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 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

The receptor for the active metabolite of vitamin D, 1,25(OH)(2)D(3), known as the vitamin D receptor (VDR), belongs to the steroid hormone nuclear receptor superfamily. We have developed novel methods for detection of VDR mRNA and protein within a human promyelomonocytic cell line, HL-60. Using the newly developed technique of in situ-reverse transcriptase-polymerase chain reaction (IS-RT-PCR), low levels of VDR mRNA could be amplified and demonstrated unequivocally within these cells, and also within a human kidney proximal tubule cell line, CL-8. Use of a novel immunogold cytochemical technique has allowed clear and sensitive detection of VDR protein expression within the HL-60 cells. Further development of IS-RT-PCR has allowed us to apply this technique to tissue sections. We have shown clear amplification of VDR transcripts within sections of formalin-fixed paraffin-embedded human kidney and liver. These techniques will be useful to localise specifically the VDR within cell types that contain low levels of mRNA and protein, and will permit further investigation of the role played by 1,25(OH)(2)D(3) in cellular regulatory mechanisms.
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PMID:Novel and sensitive detection systems for the vitamin D receptor--in situ-reverse transcriptase-polymerase chain reaction and immunogold cytochemistry. 915 21

We investigate the intrarenal expression of two recently cloned chloride channels, rClC-K1 and rClC-K2, by reverse transcriptase-polymerase chain reaction on single microdissected tubules from the rat kidney and by immunohistochemistry using a polyclonal antibody that recognizes both highly homologous channels. Both rClC-K1 and rClC-K2 mRNAs were detected in outer medullary late proximal tubules (S3), papillary ascending thin limbs (ATL), and outer medullary (MTAL) and cortical (CTAL) thick ascending limbs, distal tubules (DCT), and cortical, outer medullary, and inner medullary collecting ducts. Indirect immunofluorescence studies demonstrated that the rClC-K proteins were restricted to the basolateral membranes from ATL, DCT, and collecting ducts cells, whereas CTAL and MTAL exhibited a more diffuse basal staining. When rats were dehydrated, a condition which increased the expression of rClC-K1 in cortex and medulla, a weak cytoplasmic staining was found in late proximal tubule cells. Thus these results demonstrate that rat kidney ClC-K channels are predominantly located in the basolateral membranes from cells of the late segments of the renal tubule where most of chloride reabsorption takes place.
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PMID:Localization and induction by dehydration of ClC-K chloride channels in the rat kidney. 917 80

Angiotensin II (ANG II), acting through angiotensin type 1A receptors (AT1A), is important in regulating proximal tubule salt and water balance. AT1A are present on apical (AP) and basolateral (BL) surfaces of proximal tubule epithelial cells (PTEC). The molecular mechanism of AT1A function in epithelial tissue is not well understood, because specific binding of ANG II to intact PTEC has not been found and because a number of isoforms of AT receptors are present in vivo. To overcome this problem, we developed a cell line from opossum kidney (OK) proximal tubule cells, which stably express AT1A (Kd = 5.27 nM, Bmax = 6.02 pmol/mg protein). Characterization of nontransfected OK cells revealed no evidence of AT1A mRNA (reverse transcriptase-polymerase chain reaction analysis) or protein (125I-labeled ANG II binding studies) expression. In cells stably expressing AT1A, ANG II binding was saturable, reversible, and regulated by G proteins. Transfected receptors were coupled to increases in intracellular calcium and inhibition of cAMP. To determine the polarity of AT1A expression and function in proximal tubules, transfected cells were grown to confluence on membrane inserts under conditions that allowed selective access to AP or BL surfaces. AT1A were expressed on both AP (Kd = 8.7 nM, Bmax = 3.33 pmol/mg protein) and BL (Kd = 10.1 nM, Bmax = 5.50 pmol/mg protein) surfaces. Both AP and BL AT1A receptors underwent agonist-dependent endocytosis (AP receptor: t1/2 = 7.9 min, Ymax = 78.5%; BL receptor: t1/2 = 2.1 min, Ymax = 86.3%). In cells transfected with AT1A, ANG II caused time- and concentration-dependent increases in transepithelial 22Na transport (2-fold over control at 20 min) by increasing Na/H exchange. In conclusion, we have established a stable proximal tubule cell line that expresses AT1A on both AP and BL surfaces, undergoes agonist-dependent receptor endocytosis, and is functional, as evidenced by inhibition of cAMP and increases in cytosolic calcium mobilization and transepithelial sodium movement. This cell line should prove useful for understanding the molecular and biochemical regulation of AT1A expression and function in PTEC.
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PMID:Angiotensin (AT1A) receptor-mediated increases in transcellular sodium transport in proximal tubule cells. 961 27

