Alcohol binding to liposomes by 2H NMR and radiolabel binding assays: does partitioning describe binding?

Implicit within the concept of membrane-buffer partition coefficients of solutes is a nonspecific solvation mechanism of solute binding. However, (2)H NMR studies of the binding of (2)H(6)-ethanol and [1-(2)H(2)] n-hexanol to phosphatidylcholine vesicles have been interpreted as evidence for two distinct alcohol binding modes. One binding mode was reported to be at the membrane surface. The second mode was reported to be within the bilayer interior. An examination of the (2)H NMR binding studies, together with direct radiolabel binding assays, shows that other interpretations of the data are more plausible. The results are entirely consistent with partitioning (nonspecific binding) as the sole mode of alcohol binding to liposomes, in accord with our previous thermodynamic interpretation of alcohol action in phosphatidylcholine liposomes.     

Detection of low density lipoprotein particle fusion by proton nuclear magnetic resonance spectroscopy

Recent evidence suggests that fusion of low density lipoprotein (LDL) particles is a key process in the initial accumulation of lipid in the arterial intima. In order to gain a better understanding of this early event in the development of atherosclerosis, it would thus be necessary to characterize the process of LDL fusion in detail. Such studies, however, pose severe methodological difficulties, such as differentiation of particle fusion from aggregation. In this paper we describe the use of novel methodology, based on 1H NMR spectroscopy, to study lipoprotein particle fusion. To test the methodology, we chose proteolytic fusion of LDL particles, an in vitro model that has been well characterized in our laboratory. The spectroscopic data suggested that proteolysis of LDL with alpha-chymotrypsin induced slow initiation of fusion, which was followed by particle fusion at an increased rate. Moreover, 1H NMR spectroscopic data on different kinds of LDL interactions, for example, when LDL formed aggregates with antibodies against human apolipoprotein B-100, were obtained and compared with the electron microscopic characteristics of these preparations. An important finding was that limited aggregation of LDL particles did not disturb the 1H NMR spectroscopic parameters used for the detection of particle fusion and preserved the physico-chemical information on the particles. The 1H NMR methodology developed is sensitive to and specific for low density lipoprotein (LDL) fusion and may also allow for studies of the fate of LDL particles in other in vitro preparations that mimic the arterial interactions in vivo.     

Catecholamine regulation of the prefrontal cortex

In order to contribute to the understanding of the biological properties of nafazatrom, an antithrombotic agent (NAP), we studied its effects on peroxidation of low density lipoproteins (LDL), lipid liposomes, heart homopgenate, and its interaction with alpha-tocopherol radical. NAP decreased the FeSO4 and H202-induced peroxidation of phosphatidylcholine liposomes and heart homogenate, and it decreased peroxidation of LDL induced by CuSO4 or 2,2'-azobis(2-amidinopropane). The antioxidant effect of NAF was about 3 times less potent than that of alpha-tocopherol (alpha-TOC) in phosphatidylcholine liposomes, and NAF was about 2-4 times more efficient to decrease peroxidation of LDL than alpha-TOC. Possible interaction of NAF with alpha-tocopherol radical (alpha-TR) was studied by EPR spectroscopy. NAF decreased the concentration of alpha-TR, but it was about 100-times less efficient than vitamin C. This may indicate that NAF does not interfere with alpha-TR formation and/or reduction of alpha-TR in biological system. The obtained results may help the explanation of biological effects of NAF.     

