Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.21.1 (DNase)
7,655 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Protein kinase A (PKA)-activation of epithelial Na+ channels requires actin filaments. Mouse mammary adenocarcinoma cells expressing the human cystic fibrosis transmembrane conductance regulator (CFTR) or mock transfectants were used to determine whether CFTR is also modulated by the actin cytoskeleton. The actin filament disrupter cytochalasin D (CD; approximately 5 micrograms/ml) readily activated whole cell currents in CFTR but not in mock-transfected (MOCK) cells. Addition of actin to the cytosolic side of quiescent excised inside-out patches of CFTR but not MOCK cells also activated CFTR. The actin-activated Cl- channels (symmetrical Cl-) had a linear conductance of 9.3 pS and were inhibited by diphenylamine-2-carboxylate and monoclonal antibodies raised against CFTR. Channel activity was also blocked by addition of the actin-binding proteins deoxyribonuclease I and filamin. Incubation of CFTR cells with CD (approximately 15 micrograms/ml) for > 6 h prevented CFTR activation by the addition of either 8-bromoadenosine 3',5'-cyclic monophosphate plus forskolin under whole cell conditions or PKA under excised inside-out conditions. However, CFTR activation was restored by subsequent addition of actin. The data indicate that CFTR is regulated by actin filaments whose effect may, in turn, be associated with the PKA-dependent pathway.
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PMID:cAMP-independent regulation of CFTR by the actin cytoskeleton. 754 42

The cystic fibrosis transmembrane conductance regulator gene (CFTR) exhibits a complex pattern of expression that shows temporal and spatial regulation, although the control mechanisms are not fully known. We have mapped DNase-I-hypersensitive sites (DHSs) flanking the CFTR gene with the aim of identifying potential regulatory elements. We previously characterized DHSs at -79.5 and -20.9 kb with respect to the CFTR translational start site and a regulatory element in the first intron of the gene at 185+10 kb. We have now mapped five DHSs lying 3' to the CFTR gene at 4574+5.4, +6.8, +7.0, +7.4 and +15.6 kb that show some degree of tissue specificity. The DHSs are seen in chromatin extracted from human primary epithelial cells and cell lines; the presence of the +15.6 kb site is tissue-specific in transgenic mice carrying a human CFTR yeast artificial chromosome. Further analysis of the 4574+15.6 kb DHS implicates the involvement of CCAAT-enhancer-binding protein (C/EBP), cAMP-response-element-binding protein (CREB)/activating transcription factor (ATF) and activator protein 1 (AP-1) family transcription factors at this regulatory element.
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PMID:Analysis of DNase-I-hypersensitive sites at the 3' end of the cystic fibrosis transmembrane conductance regulator gene (CFTR). 1041 23

Cystic fibrosis (CF) is characterized by the presence of a viscoelastic mucus layer in the upper airways and bronchi. The underlying problem is a mutation in the gene encoding the cystic fibrosis transmembrane conductance regulator protein. Clinical studies of gene transfer for CF are ongoing. For gene delivery to the airways of CF patients to be effective, the mucus covering the target cells must be overcome. We therefore examined the extent to which CF sputum presents a physical barrier to the transport of nanospheres of a size comparable to that of lipoplexes and other transfection systems currently being clinically evaluated for CF gene therapy. We observed that an extremely low percentage of nanospheres (< 0.3%) moved through a 220-microm-thick CF sputum layer after 150 min. The largest nanospheres studied (560 nm) were almost completely blocked by the sputum, whereas the smaller nanospheres (124 nm) were retarded only by a factor of 1.3 as compared with buffer. Surprisingly, the nanospheres diffused significantly more easily through the more viscoelastic sputum samples. We hypothesize that the structure of the network in sputum becomes more macroporous when the sputum becomes more viscoelastic. Sputum from a patient with chronic obstructive pulmonary disease retarded the transport of nanospheres to the same extent as did CF sputum. When directly mixed with CF sputum, recombinant human deoxyribonuclease I moderately facilitated the transport of nanospheres through CF sputum.
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PMID:Cystic fibrosis sputum: a barrier to the transport of nanospheres. 1106 33

