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:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cystic fibrosis transmembrane conductance regulator (CFTR) is expressed at low levels in nonepithelial cells. Recently, we demonstrated that CFTR is responsible for cell cycle-dependent adenosine 3',5'-cyclic monophosphate-responsive Cl- permeability in lymphocytes. Agonist responsiveness of cystic fibrosis (CF) lymphocytes was restored by transfection with plasmid containing wild type CFTR cDNA. CFTR mRNA is expressed in the B lymphoid cell line GM03299; however, quantitative reverse transcriptase-polymerase chain reaction indicates that the level of CFTR mRNA is at least 1,000 times lower than in T84 cells. CFTR protein could not be detected by Western blot or by immunoprecipitation of in vitro phosphorylated protein. However, antisense oligonucleotides representing codons 1-12 of CFTR caused a complete inhibition of cell cycle-dependent Cl-permeability [as determined by 6-methoxy-N-(3-sulfopropyl)-quinolinium fluorescence digital-imaging microscopy], thereby inducing normal cells to acquire a "CF phenotype." These studies provide direct evidence that a CFTR-associated Cl- permeability is present and measurable in lymphocytes, even though CFTR mRNA and protein are expressed at low levels.
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PMID:Antisense oligonucleotides to CFTR confer a cystic fibrosis phenotype on B lymphocytes. 128 96

The haploid nucleus of a human cell contains 3 X 10(9) base pairs. Organized in linear duplex, this DNA would stretch out to a length of some 90 cm. Thus, organization of chromosomes has been a major subject for pioneer cytogenetists. Long lasting controversies on the strandedness of chromosomes, together with newly developed banding techniques, led us to molecular cytogenetics. Next, the discovery of reverse transcriptase, restriction endonucleases, and other recombinant DNA methods have enabled us to isolate and characterize genes from any organism and to determine the DNA sequences and any encoded protein sequences. These new technologies have already helped us to understand many inherited diseases at a molecular level. In sickle cell anemia, thalassemia and in other mendelian disorders we can know their molecular defects by examining the DNA from peripheral leukocytes, without the need for complex biochemical assays or biopsies. Southern blot analysis using restriction endonuclease and a probe is a basic tool for molecular diagnosis. cDNA or DNA fragments are used as probes. Recently, synthesized oligonucleotide probes are available, if the DNA sequence of a gene is determined. In addition, restriction fragment length polymorphisms (RFLPs), play a very important role in the molecular diagnosis. Linkage analysis using RFLPs linked to the gene locus of a certain disease also permits the detection of the patients and carriers within families with genetic diseases of unknown cause. Starting with the genetic map and physical map, genes for cystic fibrosis and Duchenne muscular dystrophy have recently been isolated and cloned.
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PMID:[Recent advances in human molecular genetics]. 197 24

Biologically active RNA has been isolated from several tissues implicated in the pathogenesis of the genetic disease cystic fibrosis. To ensure that mRNA from stomach and pancreas has not been degraded, it must be isolated within 2 h postmortem, while intact RNA can be isolated from lung material up to 20 h postmortem. It is imperative that tissue removed during autopsy be dispersed in a strong denaturant buffer (guanidine isothiocyanate) to inactivate nucleases. While stomach and pancreas yields of RNA are low, relative to the amount of gland material, yields of RNA from lung samples are sufficient for the further isolation of mRNA by oligo(dT)-cellulose chromatography. The biological integrity of subsequently isolated mRNA has been assessed by AMV-reverse transcriptase cDNA synthesis as well as by in vitro translation. Protein products generated in this manner show distinct differences from mRNA translation patterns of age- and sex-matched controls when compared with mRNA from CF samples. R0t analysis of cDNA libraries thus generated indicates that the complexity of such libraries is representative of the starting RNA species.
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PMID:Isolation of biologically active RNA from human autopsy for the study of cystic fibrosis. 376 47

The effect of nonsense mutations on mRNA levels is variable. The levels of some mRNAs are not affected and truncated proteins are produced, while the levels of others are severely decreased and null phenotypes are observed. The effect on mRNA levels is important for the understanding of phenotype-genotype association. Cystic fibrosis (CF) is a lethal autosomal recessive disease with variable clinical presentation. Recently, two CF patients with mild pulmonary disease carrying nonsense mutations (R553X, W1316X) were found to have severe deficiency of mRNA. In the Jewish Ashkenazi CF patient population, 60% of the chromosomes carry a nonsense mutation, W1282X. Patients homozygous for this mutation have severe disease presentation with variable pulmonary disease. The presence of CF transcripts in a group of patients homozygous and heterozygous for this mutation was studied by reverse transcriptase PCR of various regions of the gene. Subsequent hybridization to specific CF PCR probes and densitometry analysis indicated that the CF mRNA levels in patients homozygous for the W1282X mutation are not significantly decreased by the mutation. mRNA levels were compared for patients heterozygous for the W1282X mutation. The relative levels of mRNA with the W1282X, and the delta F508 or the normal alleles, were similar in each patient. These results indicate that the severe clinical phenotype of patients carrying the W1282X mutation is not due to a severe deficiency of mRNA. In addition, the severity, progression, and variability of the pulmonary disease are affected by other, as yet unknown factors.
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PMID:Similar levels of mRNA from the W1282X and the delta F508 cystic fibrosis alleles, in nasal epithelial cells. 751 81

