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

Pluripotent hematopoietic stem cells (PHSCs) were highly enriched from mouse bone marrow by counterflow centrifugal elutriation, lineage subtraction, and fluorescence-activated cell sorting based on high c-kit receptor expression (c-kitBR). We used reverse transcriptase polymerase chain reaction to assay the c-kitBR subset and the subsets expressing low (c-kitDULL) and no (c-kitNEG) c-kit receptor for expression of mRNA encoding hematopoietic growth factor receptors and transcription factors. The c-kitBR cells had approximately 3.5-fold more c-kit mRNA than unfractionated bone marrow cells. The c-kitDULL cells had 47-58% of the c-kit mRNA found in c-kitBR cells and the c-kitNEG cells had 4-9% of the c-kit mRNA present in c-kitBR cells. By comparing mRNA levels in c-kitBR cells (enriched for PHSCs) with those of unfractionated bone marrow, we demonstrated that c-kitBR cells contained low or undetectable levels of mRNA for c-fms, granulocyte colony-stimulating factor receptor, interleukin 5 receptor (IL-5R), and IL-7R. These same cells had moderate levels of mRNA for erythropoietin receptor, IL-3R subunits IL-3R alpha (SUT-1), AIC-2A, and AIC-2B, IL-6R and its partner gp-130, and the transcription factor GATA-1 and high levels of mRNA for transcription factors GATA-2, p45 NF-E2, and c-myb. We conclude from these findings that PHSCs are programmed to interact with stem cell factor, IL-3, and IL-6 but not with granulocyte or macrophage colony-stimulating factor. These findings also indicate that GATA-2, p45 NF-E2, and c-myb activities may be involved in PHSC maintenance or proliferation.
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PMID:Pluripotent hematopoietic stem cells contain high levels of mRNA for c-kit, GATA-2, p45 NF-E2, and c-myb and low levels or no mRNA for c-fms and the receptors for granulocyte colony-stimulating factor and interleukins 5 and 7. 753 77

By using the reverse transcriptase (RT)-PCR and in situ hybridization we have studied the expression of mRNA for IL-5 and IL-4 in human lung mast cells induced by cross-linkage of high affinity Fc epsilon Rs. Lung mast cells were purified using affinity magnetic selection with mAb YB5.B8 against c-kit to achieve a final mast cell purity > 93%. Purified mast cells were precultured with stem cell factor (SCF) (10 ng/ml) and myeloma IgE (3 micrograms/ml) for 16 h before challenge with anti-IgE (1 or 10 micrograms/ml). IgE-dependent activation of lung mast cells caused expression of IL-5 mRNA, which was evident by 2 h and persisted for up to 48-72 h in all of 12 experiments, whereas IL-4 mRNA expression was of a shorter duration and was demonstrable in 6 of 13 experiments. We confirmed that mast cells, and not T cells, were the source of these cytokine messages by using reverse transcriptase-PCR in cell preparations containing known numbers of mast cells and T cells, in situ hybridization in enriched mast cell preparations, and double in situ hybridization-immunocytochemical staining. IL-5 mRNA expression did not require the pretreatment of cells with SCF, whereas expression of IL-4 mRNA seemed to require both anti-IgE and SCF. The strength of IL-5 mRNA signal was related to anti-IgE concentration. Immunoreactive IL-5 was detectable 8 h after anti-IgE challenge, and 10(6) mast cells generated a mean of 731 +/- 400 pg of IL-5 into the supernatant during 48-h culture, but no IL-4 product was detectable. These findings demonstrate the capacity of human lung mast cells to transcribe IL-4 and IL-5 after IgE-dependent activation and to synthesize and release immunoreactive IL-5.
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PMID:IgE-dependent expression of mRNA for IL-4 and IL-5 in human lung mast cells. 754 33

