Immunopurification of Pathological Prion Protein Aggregates

Background: Prion diseases are fatal neurodegenerative disorders that can arise sporadically, be genetically inherited or acquired through infection. The key event in these diseases is misfolding of the cellular prion protein (PrP) into a pathogenic isoform that is rich in β-sheet structure. This conformational change may result in the formation of PrP, the prion isoform of PrP, which propagates itself by imprinting its aberrant conformation onto PrP molecules. A great deal of effort has been devoted to developing protocols for purifying PrP for structural studies, and testing its biological properties. Most procedures rely on protease digestion, allowing efficient purification of PrP27-30, the protease-resistant core of PrP. However, protease treatment cannot be used to isolate abnormal forms of PrP lacking conventional protease resistance, such as those found in several genetic and atypical sporadic cases. Principal Findings: We developed a method for purifying pathological PrP molecules based on sequential centrifugation and immunoprecipitation with a monoclonal antibody selective for aggregated PrP. With this procedure we purified full-length PrP and mutant PrP aggregates at electrophoretic homogeneity. PrP purified from prion-infected mice was able to seed misfolding of PrP in a protein misfolding cyclic amplification reaction, and mutant PrP aggregates from transgenic mice were toxic to cultured neurons. Significance: The immunopurification protocol described here isolates biologically active forms of aggregated PrP. These preparations may be useful for investigating the structural and chemico-physical properties of infectious and neurotoxic PrP aggregates

Coupling between precipitation and plastic deformation during electromigration in a passivated Al (0.5wt%Cu) interconnect

In the present paper the evolution of the dislocation structure during electromigration in different regions along the Al(Cu) interconnect line is considered. It is shown that plastic deformation increases in the regions close to cathode end of the interconnect line. A coupling between the dissolution, growth and re-precipitation of Al2Cu precipitates and the electromigration-induced plastic deformation of grains in interconnects is observed. Possible mechanism of the Cu doping effect on the improved electromigration resistance of the Al(Cu) interconnects is discussed

Etudes génétiques de la symbiose Medicago truncatula-Sinorhizobium melitoti (utilisation d'approches de génétique inverse pour l'étude de la fonction biologique d'ENOD40)

Les légumineuses sont capables de s associer avec des bactéries du sol pour former un nouvel organe: la nodosité. Le modèle utilisé au laboratoire pour l étude de cette symbiose correspond au couple Medicago truncatula-Sinorhizobium meliloti. Afin de tenter de mieux comprendre la fonction du gène ENOD40, des approches par silencing ont été initiées. Des lignées transgéniques de M. truncatula, qui expriment une construction ARNi (ARN interference) dirigée contre ENOD40, ont été produites. Les analyses phénotypiques et moléculaires de ces lignées montrent que l induction de ce gène n est pas requise pour cette association symbiotique. De plus, au cours de ce travail, un deuxième gène ENOD40, MtENOD40-2, a été identifié chez M. truncatula. MtENOD40-2 se différencie de MtENOD40-1 par son profil de transcription mais également par la séquence du peptide I putativement codé par la majorité des gènes ENOD40 identifiés. En parallèle, afin de découvrir de nouveaux mutants de nodulation et les gènes correspondants, le criblage de deux collections de lignées d insertion, soit ADN-T soit Tnt1, de M. truncatula a été initié. Sur 271 lignées ADN-T criblées, quatre présentent des phénotypes symbiotiques qui ne co-ségrègent pas avec l étiquette mutagène ; tandis que deux mutants symbiotiques étiquetés ont été identifiés parmi 200 lignées Tnt1. La production ainsi que le criblage de nouvelles lignées Tnt1 semblent constituer un outil plus efficace pour la découverte, chez M. truncatula, de nouveaux gènes symbiotiques, par génétique directe, et de nouveaux mutants d intérêt, par génétique inverse.The symbiotic interaction between leguminous plants and soil bacteria leads to the formation of a new organ : the nodule. The molecular mechanisms controlling this interaction are not yet well understood. The interaction between Medicago truncatula and Sinorhizobium meliloti represents an interesting model to study this symbiotic interaction. We have used a silencing approach with RNAi to investigate the function of the ENOD40 gene that is induced during the symbiotic interaction and encodes a short ORFs-containing RNA. Because of the lack of an appropriated viral vector for Virus-induced gene silencing (VIGS) in M. truncatula, transgenic lines expressing an RNAi construct directed against ENOD40 were produced. The phenotype and the molecular analysis of these transgenic lines, analyzed under three different growth conditions indicated that the ENOD40 induction is not required for the symbiotic interaction. Moreover, a second MtENOD40 gene, MtENOD40-2, was discovered during this study. These MTENOD40 genes differ by their transcription patterns and their putative peptide I sequences. In addition, in order to discover new nodulation mutants and the corresponding genes, we initiated the screening of two insertion mutant collections (T-DNA or Tnt1). Four untagged and two tagged symbiotic mutants were isolated using respectively 271 T-DNA lines and 200 Tnt1 lines. Thus, as a result of the multicopy nature of the Tnt1 element in the Tnt1 lines, screening of these lines seems to be more efficient for the discovery of new symbiotic mutants in M. truncatula by forward and reverse genetic approaches.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF