RESUMEN
The signaling lipids phosphatidic acid (PA) and diacylglycerol pyrophosphate (DGPP) are involved in regulating the stress response in plants. PA and DGPP are anionic lipids consisting of a negatively charged phosphomonoester or pyrophosphate group attached to diacylglycerol, respectively. Changes in the pH modulate the protonation of their head groups modifying the interaction with other effectors. Here, we examine in a controlled system how the presence of Ca2+ modulates the surface organization of dioleyl diacylglycerol pyrophosphate (DGPP) and its interaction with dioleoyl phosphatidic acid (DOPA) at different pHs. Both lipids formed expanded monolayers at pH 5 and 8. At acid and basic pHs, monolayers formed by DOPA or DGPP became denser when Ca2+ was added to the subphase. At pH 5, Ca2+ also induced an increase of surface potential of both lipids. Conversely, at pH 8 the effects induced by the presence of Ca2+ on the surface potential were reversed. Mixed monolayers of DOPA and DGPP showed a non-ideal behavior. The addition of even tiny amounts of DGPP to DOPA films caused a reduction of the mean molecular area. This effect was more evident at pH 8 compared to pH 5. Our finding suggests that low amounts of DGPP in an film enriched in DOPA could lead to a local increase in film packing with a concomitant change in the local polarization, further regulated by local pH. This fact may have implications for the assigned role of PA as a pH-sensing phospholipid or during its interaction with proteins.
Asunto(s)
Calcio/química , Difosfatos/química , Glicerol/análogos & derivados , Concentración de Iones de Hidrógeno , Ácidos Fosfatidicos/química , Glicerol/química , Electricidad Estática , Propiedades de SuperficieRESUMEN
Systemic lupus erythematosus is characterized by dysregulated activation of T and B cells and autoantibodies to nuclear antigens and, in some cases, lipid antigens. Liposomes with nonbilayer phospholipid arrangements induce a disease resembling human lupus in mice, including IgM and IgG antibodies against nonbilayer phospholipid arrangements. As the effect of these liposomes on the innate immune response is unknown and innate immune system activation is necessary for efficient antibody formation, we evaluated the effect of these liposomes on Toll-like receptor (TLR) signaling, cytokine production, proinflammatory gene expression, and T, NKT, dendritic, and B cells. Liposomes induce TLR-4- and, to a lesser extent, TLR-2/TLR-6-dependent signaling in TLR-expressing human embryonic kidney (HEK) cells and bone marrow-derived macrophages. Mice with the lupus-like disease had increased serum concentrations of proinflammatory cytokines, C3a and C5a; they also had more TLR-4-expressing splenocytes, a higher expression of genes associated with TRIF-dependent TLR-4-signaling and complement activation, and a lower expression of apoptosis-related genes, compared to healthy mice. The percentage of NKT and the percentage and activation of dendritic and B2 cells were also increased. Thus, TLR-4 and TLR-2/TLR-6 activation by nonbilayer phospholipid arrangements triggers an inflammatory response that could contribute to autoantibody production and the generation of a lupus-like disease in mice.