To analyze the regulation of water channels in the peritoneum, we tried to establish a primary mesothelial cell culture system. Male Sprague-Dawley rats weighing about 250 g were anesthetized, and 10 mL of phosphate-buffered saline (PBS) containing 0.25% trypsin and 1 mmol/L ethylenediamine tetraacetic acid (EDTA) was infused into the peritoneal cavity for 15 minutes. Sediments from the recovered fluid were cultured in medium M199 supplemented with 10% fetal bovine serum (FBS). The culture was succeeded 4-6 times before experiments commenced. After exposure to the test medium, RNA was extracted and subjected to reverse transcriptase polymerase chain reaction (RT-PCR) for 10-19 cycles, then was measured by Southern blot analysis with a digoxin-labeled probe. Cultured cells were positively stained with mouse monoclonal anti-cytokeratin antibody, confirming their characteristics as mesothelial cells. Aquaporin-1 (AQP-1) message in the cultured cells increased with increases in glucose and mannitol concentrations when beta-actin message was used as an internal control. Tranexamic acid effected no change in AQP-1 message in the cultured mesothelial cells. This system offers potential as a simple approach to test the effects of osmolytes, cytokines, and vasoactive hormones on aquaporin expression and water transport in the peritoneum.
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PMID:Water channel AQP-1 in the primary cell culture of rat peritoneum. 1068 62

Renin secretion can be stimulated by ATP via purinergic P2Y receptors. ATP is a cotransmitter with norepinephrine and is released from the cytosol during cell damage. Such release could account for the de novo renin expression seen in the proximal tubule in renal disease and in myocardial infarct borders. Whereas most P2Y purinoceptor subtypes utilize phosphoinositide signal-transduction pathways, the effector mechanisms of the subtype P2Y(11) also involve increases in cAMP, a well-known renin secretagogue and stimulus to renin production. The present study tested the effect of ATP on human renin gene (REN) promoter activity and the role of P2Y(11). By means of reverse transcriptase-polymerase chain reaction, we found that renin-expressing Calu-6 cells express P2Y(11) mRNA. Expression was also detected in the brain, kidney, testis, muscle, liver, and spleen. We made a novel cell line (Calu-6/P2Y11) in which P2Y(11) cDNA, under the control of a strong promoter, was stably integrated into genomic DNA. These cells produced P2Y(11) mRNA during culture. Treatment of Calu-6/P2Y11 cells with 1 mmol/L ATP caused a 3-fold increase in renin mRNA and protein over 36 hours. Transient transfection of Calu-6/P2Y11 cells with constructs containing 896 bp of human REN 5'-flanking DNA linked to the luciferase reporter gene led to a 5.8+/-0.6-fold increase (mean+/-SEM) in reporter activity in response to ATP (P=0.0015). In contrast, UTP produced only a 1.4+/-0.1-fold increase (P=0.016). For ADP, it was 1.7+/-0.1-fold (P=0.011). The response profile was ATP>ADP>AMP=adenosine=0, consistent with a P2Y(11) effect. Mutation of the cAMP response element (CRE) located at -222 in the REN promoter DNA abolished the effect of ATP. Furthermore, ATP induced a rapid, time-dependent increase in the phosphorylation of CRE binding protein (CREB) and activating transcription factor-1. These data implicate a cAMP pathway in mediation of the P2Y(11) effect. In conclusion, we have made a novel cell line that overexpresses the P2Y(11) purinoceptor. Stimulation of these cells by ATP activates a cAMP signal-transduction pathway that phosphorylates CREB and stimulates renin promoter activity via the CRE at -222. The data raise the possibility of a contribution of ATP/P2Y(11) effects to sympathetic stimulation of renin, as well as to responses in renin seen after tissue damage, such as in kidney disease and myocardial infarction.
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PMID:Capacity for purinergic control of renin promoter via P2Y(11) receptor and cAMP pathways. 1111 31