1H MAS and 1H[23Na] double resonance NMR studies on the modification of surface hydroxyl groups of gamma-alumina by sodium

The modification of surface hydroxyl groups with sodium in a series of Na2CO3-gamma-Al2O3 catalysts was investigated as a function of both the Na2CO3 loading and the calcination temperature by means of 1H magic angle spinning (MAS) and 1H(23Na) spin-echo double resonance NMR techniques. The 1H NMR experiments revealed that sodium ions are homogeneously distributed over the alumina surface and closely coordinated with the surface hydroxyl groups. In the catalysts calcined at 250 degrees C, the acidic hydroxyl groups (with a chemical shift of 2.0 ppm) are preferentially associated with sodium ions at low Na2CO3 coverages (5 and 10%), while both the acidic and the basic (0 ppm) hydroxyl groups are accessible for sodium ions at high coverages (15 and 20%). The coordination causes a low-field shift of about 2 ppm in the 1H MAS spectra, and a broad signal at 4.5 ppm appears. It is interesting that the 4.5 ppm signal is completely suppressed in the 1H(23Na) MAS experiments, providing direct evidence that a strong interaction exists between adsorbed sodium ions and the surface hydroxyl groups. Increasing the calcination temperature to 450 degrees C results in preferential removal of the acidic hydroxyl groups, and only the most basic hydroxyl groups remain when the calcination temperature is raised to 600 degrees C. This is attributed to the formation of the coordinated species. [formula: see text] which enhances the acidity of the surface hydroxyl groups and prompts their dehydroxylation, especially at high calcination temperature. Correlation of the 1H MAS NMR results and catalytic activity measurements indicates that the basic hydroxyl groups are essential for the carbonyl sulfide hydrolysis reaction.     

Phospholipid head-group conformations; intermolecular interactions and cholesterol effects

The predominant orientation of the phosphorylcholine polar head group in phosphatidylcholine and sphingomyelin bilayers and cholesterol perturbations of that orientation have been identified by exploiting the 31P (1H) nuclear Overhauser effect (NOE) in the 31P NMR spectra of phospholipid bilayers. In pure egg phosphatidylcholine bilayers, a NOE of 40% is observed. The magnitude of the NOE has been measured as a function of continuous-wave proton-decoupler frequency in order to identify the proton source of the NOE. In pure egg phosphatidylcholine bilayers, the maximum NOE occurs at the N-methyl proton resonance position of the choline moiety. In a modified phosphatidylcholine in which all the N-methyl protons were replaced by deuterium, the NOE arose from methylene protons next to the phosphate. In mixed systems of phosphatidylcholine and phosphatidylethanolamine, and phosphatidylcholine and diphosphatidylglycerol, both phospholipid resonances attained maximum NOE at the position of the N-methyl proton resonance of phosphatidylcholine. An analogous result was obtained with pure sphingomyelin. These results are explained by orienting the phosphorylcholine portion of the molecule parallel to the surface of the bilayer so that the positively charged N-methyl moiety is located close to the negatively charged phosphate on a neighboring phospholipid in an intermolecular interaction. Addition of cholesterol is shown to disrupt the intermolecular interaction in phosphatidylcholine bilayers.     

31P NMR studies of unsonicated aqueous dispersions of neutral and acidic phospholipids. Effects of phase transitions, p2H and divalent cations on the motion in the phosphate region of the polar headgroup

