Biophysical characterization of interaction between apolipoprotein A-I and bacterial lipopolysaccharide.

We studied the effect of bacterial lipopolysaccharide (LPS)-apolipoprotein A-I (apo A-I) interaction on the structure and function of this protein. The micellization process of dimirystoil phosphatidylcholine liposomes (MLV-DMPC) by apo A-I in the presence of LPS was characterized. Apo A-I may interact with MLV-DMPC at the lipid transition temperature, forming micellar complexes. The kinetics of MLV-DMPC micellization was studied by turbidimetry. In the absence of LPS, a monoexponential decrease in turbidity is observed. Preincubation of apo A-I with LPS impairs the micellization reaction, resulting in biphasic kinetics. The amplitude of the fast phase decreases with increasing concentrations of LPS. In the absence or in the presence of low amounts of LPS (1:0.1 protein:LPS weight ratio), two major micellization products-containing two and three apo A-I molecules per particle-were observed. However, in the presence of higher amounts of LPS (1:1 protein:LPS weight ratio), particles mainly contained two apo A-I molecules. In contrast, a decrease in intrinsic fluorescence intensity of the protein was observed in the presence of an increasing LPS concentration. Finally, we studied the effect of LPS on the transition temperature (Tt) of MLV-DMPC without detecting changes in Tt. In conclusion, the changes found in the micellization process are likely to be mainly caused by changes in the apo A-I conformation by LPS interaction in solution.

Location of tryptophan residues in free and membrane bound Escherichia coli alpha-hemolysin and their role on the lytic membrane properties.

alpha-hemolysin (HlyA) is an extracellular protein toxin secreted by Escherichia coli that acts at the level of plasma cell membranes of target eukaryotic cells. Previous studies showed that toxin binding to the bilayers occurs in at least two ways, a reversible adsorption and an irreversible insertion. Studies of HlyA insertion into bilayers formed from phosphatidylcholine show that insertion is accompanied by an increase in the protein intrinsic fluorescence. In order to better define structural parameters of the membrane-bound form, the location of tryptophan residues was studied by means of quenchers of their intrinsic fluorescence located at 7, 12 and 16 positions of the acyl chain of phosphatidylcholine. The quenching was progressively weaker suggesting an interfacial location of the Trp. In parallel, HlyA was subjected to oxidation with N-bromosuccinimide to study the role of Trp residues exposed to aqueous media in its structure-function relationship. In the folded toxin molecule, a single residue was susceptible to oxidation with NBS, whereas incubation with LUV of the toxin prior modification prevents its oxidation, suggesting that Trp residue(s) are directly involved in toxin binding and insertion. Finally, the modification of residues exposed to solvent resulted in a complete impairment of the lytic activity. It was concluded that the modification-sensitive Trp residues are essential for the structure and function of native HlyA. These results are consistent with the model proposed by Soloaga et al. (Mol. Microbiol. 31 (1999) 1013-1024) according to which HlyA is bound to a single monolayer through a number of amphipathic instead of inserted transmembrane helices.

Effect of encapsulated Ca2+ on the surface properties of curved phosphatidylcholine bilayers.

In this paper, the influence of Ca2+ on the adsorption properties of 1,8-anilinonaphthalenesulfonate (ANS) and analogous probes to sonicated vesicles of phosphatidylcholine was studied by means of spectrofluorometry. The fluorescence of ANS added to the vesicle dispersion increases with the Ca2+ concentration in the inner media but remains constant when Ca2+ concentration is changed in the outside solution. However, the fluorescence decreases when large anions such as ClO4- are present in the external solution. Ca2+ inside large liposomes promotes a similar behaviour to that found in sonicated vesicles when they are osmotically contracted in hypertonic media. The results can be interpreted in terms of Ca2+ adsorption on the inner interface and a cooperative interaction between the monolayers.

Ca2+-induced phosphatidylcholine vesicle aggregation in the presence of ferricyanide.

