RESUMEN
Mitochondria are energy factories of cells and important targets for methylmercury chloride (MgHgCl). Methylmercury (MeHg) is a well-known environmental toxicant that bioaccumulates in fish and shellfish. It readily crosses the placental barrier, making it a threat to correct fetal development. Despite being comprehensively investigated for years, this compound has not been assessed for its in vitro mitochondrial toxicity under different oxygen conditions. In this study, human induced pluripotent stem cells (hiPSCs) were used to evaluate the dependence of the expression of genes associated with pluripotency and mitochondria on atmospheric (21% O2) and low (5% O2) oxygen concentrations upon MeHgCl treatment. We showed that the toxicity of MeHgCl was strongly related to an increased mtDNA copy number and downregulation of the expression of an mtDNA replication and damage repair-associated gene POLG1 (Mitochondrial Polymerase Gamma Catalytic Subunit) in both tested oxygen conditions. In addition, the viability and mitochondrial membrane potential of hiPSCs were significantly lowered by MeHgCl regardless of the oxygen concentration. However, reactive oxygen species accumulation significantly increased only under atmospheric oxygen conditions; what was associated with increased expression of TFAM (Transcription Factor A, Mitochondrial) and NRF1 (Nuclear Respiratory Factor 1) and downregulation of PARK2 (Parkin RBR E3 Ubiquitin Protein Ligase). Taken together, our results demonstrated that MeHgCl could induce in vitro toxicity in hiPSCs through altering mitochondria-associated genes in an oxygen level-dependent manner. Thus, our work suggests that oxygen should be considered a factor was modulating the in vitro toxicity of environmental pollutants. Typical atmospheric conditions of in vitro culture significantly lower the predictive value of studies of such toxicity.
Asunto(s)
Células Madre Pluripotentes Inducidas , Compuestos de Metilmercurio , Animales , ADN Mitocondrial , Femenino , Genes Mitocondriales , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Compuestos de Metilmercurio/toxicidad , Oxígeno , Placenta/metabolismo , Embarazo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Pyrroloquinoline quinone (PQQ) is a factor influencing on the mitochondrial biogenesis. In this study the PQQ effect on viability, total cell number, antioxidant capacity, mitochondrial biogenesis and differentiation potential was investigated in human induced Pluripotent Stem Cells (iPSC) - derived: neural stem cells (NSC), early neural progenitors (eNP) and neural progenitors (NP). Here we demonstrated that sensitivity to PQQ is dependent upon its dose and neural stage of development. Induction of the mitochondrial biogenesis by PQQ at three stages of neural differentiation was evaluated at mtDNA, mRNA and protein level. Changes in NRF1, TFAM and PPARGC1A gene expression were observed at all developmental stages, but only at eNP were correlated with the statistically significant increase in the mtDNA copy numbers and enhancement of SDHA, COX-1 protein level. Thus, the "developmental window" of eNP for PQQ-evoked mitochondrial biogenesis is proposed. This effect was independent of high antioxidant capacity of PQQ, which was confirmed in all tested cell populations, regardless of the stage of hiPSC neural differentiation. Furthermore, a strong induction of GFAP, with down regulation of MAP2 gene expression upon PQQ treatment was observed. This indicates a possibility of shifting the balance of cell differentiation in the favor of astroglia, but more research is needed at this point.
