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)

Vasoactive intestinal peptide receptor I (VIPRI) expression was examined in megakaryocytes using reverse transcriptase-polymerase chain reaction (RT-PCR). VIPRI protein was characterized in platelet membranes using covalent crosslinking techniques. Human megakaryocytes were isolated from suspension cultures of cord blood and adult bone marrow mononuclear cells using a murine monoclonal antibody to human platelet glycoprotein IIB/IIIA (CD41) and immunomagnetic beads. RT-PCR primers were constructed for the VIP, VIPRI, and VIPRII genes as well as for megakaryocyte specific genes, c-mpl and platelet factor 4 (PF-4). VIP, VIPRI, c-mpl, and PF-4 were coexpressed in megakaryocyte mRNA. Southern blot analysis confirmed the expression of VIPRI. 125I-VIP was covalently cross-linked to human platelet membranes using the homobifunctional reagent disuccinimidyl suberate, followed by polyacrylamide gel electrophoresis and autoradiography. A 125I-VIP-protein complex of Mr = 50,000 was identified. Labeling of the Mr = 50,000 component was completely abolished by unlabeled VIP, but not by peptide histidine methionine or growth hormone releasing factor, indicating specific binding of VIP to the platelet membranes. Taken together, these results suggest that VIP may have direct effects on megakaryocytopoiesis and support our earlier observations of VIP modulation of platelet aggregation.
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PMID:Characterization of vasoactive intestinal peptide receptors on human megakaryocytes and platelets. 863 31

The leukemia-specific AML1/ETO fusion gene has been shown to be detected by reverse transcriptase polymerase chain reaction (RT-PCR) analysis in patients with t(8;21) acute myelogenous leukemia (AML) in long-term remission. In the present study, the AML1/ETO mRNA could be detected by RT-PCR in bone marrow (BM) and/or peripheral blood (PB) samples from all 18 patients who had been maintaining complete remission for 12 to 150 months (median, 45 months) following chemotherapy or PB stem cell transplantation (PBSCT), whereas it could not be detected in four patients who had been maintaining remission for more than 30 months following allogeneic BM transplantation (BMT). We surveyed the expression of AML1/ETO mRNA in clonogenic progenitors from BM in these cases. Notably, 51 of 2,469 colonies from clonogenic progenitors (2.1%) expressed the AML1/ETO mRNA in 18 cases who were RT-PCR+ in BM and/or PB samples. Expression was observed in various clonogenic progenitors, including granulocyte-macrophage colonies, mixed colonies, erythroid colonies, and megakaryocyte colonies. Furthermore, we analyzed the clonality of these progenitors by X-chromosome inactivation patterns of the phosphoglycerate kinase (PGK) gene in four female patients. The AML1/ETO mRNA+ progenitors showed the PGK allele identical to that detected in the leukemic blasts from the time of initial diagnosis. Normal constitutive hematopoiesis was sustained by polyclonal BM reconstitution in these patients. Accordingly, these committed progenitor cells that express AML1/ETO mRNA during remission likely have arisen from common t(8;21)+ pluripotent progenitor cells with at least trilineage differentiation potential. These data strongly suggest that the origin of the clonogenic leukemic progenitors of t(8;21) AML may be multipotent hematopoietic progenitors that acquired the t(8;21) chromosomal abnormality.
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PMID:Persistence of multipotent progenitors expressing AML1/ETO transcripts in long-term remission patients with t(8;21) acute myelogenous leukemia. 863 50

