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)

We report identification of a new human nucleoside transporter protein by molecular cloning and functional expression of its cDNA. Previously, we used expression selection in Xenopus oocytes to isolate a cDNA from rat jejunal epithelium encoding the pyrimidine-selective Na+-dependent nucleoside transporter rCNT1 (Q.-Q. Huang, S. Y. M. Yao, M. W. L. Ritzel, A. R. P. Paterson, C. E. Cass, and J. D. Young. J. Biol. Chem. 269: 17757-17760, 1994). cDNAs for a human homologue of rCNT1, designated hCNT1, have been isolated from human kidney by hybridization cloning and reverse transcriptase polymerase chain reaction amplification strategies. hCNT1 was 83% identical to rCNT1 in amino acid sequence and exhibited the transport characteristics of an Na+-dependent nucleoside transporter with selectivity for pyrimidine nucleosides and adenosine when expressed in Xenopus oocytes. Deoxyadenosine, which undergoes net renal secretion, and guanosine were poor permeants. hCNT1 did, however, transport 3'-azido-3'-deoxythymidine. This is the first demonstration that members of the CNT family exist in human cells and provides evidence of their involvement in the renal transport of physiological nucleosides and nucleoside drugs. The hCNT1 gene was mapped to chromosome 15q25-26.
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PMID:Molecular cloning and functional expression of cDNAs encoding a human Na+-nucleoside cotransporter (hCNT1). 912 15

The P-type ATPases (e.g., Na+-K+-ATPase and Ca2+-ATPase) occur widely in living cells of fungi, Protozoa, plants, and animals. These ion pumps show a high degree of divergence in their primary structures but share a limited number of common amino acid residues for their ATP-catalytic function. Particularly, the amino acid sequences for the phosphorylation site (DKTGTLT) and the binding site for ATP (and its analogs; GDGVND) are conserved throughout evolution. Using two degenerate oligonucleotides corresponding to these regions, we applied a polymerase chain reaction (PCR) technique to the search for P-type ATPase isoforms, which will provide a clue to the evolutionary mechanisms of ion pumps in Tetrahymena thermophila. A total of 12 distinct P-type ATPase genes were identified. Sequence comparisons revealed that seven of them can be compiled into a multigene family, which is similar to animal Na+-K+- and H+-K+-ATPase genes. One of them is close to the sarco(endo)plasmic reticulum Ca2+-ATPase gene, and the other four share a significant homology with the gene encoding Plasmodium ATPase-1 whose function is unknown. A Northern blot analysis and reverse transcriptase-PCR demonstrated that all identified genes are expressed, but the expression levels vary widely under different culture conditions. A Southern blot analysis after pulse-field gel electrophoresis showed that all of these genes exist in T. thermophila macronuclei. The Na+-K+- and H+-K+-ATPase gene family has a high multiplicity (at least 10 different genes detected on genomic Southern blot analysis) and is distributed on four different macronuclear chromosomes. On the basis of a calculation with the amino acid sequences of the cloned cytoplasmic loop region (between the phosphorylation and the gamma-[4-(N-2-chloroethyl-N-methylamino)]-benzylamido ATP sites), the genes with >80% identity form a cognate linkage group within the same macronuclei chromosome, whereas the genes with <70% identity are separated in different chromosomes. The phylogenetic analysis showed that this multigene family is the result of a series of gene duplications.
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PMID:Primary structure and evolution of the ATP-binding domains of the P-type ATPases in Tetrahymena thermophila. 912 16

Evidence for an H+-K+-adenosinetriphosphatase (HKA) in vascular smooth muscle cells has recently been demonstrated in the rat aorta and a rat smooth muscle cell line (A(7)r(5)) by functional studies. Our laboratory has used reverse transcriptase (RT)-polymerase chain reaction (PCR) and Northern hybridization techniques to define the type of HKA present in canine whole carotid artery and confluent carotid artery cells. PCR primers to detect an HKA message of unknown isoform were designed on the basis of homology to known sequences of gastric, colonic, and toad bladder HKA isoforms and dissimilarity to the Na+-K+-adenosinetriphosphatase isoforms. A PCR product of predicted size was generated from cDNAs of both confluent cells and whole vessel carotid artery that possess 91% nucleotide sequence identity to the canine gastric HKA. Northern hybridization with the PCR product as a probe revealed hybridization to whole vessel carotid artery, cultured confluent carotid artery cell, kidney, and stomach canine total RNA at the known message size for the gastric HKA isoform. These data suggest that vascular smooth muscle cells express an HKA mRNA that may be identical to the gastric isoform.
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PMID:Molecular evidence for a vascular smooth muscle H+-K+-ATPase. 912 50

