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 tested the hypothesis that estrogen acutely stimulates constitutive nitric oxide synthase activity in human granulocytes by acting on a cell surface estrogen receptor (ER). The release of nitric oxide was measured in real time with an amperometric probe. Exposure of granulocytes to 17beta-estradiol stimulated NO release within seconds in a concentration-dependent manner. The NO release was also stimulated by 17beta-estradiol conjugated to bovine serum albumin (E(2)-BSA), which suggests mediation by a cell surface receptor. Tamoxifen, an ER inhibitor, antagonized the action of both 17beta-estradiol and E(2)-BSA, whereas ICI 182,780, an inhibitor of the nuclear ER, had no effect. Using dual emission microfluorometry in a calcium-free medium, the 17beta-estradiol-stimulated release of NO from granulocytes was shown to be dependent on intracellular calcium ([Ca(2+)]i) transients in a tamoxifen-sensitive process. Exposure to BAPTA-AM (1,2bis-(-aminophenoxy)ethans-N,N,N', N'-tetraacetic acid tetra(acetoxyymethyl) ester), a [Ca(2+)]i chelator, reduced [Ca(2+)]i in response to E(2)-BSA, and depleting [Ca(2+)]i stores abolished the effect of 17beta-estradiol on NO release. Confocal photomicrographs using E(2)-BSA-FITC (fluorescein isothiocyanate) revealed cell membrane reactivity. Estrogen-stimulated NO release had an immunosuppressive effect, and it initiated granulocyte rounding and loss of adherence in a tamoxifen-sensitive manner. Finally, using reverse transcriptase-polymerase chain reaction, human neutrophil granulocytes expressed ERalpha but not ERbeta, suggesting that ERalpha may be the membrane receptor for 17beta-estradiol. The study demonstrated that a physiological dose of estrogen down-regulates granulocyte activity by acutely stimulating NO release via the activation of a cell surface ER which is coupled to increases in [Ca(2+)]i. (Blood. 2000;95:3951-3958)
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PMID:Estradiol-stimulated nitric oxide release in human granulocytes is dependent on intracellular calcium transients: evidence of a cell surface estrogen receptor. 1084 33

The specificity and sensitivity of the nested reverse transcriptase polymerase chain reaction (RT-PCR) on tyrosinase was studied, for the detection of micrometastases of malignant melanoma. The specificity was assessed in the blood of six healthy donors, four patients with non-melanoma cancers of which one patient was treated with granulocyte-colony stimulating factor. Lymph nodes of nine patients without malignant melanoma were tested and four cell lines of various other tumours. Six of the nine non-melanoma lymph nodes were positive in this assay. The sensitivity was tested in a spike experiment in vitro, using a melanoma cell line. The detection limit was ten melanoma cells per 10(7) peripheral blood lymphocytes.
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PMID:Limitations of the nested reverse transcriptase polymerase chain reaction on tyrosinase for the detection of malignant melanoma micrometastases in lymph nodes. 1090 68

It has been reported previously that in vitro treatment of human blood derived dendritic cells (DC) with contact allergens provokes the elevated expression of mRNA for interleukin (IL) 1beta, under conditions where similar treatment of cells with the non-sensitizing skin irritant sodium lauryl sulfate (SLS) did not alter IL-1beta mRNA levels (Reutter et al., 1997). The purpose of the present investigation was to evaluate further this phenomenon and to explore the potential utility of this approach for the purpose of skin sensitization testing. Human peripheral blood progenitor cells prepared from healthy adult volunteers were cultured in the presence of IL-4 and granulocyte/macrophage colony stimulating factor. After 5 days of culture, the majority of cells had a Langerhans cell-like phenotype, with characteristic dendritic morphology and cell surface expression of CD83, major histocompatibility complex class II and CD1a determinants. These blood-derived DC were cultured in the presence of the contact allergen 2,4-dinitrofluorobenzene (DNFB), SLS or vehicle alone and mRNA expression for IL-1beta, IL-6 and IL-18 was analysed by semiquantitative reverse transcriptase polymerase chain reaction. Constitutive expression of all three cytokines was observed for DC isolated from all donors examined. Exposure to DNFB resulted in upregulation of IL-1beta mRNA (two- to threefold) in cells derived from three out of eight donors whereas IL-6 and IL-18 were largely unaffected by allergen exposure. In contrast, SLS treatment did not induce IL-1beta mRNA expression in any of the donors investigated. Analysis of cytokine mRNA expression using the protocol described by Reutter et al. (1997), did not increase the sensitivity of measurement of induced cytokine expression. Although selected upregulation of IL-1beta by blood derived DC has been confirmed, a wider range of contact allergens and irritants need to be assessed before this approach could be considered for hazard identification.
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PMID:Investigation of induced changes in interleukin 1beta mRNA expression by cultured human dendritic cells as an in vitro approach to skin sensitization testing. 1090 42

