ABSTRACT
Real-time quaking-induced conversion (RT-QuIC) assay is a newly established PrP -detecting method. The development of RT-QuIC improves the diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD), showing good sensitivity and specificity in many countries when the method was used in cerebrospinal fluid (CSF) samples. However, in China, the sensitivity and specificity of RT-QuIC has yet to be determined due to the lack of definitive diagnosis samples. Recently, 30 definitive sCJD and 30 non-CJD diagnoses were evaluated by RT-QuIC assay. In the 30 sCJD CSF samples, 29 showed positive results. By contrast, all the non-CJD samples were negative. The sensitivity and specificity of our RT-QuIC assay were 96.67% and 100%, respectively, and are comparable to other published data. Results can provide a fundamental basis for the usage of RT-QuIC assay in CJD surveillance in China.
Subject(s)
Humans , China , Creutzfeldt-Jakob Syndrome , Diagnosis , Diagnostic Tests, Routine , Methods , PrPSc Proteins , Cerebrospinal Fluid , Sensitivity and SpecificityABSTRACT
Objective@#The definite diagnosis of human and animal prion diseases depends on the examination of special pathological changes and/or detection of PrP in the brain tissues of suspected cases. Thus, developing methods to obtain PrP antibody with good specificity and sensitivity is fundamental for prion identification.@*Methods@#We prepared a PrP-specific polyclonal antibody (pAb P54) in a -knockout mouse model immunization with recombinant full-length human PrP protein residues 23-231. Thereafter, we verified that pAb in Western blot, immunohistochemistry (IHC), and immunofluorescent (IFA) assays.@*Results@#Western blot illustrated that the newly prepared pAb P54 could react with recombinant PrP protein, normal brain PrP from healthy rodents and humans, and pathological PrP in the brains of experimental rodents infected with scrapie and humans infected with different types of prion diseases. The electrophoretic patterns of brain PrP and PrP observed after their reaction with pAb P54 were nearly identical to those produced by commercial PrP monoclonal antibodies. Three glycosylated PrP molecules in the brain homogenates were clearly demonstrated in the reactions of these molecules with pAb P54. IHC assay revealed apparent PrP deposits in the GdnCl-treated brain slices of 139A-infected mice and 263K-infected hamsters. IFA tests with pAb P54 also showed clear green signals surrounding blue-stained cell nuclei.@*Conclusion@#The newly prepared pAb P54 demonstrated reliable specificity and sensitivity and, thus, may have potential applications not only in studies of prion biology but also in the diagnosis of human and experimental rodent prion diseases.
Subject(s)
Animals , Mice , Antibodies , Allergy and Immunology , Blotting, Western , Fluorescent Antibody Technique , Immunization , Immunohistochemistry , Mice, Knockout , PrPC Proteins , Allergy and Immunology , PrPSc Proteins , Allergy and Immunology , Prion Proteins , Allergy and Immunology , Recombinant Proteins , Allergy and ImmunologyABSTRACT
Scrapie is a mammalian transmissible spongiform encephalopathy or prion disease that predominantly affects sheep and goats. Scrapie has been shown to overcome the species barrier via experimental infection of other rodents. To confirm the re-transmissibility of the mouse-adapted ME7 scrapie strain to ovine prion protein (PrP) transgenic mice, mice of an ovinized transgenic mouse line carrying the Suffolk sheep PrP gene that contained the A₁₃₆ R₁₅₄ Q₁₇₁/ARQ allele were intracerebrally inoculated with brain homogenates obtained from terminally ill ME7-infected C57BL/6J mice. Herein, we report that the mouse-adapted ME7 scrapie strain was successfully re-transmitted to the transgenic mice expressing ovine PrP. In addition, we observed changes in the incubation period, glycoform profile, and pattern of scrapie PrP (PrP(Sc)) deposition in the affected brains. PrP(Sc) deposition in the hippocampal region of the brain of 2nd-passaged ovine PrP transgenic mice was accompanied by plaque formation. These results reveal that the mouse-adapted ME7 scrapie strain has the capacity to act as a template for the conversion of ovine normal monomeric precursors into a pathogenic form in ovine PrP transgenic mice. The change in glycoform pattern and the deposition of plaques in the hippocampal region of the brain of the 2nd-passaged PrP transgenic mice are most likely cellular PrP species dependent rather than being ME7 scrapie strain encoded.
