Limits...
Molecular model of prion transmission to humans.

Jones M, Wight D, Barron R, Jeffrey M, Manson J, Prowse C, Ironside JW, Head MW - Emerging Infect. Dis. (2009)

Bottom Line: To assess interspecies barriers to transmission of transmissible spongiform encephalopathies, we investigated the ability of disease-associated prion proteins (PrPd) to initiate conversion of the human normal cellular form of prion protein of the 3 major PRNP polymorphic variants in vitro.Protein misfolding cyclic amplification showed that conformation of PrPd partly determines host susceptibility.

View Article: PubMed Central - PubMed

Affiliation: National CJD Surveillance Unit, School of Molecular and Clinical Medicine, University of Edinburgh, Western General Hospital, Edinburgh, Scotland EH4 2XU, UK. mjones1@staffmail.ed.ac.uk

ABSTRACT
To assess interspecies barriers to transmission of transmissible spongiform encephalopathies, we investigated the ability of disease-associated prion proteins (PrPd) to initiate conversion of the human normal cellular form of prion protein of the 3 major PRNP polymorphic variants in vitro. Protein misfolding cyclic amplification showed that conformation of PrPd partly determines host susceptibility.

Show MeSH

Related in: MedlinePlus

A) Semiquantitative densitometric analysis (optical density × area in mm2) of Western blot data (Figure 1, panel A, top panel), showing the amplification factors (+PMCA/−PMCA) obtained for all 4 seeds (bovine BSE, ovine scrapie, ovine BSE, and human vCJD in the PRNP-129MM substrate. B) Amplification of PrPd associated with ovine BSE (left) and ovine scrapie (right) from each of 3 different sheep in PRNP-129MM substrate as determined by Western blotting using MAb 3F4 to detect PrPres after limited proteinase K digestion. Substrate was seeded with brain homogenates prepared from sheep with confirmed scrapie and BSE such that each PMCA reaction mix contained an equivalent amount of PrPd according to detection of PrPres by Western blot titration after limited proteinase K digestion. PRNP-129MM substrate seeded with vCJD brain homogenate was included as a positive control in each experiment. C) Amplification of PrPd associated with ovine scrapie and BSE in substrates prepared from PRNP-129 methionine homozygous humanized transgenic mouse brain tissue (MM substrate) and NSB substrate. Substrates were prepared as 10% (wt/vol) homogenates in PMCA conversion buffer (10). Each substrate was seeded with brain homogenates prepared from sheep with confirmed scrapie and BSE so that each PMCA reaction mix contained an equivalent amount of PrPd as determined by detection of PrPres by Western blot titration after limited proteinase K digestion. Reaction mixes were divided into 2 lots: 1 was stored immediately at –80°C (−PMCA) and the other was subjected to 48 cycles of PMCA (+PMCA) by using standard conditions (10). After limited proteinase K digestion, PrPres in samples −/+PMCA was detected by Western blotting using MAb 6H4. PMCA, protein misfolding cyclic amplification; BSE, bovine spongiform encephalopathy; vCJD, variant Creutzfeldt-Jakob disease; MM, methionine homozygous; PrPd, disease-associated prion protein; MAb, monoclonal antibody; PrPres, protease-resistant prion protein; NSB, normal ARQ/ARQ sheep brain tissue. Values on the left in panels B and C are in kilodaltons.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3044515&req=5

Figure 2: A) Semiquantitative densitometric analysis (optical density × area in mm2) of Western blot data (Figure 1, panel A, top panel), showing the amplification factors (+PMCA/−PMCA) obtained for all 4 seeds (bovine BSE, ovine scrapie, ovine BSE, and human vCJD in the PRNP-129MM substrate. B) Amplification of PrPd associated with ovine BSE (left) and ovine scrapie (right) from each of 3 different sheep in PRNP-129MM substrate as determined by Western blotting using MAb 3F4 to detect PrPres after limited proteinase K digestion. Substrate was seeded with brain homogenates prepared from sheep with confirmed scrapie and BSE such that each PMCA reaction mix contained an equivalent amount of PrPd according to detection of PrPres by Western blot titration after limited proteinase K digestion. PRNP-129MM substrate seeded with vCJD brain homogenate was included as a positive control in each experiment. C) Amplification of PrPd associated with ovine scrapie and BSE in substrates prepared from PRNP-129 methionine homozygous humanized transgenic mouse brain tissue (MM substrate) and NSB substrate. Substrates were prepared as 10% (wt/vol) homogenates in PMCA conversion buffer (10). Each substrate was seeded with brain homogenates prepared from sheep with confirmed scrapie and BSE so that each PMCA reaction mix contained an equivalent amount of PrPd as determined by detection of PrPres by Western blot titration after limited proteinase K digestion. Reaction mixes were divided into 2 lots: 1 was stored immediately at –80°C (−PMCA) and the other was subjected to 48 cycles of PMCA (+PMCA) by using standard conditions (10). After limited proteinase K digestion, PrPres in samples −/+PMCA was detected by Western blotting using MAb 6H4. PMCA, protein misfolding cyclic amplification; BSE, bovine spongiform encephalopathy; vCJD, variant Creutzfeldt-Jakob disease; MM, methionine homozygous; PrPd, disease-associated prion protein; MAb, monoclonal antibody; PrPres, protease-resistant prion protein; NSB, normal ARQ/ARQ sheep brain tissue. Values on the left in panels B and C are in kilodaltons.

