Mechanism of tocopherol transfer by human α-tocopherol transfer protein (α-hTTP)

Vitamin E is a well known fat soluble chain breaking antioxidant. It is a general tenn used to describe a family of eight stereoisomers of tocopherols. Selective retention of a-tocopherol in the human circulation system is regulated by the a -Tocopherol Transfer Protein (a-TIP). Using a fluorescently labelled a-tocopherol (NBD-a-Toc) synthesized in our laboratory, a fluorescence resonance energy transfer (FRET) assay was developed to monitor the kinetics of ligand transfer by a-hTTP in lipid vesicles. Preliminary results implied that NBD-a-Toe simply diffused from 6-His-a-hTTP to acceptor membranes since the kinetics of transfer were not responsive to a variety of conditions tested. After a series of trouble shooting experiments, we identified a minor contaminant, E coli. outer membrane porin F (OmpF) that co-purified with 6-His-a-hTTP from the metal affinity column as the source of the problem. In order to completely avoid OmpF contamination, a GST -a-hTTP fusion protein was purified from a glutathione agarose column followed by an on-column thrombin digestion to remove the GST tag. We then demonstrated that a-hTTP utilizes a collisional mechanism to deliver its ligand. Furthennore, a higher rate of a-tocopherol transfer to small unilamellar vesicles (SUV s) versus large unilamellar vesicles (LUV s) indicated that transfer is sensitive to membrane curvature. These findings suggest that ahTTP mediated a-Toc transfer is dominated by the hydrophobic nature of a-hTTP and the packing density of phospholipid head groups within acceptor membranes. Based on the calculated free energy change (dG) when a protein is transferred from water to the lipid bilayer, a model was generated to predict the orientation of a-hTTP when it interacts with lipid membranes. Guided by this model, several hydrophobic residues expected to penetrate deeply into the bilayer hydrophobic core, were mutated to either aspartate or alanine. Utilizing dual polarization interferometry and size exclusion vesicle binding assays, we identified the key residues for membrane binding to be F 165, F 169 and 1202. In addition, the rates of ligand transfer of the u-TTP mutants were directly correlated to their membrane binding capabilities, indicating that membrane binding was likely the rate limiting step in u-TTP mediated transfer of u-Toc. The propensity of u-TTP for highly curved membrane provides a connection to its colocalization with u-Toc in late endosomes.

Canada/U.S. border crossing: facilitation and constraint

Abstract The aim of this research project is to draw on accounts of experiences ofborder crossing and regulation at the Canada/U.S. border at Niagara in order to illuminate the dynamics of differentiation and inequality at this site. The research is informed by claims that the world is turning into a global village due to transnational flows oftechnology, infonnation, capital and people. Much of the available literature on globalization shows that while the transfer of technology, information, and capital are enhanced, the transnational movement of people is both facilitated and constrained in complex and unequal ways. In this project, the workings of facilitation and constraint were explored through an analysis often interviews with people who had spent a substantial portion oftheir childhood (e.g. 5 years) in a Canadian border community. The interviewees were at the time ofthe research between the ages of 19 and 25. Because most ofthe respondents were 'white' Canadians of working to upper middle class status, my focus was to explore how 'whiteness' as privilege may translate into enhanced movement across borders and how 'white' people may internalize and enjoy this privilege but may often deny its reality. I was also interested in how inequality is perceived, understood, and legitimated by these relatively privileged people. My analysis ofthe ten accounts ofborder crossing and regulation suggests that differentially situated people experience border crossing differently. An important finding is that while relatively privileged border crossers perceived and often problernatized differential treatment based on external factors such as physical appearance, and especially race, most did not challenge such treatment but rather saw it as acceptable. These findings are located within newer literature that addresses the increasing securitization ofborders and migration in western societies.

Special Orders No. 18 of the Office U.S. Army Hospital, Camp Parole

Special Orders No. 18 of the Office U.S. Army Hospital, Camp Parole, near Annapolis, MD., March 14, 1865 to W.K. Cleveland assigning him to duty as pathologist and consulting surgeon for the hospital. This is signed by W. Stewart, Surgeon U.S. Vol’s, in charge, March 14, 1865.