12 research outputs found
PMCA analysis of white blood cells and platelets samples.
<p>Platelets (A) and white blood cells (WBC) (B) from sheep D2 collected at the indicated time points (dpi) were subjected to two successive rounds of PMCA. Unseeded reactions were run in parallel. Samples were processed for PrP<sup>res</sup> isolation and analyzed by immunoblotting. A western-blotting positive control (cont) is included in each gel.</p
Evaluation of the infectivity present in platelets prepared from scrapie infected sheep by two different methods: PMCA and inoculation into tg338 mice (bioassay).
<p>Five susceptible VRQ/VRQ sheep were orally challenged with 2 g of brain homogenate (10<sup>6.6</sup> ID<sub>50</sub>/g IC in tg<i>338</i> mice) between 6 and 10 months of age. The five VRQ/VRQ sheep respectively died at 198, 193, 198, 194 and 191days post inoculation (dpi). Classical scrapie was confirmed by histopathology (vacuolar change in central nervous system) and detection of abnormal PrP deposit in central nervous system and lymphoid tissues. At different time points, whole blood was collected from each donor and aliquots of platelets corresponding to 15 mL of plasma were prepared. Platelet homogenates (in 200ÎŒL) were inoculated in groups of six tg338 mice. Mice were monitored up to occurrence of TSE compatible clinical sign onset or killed at 250 days post inoculation. All mice were tested for presence of abnormal PrP deposition in brain. Incubation period in mice are presented in days (+/âSD). When less than 3 mice were positive, individual incubation period are given. In parallel the same homogenates were tested by PMCA (using tg338 mice brain homogenate as substrate). Each sample was run in 5 replicates for 2 successive rounds and the number of positive reactions is presented.</p
Cell-based assay of white blood cells infectivity from asymptomatic scrapie sheep.
<p>White blood cells from 5 infected sheep (D1 to D5) were isolated 80 days and 130 days post inoculation (dpi) when sheep were still asymptomatic. White blood cell homogenates (4Ă10<sup>7</sup> cells) were inoculated to recipient ovRK13 cells. After 2 successive rounds of cell assay, the cultures were assayed for PrP<sup>res</sup> by immunoblotting. PrP<sup>res</sup> level is higher in cells infected with D3 130 dpi sample but its banding pattern is similar to that in cells infected with the other samples. M are molecular mass marker proteins (20, 30 and 40 kDa).</p
Evaluation of the infectivity present in white blood cells prepared from scrapie infected sheep by Cerebellar Organotypic Slice Culture Assay.
<p>Immunoblots of PK-treated slice culture homogenates probed with anti-PrP antibody Sha31, showing PrP<sup>res</sup> accumulation in slice culture. (A) Cerebellar organotypic slices were prepared from tg338 pups and maintained in culture during 42 days <i>in vitro</i> after exposure to white blood cells prepared from blood collected from five scrapie infected sheep (D1, D2, D3, D4 and D5) at different times: 50 days post inoculation (dpi), 80 dpi, 130 dpi and at the terminal stage (180 dpi). For quantification purposes, slice cultures were also exposed to serial dilutions of PG127 scrapie-infected brain stock prepared from terminally ill tg338 mice, previously used <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104287#pone.0104287-ArellanoAnaya1" target="_blank">[31]</a>. To visualize low levels of PrP<sup>res</sup>, membranes were exposed over-night (B).</p
StructureâActivity Relationship Study around Guanabenz Identifies Two Derivatives Retaining Antiprion Activity but Having Lost α2-Adrenergic Receptor Agonistic Activity
Guanabenz
(GA) is an orally active α2-adrenergic agonist
that has been used for many years for the treatment of hypertension.
We recently described that GA is also active against both yeast and
mammalian prions in an α2-adrenergic receptor-independent manner.
These data suggest that this side-activity of GA could be explored
for the treatment of prion-based diseases and other amyloid-based
disorders. In this perspective, the potent antihypertensive activity
of GA happens to be an annoying side-effect that could limit its use.