Drug-associated dysfunction of mitochondria is believed to play a role in the etiology of the various adverse symptoms that occur in human immunodeficiency virus (HIV)-infected patients treated with the nucleoside reverse transcriptase inhibitors (NRTIs). Tenofovir, a nucleotide analog recently approved for use in the treatment of HIV infection, was evaluated in vitro for its potential to cause mitochondrial toxicity and was compared to currently used NRTIs. Treatment with tenofovir (3 to 300 microM) for up to 3 weeks produced no significant changes in mitochondrial DNA (mtDNA) levels in human hepatoblastoma (HepG2) cells, skeletal muscle cells (SkMCs), or renal proximal tubule epithelial cells. The potencies of inhibition of mtDNA synthesis by the NRTIs tested were zalcitabine (ddC) > didanosine (ddI) > stavudine > zidovudine (ZDV) > lamivudine = abacavir = tenofovir, with comparable relative effects in the three cell types. Unlike ddC and ddI, tenofovir did not affect cellular expression of COX II and COX IV, two components of the mitochondrial cytochrome c oxidase complex. Lactate production was elevated by less than 20% in HepG2 cells or SkMCs following treatment with 300 microM tenofovir. In contrast, lactate synthesis increased by >200% in the presence of 300 microM ZDV. Thus, treatment of various human cell types with tenofovir at concentrations that greatly exceed those required for it both to have in vitro anti-HIV type 1 activity in peripheral blood mononuclear cells (50% effective concentration, 0.2 microM) and to achieve therapeutically relevant levels in plasma (maximum concentrations in plasma, 0.8 to 1.3 microM) is not associated with mitochondrial toxicity.
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PMID:Assessment of mitochondrial toxicity in human cells treated with tenofovir: comparison with other nucleoside reverse transcriptase inhibitors. 1185 Feb 53

Clinical studies with tenofovir disoproxil fumarate, an oral prodrug of the nucleotide analog tenofovir, recently approved for the treatment of HIV, have demonstrated antiviral activity and good tolerability in HIV-infected patients. In order to better understand the cytotoxicity profile of tenofovir relative to the other nucleoside reverse transcriptase inhibitors (NRTIs), the in vitro effects of these agents were evaluated in various human cell types. Tenofovir inhibited the proliferation of liver-derived HepG2 cells and normal skeletal muscle cells with CC(50) values of 398 and 870 microM, respectively. In comparison, ZDV, ddC, ddI, d4T, and abacavir all showed lower CC(50) values in these two cell types. Evaluation of hematopoietic toxicity revealed that tenofovir was less cytotoxic towards erythroid progenitor cells (CC(50)>200 microM) than ZDV, d4T, and ddC (CC(50)=0.06-5 microM). Despite some degree of donor-to-donor variability, the inhibitory activity of the tested NRTIs against myeloid cell lineage, in the order of decreasing severity, was consistently ddC>ZDV>d4T>tenofovir>3TC. Finally, tenofovir showed substantially weaker effects on proliferation and viability of renal proximal tubule epithelial cells than cidofovir, a related nucleotide analog with the potential to induce renal tubular dysfunction. In conclusion, tenofovir exhibited weak cytotoxic effects in all cell types tested with less in vitro cytotoxicity than the majority of NRTIs currently used for the treatment of HIV disease.
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PMID:Tenofovir exhibits low cytotoxicity in various human cell types: comparison with other nucleoside reverse transcriptase inhibitors. 1188 56