Subject(s)
Animals , Humans , Mice , Alleles , Brain , Gliosis , Goats , Mice, Transgenic , Plaque, Amyloid , Prion Diseases , PrPSc Proteins , Rodentia , Scrapie , Sheep , Terminally IllABSTRACT
Scrapie is a fatal and progressive transmissible spongiform encephalopathy (TSE) of natural occurrence in sheep and goats. The suspicion of scrapie may be based on clinical signs; however, the detection of pathological features of the prionic protein (PrP) in target tissues is necessary to diagnose the disease. The presence of an abnormal protein form (PrPSc) in lymphoreticular and nervous tissues is an important characteristic in diagnosis. This paper reports a case of scrapie in a flock of 55 Suffolk crossbred sheep, 19 Santa Inês sheep and 21 goats in the Mato Grosso state, midwestern Brazil. The animals were euthanized after the confirmation of a scrapie case with clinical signs in a Suffolk sheep in the same farm...
Scrapie é uma encefalopatia espongiforme transmissível (EET) progressiva e fatal de ocorrência natural em ovinos e caprinos. A suspeita de scrapie é baseada nos sinais clínicos, porém a manifestação patológica da proteína priônica (PrP) nos tecidos-alvo é necessária para a confirmação da doença. A presença de uma forma anormal da proteína (PrPSc) em tecido linforreticular e tecido nervoso constitui uma característica importante para o diagnóstico. Este trabalho é o relato de um foco de scrapie ocorrido em rebanho com 55 ovinos mistos Suffolk, 21 caprinos e 19 ovinos Santa Inês, na região Centro-Oeste do Brasil. Os animais foram eutanasiados após a confirmação de um caso de scrapie com sinais clínicos em um ovino Suffolk nessa propriedade...
Subject(s)
Animals , Sheep/virology , Prions/isolation & purification , PrPSc Proteins/analysis , Ruminants , Scrapie/virology , Lymphoid Tissue/pathology , Immunohistochemistry/veterinary , Histological Techniques/veterinaryABSTRACT
Mammalian cells remove misfolded proteins using various proteolytic systems, including the ubiquitin (Ub)-proteasome system (UPS), chaperone mediated autophagy (CMA) and macroautophagy. The majority of misfolded proteins are degraded by the UPS, in which Ub-conjugated substrates are deubiquitinated, unfolded and cleaved into small peptides when passing through the narrow chamber of the proteasome. The substrates that expose a specific degradation signal, the KFERQ sequence motif, can be delivered to and degraded in lysosomes via the CMA. Aggregation-prone substrates resistant to both the UPS and the CMA can be degraded by macroautophagy, in which cargoes are segregated into autophagosomes before degradation by lysosomal hydrolases. Although most misfolded and aggregated proteins in the human proteome can be degraded by cellular protein quality control, some native and mutant proteins prone to aggregation into beta-sheet-enriched oligomers are resistant to all known proteolytic pathways and can thus grow into inclusion bodies or extracellular plaques. The accumulation of protease-resistant misfolded and aggregated proteins is a common mechanism underlying protein misfolding disorders, including neurodegenerative diseases such as Huntington's disease (HD), Alzheimer's disease (AD), Parkinson's disease (PD), prion diseases and Amyotrophic Lateral Sclerosis (ALS). In this review, we provide an overview of the proteolytic pathways in neurons, with an emphasis on the UPS, CMA and macroautophagy, and discuss the role of protein quality control in the degradation of pathogenic proteins in neurodegenerative diseases. Additionally, we examine existing putative therapeutic strategies to efficiently remove cytotoxic proteins from degenerating neurons.