Cystic fibrosis (CF) is one of the most common autosomal recessive disorders in North America, leading to significant morbidity and early mortality. The defect in the cystic fibrosis transmembrane conductance regulator protein (CFTR) function can be corrected in vitro by gene replacement with a wild-type gene. A Phase I, single administration, dose escalation trial was designed and executed to assess safety and delivery of tgAAVCF, an adeno-associated virus (AAV) vector encoding the human CFTR cDNA, by nebulization to the lungs of CF subjects. Four cohorts of three subjects each were administered increasing doses of the study agent, beginning with 10(10) DNase-resistant particles (DRP) and escalating in log increments up to 10(13) DRP. Sequential bronchoscopies were performed to gather analytical samples throughout the study. All 12 subjects completed the study. There were a total of 242 adverse events (AEs), six of which were defined as serious and three of which were defined as possibly being related to the study drug. A clear dose-response relationship was observed in vector gene transfer. A maximum of 0.6 and 0.1 vector copies per brushed cell were observed 14 days and 30 days, respectively, following nebulization of 10(13) DRP tgAAVCF, and this declined to nearly undetectable levels by day 90. Vector gene transfer was evenly distributed throughout the fourth airway generation following single-dose administration. RNA-specific PCR did not detect vector-derived mRNA. This Phase I trial shows that aerosolized tgAAVCF is safe and widely delivered to the proximal airways of CF subjects by nebulization.
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PMID:A phase I study of aerosolized administration of tgAAVCF to cystic fibrosis subjects with mild lung disease. 1158 32

The median estimated life expectancy of children with cystic fibrosis (CF) born in 1990 is 40 years which represents a doubling in the last 20 years, and nearly half of all patients are now adults. Since the identification of the gene, more than 1000 gene mutations have been discovered. This gene encodes for the cystic fibrosis transmembrane conductance regulator (CFTR), a protein that is thought to have a role in ion transport, mucus rheology, inflammation and bacterial adherence. Various therapeutic mechanisms are currently being investigated in an attempt to overcome these abnormalities. Recombinant human DNase is beneficial in many patients and the use of anti-inflammatory agents such as steroids, ibuprofen and macrolides have potential. Despite these advances in treatment it is essential that these patients are diagnosed early. Whilst the case for neonatal screening is not absolutely conclusive the evidence is highly suggestive that it would be beneficial. It may be that, in the future, new treatments such as gene therapy will be more effective in those patients who have not yet developed lung disease. Whilst gene therapy and other new treatments such as bilateral living lobar lung donation give our patients optimism for the future it is important to remember that the increase in survival is a result of good physiotherapy, nutrition, aggressive antibiotic use and an increase in our understanding of the disease. It is important that patients continue to be referred early to tertiary CF centres.
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PMID:Cystic fibrosis: review of the decade. 1166 4

Recombinant adeno-associated serotype 2-based vectors (rAAV2) possess a number of theoretical advantages for cystic fibrosis (CF) gene therapy because they elicit little or no inflammatory response and generally result in stable expression. rAAV2 vectors expressing the cystic fibrosis transmembrane conductance regulator (CFTR) gene have previously been shown to mediate stable correction of the CF defect in CF bronchial epithelial cells and stable expression of CFTR in rabbit and nonhuman primate models. Here we report the results of the first trial initiated with rAAV in humans, a phase I study in 25 adult and adolescent CF patients with mild to moderate lung disease. Doses of the rAAV-CFTR vector (tgAAVCF) ranging from 3 x 10(1) to 1 x 10(9) replication units (RU), which is equivalent to approximately 6 x 10(4) to 2 x 10(12) DNase resistant particles (DRP), were administered to one side of the nose and to the superior segment of the lower lobe of the right lung. Several adverse events were noted prior to and/or after vector delivery, but most of them appeared to be related to the endogenous CF lung disease or a result of the bronchoscopic procedures. Only one of the serious events was judged to be possibly vector-related (based on temporal association), and this event was a pulmonary exacerbation very similar to several others experienced by the same subject in the three months preceding vector delivery. Vector shedding was minimal throughout the study, and serum-neutralizing antibodies were detected after vector delivery to subjects in the highest dosage cohorts. Gene transfer as measured by DNA polymerase chain reaction (PCR) was not observed until cohort 10 in nasal and bronchial epithelia. Sporadic low-level copy numbers suggested gene transfer of anywhere from 0.002 copies per cell up to 0.5 copies per cell was possible; however, DNA PCR was positive in lungs prior to direct dosing suggesting aspiration from the nasal dosing. These data indicate the need for continued evaluation of rAAV-CFTR vectors in additional clinical trials.
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PMID:Phase I trial of intranasal and endobronchial administration of a recombinant adeno-associated virus serotype 2 (rAAV2)-CFTR vector in adult cystic fibrosis patients: a two-part clinical study. 1288 47

The CFTR (cystic fibrosis transmembrane conductance regulator) gene shows a complex pattern of expression with tissue-specific and temporal regulation. However, the genetic elements and transcription factors that control CFTR expression are largely unidentified. The CFTR promoter does not confer tissue specificity on gene expression, suggesting that there are regulatory elements outside the upstream region. Analysis of potential regulatory elements defined as DNase 1-hypersensitive sites within introns of the gene revealed multiple predicted binding sites for the HNF1alpha (hepatocyte nuclear factor 1alpha) transcription factor. HNF1alpha, which is expressed in many of the same epithelial cell types as CFTR and shows similar differentiation-dependent changes in gene expression, bound to these sites in vitro. Overexpression of heterologous HNF1alpha augmented CFTR transcription in vivo. In contrast, antisense inhibition of HNF1 alpha transcription decreased the CFTR mRNA levels. Hnf1 alpha knockout mice showed lower levels of CFTR mRNA in their small intestine in comparison with wild-type mice. This is the first report of a transcription factor, which confers tissue specificity on the expression of this important disease-associated gene.
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PMID:HNF1alpha is involved in tissue-specific regulation of CFTR gene expression. 1465 22