Asunto(s)
Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células-Madre Neurales/efectos de los fármacos , Cofactor PQQ/farmacología , Antioxidantes/metabolismo , Recuento de Células , Diferenciación Celular , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Dosificación de Gen , Proteína Ácida Fibrilar de la Glía/biosíntesis , Humanos , Potencial de la Membrana Mitocondrial , Proteínas Asociadas a Microtúbulos/biosíntesis , Proteínas Asociadas a Microtúbulos/genética , Mitocondrias , Proteínas Mitocondriales/biosíntesis , Proteínas Mitocondriales/genética , Factor Nuclear 1 de Respiración/biosíntesis , Factor Nuclear 1 de Respiración/genética , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/biosíntesis , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Especies Reactivas de Oxígeno/metabolismo , Factores de Transcripción/biosíntesis , Factores de Transcripción/genéticaRESUMEN
BACKGROUND: Interferon alpha (IFN-alpha) activated cellular signalling is negatively regulated by inhibitory factors, including the suppressor of cytokine signalling (SOCS) family. The effects of host factors such as obesity on hepatic expression of these inhibitory factors in subjects with chronic hepatitis C virus (HCV) are unknown. OBJECTIVES: To assess the independent effects of obesity, insulin resistance, and steatosis on response to IFN-alpha therapy and to determine hepatic expression of factors inhibiting IFN-alpha signalling in obese and non-obese subjects with chronic HCV. METHODS: A total of 145 subjects were analysed to determine host factors associated with non-response to antiviral therapy. Treatment comprised IFN-alpha or peginterferon alpha, either alone or in combination with ribavirin. In a separate cohort of 73 patients, real time-polymerase chain reaction was performed to analyse hepatic mRNA expression. Immunohistochemistry for SOCS-3 was performed on liver biopsy samples from 38 patients with viral genotype 1 who had received antiviral treatment. RESULTS: Non-response (NR) to treatment occurred in 55% of patients with HCV genotypes 1 or 4 and 22% with genotypes 2 or 3. Factors independently associated with NR were viral genotype 1/4 (p < 0.001), cirrhosis on pretreatment biopsy (p = 0.025), and body mass index > or = 30 kg/m2 (p = 0.010). Obese subjects with viral genotype 1 had increased hepatic mRNA expression of phosphoenolpyruvate carboxy kinase (p = 0.01) and SOCS-3 (p = 0.047), in comparison with lean subjects. Following multivariate analysis, SOCS-3 mRNA expression remained independently associated with obesity (p = 0.023). SOCS-3 immunoreactivity was significantly increased in obesity (p = 0.013) and in non-responders compared with responders (p = 0.014). CONCLUSIONS: In patients with chronic HCV viral genotype 1, increased expression of factors that inhibit interferon signalling may be one mechanism by which obesity reduces the biological response to IFN-alpha.
Asunto(s)
Antivirales/uso terapéutico , Hepatitis C Crónica/tratamiento farmacológico , Interferón-alfa/uso terapéutico , Hígado/química , Obesidad/complicaciones , Polietilenglicoles/uso terapéutico , Proteínas Supresoras de la Señalización de Citocinas/análisis , Adulto , Quimioterapia Combinada , Hígado Graso/complicaciones , Femenino , Genotipo , Hepacivirus/genética , Hepatitis C Crónica/virología , Humanos , Inmunohistoquímica/métodos , Resistencia a la Insulina/fisiología , Interferón alfa-2 , Masculino , Persona de Mediana Edad , Fosfoenolpiruvato Carboxiquinasa (GTP)/análisis , Proteínas Recombinantes , Estudios Retrospectivos , Ribavirina/uso terapéutico , Transducción de Señal , Proteína 3 Supresora de la Señalización de Citocinas , Resultado del TratamientoRESUMEN
The behavior of mixed bile salt micelles consisting of sodium taurocholate, egg phosphatidylcholine, and cholesterol has been studied by ESR spin labeling and synchrotron x-ray scattering. Consistent with published phase diagrams, pure and mixed bile salt micelles have a limited capacity to incorporate and, hence, solubilize cholesterol. Excess cholesterol crystallizes out, a process that is readily detected both by ESR spin labeling using 3-doxyl-5 alpha-cholestane as a probe for cholesterol and synchrotron x-ray scattering. Both methods yield entirely consistent results. The crystallization of cholesterol from mixed bile salt micelles is indicated by the appearance of a magnetically dilute powder spectrum that is readily detected by visual inspection of the ESR spectra. Both the absence of Heissenberg spin exchange and the observation of a magnetically dilute powder spectrum provide evidence for the spin label co-crystallizing with cholesterol. In mixed bile salt micelles containing egg phosphatidylcholine, the solubility of cholesterol is increased as detected by both methods. With increasing content of phosphatidylcholine and increasing mole ratio cholesterol/phosphatidylcholine, the anisotropy of motion of the spin probe increases. The spin label 3-doxyl-5 alpha-cholestane is a useful substitute for cholesterol provided that it is used in dilute mixtures with excess cholesterol: the cholesterol/spin label mole ratio in these mixtures should be greater than 100. Despite the structural similarity between the two compounds, there are still significant differences in their physico-chemical properties. These differences come to the fore when cholesterol is totally replaced by the spin-label: 3-doxyl-5a-cholestane is significantly less soluble in bile salt and mixed bile salt micelles than cholesterol and, in contrast with cholesterol, it interacts only very weakly, if at all,with phosphatidylcholine. The potential of the ESR method for detecting cholesterol crystal growth in human bile is discussed.
