Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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In order to identify a possible hereditary predisposition to the development of obstructive pulmonary disease of unknown origin, we have looked for the presence of Cystic Fibrosis Transmembrane Regulator (CFTR) gene mutations in unrelated patients with no signs of Cystic Fibrosis (CF). We screened for 70 common mutations, and also for rare mutations by denaturing gradient gel electrophoresis analysis. In this search, different CFTR gene mutations (R75Q, delta F508, R1066C, M1137V and 3667ins4) were found in five out of 16 adult Italian patients with disseminated bronchiectasis, a significant increase over the expected frequency of carriers. Moreover, three rare CFTR gene DNA polymorphisms (G576A, R668C, and 2736 A-->G), not deemed to be the cause of CF, were found in two patients, one of which was a compound heterozygote with R1066C. These results indicate that CFTR gene mutations, and perhaps also DNA polymorphisms, may be involved in the etiopathogenesis of at least some cases of bronchiectasis.
Hum Mol Genet 1995 Apr
PMID:Increased incidence of cystic fibrosis gene mutations in adults with disseminated bronchiectasis. 754 17

Hypothetical Products from Noncoding Frames (i.e., HyPNoFs) are hypothetical, not-coded proteins, translated from alternate reading frames (i.e., coding + 1 and coding + 2) of cDNAs. HyPNoFs of CD4, PKC, oncostatin, bcl-2 proto-oncogene, tumor suppressor p53, cystic fibrosis transmembrane regulator (CFTR), and tumor necrosis factors alpha and beta were searched as query sequences vs the SWISS-PROT data bank. Homology searchers carried out revealed that hypothetical products (i.e., HyPNoFs) may share high similarity with real protein products actually coded. Sequence similarity of hypothetical products to real proteins is sometimes very high, suggesting common conformational features, according to the Sander and Schneider cutoff value. This finding supports the hypothesis that eukaryotic DNA, currently considered to be monocistronic, might occasionally have polycistronic regions, carrying different protein messages on overlapping frames. As yet, polycistronic genes have been observed in viral genomes only. The presence of polycistronic regions in eukaryotic genes is likely reminiscent of an ancient strategy, rather than a present feature of the genome in eukaryotes. These data suggest that thorough investigation of HyPNoFs is likely to improve our ability to trace genes' evolution and to investigate structure-function relationships of protein and DNA sequences.
J Mol Evol 1995 Jun
PMID:Investigating hypothetical products from noncoding frames (HyPNoFs). 754 50

Interleukin-10 (IL-10) is a potent regulatory cytokine that decreases inflammatory responses and T-cell stimulation. We have found that respiratory epithelial lining fluid (ELF) from patients with cystic fibrosis (CF) contains significantly less soluble IL-10 than ELF of healthy control subjects. Although macrophages from the chronically infected lungs of CF patients appear to be one source of IL-10, little or no intracellular IL-10 was found in bronchoalveolar lavage macrophages from healthy control subjects, suggesting that there must be another source of this cytokine in healthy lungs. We found that bronchial epithelial cells from healthy control subjects constitutively produce IL-10, which appears to be downregulated in CF patients. It is thus likely that the bronchial epithelium plays an important role in regulating the local immune response, producing IL-10 to decrease inflammation in the healthy lung. Conversely, downregulation of epithelial IL-10 production in CF airways may contribute to enhancing local inflammation and tissue damage.
Am J Respir Cell Mol Biol 1995 Sep
PMID:Normal bronchial epithelial cells constitutively produce the anti-inflammatory cytokine interleukin-10, which is downregulated in cystic fibrosis. 754 94

The absence of pathologic changes in newborn cystic fibrosis (CF) lung suggests that the fetal CF lung is inflated with a normal volume of liquid and that Cl- is secreted through paths other than the cystic fibrosis transmembrane conductance regulator (CFTR)-associated Cl- channel. We studied liquid content of distal lung and transepithelial electrical potential difference (PD) of cultured cystic tracheal explants from 16 to 19 day gestation fetal mice of CFTR (+/-)(heterozygous) females that were mated with CFTR (-/-) "knockout" males. Distal lung water content was not affected by fetal genotype. Basal PDs were not different (CFTR (+/-), 8.6 mV, and CFTR (-/-), 9.1 mV), and PDs of both groups were inhibited by intraluminal injection of amiloride (10(-4) M) (-25%) and after addition of bumetanide (10(-4) M) to the bath (-40%). Terbutaline (3 x 10(-5) M) induced a similar increase in PD (about 65%) in both groups. Intraluminal injection of ionomycin (2 x 10(-5) and 5 x 10(-6) M) raised PD in both groups (CFTR (+/-) by 32 and 27% and CFTR (-/-) by 41 and 11%). All of the increase in PD induced by terbutaline and ionomycin was inhibited by bumetanide. The PD response to terbutaline was not attenuated by pretreatment with ionomycin or the Ca2+ chelator BAPTA (10(-4) M). Ionomycin or ATP, but not terbutaline, increased intracellular Ca2+ concentration of isolated cultured tracheal epithelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Am J Respir Cell Mol Biol 1995 Sep
PMID:Cl- secretion by trachea of CFTR (+/-) and (-/-) fetal mouse. 754 95

