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)

The c-kit ligand (KL; Steel factor, mast cell growth factor, stem cell factor) is a hematopoietic factor that has been shown to act as a potent cofactor for hematopoietic growth and differentiation in vitro. The in vivo effects of KL, however, have been variable. To study the hematopoietic role of KL in vivo, we evaluated KL gene expression in both normal mice and mice recovering from myelosuppressive radiation exposure using the reverse transcriptase-polymerase chain reaction (RT-PCR) technique. In a single RNA sample, we found that the RT-PCR technique has high precision (co-efficient of variation, 15.7%). Amplifications of serial 1:2 dilutions of template RNA precisely correlated with starting RNA concentrations at 20 cycles or at 25 cycles, depending on the level of expression. Amplification of individual normal bone marrow and spleen cell RNA showed basal expression in all normal bone marrows but irregular expression in normal spleens. On day 2 after a sublethal 7.75-Gy (0.4 Gy/min) 60Co irradiation, splenic KL gene expression increased approximately 2.5-fold (P = .011), and bone marrow expression increased 15-fold (P = .004). During a 28-day postirradiation recovery period, KL expression increased in bone marrow on days 2 through 7. Splenic expression during the same period was more variable. In conclusion, the KL gene is invariably expressed in normal murine spleens. Postirradiation, recovering bone marrow and spleen both express increased levels of KL mRNA at day 2 and continue to express increased levels for several days postexposure. These data support a role for KL in the endogenous recovery of hematopoiesis after hypoplastic injury.
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PMID:c-kit ligand gene expression in normal and sublethally irradiated mice. 753 11

A number of cytokines have been shown to have stimulatory activity on multipotent haematopoietic precursors. These include kit ligand (KL), interleukins (IL) 1, 3 and 6 and granulocyte macrophage-colony stimulating factor (GM-CSF). Using reverse transcriptase/polymerase chain reaction method (RT/PCR) we have examined the expression of these cytokines, the c-kit and IL-6 receptors, in long-term bone marrow culture (LTC) adherent layer cells in human bone marrow hypoplasia syndromes. Disorders studied include Fanconi's anaemia (FA, n = 16), idiopathic aplastic anaemia (AA, n = 11), Seckel's syndrome (n = 2), dyskeratosis congenita (n = 2), Shwachman-Diamond syndrome (n = 1), thrombocytopenia with absent radii syndrome (n = 1), acquired amegakaryocytosis (n = 1), paroxysmal nocturnal haemoglobinuria (n = 1) and acquired agranulocytosis (n = 1). IL-6 and GM-CSF expression appeared reduced in most patients with FA, suggesting that impaired production of these cytokines may contribute to the bone marrow failure seen in most patients with FA. In contrast, abundant IL-6 and GM-CSF expression were seen in most patients with AA when compared with the FA group and controls; these may be mediators of a stromal response in this disorder. No obvious differences were seen between the different patients' groups and controls in expression of the other cytokines or cytokine receptors studied.
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PMID:The expression of cytokine and cytokine receptor genes in long-term bone marrow culture in congenital and acquired bone marrow hypoplasias. 751 72

To determine whether c-kit and kit ligand (KL) mRNAs could be expressed in human epithelial tumors, reverse transcriptase-polymerase chain reaction and Northern blot analysis were performed. KL mRNA was shown to be expressed in a variety of epithelial tissues and cell lines. The expression of c-kit mRNA was then examined in hepatocellular and colon carcinoma cell lines. While hepatocellular carcinoma cell lines did not express c-kit mRNA as far as we could ascertain, 2 of 5 colon carcinoma cell lines showed the expression of both c-kit and KL mRNAs. Furthermore, the expression of c-kit in these cells was demonstrated at the protein level by flow cytometry. These data suggest that c-kit and KL may play an important role as an autocrine loop in the proliferation of some colon carcinoma cells.
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PMID:Expression of c-kit and kit ligand in human colon carcinoma cells. 769 50