Mentions: Using MAb 6H4 to probe Western blots, we noted amplification of vCJD, bovine BSE, and ovine BSE PrPres in the PRNP-129MM substrate (Figure 1, panel A, top) but not in the PRNP-129VV substrate (Figure 1, panel A, bottom). Semiquantitative assessment of these Western blots by densitometry showed that the degree of amplification of vCJD PrPres was considerably greater than that of bovine or ovine BSE in the PRNP-129MM substrate (Figure 2, panel A). A more sensitive and discriminatory Western blot conducted by using MAb 3F4 confirmed efficient amplification of vCJD, bovine BSE, and ovine BSE PrPres in the PRNP-129MM substrate (Figure 1, panel B, top), weaker amplification in the PRNP-129MV substrate (Figure 1, panel B, middle), and little, if any, amplification in the PRNP-129VV substrate (Figure 1, panel B, bottom). In all substrates, the amplified PrPres retained the electrophoretic mobility and glycoform ratio associated with BSE-related PrPres. No amplification of ovine scrapie PrPres was evident after PMCA in any of the PRNP humanized transgenic mouse brain substrates (Figure 1, panels A, B). The difference between ovine scrapie and ovine BSE in ability to seed amplification in PRNP-129MM substrate was a robust phenomenon evident in brain samples from 3 different ARQ/ARQ sheep with each disease (Figure 2, panel B). However, failure of the ovine scrapie seed to amplify was not caused by a general lack of competence to do so or by inappropriate amplification conditions because robust amplification of ovine scrapie PrPres was evident after PMCA in a substrate prepared from normal ARQ/ARQ sheep brain (Figure 2, panel C).


Molecular model of prion transmission to humans.

Jones M, Wight D, Barron R, Jeffrey M, Manson J, Prowse C, Ironside JW, Head MW - Emerging Infect. Dis. (2009)

A) Semiquantitative densitometric analysis (optical density × area in mm2) of Western blot data (Figure 1, panel A, top panel), showing the amplification factors (+PMCA/−PMCA) obtained for all 4 seeds (bovine BSE, ovine scrapie, ovine BSE, and human vCJD in the PRNP-129MM substrate. B) Amplification of PrPd associated with ovine BSE (left) and ovine scrapie (right) from each of 3 different sheep in PRNP-129MM substrate as determined by Western blotting using MAb 3F4 to detect PrPres after limited proteinase K digestion. Substrate was seeded with brain homogenates prepared from sheep with confirmed scrapie and BSE such that each PMCA reaction mix contained an equivalent amount of PrPd according to detection of PrPres by Western blot titration after limited proteinase K digestion. PRNP-129MM substrate seeded with vCJD brain homogenate was included as a positive control in each experiment. C) Amplification of PrPd associated with ovine scrapie and BSE in substrates prepared from PRNP-129 methionine homozygous humanized transgenic mouse brain tissue (MM substrate) and NSB substrate. Substrates were prepared as 10% (wt/vol) homogenates in PMCA conversion buffer (10). Each substrate was seeded with brain homogenates prepared from sheep with confirmed scrapie and BSE so that each PMCA reaction mix contained an equivalent amount of PrPd as determined by detection of PrPres by Western blot titration after limited proteinase K digestion. Reaction mixes were divided into 2 lots: 1 was stored immediately at –80°C (−PMCA) and the other was subjected to 48 cycles of PMCA (+PMCA) by using standard conditions (10). After limited proteinase K digestion, PrPres in samples −/+PMCA was detected by Western blotting using MAb 6H4. PMCA, protein misfolding cyclic amplification; BSE, bovine spongiform encephalopathy; vCJD, variant Creutzfeldt-Jakob disease; MM, methionine homozygous; PrPd, disease-associated prion protein; MAb, monoclonal antibody; PrPres, protease-resistant prion protein; NSB, normal ARQ/ARQ sheep brain tissue. Values on the left in panels B and C are in kilodaltons.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3044515&req=5

