Phospholipid patterns of erythrocytes in schizophrenia: relationships to symptomatology.

The phospholipid composition of red blood cells (RBC) from 32 haloperidol-treated schizophrenic patients, classified according to the positive and negative syndrome scale (PANSS) as showing either predominantly positive or predominantly negative symptoms, was determined and compared with that of normal controls. While the levels of phosphatidylcholine and phosphatidylserine were similar in all three groups, sphingomyelin (SM) and phosphatidylethanolamine (PE) were, respectively, increased and decreased in RBCs of schizophrenic patients. In both patient groups, the SM/PE ratios correlated directly with the PANSS negative symptom scale scores and inversely with the positive symptom scale scores. However, the inverse changes in the contents of SM and PE were much more expressed in the negative group. It is suggested that a main source of that difference is a higher activity of the polyunsaturated acid-selective phospholipase A(2) in the negative syndrome patients than in the positive syndrome and control groups.

The effect of lipid composition and physical state of phospholipid monolayer on the binding and incorporation of a basic amphipathic peptide from the C-terminal region of the HIV envelope protein gp41.

The interaction of a peptide identical to the carboxy terminal region of the envelope glycoprotein gp41(828) of HIV with negatively charged phospholipids in a monolayer was studied by a Wilhelmy film balance. No significant interaction of the peptide with a monolayer composed of pure neutral but a strong affinity to negatively charged phospholipids could be observed. In mixed phospholipid monolayers the binding of the gp41(828) is primarily limited by the amount of acidic phospholipids. The physical state of the monolayer is another important parameter for binding. Clustering of negatively charged phospholipids and the surface pressure are crucial. Ca(2+) ions strongly interfere with the peptide-lipid interaction up to complete abolishment. The effects observed are dependent on the nature of the acidic lipid. Phosphatidylglycerol was found to be more sensitive than phosphatidylserine. The significance of the results for processes like virus assembly and budding will be discussed.

The down-modulation of CD4 induced by the GM1 ganglioside is regulated by phosphatases and kinases: evidence from enzyme inhibitors and anti-CD45 antibodies.

The involvement of protein kinases and phosphatases in the down-modulation of expression of CD4 molecules on peripheral blood lymphocytes (PBL) by gangliosides was studied. Exposure of PBL either to genistein or to H7 practically abolished the down-modulation of CD4 induced by GM1 and diminished their susceptibility to CD4+ down-modulation by exposure to GD1a. Staurosporine had no effect on the down-modulation of CD4 by either GM1 or GD1a. Orthovanadate treatment drastically inhibited the down-regulation of CD4 induced by GM1 but had no effect on down-modulation of CD4 induced by GD1a. Exposure to monoclonal antibodies (mAbs) against CD45 and CD45RA but not against CD45RO abrogated the down-modulation of CD4 by GM1. The down-modulation of CD4 elicited by GD1a, GD1b, or GT1b was not inhibited by anti-CD45RA and anti-CD45RO mAbs. MAbs against CD3, CD2, or HLA-DR had no effect on the GM1-induced down-modulation of CD4. In view of the differences obtained between GM1 and GD1a it was of interest to check whether these gangliosides competed for cellular binding sites. When PBL were first treated with anti-CD45RA and GM1 or with orthovanadate and GM1, which had a negligible effect on CD4 expression, and subsequently treated with GD1a the expression of CD4 was down-modulated. This demonstrated that GD1a binds to sites on the cell membrane to which GM1 does not bind. The present study indicates that the capacity of GM1 to down-modulate CD4 depended on the CD45 and particularly CD45RA molecules, while other gangliosides may utilize different cell surface structures to down-modulate the expression of CD4.

Autoantibodies to gangliosides in sera of atherosclerotic patients.