Subject(s)
Animals , Humans , Alzheimer Disease/drug therapy , Amyloid beta-Peptides/metabolism , Amyotrophic Lateral Sclerosis/drug therapy , Autophagy/drug effects , DNA-Binding Proteins/metabolism , Huntington Disease/drug therapy , Lysosomes/metabolism , Molecular Targeted Therapy , Mutation , Nerve Tissue Proteins/genetics , Neurodegenerative Diseases/drug therapy , Parkinson Disease/drug therapy , PrPSc Proteins/metabolism , Prion Diseases/drug therapy , Proteasome Endopeptidase Complex/metabolism , Proteolysis , Proteostasis Deficiencies/metabolism , Superoxide Dismutase/metabolism , Ubiquitin/metabolism , alpha-Synuclein/metabolism , tau Proteins/metabolismABSTRACT
Breast cancer is a major public health problem in Latin America (LA) and the most common form of cancer among women. An important variability according to ethnicity/race with respect to incidence/mortality, clinical characteristics, and prognosis is observed throughout LA. In addition, women are more likely to develop breast cancer (BC) at younger age and to be diagnosed at an advanced stage compared to western women. While little is known about specific risk factors, changes in reproductive pattern (parity, breastfeeding) and lifestyle factors including sedentary behaviours, unhealthy diet, and alcohol intake may contribute to the increase of BC incidence. In this paper we give an overview of the burden and patterns of BC, review the leading causes of BC and discuss the possible ways to improve BC prevention and control in LA.
El cáncer de mama (CaMa) es uno de los mayores problemas de salud pública en América Latina (AL) y el cáncer más frecuente en mujeres. Se observa una importante variabilidad en la incidencia/mortalidad, las características clínicas y el pronóstico según la etnia/raza a lo largo de AL. Además, las mujeres latinoamericanas son más propensas a desarrollar CaMa en edades más tempranas y a ser diagnosticadas en una etapa más avanzada, comparando con mujeres occidentales. Aunque poco se sabe sobre sus factores de riesgo específicos, cambios en los patrones reproductivos (paridad y lactancia) y estilos de vida, incluyendo los hábitos sedentarios, las dietas poco saludables y el consumo de alcohol, podrían contribuir al incremento de la incidencia del CaMa. En este artículo se da una visión general de la carga y los patrones del CaMa, se revisan las causas principales del CaMa y se discuten posibles vías para mejorar la prevención y el control del CaMa en AL.
Subject(s)
Animals , Mice , Collagenases/chemistry , Detergents/chemistry , PrPSc Proteins/isolation & purification , Sarcosine/analogs & derivatives , Scrapie/etiology , Sodium Chloride/chemistry , Chromatography, Affinity , Mice, Inbred ICR , Octoxynol/chemistry , Sarcosine/chemistry , SpleenABSTRACT
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.
Subject(s)
Animals , Mice , Rabbits , Cell Line , Kidney/metabolism , PrPSc Proteins/metabolism , Prion Diseases/metabolism , Prions/metabolism , Recombinant Proteins/metabolism , Up-RegulationABSTRACT
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.
Subject(s)
Animals , Cricetinae , Humans , Brain , Metabolism , Pathology , PrPSc Proteins , Metabolism , Prion Diseases , Genetics , Metabolism , Pathology , Up-Regulation , alpha-Crystallin B Chain , Genetics , MetabolismABSTRACT
<p><b>UNLABELLED</b>OBJECTIVE To study the potential interaction between PrP protein.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>Both 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).</p><p><b>CONCLUSION</b>The 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.</p>
Subject(s)
Animals , Cricetinae , 14-3-3 Proteins , Metabolism , Binding Sites , Brain Chemistry , Endopeptidases , Metabolism , PrPSc Proteins , Metabolism , Prion Diseases , Pathology , Prions , Metabolism , ScrapieABSTRACT
