RESUMO
The origin recognition complex (ORC) regulates DNA replication. However, some members of the ORC core, such as ORC3 and ORC5, have been implicated in neuronal maturation. In cultured cerebellar granule cells (CGCs), ORC3 mRNA and protein levels increased from 6 to 8days in vitro, a time that coincided with the maximal development of the dendritic arbor. In contrast, expression of ORC5 remained low throughout CGC maturation. Activation of type-4 metabotropic glutamate receptors with the selective enhancer, PHCCC, during a critical time-window (from 4 to 6days in vitro) anticipated the developmental peak of ORC3, increased the expression of two proteins associated with neuronal maturation, i.e. the mitogen-associated protein-2 (MAP-2) and postsynaptic density-95 (PSD-95), as well as dendritic length. siRNA-induced ORC3 knockdown reduced MAP-2 and PSD-95 expression on its own and abrogated the action of PHCCC. We examined whether the maturational effects of ORC3 were mediated by changes in the activity of the monomeric GTP-binding protein, Rho, which is known to regulate granule cell morphology. ORC3 knockdown increased the levels of the GTP-bound active form of Rho, whereas exposure to PHCCC reduced Rho activation. The action of PHCCC was largely attenuated in cultures deprived of ORC3. Finally, granule cell exposure to the Rho-associated kinase inhibitor, Y-27632, abolished the lowering effect of ORC3 knockdown on MAP-2 expression, and increased dendritic length. These data suggest that ORC3 supports neuronal maturation by inhibiting the Rho signaling pathway, and mediates the differentiating activity of mGlu4 receptors in cultured cerebellar granule cells.
Assuntos
Cerebelo/citologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios/fisiologia , Complexo de Reconhecimento de Origem/metabolismo , Fatores Etários , Amidas/farmacologia , Análise de Variância , Animais , Animais Recém-Nascidos , Benzopiranos/farmacologia , Dendritos/fisiologia , Proteína 4 Homóloga a Disks-Large , Inibidores Enzimáticos/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Proteínas Associadas aos Microtúbulos/genética , Neurônios/citologia , Neurônios/efeitos dos fármacos , Complexo de Reconhecimento de Origem/genética , Piridinas/farmacologia , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Ratos , Ratos Sprague-Dawley , Fator Rho/genética , Fator Rho/metabolismo , Fatores de Tempo , Transfecção/métodosRESUMO
Two new ISFETs recently developed by us have now been applied to some pharmaceutical determinations in real matrices; the first device, responsive to cationic surfactants, was employed in the determination of benzalkonium chloride contained in two different disinfectant solutions and in three types of commercial collyrium; the second device, responsive to cocaine hydrochloride, showed an appreciable response also to lidocaine hydrochloride and was used in the determination of lidocaine hydrochloride contained in some injectable antibiotics. The repeatability and accuracy of measurements performed in the analysis of these pharmaceutical matrices using new solid state sensors were evaluated. A further aspect of the research involved the use of two sensors to record complete titration curves for the determination of benzalkonium chloride, cocaine hydrochloride and lidocaine hydrochloride, respectively. Applications to real matrices were also performed by analysing by titration pharmaceutical formulations containing benzalkonium chloride, or lidocaine hydrochloride and an illicit powder containing cocaine hydrochloride and sugars.
Assuntos
Compostos de Benzalcônio/análise , Técnicas Biossensoriais/instrumentação , Cocaína/análise , Lidocaína/análise , Titulometria/métodos , Química Farmacêutica/instrumentação , Química Farmacêutica/métodos , Técnicas In Vitro , Reprodutibilidade dos Testes , Tensoativos/química , Titulometria/instrumentaçãoRESUMO
A new cocaine-sensitive ISFET device based on a cocaine-reineckate ion-pair complex dispersed in a PVC--sebacate matrix has been fabricated. this sensor displays a linear range for cocaine hydrochloride between about 3 x 10(-6) and 2 x 10(-2) M and a fast response (< or = 25 s), which remains almost constant over the pH range 3-7. The sensor has been applied to the analysis of authentic illicit powders containing cocaine hydrochloride and other substances commonly associated with it, or of cocaine free base (crack). Experimental results were compared with those obtained employing two more common instrumental methods of analysis: gas chromatography (GC), and UV absorption spectrometry (the latter applied directly or based on second derivative absorption spectroscopy). Good agreement was found between results obtained by ISFET and GC methods, while UV absorption spectrophotometry proved suitable only in the case of pure cocaine hydrochloride and free base, or in samples also containing lidocaine, but using second derivative absorption spectroscopy.
Assuntos
Cocaína/análise , Drogas Ilícitas/análise , Entorpecentes/análise , Cromatografia Gasosa , Cromatografia por Troca Iônica/instrumentação , Indicadores e Reagentes , Eletrodos Seletivos de Íons , Pós/análise , Espectrofotometria Ultravioleta , TiocianatosRESUMO
An ISFET device selective for cholanic acids, based on a PVC-sebacate membrane, containing benzyldimethylcetylammoniumcholate as exchanger, has been prepared, characterized and applied to the determination of cheno or ursodeoxycholic acid content of commercial pharmaceutical drugs and critical micellar concentration (CMC) values for cholate, deoxycholate and chenodeoxycholate. The results are compared with those obtained using previously described polymeric membrane sensors based on the same exchanger.
