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)

Morphiceptin (Tyr-Pro-Phe-Pro-NH(2)) and its analogs modified at position 3: [D-Phe(3)]morphiceptin, [D-ClPhe(3)]morphiceptin and [D-Cl(2)Phe(3)]morphiceptin were synthesized and labeled with [(125)I] or [(131)I]. Their binding to membranes isolated from experimental adenocarcinoma was examined in vitro with the use of a cross-linking assay followed by the Western blot technique. The radioactive complex had molecular weight of about 65 kDa and was detectable by anti-mu-opioid receptor polyclonal antibody. Expression of the mu-opioid receptor in mouse mammary adenocarcinoma was confirmed by reverse transcriptase-polymerase chain reaction. The binding studies showed the highest affinity and capacity for [D-Phe(3)]morphiceptin (K(d) 0.39 and B(max) 1112) and [D-ClPhe(3)]morphiceptin (K(d) 1.8 and B(max) 220). Morphiceptin and its D-Cl(2)Phe analog had significantly lower B(max) values (131 and 83, respectively). Biodistribution experiments in tumor-bearing C3H/Bi mice with the use of the (131)I-labeled peptides confirmed the results of our in vitro studies. The highest accumulation of radioactive peptides in the tumor tissue was also found for peptides with D-Phe and D-ClPhe.
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PMID:Uptake of radiolabeled morphiceptin and its analogs by experimental mammary adenocarcinoma: in vitro and in vivo studies. 1509 15

The effects on virus viability and reverse transcriptase (RT) function of substituting Trp for Tyr or Phe residues within the polymerase domain of human immunodeficiency virus type 1 (HIV-1) RT have been analyzed with an infectious HIV-1 clone. Viruses containing mutations Y56W, F61W, F87W, F116W, Y127W, Y144W, F171W, Y181W, Y183W, Y188W, F227W, or Y232W in their RT-coding regions were viable and showed replication capacities similar or slightly reduced in comparison with the wild-type HIV-1. However, RTs bearing mutations F77W or Y146W had a dNTP-binding defect, rendering nonviable viruses. HIV-1 carrying RT mutations F124W or F130W replicated very poorly, but compensatory changes (K83R for F124W, and T58S for F130W) were selected upon passaging the virus in cell culture. The amino acid substitution F130W diminishes the stability of the 51-kDa subunit of the RT (p51) and impairs polyprotein processing in virus-infected cells, an effect that can be mitigated when T58S is found in p51.
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PMID:Tryptophan scanning mutagenesis of aromatic residues within the polymerase domain of HIV-1 reverse transcriptase: critical role of Phe-130 for p51 function and second-site revertant restoring viral replication capacity. 1520 25

We recently identified a novel hypothalamic dodecapeptide inhibiting gonadotropin release in the Japanese quail (Coturnix japonica). This novel peptide was therefore named gonadotropin-inhibitory hormone (GnIH). The GnIH precursor encoded one GnIH and two GnIH-related peptides (GnIH-RP-1 and GnIH-RP-2) that shared the same C-terminal motif, Leu-Pro-Xaa-Arg-Phe-NH(2) (Xaa=Leu or Gln; LPXRF-amide peptides). Identification of the receptor for GnIH is crucial to elucidate the mode of action of GnIH. We therefore identified the receptor for GnIH in the quail diencephalon and characterized its expression and binding activity. We first cloned a cDNA encoding a putative GnIH receptor by a combination of 3' and 5' rapid amplification of cDNA ends (RACE) using PCR primers designed from the sequence for the receptor for rat RF-amide-related peptide (RFRP), an orthologous peptide of GnIH. Hydrophobic analysis revealed that the putative GnIH receptor possessed seven transmembrane domains, indicating a new member of the G protein-coupled receptor superfamily. The crude membrane fraction of COS-7 cells transfected with the putative GnIH receptor cDNA specifically bound to GnIH and GnIH-RPs in a concentration-dependent manner. Scatchard plot analysis of the binding showed that the identified GnIH receptor possessed a single class of high-affinity binding sites (K(d)=0.752 nM, B(max)=24.8 fmol/mg protein). Southern blotting analysis of reverse transcriptase-mediated PCR products revealed the expression of GnIH receptor mRNA in the pituitary gland and several brain regions including diencephalon in the quail. These results suggest that GnIH acts directly on the pituitary via GnIH receptor to inhibit gonadotropin release. GnIH may also act on the hypothalamus to inhibit gonadotropin-releasing hormone release.
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PMID:A novel G protein-coupled receptor for gonadotropin-inhibitory hormone in the Japanese quail (Coturnix japonica): identification, expression and binding activity. 1564 2

