Philip J. Davis

Cholesterol in condensed and fluid phosphatidylcholine monolayers studied by epifluorescence microscopy

Epifluorescence microscopy was used to investigate the effect of cholesterol on monolayers of dipalmitoylphosphatidylcholine (DPPC) and 1 -palmitoyl-2-oleoyl phosphatidylcholine (POPC) at 21 +/- 2 degrees C using 1 mol% 1-palmitoyl-2-[12-[(7-nitro-2-1, 3-benzoxadizole-4-yl)amino]dodecanoyl]phosphatidylcholine (NBD-PC) as a fluorophore. Up to 30 mol% cholesterol in DPPC monolayers decreased the amounts of probe-excluded liquid-condensed (LC) phase at all surface pressures (pi), but did not effect the monolayers of POPC, which remained in the liquid-expanded (LE) phase at all pi. At low pi (2-5 mN/m), 10 mol% or more cholesterol in DPPC induced a lateral phase separation into dark probe-excluded and light probe-rich regions. In POPC monolayers, phase separation was observed at low pi when > or =40 mol% or more cholesterol was present. The lateral phase separation observed with increased cholesterol concentrations in these lipid monolayers may be a result of the segregation of cholesterol-rich domains in ordered fluid phases that preferentially exclude the fluorescent probe. With increasing pi, monolayers could be transformed from a heterogeneous dark and light appearance into a homogeneous fluorescent phase, in a manner that was dependent on pi and cholesterol content. The packing density of the acyl chains may be a determinant in the interaction of cholesterol with phosphatidylcholine (PC), because the transformations in monolayer surface texture were observed in phospholipid (PL)/sterol mixtures having similar molecular areas. At high pi (41 mN/m), elongated crystal-like structures were observed in monolayers containing 80-100 mol% cholesterol, and these structures grew in size when the monolayers were compressed after collapse. This observation could be associated with the segregation and crystallization of cholesterol after monolayer collapse.

Exclusion of fluid lipid during compression of monolayers of mixtures of dipalmitoylphosphatidylcholine with some other phosphatidylcholines.

Monolayers composed of dipalmitoylphosphatidylcholine and one of four fluid phosphatidylcholines have been studied for their ability to attain low minimum surface tension during compression at two different speeds. The minimum surface tension depended on the compression rate and the proportions of the fluid and rigid lipid in the monolayer. The type of fluid lipid used in the monolayer also affected the minimum surface tension.

N-3 polyunsaturated fatty acids enhance cholesterol efflux from human fibroblasts in culture

Normal human skin fibroblasts were incubated in medium supplemented with 60 micrograms/ml linoleic acid (18:2n6) or eicosapentaenoic acid (20:5n3). After five days, cells lipids were enriched with linoleic acid or with docosapentaenoic acid (22:5n3). The HDL-mediated efflux of cholesterol from cells enriched with n-3 polyunsaturated fatty acids (PUFAs) was twice as fast as the rate of efflux of cholesterol from cells enriched with n-6 PUFAs. This suggests that the fatty acid composition of cellular lipids affects cholesterol efflux. The faster efflux when cells contain n-3 PUFAs may account for part of the reduction in risk of coronary disease with increases in dietary n-3 PUFAs (fish oils).

Scanning calorimetric studies of aqueous dispersions of bilayers made with cholesterol and a pair of positional isomers of 3-sn-phosphatidylcholine

Abstract Differential scanning calorimetric studies have been carried out on aqueous dispersions of cholesterol plus one of the pair of positional isomers, 1-arachidoyl-2-oleoylphosphatidylcholine (AOPC) or 1-oleoyl-2-arachidoylphosphatidylcholine (OAPC). There were some differences in the shapes of the endotherms obtained from the dispersions with the two positional isomers. These observations confirm the previous finding (Davis, P.J. and Keough, K.M.W. (1984) Biochemistry 22, 6334–6340) that positional isomers of unsaturated phosphatidylcholines may interact differently with cholesterol, at least in the gel state. The shapes of endotherms obtained here and in the previous study are consistent with the suggestion that the position of the unsaturated chain on the glycerol has a role in determining the nature of the phosphatidylcholine-cholesterol interaction. Certain features of the endotherms seen here and previously (op. cit.) suggest that factors such as effective chain depth in the bilayer or efficiency of chain packing in the bilayer may also influence this interaction.

Biochemical analysis of the interaction of calcium with toposome: A major protein component of the sea urchin egg and embryo

We have investigated the biochemical and functional properties of toposome, a major protein component of sea urchin eggs and embryos. Atomic force microscopy was utilized to demonstrate that a Ca2+‐driven change in secondary structure facilitated toposome binding to a lipid bilayer. Thermal denaturation studies showed that toposome was dependent upon calcium in a manner paralleling the effect of this cation on secondary and tertiary structure. The calcium‐induced, secondary, and tertiary structural changes had no effect on the chymotryptic cleavage pattern. However, the digestion pattern of toposome bound to phosphatidyl serine liposomes did vary as a function of calcium concentration. We also investigated the interaction of this protein with various metal ions. Calcium, Mg2+, Ba2+, Cd2+, Mn2+, and Fe3+ all bound to toposome. In addition, Cd2+ and Mn2+ displaced Ca2+, prebound to toposome, while Mg2+, Ba2+, and Fe3+ had no effect. Collectively, these results further enhance our understanding of the role of Ca2+ in modulating the biological activity of toposome. J. Cell. Biochem. 103: 1464–1471, 2008.

