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)

In this paper, we have characterized the structure, evolutionary origin, and function of rat and human carcinoembryonic antigen-related cell adhesion molecule1 (CEACAM1) multifunctional Ig-like cell adhesion proteins that are expressed by many epithelial tissues. Restriction enzyme digestion reverse transcriptase-PCR analysis identified three cDNAs encoding novel CEACAM1 N-domains. Comparative sequence analysis showed that human and rat CEACAM1 N-domains segregated into two groups differing in similarity to rat CEACAM1(a)-4L and human CEACAM1. Sequence variability analysis indicated that both human and rat N-domains possessed two variable regions, and one contained a major adhesive epitope. Recombination analysis showed that the group of rat but not human N-domains with high sequence similarity was derived at least in part by recombination. Binding assays revealed that three monoclonal antibodies with strong reactivity for the CEACAM1(a)-4L N-domain showed no reactivity with CEACAM1(b)-4S, an allele with a different N-domain sequence. CEACAM1(b)-4S displayed adhesive activity efficiently blocked by a synthetic peptide corresponding to the adhesive epitope in CEACAM1(a)-4L. Blocking analysis also showed that the adhesive epitope for rat CEACAM1 was located downstream from the equivalent human and mouse epitopes. Glycosylation analysis demonstrated O-linked sugars on rat CEACAM1(b)-4S from COS-1 cells. However, this was not the alteration responsible for the lack of monoclonal antibody reactivity. When considered together with previous studies, our findings suggest an inverse relationship between functionality and amino acid sequence similarity to CEACAM1. Like IgG, the N-domain of CEACAM1 appears to tolerate 10-15% sequence diversification without loss of function but begins to show either altered specificity or diminished functionality at higher levels.
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PMID:Two variable regions in carcinoembryonic antigen-related cell adhesion molecule1 N-terminal domains located in or next to monoclonal antibody and adhesion epitopes show evidence of recombination in rat but not in human. 1518 66

Estrogen has been postulated to be involved in inhibition of vascular smooth muscle cell (VSMC) proliferation mainly via estrogen receptor (ER), but the detailed mechanism has remained primarily unknown. Therefore, in this study, microarray analysis was used in two types of cultured human VSMCs: one positive for ER alpha, and the other for ER beta, which were treated by estrogens to detect the estrogen-responsive genes. We also used quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to evaluate mRNA levels of selective target gene (TG) in these cells. We further studied whether the TG product was involved in inhibition of proliferation using small interfering RNA (siRNA) of the TG transfection. We subsequently used quantitative RT-PCR and in situ hybridization analysis to evaluate the expression of these gene products in human aorta. E4F1, a possible inducer of cell growth arrest, was markedly increased only in ER alpha-positive VSMCs by estrogens in both microarray and RT-PCR analyses. Blocking of E4F1 using siRNA suppressed estrogenic inhibition of ER alpha-positive VSMC proliferation. E4F1 mRNA was abundant in premenopausal female aorta with mild atherosclerotic changes. E4F1 is therefore considered one of the estrogen-responsive genes involving ER alpha-mediated inhibition of VSMC proliferation and may play an important role in estrogen-related atheroprotection of human aorta.
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PMID:E4F1, a novel estrogen-responsive gene in possible atheroprotection, revealed by microarray analysis. 1557 45