1. The 129 MHz (non-proton decoupled) and 36.4 MHz (proton decoupled) 31P NMR spectra arising from unsonicated aqueous dispersions of well defined species of phospholipid have been investigated. The phospholipids employed (and the parameters varied) include phosphatidylcholine (temperature), phosphatidylethanolamine (temperature), phosphatidic acid (temperature and p2H) and phosphatidylglycerol (temperature, p2H and Ca2+ (or Mg2+)) concentration. 2. At p2H = 7 the 31 P MNR spectra arising from saturated species of phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol become progressively broader as the temperature is reduced below the phase transition, demonstrating reduced motion in the phosphate region of the polar headgroup. 3. In the liquid crystalline state at p2H = 7 the molecular dipolar order parameters obtained for saturated species of phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol and very similar, and are independent of the acyl chain length for species derived from lauric and myristic acid. Thus the motion in the methylene-phosphate-methylene region is similar for these different liquid crystaline phospholipid species. 4. The 31 P NMR spectra of aqueous dispersions of 14:0/14:0 phosphatidic acid display anomalous temperature and p2H dependences. The effective chemical shift anistropy (delta v CSA EFF) at 5 degrees C varies from 71 ppm at p2H = 8.5 to 38 ppm at p2H = 2.5. Further, the motion in the phosphate region is relatively insensitive to the gel or liquid crystalline nature of the hydrocarbon chains. 5. The addition of 40 mol% Ca2+ (or Mg2+) to saturated species of phosphatidylglycerol causes an increase of approx. 20 degrees C in the hydrocarbon phase transition temperature as indicated by 31 P NMR. Equimolar concentrations of Ca2+ increase the transition temperature by approx. 70 degrees C, and no 31P NMR signal could be observed for the very condensed precipitate formed below this temperature. In the liquid crystalline state the motion in the phosphate region of the polar headgroup is not significantly affected by the presence of Ca+ or Mg2+. 6. The 31P NMR spectra obtained from 18 : 1c/18 : 1c phosphatidylethanolamine are consistent with a phase transition from a lamellar to an hexagonal (HII) phase in the region 10-15 degrees C. 7. The observed narrowing of the 31 P NMR spectra of aqueous dispersions of phospholipids as the temperature is raised toward the hydrocarbon transition temperature is discussed in terms of the "pretransition" observed in calorimetric studies.     

TNF-induced modulations of phospholipid metabolism in human breast cancer cells

Tumor necrosis factor alpha (TNF) is a cytokine that is cytocidal for certain tumor cells and induces necrotic and apoptotic forms of cell death. Flow cytometry and transmission electron microscopy analysis demonstrated that in human breast cancer cells (MCF7) TNF induces cell cycle arrest in G0+G1/S, accompanied by apoptosis. 31P and 13C NMR spectroscopy was applied to study cellular metabolism of MCF7 cells during TNF-induced signal to apoptosis. Deuterated choline and 2H NMR spectroscopy were utilized to monitor the kinetics of the rate limiting reactions in phosphocholine metabolism. The NMR measurements revealed that immediately after administration of TNF, choline transport was inhibited by 52+/-6%. Later (approximately 15 h), the activity of phosphocholine:cytidine triphosphate cytidylyltransferase, a key enzyme in the biosynthesis of phosphatidylcholine, was enhanced two-fold. These two opposing changes led to a decrease in the level of phosphocholine. Throughout these changes the energetic state of the cells, determined by the level of nucleoside triphosphates and the rate of glucose metabolism via glycolysis, remained constant. The results indicate that TNF specifically modulates the kinetics of membrane-bound enzymes of the rate determining steps in phosphatidylcholine biosynthesis, possibly as part of early events involved in apoptosis.     

Acetylated low density lipoprotein inhibits the incorporation of arachidonic acid in phospholipids with a concomitant increase of cholesterol arachidonate in rat peritoneal macrophages

The aim of our work was to evaluate the influence of native low density lipoproteins (LDL) and LDL chemically modified by acetylation (acLDL) on incorporation and release of arachidonic acid (AA) in rat peritoneal macrophages. Compared to a control group without treatment, 100 micrograms/ml of acLDL for 15 h considerably increased the incorporation of [3H]AA in cholesterol-ester (CE) of rat peritoneal macrophages and induced a decrease of 3H-labeled membrane phospholipids (PL). No effect was shown with LDL treatment. In the presence of acLDL, LS3251 (100 nM), an acyl-coenzyme A:cholesterol acyltransferase (ACAT) inhibitor, inhibited the [3H]AA incorporation into CE in macrophages. [3H]AA-prelabeled macrophages cultured for 15 h with acLDL (compared to macrophages untreated or treated with LDL) showed an increase of labeled CE and a decrease of labeled PL and of cyclooxygenase and lipoxygenase eicosanoid production. After zymosan stimulation of macrophages prelabeled with [3H]AA and treated with or without LDL or acLDL, AA release and eicosanoid production increased in all groups of macrophages. The inhibition of eicosanoid production in foam cells does not seem to be linked to an inhibition of phospholipase but rather paralleled to an increase of the cholesterol [3H]arachidonate. A significant portion of cellular arachidonate released from phospholipids, in particular from phosphatidylcholine, could serve as a substrate to ACAT in this foam cell.     

