Efficiency of Western blotting for the specific immunodetection of proteinase K-resistant prion protein in BSE diagnosis in France

2000 ◽  
Vol 146 (3) ◽  
pp. 74-76 ◽  
Author(s):  
J.-Y. Madec ◽  
P. Belli ◽  
D. Calavas ◽  
T. Baron
Keyword(s):  
Prion Protein ◽  
Western Blotting ◽  
Proteinase K

scholarly journals Efficient Conversion of Normal Prion Protein (PrP) by Abnormal Hamster PrP Is Determined by Homology at Amino Acid Residue 155

2001 ◽  
Vol 75 (10) ◽  
pp. 4673-4680 ◽  
Author(s):  
Suzette A. Priola ◽  
Joëlle Chabry ◽  
Kaman Chan
Keyword(s):  
Amino Acid ◽  
Prion Protein ◽  
Amino Acid Residue ◽  
Animal Species ◽  
Alpha Helix ◽  
Proteinase K ◽  
Transmissible Spongiform Encephalopathies ◽  
Spongiform Encephalopathies ◽  
A Cell ◽  
Resistant Product

ABSTRACT In the transmissible spongiform encephalopathies, disease is closely associated with the conversion of the normal proteinase K-sensitive host prion protein (PrP-sen) to the abnormal proteinase K-resistant form (PrP-res). Amino acid sequence homology between PrP-res and PrP-sen is important in the formation of new PrP-res and thus in the efficient transmission of infectivity across species barriers. It was previously shown that the generation of mouse PrP-res was strongly influenced by homology between PrP-sen and PrP-res at amino acid residue 138, a residue located in a region of loop structure common to PrP molecules from many different species. In order to determine if homology at residue 138 also affected the formation of PrP-res in a different animal species, we assayed the ability of hamster PrP-res to convert a panel of recombinant PrP-sen molecules to protease-resistant PrP in a cell-free conversion system. Homology at amino acid residue 138 was not critical for the formation of protease-resistant hamster PrP. Rather, homology between PrP-sen and hamster PrP-res at amino acid residue 155 determined the efficiency of formation of a protease-resistant product induced by hamster PrP-res. Structurally, residue 155 resides in a turn at the end of the first alpha helix in hamster PrP-sen; this feature is not present in mouse PrP-sen. Thus, our data suggest that PrP-res molecules isolated from scrapie-infected brains of different animal species have different PrP-sen structural requirements for the efficient formation of protease-resistant PrP.

scholarly journals Genetic human prion disease modelled in PrP transgenic Drosophila

2017 ◽  
Vol 474 (19) ◽  
pp. 3253-3267 ◽  
Author(s):  
Alana M. Thackray ◽  
Alzbeta Cardova ◽  
Hanna Wolf ◽  
Lydia Pradl ◽  
Ina Vorberg ◽  
...  
Keyword(s):  
Prion Protein ◽  
Prion Disease ◽  
Prion Diseases ◽  
Principal Component ◽  
Site Directed Mutagenesis ◽  
Host Protein ◽  
Therapeutic Interventions ◽  
Proteinase K ◽  
Human Prion Disease ◽  
Transgenic Drosophila

Inherited human prion diseases, such as fatal familial insomnia (FFI) and familial Creutzfeldt–Jakob disease (fCJD), are associated with autosomal dominant mutations in the human prion protein gene PRNP and accumulation of PrPSc, an abnormal isomer of the normal host protein PrPC, in the brain of affected individuals. PrPSc is the principal component of the transmissible neurotoxic prion agent. It is important to identify molecular pathways and cellular processes that regulate prion formation and prion-induced neurotoxicity. This will allow identification of possible therapeutic interventions for individuals with, or at risk from, genetic human prion disease. Increasingly, Drosophila has been used to model human neurodegenerative disease. An important unanswered question is whether genetic prion disease with concomitant spontaneous prion formation can be modelled in Drosophila. We have used pUAST/PhiC31-mediated site-directed mutagenesis to generate Drosophila transgenic for murine or hamster PrP (prion protein) that carry single-codon mutations associated with genetic human prion disease. Mouse or hamster PrP harbouring an FFI (D178N) or fCJD (E200K) mutation showed mild Proteinase K resistance when expressed in Drosophila. Adult Drosophila transgenic for FFI or fCJD variants of mouse or hamster PrP displayed a spontaneous decline in locomotor ability that increased in severity as the flies aged. Significantly, this mutant PrP-mediated neurotoxic fly phenotype was transferable to recipient Drosophila that expressed the wild-type form of the transgene. Collectively, our novel data are indicative of the spontaneous formation of a PrP-dependent neurotoxic phenotype in FFI- or CJD-PrP transgenic Drosophila and show that inherited human prion disease can be modelled in this invertebrate host.

scholarly journals Effect of Non-Concentrated and Concentrated Vaporized Hydrogen Peroxide on Scrapie Prions

Pathogens ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 947
Author(s):  
Akikazu Sakudo ◽  
Risa Yamashiro ◽  
Chihiro Harata
Keyword(s):  
Hydrogen Peroxide ◽  
Prion Protein ◽  
Protein Misfolding ◽  
Soft Mode ◽  
Brain Homogenate ◽  
Proteinase K ◽  
Mouse Bioassay ◽  
Half Cycle ◽  
Cover Glass ◽  
Standard Mode

