A sputum bioassay for airway eosinophilia using an eosinophil peroxidase aptamer.

Eosinophils are granulocytes that play a significant role in the pathogenesis of asthma and other airway diseases. Directing patient treatment based on the level of eosinophilia has been shown to be extremely effective in reducing exacerbations and therefore has tremendous potential as a routine clinical test. Herein, we describe the in vitro selection and optimization of DNA aptamers that bind to eosinophil peroxidase (EPX), a protein biomarker unique to eosinophils.

Functional dynamics in cyclic nucleotide signaling and amyloid inhibition.

It is now established that understanding the molecular basis of biological function requires atomic resolution maps of both structure and dynamics. Here, we review several illustrative examples of functional dynamics selected from our work on cyclic nucleotide signaling and amyloid inhibition. Although fundamentally diverse, a central aspect common to both fields is that function can only be rationalized by considering dynamic equilibria between distinct states of the accessible free energy landscape.

Mapping the interactions between the Alzheimer's Aβ-peptide and human serum albumin beyond domain resolution.

Human serum albumin (HSA) is a potent inhibitor of Aβ self-association and this novel, to our knowledge, function of HSA is of potential therapeutic interest for the treatment of Alzheimer's disease. It is known that HSA interacts with Aβ oligomers through binding sites evenly partitioned across the three albumin domains and with comparable affinities. However, as of this writing, no information is available on the HSA-Aβ interactions beyond domain resolution.

In vitro amyloid-β binding and inhibition of amyloid-β self-association by therapeutic albumin.

Background: A promising approach for treating Alzheimer's disease relies on the net efflux of the amyloid-β (Aβ) peptide from the brain to peripheral plasma, as a result of plasma Aβ clearance promoted by plasma removal and therapeutic albumin replacement.

Objective: To assess the binding of therapeutic albumin (Albutein, Grifols) to monomeric and aggregated Aβ according to methods previously tested on the interactions between Aβ and research-grade albumin.

Methods: Albumin integrity and the interactions with albumin stabilizers (octanoic acid and N-Ac-Trp) were assessed through one-dimensional (1D) 1H-NMR and saturation transfer difference (STD) NMR spectra.

Aβ association inhibition by transferrin.

The iron-transport glycoprotein transferrin has recently been shown to serve as a potent inhibitor of Aβ self-association. Although this novel, to our knowledge, inhibitory function of transferrin is of potential therapeutic interest for the treatment of Alzheimer's disease, the underlying mechanism is still not fully understood. Although it has been shown that the Fe(III) sequestration by transferrin reduces oxidative damage and Aβ aggregation, it is not clear whether transferrin is also able to inhibit Aβ self-association through direct binding of Aβ.

Stoichiometry and affinity of the human serum albumin-Alzheimer's Aβ peptide interactions.

A promising strategy to control the aggregation of the Alzheimer's Aβ peptide in the brain is the clearance of Aβ from the central nervous system into the peripheral blood plasma. Among plasma proteins, human serum albumin plays a critical role in the Aβ clearance to the peripheral sink by binding to Aβ oligomers and preventing further growth into fibrils. However, the stoichiometry and the affinities of the albumin-Aβ oligomer interactions are still to be fully characterized.

Human serum albumin inhibits Abeta fibrillization through a "monomer-competitor" mechanism.

Human serum albumin (HSA) is not only a fatty acid and drug carrier protein, it is also a potent inhibitor of Abeta self-association in plasma. However, the mechanism underlying the inhibition of Abeta fibrillization by HSA is still not fully understood. We therefore investigated the Abeta-HSA system using a combined experimental strategy based on saturation transfer difference (STD) NMR and intrinsic albumin fluorescence experiments on three Abeta peptides with different aggregation propensities (i.

A new function of human HtrA2 as an amyloid-beta oligomerization inhibitor.

Human HtrA2 is part of the HtrA family of ATP-independent serine proteases that are conserved in both prokaryotes and eukaryotes and localizes to the intermembrane space of the mitochondria. Several recent reports have suggested that HtrA2 is important for maintaining proper mitochondrial homeostasis and may play a role in Alzheimer's disease (AD), which is characterized by the presence of aggregates of the amyloid-beta peptide 1-42 (Abeta1-42). In this study, we analyzed the ability of HtrA2 to delay the aggregation of the model substrate citrate synthase (CS) and of the toxic Abeta1-42 peptide.

Analysis and optimization of saturation transfer difference NMR experiments designed to map early self-association events in amyloidogenic peptides.

Saturation transfer difference (STD) methods recently have been proposed to be a promising tool for self-recognition mapping at residue and atomic resolution in amyloidogenic peptides. Despite the significant potential of the STD approach for systems undergoing oligomer/monomer (O/M) equilibria, a systematic analysis of the possible artifacts arising in this novel application of STD experiments is still lacking. Here, we have analyzed the STD method as applied to O/M peptides, and we have identified three major sources of possible biases: offset effects, intramonomer cross-relaxation, and partial spin-diffusion within the oligomers.

Understanding the molecular basis for the inhibition of the Alzheimer's Abeta-peptide oligomerization by human serum albumin using saturation transfer difference and off-resonance relaxation NMR spectroscopy.

Human serum albumin (HSA) inhibits the formation of amyloid beta-peptide (Abeta) fibrils in human plasma. However, currently it is not known how HSA affects the formation of the highly toxic soluble diffusible oligomers that occur in the initial stages of Abeta fibrillization. We have therefore investigated by solution NMR the interaction of HSA with the Abeta(12-28) peptide, which has been previously shown to provide a reliable and stable model for the early prefibrillar oligomers as well as to contain key determinants for the recognition by albumin.

Analysis and parametric optimization of 1H off-resonance relaxation NMR experiments designed to map polypeptide self-recognition and other noncovalent interactions.

The measurement of 1H off-resonance nonselective relaxation rates (R(theta,ns)) has been recently proposed as an effective method to probe peptide self-recognition, opening new perspectives in the understanding of the prefibrillization oligomerization processes in amylodogenesis. However, a full analysis and parametric optimization of the NMR experiments designed to measure R(theta,ns) relaxation rates is still missing. Here we analyze the dependence of the R(theta,ns) rates upon three critical parameters: the tilt angle of the effective field during the spin lock, the static magnetic field, and finally the repetition delay.