RESUMEN

Fluralaner is a relatively new insecticide belonging to the isoxazoline group, whose action mechanism involves the blocking of GABAA-receptors in the insect nervous system. Because of its high hydrophobicity, fluralaner could bioaccumulate and reach toxic local concentrations. Since there are no data available about the penetration and persistence of isoxazolines in biological membranes, we intend to evaluate fluralaner permanence as a pollutant by using model membranes. We used experimental and in silico models to characterize the incorporation of fluralaner into the lipid phase at different packing states. We determined its impact in the membrane structure and organization. Our results confirm that fluralaner is capable of penetrating, holding, and accumulating in the lipid membrane and provide details on its precise location and orientation. These properties would allow fluralaner to reach high local concentrations in different membranes and organs, which could be dangerous for vertebrate organisms if its handling is not properly controlled.

Asunto(s)

Insecticidas , Insecticidas/química , Isoxazoles , Receptores de GABA-A , Lípidos

RESUMEN

The spread of beta-lactamase-producing bacteria is of great concern and the environment has been found to be a main source of contamination. Herein, it was proposed to determine the frequency of antimicrobial-resistant-Gram-negative bacteria throughout the Lerma River basin using phenotypic and molecular methods. Resistant bacteria were isolated with chromogenic media and antimicrobial susceptibility tests were used to characterize their resistance. ARGs for beta-lactams, aminoglycosides, and quinolones were detected by PCR. Species were identified by Sanger sequencing the 16S rRNA gene and the representative genomes of MDR strains were sequenced by NGS. A high variation in the number of isolates was observed in the 20 sampled sites, while observing a low diversity among the resistant bacteria. Of the 12 identified bacterial groups, C. freundii, E. coli, and S. marcescens were more predominant. A high frequency of resistance to beta-lactams, quinolones, and aminoglycosides was evidenced, where the blaCTX,qnrB, qnrS y, and aac(6')lb-cr genes were the most prevalent. C. freundii showed the highest frequency of MDR strains. Whole genome sequencing revealed that S. marcescens and K. pneumoniae showed a high number of shared virulence and antimicrobial resistance genes, while E. coli showed the highest number of unique genes. The contamination of the Lerma River with MDR strains carrying various ARGs should raise awareness among environmental authorities to assess the risks and regulations regarding the optimal hygienic and sanitary conditions for this important river that supports economic activities in the different communities in Mexico.

Asunto(s)

Antibacterianos , Quinolonas , Antibacterianos/farmacología , Ríos/microbiología , Escherichia coli , Pruebas de Sensibilidad Microbiana , ARN Ribosómico 16S , México , beta-Lactamasas/genética , Farmacorresistencia Microbiana , Klebsiella pneumoniae/genética , beta-Lactamas , Aminoglicósidos/farmacología , Quinolonas/farmacología , Farmacorresistencia Bacteriana Múltiple/genética

RESUMEN

The RDL receptor is one of the most relevant protein targets for insecticide molecules. It belongs to the pentameric ligand-gated ion channel (pLGIC) family. Given that the experimental structures of pLGICs are difficult to obtain, homology modeling has been extensively used for these proteins, particularly for the RDL receptor. However, no detailed assessments of the usefulness of homology models for virtual screening (VS) have been carried out for pLGICs. The aim of this study was to evaluate which are the determinant factors for a good VS performance using RDL homology models, specially analyzing the impact of the template conformational state. Fifteen RDL homology models were obtained based on different pLGIC templates representing the closed, open, and desensitized states. A retrospective VS process was performed on each model, and their performance in the prioritization of active ligands was assessed. In addition, the three best-performing models among each of the conformations were subjected to molecular dynamics simulations (MDS) in complex with a representative active ligand. The models showed variations in their VS performance parameters that were related to the structural properties of the binding site. VS performance tended to improve in more constricted binding cavities. The best performance was obtained with a model based on a template in the closed conformation. MDS confirmed that the closed model was the one that best represented the interactions with an active ligand. These results imply that different templates should be evaluated and the structural variations between their channel conformational states should be specially examined, providing guidelines for the application of homology modeling for VS in other proteins of the pLGIC family.