Asunto(s)
Liposomas/farmacología , Lupus Eritematoso Sistémico/inmunología , Receptor Toll-Like 2/inmunología , Receptor Toll-Like 4/inmunología , Receptor Toll-Like 6/inmunología , Animales , Autoanticuerpos/biosíntesis , Autoanticuerpos/sangre , Clorpromazina/farmacología , Citocinas/biosíntesis , Citocinas/sangre , Diglicéridos/farmacología , Modelos Animales de Enfermedad , Femenino , Flagelina/farmacología , Regulación de la Expresión Génica , Células HEK293 , Humanos , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/sangre , Inmunoglobulina M/biosíntesis , Inmunoglobulina M/sangre , Inflamación , Lipopolisacáridos/farmacología , Liposomas/química , Lupus Eritematoso Sistémico/inducido químicamente , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/patología , Ratones , Ratones Endogámicos BALB C , Oligopéptidos/farmacología , Ácidos Fosfatidicos/química , Ácidos Fosfatidicos/farmacología , Fosfatidilcolinas/química , Fosfatidilcolinas/farmacología , Fosfatidilserinas/química , Fosfatidilserinas/farmacología , Receptor Toll-Like 2/agonistas , Receptor Toll-Like 2/genética , Receptor Toll-Like 4/agonistas , Receptor Toll-Like 4/genética , Receptor Toll-Like 6/agonistas , Receptor Toll-Like 6/genéticaRESUMEN
BACKGROUND: Low plasma levels of high-density lipoprotein-cholesterol (HDL-C) are typical of acute myocardial infarction (MI) and predict risk of recurrent cardiovascular events. The potential relationships between modifications in the molecular composition and the functionality of HDL subpopulations in acute MI however remain indeterminate. METHODS AND RESULTS: ST segment elevation MI (STEMI) patients were recruited within 24h after diagnosis (n=16) and featured low HDL-C (-31%, p<0.05) and acute-phase inflammation (determined as marked elevations in C-reactive protein, serum amyloid A (SAA) and interleukin-6) as compared to age- and sex-matched controls (n=10). STEMI plasma HDL and its subpopulations (HDL2b, 2a, 3a, 3b, 3c) displayed attenuated cholesterol efflux capacity from THP-1 cells (up to -32%, p<0.01, on a unit phospholipid mass basis) vs. CONTROLS: Plasma HDL and small, dense HDL3b and 3c subpopulations from STEMI patients exhibited reduced anti-oxidative activity (up to -68%, p<0.05, on a unit HDL mass basis). HDL subpopulations in STEMI were enriched in two proinflammatory bioactive lipids, lysophosphatidylcholine (up to 3.0-fold, p<0.05) and phosphatidic acid (up to 8.4-fold, p<0.05), depleted in apolipoprotein A-I (up to -23%, p<0.05) and enriched in SAA (up to +10.2-fold, p<0.05); such changes were most marked in the HDL3b subfraction. In vitro HDL enrichment in both lysophosphatidylcholine and phosphatidic acid exerted deleterious effects on HDL functionality. CONCLUSIONS: In the early phase of STEMI, HDL particle subpopulations display marked, concomitant alterations in both lipidome and proteome which are implicated in impaired HDL functionality. Such modifications may act synergistically to confer novel deleterious biological activities to STEMI HDL. SIGNIFICANCE: Our present data highlight complex changes in the molecular composition and functionality of HDL particle subpopulations in the acute phase of STEMI, and for the first time, reveal that concomitant modifications in both the lipidome and proteome contribute to functional deficiencies in cholesterol efflux and antioxidative activities of HDL particles. These findings may provide new biomarkers and new insights in therapeutic strategy to reduce cardiovascular risk in this clinical setting where such net deficiency in HDL function, multiplied by low circulating HDL concentrations, can be expected to contribute to accelerated atherogenesis.
Asunto(s)
Lipoproteínas HDL3/sangre , Lisofosfatidilcolinas/sangre , Infarto del Miocardio/sangre , Ácidos Fosfatidicos/sangre , Proteína Amiloide A Sérica/metabolismo , Adulto , Anciano , Apolipoproteína A-I/química , Apolipoproteína A-I/deficiencia , Apolipoproteína A-I/metabolismo , Proteína C-Reactiva/metabolismo , Estudios de Casos y Controles , Línea Celular , Femenino , Humanos , Interleucina-6/sangre , Lipoproteínas HDL3/química , Lisofosfatidilcolinas/química , Masculino , Persona de Mediana Edad , Monocitos/metabolismo , Infarto del Miocardio/patología , Ácidos Fosfatidicos/química , Proteoma/química , Proteoma/metabolismoRESUMEN
The interaction of Co hexagonal magnetic nanoparticles (MNPs) with distearoyl phosphatidyl glycerol (DSPG) and distearoyl phosphatidic acid (DSPA) films adsorbed at a water/1,2-dichloroethane interface is studied employing cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), capacity curves and interfacial pressure-area isotherms. DSPA and DSPG adsorb at the interface forming homogenous films and producing a blocking effect on the transfer process of tetraethyl ammonium (TEA(+)), used as a probe cation. In the presence of Co NPs this effect is reversed and the reversible transfer process for TEA(+) is reestablished, to a greater or lesser extent depending on the structuration of the film. Co-DSPA hybrid films have a homogeneous structure while Co-DSPG films present different domains. Moreover, the presence of Co on DSPA film modifies the partition coefficient of the organic electrolyte into the hydrocarbon layer.