Renal transplantation in children with augmentation cystoplasty: long-term results

Inflammatory processes have been hypothesized to mediate some of the clinical sequelae associated with endometriosis. The peritoneal fluid (PF) of women with endometriosis is known to contain more inflammatory cells and their associated cytokines, chemokines, and growth factors. This work provides strong evidence for oxidative stress in the PF of women with endometriosis. 1) The low density lipoprotein (LDL) isolated from the PF of subjects with endometriosis shows a small but detectable increase in electrophoretic mobility compatible with mildly oxidized LDL compared with LDL isolated from the plasma of the same subjects and PF of controls. 2) Isolated PF-LDL of endometriosis subjects is more readily oxidized in vitro than PF-LDL of controls, or LDL isolated from plasma. 3) Vitamin E content is significantly lower in endometriosis PF compared with controls, and compared with plasma of women with endometriosis and controls. No difference is seen between plasma and PF of control subjects. 4) The ratio of phosphatidylcholine/lyso phosphatidylcholine (Ptd/lyso PtdCho) in the PF of endometriosis subjects is significantly lower compared with PF of controls. Taken together, these data provide strong evidence for a pro-oxidant environment in the peritoneal cavity of women with endometriosis. Lyso PtdCho, a product derived from phospholipase A2 action on peroxidized phosphatidylcholine and a potent chemotactic factor for monocytes and T-lymphocytes, is elevated in endometriosis. We hypothesize that the increased presence of lipid peroxidation products in the PF of endometriosis subjects may, at least partly, account for the recruitment of leukocytes, the increase in macrophage activation, the secretion of monocyte--macrophage-derived cytokines, and the endometrial growth-promoting activity associated with endometriosis.     

Host plasma low density lipoprotein particles as an essential source of lipids for the bloodstream forms of Trypanosoma brucei

In contrast to mammalian cells, bloodstream forms of Trypanosoma brucei show no activity for fatty acid and sterol synthesis and critically depend on plasma low density lipoprotein (LDL) particles for their rapid growth. We report here that these parasites acquire such lipids by receptor-mediated endocytosis of LDL, subsequent lysosomal degradation of apoprotein B-LDL, and utilization of these lipids. Uptake of LDL-associated [3H]sphingomyelin and of LDL-associated [3H]cholesteryl oleate paralleled each other, and that of 125I-apoprotein B-LDL showed saturation and could be inhibited by unlabeled LDL or by anti-LDL receptor antibodies. Metabolism of lipids carried by LDL was abolished by chloroquine and by the thiol protease inhibitor, leupeptin. Sphingomyelin was cleaved by an acid sphingomyelinase to yield ceramide, which was itself split up into sphingosine and fatty acids. The latter were further incorporated into phosphatidylcholine, triacylglycerols, or cholesteryl esters. Similarly, cholesteryl oleate was hydrolyzed by an acid lipase to yield free cholesterol, which was reesterified with fatty acids, presumably in the cytosol. Like free cholesterol, LDL provided substrate for cholesterol esterification. In the culture-adapted procyclic form of T. brucei, which is capable of sterol synthesis, exogenous LDL-cholesterol rather than endogenously synthesized sterol was utilized for sterol esterification. Interference with exogenous supply of lipids via receptor-mediated endocytosis of LDL should be explored to fight against trypanosomiasis.     

