RESUMEN
Genomic expression signatures provide high-content biomarkers of cellular physiology, including the diverse responses to therapeutic drugs. To recognize these signatures, we devised a method of biomarker evaluation called 'sampling over gene space' (SOGS) that imparts superior predictive performance to existing supervised classification algorithms. Applied to microarray data from drug-treated human cortical neuron 1A cell cultures, this method predicts whether individual compounds possess anticonvulsant, antihypertensive, cyclooxygenase inhibitor, or opioid action. Thus, stable cell lines can be suitable for expression signature-based screening of a diverse range of activities. A SOGS-based system also discriminates physiologically active from inactive compounds, identifies drugs with off-target side effects, and incorporates a quantitative method for assigning confidence to individual predictions that, at its most stringent, approaches 100% accuracy. The capacity to resolve multiple distinct drug activities while simultaneously discriminating inactive and potential false-positive compounds in a cell line presents a unified framework for streamlined chemical genomic drug discovery.
Asunto(s)
Expresión Génica , Farmacología/métodos , Algoritmos , Inteligencia Artificial , Línea Celular , ADN Complementario/biosíntesis , ADN Complementario/genética , Interpretación Estadística de Datos , Marcadores Genéticos , Humanos , Modelos Estadísticos , Neuronas/efectos de los fármacos , Neuronas/fisiología , Análisis de Secuencia por Matrices de Oligonucleótidos , Preparaciones Farmacéuticas/clasificación , Valor Predictivo de las PruebasRESUMEN
Secretion of neurotrophins is critical for the delivery of neurotrophic support. Brain-derived neurotrophic factor is targeted to a regulated secretory pathway in neurons as well as the neurosecretory AtT-20 cells. Here, we show that pertussis toxin, which inactivates Gi and Go G proteins, inhibits up to 50% of the regulated release of brain derived neurotrophic factor by AtT-20 cells. To determine whether pertussis toxin-sensitive G proteins may regulate brain-derived neurotrophic factor release in vivo, the effect of intraocular pertussis toxin was assessed on the isthmo-optic nucleus in the developing chick visual system. The isthmo-optic nucleus projects axons from the midbrain to innervate retinal amacrine cells and depends on target-derived brain-derived neurotrophic factor between embryonic days 13 and 17 (E13-17). During this period approximately 50% of isthmo-optic neurons are eliminated by programmed cell death. Intraocular pertussis toxin administered at E13 increased cell death of isthmo-optic neurons by 42%, whereas injections at E19 had no effect. Co-injection of brain-derived neurotrophic factor with pertussis toxin rescued approximately 50% of isthmo-optic neurons from enhanced cell death, although overall retinal brain derived neurotrophic factor protein levels were unaffected by pertussis toxin. Retrograde transport of exogenous 125I-labeled brain derived neurotrophic factor from the retina to the midbrain was increased by co-administration of pertussis toxin, possibly owing to diminished competition from endogenously released brain-derived neurotrophic factors for the receptors that mediate retrograde axonal transport. These data suggest that the release of a major fraction of brain-derived neurotrophic factor in the secretory pathway in vitro and in vivo is regulated by the activity of pertussis toxin-sensitive G proteins.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/antagonistas & inhibidores , Subunidades alfa de la Proteína de Unión al GTP Gi-Go , Proteínas de Unión al GTP/antagonistas & inhibidores , Toxina del Pertussis , Factores de Virulencia de Bordetella/farmacología , Animales , Transporte Biológico Activo , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Muerte Celular/fisiología , Línea Celular , Embrión de Pollo , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Humanos , Ratones , Neuronas/efectos de los fármacos , Neuronas/fisiología , Concentración Osmolar , Cloruro de Potasio/farmacología , Retina/fisiología , Distribución Tisular , Vías Visuales/citología , Vías Visuales/efectos de los fármacos , Vías Visuales/fisiologíaRESUMEN
NIH-3T3 cells stably transfected with TrkB, the receptor for brain-derived neurotrophic factor (BDNF), were used to study the effects of NO and peroxynitrite on TrkB. 3-Morpholinosydnonimine (SIN-1), a donor of NO and O2- which immediately react to form peroxynitrite, induced TrkB tyrosine phosphorylation in a dose-dependent relationship from 2 to 40 mM. TrkB phosphorylation by SIN-1 was blocked by superoxide dismutase, which converts O2 to H2O2 and prevents its reaction with NO to form peroxynitrite, and by K252a, an inhibitor of TrkB phosphorylation by BDNF. Treatment with NO or O2- alone did not activate TrkB. Treatment directly with 1-4 mM peroxynitrite resulted in a dose-dependent increase in tyrosine phosphorylation of TrkB. SIN-1 treatment induced tyrosine phosphorylation of phospholipase C-gamma1 (PLC-gamma1) and induced its binding with activated TrkB, similar to that seen with BDNF downstream signaling pathways. These studies demonstrate activation of TrkB through peroxynitrite.
