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Immobilization of homogeneous monomeric, oligomeric and fibrillar A? species for reliable SPR measurements.

ABSTRACT: There is strong evidence that the amyloid-beta peptide (A?) plays a central role in the pathogenesis of Alzheimer's disease (AD). In this context, a detailed quantitative description of the interactions with different A? species is essential for characterization of physiological and artificial ligands. However, the high aggregation propensity of A? in concert with its susceptibility to structural changes due to even slight changes in solution conditions has impeded surface plasmon resonance (SPR) studies with homogeneous A? conformer species. Here, we have adapted the experimental procedures to state-of-the-art techniques and established novel approaches to reliably overcome the aforementioned challenges. We show that the application of density gradient centrifugation (DGC) for sample purification and the use of a single chain variable fragment (scFv) of a monoclonal antibody directed against the amino-terminus of A? allows reliable SPR measurements and quality control of the immobilized A? aggregate species at any step throughout the experiment.

SUBMITTER: Frenzel D

PROVIDER: S-EPMC3940443 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Immobilization of homogeneous monomeric, oligomeric and fibrillar Aβ species for reliable SPR measurements.

Frenzel Daniel D Glück Julian M JM Brener Oleksandr O Oesterhelt Filipp F Nagel-Steger Luitgard L Willbold Dieter D

PloS one 20140303 3

There is strong evidence that the amyloid-beta peptide (Aβ) plays a central role in the pathogenesis of Alzheimer's disease (AD). In this context, a detailed quantitative description of the interactions with different Aβ species is essential for characterization of physiological and artificial ligands. However, the high aggregation propensity of Aβ in concert with its susceptibility to structural changes due to even slight changes in solution conditions has impeded surface plasmon resonance (SPR ...[more]

PMID: 24594736

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Project description:Background: In a recent intervention study, the daily supplementation with 200mg monomeric and oligomeric flavanols (MOF) from grape seeds for 8 weeks revealed a vascular health benefit in male smokers. The objective of the present study was to determine the impact of MOF consumption on the gene expression profile of leukocytes and to assess changes in DNA methylation. Methodology/Principal Findings: Gene expression profiles were determined using whole genome microarrays (Agilent) and DNA methylation was assessed using HumanMethylation450 BeadChips (Illumina). MOF significantly modulated the expression of 869 genes. The majority of the affected genes are involved in chemotaxis, cell adhesion, cell infiltration or cytoskeleton organisation, suggesting lower immune cell adhesion to endothelial cells. This was corroborated by in vitro experiments showing that MOF exposure of monocytes attenuates their adhesion to TNFα-stimulated endothelial cells. Nuclear factor-kappa B reporter gene assays confirmed that MOF decrease the activity of NF-κB. Strong inter-individual variability in the leukocytes DNA methylation was observed. As a consequence, on group level, changes due to MOF supplementation could not be found. However, in individuals, significant changes in DNA methylation of genes involved in leukocyte rolling, adhesion and cytoskeleton remodelling were seen. At the group level, the variation in DNA methylation related to life style and clinical parameters appeared to overrule the magnitude of effects of supplementation with MOF. Conclusion: Our study revealed that regular consumption of MOF modulates the expression of genes in immune cells which, however, cannot be correlated to alterations in the DNA methylome. Remarkably, at the individual level, genes related to leukocyte adhesion pathways present complex variation in DNA methylation. As such, the individual transcriptional and epigenetic modulation of genes involved in early manifestation of cardiovascular diseases constitutes important subcellular mechanisms by which MOF promote vascular health in humans.

Dataset Information

Immobilization of homogeneous monomeric, oligomeric and fibrillar A? species for reliable SPR measurements.

Publications

Immobilization of homogeneous monomeric, oligomeric and fibrillar Aβ species for reliable SPR measurements.

Similar Datasets

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