Asunto(s)
Ácidos y Sales Biliares/química , Colesterol/química , Óxidos N-Cíclicos , Fosfatidilcolinas/química , Ácido Taurocólico/química , Cristalización , Espectroscopía de Resonancia por Spin del Electrón/métodos , Cinética , Micelas , Microscopía de Polarización , Marcadores de Spin , Sincrotrones , Difracción de Rayos X/métodosRESUMEN
Subjecting rabbit small intestinal brush border membrane vesicles (BBMV) to freeze-thaw cycles releases water-soluble lipid exchange (transfer) proteins into the supernatant. They differ widely in apparent molecular weight and catalyze cholesterol, phosphatidylcholine, and phosphatidylinositol exchange between two populations of small unilamellar lipid vesicles. In order to determine their interrelations, the smallest water-soluble lipid exchange protein was purified to homogeneity by gel filtration on Sephadex G-75 and cation exchange chromatography on Mono S. It is a basic protein of apparent molecular mass of 13 +/- 0.5 kDa. The purified protein was used to raise polyclonal antibodies. Polyclonal antibodies were also produced against a lipid exchange protein of apparent molecular mass of 100-120 kDa. By comparing lipid exchange, lipid binding, and immunological properties of the water-soluble lipid exchange proteins it can be shown that the 13-kDa (peak 3) protein is related to the 100-120 kDa (peak 1) protein; the properties of these two proteins are different from those of the peak 2 lipid exchange protein of apparent molecular mass of 22 kDa. Based on the immunological cross-reactivity observed between the 13 and 100-120 kDa and the lipid binding properties of these two proteins, a working hypothesis is proposed: both proteins are probably part of an integral membrane protein of the brush border membrane that facilitates cholesterol and phosphatidylcholine absorption in this membrane. Evidence derived from immunogold labeling of BBMV supports the notion that this protein is located on the external (luminal) side of the brush border membrane. The analogous behavior of rabbit and human small intestinal brush border membrane in terms of lipid absorption and the release of water-soluble lipid exchange proteins is discussed.
Asunto(s)
Proteínas Portadoras/aislamiento & purificación , Proteínas Portadoras/metabolismo , Intestino Delgado/metabolismo , Microvellosidades/metabolismo , Animales , Anticuerpos , Proteínas Portadoras/química , Cromatografía de Afinidad , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Espectroscopía de Resonancia por Spin del Electrón , Electroforesis en Gel de Poliacrilamida , Humanos , Immunoblotting , Inmunoglobulina G/aislamiento & purificación , Cinética , Microscopía Inmunoelectrónica , Microvellosidades/ultraestructura , Peso Molecular , Fosfatidilcolinas/metabolismo , Conejos , Marcadores de SpinRESUMEN
The absorption of monoacylglycerol by small intestinal brush border membrane is a passive process, i.e., the movement of monoacylglycerol from small unilamellar phospholipid vesicles as donor particles through the aqueous medium and the incorporation into the outer monolayer of the lipid bilayer of the brush border membrane are passive processes involving diffusion of the lipid along a concentration gradient. Small unilamellar vesicles of egg phosphatidylcholine containing 1 mol% of radiolabeled hexadecylglycerol were used as donor, and rabbit small intestinal brush border membrane vesicles or intact enterocytes isolated from pig jejunum, as acceptor. Hexadecylglycerol was employed as a lipase-resistant model compound for monoacylglycerols. Both acceptor membranes behave similarly in terms of hexadecylglycerol absorption: the kinetics of hexadecylglycerol absorption are biphasic. The initial fast phase is due to the movement of hexadecylglycerol from the donor particle through the aqueous medium to the outer lipid monolayer of the acceptor membrane, and the second slow phase probably involves the flip-flop motion of hexadecylglycerol from the outer to the inner monolayer of the acceptor membrane. The values for the pseudo-first-order rate constants of the initial fast phase for hexadecylglycerol absorption are relatively large and primarily determined by the high solubility (cmc) of hexadecylglycerol in aqueous media. The pseudo-first-order rate constants depend linearly on the protein (lipid) concentration of the acceptor membrane, indicating that the on rate of the hexadecylglycerol into the brush border membrane is rate limiting. The mechanism of the hexadecylglycerol absorption involves mainly monomer diffusion and probably collision-induced transfer.
