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:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An alternatively spliced isoform of the P2X1 receptor (P2X1a) was cloned from rat mesenteric artery. The spliced isoform does not have the 27 amino acids that are in the middle of the putative extracellular loop domain of the P2X1 original subunit (P2X1). Reverse transcriptase polymerase chain reaction (RT-PCR) revealed co-localization of P2X1a mRNA and P2X1 mRNA in vascular and other smooth muscle tissues and heart, but not in the spinal cord. In HEK293 cells transfected with P2X1 cDNA, ATP (1 microM) evoked an inward current which strongly desensitized, and an intense signal for GFP (green fluorescent protein) fused with P2X1 was detected at the membrane. Neither of these results was obtained in HEK293 cells expressing P2X1a alone. The fluorescent GFP signal was detected at the membrane when GFP-fused P2X1a was co-expressed with P2X1, and no significant difference in the ATP-activated current was noted between cells expressing P2X1 and those coexpressing P2X1 and P2X1a. These results indicate that the 27-amino-acid sequence (175-201) is important for protein trafficking to the membrane and for the formation of a functional P2X1 receptor. Our results also show that P2X1a is transported to the membrane when P2X1a is co-expressed with P2X1, although the co-expression of P2X1a does not modify the channel's current properties.
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PMID:Presence and possible role of the spliced isoform of the P2X1 receptor in rat vascular smooth muscle cells. 1120 62

Insulin-secreting pancreatic islet beta-cells possess anion-permeable Cl- channels (I(Cl,islet)) that are swelling-activated, but the role of these channels in the cells is unclear. The Cl- channel blockers 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and niflumic acid were evaluated for their ability to inhibit I(Cl,islet) in clonal beta-cells (HIT cells). Both drugs blocked the channel, but the blockade due to niflumic acid was less voltage-dependent than the blockade due to DIDS. HIT cell volume initially increased in hypotonic solution and was followed by a regulatory volume decrease (RVD). The addition of niflumic acid and, to a lesser extent, DIDS to the hypotonic solution potentiated swelling and blocked the RVD. In isotonic solution, niflumic acid produced swelling, suggesting that islet Cl- channels are activated under basal conditions. The channel blockers glyburide, gadolinium, or tetraethylammonium-Cl did not alter hypotonic-induced swelling or volume regulation. The Na/K/2Cl transport blocker furosemide produced cell shrinkage in isotonic solution and blocked cell swelling normally induced by hypotonic solution. Perifused HIT cells secreted insulin when challenged with hypotonic solutions. However, this could not be completely attributed to I(Cl,islet)-mediated depolarization, because secretion persisted even when Cl- channels were fully blocked. To test whether blocker-resistant secretion occurred via a distal pathway, distal secretion was isolated using 50 mmol/l potassium and diazoxide. Under these conditions, glucose-dependent secretion was blunted, but hypotonically induced secretion persisted, even with Cl- channel blockers present. These results suggest that beta-cell swelling stimulates insulin secretion primarily via a distal I(Cl,islet)-independent mechanism, as has been proposed for K(ATP)-independent glucose- and sulfonylurea-stimulated insulin secretion. Reverse transcriptase-polymerase chain reaction of HIT cell mRNA identified a CLC-3 transcript in HIT cells. In other systems, CLC-3 is believed to mediate swelling-induced outwardly rectifying Cl- channels. This suggests that the proximal effects of swelling to regulate cell volume may be mediated by CLC-3 or a closely related Cl- channel.
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PMID:Chloride channels regulate HIT cell volume but cannot fully account for swelling-induced insulin secretion. 1133 43