Asunto(s)
Nanopartículas de Magnetita/química , Fosfolípidos/química , Adsorción , Dicloruros de Etileno/química , Ácidos Fosfatidicos/química , Fosfatidilgliceroles/química , Propiedades de Superficie , Agua/químicaRESUMEN
We examined the interaction of the cationic antimicrobial peptide (AMP) tritrpticin (VRRFPWWWPFLRR, TRP3) with Langmuir monolayers of zwitterionic (dipalmitoyl phosphatidylcholine, DPPC, and dipalmitoyl phosphatidylethanolamine, DPPE) and negatively charged phospholipids (dipalmitoyl phosphatidic acid, DPPA, and dipalmitoyl phosphatidylglycerol, DPPG). Both surface pressure and surface potential isotherms became more expanded upon addition of TRP3 (DPPE~DPPC<Asunto(s)
1,2-Dipalmitoilfosfatidilcolina/química
, Péptidos Catiónicos Antimicrobianos/química
, Oligopéptidos/química
, Ácidos Fosfatidicos/química
, Fosfatidiletanolaminas/química
, Fosfatidilgliceroles/química
, Antibacterianos/química
, Bacterias/química
, Membrana Celular/química
, Células Eucariotas/química
, Membranas Artificiales
, Modelos Moleculares
, Electricidad Estática
, Propiedades de Superficie
, Termodinámica
RESUMEN
BACKGROUND: Fungal extracellular vesicles are able to cross the cell wall and transport molecules that help in nutrient acquisition, cell defense, and modulation of the host defense machinery. METHODOLOGY/PRINCIPAL FINDINGS: Here we present a detailed lipidomic analysis of extracellular vesicles released by Paracoccidioides brasiliensis at the yeast pathogenic phase. We compared data of two representative isolates, Pb3 and Pb18, which have distinct virulence profiles and phylogenetic background. Vesicle lipids were fractionated into different classes and analyzed by either electrospray ionization- or gas chromatography-mass spectrometry. We found two species of monohexosylceramide and 33 phospholipid species, including phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylserine, phosphatidylinositol, and phosphatidylglycerol. Among the phospholipid-bound fatty acids in extracellular vesicles, C181 predominated in Pb3, whereas C18:2 prevailed in Pb18. The prevalent sterol in Pb3 and Pb18 vesicles was brassicasterol, followed by ergosterol and lanosterol. Inter-isolate differences in sterol composition were observed, and also between extracellular vesicles and whole cells. CONCLUSIONS/SIGNIFICANCE: The extensive lipidomic analysis of extracellular vesicles from two P. brasiliensis isolates will help to understand the composition of these fungal components/organelles and will hopefully be useful to study their biogenesis and role in host-pathogen interactions.