Template switching during reverse transcription contributes to recombination in human immunodeficiency virus type 1 (HIV-1). Our recent studies suggest that the process can occur through a multi-step mechanism involving RNase H cleavage, acceptor invasion, branch migration, and finally primer terminus transfer. In this study, we analyzed the effects of reverse transcriptase (RT)-pausing, RNase H cleavages and template structure on the transfer process. We designed a series of donor and acceptor template pairs with either minimal pause sites or with pause sites at various locations along the template. Restriction sites within the region of homology allowed efficient mapping of the location of primer terminus transfer. Blocking oligomers were used to probe the acceptor invasion site. Introduction of strong pause sites in the donor increased transfer efficiency. However, the new pauses were not necessarily associated with effective invasion. In this system, the primary invasion occurred at a region of donor cleavage associated with weak pausing. These results together with acceptor structure predictions indicated that a potential invasion site is used only in conjunction with a favorable acceptor structure. Stabilizing acceptor structure at the predicted invasion region lowered the transfer efficiency, supporting this conclusion. Differing from previous studies, terminus transfer occurred at a short distance from the invasion site. Introduction of structure into the acceptor template shifted the location of terminus transfer. Nucleocapsid protein, which can improve cDNA-acceptor interactions, increased transfer efficiency with some shift of terminus transfer closer to the invasion site. Overall results support that the acceptor structure has a major influence on the efficiency and position of the invasion and terminus transfer steps.
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PMID:Effects of donor and acceptor RNA structures on the mechanism of strand transfer by HIV-1 reverse transcriptase. 1621 74

Interaction between spontaneous and neurally mediated regulation of tone in the corpus cavernosum smooth muscle (CCSM) of the rabbit was investigated. Changes in isometric muscle tension, intracellular Ca2+ concentration ([Ca2+]i) and membrane potential were recorded. CCSM developed spontaneous contractions, transient increases in [Ca2+]i (Ca2+ transients) and depolarizations. This spontaneous activity was abolished by blocking L-type Ca2+ channels (nicardipine, 1 mum), sarcoplasmic reticulum Ca2+ pump activity (cyclopiazonic acid, 10 microm), Ca2(+)-activated Cl- channels (niflumic acid, 10 mum) or cyclooxygenase-2 (COX-2; NS-398, 1 microm). Transmural nerve stimulation initiated either alpha-adrenergic contractions or nitrergic relaxations of CCSM depending on the level of muscle tone. NS-398 suppressed nerve-evoked contractions by about 70% but caused only a 40% reduction in the corresponding Ca2+ transient. Blocking nitric oxide synthase with N(omega)-nitro-l-arginine (LNA, 100 microm) reinforced nerve-evoked Ca2+ transients by about 150%, whilst increasing the corresponding Ca2+ transients by only 20%. In CCSM preparations that had been pre-contracted with either noradrenaline (0.3 microm) or prostaglandin F(2alpha) (0.1 microm), nerve stimulation inhibited about 70% of the contraction and caused only a 20% decrease in [Ca2+]i. Fluorescent immunohistochemistry with COX-2 antibodies and the reverse transcriptase-polymerase chain reaction (RT-PCR) method showed that the enzyme and its mRNA were highly expressed in the CCSM. These results suggest that spontaneously produced prostaglandins (PGs) not only contribute to the generation of spontaneous contractions but also facilitate nerve-evoked contractions. Conversely, spontaneously released nitric oxide (NO) suppresses excitation. Thus, interaction between spontaneous and neurally mediated regulation of CCSM tone may be fundamental to maintaining the muscle contractility. In addition, both PGs and NO appear to alter CCSM tone with only small changes in [Ca2+]i.
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PMID:Interaction between spontaneous and neurally mediated regulation of smooth muscle tone in the rabbit corpus cavernosum. 1623 65

Embryonic stem (ES) cells are pluripotent cells that can differentiate into a wide variety of cell types. This has made them an attractive source of donor cells for developmental studies and cell therapy. Blocking the differentiation of ES cells in culture and using them in clinical applications requires genetic manipulation of the cells. The aim of the present study was to transfect CCE ES cells with EGFP and BDNF genes, in which pcDNA3-hBDNF-v5 and pIRES2-EGFP plasmids were used, respectively. Transformation of DH5alpha competent bacteria by the plasmids was done. Then the plasmids were purified and transfected into ES cells using the electroporation method. The expression of the EGFP gene was confirmed using invert fluorescent microscopy; accordingly, RT-PCR was used for the BDNF gene. The latter was evaluated by extracting the total RNA from the transfected cells. cDNA was obtained by using reverse transcriptase and was amplified by specific primers. The products of the PCR were separated and visualized by agarose gel electrophoresis. Both techniques revealed a successful transfection of CCE ES cells by both plasmids. The obtained data indicated that electroporation is an efficient method for transfection of CCE ES cells.
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PMID:Transfection of CCE mouse embryonic stem cells with EGFP and BDNF genes by the electroporation method. 1660 92

