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Biomedical subjects

E Freire

Publications and source records attributed to E Freire.

At least 145 records · Page 8Linked to original sources
PubMed1984

Thermotropic and dynamic characterization of interactions of acylated alpha-bungarotoxin with phospholipid bilayer membranes.

The interactions of palmitoyl-alpha-bungarotoxin (PBGT) with dipalmitoylphosphatidylcholine (DPPC) bilayers have been studied by using high-sensitivity differential scanning calorimetry together with steady-state and time-resolved phosphorescence and fluorescence spectroscopy. The incorporation of PBGT into large single lamellar vesicles causes a decrease in the phospholipid phase transition temperature (Tm), a broadening of the heat capacity function, and a decrease in the enthalpy change associated with the phospholipid gel to liquid-crystalline transition. Analysis of the dependence of this decreased enthalpy change on the protein/lipid molar ratio indicates that each PBGT molecule exhibits a localized effect upon the bilayer, preventing approximately six lipid molecules from participating in the lipid phase transition. Additional calorimetric experiments indicate that binding to acetylcholine receptor enriched membranes causes a small increase in the Tm of the PBGT/DPPC vesicles. Steady-state fluorescence depolarization measurements employing 1,6-diphenyl-1,3,5-hexatriene (DPH) indicate that the association of PBGT with the phospholipid bilayer decreases the apparent order of the bulk lipid below Tm while increasing the order above Tm. These results have been further supported by rotational mobility measurements of erythrosin-labeled PBGT associated with giant (about 2-micron) unilamellar vesicles composed of dielaidoylphosphatidylcholine or dioleoylphosphatidylcholine using the time-dependent decay of delayed fluorescence/phosphorescence emission anisotropy. Rotational correlation times in the submillisecond time scale (about 30 microseconds) indicate that the protein is highly mobile in the fluid phase and that below Tm the rotational mobility is only slightly restricted.(ABSTRACT TRUNCATED AT 250 WORDS)

Bungarotoxins↗
PubMed1984

Modulation of neuraminidase activity by the physical state of phospholipid bilayers containing gangliosides Gd1a and Gt1b.

The thermotropic behavior of large unilamellar dipalmitoylphosphatidylcholine vesicles containing the disialoganglioside Gd1a and the trisialoganglioside Gt1b on their outer surface has been studied as a function of the ganglioside molar fraction and Ca2+ concentration by using high-sensitivity differential scanning calorimetry and steady-state fluorescence spectroscopy. These studies indicate that both gangliosides have an ordering effect on the hydrocarbon region of the bilayer and that this effect is enhanced by the presence of Ca2+ ions. The calorimetric experiments also indicate that ganglioside Gt1b has an intrinsic tendency to phase separate into compositional-rich ganglioside domains even in the absence of Ca2+. Ganglioside Gd1a, on the other hand, only phase separates at Ca2+ concentrations equal to or higher than 10 mM. These studies have allowed us to identify and evaluate the factors affecting the rates of hydrolysis of gangliosides by the soluble neuraminidase from Clostridium perfringens. The data presented in this paper indicate that the rates of hydrolysis of membrane-bound gangliosides are correlated to the physical state of the membrane and the state of aggregation of the ganglioside molecules within the lipid bilayer. For membrane-bound gangliosides, maximal activation energies were found at temperatures slightly below the lipid phase transition temperature. The rates of hydrolysis of the soluble substrate sialyllactose or that of the micellar ganglioside is independent of Ca2+ concentration, whereas the rates of hydrolysis of membrane-bound ganglioside are inhibited by Ca2+ especially under conditions in which the clustering effect of Ca2+ is maximal. These studies suggest that the soluble neuraminidases from Clostridium perfringens prefer ganglioside substrates that are dispersed within the membrane and not forming part of largely aggregated clusters.

Calcium↗
PubMed1983

Calorimetric and fluorescence characterization of interactions between cytochrome b5 and phosphatidylcholine bilayers.

