacent to branch points. Asterisks denote bootstraps below 50%. Filled circles denote bootstraps at 100%. Scale-bar represents evolutionary distance (estimated numbers of amino-acid substitutions per site).</p><p>(A) Cytoplasmic dynein heavy chain family. <i>Chlamydomonas</i> outer arm heavy chain <i>(ODA11)</i> is used as the outgroup. DNAH12frag is the partial axonemal heavy chain fragment taken from [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020001#pgen-0020001-b023" target="_blank">23</a>]. For mouse DYNC2H1, XP_35830, only partial protein sequence (336aa) was available in the GenBank database. Adding this partial sequence to our analysis resulted in spurious clustering, therefore we obtained an extended, putative sequence by using BLAST (TBLASTN) against the mouse genome (Build 32) with human and rat sequences XP_370652 and NP_075413, respectively. Incomplete mouse genomic assembly at the <i>DYNC2H1</i> locus yielded a truncated sequence 3455 amino acids in length, 85% the length of human DYNC2H1.</p><p>(B) Cytoplasmic dynein intermediate chain family. The <i>Chlamydomonas</i> IC2 <i>(ODA6)</i> is used as the outgroup.</p><p>(C) Cytoplasmic dynein light intermediate chain family. There does not appear to be a sufficiently distant homolog in <i>Chlamydomonas</i> to be used as an outgroup in this analysis, therefore ODA11 (Q39610, a heavy chain protein) was chosen as the outgroup for this tree.</p><p>(D) Cytoplasmic dynein light chain Tctex1 family. The <i>Chlamydomonas</i> LC2 light chain is used as the outgroup.</p><p>(E) Cytoplasmic dynein light chain Roadblock family. The <i>Chlamydomonas</i> outer arm dynein LC7a, is used as the outgroup.</p><p>(F) Cytoplasmic dynein light chain LC8 family. The <i>Chlamydomonas</i> Q39579 sequence is used as an outgroup. This phylogeny is poorly resolved, with low bootstrap support values and posterior clade probabilities, most likely due to there being little variation amongst the ingroup sequences. We found good support for the LC8 light chain 1 clade, and some support for the LC8 light chain 2 clade, of four vertebrate sequences. The relationships of the two sequences, C. elegans and <i>Takifugu</i> were poorly resolved, and therefore we have not included these in the LC8 light chain 2 clade.</p></div

    The Mammalian Cytoplasmic Dynein Complexes

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    <div><p>(A) Cytoplasmic dynein. (Left panel) Polypeptides of immunoaffinity-purified rat brain cytoplasmic dynein. Polypeptide mass (in kDa) is indicated on the right side of the gel, and the consensus family names are indicated on the left. (Right panel) Structural model for the association of the cytoplasmic dynein complex subunits. The core of the cytoplasmic dynein complex is made of two DYNC1H1 heavy chains which homodimerize via regions in their N-termini. The motor domains are at the C-termini of the heavy chains, the large globular heads of ~350 kDa that are composed of a ring of seven densities surrounding a central cavity; six of the densities are AAA domains (numbered 1–6). AAA domain 1 is the site of ATP hydrolysis. The microtubule-binding domain is a projection found on the opposite side of the ring between AAA domains 4 and 5. C is the C-terminus of the heavy chain that would form the 7th density. Two DYNC1I intermediate chains (IC74) and DYNC1LI light intermediate chains bind at overlapping regions of the N-terminus of the heavy chain, overlapping with the heavy chain dimerization domains. Dimers of the three light chain families; DYNLT, the Tctex1 light chains; DYNLRB, the Roadblock light chains; and DYNLL, the LC8 light chains, bind to the intermediate chain dimers.</p><p>(B) Cytoplasmic dynein 2 complex, structural model for subunit association. This dynein complex has a unique role in IFT and is sometimes known as IFT dynein. Structural predictions indicate that the heavy chain, DYNC2H1, is similar to the cytoplasmic and axonemal dyneins. The only known subunit of this complex is a 33- to 47-kDa polypeptide, DYNC2LI1, which is related to the cytoplasmic dynein light intermediate chains. No intermediate chain or light chains have yet been identified [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020001#pgen-0020001-b016" target="_blank">16</a>].</p></div

    Exosomes are released from scrapie-infected B cells <i>ex vivo</i>.

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    <p>Spleens were dissected from 129/Sv×C57BL/6 mice 30 days after i.p. inoculation with 1% (w/v) RML I6200. MACS-isolated B lymphocytes were cultured under passive leakage (A) and basal (B) conditions essentially as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002538#ppat-1002538-t004" target="_blank">Table 4</a> and tissue culture supernatants were isolated by sequential centrifugation (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002538#s4" target="_blank">Materials and Methods</a>). After centrifugation at 100,000× g for 2 h pellets were resuspended in PBS, absorbed onto carbon-coated grids and negatively stained with 1% uranyl acetate. Cup-shaped exosome-like membrane particles of different sizes (see arrows) are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002538#ppat-1002538-g001" target="_blank">Figure 1B</a>. Twenty randomly recorded images (surface area: 2.82 µm<sup>2</sup>) from each condition were counted and the number of exosome-like particles (1.7±1.2 (A) and 22.8±6.5 (B) per surface area, p≪0.001) determined in a blinded manner. Scale bar: 0.2 µm.</p
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