The in vivo effect of G-CSF on the maturation of mouse bone marrow megakaryocytes was studied by monitoring the DNA contents. Megakaryocytes were first identified by a specific 1C2 monoclonal antibody against mouse platelets and megakaryocytes and DNA contents of these cells were measured by propidium iodine. Megakaryocytes of mice transgenic for human G-CSF had a modal DNA class of 8N, showing a striking contrast to the previous reports that normal mouse megakaryocytes from most strains have 16N DNA content as a modal class. Daily 10 micrograms administration of G-CSF to mice for three to five days affected the DNA distribution pattern of bone marrow megakaryocytes, with a higher proportion of cells having 8N DNA contents. This G-CSF treatment, however, did not influence the peripheral blood platelet count or bone marrow megakaryocyte number. Administration of G-CSF along with thrombopoietin (TPO) reduced the proportion of megakaryocytes, with 32N DNA, the DNA class that was increased by TPO. Finally, the presence of mRNA for the mouse G-CSF receptor was demonstrated in two megakaryoblastic cell lines by reverse transcriptase polymerase chain reaction. These results indicated that G-CSF may have a suppressive effect on the maturation of mouse bone marrow megakaryocytes when monitored by the DNA polyploidy. Although further study is clearly necessary, the presence of mRNA for the G-CSF receptor in megakaryocytic lineage strongly suggests the direct action of G-CSF on this cell lineage.
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PMID:Overexpression of granulocyte colony-stimulating factor in vivo decreases the level of polyploidization of mouse bone marrow megakaryocytes. 882 Sep 58

Interleukin-11 (IL-11) is a pleiotropic growth factor with a prominent effect on megakaryopoiesis and thrombopoiesis. The receptor for IL-11 is a heterodimer of the signal transduction unit gp130 and a specific receptor component, the alpha-chain (IL-11R alpha). Two genes potentially encode the IL-11R alpha: the IL11Ra and IL11Ra2 genes. The IL11Ra gene is widely expressed in hematopoietic and other organs, whereas the IL11Ra2 gene is restricted to only some strains of mice and its expression is confined to testis, lymph node, and thymus. To investigate the essential actions mediated by the IL-11R alpha, we have generated mice with a null mutation of IL11Ra (IL11Ra-/-) by gene targeting. Analysis of IL11Ra expression by Northern blot and reverse transcriptase-polymerase chain reaction, as well as the absence of response of IL11Ra-/- bone marrow cells to IL-11 in hematopoietic assays, further confirmed the null mutation. Compensatory expression of the IL11Ra2 in bone marrow cells was not detected. IL11Ra-/- mice were healthy with normal numbers of peripheral blood white blood cells, hematocrit, and platelets. Bone marrow and spleen contained normal numbers of cells of all hematopoietic lineages, including megakaryocytes. Clonal cultures did not identify any perturbation of granulocyte-macrophage (GM), erythroid, or megakaryocyte progenitors. The number of day-12 colony-forming unit-spleen progenitors were similar in wild-type and IL11Ra-/- mice. The kinetics of recovery of peripheral blood white blood cells, platelets, and bone marrow GM progenitors after treatment with 5-flurouracil were the same in IL11Ra-/- and wild-type mice. Acute hemolytic stress was induced by phenylhydrazine and resulted in a 50% decrease in hematocrit. The recovery of hematocrit was comparable in IL11Ra-/ - and wild-type mice. These observations indicate that IL-11 receptor signalling is dispensable for adult hematopoiesis.
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PMID:Adult mice with targeted mutation of the interleukin-11 receptor (IL11Ra) display normal hematopoiesis. 931 Apr 65

