Combined in-silico and in-vitro experiments support acid-base equilibrium as a tool to estimate the localization depth of 4-nitrophenol within a phospholipid bilayer.

The interaction and location of 4-nitrophenol (PNP) in biomembranes are relevant in the bioaccumulation and potentiation of the intensive toxic effects of this persistent organic pollutant. In this work, in-silico analyses predicted that, in a fluid phospholipid bilayer, the minimum energy of the protonated (PNPH) and deprotonated (PNP-) species is located within the glycerol and choline region, respectively. This was experimentally confirmed by acid-base equilibrium experiments and theory, allowing the estimation of the mean location of PNP within a bilayer region with a dielectric constant D = 50.6 compatible with the phosphate/choline moiety of egg-yolk phosphatidylcholine unilamellar (EPC) vesicles. The comparison with the D = 43.2 value obtained in Triton X-100 micelles allow predicting a mean surface potential of ψ = 25.37 mV for the EPC-water interface. Changes in the chemical shifts and longitudinal relaxation times of EPC hydrogens by 1H NMR confirm the deeper location of the PNPH within the glycerol region and at the choline region (PNP-) at higher pH. Intermolecular PNP-EPC dipolar interactions within the choline region was also demonstrated at pH 10.2 using ROESY experiments. Additional information was obtained trough 31P NMR, that detected an increase in the anisotropy at the membrane interface after insertion of PNP which probably act as a bridge between choline moieties rigidizing the crystalline structure at that spot. Concluding, here we provide experimental support to the "pH-piston hypothesis" proposed some decades ago in the pharmaceutical field, and that reinforce the importance of the environmental conditions (e.g. pH) to modulate the bioavailability of this highly toxic pollutant.

Membrane Homeoviscous Adaptation in Sinorhizobium Submitted to a Stressful Thermal Cycle Contributes to the Maintenance of the Symbiotic Plant-Bacteria Interaction.

Here, we estimate fast changes in the fluidity of Sinorhizobium meliloti membranes submitted to cyclic temperature changes (10°C-40°C-10°C) by monitoring the fluorescence polarization (P) of DPH and TMA-DPH of the whole cell (WC) as well as in its outer (OM) and inner (IM) membranes. Additionally, the long-term response to thermal changes is demonstrated through the dynamics of the phospholipid and fatty acid composition in each membrane. This allowed membrane homeoviscous adaptation by the return to optimal fluidity levels as measured by the PDPH/TMA-DPH in WC, OM, IM, and multilamellar vesicles of lipids extracted from OM and IM. Due to probe-partitioning preferences and membranes' compositional characteristics, DPH and TMA-DPH exhibit different behaviors in IM and OM. The rapid effect of cyclic temperature changes on the P was the opposite in both membranes with the IM being the one that exhibited the thermal behavior expected for lipid bilayers. Interestingly, only after the incubation at 40°C, cells were unable to recover the membrane preheating P levels when cooled up to 10°C. Solely in this condition, the formation of threads and nodular structures in Medicago sativa infected with S. meliloti were delayed, indicating that the symbiotic interaction was partially altered but not halted.

Molecular features of nonionic detergents involved in the binding kinetics and solubilization efficiency, as studied in model (Langmuir films) and biological (Erythrocytes) membranes.

The effect of the nonionic detergents Brij-98 and Brij-58 over human erythrocytes was studied through quantitative hemolysis and in Langmuir films. Hemolytic tests revealed that Brijs are stronger membrane solubilizers than Triton X-100 (TX-100), with effective detergent/lipid ratios of 0.18 and 0.37 for Brij-98 and Brij-58, respectively. Experiments with Langmuir films provided significant information on the kinetics and thermodynamics of detergent-membrane interaction. The adsorption (ka) and desorption (kd) rate constants of Brijs were lower than those of TX-100. In the case of ka, that is probably due to their larger hydrophilic head (with twice (20) the oxyethylene units of TX-100). As for the thermodynamic binding constant, the linear and longer hydrophobic acyl chains of Brijs favor their stabilization in-between the lipids, through London van der Waals forces. Consequently, Kb,m values of Brij-98 (12,500 M-1) and Brij-58 (19,300 M-1) resulted higher than TX-100 (7500 M-1), in agreement with results from the hemolytic tests. Furthermore, Brij-58 binds with higher affinity than Brij-98 to bilayers and monolayers, despite its shorter (palmitic) hydrocarbon chain, showing that unsaturation restrains the detergent insertion into these environments. Our results provide significant information about the mechanism of interaction between Brijs and membranes, supporting their distinct solubilization effect.

Hydroxyzine, promethazine and thioridazine interaction with phospholipid monomolecular layers at the air-water interface.