We report that embryonic stem cells efficiently undergo differentiation in vitro to mesoderm and hematopoietic cells and that this in vitro system recapitulates days 6.5 to 7.5 of mouse hematopoietic development. Embryonic stem cells differentiated as embryoid bodies (EBs) develop erythroid precursors by day 4 of differentiation, and by day 6, more than 85% of EBs contain such cells. A comparative reverse transcriptase-mediated polymerase chain reaction profile of marker genes for primitive endoderm (collagen alpha IV) and mesoderm (Brachyury) indicates that both cell types are present in the developing EBs as well in normal embryos prior to the onset of hematopoiesis. GATA-1, GATA-3, and vav are expressed in both the EBs and embryos just prior to and/or during the early onset of hematopoiesis, indicating that they could play a role in the early stages of hematopoietic development both in vivo and in vitro. The initial stages of hematopoietic development within the EBs occur in the absence of added growth factors and are not significantly influenced by the addition of a broad spectrum of factors, including interleukin-3 (IL-3), IL-1, IL-6, IL-11, erythropoietin, and Kit ligand. At days 10 and 14 of differentiation, EB hematopoiesis is significantly enhanced by the addition of both Kit ligand and IL-11 to the cultures. Kinetic analysis indicates that hematopoietic precursors develop within the EBs in an ordered pattern. Precursors of the primitive erythroid lineage appear first, approximately 24 h before precursors of the macrophage and definitive erythroid lineages. Bipotential neutrophil/macrophage and multilineage precursors appear next, and precursors of the mast cell lineage develop last. The kinetics of precursor development, as well as the growth factor responsiveness of these early cells, is similar to that found in the yolk sac and early fetal liver, indicating that the onset of hematopoiesis within the EBs parallels that found in the embryo.
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PMID:Hematopoietic commitment during embryonic stem cell differentiation in culture. 841 45

Highly purified progenitors (including erythroid [BFU-E], granulo-monocytic [CFU-GM], multipotent [CFU-GEMM] progenitors, as well as multipotent progenitors with self-renewal capacity [CFU-B]) express high-affinity growth factor receptors (GFRs), with prevalent interleukin-3 receptors (IL-3Rs) (2,700/cell), a > or = 10-fold lower number of IL-6Rs (145/cell) and granulocyte-macrophage colony-stimulating factor receptors (GM-CSFRs) (300/cell), and a barely detectable level of erythropoietin (Ep) receptors (75/cell). Hematopoietic growth factor (HGF) dosages inducing peak clonogenetic effects are associated with partial/subtotal occupancy of the homologous HGF receptor (HGFR). Cross-reactivity between GFRs and heterologous GFs (including IL-6, IL-3, GM-CSF, Ep, and the kit ligand [KL]) was explored by competition experiments on purified progenitors with radiolabeled and excess cold HGFs at +4 degrees C. No cross-reaction was observed between IL-6R, IL-3R, EpR, and the heterologous GFs, whereas the GM-CSFR showed cross-reactivity with IL-3 and, to a lesser extent, KL. Modulation of GFRs was examined after 18 or 40 hours of incubation with GF(s) at 37 degrees C, followed by ligand-binding assay at 20 degrees C. IL-6, IL-3, GM-CSF, and Ep induce a marked down-modulation of their own receptors. Interestingly, each GF induces the transactivation of the R(s) for the "distal" GF(s): (1) IL-6 induces transactivation of IL-3R, but not of GM-CSFR/EpR; (2) IL-3 causes a rapid upmodulation of GM-CSFR/EpR ("pure" progenitors treated with IL-3 show upmodulation of GM-CSFR alpha-chain mRNA by reverse transcriptase-polymerase chain reaction); whereas (3) GM-CSF induces the transactivation of the EpR. This chain upmodulation of HGFRs may underlie the synergistic interactions between the HGFs in clonogenetic culture. It is emphasized that KL does not induce upmodulation of the other GFRs. Finally, Ep, GM-CSF, and IL-3 do not modulate the expression of the "proximal" HGFRs (ie, GM-CSFR/IL-3R/IL-6R, IL-3R/IL-6R, and IL-6R, respectively). These results allow insight into the cellular basis of hematopoiesis, ie, the complex and coordinate interactions between HGFs and their receptors. They are compatible with a model of cascade transactivation via upmodulation of GFRs in the initial key steps of hematopoietic differentiation, whereby the action of each GF enhances the effect of the distal GF(s) by a multistep chain-potentiation mechanism.
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PMID:Cascade transactivation of growth factor receptors in early human hematopoiesis. 845 93