Platelet basic protein (PBP) is a chemokine family member that is only found in platelets and their precursors megakaryocytes. The PBP gene is physically linked to the gene for another platelet-specific chemokine, platelet factor 4. While the biological basis of platelet factor 4 expression has been pursued by others, the regulatory features controlling the platelet-specific expression of PBP have not been investigated. In this article, we examined the molecular basis by which this megakaryocyte-specific gene is regulated. Transient expression studies of truncated reporter constructs containing from 4.5 to 0.1 kilobases of the functional PBP gene 5'-flanking region, demonstrated that the proximal 0.1 kilobases of the promoter was sufficient for high levels of expression in human erythroleukemia and CHRF-288 cells, two megakaryocytic cell lines. However, none of these constructs was expressed above background levels in HeLa and 293 cells, two non-megakaryocytic cell lines. Further truncation of this promoter suggested that there was an important regulatory element(s) within a pyrimidine-rich tract. Mobility shift analysis of the pyrimidine-rich tract defined a region between -85 and -64 which bound to a nuclear factor(s). This region contains sequences matching the consensus Ets-binding site from -78 to -75 base pairs. In particular, we noted that this site matched a PU.1 consensus sequence known as a PU box. Mobility shift and supershift studies with nuclear extracts as well as recombinant PU.1 protein and anti-PU.1 antibody further confirmed that PU.1 was the specific Ets family factor that bound to this site. Transient expression assays using reporter constructs which contained point mutations that abrogated PU.1 binding also significantly reduced PBP promoter activity in human erythroleukemia and CHRF cells. In addition, while all reporter gene constructs containing PBP promoters were completely inactive in HeLa cells, transactivation experiments using a PU.1 expression construct demonstrated that exogenous expression of PU.1 could increase reporter gene expression up to 8-fold in these cells. Finally, the role of PU.1 in PBP gene expression was compared between wild-type and PU.1-null embryonic stem (ES) cells that were differentiated in vitro into cells that resembled megakaryocytes both morphologically and immunologically. We found that PBP gene expression in the differentiated PU.1(-/-) null ES cells (as determined by semi-quantitative reverse transcriptase-polymerase chain reaction) was more than four times lower than that in the wild-type ES cells, while other platelet-specific genes were expressed equally or similarly in the two ES cell lines. Previous reports have shown that PU.1 is expressed in several hematopoietic lineages, including megakaryocytes. However, the functional role of PU.1 has only been previously demonstrated in the myeloid and lymphoid lineages. Therefore, our studies are the first to show the biological importance of this nuclear factor in the regulated expression of a megakaryocyte-specific gene.
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PMID:Activation of the megakaryocyte-specific gene platelet basic protein (PBP) by the Ets family factor PU.1. 933 92

Signals from transforming growth factor-beta (TGF-beta), a bifunctional regulator of the proliferation of hematopoietic progenitor cells, have been recently shown to be transduced by five novel human genes related to a Drosophila gene termed MAD (mothers against the decapentaplegic gene). We showed by reverse transcriptase polymerase chain reaction that the RNA from one homologue gene, Smad5, was present in the immortalized myeloid leukemia cell lines, KG1 and HL60, in bone marrow mononuclear and polymorphonuclear cells, as well as in purified CD34+ bone marrow cells. Therefore, we studied the role of this gene in the regulation of human hematopoiesis by TGF-beta. TGF-beta1 and TGF-beta2 significantly inhibited myeloid, erythroid, megakaryocyte, and multilineage colony formation as assayed in semisolid culture systems. The levels of Smad5 mRNA in CD34+ cells were decreased by antisense but not sense oligonucleotides to Smad5. Preincubation of CD34+ marrow cells with two sense oligonucleotides to Smad5 did not reverse the inhibitory effects of TGF-beta on hematopoietic colony formation. However, preincubation with two antisense oligonucleotides to Smad5 reversed the inhibitory effects of TGF-beta. These data show that the Smad5 gene is involved in the signaling pathway by which TGF-beta inhibits primitive human hematopoietic progenitor cell proliferation and that Smad5 antisense oligonucleotides can interrupt this signal.
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PMID:The Smad5 gene is involved in the intracellular signaling pathways that mediate the inhibitory effects of transforming growth factor-beta on human hematopoiesis. 949 Jun 74

Platelet factor XI is associated with the platelet plasma membrane and has an apparent Mr (220,000 nonreduced, 55,000 reduced) different from that of plasma factor XI. However, the site of synthesis and the nature of platelet factor XI are not known. Using reverse transcriptase polymerase chain reaction, 12 out of 13 exons (all except exon V) coding for mature plasma factor XI were amplified from human platelet mRNA. The sequence of each of these exons was identical to that of plasma factor XI. In situ amplification and hybridization of factor XI mRNA was positive for exon III and negative for exon V in platelets and negative for both exons in other blood cells. By Northern hybridization, a factor XI mRNA transcript of approximately 1.9 kilobases was detected in megakaryocytic cells, and one of approximately 2.1 kilobases was detected in liver cells. Factor XI cDNA was cloned from a megakaryocyte library and sequenced. Exon V was absent, and the splicing of exon IV to exon VI maintained the open reading frame without alteration of the amino acid sequence except for the deletion of amino acids Ala91-Arg144 within the amino-terminal portion of the Apple 2 domain. Thus, platelet factor XI is an alternative splicing product of the factor XI gene, localized to platelets and megakaryocytes but absent from other blood cells.
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PMID:Molecular cloning of platelet factor XI, an alternative splicing product of the plasma factor XI gene. 959 22