The granulocyte-derived hemoregulatory peptide pyroGlu-Glu-Asp-Cys-Lys = pEEDCK is known to keep hematopoietic cells quiescent. When oxidized to its dimeric form (pEEDCK)2, it activates growth of hematopoietic progenitors in association with stroma-derived cytokines. (pEEDCK)2 has a Cys-Cys motif which is also a typical feature of the macrophage inflammatory protein (MIP-1alpha). The present study was designed to analyze differences between the response of normal and leukemic progenitor cells to (pEEDCK)2 or MIP-1alpha. When long-term bone marrow cultures (LTBMCs) were incubated with (pEEDCK)2 or MIP-1alpha and/or cytokines, the stimulatory effect on colony-forming units-granulocyte/erythroid/macrophage/megakaryocyte of LTBMC from chronic myeloid leukemia (CML) patients was less than 50% compared to LTBMC from healthy humans. No difference in oncogene expression could be observed in LTBMC from CML patients regarding reduction of Philadelphia chromosome-associated transcription of the BCR-ABL gene. With respect to the expression of growth and differentiation-associated genes (Galpha16, 5-lipoxygenase, phospholipaseA2, c-kit, and CD34), which were analyzed from LTBMC by semiquantitative reverse transcriptase-polymerase chain reaction, the same transcription rate was observed in CML patients and healthy donors. However, two isoforms of a key enzyme of oxidative metabolism, carnitine palmitoyltransferase (CPT1A and CPT1B), showed 50-fold higher expression rates in LTBMC cells of healthy donors compared to CML patients. It is known that a decrease in oxidative metabolism is associated with an increase in redox equivalents in malignancy. This might result in a reduction of disulphide bonds in (pEEDCK)2 or MIP-1alpha, thus inducing a downregulation of these factors in bone marrow from CML patients.
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PMID:Effect of the hemoregulatory peptide (pEEDCK)2 (pyroGlu-Glu-Asp-Cys-Lys)2 and MIP-1alpha is reduced in bone marrow cultures from patients with chronic myeloid leukemia (CML). 1146 52

We examined the expression of mRNAs for inflammatory cytokines and Fas in cultured human fetal membrane cells responding to influenza virus (IV) infection using the reverse transcriptase-polymerase chain reaction (RT-PCR). Primary cultured chorion and amnion cells prepared from human fetal membranes were infected with IV. Chorion cells expressed significant amounts of interleukin (IL)-1beta, IL-6, tumor necrosis factor (TNF)-alpha, interferon (IFN)-beta, IFN-gamma and granulocyte macrophage colony-stimulating factor (GM-CSF) mRNAs and small amounts of Fas mRNA in response to IV infection. Amnion cells expressed TNF-alpha and IFN-beta mRNAs in response to IV infection, while expression of the other mRNAs was not altered. We also examined whether or not TNF-alpha, IFN-beta, IFN-gamma and Fas participated in IV infection-induced apoptotic DNA fragmentation in chorion cells. Neutralizing antibodies against them did not inhibit DNA fragmentation. These results suggested that chorion cells expressed significant amounts of mRNAs for inflammatory cytokines in response to IV infection, and that, in contrast, mRNA expression was quiescent in amnion cells. Moreover, TNF-alpha, IFN-beta, IFN-gamma and Fas do not appear to be directly involved in the apoptosis induction of IV-infected chorion cells. The results indicated that chorion cells may play a role in defense against IV through an antiviral immune response and apoptosis to eliminate own cells and viral pathogens in infected organs, whereas amnion cells do not play such a role.
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PMID:Differential mRNA expression of inflammatory cytokines in cultured human fetal membrane cells responding to influenza virus infection. 1185 74