We reported previously that NO is responsible for the angiogenesis produced by endothelium-dependent vasodilating peptides. To investigate the mechanisms by which NO controls angiogenesis, NO was assessed for the ability to affect cell proliferation and upregulation of urokinase-type plasminogen activator (uPA) induced by basic fibroblast growth factor (bFGF) when added exogenously to or when produced endogenously by coronary venular endothelial cells (CVECs). The treatment of the cells with the NO donor sodium nitroprusside (NaNp) induced uPA upregulation and cell proliferation, which were prevented by anti-bFGF antibodies. Similarly, the NO-dependent mitogenic activity of the vasodilating peptide substance P (SP) was blocked by anti-bFGF antibodies, thus implicating endogenous bFGF in the NO-induced response. NaNp and SP induced bFGF expression as measured by Western blot analysis of CVEC extracts and by differential reverse transcriptase-polymerase chain reaction of bFGF mRNA. SP-induced upregulation of bFGF was prevented by the NO synthase inhibitor N omega-monomethyl-L-arginine. We conclude that NO promotes cell proliferation and uPA upregulation in CVECs by inducing endogenous bFGF and that this pathway mediates the angiogenetic response to the vasoactive neuropeptide SP. This signaling paradigm may provide an important link between shear rate, NO, bFGF, and coronary angiogenesis.
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PMID:Nitric oxide promotes proliferation and plasminogen activator production by coronary venular endothelium through endogenous bFGF. 916 87

To develop a rapid and sensitive means of detecting cell-associated human immunodeficiency virus (HIV), donor cells from HIV seropositive patients were treated with the potent viral activator sodium-n-butyrate (NaB) and subsequently assayed by both in situ RNA hybridization and a reverse transcriptase polymerase chain reaction (RT-PCR). The sensitivity of RT-PCR was estimated to be equivalent to 1 x 10(-16) grams (0.1 fg) or approximately 64 copies of the input standard viral RNA per reaction. The present study takes advantage of the ability of NaB to introduce changes in chromatin structure of latently infected cells, leading to increased HIV gene expression. Human ACH-2 and U1 cell lines were used as representatives of T-lymphocytic and monocytoid cells harboring latent inducible proviruses. HIV gene expression was readily detected when these cells were treated with NaB. Viral gag RNA was detected by both in situ and RT-PCR assays. When peripheral blood mononuclear cells (PBMCs) from acquired immunodeficiency syndrome (AIDS) patients, who were all negative for in situ hybridization and serum/plasma p24 assays, were used for detection of viral gene expression, four categories with distinct patterns of induction were observed. The first set of patients showed HIV-positive PBMCs by RT-PCR without any added NaB, and suppression by added NaB or PHA. The second set of samples showed induction of viral RNA by NaB alone. The third set could be induced with PHA, but not NaB, and the fourth set required both NaB and PHA for induction of HIV gene expression. Our results suggest that direct treatment of the cells with HIV activators may be useful in increasing sensitivity of the RT-PCR intended to be used for detection of cell-associated viral RNAs. This approach may be used to confirm true status of the HIV infection when p24 results are negative or HIV RNAs in serum/plasma are below the threshold of detection. Moreover, this method may identify the presence of latent proviral genomes possibly reflecting the true rate of cell-associated viral load in vivo and without possible mutations brought about by long-term co-cultivation assays with cells from seronegative donors.
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PMID:Rapid and sensitive detection of cell-associated HIV-1 in latently infected cell lines and in patient cells using sodium-n-butyrate induction and RT-PCR. 917 66

Events controlling differentiation to insulin-secreting beta-cells in the pancreas are not well understood, although beta-cells are thought to arise from pluripotent ductal precursor cells. To search for signaling proteins that might be involved in beta-cell maturation, we analyzed protein kinase expression in two developmentally and functionally distinct pancreatic beta-cell lines, RIN-5AH and RIN-A12, by reverse transcriptase polymerase chain reaction. A number of tyrosine and serine/threonine kinases were identified in both lines. One protein kinase, mixed lineage kinase-1 (MLK-1), was expressed at both the RNA and protein levels in RIN-5AH cells, which display an immature beta-cell phenotype, but was not detected in the more mature RIN-A12 cells. Furthermore, levels of MLK-1 mRNA and protein were increased after brief stimulation of RIN-5AH cells with either the differentiation inducer, sodium butyrate, or with serum after serum starvation. These increases in expression were independent of phenotypic markers such as insulin secretion or surface expression of major histocompatibility class I- and A2B5-reactive ganglioside. In addition, increases in MLK-1 expression in the stimulated RIN-5AH cells were accompanied by phosphorylation of MLK-1 on serine but not tyrosine. Antisense oligonucleotides to two distinct regions of MLK-1 caused RIN-5AH cells, but not RIN-A12 cells, to adopt a highly undifferentiated morphology, with a reduction in DNA synthesis and MLK-1 protein levels and elevated glucagon mRNA levels, but with no effect on insulin mRNA. In an immunohistochemical survey of embryonic mouse tissues, we found that temporal expression of MLK-1 was regulated in a tissue-specific manner. In the embryonic pancreas, MLK-1 expression was evident in ductal cells from day 13 to 16 but was not detected in late stage gestation or neonatal pancreas. These data suggest that MLK-1 is regulated in immature pancreatic beta-cells and their ductal precursors at the level of functional maturity and may therefore play a role in beta-cell development.
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PMID:Expression of mixed lineage kinase-1 in pancreatic beta-cell lines at different stages of maturation and during embryonic pancreas development. 919 43