RESUMEN

The use of chitosan to harvest microalgae is a strategic step that seeks to reach an economically competitive price to recover lipids, proteins, and pigments. The aim of the present work was to design low-molecular-weight chitosan from shrimp shells and its physicochemical characterization, to be used for the harvesting of wild microalgae consortia. The chitosan was obtained by chemical deacetylation of shrimp shells, and physicochemical characterization was made using the instrumental methods DSC, TGA, X-ray, FTIR, and SEM. The harvesting of wild microalgae consortia was performed by the jar test method. The obtained chitosan had a low molecular weight (169 KDa), a deacetylation degree of 83 %, a decomposition temperature (TD) of 280 °C, and a crystallinity of 38.2 %. The microalgae genera found in the consortium were Scenedesmus sp., Chlorella sp., Schroderia sp., and Chlamydomonas sp. The microalgae removal efficiency of the chitosan was 99.2 % with 20 mg L-1.

RESUMEN

γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the vertebrate and invertebrate nervous system. GABAA receptors are activated by GABA and their agonists, and modulated by a wide variety of recognized drugs, including barbiturates, anesthetics, and benzodiazepines. The phenols propofol, thymol, chlorothymol, carvacrol and eugenol act as positive allosteric modulators on GABAA-R receptor. These GABAergic phenols interact with the lipid membrane, therefore, their anesthetic activity could be the combined result of their specific activity (with receptor proteins) as well as nonspecific interactions (with surrounding lipid molecules) modulating the supramolecular organization of the receptor environment. Therefore, we aimed to contribute to a description of the molecular events that occur at the membrane level as part of the mechanism of general anesthesia, using a molecular dynamic simulation approach. Equilibrium molecular dynamics simulations indicate that the presence of GABAergic phenols in a DPPC bilayer orders lipid acyl chains for carbons near the interface and their effect is not significant at the bilayer center. Phenols interacts with the polar interface of phospholipid bilayer, particularly forming hydrogen bonds with the glycerol and phosphate group. Also, potential of mean force calculations using umbrella sampling show that propofol partition is mainly enthalpic driven at the polar region and entropic driven at the hydrocarbon chains. Finally, potential of mean force indicates that propofol partition into a gel DPPC phase is not favorable. Our in silico results were positively contrasted with previous experimental data.

Asunto(s)

Membrana Dobles de Lípidos/química , Simulación de Dinámica Molecular , Fenoles/farmacología , Ácido gamma-Aminobutírico/química , 1,2-Dipalmitoilfosfatidilcolina/química , Entropía , Enlace de Hidrógeno , Propofol/farmacología , Termodinámica , Factores de Tiempo

RESUMEN

γ-Aminobutyric-acid receptor (GABAA-R), a membrane intrinsic protein, is activated by GABA and modulated by a wide variety of recognized drugs. GABAA-R is also target for several insecticides which act by recognition of a non-competitive blocking site. Mentha oil is rich in several ketones with established activity against various insects/pests. Considering that mint ketones are highly lipophilic, their action mechanism could involve, at least in part, a non-specific receptor modulation by interacting with the surrounding lipids. In the present work, we studied in detail the effect on membranes of five cyclic ketones present in mint plants, with demonstrated insecticide and gabaergic activity. Particularly, we have explored their effect on the organization and dynamics of the membrane, by using Molecular Dynamics (MD) Simulation studies in a bilayer model of DPPC. We performed free diffusion MD and obtained spatially resolved free energy profiles of ketones partition into bilayers based on umbrella sampling. The most favored location of ketones in the membrane corresponded to the lower region of the carbonyl groups. Both hydrocarbon chains were slightly affected by the presence of ketones, presenting an ordering effect for the methylene groups closer to the carbonyl. MD simulations results were also contrasted with experimental data from fluorescence anisotropy studies which evaluate changes in membrane fluidity. In agreement, these assays indicated that the presence of ketones between lipid molecules induced an enhancement of the intermolecular interaction, increasing the molecular order throughout the bilayer thickness.