Asunto(s)
Paracoccidioides/química , Fosfolípidos/química , Vesículas Secretoras/química , Cerebrósidos/química , Cromatografía de Gases y Espectrometría de Masas , Ácidos Fosfatidicos/química , Fosfatidilcolinas/química , Fosfatidilgliceroles/química , Fosfatidilinositoles/química , Fosfatidilserinas/químicaRESUMEN
Non-bilayer phospholipid arrangements are three-dimensional structures that can form when anionic phospholipids with an intermediate form of the tubular hexagonal phase II (H(II)), such as phosphatidic acid, phosphatidylserine or cardiolipin, are present in a bilayer of lipids. The drugs chlorpromazine and procainamide, which trigger a lupus-like disease in humans, can induce the formation of non-bilayer phospholipid arrangements, and we have previously shown that liposomes with non-bilayer arrangements induced by these drugs cause an autoimmune disease resembling human lupus in mice. Here we show that liposomes with non-bilayer phospholipid arrangements induced by Mn²âº cause a similar disease in mice. We extensively characterize the physical properties and immunological reactivity of liposomes made of the zwitterionic lipid phosphatidylcholine and a H(II)-preferring lipid, in the absence or presence of Mn²âº, chlorpromazine or procainamide. We use an hapten inhibition assay to define the epitope recognized by sera of mice with the disease, and by a monoclonal antibody that binds specifically to non-bilayer phospholipid arrangements, and we report that phosphorylcholine and glycerolphosphorylcholine, which form part of the polar region of phosphatidylcholine, are the only haptens that block the binding of the tested antibodies to non-bilayer arrangements. We propose a model in which the negatively charged H(II)-preferring lipids form an inverted micelle by electrostatic interactions with the positive charge of Mn²âº, chlorpromazine or procainamide; the inverted micelle is inserted into the bilayer of phosphatidylcholine, whose polar regions are exposed and become targets for antibody production. This model may be relevant in the pathogenesis of human lupus.
Asunto(s)
Lupus Eritematoso Sistémico/metabolismo , Liposomas Unilamelares/metabolismo , Animales , Anticuerpos/sangre , Anticuerpos/metabolismo , Anticuerpos Monoclonales/metabolismo , Cardiolipinas/química , Cardiolipinas/inmunología , Bovinos , Clorpromazina/toxicidad , Glomérulos Renales/patología , Lupus Eritematoso Sistémico/inducido químicamente , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/patología , Manganeso/toxicidad , Ratones , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Ácidos Fosfatidicos/química , Ácidos Fosfatidicos/inmunología , Fosfatidilcolinas/química , Fosfatidilcolinas/inmunología , Fosfatidilserinas/química , Fosfatidilserinas/inmunología , Procainamida/toxicidad , Piel/patología , Liposomas Unilamelares/química , Liposomas Unilamelares/inmunologíaRESUMEN
The exposure of the plasma membrane calcium pump (PMCA) to the surrounding phospholipids was assessed by measuring the incorporation of the photoactivatable phosphatidylcholine analog [(125)I]TID-PC/16 to the protein. In the presence of Ca(2+) both calmodulin (CaM) and phosphatidic acid (PA) greatly decreased the incorporation of [(125)I]TID-PC/16 to PMCA. Proteolysis of PMCA with V8 protease results in three main fragments: N, which includes transmembrane segments M1 and M2; M, which includes M3 and M4; and C, which includes M5 to M10. CaM decreased the level of incorporation of [(125)I]TID-PC/16 to fragments M and C, whereas phosphatidic acid decreased the incorporation of [(125)I]TID-PC/16 to fragments N and M. This suggests that the conformational changes induced by binding of CaM or PA extend to the adjacent transmembrane domains. Interestingly, this result also denotes differences between the active conformations produced by CaM and PA. To verify this point, we measured resonance energy transfer between PMCA labeled with eosin isothiocyanate at the ATP-binding site and the phospholipid RhoPE included in PMCA micelles. CaM decreased the efficiency of the energy transfer between these two probes, whereas PA did not. This result indicates that activation by CaM increases the distance between the ATP-binding site and the membrane, but PA does not affect this distance. Our results disclose main differences between PMCA conformations induced by CaM or PA and show that those differences involve transmembrane regions.