<p><b>OBJECTIVE</b>To establish a stable PrP(Sc) panel from brain tissues of experimental hamsters infected with scrapie agent 263K for evaluating diagnostic techniques of human and animals' prion diseases.</p><p><b>METHODS</b>Thirty brain tissue samples from hamsters intracerebrally infected with scrapie strain 263K and another 30 samples from normal hamsters were selected to prepare 10%, 1%, and 0.5% brain homogenates, which were aliquoted into stocks. PrP(Sc) in each brain homogenate was determined by proteinase K digestions followed by Western blot assay and partially by immunohistochemistry. Stability and glycoforms of PrP(Sc) were repeatedly detected by PrP(Sc)-specific Western blots in half a year and 3 years later.</p><p><b>RESULTS</b>PrP(Sc) signals were observed in all 10% brain homogenates of infected hamsters. Twenty out of 30 stocks and 19 out of 30 stocks were PrP(Sc) positive in 1% and 0.5% brain homogenatesof infected hamsters, respectively. Twenty-seven out of 30 stocks presented three positive bands in 10% brain homogenates, whereas none of 1% and 0.5% homogenates contained 3 bands. The detection of PrP(Sc)-specific signals stored in half a year and 3 years later demonstrated that the ratio of PrP(Sc) positive samples and glycoforms was almost unchanged. All normal hamsters' brain homogenates were PrP(Sc) negative.</p><p><b>CONCLUSION</b>A PrP(Sc) panel of prion disease can be established, which displays reliably stable PrP(Sc)-specific signals and glycoforms.</p>
Subject(s)
Animals , Cricetinae , Male , Brain , Immunohistochemistry , PrPSc Proteins , Classification , ScrapieABSTRACT
In human prion diseases, phosphorylated-tau deposition has been described in a rare genetic form, Gerstmann-Straussler-Scheinker disease, but is not considered part of the neuropathological picture of Creutzfeldt-Jakob disease. To investigate the possible changes of tau and phosphorylated tau (Ser396/Ser404) in transmissible spongiform encephalopathies (TSEs), the expressions and transcriptions of above biological factors in the brain tissues of 263K- and 139A-infected hamsters were evaluated by Western blots and Real Time PCR, respectively, followed by quantitative analyses of immunoblot images and relative transcriptional levels compared with normal animals. The contents of total tau increased, but phosphorylated tau at Ser396 and Ser404 decreased, regardless of the types of scrapie agents and clinical incubations. Transcriptions of two tau isoforms were also markedly increased. These findings suggested that dephosphorylation of tau at Ser396/Ser404 was a illness-correlative phenomenon in TSEs. Alterations of tau and phosphorylated tau (Ser396/Ser404) were either intermediate or consequent events in TSE pathogenesis and proposed the potential linkage of these bioactive proteins with the pathogenesis of prion diseases.
Subject(s)
Animals , Cricetinae , Blotting, Western , Brain , Metabolism , Gene Expression Regulation , Physiology , Phosphorylation , Polymerase Chain Reaction , PrPSc Proteins , Virulence , Prion Diseases , Metabolism , tau Proteins , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To report and study a case of sporadic family fatal insomnia (SFFI) on its.</p><p><b>METHODS</b>Investigate clinical characteristics and family disease history of a suspect FFI patient. His clinical characteristic was analyzed, he and his 14 family members genomic DNA was extracted by standard techniques from their and blood detected with polymerase chain reaction (PCR) method and DNA sequencing to find out his prion protein (PrP) gene mutation. The patient's CSF was detected with Western-Blot method for 14-3-3 brain protein.</p><p><b>RESULTS</b>The patient was diagnosed as an sporadic FFI by his developed sleep disturbance and changes in sleep-awake rhythm, motor abnormalities, mental disorder, dementia, autonomic dysfunction; his family history; his 14-3-3 brain protein-positive (CSF) and analysis results of his PrP gene (codon point mutation D178N and methionine homozygosity at position 129M/M). Suggesting that in the future to identify CJD and FFI patients, screening should focus on clinical symptoms and laboratory results. The PrP gene of 14 family members did not appear Mutation, and there is no person suffering from the same disease.</p><p><b>CONCLUSIONS</b>The case was diagnosed as a sporadic familial fatal insomnia. Analysis of suspicious patients' genomic DNA for PrP gene mutation might be very important for FFI diagnosis because there exist many difficulties in clinical laboratory evaluation. This patient might be the first SFFI patient reported in China and the case finding might have momentousness in clinical and basical study.</p>
