RESUMEN
ATP-Binding Cassette (ABC) transporters are the main class of transmembrane transporters involved in pathogenic fungal resistance against chemotherapeutic agents. Herein we report results which show that batzelladine D (1) and norbatzelladine L (2) reverse the fluconazole resistance phenotype mediated by Pdr5p transporter on Saccharomyces cerevisiae. Both alkaloids were able to chemosensitize the Pdr5p-overexpressing strain by synergistic interaction with fluconazole. Both compounds also showed an inhibitory effect on the catalytic activity and on the intracellular accumulation of rhodamine 6G, and did not show significant in vitro mammalian cells toxicity.
Asunto(s)
Alcaloides/farmacología , Fluconazol/farmacología , Poríferos/química , Pirimidinas/farmacología , Rodaminas/antagonistas & inhibidores , Transportadoras de Casetes de Unión a ATP/metabolismo , Alcaloides/química , Alcaloides/aislamiento & purificación , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Ratones , Estructura Molecular , Pirimidinas/química , Pirimidinas/aislamiento & purificación , Rodaminas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Relación Estructura-ActividadRESUMEN
A high-speed and hardware-only algorithm using a center of mass method has been proposed for single-detector fluorescence lifetime sensing applications. This algorithm is now implemented on a field programmable gate array to provide fast lifetime estimates from a 32 × 32 low dark count 0.13 µm complementary metal-oxide-semiconductor single-photon avalanche diode (SPAD) plus time-to-digital converter array. A simple look-up table is included to enhance the lifetime resolvability range and photon economics, making it comparable to the commonly used least-square method and maximum-likelihood estimation based software. To demonstrate its performance, a widefield microscope was adapted to accommodate the SPAD array and image different test samples. Fluorescence lifetime imaging microscopy on fluorescent beads in Rhodamine 6G at a frame rate of 50 fps is also shown.
Asunto(s)
Algoritmos , Procesamiento de Imagen Asistido por Computador/métodos , Microscopía Fluorescente/instrumentación , Microscopía por Video/instrumentación , Diseño de Equipo , Análisis de los Mínimos Cuadrados , Microscopía Fluorescente/métodos , Microesferas , Fotones , Rodaminas/antagonistas & inhibidores , SemiconductoresRESUMEN
The ability of phenothiazine derivatives to inhibit the transport activity of P-glycoprotein in resistant mouse lymphoma and MDR/COLO 320 cells was studied. A rhodamine 123 efflux from the above-mentioned neoplastic cells in the presence of tested compounds was examined by flow cytometry. Two of the phenothiazine derivatives, namely perphenazine and prochlorperazine dimaleate, proved to be effective inhibitors of the rhodamine efflux. Other tested phenothiazine derivatives (promethazine hydrochloride, oxomemazine, methotrimeprazine maleate, trifluoropromazine hydrochloride, trimeprazine) also modulated the intracellular drug accumulation in both resistant cell lines, however, they exerted additional cytotoxic effects. The differences observed between the effects of the test compounds on intracellular drug accumulation could be the outcome of differences in phenothiazine's chemical structure, which is crucial for drug-cell membrane interactions. The results of this study provide information about a new group of compounds that offer promise in multidrug resistance reversal in tumor cells.
Asunto(s)
Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/patología , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Genes MDR/efectos de los fármacos , Linfoma de Células T/tratamiento farmacológico , Linfoma de Células T/patología , Fenotiazinas/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Animales , Transporte Biológico/efectos de los fármacos , Línea Celular Tumoral , Neoplasias del Colon/genética , Resistencia a Múltiples Medicamentos/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Citometría de Flujo , Colorantes Fluorescentes , Humanos , Linfoma de Células T/genética , Ratones , Estructura Molecular , Perfenazina/química , Perfenazina/farmacología , Fenotiazinas/química , Proclorperazina/química , Proclorperazina/farmacología , Rodaminas/antagonistas & inhibidoresRESUMEN
The yeast ABC (ATP-binding cassette protein) multidrug transporter Pdr5p transports a broad spectrum of xenobiotic compounds, including antifungal and antitumor agents. Previously, we demonstrated that substrate size is an important factor in substrate-transporter interaction and that Pdr5p has at least three substrate-binding sites. In this study, we use a combination of whole cell transport assays and photoaffinity labeling of Pdr5p with [(125)I]iodoarylazidoprazosin in purified plasma membrane vesicles to study the behavior of two series of novel substrates: trityl (triphenylmethyl) and carbazole derivatives. The results indicate that site 2, defined initially by tritylimidazole efflux, requires at least a single hydrogen bond acceptor group (electron pair donor). In contrast, complete inhibition of rhodamine 6G efflux and [(125)I]iodoarylazidoprazosin binding at site 1 requires substrates with three electronegative groups. Carbazole and trityl substrates with two groups show saturating, incomplete inhibition at this site. This type of inhibition is frequently observed in bacterial multidrug-binding proteins that use a pocket with multiple binding sites. The presence of multiple sites with different requirements for substrate-Pdr5p interaction may explain the broad specificity of xenobiotic compounds transported by this protein.