Asunto(s)
Calmodulina/metabolismo , Membrana Eritrocítica/enzimología , Ácidos Fosfatidicos/metabolismo , ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismo , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Sitios de Unión , Calmodulina/química , Activación Enzimática , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Micelas , Ácidos Fosfatidicos/química , ATPasas Transportadoras de Calcio de la Membrana Plasmática/química , ATPasas Transportadoras de Calcio de la Membrana Plasmática/aislamiento & purificación , Estructura Terciaria de ProteínaRESUMEN
Diacylglycerol pyrophosphate (DGPP), a phosphorylated form of phosphatidic acid (PA), gained attention recently due to its role as signaling lipid. However, little is known about its surface organization and potential impact on membrane-mediated function. In this work we investigated the interfacial behavior of Langmuir monolayers formed with pure DGPP and of its mixtures with PA. We found that changes of the subphase pH affect the surface behavior of DGPP. At pH 8, DGPP forms liquid expanded monolayers with a compressibility modulus of about 60mNm⻹ at collapse. On acidic solutions, the compressibility modulus increases to 90mNm⻹ and the average molecular area is smaller. At pH 8, DGPP and its precursor PA form thermodynamically favored topographically homogeneous non-ideal mixtures. The interaction among these lipids leads to a non-ideal diminution of the mean molecular area and consequently, to an increase of the compressibility modulus, with variations of the surface electrostatics. The favorable interaction of PA and DGPP, leading to changes of the film packing suggest that DGPP may act as a structural signal transducer in membrane-mediated cellular processes.
Asunto(s)
Aire , Difosfatos/química , Glicerol/análogos & derivados , Ácidos Fosfatidicos/química , Agua/química , Glicerol/química , Concentración de Iones de Hidrógeno , Propiedades de SuperficieRESUMEN
The polysaccharide chitosan has been largely used in many biological applications as a fat and cholesterol reducer, bactericide agent, and wound healing material. While the efficacy for some of such uses is proven, little is known about the molecular-level interactions involved in these applications. In this study, we employ mixed Langmuir and Langmuir-Blodgett (LB) films of negatively charged dimyristoyl phosphatidic acid (DMPA) and cholesterol as cell membrane models to investigate the role of cholesterol in the molecular-level action of chitosan. Chitosan does not remove cholesterol from the monolayer. The interaction with chitosan tends to expand the DMPA monolayer due to its interpenetration within the film. On the other hand, cholesterol induces condensation of the DMPA monolayer. The competing effects cause the surface pressure isotherms of mixed DMPA-cholesterol films on a chitosan subphase to be unaffected by the cholesterol mole fraction, due to distinct degrees of chitosan penetration into the film in the presence of cholesterol. By combining polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS) and sum-frequency generation spectroscopy (SFG), we showed that chitosan induces order into negatively charged phospholipid layers, whereas the opposite occurs for cholesterol. In conclusion, chitosan has its penetration in the film modulated by cholesterol, and electrostatic interactions with negatively charged phospholipids, such as DMPA, are crucial for the action of chitosan.
Asunto(s)
Quitosano/química , Colesterol/química , Fosfolípidos/química , Compuestos de Anilina/química , Concentración de Iones de Hidrógeno , Membranas Artificiales , Ácidos Fosfatidicos/química , Presión , Espectrofotometría/métodos , Espectrofotometría Infrarroja/métodos , Electricidad Estática , Propiedades de Superficie , Tensión SuperficialRESUMEN
In this work we have investigated the influence of NaCl on the adsorption of the antimicrobial cationic peptide bactenecin in the monolayer of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) at the air-water interface, as a function of NaCl concentrations in the subphase. We show that the effect of the salt concentration on DPPC monolayers is a monotonic decrease of the liquid-condensed-liquid-expanded (LC-LE) coexistence region. By contrast, the effect of the bactenecin adsorption at the DPPC monolayer not only removed the LC-LE coexistence region plateau, but also shifted the DPPC isotherms to higher pressures and increased the compressibility of the DPPC/bactenecin monolayers with respect to the pure DPPC monolayer around the LC phase. Analysis of the domain structure, obtained by Brewster angle and atomic force microscopes, indicates that the salt concentration in the subphase builds an electrostatic barrier, increasing the rigidity of DPPC monolayers and limiting the bactenecin adsorption at the LC-LE phase coexistence.
