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)

A new high-performance liquid chromatography (HPLC) with UV detection assay was developed for the simultaneous determination of protease inhibitors (PIs), nucleoside and non-nucleoside reverse transcriptase inhibitors (NRTIs, NNRTIs) using a single 1-ml plasma samples. A solid-liquid extraction procedure without internal standard was coupled with two separate reversed-phase HPLC systems; one for the determination of amprenavir, efavirenz, indinavir, nelfinavir, ritonavir, saquinavir (run time=32 min) and one for the determination of abacavir, didanosine, lamivudine, stavudine, nevirapine, zidovudine (run time=40 min). The first requires a mobile phase containing sodium phosphate buffer+ion pair-acetonitrile (50:50, v/v) through a C18 Symmetry column (250x4.6 mm I.D., 5 microm particle size), using variable wavelengths (241, 254 and 261 nm). The second system requires three mobile phases (potassium phosphate buffer+ion pair-acetonitrile) for different elution through a C18 Symmetry Shield column (250x4.6 mm I.D., 5 microm), using a single wavelength (260 nm). Peak-areas are linear; correlation coefficients are better than 0.998 for all compounds, with both inter- and intra-day relative standard deviations lower than 12%. Extraction recoveries are higher than 93% for PIs and NNRTIs and higher than 70% for NRTIs. The method is specific and sensitive and was used to determine trough and peak levels of antiretroviral drugs in HIV infected patients under various combinations of RTIs and PIs.
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PMID:Determination of twelve antiretroviral agents in human plasma sample using reversed-phase high-performance liquid chromatography. 1099 10

Angiotensin II (Ang II) is one of the most potent vasoconstrictor substances, yet paradoxically, Ang II may dilate certain vascular beds via an undefined mechanism. Ang II-induced vasoconstriction is mediated by the AT(1) receptor, whereas the relative expression and functional importance of the AT(2) receptor in regulating vascular resistance and blood pressure are unknown. We now report that Ang II induces relaxation of mesenteric microvessels and that this vasodilatory response was unaffected by losartan, an AT(1) receptor antagonist, but was inhibited by PD123,319, a selective antagonist of AT(2) receptors. In addition, reverse transcriptase-polymerase chain reaction studies revealed high amounts of AT(2) receptor mRNA in smooth muscle from these same microvessels. Ang II-induced relaxation was inhibited by either tetraethylammonium or iberiotoxin, suggesting involvement of the large-conductance, calcium- and voltage-activated potassium (BK(Ca)) channel. Subsequent whole-cell and single-channel patch-clamp studies on single myocytes demonstrated that Ang II increases the activity of BK(Ca) channels. As in our tissue studies, the effect of Ang II on BK(Ca) channels was inhibited by PD123,319, but not by losartan. In light of these consistent findings from tissue physiology, molecular studies, and cellular/molecular physiology, we conclude that Ang II relaxes microvessels via stimulation of the AT(2) receptor with subsequent opening of BK(Ca) channels, leading to membrane repolarization and vasodilation. These findings provide evidence for a novel endothelium-independent vasodilatory effect of Ang II.
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PMID:Angiotensin II relaxes microvessels via the AT(2) receptor and Ca(2+)-activated K(+) (BK(Ca)) channels. 1123 Feb 89

