| For medical practitioners and the general public - Cholesterol Journal Article Catalog. |
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| Differential lipid packing abilities and dynamics in giant unilamellar vesicles composed of short-chain saturated glycerol-phospholipids, sphingomyelin and cholesterol Kahya, N., D. Scherfeld, et al. (2005), Chem Phys Lipids 135(2): 169-80. Abstract: The ability of membrane components to arrange themselves heterogeneously within the bilayer induces the formation of microdomains. Much work has been devoted to mimicking domain-assembly in artificial bilayers and characterizing their physico-chemical properties. Ternary lipid mixtures composed of unsaturated phospholipids, sphingomyelin and cholesterol give rise to large, round domains. Here, we replaced the unsaturated phospholipid in the ternary mixture with sphingomyelin and cholesterol by saturated glycero-phospholipids of different chain length and characterized the critical role of cholesterol in sorting these lipids by confocal imaging and fluorescence correlation spectroscopy (FCS). More cholesterol is needed to obtain phase segregation in ternary mixtures, in which the unsaturated phospholipid is replaced by a saturated one. Finally, lipid dynamics in distinct phases is very low and astonishingly similar, thereby suggesting the poor ability of cholesterol in sorting short-chain saturated glycero-phospholipids and sphingomyelin. |
| Differential mobilization of newly synthesized cholesterol and biosynthetic sterol precursors from cells Lusa, S., S. Heino, et al. (2003), J Biol Chem 278(22): 19844-51. Abstract: Previous work demonstrates that the biosynthetic precursor of cholesterol, desmosterol, is released from cells and that its efflux to high density lipoprotein or phosphatidylcholine vesicles is greater than that of newly synthesized cholesterol (Johnson, W. J., Fischer, R. T., Phillips, M. C., and Rothblat, G. H. (1995) J. Biol. Chem. 270, 25037-25046). Here we report that the release of individual precursor sterols varies with the efflux of newly synthesized zymosterol being greater than that of lathosterol and both exceeding that of newly synthesized cholesterol when using either methyl-beta-cyclodextrin or complete serum as acceptors. The transfer of newly synthesized lathosterol to methyl-beta-cyclodextrin was inhibited by actin polymerization but not by Golgi disassembly whereas that of newly synthesized cholesterol was inhibited by both conditions. Newly synthesized lathosterol associated with cellular detergent-resistant membranes more rapidly than newly synthesized cholesterol. Upon efflux to serum, newly synthesized cholesterol precursors associated with both high and low density lipoproteins. Stimulation of the formation of direct endoplasmic reticulum-plasma membrane contacts was accompanied by enhanced efflux of newly synthesized lathosterol but not of newly synthesized cholesterol to serum acceptors. The data indicate that the efflux of cholesterol precursors differs not only from that of cholesterol but also from each other, with the more polar zymosterol being more avidly effluxed. Moreover, the results suggest that the intracellular routing of cholesterol precursors differs from that of newly synthesized cholesterol and implicates a potential role for the actin cytoskeleton and endoplasmic reticulum-plasma membrane contacts in the efflux of lathosterol. |
| Differential modulation of the antifungal activity of amphotericin B by natural and ent-cholesterol Richter, R. K., D. E. Mickus, et al. (2004), Bioorg Med Chem Lett 14(1): 115-8. Abstract: The addition of exogenous ent-cholesterol suppressed the antifungal activity of the amphotericin B when added to cultures of Candida albicans, but to a lesser extent than natural cholesterol. There were no detectable differences between added 2a or 2b on the antifungal activities of jaspamide or bengazole A, two unrelated antifungal natural products. |