Asunto(s)
Membranas Artificiales , Péptidos Cíclicos/química , Ácidos Fosfatidicos/química , Cloruro de Sodio/química , Agua/química , Adsorción , Aire , Cationes/química , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
The kinetics and the thermodynamics of melanin concentrating hormone (MCH) adsorption, penetration, and mixing with membrane components are reported. MCH behaved as a surface active peptide, forming stable monolayers at a lipid-free air-water interface, with an equilibrium spreading pressure, a collapse pressure, and a minimal molecular area of 11 mN/m, 13 mN/m, and 140 A (2), respectively. Additional peptide interfacial stabilization was achieved in the presence of lipids, as evidenced by the expansion observed at pi > pi sp in monolayers containing premixtures of MCH with zwitterionic or charged lipids. The MCH-monolayer association and dissociation rate constants were 9.52 x 10 (-4) microM (-1) min (-1) and 8.83 x 10 (-4) min (-1), respectively. The binding of MCH to the dpPC-water interface had a K d = 930 nM at 10 mN/m. MCH penetration in lipid monolayers occurred even up to pi cutoff = 29-32 mN/m. The interaction stability, binding orientation, and miscibility of MCH in monolayers depended on the lipid type, the MCH molar fraction in the mixture, and the molecular packing of the monolayer. This predicted its heterogeneous distribution between different self-separated membrane domains. Our results demonstrated the ability of MCH to incorporate itself into biomembranes and supports the possibility that MCH affects the activity of mechanosensitive membrane proteins through mechanisms unrelated with binding to specific receptors.
Asunto(s)
Hormonas Hipotalámicas/química , Lípidos/química , Melaninas/química , Hormonas Hipofisarias/química , 1,2-Dipalmitoilfosfatidilcolina/química , Cetrimonio , Compuestos de Cetrimonio/química , Cinética , Membranas Artificiales , Palmitatos/química , Ácidos Fosfatidicos/química , Electricidad Estática , Propiedades de Superficie , Tensión Superficial , Tensoactivos/química , Termodinámica , Agua/químicaRESUMEN
In this work the interaction of Hydroxyzine, Promethazine and Thioridazine with Langmuir films of dipalmitoylphosphatidylcholine (dpPC) and dipalmitoylphosphatidic acid (dpPA), is studied. Temporal variations in lateral surface pressure (pi) were measured at different initial pi (pi(i)), subphase pH and drug-concentration. Drugs with the smallest (PRO) and largest (HYD) molecular size exhibited the lowest adsorption (k(a)) and the highest desorption (k(d)) rate constant values, respectively. The affinity binding constants (K(b)) obtained in monolayers followed the same profile (K(b,PRO) < K(b,HYD) < K(b,THI)) of the egg-PC/water partition coefficients (P) determined in bilayers. The drug concentration required to reach the half-maximal Deltapi at pi(i) = 14 mN/m (K(0.5)), was very sensitive to pH. The maximal increment in pi upon drug incorporation into the monolayer (deltapi(max)) will depend on the phospholipid collapse pressure (pi(c)), the monolayers's compressibility and drug's size, shape and charge. The higher pi(c) of dpPC lead to higher pi(cut-off) values (maximal pi allowing drug penetration), if compared with dpPA. In dpPC and dpPA pi(cut-off) decreased as a function of the molecular size of the uncharged drugs. In dpPA, protonated drugs became electrostatically trapped at the monolayer surface hence drug penetration, monolayer deformation and pi increase were impaired and the correlation between pi(cut-off) and drug molecular size was lost.
