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)

We evaluated the effects of natural purine and pyrimidine nucleosides on protection from or reversal of 3'-azido-3'-deoxythymidine (AZT) cytotoxicity in human bone marrow progenitor cells by using clonogenic assays. The selectivity of the "protection" or "rescue" agents was examined in evaluating the antiretroviral activity of AZT in combination with these modulating agents and of AZT alone. Following exposure of human granulocyte-macrophage progenitor cells for 2 h to 5 microM AZT (70% inhibitory concentration), increasing concentrations of potential rescue agents were added. Cells were cultured, and colony formation was assessed after 14 days. At concentrations of up to 50 microM no natural 2'-deoxynucleosides, including thymidine, were able to reverse the toxic effects of AZT. Dose-dependent reversal was observed with uridine and cytidine, and essentially complete reversal was achieved with 50 microM uridine. In the protection studies, 100 microM thymidine almost completely antagonized the inhibition of granulocyte-macrophage colony formation produced by 1 microM AZT (50% inhibitory concentration), and 50 microM uridine effected 60% protection against a toxic concentration of AZT (5 microM) (70% inhibitory concentration). The antiretroviral activity of AZT in human peripheral blood mononuclear cells, assessed by revere transcriptase assays, was substantially decreased in the presence of thymidine, whereas no impairment of suppression of viral replication was observed in the presence of uridine in combination with AZT at a molar ratio (uridine/AZT) as high as 10,000. This demonstration of the capacity of uridine to selectively rescue human bone marrow progenitor cells from the cytotoxicity of AZT suggests that use of uridine rescue regimen with AZT may have potential therapeutic benefit in the treatment of acquired immunodeficiency syndrome.
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PMID:Uridine reverses the toxicity of 3'-azido-3'-deoxythymidine in normal human granulocyte-macrophage progenitor cells in vitro without impairment of antiretroviral activity. 319 Feb 1

Lymphohematopoiesis occurs in the densely packed environment of the intramedullary spaces. Primitive lymphohematopoietic stem cells exist in close apposition to a variety of supportive cells including both hemopoietic and nonhemopoietic lineages. Using an in vitro long-term Dexter liquid culture system, we have established that a variety of cytokines are produced constitutively by such stromal cells in culture. These cytokines include Steel factor, interleukin-6 (IL-6), and colony-stimulating factor (CSF-1). Granulocyte-CSF and granulocyte-macrophage-CSF mRNA can be detected after refeeding of cultures, although in quiescent cultures message for these factors is difficult to detect. Interleukin-3, IL-4, and IL-5 are not detectable by standard Northern blot analysis or bioassay of condition media. However, IL-3--detectable by reverse-transcriptase PCR and biologic activity--was confirmed by growth of factor-dependent cells on stromal cells with IL-3 antibody blocking of such growth. Stem cells resident on such stromal cells are mirrored by the high proliferative potential colony-forming cell assay and are responsive to a relatively large number of cytokines, with Steel factor being of central importance, appearing to be a critical component of various synergistic combinations. Steel factor allows reduced levels of other factors in such combinations and works early in a temporal sequence. Hematopoietic stem cells can engraft in normal nonmyeloablated hosts. Using a male/female BALB/c transplantation model, we have shown high rates of engraftment into normal animals, out after marrow infusion to 25 months, after marrow infusion and that post-5-fluorouracil bone marrow is quite deficient in such engraftment.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:In vitro and in vivo studies of stromal niches. 799 65

