High speed atomic force microscopy to investigate the interactions between toxic Aß1-42 peptides and model membranes in real time: impact of the membrane composition.

Due to an aging population, neurodegenerative diseases have become a major health issue, the most common being Alzheimer's disease. The mechanisms leading to neuronal loss still remain unclear but recent studies suggest that soluble Aß oligomers have deleterious effects on neuronal membranes. Here, high-speed atomic force microscopy was used to assess the effect of oligomeric species of a variant of Aß1-42 amyloid peptide on model membranes with various lipid compositions. Results showed that the peptide does not interact with membranes composed of phosphatidylcholine and sphingomyelin. Ganglioside GM1, but not cholesterol, is required for the peptide to interact with the membrane. Interestingly, when they are both present, a fast disruption of the membrane was observed. It suggests that the presence of ganglioside GM1 and cholesterol in membranes promotes the interaction of the oligomeric Aß1-42 peptide with the membrane. This interaction leads to the membrane's destruction in a few seconds. This study highlights the power of high-speed atomic force microscopy to explore lipid-protein interactions with high spatio-temporal resolution.

Interaction of Aß1-42 peptide or their variant with model membrane of different composition probed by infrared nanospectroscopy.

Toxicity of Aß peptides involved in Alzheimer's disease is linked to the interaction of intermediate species with membranes. Nanoscale Infrared Spectroscopy enhances the study of the morphology and the secondary structure of the peptides as fibers or oligomers interacting with membranes of different compositions, with nanometer scale resolution. Membrane models are used to investigate the role of different lipids in their interactions with Aß peptides. This work clearly brings to light that the presence of cholesterol in membranes is favorable to the interaction with Aß peptides in oligomers or aggregates.

Cation diffusion selectivity in a pore model. The phosphatidylcholine/water lamellar phase.

The diffusion coefficients D (cm2/s), of four monovalent cations K+, Na+, Rb+ and Cs+ and of Ca2+ have been measured in phosphatidylcholine/water lamellar phase as a function of phase hydration and temperature and in the presence of divalent cations. Diffusion rates vary strongly with phase hydration, between 10(-7) and 10(-6) cm2/s for monovalent and 10(-8) and 10(-7) for Ca2+. The activation energies obtained are relatively small (5--10 kcal/mol). As the phase water content increases, a series of diffusion sequences is obtained, corresponding to the sequences predicted by Eisenman's theory of alkali ion equilibrium selectivity. This diffusional selectivity, which depends exclusively upon non-equilibrium parameters (mobility) within the hydrophilic path is discussed in respect to current theories of pore selectivity.

Interaction of 14C-labelled amphotericin B derivatives with human erythrocytes: relationship between binding and induced K+ leak.

Four 14C-labelled amphotericin B (Am B) derivatives with different net electric charges were examined: zwitterionic N-fructosyl Am B, positively charged N-fructosyl Am B methyl ester, negatively charged N-acetyl Am B and neutral N-acetyl Am B methyl ester. The binding of these four derivatives to human red cells and their octanol-water partition coefficients were measured. Simple partitioning between red cells and buffer was found for the four compounds, regardless of concentration, within a range of 10(-8) and 10(-4) M. This indicates the absence of cooperativity and saturability of binding at least in this concentration range. The constant partition coefficients were found to be three to five times higher for the two methyl ester derivatives than for the two non-esterified compounds. All partition coefficients were proportional to those found for the octanol-water system. Efficiency in inducing K+ leak from red cells was measured during the binding experiments. Despite the higher partition coefficients of the two methyl ester derivatives, they were found to have much lower ionophoric efficiency than the two non-esterified compounds. These results are discussed in terms of the mechanism of permeability pathway formation by polyene antibiotics.

Amphotericin B-sterol complex formation and competition with egg phosphatidylcholine: a monolayer study.

The conditions of formation of amphotericin B-cholesterol or -ergosterol complexes in monolayers are investigated by the penetration into a monolayer of egg phosphatidylcholine/sterol of 14C-labelled N-fructosyl-amphotericin B dissolved in the aqueous subphase. An increase of both surface pressure and radioactivity as a function of concentration are observed simultaneously while a 'saturation' effect occurs only for the surface pressure. The experiments are not accurate enough to make conclusions about the number of actually penetrated amphotericin B molecules. Therefore, the existence of an amphotericin B-sterol complex was evidenced from a study of surface pressure area per molecule isotherm. The results indicate that a complex with a 2:1 stoichiometry is formed and that the amphotericin B-ergosterol interaction is larger than the amphotericin B-cholesterol interaction. The complex is dissociated by addition of egg phosphatidylcholine due to a competition between egg phosphatidylcholine and amphotericin B for sterol.