In order to get rid of GA agonist activity at α2-adrenergic
receptors, we performed a structureâactivity relationship study
around GA based on changes of the chlorine positions on the benzene
moiety and then on the modifications of the guanidine group. Hence,
we identified the two derivatives <b>6</b> and <b>7</b> that still possess a potent antiprion activity but were totally
devoid of any agonist activity at α2-adrenergic receptors. Similarly
to GA, <b>6</b> and <b>7</b> were also able to inhibit
the protein folding activity of the ribosome (PFAR) which has been
suggested to be involved in prion appearance/maintenance. Therefore,
these two GA derivatives are worth being considered as drug candidates
Effect of ShRNA-mediated <i>Sprn</i> knockdown on embryo resorption at E11.5.
*<p>p<0.05 (x<sup>2</sup> test) when compared to any of the FoxL2 results and to LS2 on P10 and FVB/N genetic backgrounds.</p><p>These data are a compilation of at least 2 independent experiments. No statistically significant variability was observed between the analyzed litters (3 for P10, more than 4 for the other lentiviral infections).</p
List of top-function clusters for differentially expressed genes in <i>Sprn</i>-knockdown embryos.
<p>Bold faced genes: upregulated genes in <i>Sprn</i>-knockdown embryos. Un-bolded genes: downregulated genes in <i>Sprn</i>-knockdown embryos.</p
Prion Protein and Shadoo Are Involved in Overlapping Embryonic Pathways and Trophoblastic Development
<div><p>The potential requirement of either the Prion or Shadoo protein for early mouse embryogenesis was recently suggested. However, the current data did not allow to precise the developmental process that was affected in the absence of both proteins and that led to the observed early lethal phenotype. In the present study, using various <em>Prnp</em> transgenic mouse lines and lentiviral vectors expressing shRNAs that target the Shadoo-encoding mRNA, we further demonstrate the specific requirement of at least one of these two PrP-related proteins at early developmental stages. Histological analysis reveals developmental defect of the ectoplacental cone and important hemorrhage surrounding the <em>Prnp</em>-knockout-<em>Sprn</em>-knockdown E7.5 embryos. By restricting the RNA interference to the trophoblastic cell lineages, the observed lethal phenotype could be attributed to the sole role of these proteins in this trophectoderm-derived compartment. RNAseq analysis performed on early embryos of various <em>Prnp</em> and <em>Sprn</em> genotypes indicated that the simultaneous down-regulation of these two proteins affects cell-adhesion and inflammatory pathways as well as the expression of ectoplacental-specific genes. Overall, our data provide biological clues in favor of a crucial and complementary embryonic role of the prion protein family in <em>Eutherians</em> and emphasizes the need to further evaluate its implication in normal and pathological human placenta biology.</p> </div
Histological analysis of E7.5 embryos.
<p>E7.5 embryos were fixed and stained by hematoxylin, eosin, and saffron. Top left: schematic representation of a mouse E7.5 embryo. 1,2: FVB/N <i>Prnp<sup>KO</sup></i> embryos. 3,4: FG12-injected FVB/N <i>Prnp<sup>KO</sup></i> embryos. 5â8: LS2-injected FVB/N <i>Prnp<sup>KO</sup></i> embryos. 3â8: embryos that were injected at the zygotic stage. 9: LS2-infected FVB/N <i>Prnp<sup>KO</sup></i> embryos. *: infection performed at the blastocyst stage. Interesting features include i) the size differences between injected and non-injected embryos, ii) the relatively important hemorrhagic tissue that is totally surrounding the LS2-injected FVB/N <i>Prnp<sup>KO</sup></i> embryos 5, 6 and 8, iii) the developmental defect of the ectoplacental cone (area surrounded using a dashed line ) of all the LS2-injected FVB/N <i>Prnp<sup>KO</sup></i> embryos (5â8) that even leads to its nearly complete disappearance in embryo 6 and iv) the important developmental delay and the total disorganization of the extra-embryonic ectoderm and of the ectoplacental cone of embryo 9. Scale: 250 ”m. Sho: <i>Sprn</i>.</p
Effect of trophoblastic-restricted ShRNA- mediated <i>Sprn</i> knockdown on embryo resorption at E13.5.
*<p>p<0.05 (x<sup>2</sup> test) when compared to any of the other results.</p><p>These data are a compilation of at least 2 independent experiments. No statistically significant variability was observed between the analyzed litters (more than 4 for each lentiviral infections).</p