Asunto(s)

Cetonas/química , Simulación de Dinámica Molecular , Liposomas Unilamelares/química , Ácido gamma-Aminobutírico/química , Polarización de Fluorescencia , Enlace de Hidrógeno , Cetonas/metabolismo , Temperatura , Termodinámica , Liposomas Unilamelares/metabolismo

RESUMEN

AIMS: Various investigations have demonstrated the protective capacity of general anesthetics as neuroprotective agents. The effects of propofol against ischemia are known to reside in its antioxidant properties and its GABAergic activity. Other aromatic alcohols have also been reported as able to protect neurons against oxidative damage. The aim of this work is to evaluate the potential neuroprotective effect of some phenols, structurally analogues of propofol, with proven GABAergic activity. These phenols include the naturally occurring compounds thymol, carvacrol and eugenol, the synthetic product chlorothymol, and the most widely used intravenous anesthetic, propofol, as a reference compound. MATERIALS AND METHODS: Taking primary cultures of cortical neurons as a suitable model to evaluate cellular protection against oxidative damage, we developed an injury model to test potential neuroprotective activity. The intracellular hydroperoxides were also determined. KEY FINDINGS: The results showed that no compound decreased cell viability at concentrations where they were active on the GABAA receptor. In neuroprotection tests, some phenols and Vit E showed a partial protective effect against the oxidative injury. These compounds induced a clear tendency to reduce H2O2 damage, comparing production of hydroperoxides, although these last changes were statistically non-significant. SIGNIFICANCE: Testing the intracellular oxidation levels suggests that this partial protection exerted by propofol, thymol and chlorothymol may be mediated in some way by their antioxidant activities. However, this neuroprotection is not completely correlated with the antioxidant capacity, but it approaches their relative pharmacological potency, which could be interpreted as a final effect that would involve both activities.

Asunto(s)

Anestésicos Intravenosos/farmacología , Antioxidantes/farmacología , Corteza Cerebral/metabolismo , Eugenol/farmacología , Monoterpenos/farmacología , Neuronas/metabolismo , Fármacos Neuroprotectores/farmacología , Timol/farmacología , Animales , Células Cultivadas , Corteza Cerebral/citología , Cimenos , Neuronas/citología , Ratas , Ratas Wistar , Receptores de GABA-A/metabolismo

RESUMEN

Two recently synthesized dihydropyrimidines (DHPMs) analogues have demonstrated larvicide and repellent activity against Anopheles arabiensis. DHPMs high lipophilicity suggests that these compounds may interact directly with the membrane and modify their biophysical properties. The purpose of the present study was to characterize the interaction of both compounds with artificial membranes. Changes on the properties of DPPC films were studied using Langmuir monolayers. The presence of DHPMs in the subphase modified the interfacial characteristics of DPPC compression isotherms, causing the expansion of the monolayer, inducing the disappearance of DPPC phase transition and increasing the molecular packing of the film. Moreover, both compounds showed ability to penetrate into the lipid monolayers at molecular pressures comparable to those in biological membranes. The effects of both DHPMs on the molecular organization of DPPC liposomes were measured by fluorescence anisotropy. The results indicate that their presence between lipid molecules would induce an increasing intermolecular interaction, diminishing the bilayer fluidity mainly at the polar region. Finally, we performed free diffusion MD simulations and obtained spatially resolved free energy profiles of DHPMs partition into a DPPC bilayer through Potential of Mean Force (PMF) calculations. In agreement with the experimental assays, PMF profiles and MD simulations showed that DHPMs are able to partition into DPPC bilayers, penetrating into the membrane and stablishing hydrogen bonds with the carbonyl moiety. Our results suggest that DHPMs bioactivity could involve their interaction with the lipid molecules that modulate the supramolecular organization of the biological membranes and consequently the membrane proteins functionality.