Kinetic study of interaction between [14C]amphotericin B derivatives and human erythrocytes: relationship between binding and induced K+ leak.

The relationship between polyene antibiotic binding to red cells and their membrane permeabilization was studied using two 14C-labelled amphotericin B (AmB) derivatives: N-fructosyl AmB and N-acetyl methyl ester AmB. The binding kinetics of both derivatives were determined on whole red cells and ghosts. The resulting experimental points were well fitted by monoexponential functions, and the characteristic t1/2 for both derivatives were calculated from these functions. At 2 X 10(-5) M, the half time t1/2 for N-acetyl methyl ester AmB (30.2 min) which forms aqueous aggregates was longer than the t1/2 for the more soluble species N-fructosyl AmB (4.5 min). At lower concentrations (10(-7) M), the t1/2 for N-acetyl methyl ester AmB (6.3 min) in a more solubilized form was close to that of N-fructosyl AmB (7.9 min). These results suggest that only solubilized species bound to red cell membranes and that disaggregation of aggregates is the limiting step in the binding process. The permeabilization of red cell membranes by N-fructosyl AmB, measured as intracellular K+ leak, was not instantaneous and at 10 degrees C external K+ was only detected 20 min after antibiotic addition. In contrast, binding occurs without lag time. Consequently, different mecanisms underlie binding and K+ permeability inducement. Absorption spectroscopy data showed that bound antibiotic is located in the hydrophobic membrane interior and that this penetration of the membrane by AmB derivatives occurs without lag time. Consequently, the lag time occurring for K+ permeability inducement would be due to some steps subsequent to binding and probably located in the hydrophobic membrane interior. This statement is further supported by the observation that the lag time is sensitive to changes in membrane fluidity as shown here by the break between 20 and 30 degrees C in the slope of the Arrhenius plot for the lag time, coinciding with the phase transition in red cell membranes.

Anion diffusion selectivity in a pore model. The phosphatidylcholine-water lamellar phase.

The diffusion coefficients D(cm2.s-1) of the sodium salts of a series of hydrophilic mono- and dicarboxylic acids, have been measured in the hydrophilic layers of phosphatidylcholine-water lamellar phases, as a function of phase hydration. At pH 9.0, the diffusion rates of the anionic (RCOO-) form of the acid exhibit a prominent increase within a narrow range of water content, specific to each anion. This high diffusion rate seems to occur when the Stokes diameter of an anion is equal to the thickness of the aqueous layer between the two planes formed by the quaternary ammonium groups of the choline phosphate dipoles of two facing layers of phosphatidylcholine molecules. This phenomenon demonstrates the importance of the spatial organization of successive binding sites in the rate constant of diffusional processes in hydrophilic channels.

Nonsolvent water in human erythrocytes and hemoglobin solutions.

Distribution ratios of water-soluble nonelectrolytes have been measured for two systems, human red bood cells/isotonic saline and hemoglobin solutions/water. The results show that for these solutes there is a significant amount of nonsolvent water associated with Hb. However, the amount of this nonsolvent water depends markedly on the temperature, as well as on the size, steric configuration, and functional groups of the probe solutes. The significance of the data is discussed.

Properties of hemoglobin solutions in red cells.

The present studies are concerned with a detailed examination of the apparent anomalous osmotic behavior of human red cells. Red cell water has been shown to behave simultaneously as solvent water for nonelectrolytes and nonsolvent water, in part, for electrolytes. The nonsolvent properties are based upon assumptions inherent in the conventional van't Hoff equation. However, calculations according to the van't Hoff equation give osmotic volumes considerably in excess of total cell water when the pH is lowered beyond the isoelectric point for hemoglobin; hence the van't Hoff equation is inapplicable for the measurement of the solvent properties of the red cell. Furthermore, in vitro measurements of osmotic and other properties of 3.7 millimolal solutions of hemoglobin have failed to reveal the presence of any salt exclusion. A new hypothesis has been developed from thermodynamic principles alone, which predicts that, at constant pH, the net charge on the hemoglobin molecule decreases with increased hemoglobin concentration. The existence of such cooperative interaction may be inferred from the effect of pH on the changes in hemoglobin net charge as the spacing between the molecules decreases. The resultant movement of counterions across the cell membrane causes the apparent anomalous osmotic behavior. Quantitative agreement has been found between the anion shift predicted by the equation and that observed in response to osmotic gradients. The proposed mechanism appears to be operative in a variety of tissues and could provide an electrical transducer for osmotic signals.

Effect of geometrical and chemical constraints on water flux across artificial membranes.