Gene transfer or gene therapy has advantages in the treatment of a variety of disorders due to its selective expression within specific mammalian cells. Interferon-alpha (IFN-alpha) has been used in the management of leukemia but its diverse adverse activities with multiple potential side effects, possibly unrelated to therapeutic targets, may negatively influence the ability of IFN-alpha to treat this disorder. Therefore, we examined the ability of adenovirus (Ad)-IFN-alpha gene construct to transfect normal (CD34+ cells) and chronic myelogenous leukemia (CML) bone marrow mononuclear cells (BMMNC) and the transient overexpression of IFN-alpha in these cells. Ad-cytomegalovirus promoter driven IFN-alpha (AdCMV-IFN-alpha) at multiple doses was assessed to transfect highly purified CD34+ cells in liquid culture, and optimal transduction of CD34+ cells was achieved using 120 plaque forming units. Flow cytometric determinations revealed that there was no significant difference in cell viability for the 4 h or 24 h transfection periods. Immunoassay of IFN-alpha produced by CD34+ cells shows that IFN-alpha levels increased several fold in transfected cells. Transient expression of the IFN-alpha gene did not suppress proliferation of CD34+ progenitors as indicated by BFU-E or colony forming units-granulocyte-macrophage (CFU-GM) growth. Reverse transcriptase/polymerase chain reaction analysis of RNA from CD34+ harvested CFU-GM progenitor cells demonstrated transient IFN-alpha mRNA expression. Similarly, CML BMMNC were transfected with AdCMV-IFN-alpha under similar conditions as described for CD34+ cells. BMMNC cells exposed to adenovirus for 24 h and 48 h were found to express IFN-alpha at a substantial level. This in vitro data suggest that Ad-mediated gene transfer of IFN-alpha into hematopoietic stem cells can be achieved and that the IFN-alpha gene can be translated into its specific mRNA in CD34 progenitor cells.
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PMID:Adenovirus mediated alpha interferon (IFN-alpha) gene transfer into CD34+ cells and CML mononuclear cells. 2739 20

The distribution of myeloid lineage-associated cytokine receptors and lysosomal proteins was analyzed in human CD34+ cord blood cell (CB) subsets at different stages of myeloid commitment by reverse-transcriptase polymerase chain reaction (RT-PCR). The highly specific granulomonocyte-associated lysosomal proteins myeloperoxidase (MPO) and lysozyme (LZ), as well as the transcription factor PU.1, were already detectable in the most immature CD34+Thy-1+ subset. Messenger RNA (mRNA) levels for the granulocyte-colony stimulating factor (G-CSF) receptor, granulocyte-macrophage (GM)-CSF receptor alpha subunit and tumor necrosis factor (TNF) receptors I (p55) and II (p75) were also detected in this subset in addition to c-kit and flt-3, receptors known to be expressed on progenitor cells. By contrast, the monocyte-macrophage colony stimulating factor (M-CSF) receptor was largely absent at this stage and in the CD34+Thy-1-CD45RA- subsets. The M-CSF receptor was first detectable in the myeloid-committed CD34+Thy-l-CD45RA+ subset. All other molecules studied were found to be expressed at this stage of differentiation. Different cocktails of the identified ligands were added to sorted CD34+Thy-1+ single cells. Low proliferative capacity was observed after 1 week in culture in the presence of stem cell factor (SCF) + Flt-3 ligand (FL) + G-CSF. Addition of GM-CSF to this basic cocktail consistently increased the clonogenic capacity of single CD34+Thy-1+ cells, and this effect was further enhanced (up to 72.3 +/- 4.3% on day 7) by the inclusion of TNF-alpha. In conclusion, the presence of myeloid-associated growth factor receptor transcripts in CD34+ CB subsets does not discriminate the various stages of differentiation, with the exception of the M-CSF receptor. In addition, we show that TNF-alpha is a potent costimulatory factor of the very immature CD34+Thy-1+ CB subset.
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PMID:Analysis of myeloid-associated genes in human hematopoietic progenitor cells. 932 52

