In nanopore analysis, peptides and proteins can be detected by the change in current when single molecules interact with an α-hemolysin pore embedded in a lipid membrane. Studies into the effects of fluorenylmethoxycarbonyl (Fmoc), acetylation or proline modification to negatively charged α-helical peptides showed that Fmoc peptides give more translocations than acetylated peptides. The addition of a proline in the middle of an acetylated peptide further reduces the number of translocations compared to Fmoc. The effect of peptide conformation on translocation or intercalation was studied with small α-helical and β-sheet hairpins. The capped β-hairpin increased translocations compared to the uncapped. The Fmoc-α-helical hairpin, containing a disulfide link, displayed both bumping and translocations whereas in the unlinked peptide the proportion of translocations was greater. Prion diseases arise from the misfolding and aggregation of the normal cellular prion protein. Nanopore analysis of prion peptides with α-helical and β-strand sequences show changes to the event parameters that help distinguish them. The interaction of bovine prion protein (bPrP), with α-hemolysin showed both bumping (type-I) and intercalation/translocation (type-II) events. There are several lines of evidence that indicate these type-II events with a blockade current of -65 pA for bPrP, represent translocations. Nanopore analysis showed that about 37% events were translocations. The interaction of metal ions with bPrP showed that Cu(II) or Zn(II) reduced translocations. Surprisingly, Mn(II) caused an increase in translocation events to about 64%. Complex formation between antibodies and prion peptides and proteins can be detected by nanopore analysis. The PrP/antibody complex is too large to translocate whereas the event parameters for unbound molecules are unchanged. In principle, a nanopore can detect a single molecule; thus, this work represents the first step towards the development of a prion detector. The nanopore will provide the sensitivity and the antibodies will provide the specificity to distinguish between PrPC and PrPSc. Also, the prion N- and C-terminal signal peptides interact with bPrP changing the event parameters, relating to a new mechanism. Finally, the folding intermediates of bPrP at 0.86 M Gdn-HCl suggests that the protein unfolds and then refolds into a different conformation with event parameters similar to those of bPrP.

In nanopore analysis, peptides and proteins can be detected by the change in current when single molecules interact with an α-hemolysin pore embedded in a lipid membrane. Studies into the effects of fluorenylmethoxycarbonyl (Fmoc), acetylation or proline modification to negatively charged α-helical peptides showed that Fmoc peptides give more translocations than acetylated peptides. The addition of a proline in the middle of an acetylated peptide further reduces the number of translocations compared to Fmoc. The effect of peptide conformation on translocation or intercalation was studied with small α-helical and β-sheet hairpins. The capped β-hairpin increased translocations compared to the uncapped. The Fmoc-α-helical hairpin, containing a disulfide link, displayed both bumping and translocations whereas in the unlinked peptide the proportion of translocations was greater. Prion diseases arise from the misfolding and aggregation of the normal cellular prion protein. Nanopore analysis of prion peptides with α-helical and β-strand sequences show changes to the event parameters that help distinguish them. The interaction of bovine prion protein (bPrP), with α-hemolysin showed both bumping (type-I) and intercalation/translocation (type-II) events. There are several lines of evidence that indicate these type-II events with a blockade current of -65 pA for bPrP, represent translocations. Nanopore analysis showed that about 37% events were translocations. The interaction of metal ions with bPrP showed that Cu(II) or Zn(II) reduced translocations. Surprisingly, Mn(II) caused an increase in translocation events to about 64%. Complex formation between antibodies and prion peptides and proteins can be detected by nanopore analysis. The PrP/antibody complex is too large to translocate whereas the event parameters for unbound molecules are unchanged. In principle, a nanopore can detect a single molecule; thus, this work represents the first step towards the development of a prion detector. The nanopore will provide the sensitivity and the antibodies will provide the specificity to distinguish between PrPC and PrPSc. Also, the prion N- and C-terminal signal peptides interact with bPrP changing the event parameters, relating to a new mechanism. Finally, the folding intermediates of bPrP at 0.86 M Gdn-HCl suggests that the protein unfolds and then refolds into a different conformation with event parameters similar to those of bPrP.

Cellular prion protein (PrPc) is a GPI–anchored protein, of unknown function, found in a number of cells throughout the body. It is now widely believed that a mis-folded, protease resistant form of this protein is responsible for a group of fatal neurodegenerative diseases called transmissible spongiform encephalopathies (TSE), including Creutzfeldt-Jakob disease (CJD) and kuru in humans, scrapie in sheep, chronic wasting disease (CWD) in deer and elk and bovine spongiform encephalopathy (BSE) in cattle. Although the exact function of PrPc is unknown it has been implicated in copper binding, signal transduction and cell adhesion. The pathogenesis of prion diseases is poorly understood, however it is known that PrPc must be present in order for the disease to progress. Platelets have been shown to be the largest reservoir of PrPc in peripheral blood cells and previous studies in animal models have suggested platelets may also be involved in TSE infectivity. In this study, we determine the exact location of PrPc within human platelets, examine the mobilization and release of PrPc from activated platelets on both microvesicles and exosomes and suggest a possible role for platelets in prion infectivity. In addition we examine the role of PrPc within normal platelet functions including aggregation, signal transduction and adhesion. / May 2005

No description available.