| Differential patterns of lipid-protein association in fast and slow cholesterol nucleating human gallbladder biles: implications for cholesterol nucleation from biliary lipid carriers Ginanni Corradini, S., D. Alvaro, et al. (1991), Biochim Biophys Acta 1086(1): 125-33. Abstract: We compared the protein/lipid structure and Ch-nucleating capacity of individual lipid carriers in two groups of human gallbladder biles: 11 with Fast cholesterol nucleation (2.2 +/- 1.3 days) and 10 with Slow cholesterol nucleation (19.2 +/- 4.4 days). The groups had comparable cholesterol-saturation (1.31 vs. 1.28), total lipids (9.9 vs. 8.5 g/dl) and proteins (8.5 vs. 7.6 mg/ml). Bile was ultracentrifuged (2 h at 150,000 x g) and the resulting isotropic phase was incubated with 3HCh and 14Clecithin and gel-chromatographed on a Superose 6 column with a buffer containing 7.0 mM sodium-taurocholate. Seven protein peaks were identified (280 nm and biochemistry), with the following molecular mass ranges (kDa): 1 (Void volume), 2 (155-205), 3 (50-79), 4 (20-29), 5 (6-15), 6 (3.5-6), 7 (2-3.5). Peaks 2 and 3 were identified as vesicles and micelles, respectively. Fast vs. Slow Ch nucleating biles had: (a) more (P less than 0.02) cholesterol coeluting with vesicles, (b) more (P less than 0.01) lecithin coeluting with low m.w. peaks (Nos. 5-6), (c) less (P less than 0.01) cholesterol and lecithin coeluting with micelles. An inverse correlation (P less than 0.001) was observed between the amount of proteins coeluting with the micellar peak and the cholesterol nucleation of both whole bile and isolated micellar fractions. A marked shift of cholesterol and lecithin from micelles to vesicles was apparent, in the whole bile, after cholesterol nucleation had occurred. Incubation and sequential analysis of isolated and radiolabeled micelles showed a progressive transfer of lecithin and cholesterol molecules to low molecular weight fractions and to vesicles before cholesterol nucleation. We conclude that pro-nucleating biliary vesicles develop from micelles, due to the phasing out and redistribution of micellar cholesterol and lecithin, which are probably induced by biliary proteins. |
| Differential phenotypic expression by three mutant alleles in familial lecithin:cholesterol acyltransferase deficiency Gotoda, T., N. Yamada, et al. (1991), Lancet 338(8770): 778-81. Abstract: Familial deficiency of lecithin:cholesterol acyltransferase (LCAT) is an autosomal recessive disorder characterised by abnormalities of all plasma lipoprotein classes and by abnormal deposition of unesterified cholesterol in tissues. To elucidate the molecular basis of the disease, the LCAT genes of three unrelated Japanese patients were amplified by means of the polymerase chain reaction. Direct sequencing of the amplified fragments covering all exons and junctions showed that the patients are homozygotes for separate gene mutations. In one patient a 3 bp insertion, which should cause a substantial change in the enzyme structure, was found in exon 4; he had near absence of LCAT mass and activity. Two separate missense mutations were identified in exon 6 of the other two patients, who produced functionally defective enzymes that differed widely in specific activity. The replacement of asparagine228 with positively charged lysine completely abolished enzyme activity, whereas the other, conservative, aminoacid substitution (methionine293----isoleucine) gave rise to a partially defective enzyme. These results show that distinct mutations cause differences in plasma LCAT activity and LCAT mass, ultimately leading to differential phenotypic expression of familial LCAT deficiency. |
| Differential pulse voltammetric measurements of cholesterol based on its oxidation after adsorption at the mercury electrode Peng, T. Z., H. P. Li, et al. (1992), Yao Xue Xue Bao 27(12): 928-33. Abstract: Cholesterol can be adsorbed on the surface of the static mercury drop electrode, and then oxidized during anodic potential scan. The stripping peak potential in differential pulse voltammetry is at -0.08 V in 0.1 mol/L K2HPO4-KH2PO4 buffer solution. The stripping current is linear with the concentration of cholesterol in the range of 10(-7)-10(-6) mol/L. The detection limit found is 8 x 10(-8) mol/L after a 5 min pre-concentration period. The adsorption and oxidation mechanisms of cholesterol at the mercury electrode were then explored. The experimental conditions for measuring free cholesterol in human sera were examined. |
