PubMed · 14472185
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P MEIISEL. 1962. [Cholesterol polyposis].. https://pubmed.ncbi.nlm.nih.gov/14472185/
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A myristoylated peptide corresponding to the N-terminus of NAP-22 (neuronal axonal myristoylated membrane protein of 22 kDa) causes the quenching of the fluorescence of BODIPY-TMR-labelled PtdIns(4,5) P2 in bilayers of 1-palmitoyl-2-oleoyl phosphatidylcholine containing 40 mol% cholesterol and 0.1 mol% BODIPY-PtdIns(4,5)2. Both fluorescence spectroscopy and total internal reflectance fluorescence microscopy revealed the cholesterol-dependent nature of PtdIns(4,5) P2-enriched membrane-domain formation.
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An assay detecting and quantifying cholesterol nucleation from low-density lipoproteins has been established. Förster resonance energy transfer between dehydroergosterol and dansylated lecithin becomes significantly alleviated as a consequence of conucleation of dehydroergosterol and cholesterol. The assay, in combination with dynamic light scattering, absorbance spectroscopy, and fluorescence microscopy, can be used to study aggregation and nucleation in model blood systems. Human plasma LDL was labeled with dehydroergosterol and dansylated lecithin by incubation with donor multilamellar liposomes and isolated by centrifugation. Exposure of labeled LDL (0.5 mg/mL of total lipids) to sphingomyelinase (0.0-0.2 unit/mL) led to modest particle aggregation but produced no changes in energy transfer and no crystallization. However, addition of sphingomyelinase produced significant particle aggregation, nucleation, and crystallization, in a dose-dependent fashion, in samples that were previously treated with the enzyme, cholesterol esterase (0.2 unit/mL). The combination of cholesterol esterase and sphingomyelinase led to a significant alleviation of energy transfer, which preceded by 24 h the appearance of fluorescent, microscopic sterol crystals. These results point to a synergistic effect between cholesterol esterase and sphingomyelinase, suggesting that mere aggregation of LDL is insufficient to promote nucleation, and crystal formation likely proceeds in the intracellular space after LDL uptake by macrophages.