Effect of anti-tumor ether lipids on ordered domains in model membranes

1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (OMPC, edelfosine) and 1-hexadecylphosphocholine (HePC, miltefosine) represent two groups of synthetic ether lipid analogues with anti-tumor activity. Because of their hydrophobic nature, they may become incorporated into plasma membranes of cells, and it has been argued that they may act via association with lipid rafts. With the quenching of steady-state fluorescence of probes preferentially partitioning into sterol-rich ordered domains (cholestatrienol and trans-parinaric acid), we showed that OMPC and HePC by themselves did not form sterol-rich domains in fluid model membranes, in contrast to the two chain ether lipid 1,2-O-dihexadecyl-sn-glycero-3-phosphocholine. Nevertheless, all three ether lipids significantly stabilized palmitoyl-sphingomyelin/cholesterol-rich domains against temperature induced melting. In conclusion, this study shows that anti-tumor ether lipids are likely to affect the properties of cholesterol-sphingomyelin domains (i.e., lipid rafts) when incorporated into cell membranes.     

Composition and physical properties of lipids from plasma membranes of dog kidney

Lipid composition, physical state of major phospholipid classes and transbilayer migration of phosphatidylcholine have been determined in plasma membranes of the dog kidney. The lipid composition of brush-border membranes markedly differs from that of antiluminal membranes with respect to: (a) the total phospholipid content; (b) the cholesterol to phospholipid ratio (C/P); (c) the distribution of the major phospholipid classes. Sphingomyelin present in large amounts in both luminal and antiluminal membranes extracts exhibits a transition of phase between 20 and 44 degrees C approximately. In the range of temperature studied (5-55 degrees C) no phase transitions were detected for the other phospholipid species. Our data suggest that: (1) at physiological temperature the higher C/P ratio of brush-border membranes is in large part responsible for their lower fluidity; (2) both the relatively low cholesterol and high sphingomyelin contents contribute to the thermotropic transitions observed in intact membranes. Finally transbilayer migration of phosphatidylcholine in brush-border membranes is a very slow process with a half time of 6.5 h at 37 degrees C which compares with that of other biological membranes.     

Effect of dietary lipids on plasma lipoproteins and fluidity of lymphoid cell membranes in normal and leukemic mice

Mice of the GR/A strain were fed four different isocaloric semipurified diets, enriched in either (1) saturated fatty acids (palm oil), or (2) polyunsaturated fatty acids (corn oil), or (3) palm oil plus cholesterol, or (4) a fat-poor diet containing only a minimal amount of essential fatty acids. We have studied the effects of these dietary lipids on the density profile and composition of the plasma lipoproteins and on the lipid composition and fluidity of (purified) lymphoid cell membranes in healthy mice and in mice bearing a transplanted lymphoid leukemia (GRSL). Tumor development in these mice occurred in the spleen and in ascites. While the fatty acid composition of the VLDL-triacylglycerols still strongly resembled the dietary lipids, the effects of the diets decreased in the order VLDL-triacylglycerols greater than HDL-phospholipids greater than plasma membrane phospholipids. Diet-induced differences in the latter fraction were virtually confined to the content of oleic acid and linoleic acid, and they were too small to affect the membrane fluidity, as measured by fluorescence polarization using the probe 1,6-diphenyl-1,3,5-hexatriene. Healthy mice were almost irresponsive to dietary cholesterol, but in the tumor bearers, where lipoprotein metabolism has been shown to be disturbed, the cholesterol diet caused a substantial increase in the low- and very-low density regions of both blood and ascites plasma lipoproteins. The cholesterol-rich diet also increased the cholesterol/phospholipid molar ratio and lipid structural order (decreased fluidity) in GRSL ascites cell membranes, but not in the splenic GRSL cell membranes. We conclude that the composition of plasma lipoproteins and cell membrane lipids in GR/A mice is subject to exquisite homeostatic control. However, in these low-responders to dietary lipids the development of an ascites tumor may lead to increased responsiveness to dietary cholesterol. The elevated level of membrane cholesterol thus obtained in GRSL ascites cells did not affect the expression of various cell surface antigens or tumor cell growth.     

Synthesis and chiroptical properties of neutral ether lipids

A variety of neutral ether lipids was synthesized. A method for the synthesis of 1,3-O-dialkyl-sn-glycerols was developed which involves selective alkylation of 3-O-alkyl-sn-glycerols. The ORD and CD curves of the various glyceryl ethers and their esters were analyzed. The correlation between the CD sign of the acyl residue and its position in the glycerol derivative was clarified.     

