Electrical resonance of Amphotericin B channel activity in lipidic membranes.

In our previous work [J. Membrane Biol. 237, 31 (2010)], we showed the dependence of the time average conductance of Nystatin channels as a function of the applied potential. Specifically, it was observed that greater potential induced enhanced channel activity. This indicates that the supramolecular structure could be stabilized by a large field, possibly by giving a preferential orientation to the monomers. In the present work, we entertain the notion that the process of pore formation in the lipidic membranes has an underlying deterministic component. To verify this hypothesis, experiments were performed under potentio-dynamic conditions, i.e., a square train of pulses of different frequencies (0.05-2 Hz) were applied to a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membrane having 30 mol. % cholesterol and the presence of 35 µM Amphotericin B. An emergence of a resonant frequency, in the present experiments, is tantamount to observing fingerprints of determinism in the activity of these channels in lipidic membranes.

Cell penetrating peptides and cationic antibacterial peptides: two sides of the same coin.

Cell penetrating peptides (CPP) and cationic antibacterial peptides (CAP) have similar physicochemical properties and yet it is not understood how such similar peptides display different activities. To address this question, we used Iztli peptide 1 (IP-1) because it has both CPP and CAP activities. Combining experimental and computational modeling of the internalization of IP-1, we show it is not internalized by receptor-mediated endocytosis, yet it permeates into many different cell types, including fungi and human cells. We also show that IP-1 makes pores in the presence of high electrical potential at the membrane, such as those found in bacteria and mitochondria. These results provide the basis to understand the functional redundancy of CPPs and CAPs.

X-ray accelerated photo-oxidation of As(III) in solution.

We performed near edge X-ray absorption spectroscopy (XANES) measurements on the arsenic K-edge of As(III) in solution under acidic and basic conditions, after exposure of the solutions to air. Spectra were recorded for increasing exposure times to the X-rays used to perform absorption spectroscopy measurements. We did not find changes for the solution under acidic conditions, whereas we observed significant changes in the case of solution under alkaline conditions. To interpret these changes, we compared the obtained spectra with XANES spectra of As(III) and As(V) solutions under alkaline conditions, not exposed to air, and used as standards. Principal component fits using these standards indicate an accelerated conversion of As(III) to As(V) due to the exposure to X-rays.

Interpretation of X-ray absorption spectra of As(III) in solution using Monte Carlo simulations.

We performed X-ray absorption spectroscopy measurements on the arsenic K-edge of As(III) in solution under acidic conditions. Extended X-ray absorption fine structure (EXAFS) and X-ray near edge structure (XANES) spectra were compared with theoretical calculations which use local atomic structure configurations, either derived from density functional theory (DFT) energy minimization (EM) calculations or based on classical Monte Carlo (MC) simulations, for a As(OH)3 cluster surrounded by water molecules. The nearest arsenic-oxygen distances obtained from the fit of the XAFS spectra are consistent with the distances present in configurations derived from Monte Carlo simulations but not with those obtained from DFT-EM calculations. Calculations of XANES using either DFT-EM or the average configuration obtained from MC simulations do not reproduce the XANES spectra in the vicinity of the absorption edge. However, specific local atomic structural configurations of the As(OH)3 and water molecules, obtained from MC simulations, which show some ordering of water molecules up to 5 Å from the arsenic, reproduce qualitatively the experimental spectra. These results highlight the capability of XANES to yield information about hydration of ions in solution.

Notable enhancement of Amphotericin B channel activity by applied pressures in the range of MS channel activation.

The mechanism of Amphotericin B at the membrane is still subject of debate, with the prevailing hypothesis being the formation of pores. The activity of these pores is influenced by various factors. Recently aggregation in solution and insertion in the membrane had been highlighted as crucial for action of the drug Here we investigated the effect of applied pressure on the activity of Amphotericin B. Our findings demonstrate that applied pressure of 50 mmHg is sufficient to enhance the activity. We interpreted the results as supporting the idea that pressure fractures the membrane and promotes the insertion of the polyene.

A Promising Amphotericin B Derivative Induces Morphological Alterations, Mitochondrial Damage, and Oxidative Stress In Vitro and Prevents Mice from Death Produced by a Virulent Strain of Trypanosoma cruzi.

