In vitro reverse cholesterol transport from THP-1-derived macrophage-like cells with synthetic HDL particles consisting of proapolipoprotein A1 or apolipoprotein A1 and phosphatidylcholine.

The human monocytic leukaemia cell line THP-1 was induced to differentiate to macrophage-like cells by the addition of phorbol myristoyl acetate (PMA). Subsequently, the cells were enriched in cholesterol and these cholesterol laden cells were used to study the capability of reconstituted discoidal complexes (RDCs), consisting of either human apolipoprotein A1 (apo A1) or recombinant human proapolipoprotein A1 (proapo A1) and phosphatidylcholine (PC), to promote cholesterol efflux. RDCs containing apo A1 and proapo A1 were both effective in the mobilization of intracellular cholesterol, whether this was measured by intracellular cholesterol mass or by the appearance of radiolabelled cholesterol in the supernatant.

Efflux of cholesterol from cholesterol loaded macrophages by incubation with synthetic HDL-particles.

Cells from the mouse monocyte/macrophage cell line J774A.1 were incubated with acetylated human low density lipoprotein for 2 days, resulting in an intracellular accumulation of mainly cholesteryl esters. These in vitro foam cell models were used to study the capability of synthetic HDL-particles to promote efflux of cholesterol.

Production of human recombinant proapolipoprotein A-I in Escherichia coli: purification and biochemical characterization.

A human liver cDNA library was used to isolate a clone coding for apolipoprotein A-I (Apo A-I). The clone carries the sequence for the prepeptide (18 amino acids), the propeptide (6 amino acids), and the mature protein (243 amino acids). A coding cassette for the proapo A-I molecule was reconstructed by fusing synthetic sequences, chosen to optimize expression and specifying the amino-terminal methionine and amino acids -6 to +14, to a large fragment of the cDNA coding for amino acids 15-243.

The role of fluorinated pyrimidine analogues in the induction of the in vitro expression of the fragile X chromosome.

The modes of action of 5-fluoro-2'-deoxyuridine (FdUrd) and 5-fluoro-2'-deoxycytidine (FdCyd) were studied in PHA-stimulated lymphocytes from normal volunteer donors and a fragile X patient. In both cell types, FdUrd and FdCyd inhibited cell proliferation at concentrations of 3 x 10(-8) M. Thymidylate synthetase was identified as the decisive target for the action of both FdUrd and FdCyd, as judged from the following observations: First, addition of thymidine to the culture medium was able to counteract both FdUrd and FdCyd toxicities, whereas addition of dCyd had no observable effect.