Asunto(s)

Insecticidas/química , Pirimidinas/química , 1,2-Dipalmitoilfosfatidilcolina/química , Animales , Anisotropía , Anopheles , Membrana Celular/química , Fuerza Compresiva , Simulación por Computador , Enlace de Hidrógeno , Membrana Dobles de Lípidos/química , Lípidos/química , Fluidez de la Membrana , Membranas Artificiales , Microscopía Fluorescente , Simulación de Dinámica Molecular , Transición de Fase , Presión , Reología , Propiedades de Superficie , Agua/química

RESUMEN

The cyclic ketones, thujone and dihydrocarvone, are lipophilic components of essential oils extracted from different plants, which have proven insecticidal activity. The GABAA receptor is activated by the neurotransmitter GABA and is the action site of widely used neurotoxic pesticides. Many compounds that regulate GABAA receptor function interact with membrane lipids, causing changes in their physical properties and consequently, in the membrane dynamic characteristics that modulate receptor macromolecules. In the present study, the biophysical effects of thujone (a gabaergic reference compound) and dihydrocarvone (structurally very similar) were explored by using monomolecular films of DPPC as a model membrane system, to gain insight into membrane-drug interaction. The compression isotherms showed that both ketones expand the DPPC isotherms and increase membrane elasticity. They penetrate the monolayer but their permanence depends on the possibility of establishing molecular interactions with the film component, favored by defects present in the membrane at the phase transition. Finally, by using Brewster angle microscopy (BAM) as a complementary technique for direct visualization of the study films, we found that incorporating ketone seems to reduce molecular repulsion among phospholipid headgroups. Our results reinforce the notion that changes in membrane mechanics may be occurring in the presence of the assayed ketones, suggesting that their interaction with the receptor's surrounding membrane may modulate or affect its functionality, possibly as part of the mechanism of the bioactivity described for thujone and DHC.

Asunto(s)

Membrana Celular/química , Membrana Celular/efectos de los fármacos , Monoterpenos/farmacología , 1,2-Dipalmitoilfosfatidilcolina/química , 1,2-Dipalmitoilfosfatidilcolina/metabolismo , Adsorción , Monoterpenos Bicíclicos , Membrana Celular/metabolismo , Monoterpenos Ciclohexánicos , Monoterpenos/química , Reología

RESUMEN

Carvone is a natural terpene which can be purified as R-(-) or S-(+) enantiomers. There are many reports about its antibacterial, antifungal, and insecticide activities, and also of some effects on the nervous system, where both enantiomers showed different potencies. Considering that the GABA(A) receptor is a major insecticide target, we studied the pharmacological activity of both carvone enantiomers, and of thujone as a reference compound acting on the receptor, on native GABA(A) by determining their effects on benzodiazepine recognition sites using primary neuronal cultures. Both isomers were able to inhibit the GABA-induced stimulation of [(3)H]flunitrazepam binding, suggesting their interaction with the GABA(A) receptor as negative allosteric modulators. Their activity was comparable to that described for thujone in the present article, with the R-(-)-carvone being the more similar and potent stereoisomer. The different configuration of the isopropenyl group in position 5 thus seems to be significant for receptor interaction and the bicycle structure not to be critical for receptor recognition. The concentrations necessary to induce negative modulation of the receptor were not cytotoxic in a murine neuron culture system. These results confirm that, at least partially, the reported insecticidal activity of carvones may be explained by their interaction with the GABA(A) receptor at its noncompetitive blocker site.

Asunto(s)

Corteza Cerebral/citología , Monoterpenos/química , Monoterpenos/farmacología , Neuronas/citología , Neuronas/efectos de los fármacos , Receptores de GABA-A/metabolismo , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Monoterpenos Ciclohexánicos , Relación Dosis-Respuesta a Droga , Femenino , Flunitrazepam/metabolismo , Antagonistas de Receptores de GABA-A/química , Antagonistas de Receptores de GABA-A/farmacología , Embarazo , Ratas , Estereoisomerismo

