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)

The oncogene-inducible secreted T1-S glycoprotein is overexpressed in invasive breast carcinomas in mice. As yet, nothing is known about the expression of T1-S in spontaneously occurring human cancers. A report follows on the overexpression of T1-S mRNA in 67% of primary invasive lymph node-negative breast carcinomas (31 of 46 patients) as determined by quantitative reverse transcriptase polymerase chain reaction. Overexpression of T1-S mRNA was independent of the tumor size, the histologic tumor type, and the estrogen-and progesterone-receptor status but was associated with high to moderate differentiation of the tumors (G , G2). T1-S mRNA levels were low to nondetectable in resting normal mammary tissue and benign fibrocystic disease of the breast. Immunohistochemistry confirmed a low to moderate T1 immunoreactivity in epithelial cells of resting mammary tissue and benign fibrocystic disease and highly variable levels of T1 immunoreactivity in breast carcinoma cells. Kaplan-Meier analysis of disease-free survival during a median observation period of 61 months revealed a trend toward a reduced relapse rate and an extended relapse-free survival period for T1-S mRNA--overexpressing breast carcinomas. It is concluded that overexpression of T1-S receptor in lymph node-negative breast cancer may be a potential indicator for tumors with a low metastatic potential.
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PMID:Tumor-associated overexpression of the soluble T1-S receptor in lymph node-negative breast cancer. 1071 10

Cystic fibrosis is the most common inherited disease in the Caucasian population. About 70% of all CF chromosomes carry the DeltaF508 mutation, a 3-bp deletion that results in the loss of a phenylalanine at amino acid 508 in the CF transmembrane conductance regulator (CFTR) protein. Direct modification of the DeltaF508 locus of endogenous CFTR was achieved by small fragment homologous replacement (SFHR). Transformed human airway epithelial cells (CFBE41o(-)), homozygous for DeltaF508 mutation, were transfected with small fragments (491-bp) of wild-type (WT) CFTR DNA comprising exon 10 and the flanking introns. The DNA fragments were in a liposome-DNA complex at a charge ratio of 6:1 (+:-), respectively). The population of transfected cells was subcloned by limiting dilution at approximately 1 cell/well in 96-well plates. Individual colonies were isolated and analyzed. The DNA from several colonies was characterized by radiolabeled, nonallele-specific and radiolabeled, allele-specific PCR amplification, as well as by genomic DNA fingerprinting. The CFTR-WT allele was detected in five of these colonies by allele-specific PCR amplification thus indicating that the cell lines carried both WT and DeltaF alleles. DNA fingerprint analysis confirmed that the colonies were isogenic and derived from the parental CFBE41o(-) cell line. Although, the WT allele was detected by allele-specific PCR, it was not detected initially when the same samples were analyzed by non allele-specific PCR. A sensitivity assay, mixing the genomic DNA of wild-type (16HBE14o(-)) and mutant (CFBE41o(-)) cell lines, indicated that the allele-specific PCR was at least 25-fold more sensitive than non allele-specific PCR. These results suggest that the colony is not yet clonal, but still contains a population of parental, CFBE41o(-) cells that have not been modified. Based on the mixing analysis, the proportion of corrected cells appears to be between 1 and 10% of the total population. Nonallele-specific reverse transcriptase PCR (RT-PCR) analysis of the CFTR mRNA indicated that two of the colonies expressed both WT and DeltaF508 CFTR mRNA, while one colony appeared to express only the WT mRNA. The mRNA results were confirmed by sequence analysis of 3' end primer extension products from the mRNA of CFTR exon 10 showing that the mRNA containing exon 10. Furthermore, a survey of primer extension products indicated no random insertion of the fragment in an expressed gene. This study demonstrates SFHR-mediated modification of the DeltaF508 allele in DeltaF508 homozygote human airway epithelial cells over multiple generations. The resultant cells express WT-CFTR mRNA and can be subcloned further to isolate isogenic clonal populations of cells.
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PMID:Isolation of CF cell lines corrected at DeltaF508-CFTR locus by SFHR-mediated targeting. 1203 87