Cellular prion protein (PrPc) is a GPI–anchored protein, of unknown function, found in a number of cells throughout the body. It is now widely believed that a mis-folded, protease resistant form of this protein is responsible for a group of fatal neurodegenerative diseases called transmissible spongiform encephalopathies (TSE), including Creutzfeldt-Jakob disease (CJD) and kuru in humans, scrapie in sheep, chronic wasting disease (CWD) in deer and elk and bovine spongiform encephalopathy (BSE) in cattle. Although the exact function of PrPc is unknown it has been implicated in copper binding, signal transduction and cell adhesion. The pathogenesis of prion diseases is poorly understood, however it is known that PrPc must be present in order for the disease to progress. Platelets have been shown to be the largest reservoir of PrPc in peripheral blood cells and previous studies in animal models have suggested platelets may also be involved in TSE infectivity. In this study, we determine the exact location of PrPc within human platelets, examine the mobilization and release of PrPc from activated platelets on both microvesicles and exosomes and suggest a possible role for platelets in prion infectivity. In addition we examine the role of PrPc within normal platelet functions including aggregation, signal transduction and adhesion.

Cellular prion protein (PrPc) is a GPI–anchored protein, of unknown function, found in a number of cells throughout the body. It is now widely believed that a mis-folded, protease resistant form of this protein is responsible for a group of fatal neurodegenerative diseases called transmissible spongiform encephalopathies (TSE), including Creutzfeldt-Jakob disease (CJD) and kuru in humans, scrapie in sheep, chronic wasting disease (CWD) in deer and elk and bovine spongiform encephalopathy (BSE) in cattle. Although the exact function of PrPc is unknown it has been implicated in copper binding, signal transduction and cell adhesion. The pathogenesis of prion diseases is poorly understood, however it is known that PrPc must be present in order for the disease to progress. Platelets have been shown to be the largest reservoir of PrPc in peripheral blood cells and previous studies in animal models have suggested platelets may also be involved in TSE infectivity. In this study, we determine the exact location of PrPc within human platelets, examine the mobilization and release of PrPc from activated platelets on both microvesicles and exosomes and suggest a possible role for platelets in prion infectivity. In addition we examine the role of PrPc within normal platelet functions including aggregation, signal transduction and adhesion.

This thesis utilizes input-output analysis to calculate the economic impacts from potential prion diseases outbreaks in Alberta and Canada. Both chronic wasting disease and bovine spongiform encephalopathy have the capacity to not only affect the farmed cervid and cattle industries, but to impact all industries with direct and indirect links to these sectors. Cervid sector shocks consistently yield small spillover effects on the economy in all models. In contrast, the cattle sector generates larger multiplier effects. A worst-case scenario that reduces cervid sector output to zero yields total economic losses of $11.5 million in Alberta, and $43.7 million in Canada. A reduction of cattle sector output to zero results in total economic losses of $6.4 billion in Alberta, and $34.9 billion in Canada. / Agricultural and Resource Economics

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19770号 / 農博第2166号 / 新制||農||1040(附属図書館) / 学位論文||H28||N4986(農学部図書室) / 32806 / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 河田 照雄, 教授 保川 清, 教授 橋本 渉 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

The human prion protein (PrP) gene (PRNP) codon 129 (M/V) polymorphism is a susceptibility factor for variant Creutzfeldt-Jakob Disease (vCJD) and a major determinant of clinico-pathological phenotype in sporadic CJD. The role of codon 129 in defining susceptibility and strain transmission properties has been investigated in three lines of transgenic mice that express human PrP. The human PRNP gene has directly replaced the murine version, by gene targeting, and variation at codon 129 has given the three genotype lines (HuMM, HuMV, and HuVV). The genetics of these three mouse lines are otherwise identical, and therefore differences in transmission properties can be directly attributable to the codon 129 genotype. vCJD inoculation has shown that all three codon 129 genotype mice are susceptible with a ranking of transmission efficiency of HuMM>HuMV>HuVV. HuMM mice develop the most widespread neuropathology with features similar to human vCJD. Subclinical infection was noted in each mouse line. These data suggest that the vCJD strain is transmissible to humans of each of the three codon 129 genotypes, implying that non-MM cases of human infection with bovine spongiform encephalopathy (BSE) may exist but with long subclinical incubation periods. Inoculation of material from blood transfusion associated vCJD showed no change in transmission properties suggesting that the threat of a future epidemic of human-to-human vCJD infection has not been increased by adaptation of the vCJD strain. However the route of infection, for example via blood transfusion or surgery, may be more efficient that the original oral route of BSE infection. sCJD is classified into six subgroups according to clinico-pathological features, and defined by codon 129 genotype and electrophoretic mobility type (1 or 2) of disease associated PrPSc (MM1, MM2, MV1, MV2, VV1, VV2). Typical cases from each subgroup have shown specific transmission properties suggesting that the subgrouping is defining separate disease strains. The commonest subgroup (MM1) was the most transmissible and the HuVV mouse line the most susceptible host. These data outline the transmission risk from all sCJD types to recipients of each codon 129 genotype should an infection event occur, and show the significant role of recipient codon 129 genotype in defining the clinical or subclinical state and the success or failure of transmission. This is important for determining individual risk following known exposure, and for modelling the potential of iatrogenic infection from sCJD patients.

No description available.