Electrostatic properties of membranes containing acidic lipids and adsorbed basic peptides: theory and experiment

The interaction of heptalysine with vesicles formed from mixtures of the acidic lipid phosphatidylserine (PS) and the zwitterionic lipid phosphatidylcholine (PC) was examined experimentally and theoretically. Three types of experiments showed that smeared charge theories (e.g., Gouy-Chapman-Stern) underestimate the membrane association when the peptide concentration is high. First, the zeta potential of PC/PS vesicles in 100 mM KCl solution increased more rapidly with heptalysine concentration (14.5 mV per decade) than predicted by a smeared charge theory (6.0 mV per decade). Second, changing the net surface charge density of vesicles by the same amount in two distinct ways produced dramatically different effects: the molar partition coefficient decreased 1000-fold when the mole percentage of PS was decreased from 17% to 4%, but decreased only 10-fold when the peptide concentration was increased to 1 microM. Third, high concentrations of basic peptides reversed the charge on PS and PC/PS vesicles. Calculations based on finite difference solutions to the Poisson-Boltzmann equation applied to atomic models of heptalysine and PC/PS membranes provide a molecular explanation for the observations: a peptide adsorbing to the membrane in the presence of other surface-adsorbed peptides senses a local potential more negative than the average potential. The biological implications of these "discreteness-of-charge" effects are discussed.     

Biophysical properties of lipids and dynamic membranes

The lipid bilayer is a 3D assembly with a rich variety of physical features that modulate cell signaling and protein function. Lateral and transverse forces within the membrane are significant and change rapidly as the membrane is bent or stretched and as new constituents are added, removed or chemically modified. Recent studies have revealed how differences in structure between the two leaflets of the bilayer and between different areas of the bilayer can interact together with membrane deformation to alter the activities of transmembrane channels and peripheral membrane binding proteins. Here, we highlight some recent reports that the physical properties of the membrane can help control the function of transmembrane proteins and the motor-dependent elongation of internal organelles, such as the endoplasmic reticulum.     

Interaction of lipoproteins with isolated ovary plasma membranes

Plasma membranes of ovarian luteal and adrenal cortical cells from "microvillar channels," a unique extracellular compartment formed by the close apposition of flattened microvillar surfaces. Microvillar channels have unusual affinity for cholesterol-rich lipoproteins, and, in vivo, may provide an increased surface area for these particles. In this research, we have isolated a plasma membrane-enriched fraction from rat luteinized ovaries, in which closely apposed membrane (i.e. microvillar channels) comprise about 30% of the preparation. Following in vitro incubations (approximately 1 h) of this plasma membrane fraction with different plasma lipoproteins, the closely apposed plasma membrane surfaces widen and become filled with lipoprotein particles (up to about 30 nm), whereas other membranes of the fraction show little binding. Competition experiments show that rat high density lipoproteins have the highest affinity for binding to the plasma membrane fraction. Radiolabeled plasma lipoprotein and the tissue-specific hormone, human chorionic gonadotropin, showed specific and saturable binding to the plasma membrane fraction, whereas other macromolecules used as controls did not. Radioautographic analyses of 125I-labeled lipoproteins and human chorionic gonadotropin indicate that binding occurs predominantly to the closely apposed plasma membranes (i.e. microvillar channels of the fraction). These studies show that microvillar channels of steroid-secreting cells entrap large numbers of plasma lipoproteins, particularly high density lipoproteins particles, presumably functioning in the delivery of cholesterol to these cells.     

Intra-individual variation of plasma lipids and lipoproteins in prepubescent children

Estimates of the average intra-individual biological variability for plasma lipids and lipoproteins differs substantially among published studies. Moreover, this topic does not appear to have received consideration in exercise and health literature with normal, healthy children as subjects. The purpose of this study was, therefore, to determine the short-term, day-to-day variability of the lipid-lipoprotein profile from 19 children [mean (SD), 11.5 (0.8) years] from 3 separate venous blood samples. Intra-individual standard deviations, variances and coefficients of variance were determined for total cholesterol (TC), triacylglycerol (TG), high-density lipoprotein-cholesterol (HDL-C), HDL-C sub-fractions HDL2 and HDL3, and low-density lipoprotein-cholesterol (LDL-C). The intra-individual variation for TC and LDL-C was used to calculate the 95% confidence intervals (CIs) around the National Cholesterol Education Programme (NCEP 1991) cut-off points. The main finding was that all of the measured blood analytes including TC, TG, HDL-C, HDL2, HDL3, and LDL-C varied considerably from day-to-day. Coefficients of total variation ranged from 3.5% for HDL3 to 25.4% for TG. Classification of individuals using NCEP guidelines was difficult based on only one or two blood samples. The magnitude of variation for LDL-C meant that a 95% CI could not be constructed around the NCEP borderline-high classification from either one or two samples. However, averaging three TC and LDL-C measurements increased the likelihood of classification within the 95% CI. The results indicate that when using the NCEP guidelines for children and adolescents, true concentrations for TC and LDL-C should be based on the mean of multiple samples.     