Human airway epithelia express Ca2+-activated Cl- channels (CaCC) that are activated by extracellular nucleotides (ATP and UTP). CaCC is preserved and seems to be up-regulated in the airways of cystic fibrosis (CF) patients. In the present study, we examined the role of basolateral K+ channels in CaCC-mediated Cl- secretion in native nasal tissues from normal individuals and CF patients by measuring ion transport in perfused micro Ussing chambers. In the presence of amiloride, UTP-mediated peak secretory responses were increased in CF compared with normal nasal tissues. Activation of the cAMP pathway further increased CaCC-mediated secretion in CF but not in normal nasal mucosa. CaCC-dependent ion transport was inhibited by the chromanol 293B, an inhibitor of cAMP-activated hKvLQT1 K+ channels, and by clotrimazole, an inhibitor of Ca2+-activated hSK4 K+ channels. The K+ channel opener 1-ethyl-2-benzimidazolinone further increased CaCC-mediated Cl- secretion in normal and CF tissues. Expression of hSK4 as well as hCACC-2 and hCACC-3 but not hCACC-1 was demonstrated by reverse transcriptase PCR on native nasal tissues. We conclude that Ca2+-activated Cl- secretion in native human airway epithelia requires activation of Ca2+-dependent basolateral K+ channels (hSK4). Co-activation of hKvLQT1 improves CaCC-mediated Cl- secretion in native CF airway epithelia, and may have a therapeutic effect in the treatment of CF lung disease.
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PMID:Modulation of Ca2+-activated Cl- secretion by basolateral K+ channels in human normal and cystic fibrosis airway epithelia. 1261 94

In this study, we utilized the reverse transcriptase component of telomerase, hTERT, and human papillomavirus type 16 (HPV-16) E6 and E7 genes to transform normal and cystic fibrosis (CF) human airway epithelial (HAE) cells. One cell line, designated NuLi-1 (normal lung, University of Iowa), was derived from HAE of normal genotype; three cell lines, designated CuFi (cystic fibrosis, University of Iowa)-1, CuFi-3, and CuFi-4, were derived from HAE of various CF genotypes. When grown at the air-liquid interface, the cell lines were capable of forming polarized differentiated epithelia that exhibited transepithelial resistance and maintained the ion channel physiology expected for the genotypes. The CF transmembrane conductance regulator defect in the CuFi cell lines could be corrected by infecting from the basolateral surface using adenoviral vectors. Using nuclear factor-kappaB promoter reporter constructs, we also demonstrated that the NuLi and CuFi cell lines retained nuclear factor-kappaB responses to lipopolysaccharide. These cell lines should therefore be useful as models for studying ion physiology, therapeutic intervention for CF, and innate immunity.
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PMID:Development of cystic fibrosis and noncystic fibrosis airway cell lines. 1267 69

Burkholderia cepacia is a prevalent pulmonary pathogen in patients with cystic fibrosis (CF). The lung pathology observed in patients with CF is postulated to be due to an overexpression of chemokines. This study investigated the induction of the neutrophil chemoattractant chemokine IL-8 and the signaling pathways activated by B. cepacia-infected human lung epithelial A549 (HLE) cells. Cells were infected with B. cepacia (genomovar III of the B. cepacia complex), and reverse transcriptase-PCR and ELISA for the cytokines were performed. B. cepacia (multiplicity of infection > or =4:1) induced HLE cells to significantly secrete IL-8 in a more potent manner than the predominant CF pathogen Pseudomonas aeruginosa (multiplicity of infection > or =64:1). IL-8 secretion by B. cepacia-infected HLE cells was abrogated by the gene transcription inhibitor actinomycin D and the protein translation inhibitor cycloheximide, confirming that B. cepacia-induced IL-8 secretion was mediated through de novo protein synthesis. Treatment of B. cepacia with proteinase K failed to down-regulate IL-8 secretion; furthermore, IL-8 secretion by B. cepacia-infected HLE cells was abrogated by > or =80% in the presence of anti-CD14 [specific lipopolysaccharide (LPS) receptor] antibody, thus suggesting that the IL-8-inducing component of B. cepacia was LPS and therefore dependent on CD14. The p38 mitogen-activated protein kinase (MAPK) inhibitor and the extracellular signal-regulated kinase MAPK inhibitor significantly abrogated IL-8 secretion by B. cepacia-infected HLE cells (SB203580, > or =80% inhibition; PD98059, > or =30% inhibition). In conclusion, B. cepacia-induced IL-8 secretion in A549 airway epithelial cells is more potent than P. aeruginosa; is mediated through LPS, which is CD14 dependent; and involves activation of the p38 and ERK MAPK pathways.
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PMID:Burkholderia cepacia-induced IL-8 gene expression in an alveolar epithelial cell line: signaling through CD14 and mitogen-activated protein kinase. 1276 57

