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)

Equilibrative nucleoside transport processes in mammalian cells are categorized as either nitrobenzylthioinosine (NBMPR)-sensitive (es) or NBMPR-insensitive (ei). Inhibition of the es process arises from binding of NBMPR to a high-affinity site(s) on the es transporter that can be identified by photoaffinity labeling with [3H]NBMPR. This study examined the equilibrative nucleoside transport processes of cultured human erythroleukemia (K562) cells. The presence of NBMPR binding sites (4.8 +/- 0.9 x 10(5)/cell, Kd = 0.3 nM), together with the identification of polypeptides by specific photolabeling of membranes with [3H]NBMPR, indicated that K562 cells possess es nucleoside transporters (ca 500,000 copies/cell). The photolabeled polypeptides of K562 cells migrated with lower relative mobility (peak M(r) value, 63,000) than did those of human erythrocytes (peak M(r) value, 53,000). This difference in apparent M(r) was abolished by prolonged treatment of membrane proteins with N-glycosidase F, suggesting that equilibrative nucleoside transport in K562 cells and erythrocytes is mediated by the same, or a closely related, es isoform. A cDNA encoding the es nucleoside transporter of human placenta (termed hENT1) was recently isolated by a strategy based on the N-terminal sequence of the es transporter of human erythrocytes. hENT-like mRNA species were detected in K562 cells, as well as in several other human cell lines of neoplastic origin (A459, G361, HeLa, HL-60, Molt-4, Raji, SW480), by high-stringency northern analysis with a placental hENT1 probe. A cDNA that encoded a protein identical to hENT1 was isolated by reverse transcriptase polymerase chain reaction with primers specific for hENT1. NBMPR inhibited zero-trans influx of 3H-labeled adenosine, uridine and thymidine by 50% (IC50 values) at 0.4-1.0 nM, confirming the presence of an NBMPR-sensitive (es) transport process, which accounted for 80-90% of total transport activity. The remaining component was identified as the equilibrative NBMPR-insensitive (ei) transport process since it: (i) exhibited low (IC50 > 1.0 microM) sensitivity to NBMPR; (ii) was not concentrative; and (iii) was unchanged by elimination of the sodium gradient. The kinetic parameters (determined at 37 degrees C) for the es- and ei-mediated processes differed markedly. Values for transport of uridine by the es- and ei-mediated processes were, respectively: K(m) = 229 +/- 39 and 1077 +/- 220 microM; Vmax, 186 +/- 31 and 40 +/- 5 pmol/microliter cell water/sec. Values for transport of adenosine by the es and ei-mediated processes were, respectively, 61 +/- 9 and 133 +/- 17 microM; Vmax, 70 +/- 5 and 23 +/- 8 pmol/microlitere cell water/sec. The ei-mediated process, although small, was of pharmacologic importance since K562 cells could not be protected by NBMPR (10 microM) from the cytotoxic effects of tubercidin (7-deazaadenosine).
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PMID:Molecular identification of the equilibrative NBMPR-sensitive (es) nucleoside transporter and demonstration of an equilibrative NBMPR-insensitive (ei) transport activity in human erythroleukemia (K562) cells. 936 72

We examined expression of the erythroid-associated genes GATA-1 and erythropoietin receptor (EPOR) in primary leukemia using the reverse transcriptase-polymerase chain reaction (RT-PCR). GATA-1 and EPOR mRNAs were detectable in all cases of erythroleukemia (French-American-British classification: M6) or early erythroblastic leukemia. In all other leukemia cases, including M2 through M5, stem cell leukemia, and adult T-cell leukemia, these gene transcripts were undetectable. GATA-2 was detectable in all the cases of primary leukemias examined in this study, except one case of M5. In one case, the phenotype switched from myeloid (M2) to erythroid (M6) and then back to myeloid. Northern blotting and RT-PCR revealed that GATA-1 and EPOR mRNAs were significantly upregulated at the M6 stage compared with the M2 stage. GATA-1 may be involved in the expression of an erythroid phenotype in acute leukemia. We generated HL-60 transfectants exogenously expressing GATA-1. The majority of HL-60 cells expressing GATA-1 lacked azurophilic granules, and electron microscopic analysis revealed that myeloperoxidase activity was negative. Platelet peroxidase activity, which was detectable in both megakaryoblasts and erythroid progenitors, was positive. However, EPOR and glycophorin A mRNAs were undetectable by RT-PCR. These findings suggest that besides GATA-1, a third factor may be required for the expression of mature erythroid phenotypes. In addition, our results indicate that GATA-1 is involved in inactivation of myeloperoxidase and activation of the platelet peroxidase.
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PMID:GATA-1 and erythropoietin receptor genes are highly expressed in erythroleukemia. 980 54

