RESUMO
BACKGROUND AND PURPOSE: Transient lower oesophageal sphincter relaxations (TLESRs) are triggered by activation of mechanosensitive gastric vagal afferents and are the major cause of gastroesophageal reflux and therefore an important target for therapeutic intervention in gastroesophageal reflux disease (GERD). Activation of the metabotropic GABA(B) receptor has shown to inhibit TLESRs. The aim of the present study was to assess the role of the ionotropic GABA(A) receptor in the regulation of TLESRs. EXPERIMENTAL APPROACH: TLESRs were quantified using Dentsleeve manometry in dogs, and GABA(A) agonists were given i.v. prior to gastric distension. Immunohistochemistry and RT-PCR were used to localize GABA(A) receptors in the dog nodose ganglion, the source of vagal afferents which initiate TLESRs. KEY RESULTS: The prototypical GABA(A) agonist muscimol produced a dose-dependent inhibition of TLESRs ranging from 19 to 56%. The two other GABA(A) agonists evaluated, isoguvacine and 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridin-3-ol (THIP), as well as the GABA(A) positive allosteric modulator diazepam, had no major effects on TLESRs. Evaluation of higher doses was limited by emesis (THIP and isoguvacine) or restlessness/sedation (diazepam). Of the predominant GABA(A) receptor subunits (alpha, beta and gamma components), alpha and beta but not gamma were detected in the dog nodose ganglion by RT-PCR, while immunohistochemistry in addition demonstrated nerve fibres expressing the gamma subunit. CONCLUSIONS AND IMPLICATIONS: The present observations demonstrate that GABA(A) receptors exert an inhibitory control of TLESRs. These results warrant further studies using GABA(A) isoform-selective agonists to define the identity of receptors involved.
Assuntos
Esfíncter Esofágico Inferior/metabolismo , Subunidades Proteicas , Receptores de GABA-A/metabolismo , Animais , Cães , Relação Dose-Resposta a Droga , Feminino , Agonistas GABAérgicos/administração & dosagem , Agonistas GABAérgicos/farmacologia , Agonistas de Receptores de GABA-A , Expressão Gênica , Imuno-Histoquímica , Masculino , Manometria , Muscimol/administração & dosagem , Muscimol/farmacologia , Gânglio Nodoso/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Considerable research has been conducted into the nature of airway inflammation in chronic obstructive pulmonary disease (COPD) but the relationship between proximal airways inflammation and both dynamic collapse of the peripheral airways and HRCT determined emphysema severity remains unknown. A number of research tools have been combined to study smokers with a range of COPD severities classified according to the GOLD criteria. METHODS: Sixty five subjects (11 healthy smokers, 44 smokers with stage 0-IV COPD, and 10 healthy non-smokers) were assessed using lung function testing and HRCT scanning to quantify emphysema and peripheral airway dysfunction and sputum induction to measure airway inflammation. RESULTS: Expiratory HRCT measurements and the expiratory/inspiratory mean lung density ratio (both indicators of peripheral airway dysfunction) correlated more closely in smokers with the severity of airflow obstruction (r = -0.64, p<0.001) than did inspiratory HRCT measurements (which reflect emphysema severity; r = -0.45, p<0.01). Raised sputum neutrophil counts also correlated strongly in smokers with HRCT indicators of peripheral airway dysfunction (r = 0.55, p<0.001) but did not correlate with HRCT indicators of the severity of emphysema. CONCLUSIONS: This study suggests that peripheral airway dysfunction, assessed by expiratory HRCT measurements, is a determinant of COPD severity. Airway neutrophilia, a central feature of COPD, is closely associated with the severity of peripheral airway dysfunction in COPD but is not related to the overall severity of emphysema as measured by HRCT.