We investigated the role of endogenous or exogenous nitric oxide (NO) on human lymphocyte function. We used sodium nitroprusside, nitroglycerine, S-nitroso-N-acetylpenicillamine, sodium nitrite and S-nitroso-L-glutathione as NO-generating compounds. All agents were used at doses that do not produce direct cytotoxicity as measured by trypan blue exclusion as well as chromium-51 release assay. The immune responses examined were peripheral blood lymphocytes (PBL) proliferation and IL-2 production after activation with OKT3 and PHA; allogeneic mediated proliferation and cell mediated cytotoxicity (CML) in MLR; IgG and IgM production after PBL activation with Con-A; proliferation and expression of IFN-gamma and IL-4 mRNA after activation of allogeneic CD4+T cell clones. Cytokine mRNA expression was measured by reverse transcriptase PCR. Our results show that proliferating lymphocytes do not produce a detectable amount of NO as measured by the Griess reaction. In separate experiments, the addition of NG-monomethyl-L-arginine (L-NMMA) did not affect lymphocyte proliferation. Sodium nitroprusside and nitroglycerine exerted a dose dependent antimitogenic effect, inhibited cytokine production and expression, CML generation and antibody production. DNA gel electrophoresis showed no evidence for enhanced programmed cell death. The antimitogenic effect could not be blocked by the NO scavengers, hemoglobin or methylene blue. In contrast, the other nitric oxide generating compounds did not inhibit lymphocyte mitogenesis. The results suggest that human lymphocytes do not produce appreciable amounts of NO to affect lymphocyte mitogenesis. Sodium nitroprusside and nitroglycerine have a potent but nonspecific immunoinhibitory effect on human lymphocyte function by a mechanism other than NO production. In addition, pharmacological levels of NO do not inhibit human lymphocyte mitogenesis.
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PMID:Analysis of the in vitro effect of exogenous nitric oxide on human lymphocytes. 920 99

Lipophilic esters of 3'-azido-3'-deoxy-5'-O-(carboxyphosphinyl)thymidine (PFA-AZT) were synthesized and tested for antiretroviral activity in CD4+ HT4-6C cells infected with either wild-type HIV-1LAI, a PFA-resistant strain encoding a single-point mutation in reverse transcriptase (E89K), or an AZT-resistant clinical isolate (A018-post). Arbuzov condensation of 1-octadecyl, 1-eicosanyl, and 1-docosanyl chloroformate with trimethyl phosphite yielded the corresponding dimethyl long-chain alkyl triesters of PFA. Selective removal of one methyl group from the triesters with sodium iodide yielded monosodium salts, whereas treatment with bromotrimethylsilane cleaved both methyl groups while leaving the long-chain alkyl group intact. Neutralization of the resulting [(alkyloxy)carbonyl]phosphonic acids with 2 equiv of sodium methoxide afforded disodium salts of the phosphonic acid moiety. Similar chemistry was used to obtain the mono- and disodium salts of the cholesterol ester of PFA. Reaction of the triesters with phosphorous pentachloride, followed by coupling with AZT and O-demethylation with sodium iodide, afforded 3'-azido-3'-deoxy-5'-O-[[(1-octadecyloxy)carbonyl]phosphinyl ]thymidine (9a), 3'-azido-3'-deoxy-5'-O-[[(1-eicosanyloxy)carbonyl]phosphinyl ]thymidine (9b), 3'-azido-3'-deoxy-5'-O-[[(1-docosanyloxy)carbonyl]phosphinyl ]thymidine (9c), and 3'-azido-3'-deoxy-5'-O-[[(3 beta-cholest-5-enyloxy)carbonyl]phosphinyl]thymidine (9d). Concentrations of 9a-d found to inhibit replication of wild-type HIV-1LAI by 50% (EC50 values) as measured in a plaque reduction assay were in the 0.1-0.3 microM range as compared with 0.013 microM for AZT and 133 microM for PFA. The concentration at which toxicity was observed in 50% of the host cells (TC50 values) as measured by a visual grading scale of cellular morphology was 10 microM for 9a and 9d, 32 microM for 9b, and 320 microM for 9c. Thus, the TC50/EC50 ratio or selectivity index (SI) was 100 for 9a, 230 for 9b, and 1000 for 9c but only 33 for 9d, suggesting that the straight-chained fatty alcohol esters were more therapeutically selective. Similar TC50 and SI values were obtained for rapidly dividing CEM lymphoblasts as for HT4-6C cells. In assays against E89K, 9a-c had mean EC50 values of 0.13, 0.009, and 0.17 microM, whereas the EC50 of PFA was > 1000 microM and that of AZT was 0.009 microM; thus, E89K was highly resistant to PFA but not cross-resistant to either AZT or the lipophilic PFA-AZT conjugates. In viral replication assays against the A018C-post isolate, the mean EC50 values of 9a-c were 0.30, 0.53, and 0.77 microM as compared with 2.9 microM for AZT and 65 microM for PFA; thus, the virus recovered from a patient pretreated with AZT was not cross-resistant to either PFA or 9a-c. A notable feature of these results was that, in addition to being > 1000-fold more potent than PFA against the PFA-resistant mutant, the lipophilic PFA-AZT conjugates were more potent than PFA, as well as AZT, against AZT-resistant HIV-1.
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PMID:Synthesis and in vitro activity of long-chain 5'-O-[(alkoxycarbonyl)phosphinyl]-3'-azido-3'-deoxythymidines against wild-type and AZT- and foscarnet-resistant strains of HIV-1. 925 55

