OBJECTIVETo investigate whether gliosis in the brain tissues of the hamsters infected with various amounts of scrapie strain 263K is correlated with the inoculation doses or the incubation times.

METHODSThe total values of glial fibrillary acidic protein (GFAP) in brains were evaluated by Western Blots and the GFAP-stained cells were detected by immunohistochemistry (IHC). The characteristics of GFAP distributions among various groups were defined by quantitive and statistic analyses.

RESULTSCompared with the brain tissues of normal hamsters, remarkably higher total GFAP levels and more GFAP-stained cells were observed in the brain tissues of infected ones, howbeit, no significant difference was addressed among the infected groups.

CONCLUSIONInoculations of various amounts of scrapie strain 263K into experimental hamsters intracerebrally induced the similar patterns of gliosis in the brains at the clinically terminal stage, regardless of infectious doses and incubation times.

Animals ; Brain ; metabolism ; pathology ; Cricetinae ; Gene Expression ; Glial Fibrillary Acidic Protein ; genetics ; metabolism ; Gliosis ; metabolism ; pathology ; Humans ; PrPSc Proteins ; metabolism ; Prion Diseases ; metabolism ; pathology

Misfolded isoform of prion protein (PrP), termed scrapie PrP (PrP(Sc)), tends to aggregate into various fibril forms. Previously, we reported various conditions that affect aggregation of recombinant PrP into amyloids. Because amyloidogenesis of PrP is closely associated with transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease in humans, we investigated infectivity of recombinant PrP amyloids generated in vitro. Using cultured cell lines which overexpress cellular PrP of different species, we measured the level of de novo synthesized PrP(Sc) in cells inoculated with recombinant mouse PrP amyloids. While PrP-overexpressing cells were susceptible to mouse-adapted scrapie prions used as the positive control, demonstrating the species barrier effect, infection with amyloids made of truncated recombinant PrP (PrP[89-230]) failed to form and propagate PrP(Sc) even in the cells that express mouse cellular PrP. This suggests that infectivity of PrP amyloids generated in vitro is different from that of natural prions. Recombinant PrP (89-230) amyloids tested in the current study retain no or a minute level, if any, of prion infectivity.
Animals ; Cell Line ; Kidney/*metabolism/*pathology ; Mice ; PrPSc Proteins/*metabolism ; Prion Diseases/*metabolism/*pathology ; Prions/*metabolism ; Rabbits ; Recombinant Proteins/*metabolism ; Up-Regulation

alphaB-crystallin is a member of the sHSP (Small heat shock protein) family, which plays an impor tant role in multiple neurodegeneration diseases. To give insight into the possible alternation and the role of aB-crystallin in prion disease, the alphaB-crystallin levels in the brain tissues of agent 263K-infected hamsters were evaluated. Western blots and IHC revealed that at the terminal stages of the diseases, the levels of alphaB-crystallin were increased up to 3-fold in the brain tissues of scrapie infected 263K hamsters compared with normal controls. Immunofluorescent assays revealed that the up-regulated alphaB-crystallin was mainly observed in astrocytes, but not in neurons. The co-localization between alphaB-crystallin and abnormal deposition of PrPsc in the brain tissues of the scrapie infected hamsters was not observed. The study may provide a foundation for further revealing the potential role of alphaB-crystallin in prion disease.
Animals ; Brain ; metabolism ; pathology ; Cricetinae ; Humans ; PrPSc Proteins ; metabolism ; Prion Diseases ; genetics ; metabolism ; pathology ; Up-Regulation ; alpha-Crystallin B Chain ; genetics ; metabolism

UNLABELLEDOBJECTIVE To study the potential interaction between PrP protein.

METHODSThe supernatant of health and scrapie-infected hamsters' brain homogenate was prepared, while various recombinant 14-3-3beta or PrP proteins were purified. The possible molecular interaction between 14-3-3beta proteins and PrP was tested by pull-down and immunoprecipitation assays.

RESULTSBoth native PrP(c) and its protease-resistant isoform (PrP(Sc)) formed complexes with 14-3-3beta. The full-length recombinant 14-3-3beta proteins interacted with PrP. The domain responsible for interacting 14-3-3beta was located at N-terminal of 14-3-3beta (residues 1 to 38).

CONCLUSIONThe studies of the association of PrP with 14-3-3beta may further provide insight into a potential role of 14-3-3beta in the biological function of PrP and the pathogenesis of prion disease.