Changes in plasma lipoproteins and liver lipids in neonatal rats

Transient increases in triglycerides and cholesterol were found in rat liver immediately after birth. Plasma VLDL and HDL increased after birth and reached a plateau after one week of life. The content of cholesterol ester was low at birth in all lipoproteins and increased in LDL and HDL during the first week of life. After birth, VLDL became enriched in apolipoproteins C and E, whereas HDL was enriched in apolipoprotein C and depressed in apolipoprotein E. The developmental changes in plasma lipoprotein levels and compositions in rats during the first week of life are comparable to those described in humans.     

Interaction of plasma lipoproteins and apolipoproteins with lipid bilayer membranes

It was shown that the interaction of lipoproteins (LP) with bilayer lipid membranes (BLM) resulted in some changes in the physical-chemical properties of the membranes. Adsorption of very low and low density lipoproteins (VLDL and LDL) at concentrations of 5-8 g protein/ml increased the surface potential difference and decreased transversal elasticity module of the bilayer. LP concentrations higher than the mentioned ones increased BLM conductance and caused instability and disruption of the membranes. The same effects were revealed for high density lipoproteins (HDL) at higher concentrations--15-20 micrograms protein/ml. The effect of apolipoproteins in the interaction of LP with BLM was investigated. It is proposed that apolipoproteins and especially apo B are the main factor which affects the nonreceptor interactions of LP with the membranes.     

Characterization of plasma lipids and lipoproteins in cholesteryl ester storage disease

Cholesteryl ester storage disease, caused by the loss of lysosomal acid ester hydrolase (EC 3.1.1.13), has been previously associated with hyperlipidemia and premature atherosclerosis. We identified a 23-month-old female with cholesteryl ester storage disease and characterized the plasma lipids and lipoproteins in the proband and her family. These studies illustrate several important points about this disease. First, a high index of suspicion is required to diagnose this disease since the major physical manifestation of the disorder, mild hepatomegaly, is subtle. Second, the Type II hyperlipoproteinemia in the proband is paralleled by a reduction in the concentration of high density lipoproteins. Third, analysis of the plasma lipids and lipoproteins in family members revealed both Type II and Type IV hyperlipoproteinemia with an inheritance pattern similar to that of familial combined hyperlipoproteinemia. Fourth, the parents and brother of this patient had 50% normal fibroblast acid ester hydrolase activity. These results raise the possibility that deficiency of the lysosomal acid ester hydrolase may be linked to familial combined hyperlipoproteinemia and that this enzyme deficiency may be more common than previously appreciated.     

Structure and interactions of lipids in human plasma low density lipoproteins.

Temperature-dependent techniques (differential scanning calorimetry, polarizing microscopy, and x-ray scattering and diffraction techniques) were used to compare the properties of human plasma low density lipoproteins (LDL) with its extracted lipid classes. Three types of thermal transitions were characterized: (a) a reversible transition in intact LDL near body temperature associated with a liquid crystalline order-disorder phase change of cholesterol esters within the particles; (b) an irreversible high temperature transition (approximately 70-90 degrees) associated with LDL denaturation and release of cholesterol esters from the disrupted particles; and (c) low temperature transitions related to liquid crystalline and crystalline phase changes in these released esters. The temperature of the reversible transition in intact LDL varies among individual donors. Correlation analysis shows that the temperature of this transition negatively correlates with the amount of triglyceride relative to cholesterol ester in LDL. Studies on mixtures of cholesterol esters and triglycerides isolated from LDL show a similar effect, increasing amounts of triglycerides decreasing the temperature of the liquid leads to smectic liquid crystalline transition of the isolated esters. Thus, the amount of triglyceride in LDL influences the fluidity of the cholesterol esters in LDL. The enthalpy of the reversible transition in intact LDL is 0.69 cal/g of LDL cholesterol ester. This compares with 0.89 cal/g for the liquid leads to liquid crystalline transition of the cholesterol esters released from denatured LDL and 1.01 cal/g for the same transition in the extracted esters. Unlike the cholesterol esters released from denatured LDL, or isolated LDL esters, cholesterol ester in the intact LDL particle does not crystallize. These findings suggest that the behavior of cholesterol esters in intact LDL is constrained relative to their behavior when freed from the restrictions of the particle. These results together with experiments on partitioning of the individual lipid classes of LDL allow us to define the distribution and interaction of lipids in the intact LDL particle.     