Studies have been made on the temperature dependence of both the hydraulic conductivity, L(p), and the THO diffusion coefficient, omega, for a series of cellulose acetate membranes (CA) of varying porosity. A similar study was also made of a much less polar cellulose triacetate membrane (CTA). The apparent activation energies, E(a), for diffusion across CA membranes vary with porosity, being 7.8 kcal/mole for the nonporous membrane and 5.5 kcal/mole for the most porous one. E(a) for diffusion across the less polar CTA membrane is smaller than E(a) for the CA membrane of equivalent porosity. Classical viscous flow, in which the hydraulic conductivity is inversely related to bulk water viscosity, has been demonstrated across membranes with very small equivalent pores. Water-membrane interactions, which depend upon both chemical and geometrical factors are of particular importance in diffusion. The implication of these findings for the interpretation of water permeability experiments across biological membranes is discussed.

Hemoglobin charge dependence on hemoglobin concentration in vitro.

Studies have been made of the dependence of the charge of the hemoglobin molecule on hemoglobin concentration in the concentration range between 3 and 11 mmolal. The charge has been determined by measuring the distribution of (42)K between a hemoglobin solution in a cellophane bag and an external solution. The pH was 6.6, the K concentration was 10 mM, and the temperature was 4 degrees C. The charge decreased along a sigmoid curve from a value of +3 in the most dilute solutions to a value of +0.5 in the most concentrated solutions. The results were in excellent agreement with earlier studies of Gary-Bobo and Solomon in which Cl distribution was measured between human red cells and external solutions and thus give added support to the conclusion that the apparent anomalous osmotic behavior of human red cells may be attributed to concentration-dependent changes in the hemoglobin net charge. The present findings also support the view that the water in the red cell is solvent water for K and Cl and differs in no quantitatively important respect from bulk water in free solution.

Role of hydrogen-bonding in nonelectrolyte diffusion through dense artificial membranes.

The diffusion of two series of alcohols and amides through complex cellulose acetate membranes was studied. The thin dense part of these membranes behaves as a nonporous layer of low water content. In this layer, called the skin, the solute diffusion coefficients, omega, depend upon size, steric configuration, and the partition coefficient, K(8), between membrane and bathing solution. From the experimental values of omega and K(8), the over-all friction, f, experienced by the solutes in the membrane was computed. It was found that f depends upon the chemical nature of the solute and is related to hydrogen-bonding ability. In the coarse, porous layer of the cellulose acetate membrane, diffusion occurs mainly through aqueous channels. In this instance also the hydrogen-bonding ability of the solute seems to exercise a smaller but significant influence.

Permeability of red cell membranes to small hydrophilic and lipophilic solutes.

The permeability coefficients of a series of amides, ureas, and diols have been measured on red cells of man and dog using the minimum volume method of Sha'afi et al. When the molecules are grouped according to their ether-water partition coefficients, k(ether), the behavior of the hydrophilic molecules, with k(ether) less than water, is different from that of the lipophilic molecules, characterized by k(ether) greater than water. The rate of permeation of the hydrophilic molecules through an aqueous pathway is determined by the molar volume, a parameter in which the geometrical measure of molecular volume is modified by hydrogen-bonding ability. This indicates the importance of chemical interactions within the aqueous path. The permeation of the lipophilic molecules is determined in the first instance by k(ether), taken as a measure of the ease with which the molecule can escape from its aqueous environment. Within the membrane, lipophilic permeability is modified both by steric factors and by the formation of hydrogen bonds with membrane components. These data allow one to infer that lipid-soluble molecules travel through an organized structure within the lipid membrane and come into contact with polar moieties.

The possible proton translocating activity of the mitochondrial uncoupling protein of brown adipose tissue. Reconstitution studies in liposomes.

Loose coupling of thermogenic mitochondria of brown adipose tissue is related to a high proton (or hydroxyl) conductance of the inner membrane and to the presence of a unique 32 kDa uncoupling protein. Reconstitution experiments of the purified protein in liposomes are reported which suggest that this component could form proton channels in the membrane.

Polyene--sterol interaction and selective toxicity.