The interactions of cytochrome b5 with dimyristoylphosphatidylcholine and dipalmitoylphosphatidylcholine lipid bilayers have been studied with high-sensitivity differential scanning calorimetry and fluorescence spectroscopy. The incorporation of cytochrome b5 into large single lamellar vesicles causes a reduction in the enthalpy change associated with the lipid phase transition. Analysis of the dependence of this enthalpy change on the protein/lipid molar ratio indicates that each cytochrome b5 molecule prevents 14 +/- 1 lipid molecules from participating in the gel to liquid-crystalline transition and that this number is independent of the phospholipid acyl chain length. Resonance energy transfer between the intrinsic tryptophan fluorescence of cytochrome b5 and pyrenedecanoic acid indicates that, in the liquid-crystalline phase, protein and lipid molecules are uniformly distributed within the bilayer plane. In the gel phase, pyrenedecanoic acid partitions into the boundary layer lipid causing a dramatic decrease in the fluorescence intensity of cytochrome b5. The excimer/monomer ratios of pyrenedecanoic acid decrease upon increasing the protein/lipid molar ratio, indicating that the presence of protein molecules within the bilayer slows down the lateral mobility of the lipid probes. The picture that emerges from this set of experiments is that cytochrome b5 perturbs one layer of lipid around the hydrophobic segment of the protein and that this layer is unable to undergo the gel-liquid-crystalline transition, remaining instead in a relatively disordered configuration above and below the transition temperature of the bulk lipid.

Animals↗
PubMed1982

Fluorescence energy transfer in two dimensions. A numeric solution for random and nonrandom distributions.

A method of Monte Carlo calculations has been applied to the problem of fluorescence energy transfer in two dimensions in order to provide a quantitative measure of the effects of nonideal mixing of lipid and protein molecules on the quenching profiles of membrane systems. These numerical techniques permit the formulation of a detailed set of equations that describes in a precise manner the quenching and depolarization properties of planar donor-acceptor distributions as a function of specific spectroscopic and organizational parameters. Because of the exact nature of the present numeric method, these results are used to evaluate critically the validity of previous approximate treatments existing in the literature. This method is also used to examine the effects of excluded volume interactions and distinct lattice structures on the expected transfer efficiencies. As a specific application, representative quenching profiles for protein-lipid mixtures, in which donor groups are covalently linked to the protein molecules and acceptor species are randomly distributed within lipid domains, have been obtained. It is found that the existence of phase-separated protein domains gives rise to a shielding effect that significantly decreases the transfer efficiencies with respect to those expected for an ideal distribution of protein molecules. The results from the present numerical study indicate that the experimental application of fluorescence energy transfer measurements in multicomponent membrane systems can be used to obtain organizational parameters that accurately reflect the lateral distribution of protein and lipid molecules within the bilayer membrane.

Energy Transfer↗
PubMed1982

Quantitative characterization of the lateral distribution of membrane proteins within the lipid bilayer.

The dependence of the lateral distribution of membrane proteins on the size, protein/lipoid molar ratio, and the magnitude of the interaction potentials has been investigated by computer modeling protein-lipid distributions with Monte Carlo calculations. These results have allowed us to develop a quantitative characterization of the distribution of membrane proteins and to correlate these distributions with experimental observables. The topological arrangement of protein domains, protein plus annular lipid domains, and free lipid domains is described in terms of radial distribution, pair connectedness, and cluster distribution functions. The radial distribution functions are used to measure the distribution of intermolecular distances between protein molecules, whereas the pair connectedness functions are used to estimate the physical extension of compositional domains. It is shown that, at characteristic protein/lipid molar ratios, previously isolated domains become connected, forming domain networks that extend over the entire membrane surface. These changes in the lateral connectivity of compositional domains are paralleled by changes in the calculated lateral diffusion coefficients and might have important implications for the regulation of diffusion controlled processes within the membrane.

