RESUMO
This work considers the estimation of dispersion in materials via an interferometric technique. At its core, the problem involves extracting the quadratic variation in phase over a range of wavelengths based on measured optical intensity. The estimation problem becomes extremely difficult for weakly dispersive materials where the quadratic nonlinearity is very small relative to the uncertainty inherent in experiment. This work provides a means of estimating dispersion in the face of such uncertainty. Specifically, we use a Markov Chain Monte Carlo implementation of Bayesian analysis to provide both the dispersion estimate and the associated confidence interval. The interplay between various system parameters and the size of the resulting confidence interval is discussed. The approach is then applied to several different experimental samples.
RESUMO
The apoptotic protein Hrk is expressed in hematopoietic progenitors after growth factor deprivation. Here we identify a silencer sequence in the 3' untranslated region of the hrk gene that binds to the transcriptional repressor DREAM in interleukin-3 (IL-3)-dependent hematopoietic progenitor cells, and abrogates the expression of reporter genes when located downstream of the open reading frame. In addition, the binding of DREAM to the hrk gene is reduced or eliminated when cells are cultured in the absence of IL-3 or treated with a calcium ionophore or a phosphatidylinositol 3-kinase-specific inhibitor, suggesting that both calcium mobilization and phosphorylation can regulate the transcriptional activity of DREAM. Furthermore, we have shown that DREAM is phosphorylated by a phosphatidylinositol 3-kinase-dependent, but Akt-independent pathway. In all cases, loss of the DREAM-DNA binding complex was correlated with increased levels of Hrk and apoptosis. These data suggest that IL-3 may trigger the activation of DREAM through different signaling pathways, which in turn binds to a silencer sequence in the hrk gene and blocks transcription, avoiding inappropriate cell death in hematopoietic progenitors.
Assuntos
Proteínas de Ligação ao Cálcio , Proteínas de Transporte/genética , Inativação Gênica/fisiologia , Células-Tronco Hematopoéticas/metabolismo , Interleucina-3/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Repressoras/metabolismo , Regiões 3' não Traduzidas/genética , Animais , Apoptose , Cálcio/metabolismo , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/metabolismo , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Células-Tronco Hematopoéticas/citologia , Humanos , Interleucina-3/farmacologia , Ionóforos/farmacologia , Proteínas Interatuantes com Canais de Kv , Camundongos , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Sequências Reguladoras de Ácido Nucleico/fisiologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Transcriptional repressor DREAM, an EF-hand containing calcium-binding protein, blocks basal expression of target genes through specific interaction with DRE sites in the DNA. The sequence GTCA forms the central core of the DRE site, whereas flanking nucleotides contribute notably to the affinity for DREAM. Release of binding of DREAM from the DRE results in derepression, a process that is regulated by Ca(2+). Change of two amino acids within an EF-hand in DREAM blocks Ca(2+)-induced derepression and results in potent dominant negative mutants of endogenous DREAM.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas de Ligação a DNA , Proteínas Repressoras/metabolismo , Fatores de Transcrição , Animais , Sítios de Ligação , Proteínas Estimuladoras de Ligação a CCAAT/química , Proteínas Estimuladoras de Ligação a CCAAT/genética , Cálcio/farmacologia , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular , Motivos EF Hand , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas Interatuantes com Canais de Kv , Mutação , Fatores de Transcrição NFI , Proteínas Nucleares , Proteínas Repressoras/química , Proteínas Repressoras/genética , Transfecção , Células Tumorais Cultivadas , Proteína 1 de Ligação a Y-BoxRESUMO
Protein kinase A-dependent derepression of the human prodynorphin gene is regulated by the differential occupancy of the Dyn downstream regulatory element (DRE) site. Here, we show that a direct protein-protein interaction between DREAM and the CREM repressor isoform, alphaCREM, prevents binding of DREAM to the DRE and suggests a mechanism for cyclic AMP-dependent derepression of the prodynorphin gene in human neuroblastoma cells. Phosphorylation in the kinase-inducible domain of alphaCREM is not required for the interaction, but phospho-alphaCREM shows higher affinity for DREAM. The interaction with alphaCREM is independent of the Ca(2+)-binding properties of DREAM and is governed by leucine-charged residue-rich domains located in both alphaCREM and DREAM. Thus, our results propose a new mechanism for DREAM-mediated derepression that can operate independently of changes in nuclear Ca(2+).
