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 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

Extracellular nucleotides are stress-responsive ligands that mediate a variety of cellular processes via purinoceptors. We hypothesized that mechanical ventilation (MV) would alter the extracellular adenyl-nucleotide profile and purinoceptor expression in lung and extrapulmonary tissues. Twenty-eight rats were randomized to: (i) unventilated control animals; (ii) tidal volume (VT; 6 ml/kg); (iii) VT (6 ml/kg) and positive end-expiratory pressure (PEEP; 5 cm H20); (iv) VT (12 ml/kg); or (v) VT (12 ml/kg) and PEEP (5 cm H20). Bronchoalveolar lavage (BAL) was analyzed for adenyl-nucleotides. Pulmonary, hepatic, and renal tissues were assessed for P2Y4, P2Y6, P2X7, A3, and A2b receptor expression by real-time reverse transcriptase-polymerase chain reaction and Fas/Fas ligand mRNA was quantified in the lung. MV produced volume-dependent changes in BAL nucleotides; AMP and adenosine increased, whereas ATP and ADP proportions decreased. Large-volume MV increased A2b mRNA and decreased P2X7 in the lung; mRNA changes in lung Fas ligand paralleled P2X7. PEEP normalized BAL nucleotide profiles and A2b expression. Injurious MV reduced hepatic and renal P2X7 mRNA; PEEP normalized these levels in both tissues. Large-volume MV also decreased renal A2b mRNA. MV alters the BAL adenyl-nucleotide profile and purinoceptor patterns in lung, liver, and kidney. PEEP normalizes the BAL nucleotide profile and receptor patterns in lung and extrapulmonary tissues.
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PMID:Mechanical ventilation alters airway nucleotides and purinoceptors in lung and extrapulmonary organs. 1538 14

Treatment of human immunodeficiency virus type 1 (HIV-1)-infected patients with 3'-azido-3'-deoxythymidine (AZT) selects for mutant forms of viral reverse transcriptase (RT) with increased ability to remove chain-terminating nucleotides from blocked DNA chains. We tested various cell extracts for the presence of endogenous acceptor substrates for this reaction. Cell extracts incubated with HIV-1 RT and [(32)P]ddAMP-terminated DNA primer/template gave rise to (32)P-labeled adenosine 2',3'-dideoxyadenosine 5',5'''-P(1),P(4)-tetraphosphate (Ap(4)ddA), ddATP, Gp(4)ddA, and Ap(3)ddA, corresponding to the transfer of [(32)P]ddAMP to ATP, PP(i), GTP, and ADP, respectively. Incubation with [(32)P]AZT monophosphate (AZTMP)-terminated primer/template gave rise to the analogous (32)P-labeled AZT derivatives. Based on the rates of formation of the specific excision products, ATP and PP(i) levels were determined: ATP was present at 1.3 to 2.2 mM in H9 cells, macrophages, and unstimulated CD4(+) or CD8(+) T cells, while PP(i) was present at 7 to 15 microM. Under these conditions, the ATP-dependent reaction predominated, and excision by the AZT-resistant mutant RT was more efficient than wild type RT. Activated CD4(+) or CD8(+) T cells contained 1.4 to 2.7 mM ATP and 55 to 79 microM PP(i). These cellular PP(i) concentrations are lower than previously reported; nonetheless, the PP(i)-dependent reaction predominated in extracts from activated T cells, and excision by mutant and wild-type RT occurred with similar efficiency. While PP(i)-dependent excision may contribute to AZT resistance in vivo, it is likely that selection of AZT-resistant mutants occurs primarily in an environment where the ATP-dependent reaction predominates.
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PMID:Intracellular substrates for the primer-unblocking reaction by human immunodeficiency virus type 1 reverse transcriptase: detection and quantitation in extracts from quiescent- and activated-lymphocyte subpopulations. 1585 93

Obesity is associated with elevated levels of leptin in the blood. Elevated leptin is a risk factor for thrombosis in humans, and leptin administration promotes platelet activation and thrombosis in the mouse. The current study examines the effect of leptin on human platelets, and provides initial insights into the nature of the leptin receptor on these platelets. Leptin potentiated the aggregation of human platelets induced by low concentrations of ADP, collagen and epinephrine. However, the response varied significantly between donors, with platelets from some donors (approximately 40%) consistently responding to leptin (responders) and those from other donors (approximately 60%) never responding (non-responders). Western blotting and reverse transcriptase-polymerase chain reaction (RT-PCR) experiments showed that platelets from both groups only express the signaling form of the leptin receptor, and that responder platelets express higher levels of this receptor than non-responders. Ligand-binding assays demonstrate specific, saturable binding of leptin to platelets from both groups with apparent K(d) values of 76 +/- 20 nM for responders and 158 +/- 46 nM for non-responders. Thus, the decreased sensitivity of non-responder platelets to leptin does not result from the absence of the signaling form of this receptor, but may reflect differences in its level of expression and/or affinity for leptin. These preliminary studies demonstrate that platelets are a major source of leptin receptor in the circulation, and suggest that leptin-responsive individuals may have a higher risk for obesity-associated thrombosis than non-responsive individuals.
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PMID:The leptin receptor system of human platelets. 1586 2