Subject(s)
Humans , Male , Middle Aged , 14-3-3 Proteins , Genetics , Insomnia, Fatal Familial , Genetics , Mutation , PrPSc Proteins , GeneticsABSTRACT
Human prian diseases are sporadic, acquired, and genetic neurodegenerative conditions characterized by brain accumulation and deposition of pathological prion protein. These disorders are highly heterogeneous and display a wide range of clinicopathological phenotypes. This well-known phenotypic heterogeneity is thought to be determined by two main disease modifiers: [i] the genotype at polymorphic codon 129 of the prion protein gene [PRNP], allowing three possible combinations, and [ii] the physicochemical properties of the pathological prion protein, or PrPSc, existing under distinct conformational variants. In addition to PrPSc conformation, glycosylation site occupancy of PrPSc at Asp 181 and Asp 197, and truncated PrP fragments may influence the biological properties of prion strains. Here we review molecular and clinical phenotypes encountered in human prion diseases
Subject(s)
Humans , Phenotype , PrPSc Proteins , Neurodegenerative Diseases , Creutzfeldt-Jakob Syndrome , Gerstmann-Straussler-Scheinker Disease , Insomnia, Fatal FamilialABSTRACT
In order to establish an amplification system in vitro with which the PrP(Sc) is able to convert PrP(C) into proteinase K-resistant isoform infinitely and whether this system is more efficient than conventional protein misfolding cyclic amplification (PMCA), scrapie strain 263K-infected hamster's brain homogenate and homologous normal brain homogenate were prepared, respectively. A new methodology, namely serial PMCA, was utilized to reveal the continuous propagation ability of PrP(Sc). Totally 8 rounds of serial PMCA were proceeded and each round contained 48 cycles of alternative sonication and incubation. Simultaneously 144 cycles of conventional PMCA was used as a control. The results showed the PrP(Sc) from hamsters' brain tissues of scrapie agent 263K could replicate efficiently and infinitely with serial PMCA compared with finite propagation of PrP(Sc) with conventional PMCA system. The study of PrP(Sc) continuous propagation in brain homogenate with serial PMCA may further provide insight into the unsettled mechanism of prions misfolding and replication and apply to detect trace amount of PrP(Sc).
Subject(s)
Animals , Cricetinae , Brain , Metabolism , PrPC Proteins , Chemistry , PrPSc Proteins , Chemistry , Protein FoldingABSTRACT
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.
Subject(s)
Animals , Cricetinae , Blotting, Western , Methods , Brain , Metabolism , Pathology , Brain Chemistry , Chemical Precipitation , PrPSc Proteins , Chemistry , Metabolism , Prion Diseases , Diagnosis , Metabolism , Reproducibility of Results , Sensitivity and Specificity , Streptomycin , ChemistryABSTRACT
<p><b>OBJECTIVE</b>To establish a prion disease PrP(Sc) panel from the brain tissues of experimental hamsters and to address the stability of the panel conserved under the specific condition, for evaluating the diagnostic techniques of human and animal's prion diseases.</p><p><b>METHODS</b>30 brain tissues of hamsters infected with scrapie strain 263K intracerebrally and 30 ones of normal hamsters were enrolled in this panel. Each brain sample was prepared to 10%, 1% and 0.5% homogenates and aliquoted into stocks. The presences of PrP(Sc) in each brain sample were evaluated with PrP-specific Western Blots and partially with immunohistochemistry, and the stability of PrP(Sc) signals in each sample were repeatedly assessed half a year later and 3 years later.</p><p><b>RESULTS</b>PrP(Sc) signals were detected in all stocks of 10% brain homogenates from infected hamsters, 26 out of 30 stocks of 1% homogenates and 19 out of 30 stocks of 0.5% homogenates. The assessments of PrP(Sc) signals in all samples half-year and three years later demonstrated almost unchanged. All homogenates of brain tissues of normal hamsters were PrP(Sc) negative.</p><p><b>CONCLUSION</b>A prion disease PrP(Sc) panel of the brain tissues, which includes 90 PrP(Sc) positive stocks and PrP(Sc) negative ones, was successfully established, with a reliable stability of PrP(Sc) signals.</p>
Subject(s)