The position-independent expression of transgenes in target cells is an essential subject for determining effective gene therapies. The chicken beta-globin insulator blocks the effects of regulatory sequences on transcriptional units at differential domains. We prepared a recombinant adeno-associated virus (rAAV) containing various combinations of the DNase I-hypersensitive site 2 (HS2), 3 (HS3), and 4 (HS4) core elements from the human beta-globin locus control region (LCR), the human beta-globin gene, and the herpes virus thymidine kinase promoter driven neomycin-resistant gene (neoR) (rHS432, rHS43, rHS42, rHS32, and rHS2), and also rAAV containing two copies of the 250-bp core sequence of the chicken beta-globin insulator on both sides of the rHS2 (rIns/HS2/2Ins). After isolating neomycin-resistant mouse erythroleukemia (MEL) cells infected with each rAAV, we analyzed the rAAV genome by Southern blots and polymerase chain reaction (PCR), using primers specific for HS core elements and the human beta-globin gene. All clones contained a single copy of the rAAV genome in the chromosome, however, some of them had a rearranged proviral genome. In five clones with a single unrearranged rAAV genome for each rAAV construct, we assayed the expression of the human b-globin gene relative to the endogenous mouse beta maj-globin gene, using quantitative reverse transcriptase (RT)-PCR. In clones infected with rHS432, the expression level of the human beta-globin gene ranged from 51.6% to 765.6% of that in the mouse beta maj-globin gene. Likewise, in rHS43, the expression level ranged from 36.7% to 259.0%; in rHS42, from 47.8% to 207.0%; in rHS32, from 47.9% to 105.4%; and in rHS2, from 6.1% to 172.1%, indicating a high variability of expression level in clones infected with recombinant virus lacking the insulator. In contrast, in clones infected with rIns/HS2/Ins, the range of expression of the human beta-globin gene ranged from 52.8% to 58.3% of that in the mouse beta maj-globin gene. These results indicate that the insulator functioned dramatically to reduce the variability of transgene expression due to the position effect. This insulator-rAAV vector system holds promise to provide a constant level of transgene expression for gene therapy, regardless of the insertion sites on the chromosome.
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PMID:Position-independent human beta-globin gene expression mediated by a recombinant adeno-associated virus vector carrying the chicken beta-globin insulator. 1031 78

In addition to its inhibitory activity against viral DNA polymerases and reverse transcriptase, the acyclic nucleoside phosphonate 9-(2-phosphonylmethoxyethyl)adenine (PMEA) also markedly inhibits the replicative cellular DNA polymerases alpha, delta, and epsilon. We have previously shown that PMEA is a strong inducer of differentiation in several in vitro tumor cell models and has marked antitumor potential in vivo. To elucidate the molecular mechanism of the differentiation-inducing activity of PMEA, we have now investigated the effects of the drug on cell proliferation and differentiation, cell cycle regulation, and oncogene expression in the human erythroleukemia K562 cell line. Terminal, irreversible erythroid differentiation of PMEA-treated K562 cells was evidenced by hemoglobin production, increased expression of glycophorin A on the K562 cell membrane, and induction of acetylcholinesterase activity. After exposure to PMEA, K562 cell cultures displayed a marked retardation of S-phase progression, leading to a severe perturbation of the normal cell cycle distribution pattern. Whereas no substantial changes in c-myc mRNA levels and p21, PCNA, cdc2, and CDK2 protein levels were noted in PMEA-treated K562 cells, there was a marked accumulation of cyclin A and, most strikingly, cyclins E and B1. A similar picture of cell cycle deregulation was also observed in PMEA-exposed human myeloid THP-1 cells. However, in contrast to the strong differentiation-inducing activity of PMEA in K562 cells, the drug completely failed to induce monocytic maturation of human myeloid THP-1 cells. On the contrary, THP-1 cells underwent apoptotic cell death in the presence of PMEA, as demonstrated by prelytic, intracellular DNA fragmentation and the binding of annexin V to the cell surface. We hypothesize that, depending on the nature of the tumor cell line, PMEA triggers a process of either differentiation or apoptosis by the uncoupling of normally integrated cell cycle processes through inhibition of DNA replication during the S phase.
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PMID:9-(2-Phosphonylmethoxyethyl)adenine induces tumor cell differentiation or cell death by blocking cell cycle progression through the S phase. 1039 5

