RESUMO
Rhythmic changes in light and temperature conditions form the primary environmental cues that synchronize the molecular circadian clock of most species with the external cycles of day and night. Previous studies established a role for the CREB-binding protein (CBP) in molecular clock function by coactivation of circadian transcription. Here, we report that moderately increased levels of CBP strongly dampen circadian behavioural rhythms without affecting molecular oscillations of circadian transcription. Interestingly, light-dark cycles as well as high temperature facilitated a circadian control of behavioural activity. Based on these observations we propose that in addition to its coactivator function for circadian transcription, CBP is involved in the regulation of circadian behaviour down-stream of the circadian clock.
Assuntos
Proteína de Ligação a CREB/metabolismo , Ritmo Circadiano/genética , Drosophila/metabolismo , Animais , Proteínas CLOCK/genética , Proteínas CLOCK/metabolismo , Proteína de Ligação a CREB/genética , Drosophila/genética , Drosophila/fisiologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismoRESUMO
Reconstituting posttranslational modification with SUMO in vitro is an essential tool in the analysis of sumoylation. In this article, we provide detailed protocols that allow to set up and perform sumoylation reactions using a purified recombinant sumoylation machinery. The protocols include purification of the SUMO E1 enzyme His-Aos1/Uba2, untagged E2 enzyme Ubc9, untagged SUMO, and the RanBP2 E3 ligase fragment IR1 + M. Using these components, we provide step-by-step instructions to set up sumoylation reactions. Two established SUMO model substrates, His-RanGAPtail and HisYFP-Sp100, complement the described tool box; these proteins serve as positive controls in E3 ligase-independent and -dependent sumoylation reactions and are valuable instruments to adjust the reaction conditions if necessary.
Assuntos
Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação , Proteínas Ativadoras de GTPase/metabolismo , Humanos , Enzimas Ativadoras de Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
BACKGROUND: The surfactant proteins B (SP-B) and C (SP-C) are important for the stability and function of the alveolar surfactant film. Their involvement and down-regulation in inflammatory processes has recently been proposed, but their level during neutrophilic human airway diseases are not yet known. METHODS: We used 1D-electrophoresis and Western blotting to determine the concentrations and molecular forms of SP-B and SP-C in bronchoalveolar lavage (BAL) fluid of children with different inflammatory airway diseases. 21 children with cystic fibrosis, 15 with chronic bronchitis and 14 with pneumonia were included and compared to 14 healthy control children. RESULTS: SP-B was detected in BAL of all 64 patients, whereas SP-C was found in BAL of all but 3 children; those three BAL fluids had more than 80% neutrophils, and in two patients, who were re-lavaged later, SP-C was then present and the neutrophil count was lower. SP-B was mainly present as a dimer, SP-C as a monomer. For both qualitative and quantitative measures of SP-C and SP-B, no significant differences were observed between the four evaluated patient groups. CONCLUSION: Concentration or molecular form of SP-B and SP-C is not altered in BAL of children with different acute and chronic inflammatory lung diseases. We conclude that there is no down-regulation of SP-B and SP-C at the protein level in inflammatory processes of neutrophilic airway disease.