Computers↗
PubMed1981

Thermal behavior of stearoylsphingomyelin-cholesterol dispersions.

The thermotropic behavior of aqueous dispersions of stearoylsphingomyelin-cholesterol mixtures was examined by high-sensitivity differential scanning calorimetry. When less than 20 mol % cholesterol was mixed with the sphingomyelin and the samples were held at room temperature for 7-9 days before the initiation of calorimetric measurements, a sharp endotherm at 56-57 degrees C and a broad endotherm at 35-50 degrees C were observed. In addition, samples containing 15-20 mol % stearol exhibited a sharp endotherm at 43-45 degrees C. If samples were held at room temperature for less than 2 h before the initiation of calorimetric analysis, the 56-57 degrees C endotherm was usually not seen. Instead, a combination of broad and sharp endotherms over the range of 35-50 degrees C was observed. Occasionally, exotherms were also observed within this temperature range. These results, along with those from previous studies, imply that a cholesterol-rich phase coexists with a cholesterol-poor phase in which the sphingomyelin molecules may exist in two distinctly different gel states.

Calorimetry, Differential Scanning↗
PubMed1981

Effect of surface curvature on stability, thermodynamic behavior, and osmotic activity of dipalmitoylphosphatidylcholine single lamellar vesicles.

The size and surface curvature dependence of the properties and stability of single lamellar vesicles have been investigated by using a variety of physicochemical techniques. Dipalmitoylphosphatidylcholine single lamellar vesicles of sizes ranging between 200 and 900 A in diameter have been prepared by the French press method and characterized with respect to their size distribution, stability, and thermotropic behavior by negative stain electron microscopy, molecular sieve chromatography, nuclear magnetic resonance spectroscopy, and differential scanning calorimetry. Vesicles with a diameter smaller than 400 A are unstable below their transition temperature and fuse spontaneously to form larger single lamellar vesicles. Correlation analysis of experimentally obtained size distributions and calorimetric phase transitions profiles allowed estimation of the size dependence of the transition temperature. The phase transition temperature depends on the vesicle size in a sigmoidal fashion. Throughout the entire 200-700 A diamter range, the phase transition parameters are sensitive to size; however, the size dependence is especially pronounced around 400 A in diameter. The anomalous size dependence of the transition temperature for vesicles smaller than 400 A in diameter has been attributed to a decrease in the effective bilayer curvature due to packing rearrangements of the lipid molecules. Changes in the fractional degree of self-quenching of trapped 6-carboxyfluorescein induced by osmotic stress indicate that large single lamellar vesicles are not spherical under isoosmotic conditions. These vesicles are relatively flexible and can sustain almost a 2-fold increase in their internal aqueous volume without any leakage of the internal content.

Drug Stability↗
PubMed1981

Asymmetric incorporation of trisialoganglioside into dipalmitoylphosphatidylcholine vesicles.

Results are presented which demonstrate that purified trisialoganglioside spontaneously incorporates into performed phospholipid vesicles. Determinations of the extent of incorporation were made by separating large unilamellar dipalmitoylphosphatidylcholine vesicles containing incorporated ganglioside from micellar ganglioside on a Sepharose-2B column. Incorporation occurs without appreciably altering the vesicular character of the phospholipid bilayer as judged by the maintenance of an outside/inside ratio, determined by 31P NMR, comparable to that of the original vesicles. All of the incorporated ganglioside ias accessible to neuraminidase, indicating that incorporation occurs only on the outer face of the bilayer. The thermotropic behavior of these asymmetric dipalmitoylphosphatidylcholine-trisialoganglioside vesicles, examined by high sensitivity scanning calorimetry, strongly suggests that the incorporated ganglioside is intercalated into the outer monolayer of the vesicle bilayer. Calorimetric studies indicate that the ganglioside stabilizes these vesicular structures by inhibiting the fusion of small vesicles that occurs below the phase-transition temperature. These structures are a representative model system, which like the mammalian plasma membrane contain an asymmetric distribution of glycosphingolipid in the outer surface.