To date, there have been no studies on the sterilization of prions by non-concentrated and concentrated vaporized hydrogen peroxide (VHP) applied by the same instrument. Here, the effect of the two types of VHP applied using an ES-700 sterilizer on prions was investigated. Brain homogenate from scrapie (Chandler) prion-infected mice was spotted on a cover glass and subjected to ES-700 treatment in soft (non-concentrated VHP from 59% hydrogen peroxide) or standard (concentrated VHP from 80% hydrogen peroxide) mode. Proteinase K-resistant prion protein (PrPres), an indicator of the abnormal isoform of prion protein (PrPSc), was reduced by ES-700 treatment under several conditions: SFT1/4 (soft mode, quarter cycle), SFT1/2 (soft mode, half cycle), SFT1 (soft mode, full cycle), and STD1/2 (standard mode, half cycle). PrPres was detected after the first and second rounds of protein misfolding cyclic amplification (PMCA) of untreated samples, but was undetectable in SFT1/4, SFT1/2, SFT1, and STD1/2 treated samples. In a mouse bioassay, SFT1/2 and STD1/2 treatment of prions significantly prolonged survival time, suggesting that prion infectivity is reduced after ES-700 treatment. In summary, both non-concentrated and concentrated VHP inactivate prions and may be useful for the low-temperature sterilization of prion-contaminated medical devices.

scholarly journals Synergistic and strain-specific effects of bovine spongiform encephalopathy and scrapie prions in the cell-free conversion of recombinant prion protein

2006 ◽  
Vol 87 (12) ◽  
pp. 3753-3761 ◽  
Author(s):  
Martin Eiden ◽  
Gottfried J. Palm ◽  
Winfried Hinrichs ◽  
Ulrich Matthey ◽  
Ralph Zahn ◽  
...  
Keyword(s):  
Bovine Spongiform Encephalopathy ◽  
Prion Protein ◽  
De Novo ◽  
Proteinase K ◽  
Spongiform Encephalopathy ◽  
Cooperative Action ◽  
Specific Effects ◽  
Scrapie Strains ◽  
Recombinant Prion Protein

This study describes the conversion of murine PrPC by PrPSc from three different mouse scrapie strains (ME7, 87V and 22A) and from a mouse-passaged bovine spongiform encephalopathy (BSE) strain (BSE/Bl6). This was demonstrated by a modified, non-radioactive, cell-free conversion assay using bacterial prion protein, which was converted into a proteinase K (PK)-resistant fragment designated PrPres. Using this assay, newly formed PrPres could be detected by an antibody that discriminated de novo PrPres and the original PrPSc seed. The results suggested that PrPres formation occurs in three phases: the first 48 h when PrPres formation is delayed, followed by a period of substantially accelerated PrPres formation and a plateau phase when a maximum concentration of PrPres is reached after 72 h. The conversion of prokaryotically expressed PrPC by ME7 and BSE prions led to unglycosylated, PK-digested, abnormal PrPres fragments, which differed in molecular mass by 1 kDa. Therefore, prion strain phenotypes were retained in the cell-free conversion, even when recombinant PrPC was used as the substrate. Moreover, co-incubation of ME7 and BSE prions resulted in equal amounts of both ME7- and BSE-derived PrPres fragments (as distinguished by their different molecular sizes) and also in a significantly increased total amount of de novo-generated PrPres. This was found to be more than twice the amount of either strain when incubated separately. This result indicates a synergistic effect of both strains during cell-free conversion. It is not yet known whether such a cooperative action between BSE and scrapie prions also occurs in vivo.

scholarly journals PrPSc in Salivary Glands of Scrapie-Affected Sheep

2007 ◽  
Vol 81 (9) ◽  
pp. 4872-4876 ◽  
Author(s):  
Marta Vascellari ◽  
Romolo Nonno ◽  
Franco Mutinelli ◽  
Michela Bigolaro ◽  
Michele Angelo Di Bari ◽  
...  
Keyword(s):  
Salivary Glands ◽  
Prion Protein ◽  
Western Blotting ◽  
Horizontal Transmission ◽  
Infected Sheep ◽  
Healthy Controls

ABSTRACT The salivary glands of scrapie-affected sheep and healthy controls were investigated for the presence of the pathological prion protein (PrPSc). PrPSc was detected in major (parotid and mandibular) and minor (buccal, labial, and palatine) salivary glands of naturally and experimentally infected sheep. Using Western blotting, the PrPSc concentration in glands was estimated to be 0.02 to 0.005% of that in brain. Immunohistochemistry revealed intracellular depositions of PrPSc in ductal and acinar epithelia and occasional labeling in the lumina of salivary ducts. The presence of PrPSc in salivary glands highlights the possible role of saliva in the horizontal transmission of scrapie.

scholarly journals Distinct Proteinase K-Resistant Prion Protein Fragment in Goats with No Signs of Disease in a Classical Scrapie Outbreak

