Filling The Holes Of Spongiform Encephalopathies

Until a few years ago it was believed that a disease could only be transmitted from one organism to another through a virus or microorganism (1). Evidence now exists that transmission of a protein may be the cause of a class of diseases known as Transmissible Spongiform Encephalopathies (TSEs). The most well known of these diseases is bovine spongiform encephalopathy or mad cow disease. Other forms include scrapie in sheep, and Creutzfeldt-Jakob Disease (CJD) in humans (2). The protein responsible for these diseases has been termed prion-related-protein or PrP. The normal, cellular protein (PrPc) is found in many animals including humans. Prions are transmissible particles that are composed exclusively of a modified protein (PrPsc) (2). Under certain conditions, not yet fully understood, a conformational shift occurs of PrPc to a disease causing prion termed prion-related protein scapies (PrPsc). This shift causes the amino-terminus of PrPc to partially fold into a ƒÒ-sheet that results in the disruption of electrical stimulation of the nervous system. The disruptions are caused by the formation of plaque bundles in the brain that give the brain a spongy

Surachai Supattapone analyzes the elimination of PrPsc by us of noncytotoxic concentrations of branched polyamines (8). Scrapie infected neuroblastoma (Sc2Na) cells stored in a mixture of polyamines and streptomycin was exposed to SuperFect. SuperFect is a mixture of branched polyamines derived from heat-induced degradation of polyamidoamide (8). The cells were then harvested by lysis to obtain a total protein concentration of 1 mg/ml. Centrifugation was then used to remove the nuclei from the lysate. Proteinase K digestion was then performed. The sample was then incubated for 1 hour at 37o C. Samples were then centrifuged at 100,000-x g for 1 hour at 4o C and the supernatants were discarded and the pellets were resuspended in 80 ƒÝl of SDS. Brain homogenate from scrapie affected mice were then prepared by repeated extrusion through syringe needles. A mixture of branched polyamines derived from heat-induced degradation. After electrophoresis, Western blot analysis indicated that the branched polyamines cleared the PrPsc from ScN2a cells within a few hours of treatment. Next Supattapone tested the cytotoxicity of the polyamine using cell growth morphology by measuring the trypan blue staining. After one week of testing, the polyamines showed no cytotoxicity on the ScN2a cells. Supattapone then tested whether polyamines could cure ScN2a cells of scrapie infection without affecting cell viability. The kinetics of prion clearance in the presence of three different branched polyamines was analyzed by exposing the ScN2a cells to SuperFect for varying periods of time. Western blot was used to assess the kinetics of PrPsc elimination and it indicated that all three polyamines reduced PrPsc levels after 8-16 hours of treatment. These results indicate that polyamines used at below toxicity levels, still to be determined, could be a future cure for TSEs.

PrP-null mice, mice without any PrPc, were immunized with full-length recombinant bovine PrP by first fusing spleen cells with my

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