Rapid flip-flop in polyunsaturated (docosahexaenoate) phospholipid membranes.

The transbilayer movement (flip-flop) of 7-nitrobenz-2-oxa-1,3-diazol-4-yl phosphatidylethanolamine (NBD-PE) in phosphatidylcholine (PC) membranes containing various acyl chains was measured by dithionite quenching of NBD fluorescence. Of specific interest was docosahexaenoic acid (DHA), the longest and most unsaturated acyl chain commonly found in membranes. This molecule represents the extreme example of a family of important fatty acids known as omega-3s and has been clearly demonstrated to alter membrane structure and function.

Cell-cycle arrest in Jurkat leukaemic cells: a possible role for docosahexaenoic acid.

Docosahexaenoic acid (DHA) is known to have anti-cancer activities by mechanisms that are not well understood. In the present study, we test one possible pathway for DHA action in Jurkat leukaemic cells. Low doses of DHA (10 microM) are shown to induce cell-cycle arrest, whereas higher doses are cytotoxic.

Synthesis of a novel phosphatidylcholine conjugated to docosahexaenoic acid and methotrexate that inhibits cell proliferation.

Here we report the synthesis and characterization of a lipophilic phosphatidylcholine containing the omega-3 fatty acid docosahexaenoic acid (DHA) and the cytotoxic drug methotrexate (MTX). This novel phospholipid combines the fatty acid's and the drug's anticancer activities in a molecule amenable to a liposome bilayer for safe, simultaneous delivery of the two agents. Two phosphatidylcholines were synthesized, from 1-stearoyl or 1-docosahexaenoyl, 2-hydroxy-sn-glycero-3-phosphocholine, to contain MTX in the sn-2 position and either stearic acid or DHA in the sn-1 position.

Prevention of docosahexaenoic acid-induced cytotoxicity by phosphatidic acid in Jurkat leukemic cells: the role of protein phosphatase-1.

The present investigation explores the role of phosphatidic acid (PA), a specific protein phosphatase-1 (PP1) inhibitor, in cytotoxicity induced by docosahexaenoic acid (DHA). The cytotoxicity of DHA was assayed by quantifying cell survival using the trypan blue exclusion method. A dose-response effect demonstrated that 5 or 10 microM DHA has no effect on Jurkat cell survival; however, 15 microM DHA rapidly decreased cell survival to 40% within 2 h of treatment.

Lipid phase separation in phospholipid bilayers and monolayers modeling the plasma membrane.

It is postulated that biological membrane lipids are heterogeneously distributed into lipid microdomains. Recent evidence indicates that docosahexaenoic acid-containing phospholipids may be involved in biologically important lipid phase separations. Here we investigate the elastic and thermal properties of a model plasma membrane composed of egg sphingomyelin (SM), cholesterol and 1-stearoyl-2-docosahexaenoyl-sn-glycerophosphoethanolamine (SDPE).

Docosahexaenoic acid induces apoptosis in Jurkat cells by a protein phosphatase-mediated process.

Docosahexaenoic acid (DHA) is an omega-3 fatty acid under intense investigation for its ability to modulate cancer cell growth and survival. This research was performed to study the cellular and molecular effects of DHA. Our experiments indicated that the treatment of Jurkat cells with DHA inhibited their survival, whereas similar concentrations (60 and 90 microM) of arachidonic acid and oleic acid had little effect.

The curvature and cholesterol content of phospholipid bilayers alter the transbilayer distribution of specific molecular species of phosphatidylethanolamine.

The curvature, cholesterol content, and transbilayer distribution of phospholipids significantly influence the functional properties of cellular membranes, yet little is known of how these parameters interact. In this study, the transbilayer distribution of phosphatidylethanolamine (PE) is determined in vesicles with large (98 nm) and small (19 nm) radii of curvature and with different proportions of PE, phosphatidylcholine, and cholesterol. It was found that the mean diameters of both types of vesicles were not influenced by their lipid composition, and that the amino-reactive compound 2,4,6-trinitrobenzenesulphonic acid (TNBS) was unable to cross the bilayer of either type of vesicle.

Docosahexaenoic acid-containing phosphatidylcholine affects the binding of monoclonal antibodies to purified Kb reconstituted into liposomes.

Class I major histocompatibility complex (MHC I) molecules are transmembrane proteins that bind and present peptides to T-cell antigen receptors. The role of membrane lipids in controlling MHC I structure and function is not understood, although membrane lipid composition influences cell surface expression of MHC I. We reconstituted liposomes with purified MHC I (Kb) and probed the effect of lipid composition on MHC I structure (monoclonal anti-MHC I antibody binding).

Liquid crystalline/gel state phase separation in docosahexaenoic acid-containing bilayers and monolayers.

