20 research outputs found

    Antibody Concentrations to A Beta 1-42 Monomer and Soluble Oligomers in Untreated and Antibody-Antigen-Dissociated Intravenous Immunoglobulin Preparations

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    Cognitive improvement in Alzheimer\u27s disease (AD) patients treated with intravenous immunoglobulin (IvIg) has been attributed to its antibodies to amyloid beta (A beta) We compared the concentrations of specific antibodies to soluble A beta 1-42 conformations, namely A beta 1-42 monomer and A beta 1-42 soluble oligomers, between three IvIg preparations. Gamunex, Gammagard. and Flebogamma. To determine specific antibody Concentrations to these A beta 1-42 conformations. nonspecific binding of the IvIg preparations to the An reverse sequence, A beta 42-1. was subtracted These antibodies were measured in untreated IvIg preparations and also after they were treated to dissociate antibody-antigen complexes. because this procedure has been reported to increase the detectable levels of serum anti-A beta antibodies. Antibody levels to A beta 1-42 monomer were significantly higher in untreated Gamunex than in the other two IvIg preparations, and antibody-antigen dissociation increased the measured anti-A beta monomer concentrations in Gamunex and Gammagard Dissociated Gamunex and Gammagard had higher anti-A beta monomer levels than Flebogamma. Generally similar results were found for antibodies to soluble A beta 1-42 oligomers. with the exception that after antibody-antigen dissociation, only Gammagard had significantly higher antibody levels than Flebogamma. These differences in antibody concentrations to A beta 1-42 conformations (particularly to A beta 1-42 soluble oligomers, thought to be the most neurotoxic conformation of soluble A beta) and the increased availability of these antibodies after antibody-antigen complex dissociation have important implications for IvIg treatment of AD patients

    Effects of External Beam Radiation on \u3ci\u3eIn Vitro\u3c/i\u3e Formation of Abeta1-42 Fibrils and Preformed Fibrils

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    Plaques containing fibrillar amyloid-beta (Abeta) are a characteristic finding in Alzheimer\u27s disease. Although plaque counts correlate poorly with the extent of cognitive deficits in this disorder, fibrillar Abeta can promote neuronal damage through a variety of mechanisms. External beam radiotherapy has been reported to be an effective treatment for tracheobronchial amyloidosis, in which amyloid is deposited as submucosal plaques and tumor-like masses in the trachea and/or bronchi. Radiotherapy\u27s effectiveness in this disorder is thought to be due to its toxicity to plasma cells, but direct effects of radiotherapy on amyloid may also be involved. On this basis, whole-brain radiotherapy has been suggested as a treatment for Alzheimer\u27s disease. The objective of this study was to determine the effects of external beam radiation on preformed Abeta1-42 fibrils and on the formation of these fibrils. Using the Thioflavin-T assay, no effects of radiation were found on either of these parameters. Our results in this in vitro study suggest that whole-brain irradiation is unlikely to directly reduce plaque counts in the Alzheimer\u27s disease brain. This treatment might still lower plaque counts indirectly, but any potential benefits would need to be weighed against its possible neurotoxic effects, which could induce further cognitive deficits

    Are Long-Range Structural Correlations Behind the Aggregration Phenomena of Polyglutamine Diseases?

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    We have characterized the conformational ensembles of polyglutamine peptides of various lengths (ranging from to ), both with and without the presence of a C-terminal polyproline hexapeptide. For this, we used state-of-the-art molecular dynamics simulations combined with a novel statistical analysis to characterize the various properties of the backbone dihedral angles and secondary structural motifs of the glutamine residues. For (i.e., just above the pathological length for Huntington's disease), the equilibrium conformations of the monomer consist primarily of disordered, compact structures with non-negligible -helical and turn content. We also observed a relatively small population of extended structures suitable for forming aggregates including - and -strands, and - and -hairpins. Most importantly, for we find that there exists a long-range correlation (ranging for at least residues) among the backbone dihedral angles of the Q residues. For polyglutamine peptides below the pathological length, the population of the extended strands and hairpins is considerably smaller, and the correlations are short-range (at most residues apart). Adding a C-terminal hexaproline to suppresses both the population of these rare motifs and the long-range correlation of the dihedral angles. We argue that the long-range correlation of the polyglutamine homopeptide, along with the presence of these rare motifs, could be responsible for its aggregation phenomena

