Abnormal isoform of prion proteins (PrP
Sc), which are infectious agents associated with prion diseases, retain infectivity after undergoing routine sterilization processes. A sensitive method to ...detect the infectivity is a bioassay, and it has been used for assessing prion inactivation. However, the result is obtained after several hundred days. Here, protein misfolding cyclic amplification (PMCA) in which PrP
Sc can be amplified
in vitro was applied for assessing prion inactivation by dry heating and autoclaving. Scrapie-infected hamster brains were inactivated under various conditions, and residual infectivity and PrP
Sc were detected by the bioassay and PMCA, respectively. The PMCA results were in good agreement with those of the bioassay. In samples autoclaved at temperatures below 150
°C, while infected mice died in the bioassay, protease-resistant PrP (PrP
res) signals were detected in the second or third round of PMCA. Three rounds of PMCA require only 6 days, which means that the PMCA method is much faster than the bioassay.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
The E200K mutation of the human prion protein (PrP) is known to cause familial Creutzfeldt-Jakob disease. In order to elucidate the effects of the mutation on the local structural stability of PrP, ...we performed ab initio fragment molecular orbital calculations for the wild-type human PrP and the E200K variant modeled under neutral and mild acidic conditions. The calculations revealed that this substitution markedly altered the intramolecular interactions in the PrP, suggesting that the local structural instabilities induced by the E200K mutation might cause initial denaturation of the PrP and its subsequent conversion to a pathogenic form. This work presents a new approach for quantitatively elucidating structural instabilities in proteins that cause misfolding diseases.
ABSTRACT
Prions, infectious agents causing TSEs, are composed primarily of the pathogenic form (PrPSc) of the PrPC. The susceptibility of sheep to scrapie is determined by polymorphisms in the coding ...region of the PRNP, mainly at codons 136, 154, and 171. The efficiency of in vitro amplification of sheep PrPSc seems to be linked also to the PrP genotype. PrPSc derived from sheep with V136R154Q171‐associated genotypes can be amplified efficiently by PMCA in the presence of additional polyanion such as poly A, but there are no reports that cite ultrasensitive detection of PrPSc derived from sheep of other PrP genotypes. We report here that sheep PrPSc derived from ARQ and AHQ homozygotes was amplified efficiently by serial PMCA using mouse brain homogenate as PrPC substrate. ARQ/ARQ PrPSc was detected in infected brain homogenates diluted up to 10−10 after five rounds of amplification, and AHQ/AHQ PrPSc was detected in samples diluted up to 10−8 after four rounds of amplification. On the other hand, amplification of PrPSc from VRQ/ARQ sheep seemed to be less efficient under the experimental conditions used. The interspecies PMCA developed in this study may be useful in the detailed analysis of PrPSc distribution in classical scrapie‐infected ARQ and AHQ homozygote sheep.
Full text
Available for:
BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
An alkaline-based chemical antigen retrieval pretreatment step was used to enhance immunolabeling of disease-associated prion protein (PrPSc) in formalin-fixed and paraffin-embedded tissue sections ...from cattle naturally affected with bovine spongiform encephalopathy (BSE). The modified chemical method used in this study amplified the PrPSc signal by unmasking PrPSc compared with the normal cellular prion protein. In addition, this method reduced nonspecific background immunolabeling that resulted from the destruction of the residual normal cellular form of prion protein, and reduced the treatment time compared with the usual autoclave pretreatment step. Immunolabeled PrPSc was thereby clearly detected in the myenteric plexus of the ileum in naturally occurring BSE cattle.
L-type bovine spongiform encephalopathy (BSE) is an atypical form of BSE. To characterize the Japanese L-type BSE prion, we conducted a comparative study of the Japanese and foreign L-type BSE ...isolates. The L-type BSE isolates of Japan, Germany, France and Canada were intracerebrally inoculated into bovinized prion protein-overexpressing transgenic mice (TgBoPrP). All the examined L-type BSE isolates were transmitted to TgBoPrP mice, and no clear differences were observed in their biological and biochemical properties. Here, we present evidence that the Japanese and Canadian L-type BSE prions are identical to those from the European cases.
An abnormal isoform of the prion protein, associated with transmissible spongiform encephalopathies, retains infectivity even after undergoing routine sterilization processes. We found that a ...formulation of iron ions combined with hydrogen peroxide effectively reduced infectivity and the level of abnormal isoforms of the prion protein in scrapie-infected brain homogenates. Therefore, the Fenton reaction has potential for prion decontamination.
Prions, infectious agents causing transmissible spongiform encephalopathy, retain infectivity even after undergoing routine sterilization processes. We found that MSK103 protease, identified in our ...previous study, effectively reduces infectivity and the level of misfolded isoform of the prion protein in scrapie-infected brain homogenates in the presence of SDS. The treatment therefore can be applied to the decontamination of thermolabile instruments.
Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative disorder of cattle characterized by accumulation of the disease-associated prion protein (PrPSc) in the central nervous system ...(CNS). The immunohistochemical patterns and distribution of PrPSc were investigated in the CNS, brains, and spinal cords of 7 naturally occurring BSE cases confirmed by the fallen stock surveillance program in Japan. No animals showed characteristic clinical signs of the disease. Coronal slices of 14 different brain areas in each case were immunohistochemically analyzed using an anti-prion protein antibody. Immunolabeled PrPSc deposition was widely observed throughout each brain and spinal cord. Intense PrPSc deposition was greater in the thalamus, brainstem, and spinal cord of the gray matter than in the neocortices. The topographical distribution pattern and severity of PrPSc accumulation were mapped and plotted as immunohistochemical profiles of the different brain areas along the caudal-rostral axis of the brain. The distribution pattern and severity of the immunolabeled PrPSc in the CNS were almost the same among the 7 cases analyzed, suggesting that the naturally occurring cases in this study were at the preclinical stage of the disease. Immunohistochemical mapping of the PrPSc deposits will be used to clarify the different stages of BSE in cattle.
The human PrP gene (PRNP) has two major polymorphic codons: 129 for methionine (M) or valine (V), and 219 for glutamate (E) or lysine (K). The PRNP heterozygotes appear to be protected from sporadic ...CJD compared to the PRNP homozygotes. The molecular mechanism responsible for these protective effects of PRNP heterozygosity has remained elusive. In this review, we describe the inhibition of PrP conversion observed in a series of transmission studies using PRNP heterozygous animal models. In vCJD infection, the conversion incompetent human PrP 129V molecules showed an inhibitory effect on the conversion of human PrP 129M molecules in the 129M/V heterozygous mice. Furthermore, though the human PrP 219E and PrP 219K were both conversion competent in vCJD infection, these conversion competent PrP molecules showed an inhibitory effect in the 219E/K heterozygous animals. To explain this heterozygous inhibition, we propose a possible mechanism designated as the stone fence model.
PDF
