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

Vi vil gerne takke Dr. Vesper Fe Marie Ramos for kritisk læsning af manuskriptet. Dette arbejde blev støttet af National Center for Forskning Ressourcer (P20 RR0115635-6, C06 RR17417-01 og G20RR024001) og Statens Institut for Neurologiske Lidelser (2R01 NS052609).

1. Klargøring af udstyr <ol><li> Brug en Misonix 3000 eller Misonix 4000 sonikator (Farmingdale, NY) forbundet med en Thermo Electron Neslab EX-7 vandbad (Newington, NH) for at holde en konstant temperatur på 37 ° C. Soniker prøverne i 200 ul tyndvæggede PCR-rør strips med hvælvede låg opnået fra Thermo Scientific (Waltham, MA). </li><li> En helt ny sonikator kræver et &quot;break-in&quot; periode med kontinuerlig drift 9. En to-måneders pause-periode består af en 40-sec sonication burst og 10-m...

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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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Udfordringer for uddybende smitsomme prionproteiner er de lange inkubationstid og udgifter i in vivo forsøg. Det PMCA teknik er en omkostningseffektiv måde at amplificere smitsomme prion agenter. Adskillige laboratorier har bekræftet evne PMCA til præcist at amplificere prion-stammer in vitro 7, 9, 12, 14, 19,24. Prionsygdomme kan overføres mellem arter. Bessen og Marsh har faktisk inokuleret hamstere med transmissibel minkencephalopati, der producerede to f...

<table border="1"><tbody><tr><td> Reagens / Udstyr </td><td> Producent </td><td> Kat. Nummer </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 vævsformaler </td><td> Kontes </td><td> 885000-0007 </td></tr><tr><td> Neslab EX-7 vandbad </td><td> Thermo Electron </td><td> Neslab EX-7 </td></tr><tr><td> 0,2 ml PCR-rør Strips </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> Complete Protease Inhibitor </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 Heater </td><td> Zoo Med Laboratories, Inc. </td><td> RH-4 </td></tr></tbody></table>

protein misfolding cyclic amplification of prions

Prioner er infektiøse stoffer, der medfører uundgåeligt fatal transmissible spongiforme encephalopatier (TSE) hos dyr og mennesker 9,18. Prionproteinet har to forskellige isoformer, de ikke-infektiøse host-kodede protein (PrP C) og smitsomt protein (PrP Sc), et unormalt-foldet isoform af PrP C 8. En af udfordringerne ved at arbejde med prion midler er den lange inkubationstid før udviklingen af kliniske symptomer efter vært podning 13. Dette traditionelt pålagt lange og dyre dyr bioassay undersøgelser. Endvidere er de biokemiske og biofysiske egenskaber af PrPsc dårligt karakteriseret på grund af deres usædvanlige konformation og aggregering tilstande. PrPsc kan frø omdannelsen af PrP C til PrP Sc in vitro 14. PMCA er en in vitro-teknik, der tager advantage af denne evne ved hjælp af sonikering og inkubering cyklusser for at producere store mængder af PrP Sc, med en accelereret hastighed, fra et system indeholdende overskydende mængder af PrP ° og små mængder af PrP Sc frø 19. Denne teknik har vist sig effektivt at rekapitulere arten og stammen specificiteten af PrPsc konvertering fra PrP C, for at emulere prion stamme indgreb, og til amplifikation af meget lave niveauer af PrP Sc fra inficerede væv, væsker, og miljøprøver 6,7,16, 23. Dette papir beskriver den PMCA protokol, herunder anbefalinger til minimering af forurening, genererer ensartede resultater, og kvantificere disse resultater. Vi diskuterer også adskillige PMCA applikationer, herunder generering og karakterisering af smittebærere prion stammer, prion stamme indgreb og påvisning af prioner i miljøet.

Protein misfoldning cyklisk amplifikation (PMCA) anvendes til at amplificere PrP Sc in vitro 7, 12, 14, 19, 24. En vellykket PrPsc amplifikation er vist med en stigning i båndintensitet på Western blots af PK-resistent prionprotein (migrerer mellem 19 og 30 kDa for hamster-afledte prion-stammer) som vist i fig. 3. Stigningen i sin båndintensitet efter PMCA indikerer amplificering af PK-resistent PrPsc materiale. Vellykkede amplifikation af hamster-...

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

Protein Misfolding Cyclic Amplification of Prions

Protein misfoldning cyklisk amplifikation (PMCA) er et in vitro assay til undersøgelse af prion konvertering og stamme og artsbarrierer. Den kan også anvendes som et prion påvisningsassay.

Protein misfoldning Cyklisk Amplifikation af Prioner

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

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