Knockout of cyclophilin D in Ppif⁻/⁻ mice increases stability of brain mitochondria against Ca²âº stress.

The mitochondrial peptidyl prolyl isomerase cyclophilin D (CypD) activates permeability transition (PT). To study the role of CypD in this process we compared the functions of brain mitochondria isolated from wild type (BMWT) and CypD knockout (Ppif(-/-)) mice (BMKO) with and without CypD inhibitor Cyclosporin A (CsA) under normal and Ca(2+) stress conditions. Our data demonstrate that BMKO are characterized by higher rates of glutamate/malate-dependent oxidative phosphorylation, higher membrane potential and higher resistance to detrimental Ca(2+) effects than BMWT. Under the elevated Ca(2+) and correspondingly decreased membrane potential the dose response in BMKO shifts to higher Ca(2+) concentrations as compared to BMWT. However, significantly high Ca(2+) levels result in complete loss of membrane potential in BMKO, too. CsA diminishes the loss of membrane potential in BMWT but has no protecting effect in BMKO. The results are in line with the assumption that PT is regulated by CypD under the control of matrix Ca(2+). Due to missing of CypD the BMKO can favor PT only at high Ca(2+) concentrations. It is concluded that CypD sensitizes the brain mitochondria to PT, and its inhibition by CsA or CypD absence improves the complex I-related mitochondrial function and increases mitochondria stability against Ca(2+) stress.

Vaccinia virus interactions with the cell membrane studied by new chromatic vesicle and cell sensor assays.

The potential danger of cross-species viral infection points to the significance of understanding the contributions of nonspecific membrane interactions with the viral envelope compared to receptor-mediated uptake as a factor in virus internalization and infection. We present a detailed investigation of the interactions of vaccinia virus particles with lipid bilayers and with epithelial cell membranes using newly developed chromatic biomimetic membrane assays. This analytical platform comprises vesicular particles containing lipids interspersed within reporter polymer units that emit intense fluorescence following viral interactions with the lipid domains. The chromatic vesicles were employed as membrane models in cell-free solutions and were also incorporated into the membranes of epithelial cells, thereby functioning as localized membrane sensors on the cell surface. These experiments provide important insight into membrane interactions with and fusion of virions and the kinetic profiles of these processes. In particular, the data emphasize the significance of cholesterol/sphingomyelin domains (lipid rafts) as a crucial factor promoting bilayer insertion of the viral particles. Our analysis of virus interactions with polymer-labeled living cells exposed the significant role of the epidermal growth factor receptor in vaccinia virus infectivity; however, the data also demonstrated the existence of additional non-receptor-mediated mechanisms contributing to attachment of the virus to the cell surface and its internalization.