The biologically active forms of human immunodeficiency viruses type 1 and 2 reverse transcriptase (RT) found in infectious virions are heterodimers. We have previously shown that the dimeric nature of reverse transcriptase represents an important target for the design of a new class of antiviral agents and have designed a short peptide (Pep-7) derived from the tryptophan-rich motif of the connection subdomain that blocks dimerization of reverse transcriptase in vitro and abolishes viral infection. In the present work, we have investigated the mechanism through which this peptide inhibits RT dimerization and consequently viral propagation. We demonstrate that Pep-7 interacts preferentially with the p51 subunit within the heterodimeric reverse transcriptase, which destabilizes reverse transcriptase dimer conformation, thereby triggering dissociation. We have identified two residues Trp(24) and Phe(61), located on the fingers subdomain of p51, required for Pep-7 binding. Selective mutation of these residues on p51 to a glycine dramatically alters the stability of the RT-heterodimer suggesting that the fingers subdomain of p51 is also involved in stabilization of reverse transcriptase. We propose that the binding site of Pep-7 is located in a cleft between the fingers and the connection subdomains of p51 that contains the two highly conserved residues Phe(61) and Trp(24).
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PMID:Insight into the mechanism of a peptide inhibitor of HIV reverse transcriptase dimerization. 1569 16

Mutations at codon 215 of HIV-1 reverse transcriptase (RT) confer resistance to nucleoside analogs through RT-catalyzed ATP-dependent phosphorolysis. We showed that mutation T215Y is predominant over T215F (respectively 38.8 vs. 7.04% of 7312 sequences from a cohort of patients receiving antiretroviral therapy in France). Ambiguous mixtures at codon 215 (e.g. TNYS and TFSI) were resolved by cloning and sequencing representative clinical samples. Mutation T215F was preferentially associated with K70R (>71%), D67N (>73%) and K219Q/E/N (>76%), whereas T215Y was associated with M41L (>84%) and L210W (>58%). A similar distribution was observed with RT sequences stored in the Stanford HIV Drug Resistance Database. The structural background of these two distinct mutational patterns was investigated by molecular modeling of ATP-mutant RT complexes, on the basis of known ATP-protein interactions. We found that the aromatic side chain of tyrosine (Y)--but not phenylalanine (F)--optimally stacked with the adenine ring of ATP. Mutation L210W further stabilized this aromatic pi-pi stacking interaction, increasing the affinity of the T215Y/L210W double mutant for ATP. Overall, this study provides a biochemical basis accounting for the evolutionary pathway of T215 mutations in HIV-1 RT, leading to the preferential selection of T215Y vs. T215F.
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PMID:Structural analysis of reverse transcriptase mutations at codon 215 explains the predominance of T215Y over T215F in HIV-1 variants selected under antiretroviral therapy. 1620 Mar 50