Calorimetry, Differential Scanning↗
PubMed1980

Monte Carlo studies of the lateral organization of molecules in two-component lipid bilayers.

The lateral organization of two-component phosphatidylcholine bilayers has been investigated using Monte Carlo calculations based upon non-ideality parameters deduced from the phase diagrams of these mixtures. The results are used to develop a quantitative description of the distribution and spatial localization of compositional regions along the bilayer plane in both the gel and liquid crystalline phases. In particular, a detailed analysis of the physical extension (lateral connectivity) and compactness of the compositional clusters is made. It is concluded that the chemical composition of the membrane, the physical state of the bilayer and the interaction energies between molecules greatly influence the lateral connectivity and compactness of compositional regions and that these parameters might play an important role in the formation of diffusional pathways along the membrane plane.

Computers↗
PubMed1980

Calorimetric investigation of the complex phase behavior of glucocerebroside dispersions.

The thermotropic behavior of aqueous dispersions of glucocerebroside from Gaucher's spleen has been investigated by different scanning calorimetry. These results indicate that glucocerebroside undergoes two distinct phase transitions centered at 47 and 83 degrees C, respectively. The high-temperature transition is associated with the main gel-liquid crystalline transition and has an enthalpy change of 13.6 kcal/mol of lipid; this transition is not rapidly reversible and the liquid crystalline phase supercools to a metastable gel phase. The low-temperature transition is exothermic with an enthalpy change of -6.3 kcal/mol and involves a transformation of the metastable gel phase to a more highly ordered gel conformation, without involving changes in the conformational state of the hydrocarbon chains. The behavior of these transitions as a function of the amount of water suggests that the origin of the metastability is related to a hydration-dehydration process of the cerebroside molecule. Experiments with synthetic-D-erythro-N-palmitoylglucocerebroside revealed the same thermotropic behavior. These glucocerebroside transitions are irreversible and together define a unidirectional cycle in which each state of the molecule can only be reached by completing the entire cycle.

Calorimetry, Differential Scanning↗
PubMed1980

Estimation of the lateral distribution of molecules in two-component lipid bilayers.

A new formalism to investigate the lateral distribution of molecules in lipid bilayers has been developed, and the results have been applied to the case of phosphatidylcholine mixtures. It is demonstrated that the experimental phase diagrams for these mixtures can provide the necessary information to generate computer-simulated bilayers with the desired molecular interactions and lattice constraints. Analysis of these computer-generated bilayers allows calculation of the number of contacts between like and unlike molecules, the average size and number of compositional clusters, and the pair correlation functions. The results of this analysis provide a full quantitative description of the molecular organization of phosphatidylcholine within the plane of the bilayer.

Computers↗
PubMed1980

Compositional domain structure in phosphatidylcholine--cholesterol and sphingomyelin--cholesterol bilayers.

the lateral distribution of cholesterol in phospholipid bilayers has been investigated through a method of Monte Carlo calculations, using interaction energies deduced from calorimetric results for cholesterol-phospholipid mixtures. Analysis of computer-generated bilayer configurations allows calculation of the spatial localization and relative abundance of distinct regions of varying cholesterol content along the plane of the bilayer. An interfacial phospholipid region between cholesterol-bound and cholesterol-free domains is found to extend one lipid beyond the cholesterol-bound domain for mixtures of cholesterol with palmitoyl sphingomyelin, lignoceroyl sphingomyelin, and dipalmitoyl phosphatidylcholine. The results indicate that the degree of nonideality in the mixing of cholesterol is dependent on fatty acid chain length and that cholesterol mixes more ideally in sphingomyelins than in phosphatidylcholines of equal chain length. It is found that at approximately 20 mol % cholesterol the cholesterol-rich areas suddenly become connected, forming a network that extends over the entire bilayer. This change in the lateral connectivity of the cholesterol-rich domains occurs over a narrow concentration interval and is presumably responsible for the abrupt change in the lateral diffusion coefficient observed at this concentration.

