RESUMEN
BACKGROUND: A plethora of biological metabolisms are regulated by the mechanisms of ubiquitination, wherein this process is balanced with the action of deubiquitination system. Dub-2 is an IL-2-inducible, immediate-early gene that encodes a deubiquitinating enzyme with growth regulatory activity. DUB-2 presumably removes ubiquitin from ubiquitin-conjugated target proteins regulating ubiquitin-mediated proteolysis, but its specific target proteins are unknown yet. METHODOLOGY/PRINCIPAL FINDINGS: To elucidate the functional role of Dub-2, we generated genetically modified mice by introducing neo cassette into the second exon of Dub-2 and then homologous recombination was done to completely abrogate the activity of DUB-2 proteins. We generated Dub-2+/- heterozygous mice showing a normal phenotype and are fertile, whereas new born mouse of Dub-2-/- homozygous alleles could not survive. In addition, Dub-2-/- embryo could not be seen between E6.5 and E12.5 stages. Furthermore, the number of embryos showing normal embryonic development for further stages is decreased in heterozygotes. Even embryonic stem cells from inner cell mass of Dub-2-/- embryos could not be established. CONCLUSIONS: Our study suggests that the targeted disruption of Dub-2 may cause embryonic lethality during early gestation, possibly due to the failure of cell proliferation during hatching process.
Asunto(s)
Pérdida del Embrión/enzimología , Pérdida del Embrión/patología , Endopeptidasas/genética , Eliminación de Gen , Marcación de Gen , Proteínas Inmediatas-Precoces/genética , Animales , Apoptosis , Masa Celular Interna del Blastocisto/metabolismo , Masa Celular Interna del Blastocisto/patología , Proliferación Celular , Supervivencia Celular , Desarrollo Embrionario , Endopeptidasas/deficiencia , Endopeptidasas/metabolismo , Fertilización In Vitro , Técnicas de Genotipaje , Heterocigoto , Proteínas Inmediatas-Precoces/deficiencia , Proteínas Inmediatas-Precoces/metabolismo , Tejido Linfoide/metabolismo , Tejido Linfoide/patología , Masculino , Ratones , Ratones Mutantes , Fenotipo , Motilidad Espermática , Espermatozoides/patología , Bazo/patología , Timo/patologíaAsunto(s)
Relojes Biológicos/genética , Ritmo Circadiano/genética , Cicloleucina/análogos & derivados , Isoenzimas/deficiencia , Neuronas/enzimología , Receptores de Glutamato Metabotrópico/metabolismo , Núcleo Supraquiasmático/enzimología , Fosfolipasas de Tipo C/deficiencia , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/genética , Animales , Relojes Biológicos/efectos de los fármacos , Proteínas de Ciclo Celular , Ritmo Circadiano/efectos de los fármacos , Cicloleucina/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Ácido Glutámico/metabolismo , Isoenzimas/genética , Ratones , Ratones Noqueados , Mutación/genética , Neuronas/citología , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Técnicas de Cultivo de Órganos , Proteínas Circadianas Period , Fosfolipasa C beta , Receptores de Glutamato Metabotrópico/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Núcleo Supraquiasmático/citología , Núcleo Supraquiasmático/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/genética , Fosfolipasas de Tipo C/genéticaRESUMEN
Transmission at the mouse neuromuscular junction normally relies on P/Q-type channels, but became jointly dependent on both N- and R-type Ca(2+) channels when the PQ-type channel alpha(1A) subunit was deleted. R-type channels lay close to Ca(2+) sensors for exocytosis and I(K(Ca)) channel activation, like the P/Q-type channels they replaced. In contrast, N-type channels were less well localized, but abundant enough to influence secretion strongly, particularly when action potentials were prolonged. Our data suggested that active zone structures may select among multiple Ca(2+) channels in the hierarchy P/Q >R >N. The alpha(1A)-/- neuromuscular junction displayed several other differences from wild-type: lowered quantal content but greater ability to withstand reductions in the Ca(2+)/Mg(2+) ratio, and little or no paired-pulse facilitation, the latter findings possibly reflecting compensatory mechanisms at individual release sites. Changes in presynaptic function were also associated with a significant reduction in the size of postsynaptic acetylcholine receptor clusters.