The p53-related genes, p51/p63 and p73, have been isolated respectively from cDNA libraries of skeletal muscle and the brain, and their structural features and biological functions have been compared. High expression of p51A (TAp63gamma) in the skeletal muscle tissue drove us to investigate a differentiation-inducible myoblastic cell line which showed increased p51A expression after differentiation induction. Tissue-specific expression was further confirmed by reverse transcriptase-polymerase chain reaction (RT - PCR) using primers specific for DeltaN (TA-domain lacking p51), p51A, and p51B expression. p51A alone induced erythrodifferentiation when expressed in the erythroleukemia line (Tg-gp55-1-2-3) expressing a temperature-sensitive mutant of p53, and induced remarkable apoptosis when wild-type p53 expression was induced by the temperature shift to 32 degrees C. Human p51A and p53 were introduced exogenously into the above erythroleukemia cells, and although their expression was rather low, both p51A and p53 proteins were induced by DNA-damaging treatment with UV and ActinomycinD. However, the protein-protein interactions analyzed by a yeast two-hybrid assay between p51 and p53, between p51 and p73, and between p51 and oncoproteins showed that p51 is functionally rather distant from p53. Extensive mutation analysis of p51/p63 in human tumors revealed only four mutations in 80 non-small cell lung carcinomas; two adenocarcinoma cases possessing Glu31His mutations in the transactivation domain (TA) domain, suggesting that p51/p63 is not a Knudson type tumor suppressor gene. Mutation and loss of heterozygosity (LOH) of p73, deregulated expression of p73 and loss of imprinting of p73 are also discussed.
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PMID:p53 family genes: structural comparison, expression and mutation. 1063 27

The Philadelphia (Ph) chromosome is observed in approximately 1% of patients with acute myeloblastic leukaemia (AML), especially subtypes M1 and M2 in the French-American-British classification. We describe here a cytogenetic and molecular investigation of a rare case with Ph-positive AML M6 (erythroleukaemia). A 63-yr-old woman was diagnosed as having erythroleukaemia. Leukaemic cells were positive for CD4 and CD7 as well as CD13, CD33, CD34 and HLA-DR. They were analyzed by G-banding, fluorescence in situ hybridization (FISH), Southern blot and reverse transcriptase polymerase chain reaction analyses. The karyotypes at diagnosis were as follows: 61, XX, -X, -1, -2, -3, -4, -5, -7, t(9;22)(q34;q11)x 2, -15, -16, -17, -18, + 19, +21, +22 [3]/61, idem, -22, +der(22)t(9;22) [36]. FISH with BCR/ABL probes showed that 39% and 57% of interphase nuclei had double and triple BCR/ABL fusion signals, respectively. Chromosome analysis in complete remission showed a normal karyotype in all 20 metaphases, confirming the diagnosis as Ph positive-acute leukaemia. FISH at relapse showed that 92% of interphase nuclei had triple fusion signals. Rearrangement of major breakpoint cluster region (M-bcr) in the BCR gene and coexpression of p210-type (b2a2) and p190-type (e1a2) BCR/ABL fusion transcripts due to alternative splicing were also detected. We conclude that clonal evolution from double to triple Ph chromosomes may be implicated in the disease progression. Considering other two reported cases, Ph-positive erythroleukaemia appears to be correlated with coexpression of myeloid/T-lymphoid markers and hyperdiploidy with double or triple Ph chromosomes, although breakpoints in the BCR gene are heterogenous.
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PMID:Triple Philadelphia chromosomes with major-bcr rearrangement in hypotriploid erythroleukaemia. 1100 54

Interleukin-13 (IL-13) regulates immune responses mediated by type 2 T helper lymphocytes (Th2) in the human and mouse. To study the function of this cytokine in the dog, we have isolated a cDNA that encodes the full-length canine IL-13 (CaIL-13) precursor polypeptide of 131 amino acids. CaIL-13 shares significant homology with the IL-13 amino acid sequences of cattle (54.1%), mouse (39.6%), and rat (36.6%) but shares the highest identity with human IL-13 (HuIL-13) (61.8%). The predicted CaIL-13 mature polypeptide of 111 residues was expressed in bacteria, and recombinant CaIL-13 (rCaIL-13) was isolated from inclusion bodies and refolded. rCaIL-13 stimulated the proliferation of TF-1 cells, which are derived from human erythroleukemia cells and respond to IL-13 as well as to a number of other human and murine cytokines. CaIL-13 mRNA was readily detectable by reverse transcriptase-polymerase chain reaction (RT-PCR) in cells from lymph nodes and peripheral blood. The gene sequence and biologically active recombinant protein for CaIL-13 will be useful reagents to determine the role of IL-13 in the regulation of canine immune responses.
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PMID:Canine interleukin-13: molecular cloning of full-length cDNA and expression of biologically active recombinant protein. 1103 97