To address the issue of melanocortin-1 receptor (MC1R) expression in non-melanocytic cells, we have quantitatively evaluated the relative expression levels of both MC1R mRNA and protein in a subset of different cell types. Using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) at high cycle numbers, we detected MC1R mRNA in all cell types examined, including human embryonic kidney-293 (HEK 293) cells, a cell type widely used as a negative control in melanocortin expression studies. Quantitative real-time PCR revealed the highest levels of MC1R transcripts were in melanocytic cells, whereas the keratinocyte and fibroblast cell cultures examined had only a low level of expression, similar to that of HEK 293 cells. Antibody mediated detection of MC1R protein in membrane extracts demonstrated exogenous receptor in MC1R transfected cell lines, as well as endogenous MC1R in melanoma cells. However, radioligand binding procedures were required to detect MC1R protein of normal human melanocytes and no surface expression of MC1R was detected in any of the non-melanocytic cells examined. This was consistent with their low level of mRNA, and suggests that, if present, the levels of surface receptor are significantly lower than that in melanocytes. The capacity of such limited levels of MC1R protein to influence non-melanocytic skin cell biology would likely be severely compromised. Indeed, the MC1R agonist [NIe(4), D-Phe(7)] alpha-melanocyte stimulating hormone (NDP-MSH) was unable to elevate intracellular cyclic adenosine monophosphate (cAMP) levels in the keratinocyte and fibroblast cells examined, whereas a robust increase was elicited in melanocytes. Although there are a variety of cell types with detectable MC1R mRNA, the expression of physiologically significant levels of the receptor may be more restricted than the current literature indicates, and within epidermal tissue may be limited to the melanocyte.
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PMID:Quantitative analysis of MC1R gene expression in human skin cell cultures. 1642 Feb 49

Kinins are biologically active peptides that are powerful mediators of cellular inflammation. They mimic the cardinal signs of inflammation by inducing vasodilatation and by increasing vascular permeability and pain. Neutrophils are chemoattracted to sites of inflammation by several stimuli. However, the evidence concerning the chemotactic effect of kinin peptides has been contradictory. We analyzed the chemotactic effect of kinin B(1) receptor agonists on neutrophils isolated from peripheral blood of human healthy subjects. Chemotaxis was performed using the migration under agarose technique. To test the effect of B(1) receptor agonists, each assay was carried out overnight at 37 degrees C in 5% CO(2)-95% air on neutrophils primed with 1 ng/ml interleukin-1beta. Simultaneous experiments were performed using unprimed cells or cells challenged with formyl-Met-Leu-Phe (fMLP). A clear chemotactic activity was observed when primed neutrophils were challenged with Lys-des[Arg(9)]-bradykinin (LDBK) or des[Arg(9)]-bradykinin at 10(-10) M but not when unprimed cells were used. A reduction in the chemotactic response was observed after priming of cells in the presence of 0.5 mM cycloheximide and 10 mug/ml brefeldin A, suggesting that some protein biosynthesis is required. Techniques such as reverse transcriptase-polymerase chain reaction and in situ hybridization confirmed the expression of the B(1) receptor mRNA, and immunocytochemistry and autoradiography demonstrated the expression of the B(1) receptor protein. In contrast to other chemoattractants such as fMLP, cytosolic intracellular calcium did not increase in response to the B(1) receptor agonist LDBK. A generation of kinin B(1) receptor agonists during the early phase of acute inflammation may favor the recruitment of neutrophils to the inflammatory site.
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PMID:Activation of kinin B1 receptors induces chemotaxis of human neutrophils. 1667 Jan 23

Previous studies using quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis have shown that the P2Y(14) receptor is expressed at high levels in human neutrophils. Therefore the primary aim of this study was to determine whether the P2Y(14) receptor is functionally expressed in human neutrophils. In agreement with previous studies RT-PCR analysis detected the expression of P2Y(14) receptor mRNA in human neutrophils. UDP-glucose (IC(50)=1 microM) induced a small but significant inhibition (circa 30%) of forskolin-stimulated cAMP accumulation suggesting functional coupling of endogenously expressed P2Y(14) receptors to the inhibition of adenylyl cyclase activity in human neutrophils. In contrast, the other putative P2Y(14) receptor agonists UDP-galactose and UDP-glucuronic acid (at concentrations up to 100 microM) had no significant effect, whereas 100 microM UDP-N-acetylglucosamine-induced a small but significant inhibition of forskolin-stimulated cAMP accumulation (20% inhibition). UDP-galactose, UDP-glucuronic acid and UDP-N-acetylglucosamine behaved as partial agonists by blocking UDP-glucose mediated inhibition of forskolin-induced cAMP accumulation. Treatment of neutrophils with pertussis toxin (G(i/o) blocker) abolished the inhibitory effects of UDP-glucose on forskolin-stimulated cAMP accumulation. UDP-glucose (100 microM) also induced a modest increase in extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, whereas the other sugar nucleotides had no effect on ERK1/2 activation. Finally, UDP-glucose and related sugar nucleotides had no significant effect on N-formyl-methionyl-leucyl-phenylalanine-induced elastase release from neutrophils. In summary, although we have shown that the P2Y(14) receptor is functionally expressed in human neutrophils (coupling to inhibition of forskolin-induced cAMP and ERK1/2 activation) it does not modulate neutrophil degranulation (assessed by monitoring elastase release). Clearly further studies are required in order to establish the functional role of the P2Y(14) receptor expressed in human neutrophils.
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PMID:Functional expression of the P2Y14 receptor in human neutrophils. 1682 Jan 47

