PrP^C expression in histoblots of P15 and P20 opossum brains.

<p>(A–B) In coronal sections of P15 and P20 a PrP^C signal was detected in the thalamus (Th), in the neocortex (NCx) and in the hippocampus (H). The pyramidal cell layer (pyr) and the granule cell layer (gr) of the hippocampus were not stained by PrP^C (Bars: A–B 4 mm).</p

Coronal sections of opossum brain at P70 (A–C).

<p>In (A) the coronal section was Nissl-stained. In the adult opossum (B) a strong signal was predominantly present in the hippocampus. Marginal signal was also detected in the caudate nucleus (C), in the internal capsule (iC) and in the putamen (Pu). A very residual signal was observed in the external capsule (eC). In (C) the specific distribution of PrP^C in the different hippocampal layers was analyzed. Staining in the <i>lacunosum</i> and <i>moleculare</i> (lac/mol) was lower than in the <i>oriens</i> (Or) and the <i>radiatum</i> (Rad). PrP^C immunoreactivity was also observed in the <i>subiculum</i> (Sb) the main output of the hippocampus. (Bars: 0.5 mm).</p

Confirmation of antibody specificity and developmental expression of PrP^C in opossum CNS.

<p>(A) Western blotting of Op, MoPrP^+/+ and MoPrP^−/− whole brain homogenates confirmed the specificity of the PrP^C signal. The blot was then reprobed with β-tubulin antibody to demonstrate equal loading of samples (50 µg per lane). (B) Western blotting analysis of equal amounts of total brain homogenate (50 µg per lane) at different developmental stages showed a major electrophoretic band at 37 kDa, which corresponds to the diglycosylated form of the protein. β-tubulin was used as loading control. (C) Western blotting of indicated brain regions at different developmental stages showed a relevant change in PrP^C expression during postnatal development. Each data point represents the mean absorbance ±SEM of 3 females from different litters. All the absorbance values were normalized against β-actin.</p

Comparison of amino acid sequences and secondary full-length prion protein structure of selected mammalian species: mouse (MoPrP; <i>Mus musculus</i>, GenBank accession number: AAA39997), human (HuPrP; <i>Homo sapiens</i>, AAA60182), Syrian hamster (ShPrP; <i>Mesocricetus auratus</i>, AAA37091), sheep (OvPrP; <i>Ovis aries</i>, ABC61639), elk (ElPrP; <i>Cervus elaphus nelsoni</i>, AAB94788), bank vole (BvPrP; <i>Clethrionomys glareolus</i>, AAL57231), horse (EcPrP; <i>Equus caballus</i>, ABL86003), rabbit (RaPrP; <i>Oryctolagus cuniculus</i>, AAD01554), tammar wallaby (TwPrP; <i>Macropus eugenii</i>, AAT68002) and opossum (OpPrP; <i>Monodelphis domestica</i>, CBY05848).

<p>Comparison of amino acid sequences and secondary full-length prion protein structure of selected mammalian species: mouse (MoPrP; <i>Mus musculus</i>, GenBank accession number: AAA39997), human (HuPrP; <i>Homo sapiens</i>, AAA60182), Syrian hamster (ShPrP; <i>Mesocricetus auratus</i>, AAA37091), sheep (OvPrP; <i>Ovis aries</i>, ABC61639), elk (ElPrP; <i>Cervus elaphus nelsoni</i>, AAB94788), bank vole (BvPrP; <i>Clethrionomys glareolus</i>, AAL57231), horse (EcPrP; <i>Equus caballus</i>, ABL86003), rabbit (RaPrP; <i>Oryctolagus cuniculus</i>, AAD01554), tammar wallaby (TwPrP; <i>Macropus eugenii</i>, AAT68002) and opossum (OpPrP; <i>Monodelphis domestica</i>, CBY05848).</p

Localization of PrP^C in P30 MoPrP^+/+ brain and control of signal specificity.

