Summary
The conformational transition of α‐helix‐rich cellular prion protein (PrPC) to an isomer with high β‐sheet content is associated with transmissible spongiform encephalopathies. With the ultimate long‐term goal of using imaging techniques to study PrP aggregation, we report the results of initial experiments to determine whether PrP molecules could be visualized as single molecules, and if the observed size corresponded to the calculated size for PrP. The investigation of single molecules, and not those embedded into larger aggregates, was the key in our experimental approach. Using atomic force microscopy (AFM) as an imaging method, the immobilization of recombinant histidine (His)10‐tagged PrP on mica was performed in the presence of different heavy metal ions. The addition of Cu2+ resulted in an enhanced PrP immobilization, whereas Ni2+ reduced coverage of the surface by PrP. High‐resolution data from dried PrP preparations provided a first approximation to geometrical parameters of PrP precipitates, which indicated that the volume of a single PrP molecule was 30 nm3. Molecular dynamics simulations performed to complement the structural aspects of the AFM investigation yielded a calculated molecular volume of 33 nm3 for PrP. These experimentally observed and theoretically expected values provide basic knowledge for further studies on the size and composition of larger amyloidal PrP aggregates, PrP isoforms or mutants such as PrP molecules without octarepeats.
Unwanted interactions of biomedical sensors with surrounding tissues, body fluids, and cells are one of the most crucial problems affecting their long-term stability. In vivo processes were simulated in a computer-controlled bioreactor connected to a flow chamber system. Optical sensor materials were inserted into a parallel-plate chamber and monitored by light microscopy in order to get information about the number of adhered cells. Tests with thrombocyte-enriched plasma show that novel phosphorylcholine (PC)-polymer-coated sensors appear to be more bioinert, and thus demonstrate better haemocopatibility in comparison with untreated glass sensors. The influence of different materials on the morphology of adhered cells was investigated by off-line methods such as scanning electron microscopy (SEM) and atomic-force microscopy (AFM). SEM showed a reduction in the number of adhered thrombocytes and the lack of any fibrin network on the PC-polymer-modified glass surface, in contrast to the pure glass surface. AFM gives topographical information, and the calculated contact areas and cell volumes indicate smaller interactions between cells and sensor material in the case of PC-polymer-coated sensors.
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