Protein Misfolding Cyclic Amplification for Diagnosis and Prion Propagation Studies

Castilla, Joaquı́n; Saá, Paula; Morales, Rodrigo; Abid, Karim; Maundrell, Kinsey; Soto, Claudio

doi:10.1016/s0076-6879(06)12001-7

Cited by 123 publications

(108 citation statements)

References 43 publications

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“…4A), well below the level of cytotoxicity. This ability of Fe(III)-TMPyP to retard prion formation was tested in a cell-free system using protein-misfolding cyclic amplification (PMCA) (42) (Fig. 4B).…”

Section: Resultsmentioning

confidence: 99%

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Pharmacological chaperone for the structured domain of human prion protein

Nicoll¹,

Trevitt

Tattum

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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In prion diseases, the misfolded protein aggregates are derived from cellular prion protein (PrP C ). Numerous ligands have been reported to bind to human PrP C (huPrP), but none to the structured region with the affinity required for a pharmacological chaperone. Using equilibrium dialysis, we screened molecules previously suggested to interact with PrP to discriminate between those which did not interact with PrP, behaved as nonspecific polyionic aggregates or formed a genuine interaction. Those that bind could potentially act as pharmacological chaperones. Here we report that a cationic tetrapyrrole [Fe(III)-TMPyP], which displays potent antiprion activity, binds to the structured region of huPrP. Using a battery of biophysical techniques, we demonstrate that Fe(III)-TMPyP forms a 1∶1 complex via the structured C terminus of huPrP with a K d of 4.5 ± 2 μM, which is in the range of its IC 50 for curing prion-infected cells of 1.6 ± 0.4 μM and the concentration required to inhibit protein-misfolding cyclic amplification. Therefore, this molecule tests the hypothesis that stabilization of huPrP C , as a principle, could be used in the treatment of human prion disease. The identification of a binding site with a defined 3D structure opens up the possibility of designing small molecules that stabilize huPrP and prevent its conversion into the disease-associated form.

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Section: Resultsmentioning

confidence: 99%

“…Protein-Misfolding Cyclic Amplification. PMCA was performed as described previously (42,59). Briefly, 10% (wt∕vol) PMCA substrate homogenates were prepared from Tg20 mice brains perfused with PBS containing 5 mM EDTA at the time of death.…”

Section: Methodsmentioning

confidence: 99%

Pharmacological chaperone for the structured domain of human prion protein

Nicoll¹,

Trevitt

Tattum

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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“…The protein misfolding cyclic amplification (PMCA) technique (3) is a powerful in vitro tool that mimics the in vivo conversion process of PrP C to PrP Sc but with accelerated kinetics, amplifying minute quantities of any PrP Sc present in a sample in an exponential fashion (4)(5)(6)(7). This technique, which considerably reduces the protracted incubation periods, is potentially very important in the prion field in general (8)(9)(10), and more specifically, for the study of the transmission barrier (5,11).…”

mentioning

confidence: 99%

Rabbits are not resistant to prion infection

Chianini

Fernández‐Borges

Vidal

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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The ability of prions to infect some species and not others is determined by the transmission barrier. This unexplained phenomenon has led to the belief that certain species were not susceptible to transmissible spongiform encephalopathies (TSEs) and therefore represented negligible risk to human health if consumed. Using the protein misfolding cyclic amplification (PMCA) technique, we were able to overcome the species barrier in rabbits, which have been classified as TSE resistant for four decades. Rabbit brain homogenate, either unseeded or seeded in vitro with disease-related prions obtained from different species, was subjected to serial rounds of PMCA. De novo rabbit prions produced in vitro from unseeded material were tested for infectivity in rabbits, with one of three intracerebrally challenged animals succumbing to disease at 766 d and displaying all of the characteristics of a TSE, thereby demonstrating that leporids are not resistant to prion infection. Material from the brain of the clinically affected rabbit containing abnormal prion protein resulted in a 100% attack rate after its inoculation in transgenic mice overexpressing rabbit PrP. Transmissibility to rabbits (>470 d) has been confirmed in 2 of 10 rabbits after intracerebral challenge. Despite rabbits no longer being able to be classified as resistant to TSEs, an outbreak of “mad rabbit disease” is unlikely.

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“…In order to promote the further development of effective disinfectants, it might therefore be helpful to compare and validate candidate formulations against human prions with a focus on the most relevant human TSEs such as sCJD/subtype MM1 or vCJD. Additionally, alternative techniques such as protein misfolding cyclic amplification (Castilla et al, 2006;Jones et al, 2007) or novel cell culture assays may be considered as potential surrogate methods for bioassays in animals.…”

Section: Discussion Efficacy Of Tested Formulations For Broad-range Dmentioning

confidence: 99%

Fast, broad-range disinfection of bacteria, fungi, viruses and prions

Beekes

Lemmer

Thomzig

et al. 2009

Journal of General Virology

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Effective disinfectants are of key importance for the safe handling and reprocessing of surgical instruments. This study tested whether new formulations containing SDS, NaOH and 1-propanol (n-propanol) are simultaneously active against a broad range of pathogens including bacteria, fungi, non-enveloped viruses and prions. Inactivation and disinfection were examined in suspension and on carriers, using coagulated blood or brain homogenate as an organic contaminant. Coomassie blue staining was used to assess whether the formulations undesirably fixed proteins to rough surfaces. A mixture of 0.2 % SDS and 0.3 % NaOH in 20 % n-propanol achieved potent decontamination of steel carriers contaminated with PrP TSE , the biochemical marker for prion infectivity, from 263K scrapie hamsters or from patients with sporadic or variant Creutzfeldt-Jakob disease. 263K scrapie infectivity on carriers was decreased by ¢5.5 logs. Furthermore, the formulation effectively inactivated poliovirus, hepatitis A virus and caliciviruses (including murine norovirus) in suspension tests. It also yielded significant titre reductions of bacteria (Enterococcus faecium, Mycobacterium avium; .6 logs), fungi (spores of Aspergillus niger; ¢5 logs) and poliovirus (.4 logs) embedded in coagulated blood on carriers. The formulation was not found to fix proteins more than was observed with water as the cleaning reagent. In conclusion, SDS, NaOH and n-propanol can synergistically achieve fast, broad-range disinfection.

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Protein Misfolding Cyclic Amplification for Diagnosis and Prion Propagation Studies

Cited by 123 publications

References 43 publications

Pharmacological chaperone for the structured domain of human prion protein

Pharmacological chaperone for the structured domain of human prion protein

Rabbits are not resistant to prion infection

Fast, broad-range disinfection of bacteria, fungi, viruses and prions

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