RESUMEN

GABA(A) receptor is the main inhibitory receptor of the central nervous system. The phenols propofol and thymol have been shown to act on this receptor. GABA(A) is an intrinsic protein, the activity of which may be affected by physical changes in the membrane. Taking into account the lipophilicity of phenols, their interaction with the membrane and a consequent non-specific receptor modulation cannot be discarded. By using Langmuir films, we analyze the comparative effects on the molecular properties of the membrane exerted by propofol, thymol and other related compounds, the activities of which on the GABA(A) are under investigation in our laboratory. All the compounds were able to expand phospholipid films, by their incorporation into the monolayer being favored by less-packed structures. Nonetheless, they were able to be incorporated at lateral pressures above the equilibrium pressure estimated for a natural membrane. Epifluorescence images revealed their presence between phospholipid molecules, probably at the head-group region. Hence, all results indicated that the phenols studied were clearly able to interact with membranes, suggesting that their anesthetic activity could be the combined result of their interaction with specific receptor proteins and with their surrounding lipid molecules modulating the supramolecular organization of the receptor environment.

Asunto(s)

Anestésicos Intravenosos/química , GABAérgicos/química , Membranas/química , Fenoles/química , Propofol/química , Receptores de GABA-A/química , 1,2-Dipalmitoilfosfatidilcolina/química , Algoritmos , Indicadores y Reactivos , Microscopía Fluorescente , Fosfolípidos/química , Presión , Termodinámica

RESUMEN

Combined effects of flunitrazepam (FNZ) and lidocaine (LDC) were studied on the thermotropic equilibrium of dipalmitoyl phosphatidylcholine (dpPC) bilayers. This adds a thermodynamic dimension to previously reported geometric analysis in the erythrocyte model. LDC decreased the enthalpy and temperature for dpPC pre- and main-transitions (ΔHp, ΔHm, Tp, Tm) and decreased the cooperativity of the main-transition (ΔT(1/2,m)). FNZ decreased ΔHm and, at least up to 59 µM, also decreased ΔHp. In conjunction with LDC, FNZ induced a recovery of ∆T(1/2,m) control values and increased ΔHm even above the control level. The deconvolution of the main-transition peak at high LDC concentrations revealed three components possibly represented by: a self-segregated fraction of pure dpPC, a dpPC-LDC mixture and a phase with a lipid structure of intermediate stability associated with LDC self-aggregation within the lipid phase. Some LDC effects on thermodynamic parameters were reverted at proper LDC/FNZ molar ratios, suggesting that FNZ restricts the maximal availability of the LDC partitioned into the lipid phase. Thus, beyond its complexity, the lipid-LDC mixture can be rationalized as an equilibrium of coexisting phases which gains homogeneity in the presence of FNZ. This work stresses the relevance of nonspecific drug-membrane binding on LDC-FNZ pharmacological interactions and would have pharmaceutical applications in liposomal multidrug-delivery.

Asunto(s)

Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Flunitrazepam/farmacología , Lidocaína/farmacología , Luz , Membrana Dobles de Lípidos/metabolismo , Dispersión de Radiación , 1,2-Dipalmitoilfosfatidilcolina/metabolismo , Rastreo Diferencial de Calorimetría , Membrana Celular/química , Interacciones Farmacológicas , Membrana Dobles de Lípidos/química , Termodinámica

RESUMEN

Some phenols, like propofol, thymol and related compounds, have been shown to act on the GABA(A) receptor. Several compounds with GABAergic activity have displayed neuroprotective effects attributed mainly to the potentiation of GABA(A)-mediated inhibition of synaptic transmission. It has also been found that compounds containing a phenolic OH group can scavenge reactive oxygen species, as in the case of propofol, among others. Thus, the neuroprotective action mechanism of GABAergic phenols would involve both effects, their pharmacological activity on GABA(A) and their intrinsic antioxidant ability. In this context, the study of the antioxidant properties of phenolic compounds included in the present work will enable these capacities to be correlated with their eventual pharmacological activities. The assays chosen in this study included determination of antioxidant ability in homogeneous isotropic systems (DPPH reduction, FRAP and hydrogen peroxide scavenging) and in heterogeneous membrane systems (inhibition of lipid peroxidation of phospholipid SUVs). The comparative evaluation of the results showed some differences between the relative order of antioxidant potency among all assayed compounds determined by using both types of systems. This analysis supports the conclusion that the antioxidant values obtained in homogeneous non-membrane systems, for phenols or other lipophilic compounds, should be revised according to their capacity of interaction with membranes (i.e. Log P in membrane-buffer system) in order to obtain antioxidant potency values more approximate to those actually occurring in biological systems. These results are essential to understand the actual neuroprotective action mechanism exerted by phenolic compounds involving a pharmacological activity, an antioxidant effect or both actions exerted mutually.