To assess the feasibility of protein replacement as a potential therapy for cystic fibrosis, we have evaluated the ability of influenza hemagglutinin (HA) to mediate the delivery of purified cystic fibrosis transmembrane conductance regulator (CFTR) to recipient cells in vitro. CFTR was purified from both CHO cells and Sf9 cells and reconstituted into two different types of vesicular delivery vehicles. In one, CFTR and HA were co-reconstituted into the same lipid vesicle. After binding to the cell surface, delivery of CFTR to the recipient cell was achieved by a transient, low-pH activation of the fusion activity of HA. A second delivery strategy used HA virosomes together with purified CFTR that had been reconstituted into vesicles containing gangliosides, a receptor for HA. After binding of the HA virosomes and CFTR-containing vesicles to the recipient cells, delivery to the plasma membrane again was achieved by a transient pH drop. Delivery of functional CFTR was assessed using the SPQ fluorescence assay. Functional CFTR was detected in a fraction (> 20%) of the recipient cells using this assay. Quantitative binding and fusion assays using radiolabeled virosomes and lipid vesicles showed that on the order of 1,000 of the added CFTR-containing vesicles bound to each C127 cell under the conditions of our delivery protocols. However, only a fraction of these vesicles fused and delivered CFTR to the cell plasma membrane. The two delivery strategies were found to be approximately equivalent in their ability to deliver active CFTR, and there were no significant differences between deliveries using purified CFTR from either cell source. These feasibility studies suggest that purified CFTR can be delivered to a recipient cell in a functional form and therefore represent a significant step in establishing the concept of protein replacement as a therapy for cystic fibrosis.
Am J Respir Cell Mol Biol 1995 Sep
PMID:Delivery of purified, functional CFTR to epithelial cells in vitro using influenza hemagglutinin. 754 96

The purpose of this study was to determine the biochemical and molecular characteristics of mucin synthesized by cystic fibrosis cells (CFPAC-1), a pancreatic cancer cell line derived from a patient with cystic fibrosis, and pancreatic cancer (SW-1990) cell lines. High molecular weight glycoproteins (HMG) were quantified by [3H]-glucosamine labeling and chromatography on sepharose CL-4B. Mucin gene expression was determined by using cDNA probes for 2 distinct intestinal mucins (MUC2 and MUC3) and one stomach mucin (MUC1). The specific mucin core epitopes were confirmed by immunoblots using antibodies that recognize T, Tn, sialosyl Tn, MUC1, MUC2, and MUC3. The results of these experiments demonstrate that CFPAC-1 cells contained 1.25 fold and 1.4 fold more HMG in the membrane and cytosolic fractions, however, secreted 4-fold more HMG into the medium compared to SW-1990 cells. The HMG of SW-1990 was found to be mucinous in nature and not proteoglycans, as it was not susceptible to hyalurinidase, heparinase and chondroitinase ABC. The HMG of CFPAC-1 was also predominantly (80%) mucinous but with small amounts of proteoglycans. mRNA and immunoblot analysis suggest that these CFPAC-1 and SW-1990 cells predominantly express MUC1 apomucin, small amounts of MUC2 apomucin, and no MUC3. Pulse chase labeling and immunoprecipitation of MUC1 type mucin using the 139H2 monoclonal antibody demonstrated that different sizes of mucin gene product were present in both cell lines, corresponding to the known length polymorphism of this mucin. Both T and Tn antigens were significantly higher in CFPAC-1 and SW-1990 cells as compared to sialosyl Tn antigen. These findings were associated with the increased activities of polypeptidyl N-acetylgalactosaminyl transferase and b1,3-galactosyltransferase. These investigations demonstrate for the first time that cystic fibrosis cells (CFPAC-1) secrete and synthesize high amounts of mucin which is associated with high levels of MUC1 mRNA, low levels of MUC2 mRNA and non detectable MUC3 mRNA.
Biochem Mol Biol Int 1995 Feb
PMID:Cystic fibrosis and pancreatic cancer cells synthesize and secrete MUC1 type mucin gene product. 754 50

We have found that preparations of DNA isolated from purulent sputum possess a novel activity which accelerates and stabilizes the binding of human leukocyte elastase to secretory leukoprotease inhibitor, a major endogenous antielastase in the respiratory tract. DNA in sputum is derived from the nuclear debris of disintegrated inflammatory leukocytes, and can attain concentrations ranging from 10(2) to 10(4) micrograms/ml, depending on the severity of pulmonary infection and inflammation. In the presence of 23 micrograms/ml DNA, a concentration lower than those found in most purulent sputa, the rate constant for association of secretory leukoprotease inhibitor with elastase is increased to 1.1 x 10(8) M-1s-1, 44-fold greater than that in the absence of DNA. The equilibrium dissociation constant for the enzyme-inhibitor complex drops to 0.7 pM, two orders of magnitude lower than that in the absence of DNA. The accelerating effect of DNA is further increased by thermal denaturation or by modification with exonuclease III, while it is significantly reduced by digestion with S1 nuclease or by binding of Escherichia coli single-stranded DNA binding protein. The results from these experiments indicate that the structural elements in sputum DNA that are responsible for the accelerating effect have the characteristics of single-stranded sites. Similar kinetic effects on elastase inhibition were also observed with human placental DNA and genomic DNAs from a variety of other species. These findings suggest that DNA in pulmonary secretions may participate in antielastase defense by promoting the binding of secretory leukoprotease inhibitor to leukocyte elastase. The results may have important implications for use of nuclease preparations in mucolytic therapy for cystic fibrosis.
Am J Respir Cell Mol Biol 1995 Dec
PMID:Accelerated binding of secretory leukoprotease inhibitor to human leukocyte elastase mediated by single-stranded sites in DNA from tracheobronchial mucus. 757 8

