Electroblotting onto activated glass. High efficiency preparation of proteins from analytical sodium dodecyl sulfate-polyacrylamide gels for direct sequence analysis

We have developed a new method for the isolation of proteins for microsequencing. It consists of electrophoretic transfer (electroblotting) of proteins or their cleavage fragments onto activated glass filter paper sheets immediately after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins are immobilized on the glass fiber sheets by ionic interactions or by covalent attachment. A wide range of proteins can be prepared in this fashion with no apparent restriction due to solubility, size, charge, or other intrinsic properties of the proteins. As little as 50 ng of the transferred proteins can be detected using Coomassie Blue or fluorescent dye staining procedures and even smaller amounts of radiolabeled proteins by autoradiography. After detection, the protein- containing bands or spots are cut out and inserted directly into a gas- phase sequenator. The piece of glass fiber sheet acts as a support for the protein during the sequencing. Amounts of protein in the 5- to 150- pmol range can be sequenced, and extended runs can be obtained from the blotted samples because of improved stepwise yields and lower backgrounds. The method has been successfully applied to the sequencing of a variety of proteins and peptides isolated from one-dimensional and two-dimensional polyacrylamide gels

Sugar and Spice, Not Everything\u27s Nice: Changing Dietary Habits in Bali

In this paper, I will explore the ways in which locals interact with new food options on the island of Bali. Specifically, I will explore dietary behaviors surrounding pre-­‐ packaged snacks and ‘fast-­‐food’ chains and determine whether the presence of these food options is affecting the Balinese’s relationship with more traditional cuisine. I will use everyday observations and conversations with locals to analyze dietary habits and the various food items that are being consumed while examining the potential dietary differentiation between two age groups. I will also consider health outcomes in Bali and discuss how changing dietary habits may be playing a role in the increasing cases of non-­‐ communicable diseases and declining dental health on the island

Activated Bone Marrow-Derived Macrophages Eradicate Alzheimer's-Related Aβ42 Oligomers and Protect Synapses.

Impaired synaptic integrity and function due to accumulation of amyloid β-protein (Aβ42) oligomers is thought to be a major contributor to cognitive decline in Alzheimer's disease (AD). However, the exact role of Aβ42 oligomers in synaptotoxicity and the ability of peripheral innate immune cells to rescue synapses remain poorly understood due to the metastable nature of oligomers. Here, we utilized photo-induced cross-linking to stabilize pure oligomers and study their effects vs. fibrils on synapses and protection by Aβ-phagocytic macrophages. We found that cortical neurons were more susceptible to Aβ42 oligomers than fibrils, triggering additional neuritic arborization retraction, functional alterations (hyperactivity and spike waveform), and loss of VGluT1- and PSD95-excitatory synapses. Co-culturing neurons with bone marrow-derived macrophages protected synapses against Aβ42 fibrils; moreover, immune activation with glatiramer acetate (GA) conferred further protection against oligomers. Mechanisms involved increased Aβ42 removal by macrophages, amplified by GA stimulation: fibrils were largely cleared through intracellular CD36/EEA1+-early endosomal proteolysis, while oligomers were primarily removed via extracellular/MMP-9 enzymatic degradation. In vivo studies in GA-immunized or CD115+-monocyte-grafted APPSWE/PS1ΔE9-transgenic mice followed by pre- and postsynaptic analyses of entorhinal cortex and hippocampal substructures corroborated our in vitro findings of macrophage-mediated synaptic preservation. Together, our data demonstrate that activated macrophages effectively clear Aβ42 oligomers and rescue VGluT1/PSD95 synapses, providing rationale for harnessing macrophages to treat AD

Single-molecule imaging reveals that small amyloid-β1-42 oligomers interact with the cellular prion protein (PrP(C)).

Oligomers of the amyloid-β peptide (Aβ) play a central role in the pathogenesis of Alzheimer's disease and have been suggested to induce neurotoxicity by binding to a plethora of cell-surface receptors. However, the heterogeneous mixtures of oligomers of varying sizes and conformations formed by Aβ42 have obscured the nature of the oligomeric species that bind to a given receptor. Here, we have used single-molecule imaging to characterize Aβ42 oligomers (oAβ42) and to confirm the controversial interaction of oAβ42 with the cellular prion protein (PrP(C)) on live neuronal cells. Our results show that, at nanomolar concentrations, oAβ42 interacts with PrP(C) and that the species bound to PrP(C) are predominantly small oligomers (dimers and trimers). Single-molecule biophysical studies can thus aid in deciphering the mechanisms that underlie receptor-mediated oAβ-induced neurotoxicity, and ultimately facilitate the discovery of novel inhibitors of these pathways.This is the final published version. It was first published by Wiley in ChemBioChem (http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1439-7633)