Asunto(s)

Antioxidantes/química , Ácidos Grasos/química , GABAérgicos/química , Fármacos Neuroprotectores/química , Fenoles/química , Propofol/química , Antioxidantes/análisis , Antioxidantes/farmacología , Compuestos de Bifenilo/metabolismo , Ácidos Grasos/análisis , Ácidos Grasos/farmacología , Depuradores de Radicales Libres/química , Depuradores de Radicales Libres/farmacología , GABAérgicos/análisis , GABAérgicos/farmacología , Peroxidación de Lípido/efectos de los fármacos , Liposomas , Fármacos Neuroprotectores/análisis , Fármacos Neuroprotectores/farmacología , Oxidación-Reducción , Fenoles/análisis , Fenoles/farmacología , Fosfolípidos , Picratos/metabolismo , Propofol/análisis , Propofol/farmacología , Especies Reactivas de Oxígeno/metabolismo

RESUMEN

Some phenolic compounds, like propofol and thymol, have been shown to act on the GABA(A) receptor. Taking into account the hydrophobicity of these compounds, their interaction with the membrane surrounding the receptor and consequent non-specific effect on receptor modulation cannot be neglected. In the present work, we determined and correlated several lipophilic parameters for both GABAergic agents and three other related phenolic compounds (eugenol, carvacrol and chlorothymol), including logP(o/w), retention data in high performance liquid chromatography (HPLC) by using C18 and immobilized artificial membrane (IAM) columns at different temperatures, and partition coefficients determined in phospholipid liposomes. The correlation results demonstrated the high capacity of the compounds assayed to interact with phospholipid membrane phases, which can be predicted by simple model systems as logP(o/w) or HPLC. The values obtained by HPLC using a fast screening IAM column were the quantitatively closest to the partition coefficients determined in liposome systems, due to the capacity of this column to permit the establishment of molecular interactions like those found in phospholipid membranes. Finally, the fact that all the compounds studied are able to interact with membranes would suggest the participation of some alteration of the GABA(A) receptor lipid environment as part of the receptor modulation exerted by phenolic compounds.

Asunto(s)

GABAérgicos/química , Fenoles/química , Tampones (Química) , Cromatografía Líquida de Alta Presión , Lípidos/química , Liposomas/química , Análisis de Componente Principal , Receptores de GABA-A/efectos de los fármacos , Solubilidad , Solventes

RESUMEN

Menthol is a naturally occurring compound, which has three chiral centers that define eight possible optically actives stereoisomers. Neuroactivity of menthol and related agents by affecting neuronal intracellular signaling or by modulation of neurotransmitter-gated currents has been reported. Furthermore, stereo-selectivity of menthol in its analgesic activity as well as in its sensory properties and other biological activities was also described. The present study is the first contribution to the description of stereo-selectivity of GABA(A) receptor against the most possible isomers of menthol, discussed in terms of their chirality. The results showed that only (+)-menthol, among the five stereoisomers analyzed, was active, stimulating in a dose-response manner the binding of an allosteric GABA(A) receptor ligand. Taking into account these results, and comparing them with those of some active phenolic compounds, it is strongly suggested that the existence of a relative spatial location of its substituents with respect to the ring (equatorial position of all substituents and (1S,2R,5S)-configuration) as well as the presence in the cyclic molecule of an aliphatic non polar group (isopropyl) with free rotation near to a polar group (hydroxyl) are crucial points to demonstrate activity on the receptor.