The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) is a cAMP-dependent Cl(-) channel that is defective in CF disease. CFTR activity has been shown to be regulated by the G(q)/phospholipase C-linked P2Y2 subtype of P2Y nucleotide receptors (P2YR) in various systems. Here, we tested whether other P2YR may exert a regulation on CFTR activity and whether CFTR may in turn exert a regulation on P2YR signaling. Using reverse transcriptase-polymerase chain reactions, antisense oligodeoxynucleotide knockdown, and measurements of intracellular calcium concentration ([Ca(2+)](i)), we showed that, in addition to P2Y2R, Chinese hamster ovary (CHO) cells also express functional P2Y1R. P2Y1R were activated by 2-methylthioadenosine 5'-diphosphate > 2-methylthioadenosine-5'-triphosphate > ADP with an EC(50) of 30 nM, 0.2 microM, and 0.8 microM, respectively. Activation of P2Y1R increased [Ca(2+)](i), which was prevented by the P2Y1R antagonists pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) (10 microM) and N6-methyl 2'-deoxyadenosine 3',5'-bisphosphate (MRS2179) (10 microM) and by pretreatment with P2Y1R antisense oligodeoxynucleotides. In CHO-K1 and CHO-KNUT (mock-transfected) cells lacking CFTR, both P2Y1R and P2Y2R caused [Ca(2+)](i) mobilization via pertussis toxin (PTX)-insensitive G(q/11)-proteins. In contrast, in CFTR-expressing CHO cells (CHO-BQ1), the P2Y1R response was completely PTX-sensitive, indicating that P2Y1R couples to G(i/o)-proteins, whereas the P2Y2R response remained PTX-insensitive. In CHO-BQ1 cells, P2Y1R activation by ADP (100 microM) failed to inhibit both forskolin (1 microM)-induced CFTR activation, measured using iodide ((125)I) efflux, and forskolin (0.1-10 microM)-evoked cAMP increase. Together, our results indicate that, in contrast to P2Y2R, P2Y1R does not modulate CFTR activity in CHO cells and that CFTR expression may alter the G-protein-coupling selectivity of P2Y1R.
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PMID:Pharmacological and signaling properties of endogenous P2Y1 receptors in cystic fibrosis transmembrane conductance regulator-expressing Chinese hamster ovary cells. 1474 36

Airway submucosal gland serous cells express the cystic fibrosis transmembrane conductance regulator (CFTR) and secrete antimicrobial, anti-inflammatory, and antioxidant molecules. In cystic fibrosis, diminished gland secretion may impair innate airway host defenses. We used Calu-3 cells as a serous cell model to study the types of proteins released, the pathways that release them, and the possible involvement of CFTR activity in protein release. Many proteins were secreted constitutively into the apical fluid and showed increased release to agonists. We identified some of them by high pressure liquid chromatography-mass spectrometry and reverse transcriptase PCR, including lysozyme, siderocalin (the protein NGAL), which inhibits bacterial growth by binding iron-containing siderophores, HSC-71, which is thought to have anti-inflammatory properties, and the serine protease inhibitors alpha-1-antitrypsin and alpha-1-antichymotrypsin, which may function as antimicrobials as well as play a potential role in diminishing the activation of epithelial Na(+) channels by serine proteases. We used an enzyme-linked immunosorbent assay to quantify lysozyme secretion by Calu-3 cells in response to various agonists and inhibitors. Forskolin increased the lysozyme secretion rate (J(lyz)) from 32 to 77 ng/hr/cm(2) (n = 36, p < 0.005). Thapsigargin increased J(lyz) from 40 to 63 ng/h/cm(2) (n = 16, p < 0.005), and forskolin plus thapsigargin further increased the forskolin-stimulated J(lyz) by 48% (n = 9, p < 0.05). 1-Ethyl-benzimidazolinone and carbachol were less effective. Glibenclamide inhibited basal and stimulated J(lyz), but clotrimazole was without effect. CFTR(inh)172 caused a small (15%) but significant inhibition of forskolin-stimulated J(lyz) without affecting basal J(lyz). Thus, Calu-3 cells secrete diverse proteins that in aggregate would be expected to suppress microbial growth, protect the airways from damage, and limit the activation of epithelial Na(+) channels via serine proteases.
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PMID:Regulation of antiprotease and antimicrobial protein secretion by airway submucosal gland serous cells. 1523 67

