Browsing by Subject "plasma membrane"

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    AFFINITY OF CHOLESTEROL FOR POLYUNSATURATED FATTY ACID-CONTAINING PHOSPHOLIPIDS
    (Office of the Vice Chancellor for Research, 2012-04-13) Kagimbi, Maureen W.; Williams, Justin A.; Wassall, Stephen R.
    A wide range of health benefits is associated with the consumption of omega-3 polyunsaturated fatty acids (PUFAs). One possible mechanism is that through our diet, they are incorporated into the phospholipids of the plasma membrane and disrupt the molecular organization of membrane do-mains due to the high disorder of PUFA. Our focus is the interaction of PUFA with cholesterol, a major component in plasma membranes. The objective here is to measure the affinity of cholesterol for PUFA-containing phospholip-ids by observing how cholesterol partitions between large unilamellar vesi-cles (LUVs) and Cyclodextrin (CD). Crucial to this determination, we need to be able to determine the concentration of cholesterol in LUVs and CD using an enzymatic colorimetric assay to create a standard curve of light absorb-ance (at 570nm wavelength) as a function of cholesterol concentration. The assay and its application to measuring binding coefficients for cholesterol will be described.
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    A Loop Region in the N-Terminal Domain of Ebola Virus VP40 Is Important in Viral Assembly, Budding, and Egress
    (Multidisciplinary Digital Publishing Institute (MDPI), 2014-10-17) Adu-Gyamfi, Emmanuel; Soni, Smita P.; Jee, Clara S.; Digman, Michelle A.; Gratton, Enrico; Stahelin, Robert V.; Department of Biochemistry & Molecular Biology, IU School of Medicine-South Bend
    Ebola virus (EBOV) causes viral hemorrhagic fever in humans and can have clinical fatality rates of ~60%. The EBOV genome consists of negative sense RNA that encodes seven proteins including viral protein 40 (VP40). VP40 is the major Ebola virus matrix protein and regulates assembly and egress of infectious Ebola virus particles. It is well established that VP40 assembles on the inner leaflet of the plasma membrane of human cells to regulate viral budding where VP40 can produce virus like particles (VLPs) without other Ebola virus proteins present. The mechanistic details, however, of VP40 lipid-interactions and protein-protein interactions that are important for viral release remain to be elucidated. Here, we mutated a loop region in the N-terminal domain of VP40 (Lys127, Thr129, and Asn130) and find that mutations (K127A, T129A, and N130A) in this loop region reduce plasma membrane localization of VP40. Additionally, using total internal reflection fluorescence microscopy and number and brightness analysis we demonstrate these mutations greatly reduce VP40 oligomerization. Lastly, VLP assays demonstrate these mutations significantly reduce VLP release from cells. Taken together, these studies identify an important loop region in VP40 that may be essential to viral egress.