Asunto(s)
1,2-Dipalmitoilfosfatidilcolina/metabolismo , Hidroxizina/metabolismo , Ácidos Fosfatidicos/metabolismo , Prometazina/metabolismo , Tioridazina/metabolismo , 1,2-Dipalmitoilfosfatidilcolina/química , Aire , Hidroxizina/química , Membrana Dobles de Lípidos , Ácidos Fosfatidicos/química , Prometazina/química , Propiedades de Superficie , Tioridazina/química , AguaRESUMEN
Membrane lipid peroxidation (LPO) induced by hydroxyl (*OH) and ascorbyl (*Asc) radicals and by peroxynitrite (ONOO-) was investigated in asolectin (ASO), egg phosphatidylcholine (PC) and PC/phosphatidic acid mixtures (PC:PA) liposomes and rat liver microsomes (MC). Enthalpy variation (DeltaH) of PC:PA at different molar ratios were obtained by differential scanning calorimetry. It was also evaluated the LPO inhibition by quercetin, melatonin and Vitamin B6. The oxidant effect power follows the order *OH approximately *Asc > ONOO- on PC and MC; whilst on ASO liposomes, it follows *Asc > *OH approximately ONOO-. Increasing amounts of PA in PC liposomes resulted in lower levels of LPO. The DeltaH values indicate a more ordered membrane arrangement as a function of PA amount. The results were discussed in order to provide a complete view involving the influence of membranes, oxidants and antioxidants intrinsic behavior on the LPO dynamics.
Asunto(s)
Antioxidantes/química , Membrana Dobles de Lípidos/química , Lípidos de la Membrana/química , Especies de Nitrógeno Reactivo/química , Especies Reactivas de Oxígeno/química , Animales , Antioxidantes/metabolismo , Rastreo Diferencial de Calorimetría , Yema de Huevo/química , Radicales Libres/química , Radicales Libres/metabolismo , Membrana Dobles de Lípidos/metabolismo , Peroxidación de Lípido/fisiología , Liposomas/química , Liposomas/metabolismo , Lípidos de la Membrana/metabolismo , Microsomas Hepáticos/química , Microsomas Hepáticos/metabolismo , Ácidos Fosfatidicos/química , Fosfatidilcolinas/química , Fosfolípidos/química , Ratas , Especies de Nitrógeno Reactivo/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Relación Estructura-ActividadRESUMEN
The interaction of dynamin II with giant unilamellar vesicles was studied using two-photon fluorescence microscopy. Dynamin II, labeled with fluorescein, was injected into a microscope chamber containing giant unilamellar vesicles, which were composed of either pure 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or a mixture of POPC and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). Binding of the fluorescent dynamin II to giant unilamellar vesicles, in the presence and absence of PI(4,5)P2, was directly observed using two-photon fluorescence microscopy. This binding was also visualized using the fluorescent N-methylanthraniloyl guanosine 5'-[gamma-thio]triphosphate analogue. The membrane probe 6-dodecanoyl-2-dimethylamine-naphthalene was used to monitor the physical state of the lipid in the giant unilamellar vesicles in the absence and presence of dynamin. A surprising finding was the fact that dynamin II bound to vesicles in the absence of PI(4,5)P2. Activation of the GTPase activity of dynamin II by pure POPC was then shown.
Asunto(s)
Dinamina II/metabolismo , Microscopía Fluorescente/métodos , Ácidos Fosfatidicos/química , Dinamina II/química , FotonesRESUMEN
Phospholipase A2 activity against mixed monolayers of dilauroylphosphatidic acid or dilauroylphosphatidylcholine with glycosphingolipids can be reversibly modulated by external constant electrostatic fields. The changes of enzymatic activity are correlated to the depolarization or hyperpolarization of the film caused by specific dipolar properties of glycosphingolipids. Hyperpolarizing fields enhance the enzymatic activity against pure dilauroylphosphatidic acid while depolarizing fields induce a decrease of activity. Compared to the pure substrate, the interface of mixed films containing neutral glycosphingolipids or gangliosides is already partially depolarized and the magnitude of activation induced by an external hyperpolarizing field is decreased; conversely, depolarizing fields cause an increased inhibition of activity. Differing from gangliosides, sulfatides bring about a hyperpolarization of the mixed lipid monolayer and external hyperpolarizing or depolarizing fields cause enhanced activation and reduced inhibition, respectively. The effects of glycosphingolipids depend on their relative proportion in the monolayer. Results were similar with dilauroylphosphosphatidylcholine but the field effects were less than half of those found with dilauroylphosphatidic acid. Our work shows that the activity of phospholipase A2 in addition to responding reversibly to external electrostatic fields, is directly modulated by the polarity and magnitude of the lipid polar head group dipole moments.