Since the role of respiratory viruses in lung exacerbations of patients with cystic fibrosis has been hampered by the difficulty of detecting viruses in viscous sputum specimens, a multiplex reverse transcriptase PCR (RT-PCR) assay combined with colorimetric amplicon detection was tested for the identification of seven common respiratory viruses in the sputa of cystic fibrosis patients. Of 52 sputa from 38 patients, 12 (23%) samples from 12 patients were positive for a respiratory virus (4 for influenza B, 3 for parainfluenza 1, 3 for influenza A and 2 for respiratory syncytial virus). These results suggest that the RT-PCR method carried out on sputum may provide a convenient means of investigating the role of virus infection in lung exacerbations of cystic fibrosis patients.
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PMID:Method for detection of respiratory viruses in the sputa of patients with cystic fibrosis. 1561 37

CFTR channels conduct HCO(3)(-) in addition to Cl(-) in airway epithelial cells. A defective HCO(3)(-)-transporting function of CFTR may underlie the pathogenesis of cystic fibrosis. In the present study, we have investigated whether a HCO(3)(-)-sensitive soluble adenylyl cyclase (sAC) is functionally coupled with CFTR and thus forms an autoregulatory mechanism for HCO(3)(-) transport in human airway epithelial Calu-3 cells. A reverse transcriptase-polymerase chain reaction showed that transcripts of both full-length and truncated sACs are present in Calu-3 cells. Truncated sAC protein is the predominant, if not the only, isoform expressed in Calu-3 cells. HCO(3)(-) stimulated a modest increase in cAMP production, and the increase was sensitive to 2-hydroxyestradiol (2-HE), a sAC inhibitor, but not to SQ22,536, a blocker of conventional transmembrane adenylyl cyclases. These results suggest that sAC is functional in Calu-3 cells. Adding 25 mM HCO(3)(-) to the bath stimulated CFTR-mediated whole cell currents in the absence, but not in the presence, of 2-HE. In cell-attached membrane patches, 25 or 50 mM HCO(3)(-) in the bath markedly increased the product of channel number and open probability of CFTR, and this activation was attenuated by 2-HE. These findings demonstrate that sAC signaling pathway is involved in the regulation of CFTR function in human airway epithelium and thereby provides a link between the level of intracellular HCO(3)(-)/CO(2) and the modulation of HCO(3)(-)-conductive CFTR function by cAMP/PKA.
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PMID:Regulation of CFTR channels by HCO(3)--sensitive soluble adenylyl cyclase in human airway epithelial cells. 1595 23

Pseudomonas aeruginosa poses a serious risk in individuals suffering from cystic fibrosis (CF). Strains colonizing the CF lung are generally motile but frequently convert to a nonmotile phenotype as the disease progresses. In many cases, this is coordinately regulated with the overproduction of the exopolysaccharide alginate. Both the expression of alginate (mucoidy) and the loss of flagellum synthesis may provide the bacterium with a selective advantage in the CF lung. Previously published data showed that the regulation of alginate production and flagellum biosynthesis in the CF isolate FRD1 is inversely controlled by the alternative sigma factor AlgT. In this study, we observed that in CF isolates, the mucoid and the nonmotile phenotypes occur predominantly together. Using microarrays, we compared the transcriptomes of isogenic AlgT(+) and AlgT(-) P. aeruginosa and discovered that AlgT significantly downregulated the majority of flagellar genes. A pronounced inhibitory effect was observed in several genes essential for proper flagellum expression, including fleQ, which encodes an essential flagellar regulator. The microarray data were confirmed by reverse transcriptase PCR analysis and promoter fusion assays in isogenic AlgT(+) and AlgT(-) strains. Transmission electron microscopy, motility assays, and Western blots showed that ectopic expression of FleQ in mucoid, nonmotile CF isolates restored flagellum biosynthesis and motility. Together, these data show that AlgT mediates the negative control of flagellum expression by inhibiting the expression of the flagellar regulator fleQ.
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PMID:The alternative sigma factor AlgT represses Pseudomonas aeruginosa flagellum biosynthesis by inhibiting expression of fleQ. 1629 68


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