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 purpose of this investigation was to determine whether mouse lung fibroblast subsets have the ability to produce nitric oxide (NO), and if so, to characterize the induction and effects of its synthesis. Previously, we isolated Thy1+ and Thy1- subpopulations of mouse lung fibroblasts, which differ in terms of cytokine production, morphology, response to cytokines and radiation, and ability to present antigen to T lymphocytes. When treated with the proinflammatory cytokines IFN-gamma, TNF-alpha, and IL-1 alpha, these fibroblast lines produce micromolar quantities of NO2- and NO3-, two stable end products of the NO pathway. A combination of all three cytokines provided the greatest induction, and there was no measurable production of NO in unstimulated cells. Thy1+ fibroblasts have fewer requirements for induction of NO production than the Thy1- line, in that NO production could be induced by only two of the above cytokines, where the Thy1- fibroblasts required all three. Inducible NO synthase (iNOS) mRNA was shown to be present by the reverse transcriptase-polymerase chain reaction as early as 2 hr after cytokine treatment in both cell lines. Addition of the NO synthase inhibitors NG-monomethyl-L-arginine and aminoguanidine inhibited production of NO2- and NO3-, but not iNOS mRNA. This inhibition was partially reversed by the addition of an excess of L-arginine. Interestingly, inhibition of NO synthesis was shown to decrease IL-6 production by more than 50% in cytokine-treated Thy1+ fibroblasts. These results indicate for the first time that Thy1+ and Thy1- mouse lung fibroblast subsets have the capability to produce NO to differing extents in response to cytokines and may therefore play an important role in the inflammatory response in the lung as well as in the progression of lung disease.
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PMID:Induction of nitric oxide synthase in subsets of murine pulmonary fibroblasts: effect on fibroblast interleukin-6 production. 751 14

Lung antiprotease activity is routinely assayed in the supernatant of bronchoalveolar lavage fluid (BALF). In this study the cellular fraction of lavages was also analyzed. Functionally active acid-resistant inhibitors with molecular masses characteristic of the mucus proteinase inhibitor (MPI, 14 kDa) and elastase-specific inhibitor (ESI, 7 kDa) were demonstrated by gel chromatography. Immunocytochemical studies of cells obtained at various postnatal time points from lavages of 10 premature infants with chronic lung disease showed that the inhibitors were confined to neutrophils and macrophages. At each time point, about 70% and 21% of these cells, respectively, stained positively. The polyclonal antibodies usually used to detect MPI did not distinguish between MPI and ESI. Because of this cross reactivity, it was not possible to differentiate between MPI and ESI. Analysis with reverse transcriptase-polymerase chain reaction (RT-PCR) of cells from lavages and of nucleated cells isolated from the peripheral blood showed the production of ESI only, but not of MPI. Nevertheless, MPI can associate with neutrophils and macrophages, as was shown in binding studies with the recombinant protein. These data suggest that when assaying bronchoalveolar lavages (BALs) for these antiproteases in the supernatant only, the total pool of inhibitors may be underestimated.
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PMID:Cellular association of antiproteases in lavages from ventilated preterm human neonates. 919 16

Patients with cystic fibrosis develop lung disease after birth, therefore CFTR gene replacement therapy should be most efficacious in the neonatal period prior to the onset of pulmonary damage. An adeno-associated virus (AAV) vector, SA306 (Flotte TR et al Proc Natl Acad Sci USA 1993; 90: 10613-10617), which contains the AAV inverted terminal repeats flanking the human CFTR cDNA linked to an amino-terminal epitope tag, was used to transduce a human CFTR fusion protein into neonatal New Zealand white rabbits. Vector inocula of 1 x 10(5) to 5 x 10(10) particles were given by intratracheal instillation on day 3 of life and the rabbit lungs were studied at 3 or 4 days, 2-6 weeks, or 6 months after infection; the 2-6 week time-point corresponds to the completion of the alveolar phase of lagomorph lung development. Vector DNA was detected by an in situ polymerase chain reaction (PCR) using vector-specific primers at up to 6 weeks after inoculation. Human CFTR mRNA was detected by Northern analysis at up to 2 weeks after vector inoculation, and by a reverse transcriptase PCR assay at up to 3 weeks after infection. Epithelial expression of the human CFTR fusion protein was detected using antisera to both the human CFTR R domain and the amino-terminal epitope at up to 6 weeks after vector inoculation. Vector DNA, mRNA, or human CFTR immunoreactivity were not observed at the 6 month time-point. Rabbits infected with SA306 were clinically indistinguishable from their uninfected litter mates. These data indicate that CFTR gene transduction using an AAV vector is feasible in the neonatal rabbit, and that expression of vector-derived CFTR persists throughout the alveolar phase of lung development. The apparent lack of vector persistence after the alveolar phase may reflect dilution of transduced cells by further lung growth or a lack of transduction of pulmonary epithelial stem cells.
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PMID:CFTR gene transduction in neonatal rabbits using an adeno-associated virus (AAV) vector. 927 14