Design and Characterization of Chemically Stabilized Aβ42 Oligomers

A popular working hypothesis of Alzheimer's disease causation is amyloid β-protein oligomers are the key neuropathogenetic agents. Rigorously elucidating the role of oligomers requires the production of stable oligomers of each size. We previously used zero-length photochemical cross-linking to allow stabilization, isolation, and determination of structure-activity relationships of pure populations of Aβ40 dimers, trimers, and tetramers. We also attempted to study Aβ42 but found that Aβ42 oligomers subjected to the same procedures were not completely stable. On the basis of the fact that Tyr is a critical residue in cross-linking chemistry, we reasoned that the chemical accessibility of Tyr10 in Aβ42 must differ from that in Aβ40. We thus chemically synthesized four singly substituted Tyr variants that placed the Tyr in different positions across the Aβ42 sequence. We then studied the stability of the resulting cross-linked oligomers as well as procedures for fractionating the oligomers to obtain pure populations of different sizes. We found that [Phe(10),Tyr(42)]Aβ42 produced stable oligomers yielding highly pure populations of dimers through heptamers. This provides the means to establish formal structure-activity relationships of these important Aβ42 assemblies. In addition, we were able to analyze the dissociation patterns of non-cross-linked oligomers to produce a model for oligomer formation. This work is relevant to the determination of structure-activity relationships that have the potential to provide mechanistic insights into disease pathogenesis

Self-assembly and anti-amyloid cytotoxicity activity of amyloid beta peptide derivatives

The self-assembly of two derivatives of KLVFF, a fragment Abeta(16-20) of the amyloid beta (Abeta) peptide, is investigated and recovery of viability of neuroblastoma cells exposed to Abeta is observed at sub-stoichiometric peptide concentrations. Fluorescence assays show that NH2-KLVFF-CONH2 undergoes hydrophobic collapse and amyloid formation at the same critical aggregation concentration (cac). In contrast, NH2-K(Boc)LVFF-CONH2 undergoes hydrophobic collapse at a low concentration, followed by amyloid formation at a higher cac. These findings are supported by the beta-sheet features observed by FTIR. Electrospray ionization mass spectrometry indicates that NH2-K(Boc)LVFF-CONH2 forms a significant population of oligomeric species above the cac. Cryo-TEM, used together with SAXS to determine fibril dimensions, shows that the length and degree of twisting of peptide fibrils seem to be influenced by the net peptide charge. Grazing incidence X-ray scattering from thin peptide films shows features of beta-sheet ordering for both peptides, along with evidence for lamellar ordering of NH2-KLVFF-CONH2. This work provides a comprehensive picture of the aggregation properties of these two KLVFF derivatives and show their utility, in unaggregated form, in restoring the viability of neuroblastoma cells against Abeta-induced toxicity

Mechanism of amyloid β-protein dimerization determined using single-molecule AFM force spectroscopy.

Aβ42 and Aβ40 are the two primary alloforms of human amyloid β-protein (Aβ). The two additional C-terminal residues of Aβ42 result in elevated neurotoxicity compared with Aβ40, but the molecular mechanism underlying this effect remains unclear. Here, we used single-molecule force microscopy to characterize interpeptide interactions for Aβ42 and Aβ40 and corresponding mutants. We discovered a dramatic difference in the interaction patterns of Aβ42 and Aβ40 monomers within dimers. Although the sequence difference between the two peptides is at the C-termini, the N-terminal segment plays a key role in the peptide interaction in the dimers. This is an unexpected finding as N-terminal was considered as disordered segment with no effect on the Aβ peptide aggregation. These novel properties of Aβ proteins suggests that the stabilization of N-terminal interactions is a switch in redirecting of amyloids form the neurotoxic aggregation pathway, opening a novel avenue for the disease preventions and treatments

Rapid Photochemical Cross-Linking — A New Tool for Studies of Metastable, Amyloidogenic Protein Assemblies

Amyloidoses comprise a class of diseases characterized pathologically by the presence of deposits of fibrillar, aberrantly folded proteins, known as amyloids. Historically, these deposits were considered the key factors causing disease. However, recent evidence suggests that soluble protein oligomers, which are precursors for amyloid fibrils, are the primary toxic effectors responsible for the disease process. Understanding the mechanism by which these oligomers exert their toxicity requires knowledge of the structure, kinetics, and thermodynamics of their formation and conversion into larger assemblies. Such studies have been difficult due to the metastable nature of the oligomers. For the amyloid beta-protein (Abeta), a consensus about the size and relative abundance of small oligomers has not been achieved. We describe here the application of the method Photoinduced Cross-Linking of Unmodified Proteins (PICUP) to the study of Abeta oligomerization. This approach distinguishes oligomerization patterns of amyloidogenic and nonamyloidogenic proteins, allows quantification of each component in oligomer mixtures, and provides a means of correlating primary structure modifications with assembly characteristics. PICUP thus is a powerful tool for the investigation of small, metastable protein oligomers. The method provides essential insights into the factors that control the assembly of pathogenic protein oligomers, facilitating efforts toward the development of therapeutic agents