Asunto(s)

Mentol/farmacología , Receptores de GABA-A/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Animales , Aves , Células Cultivadas , Pollos , Femenino , Masculino , Mentol/química , Ratas , Receptores de GABA-A/química , Receptores de GABA-A/metabolismo , Estereoisomerismo , Relación Estructura-Actividad

RESUMEN

Previous studies of our group demonstrated that flunitrazepam is a lipophilic drug capable of interacting with membranes through a partition equilibrium phenomenon. Its localization at the phospholipid polar head region could explain the decrease in the size of dipalmitoylphosphatidylcholine (dpPC) vesicles, through a mechanism that involves the increment in the relative volume of this region with a subsequent increase in the vesicle's surface curvature. In the present work, we investigated if flunitrazepam can affect the L(alpha)-H(II) phase transition of phosphatidylethanolamine through a similar mechanism. This study was approached by using merocyanine 540, a dye sensitive to the molecular packing of membrane lipids. A detailed analysis of merocyanine absorption and fluorescence emission and excitation spectra was performed. The results indicated that the fluorescence emitted came mainly from the monomeric form of merocyanine and that it resulted a good indicator of this phase transition, as was previously described. Flunitrazepam did not affect significantly the onset of the phase transition but showed a tendency to diminish the dye fluorescence emission intensity, which could involve a lower partition of merocyanine in the vesicles. Moreover, the results suggest that this drug produced a delay in the completeness of the phase transition and a decrement in the cooperativity of this phenomenon.

Asunto(s)

Flunitrazepam/química , Colorantes Fluorescentes/química , Transición de Fase/efectos de los fármacos , Fosfatidiletanolaminas/química , Pirimidinonas/química , Dimerización , Flunitrazepam/farmacología , Indicadores y Reactivos/química , Lípidos de la Membrana/química , Propiedades de Superficie , Temperatura

RESUMEN

In the present work, we studied the ability of thymol to affect the organization of model membranes and the activity of an intrinsic membrane protein, the GABA(A) receptor (GABA(A)-R). In this last aspect, we tried to elucidate if the action mechanism of this terpene at the molecular level, involves its binding to the receptor protein, changes in the organization of the receptor molecular environment, or both. The self-aggregation of thymol in water with a critical micellar concentration approximately = 4 microM and its ability to penetrate in monomolecular layers of soybean phosphatidylcholine (sPC) at the air-water interface, even at surface pressures above the equilibrium, lateral pressure of natural bilayers were demonstrated. Thymol affected the self-aggregation of Triton X-100 and the topology of sPC vesicles. It also increased the polarity of the membrane environment sensed by the electrochromic dye merocyanine. A dipolar moment of 1.341 Debye was calculated from its energy-minimized structure. Its effect on the binding of [3H]-flunitrazepam ([3H]-FNZ) to chick brain synaptosomal membranes changed qualitatively from a tendency to the inhibition to a clear activatory regime, up on changing the phase state of the terpene (from a monomeric to a self-aggregated state). Above its CMC, thymol increased the affinity of the binding of [3H]-FNZ (K(d-control)= 2.9, K(d-thymol)= 1.7 nM) without changing the receptor density (B(max-control)= 910, B(max-thymol)= 895 fmol/mg protein). The activatory effect of thymol on the binding of [ [3H]-FNZ was observed even in the presence of the allosteric activator gamma-aminobutyric acid (GABA) at a concentration of maximal activity, and was blocked by the GABA antagonist bicuculline. Changes in the dipolar arrangement and in the molecular packing of GABA(A)-R environment are discussed as possible mediators of the action mechanism of thymol.

Asunto(s)

Tensoactivos/química , Timol/química , Aire , Animales , Bicuculina/química , Membrana Celular/metabolismo , Pollos , Flunitrazepam/química , Antagonistas del GABA/química , Cinética , Membrana Dobles de Lípidos/química , Micelas , Octoxinol/química , Fosfatidilcolinas/química , Unión Proteica , Pirimidinonas/química , Glycine max/metabolismo , Espectrofotometría , Factores de Tiempo , Agua/química