In this work the interaction of Hydroxyzine, Promethazine and Thioridazine with Langmuir films of dipalmitoylphosphatidylcholine (dpPC) and dipalmitoylphosphatidic acid (dpPA), is studied. Temporal variations in lateral surface pressure (pi) were measured at different initial pi (pi(i)), subphase pH and drug-concentration. Drugs with the smallest (PRO) and largest (HYD) molecular size exhibited the lowest adsorption (k(a)) and the highest desorption (k(d)) rate constant values, respectively. The affinity binding constants (K(b)) obtained in monolayers followed the same profile (K(b,PRO) < K(b,HYD) < K(b,THI)) of the egg-PC/water partition coefficients (P) determined in bilayers. The drug concentration required to reach the half-maximal Deltapi at pi(i) = 14 mN/m (K(0.5)), was very sensitive to pH. The maximal increment in pi upon drug incorporation into the monolayer (deltapi(max)) will depend on the phospholipid collapse pressure (pi(c)), the monolayers's compressibility and drug's size, shape and charge. The higher pi(c) of dpPC lead to higher pi(cut-off) values (maximal pi allowing drug penetration), if compared with dpPA. In dpPC and dpPA pi(cut-off) decreased as a function of the molecular size of the uncharged drugs. In dpPA, protonated drugs became electrostatically trapped at the monolayer surface hence drug penetration, monolayer deformation and pi increase were impaired and the correlation between pi(cut-off) and drug molecular size was lost.

Un nuevo método radiorreceptor para la cuantificación de benzodiazepinas en fluidos biológicos / A new radioreceptor method for the quantification of benzodiazepines in biological fluids

Se desarrolló un método radiorreceptor, relativamente rápido y de bajo costo, para determinar concentraciones de benzodiazepinas en fluidos biológicos. El método se basa en el hecho que la cantidad de [3H]-flunitrazepam incorporada específicamente, por fotomarcado, en receptores de membranas sinaptosomales, está relacionada cuantitativamente con la cantidad de benzodiazepina no radiactiva presente en el medio de incubación. Después del fotomarcado, las membranas fueron tratadas con ácido tricloroacético, y el sedimento obtenido fue lavado con acetona para eliminar el [3H]-flunitrazepam remanente, no unido. La preparación de receptor, constituida por membranas liofilizadas obtenidas a partir de corteza de cerebro bovino, fue estable durante seis meses por lo menos. El análisis estadístico de los gráficos logit-log de las curvas de desplazamiento con varias benzodiazepinas, mostró los siguientes valores de IC50: clonazepam, 1,6 nM; flunitrazepam, 3,8 nM; nitrazepam, 15,3 nM; diazepam, 43,2 nM; y clorodiazepóxido, 7,4 micronM, valores que se correlacionaron significativamente con los correspondientes valores determinados por otro método. Los valores de concentración correspondientes a benzodiazepinas no identificadas, extraídas de sangre, fueron expresados en equivalentes de diazepam. La sensibilidad para diazepam fue 3,3 nM, y los niveles de dosis entre 15 y 138 nM, mostraron valores de coeficiente de variación intra e inter ensayo, variando desde 3,4 a 12,2%, y desde 13 a 30,7%, respectivamente

Un nuevo método radiorreceptor para la cuantificación de benzodiazepinas en fluidos biológicos / A new radioreceptor method for the quantification of benzodiazepines in biological fluids

Se desarrolló un método radiorreceptor, relativamente rápido y de bajo costo, para determinar concentraciones de benzodiazepinas en fluidos biológicos. El método se basa en el hecho que la cantidad de [3H]-flunitrazepam incorporada específicamente, por fotomarcado, en receptores de membranas sinaptosomales, está relacionada cuantitativamente con la cantidad de benzodiazepina no radiactiva presente en el medio de incubación. Después del fotomarcado, las membranas fueron tratadas con ácido tricloroacético, y el sedimento obtenido fue lavado con acetona para eliminar el [3H]-flunitrazepam remanente, no unido. La preparación de receptor, constituida por membranas liofilizadas obtenidas a partir de corteza de cerebro bovino, fue estable durante seis meses por lo menos. El análisis estadístico de los gráficos logit-log de las curvas de desplazamiento con varias benzodiazepinas, mostró los siguientes valores de IC50: clonazepam, 1,6 nM; flunitrazepam, 3,8 nM; nitrazepam, 15,3 nM; diazepam, 43,2 nM; y clorodiazepóxido, 7,4 micronM, valores que se correlacionaron significativamente con los correspondientes valores determinados por otro método. Los valores de concentración correspondientes a benzodiazepinas no identificadas, extraídas de sangre, fueron expresados en equivalentes de diazepam. La sensibilidad para diazepam fue 3,3 nM, y los niveles de dosis entre 15 y 138 nM, mostraron valores de coeficiente de variación intra e inter ensayo, variando desde 3,4 a 12,2%, y desde 13 a 30,7%, respectivamente (AU)