FIGURE 5.

Effects of oxysterols on unilamellar liposomes. A, oxysterol-induced dequenching of CF-containing liposomes. Liposomes containing 20 mm CF were maintained at room temperature followed by the addition of 0-50 μm 25-HC to the solution. Fluorescence was normalized to the total dequenching, following the addition of 1% Triton X-100. B, concentration dependence of 25-HC on fluorescence dequenching in liposomes. A dequenching curve was fitted to a linear function using standard least-squares methods (95% confidence limits are plotted). C, comparison of the effect of oxysterols (50 μm) on fluorescence dequenching of liposomes. Oxysterol dequenching was normalized to complete dequenching by Triton X-100 and is corrected for solvent effects on the fluorescence base line. Data shown are means ± S.E. ^*, p < 0.05 for 27-HC versus 7β-HC; ^**, p < 0.01 for 25-HC versus 7α-HC, 7β-HC, or 7-KC treatment and 27-HC versus 7α-HC or 7-KC treatment. Data shown are the mean of three experiments. D, comparison of the incorporation of 25-HC and 7-KC in DOPC/dioleoylphosphatidic acid (70:30) liposomes as determined by SPR spectroscopy. The liposome lipid and the oxysterol in the liposomes were determined by the SPR signal and used to calculate the mol % sterol in the liposome bilayer. Data were acquired at a rate of 10 data points/s and plotted as one data point/s. E, inhibition of oxysterol-induced fluorescence dequenching by CD. CF-containing liposomes were monitored for fluorescence and maintained at room temperature. At time 0, 0 μm (black line), 7.3 μm (blue line), or 50 μm (red line) 25-HC was added to the liposomes. At 300 s, 5 μm CD was added (black arrow) to the 25-HC liposome mixture, and subsequent fluorescence changes were plotted. Values are normalized to fluorescence measured following Triton X-100 addition at the end of each time series. Data shown are the mean of three experiments.