Asunto(s)
Glicoesfingolípidos/metabolismo , Fosfolipasas A/metabolismo , Animales , Bovinos , Gangliósidos/química , Gangliósidos/metabolismo , Glicoesfingolípidos/química , Técnicas In Vitro , Potenciales de la Membrana , Modelos Moleculares , Conformación Molecular , Ácidos Fosfatidicos/química , Ácidos Fosfatidicos/metabolismo , Fosfolipasas A2 , Electricidad Estática , Sulfoglicoesfingolípidos/química , Sulfoglicoesfingolípidos/metabolismo , PorcinosRESUMEN
Chemically modified lysozymes, namely: N-succinyl lysozyme, glycine methyl ester of N-succinyl lysozyme and oxoindole lysozyme have been prepared. Aggregation, fusion and leakage of phospholipid vesicles induced by these derivatives have been studied in comparison with the effect of the unmodified protein. The experiments were carried out with negatively charges 9PC/PA, 9:1) and uncharged (PC and PC/DOPE/Chol (10:5:5)) lipid vesicles of different packing. Fusion and aggregation of negatively charged phospholipid vesicles in induced by proteins positively charged at pH 7.0 involving electrostatic interactions, a similar pattern on fusion and aggregation of the least stably packed lipid vesicles points also to hydrophobic forces playing a role in the lipid-protein interaction. A conformational change of the protein involved increasing beta-turns, loops and unordered structure at the expenses of beta-sheet without affecting alpha helix content. The conformational effect is necessary to provoke the effects studied, since one of the derivatives (N-succinyl lysozyme) neither changes conformation nor causes aggregation and fusion of vesicles. However, there is no relationship between lysozyme activity and fusion or aggregation of lipid vesicles that catalytic and fusogenci sites of, indicating lysozyme are topographically different.
Asunto(s)
Liposomas/metabolismo , Muramidasa/farmacología , Animales , Fenómenos Químicos , Química Física , Colesterol/química , Glicina/análogos & derivados , Glicina/farmacología , Fusión de Membrana/efectos de los fármacos , Muramidasa/química , Permeabilidad/efectos de los fármacos , Ácidos Fosfatidicos/química , Fosfatidilcolinas/química , Fosfatidiletanolaminas/química , Conformación Proteica , Succinatos/farmacologíaRESUMEN
The aim of this study was to compare the effects of chronic (0.1 mol/L ethanol exposure during 30 days) and acute (0.5 mol/L ethanol exposure during 24 h) ethanol treatment on the physical properties and the lipid composition of plasma membranes of the WRL-68 cells (fetal human hepatic cell line). Using fluorescence polarization we found that ethanol treatment reduced membrane anisotropy due to disorganization of acyl chains in plasma membranes and consequently increased fluidity, as measured with the diphenylhexatriene probe. Addition of ethanol in vitro reduced anisotropy in control plasma membranes, whereas chronically ethanol-treated plasma membranes were relatively tolerant to the in vitro addition of ethanol. Acutely ethanol-treated plasma membranes exhibited a smaller anisotropy parameter value than control plasma membranes. We found a decrease in total phospholipid content in acute ethanol WRL-68 plasma membranes. Cholesterol content was increased in both ethanol treatments, and we also found a significant decrease in phosphatidylinositol and phosphatidylcholine and an increase in phosphatidylethanolamine content in ethanol-treated plasma membranes. Our data showed that ethanol treatment decreased the anisotropy parameter consistently with increased fluidity, while increasing the cholesterol/phospholipid ratio of plasma membranes of WRL-68 cells, but only chronically ethanol-treated plasma membranes exhibited tolerance to the in vitro addition of ethanol. It is important to note that some changes that were interpreted as a result of chronic ethanol treatment were also present in short-period ethanol treatments.