Cholesterol↗
PubMed1980

Factors affecting glucose turnover and utilization in the neonatal subhuman primate (Macaca mulatta).

The rate of glucose turnover (RT) has been studied in 33 subhuman primate newborn (Macaca mulatta). There appears to be a linear relationship between RT and plasma glucose (G) such that RT = 1.053 G + 0.242. Liver enzymes show a change in specificity such that hexokinase is predominant in the preterm animals and glucokinase has little activity, while in the term animal these enzymes of of equal activity. In the 1-year-old animal glucokinase predominates. Hexokinase activity in the brain remains constant throughout development. These results imply that low blood glucose in the neonate is not associated with increased glucose utilization.

Aging↗
PubMed1980

Factors affecting gluconeogenesis in the neonatal subhuman primate (Macaca mulatta).

The capacity for gluconeogenesis has been studied in 33 subhuman primate newborn (Macaca mulatta) in the basal steady state. Basal blood glucose levels were seen to rise with increasing postnatal age. Availability of the major gluconeogenic substrates, alanine and lactate, was adequate at times when blood glucose and the rate of gluconeogenesis were low. Hepatic and renal cortical content of the four key gluconeogenic enzymes was low during the 1st week of life compared to adult levels. Diminished induction of the gluconeogenic enzymes did not appear to be the cause of low blood glucose levels. The serum-free fatty levels were directly correlated to both the basal glucose levels and to the rate of gluconeogenesis.

Aging↗
PubMed1979

Thermotropic behavior of monoglucocerebroside--dipalmitoylphosphatidylcholine multilamellar liposomes.

The thermotropic behavior of multilamellar liposomes prepared from mixtures of glucocerebroside and dipalmitoylphosphatidylcholine has been studied by high-sensitivity scanning calorimetry. It is shown that glucocerebroside has a marked effect on the gel--liquid crystalline transition of dipalmitoylphosphatidylcholine. The pretransition seen in pure samples of dipalmitoylphosphatidylcholine is undetectable at small mode fractions of glucocerebrosides (less than 10%). The main transition is shifted to higher temperatures and becomes broader and less cooperative in the presence of glucocerebroside. The enthalpy change of the main transition decreases with increasing the glucocerebroside content. However, this decrease is not linear with the glucocerebroside/phospholipid mole ratio. Glucocerebroside itself does not show a separate transition in the temperature range of these studies (10--75 degree C). The origin of these effects and their dependence on the glucocerebroside content suggest that the in-plane distribution of glucocerebroside molecules is affected by the physical state of the lipid bilayer and by the glucocerebroside/phospholipid mole ratio.

Calorimetry, Differential Scanning↗
PubMed1978

Estimation of molecular averages and equilibrium fluctuations in lipid bilayer systems from the excess heat capacity function.

It is demonstrated that the bilayer partition function can be numerically obtained from scanning calorimetric data without assuming a particular model for the gel-liquid crystalline transition. From this partition function, the enthalpy, entropy and volume changes accompanying the transition can be calculated. In the limit of very large systems, the method of the grand partition function allows calculation of cluster model distribution functions from which average sizes of gel and liquid-crystal clusters, cluster densities and equilibrium fluctuations are obtained. These results indicate that the main transition in phospholipid bilayers proceeds through the formation of clusters and that these clusters are not static domains but highly fluctuating entities. These fluctuations in cluster size are approximately equal to the average cluster size and give rise to localized density and volume fluctuations. The magnitude of these fluctuations is affected by the radius of curvature of the bilayer and by the addition of small molecular weight compounds to the system.

Calorimetry, Differential Scanning↗