An airway-selective DNase-hypersensitive site (DHS) at kb -35 (DHS-35kb) 5' to the cystic fibrosis transmembrane conductance regulator (CFTR) gene is evident in many lung cell lines and primary human tracheal epithelial cells but is absent from intestinal epithelia. The DHS-35kb contains an element with enhancer activity in 16HBE14o- airway epithelial cells and is enriched for monomethylated H3K4 histones (H3K4me1). We now define a 350-bp region within DHS-35kb which has full enhancer activity and binds interferon regulatory factor 1 (IRF1) and nuclear factor Y (NF-Y) in vitro and in vivo. Small interfering RNA (siRNA)-mediated depletion of IRF1 or overexpression of IRF2, an antagonist of IRF1, reduces CFTR expression in 16HBE14o- cells. NF-Y is critical for maintenance of H3K4me1 enrichment at DHS-35kb since depletion of NF-YA, a subunit of NF-Y, reduces H3K4me1 enrichment at this site. Moreover, depletion of SETD7, an H3K4 monomethyltransferase, reduces both H3K4me1 and NF-Y occupancy, suggesting a requirement of H3K4me1 for NF-Y binding. NF-Y depletion also represses Sin3A and reduces its occupancy across the CFTR locus, which is accompanied by an increase in p300 enrichment at multiple sites. Our results reveal that the DHS-35kb airway-selective enhancer element plays a pivotal role in regulation of CFTR expression by two independent regulatory mechanisms.
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PMID:Immune mediators regulate CFTR expression through a bifunctional airway-selective enhancer. 2368 37

The epithelium lining the epididymis has a pivotal role in ensuring a luminal environment that can support normal sperm maturation. Many of the individual genes that encode proteins involved in establishing the epididymal luminal fluid are well characterized. They include ion channels, ion exchangers, transporters, and solute carriers. However, the molecular mechanisms that coordinate expression of these genes and modulate their activities in response to biological stimuli are less well understood. To identify cis-regulatory elements for genes expressed in human epididymis epithelial cells, we generated genome-wide maps of open chromatin by DNase-seq. This analysis identified 33,542 epididymis-selective DNase I hypersensitive sites (DHS), which were not evident in five cell types of different lineages. Identification of genes with epididymis-selective DHS at their promoters revealed gene pathways that are active in immature epididymis epithelial cells. These include processes correlating with epithelial function and also others with specific roles in the epididymis, including retinol metabolism and ascorbate and aldarate metabolism. Peaks of epididymis-selective chromatin were seen in the androgen receptor gene and the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which has a critical role in regulating ion transport across the epididymis epithelium. In silico prediction of transcription factor binding sites that were overrepresented in epididymis-selective DHS identified epithelial transcription factors, including ELF5 and ELF3, the androgen receptor, Pax2, and Sox9, as components of epididymis transcriptional networks. Active genes, which are targets of each transcription factor, reveal important biological processes in the epididymis epithelium.
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PMID:A genome-wide analysis of open chromatin in human epididymis epithelial cells reveals candidate regulatory elements for genes coordinating epididymal function. 2400 78

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF), but are not good predictors of lung phenotype. Genome-wide association studies (GWAS) previously identified additional genomic sites associated with CF lung disease severity. One of these, at chromosome 11p13, is an intergenic region between Ets homologous factor (EHF) and Apaf-1 interacting protein (APIP). Our goal was to determine the functional significance of this region, which being intergenic is probably regulatory. To identify cis-acting elements, we used DNase-seq and H3K4me1 and H3K27Ac ChIP-seq to map open and active chromatin respectively, in lung epithelial cells. Two elements showed strong enhancer activity for the promoters of EHF and the 5' adjacent gene E47 like ETS transcription factor 5 (ELF5) in reporter gene assays. No enhancers of the APIP promoter were found. Circular chromosome conformation capture (4C-seq) identified direct physical interactions of elements within 11p13. This confirmed the enhancer-promoter associations, identified additional interacting elements and defined topologically associating domain (TAD) boundaries, enriched for CCCTC-binding factor (CTCF). No strong interactions were observed with the APIP promoter, which lies outside the main TAD encompassing the GWAS signal. These results focus attention on the role of EHF in modifying CF lung disease severity.
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PMID:Regulatory dynamics of 11p13 suggest a role for EHF in modifying CF lung disease severity. 2854 69


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