Asunto(s)
Canales de Calcio Tipo P/deficiencia , Canales de Calcio Tipo Q/deficiencia , Placa Motora/metabolismo , Neurotransmisores/metabolismo , 4-Aminopiridina/farmacología , Animales , Canales de Calcio Tipo N/fisiología , Canales de Calcio Tipo P/genética , Canales de Calcio Tipo P/fisiología , Canales de Calcio Tipo Q/genética , Canales de Calcio Tipo Q/fisiología , Canales de Calcio Tipo R/fisiología , Ratones , Ratones Noqueados , Modelos Neurológicos , Placa Motora/efectos de los fármacos , Plasticidad Neuronal , Transmisión Sináptica/efectos de los fármacosRESUMEN
To investigate the role of P/Q type Ca(2+) channels in determining the firing pattern of Purkinje cells (PCs) we compared the somatically evoked discharge of action potentials (APs) in PCs from 3 to 4 week old cerebellar slice cultures obtained with ataxic mice lacking alpha(1A)-subunit (alpha(-/-)) and with normal mice (non-ataxic alpha(+/-) or alpha(+/+)) using the whole-cell configuration of the patch-clamp recording method. Whereas evoked responses of PCs in normal mice were mainly fast APs, those of PCs from ataxic mice were mainly low-threshold Ca(2+) spikes (LTS). Furthermore, a sustained plateau potential due to the activation of cadmium sensitive Ca(2+) conductances was not observed in PCs from ataxic mice by blocking K(+) channels. These results confirm that P/Q Ca(2+) channels elicit Ca(2+)-dependent plateau potentials and control the propagation of the dendritic LTS to the soma.
Asunto(s)
Potenciales de Acción/fisiología , Canales de Calcio Tipo N/deficiencia , Canales de Calcio Tipo N/genética , Células de Purkinje/fisiología , Animales , Canales de Calcio Tipo N/fisiología , Cerebelo/fisiología , Ratones , Ratones Noqueados , Ratones Transgénicos , Técnicas de Cultivo de Órganos , PerfusiónRESUMEN
Simultaneous recordings of inward whole-cell Ca(2+) channel currents (I(Ca) ) and increments of capacitance as an indication of exocytosis (Delta(Cm)), were performed in voltage-clamped single adrenal chromaffin cells from wild-type and alpha(1A) subunit deficient mice, using the perforated-patch configuration of the patch-clamp technique. Using protocol #1 (one single Ca(2+) channel blocker per cell), to dissect the components of I(Ca), L channels contributed 43%, N channels 35% and P/Q channels 30% to the total I(Ca) of wild-type cells. Using protocol #2 (cumulative sequential addition of 3 microm nifedipine, 1 microm omega-conotoxin GVIA, and 1 microm omega-agatoxin IVA), L, N and P/Q channels contributed 40%, 34% and 14%, respectively, to I(Ca); an R component of around 11% remained. In wild-type mice the changes of Delta(Cm) paralleled those of I(Ca). In alpha(1A) deficient mice the L component of I(Ca) rose to 53% while the P/Q disappeared; the N and R components were similar. In these mice, Delta(Cm) associated to N and R channels did not vary; however, the P/Q component was abolished while the L component increased by 20%. In conclusion, exocytosis was proportional to the relative density of each Ca(2+) channel subtype, L, N, P/Q, R. Ablation of the alpha(1A) gene led to a loss of P/Q channel current and to a compensatory increase of L channel-associated secretion; however, this compensation was not sufficient to maintain the overall exocytotic response, that was diminished by 35% in alpha(1A) -deficient mice. This may be due to altered Ca(2+) homeostasis in these mice, as compared to wild mouse chromaffin cells.