The expression of c-kit and flk-2/flt3 was analyzed in various stages of mast cell differentiation using reverse transcriptase polymerase chain reaction (RT-PCR). Mouse fetal liver cells were sorted using antibodies for Sca-1 (Ly6A/E) and CD43 to obtain a population enriched for early progenitors; committed mast cell progenitors were absent from this population. Mouse fetal liver-derived, IL-3-dependent blast cell colonies provided a source of committed mast cell progenitors, and mast cell colonies provided mature mast cells. Comparison of these populations showed that some uncommitted cells express both c-kit and flk-2/flt3. At the time of commitment to the mast cell lineage, the expression of c-kit increases compared to that of uncommitted progenitors, and the expression of flk-2/flt3 becomes undetectable. Previous studies have shown that steel factor, the ligand for c-kit, supports mast cell differentiation in vivo and in vitro. In contrast, the ligand for flk-2/flt3 is inactive on mast cells. Thus, receptor gene expression appears to be an important determinant of the response or lack of response of mast cells to the ligands for these two homologous receptors.
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PMID:Loss of flk-2/flt3 expression during commitment of multipotent mouse hematopoietic progenitor cells to the mast cell lineage. 921 38

CXCR4 is the receptor for the alpha-chemokine stromal cell-derived factor 1 (SDF-1) and has been shown to be expressed on a diversity of leukocytes. In this report, the expression of the CXCR4 receptor in cells of megakaryocytic lineage and the role of SDF-1 in megakaryocytopoiesis were investigated. Using flow cytometry in combination with reverse transcriptase-polymerase chain reaction (RT-PCR), we observed that bone marrow CD34(+), CD61(+) cells, blood platelets, and megakaryocytic leukemia cell lines all expressed the CXCR4 receptor. To examine the expression of the CXCR4 receptor on megakaryocyte progenitors (colony-forming units-megakaryocyte [CFU-Meg]), CXCR4-positive and -negative CD34(+) populations were separated from bone marrow and cultured in a plasma clot culture system. A subpopulation of the CFU-Meg was found in the CXCR4-positive fraction. The functional significance of CXCR4 expression on cells of the megakaryocytic lineage was examined by studying the effects of SDF-1alpha on migration and proliferation of megakaryocyte progenitor cells in vitro. We found that SDF-1alpha potently induced megakaryocyte progenitor migration and significantly enhanced adhesion of mature marrow megakaryocytes to endothelium. No marked effects of SDF-1alpha alone or in combination with thrombopoietin and stem cell factor/kit ligand on megakaryocyte production in vitro were noted. These results demonstrate for the first time that the CXCR4 alpha-chemokine receptor is expressed on cells of the megakaryocytic lineage from progenitors to platelets and that its ligand SDF-1alpha may modulate several aspects of megakaryocytopoiesis.
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PMID:The alpha-chemokine receptor CXCR4 is expressed on the megakaryocytic lineage from progenitor to platelets and modulates migration and adhesion. 968 Mar 41

The c-kit receptor and its ligand, steel factor (SLF), are critical for optimal hematopoiesis. We evaluated effects of transducing cord blood (CB) progenitor cells with a retrovirus encoding human c-kit cDNA. CD34(+) cells were sorted as a population or as 1 cell/well for cells expressing high levels of CD34 and different levels of c-kit (++,+,Lo/-), transduced and then cultured in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), IL-6, erythropoietin (Epo) +/- SLF in the absence of serum. At a single-cell level, transduction with c-kit, but not with control (neo only), virus significantly increased colony formation, especially by erythroid and multipotential progenitors. The enhancing effect of c-kit transduction was inversely correlated with expression of c-kit protein before transduction. The greatest enhancing effects were noted in CD34KitLo+/- cells transduced with c-kit. The stimulating effect was apparent even in the absence of exogenously added SLF, but in the presence of GM-CSF, IL-3, IL-6, and Epo. Enzyme-linked immunosorbent assay (ELISA) of SLF protein, reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of SLF mRNA expression in CD34+ cells, and use of neutralizing antibodies to SLF and/or c-kit suggested the presence of endogenous, although probably very low level, expression of SLF by these progenitor cells. Transduction of c-kit significantly decreased sensitivity of progenitor cells to the inhibitory effects of transforming growth factor-beta1 and tumor necrosis factor-alpha. c-kit-transduced cells had increased expression of c-kit protein and decreased spontaneous or cytokine-induced apoptosis. Our results suggest that transduced c-kit into selected progenitor cells can enhance proliferation and decrease apoptosis and that endogenous SLF may mediate this effect.
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PMID:Retroviral-mediated gene transduction of c-kit into single hematopoietic progenitor cells from cord blood enhances erythroid colony formation and decreases sensitivity to inhibition by tumor necrosis factor-alpha and transforming growth factor-beta1. 1049 4