    Conformations within soluble oligomers and insoluble aggregates revealed by resonance energy transfer

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    A fluorescently labeled 20-residue polyglutamic acid (polyE) peptide 20 amino acid length polyglutamic acid (E(20)) was used to study structural changes which occur in E(20) as it co-aggregates with other unlabeled polyE peptides. Resonance energy transfer (RET) was performed using an o-aminobenzamide donor at the N-terminus and 3-nitrotyrosine acceptor at the C-terminus of E(20). PolyE aggregates were not defined as amyloid, as they were nonfibrillar and did not bind congo red. Circular dichroism measurements indicate that polyE aggregation involves a transition from alpha-helical monomers to aggregated beta-sheets. Soluble oligomers are also produced along with aggregates in the reaction, as determined through size exclusion chromatography. Time-resolved and steady-state RET measurements reveal four dominant E(20) conformations: (1) a partially collapsed conformation (24 A donor-acceptor distance) in monomers, (2) an extended conformation in soluble oligomers (\u3e29 A donor-acceptor distance), (3) a minor partially collapsed conformation (22 A donor-acceptor distance) in aggregates, and (4) a major highly collapsed conformation (13 A donor-acceptor distance) in aggregates. These findings demonstrate the use of RET as a means of determining angstrom-level structural details of soluble oligomer and aggregated states of proteins

    Antibody Concentrations to Abeta1-42 Monomer and Soluble Oligomers in Untreated and Antibody-Antigen-Dissociated Intravenous Immunoglobulin Preparations

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    Cognitive improvement in Alzheimer\u27s disease (AD) patients treated with intravenous immunoglobulin (IvIg) has been attributed to its antibodies to amyloid beta (Abeta). We compared the concentrations of specific antibodies to soluble Abeta1-42 conformations, namely Abeta1-42 monomer and Abeta1-42 soluble oligomers, between three IvIg preparations, Gamunex, Gammagard, and Flebogamma. To determine specific antibody concentrations to these Abeta1-42 conformations, nonspecific binding of the IvIg preparations to the Abeta reverse sequence, Abeta42-1, was subtracted. These antibodies were measured in untreated IvIg preparations and also after they were treated to dissociate antibody-antigen complexes, because this procedure has been reported to increase the detectable levels of serum anti-Abeta antibodies. Antibody levels to Abeta1-42 monomer were significantly higher in untreated Gamunex than in the other two IvIg preparations, and antibody-antigen dissociation increased the measured anti-Abeta monomer concentrations in Gamunex and Gammagard. Dissociated Gamunex and Gammagard had higher anti-Abeta monomer levels than Flebogamma. Generally similar results were found for antibodies to soluble Abeta1-42 oligomers, with the exception that after antibody-antigen dissociation, only Gammagard had significantly higher antibody levels than Flebogamma. These differences in antibody concentrations to Abeta1-42 conformations (particularly to Abeta1-42 soluble oligomers, thought to be the most neurotoxic conformation of soluble Abeta) and the increased availability of these antibodies after antibody-antigen complex dissociation have important implications for IvIg treatment of AD patients