The obligate intracytoplasmic pathogen Rickettsia prowazekii relies on the transport of many essential compounds from the cytoplasm of the eukaryotic host cell in lieu of de novo synthesis, an evolutionary outcome undoubtedly linked to obligatory growth in this metabolite-replete niche. The paradigm for the study of rickettsial transport systems is the ATP/ADP translocase Tlc1, which exchanges bacterial ADP for host cell ATP as a source of energy, rather than as a source of adenylate. Interestingly, the R. prowazekii genome encodes four open reading frames that are highly homologous to the well-characterized ATP/ADP translocase Tlc1. Therefore, by annotation, the R. prowazekii genome encodes a total of five ATP/ADP translocases: Tlc1, Tlc2, Tlc3, Tlc4, and Tlc5. We have confirmed by quantitative reverse transcriptase PCR that mRNAs corresponding to all five tlc homologues are expressed in R. prowazekii growing in L-929 cells and have shown their heterologous protein expression in Escherichia coli, suggesting that none of the tlc genes are pseudogenes in the process of evolutionary meltdown. However, we demonstrate by heterologous expression in E. coli that only Tlc1 functions as an ATP/ADP transporter. A survey of nucleotides and nucleosides has determined that Tlc4 transports CTP, UTP, and GDP. Intriguingly, although GTP was not transported by Tlc4, it was an inhibitor of CTP and UTP uptake and demonstrated a K(i) similar to that of GDP. In addition, we demonstrate that Tlc5 transports GTP and GDP. We postulate that Tlc4 and Tlc5 serve the primary function of maintaining intracellular pools of nucleotides for rickettsial nucleic acid biosynthesis and do not provide the cell with nucleoside triphosphates as an energy source, as is the case for Tlc1. Although heterologous expression of Tlc2 and Tlc3 was observed in E. coli, we were unable to identify substrates for these proteins.
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PMID:Study of the five Rickettsia prowazekii proteins annotated as ATP/ADP translocases (Tlc): Only Tlc1 transports ATP/ADP, while Tlc4 and Tlc5 transport other ribonucleotides. 1692 93

Histaminergic neurons located in the posterior hypothalamus (tuberomamillary nucleus, TMN) project widely through the whole brain controlling arousal and attention. They are tonically active during wakefulness but cease firing during sleep. As a homeostatic theory of sleep involves ATP depletion and adenosine accumulation in the brain, we investigated the role of ATP and its analogues as well as adenosine on neuronal activity in the TMN. We show increased firing of rat TMN neurons by ATP, ADP, UTP and 2meSATP, indicating activation of receptors belonging to the P2Y family. Adenosine affected neither membrane potential nor firing of these cells. Single-cell reverse transcriptase-polymerase chain reaction revealed that P2Y1 and P2Y4 are prevailing receptors in TMN neurons. P2Y1 receptor mRNA was detected with a higher frequency in 2-week-old than in 4-week-old rats; in accordance, 2meSATP was more potent than ATP. Semi-quantitative real-time polymerase chain reaction revealed a developmental downregulation of mRNA levels for P2Y1 and P2Y4 receptors. Immunocytochemistry demonstrated neuronal and glial localization of the P2Y1 receptor protein. Network activity measured with multielectrode arrays in primary cultures made from the posterior hypothalamus was enhanced by UTP and 2meSATP (P2Y4 and P2Y1 agonists, respectively). ATP caused an inhibition of firing, which was reversed in the presence of suramin or gabazine [gamma-aminobutyric acid (GABA)A receptor antagonist], indicating that GABAergic neurons are preferentially activated by ATP in this network. Excitation of the wake-active TMN neurons by nucleotides and the lack of adenosine action may be important factors in sleep-wake regulation.
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PMID:P2Y receptor-mediated excitation in the posterior hypothalamus. 1696 43

Antiviral alpha-P-borano substituted NTPs are promising chain terminators targeting HIV reverse transcriptase (RT). Activation of antiviral nucleoside diphosphates (NDPs) to NTPs may be carried out by pyruvate kinase (PK) and creatine kinase (CK). Herein, are presented the effects of nucleobase, ribose, and alpha-phosphate substitutions on substrate specificities of CK and PK. Both enzymes showed two binding modes and negative cooperativity with respect to substrate binding. The stereospecificity and inhibition of ADP phosphorylation by alpha-P-borano substituted NDP (NDPalphaB) stereoisomers were also investigated. The Sp-ADPalphaB isomer was a 70-fold better substrate for CK than the Rp isomer, whereas PK preferred the Rp isomer of NDPalphaBs. For CK, the Sp-ADPalphaB isomer was a competitive inhibitor; for PK, the Rp-ADPalphaB isomer was a poor competitive inhibitor and the Sp-ADPalphaB isomer was a poor non-competitive inhibitor. Taken together, these data suggest that, although the Rp-NDPalphaB isomer would be minimally phosphorylated by CK or PK, it should not inhibit either enzyme.
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PMID:Stereospecificity, substrate, and inhibitory properties of nucleoside diphosphate analogs for creatine and pyruvate kinases. 1843 30