| Differential rate of cholesterol efflux from the apical and basolateral membranes of MDCK cells Remaley, A. T., B. D. Farsi, et al. (1998), J Lipid Res 39(6): 1231-8. Abstract: Epithelial cells contain two distinct membrane surfaces, the apical and basolateral plasma membranes, which have different lipid and protein compositions. In order to assess the effect of the compositional differences of the apical and basolateral membranes on their ability to undergo cholesterol efflux, MDCK cells were radiolabeled with 3Hcholesterol and grown as a polarized monolayer on filter inserts, that separate the upper apical compartment from the lower basolateral compartment. The rate of cholesterol efflux from the basolateral membrane into media containing HDL in the basolateral compartment was 6.3%/h +/-0.7, whereas HDL-mediated efflux from the apical membrane was approximately 3-fold slower (1.9%/h +/-0.3). In contrast, Fu5AH cells, which do not form distinct polarized membrane domains, had a similar rate of HDL-mediated cholesterol efflux into the apical and basolateral compartments. Similar to HDL, other cholesterol acceptors, namely LDL, bovine serum albumin, and a lipid emulsion, also showed a decreased rate of cholesterol efflux from the apical membrane surface versus the basolateral membrane. Compared to the basolateral membrane, the apical membrane was also found to be more resistant to cholesterol oxidase treatment, to bind less HDL, and to take up less cholesterol from the medium. In conclusion, cholesterol efflux occurred less readily from the apical membrane than from the basolateral membrane for all types of acceptors tested. These results suggest that differences in the composition of the apical and basolateral membrane lead to a relative decrease in cholesterol desorption from the apical membrane and hence a reduced rate of cholesterol efflux. |
| Differential reactivity of two homogeneous LDL-cholesterol methods to LDL and VLDL subfractions, as demonstrated by ultracentrifugation and HPLC Usui, S., H. Kakuuchi, et al. (2002), Clin Chem 48(11): 1946-54. Abstract: BACKGROUND: The analytical and clinical performance of homogeneous LDL-cholesterol assays has been reported, but their reactions with subfractions of LDL and VLDL have not been described in detail. METHODS: We evaluated reaction selectivity of two homogeneous LDL-cholesterol assays, LDLk (Kyowa Medex) and LDLd (Daiichi Pure Chemical), with ultracentrifugally isolated VLDL and LDL subfractions to identify the lipoprotein particles from which the cholesterol recognized by these assays originates. RESULTS: The LDLd (y) and LDLk (x) methods correlated highly for whole serum samples: y = 0.986x - 39.5 mg/L (r = 0.966; n = 34). In isolated VLDL, the LDLk and the LDLd methods recovered 17.3% and 23.8% of cholesterol, respectively; but correlation analysis revealed differential reactivity to small and large VLDL particles. For the isolated LDL subfraction of density 1.019-1.040 kg/L, the LDLd method had significantly higher reactivity (95.6-98.7%) than the LDLk (88.4-92.0%). Both methods, however, demonstrated poor recovery (approximately 50%) for the 1.050-1.063 kg/L fraction, indicating incomplete reactivity with small, dense LDL. Reactivity with lipoprotein(a) was better (71.2-90.8%) for both methods than with small LDL. For intermediate-density lipoprotein (IDL), there was no significant difference in recovery between the two methods (71.7% for LDLk and 68.9% for LDLd), but the LDLk method appeared to be more sensitive to IDL particle size. CONCLUSIONS: The two homogeneous assays for LDL-cholesterol demonstrate only partial reactivity to small, dense LDL and nonspecific reactions to VLDL particles. Modification will be required in the homogeneous methods to obtain LDL-cholesterol values equivalent to those obtained by ultracentrifugation. |
| Differential reduction of plasma cholesterol by the American Heart Association Phase 3 Diet in moderately hypercholesterolemic, premenopausal women with different body mass indexes Cole, T. G., P. E. Bowen, et al. (1992), Am J Clin Nutr 55(2): 385-94. Abstract: The ability of a low-fat, low-cholesterol diet to improve the risk-factor profiles of moderately hypercholesterolemic, premenopausal women was evaluated. Nineteen women were fed a typical American diet for 1 mo, after which a low-fat diet consisting of 21% of total energy (en%) as fat, 59 en% carbohydrates, 19 en% protein, and 96 mg cholesterol/d (P:S 1.8) was given. After 5 months, total and low-density lipoprotein (LDL) cholesterol was decreased by 7% and 11%, respectively, and total triglycerides increased by approximately 30%. High-density-lipoprotein (HDL) cholesterol was decreased by 12% at month 2 and 5% at month 5 (P less than 0.05). Although HDL2 cholesterol decreased progressively throughout the diet period to -35% by month 5, HDL3 cholesterol, which decreased to -5% at month 1, increased to +7% by month 5. Of the plasma apolipoproteins only apo A-II was altered (+15%) by the diet. Body mass index correlated to baseline values and affected response to diet; only the leanest women had significant decreases in total, LDL, and HDL2 cholesterol in response to the low-fat diet. |
