Surface Assay for Specific Detection of Soluble Amyloid Oligomers Utilizing Pronucleon Peptides Instead of Antibodies.

Immunoassays such as enzyme-linked immunosorbent assays (ELISAs) are widely used for diagnostics; however, antibodies as detection reagents may be insufficiently selective and have other shortcomings. We present a novel non-antibody-based detection method based on binding target molecules to peptides (used as recognition molecules): a surface assay for A-β oligomers employing a peptide comprising amino acid residues of the human β-amyloid protein (Pronucleon peptide) as the capture agent. For the sake of convenience, we term this the "Pronucleon peptide-linked immunosorbent assay", or PLISA.

Amyloid beta modulation of neuronal network activity in vitro.

In vitro assays offer a means of screening potential therapeutics and accelerating the drug development process. Here, we utilized neuronal cultures on planar microelectrode arrays (MEA) as a functional assay to assess the neurotoxicity of amyloid-β 1-42 (Aβ42), a biomolecule implicated in the Alzheimer׳s disease (AD). In this approach, neurons harvested from embryonic mice were seeded on the substrate-integrated microelectrode arrays.

In vivo and ex vivo imaging of amyloid-β cascade aggregates with a Pronucleon™ peptide.

Accumulation of amyloid-β (Aβ) cascade aggregates is considered a hallmark of Alzheimer's disease (AD). Current dogma holds that the appearance of Aβ oligomers and larger aggregates occur many years prior to plaque formation associated with the advanced and irreparable neurocognitive decline characteristic of AD. This premise is the impetus to identify these Aβ precursor structures prior to advanced plaque development.

Structure-selective anisotropy assay for amyloid Beta oligomers.

Amyloid β (Abeta) peptides in their oligomeric form have been proposed as major toxic species in Alzheimer's disease (AD). There are a limited number of anti-Abeta antibodies specific to oligomeric forms of Abeta compared to the monomeric form, and accurate measurement of oligomeric forms in biological samples, cerebrospinal fluid (CSF), or brain extracts remains challenging. We introduce an oligomer-specific (in preference to monomers or fibrils) fluorescence assay based on a conformationally sensitive bis-pyrene-labeled peptide that contains amino acid residues 16-35 of the human amyloid beta protein (pronucleon peptide, PP).

A heterodimerizing leucine zipper coiled coil system for examining the specificity of a position interactions: amino acids I, V, L, N, A, and K.

We use a heterodimerizing leucine zipper system to examine the contribution of the interhelical a-a' interaction to dimer stability for six amino acids (A, V, L, I, K, and N). Circular dichroism (CD) spectroscopy monitored the thermal denaturation of 36 heterodimers that generate six homotypic and 30 heterotypic a-a' interactions. Isoleucine (I-I) is the most stable homotypic a-a' interaction, being 9.

B-ZIP proteins encoded by the Drosophila genome: evaluation of potential dimerization partners.

The basic region-leucine zipper (B-ZIP) (bZIP) protein motif dimerizes to bind specific DNA sequences. We have identified 27 B-ZIP proteins in the recently sequenced Drosophila melanogaster genome. The dimerization specificity of these 27 B-ZIP proteins was evaluated using two structural criteria: (1) the presence of attractive or repulsive interhelical g<-->e' electrostatic interactions and (2) the presence of polar or charged amino acids in the 'a' and 'd' positions of the hydrophobic interface.

Magnesium is required for specific DNA binding of the CREB B-ZIP domain.

We have examined binding of the CREB B-ZIP protein domain to double-stranded DNA containing a consensus CRE sequence (5'-TGACGTCA-3'), the related PAR, C/EBP and AP-1 sequences and the unrelated SP1 sequence. DNA binding was assayed in the presence or absence of MgCl2 and/or KCl using two methods: circular dichroism (CD) spectroscopy and electrophoretic mobility shift assay (EMSA). The CD assay allows us to measure equilibrium binding in solution.