Chagas Disease is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi, affecting 6-8 million people, mainly in Latin America. The medical treatment is based on two compounds, benznidazole and nifurtimox, with limited effectiveness and that produce severe side effects; consequently, there is an urgent need to develop new, safe, and effective drugs. Amphotericin B is the most potent antimycotic known to date. A21 is a derivative of this compound with the property of binding to ergosterol present in cell membranes of some organisms. In the search for a new therapeutic drug against T. cruzi, the objective of this work was to study the in vitro and in vivo effects of A21 derivative on T. cruzi. Our results show that the A21 increased the reactive oxygen species and reduced the mitochondrial membrane potential, affecting the morphology, metabolism, and cell membrane permeability of T. cruzi in vitro. Even more important was finding that in an in vivo murine model of infection, A21 in combination with benznidazole was able to reduce blood parasitemia, diminish the immune inflammatory infiltrate in skeletal muscle and rescue all the mice from death due to a virulent T. cruzi strain.

Polyene Antibiotics Physical Chemistry and Their Effect on Lipid Membranes; Impacting Biological Processes and Medical Applications.

This review examined a collection of studies regarding the molecular properties of some polyene antibiotic molecules as well as their properties in solution and in particular environmental conditions. We also looked into the proposed mechanism of action of polyenes, where membrane properties play a crucial role. Given the interest in polyene antibiotics as therapeutic agents, we looked into alternative ways of reducing their collateral toxicity, including semi-synthesis of derivatives and new formulations. We follow with studies on the role of membrane structure and, finally, recent developments regarding the most important clinical applications of these compounds.

Formation and Nanoscale Characterization of Asymmetric Supported Lipid Bilayers Containing Raft-Like Domains.

The development of new strategies for achieving stable asymmetric membrane models has turned interleaflet lipid asymmetry into a topic of major interest. Cyclodextrin-mediated lipid exchange constitutes a simple and versatile method for preparing asymmetric membrane models without the need for sophisticated equipment. Here we describe a protocol for preparing asymmetric supported lipid bilayers mimicking membrane rafts by cyclodextrin-mediated lipid exchange and the main guidelines for obtaining structural information and quantitative measures of their mechanical properties using Atomic force microscopy and Force spectroscopy; two powerful techniques that allow membrane characterization at the nanoscale.

A refined potential for hydroxylamine clusters and the liquid phase.

A detailed study including ab initio calculations and classic Monte-Carlo simulations of hydroxylamine in the gas and liquid phases is presented. A classical interaction potential for hydroxylamine, which includes polarizability, many-body effects, and intramolecular relaxation, was constructed. The results of the simulation were compared to the available experimental data in order to validate the model. We conclude that liquid hydroxylamine has a multitude of hydrogen bonds leading to a large density where the existence of cis conformers and clusters of these conformers is possible. This explains the occurrence of the classical [R. Nast and I. Z. Foppl, Z. Anorg. Allg. Chem. 263, 310 (1950)] scheme for the molecule's decomposition at room temperature and its large exothermicity and instability.

Asymmetric bilayers mimicking membrane rafts prepared by lipid exchange: Nanoscale characterization using AFM-Force spectroscopy.

Sphingolipids-enriched rafts domains are proposed to occur in plasma membranes and to mediate important cellular functions. Notwithstanding, the asymmetric transbilayer distribution of phospholipids that exists in the membrane confers the two leaflets different potentials to form lateral domains as next to no sphingolipids are present in the inner leaflet. How the physical properties of one leaflet can influence the properties of the other and its importance on signal transduction across the membrane are questions still unresolved. In this work, we combined AFM imaging and Force spectroscopy measurements to assess domain formation and to study the nanomechanical properties of asymmetric supported lipid bilayers (SLBs) mimicking membrane rafts. Asymmetric SLBs were formed by incorporating N-palmitoyl-sphingomyelin (16:0SM) into the outer leaflet of preformed 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC)/Cholesterol SLBs through methyl-ß-cyclodextrin-mediated lipid exchange. Lipid domains were detected after incorporation of 16:0SM though their phase state varied from gel to liquid ordered (Lo) phase if the procedure was performed at 24 or 37 °C, respectively. When comparing symmetric and asymmetric Lo domains, differences in size and morphology were observed, with asymmetric domains being smaller and more interconnected. Both types of Lo domains showed similar mechanical stability in terms of rupture forces and Young's moduli. Notably, force curves in asymmetric domains presented two rupture events that could be attributed to the sequential rupture of a liquid disordered (Ld) and a Lo phase. Interleaflet coupling in asymmetric Lo domains could also be inferred from those measurements. The experimental approach outlined here would significantly enhance the applicability of membrane models.