Mast cell hyperplasia is observed in various inflammatory skin diseases. Although the mechanisms involved in the pathogenesis of these conditions remains largely uninvestigated, it is speculated that mediators produced in the lesional skin provide a favorable microenvironment for mast cell growth. Among the proinflammatory mediators, leukemia inhibitory factor (LIF), which shares a receptor component (gp130 subunit) with interleukin-6 (IL-6), has been identified as a mast cell growth-enhancing factor produced by cells of the keratinocyte-derived cell line (KCMH-1). In this study, we investigated the effect of four IL-6 family cytokines, IL-6, IL-11, oncostatin M (OSM) and LIF on mast cell growth in a mast cell/fibroblast co-culture system. When mouse bone marrow-derived cultured mast cells (BMMC) were maintained on a NIH/3T3 fibroblast monolayer, these cytokines induced proliferation of the mast cells, but none of the cytokines had any effect on mast cell proliferation in the absence of fibroblasts. mRNA for gp130 and receptors for the four IL-6 family cytokines were detected in NIH/3T3 fibroblasts by reverse transcriptase-mediated polymerase chain reaction. In contrast, only mRNA for the IL-11 receptor and gp130 were detected in BMMC. Tyrosine phosphorylation of gp130 was observed in NIH/3T3 fibroblasts after stimulation with all the cytokines. Some IL-6 family cytokines enhanced the production of stem cell factor (SCF), a potent mast cell growth factor, from NIH/3T3 fibroblasts, but the amount of SCF produced by NIH/3T3 fibroblasts was not paralleled by the mast cell growth-enhancement induced by the IL-6 family cytokines. When anti-SCF antibody was added with the IL-6 family cytokines in the BMMC/fibroblast coculture system, a significant effect of these cytokines remained, although the growth-enhancing activity was markedly reduced. A similar result was obtained when BMMC were prepared from W/W(V)-mice, which lack functional c-kit, in the BMMC/ fibroblast coculture system. These results suggest that IL-6 family cytokines stimulate mast cell growth by a fibroblast-dependent mechanism, and also suggest the existence of another pathway between BMMC and NIH/3T3 fibroblasts cooperating with the SCF/c-kit pathway. IL-6 family cytokines may thus contribute to mast cell hyperplasia in skin diseases.
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PMID:The IL-6 family cytokines, interleukin-6, interleukin-11, oncostatin M, and leukemia inhibitory factor, enhance mast cell growth through fibroblast-dependent pathway in mice. 1182 Jul 27

Relatively little information is available on the local factors that regulate folliculogenesis in goats. To examine the possibility that the Kit ligand (KL) system is expressed throughout the folliculogenesis, we studied the presence and distribution of KL and its receptor, c-Kit, in goat ovaries. Ovaries of goats were collected and either fixed in paraformaldehyde for immunohistochemical localization of KL and c-Kit proteins, or used for the isolation of follicles, luteal cells, surface epithelium and medullary samples to study mRNA expression for KL and c-Kit, using the reverse transcriptase polymerase chain reaction (RT-PCR). KL protein and mRNA were found in follicles at all stages of development, i.e. primordial, primary, secondary, small and large antral follicles, as well as in corpora lutea, surface epithelium and medullary tissue. Antral follicles expressed both KL-1 and KL-2 mRNAs, while earlier staged follicles expressed KL-1 transcript only. KL protein was demonstrated in granulosa cells from the primordial follicle onward. Its mRNA could be detected in granulosa cells isolated from antral follicles and occasionally in their theca cells. c-Kit mRNA was expressed in all antral follicular compartments and at all stages of follicular development. c-Kit protein was predominantly found in oocytes from the primordial follicle stage onwards, in theca cells of antral follicles, as well as in corpora lutea, surface epithelium and medullary tissue, particularly in the wall of blood vessels, which may indicate these cells as the main sites of action of KL. It is concluded that the KL/c-Kit system, in goat ovaries, is widespread and that it may be involved in the regulation of various local processes, including folliculogenesis and luteal activity.
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PMID:The Kit ligand/c-Kit receptor system in goat ovaries: gene expression and protein localization. 1726 90


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