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Xenobióticos/metabolismo , Antifúngicos/metabolismo , Antineoplásicos/metabolismo , Azidas/metabolismo , Sitios de Unión/efectos de los fármacos , Transporte Biológico/efectos de los fármacos , Carbazoles/química , Carbazoles/metabolismo , Clotrimazol/análogos & derivados , Clotrimazol/antagonistas & inhibidores , Clotrimazol/metabolismo , Reactivos de Enlaces Cruzados/metabolismo , Elipticinas/química , Elipticinas/metabolismo , Enlace de Hidrógeno/efectos de los fármacos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/química , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Prazosina/análogos & derivados , Prazosina/metabolismo , Rodaminas/antagonistas & inhibidores , Rodaminas/metabolismo , Especificidad por Sustrato/efectos de los fármacos , Tritio , Compuestos de Tritilo/química , Compuestos de Tritilo/metabolismo , Xenobióticos/químicaRESUMEN
Genome sequencing projects have uncovered many novel enzymes and enzyme classes for which knowledge of active site structure and mechanism is limited. To facilitate mechanistic investigations of the numerous enzymes encoded by prokaryotic and eukaryotic genomes, new methods are needed to analyze enzyme function in samples of high biocomplexity. Here, we describe a general strategy for profiling enzyme active sites in whole proteomes that utilizes activity-based chemical probes coupled with a gel-free analysis platform. We apply this gel-free strategy to identify the sites of labeling on enzymes targeted by sulfonate ester probes. For each enzyme examined, probe labeling was found to occur on a conserved active site residue, including catalytic nucleophiles (e.g., C32 in glutathione S-transferase omega) and bases/acids (e.g., E269 in aldehyde dehydrogenase-1; D204 in enoyl CoA hydratase-1), as well as residues of unknown function (e.g., D127 in 3 beta-hydroxysteroid dehydrogenase/isomerase-1). These results reveal that sulfonate ester probes are remarkably versatile activity-based profiling reagents capable of labeling a diversity of catalytic residues in a range of mechanistically distinct enzymes. More generally, the gel-free strategy described herein, by consolidating into a single step the identification of both protein targets of activity-based probes and the specific residues labeled by these reagents, provides a novel platform in which the proteomic comparison of enzymes can be accomplished in unison with a mechanistic analysis of their active sites.
Asunto(s)
Enzimas/química , Enzimas/metabolismo , Proteoma/química , Proteoma/metabolismo , Aldehído Deshidrogenasa/química , Aldehído Deshidrogenasa/metabolismo , Familia de Aldehído Deshidrogenasa 1 , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células COS , Catálisis , Chlorocebus aethiops , Enoil-CoA Hidratasa/química , Enoil-CoA Hidratasa/metabolismo , Glutatión Transferasa/química , Glutatión Transferasa/metabolismo , Isoenzimas/química , Isoenzimas/metabolismo , Datos de Secuencia Molecular , Mapeo Peptídico , Retinal-Deshidrogenasa , Rodaminas/antagonistas & inhibidores , Rodaminas/química , Rodaminas/metabolismoRESUMEN
It is commonly assumed that human neutrophils possess few, if any, functional mitochondria and that they do not depend on these organelles for cell function. We have used the fluorescent mitochondrial indicators, JC-1, MitoTracker Red, and dihydrorhodamine 123 to show that live neutrophils possess a complex mitochondrial network that extends through the cytoplasm. The membrane potential of these mitochondria was rapidly (within 2 min) disrupted by the addition of FCCP (IC(50) = 20 nM), but not by the Fo-ATPase inhibitor, oligomycin (at up to 7 microg/ml). However, inhibition of mitochondrial function with both agents resulted in cell shape changes. Neither activation of the respiratory burst nor phagocytosis of either latex particles or serum-opsonized Staphylococcus aureus was affected by the addition of FCCP or oligomycin. However, FCCP inhibited chemotaxis at concentrations that paralleled disruption of mitochondrial membrane potential. Furthermore, prolonged (2-h) incubation with oligomycin resulted in an impaired ability to activate a respiratory burst and also inhibited chemotaxis. These observations indicate that intact mitochondrial function is required to sustain some neutrophil functions, but not for the rapid initiation of the respiratory burst or phagocytosis. Loss of mitochondrial membrane potential was a very early marker for commitment of neutrophils into apoptosis and preceded the appearance of phosphatidylserine on the cell surface. However, inhibition of mitochondrial function did not accelerate the rate of neutrophil apoptosis. These data shed important insights into the hitherto unrecognized importance of mitochondria in the function of neutrophils during infection and inflammation.