The effects of extracellular ATP, ADP, AMP and adenosine on cAMP accumulation have been studied in freshly isolated B-lymphocytes from patients with chronic lymphocytic leukemia. Extracellular ATP and several nucleotide analogs stimulated cAMP accumulation with the following order of potency: ATP (EC(50)=120+/-20 microM)>ADP>>AMP. ADP was less effective than ATP and may be a partial agonist. AMP exhibited variable but generally weak activity. The stable analog of ATP, alpha,beta-methylene ATP (EC(50)=110+/-15 microM) also stimulated cAMP accumulation and exhibited similar efficacy to ATP. The P2Y(2) receptor agonist, UTP had no effect on intracellular cAMP levels. Adenosine and the A(2A)/A(2B) receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA) also stimulated cAMP accumulation in CLL lymphocytes. Adenosine deaminase inhibited the cAMP response to adenosine but had no effect on the ATP-induced cAMP response. On the other hand, the AMP analog, adenosine 5'-thiomonophosphate, (AMPS; 1.0 mM) inhibited ATP-induced and alpha,beta-methylene ATP-induced cAMP production but had no effect on adenosine-induced cAMP production. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed the presence of P2Y(11) receptor as well as A(2A) and A(2B) receptor mRNA in chronic lymphocytic leukemia lymphocytes. However, A(2B) receptors would appear to be relatively ineffective because the A(2A) selective agonist, CGS-21680 exhibited comparable efficacy to NECA. Furthermore, the A(2A)-selective antagonist 8-(3-chlorostyryl)-caffeine (CSC) right-shifted the concentration-response curve for NECA. Taken together, the data indicate that ATP induces cAMP accumulation via the activation of P2Y(11) receptors whereas adenosine induces cAMP accumulation via the activation of A(2A) receptors. Coordinate activation of P2Y(11) and A(2A) receptors may influence the developmental fate of normal B-lymphocytes.
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PMID:P2Y(11) receptor expression by human lymphocytes: evidence for two cAMP-linked purinoceptors. 1152 39

The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), represented by nonstructural protein 5B (NS5B), is believed to form a membrane-associated RNA replication complex together with other nonstructural proteins and as yet unidentified host components. However, the determinants for membrane association of this essential viral enzyme have not been defined. By double label immunofluorescence analyses, NS5B was found in the endoplasmic reticulum (ER) or an ER-like modified compartment both when expressed alone or in the context of the entire HCV polyprotein. The carboxyl-terminal 21 amino acid residues were necessary and sufficient to target NS5B or a heterologous protein to the cytosolic side of the ER membrane. This hydrophobic domain is highly conserved among 269 HCV isolates analyzed and predicted to form a transmembrane alpha-helix. Association of NS5B with the ER membrane occurred by a posttranslational mechanism that was ATP-independent. These features define the HCV RdRp as a new member of the tail-anchored protein family, a class of integral membrane proteins that are membrane-targeted posttranslationally via a carboxyl-terminal insertion sequence. Formation of the HCV replication complex, therefore, involves specific determinants for membrane association that represent potential targets for antiviral intervention.
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PMID:Determinants for membrane association of the hepatitis C virus RNA-dependent RNA polymerase. 1155 52

In the central nervous system, the primary targets of the human immunodeficiency virus-1 (HIV-1) are microglia, resulting in a disorder called HIV-1 dementia. P-glycoprotein (P-gp), a membrane-associated ATP-dependent efflux transporter, limits entry into the brain of numerous xenobiotics, including anti-HIV drugs (i.e., protease inhibitors). This project investigates the functional expression of P-gp in the endogenous immune cells of the brain, a parenchymal compartment not previously studied. We used a cell line (MLS-9) derived from rat microglia to study the transport of digoxin, a known P-gp substrate. Reverse transcriptase-polymerase chain reaction analysis detected mRNA for only mdr1b in MLS-9 cells, whereas both mdr1a and mdr1b mRNA were expressed in primary cultured microglia from which they were derived. Western blot analysis with the C219 antibody detected a single band at ~170 to 180 kDa in MLS-9 cells, which is the size previously reported for P-gp. Immunocytochemical analysis with the monoclonal antibodies C219, MRK16, and MAB-448 labeled P-gp protein along the plasma membrane and nuclear envelope of MLS-9 cells. [3H]Digoxin accumulation by monolayers of MLS-9 cells was significantly enhanced in the presence of any of several P-gp inhibitors (verapamil, cyclosporin A, quinidine, PSC 833), protease inhibitors (i.e., saquinavir, indinavir, and ritonavir), and sodium azide, an ATPase inhibitor. These results provide the first evidence for the functional expression of P-gp in microglia and imply that entry of pharmacological agents, including protease inhibitors, may be prevented within the brain parenchyma, as well as at the blood-brain barrier.
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PMID:Functional expression of P-glycoprotein in rat brain microglia. 1156 Oct 81