In the present study, we have analyzed the pattern of cytokines expressed by two independent dendritic cell (DC) subpopulations generated in vitro from human cord blood CD34+ progenitors cultured with granulocyte-macrophage CSF and TNF-alpha. Molecularly, we confirmed the phenotypic differences discriminating the two subsets: E-cadherin mRNA was only detected in CD1a+-derived DC, whereas CD68 and factor XIIIa mRNAs were observed exclusively in CD14+-derived DC. Semiquantitative reverse-transcriptase PCR analysis revealed that both DC subpopulations spontaneously expressed IL-1alpha, IL-1beta, IL-6, IL-7, IL-12 (p35 and p40), IL-15, IL-18, TNF-alpha, TGF-beta, macrophage CSF, and granulocyte-macrophage CSF, but not IL-2, IL-3, IL-4, IL-5, IL-9, and IFN-gamma transcripts. Both subpopulations were shown to secrete IL-12 after CD40 triggering. Interestingly, only the CD14+-derived DC secreted IL-10 after CD40 activation, strengthening the notion that the two DC subpopulations indeed represent two independent pathways of DC development. Furthermore, both DC subpopulations expressed IL-13 mRNA and protein following activation with PMA-ionomycin, but not with CD40 ligand, in contrast to IL-12 and IL-10, revealing the existence of different pathways for DC activation. Finally, we confirmed the expression of IL-7, IL-10, and IL-13 mRNA by CD4+ CD11c+ CD3- DC isolated ex vivo from tonsillar germinal centers. Thus, CD14+-derived DC expressing IL-10 and factor XIIIa seemed more closely related to germinal center dendritic cellsGCDC than to Langerhans cells.
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PMID:The cytokine profile expressed by human dendritic cells is dependent on cell subtype and mode of activation. 946 23

The recent discovery of chemokine receptors as coreceptors for human immunodeficiency virus-type 1 (HIV-1) entry offers new avenues for investigating the pathogenesis of acquired immunodeficiency syndrome (AIDS)-related cytopenias. To this end, we sought to (1) phenotype human hematopoietic cells for CD4 and the HIV-1 coreceptors CXCR4, CCR5, CCR3, and CCR2b; (2) correlate CD4 and chemokine receptor expression with their susceptibility to HIV-1 infection; and (3) examine any potential interplay between inflammatory cytokines released during HIV-1 infection and regulation of chemokine receptor expression. Fluorescence-activated cell sorting (FACS) analysis of bone marrow mononuclear cells (BMMNC), cells derived from serum-free expanded hematopoietic lineages (colony-forming unit-granulocyte-macrophage [CFU-GM], colony-forming unit-megakaryocyte [CFU-Meg], and burst-forming unit-erythroid [BFU-E]), and CD34(+) cells showed differential expression of chemokine receptors and CD4 with some lineage specificity. Significantly, FACS-sorted CXCR4(+)/CD34(+) cells had the same clonogeneic potential as CXCR4(-)/CD34(+) cells. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of FACS-sorted human candidate stem cells (HSC; CD34(+), c-kit+, Rho123(low)) showed the presence of CXCR4 mRNA but not CD4 mRNA. Infection studies with HIV-1 Env-pseudotyped luciferase reporter viruses indicated that X4 Env (CXCR4-using) pseudotypes infected megakaryocytic cells, whereas R5 Env (CCR5-using) pseudotypes did not. Similarly, R5 but not X4 Env-pseudotyped viruses infected granulocyte-macrophage cells in a CD4/CCR5-dependent manner. Erythroid cells were resistant to R5 or X4 viral infection. Finally, we found that gamma-interferon treatment upregulated CXCR4 expression on primary hematopoietic cells. In summary, the delineation of chemokine receptor expression on primary hematopoietic cells is a first step towards dissecting the chemokine-chemokine receptor axes that may play a role in hematopoietic cell proliferation and homing. Furthermore, susceptibility of hematopoietic cells to HIV-1 infection is likely to be more complicated than the mere physical presence of CD4 and the cognate chemokine receptor. Lastly, our results suggest a potential interplay between gamma-interferon secretion and CXCR4 expression.
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PMID:Coreceptor/chemokine receptor expression on human hematopoietic cells: biological implications for human immunodeficiency virus-type 1 infection. 994 56