Assuntos
Motivos de Aminoácidos/genética , Proteínas de Ligação ao Cálcio , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Encefalinas/genética , Genes Reguladores/genética , Precursores de Proteínas/genética , Proteínas Repressoras/metabolismo , Sequência de Aminoácidos , Cálcio/metabolismo , Linhagem Celular , Colforsina/farmacologia , Modulador de Elemento de Resposta do AMP Cíclico , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Encefalinas/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter/genética , Humanos , Proteínas Interatuantes com Canais de Kv , Dados de Sequência Molecular , Mutação/genética , Neuroblastoma , Fosforilação , Precursores de Proteínas/efeitos dos fármacos , RNA Mensageiro/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Alinhamento de Sequência , Transfecção , Células Tumorais CultivadasRESUMO
Using mRNA differential display, we found that the gene for NAD(+)-dependent glycerol phosphate dehydrogenase (GPDH; EC 1.1.1.8) is induced in rat brain following seizure activity. Northern blot and in situ hybridization analysis confirmed the differential display results; they also showed, in a separate model of neuronal activation, that after thermal noxious stimulation of the hind-paws, a similar increase in GPDH mRNA occurs in the areas of somatotopic projection in the lumbar spinal cord. Surprisingly, administration of analgesic doses of morphine or the nonsteroidal antiinflammatory drugs aspirin, metamizol (dipyrone), and indomethacin also increased GPDH mRNA levels in rat spinal cord. The opioid receptor antagonist naloxone completely blocked morphine induction of GPDH but had no effect on GPDH induction by noxious heat stimulation or metamizol treatment, implicating different mechanisms of GPDH induction. Nevertheless, in all cases, induction of the GPDH gene requires adrenal steroids and new protein synthesis, as the induction was blocked in adrenalectomized rats and by cycloheximide treatment, respectively. Our results suggest that the induction of the GPDH gene upon peripheral noxious stimulation is related to the endogenous response to pain as it is mimicked by exogenously applied analgesic drugs.
Assuntos
Analgesia , Regulação da Expressão Gênica/genética , Expressão Gênica , Glicerolfosfato Desidrogenase/genética , Convulsões/enzimologia , Adrenalectomia , Analgésicos Opioides/farmacologia , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glucocorticoides/metabolismo , Glucocorticoides/farmacologia , Glicerolfosfato Desidrogenase/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/enzimologia , Masculino , Antagonistas de Entorpecentes/farmacologia , Fibras Nervosas Mielinizadas/enzimologia , Medição da Dor/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Convulsões/induzido quimicamente , Medula Espinal/efeitos dos fármacos , Medula Espinal/enzimologiaRESUMO
Fluxes in amounts of intracellular calcium ions are important determinants of gene expression. So far, Ca2+-regulated kinases and phosphatases have been implicated in changing the phosphorylation status of key transcription factors and thereby modulating their function. In addition, direct effectors of Ca2+-induced gene expression have been suggested to exist in the nucleus, although no such effectors have been identified yet. Expression of the human prodynorphin gene, which is involved in memory acquisition and pain, is regulated through its downstream regulatory element (DRE) sequence, which acts as a location-dependent gene silencer. Here we isolate a new transcriptional repressor, DRE-antagonist modulator (DREAM), which specifically binds to the DRE. DREAM contains four Ca2+-binding domains of the EF-hand type. Upon stimulation by Ca2+, DREAM's ability to bind to the DRE and its repressor function are prevented. Mutation of the EF-hands abolishes the response of DREAM to Ca2+. In addition to the prodynorphin promoter, DREAM represses transcription from the early response gene c-fos. Thus, DREAM represents the first known Ca2+-binding protein to function as a DNA-binding transcriptional regulator.