Membrane structure and dynamics as viewed by solid-state NMR spectroscopy

The purpose of the present study is the investigation of the structure and dynamics of biological membranes using solid-state nuclear magnetic resonance (NMR) spectroscopy. Two approaches are used in our laboratory. The first involves the measurement of high-resolution 13C and 1H spectra obtained by the magic angle spinning (MAS) technique while the second approach involves the measurement of 31P and 2H powder spectra in static samples. This paper will present some recent results obtained by high-resolution solid-state 1H NMR on the conformation of gramicidin A incorporated in a phosphatidylcholine bilayers. More specifically, we were able to observe changes in the gramicidin spectra as a function of the cosolubilization solvent initially used to prepare the samples. The interaction between lipid bilayers and an anticancer drug derived from chloroethylurea was also investigated using proton NMR spectroscopy. Finally, we have studied the interaction between cardiotoxin, a toxic protein extracted from snake venom, and negatively charged lipid bilayers using 31P solid-state NMR spectroscopy.     

Study of the metabolism of choline and phosphatidylcholine in tumors in vivo using phosphonium-choline

The results of an initial study on the feasibility of using the phosphonium analog of choline to follow the metabolism of phosphatidylcholine in tumors in vivo using 31P NMR are reported. C3H/He mice bearing a mammary carcinoma tumor on the foot pad were fed a choline-free diet supplemented with the phosphonium analog of choline. Metabolites of this compound, including the phosphonium analogs of phosphatidylcholine, phosphocholine, glycerophosphocholine, and betaine were observed noninvasively in vivo in tumors by 31P NMR after 2-3 weeks of feeding. Clearance of these phosphonium-labeled metabolites from tumors was measured after a change to a choline-containing diet. Significant decreases were seen in the levels of the analogs of betaine (P < 0.003) and phosphatidylcholine (P < 0.004) by Day 4. A significant increase in the level of authentic phosphocholine (P < 0.003) occurred over the same time period.     

Induced and synchronized estrus in cattle: dose titration of estradiol benzoate in peripubertal heifers and postpartum cows after treatment with an intravaginal progesterone-releasing insert and prostaglandin F2alpha

Seven cysteine-rich repeats form the ligand-binding region of the low-density lipoprotein (LDL) receptor. Each of these repeats is assumed to bind a calcium ion, which is needed for association of the receptor with its ligands, LDL and beta-VLDL. The effects of metal ions on the folding of the reduced N-terminal cysteine-rich repeat have been examined by using reverse-phase high-performance liquid chromatography to follow the formation of fully oxidized isomers with different disulfide connectivities. In the absence of calcium many of the 15 possible isomers formed on oxidation, whereas in its presence the predominant product at equilibrium had the native disulfide bond connectivities. Other metals were far less effective at directing disulfide bond formation: Mn2+ partly mimicked the action of Ca2+, but Ba2+, Sr2+, and Mg2+ had little effect. This metal-ion specificity was also observed in two-dimensional 1H NMR spectral studies; only Ca2+ induced the native three-dimensional fold. The two paramagnetic ions, Gd3+ and Mn2+, and Cd2+ did not promote adoption of a well-defined structure, and the two paramagnetic ions did not displace calcium ions. The location of calcium ion binding sites in the repeat was also explored by NMR spectroscopy. The absence of chemical shift changes for the side chain proton resonances of Asp26, Asp36, and Glu37 from pH 3.9 to 6.8 in the presence of calcium ions and their proximal location in the NMR structures implicated these side chains as calcium ligands. Deuterium exchange NMR experiments also revealed a network of hydrogen bonds that stabilizes the putative calcium-binding loop.     

Potential pathogenicity of deglycosylated IgG cross reactive with streptokinase and fibronectin in the serum of patients with rheumatoid arthritis