Sonodynamic therapy employs a combination of ultrasound and a sonosensitizer to enhance the cytotoxic effect of ultrasound and promote apoptosis. However, the mechanism underlying the synergistic effect of ultrasound and hematoporphyrin is still unclear. In this study, we investigated mechanism of the induction of apoptosis by sonodynamic therapy in Sarcoma 180 cells. The cell suspension was treated by 1.75-MHz focused continuous ultrasound at an acoustic power (I(SATA)) of 1.4+/-0.07 W/cm(2) for 3 min in the absence or presence of 20 microg/ml hematoporphyrin. The proportion of apoptotic cells was determined by flow cytometry. We then analyzed the reactive oxygen species generation and localization by confocal microscopy. Western blotting and reverse transcriptase-polymerase chain reaction were used to analyze the expression of caspase-8, caspase-9, poly(ADP)-ribose polymerase, and nuclear factor-kappaB. The findings of our study indicate that ultrasound treatment induced the activation of nuclear factor-kappaB as an early stress response. When cells were pretreated with hematoporphyrin, the initial response to the therapy was the formation of (1)O(2) in the mitochondria. Our results primarily demonstrate that the mechanisms of induction of apoptosis by ultrasound and hematoporphyrin-sonodynamic therapies are very different. Our findings can provide a basis for explaining the synergistic effect of ultrasound and hematoporphyrin.
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PMID:Potential mechanism in sonodynamic therapy and focused ultrasound induced apoptosis in sarcoma 180 cells in vitro. 1964 May 55

Trichomonas vaginalis is a parasite of the human urogenital tract that causes trichomonosis, the most prevalent non-viral sexually transmitted disease. Ectonucleoside triphosphate diphosphohydrolase (NTPDase) family members, which hydrolyse extracellular ATP and ADP and ecto-5'-nucleotidase, which hydrolyses AMP, have been characterised in T. vaginalis. For trichomonad culture, the growth medium is supplemented with 10% serum, which is an important source of nutrients, such as adenosine. Here, we investigated the ATP metabolism of T. vaginalis trophozoites from long-term cultures and clinical isolates under limited bovine serum conditions (1% serum). The specific enzymatic activities were expressed as nmol inorganic phosphate (Pi) released/min/mg protein, the gene expression patterns were determined by reverse transcriptase-polymerase chain reaction, the extracellular adenine nucleotide hydrolysis was analysed by high performance liquid chromatography and the cell cycle analysis was assessed by flow cytometry. Serum limitation led to the profound activation of NTPDase and ecto-5'-nucleotidase activities. Furthermore, the levels of NTPDase A and B transcripts increased and extracellular ATP metabolism was activated, which led to enhanced ATP hydrolysis and the formation of ADP and AMP. Moreover, the cell cycle was arrested at the G0/G1 stage, which suggested adenosine uptake. Our data suggest that under conditions of serum limitation, NTPDase and ecto-5'-nucleotidase play a role in providing the adenosine required for T. vaginalis growth and that this process contributes to the establishment of parasitism.
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PMID:Analysis of the NTPDase and ecto-5'-nucleotidase profiles in serum-limited Trichomonas vaginalis. 2241 54

The sirtuin/Sir2 (Silent information regulator 2) family of NAD+-dependent deacetylases and mono-ADP-ribosyltransferases plays an important role in several cellular processes including gene silencing, cell cycle regulation and life span extension in yeast and animals. Compared to other eukaryotes, plants have relatively fewer SIR2 related genes encoding only two putative SIR2 family proteins. Recently, two putative sirtuin genes were identified also in the grapevine genome. Starting from the predicted coding sequences present in the database, we have been able to obtain two truly expressed coding sequences from the start to the stop codon for both sirtuin genes that were named VvSRT1 and VvSRT2. The search for the expressed coding sequences was performed by comparing the predicted sequences with the recently available grape RNA seq database with the aim to develop the primers to be used in reverse transcriptase PCR reactions to amplify the genes of interest. Finally, in order to better understand the physiological role of both sirtuins, we investigated the expression of these genes in young leaves, mature leaves, and berries sampled at different growing stages. In leaves, usually it has been observed that VvSRT1 is less expresses than VvSRT2, moreover in young leaves VvSRT2 showed the higher expression during setting while in mature leaves during the flowering time. No particular variations have been observed concerning VvSRT1. In berries the two genes showed more similar expression level, and they showed the highest expression during the flowering time. Finally, the expression of VvSRT2 in berries is smaller than in leaves.
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PMID:Characterization and differential expression analysis of complete coding sequences of Vitis vinifera L. sirtuin genes. 2244 84


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