Using ELISA we studied the levels and clinical correlation of serum antibodies against gangliosides and 5-hydroxytryptamine (5-HT) in patients with atherosclerosis and clinical manifestations of cardiovascular disease. A range of 70-80% of the patients showed higher titers of anti-GM3(L) and anti-5HT as compared to normal serum. The anti-GM3(L) antibodies appeared to be directed mainly against GM3 present in platelets and were much less reactive against GM3 isolated from the aorta. We concluded that the antigens responsible for the elevated anti-GM3(L) and anti 5-HT levels in atherosclerotic sera are released by vessel-wall activated platelets. These results provide further evidence of on-going autoimmune processes in atherosclerosis. The content of total sialic (TS) and lipid-bound sialic acid (LBS) was measured in sera of patients with IHD and of similar numbers of healthy donors. In the patient groups the average TS and LBS concentration was about 25% higher than in the control group. These changes appeared to be associated with higher degrees of protein sialylation and larger amounts of LDL in the patient sera than in those of healthy controls.

Nanomolar concentrations of gangliosides stimulate primary humoral response.

Exogenous gangliosides act as immunosuppressors when applied at micromolar concentrations corresponding to their average level in human plasma. Here we show that at nanomolar concentrations the gangliosides GD3, GD1a and GM1 can act as immunostimulators markedly enhancing the number of plaque-forming cells in mouse splenocyte culture responding to sheep erythrocytes. At such low concentration these gangliosides as well as GM3 were not able to influence significantly proliferative responses of splenic B and T lymphocytes or of cytotoxic T-cells. Neither did they change significantly the production of IL-1 by antigen- representing cells, or of IL-2 by Con A-induced blasts in the splenocyte culture. It is suggested that the stimulatory effect of low ganglioside concentrations on humoral response is due to their influence on cooperative cell-cell interactions required for the differentiation of B-cells into Ig-secreting cells.

Serum gangliosides as endogenous immunomodulators.

Gangliosides suppress various immune activities in vitro and in vivo. Their level is significantly elevated in tumors and atherosclerotic aorta tissue, as well as in the sera of patients with tumors or atherosclerosis. Here, Lev Bergelson suggests that ganglioside-induced immunomodulation might be involved in atherogenesis and carcinogenesis, and describes a hypothesis that cites gangliosides as a factor interfering with the clearance of low-density lipoproteins (LDLs) and promoting the formation of atherosclerotic plaques.

Dynamic lipid heterogeneity and receptor events.

Receptor occupation by specific ligands induces changes in the dynamic domain organization of surrounding lipids. Such changes were observed by measuring changes in the fluorescence parameters of fluorescent-labelled lipids incorporated into plasma membranes of intact cells, membrane vesicles or lipoprotein particles in response to specific binding of a broad range of biologically active agents, including drugs, prostaglandins, neuropeptides, antibodies and viruses. The high sensitivity of the fluorescence response allowed us to register changes in lipid heterogeneity induced in a multitude of discrete targets by transient weak binding of a single rapidly translocating molecule. To explain these observations a non-equilibrium model of ligand-receptor interaction based on low relaxation phenomena in heterogeneous lipid matrixes is proposed.

Transient domains induced by influenza haemagglutinin during membrane fusion.

During low pH-induced fusion of influenza virus with erythrocytes we have observed differential dispersion of viral lipid and haemagglutinin (HA) into the erythrocyte membrane, and viral RNA into the erythrocyte using fluorescence video microscopy. The movement of both viral lipid and HA from virus to cell was restricted during the initial stages of fusion relative to free diffusion. This indicates the existence of relatively long-lived barriers to diffusion subsequent to fusion pore formation. Fluorescence anisotropy of phospholipid analogues incorporated into the viral membrane decreased when the pH was lowered to levels required for optimum fusion. This indicates that the restricted motion of viral membrane components was not due to rigidification of membrane lipids. The movement of HA from the fusion site was also assessed by photosensitized labelling by means of a fluorescent substrate (NBD-taurine) passing through the band 3 sialoglycoprotein (the erythrocyte anion transporter). We also examined the flow of lipid and aqueous markers during fusion of HA-expressing cells with labelled erythrocytes. During this cell-cell fusion, movement of lipid between fusing membranes begins before the fusion pore is wide enough to allow diffusion of aqueous molecules (M(r) > 500). The data indicate that HA is capable of creating domains in the membrane and controlling continuity of aqueous compartments which are bounded by such domains.

Role of interactions at the lipid-water interface for domain formation.