Asunto(s)
Glicéridos/metabolismo , Absorción Intestinal , Microvellosidades/metabolismo , Animales , Difusión , Éteres de Glicerilo/metabolismo , Intestino Delgado/metabolismo , Intestino Delgado/ultraestructura , Cinética , Membrana Dobles de Lípidos/metabolismo , Liposomas/metabolismo , Micelas , Papaína/metabolismo , Fosfatidilcolinas/metabolismo , Fosfolípidos/metabolismo , ConejosRESUMEN
All classes of phospholipids present in brush border membrane are exchanged in a 1:1 ratio for egg phosphatidylcholine when brush border membrane vesicles from rabbit small intestine are incubated with small unilamellar vesicles of egg phosphatidylcholine. The exchange reaction exhibits biphasic kinetics similar to those of the hydrolysis of brush border membrane phospholipids by phospholipase A2 and sphingomyelinase C. In both reactions there is an initial fast phase followed by a markedly slower one. The phospholipid exchange appears to be catalyzed by intrinsic brush border membrane protein(s), while the digestion by phospholipases is mediated by externally added enzymes. From a comparison of the kinetics of phospholipid exchange and phospholipid hydrolysis, the following conclusions can be drawn: Both sets of experiments indicate the presence of two phospholipid pools differing in the rate of phospholipid exchange and hydrolysis. Except for sphingomyelin, the size of the two phospholipid pools derived from phospholipid exchange is in good agreement with that derived from phospholipid hydrolysis. This is the main finding of this work, and on the basis of this result the two lipid pools are tentatively assigned to phospholipid molecules located on the outer and inner layer of the brush border membrane. The slow rate of phospholipid exchange reflects the rate of transverse or flip-flop movement of phospholipids. The half-time of this motion is approximately 8 h for isoelectric (neutral) phospholipids such as phosphatidylethanolamine and approximately 80 h for negatively charged phosphatidylserine and phosphatidylinositol. Isoelectric phospholipids (phosphatidylcholine, phosphatidylethanolamine) are preferentially located on the inner (cytoplasmic) side (to about 70%) while the negatively charged phospholipids are more evenly distributed: 55-60% are located on the inner side.
Asunto(s)
Proteínas Portadoras/metabolismo , Intestino Delgado/metabolismo , Lípidos de la Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Microvellosidades/metabolismo , Proteínas de Transferencia de Fosfolípidos , Fosfolípidos/metabolismo , Animales , Proteínas Portadoras/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Cinética , Liposomas , Proteínas de la Membrana/aislamiento & purificación , Microvellosidades/química , Fosfatidilcolinas/química , ConejosRESUMEN
The intestinal brush-border membrane contains one or several membrane proteins that mediate fusion and/or aggregation of small unilamellar egg phosphatidylcholine vesicles. The fusion is accompanied by a partial loss of vesicle contents. Proteolytic treatment of the brush-border membrane with proteinase K abolishes the fusogenic property. This finding suggests that the fusogenic activity is associated with a membrane protein exposed on the external or luminal side of the brush-border membrane. Activation of intrinsic proteinases of the brush-border membrane liberates water-soluble proteins (supernate proteins). These proteins behave in an analogous way to intact brush-border membrane vesicles; they induce fusion of egg phosphatidylcholine vesicles and render the egg phosphatidylcholine bilayer permeable to ions and small molecules (Mr less than or equal to 5000). Furthermore, supernate proteins mediate phosphatidylcholine and cholesterol exchange between two populations of small, unilamellar phospholipid vesicles. Supernate proteins are fractionated on Sephadex G-75 SF yielding three protein peaks of apparent Mr greater than or equal to 70,000, Mr = 22,000 and Mr = 11,500. All three protein fractions show similar phosphatidylcholine-exchange activity, but they differ in their effects on the stability of egg phosphatidylcholine vesicles. The protein fraction with an apparent Mr greater than or equal to 70,000 has the highest fusogenic activity while the protein fraction of apparent Mr = 11,500 appears to be most effective in rendering the egg phosphatidylcholine bilayer permeable.