In the surfactant protein C/tumor necrosis factor (SP-C/TNF) transgenic mouse, the TNF-alpha transgene is overexpressed in type II pneumocytes. Pulmonary lymphocytic infiltration develops which is followed by fibrotic changes including accumulation of fibroblasts and deposition of extracellular matrix. We hypothesized that lymphocytes played a role in the development of pulmonary fibrosis in this model. Lymphocytes were recovered from the interstitium of the lung and analyzed by flow cytometry. The absolute number of lymphocytes recovered from transgenic mice were approximately four times of that in littermates. Flow cytometric analysis showed the presence of gamma delta T cells and B1 cells in the former group but these cells were almost absent in the lung of non-transgenic littermates. We also studied lymphocytes accumulating in the lung during bleomycin (BLM)-induced pneumopathy. Serial analyses showed a progressive increase of CD4/CD8 ratio after injection of BLM, reaching a peak at day 14, then decreased to the normal level by day 48. Northern blot analysis of the lung showed an enhanced expression of interleukin (IL)-2 and osteopontin (OPN) mRNA in those two models of pulmonary fibrosis. Expansion of clonal alpha beta T cells as detected by reverse transcriptase-polymerase chain reaction/single strand conformation polymorphism (RT-PCR/SSCP) suggests involvement of antigen-driven mechanisms in the development of pulmonary fibrosis.
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PMID:Immunophenotyping of lymphocytes in the lung interstitium and expression of osteopontin and interleukin-2 mRNAs in two different murine models of pulmonary fibrosis. 945 69

EWS/ets-oncogene fusion transcripts can be detected in at least 98% of Ewing tumors [(ET) Ewing sarcoma and peripheral primitive neuroectodermal tumor] by reverse transcriptase-polymerase chain reaction (RT-PCR), thus confirming the histopathologic diagnosis. To detect minimal amounts of tumor cells in the bone marrow (BM), we used an RT-PCR assay with a high sensitivity, revealing one tumor cell in a background of 10(6) normal cells. We examined BM samples from 35 newly diagnosed ET patients (23 with localized and 12 with metastatic disease). At diagnosis, tumor cells in the BM were detected in 7/23 patients with localized disease (30%). Fifty percent of patients with isolated lung metastasis were RT-PCR positive (3/6), whereas 6/6 patients with bone metastases showed positive signals (100%). All patients with initial PCR positivity in the BM became negative during treatment. After a median follow-up of 30 months, relapses were observed in both groups of patients with localized disease (3/7 RT-PCR positive and 2/16 RT-PCR negative). The only recurrence in the group with isolated lung metastases occurred as progressive lung disease in 1 of the 2 RT-PCR-negative patients, whereas among the 6 patients with bone metastases 2 remain in complete remission. So far, RT-PCR screening for BM involvement did not allow prediction of early relapse in ET. To assess better the significance of this test in the evaluation of long-term prognosis and in monitoring the effectiveness of systemic therapy, long observation periods are warranted before it becomes a tool for treatment stratification.
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PMID:Predictive potential of testing for bone marrow involvement in Ewing tumor patients by RT-PCR: a preliminary evaluation. 949 59

Ureaplasma urealyticum is relatively common in the respiratory tract of very low birth weight infants and has been hypothesized to be involved in the development of chronic lung disease. The purpose of this study was to investigate whether U. urealyticum could stimulate macrophages to produce proinflammatory cytokines in vitro, which are early pathologic changes in the lung during the development of chronic lung disease. A human monocytic cell line (THP-1) differentiated to macrophages, a rat alveolar macrophage cell line (Nr8383), and human lung macrophages from tracheobronchial aspirate fluid in preterm infants were exposed to U. urealyticum antigen for 24 h. The protein levels of human IL-6, tumor necrosis factor-alpha (TNF-alpha), and rat TNF-alpha were measured with ELISA. Rat IL-6 was analyzed with a specific bioassay. The mRNA levels of these cytokines were detected by reverse transcriptase-PCR. The production of TNF-alpha and IL-6 increased after stimulation with U. urealyticum in both the human and rat macrophage cell lines. In tracheobronchial aspirate fluid macrophages, U. urealyticum increased the production of TNF-alpha from 14 to 84% and IL-6 from 46 to 268% above control levels. U. urealyticum also induced gene expression of TNF-alpha and IL-6. In conclusion, U. urealyticum could be an important factor in the development of chronic lung disease because of its ability to induce alveolar macrophage proinflammatory cytokine production.
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PMID:Ureaplasma urealyticum-induced production of proinflammatory cytokines by macrophages. 1087 9