Crystallographic studies have implicated several residues of the p66 fingers subdomain of human immunodeficiency virus type-1 reverse transcriptase in contacting the single-stranded template overhang immediately ahead of the DNA polymerase catalytic center. This interaction presumably assists in inducing the appropriate geometry on the template base for efficient and accurate incorporation of the incoming dNTP. To investigate this, we introduced nucleoside analogs either individually or in tandem into the DNA template ahead of the catalytic center and investigated whether they induce pausing of the replication machinery before serving as the template base. Analogs included abasic tetrahydrofuran linkages, neutralizing methylphosphonate linkages, and conformationally locked nucleosides. In addition, several Phe-61 mutants were included in our analysis, based on previous data indicating that altering this residue affects both strand displacement synthesis and the fidelity of DNA synthesis. We demonstrate here that altering the topology of the template strand two nucleotides ahead of the catalytic center can interrupt DNA synthesis. Mutating Phe-61 to either Ala or Leu accentuates this defect, whereas replacement with an aromatic residue (Trp) allows the mutant enzyme to bypass the template analogs with relative ease.
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PMID:Examining interactions of HIV-1 reverse transcriptase with single-stranded template nucleotides by nucleoside analog interference. 1686 79

Molt-inhibiting hormone (MIH), a member of the crustacean hyperglycemic neuropeptide hormone family, inhibits ecdysteroidogenesis in the molting gland or Y-organ (YO). A cDNA encoding MIH of the land crab (Gel-MIH) was cloned from eyestalk ganglia (EG) by a combination of reverse transcriptase polymerase chain reaction (RT-PCR) and 3'- and 5'-rapid amplification of cDNA ends (RACE). The cDNA (1.4 kb) encoded MIH prohormone containing a 35 amino acid signal peptide and a 78 amino acid mature peptide. The mature peptide had the six cysteines, one glycine, two arginines, one aspartate, one phenylalanine, and one asparagine in identical positions in the highly conserved sequence characteristic of other crustacean MIHs. Gel-MIH was expressed only in the EG, as determined by RT-PCR; it was not detected in Y-organ, heart, integument, gill, testis, ovary, hepatopancreas, thoracic ganglion, or skeletal muscle. A cDNA encoding the mature peptide was used to express recombinant MIH (rMIH) using a yeast (Pichia pastoris) expression system. Two constructs were designed to yield either a mature MIH fusion protein with a c-myc epitope and histidine (His) tag at the carboxyl terminus or an untagged mature protein without the c-myc and His sequences. Immunoreactive peptides were detected in Western blots of the cell culture media with both MIH constructs, indicating secretion of the processed rMIH into the medium. Culture media containing the untagged mature peptide significantly inhibited ecdysteroid secretion by YOs from land crab and green crab (Carcinus maenas) cultured in vitro, indicating that the Gel-rMIH was biologically active.
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PMID:Molt-inhibiting hormone from the tropical land crab, Gecarcinus lateralis: cloning, tissue expression, and expression of biologically active recombinant peptide in yeast. 1709 91


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