Assuntos
Bronquite/patologia , Doença Pulmonar Obstrutiva Crônica/patologia , Obstrução das Vias Respiratórias/patologia , Obstrução das Vias Respiratórias/fisiopatologia , Bronquite/fisiopatologia , Volume Expiratório Forçado/fisiologia , Humanos , Contagem de Leucócitos , Pessoa de Meia-Idade , Neutrófilos/patologia , Doença Pulmonar Obstrutiva Crônica/diagnóstico por imagem , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Tomografia Computadorizada por Raios X/métodos , Capacidade Vital/fisiologiaRESUMO
Oral N-acetyl-L-cysteine (NAC) is used clinically for treatment of chronic obstructive pulmonary disease. NAC is easily oxidized to its disulfide. We show here that N,N'-diacetyl-L-cystine (DiNAC) is a potent modulator of contact sensitivity (CS)/delayed type hypersensitivity (DTH) reactions in rodents. Oral treatment of BALB/c mice with 0.003 to 30 micromol/kg DiNAC leads to enhancement of a CS reaction to oxazolone; DiNAC is 100 to 1000 times more potent than NAC in this respect, indicating that it does not act as a prodrug of NAC. Structure-activity studies suggest that a stereochemically-defined disulfide element is needed for activity. The DiNAC-induced enhancement of the CS reaction is counteracted by simultaneous NAC-treatment; in contrast, the CS reaction is even more enhanced in animals treated with DiNAC together with the glutathione-depleting agent buthionine sulfoximine. These data suggest that DiNAC acts via redox processes. Immunohistochemically, ear specimens from oxazolone-sensitized and -challenged BALB/c mice treated with DiNAC display increased numbers of CD8(+) cells. DiNAC treatment augments the CS reaction also when fluorescein isothiocyanate is used as a sensitizer in BALB/c mice; this is a purported TH2 type of response. However, when dinitrofluorobenzene is used as a sensitizer, inducing a purported TH1 type of response, DiNAC treatment reduces the reaction. Treatment with DiNAC also reduces a DTH footpad-swelling reaction to methylated BSA. Collectively, these data indicate that DiNAC in vivo acts as a potent and effective immunomodulator that can either enhance or reduce the CS or DTH response depending on the experimental conditions.
Assuntos
Adjuvantes Imunológicos/farmacologia , Cistina/análogos & derivados , Dermatite de Contato/imunologia , Hipersensibilidade Tardia/imunologia , Acetilcisteína/análogos & derivados , Animais , Linfócitos T CD8-Positivos , Cistina/farmacologia , Dermatite de Contato/etiologia , Dinitrofluorbenzeno , Orelha , Feminino , Fluoresceína-5-Isotiocianato , Pé , Granuloma/etiologia , Granuloma/imunologia , Hipersensibilidade Tardia/etiologia , Imuno-Histoquímica , Contagem de Linfócitos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos , Oxazolona , Coelhos , Soroalbumina BovinaRESUMO
We describe the cloning and sequence analysis of a nearly full-length cDNA as well as a corresponding 5.2-kilobase pair genomic fragment encoding FREAC-4, a member of the forkhead family of transcription factors. The cDNA is collinear with respect to the coding region of the intronless genomic clone. The conceptual translation product predicts a protein of 465 amino acids with a hyperacidic amino-terminal end, a DNA binding forkhead domain and a carboxyl-terminal part that is rich in homopolymeric runs of prolines and alanines. The transcription start is identified using an RNase protection assay. A 2.7-kilobase pair genomic DNA fragment, located immediately upstream of the translation start, was fused to a luciferase reporter gene. Significant levels of luciferase activity were detected when this construct was transfected into two kidney-derived cell lines, 293 and COS-7 cells, whereas only background reporter gene expression was observed in a cell line of nonkidney origin. Cotransfections with plasmids expressing WT-1, WTAR (a mutated form of WT-1), p53, and a mutated form of p53 revealed a complex pattern of regulation with a 3-fold induction with WT-1, a 7-fold induction with mutated p53, and a 4-fold repression with wild-type p53. A 5'-promoter deletion series delimits a DNA fragment necessary for WT-1 inducibility in cotransfection experiments. This fragment is shown to contain at least one cis-element that is capable of interacting with recombinant WT-1.