Hereditary surfactant protein B (SP-B) deficiency has been lethal in the first year of life without lung transplantation. We tested the hypothesis that SP-B gene mutations may result in milder phenotypes by investigating the mechanisms for lung disease in two children with less severe symptoms than have been previously observed in SP-B deficiency. Immunostaining patterns for pulmonary surfactant proteins were consistent with SP-B deficiency in both children. DNA sequence analysis indicated that both children were homozygous for a mutation in exon 5 that created an alternative splice site. Reverse transcriptase PCR and sequence analysis confirmed use of this splice site, which resulted in a frameshift and a premature termination codon in exon 7. The predominant reverse transcriptase PCR product, however, lacked exon 7, which restored the reading frame but would not allow translation of the exons that encode mature SP-B. Western blot analysis detected reduced amounts of mature SP-B as well as an aberrant SP-B proprotein that corresponded to the size expected from translation of the abnormal transcript. We conclude that a novel splicing mutation was the cause of lung disease in these children and that hereditary SP-B deficiency can be the cause of lung disease in older children.
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PMID:Prolonged survival in hereditary surfactant protein B (SP-B) deficiency associated with a novel splicing mutation. 1096 Apr 90

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disorder characterized by fibroblast proliferation and extracellular matrix accumulation. However, studies on fibroblast growth rate and collagen synthesis have given contradictory results. Here we analyzed fibroblast growth rate by a formazan-based chromogenic assay; fibroblast apoptosis by in situ end labeling (ISEL) and propidium iodide staining; percent of alpha-smooth muscle actin (alpha-SMA) positive cells by fluorescence-activated cell sorter; and alpha1-(I) collagen, transforming growth factor (TGF)-beta1, collagenase-1, gelatinases A and B, and tissue inhibitor of metalloproteinase (TIMP)-1, -2, -3, and -4 expression by reverse transcriptase/polymerase chain reaction in fibroblasts derived from IPF and control lungs. Growth rate was significantly lower in IPF fibroblasts compared with controls (13.3 +/- 38.5% versus 294.6 +/- 57%, P < 0.0001 at 13 d). Conversely, a significantly higher percentage of apoptotic cells was observed in IPF-derived fibroblasts (ISEL: 31.9 +/- 7.0% versus 15.5 +/- 7.6% from controls; P < 0.008). alpha-SMA analysis revealed a significantly higher percentage of myofibroblasts in IPF samples (62.8 +/- 25.2% versus 14.8 +/- 11.7% from controls; P < 0.01). IPF fibroblasts were characterized by an increase in pro-alpha1-(I) collagen, TGF-beta1, gelatinase B, and all TIMPs' gene expression, whereas collagenase-1 and gelatinase A expression showed no differences. These results suggest that fibroblasts from IPF exhibit a profibrotic secretory phenotype, with lower growth rate and increased spontaneous apoptosis.
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PMID:Fibroblasts from idiopathic pulmonary fibrosis and normal lungs differ in growth rate, apoptosis, and tissue inhibitor of metalloproteinases expression. 1135 Aug 29

Interleukin-4 (IL-4) is known to activate mononuclear cells as well as fibroblasts, all of which are important in the pathogenesis of pulmonary fibrosis. To investigate the potential role of this cytokine, lung IL-4 expression was examined in a murine model of bleomycin-induced pulmonary fibrosis. Lung fibrosis was induced in CBA/J mice by endotracheal injection of bleomycin on day 0. On selected days after treatment, lungs were harvested for reverse transcriptase polymerase chain reaction (RT-PCR), Northern, in-situ hybridization and immunohistochemical analyses. RT-PCR and Northern analyses revealed significant increases in lung IL-4 mRNA content between days 3 and 14 after induction of lung injury, which decreased toward control level after day 21. Both in-situ hybridization and immunohistochemistry showed low or undetectable IL-4 expression in control lungs and in injured lungs before day 3 after bleomycin injection. There was however elevated expression in mononuclear cells and macrophages between days 3 and 14, localized to areas of active fibrosis. These results demonstrate that IL-4 is upregulated significantly in this model. They suggest a potential role for this cytokine in pulmonary fibrosis, perhaps via its ability to stimulate and amplify the inflammatory response, stimulate collagen synthesis in fibroblasts, and thus promote the progression to fibrosis and end stage lung disease.
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PMID:Lung interleukin-4 gene expression in a murine model of bleomycin-induced pulmonary fibrosis. 1155 83

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


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