    An Accurate Model of Polyglutamine

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    Polyglutamine repeats in proteins are highly correlated with amyloid formation and neurological disease. To better understand the molecular basis of glutamine repeat diseases, structural analysis of polyglutamine peptides as soluble monomers, oligomers, and insoluble amyloid fibrils is necessary. In this study, fluorescence resonance energy transfer (FRET) experiments and molecular dynamics simulations using different theoretical models of polyglutamine were conducted. This study demonstrates that a previously proposed simple CαCβ model of polyglutamine, denoted as FCO, accurately reproduced the present FRET results and the results of previously published FRET, triplet-state quenching, and fluorescence correlation studies. Other simple CαCβ models with random coil and extended β-strand parameters, and all-atom models with parm96 and parm99SB force fields, did not match the FRET result well. The FCO is an intrinsically disordered model with a high-effective persistence length producing extended peptides at short lengths (QN \u3c 10). Because of an increasing number of attractive Q–Q interactions at longer lengths, the FCO model becomes increasingly more compact at lengths between QN ∼ 10–16 and is as compact as many folded proteins at QN \u3e 16

    Kinetic analysis of IgG antibodies to beta-amyloid oligomers with surface plasmon resonance

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    Surface plasmon resonance was used to investigate the kinetics, affinity, and specificity of binding between anti-Aβ (beta-amyloid) IgG antibodies and oligomeric Aβ. Two factors were needed to accurately characterize the IgG binding kinetics. First, a bivalent model was necessary to properly fit the kinetic association and dissociation sensograms. Second, a high concentration of IgG was necessary to overcome a significant mass transport limitation that existed regardless of oligomer density on the sensor surface. Using high IgG concentrations and bivalent fits, consistent kinetic parameters were found at varying sensor surface ligand densities. A comparison of binding specificity, affinity, and kinetic flux between monoclonal and natural human anti-Aβ IgG antibodies revealed the following findings. First, monoclonal antibodies 6E10 and 4G8 single-site binding affinity is similar between Aβ oligomers and monomers. Second, natural human anti-Aβ IgG binding readily binds Aβ oligomers but does not bind monomers. Third, natural human anti-Aβ IgG binds Aβ oligomers with a higher affinity and kinetic flux than 6E10 and 4G8. Both the current analytical methodology and antibody binding profiles are important for advances in antibody drug development and kinetic biomarker applications for Alzheimer’s disease

    Biophysical characterization data on Aβ soluble oligomers produced through a method enabling prolonged oligomer stability and biological buffer conditions

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    The data here consists of time-dependent experimental parameters from chemical and biophysical methods used to characterize Aβ monomeric reactants as well as soluble oligomer and amyloid fibril products from a slow (3–4 week) assembly reaction under biologically-relevant solvent conditions. The data of this reaction are both of a qualitative and quantitative nature, including gel images from chemical cross-linking and Western blots, fractional solubility, thioflavin T binding, size exclusion chromatograms, transmission electron microscopy images, circular dichroism spectra, and fluorescence resonance energy transfer efficiencies of donor–acceptor pair labels in the Aβ chain. This data enables future efforts to produce the initial monomer and eventual soluble oligomer and amyloid fibril states by providing reference benchmarks of these states pertaining to physical properties (solubility), ligand-binding (thioflavin T binding), mesoscopic structure (electron microscopy, size exclusion chromatography, cross-linking products, SDS and native gels) and molecular structure (circular dichroism, FRET donor-acceptor distance). Aβ1-40 soluble oligomers are produced that are suitable for biophysical studies requiring sufficient transient stability to exist in their “native” conformation in biological phosphate-saline buffers for extended periods of time. The production involves an initial preparation of highly monomeric Aβ in a phosphate saline buffer that transitions to fibrils and oligomers through time incubation alone, without added detergents or non-aqueous chemicals. This criteria ensures that the only difference between initial monomeric Aβ reactant and subsequent Aβ oligomer products is their degree of peptide assembly. A number of chemical and biophysical methods were used to characterize the monomeric reactants and soluble oligomer and amyloid fibril products, including chemical cross-linking, Western blots, fraction solubility, thioflvain T binding, size exclusion chromatography, transmission electron micrscopy, circular dichroism spectroscopy, and fluorescence resonance energy transfer
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