Bone marrow endothelial cells are the essential component of the bone marrow microenvironment. They produce many kinds of cytokines, including stimulators and inhibitors. Many researchers have suggested that in the presence of endothelial cell layer, CD34+CD38- cells are capable of expansion. The ability of the endothelial cell layer to protect hematopoietic stem cells from extensive differentiation may be related to the inhibitors derived from endothelial cells. The aim of the present study was to determine whether the inhibitors thymosin beta4 and AcSDKP are elaborated by murine bone marrow endothelial cells. Murine bone marrow endothelial cells (mBMECs) were cultured in serum-free conditioned medium. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to analyze the differential expression of the thymosin-beta gene, and reverse phase high-performance chromatography (HPLC) and mass spectroscopy were used to determine the concentration of thymosin beta4 (Tbeta4) and AcSDKP in EC lysate and in the medium (mBMEC-CM). Colony-forming unit granulocyte-macrophage (CFU-GM) colony assays were used to examine the effect of components (mw 3-10 kD, <3 kD) of mBMEC-CM, thymosin beta4, and AcSDKP on the proliferation of hematopoietic cells.mBMECs expressed Tbeta4 mRNA. In EC lysate and mBMEC-CM, Tbeta4 and AcSDKP were detected. After adding protease inhibitors, the concentration of Tbeta4 in EC lysate increased significantly, while the concentration of AcSDKP decreased. mBMEC-CM (mw 3-10 kD) had no effect on the formation of CFU-GM. However, mBMEC-CM (mw <3 kD) could inhibit the growth of CFU-GM. Tbeta4 (10(-11) approximately 10(-7)mol/L) and AcSDKP (10(-11) approximately 10(-5)mol/L) had dose-dependent inhibitory effects on the growth of CFU-GM. Angiotensin converting enzyme (ACE), the enzyme degrading AcSDKP, could partially eliminate the inhibitory effect of mBMEC-CM (mw <3 kD) on CFU-GM.BMECs express and secrete Tbeta4 and AcSDKP. Tbeta4 exists in the 3-10 kD component of mBMEC-CM, while AcSDKP exists in the <3 kD component of ECCM. Both components exert inhibitory effects on the proliferation of hematopoietic progenitors.
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PMID:Bone marrow endothelial cells secrete thymosin beta4 and AcSDKP. 1116 1

The authors studied mineralocorticoid receptor (MCR)-mediated effects of steroids on CD34(+) progenitor cells. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed the presence of mRNA for both the MCR and the alpha subunit of the epithelial sodium channel, a member of the amiloride-sensitive sodium channel (ASSC) superfamily, in human CD41(+) megacaryoblastic cells derived from cultured bone marrow CD34(+) isolates, as well as in the human erythromegakaryoblastic leukemia (HEL) cell line. Immunofluorescence also revealed the presence of both the MCR and ASSC in circulating CD34(+) and medullar CD41(+) megacaryoblastic cells, the former as a nucleocytoplasmic protein and the latter as a membrane-bound protein, as expected from earlier studies using MCR-specific targets. In a selective medium, the formation of erythrocyte burst-forming units, and of the granulocyte-macrophage colony-forming units, by circulating CD34(+) cells was influenced by the agonists deoxycorticosterone and aldosterone, as well as by the antagonists RU 26752 and ZK 91587, targeted for the MCR. The multiplication of the leukemic HEL progeny, derived from CD41(+) cells, was similarly altered by these steroids targeted for the MCR. In contrast, in the optimal growth medium, the multiplication, and colony formation by bone marrow CD34(+) progenitor cells were not altered by either aldosterone or ZK 91587. These and other studies reveal that the receptor-mediated action of mineralocorticoids may influence the functional maturation of the hematopoietic progenitor lineage, contrary to the classical notion where the mineralotropic effect would be a unique feature of the epithelial cell.
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PMID:Mineralocorticoid hormones exert dramatic effects on pluripotent human stem cell progeny. 1293 24

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