<p>(A) At P30 a well-defined signal was present in the hippocampal <i>stratum lacunosum-moleculare</i> layer (lac/mol) and in the alveus (alv) lying just deep to the <i>stratum oriens</i> layer (or). (B) In the septum-caudatum, PrP^C signal was detected predominantly in white matter fiber bundles, such as the anterior commissure (ac) and the <i>corpus callosum</i> (cc). The dark dots observed in the caudate-putamen (CPu) are fiber fascicles cut on end. (C) At P30, the lack of IR in MoPrP^−/− coronal section confirmed the signal specificity. (D) The presence of the brain section on the nitrocellulose membrane was confirmed by Ponceau staining. (Bars: A–D 0.5 mm).</p

PrP^C distribution in a P37 opossum brain.

<p>(A–B) Coronal sections in the caudal diencephalon from a P37 opossum were stained for PrP^C in the thalamus (Th) and in the parenchyma of the hippocampus. (A) In the ventral thalamus, a strong IR was observed in the reticular nucleus (Rt) and in the nucleus reuniens (Re). No IR was observed in the optic tract (Ot). In the hypothalamic area (H) the immunoreactivity was lower than in the thalamus. (B) A strong PrP^C signal was observed in the <i>stratum oriens</i> (or) and in the <i>stratum radiatum</i> (rad) of the hippocampus. A low PrP^C immunoreactivity was observed in the hilar region (h), while the pyramidal (pyr) and granule (gr) cell layers of the CA1-CA3 and DG were devoid of immunostaining (Bars: 1 mm).</p

PrP^C co-immunolabeling with Tau and MAP2.

<div><p>Confocal images of 21 DIV hippocampal primary neurons. Surface immunolabeling of PrP^C (green) coupled with Tau staining (red) (left) or MAP2 staining (red) (right). Antibodies used: see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074244#pone-0074244-g006" target="_blank">figures 6</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074244#pone-0074244-g007" target="_blank">7</a>.</p> <p>Scale bar = 10 µm.</p></div

PrP^C in DRMs from hippocampal membrane of young wild-type mice compared with age-matched ASMKO mice.

<div><p>Western blot analysis of DRMs prepared from equal amounts of hippocampal extracts (100 µg of protein) from young (4-5 months old) wild-type and ASMKO mice. Each lane corresponds to a single animal. Antibodies used: D18 (1:1,000; InPro Biotechnology, Inc, South San Francisco), mouse monoclonal anti flotillin1 (1:1,000; BD Biosciences). Quantification of relative PrP^C amounts from 3 control mice and 6 ASMKO mice. Each data point represents the relative PrP level normalized over flotillin1 ± SD. PrP^C levels are 20% higher in ASMKO mice compared to age-matched wild-type mice.</p> <p>**: <i>p</i><0.01.</p></div

On the Examination of the Strong Discontinuity Analysis for a Kinking Discontinuous Surface

The purpose of this paper is to examine the accuracy of the analysis of the strong discontinuity with a kinking discontinuous surface. We first examine the path independent J-integral and the E-integral formula for a damage model with a kinking cohesive region. As a result, we find that the strong discontinuity analysis based on the E-integral has a high accuracy in view of the energy release rate even when the discontinuous surface kinks

PrP^C in DRM preparation of young (3-4 months) vs. old (20-21 months) mice.

<p>Western blot analysis of DRMs prepared from equal amounts of total hippocampal protein extracts (100 µg of total protein) at the indicated ages. Young = 3-4 months old; old = 21-22 months old. Each lane corresponds to a single animal. Antibodies used: D18 (1:1,000; InPro Biotechnology, Inc, South San Francisco), mouse monoclonal anti flotillin1 (1:1,000; BD Biosciences). Relative PrP^C amounts from 3 mice per time point were analyzed. Each data point represents the relative protein level normalized over flotillin1 ± SD.</p