Background: Nonfibrillar amyloid oligomers are cytotoxic and may act through physical disruption of cell membranes. Results: Cytotoxic oligomers of the amyloid peptide PrP(106 -126) disrupt membranes through distinct mechanisms, depending on lipid composition. Conclusion: Cytotoxicity of PrP(106 -126) oligomers can occur through at least two different physical processes. Significance: Mechanisms for the membrane disruption of amyloid oligomers are proposed, providing new insight into their cytotoxicity.

cells. Using surface plasmon resonance and centrifugation assays, we have found that the Smurf1 C2 domain binds to phosphoinositides and phosphatidylserine in an synergistic fashion. Confocal images of Smurf1 C2-GFP demonstrate that the domain localizes to the plasma membrane as well as intracellular vesicles in cells. Site-directed mutagenesis has shown the specific residues in the loop region of the protein involved in its cellular membrane localization. In addition, we have used a rapamycin-inducible phosphoinositide phosphatase system to demonstrate that this domain binds phosphoinositides at the plasma membrane. We conclude that the unique properties of the Smurf1 C2 domain to sense specific lipids in addition to anionic charge enable it to target multiple subcellular locations.

Understanding the mechanisms by which membrane-active molecules exert their action would benefit greatly from direct observation of the dynamics of the interactions and quantitative insights into the degree of physical restructuring and reordering. We previously developed a correlated total-internal reflectance fluorescence-atomic force microscopy (TIRF-AFM) platform that enabled direct determination of local order within the membrane [Oreopoulos and Yip, 2009] We report here the results of a study into the interactions of a series of variants of alpha-synuclein, a presynaptic protein that plays a key role in the pathogenesis of Parkinson's disease, with model membranes. These studies illustrate the role of membrane composition and fluidity on alpha-synuclein self-association and provide direct evidence of membrane reordering. This studies portend the application of related correlated approaches, including coupled ATR-FTIR-AFM for examining conformational changes upon membrane-induced aggregation.

Acyl group transfer from glycerophospholipids to melittin has recently been demonstrated using MS, LC-MS and LC-MS n methods (doi: c2ob07113d). This transfer is not mediated by enzyme catalysis, but rather is a consequence of the innate reactivity of the peptide toward lipids. Transfer from phosphatidylcholines (PCs) to melittin has been observed in a range of conditions of salt (from water to physiological concentrations), temperature (20 C to 37 C) and peptide to lipid ratio (P:L = 1:100 to 1:5). Lipidated peptides may be detected after 2-3 h, with a half-life for acyl transfer of~24 h for melittin. Other peptides that have been found to undergo acyl transfer in this manner include magainin II, PGLa, LAK1, LAK3 and penetratin. In mixed membranes (PC þ PE, PG, or PS), this intrinsic lipidation exhibits selectivity in respect of lipid type and the acyl chain composition of the individual components. This process is of high relevance for peptide and protein turnover in vivo, as well as an important factor to consider when interpreting data accumulated over significant time periods in vitro.

Background and Objectives: The growing demand for immunoglobulin (IG) requires development of improved plasma fractionation methods to provide higher yields in a cost effective, scalable manner without compromising product purity and efficacy.A novel protein extraction method, utilizing expanded bed adsorption (EBA) chromatography, has been developed. PlasmaCap IG (10% liquid formulation intravenous IG [IVIG]) is the first plasma-derived product manufactured using PlasmaCap EBA technology. Materials and Methods:The PlasmaCap EBA platform consists of a series of consecutive columns which bind a target protein, or group of proteins, in their native state directly from cryo-poor plasma. EBA chromatography includes five key steps:(1) expand, (2) sanitize and equilibrate, (3) load, (4) wash and (5) elute. These steps are made possible using high-density tungsten-carbide agarose beads, suspended by upward flow. The PlasmaCap EBA process was evaluated during Evolve's clinical campaign for scalability, product quality and yield.Results: PlasmaCap EBA technology can be predictably scaled by maintaining the minimum residence time and residence time distribution for EBA columns of different diameters. Scalability of the manufacturing process was demonstrated by the 50-fold volumetric increase from laboratory-scale lots to clinical-scale lots.The process is also associated with enhanced product purity, such as lower aggregates. The PlasmaCap EBA process is expected to have the same or better yield and purity at commercial scale production compared to the clinical campaign. Conclusion:The PlasmaCap EBA platform was used to successfully develop PlasmaCap IG (10% liquid formulation IVIG) with proven scalability, product quality and yield.