RESUMO
Multiple species of G1 cyclins and cyclin-dependent kinases are induced sequentially during G1 phase, and the expression of cyclin A and cdc2 genes is subsequently induced at the G1/S boundary. To analyze the mechanism of cdc2 promoter activation, the 5'-flanking region of the rat cdc2 gene was isolated and its structural features were characterized. The highly conserved sequence between human and rat cdc2 genes is present in the basal promoter region from positions -183 to -122, which contains the E box, SpI, and E2F motifs. The expression of 5' sequential deletion derivatives of the promoter fused to luciferase cDNA in rat 3Y1 cells revealed the presence of the enhancer element. The presumed enhancer region was further analyzed by the introduction of base substitutions and by the formation of DNA-protein complexes with cell extracts prepared at various times during the G1-to-S-phase progression. These analyses revealed that the enhancer sequence, AAGTTACAAATA, located from -276 to -265, confers strong inducibility on the basal promoter at the G1/S boundary. The base substitutions introduced into the motifs of transcription factors indicated that the E2F motif is essential for the enhancer-dependent activation of the cdc2 promoter at the G1/S boundary. Electrophoretic mobility shift assays and DNase I footprinting showed that a factor which interacts with the enhancer element is induced late in G1 phase.
Assuntos
Proteína Quinase CDC2/genética , Ciclo Celular/genética , Elementos Facilitadores Genéticos , Regiões Promotoras Genéticas , Animais , Sequência de Bases , Linhagem Celular , DNA/genética , Fase G1/genética , Regulação da Expressão Gênica , Genes , Dados de Sequência Molecular , Mutação , Ratos , Fase S/genéticaRESUMO
Pituitary adenylate cyclase activating polypeptide (PACAP)-like immunoreactivity and its receptor mRNA have been reported in the supraoptic and the paraventricular nucleus (SON and PVN, respectively) and PACAP has been implicated in the regulation of magnocellular neurosecretory cell function. To examine the site and the mechanism of the action of PACAP in the neurosecretory cells, we measured AVP release from SON slice preparations and the cytosolic Ca2+ concentration ([Ca2+]i) from single dissociated SON neurons. PACAP at concentrations from 10(-12) to 10(-7) M increased [Ca2+]i in dissociated SON neurons in a dose-dependent manner. The patterns of the PACAP-induced [Ca2+]i increase were either sustained increase or cytosolic Ca2+ oscillations. PACAP (10[-7] M) increased [Ca2+]i in 27 of 27 neurons and glutamate (10[-4] M) increased [Ca2+]i in 19 of 19 SON neurons examined, whereas angiotensin II (10[-7] M) increased [Ca2+]i in only 15 of 60 SON neurons examined. PACAP at lower concentrations (10[-10] to 10[-8] M) increased [Ca2+]i in 70-80% of neurons examined. Although the onset and recovery of the PACAP-induced [Ca2+]i increase were slower than those observed with glutamate, the spatial distribution of the [Ca2+]i increases in response to the two ligands were similar: [Ca2+]i increase at the proximal dendrites was larger and faster and that at the center of the soma was smaller and slower. The PACAP-induced [Ca2+]i responses were abolished by extracellular Ca2+ removal, the L-type Ca2+-channel blocker, nicardipine, or by replacement of extracellular Na+ with N-methyl D-glucamine, and were partially inhibited by the Na+-channel blocker, tetrodotoxin. The N-type Ca2+-channel blocker, omega-conotoxin GVIA did not significantly inhibit the PACAP-induced [Ca2+]i responses. Furthermore, PACAP (10[-7] M) as well as glutamate (10[-4] M) increased AVP release from SON slice preparations, and extracellular Ca2+ removal or nicardipine inhibited the AVP release in response to PACAP. These results indicate that PACAP enhances Ca2+ entry via voltage-gated Ca2+ channels and increases [Ca2+]i, which, in turn, stimulates somatodendritic vasopressin release by directly activating PACAP receptors on SON neurons. The results also suggest that PACAP in the SON may play a pivotal role in the control of the neurohypophyseal function at the level of the soma or the dendrites.
Assuntos
Cálcio/metabolismo , Citosol/metabolismo , Dendritos/metabolismo , Neurônios/metabolismo , Neuropeptídeos/farmacologia , Fármacos Neuroprotetores/farmacologia , Núcleo Supraóptico/metabolismo , Vasopressinas/metabolismo , Angiotensina II/metabolismo , Animais , Arginina Vasopressina/metabolismo , Citosol/efeitos dos fármacos , Dendritos/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ácido Glutâmico/metabolismo , Técnicas In Vitro , Masculino , Neurônios/efeitos dos fármacos , Sistemas Neurossecretores/citologia , Sistemas Neurossecretores/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Ratos , Ratos Wistar , Estimulação Química , Núcleo Supraóptico/citologia , Núcleo Supraóptico/efeitos dos fármacosRESUMO
Molar volumes (Vm) of vapor-deposited ethylcyclohexane (ECH) and butyronitrile (BN, sometimes called butanenitrile) glasses were studied as a function of deposition temperature (Td). ECH glasses deposited at Td sufficiently below their glass-transition temperature (Tg) exhibited changes in Vm on heating similarly to alkylbenzenes. At Td close to Tg, ECH formed dense glasses as alkylbenzenes do, although these glasses were only slightly more dense than its supercooled liquid (SCL) states at the same temperatures. For BN, no indication of the formation of dense glasses was observed even at Td close to Tg, and the variations in Vm with the temperature elevation were different from those of alkylbenzenes. Analysis of the initial Vm of the deposited glasses of different compounds demonstrated that its Td-dependence was well correlated with the steepness index (m) of the corresponding SCL. Quantum-chemical calculations concerning dimer formation by the studied compounds showed that the hydrogen bond between a C-H bond in the alkyl group and π-electrons in the phenyl ring stabilizes the alkylbenzene dimers, suggesting the possibility of the dense glass formation and large m of these compounds. The small m value of BN was also discussed on the basis of the calculation results.