2011 ◽  
Vol 49 (6) ◽  
pp. 2109-2115 ◽  
Author(s):  
Ilias G. Bouzalas ◽  
Florian Lörtscher ◽  
Chrysostomos I. Dovas ◽  
Anna Oevermann ◽  
Jan P. M. Langeveld ◽  
...  
Keyword(s):  
Prion Protein ◽  
Classical Scrapie ◽  
Proteinase K ◽  
Protein Fragment

3-Methyl-4-Chlorophenol for Prion Decontamination of Medical Devices

2006 ◽  
Vol 27 (7) ◽  
pp. 778-780 ◽  
Author(s):  
Constanze Riemer ◽  
Theresa Bamme ◽  
Simon Wing Fai Mok ◽  
Michael Baier
Keyword(s):  
Prion Protein ◽  
Medical Devices ◽  
Limit Of Detection ◽  
Terminally Ill ◽  
Infectious Agent ◽  
Proteinase K

Disinfectants containing 3-methyl-4-chlorophenol were tested for their capacity to inactivate the infectious agent of scrapie. Coincubation of brain homogenates prepared from terminally ill scrapie-infected hamsters with the disinfectants rendered the prion protein PrPSc sensitive to proteinase K digestion. Inoculation of hamsters with disinfectant-treated samples indicated a reduction in infectivity levels to below the limit of detection.

scholarly journals Proteinase K-Resistant Material in ARR/VRQ Sheep Brain Affected with Classical Scrapie Is Composed Mainly of VRQ Prion Protein

2011 ◽  
Vol 85 (23) ◽  
pp. 12537-12546 ◽  
Author(s):  
J. G. Jacobs ◽  
A. Bossers ◽  
H. Rezaei ◽  
L. J. M. van Keulen ◽  
S. McCutcheon ◽  
...  
Keyword(s):  
Prion Protein ◽  
Classical Scrapie ◽  
Proteinase K ◽  
Resistant Material ◽  
Sheep Brain

Sanitization of Equipment and Material Used in the Production of Plasma Derivatives by Standard Procedures Results in Removal and Inactivation of Prions.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3641-3641
Author(s):  
Albrecht Groener ◽  
Waltraud Seyfert-Brandt ◽  
Wolfram Schäfer
Keyword(s):  
Prion Protein ◽  
Sandwich Elisa ◽  
Reduction Factor ◽  
Proteinase K ◽  
Spongiform Encephalopathy ◽  
Infectious Agents ◽  
Medicinal Products ◽  
Cleaning Procedures ◽  
The Difference ◽  
Alkaline Cleaning

Abstract Prions are novel infectious agents causing neurodegenerative disorders such as scrapie, bovine spongiform encephalopathy (BSE), and (variant) Creutzfeldt-Jakob disease ((v)CJD). The infectious agents consist mainly, if not exclusively, of a malformed protein termed PrPSc, which accumulates in the brain of infected individuals. This PrPSc is resistant to proteinase K treatment. As there are concerns that minute amounts of prions from a donor in the incubation period for CJD / vCJD may contaminate the equipment and material used for the production of plasma-derived medicinal products and, therefore, may taint subsequently produced batches of these products, we evaluated the cleaning capacity of selected cleaning procedures used within the production of plasma proteins: NaOH and commercial alkaline cleaning solutions at different concentrations to clean and sanitize equipment and materials (e.g., resin). Sanitization of equipment was evaluated employing microsomal preparations from brains of Sc237-infected hamsters dried onto stainless steel coupons for 1 day, subsequently placed in 0.1 N NaOH, various concentrations of commercial cleaning solutions CIP-100 / CIP-150, and water as a control, respectively, followed by rinsing in water, and recovered from the coupons by wiping off with swabs. The cleaning efficacy was calculated as the difference of the recovered prion load (in log10) of a coupon with dried-on PrPSc without any further treatment and after treatment with 0.1 N NaOH or commercial cleaning solutions. PrPSc was quantified utilizing a Conformation Dependent Immunoassay (CDI) [Safar et al., Nat Med1998; 4: 1157–1165] in a sandwich configuration. In addition, prion inactivation by NaOH and commercial alkaline cleaning solutions could be demonstrated when prions were incubated in these solutions and treated with proteinase K. Furthermore, the removal of prions from chromatographic resins was studied by incubation of resin with microsomes from Sc237 hamster brain, extensive washing of the resin and subsequently incubating the resin in either water for injection or in 0.1 N NaOH followed by a treatment of both samples with or without proteinase K (PK). In order to extract all residual prion protein from the resin, all four samples were incubated at elevated temperature in guanidinium-HCl and residual prions were quantified utilizing a sandwich ELISA format according to the CDI. From the PK treated sanitized resin no residual prion protein could be detected resulting in a reduction factor of ≥3 log10 compared to non-sanitized (WFI incubated) resin. The experimental data clearly demonstrate that prions, if they were in plasma, would be removed from the surface of equipment and resins used in the production of plasma-derived products and furthermore inactivated by standard cleaning procedures according to cGMP. Therefore, a potential risk for batches of plasma-derived medicinal products produced in succession due to cross-contamination by carry-over of prions can be excluded.

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