Complex folding and misfolding effects of deer-specific amino acid substitutions in the β2-α2 loop of murine prion protein

The β2–α2 loop of PrPC is a key modulator of disease-associated prion protein misfolding. Amino acids that differentiate mouse (Ser169, Asn173) and deer (Asn169, Thr173) PrPC appear to confer dramatically different structural properties in this region and it has been suggested that amino acid sequences associated with structural rigidity of the loop also confer susceptibility to prion disease. Using mouse recombinant PrP, we show that mutating residue 173 from Asn to Thr alters protein stability and misfolding only subtly, whilst changing Ser to Asn at codon 169 causes instability in the protein, promotes oligomer formation and dramatically potentiates fibril formation. The doubly mutated protein exhibits more complex folding and misfolding behaviour than either single mutant, suggestive of differential effects of the β2–α2 loop sequence on both protein stability and on specific misfolding pathways. Molecular dynamics simulation of protein structure suggests a key role for the solvent accessibility of Tyr168 in promoting molecular interactions that may lead to prion protein misfolding. Thus, we conclude that ‘rigidity’ in the β2–α2 loop region of the normal conformer of PrP has less effect on misfolding than other sequence-related effects in this region

Genetic Predictions of Prion Disease Susceptibility in Carnivore Species Based on Variability of the Prion Gene Coding Region

Mammalian species vary widely in their apparent susceptibility to prion diseases. For example, several felid species developed prion disease (feline spongiform encephalopathy or FSE) during the bovine spongiform encephalopathy (BSE) epidemic in the United Kingdom, whereas no canine BSE cases were detected. Whether either of these or other groups of carnivore species can contract other prion diseases (e.g. chronic wasting disease or CWD) remains an open question. Variation in the host-encoded prion protein (PrP(C)) largely explains observed disease susceptibility patterns within ruminant species, and may explain interspecies differences in susceptibility as well. We sequenced and compared the open reading frame of the PRNP gene encoding PrP(C) protein from 609 animal samples comprising 29 species from 22 genera of the Order Carnivora; amongst these samples were 15 FSE cases. Our analysis revealed that FSE cases did not encode an identifiable disease-associated PrP polymorphism. However, all canid PrPs contained aspartic acid or glutamic acid at codon 163 which we propose provides a genetic basis for observed susceptibility differences between canids and felids. Among other carnivores studied, wolverine (Gulo gulo) and pine marten (Martes martes) were the only non-canid species to also express PrP-Asp163, which may impact on their prion diseases susceptibility. Populations of black bear (Ursus americanus) and mountain lion (Puma concolor) from Colorado showed little genetic variation in the PrP protein and no variants likely to be highly resistant to prions in general, suggesting that strain differences between BSE and CWD prions also may contribute to the limited apparent host range of the latter

Characterizaion of conformation-dependent prion protein epitopes

Whereas prion replication involves structural rearrangement of cellular prion protein (PrP(C)), the existence of conformational epitopes remains speculative and controversial, and PrP transformation is monitored by immunoblot detection of PrP(27–30), a protease-resistant counterpart of the pathogenic scrapie form (PrP(Sc)) of PrP. We now describe the involvement of specific amino acids in conformational determinants of novel monoclonal antibodies (mAbs) raised against randomly chimeric PrP. Epitope recognition of two mAbs depended on polymorphisms controlling disease susceptibility. Detection by one, referred to as PRC5, required alanine and asparagine at discontinuous mouse PrP residues 132 and 158, which acquire proximity when residues 126–218 form a structured globular domain. The discontinuous epitope of glycosylation-dependent mAb PRC7 also mapped within this domain at residues 154 and 185. In accordance with their conformational dependence, tertiary structure perturbations compromised recognition by PRC5, PRC7, as well as previously characterized mAbs whose epitopes also reside in the globular domain, whereas conformation-independent epitopes proximal or distal to this region were refractory to such destabilizing treatments. Our studies also address the paradox of how conformational epitopes remain functional following denaturing treatments and indicate that cellular PrP and PrP(27–30) both renature to a common structure that reconstitutes the globular domain

Atomic Energy Commission Report AECU-3777

"For presentation at the 1958 Nuclear Congress, Nuclear Science and Engineering Conference, March 17The experimental program was focused on the problems of containing the Na, of providing a barrier material with low neutron absorption cross section and an ability to withstand the action of hot Na, of developing an arrangement of this material which is mechanically adequate, and of demonstrating that an integrated barrier system can be safety operated under reactor temperature and pressure conditions. Possible barrier materials were subjected to hot Na jets. Stainless steel, Zr, and Al withstood the jet very well- The experimental arrangements of other tests are shown.

Ranked Matching for Service Descriptions using OWL-S

Abstract. Semantic Web services envision the automated discovery and selection of Web services. This can be realised by adding semantic information to advertised services and service requirements. The discovery and selection process finds matches between requirements and advertisements according to their semantic description. Based on the Web Ontology Language (OWL) an ontology for Web services (OWL-S) was introduced to standardise their semantic description. There are already some approaches available for matching of service requirements with service advertisements according to such an ontology. We propose an algorithm, which ranks the matching degree of service descriptions according to OWL-S. Different matching degrees are achieved based on the contravariance of the input and output types for requested and advertised services. Furthermore, additional elements of the service description, such as the service category, are either covered by reasoning processes or, such as quality of service constraints, by custom matching rules. Contrary to mechanisms that return only success or fail, ranked results provide criteria for the selection of a service among a large set of results. With such a discovery mechanism additional Web services can be found that might have normally been ignored.

Mycobacteria infect different cell types in the human lung and cause species dependent cellular changes in infected cells

BACKGROUND: Mycobacterial infections remain a significant cause of morbidity and mortality worldwide. Due to limitations of the currently available model systems, there are still comparably large gaps in the knowledge about the pathogenesis of these chronic inflammatory diseases in particular with regard to the human host. Therefore, we aimed to characterize the initial phase of mycobacterial infections utilizing a human ex vivo lung tissue culture model designated STST (Short-Term Stimulation of Tissues). METHODS: Human lung tissues from 65 donors with a size of 0.5–1 cm 3 were infected each with two strains of three different mycobacterial species (M. tuberculosis, M. avium, and M. abscessus), respectively. In order to preserve both morphology and nucleic acids, the HOPE® fixation technique was used. The infected tissues were analyzed using histo- and molecular-pathological methods. Immunohistochemistry was applied to identify the infected cell types. RESULTS: Morphologic comparisons between ex vivo incubated and non-incubated lung specimens revealed no noticeable differences. Viability of ex vivo stimulated tissues demonstrated by TUNEL-assay was acceptable. Serial sections verified sufficient diffusion of the infectious agents deep into the tissues. Infection was confirmed by Ziel Neelsen-staining and PCR to detect mycobacterial DNA. We observed the infection of different cell types, including macrophages, neutrophils, monocytes, and pneumocytes-II, which were critically dependent on the mycobacterial species used. Furthermore, different forms of nuclear alterations (karyopyknosis, karyorrhexis, karyolysis) resulting in cell death were detected in the infected cells, again with characteristic species-dependent differences. CONCLUSION: We show the application of a human ex vivo tissue culture model for mycobacterial infections. The immediate primary infection of a set of different cell types and the characteristic morphologic changes observed in these infected human tissues significantly adds to the current understanding of the initial phase of human pulmonary tuberculosis. Further studies are ongoing to elucidate the molecular mechanisms involved in the early onset of mycobacterial infections in the human lung