PURPOSE: Drug free and drug loaded protein-free low density lipoprotein (LDL) models consisting mainly of phospholipids, cholesterol, cholesterol esters, and triglycerides in ratios found for physiological LDL have been prepared. Their physicochemical characteristics were compared with those of physiological LDL. METHODS: Different characterization methods were used: photon correlation spectroscopy, transmission electron microscopy, X-ray solution scattering, and 1H nuclear magnetic resonance spectroscopy (NMR). RESULTS: Particle sizes are highly dependent on the preparation method and in particular on the homogenization conditions. Electron microscopy indicates that the size distributions of model systems are much broader than those of physiological LDL. The X-ray solution scattering patterns of the model systems display a temperature dependent maximum near 3.8 nm similar to that found in the patterns of physiological LDL. NMR indicates a comparable mobility of the lipid molecules in model particles and in physiological LDL. The influence of drug loading is similar to that found earlier for physiological LDL. In particular, the incorporation of the anti-cancer drug WB 4291 seems to have a fluidizing effect on the lipids in the core region of the particles. CONCLUSIONS: The preparation method of LDL model systems is of crucial importance as only the solvent evaporation method yielded systems in the size range of physiological LDL with acceptable high lipid concentrations. The fluidizing influence of temperature and drug incorporation (WB 4291) may be disadvantage in drug targeting.     

Calorimetry of apolipoprotein-A1 binding to phosphatidylcholine-triolein-cholesterol emulsions

The thermotropic properties of triolein-rich, low-cholesterol dipalmitoyl phosphatidylcholine (DPPC) emulsion particles with well-defined chemical compositions (approximately 88% triolein, 1% cholesterol, 11% diacyl phosphatidylcholine) and particle size distributions (mean diameter, approximately 1000-1100 A) were studied in the absence and presence of apolipoprotein-A1 by a combination of differential scanning and titration calorimetry. The results are compared to egg yolk PC emulsions of similar composition and size. Isothermal titration calorimetry at 30 degrees C was used to saturate the emulsion surface with apo-A1 and rapidly quantitate the binding constants (affinity Ka = 11.1 +/- 3.5 x 10(6) M-1 and capacity N = 1.0 +/- 0.09 apo-A1 per 1000 DPPC) and heats of binding (enthalpy H = -940 +/- 35 kcal mol-1 apo-A1 or -0.92 +/- 0.12 kcal mol-1 DPPC). The entropy of association is -3070 cal deg-1 mol-1 protein or -3 cal deg-1 mol-1 DPPC. Without protein on the surface, the differential scanning calorimetry heating curve of the emulsion showed three endothermic transitions at 24.3 degrees C, 33.0 degrees C, and 40.0 degrees C with a combined enthalpy of 1.53 +/- 0.2 kcal mol-1 DPPC. With apo-A1 on the surface, the heating curve showed the three transitions more clearly, in particular, the second transition became more prominent by significant increases in both the calorimetric and Van't Hoff enthalpies. The combined enthalpy was 2.70 +/- 0.12 kcal mol-1 DPPC and remained constant upon repeated heating and cooling. Indicating that the newly formed DPPC emulsion-Apo-A1 complex is thermally reversible during calorimetry. Thus there is an increase in delta H of 1.17 kcal mol-1 DPPC after apo-A1 is bound, which is roughly balanced by the heat released during binding (-0.92 kcal) of apo-A1. The melting entropy increase, +3.8 cal deg-1 mol-1 DPPC of the three transitions after apo-A1 binds, also roughly balances the entropy (-3 cal deg-1 mol-1 DPPC) of association of apo-A1. These changes indicate that apo-A1 increases the amount of ordered gel-like phase on the surface of DPPC emulsions when added at 30 degrees C. From the stoichiometry of the emulsions we calculate that the mean area of DPPC at the triolein/DPPC interface is 54.5 A2 at 41 degrees C and 54.2 A2 at 30 degrees C. The binding of apo-A1 at 30 degrees C to the emulsion reduces the surface area per DPPC molecule from 54.2 A2 to 50.8 A2. At 30 degrees apo-A1 binds with high affinity and low capacity to the surface of DPPC emulsions and increases the packing density of the lipid domain to which it binds. Apo-A1 was also titrated onto DPPC emulsions at 45 degrees C. This temperature is above the gel liquid crystal transition. No heat was released or adsorbed. Furthermore, egg yolk phosphatidylcholine emulsions of nearly identical composition were also titrated at 30 degrees C with apo-A1 and were euthermic. Association constants were previously measured using a classical centrifugation assay and were used to calculate the entropy of apo-A1 binding (+28 cal deg-1 mol-1 apo-A1). This value indicates that apo-A1 binding to a fluid surface like egg yolk phosphatidylcholine or probably DPPC at 45 degrees C is hydrophobic and is consistent with hydrocarbon lipid or protein moities coming together and excluding water. Thus the binding of apo-A1 to partly crystalline surfaces is entropically negative and increases the order of the already partly ordered phases, whereas binding to liquid surfaces is mainly an entropically driven hydrophobic process.     