Unlike most DNA polymerases, reverse transcriptases can initiate DNA synthesis at a single-strand break and displace the downstream non- template strand simultaneously with extension of the primer. This reaction is important for generation of the long terminal repeat sequences in the duplex DNA product of retroviral reverse transcription. Oligonucleotide-based model displacement constructs were used to study the interaction of human immunodeficiency virus type 1 and Moloney murine leukemia virus reverse transcriptases with the DNA. Under conditions where the DNA is saturated with enzyme, there is no protection against DNase I cleavage of the 5' single-stranded extension that would correspond to the already-displaced strand. However, the DNase I footprint on the non-template strand extends from the +1 to the +9 position for the human immunodeficiency virus type 1 enzyme and from +1 to +7 or +8 for the Moloney enzyme. This extent of protection on the non-template strand is similar to what was observed previously for the template strand downstream from the primer terminus. Use of potassium permanganate as a probe for unpaired bases in the region ahead of the primer terminus reveals that the two base-pairs immediately in front of the enzyme are melted by the bound enzyme. These findings are consistent with a displacement mechanism in which the reverse transcriptase plays an active role in unpairing the DNA ahead of the translocating polymerase. The results are interpreted in light of a recent crystal structure showing the nature of the protein-DNA contacts with the template strand ahead of the primer terminus.
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PMID:Structural alterations in the DNA ahead of the primer terminus during displacement synthesis by reverse transcriptases. 1123 9

Expression of the voltage-gated K(+) channel Kv2.1, a possible molecular correlate for the cardiac delayed rectifier current (I(K)), has recently been shown to vary between individual ventricular myocytes. The functional consequences of this cell-to-cell heterogeneity in Kv2.1 expression are not known. Using multiplex single-cell reverse transcriptase-polymerase chain reaction (RT-PCR), we detected Kv2.1 mRNA in 47% of isolated midmyocardial myocytes from the rat left ventricular free wall that were positive for alpha-myosin heavy chain mRNA (n=74). Whole-cell patch-clamp recordings demonstrated marked differences in the magnitude of I(K) (200 to 1450 pA at V(Pip)=40 mV) between individual myocytes of the same origin. Furthermore, the tetraethylammonium (TEA)-sensitive outward current (I(TEA)), known to be partly encoded by Kv2.1 in mice, revealed a wide range of current magnitudes between single cells (150 to 1130 pA at V(Pip)=40 mV). Combined patch-clamp recordings and multiplex single-cell RT-PCR analysis of the same myocytes, however, showed no differences in I(K) or I(TEA) magnitude or inactivation kinetics between myocytes expressing Kv2.1 mRNA and those that did not express Kv2.1 mRNA. In contrast, in all midmyocardial myocytes expressing the transient outward potassium current (I(to1)), Kv4 mRNA, which has been shown to underlie I(to1), was detected (n=10). These results indicate that I(K) heterogeneity among individual left ventricular myocytes cannot be explained by the distribution pattern of Kv2.1 mRNA. Other mechanisms besides Kv2.1 mRNA expression appear to determine magnitude and kinetics of I(K) in rat ventricular myocytes.
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PMID:Heterogeneity of Kv2.1 mRNA expression and delayed rectifier current in single isolated myocytes from rat left ventricle. 1124 65

Cortical spreading depression (CSD) is characterized by reversible neuronal dysfunction in the absence of cell death. Preconditioning by CSD induces tolerance against subsequent lethal ischemia. In this study, we used quantitative reverse transcriptase-polymerase chain reaction and immunocytochemistry to analyze proinflammatory cytokine expression after CSD induced by topical application of potassium chloride (KCl) to the cortical surface of rat brains. Relative to control cortex, we found an increase of tumor necrosis factor-alpha (mean 62-fold, P < 0.001) and interleukin (IL)-1beta (mean 24-fold, P < 0.001) mRNA levels within 4 hours ipsilateral to the site of KCl application. At 16 hours cytokine expression was decreasing toward baseline levels. Ipsilateral cytokine induction was abolished by pretreatment with the noncompetitive N-methyl-d-aspartate antagonist, MK-801. In contrast to focal cortical infarction, cytokine induction in CSD was not accompanied by the expression of inducible nitric oxide synthase mRNA. In immunocytochemical studies, expression of IL-1beta protein was localized to ramified microglia in cortical layers I to III of the ipsilateral hemisphere. Our finding that NMDA receptor signaling without subsequent neuronal cell death is sufficient to induce inflammatory cytokine expression in the brain has basic implications for central nervous system immunoregulation. We postulate that cytokine expression in CSD forms part of a physiologic stress response that contributes to the development of ischemic tolerance in this and other preconditioning paradigms.
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PMID:Cortical spreading depression induces proinflammatory cytokine gene expression in the rat brain. 1129 76