14-3-3 Proteins ; metabolism ; Animals ; Binding Sites ; Brain Chemistry ; Cricetinae ; Endopeptidases ; metabolism ; PrPSc Proteins ; metabolism ; Prion Diseases ; pathology ; Prions ; metabolism ; Scrapie ; physiopathology

To establish a new Western blotting assay for PrP(Sc) detection, we optimized the Western blotting assay with a precipitation procedure of streptomycin sulfate. After digestion with PK, 10% scrapie infected hamster brain homogenates were incubated with 60 mmol/L streptomycin and the precipitated PrP(Sc) was recovered by centrifugation. The enrichment of PrP(Sc) by streptomycin sulfate precipitation was evaluated using Western blotting assay. The results showed streptomycin could bind to PK-treated PrP(Sc), forming high molecular masses, but not influence the glycosylated patterns on SDS-PAGE. Western blot assay revealed that the detective sensitivity of the streptomycin-precipitation PrP(Sc) was remarkably improved. As a sensitive, specific, rapid and flexible protocol for PrP(Sc), the protocol in this study has the potential utility, alone or combined with other techniques, for the detection of low level PrP(Sc) in the specimens from central nerve system, or from peripheral organs or body fluids.
Animals ; Blotting, Western ; methods ; Brain ; metabolism ; pathology ; Brain Chemistry ; Chemical Precipitation ; Cricetinae ; PrPSc Proteins ; chemistry ; isolation & purification ; metabolism ; Prion Diseases ; diagnosis ; metabolism ; Reproducibility of Results ; Sensitivity and Specificity ; Streptomycin ; chemistry

OBJECTIVETo establish a methodology of protein misfolding cyclic amplification (PMCA) and utilize in the detection of PrP(Sc) in brain tissues from prion diseases.

METHODSDifferent amounts of Scrapie 263K agent bulk were mixed with brain homogenates of health hamsters and treated with repeated incubation/sonication for 10 to 15 cycles. The proteinase K-resistant PrP(Sc) was evaluated with Western Blot.

RESULTSIn this experimental situation, 263K agent replicated rapidly in vitro, utilizing hamsters' brains as the medium. With the established PrP(Sc)-PMCA technique, PrP(Sc) signals in the preparations containing less than 10(-5) diluted 263K bulk could be detected. Compared with conveniently used immuno-blot assay, the sensitivity of PrP(Sc)-PMCA for PrP(Sc) was 10(5) to 10(6)-fold increased. It has been also shown that homogenates of cerebellar and brain stem could be used as the medium for PrP(Sc) replication.

CONCLUSIONA rapidly replicating method for PrP(Sc), PrP(Sc)-PMCA, was successfully established, providing a new approach for early diagnosis of prion diseases and research on the biological features of prion.

Animals ; Biochemistry ; methods ; Blotting, Western ; Brain ; metabolism ; pathology ; Cricetinae ; PrPSc Proteins ; analysis ; chemistry ; Prion Diseases ; diagnosis ; metabolism ; Protein Folding ; Reproducibility of Results ; Sensitivity and Specificity

14-3-3 Proteins ; genetics ; metabolism ; Animals ; Brain ; pathology ; Cricetinae ; Internal Ribosome Entry Sites ; Open Reading Frames ; Prion Diseases ; genetics ; metabolism

OBJECTIVETo report a protocol using biotin-labelled PrP protein in cell free conversion assay instead of isotope.

METHODSA hamster PrP protein (HaPrP) was expressed in E. coli and purified with HIS-tag affinity chromatograph. After being labelled with biotin, HaPrP was mixed with PrPSc preparation from scrapie strain 263K.

RESULTSProtease-resistant bands were detected after four-day incubation.

CONCLUSIONThe new conversion model provides a reliable, easily handling, and environment-friendly method for studies of prion and transmissible spongiform encephalopathies.

Animals ; Biotin ; Blotting, Western ; Cell-Free System ; Chromatography, Affinity ; methods ; Cricetinae ; Escherichia coli ; genetics ; Gene Expression Regulation ; Molecular Weight ; Peptide Hydrolases ; analysis ; metabolism ; PrPSc Proteins ; genetics ; metabolism ; Prion Diseases ; genetics ; pathology ; Recombinant Proteins ; genetics ; metabolism ; Staining and Labeling

A 68-year-old man presented with a three-week history of rapidly progressive dementia, gait ataxia and myoclonus. Subsequent electroencephalography showed periodic sharp wave complexes, and cerebrospinal fluid assay revealed the presence of a 14-3-3 protein. A probable diagnosis of sporadic Creutzfeldt-Jakob disease was made, which was further supported by magnetic resonance (MR) imaging of the brain showing asymmetric signal abnormality in the cerebral cortices and basal ganglia. The aetiology, clinical features, diagnostic criteria, various MR imaging patterns and radiologic differential diagnosis of sporadic Creutzfeldt-Jakob disease are discussed in this article.
Aged ; Brain ; pathology ; Cerebral Cortex ; Cerebrospinal Fluid ; metabolism ; Creutzfeldt-Jakob Syndrome ; diagnostic imaging ; Dementia ; physiopathology ; Diagnosis, Differential ; Diffusion Magnetic Resonance Imaging ; Electroencephalography ; Humans ; Hypoxia-Ischemia, Brain ; diagnostic imaging ; Male ; Prion Diseases ; physiopathology