Low- and high-density lipoproteins as mitogenic factors for vascular smooth muscle cells: individual, additive and synergistic effects

The mitogenic activities of low (LDL)- and high (HDL)-density lipoproteins have been examined in cultures of human vascular smooth muscle cells (VSMC). LDL and HDL3 dose-dependently (EC50 values approximately 50 micrograms/ml) stimulated DNA and protein synthesis ([3H]-thymidine and [3H]-leucine incorporation, respectively) in the absence of exogenously added mitogens. The synthetic responses of VSMC to combinations of LDL and HDL3 were additive, indicating that each lipoprotein mediates discrete effects. LDL or HDL3 promoted VSMC proliferation under strict mitogen-free conditions, but this growth response was not sustained. VSMC exposed to combinations of lipoproteins (either LDL or HDL3) and growth factors (either PDGF-BB, EGF, bFGF or IGF) exhibited synergistic DNA synthesis responses. In the combined presence of PDGF-BB and either LDL or HDL3, VSMC proliferation was sustained. Anionized lipoprotein preparations (oxidized, acetylated, carbamylated or malonimylated) also stimulated DNA and protein synthesis. Since the antioxidant beta-hydroxylated toluene did not block the effect of native LDL on DNA synthesis, and fucoidin, a specific competitor for the 'scavenger' receptor, did not inhibit oxidized LDL-induced DNA synthesis, activation of mitogenic signals by lipoproteins does not depend on lipid peroxidation. Rather, the apparent intrinsic mitogenic potential of lipoproteins may depend upon their direct activation of replication-coupled signal transduction systems.     

Oxidation and antioxidation of human low-density lipoprotein and plasma exposed to 3-morpholinosydnonimine and reagent peroxynitrite

As peroxynitrite is implicated as an oxidant for low-density lipoprotein (LDL) in atherogenesis, we investigated this process using reagent peroxynitrite (ONOO-) and 3-morpholinosydnonimine (SIN-1, which produces peroxynitrite via generation of NO. and O2.-). LDL oxidation was assessed by the consumption of ubiquinol-10 (CoQ10H2) and alpha-tocopherol (alpha-TOH), the accumulation of cholesteryl ester hydro(pero)xides, the loss of lysine (Lys) and tryptophan (Trp) residues, and the change in relative electrophoretic mobility. Exposure to ONOO- or SIN-1 resulted in rapid (<1 min) and time-dependent oxidation, respectively, of LDL's lipids and protein. Manipulating the alpha-TOH content by in vivo or in vitro means showed that when ONOO- or SIN-1 was used at oxidant-to-LDL ratios of <100:1 the extent of LDL lipid peroxidation increased with increasing initial alpha-TOH content. In contrast, in vivo enrichment with the co-antioxidant CoQ10H2 decreased LDL lipid peroxidation induced by SIN-1. At oxidant-to-LDL ratios of >200:1, alpha-TOH enrichment decreased LDL lipid peroxidation for both SIN-1 and ONOO-. In contrast to lipid peroxidation, altering the alpha-TOH content of LDL did not affect Trp or Lys loss, independent of the amounts of either oxidant added. Aqueous antioxidants inhibited ONOO--induced lipid and protein oxidation with the order of efficacy: 3-hydroxyanthranilate (3-HAA) > urate > ascorbate. With SIN-1, these antioxidants inhibited Trp consumption, while only the co-antioxidants ascorbate and 3-HAA prevented alpha-TOH consumption and lipid peroxidation. Exposure of human plasma to SIN-1 resulted in the loss of ascorbate followed by loss of CoQ10H2 and bilirubin. Lipid peroxidation was inhibited during this period, though proceeded as a radical-chain process after depletion of these antioxidants and in the presence of alpha-TOH and urate. Bicarbonate at physiological concentrations decreased ONOO--induced lipid and protein oxidation, whereas it enhanced SIN-1-induced lipid peroxidation, Trp consumption, and alpha-tocopheroxyl radical formation in LDL. These results indicate an important role for tocopherol-mediated peroxidation and co-antioxidation in peroxynitrite-induced lipoprotein lipid peroxidation, especially when peroxynitrite is formed time-dependently by SIN-1. The studies also highlight differences between ONOO-- and SIN-1-induced LDL oxidation with regards to the effects of bicarbonate, ascorbate, and urate.     

