Molecular Interactions between Prions as Seeds and Recombinant Prion Proteins as Substrates Resemble the Biological Interspecies Barrier In Vitro

Prion diseases like Creutzfeldt-Jakob disease in humans, Scrapie in sheep or bovine spongiform encephalopathy are fatal neurodegenerative diseases, which can be of sporadic, genetic, or infectious origin. Prion diseases are transmissible between different species, however, with a variable species barrier. The key event of prion amplification is the conversion of the cellular isoform of the prion protein (PrPC) into the pathogenic isoform (PrPSc). We developed a sodiumdodecylsulfate-based PrP conversion system that induces amyloid fibril formation from soluble α-helical structured recombinant PrP (recPrP). This approach was extended applying pre-purified PrPSc as seeds which accelerate fibrillization of recPrP. In the present study we investigated the interspecies coherence of prion disease. Therefore we used PrPSc from different species like Syrian hamster, cattle, mouse and sheep and seeded fibrillization of recPrP from the same or other species to mimic in vitro the natural species barrier. We could show that the in vitro system of seeded fibrillization is in accordance with what is known from the naturally occurring species barriers

Kinetics of Advanced Glycation End Products Formation on Bovine Serum Albumin with Various Reducing Sugars and Dicarbonyl Compounds in Equimolar Ratios

Reducing sugars and reactive dicarbonyl compounds play a major role in glycation of proteins in vivo. Glycation of proteins is the first step in of a nonenzymatic reaction, resulting in advanced glycation end products (AGEs). AGEs can inactivate proteins or modify their biological activities. Therefore, it is important to understand the mechanism of AGE formation. Here, we systematically analyzed the kinetics of AGE formation in vitro by fluorescence and absorption measurements utilizing a microplate reader system and bovine serum albumin (BSA) as a model protein. Comparing different concentrations of BSA, we applied various reducing sugars and reactive dicarbonyl compounds as AGE-inducing agents at different concentrations. In summary, this experimental setup enabled us to measure the kinetics of AGE formation in an efficient and defined way

Seeded fibrillation of huPrP.

<p>Different combinations of seeds from NaPTA precipitated brain homogenates of either diseased or control animals and 3 µM huPrP as substrate within ThT amyloid seeding assay. Time dependent ThT fluorescence is shown for 3 µM huPrP in combination with seeds from CJD-affected human brain (<b>A</b>), scrapie-affected sheep brain (<b>B</b>), BSE-affected bovine brain (<b>C</b>), and CWD-affected deer brain (<b>D</b>) (closed circles). The controls (open rectangles) were prepared from the respective non-diseased brain material from human (A) and animals (<b>B; C and D</b>).</p

Integrating ecosystem services and climate change responses in coastal wetlands development plans for Bangladesh

This study explores the integration of ecosystem services and climate change adaptation in development plans for coastal wetlands in Bangladesh. A new response framework for adaptation is proposed, based on an empirical analysis and consultations with stakeholders, using a modified version of the DPSIR (Driver-Pressure-State-Impact-Response) framework. The framework is tested in the Narail district of Bangladesh, where temperature has increased by about 1 0C in the summer in combination with an increase in rainfall of 0.70 mm day-1 yr-1 in the last decade. Calibrated model (MAGICC/SENGEN) projections forecast, on average, a temperature increase of up to 5 0C and an increase in rainfall of 25% by the end of this century. Water diversion in the upstream regions of the Ganges delta contributes to increase water scarcity in the dry season. Enhanced rainfall and the immense pressure of water discharges from upstream water sources are increasing the risk of floods and river erosion in the dry season. An increase in the water holding capacity of rivers, wetlands and canals by dredging is urgently required. The empirical model of this study is intended to support adaptation planning in Bangladesh and can be used in other data-poor areas which will suffer from climate change

Seeding activity of PrP^Sc seeds of different species in huPrP.

<p>The box plot shows seeding activity of seeds from the species human, cattle, sheep and deer in huPrP obtained from identical replicates of single seeding experiments. Seeding activity was calculated by dividing the average of relative fluorescence of samples with PrP^Sc positive seeds and PrP^Sc negative seeds within 80-100 h (plateau phase). For seeds from CJD (N = 7) or BSE (N = 8) material a high seeding activity is observable. For seeds from scrapie (N = 10) or CWD (N = 8) a strong species barrier is apparent.</p

Pre-amyloid and amyloid state of cerPrP.

<p>CD spectra (<b>A</b>) of cerPrP with different concentrations of SDS (0.02–0.2%) have been recorded directly after adapting the SDS buffer conditions. CD spectra show a shift from α-random dominated towards β-sheet rich secondary structure with decreasing concentration of SDS. (<b>B</b>) <i>De novo</i> fibrillation of cerPrP, an amyloid specific increase in ThT fluorescence is observable at 0.02% SDS and 9 µM cerPrP. (<b>C</b>) cerPrP (3 µM) in combination with seeds from CWD brain or control brain.</p

Pre-amyloid state of huPrP – CD spectroscopy and sedimentation velocity analysis of huPrP.

<p>CD spectra (<b>A</b>) of huPrP with varying concentrations of SDS (0.02–0.2%) have been recorded directly after adapting the SDS buffer conditions. CD spectra show a shift from α-random dominated towards β-sheet rich secondary structure with decreasing concentration of SDS. (<b>B and C</b>) A sedimentation velocity experiment at 20°C and 40.000 rpm was performed to determine the state of oligomerisation of huPrP. Fig. 1 <b>B</b> shows the successive radial absorbance scans of 5 µM huPrP, which have been corrected for time and radially invariant noise. For clarity only every 3^rd scan was plotted. Fig. 1 <b>C</b> shows the deviation of measured data points from the fitted sedimentation boundary after the fitting procedure using Ultrascan 3. The absorption difference at 230 nm is plotted in relation to the distance from the rotor axis. The RMSD of the fit was 0.00322 A₂₃₀.</p

<i>De novo</i> fibrillation of huPrP – ThT kinetics and TIRF micrographs.

<p><i>De novo</i> fibrillation is shown in (<b>A</b>), an amyloid specific increase in ThT fluorescence is only observable at 0.02% SDS and 9 µM huPrP. (<b>B</b>) <i>De novo</i> generated amyloid fibrils (plateau phase) have been stained by ThT and visualised by TIRF microscopy. (<b>C</b>) Ultrastructure of negative-stained <i>de novo</i> huPrP fibrils obtained by transmission electron microscopy. Bar equals 100 nm.</p