The phase behavior of lipid mixtures containing 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (18:0, 22:6 PC) with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was studied with bilayers using differential scanning calorimetry (DSC), and with monolayers monitoring pressure/area isotherms and surface elasticity, and lipid domain formation followed by epifluorescence microscopy. From DSC studies it is concluded that DPPC/18:0, 22:6 PC phase separates into DPPC-rich and 18:0, 22:6 PC-rich phases. In monolayers, phase separation is indicated by changes in pressure-area isotherms implying phase separation where 18:0, 22:6 PC is 'squeezed out' of the remaining DPPC monolayer.

Detection of lipid domains in docasahexaenoic acid-rich bilayers by acyl chain-specific FRET probes.

A major problem in defining biological membrane structure is deducing the nature and even existence of lipid microdomains. Lipid microdomains have been defined operationally as heterogeneities in the behavior of fluorescent membrane probes, particularly the fluorescence resonance energy transfer (FRET) probes 7-nitrobenz-2-oxa-1,3-diazol-4-yl-diacyl-sn-glycero-3-phosphoethan olamine (N-NBD-PE) and (N-lissamine rhodamine B sulfonyl)-diacyl-snglycero-3-phosphoethanolamine (N-Rh-PE). Here we test a variety of N-NBD-PEs and N-Rh-PEs containing: (a) undefined acyl chains, (b) liquid crystalline- and gel-state acyl chains, and (c) defined acyl chains matching those of phase separated membrane lipids.

Anomalous changes in forward scatter of lymphocytes with loosely packed membranes.

Background: Forward scatter (FSC) is generally associated with cell size and has been suggested as a way to differentiate apoptotic from viable cells. Among spleen cells cultured for 48 h, a population of cells (population B) was found to have decreased forward and increased side scatter relative to freshly purified cells (population A). Interestingly, population B was not present early in analysis; this report explores the change in FSC of population B.

Docosahexaenoic acid in phosphatidylcholine mediates cytotoxicity more effectively than other omega-3 and omega-6 fatty acids.

We reported previously that docosahexaenoic acid (22:6)-containing phosphatidylcholine (PC), but not oleic acid-containing PC nor 22:6-containing phosphatidylethanolamine, is toxic to tumor cells in vitro. To test whether other polyunsaturated fatty acids share 22:6's cytotoxic activity, we treated cultured T27A murine leukemia cells with PC liposomes composed of stearic acid in the sn-1 position and alpha-linolenic acid (alpha-18:3), arachidonic acid (20:4), or eicosapentaenoic acid (20:5) in the sn-2 position. PC containing 22:6 in both positions was also tested.

Use of merocyanine 540 for the isolation of quiescent, primitive human bone marrow hematopoietic progenitor cells.

Merocyanine 540 (MC540) is a membrane probe that inserts preferentially into loosely packed domains in the phospholipid bilayer of intact cells. Previous experiments have demonstrated that MC540 will bind to human bone marrow (BM) hematopoietic progenitor cells (HPC). Fractions of mononuclear BM cells expressing high MC540 fluorescence have been shown to be enriched for myeloid progenitors and cells residing in the S/G2 + M phases of the cell cycle.

Docosahexaenoic acid (DHA) alters the phospholipid molecular species composition of membranous vesicles exfoliated from the surface of a murine leukemia cell line.

Previously, we presented evidence that the vesicles routinely exfoliated from the surface of T27A tumor cells arise from vesicle-forming regions of the plasma membrane and possess a set of lateral microdomains distinct from those of the plasma membrane as a whole. We also showed that docosahexaenoic acid (DHA, or 22:6n-3), a fatty acyl chain known to alter microdomain structure in model membranes, also alters the structure and composition of exfoliated vesicles, implying a DHA-induced change in microdomain structure on the cell surface. In this report we show that enrichment of the cells with DHA reverses some of the characteristic differences in composition between the parent plasma membrane and shed microdomain vesicles, but does not alter their phospholipid class composition.

Cholesterol versus cholesterol sulfate: effects on properties of phospholipid bilayers containing docosahexaenoic acid.

The important omega-3 fatty acid docosahexaenoic acid (DHA) is present at high concentration in some membranes that also contain the unusual sterol cholesterol sulfate (CS). The association between these lipids and their effect on membrane structure is presented here. Differential scanning calorimetry (DSC), MC540 fluorescence, erythritol permeability, pressure/area isotherms on lipid monolayers and molecular modeling are used to compare the effect of CS and cholesterol on model phospholipid membranes.

The triggering signal dictates the effect of docosahexaenoic acid on lymphocyte function in vitro.