Localization of platelet-derived growth factor beta receptor expression in the periepithelial stroma of human breast carcinoma

The ability of water-insoluble molecules such as triacylglycerols to partition from oil phases into phospholipid interfaces may be crucial to their hydrolysis by lipases in the aqueous environment of plasma and cells. This study uses high resolution and magic angle spinning 13C NMR spectros-copy to measure the solubility of the 8-carbon medium chain triacylglycerol, trioctanoin, in the lamellar structure of phospholipids (vesicles and multilayers) in the presence of other neutral lipids that may compete for an interfacial location (long chain triacylglycerol, cholesteryl ester, and cholesterol). In the presence of a saturating concentration of triolein (approximately 3 mole%), the solubility of trioctanoin in egg phosphatidylcholine vesicles decreased from 10 mole% to 7 mole%. The presence of a saturating concentration of trioctanoin (approximately 10 mole%) decreased the interfacial solubility of long chain triolein to approximately 1 mole%. Cholesteryl oleate in phospholipid vesicles slightly diminished the incorporation of trioctanoin into the surface. The presence of cholesterol reduced the interfacial solubility of trioctanoin, but at a high level of cholesterol (30 mole%), trioctanoin had a solubility of 3 mole%. Thus, even in the presence of other competing neutral lipids, medium chain triacylglycerol retains a favorable location and surface concentration for efficient hydrolysis. 13C NMR analysis thus provides an explanation for preferential hydrolysis of medium, compared to long chain triacylglycerol, in a physical blend of medium and long chain triacylglycerol in a single emulsion particle, and in general, a valuable approach to determine substrate availability at phospholipid surfaces.     

Synthesis and intracellular transport of aminoglycerophospholipids in permeabilized cells of the yeast, Saccharomyces cerevisiae

The sequence of biosynthetic steps from phosphatidylserine to phosphatidylethanolamine (via decarboxylation) and then phosphatidylcholine (via methylation) is linked to the intracellular transport of these aminoglycerophospholipids. Using a [3H]serine precursor and permeabilized yeast cells, it is possible to follow the synthesis of each of the aminoglycerophospholipids and examine the requirements for their interorganelle transport. This experimental approach reveals that in permeabilized cells newly synthesized phosphatidyl-serine is readily translocated to the locus of phosphatidylserine decarboxylase 1 in the mitochondria but not to the locus of phosphatidylserine decarboxylase 2 in the Golgi and vacuoles. Phosphatidylserine transport to the mitochondria is ATP independent and exhibits no requirements for cytosolic factors. The phosphatidylethanolamine formed in the mitochondria is exported to the locus of the methyltransferases (principally the endoplasmic reticulum) and converted to phosphatidylcholine. The export of phosphatidylethanolamine requires ATP but not any other cytosolic factors and is not obligately coupled to methyltransferase activity. The above described lipid transport reactions also occur in permeabilized cells that have been disrupted by homogenization, indicating that the processes are extremely efficient and may be dependent upon stable structural elements between organelles.