From permeability experiments carried out with series of amphotericin B derivatives in both biological and model membranes, it was concluded that derivatives, whose carboxyl group at the C18 position is blocked by substitution, are much more efficient at inducing permeability in ergosterol-containing than in cholesterol-containing membranes, whereas derivatives whose carboxyl group is free and ionizable are equally efficient in both membranes types. Binding measurements on erythrocyte membranes showed that all amphotericin B derivatives simply partition between membrane lipids and aqueous medium, according to their lipid solubility. There is no relationship between binding and efficiency in inducing permeability. Permeability studies carried out on lipidic vesicles containing various sterols showed that: 1) derivatives having their carboxyl free induced permeability of the 'channel' type, regardless of the sterol present, and no detectable permeability in sterol-free membranes; 2) derivatives whose carboxyl group is blocked induce channels only in membranes containing ergosterol or sterols having an alkyl side chain identical to that of ergosterol. In the presence of other sterols or in sterol-free membranes, their ionophoric activity is poor and always of the 'mobile-carrier' type. A model of polyene-sterol interaction is proposed, accounting for the data obtained with both biological and model membranes.

A randomized study of etoposide and carboplatin with or without paclitaxel in the treatment of small cell lung cancer.

Small cell lung cancer accounts for 20% to 25% of all lung cancer cases and is initially responsive to combination chemotherapy. However, the majority of patients relapse, and at that point their disease is highly resistant to chemotherapy. The combination of etoposide with either cisplatin or carboplatin is regarded as the standard of care for these patients. Previous studies have documented the activity of paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ) at doses of 135 to 250 mg/m2 administered over 1, 3, or 24 hours as either a single agent or in combination with etoposide and a platinum compound. Studies adding paclitaxel to etoposide/carboplatin (EP) have demonstrated complete responses in both limited and extensive disease, but all have been in single-arm phase II studies. Preliminary data also suggest the possibility of a dose-response curve for the combination. We recently began a randomized phase II/III comparison of the standard EP to EP plus paclitaxel for newly diagnosed patients with limited or extensive small cell lung cancer. Carboplatin in this study is dosed according to area under the concentration-time curve as calculated by the Calvert formula. The study compares EP (carboplatin area under the concentration-time curve of 6 intravenously [IV] over 30 to 60 minutes on day 1, with etoposide 120 mg/m2 IV days 1 to 3) versus EP plus paclitaxel (paclitaxel 200 mg/m2 IV 1-hour infusion on day 1, carboplatin area under the concentration-time curve of 6 IV over 30 to 60 minutes on day 1, and etoposide 50/100 mg orally on alternating days 1 to 10). The design, inclusion criteria, and staging of patients in this study will be presented with initial accrual and patient characteristics. Randomized studies of this type are essential if the true role of this new combination is to be fully evaluated.

Relationship between ionophoric and haemolytic activities of perimycin A and vacidin A, two polyene macrolide antifungal antibiotics.

The ionophoric and hemolytic activities of two antifungal aromatic heptaenes: vacidin A and perimycin A, were studied on human red blood cells. Measurements of hemolysis, K+ influx and efflux, H+ movement and potential difference across the cell membrane, show that the hemolytic activity, being related to the K+ permeability induced by the polyene, is strongly dependent on the ability of this polyene to induce H+ movement. It was shown that: (1) both antibiotics have approximately the same efficiency in inducing K+ permeability, but a 100-fold difference in their hemolytic activity; (2) their hemolytic activity is related to their ability to induce H+ movement; (3) the protonophoric activity requires the existence of a free carboxyl group in the macrolide ring, as in vacidin A. The hemolytic activity is determined by the intrinsic efficiency of a K+/H+ exchange induced by this polyene. With perimycin A, which lacks the free carboxyl group, the hemolytic activity is dependent on the Cl- conductive flux which slows down the K+ flux.

Haemolytic activity of aromatic heptaenes. A group of polyene macrolide antifungal antibiotics.

The aromatic heptaene vacidin A induces ion selective channels in human red blood cells. The ion flux induced leads to a secondary effect--colloid osmotic haemolysis. Molecular variations at ionizable polar groups of the antibiotic modify the properties of the permeability pathway concerning intercationic selectivity and the symmetry of ion flux.

Myopic photorefractive keratectomy in eyes with atypical inferior corneal steepening.

PURPOSE: To evaluate the visual and refractive results of photorefractive keratectomy (PRK) in eyes with atypical inferior corneal steepening (AICS). SETTING: Department of Ophthalmology, Hôpital Morvan, University of Breast, France. METHODS: Using videokeratopography, we screened 310 eyes that had PRK from November 1992 through November 1993 and found that 35 eyes exhibited topographic patterns consistent with AICS with no clinical findings. The results at 6 months and 1 year were compared with those of 185 eyes with normal topography treated concurrently. RESULTS: There were no statistically significant differences between the two groups in mean spherical equivalent, mean uncorrected visual acuity, and mean best spectacle-corrected visual acuity 6 months and 1 year after PRK. CONCLUSION: After 1 year, PRK in eyes with AICS appeared to give results similar to those in eyes with normal topography. Further follow-up is needed.