Animals , Humans , Male , Brain , Metabolism , Disease Models, Animal , PrPC Proteins , Pharmacokinetics , PrPSc Proteins , Prion Diseases , Metabolism , Scrapie , Metabolism , Tissue DistributionABSTRACT
<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>Compared 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.</p><p><b>CONCLUSION</b>Inoculations 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.</p>
Subject(s)
Animals , Cricetinae , Humans , Brain , Metabolism , Pathology , Gene Expression , Glial Fibrillary Acidic Protein , Genetics , Metabolism , Gliosis , Metabolism , Pathology , PrPSc Proteins , Metabolism , Prion Diseases , Metabolism , PathologyABSTRACT
<p><b>OBJECTIVE</b>To establish a methodology of protein misfolding cyclic amplification (PMCA) and utilize in the detection of PrP(Sc) in brain tissues from prion diseases.</p><p><b>METHODS</b>Different 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.</p><p><b>RESULTS</b>In 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.</p><p><b>CONCLUSION</b>A 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.</p>
Subject(s)
Animals , Cricetinae , Biochemistry , Methods , Blotting, Western , Brain , Metabolism , Pathology , PrPSc Proteins , Chemistry , Prion Diseases , Diagnosis , Metabolism , Protein Folding , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
<p><b>OBJECTIVE</b>To study the possible effect of tetracycline on protease-resistant activity in vitro and infectivity in vivo of a scrapie strain 263K.</p><p><b>METHODS</b>Scrapie pathogens were incubated with tetracycline at different concentrations for various periods of time and protease-resistant PrP signals were evaluated with proteinase K-treatment and Western blots. The preparations treated with tetracycline were intracerebrally inoculated into golden hamsters and typical TSE manifestations were noted. PrPSc in brain tissues of the infected animals was detected by PrP specific Western blot assays.</p><p><b>RESULTS</b>Protease-resistant PrP was significantly reduced in or removed from the preparations treated with tetracycline in a dose-dependant manner. Compared with the control group after incubated for 53.75 +/- 0.50 days, the preparations treated with 5 mmol/L and 20 mmol/L tetracycline prolonged the incubation time of 61.5 +/- 1.73 and 59.5 +/- 0.58 days (P < 0.05).</p><p><b>CONCLUSION</b>Treatment of scrapie pathogen 263K with tetracycline reduces or removes its protease-resistant activity in vitro.</p>
Subject(s)
Animals , Cricetinae , Brain , Pathology , Peptide Hydrolases , Metabolism , PrPSc Proteins , Metabolism , Virulence , Scrapie , Pathology , Tetracycline , Pharmacology , Time FactorsABSTRACT
Background: Creutzfeldt-Jakob disease (CJD) is a form of transmissible spongiform encephalopathy, in which a prion protein (PrP Sc) accumulates in the brain of affected individuals. Chile has a prevalence of CJD that is more than twice than in the rest of the world and has the highest rate of familial forms. These later forms are associated with the heterozygocity of codon 200 of PrP protein gene. Aim: To search susceptibility genetic markers of CJD in members of families affected by CJD. Material and methods: A blood sample was obtained from 50 individuals pertaining to four families affected by CJD. DNA from peripheral mononuclear cells was amplified by polymerase chain reaction and sequenced for the gene that codifies PrP protein. Results: In family A, 21 of 23 members were homozygotes for codon 129 (Met/Met) and eight were simultaneously heterozygotes for codon 200 (Glu/Lys). In family B, six of nine members were homozygotes for codon 129, five were heterozygotes for codon 200 and four had both mutations. In family C, the four analyzed subjects were homozygotes for codon 129 and two were simultaneously heterozygotes for codon 200. In family D, nine of 14 members were homozygotes for codon 129 and two were simultaneously homozygotes for codon 200. No family had polymorphisms for codon 219. Conclusions: Thirty two percent of analyzed subjects were homozygotes for codon 129 and heterozygotes for codon 200, condition that defines the genetic susceptibility to acquire CJD. The dominant tendency of these genotypes could explain the higher incidence of CJF in Chile.