Diagnosis of Creutzfeldt-Jakob Disease by Western Blot Identification of Marker Protein in Human Brain Tissue

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138

Prions are unprecedented infectious pathogens that cause a group of invariably fatal neurodegenerative diseases by an entirely novel mechanism. Prion diseases may present as genetic, infectious, or sporadic disorders, all of which involve modification of the prion protein (PrP). Bovine spongiform encephalopathy (BSE), scrapie of sheep, and Creutzfeldt-Jakob disease (CJD) of humans are among the most notable prion diseases. Prions are transmissible particles that are devoid of nucleic acid and seem to be composed exclusively of a modified protein (PrP Sc ). The normal, cellular PrP (PrP C ) is converted into PrP Sc through a posttranslational process during which it acquires a high ␤-sheet content. The species of a particular prion is encoded by the sequence of the chromosomal PrP gene of the mammals in which it last replicated. In contrast to pathogens carrying a nucleic acid genome, prions appear to encipher strain-specific properties in the tertiary structure of PrP Sc . Transgenetic studies argue that PrP Sc acts as a template upon which PrP C is refolded into a nascent PrP Sc molecule through a process facilitated by another protein. Miniprions generated in transgenic mice expressing PrP, in which nearly half of the residues were deleted, exhibit unique biological properties and should facilitate structural studies of PrP Sc . While knowledge about prions has profound implications for studies of the structural plasticity of proteins, investigations of prion diseases suggest that new strategies for the prevention and treatment of these disorders may also find application in the more common degenerative diseases.

supporting

confidence: 58%

Molecular characterization of proteolytic cleavage sites of the Pseudomonas syringae effector AvrRpt2

Chisholm

Dahlbeck

Krishnamurthy

et al. 2005

137

138

“…Finally, the grids were fixed in 2% glutaraldehyde with 0.05% ruthenium red and negatively stained as above. SAF were obtained by detergent extraction of hamster brain suspension as previously described (16,18,19), and sedimented onto Formvar/carbon-coated nickel grids by low-speed centrifugation (20). The grids were fixed in paraformaldehyde, washed in PBS/Tween, and then immunostained as above.…”

Section: Methodsmentioning

confidence: 99%

Tubulofilaments in negatively stained scrapie-infected brains: relationship to scrapie-associated fibrils.

Narang¹,

Asher²,

Gajdusek³

1987

A simple method was devised for negativestain transmission electron microscopy of brain infected with the agent of scrapie. Brains of infected' hamsters contained large masses of tubuloframentous structures with irregular fuzzy surfaces. Brains of mice infected with Creutzfeldt-Jakob disease agent contained similar tubulofilaments in smaller numbers. The abnormal tubulofilaments resembled but were distinguished from normal microtubules. On grids soaked in sodium dodecyl sulfate the abnormal tubuloframents were found in stages of fragmentation, an outer coat appearing to strip from the surface to reveal thinner fibrillary structures resembling scrapie-associated fibrils (SAF). The unmasked fibrils were identified as SAF by immunogold labeling, while the larger tubulofilaments were not labeled. The findings indicate that in infected brain tissue SAF may occur as an internal part of a larger structure that is disrupted by detergent and are not likely to be an artifact formed during extraction procedures.Unique "virus-like" tubulovesicular particles (Fig. 1) have been consistently observed by electron microscopy (EM) in thin sections of plastic-embedded brain tissues from several types of animals and from humans infected with the agents of scrapie and Creutzfeldt-Jakob disease (CJD) (1-6). However, progress in characterizing those particles or investigating their relationship to the etiological agents of the spongiform encephalopathies has been impeded by failure to identify them except in plastic sections, where they are relatively inaccessible to studies other than morphological analyses. We studied unembedded preparations of brain tissue by negative staining (7) with the goal of obtaining such particles in a state in which they could be concentrated and purified. Not only were abnormal tubulofilaments identified in brains of rodents with spongiform encephalopathies but also an unanticipated association was observed between those structures and filaments of the amyloid-like protein variously designated as scrapie-associated fibrils (SAF) (8) and prion protein 27-30 (PrP 27-30) (9, 10). MATERIALS AND METHODSScrapie and CJD Agents. The strains of agent used have been previously described. The 263K strain of hamsteradapted scrapie agent (11, 12) was used at the 4th passage level (13). The KFu strain of mouse-adapted CJD agent (14) was used at our 3rd passage level (15).Animals and Inoculations. Weanling female LGV/LAK golden Syrian hamsters were inoculated intracerebrally with 0.03-ml aliquots of 10% suspension of scrapie-infected brain as previously described. The inoculum of scrapie agent contained at least 107 LD50 units per 0.03 ml; animals became ill as early as 65 days later, and all had died by 90 days after inoculation. Weanling female National Institutes of Health white Swiss mice were inoculated intracerebrally with 0.03 ml of a 10-3 dilution of CJD-agent infected brain suspension; the inoculum contained at least 50 LD50 units per 0.03 ml.Mice became ill 141 days later and were killed 149 days after...

“…PrPsc is encoded by a single-copy chromosomal gene and not by a putative nucleic acid carried within the infectious scrapie prion particle (13)(14)(15). In humans dying of prion diseases, PrPSc is often found in brain (16)(17)(18)(19)(20), but in some cases it has been difficult to detect, especially in the inherited prion diseases (21,22). PrPsc has been generally distinguished from cellular PrP (PrPC) by its relative protease resistance, its insolubility in nondenaturing detergents, its enhanced antigenicity after denaturation, and its posttranslational biogenesis; spectroscopic studies show that the two proteins differ in their conformations (23).…”

mentioning

confidence: 99%

Serial transmission in rodents of neurodegeneration from transgenic mice expressing mutant prion protein.

Hsiao

Groth

Scott

et al. 1994

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