The titration of sonicated vesicles of egg phosphatidylcholine with ferricyanide in the presence of Ca2+ results in the formation of aggregates. The turbidity increase caused by these aggregates cannot be reversed by EDTA treatment. In addition, no rearrangement of the bilayer structure has been found in this process, either measuring leakage of vesicle content or exchange of lipids among the bilayers themselves. The aggregation is dependent on the Ca2+ content of the vesicles, the outer Ca2+ and Fe(CN)3-(6) concentration and the order of addition of Ca2+ and ferricyanide. The results can be explained by a specific adsorption of Fe(CN)3-(6) to bilayers of sonicated vesicles, in contrast to other multivalent anions. In contrast to the stability found with sonicated vesicles, the aggregation causes a leakage of the internal solution when multilamellar liposomes are titrated with Fe(CN)3-(6).

Effect of the asymmetric Ca2+ distribution on the bilayer properties of phosphatidylcholine-sonicated vesicles.

The incorporation of Ca2+ in the inner volume of egg phosphatidylcholine vesicles increases the fluorescence anisotropy of a diphenylhexatriene probe. This increase is higher than for Na+ at the same normality. An effect of the same magnitude is induced by Ca2+ when using binary lipid mixture (dioleoylphosphatidylcholine and dipalmitoylphosphatidylcholine) as long as the mixture is maintained below the phase-transition temperature of the saturated species. The influence of Ca2+ may be explained by an asymmetric distribution of the saturated and unsaturated lipids between the internal and the external monolayers.

Calcium-dependent conformation of E. coli alpha-haemolysin. Implications for the mechanism of membrane insertion and lysis.

Previous studies from this laboratory had shown that calcium ions were essential for the membrane lytic activity of E. coli alpha-haemolysin (HlyA), while zinc ions did not sustain such a lytic activity. The present data indicate that calcium-binding does not lead to major changes in the secondary structure, judging from circular dichroism spectra. However binding to Ca2+ exposes new hydrophobic residues at the protein surface, as indicated by the increased binding of the fluorescent probe aniline naphtholsulphonate (ANS), and by the increased tendency of the Ca2+-bound protein to self-aggregate. In addition zinc ions are seen to decrease the thermal stability of HlyA which, according to intrinsic fluorescence and differential scanning calorimetry data, is stable below 95 degrees C when bound to calcium, while it undergoes irreversible denaturation above 60 degrees C in the zinc-bound form. Binding to phosphatidylcholine bilayers is quantitatively similar in the presence of both cations, but about one-third of the zinc-bound HlyA is released in the presence of 2 M NaCl. Differential scanning calorimetry of dimyristoylglycerophosphocholine large unilamellar vesicles reveals that Zn2+-HlyA interaction with the lipid bilayer has a strong polar component, while Ca2+-HlyA appears to interact mainly through hydrophobic forces. Experiments in which HIyA transfer is measured from phospholipid vesicles to red blood cells demonstrate that Ca2+ ions promote the irreversible binding of the toxin to bilayers. All these data can be interpreted in terms of a specific Ca2+ effect that increases the surface hydrophobicity of the protein, thus facilitating its irreversible bilayer insertion in the fashion of intrinsic membrane proteins.

Role of lipopolysaccharide on the structure and function of alpha-hemolysin from Escherichia coli.

alpha-Hemolysin (HlyA) is a protein toxin (107 kDa) secreted by some pathogenic strains of E. coli. Several studies suggested the relationship between HlyA and lipopolysaccharide (LPS). We have studied experimentally the role of LPS on the stability and function of this toxin. The HlyA conformation in both, LPS-free and LPS-bound forms was investigated by tryptophan fluorescence. Studies about HlyA thermal and chemical denaturation indicated that its stability increased in the presence of LPS. On the other hand, the presence of negative and polar residues on the LPS reduced the tendency of HlyA to self-aggregation, and they may be the reservoir of calcium, cation essential for the lytic action of this toxin on red blood cells. These results suggest that HlyA and LPS are combined mainly via hydrophobic force to form an active toxin which stability is favored by the LPS.

Acyl chains are responsible for the irreversibility in the Escherichia coli alpha-hemolysin binding to membranes.

Hemolysin (HlyA) is an extracellular protein secreted by uropathogenic strains of Escherichia coli. The mature HlyA is able to bind to mammalian target cell membranes including those of the immune system, causing lysis. The lytic activity is absolutely dependent upon the Hlyc-dependent acylation of Prohemolysin. In this paper we show, through Trp fluorescence studies and denaturation in Guanidine hydrochloride, that the acylation is responsible for the loose conformation of the active protein, necessary to transform it from soluble to membrane-bound form. Previous studies showed that toxin binding to the bilayers occurs in, at least two ways, a reversible adsorption and irreversible insertion. We demonstrated that the irreversibility is due to the acyl chains in the HlyA, as shown by the protein transfer from multilamellar liposomes composed of palmitoyl-oleoyl-phosphatidylcholine (POPC) to large unilamellar vesicles containing POPC-doxyl as protein fluorescence quencher.