Serum soluble interleukin-6 receptors (sIL-6R) have been demonstrated to play an important role in hematopoiesis. We report here that serum sIL-6R levels reflect proliferative kinetics of the progenitors after stimulation by chemotherapy plus granulocyte colony-stimulating factor. Serum sIL-6R were serially evaluated in 26 courses of peripheral blood (PB) stem cell collections in 16 patients using enzyme-linked immunosorbent assay. Expressions of IL-6R and CD34 on PB mononuclear cells were examined by flow cytometric analysis and expressions of IL-6R mRNA were examined by reverse transcriptase polymerase chain reaction. There were no significant differences between the serum sIL-6R levels on day 0 in patients (27.8+/-2.1 ng/ml, mean +/- SEM) and those in controls (27.5+/-1.5 ng/ml). Following chemotherapy the serum sIL-6R levels were significantly decreased, reaching a minimal level on day 14 (22.3+/-1.2 ng/ml, p < 0.01) and then significantly increased to above the baseline levels on day 21 (32.0+/-2.1 ng/ml, p < 0.01). Similar oscillations in the number of white blood cells, IL6R+ cells, CD34+ cells and colony-forming unit-granulocyte/macrophage (CFU-GM) in PB could be observed and the peak expression of mRNA was compatible with the expression of antigen. Serum sIL-6R levels on day 17 and 19 were positively correlated with the number of CD34+ cells, IL-6R+ cells, CFU-GM in PB and the number of collected CD34+ cells in leukapheresis products. In addition, when comparing the 2 groups divided by the number of prior chemotherapies, the status of disease or dose of the mobilizing regimen, the serum sIL-6R levels were significantly increased after day 17 in the group that received fewer courses of prior chemotherapy, the group in complete remission and the group of high-dose chemotherapy. These findings indicated that sIL-6R levels do not reflect the hematopoietic ability in the steady state, or the capability of the hematopoiesis after stimulation. Thus, sIL-6R levels may be a marker for the timing of PBSC collection or the prediction of the number of collected CD34+ cells.
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PMID:Serum soluble IL-6 receptor levels during the mobilization of stem cells to peripheral blood. 1200 69

Data concerning the specificity of cytokeratin 20 (CK20) as a reverse transcriptase-polymerase chain reaction analysis (RT-PCR) marker to detect disseminated tumor cells in blood are conflicting. Underlying causes for these discrepancies need to be determined to clarify the significance of CK20 detection. Because differences in RT-PCR assays and blood sample handling may be important, their influence on CK20 detection was studied. Using a series of healthy donor blood samples spiked with colon tumor cells, the authors compared the sensitivities of two conventional PCRs with different primer sets and a quantitative LightCycler PCR (Roche Diagnostics GmbH, Penzberg, Germany). Additionally, the influence of sample collection and preparation on assay specificity was studied by examining CK20 expression in the mononuclear cell fraction (MNC) of the first and the second aliquot of blood drawn from healthy donors and in the granulocyte cell fraction. At the concentration of one spiked tumor cell/mL blood, the CK20 detection frequency varied from 17% and 67% for the conventional to 78% for the LightCycler PCR. In the unspiked samples, CK20 was detected in 0% and 8% of the conventional and in 11% of the LightCycler PCR tests. Quantitative analysis revealed that CK20 was expressed at a high level in the granulocyte samples. The results demonstrate that differences in assay sensitivity and sample handling influence CK20 detection in blood.
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PMID:Effect of blood sample handling and reverse transcriptase-polymerase chain reaction assay sensitivity on detection of CK20 expression in healthy donor blood. 1204 12

