Name: protein misfolding cyclic amplification of prions
Uploaded: 2012-11-07T12:00:00
Description: Watch this Scientific Journal Video about Protein Misfolding Cyclic Amplification of Prions at JoVE.com

Gostaríamos de agradecer ao Dr. Vesper Fe Marie Ramos para a leitura crítica do manuscrito. Este trabalho foi financiado pelo Centro Nacional de Pesquisa de Recursos (P20 RR0115635-6, C06 RR17417-01 e G20RR024001) e do Instituto Nacional de Distúrbios Neurológicos e Derrame (2R01 NS052609).

1. Preparando o Equipamento <ol><li> Usar um 3000 Misonix ou sonicador Misonix 4000 (Farmingdale, NY) ligado a um banho de Thermo Electron Neslab EX-7 água (Newington, NH) para manter uma temperatura constante de 37 ° C. Sonicar as amostras em 200 uL de paredes finas tiras de tubos PCR com tampas abobadadas obtidos a partir de Thermo Scientific (Waltham, MA). </li><li> A sonicador novo requer um &quot;break-in&quot; período de operação contínua 9. Uma quebra-in período de dois meses é composto por u...

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+proteinaceous+infectious+particles+cause+scrapie.">Novel proteinaceous infectious particles cause scrapie.</a> Science. 216, 136-144 (1982).</li><li>Saborio, G. P., Permanne, B., Soto, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+detection+of+pathological+prion+protein+by+cyclic+amplification+of+protein+misfolding.">Sensitive detection of pathological prion protein by cyclic amplification of protein misfolding.</a> Nature. 411, 810-813 (2001).</li><li>Saunders, S. E., Bartz, J. C., Vercauteren, K. C., Bartelt-Hunt, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+enzymatic+treatment+of+soil-bound+prions+effectively+inhibits+replication.">An enzymatic treatment of soil-bound prions effectively inhibits replication.</a> Appl. Environ. Microbiol. 77, 4313-4317 (2011).</li><li>Saunders, S. E., Shikiya, R. A., Langenfeld, K., Bartelt-Hunt, S. L., Bartz, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+efficiency+of+soil-bound+prions+varies+with+soil+type.">Replication efficiency of soil-bound prions varies with soil type.</a> Journal of virology. , (2011).</li><li>Schutt, C. R., Bartz, J. 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Desafios da amplificação príons infecciosos são os longos períodos de incubação e as despesas de experimentos in vivo. A técnica PMCA é um meio rentável para ampliar agentes príon infeccioso. Vários laboratórios confirmaram a capacidade de amplificar PMCA com precisão as estirpes de priões in vitro 7, 9, 12, 14, 19,24. Doenças de priões, pode ser transmitida entre as espécies. Bessen e Marsh efetivamente inoculados hamsters com encefalo...

<table border="1"><tbody><tr><td> Reagente / Equipamento </td><td> Fabricante </td><td> Gato. Número </td></tr><tr><td> Misonix 3000 </td><td> Misonix </td><td> S-3000 </td></tr><tr><td> Misonix 4000 </td><td> Misonix </td><td> S-4000 </td></tr><tr><td> Tenbroeck Grinder Tissue </td><td> Kontes </td><td> 885000-0007 </td></tr><tr><td> Neslab Bath EX-7 Água </td><td> Thermo Electron </td><td> Neslab EX-7 </td></tr><tr><td> Tubo 0,2 ml Tiras de PCR </td><td> Thermo Scientific </td><td> AB-0451 </td></tr><tr><td> Triton X-100 </td><td> Sigma Aldrich </td><td> T9284-100ML </td></tr><tr><td> Inibidor de Protease Completo </td><td> Roche </td><td> 11 697 498 001 </td></tr><tr><td> EDTA </td><td> JT Baker </td><td> 4040-00 </td></tr><tr><td> DPBS </td><td> Mallinckrodt Baker Mediatech </td><td> 21-031-CV </td></tr><tr><td> Versi-Dry Soakers Lab </td><td> Fisher Scientific </td><td> 14 206 28 </td></tr><tr><td> Aquecedor Repti Therm </td><td> Zoo Med Laboratories, Inc. </td><td> RH-4 </td></tr></tbody></table>

protein misfolding cyclic amplification of prions

Os príons são os agentes infecciosos que causam a encefalopatia espongiforme inevitavelmente fatal transmissível (EET) em animais e seres humanos 9,18. A proteína de prião tem duas isoformas distintas, a não infecciosa da proteína codificada hospedeira (PrP C) e a proteína infecciosa (PrP Sc), uma isoforma anormal de dobrado PrP C 8. Um dos desafios de trabalhar com agentes prião é o longo período de incubação antes do desenvolvimento de sinais clínicos após a inoculação de acolhimento 13. Isto tradicionalmente, imposto estudos bioensaio longas e caras animais. Além disso, as propriedades bioquímicas e biofísicas da PrP Sc ainda são pouco caracterizados devido à sua conformação invulgar e estados de agregação. PrPSc pode semear a conversão da PrP C e PrP Sc in vitro 14. PMCA é uma técnica in vitro, em que toma advantage desta capacidade utilizando ciclos de sonicação e a incubação para a produção de grandes quantidades de PrP Sc, a uma taxa acelerada, a partir de um sistema contendo quantidades em excesso de PrP C e pequenas quantidades de sementes a PrP Sc 19. Esta técnica provou ser eficaz para recapitular a especificidade de espécie e estirpe de PrP Sc conversão da PrP C, para emular interferência estirpe do prião, e para amplificar os níveis muito baixos de PrP Sc em tecidos infectados, fluidos e amostras ambientais 6,7,16, 23. Este trabalho detalha o protocolo PMCA, incluindo recomendações para minimizar a contaminação, gerando resultados consistentes, e quantificar esses resultados. Também discutimos várias aplicações PMCA, incluindo a geração e caracterização de cepas de príon infeccioso, interferência tensão prião, e na detecção de príons no ambiente.

Proteína de amplificação cíclica misfolding (PMCA) é usado para amplificar a PrP Sc in vitro 7, 12, 14, 19, 24. Uma bem sucedida PrP Sc amplificação é mostrado por um aumento na intensidade da banda em Western blots da proteína do prião PK-resistente (migrando entre 19 e 30 kDa para as estirpes derivadas do hamster prion), como mostrado na Figura 3. O aumento da intensidade da banda após a sua PMCA indica a amplificação do material resistente ao PK ...

Watch this Scientific Journal Video about Protein Misfolding Cyclic Amplification of Prions at JoVE.com

Protein Misfolding Cyclic Amplification of Prions

Proteína de amplificação cíclica misfolding (PMCA) é um ensaio in vitro em para o estudo da conversão de prião e as barreiras de tensão e espécies. Também pode ser utilizado como um ensaio de detecção de prião.

Proteína amplificação cíclica misfolding de príons

Prions  are infectious agents that cause the inevitably fatal transmissible spongiform  encephalopathy (TSE) in animals and humans9,18. The prion protein ...

Research

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