In previous studies we have characterized the expression of the cystic fibrosis transmembrane conductance regulator (CFTR) protein in clathrin-coated vesicles derived from bovine brain and in neurons of rat brain. In this study we have further characterized the expression of the CFTR protein mRNA and protein in rat brain with reverse transcriptase polymerase chain reaction amplification (RT-PCR), in situ hybridization, and immunocytochemistry. The expression of CFTR mRNA and protein in discrete areas of brain, including the hypothalamus, thalamus, and amygdaloid nuclei, which are involved in regulation of appetite and resting energy expenditure, is identical. The presence of CFTR in neurons localized to these regions of brain controlling homeostasis and energy expenditure may elucidate the pathogenesis of other nonpulmonary and gastrointestinal manifestations which commonly are observed in children with cystic fibrosis. Dysregulation of normal neuropeptide vesicle trafficking by mutant CFTR in brain may serve as a pathogenic mechanism for disruption of homeostasis.
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PMID:Expression and localization of the cystic fibrosis transmembrane conductance regulator mRNA and its protein in rat brain. 754 88

A role for adenosine in the regulation of ion transport in pulmonary epithelial cells has recently been proposed. Although evidence exists documenting the presence and function of adenosine A2 receptors in airway epithelia, the presence of adenosine A1 receptors remains controversial. The present study used reverse transcriptase-polymerase chain reaction (PCR) and whole cell patch-clamp analysis to investigate A1 receptor presence and function in normal and cystic fibrosis (CF) human airway epithelial cells. Oligonucleotide primers complementary to the human brain A1 receptor sequence generated a PCR product of the predicted size (311 bp) in normal tracheal (9HTEo-) and CF submucosal (2CFSMEo-) airway cell lines and in primary cultures of CF nasal polyp epithelial cells. An oligonucleotide probe internal to the PCR primers hybridized with the 311-bp cDNAs by Southern blot analysis. cDNA sequencing demonstrated that the normal and CF airway cell PCR products are 100% identical to the corresponding sequence of the human brain adenosine A1 receptor. Northern blot analysis of 9HTEo-and 2CFSMEo- poly(A)+ RNA revealed the presence of two bands of approximately 3.0 and approximately 5.5 kb corresponding to the A1 receptor. Whole cell patch-clamp analyses demonstrated that 8-cyclopentyl-1,3-dipropylxanthine, a specific A1 receptor antagonist, increases adenosine 3',5'-cyclic monophosphate (cAMP)-activated Cl- conductance in 9HTEo-airway cells and allows cAMP to increase Cl- conductance in 2CFSMEo- CF airway cells and CF nasal polyp epithelial cells in primary culture. These results provide evidence for the presence and function of A1 receptors in normal and CF airway epithelial cells and provide support for a role of adenosine A1 receptors in modulating airway epithelial cell Cl- transport.
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PMID:Identification and function of A1 adenosine receptors in normal and cystic fibrosis human airway epithelial cells. 761 73

Human neutrophil elastase (NE) stimulates release of neutrophil chemotactic activity by a bronchial epithelial cell line and from nasal epithelial cells. In this article, we show that NE stimulates the production of neutrophil chemotactic activity by 2CFSMEo-cells, a transformed cystic fibrosis bronchial epithelial cell line. The production of chemotactic activity is dose- and time-dependent and can be blocked by preincubation of NE with alpha 1 antitrypsin (alpha1AT). Incubation of the NE-stimulated culture supernatant with neutralizing concentrations of rabbit anti-human interleukin 8 antibody completely neutralizes the chemotactic activity. Transfection of 2CFSMEo- cells with the eukaryotic expression vector pCMV4alpha1AT, complexed to cationic liposomes in a 1:3 wt/wt ratio, results in at least a 10-fold increase in measured human alpha1AT protein in culture supernatant. Detection of human alpha1AT mRNA by reverse transcriptase polymerase chain reaction in total RNA from transfected, but not untransfected cells, confirms successful gene transfer. Compared with untransfected cells, transfer of the human alpha1AT gene decreases chemotactic activity in culture supernatant and prevents cell detachment after NE exposure. Our data indicate that alpha1AT gene transfer is capable of blocking at least some of the biological effects of free elastase on cultured epithelial cells.
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PMID:Plasmid-liposome transfer of the alpha 1 antitrypsin gene to cystic fibrosis bronchial epithelial cells prevents elastase-induced cell detachment and cytokine release. 860 Sep 39