| Differential regulation of apolipoprotein A-I/ATP binding cassette transporter A1-mediated cholesterol and phospholipid release Yamauchi, Y., S. Abe-Dohmae, et al. (2002), Biochim Biophys Acta 1585(1): 1-10. Abstract: We compared apolipoprotein A-I (apoA-I)-mediated release of cellular cholesterol and phospholipid among several fibroblast cell lines. ApoA-I induced phospholipid release from WI-38, MRC-5, BALB/3T3, L929 and CHO-K1, but not from COS-7, while cholesterol was released only from WI-38, MRC-5 and BALB/3T3 without correlation to the cellular cholesterol content. The reaction consequently generated cholesterol-rich high density lipoprotein (HDL) with WI-38, MRC-5 and BALB/3T3, cholesterol-poor HDL with L929 and CHO-K1, and no HDL from COS-7. In contrast, diffusion-mediated cholesterol efflux to cyclodextrin took place with all the cell lines tested in proportion to the cellular free cholesterol content. While caveolin-1 was expressed in all of these cell lines, ATP-binding cassette transporter (ABC) A1 was detected in all but COS-7. We concluded that (1) fibroblasts were categorized into three groups with respect to the interaction with apoA-I, (i) those to produce cholesterol-rich HDL, (ii) cholesterol-poor HDL and (iii) no HDL; (2) ABCAl was required for the assembly of HDL from phospholipid among the cell lines tested; (3) caveolin-1 alone did not induce cholesterol incorporation into the HDL generated. |
| Differential regulation of extracellular matrix metalloproteinase and tissue inhibitor by heparin and cholesterol in fibroblast cells Tyagi, S. C., S. Kumar, et al. (1997), J Mol Cell Cardiol 29(1): 391-404. Abstract: Heparin has been shown to stimulate angiogenesis in the border zones surrounding infarcted myocardium. Matrix metalloproteinases (MMP), which are involved in extracellular matrix (ECM) organization, have also been shown to be activated. Cholesterol is required for receptor signaling in the plasma membrane, but a role of MMPs for cholesterol in ECM remodeling has not yet been shown. To examine whether heparin and cholesterol induce MMP and tissue inhibitor of metalloproteinase (TIMP) in human heart fibroblast (HHF) cells, confluent HHF cells were treated with cholesterol (100 microM) or heparin (20 microM). MMP activity was measured using zymography and TIMP was measured by Western blot analysis. The number of HHF cells, measured by a hemocytometer, increased after heparin or cholesterol treatment. Gelatinase A (MMP-2) activity increased in heparin treated cells, and the TIMP-1 level increased in cholesterol-treated cells. Based on Northern blot analysis, we observed that both MMP-1 and MMP-2 were induced at the gene transcription level by heparin and that TIMP-1 was induced by cholesterol. To examine whether the effects of heparin and cholesterol were due to Ca2+ mobilization, we carried out Ca2+ transient assays using FURA-2/AM as a fluorescence probe in HHF cells. Heparin induced a slow rise in the Ca2+ transient with a slow decay, and cholesterol induced a rapid rise with a slow reversal to the baseline calcium level. This suggested that the effect of heparin on Ca2+ release from HHF may be secondary to the receptor binding on the cell membrane but that cholesterol may have a direct effect. Protein kinase inhibitor and Ca2+-channel blocker have been shown to inhibit MMP expression. To examine whether the effect of heparin on MMP expression is mediated through the collagenase promoter activity, we carried out gel-shift assays using a 21-oligonucleotide analogue to the MMP-1 promoter sequence. Results suggested that the increase in MMP promoter activity by heparin is due to a specific transcription factor binding to MMP-1 promoter sequence. The effect of cholesterol on fibroblast cell proliferation is due in part to the tissue inhibitor. This study demonstrated the role of heparin and cholesterol in ECM remodeling and has implications for angiogenesis and athersclerosis, respectively. |