One important question in stem cell biology of childhood acute lymphoblastic leukemia (ALL) is whether immature CD34+CD19- cells are part of the leukemic cell clone. CD34+CD19- cells from the bone marrow of 9 children with TEL/AML1-positive ALL were purified by flow sorting and subjected to reverse transcriptase-polymerase chain reaction (RT-PCR), fluorescence in situ hybridization, and methylcellulose cultures. In 3 of 8 patients analyzed by RT-PCR, no TEL/AML1-positive cells could be detected in the CD34+CD19- cell fraction. Altogether, the percentage of TEL/AML1-positive cells was low: 1.6% (n = 8; SD 2.2%) by nested real-time RT-PCR and 2.5% (n = 5; SD 2.6%) by fluorescence in situ hybridization. This correlated with the percentage of contaminating CD19+ leukemic cells in the CD34+CD19- cell fraction in 6 control sorts (mean 4.6%, SD 3.6%), indicating that the low levels of leukemic cells detected in the CD34+CD19- cell fraction could be attributed to sorter errors. Methylcellulose cultures in 3 patients provided further evidence that CD34+CD19- cells represent a candidate normal cell population. The clonogenicity of the CD34+CD19- cell fraction was similar to normal progenitors, including growth of primitive granulocyte, erythroid, macrophage, megakaryocyte colony-forming units. Each of 92 colonies from cultures with CD34+CD19- cells tested negative for TEL/AML1. In conclusion, our data support the hypothesis that the leukemia in TEL/AML1-positive childhood ALL originates in a CD19+ lymphoid progenitor. This has many therapeutic implications, eg, for purging of autologous stem cell products, flow cytometric monitoring of minimal residual disease, and targeting immunotherapy against the leukemic cell clone.
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PMID:Immature CD34+CD19- progenitor/stem cells in TEL/AML1-positive acute lymphoblastic leukemia are genetically and functionally normal. 1209 59

An earlier study reported that human gingival epithelial cells in primary culture and oral epithelial cell lines KB and HSC-2 cells were devoid of membrane CD14 (mCD14) and did not show enhanced production of interleukin (IL)-8 or granulocyte macrophage-colony stimulating factor (GM-CSF) upon stimulation with bacterial cell-surface components such as lipopolysaccharide (LPS), lipoteichoic acid (LTA), peptidoglycan (PGN) and synthetic muramyldipeptide (MDP) even in the presence of serum. The present study demonstrated that after treatment with interferon (IFN)-gamma for 3 days, these cells secreted IL-8 and GM-CSF in response to the bacterial components. Treatment with IFN-gamma enhanced Toll-like receptor (TLR) 2, TLR4, MD-2 and MyD88 mRNA expression as determined by reverse transcriptase PCR. Anti-TLR2 and anti-TLR4 monoclonal antibodies (MAbs) inhibited the IL-8 production induced by PGN and LTA as well as LPS, respectively, in IFN-gamma-primed oral epithelial cells, whereas neither MAb inhibited IL-8 production induced by MDP. These findings suggested that IFN-gamma primed oral epithelial cells to produce cytokines upon stimulation with various bacterial components by up-regulation of the TLR system.
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PMID:Priming of human oral epithelial cells by interferon-gamma to secrete cytokines in response to lipopolysaccharides, lipoteichoic acids and peptidoglycans. 1217 Dec 92

Using a combination of cross species reverse transcriptase-polymerase chain reaction and 3' rapid amplification of cDNA ends techniques, we cloned the cDNA encoding gerbil granulocyte/macrophage colony-stimulating factor (GM-CSF). The open reading frame had 81% nucleotide identity with its mouse counterpart, while the mature protein had 80% homology with mature mouse GM-CSF. COS-7 cells transfected with gerbil GM-CSF cDNA secreted high levels of bioactive GM-CSF, as their supernatant stimulated gerbil bone-marrow cell proliferation and colony formation in semi-solid medium.
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PMID:Molecular cloning and expression of gerbil granulocyte/macrophage colony-stimulating factor. 1223 85


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