In this paper we describe the synthesis of new copper complexes with alpha-ketoglutaric acid thiosemicarbazone. The crystal structures of the two compounds: [Cu(H(2)ct)Cl](n) [(Cu(H(2)ct)Cl)(2)] (1) and [Cu(Hct)](n).3nH(2)O (2) (H(3)ct=alpha-ketoglutaric acid thiosemicarbazone) have been determined by X-ray and spectroscopic methods. In 1 two independent copper atoms are present. Cu(1), in a nitrogen- and oxygen-bridged polymer, is a six-coordinated (4+2), Cu(2), five coordinated (4+1), is a chlorine-bridged dimer. In 2 the copper atom presents a penta-coordination, polymeric chains form layers and the -CH(2)CH(2)COO(-) groups bridge copper atoms. In 1 a monodentate and in 2 a syn-anti bidentate bridging carboxylate are present. The biological properties of 1 and 2 and also of the free ligand (H(3)ct) were tested in vitro and compared on Friend erythroleukemia cells (FLC) and on human leukemia cell lines K562 and U937. On the FLC cells the free ligand does not inhibit cell growth, but increases the DNA synthesis; complex 1 inhibits cell proliferation and increases the DNA synthesis; complex 2 inhibits cell growth, but induces a decrement of DNA synthesis and increases the reverse transcriptase activity. Regarding the human cell lines, both complexes show proliferation inhibition through an apoptosis mechanism on cell line U937, while they have no effects on the K562 line.
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PMID:Synthesis, characterization and biological activity of two new polymeric copper(II) complexes with alpha-ketoglutaric acid thiosemicarbazone. 1193 61

TRPM2 is a Ca(2+)-permeable channel that is activated by oxidative stress and confers susceptibility to cell death. Here, an isoform of TRPM2 was identified in normal human bone marrow that consists of the TRPM2 N terminus and the first two predicted transmembrane domains. Because of alternative splicing, a stop codon (TAG) is located at the splice junction between exons 16 and 17, resulting in deletion of the four C-terminal transmembrane domains, the putative calcium-permeable pore region, and the entire C terminus. This splice variant was found in other hematopoietic cells including human burst forming unit-erythroid-derived erythroblasts and TF-1 erythroleukemia cells. Endogenous expression of both the short form of TRPM2 (TRPM2-S) and the full length (TRPM2-L) was determined by reverse transcriptase-PCR, and localization of endogenous TRPM2 to the plasma membrane was demonstrated by confocal microscopy. Heterologous expression of TRPM2-S in HEK 293T cells demonstrated similar membrane localization as TRPM2-L, and coexpression of TRPM2-S did not alter the subcellular localization of TRPM2-L. The direct interaction of TRPM2-S with TRPM2-L was demonstrated with immunoprecipitation. H(2)O(2) induced calcium influx through TRPM2-L expressed in 293T cells. Coexpression of TRPM2-S suppressed H(2)O(2)-induced calcium influx through TRPM2-L. Furthermore, expression of TRPM2-S inhibited susceptibility to cell death and onset of apoptosis induced by H(2)O(2) in cells expressing TRPM2-L. These data demonstrate that TRPM2-S is an important physiologic isoform of TRPM2 and modulates channel activity and induction of cell death by oxidative stress through TRPM2-L.
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PMID:A novel TRPM2 isoform inhibits calcium influx and susceptibility to cell death. 1259 22

Lysophosphatidic acid (LPA) is a lipid mediator that, among several other cellular responses, can stimulate cells to mobilize calcium (Ca2+). LPA is known to activate at least three different subtypes of G protein-coupled receptors. These receptors can then stimulate different kinds of G proteins. In the present study, LPA and LPA analogs were synthesized from (R)- and (S)-glycidol and used to characterize the ability to stimulate Ca2+ mobilization. The cytosolic Ca2+ concentration ([Ca2+]i) was measured in fura-2-acetoxymethylester-loaded human erythroleukemia (HEL) cells. Furthermore, a reverse transcriptase polymerase chain reaction was used to characterize LPA receptor subtypes expressed in HEL cells. The results show that HEL cells mainly express LPA1 and LPA2, although LPA3 might possibly be expressed as well. Moreover, LPA and its analogs concentration-dependently increased [Ca2+]i in HEL cells. The response involved both influx of extracellular Ca2+ and release of Ca2+ from intracellular stores. This is the first time the unnatural (S)-enantiomer of LPA, (S)-3-O-oleoyl-1-O-phosphoryl-glycerol, has been synthesized and studied according to its ability to activate cells. The results indicate that this group of receptors does not discriminate between (R)- and (S)-enantiomers of LPA and its analogs. When comparing ether analogs having different hydrocarbon chain lengths, the tetradecyl analog (14 carbons) was found to be the most effective in increasing [Ca2+]i. Pertussis toxin treatment of the HEL cells resulted in an even more efficient Ca2+ mobilization stimulated by LPA and its analogs. Furthermore, at repeated incubation with the same ligand no further increase in [Ca2+]i was obtained. When combining LPA with the ether analogs no suppression of the new Ca2+ signal occurred. All these findings may be of significance in the process of searching for specific agonists and antagonists of the LPA receptor subtypes.
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PMID:Lack of stereospecificity in lysophosphatidic acid enantiomer-induced calcium mobilization in human erythroleukemia cells. 1466 71


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