Asunto(s)
Apoptosis/fisiología , Quimiotaxis de Leucocito/fisiología , Mitocondrias/fisiología , Activación Neutrófila/fisiología , Neutrófilos/fisiología , Fagocitosis/fisiología , Estallido Respiratorio/fisiología , Apoptosis/efectos de los fármacos , Carbonil Cianuro p-Trifluorometoxifenil Hidrazona/farmacología , Células Cultivadas , Quimiotaxis de Leucocito/efectos de los fármacos , Colorantes Fluorescentes/análisis , Humanos , Membranas Intracelulares/efectos de los fármacos , Membranas Intracelulares/fisiología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Microscopía Confocal , Microscopía Fluorescente , Mitocondrias/química , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Neutrófilos/citología , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Oligomicinas/farmacología , Estallido Respiratorio/efectos de los fármacos , Rodaminas/antagonistas & inhibidores , Desacopladores/farmacologíaRESUMEN
P-Glycoprotein and homologous multidrug transporters contain a phosphorylatable linker sequence that was proposed to control drug efflux on the basis that it was indeed phosphorylated in vitro and in vivo, and that inhibitors of protein kinase C (PKC) inhibited both P-glycoprotein phosphorylation and activity. However, site-directed mutagenesis of all phosphorylatable residues did not alter the drug resistance. The present work shows that PKC effectors are able to bind directly to multidrug transporters, from either cancer cells (mouse P-glycoprotein), yeast (Saccharomyces cerevisiae Pdr5p), or protozoan parasite (Leishmania tropica ltmdr1), and to inhibit their energy-dependent drug-efflux activity. The binding of staurosporine and derivatives such as CGP 41251 is prevented by preincubation with ATP, suggesting at least partial interaction at the ATP-binding site. In contrast, more hydrophobic compounds such as calphostin C and CGP 42700 bind outside the ATP-binding site and strongly interfere with drug interaction. A direct correlation is obtained between the efficiencies of PKC effectors to inhibit energy-dependent interaction of rhodamine 6G with yeast Pdr5p, to promote intracellular drug accumulation in various multidrug resistant cells, and to chemosensitize growth of resistant cells. The noncompetitive inhibition by PKC effectors of rhodamine 6G interaction with Pdr5p suggests that the binding might interfere with signal transduction between nucleotide hydrolysis and drug interaction. The overall results indicate that the multidrug transporters from different species display common features for interaction with PKC inhibitors. The hydrophobic derivative of staurosporine, CGP 42700, constitutes a potentially powerful modulator of P-glycoprotein-mediated multidrug resistance.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Resistencia a Múltiples Medicamentos , Inhibidores Enzimáticos/metabolismo , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Proteínas de Saccharomyces cerevisiae , Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Antiprotozoarios/farmacología , Sitios de Unión , Citosol/metabolismo , Daunorrubicina/farmacología , Metabolismo Energético/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Colorantes Fluorescentes/metabolismo , Proteínas Fúngicas/metabolismo , Leishmania tropica/efectos de los fármacos , Leishmania tropica/crecimiento & desarrollo , Leishmania tropica/metabolismo , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/metabolismo , Ratones , Naftalenos/metabolismo , Naftalenos/farmacología , Estructura Terciaria de Proteína , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/metabolismo , Pirofosfatasas/antagonistas & inhibidores , Pirofosfatasas/metabolismo , Rodaminas/antagonistas & inhibidores , Rodaminas/metabolismo , Saccharomyces cerevisiae , Estaurosporina/metabolismo , Estaurosporina/farmacologíaRESUMEN
We have studied gap junctional communication in the anterior subventricular zone (SVZa) of postnatal rodents, revealed by intercellular diffusion of dyes in brain slices. Extensive intercellular dye spread was evident in the SVZa. Coupling was not uniform, being characteristically larger in the outer borders of this layer, overlapping the previously described peripheral zone of concentration of S-phase cells. Intercellular spread of the dye was unaffected by acidification, but totally blocked by high Ca(2+) concentrations. In addition, application of some known uncoupling agents as carbenoxolone and halothane led to a marked reduction of dye spread in the SVZa. Our results demonstrate the presence of dye coupling mediated by gap junctions in the SVZa. Furthermore, the spatial organization of dye coupling in these slices strongly suggests the existence of cell compartments in the postnatal SVZa.