A novel approach to quantitative reverse transcriptase polymerase chain reaction (QC RT-PCR) using real time detection and the 5' nuclease assay has been developed. Cystic fibrosis transmembrane transductance regulator (CFTR) target mRNA is reverse transcribed, amplified, detected, and quantitated in real time. A fluorogenic probe was designed to detect the CFTR amplicon. Relative increase in 6-carboxy-fluorescein reporter fluorescent emission is monitored during PCR amplification using an analytical thermal cycler. An internal control template containing the same primer sequences as the CFTR amplicon, but a different internal sequence, has been designed as a control. An internal control probe with a reporter fluorescent dye tetrachloro-6-carboxy-fluorescein was designed to hybridize to the internal control amplicon. The internal control template is placed in each reaction tube and is used for quantitative analysis of the CFTR mRNA. This method provides a convenient and high-throughput format for QC RT-PCR.
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PMID:A novel method for real time quantitative RT-PCR. 890 19

Mutations in a Cl- channel (cystic fibrosis transmembrane conductance regulator or CFTR) are responsible for the cystic fibrosis (CF) phenotype. Increased Na+ transport rates are observed in CF airway epithelium, and recent studies suggest that this is due to an increase in Na+ channel open probability (Po). The Xenopus renal epithelial cell line, A6, expresses both cAMP-activated 8-picosiemen (pS) Cl- channels and amiloride-sensitive 4-pS Na+ channels, and provides a model system for examining the interactions of CFTR and epithelial Na+ channels. A6 cells express CFTR mRNA, as demonstrated by reverse transcriptase-polymerase chain reaction and partial sequence analysis. A phosphorothioate antisense oligonucleotide, complementary to the 5' end of the open reading frame of Xenopus CFTR, was used to inhibit functional expression of CFTR in A6 cells. Parallel studies utilized the corresponding sense oligonucleotide as a control. CFTR protein expression was markedly reduced in cells incubated with the antisense oligonucleotide. Incubation of A6 cells with the antisense oligonucleotide led to inhibition of forskolin-activated amiloride-insensitive short circuit current (Isc). After a 30-min exposure to 10 microM forskolin, 8-pS Cl- channel activity was detected in only 1 of 31 (3%) cell-attached patches on cells treated with antisense oligonucleotide, compared to 5 of 19 (26%) patches from control cells. A shift in the single-channel current-voltage relationship derived from antisense-treated cells was also consistent with a reduction in Cl- reabsorption. Both amiloride-sensitive Isc and Na+ channel Po were significantly increased in antisense-treated, forskolin-stimulated A6 cells, when compared with forskolin-stimulated controls. These data suggest that the regulation of Na+ channels by CFTR is not limited to respiratory epithelia and to epithelial cells in culture overexpressing CFTR and epithelial Na+ channels.
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PMID:Expression of the cystic fibrosis phenotype in a renal amphibian epithelial cell line. 899 2

The gene product affected in cystic fibrosis, the cystic fibrosis transmembrane conductance regulator (CFTR), is a chlorideselective ion channel that is regulated by cAMP-dependent protein kinase-mediated phosphorylation, ATP binding and ATP hydrolysis. Mutations in the CFTR gene may result in cystic fibrosis characterized by severe pathology (e.g. recurrent pulmonary infection, male infertility and pancreatic insufficiency) involving organs expressing the CFTR. Interestingly, in the kidney, where expression of the CFTR has been reported, impaired ion transport in patients suffering from cystic fibrosis could not be observed. To understand the role of the CFTR in chloride transport in the kidney, we attempted to identify an epithelial cell line that can serve as a model. We demonstrate that the CFTR is expressed constitutively in Madine-Darby canine kidney (MDCK) type I cells, which are thought to have originated from the distal tubule of the dog nephron. We show expression at the mRNA level, using reverse transcriptase-PCR, and at the protein level, using Western blot analysis with three different monoclonal antibodies. Iodide efflux measurements indicate that CFTR expression confers a plasma membrane anion conductance that is responsive to stimulation by cAMP. The cAMP-stimulated iodide release is sensitive to glybenclamide, diphenylamine carboxylic acid and 5-nitro-2-(3-phenylpropylamino)benzoic acid, but not to 4,4'-di-isothiocyanostilbene-2,2'-disulphonic acid, an inhibitor profile characteristic of the CFTR chloride channel. Finally, the polarized localization of the CFTR to the apical plasma membrane was established by iodide efflux measurements and cell-surface biotinylation on MDCK I monolayers. Interestingly, MDCK type II cells, which are thought to have originated from the proximal tubule of the kidney, lack CFTR protein expression and cAMP-stimulated chloride conductance. In conclusion, we propose that MDCK type I and II cells can serve as convenient model systems to study the physiological role and differential expression of CFTR in the distal and proximal tubule respectively.
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PMID:Functional expression and apical localization of the cystic fibrosis transmembrane conductance regulator in MDCK I cells. 907 71


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