Human immunodeficiency virus (HIV) protease inhibitors (PIs) recently have been reported to be active against Pneumocystis carinii in cell culture. Twelve anti-HIV drugs were analyzed for their effects against rat P. carinii by an ATP cytotoxicity assay. Indinavir and saquinavir exhibited slight anti-P. carinii activity at concentrations above those that can be clinically achieved in serum; other PIs and nucleoside and nonnucleoside reverse-transcriptase inhibitors were inactive against the organism. Anti-HIV drugs, alone or in combination, did not materially reduce the organism count in the treatment of P. carinii pneumonia in immunosuppressed mice. Thus, anti-HIV drugs have little or no activity against P. carinii in these in vitro and in vivo systems. Caution should be used when interpreting reports of the susceptibility of P. carinii to anti-HIV drugs on the basis of in vitro testing only.
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PMID:Anti-human immunodeficiency virus drugs are ineffective against Pneumocystis carinii in vitro and in vivo. 1202 83

1. The effects of ZD6169, a novel ATP-sensitive K(+) channel (K(ATP) channel) opener, were investigated on membrane currents in isolated myocytes using patch-clamp techniques. Tension measurement was also performed to study the effects of ZD6169 on the resting tone of pig urethral smooth muscle. 2. Levcromakalim was more potent than ZD6169 in lowering the resting urethral tone. Relaxation induced by low concentrations of ZD6169 (< or =3 microM) was completely suppressed by additional application of glibenclamide (1 microM). In contrast, glibenclamide (1-10 microM) only partially inhibited the relaxation induced by higher concentrations of ZD6169 (> or = microM). 3. Bay K8644 (1 microM) reduced the maximum relaxation produced by ZD6169 (> or =10 microM). 4. In whole-cell configuration, ZD6169 suppressed the peak amplitude of voltage-dependent Ba(2+) currents in a concentration- and voltage-dependent manner, and at 100 microM, shifted the steady-state inactivation curve of the voltage-dependent Ba(2+) currents to the left at a holding potential of -90 mV. 5. In cell-attached configuration, open probability of unitary voltage-dependent Ba(2+) channels (27 pS, 90 mM Ba(2+)) was inhibited by 100 microM ZD6169 and by 10 microM nifedipine. 6. Reverse transcriptase-polymerase chain reaction (RT - PCR) analysis revealed the presence of the transcript of the alpha(1C) subunit of L-type Ca(2+) channels in pig urethra. 7. These results demonstrate that ZD6169 causes urethral relaxation through two distinct mechanisms, activation of K(ATP) channels at lower concentrations and inhibition of voltage-dependent Ca(2+) channels at higher concentrations (about 10 microM).
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PMID:The involvement of L-type Ca(2+) channels in the relaxant effects of the ATP-sensitive K(+) channel opener ZD6169 on pig urethral smooth muscle. 1172 57