Precise cleavage at the polypurine tract (PPT)/U3 junction by human immunodeficiency virus type 1 (HIV-1) reverse transcriptase RNase H is critical for generating a correct viral DNA end for subsequent integration. Using potassium permanganate (KMnO(4)) modification, we have identified a significant distortion in the nucleic acid structure at the HIV-1 PPT/U3 junction in the absence of trans-acting factors. Unusually high reactivity of template thymine +1 is detected when the PPT primer is extended by DNA or RNA at its 3' terminus. Chemical footprinting suggests that the extent of base unstacking in the wild-type species is comparable when the +1 A:T base pair is replaced by a C:T mismatch. However, reactivity of this template base is diminished after alterations to upstream (rA)(4):(dT)(4) or (rG)(6):(dC)(6) tracts. Importantly, there is a correlation between the structural deformation at base pair +1 and precise cleavage at the PPT/U3 junction by HIV-1 reverse transcriptase/RNase H. KMnO(4) modification also revealed unusually high reactivity for one of two (dT)(4):(rA)(4) duplexes upstream of the PPT/U3 junction, suggesting a significant structural distortion within the PPT itself in the absence of the retroviral polymerase. Structural abnormalities in this region are not only essential for resistance of the PPT to hydrolysis but also significantly impact the conformation of the PPT/U3 junction. Our data collectively suggest that the entire PPT sequence contributes to the structural distortion at the PPT/U3 junction, potentially providing a mechanism for its selective processing.
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PMID:Pre-existing distortions in nucleic acid structure aid polypurine tract selection by HIV-1 reverse transcriptase. 1187 59

By combining a Drosophila genome data base search and reverse transcriptase-PCR-based cDNA isolation, two G-protein-coupled receptors were cloned, which are the closest known invertebrate homologs of the mammalian opioid/somatostatin receptors. However, when functionally expressed in Xenopus oocytes by injection of Drosophila orphan receptor RNAs together with a coexpressed potassium channel, neither receptor was activated by known mammalian agonists. By applying a reverse pharmacological approach, the physiological ligands were isolated from peptide extracts from adult flies and larvae. Edman sequencing and mass spectrometry of the purified ligands revealed two decapentapeptides, which differ only by an N-terminal pyroglutamate/glutamine. The peptides align to a hormone precursor sequence of the Drosophila genome data base and are almost identical to allatostatin C from Manduca sexta. Both receptors were activated by the synthetic peptides irrespective of the N-terminal modification. Site-directed mutagenesis of a residue in transmembrane region 3 and the loop between transmembrane regions 6 and 7 affect ligand binding, as previously described for somatostatin receptors. The two receptor genes each containing three exons and transcribed in opposite directions are separated by 80 kb with no other genes predicted between. Localization of receptor transcripts identifies a role of the new transmitter system in visual information processing as well as endocrine regulation.
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PMID:Functional annotation of two orphan G-protein-coupled receptors, Drostar1 and -2, from Drosophila melanogaster and their ligands by reverse pharmacology. 1216 55