The lipid-water interface is critical for the packing of lipid molecules in membranes. We have demonstrated that lateral phase separation in membranes can be driven by electrostatic interactions such as those involving charged lipid species and oppositely charged peptides, in addition to hydration effects at the lipid-water interface. By using nuclear magnetic resonance (NMR), circular dichroism and fluorescence spectroscopy we have shown that binding of a 21-amino acid peptide containing six positively charged arginine residues to mixed phosphatidylcholine (PC)/phosphatidylglycerol (PG) membranes results in a conformational change in the peptide from a random coil to a helical structure and causes the formation of domains of negatively charged PG. Binding of the peptide to PG membranes disorders the lipid hydrocarbon chains. The strength of lipid-peptide binding at the interface, the conformational change in the peptide, and domain formation with the negatively charged lipid are coupled energetically. The lipid-peptide association constant is lower for membranes containing 20 mol% PG in PC/PG mixtures than for 100% PG membranes. We suggest that one of the factors that lower the association constant in PC/PG membranes is entropic energy of formation of PG domains. Besides electrostatic interactions, hydration of lipids is important for domain formation. We have shown that dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylethanolamine separate under conditions of decreased water activity. Furthermore, water activity controls lipid packing stress in the hydrocarbon core and the headgroups of membranes as demonstrated by induction of an inverse-hexagonal-to-lamellar phase transition in dioleoylphosphatidylethanolamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of the conformation of a peptide from gp41 on binding and domain formation in model membranes.

Binding of the peptide fragment 828-848 (P828), amino acid sequence RVIEVVQGACRAIRHIPRRIR, from the carboxy-terminal region of the envelope glycoprotein gp41 of human immunodeficiency virus type 1 (HIV-1) to membranes composed of a mixture of neutral and negatively charged phospholipids results in domain or cluster formation of the charged lipid. The conformation and dynamics of the peptide are investigated in solution and in the presence of sodium dodecyl sulphate (SDS) micelles using high resolution nuclear magnetic resonance (NMR) spectroscopy and circular dichroism (CD) spectropolarimetry. The CD results demonstrate that addition of either SDS, negatively charged phospholipid liposomes, or trifluoroethanol (TFE) induces a conformational transition of the peptide from a random coil or an extended chain in water to a more ordered structure with an estimated helical content of up to 60%. The structure of the peptide in a membrane mimetic SDS solution was investigated in detail using two-dimensional NMR. The measurements demonstrate the existence of a helical component in the peptide conformation in the SDS-bound state. The peptide most likely exists as an ensemble of conformations with exchange times between them which are fast on the chemical shift NMR time scale (10(-3) s). Simple neutralization of the six arginine sidechain charges does not cause the peptide to adopt an ordered structure. Thus, there is an additional requirement for the structural transition such as that resulting from constraint of the peptide on a surface, or localization of the peptide at the lipid-water interface where the polarity is lower.(ABSTRACT TRUNCATED AT 250 WORDS)

Anthrylvinyl-labeled phospholipids as membrane probes: the phosphatidylcholine-phosphatidylethanolamine system.

The phase behavior of mixtures of phosphatidylcholine (PC) with phosphatidylethanolamine (PE) identical or differing in their fatty acid composition has been investigated by using the steady-state fluorescence anisotropy of anthrylvinyl-labeled PC and PE (APC and APE) as well as of the non-lipid probe 1,6-diphenyl-1,3,5-hexatriene (DPH) to detect temperature-dependent changes in multilayer liposomes. APC, but not APE, was able to detect the pretransition of dimyristoyl-PC. The phospholipid probes APC and APE showed the main phase transition of their unlabeled disaturated analogues at temperatures almost identical with those revealed by differential scanning calorimetry, whereas the onset of the PE phase transition recorded by DPH was several degrees higher. In PC-PE mixtures with high content of PE the phase transitions shown by APC and APE were broader than those recorded by DPH. Comparison of phase diagrams constructed on the basis of fluorescence anisotropy and calorimetric data led to the conclusion that in biphasic PE and PC-PE systems DPH tends to partition into solid regions, whereas the anthrylvinyl-labeled phospholipids distribute more evenly between coexisting phases or prefer fluid domains. The use of anthrylvinyl phospholipid probes made it possible to demonstrate that PEs and PCs identical in their fatty acids are not miscible completely, not only below but also well above Tm of the higher melting component. Generally, APC and APE fluorescence anisotropy measurements correctly reflect headgroup-dependent phase segregations in mixtures of PC with PE, but may lead to ambiguous conclusions if demixing is caused by differences in the hydrocarbon chains.

