3 research outputs found
Non-denaturing polyacylamide gel electrophoresis (N-PAGE) of normal and heparin treated HDLs.
<p>AP-HDL, S-HDL-SAA and HDL were resolved by N-PAGE using 4–20% gradient gels with a continuous buffer system consisting of 0.1 M glycine-Tris-HCl, pH 8.6. Samples (30 µg) were loaded onto the gel and electrophoresis carried out at 4°C for 6 h at 150 volts. Bands were detected by staining with Coomassie Blue R250. A), lane 1, AP-HDL, lane 2, S-HDL-SAA, lane 3, float sample after re-centrifugation of S-HDL-SAA at d = 1.25 g/ml. Molecular weight markers are indicated on the left; B), lane 1, HDL, lane 2, S-HDL, lane 3, delipidated apoA-I.</p
The aggregation of AP-HDL at mildly acid pH when incubated with heparin or HS.
<p>A) HDL or AP-HDL at pH 5.2 or 7.2 were incubated for 30 min with increasing concentrations of heparin, HS+1 mM Ca<sup>2+</sup>, CS (chondroitin sufate) or DS (dermatan sulfate). The absorbance at 400 nm was then plotted against glycosaminoglycan concentration. B) AP-HDL (1 mg/ml) at pH 5.2 in 125 mM NaCl, 25 mM Na acetate, was incubated with 0.2 mg/ml heparin and absorbance at 400 nm read at intervals to 60 min. C) AP-HDL (1 mg/ml) incubated for 20 min at 37°C with heparin, or different chemically modified heparins, at 0.1 mg/ml as described in Panel B; CR-hep. = carboxy-reduced heparin; de-NS-hep. = N-desulfated heparin; de-2-O-hep. = de-2-O-sulfated heparin; de-6-O-hep. = de-6-O-sulfated heparin; de-OS-hep. = de-2,6-O-sulfated heparin. D) Incubations, supplemented with 1 mM Ca<sup>2+</sup>, greatly increased the AP-HDL aggregation activity of HS. AP-HDL was incubated at pH 5.2 with heparin, HS, CS, DS with or without Ca<sup>2+</sup>, as in C). The results in C) and D) are the mean±SEM of 3 experiments. The results with CS and DS were virtually identical.</p
Cholesterol efflux from J774 monocytes incubated with different lipoproteins.
<p>Monolayer cultures of J774 monocytes were loaded with cholesterol (22.7 µg/well) and [<sup>3</sup>H]-cholesterol (0.45 µCi) overnight, washed twice with DMEM in 0.2% BSA (1 ml) and once with DMEM alone. Cholesterol-loaded cells were then incubated with different lipoprotein preparations (50 µg/ml) in 1 ml DMEM, 0.2% BSA; A) S-HDL-SAA prepared with heparin; S-HDL-SAA prepared with HS+1 mM Ca2+; A-HDL-SAA; AP-HDL; HDL; S-HDL prepared with heparin. B) Lipid was extracted from cell and analyzed by thin-layer chromatography as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003867#pone.0003867-Kisilevsky2" target="_blank">[35]</a> to determine the amount of [3H]-cholestryl ester remaining in the cells relative to cholesterol-loaded cells. C) S-HDL-SAA; S-HDL-SAA preincubated with DEAE-Sepharose to remove any heparin; S-HDL-SAA recentrifuged at 1.25 g/ml to remove lipid poor species, A-HDL-SAA and delipidated apoA-I. Aliquots of medium were taken at the time intervals shown and the quantity of [<sup>3</sup>H]-cholesterol exported from the cells determined and expressed as a percentage of total [<sup>3</sup>H]-cholesterol in loaded cells before incubations.</p