The purpose of the present study was to determine whether the renal inner medulla expresses mRNA for the rat epithelial Na+ channel (rENaC) and, if so, to define its regulatory properties using a low-Na+ diet model. We detected alpha, beta and gamma subunit mRNA in rat renal inner medulla using reverse transcriptase-polymerase chain reaction (RT-PCR) with primers specific for rENaC alpha, beta and gamma subunits. Moreover, we have developed a specific probe for the alpha subunit using RT-PCR with rENaC alpha-subunit-specific primers. The resulting cDNA was verified by sequencing and was then used in Northern blot analysis of distal colon, whole kidney and inner medulla. The probe for the rENaC alpha subunit hybridized not only to distal colon RNA but also to inner medulla RNA derived from rats fed a normal diet. Furthermore, we examined the effect of a low-Na+ diet on alpha, beta and gamma subunit mRNA expression of rENaC using full-length cDNA as a probe. A marked elevation of rENaC alpha subunit mRNA abundance in the inner medulla was observed in response to a high plasma aldosterone concentration induced by dietary Na+ deprivation. On the other hand, neither beta nor gamma subunit mRNA expression was enhanced by a low-Na+ diet. From these results, it is suggested that rENaC is responsible for Na+ transport in the renal inner medulla and that is probably regulated via transcriptional control of the alpha subunit of ENaC.
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PMID:A low-Na+ diet enhances expression of mRNA for epithelial Na+ channel in rat renal inner medulla. 930 9

We have characterized the Na+/H+ exchanger (NHE) isoforms expressed in rat renal cortical tubule fragments. Amiloride sensitivity of the Na(+)-dependent intracellular pH (pHi) recovery in suspended tubules that had been acid loaded by an NH4+ prepulse was determined in nominally CO2/HCO3(-)-free solution, using the fluorescent pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. In the presence of 140 mM extracellular Na+, 800 microM amiloride inhibited the rate of Na(+)-dependent pHi recovery by only 65%, demonstrating the presence of a Na(+)-dependent amiloride-insensitive H+ extrusion system. This system was not affected by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid but was activated by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Lowering extracellular Na+ concentration permitted 300 microM amiloride to completely inhibit Na(+)-dependent pHi recovery. These results can be explained by the expression of a Na+/H+ exchange with the pharmacological properties of NHE4. Using reverse transcriptase-polymerase chain reaction, we found specific mRNA for NHE1, NHE2, NHE3, and NHE4 isoforms in the renal cortex. Immunohistochemical studies using polyclonal antibodies against rat NHE4 peptide demonstrated that NHE4 is heterogeneously expressed on basolateral membrane domains of cortical tubules. These results strongly suggest that amiloride-insensitive Na+/H+ exchange expressed in renal cortical tubule suspensions is mediated by NHE4.
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PMID:Evidence for an amiloride-insensitive Na+/H+ exchanger in rat renal cortical tubules. 931 28


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