| Differential regulation of low density lipoprotein suppression of HMG-CoA reductase activity in cultured cells by inhibitors of cholesterol biosynthesis Gupta, A. K., R. C. Sexton, et al. (1990), J Lipid Res 31(2): 203-15. Abstract: Treatment of rat intestinal epithelial cells (IEC-6 cells) with lanosterol 14 alpha-demethylase inhibitors, ketoconazole and miconazole, had similar effects on 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity and cholesterol biosynthesis but the drugs differed in their ability to prevent the low density lipoprotein (LDL) suppression of reductase activity. Miconazole, at concentrations that inhibited the metabolism of lanosterol and epoxylanosterol to the same degree as ketoconazole, did not prevent low density lipoprotein action on reductase activity, whereas ketoconazole totally abolished the low density lipoprotein action on reductase activity. Both drugs caused: 1) a biphasic response in reductase activity such that at low concentrations (less than 2 microM) reductase activity was inhibited and at high concentrations (greater than 5 microM) the activity returned to control or higher than control levels; 2) an inhibition of metabolism of lanosterol to cholesterol, and 24(S), 25-epoxylanosterol to 24(S), 25-epoxycholesterol. Neither drug prevented suppression of reductase activity by 25-hydroxylanosterol, 25-hydroxycholesterol, or mevalonolactone added to the medium. Each drug increased the binding, uptake, and degradation of 125I-labeled LDL and inhibited the re-esterification of free cholesterol to cholesteryl oleate and cholesteryl palmitate. The release of free cholesterol from 3Hcholesteryl linoleate LDL could not account for the differential effect of ketoconazole and miconazole on the prevention of low density lipoprotein suppression of reductase activity. The differential effect of the drugs on low density lipoprotein suppression of reductase activity was not unique to IEC-6 cells, but was also observed in several cell lines of different tissue origin such as human skin fibroblast cells (GM-43), human hepatoblastoma cells (HepG2), and Chinese hamster ovary cells (wild type, K-1; 4 alpha-methyl sterol oxidase mutant, 215). These observations suggest that the suppressive action of low density lipoprotein on reductase activity 1) does not require the de novo synthesis of cholesterol, or 24(S), 25-epoxysterols; 2) is not mediated via the same mechanism as that of mevalonolactone; and 3) does not involve cholesteryl reesterification. Ketoconazole blocks a site in the process of LDL suppression of reductase activity that is not affected by miconazole. |
| Differential response to low-fat diet between low and normal HDL-cholesterol subjects Asztalos, B., M. Lefevre, et al. (2000), J Lipid Res 41(3): 321-8. Abstract: Heart attacks frequently occur in normolipidemic subjects with low concentration of high density lipoproteins (35 mg/dL). We hypothesized that as subjects with low HDL-C already have low HDL concentrations, the major decrease of HDL-C will occur in subjects with normal HDL-C when a low-fat diet is consumed. Normolipidemic male subjects consumed three diets differing in total fat and saturated fat composition (AAD: 37%, Step-1: 28%, Step-2: 24% total fat) for 6 weeks in a three-period double-blind randomized crossover design. Plasma lipids and apolipoproteins were determined and changes in distribution of HDL subpopulations were evaluated. As a result of a low-fat diet, low HDL-C individuals slightly decreased their HDL-C, but substantially decreased their LDL-C resulting in a significant improvement in the LDL-C/HDL-C ratio. However, subjects with normal HDL-C levels decreased both their LDL-C and HDL-C resulting in an unchanged LDL-C/HDL-C ratio. We also observed significant differences in response to low-fat diets in HDL-C and alpha(1) concentrations between low and normal HDL-C subjects. In the normal HDL-C group, consumption of a low-fat diet also resulted in redistribution of apoA-I-containing HDL subpopulations, indicated by a decrease in the large apoA-I-only alpha(1) subpopulation. These data demonstrate that male subjects with low HDL-C respond to a low-fat diet differently than individuals with normal HDL-C. |
| Differential role of apolipoprotein AI-containing particles in cholesterol efflux from adipose cells Barkia, A., P. Puchois, et al. (1991), Atherosclerosis 87(2-3): 135-46. Abstract: Cholesterol efflux was studied in cultured Ob1771 adipose cells after preloading with LDL cholesterol. Exposure to particles containing apo AII (LpAI) and particles containing apo AI and apo AII (LpAI:AII) isolated from native human plasma, and from HDL2 or HDL3, showed that only LpAI were able to promote cholesterol efflux, despite the fact that both kinds of particles were able to bind to receptor sites within the same range of concentrations (apparent Kd values between 10 and 25 micrograms/ml). During this long-term exposure, LpAI:AII demonstrated a concentration-dependent inhibition (10-60 micrograms/ml) of LpAI-mediated cholesterol efflux from adipose cells under conditions where LpAI:AII did not deliver cholesterol to the cells and where no net change in the distribution of apo AI between LpAI and LpAI:AII was observed. The antagonizing and modulating role of LpAI:AII in preventing cholesterol efflux mediated by LpAI appears not to be related to the lipid composition and cholesterol content of the particles but, rather, appears dependent upon the presence of apo AI in LpAI particles and apo AII in LpAI:AII particles. The actual concentrations of these particles in the interstitial fluid bathing peripheral cells might be critical for the in vivo occurrence of cholesterol efflux. |