Figure 2: A) Semiquantitative densitometric analysis (optical density × area in mm2) of Western blot data (Figure 1, panel A, top panel), showing the amplification factors (+PMCA/−PMCA) obtained for all 4 seeds (bovine BSE, ovine scrapie, ovine BSE, and human vCJD in the PRNP-129MM substrate. B) Amplification of PrPd associated with ovine BSE (left) and ovine scrapie (right) from each of 3 different sheep in PRNP-129MM substrate as determined by Western blotting using MAb 3F4 to detect PrPres after limited proteinase K digestion. Substrate was seeded with brain homogenates prepared from sheep with confirmed scrapie and BSE such that each PMCA reaction mix contained an equivalent amount of PrPd according to detection of PrPres by Western blot titration after limited proteinase K digestion. PRNP-129MM substrate seeded with vCJD brain homogenate was included as a positive control in each experiment. C) Amplification of PrPd associated with ovine scrapie and BSE in substrates prepared from PRNP-129 methionine homozygous humanized transgenic mouse brain tissue (MM substrate) and NSB substrate. Substrates were prepared as 10% (wt/vol) homogenates in PMCA conversion buffer (10). Each substrate was seeded with brain homogenates prepared from sheep with confirmed scrapie and BSE so that each PMCA reaction mix contained an equivalent amount of PrPd as determined by detection of PrPres by Western blot titration after limited proteinase K digestion. Reaction mixes were divided into 2 lots: 1 was stored immediately at –80°C (−PMCA) and the other was subjected to 48 cycles of PMCA (+PMCA) by using standard conditions (10). After limited proteinase K digestion, PrPres in samples −/+PMCA was detected by Western blotting using MAb 6H4. PMCA, protein misfolding cyclic amplification; BSE, bovine spongiform encephalopathy; vCJD, variant Creutzfeldt-Jakob disease; MM, methionine homozygous; PrPd, disease-associated prion protein; MAb, monoclonal antibody; PrPres, protease-resistant prion protein; NSB, normal ARQ/ARQ sheep brain tissue. Values on the left in panels B and C are in kilodaltons.
Mentions: Using MAb 6H4 to probe Western blots, we noted amplification of vCJD, bovine BSE, and ovine BSE PrPres in the PRNP-129MM substrate (Figure 1, panel A, top) but not in the PRNP-129VV substrate (Figure 1, panel A, bottom). Semiquantitative assessment of these Western blots by densitometry showed that the degree of amplification of vCJD PrPres was considerably greater than that of bovine or ovine BSE in the PRNP-129MM substrate (Figure 2, panel A). A more sensitive and discriminatory Western blot conducted by using MAb 3F4 confirmed efficient amplification of vCJD, bovine BSE, and ovine BSE PrPres in the PRNP-129MM substrate (Figure 1, panel B, top), weaker amplification in the PRNP-129MV substrate (Figure 1, panel B, middle), and little, if any, amplification in the PRNP-129VV substrate (Figure 1, panel B, bottom). In all substrates, the amplified PrPres retained the electrophoretic mobility and glycoform ratio associated with BSE-related PrPres. No amplification of ovine scrapie PrPres was evident after PMCA in any of the PRNP humanized transgenic mouse brain substrates (Figure 1, panels A, B). The difference between ovine scrapie and ovine BSE in ability to seed amplification in PRNP-129MM substrate was a robust phenomenon evident in brain samples from 3 different ARQ/ARQ sheep with each disease (Figure 2, panel B). However, failure of the ovine scrapie seed to amplify was not caused by a general lack of competence to do so or by inappropriate amplification conditions because robust amplification of ovine scrapie PrPres was evident after PMCA in a substrate prepared from normal ARQ/ARQ sheep brain (Figure 2, panel C).

Bottom Line: To assess interspecies barriers to transmission of transmissible spongiform encephalopathies, we investigated the ability of disease-associated prion proteins (PrPd) to initiate conversion of the human normal cellular form of prion protein of the 3 major PRNP polymorphic variants in vitro.Protein misfolding cyclic amplification showed that conformation of PrPd partly determines host susceptibility.

View Article: PubMed Central - PubMed

Affiliation: National CJD Surveillance Unit, School of Molecular and Clinical Medicine, University of Edinburgh, Western General Hospital, Edinburgh, Scotland EH4 2XU, UK. mjones1@staffmail.ed.ac.uk

ABSTRACT
To assess interspecies barriers to transmission of transmissible spongiform encephalopathies, we investigated the ability of disease-associated prion proteins (PrPd) to initiate conversion of the human normal cellular form of prion protein of the 3 major PRNP polymorphic variants in vitro. Protein misfolding cyclic amplification showed that conformation of PrPd partly determines host susceptibility.

Show MeSH
Related in: MedlinePlus