RESUMEN

The molecular basis underlying the stress-induced increment in the density of central benzodiazepine receptor from chick forebrain, observed previously at 4 degrees C, was studied from a biophysical perspective. The thermal dependence of [3H]flunitrazepam binding to the central benzodiazepine receptor and the supramolecular organization were studied in forebrain membranes from chicks submitted to partial water immersion. The equilibrium dissociation constants increased with temperature in membrane from both control and stressed chicks. The heat capacity values in control samples (deltaC(p, CON)) were significantly less negative than deltaC(p STR). Changes in deltaH and deltaS between 4-37 degrees C were greater in stressed chicks compared to control; however, the binding was exothermic and driven by enthalpy in both conditions. At 4 degrees C, the receptor density (B(max)) was higher in stressed chicks compared to control. Such a difference was lost irreversibly upon temperature elevation, possibly due to the hysteresis between the heating and cooling behaviour of B(max, CON) and the constancy in B(max, STR). The fluorescence anisotropy of diphenylhexatriene was higher in control samples with respect to stressed chicks below 10 degrees C. A temperature-induced increment in protein intrinsic-fluorescence was observed only in control, and was quenched by acrylamide more easily at 4 degrees C than at 25 degrees C. A higher microviscosity at 4 degrees C in control favoured more external localizations of integral proteins; at higher temperatures, tryptophan residues moved to hydrophobic membrane-regions. Changes in the membrane-organization towards more fluid states favoured the accessibility of benzodiazepine to the central benzodiazepine receptor, expressed by the higher values of B(max) found in stressed samples at low temperatures with respect to control samples.

Asunto(s)

Membrana Celular/química , Plasticidad Neuronal/fisiología , Prosencéfalo/química , Prosencéfalo/citología , Estrés Fisiológico , Animales , Unión Competitiva , Pollos , Ácidos Grasos/análisis , Ácidos Grasos/química , Femenino , Flunitrazepam/metabolismo , Polarización de Fluorescencia , Interacciones Hidrofóbicas e Hidrofílicas , Técnicas In Vitro , Masculino , Física/métodos , Prosencéfalo/fisiología , Receptores de GABA-A/metabolismo , Temperatura , Termodinámica

RESUMEN

The effect of molecular packing on flunitrazepam's ability to interact with bio-membranes was studied using dipalmitoylphosphatidylcholine monomolecular layers at the air-water interface as a model membrane. Flunitrazepam penetrated from the subphase into monolayers at lateral pressures below 44.8 mN/m and induced their concentration-dependent expansion. As inferred from the values of compressibility modulus, the elasticity of the liquid-condensed phase decreased in the presence of flunitrazepam. Although this drug hardly penetrated into high-packed monolayers, it was easily incorporated in the low-packed ones at an extent sufficient to reach the partition equilibrium. Below a molecular area of 75 A(2), contrary to what would be expected, the drug surface concentration increased as a function of surface pressure, suggesting that after its penetration in disordered phases, it became energetically or physically trapped in newly-formed liquid condensed clusters. The phenomenon of flunitrazepam penetration and release would have different energy barriers depending on the membrane phase-state.

Asunto(s)

Flunitrazepam/química , Fenómenos Químicos , Química Física , Indicadores y Reactivos , Membranas Artificiales , Propiedades de Superficie , Termodinámica

RESUMEN

Se presentan una serie de tumores neurogénicos, de cabeza y cuello; que los clasificamos según los pares craneanos en que se presentan; sintolatología muy variada de la que deberemos tenerlos en cuenta a la hora del diagnóstico. (AU)

Asunto(s)

Humanos , Masculino , Femenino , Adolescente , Adulto , Persona de Mediana Edad , Anciano , Neoplasias de Cabeza y Cuello/diagnóstico , Neoplasias de Cabeza y Cuello/cirugía , Neoplasias de Tejido Nervioso/cirugía , Neoplasias de Tejido Nervioso/diagnóstico , Estesioneuroblastoma Olfatorio , Cavidad Nasal , Neoplasias Nasales , Sarcoma , Neoplasias Gingivales , Neurofibroma , Neurofibromatosis 1 , Maxilares , Neuroma Acústico , Neurilemoma , Neoplasias Faríngeas