| Differential scanning calorimetric and Fourier transform infrared spectroscopic studies of the effects of cholesterol on the thermotropic phase behavior and organization of a homologous series of linear saturated phosphatidylserine bilayer membranes McMullen, T. P., R. N. Lewis, et al. (2000), Biophys J 79(4): 2056-65. Abstract: We have examined the effects of cholesterol on the thermotropic phase behavior and organization of aqueous dispersions of a homologous series of linear disaturated phosphatidylserines by high-sensitivity differential scanning calorimetry and Fourier transform infrared spectroscopy. We find that the incorporation of increasing quantities of cholesterol progressively reduces the temperature, enthalpy, and cooperativity of the gel-to-liquid-crystalline phase transition of the host phosphatidylserine bilayer, such that a cooperative chain-melting phase transition is completely or almost completely abolished at 50 mol % cholesterol, in contrast to the results of previous studies. We are also unable to detect the presence of a separate anhydrous cholesterol or cholesterol monohydrate phase in our binary mixtures, again in contrast to previous reports. We further show that the magnitude of the reduction in the phase transition temperature induced by cholesterol addition is independent of the hydrocarbon chain length of the phosphatidylserine studied. This result contrasts with our previous results with phosphatidylcholine bilayers, where we found that cholesterol increases or decreases the phase transition temperature in a chain length-dependent manner (1993. Biochemistry, 32:516-522), but is in agreement with our previous results for phosphatidylethanolamine bilayers, where no hydrocarbon chain length-dependent effects were observed (1999. Biochim. Biophys. Acta, 1416:119-234). However, the reduction in the phase transition temperature by cholesterol is of greater magnitude in phosphatidylethanolamine as compared to phosphatidylserine bilayers. We also show that the addition of cholesterol facilitates the formation of the lamellar crystalline phase in phosphatidylserine bilayers, as it does in phosphatidylethanolamine bilayers, whereas the formation of such phases in phosphatidylcholine bilayers is inhibited by the presence of cholesterol. We ascribe the limited miscibility of cholesterol in phosphatidylserine bilayers reported previously to a fractional crystallization of the cholesterol and phospholipid phases during the removal of organic solvent from the binary mixture before the hydration of the sample. In general, the results of our studies to date indicate that the magnitude of the effect of cholesterol on the thermotropic phase behavior of the host phospholipid bilayer, and its miscibility in phospholipid dispersions generally, depend on the strength of the attractive interactions between the polar headgroups and the hydrocarbon chains of the phospholipid molecule, and not on the charge of the polar headgroups per se. |
| Differential scanning calorimetric studies of the interaction of cholesterol with distearoyl and dielaidoyl molecular species of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine McMullen, T. P. and R. N. McElhaney (1997), Biochemistry 36(16): 4979-86. Abstract: We have carried out a comparative study of the effect of cholesterol on the thermotropic phase behavior of the distearoyl and dielaidoyl molecular species of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine using high-sensitivity differential scanning calorimetry. For both molecular species of phosphatidylcholine, cholesterol incorporation produces bimodal endotherms at lower and unimodal endotherms at higher sterol concentrations. In both cases, heating and cooling endotherms are identical, and high concentrations of cholesterol (50 mol %) completely abolish the gel to liquid-crystalline phase transition. For the distearoyl molecular species of phosphatidylserine and phosphatidylethanolamine, heating and cooling endotherms are not identical, and cholesterol