Syntheses of [carbonyl-11C]2-(2-benzoylphenoxy)-N-phenylacetamide, a radiolabeled inhibitor of human immunodeficiency virus type 1 (HIV 1) reverse transcriptase, were achieved by applying palladium-mediated cross-coupling reactions with insertion of [11C]carbon monoxide. Our interest was focused for the present on a comparison of the Stille and Suzuki methods, using trimethylphenylstannane or phenylboronic acid as alternative coupling reagents, respectively. The Suzuki variant gave a much higher amount of [11C]CO radioactivity trapped in the reaction mixture, but a significant loss of product occurred due to adsorption phenomena on the potassium carbonate present in the heterogeneous reaction mixture. The labeled product was isolated in only 20% yield (based on trapped [11C]CO, not corrected for decay). According to Stille, the reaction provided a product that could be isolated more easily but it did not increase the final yield of the target compound due to a low trapping efficiency for [11C]CO. Both methods were performed in an overall synthesis time of 30min, starting from [11C]CO2, and gave a product with a specific radioactivity of at least 30GBq/micromol. The Stille method as well as the Suzuki reaction allowed the synthesis of a radiochemically pure product in aqueous acetonitrile.
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PMID:Syntheses of [carbonyl-11C]2-(2-benzoylphenoxy)-N-phenylacetamide from [11C]carbon monoxide by the Suzuki and the Stille reactions. 1243 42

Stretches of guanines can associate in vitro through Hoogsteen hydrogen bonding to form four-stranded structures. In the HIV-1 central DNA flap, generated by reverse transcriptase at the end of retrotranscription, both the two 99 nt-long overlapping (+) strands contain two adjacent tracts of guanines. This study demonstrates that oligonucleotides containing these G-clusters form highly stable G-quadruplexes of various structures in vitro, whose formation was controlled by an easy and reversible protocol using sodium hydroxide. Among these sequences, a G'2 hairpin dimer was the most stable structure adopted by the 5'-tail of the (+) downstream strand. Since the two (+) strands of the HIV-1 central DNA flap hold these G-clusters, and based on the properties of reverse branch migration in DNA flaps, constructions using HIV-1 sequences were assembled to mimic small DNA flaps where the G-clusters are neighbors. G-quartets were successfully probed in such flaps. They were induced by potassium and by a dibenzophenanthroline derivative already known to stabilize them. Such results suggest some function(s) for G-quartets associated with a DNA flap in the HIV-1 pre-integration steps, and argue for their transient formation during the processing of G-rich DNA flaps at the time of replication and/or repair.
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PMID:G-quartets assembly within a G-rich DNA flap. A possible event at the center of the HIV-1 genome. 1246 53

In search of K(+) channel genes expressed in the leaf of the C(4) plant Zea mays, we isolated the cDNA of KZM1 (for K(+) channel Zea mays 1). KZM1 showed highest similarity to the Arabidopsis K(+) channels KAT1 and KAT2, which are localized in guard cells and phloem. When expressed in Xenopus oocytes, KZM1 exhibited the characteristic features of an inward-rectifying, potassium-selective channel. In contrast to KAT1- and KAT2-type K(+) channels, however, KZM1 currents were insensitive to external pH changes. Northern blot analyses identified the leaf, nodes, and silks as sites of KZM1 expression. Following the separation of maize leaves into epidermal, mesophyll, and vascular fractions, quantitative real-time reverse transcriptase-PCR allowed us to localize KZM1 transcripts predominantly in vascular strands and the epidermis. Cell tissue separation and KZM1 localization were followed with marker genes such as the bundle sheath-specific ribulose-1,5-bisphosphate carboxylase, the phloem K(+) channel ZMK2, and the putative sucrose transporter ZmSUT1. When expressed in Xenopus oocytes, ZmSUT1 mediated proton-coupled sucrose symport. Coexpression of ZmSUT1 with the phloem K(+) channels KZM1 and ZMK2 revealed that ZMK2 is able to stabilize the membrane potential during phloem loading/unloading processes and KZM1 to mediate K(+) uptake. During leaf development, sink-source transitions, and diurnal changes, KZM1 is constitutively expressed, pointing to a housekeeping function of this channel in K(+) homeostasis of the maize leaf. Therefore, the voltage-dependent K(+)-uptake channel KZM1 seems to mediate K(+) retrieval and K(+) loading into the phloem as well as K(+)-dependent stomatal opening.
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PMID:The K+ channel KZM1 mediates potassium uptake into the phloem and guard cells of the C4 grass Zea mays. 1261 1


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