Asunto(s)
Nitratos/farmacología , Óxido Nítrico/fisiología , Receptor trkB/fisiología , Células 3T3 , Animales , Factor Neurotrófico Derivado del Encéfalo/farmacología , Carbazoles/farmacología , Humanos , Alcaloides Indólicos , Isoenzimas/metabolismo , Ratones , Molsidomina/análogos & derivados , Molsidomina/farmacología , Donantes de Óxido Nítrico/farmacología , Oxidantes/farmacología , Fosfolipasa C gamma , Fosforilación , Fosfotirosina/metabolismo , Ratas , Receptor trkB/efectos de los fármacos , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Superóxido Dismutasa/farmacología , Transfección , Fosfolipasas de Tipo C/metabolismoRESUMEN
Neurons in both vertebrate and invertebrate eyes are organized in regular arrays. Although much is known about the mechanisms involved in the formation of the regular arrays of neurons found in invertebrate eyes, much less is known about the mechanisms of formation of neuronal mosaics in the vertebrate eye. The purpose of these studies was to determine the cellular mechanisms that pattern the first neurons in vertebrate retina, the retinal ganglion cells. We have found that the ganglion cells in the chick retina develop as a patterned array that spreads from the central to peripheral retina as a wave front of differentiation. The onset of ganglion cell differentiation keeps pace with overall retinal growth; however, there is no clear cell cycle synchronization at the front of differentiation of the first ganglion cells. The differentiation of ganglion cells is not dependent on signals from previously formed ganglion cells, since isolation of the peripheral retina by as much as 400 microm from the front of ganglion cell differentiation does not prevent new ganglion cells from developing. Consistent with previous studies, blocking FGF receptor activation with a specific inhibitor to the FGFRs retards the movement of the front of ganglion cell differentiation, while application of exogenous FGF1 causes the precocious development of ganglion cells in peripheral retina. Our observations, taken together with those of previous studies, support a role for FGFs and FGF receptor activation in the initial development of retinal ganglion cells from the undifferentiated neuroepithelium peripheral to the expanding wave front of differentiation.
Asunto(s)
Factor 2 de Crecimiento de Fibroblastos/fisiología , Receptores de Factores de Crecimiento de Fibroblastos/fisiología , Retina/embriología , Células Ganglionares de la Retina/citología , Células 3T3 , Animales , Diferenciación Celular , Embrión de Pollo , Inhibidores Enzimáticos/farmacología , Factor 1 de Crecimiento de Fibroblastos , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Factor 2 de Crecimiento de Fibroblastos/farmacología , Factor 8 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/metabolismo , Factores de Crecimiento de Fibroblastos/farmacología , Factores de Crecimiento de Fibroblastos/fisiología , Ratones , Mitosis , Proteínas de Neurofilamentos/biosíntesis , Pirroles/farmacología , Receptores de Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Retina/citología , Células Ganglionares de la Retina/efectos de los fármacos , Células Madre , Factores de Tiempo , Tubulina (Proteína)/biosíntesisRESUMEN
The expression of the cm2 muscarinic acetylcholine receptor gene increases dramatically in chick retina during embryonic development in vivo. A similar developmental increase in cm2 expression occurs in embryonic chick retinal cells in culture. Conditioned medium from mature, but not young, retinal cultures contains a secreted factor that causes a selective increase in expression of cm2, but not cm3 or cm4, receptors. The secreted factor has been partially purified from serum-free medium, is protease-sensitive, and has a molecular weight >10 kDa. The cm2-inducing factor stimulates expression of a cm2 promoter/luciferase reporter gene, demonstrating that the increase in cm2 expression is attributable to increased gene transcription. Incubation of retinal cells with 14 identified neurotrophic and growth factors did not increase cm2 expression, suggesting that a novel developmentally regulated secreted factor mediates the subtype-specific induction of the cm2 receptor gene in retina.