Thermal history alters cholesterol effect on transition of 1-palmitoyl-2-linoleoyl phosphatidylcholine

The effect of cholesterol on the bilayer phase behavior of heteroacid phosphatidylcholines with one unsaturated fatty acid depends on the nature of the unsaturated chain. Previous differential scanning calorimetry (DSC) studies showed that 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (16:0-18:2 PC) had a broad, weak transition at about -18 degrees C, which was effectively eliminated by less than 15 mol% cholesterol. Phospholipids with greater and lesser degrees of unsaturation displayed stronger phase transitions and less sensitivity to cholesterol. In this work, deuterium nuclear magnetic resonance has been used to examine the phase behavior of 1-perdeuteriopalmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (16:0-18:2 PC-d31) alone, and with 15 mol % cholesterol. The behavior is found to be sensitive to sample thermal history. Moderately fast cooling (1 degree/h) results in a continuous phase change from a fluid to an ordered phase in the pure lipid. Under similar cooling conditions, the sample containing cholesterol displays increased chain order and a continuous phase change with no apparent isothermal transition. However, when these systems are cooled at a reduced rate (0.3 degree/h), the continuous phase change is pre-empted by a sharp transition into a more ordered phase that gives a deuterium spectrum having intensity at a value of the quadrupole-splitting characteristic of a rigid lattice system. In the pure lipid, this transition effectively coincides with the center of the continuous phase change. Addition of 15 mol % cholesterol lowers the temperature of this sharp transition by about 3 degrees C. These observations provide some insights into the behavior of this system seen using differential scanning calorimetry. Results of deuteron transverse relaxation measurements under these conditions are also reported.

Functional role of a high mol mass protein complex in the sea urchin yolk granule

We have investigated the biochemical and functional characteristics of the major protein constituents of the yolk granule organelle present in sea urchin eggs and embryos. Compositional analysis, using sodium dodecyl sulfate polyacrylamide gel electrophoresis, revealed distinctly different polypeptide patterns under reducing and non‐reducing conditions. In the presence of reducing agent, a 240 kDa species dissociated into polypeptides of apparent mol mass 160, 120 and 90 k. The relatedness of these polypeptides to the 240 kDa species was demonstrated in protein gel blot and peptide mapping analyses. The profile of yolk granule polypeptides was dynamic during embryonic development with the disappearance of the 160 kDa species and the coincidental appearance of lower mol mass polypeptides. However, the 240 kDa complex was detected even after the disappearance of the 160 kDa polypeptide. The 240 kDa complex was released from yolk granules in the absence of calcium and the purified species was shown to bind liposomes in a calcium‐dependent manner. In addition, the 240 kDa complex possessed a calcium‐dependent, liposome aggregating activity. The 240 kDa species could also induce the aggregation of yolk granules, previously denuded of the complex following treatment with either ethylenediaminetetraacetic acid or trypsin. Collectively, these results demonstrate the dynamic characteristics of the yolk granule 240 kDa protein complex and offer insights into a possible functional role.

Seasonal changes in oleosomic lipids and fatty acids of perennial root nodules of beach pea.

Seasonal changes in the fatty acid composition of phospholipids (PL), monoglycerides (MG), diglycerides (DG), free fatty acids (FA) and triglycerides (TG) separated from oleosomes (lipid bodies) of perennial root nodules of beach pea (Lathyrus maritimus) were analysed. Thin layer chromatography (TLC) revealed that PL and MG are the major lipids in nodule oleosomes. The fatty acid profile and overall double bond index (DBI) varied among lipid classes depending upon the season. High DBI in PL and MG found during late winter and early spring indicated that they may play a major role in winter survival and regeneration of perennial nodules. The DBI of DG was high at the end of the fall season and the DBI of FA and TG was high in summer months. The dominant fatty acids are C16:0 followed by C18:0 and C18:1. The levels of many unsaturated fatty acids such as C18:1, C18:2 and C18:3 increased while saturated fatty acid C18:0 decreased during winter. These unsaturated fatty acids possibly play an important role in the protection of nodule cells from cold stress. Nodules seem to retain some fatty acids and selectively utilize specific fatty acids to survive the winter and regenerate in spring.

Gel to liquid-crystalline transitions of aqueous dispersions of positional isomers of a heteroacid unsaturated phosphatidylcholine mixed with epicholesterol and cholesterol

The gel to liquid-crystalline phase transitions of dispersions of two positional isomers of a phosphatidylcholine, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) and 1-oleoyl-2-stearoyl-sn-glycero-3-phosphocholine (OSPC) mixed with 13 and 17 mol% cholesterol or epicholesterol were observed by differential scanning calorimetry. Cholesterol caused a greater reduction in the enthalpy change of the transitions than did an equal amount of epicholesterol. Both sterols had a more pronounced influence on OSPC than on SOPC. Endotherms of PC plus sterol were resolvable into two components whose properties were similar for both sterols. The results suggest that the sterols interact differently with positional isomers of PC, and that the interaction is not strongly influenced by the orientation of the hydroxyl group of the sterol.