Aminoglycoside antibiotics (AGAs) are nephrotoxic, with most of the damage confined to the proximal tubule, but the mechanism for cellular toxicity is not clear. It has been previously shown that the extracellular-calcium sensing receptor (CaR) is expressed in intact rat proximal tubule and can be stimulated by the AGA neomycin. To investigate whether CaR could contribute to AGA-induced nephrotoxicity, the acute responses to various AGAs in the proximal tubule-derived opossum kidney (OK) cell line were examined. The presence in OK cells of CaR-related transcripts and protein was demonstrated by northern analyses, reverse transcriptase-PCR, immunocytochemistry, and immunoblotting. OK cells responded to elevated extracellular calcium (Ca(2+)(o)) and neomycin but also to gentamicin and tobramycin with an increase in cytosolic [Ca(2+)]. Ca(2+)(o), neomycin, and gentamicin also activated the extracellular signal-regulated kinases, ERK1 and ERK2. Neomycin-induced ERK activation was both dose- and time-dependent and was attenuated by inhibitors of phosphatidylinositol 3-kinase, phosphatidylinositol bisphosphate (PIP(2))-specific phospholipase C, and MEK1, but not of protein kinase C. Thus, proximal tubular OK cells express a CaR that mediates Ca(2+)(i) mobilization and PIP(2)-PLC-dependent ERK activation in response to AGAs and thus could play a role in AGA-induced nephrotoxicity.
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PMID:Aminoglycosides increase intracellular calcium levels and ERK activity in proximal tubular OK cells expressing the extracellular calcium-sensing receptor. 1203 77

Human proximal tubule epithelial cell lines are potentially useful models to elucidate the complex cellular and molecular details of water and electrolyte homeostasis in the kidney. Samples of normal adult human kidney tissue were obtained from surgical specimens, and S1 segments of proximal convoluted tubules were microdissected, placed on collagen-coated culture plate inserts, and cocultured with lethally irradiated 3T3 fibroblasts. Primary cultures of proximal tubule epithelial cells were infected with a replication-defective retroviral construct encoding either wild-type or temperature-sensitive simian virus 40 large T-antigen. Cells forming electrically resistive monolayers were selected and expanded in culture. Three cell lines (HPCT-03-ts, HPCT-05-wt, and HPCT-06-wt) were characterized for proximal tubule phenotype by electron microscopy, electrophysiology, immunofluorescence, Southern hybridization, and reverse transcriptase-polymerase chain reaction. Each of the three formed polarized, resistive epithelial monolayers with apical microvilli, tight junctional complexes, numerous mitochondria, well-developed Golgi complexes, extensive endoplasmic reticulum, convolutions of the basolateral plasma membrane, and a primary cilium. Each exhibited succinate, phosphate, and Na,K- adenosine triphosphatase (ATPase) transport activity, as well as acidic dipeptide- and adenosine triphosphate-regulated mechanisms of ion transport. Transcripts for Na(+)-bicarbonate cotransporter, Na(+)-H(+) exchanger isoform 3, Na,K-ATPase, parathyroid hormone receptor, epidermal growth factor receptor, and vasopressin V2 receptor were identified. Furthermore, immunoreactive sodium phosphate cotransporter type II, vasopressin receptor V1a, and CLIC-1 (NCC27) were also identified. These well-differentiated, transport-competent cell lines demonstrated the growth, immortalization, and differentiation potential of normal, adult, human proximal tubule cells and consequently have wide applicability in cell biology and renal physiology.
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PMID:Growth, immortalization, and differentiation potential of normal adult human proximal tubule cells. 1474 22


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