exhibits a considerably reduced miscibility in the gel as compared to the liquid-crystalline phase, particularly in the latter case. Thus, in neither case does the addition of 50 mol % cholesterol completely abolish the cooperative hydrocarbon chain-melting phase transition. However, the dielaidoyl molecular species of phosphatidylserine and phosphatidylethanolamine exhibit much closer correspondence in the heating and cooling modes than do the distearoyl species, and 50 mol % cholesterol is sufficient to almost or completely abolish the gel to liquid-crystalline phase transition of dielaidoylphosphatidylethanolamine and dielaidoylphosphatidylserine. In general, there is an inverse correlation between the strength of intermolecular phospholipid-phospholipid interactions, as manifested by the relative gel to liquid-crystalline phase transition temperatures of the pure phospholipids, and the miscibility of cholesterol in bilayers, particularly gel-state bilayers, formed from these phospholipids. These results indicate that the nature of cholesterol-phospholipid interactions, and thus the miscibility of cholesterol in the bilayer, depends on both the structure of the phospholipid polar headgroup and the hydrocarbon chains, as well as on the temperature and phase state of the phospholipid bilayer. |
| Differential scanning calorimetric study of the effect of cholesterol on the thermotropic phase behavior of a homologous series of linear saturated phosphatidylcholines McMullen, T. P., R. N. Lewis, et al. (1993), Biochemistry 32(2): 516-22. Abstract: We have studied the effects of cholesterol on the thermotropic phase behavior of aqueous dispersions of a homologous series of linear saturated phosphatidylcholines, using high-sensitivity differential scanning calorimetry and an experimental protocol which ensures that broad, low-enthalpy phase transitions are accurately monitored. We find that the incorporation of small amounts of cholesterol progressively decreases the temperature and the enthalpy, but not the cooperativity, of the pretransition of all phosphatidylcholines exhibiting such a pretransition and that the pretransition is completely abolished at cholesterol concentrations above 5 mol % in all cases. The incorporation of increasing quantities of cholesterol also alters the main or chain-melting phase transition of these phospholipid bilayers in both hydrocarbon chain length-dependent and hydrocarbon chain length-independent ways. At cholesterol concentrations of from 1 to 20-25 mol %, the DSC endotherms of all phosphatidylcholines studied consist of a superimposed sharp and broad component, the former ascribed to the melting of cholesterol-poor and the latter to the melting of the cholesterol-rich phosphatidylcholine domains. The temperature and cooperativity of the sharp component are reduced only slightly and in a chain length-independent manner with increasing cholesterol concentration, an effect we ascribe to the colligative effect of the presence of small quantities of cholesterol at the domain boundaries. Moreover, the enthalpy of the sharp component decreases and becomes zero at 20-25 mol % cholesterol for all of the phosphatidylcholines examined.(ABSTRACT TRUNCATED AT 250 WORDS) |
| Differential scanning calorimetric study of the interaction of cholesterol with the major lipids of the Acholeplasma laidlawii B membrane McMullen, T. P., B. C. Wong, et al. (1996), Biochemistry 35(51): 16789-98. Abstract: It has been proposed that the lower levels of exogenous cholesterol incorporation into the membranes of the sterol-non-requiring as compared to the sterol-requiring mycoplasmas may be due to the much higher glycolipid content of the former and to the reduced ability of glycolipids, as opposed to phospholipids, to incorporate sterols Efrati et al. (1986) Arch. Biochem. Biophys. 248, 282-288. In order to test this hypothesis, we have investigated the interaction of cholesterol with the major membrane glyco- and phospholipids of the sterol-non-requiring mycoplasma Acholeplasma laidlawii B, utilizing elaidic acid-homogenous membranes in order to obviate any differences in the nature of cholesterol-lipid interactions due to variations in the fatty acid composition of the different membrane components. Specifically, we have studied the effect of increasing quantities of cholesterol on the thermotropic phase behavior of aqueous dispersions of phosphatidylglycerol, diglucosyl diacylglycerol, and monoglucosyl diacylglycerol, as well as the total membrane polar lipids of this organism, using high-sensitivity