Double-stranded RNA (dsRNA) viruses in some fungi are associated with hypovirulence and have been used or proposed as biological control agents. We isolated 7.5-kb dsRNAs from 13 of 286 field strains of Fusarium graminearum isolated from maize in Korea. One of these strains, DK21, was examined in more detail. This strain had pronounced morphological changes, including reduction in mycelial growth, increased pigmentation, reduced virulence towards wheat, and decreased (60-fold) production of trichothecene mycotoxins. The presence or absence of the 7.5-kb dsRNA was correlated with the changes in pathogenicity and morphology. The dsRNA could be transferred to virus-free strains by hyphal fusion, and the recipient strain acquired the virus-associated phenotype of the donor strain. The dsRNA was transmitted to approximately 50% of the conidia, and only colonies resulting from conidia carrying the mycovirus had the virus-associated phenotype. Partial nucleotide sequences of the purified dsRNA identify an RNA-dependent RNA polymerase sequence and an ATP-dependent helicase that are closely related to those of Cryphonectria hypovirus and Barley yellow mosaic virus. Collectively, these results suggest that this dsRNA isolated from F. graminearum encodes traits for hypovirulence.
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PMID:Double-stranded RNA mycovirus from Fusarium graminearum. 1197 30

P-ATPases are transmembrane proteins that hydrolyse ATP to drive cations or other substances across biomembranes. In this study we present the characterisation of a novel P-ATPase from the apicomplexan parasite Cryptosporidium parvum (CpATPase3), an opportunistic pathogen in autoimmune deficiency syndrome patients, for which no treatment is available. The single copy gene encodes 1488 amino acids, predicting a protein of 169.7 kDa. Primary sequence analysis, as well as an extensive phylogenetic reconstruction, indicated CpATPase3 belongs to a novel class of eukaryotic-specific P-ATPases (Type V) with undefined substrate preferences. Transcription and translation of the gene were confirmed by reverse-transcriptase polymerase chain reaction, and Western blot analysis of sporozoite protein extracts. Immunofluorescent microscopy of C. parvum sporozoites using rabbit antiserum raised against a glutathione-S-transferase-CpATPase3 (GST-ATP3) fusion protein showed that the parasite transporter was located within the apical complex associated with the parasite host-invasion machinery. Overall, these data demonstrate the diversity of C. parvum transporters, and raise the potential of Type V P-ATPases as apicomplexan-specific drug targets.
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PMID:Characterisation of a novel transporter from Cryptosporidium parvum. 1206 59

The contribution of P2 receptors to vasoconstriction of mouse mesenteric arteries was determined using wild-type (WT) and P2X(1) receptor-deficient (KO) animals. alpha,beta-methylene ATP (alpha,beta-meATP) and ATP evoked transient inward currents and constrictions of WT mesenteric arteries. In contrast, alpha,beta-meATP (100 microM) and ATP (100 microM) failed to evoke responses in KO arteries from a range of vascular beds. Nerve stimulation (100 pulses at 10 Hz) evoked constrictions of mesenteric arteries. For WT arteries, the P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2'-5'-disulfonate (PPADS) (30 microM) reduced the amplitude of response by approximately 50%; the residual constriction was abolished by prazosin (0.1 microM). In KO mice, vasoconstriction induced by nerve stimulation was reduced in amplitude by approximately 50%, unaffected by PPADS, but was abolished by prazosin. ADP (1 mM) (a P2Y(1), P2Y(12), and P2Y(13) receptor agonist) was ineffective. Because ATP had no effect on mesenteric artery tone from KO mice, this rules out the contribution of P2Y(2) receptors. The P2Y(4) receptor agonist ITP also failed to contract mesenteric arteries. However, UTP and UDP evoked sustained contractions of mesenteric arteries with similar potency (EC(50) approximately 10 microM). Complementary studies using reverse-transcriptase polymerase chain reaction showed that mesenteric arteries express P2Y(1), P2Y(2), and P2Y(6) receptors. These results demonstrate that homomeric P2X(1) receptors underlie the artery smooth muscle P2X receptor phenotype and contribute approximately 50% to sympathetic neurogenic vasoconstriction and indicate the presence of a UTP- and UDP-sensitive P2Y(6)-like receptor, but not vasoconstrictor P2Y(2) or P2Y(4) receptors, on mouse mesenteric arteries.
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PMID:P2X(1) receptor-deficient mice establish the native P2X receptor and a P2Y6-like receptor in arteries. 1243 12


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