The telomerase RNA (hTR) and reverse transcriptase (hTERT) promoters are active in most cancer cells, but not in normal cells, and are useful for transcriptional targeting in gene therapy models. Telomerase-specific conditionally replicating adenoviruses (CRAd) are attractive vectors because they should selectively lyse tumor cells. Here, we compare CRAds, in which either the hTR or hTERT promoter controls expression of the adenovirus E1A gene. In replication-defective reporter adenoviruses, the hTR promoter was up to 57-fold stronger in cancer cells than normal cells and up to 49-fold stronger than hTERT. In normal cells, hTERT promoter activity was essentially absent. Doses of telomerase-specific CRAds between 1.8 and 28 infectious units per cell efficiently killed cancer cells, but normal cells required higher doses. However, CRAd DNA replication and E1A expression were detected in both cancer and normal cells. Overall, tumor specificity of the CRAds was limited compared with nonreplicating vectors. Surprisingly, both CRAds expressed similar E1A levels and functional behavior, despite known differentials between hTR and hTERT promoter activities, suggesting that the promoters are deregulated. Rapid amplification of cDNA ends analysis of hTR-/hTERT-E1A transcripts ruled out cryptic transcription from the vector backbone. Blocking E1A translation partially restored the hTR-/hTERT-E1A mRNA differential, evidencing feedback regulation by E1A.
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PMID:Modulation of telomerase promoter tumor selectivity in the context of oncolytic adenoviruses. 1728 67

Increased tissue factor (TF)-dependent procoagulant activity in sepsis may be partly due to decreased expression or function of tissue factor pathway inhibitor (TFPI). To test this hypothesis, baboons were infused with live Escherichia coli and sacrificed after 2, 8, or 24 hours. Confocal and electron microscopy revealed increased leukocyte infiltration and fibrin deposition in the intravascular and interstitial compartments. Large amounts of TF were detected by immunostaining in leukocytes and platelet-rich microthrombi. TF induction was documented by quantitative reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, and coagulation assays. Lung-associated TFPI antigen and mRNA decreased during sepsis, and TFPI activity diminished abruptly at 2 hours. Blocking antibodies against TFPI increased fibrin deposition in septic baboon lungs, suggesting that TF-dependent coagulation might be aggravated by reduced endothelial TFPI. Decreased TFPI activity coincided with the release of tissue plasminogen activator and the peak of plasmin generation, suggesting that TFPI could undergo proteolytic inactivation by plasmin. Enhanced plasmin produced in septic baboons by infusion of blocking antibodies against plasminogen activator inhibitor-1 led to decreased lung-associated TFPI and unforeseen massive fibrin deposition. We conclude that activation of TF-driven coagulation not adequately countered by TFPI may underlie the widespread thrombotic complications of sepsis.
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PMID:Sepsis-induced coagulation in the baboon lung is associated with decreased tissue factor pathway inhibitor. 1764 Sep 67

The serine threonine kinase Raf-1 plays a protective role in many cell types, but its function in pancreatic beta-cells has not been elucidated. In the present study, we examined whether primary beta-cells possess Raf-1 and tested the hypothesis that Raf-1 is critical for beta-cell survival. Using reverse transcriptase-PCR, Western blot, and immunofluorescence, we identified Raf-1 in human islets, mouse islets, and in the MIN6 beta-cell line. Blocking Raf-1 activity using a specific Raf-1 inhibitor or dominant-negative Raf-1 mutants led to a time- and dose-dependent increase in cell death, assessed by real-time imaging of propidium iodide incorporation, TUNEL, PCR-enhanced DNA laddering, and Caspase-3 cleavage. Although the rapid increase in apoptotic cell death was associated with decreased Erk phosphorylation, studies with two Mek inhibitors suggested that the classical Erk-dependent pathway could explain only part of the cell death observed after inhibition of Raf-1. An alternative Erk-independent pathway downstream of Raf-1 kinase involving the pro-apoptotic protein Bad has recently been characterized in other tissues. Inhibiting Raf-1 in beta-cells led to a striking loss of Bad phosphorylation at serine 112 and an increase in the protein levels of both Bad and Bax. Together, our data strongly suggest that Raf-1 signaling plays an important role regulating beta-cell survival, via both Erk-dependent and Bad-dependent mechanisms. Conversely, acutely inhibiting phosphatidylinositol 3-kinase Akt had more modest effects on beta-cell death. These studies identify Raf-1 as a critical anti-apoptotic kinase in pancreatic beta-cells and contribute to our understanding of survival signaling in this cell type.
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PMID:Inhibition of Raf-1 alters multiple downstream pathways to induce pancreatic beta-cell apoptosis. 1800 2