Maternal uniparental disomy for the entire chromosome 7 has so far been reported in three patients with intrauterine and postnatal growth retardation. Two were detected because they were homozygous for a cystic fibrosis mutation for which only the mother was heterozygous, and one because he was homozygous for a rare COL1A2 mutation. We investigated 35 patients with either the Silver-Russell syndrome or primordial growth retardation and their parents with PCR markers to search for uniparental disomy 7. Four of 35 patients were found to have maternal disomy, including three with isodisomy and one with heterodisomy. The data confirm the hypothetical localization of a maternally imprinted gene (or more than one such gene) on chromosome 7. It is suggested to search for UPD 7 in families with an offspring with sporadic Silver-Russell syndrome or primordial growth retardation.
Hum Mol Genet 1995 Apr
PMID:Uniparental disomy 7 in Silver-Russell syndrome and primordial growth retardation. 763 7

It has been proposed that a combination of an activated adenylyl cyclase and a high concentration of a phosphodiesterase inhibitor (isobutylmethylxanthine [IBMX], 5 mM) stimulates Cl- secretion mediated by the heterologously expressed cystic fibrosis transmembrane regulator protein carrying the most common cystic fibrosis (CF) mutation (delta F508). We tested whether Cl- secretion could be stimulated by this protocol in vitro and in vivo in CF airway epithelia expressing endogenous delta F508 CFTR protein. In cultured airway preparations, forskolin (a direct adenylyl cyclase activator) stimulated Cl- secretion in amiloride-pretreated normal (delta Isc = 7.1 +/- 1.7 microA.cm-2) but not CF tissues (delta Isc = -02 +/- 0.1 microA.cm-2). Unexpectedly, IBMX partially inhibited the forskolin-induced Cl- secretion in normal tissues; IBMX addition had no effect on CF tissues. Direct measurements of cell cAMP concentrations revealed that 0.1 mM IBMX and forskolin produced the maximum levels of cell cAMP levels attainable with this drug combination, and 5 mM IBMX was without further effect. The combination of forskolin (10(-5) M) and isoproterenol, an adenylyl cyclase activator (10(-5) M), produced approximately 3 times higher levels of cAMP than forskolin/IBMX but also did not induce Cl- secretion in CF tissues. Studies of Cl- secretion in vivo, assessed by the transepithelial electric potential difference (PD), showed that isoproterenol (10(-5) M) stimulated Cl- secretion (delta PD = -16.3 +/- 4.3 mV; n = 4) in nasal epithelia of normal subjects but not in CF patients homozygous for the delta F508 mutation (delta PD = -2.6 +/- 1.9 mV; n = 5).(ABSTRACT TRUNCATED AT 250 WORDS)
Am J Respir Cell Mol Biol 1993 Apr
PMID:Isobutylmethylxanthine fails to stimulate chloride secretion in cystic fibrosis airway epithelia. 768 24

The development of transformed human airway epithelial cell lines has been important in advancing the understanding of the biochemical and genetic mechanisms underlying the cystic fibrosis (CF) defect. Since the most common mutation associated with CF is a phenylalanine deletion at position 508 (delta F508) in the CF transmembrane conductance regulator (CFTR) gene, a transformed airway epithelial cell line homozygous for this mutation will be important for determining the biologic significance of this mutation in the airways. We report the genotypic and phenotypic characterization of a delta F508 homozygote cell line derived from luminal epithelium in the trachea. The cells were transformed with a plasmid containing an origin of replication defective SV40 genome and have progressed through crisis. Immunocytochemical characterization of the cells shows that they express keratin, indicating epithelial cell origin, and that a calcium-dependent cell adhesion molecule, cellCAM 120/80, is present at plasma membrane junctions between cells. Electrophysiologically, the cells show no cAMP-dependent Cl transport. However, after treatment with the calcium ionophore, ionomycin, cells secrete Cl, albeit at a lower level than that observed in normal cells. Genetically, the cells express CFTR mRNA as determined by polymerase chain reaction amplification and CFTR protein as determined by Western hybridization analysis. Karyotypic analysis shows that 70% of the cells contain two copies of chromosome 7.
Am J Respir Cell Mol Biol 1993 May
PMID:An immortalized cystic fibrosis tracheal epithelial cell line homozygous for the delta F508 CFTR mutation. 768 97


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