Interaction of phosphatidylcholine liposomes and plasma lipoproteins with sheep erythrocyte membranes: Preferential transfer of phosphatidylcholine containing unsaturated fatty acids

The interaction of sheep erythrocyte membranes with phosphatidylcholine vesicles (liposomes) or human plasma lipoproteins is described. Isolated sheep red cell membranes were incubated with liposomes containing [¹⁴C]phosphatidylcholine or [^3H]phosphatidylcholine in the presence of EDTA. A time-dependent uptake of phosphatidylcholine into the membranes could be observed. The content of this phospholipid was increased from 2 to 5%. The rate of transfer was dependent on temperature, the amount of phosphatidylcholine present in the incubation mixture and on the fatty acid composition of the liposomal phosphatidylcholine. A possible adsorption of lipid vesicles to the membranes could be monitored by adding cholesteryl [¹⁴C]oleate to the liposomal preparation. As cholesterylesters are not transferred between membranes [1], it was possible to differentiate between transfer of phosphatidylcholine molecules from the liposomes into the membranes and adsorption of liposomes to the membranes. The phosphatidylcholine incorporated in the membranes was isolated, and its fatty acids were analysed by gas chromatography. It could be shown that there was a preferential transfer of phosphatidylcholine molecules containing two unsaturated fatty acids.     

Interfacial properties of phosphatidylcholine bilayers containing vitamin E derivatives

alpha-Tocopherol and alpha-tocopheryl succinate are biologically active lipids. The activity of these lipids may be related to how they affect membrane physical-chemical properties. Utilizing fluorescence methods, we have investigated the effect of alpha-tocopherol, alpha-tocopheryl succinate, and alpha-tocopheryl acetate on the properties of model membranes consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine. In liquid-crystalline phase phospholipid bilayers, alpha-tocopherol decreased acyl chain mobility and decreased the interfacial polarity, but had no effect on the interfacial surface charge. In contrast, alpha-tocopheryl succinate had little effect on acyl chain motion or interfacial hydration, but increased the interfacial surface charge. alpha-Tocopheryl acetate had very little effect on any of the measurements of these bilayer properties. In a gel phase bilayer, alpha-tocopherol decreased acyl chain order, whereas alpha-tocopheryl succinate and alpha-tocopheryl acetate did not. Each alpha-tocopheryl derivative had a different effect on interfacial polarity, however, only alpha-tocopheryl succinate increased the interfacial surface charge. The acylation of alpha-tocopherol abolishes its antioxidant activity and generates molecules with different membrane physical properties. The non-polar acetate group of alpha-tocopheryl acetate locates this compound in a region of the bilayer where it has little effect on bilayer interfacial properties. The free carboxyl group of alpha-tocopheryl succinate is located in the interfacial region of the bilayer where it increases the membrane surface charge.     

Self-segregation of myelin membrane lipids in model membranes

Rapid conduction of nerve impulses requires coating of axons by myelin sheaths, which are multilamellar, lipid-rich membranes produced by oligodendrocytes in the central nervous system. To act as an insulator, myelin has to form a stable and firm membrane structure. In this study, we have analyzed the biophysical properties of myelin membranes prepared from wild-type mice and from mouse mutants that are unable to form stable myelin. Using C-Laurdan and fluorescence correlation spectroscopy, we find that lipids are tightly organized and highly ordered in myelin isolated from wild-type mice, but not from shiverer and ceramide synthase 2 null mice. Furthermore, only myelin lipids from wild-type mice laterally segregate into physically distinct lipid phases in giant unilamellar vesicles in a process that requires very long chain glycosphingolipids. Taken together, our findings suggest that oligodendrocytes exploit the potential of lipids to self-segregate to generate a highly ordered membrane for electrical insulation of axons.