Asunto(s)
Intestinos/ultraestructura , Fusión de Membrana , Proteínas de la Membrana/fisiología , Microvellosidades/química , Animales , Cromatografía en Gel , Endopeptidasa K , Fluoresceínas/metabolismo , Técnica de Fractura por Congelación , Cinética , Membrana Dobles de Lípidos/metabolismo , Liposomas/metabolismo , Microscopía Electrónica , Fosfatidilcolinas/metabolismo , Conejos , Serina Endopeptidasas/farmacología , Espectrometría de FluorescenciaRESUMEN
Cholesterol absorption by small intestinal brush border membrane vesicles from taurocholate mixed micelles is a second-order reaction. From a comparison of reaction rates and order before and after proteinase K treatment of brush-border membrane vesicles, it is concluded that cholesterol absorption is protein-mediated. It is shown that the desorption of cholesterol from taurocholate mixed micelles is by a factor of about 10(4) faster than that from egg phosphatidylcholine bilayers. When brush border membrane vesicles are stored at room temperature, intrinsic proteinases are activated and proteins are liberated from the brush border membrane. These proteins collected in the supernatant catalyze cholesterol and phosphatidylcholine exchange between two populations of small unilamellar phospholipid vesicles. One of the active proteins present in the supernatant is purified by a two-step procedure involving gel filtration on Sephadex G-75 SF and affinity chromatography on a Nucleosil-phosphatidylcholine column. The protein thus obtained is pure by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. It has an apparent molecular weight of slightly less than 14,000 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and a value of 11,500 determined by gel filtration on Sephadex G-75 SF.
Asunto(s)
Proteínas Portadoras/aislamiento & purificación , Glicoproteínas , Intestinos/química , Microvellosidades/química , Animales , Proteínas Portadoras/química , Colesterol/metabolismo , Proteínas de Transferencia de Ésteres de Colesterol , Cromatografía , Electroforesis en Gel de Poliacrilamida , Hidrólisis , Absorción Intestinal , Intestinos/ultraestructura , Cinética , Lisofosfatidilcolinas , Micelas , Conejos , Ácido TaurocólicoRESUMEN
The effect of increasing concentrations of lipid X (2,3-bis(3-hydroxymyristoyl)-alpha-D-glucosamine 1-phosphate) on the phase behaviour of EPC (egg phosphatidylcholine) and EPE (egg phosphatidylethanolamine) is studied at a pH greater than or equal to 7 where lipid X carries one to two negative charges. Small amounts of lipid X (molar ratio approximately 0.01) induce continuous swelling of EPC and EPE bilayers and consequently the formation of large unilamellar vesicles in excess water. In many respects, the effect of lipid X on EPC and EPE bilayers is similar to that of phosphatidic acid. However, lipid X/EPC mixtures form micelles in excess lipid X whereas mixtures of phosphatidic acid/EPC vesiculate at all ratios. The same is true for lipid X/EPE mixtures. Small unilamellar vesicles of an average diameter of 40 nm form spontaneously upon dispersion of a dry lipid X/EPE film (molar ratio = 10). Unsonicated dispersions of lipid X/EPC (molar ratio = 1) are subjected to pH-jump treatment which involves raising of the pH to 11-12 and subsequent lowering of the pH to between 7.5 and 8.5. Such a treatment has little effect on the vesicle size and size distribution as compared to a control dispersion at pH 8.2. The mean size is determined to be 92 +/- 60 nm. Electron micrographs of freeze-fractured samples of lipid X/EPC (molar ratio = 1) reveal the presence of mainly micelles at pH 12. Upon lowering the pH to neutrality these micelles become unstable and aggregate/fuse rapidly to unilamellar vesicles (average diameter 95 +/- 40 nm). Sonication of equimolar mixtures of lipid X and EPC at pH 7 yields small unilamellar vesicles of a diameter of 20-25 nm as well as mixed micelles of a size between 15 and 17 nm. This behaviour is again different from that of mixed EPC/phosphatidic acid dispersions which form small unilamellar vesicles. The presence of lipid X in such mixtures does not prevent the aggregation/fusion to larger vesicles during freezing of the dispersion. As with pure EPC bilayers, stabilization is, however, achieved in the presence of 10% sucrose. This indicates that the covalently bonded glucosamine group of lipid X cannot substitute water of hydration in neighbouring EPC molecules.