Development and in vitro characterisation of a benznidazole liposomal formulation.

The purpose of this study was to find a multilamellar liposomal formulation for the antichagasic drug Benznidazole (BNZ). Different lipid matrices and organic solvents for BNZ were tested in order to obtain the liposomes with the highest g BNZ/100 g total lipid (D/TL) ratio. The best lipid matrices resulted from hydrogenated phosphatidylcholine from soybean (HSPC): Cholesterol (Chol): distearoyl-phosphatidylglycerol (DSPG) (molar ratio 2:2:1) prepared with BNZ dissolved in DMSO. Drug loading of 2 g BNZ/100 g total lipids at a total lipid concentration of 20-30 mM was obtained. Two in vitro assays on the HSPC:Chol:DSPG formulation to predict its in vivo behaviour were performed. In the first experiments, after 60 min at 1-450-fold dilution in buffer at 37 degrees C, the amount of drug associated to liposomes was reduced from 2 to 0.25 g BNZ/100 g total lipids at a rate of 65% (drug lost) min(-1) at the first minute followed by 0.4% (drug lost) min(-1) during the next hour. When incubated in plasma at 37 degrees C, the HSPC:Chol:DSPG formulations bounded a high amount of plasma proteins: r=2400 microg plasma protein per micromol total lipid.

Effect of vanadium compounds on the lipid organization of liposomes and cell membranes.

The influence of vanadate on the adsorption properties of Merocyanine 540 (MC540) to UMR cells was studied by means of specrofluorometry. An increment in the fluorescence was observed in the osteoblasts incubated with 0.1 mM vanadate. This effect could be interpreted in terms of vanadate inhibitory effects on aminotraslocase activity. However, vanadate promotes a similar behavior to that found in UMR 106 cells when it was added to lipid vesicles composed of phosphatidylcholine. The effect of vanadium in different oxidation states, such as vanadate(V) and vanadyl(IV) on lipid membrane properties was examined in large unilamellar vesicles by means of spectrofluorometry employing different probes. Merocyanine 540 and 1,6-diphenylhexatriene were used in order to sense the changes at interfacial and hydrophobic core of membranes, respectively. In contrast to vanadate, vanadyl decreased the fluorescence of MC540. Both vanadium compounds slightly perturbed the hydrocarbon core. The results can be interpreted by the specific adsorption of both compounds on the polar head groups of phospholipid and suggest a possible influence of vanadium compounds on the lipid organization of cell membranes.

The European Intra-Inter Theater Aeromedical Evacuation System:--our mission--our attempt.

The logistics of aeromedical evacuation are discussed. The order of magnitude of the problem and detailed analyses are presented. Examples are presented for patient transfer from point of injury in areas remote from hospitalization through successive installations to the hospital for extended treatment and convalescence.

[Lipid vectors. New strategies for gene therapy]. / Vectores lipidicos. Nuevas estrategias aplicadas a la terapia génica.

Phospholipids are capable of spontaneous self-assembling, a remarkable differential property if compared with the rest of biological molecules. By their means it is relatively easy to generate extremely stable sealed structures, with controlled shape, size and packing, known as liposomes. In this article, we review the use of liposomes to improve the transfection process in eucaryotic cells, in vitro as well as in vivo. By employing lipid vectors, it is feasible to selectively transport a DNA segment to any target of the body, to force it to enter a cell and once inside it, to exert a control on its ultimate intracellular fate. The goal of lipid vectors to successfully transfect a cell in vivo, lies on the provision of a mechanical protection for DNA against plasma degradation, together with the possibility of controlling DNA biodistribution, independently of its size and sequence. Moreover, lipid vectors are not carcinogenic and are poorly immunogenic. Current challenge in lipid synthesis allows for a vector design which should be efficient enough to compete with high transfection levels of a viral vector, but with the extreme versatility, simplicity and biosafety characteristic of self assembling molecules.