Gangliosides and atherosclerosis.

The ganglioside levels in atherosclerotic lesions of human aorta are considerably higher than those in unaffected areas of aorta, and atherosclerotic patients frequently have increased concentrations of serum gangliosides. The present review summarizes recent findings that suggest the possible involvement of aortic gangliosides in platelet activation and adhesion of platelets to the vessel wall. The effect of gangliosides on the structure of low density lipoproteins (LDL), on the interaction of LDL with macrophages and hepatic cells and on the LDL-regulated biosynthesis of cholesterol is also discussed. In vitro experiments have demonstrated that a major ganglioside of the intima of atherosclerotic aorta induces rapid adhesion, aggregation and spreading of platelets. Moreover, gangliosides present in elevated amounts in the intercellular space of atherosclerotic aortic tissue modify the surface structure and stimulate aggregation of LDL. Ganglioside-modified LDL are readily recognized and taken up by macrophages, while preincubation of LDL with low concentrations of gangliosides inhibits LDL binding to hepatic cells. Thus, ganglioside enrichment of LDL is likely to interfere with LDL clearance via the hepatic cells. Thus, ganglioside enrichment of LDL is likely to interfere with LDL clearance via the hepatic LDL receptor, and to stimulate binding of LDL to the scavenger receptor of macrophages. It is postulated that high ganglioside levels in the aorta and serum may be an additional risk factor in atherosclerosis.

Gangliosides and antitumor immunity.

To elucidate the possible influence on the host's immune defense of circulating gangliosides released from tumor cells, the effects of exogeneous gangliosides on the activities of some lymphocyte subpopulations were examined. The mono- and disialyllactosylceramides GM3 and GD3, which frequently are present in elevated amounts in sera of tumor-bearing hosts, were found to inhibit strongly the cytotoxicity of natural killer cells, to stimulate T-suppressor activity of peripheral blood lymphocytes, and to inhibit their phytohemagglutinin-induced blast transformation. All these effects may be linked to the ability of gangliosides to modulate the arachidonic acid cascade in lymphoid cells, which for the first time was demonstrated in the course of our studies. Possible mechanisms underlying the immunomodulatory effects of serum gangliosides as well as their role in the escape of tumor cells from immune surveillance and inhibition of the hosts immune system are discussed.

Nanomolar concentrations of prostaglandin E1 induce changes in the surface organization of high-density lipoproteins.

Incubation of high-density lipoproteins (HDL) with small amounts of the prostaglandin E1 (PGE1) results in mobilization of the spin-label 5-doxylstearic acid incorporated into the HDL surface monolayer as well as in decreased binding of apoprotein A1 antibodies to the HDL surface. These effects are manifested at strikingly low PGE1 concentrations, corresponding to one prostaglandin molecule per 10(2)-10(3) lipoprotein particles. At the same time, the flotation properties of HDL are not changed in the presence of PGE1. The data are interpreted on the basis of a nonequilibrium model proposed earlier for prostaglandin-lipoprotein interactions (Bergelson, L. D., et al. (1987) Biochim. Biophys. Acta 921, 182-190).

Interaction of peptide fragment 828-848 of the envelope glycoprotein of human immunodeficiency virus type I with lipid bilayers.

The interaction of the peptide fragment 828-848, called P828, from the carboxy-terminal region of the envelope glycoprotein gp41 of HIV-I with model membranes composed of phosphatidylcholine (PC) and phosphatidylglycerol (PG) was investigated using microelectrophoretic mobility of liposomes, fluorescence polarization of labeled lipids, NMR, and differential scanning calorimetry. The peptide binds to negatively charged lipid surfaces. No interaction between P828 and neutral PC surfaces is observed. The interaction between the peptide and the lipid is exclusively electrostatic with the six positively charged arginines of P828 acting as binding sites for PG. Circular dichroism measurements of P828 indicate that the peptide undergoes a transition from a random coil to an ordered conformation upon binding to negatively charged PG bilayers or SDS micelles, but not in the presence of neutral PC bilayers. The ordered structure has an apparent helical content of 60%. IN DOPG/DOPC mixtures containing 20 mol % DOPG, the peptide causes the formation of lipid domains enriched in DOPG, as assessed by measurement of fluorescence energy transfer between labeled PG and PC. The formation of these domains requires energy and therefore reduces the strength of peptide binding to the lipid matrix. Our data support and quantitate the results from antibody binding studies [Haffar, O.K., Dowbenko, D. J., & Berman, P. W. (1988) J. Cell Biol. 107, 1677-1687] that the carboxy-terminal segment of the envelope glycoprotein gp41 interacts with microsomal membranes.