Docosahexaenoic acid (DHA) is an n-3 fatty acid beneficial to several human conditions including inflammation and autoimmune disease. To better understand the effect of DHA on immunity, we monitored the rise in cytosolic free calcium, interleukin 2 receptor (IL2R) expression, and proliferation of splenic lymphocytes triggered with three different stimuli in the presence or absence of DHA. We found that 10 microg DHA/mL suppressed concanavalin A-induced mitogenesis and the mixed lymphocyte reaction while concurrently enhancing proliferation stimulated with anti-Thy-1 antibodies.

Docosahexaenoic acid (DHA) alters the structure and composition of membranous vesicles exfoliated from the surface of a murine leukemia cell line.

Membrane lipid microdomains are regions of the membrane thought to be functionally important, but which have remained poorly characterized because they have proven to be difficult to isolate. The exfoliation of small membranous vesicles from the cell surface is a continuous and normal activity in many cells. If microdomains are relatively large or stable, they may influence the structure and composition of exfoliated vesicles, which are easy to isolate.

Can docosahexaenoic acid inhibit metastasis by decreasing deformability of the tumor cell plasma membrane?

Murine leukemia cells were fused with small unilamellar vesicles composed of 1-stearoyl, 2-docosahexaenoylphosphatidylcholine. The docosahexaenoic acid (DHA)-modified cells were tested for deformability by forcing them through 5.0-microm Nucleopore filters.

Spleen cell survival and proliferation are differentially altered by docosahexaenoic acid.

Omega-3 fatty acids have diverse health benefits that are not clearly understood. In this study we have examined the effects of the omega-3 fatty acid docosahexaenoic acid (DHA) on mitogen-activated and resting splenic lymphocytes. DHA inhibited lymphocyte proliferation, producing an apparent block or prolongation of S phase, without evidence for direct cytotoxicity.

Effect of docosahexaenoic acid on mouse mitochondrial membrane properties.

Long-chain polyunsaturated (n-3) fatty acids have been proposed to be involved in a wide variety of biological activities. In this study, mitochondrial docosahexaenoic acid (DHA) levels were increased by either dietary manipulation or by fusing the mitochondria with phospholipid vesicles made from 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (18:0/22:6 PC). The fused mitochondria exhibited a DHA-induced decrease in respiratory control index (RCI) and membrane potential and an increase in proton movement.

Cholesterol versus alpha-tocopherol: effects on properties of bilayers made from heteroacid phosphatidylcholines.

The techniques of differential scanning calorimetry, fluorescence of merocyanine 540, fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene, proton permeability, and lipid peroxidation are used to compare the perturbations of cholesterol and alpha-tocopherol on lipid bilayer membranes composed of different phosphatidylcholines containing stearic acid in the sn-1 position and an unsaturated fatty acid (either oleic, alpha-linolenic, gamma-linolenic, or docosahexaenoic acid) in the sn-2 position. It is concluded that the structural roles of cholesterol and alpha-tocopherol may be similar with membranes composed of some phosphatidylcholines but are clearly different with membranes composed of other related phosphatidylcholines. alpha-Tocopherol exerts a much larger effect than cholesterol on membranes rich in polyunsaturated fatty acids that have their initial double bond before the delta 9 position.

Phospholipid class as a determinant in docosahexaenoic acid's effect on tumor cell viability.

Here we explore how incorporation of the omega-3 fatty acid docosahexaenoic acid (DHA) into murine leukemia cells (T27A) may alter membrane structure and function. When cells were cultured in DHA-supplemented medium, DHA incorporated rapidly and preferentially into phosphatidyl-ethanolamine (PE), with lesser and slower incorporation into phosphatidylcholine (PC). DHA at low concentrations preferred PE over neutral lipids, but in DHA excess accumulation in neutral lipids outstripped that of phospholipids.

Omega-3 fatty acid-containing liposomes in cancer therapy.

omega-3 fatty acids are associated with reduced growth and incidence of certain cancers, and in this report we demonstrate that a fish oil diet (rich in omega-3 fatty acids) enhances the longevity of mice bearing the myeloid leukemia T27A. We have proposed that the omega-3 fatty acid docosahexaenoic acid (DHA, 22:6 delta 4,7,10,13,16,19) may induce structural changes in tumor cell plasma membranes resulting in reduced tumor growth in vitro. Here, we test whether liposomes containing DHA (18:0, 22:6 PC) have antitumor effects in vivo, leading to enhanced longevity of the tumor-bearing host.

Comparison of phosphatidylcholines containing one or two docosahexaenoic acyl chains on properties of phospholipid monolayers and bilayers.

Docosahexaenoic acid (DHA) is the longest and most unsaturated of the n - 3 fatty acids found in membranes. Although a number of membrane properties have been demonstrated to be affected by the presence of this fatty acid, its mode of action has yet to be clearly elucidated. Prior reports on biological membranes have not distinguished the effect of mono-docosahexaenoyl phospholipids from those caused by phospholipids containing docosahexaenoic acid in both chains.