Assuntos
Regulação da Expressão Gênica/genética , Genes do Tumor de Wilms , Genes p53 , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , DNA Complementar , Fatores de Transcrição Forkhead , Células HeLa , Humanos , Rim/citologia , Rim/metabolismo , Dados de Sequência Molecular , Regiões Promotoras Genéticas , RNA Mensageiro/genética , Proteínas Recombinantes/genética , Ribonucleases/metabolismo , Deleção de Sequência , Transcrição Gênica , TransfecçãoRESUMO
The forkhead family of transcription factors has been shown to be involved in the regulation of embryonic development in organisms from Drosophila to humans. Genes encoding forkhead proteins are also emerging as a new class of oncogenes with relevance for human cancer. We have used a combination of fluorescence in situ hybridization and somatic cell hybrids to map the chromosomal localizations of five previously cloned human forkhead genes, freac-1, -3, -4, -5, and -6. (HGMW-approved symbols FKHL5, FKHL7, FKHL8, FKHL9 and FKHL10). We also report the sequence and the chromosomal position of a novel human forkhead gene, freac-8 (FKHL12). The freac genes have been localized to the following positions: freac-1, 16q24; freac-3, 6p25; freac-4, 5q12-q13; freac-5, 9 pericen; freac-6, 5q34, and freac-8, 1p32.
Assuntos
Proteínas Nucleares/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , Cosmídeos/genética , Cricetinae , DNA/genética , Fatores de Transcrição Forkhead , Humanos , Células Híbridas , Camundongos , Dados de Sequência MolecularRESUMO
We describe a rapid and sensitive method to isolate sets of high-affinity binding sites for sequence-specific DNA binding proteins. The DNA binding domain of the protein is expressed in Escherichia coli as a fusion with glutathione S-transferase (GST). Binding reactions are set up with total soluble extract from induced bacteria and a double-stranded oligonucleotide for which the central 32 bp have been randomized. To ensure stringent conditions, binding is done in the presence of high levels of poly(dI:C). The GST fusion protein is recovered by the addition of glutathione-Sepharose. Following extensive washing of the Sepharose beads, the bound oligonucleotides are rescued by polymerase chain reaction amplification. The amplified material is used in the next cycle of selection and amplification. Approximately five cycles are needed to obtain a pure population of high-affinity sites, which are then cloned and sequenced. This procedure should be applicable to any sequence-specific DNA binding protein for which the cDNA is available and which can be expressed in bacteria in a functional form.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Clonagem Molecular , DNA/genética , DNA/metabolismo , Escherichia coli/genética , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Humanos , Técnicas In Vitro , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The forkhead domain is a monomeric DNA binding motif that defines a rapidly growing family of eukaryotic transcriptional regulators. Genetic and biochemical data suggest a central role in embryonic development for genes encoding forkhead proteins. We have used PCR and low stringency hybridization to isolate clones from human cDNA and genomic libraries that represent seven novel forkhead genes, freac-1 to freac-7. The spatial patterns of expression for the seven freac genes range from specific for a single tissue to nearly ubiquitous. The DNA binding specificities of four of the FREAC proteins were determined by selection of binding sites from random sequence oligonucleotides. The binding sites for all four FREAC proteins share a core sequence, RTAAAYA, but differ in the positions flanking the core. Domain swaps between two FREAC proteins identified two subregions within the forkhead domain as responsible for creating differences in DNA binding specificity. Applying a circular permutation assay, we show that binding of FREAC proteins to their cognate sites results in bending of the DNA at an angle of 80-90 degrees.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição Forkhead , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Conformação de Ácido Nucleico , Homologia de Sequência de Aminoácidos , Distribuição Tecidual , Fatores de Transcrição/metabolismoRESUMO
The binding of the intercalating dye methylene blue (MB) to chromatin and to free DNA has been studied as a function of ionic strength at very low binding ratios (1 MB/400 DNA bases) using absorption spectroscopy. With increasing salt concentration MB is displaced from chromatin to a higher extent than from DNA. The free energy change for MB binding to chromatin is found to be approximately 5 kJ/mole lower than for binding to DNA. This difference can be explained by the reduced number of high affinity binding sites in chromatin due to the presence of histone proteins. The difference in binding energy is virtually independent of the degree of chromatin condensation and also of the valence of counter ions, suggesting that neither the affinity for, nor the number of intercalation sites in the linker DNA is markedly changed upon the salt-induced condensation. The unaffected thermodynamics of the linker binding suggests that factors such as DNA superhelicity and the electrostatic influence from the chromatosomes remain unchanged during chromatin condensation.