Asunto(s)
Glucolípidos , Membrana Dobles de Lípidos , Fosfatidilcolinas , Fosfatidiletanolaminas , Cromatografía en Gel , Técnica de Fractura por Congelación , Congelación , Calor , Concentración de Iones de Hidrógeno , Lípido A , Liposomas , Espectroscopía de Resonancia Magnética , Microscopía Electrónica , Tamaño de la Partícula , Sonicación , EspectrofotometríaRESUMEN
The phase behaviour of aqueous dispersions of lipid X, a precursor of bacterial lipopolysaccharides has been investigated by a variety of physico-chemical techniques. The results are consistent with the presence of disk-shaped micelles with an average diameter of 13 +/- 1.8 nm. The critical micellar concentration in water and physiological saline is 4 x 10(-5) M. Consistent with the formation of micelles in water and physiological saline is the finding that lipid X is in the liquid-crystalline state at temperatures higher than 0 degrees C. The packing and the dynamics of lipid X are characteristic of micelles. Close to the polar group the hydrocarbon chains are significantly more mobile and disordered than in the corresponding region of lipid bilayers. From monolayer studies an estimate of the molecular area of lipid X is derived; under physiological conditions the area/molecule is about 0.50 nm2 at 30 mN/m indicating that lipid X has a wedge-like shape. The two pK values of the primary phosphate group of lipid X are pK1 approximately 1.3 and pK2 = 8.2. At pH values less than 7, the area/molecule decreases, i.e. the packing of the lipid X molecules becomes tighter, and there is also a decrease in the solubility of lipid X. As is characteristic of charged lipids, the state of aggregation (phase behaviour) of lipid X depends on pH, the ionic strength and the nature of the counterion.
Asunto(s)
Glucolípidos/análisis , Lípido A/análisis , Rastreo Diferencial de Calorimetría , Fenómenos Químicos , Química , Cromatografía en Gel , Densitometría , Micelas , Microscopía Electrónica , Tamaño de la Partícula , Solubilidad , Espectrometría de FluorescenciaRESUMEN
Two independent parameters, two characteristic temperatures, one indicating the change in the molecular organization of the core, Tc, and the other in the surface layer, Ts, were measured for a number of natural and triglyceride-enriched porcine low-density lipoprotein (LDL1 (buoyant density 1.020--1.063 g/ml) and LDL2 (buoyant density 1.063--1.080 g/ml) samples. Tc was determined by differential scanning calorimetry (DSC), whereas Ts was measured by Mn(II) binding to the lipoprotein surface followed by electron spin resonance (ESR) spectroscopy. A significant causal relationship between Tc and Ts in both LDL subfractions demonstrates the surface-core interaction in LDL. The significance of that interaction is emphasized as a possible link in the chain diet----lipoprotein changes----atherosclerosis.