Ca2+ action on the stability of egg phosphatidylcholine sonicated vesicles during freeze-thaw cycles.

The stability of unilamellar vesicles during freeze-thaw cycles strongly depends on the Ca2+ concentration in the aqueous solution. Experiments performed at equal ionic strengths with Na+ and Ca2+ solutions indicate that the effect observed is specific for Ca2+. This is interpreted to be a consequence of the adsorption of Ca2+ on the vesicle bilayers. The variation of lipid and Ca2+ concentrations indicates that stability is achieved at a particular Ca2+/lipid ratio of 8 mol/mol above which vesicles are stable. The stability appears to be mainly conferred by the external Ca2+ in both slow and rapid cycles, independent of the ionic vesicle content. However, internal Ca2+ seems to increase the stability according to the F/T cycle rate to some extent in the absence of Ca2+ in the external solution.

Effect of Ca2+ on the cryoprotective action of trehalose.

The competitive effect of Ca2+ on the cryoprotective action of carbohydrates has been investigated during freeze-thaw processes of unilamellar egg phosphatidylcholine vesicles. Ca2+ inhibits the cryoprotection achieved by trehalose to a greater extent than other sugars such as galactose, sucrose, and fructose. The cryoprotection by trehalose is also dependent on the Ca2+ concentration in the inside solution of the vesicle, even in the absence of external Ca2+. The competitive effect of Ca2+/trehalose is interpreted as a consequence of the different amount of interfacial water displaced by each compound in their adsorption on the water/lipid interface.

Balance of electrostatic and hydrophobic interactions in the lysis of model membranes by E. coli alpha-haemolysin.

The relative weight of electrostatic interactions and hydrophobic forces in the process of membrane disruption caused by E. coli alpha-haemolysin (HlyA) has been studied with a purified protein preparation and a model system consisting of large unilamellar vesicles loaded with water-soluble fluorescent probes. Vesicles were prepared in buffers of different ionic strengths, or pHs, and the net surface charge of the bilayers was also modified by addition of negatively (e.g., phosphatidylinositol) or positively (e.g., stearylamine) charged lipids. The results can be interpreted in terms of a multiple equilibrium in which alpha-haemolysin may exist: aggregated HlyA <==> monomeric HlyA <==> membrane-bound HlyA. In these equilibria both electrostatic and hydrophobic forces are significant. Electrostatic forces become substantial under certain circumstances, e.g., membrane binding when bilayer and protein have opposite electric charges. Protein adsorption to the bilayer is more sensitive to electrostatic forces than membrane disruption itself. In the latter case, the irreversible nature of protein insertion may overcome electrostatic repulsions. Also of interest is the complex effect of pH on the degree of aggregation of an amphipathic toxin like alpha-haemolysin, since pH changes are not only influencing the net protein charge but may also be inducing protein conformational transitions shown by changes in the protein intrinsic fluorescence and in its susceptibility to protease digestion, that appear to regulate the presence of hydrophobic patches at the surface of the molecule, thus modifying the ability of the toxin to either aggregate or become inserted in membranes.

Reversible adsorption and nonreversible insertion of Escherichia coli alpha-hemolysin into lipid bilayers.

Alpha-Hemolysin is an extracellular protein toxin (107 kDa) produced by some pathogenic strains of Escherichia coli. Although stable in aqueous medium, it can bind to lipid bilayers and produce membrane disruption in model and cell membranes. Previous studies had shown that toxin binding to the bilayer did not always lead to membrane lysis. In this paper, we find that alpha-hemolysin may bind the membranes in at least two ways, a reversible adsorption and an irreversible insertion. Reversibility is detected by the ability of liposome-bound toxin to induce hemolysis of added horse erythrocytes; insertion is accompanied by an increase in the protein intrinsic fluorescence. Toxin insertion does not necessarily lead to membrane lysis. Studies of alpha-hemolysin insertion into bilayers formed from a variety of single phospholipids, or binary mixtures of phospholipids, or of phospholipid and cholesterol, reveal that irreversible insertion is favored by fluid over gel states, by low over high cholesterol concentrations, by disordered liquid phases over gel or ordered liquid phases, and by gel over ordered liquid phases. These results are relevant to the mechanism of action of alpha-hemolysin and provide new insights into the membrane insertion of large proteins.