Endotoxin, tumor necrosis factor (TNF), and organic dust constitute proinflammatory stimuli involved in the initiation of inflammation. The major receptor for endotoxin (lipopolysaccharide [LPS]) is Toll-like receptor 4 (TLR4), whereas TLR2 binds to agents from gram-positive bacteria. The aim of the study was to elucidate whether TLR2 and TLR4, expressed on primary bronchial epithelial cells (PBECs), are influenced by exogenous (organic dust and LPS) and endogenous (TNF) stimuli and whether this interaction is influenced by a glucocorticosteroid. The cells were exposed to LPS (10 microg/ml), TNF (10 ng/ml), or dust (100 microg/ml) 1.5 and 6 h, in the presence or absence of budesonide (10(-6) M) in vitro. The mRNA expression of interleukin (IL)-6, IL-8, TLR2, and TLR4 were measured with real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and IL-6 and IL-8 release was assessed with enzyme-linked immunosorbent assay (ELISA). To elucidate the importance of TLR-signaling for IL-6 and IL-8 secretion, the effect of TLR-blockers was studied. Endotoxin, TNF, and dust stimulated the release of IL-6 and IL-8 in a time-dependent manner. Budesonide significantly attenuated the release and expression of IL-6 and IL-8 after exposure. Budesonide did not influence TLR expression, but costimulation with LPS, TNF, or dust together with budesonide increased TLR2 expression synergistically. Blocking of TLR2 and TLR4 reduced cytokine secretion in stimulated cells. Budesonide reduced IL-6 and IL-8 production and enhanced expression of TLR2 in PBECs only in the presence of a proinflammatory stimulus. These findings contribute to our understanding of the beneficial effects of glucocorticosteroids during chronic obstructive pulmonary disease (COPD) exacerbations and asthma, which are frequently caused by microorganisms.
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PMID:Budesonide enhances Toll-like receptor 2 expression in activated bronchial epithelial cells. 2038 3

The mechanism of testosterone inducing the tissue factor pathway inhibitor (TFPI) in protecting against thrombosis is unknown. We aimed to elucidate the mechanisms involved in the induction by observing, in human umbilical vein endothelial cells (HUVECs), the phosphorylation of mitogen-activated protein kinases (MAPKs), a major cell signaling system. The level of testosterone regulating several signaling pathways, including extracellular signal-regulated kinases 1/2 (ERK1/2), c-Jun-N-terminal kinase (JNK), and p38 MAPK, was measured by western blot in HUVECs. ELISA and quantitative real-time reverse transcriptase-PCR were used to analyze TFPI expression after blocking ERK1/2 (with PD98059) or JNK (with SP600125) pathway in HUVECs. Testosterone-induced a rapid phosphorylation of ERK1/2, JNK and p38 MAPK in HUVECs, which could not be inhibited by androgen receptor antagonist flutamide. Blocking ERK1/2 or JNK pathway could significantly impair testosterone-induced TFPI at both translational and transcriptional levels in HUVECs. Testosterone at a physiological concentration may help to prevent thrombosis development by stimulating TFPI expression in HUVECs, partly through the ERK1/2 and JNK MAPK pathway.
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PMID:Mitogen-activated protein kinases pathway is involved in physiological testosterone-induced tissue factor pathway inhibitor expression in endothelial cells. 2044 53


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