Asunto(s)
Lipoproteínas LDL/sangre , Animales , Rastreo Diferencial de Calorimetría , Espectroscopía de Resonancia por Spin del Electrón , Porcinos , Temperatura , Triglicéridos/sangreRESUMEN
The effect of 0-1.0 M sucrose on the phase-transition properties of 1,2-dipalmitoyl-3-sn-phosphatidylcholine (1,2-DPPC) was examined by high-sensitivity differential scanning calorimetry at a scan rate of 0.1 K min-1. Increasing the concentration of sucrose caused a small, but experimentally significant, increase in the temperature (Tm) of maximal excess apparent specific heat (Cmax) and in delta T 1/2 (the transition width at 1/2 Cmax), a reduction in Cmax, and a small decrease (approximately 8-10% at 1.0 M sucrose compared with 0 M sucrose) in the calorimetric enthalpy (delta Hcal) of the gel-to-liquid crystalline transition. The calorimetric parameters of the pretransition of 1,2-DPPC were not significantly affected by sucrose in the concentration range examined, except there was a 1.0 degree C increase in the temperature (Tp) of maximal excess apparent specific heat in the presence of 1.0 M sucrose. The results are discussed in terms of the possible molecular mechanisms that could have caused the observed changes and are contrasted with the results obtained by C. -H. Chen et al. (1981, Biophys. J., 36:359-367).
Asunto(s)
Surfactantes Pulmonares , Sacarosa , Calorimetría , Geles , TermodinámicaRESUMEN
The B, or binding, subunit of cholera enterotoxin forms a pentameric ring structure in the intact toxin, and also when the subunit is isolated from the A subunit. The thermal denaturation of the B subunit ring was examined by differential scanning calorimetry in the presence and absence of ganglioside GM1, its natural 'receptor'. In the absence of ganglioside an irreversible endotherm was observed with maximal excess apparent heat capacity, Cmax, at 74.6 degrees C. When the ganglioside was added in increasing amounts, multiple transitions were observed at higher temperatures, the most prominent having a Cmax at 90.8 degrees C. At high ganglioside concentrations, the 74.6 degrees C transition was not observed. In addition to the thermodynamic results a model is proposed for the interaction of GM1 and B subunit pentamer. This model is derived independently of the calorimetric results (but is consistent with such data) and is based upon considerations of the geometry of the GM1 micelle B subunit pentamer.
Asunto(s)
Toxina del Cólera/metabolismo , Gangliósido G(M1)/metabolismo , Gangliósidos/metabolismo , Sitios de Unión , Rastreo Diferencial de Calorimetría , Técnicas In Vitro , Modelos Químicos , Conformación Molecular , TermodinámicaRESUMEN
The phase transition properties of aqueous suspensions of a series of nonhydrated (not heated above room temperature) and hydrated 1,2 diacylphosphatidylethanolamines (PE's) have been examined by high sensitivity differential scanning calorimetry at scan rates of 0.02-1.0 K min-1. At all scan rates nonhydrated PE's show a single asymmetric transition curve of excess heat capacity as a function of temperature. Multilamellar dispersions of hydrated PE's, however, exhibit transitions with fine structure, which can be fitted as the sum of three two-state component transitions, at scan rates of 0.02-0.1 K min-1, but give only a single asymmetric transition at 1.0 K min-1. At all scan rates the transition(s) of hydrated samples occur at lower temperatures than those of nonhydrated samples. One of the component transitions of hydrated PE's may be analogous to the pretransition that occurs in 1,2 diacylphosphatidylcholines.
Asunto(s)
Liposomas , Fosfatidiletanolaminas , Fenómenos Biofísicos , Biofisica , Rastreo Diferencial de Calorimetría , TermodinámicaRESUMEN
Interaction of lanthanum ions (La3+) with 1,2 dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC) causes an increase in Tc, the temperature of maximal excess heat capacity, and the width of the gel-to-liquid crystalline transition. At a mole ratio of La3+ to DPPC sufficient to remove the hydrocarbon chain tilt angle of DPPC, the changes in the thermodynamic parameters of the pretransition are minor, Tc and the width were unaltered and the enthalpy was reduced by only 10%. This suggests that the change in tilt angle is not a necessary concomitant of the pretransition.