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.

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.

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.

[Amebic keratitis]. / Les kératites amibiennes.

Amoebic keratitis is an uncommon pathology mainly implicated in eye infection in contact lens wearers. The offending parasite is a free-living amoeba of the genus Acanthamoeba. Diagnosis is often delayed because clinical signs are confusing especially at the onset. Samples for parasitic tests should be taken not only for the infection site but also from the lenses and cleaning products. A standard treatment of amoebic keratitis has not been established. Several drugs are theoretically active but achieve inconsistent results. Recently keratitis has been observed at a seemingly high incidence in developing countries but epidemiologic data is still scarce.

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.

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.

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.

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.

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.

Effect of the polar head structure of polyene macrolide antifungal antibiotics on the mode of permeabilization of ergosterol- and cholesterol-containing lipidic vesicles studied by 31P-NMR.

Natural polyene macrolide antibiotics and their N-acyl and methyl ester derivatives, which differ mainly in their electric net charge, were compared for their ability to increase the ionic permeability of large unilamella vesicles, using the proton-cation exchange method and 31P-NMR spectroscopy. The zwitterionic (amphotericin B, vacidin A) and negatively charged (N-N'-diacetyl vacidin) compounds induced permeability according to an all-or-none process on both cholesterol- and ergosterol-containing membranes. The same mechanism of permeability induction is obtained only on ergosterol-containing vesicles for positively charged antibiotics (perimycin A, vacidin A methyl ester, amphotericin B methyl ester). A different type of action is observed for the latter group of ionophores in cholesterol-containing vesicles. In this case, a progressive proton efflux occurs in which all of the vesicle population is involved. This qualitative difference in the kinetics of ionic fluxes induced by antibiotics without a free carboxyl group in cholesterol-containing as compared to ergosterol-containing membranes was ascribed to differences in polyene-sterol interactions as well as in the life time of the ionic path formed. This difference may provide a basis for the improvement of selective toxicity of this group of antifungal agents by rational modifications.

Transfer of amphotericin B from gel state vesicles to mycoplasma cells: biphasic action on potassium transport and permeability.

The action of amphotericin B on the K+ permeability of Mycoplasma mycoides var. capri cells, containing either cholesterol or ergosterol in their membranes, was studied. When the drug, solubilized in dimethyl sulfoxide, was added directly to the cell suspension, a slightly greater sensitivity to permeabilization was observed for ergosterol-containing cells, confirming the data reported in the literature. When amphotericin B bound to gel state phospholipid vesicles was added to the cell suspension, two effects on cholesterol-containing cells were observed. First, the K+ active transport rates increased; membrane permeabilization and K+ leakage were subsequently detected. For ergosterol-containing cells these sequential events were observed only at amphotericin B concentrations below 10(-6) M. At higher concentrations only K+ leakage was observed. The second permeabilization effect varied with the amphotericin B concentration in different ways in the two types of cells. The permeabilization of ergosterol-containing membranes depended on the amphotericin B/phospholipid molar ratio, whereas the permeabilization of cholesterol-containing membranes did not. In general, the latter remained fairly constant when the total amphotericin B concentration in the medium varied.

Enhancement of amphotericin B selectivity by antibiotic incorporation into gel state vesicles. A circular dichroism and permeability study.

The permeability induced in cholesterol-or ergosterol-containing phospholipid vesicles by Amphotericin B incorporated into Dipalmitoyl Phosphatidylcholine vesicles has been studied in parallel with Circular Dichroism spectroscopy measurements. In our conditions, Amphotericin B is 5 to 10 times more selective to ergosterol- than to cholesterol-containing vesicles. Such a large difference is not observed when Amphotericin B is directly added to the vesicles suspension as its solution in organic solvent.

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.

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.

Cation permeability induced by two 15-O5 macrocyclic polyether carriers in phospholipidic large unilamellar vesicles.

Sodium permeability in phospholipidic large unilamellar vesicles induced by two 15-O5 macrocyclic polyether derivatives, a carboxylic one 1a and a neutral one 1b, has been studied. The neutral derivative, 1b, appears to behave as a classical mobile carrier such as valinomycin and the carboxylic carrier, 1a, as an ionophore of the nigericin group.

The influence of electric charge of aromatic heptaene macrolide antibiotics on their activity on biological and lipidic model membranes.

Natural aromatic heptaene macrolide antibiotics and their N-acyl and methyl ester derivatives, which differ mainly in their electric net charge, were compared for their efficiency in inducing yeast growth inhibition, red blood cell lysis, and increase in the ionic permeability of large unilamellar lipidic vesicles. Antifungal activity was found to decrease in the following order: neutral congruent to positively charged greater than negatively charged compounds. Hemolytic activity decreased in the order: neutral greater than negatively charged much greater than positively charged compounds. On lipidic model membranes, themselves either positively or negatively charged, electrostatic interaction was shown to have practically no influence on the efficiency of the differently charged antibiotics. On both biological and model systems, positively charged antibiotics consistently were found to be more active on ergosterol-containing than on cholesterol-containing membranes, and were therefore considered as potentially good candidates for specific antifungal agents.