Anthrylvinyl-labeled phospholipids as fluorescent membrane probes. The action of melittin on multilipid systems.

The interaction of melittin with multicomponent lipid mixtures composed of phosphatidylcholine, sphingomyelin and phosphatidylserine or phosphatidylglycerol was investigated by measuring the intrinsic fluorescence of the peptide, steady state fluorescence anisotropy of, and Trp-fluorescence energy transfer to fluorescent analogs of the same phospholipids bearing the anthrylvinyl fluorophore in one of the aliphatic chains at various distances from the polar head group. Based on the finding that at high lipid/peptide ratio the peptide induces unequal changes in the fluorescence parameters of phospholipid probes differing structurally only in their polar head groups, it is concluded that melittin induces lipid demixing in its nearest environment. Comparison of the fluorescence energy transfer from Trp to different lipid probes indicates that the depth of penetration of melittin into the bilayer depends on the polar head group composition of the phospholipid matrix and that certain segments of the melittin chain display a specific affinity for a given lipid head group.

Interaction of low-density lipoproteins with gangliosides.

The ganglioside uptake capacity of human serum low-density lipoproteins (LDL), the mode of ganglioside-LDL binding, and the influence of gangliosides on the floatation properties, size distribution, stability and fluorescence of LDL were investigated. The data obtained suggest that both hydrophobic and electrostatic forces are involved in formation of ganglioside-LDL complexes, but the former appear to be more important. Although association of gangliosides with LDL is predominantly unspecific, nonsaturable, and weak, a small saturable component due to specific ganglioside-apolipoprotein binding, also appears to be involved. In the presence of gangliosides the lipoprotein particles aggregate, the intrinsic fluorescence of LDL and their interaction with antibodies against apo-B change indicating that the state of apo-B [corrected] is modified by gangliosides.

Influence of ganglioside GM3 and its breakdown products on lymphoblastic transformation and T-suppressor activity.

The influence of ganglioside GM3 and some of its breakdown products on phytohemagglutinin-induced blast transformation of human lymphocytes and concanavalin-A-induced T-suppressor activity was studied. The structures of two major hydrolysis products of GM3 were established by negative-ion fast-atom-bombardment mass spectrometry as neuraminyllactosylsphingosine (NeuLacSph) and neuraminyllactosylceramide (NeuLacCer). Both substances were shown to be potent inhibitors of mitogen-induced lymphoblastic transformation whereas their acetylation products NeuAcLacSphAc and GM3 did not affect the proliferative response of lymphocytes to phytohemagglutinin. On the other hand, only GM3 and NeuLacSph were able to enhance concanavalin-A-induced T-suppressor activity. On the basis of these data, it is suggested that the effects of GM3 and its breakdown products on lymphoblastic transformation and T-suppressor activity must rest on different mechanisms and that N-deacylation of GM3 appears to be an essential step in conversion of the ganglioside into an inhibitor of lymphocyte blast transformation.

Low-density lipoproteins interact with liposome-binding sites on the cell surface.

Under physiological conditions significant amounts of low-density lipoprotein LDL particles ar taken up by cells independently of specific high-affinity LDL receptors (apo-B receptors). Previously it was established that some cells contain surface sites capable of binding liposomes. We proposed that liposome-binding sites could contribute to LDL interaction with the cell surface via phospholipid molecules of LDL particles. To check this hypothesis we studied the competitive interaction of human LDL and DPPC liposomes with mouse embryo fibroblasts depleted of apo-B receptors by preliminary incubation with LDL. We have found that after removal of the liposome-binding sites from cell lamellae these areas of the cell surface lose their ability to bind LDL.