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

Vorremmo ringraziare il Dott. Vesper Fe Marie Ramos per la lettura critica del manoscritto. Questo lavoro è stato sostenuto dal National Center for Research Resources (P20 RR0115635-6, C06-01 e RR17417 G20RR024001) e l&#39;Istituto Nazionale per la Neurological Disorder and Stroke (2R01 NS052609).

1. Preparazione dell&#39;apparecchiatura <ol><li> Utilizzare un 3000 Misonix o Misonix 4000 sonicatore (Farmingdale, NY) collegato ad un bagno di Thermo Electron Neslab EX-7 acqua (Newington, NH) per mantenere una temperatura costante di 37 ° C. Sonicare i campioni in 200 ml a pareti sottili strisce di provette PCR con tappi a cupola ottenuti da Thermo Scientific (Waltham, MA). </li><li> Un sonicatore nuovo richiede un periodo di &quot;rodaggio&quot; di funzionamento continuo 9. A due mesi il periodo di ro...

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+scrapie-associated+form+of+PrP+is+made+from+a+cell+surface+precursor+that+is+both+protease-+and+phospholipase-sensitive.">The scrapie-associated form of PrP is made from a cell surface precursor that is both protease- and phospholipase-sensitive.</a> J. Biol. Chem. 266, 18217-18223 (1991).</li><li>Deleault, N. R., Harris, B. T., Rees, J. R., Supattapone, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+native+prions+from+minimal+components+in+vitro.">Formation of native prions from minimal components in vitro.</a> Proceedings of the National Academy of Sciences of the United States of America. 104, 9741-9746 (2007).</li><li>Dickinson, A. G., Fraser, H., Meikle, V. M., Outram, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Competition+between+different+scrapie+agents+in+mice.">Competition between different scrapie agents in mice.</a> Nat. New Biol. 237, 244-245 (1972).</li><li>Gonzalez-Romero, D., Barria, M. A., Leon, P., Morales, R., 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=Detection+of+infectious+prions+in+urine.">Detection of infectious prions in urine.</a> FEBS Lett. 582, 3161-3166 (2008).</li><li>Green, K. M., Castilla, J., Seward, T. S., Napier, D. L., Jewell, J. E., Soto, C., Telling, G. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+sources+of+scrapie+prions.">Environmental sources of scrapie prions.</a> J. Virol. 84, 11560-11562 (2010).</li><li>Nichols, T. A., Pulford, B., Wyckoff, A. C., Meyerett, C., Michel, B., Gertig, K., Hoover, E. A., Jewell, J. E., Telling, G. C., Zabel, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+protease-resistant+cervid+prion+protein+in+water+from+a+CWD-endemic+area.">Detection of protease-resistant cervid prion protein in water from a CWD-endemic area.</a> Prion. 3, 171-183 (2009).</li><li>Prusiner, S. 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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Le sfide di amplificazione proteine ​​prioniche infettive sono i periodi di incubazione lunghi e le spese in esperimenti in vivo. La tecnica PMCA è una soluzione economica per amplificare agenti prioni infettivi. Diversi laboratori hanno confermato la capacità di PMCA di amplificare accuratamente ceppi di prioni in vitro 7, 9, 12, 14, 19,24. Malattie da prioni possono essere trasmessi tra le specie. Bessen e Marsh sono effettivamente inoculato cric...

<table border="1"><tbody><tr><td> Reagente / attrezzature </td><td> Fabbricante </td><td> Cat. Numero </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 Tissue Grinder </td><td> Kontes </td><td> 885000-0007 </td></tr><tr><td> Neslab Bagno EX-7 Acqua </td><td> Thermo Electron </td><td> Neslab EX-7 </td></tr><tr><td> PCR da 0,2 ml tubo strisce </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> Proteasi 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 Lab Soakers </td><td> Fisher Scientific </td><td> 14 206 28 </td></tr><tr><td> Repti Therm Riscaldatore </td><td> Zoo Med Laboratories, Inc. </td><td> RH-4 </td></tr></tbody></table>

protein misfolding cyclic amplification of prions

I prioni sono gli agenti infettivi che causano l&#39;encefalopatia inevitabilmente fatale spongiforme trasmissibile (TSE) negli animali e nell&#39;uomo 9,18. La proteina prionica ha due isoforme distinte, non infettiva ospitante proteina codificata (PrP C) e la proteina infettiva (PrP Sc), una isoforma anormalmente piegato PrP-C 8. Una delle sfide di lavorare con agenti prioni è il lungo periodo di incubazione prima dello sviluppo dei sintomi clinici dopo l&#39;inoculazione ospite 13. Questo mandato tradizionalmente lunghi e costosi studi di saggi biologici animali. Inoltre, le proprietà biochimiche e biofisiche di PrP Sc sono poco caratterizzati per la loro particolare conformazione e gli stati di aggregazione. PrPSc può seme della conversione della PrP C e PrP Sc in vitro 14. PMCA è una tecnica in vitro che prende ADVAntage di questa capacità con cicli di sonicazione e di incubazione per la produzione di grandi quantità di PrP Sc, ad un ritmo accelerato, da un sistema che contiene una quantità eccessiva di PrP C e piccole quantità di seme PrP Sc 19. Questa tecnica ha dimostrato di ricapitolare in modo efficace la specificità di specie e la tensione di PrP Sc conversione da PrP C, per emulare ceppo interferenze prioni, e di amplificare livelli molto bassi di PrP Sc da tessuti infetti, fluidi, e campioni ambientali 6,7,16, 23. Questa carta dettagli del protocollo di PMCA, comprese le raccomandazioni per minimizzare la contaminazione, la generazione di risultati coerenti, e quantificare tali risultati. Discutiamo anche diverse applicazioni PMCA, compresa la produzione e la caratterizzazione dei ceppi di prioni infettivi, ceppo interferenza prione, e l&#39;individuazione dei prioni nell&#39;ambiente.

Misfolding amplificazione proteina ciclica (PMCA) è utilizzato per amplificare PrP Sc in vitro 7, 12, 14, 19, 24. Un successo PrP Sc amplificazione è indicato da un aumento di intensità banda su Western blot della PK-resistente proteina prionica (migrazione tra 19 e 30 kDa per criceto derivati ​​ceppi di prioni) come mostrato in Figura 3. L&#39;aumento della sua intensità band dopo PMCA indica amplificazione del PK-materiale resistente PrPSc. ...

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

Protein Misfolding Cyclic Amplification of Prions

Misfolding amplificazione proteina ciclica (PMCA) è un test in vitro per lo studio della conversione prionica e barriere deformazione e specie. Può anche essere usato come un test di rilevazione prione.

Proteine ​​ciclica Amplificazione misfolding di prioni

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

Research

JoVE Journal

Immunology and Infection

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

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Significant efforts were gathered to generate  large-scale comprehensive protein-protein interaction network maps. This is  instrumental to understand the ...

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Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission ...

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Dimostrazione di un multi-resistente ai farmaci<em&gt; Mycobacterium tuberculosis</em&gt; Amplificazione Microarray

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In tempo reale tremare-indotta conversione analisi per rilevazione di CWD prioni nel materiale fecale

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant ...

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Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the ...

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Proteine ​​ciclica Amplificazione misfolding di prioni

Proteine ciclica Amplificazione misfolding di prioni