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

Megakaryocytic differentiation of progenitor cells was investigated in nine patients with low-risk myelodysplastic syndromes (MDS) (eight refractor anemia [RA] and one RA with ringed sideroblasts [RARS] and five patients with high-risk MDS (two RA with excess of blasts [RAEB] and three RAEB in transformation [RAEB-T]). Bone marrow-derived CD34+ cells were enriched to a purity of 87% +/- 2% (mean +/- SEM) and assayed in short-term suspension cultures in the presence of 10 ng/mL of PEGylated recombinant human megakaryocyte (MK) growth and development factor (PEG-rHuMGDF) and in addition to 50 ng/mL stem cell factor and 10 ng/mL interleukin-3. Cells of the megakaryocytic lineage were identified by flow cytometric analysis of CD42b (GP1b) and mature MKs by morphologic criteria. Transcription of c-mpl receptor-specific mRNA in the CD34+ cells of these patients was investigated by full-length reverse transcriptase polymerase chain reaction of the p form of c-mpl as well as of the alternative splice product c-mpl k. CD34+ cells from seven healthy bone marrow donors served as controls. Differentiation along the MK pathway was stimulated in five patients with RA. C-mpl mRNA was expressed in the CD34+ cells in all cases. In three low-risk patients the capacity for in vitro MK growth was absent or minimal even though mRNA for c-mpl receptor was detected in the CD34+ cells of this group as well. In patients with high-risk MDS, PEG-rHuMGDF stimulated in vitro MK growth from CD34+ cells in only one of five cases. As in the patients with low-risk MDS, c-mpl mRNA for both c-mpl p and c-mpl k splicing products was detected. These results indicate that the in vitro response to stimulation with c-mpl ligand discriminates between two groups of patients with low-risk MDS and that the observed defect in megakaryocytic development is unrelated to the level of c-mpl expression in both low-risk and high-risk MDS.
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PMID:Characterization of defective megakaryocytic development in patients with myelodysplastic syndromes. 1008

CD34(+) cells and megakaryocyte progenitors were lower in marrow from patients after hematological recovery from the first cycle of 5-fluorouracil, leucovorin, adriamycin, cyclophosphamide (FLAC) chemotherapy plus PIXY321 (GM-CSF/interleukin 3; IL-3 hybrid) than in FLAC + GM-CSF or pre-FLAC marrows. Marrow stromal layers, an in vitro model of the marrow microenvironment, express a combination of stimulatory and inhibitory factors that modulate hematopoietic progenitor cell proliferation and differentiation. The TaqMan assay and quantitative reverse transcriptase-polymerase chain reaction were used to measure monocyte chemoattractant protein-1 (MCP-1), melanoma stimulatory growth activity, and monokine inducible by interferon-gamma (Mig) (inhibitory chemokines for primitive or megakaryocyte progenitors) mRNA levels in in vitro PIXY and GM-CSF-treated and patient post-FLAC marrow stromal layers. Chemokine mRNA was increased after in vitro GM-CSF and to a lesser extent after PIXY treatment. MCP-1 mRNA levels were fivefold higher in FLAC + PIXY than in FLAC + GM-CSF layers, and Mig mRNA was elevated in FLAC + GM-CSF layers. Thrombopoietin (TPO), insulin-like growth factor I (IGF-I), and IGF-II (stimulatory factors for primitive and megakaryocyte progenitors) mRNA were also measured. TPO mRNA levels were 30% lower in GM-CSF and PIXY-pretreated than in control layers with no decrease in IGF mRNA. TPO mRNA in stromal layers of patients who developed grade 3 thrombocytopenia (platelets < 20 x 10(9)/l) during the third cycle of FLAC was only 24% of levels in stromal layers of marrow from other post-FLAC patients. Results demonstrate that patient and in vitro treatment had modulatory effects on TPO and chemokine mRNA expression in marrow stromal layers.
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PMID:Thrombopoietin and chemokine mRNA expression in patient post-chemotherapy and in vitro cytokine-treated marrow stromal cell layers. 1100 17


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