differential scanning calorimetry. We find that cholesterol is highly miscible in both the lamellar gel and liquid-crystalline states of phosphatidylglycerol but exhibits limited miscibility in the two neutral glycolipids, particularly in their lamellar gel and crystalline states. We also demonstrate that cholesterol has a limited miscibility in both the lamellar gel and liquid-crystalline states of bilayers composed of the total A. laidlawii B membrane polar lipids. These results demonstrate that the nature of cholesterol-lipid interactions depends markedly on the structure of the glycerolipid polar headgroup and suggests that the incorporation of lower levels of cholesterol into the membranes of the sterol-non-requiring mycoplasmas may indeed be due, at least in part, to their high glycolipid contents. We also show that cholesterol stabilizes the lamellar liquid-crystalline phase of the monoglucosyl diacylglycerol relative to the inverted hexagonal phase at all sterol concentrations, in contrast to the effects of cholesterol on dielaidoylphosphatidylethanolamine, which destabilizes the lamellar liquid-crystalline phase at low concentrations. |
| Differential sensitivity to acute cholesterol lowering of activation mediated via the high-affinity IgE receptor and Thy-1 glycoprotein Surviladze, Z., L. Draberova, et al. (2001), Eur J Immunol 31(1): 1-10. Abstract: Lateral cross-linking of transmembrane high-affinity IgE receptors (FcepsilonRI) or glycosylphosphatidylinositol-anchored Thy-1 glycoproteins on the surface of rat mast cells and rat basophilic leukemia (RBL) cells triggers the signaling pathways that lead to the release of allergy mediators. Although both of these pathways are initiated by an increased activity of Lyn kinase, the exact mechanism by which Lyn kinase interacts with aggregated FcepsilonRI and Thy-1 is not completely understood. Here we demonstrate that pretreatment of RBL cells with methyl-beta-cyclodextrin (MBCD) resulted in a dose- and time-dependent decrease in cellular cholesterol, increased detergent solubilization of Thy-1 and Lyn kinase, and a transient increase in tyrosine phosphorylation of several proteins. Acute lowering of cholesterol suppressed the activation through Thy-1, as determined by tyrosine phosphorylation of Syk kinase and some other proteins, and modulation of free cytoplasmic calcium. In contrast, the FcepsilonRI-mediated activation events were more resistant. Thy-1 and FcepsilonRI in MBCD-pretreated cells also differed in the extent of aggregation after cross-linking: Thy-1 formed large caps, whereas FcepsilonRI accumulated in small patches. MBCD treatment induced an increased release of secretory components in both Thy-1- and FcepsilonRI-activated cells. The combined data indicate that cholesterol depletion does not merely block receptor signaling but has more complex consequences. |
| Differential stimulation of cholesterol and unsaturated fatty acid biosynthesis in cells expressing individual nuclear sterol regulatory element-binding proteins Pai, J. T., O. Guryev, et al. (1998), J Biol Chem 273(40): 26138-48. Abstract: Three sterol regulatory element-binding proteins (SREBP-1a, -1c, and -2) stimulate transcription of genes involved in synthesis and receptor-mediated uptake of cholesterol and fatty acids. Here, we explore the individual roles of each SREBP by preparing lines of Chinese hamster ovary (CHO) cells that express graded amounts of nuclear forms of each SREBP (designated nSREBPs) under control of a muristerone-inducible nuclear receptor system. The parental hamster cell line (M19 cells) lacks its own nSREBPs, owing to a deletion in the gene encoding the Site-2 protease, which releases nSREBPs from cell membranes. By varying the concentration of muristerone, we obtained graded expression of individual nSREBPs in the range that restored lipid synthesis to near physiologic levels. The results show that nSREBP-2 produces a higher ratio of synthesis of cholesterol over fatty acids than does nSREBP-1a. This is due in part to a selective ability of low levels of nSREBP-2, but not nSREBP-1a, to activate the promoter for squalene synthase. nSREBP-1a and -2 both activate transcription of the genes encoding stearoyl-CoA desaturase-1 and -2, thereby markedly enhancing the production of monounsaturated fatty acids. nSREBP-1c was inactive in stimulating any transcription at the concentrations achieved in these studies. The current data support the emerging view that the nSREBPs act in complementary ways to modulate the lipid composition of cell membranes. |