Ws/Ws rats have a small deletion at the tyrosine kinase domain of the c-kit gene, and practically no mast cells were detectable when the tissues were stained with alcian blue. Because alcian blue stains proteoglycans, there is a possibility that immature mast cells that do not contain a sufficient amount of proteoglycans are not detectable by this method. We examined this possibility by using other markers of mast cells. The histamine content in the skin of Ws/Ws rats was 0.3% that of control normal (+/+) rats. Because the number of alcian blue-positive mast cells in the skin of Ws/Ws rats was also 0.3% that of +/+ rats, histamine in the skin seemed to be concentrated to alcian blue-positive mast cells. Mast cells in the skin of +/+ rats express messenger RNA of Fc epsilon RI beta-subunit and c-kit protein. Because c-kit messenger RNA was normally expressed at least in the brain of Ws/Ws rats despite the small deletion, we examined the expression of Fc epsilon RI beta-subunit and c-kit messenger RNA in the skin and stomach of Ws/Ws rats by reverse transcriptase modification of polymerase chain reaction. Expression of either Fc epsilon RI beta-subunit or c-kit messenger RNA in the skin and stomach of Ws/Ws rats was estimated to be less than 1% that of +/+ rats. Moreover no Fc epsilon RI beta-subunit-expressing and no c-kit-expressing cells were detectable in the skin of Ws/Ws rats by in situ hybridization histochemistry. The present result suggests the absence of immature mast cells in tissues of Ws/Ws rats.
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PMID:Absence of immature mast cells in the skin of Ws/Ws rats with a small deletion at tyrosine kinase domain of the c-kit gene. 768 55

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

There is considerable evidence to suggest that polypeptide growth factors from either the oviduct or the endometrium can control preimplantation development of the mammalian embryo. These act directly through receptors expressed on the embryo. In addition, embryos also produce growth factors. The reverse transcriptase-polymerase chain reaction (RT-PCR) was used to determine the pattern of expression of mRNAs encoding several growth factor ligand and receptor genes throughout preimplantation development of cryopreserved human embryos. Transcripts encoding the receptor for c-fms, the receptor for colony-stimulating factor-1 (CSF-1), and c-kit (the receptor for stem cell factor [SCF]) were expressed throughout preimplantation development. Other growth factor ligand and receptor transcripts were expressed in a stage-specific manner: these included receptors for interleukin (IL)-6 (IL-6R), leukemia inhibitory factor (LIFR), tumor necrosis factor alpha (TNF alpha) (TNFRp80 and TNFRp60), and gp130. The transcripts for gp130 and the ligand SCF showed stage-specific splice variants. Blastocysts expressed a novel cDNA encoding gp130, which predicts a truncated form lacking the intracellular signaling domain. No expression of mRNAs encoding LIF, CSF-1, or the cloned receptor for platelet-activating factor was seen in any embryonic stage studied. We have shown that RT-PCR provides a sensitive and powerful method for identifying transcripts encoding growth factors and their receptors in single human embryos. The method is economical, allowing the expression pattern of many genes to be determined from a single embryo. These data are important in defining which cytokines may be involved in regulating human preimplantation development and when they may act.
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PMID:Stage-specific expression of cytokine and receptor messenger ribonucleic acids in human preimplantation embryos. 854 94

Expression of antigens coexpressed on cord blood (CB) CD34+ cells was evaluated by flow cytometry analysis and reverse transcriptase polymerase chain reaction (RT-PCR). Antigen expression was also comparatively analyzed by flow cytometry and limiting dilution (LD) RT-PCR to investigate effects of chymopapain on epitopes of several cell surface markers: LD RT-PCR allows detection of the expression of antigens degraded by chymopapain which are not identified by flow cytometry. Monoclonal antibodies (MoAbs) that recognize chymopapain resistant epitopes on several coexpressed cell surface markers were identified: these included MoAbs directed against CD11a, CD13, CD18, CD38, CD45RO, CD51, HLA-DR, Thy-1, c-kit, flt-3 (STK-1), and mdr-1. Interestingly, chymopapain treatment caused enhanced staining with MoAbs against HLA-DR, Thy-1, flt-3, mdr-1, and CD51. The frequency (LD RT-PCR) of CD18, CD38, Thy-1, and c-kit RT-PCR signals on pure sorted CD34+ CD18-, CD34+ CD38-, CD34+ Thy-1-, and CD34+ c-kit- cells, respectively, was similar in corresponding subsets treated or not with chymopapain. In contrast, the frequency of CD33 RT-PCR signals on sorted CD34+ CD33- cells was higher in chymopapain-treated samples than in untreated samples and thus confirmed at the transcriptional level that the epitope recognized by anti-CD33 is chymopapain sensitive. Our findings extend data on the phenotypic profile of CB CD34+ cells and show that several key cell surface markers of hematopoietic progenitor cells are chymopapain resistant. In addition, the results of the present study demonstrate that the RT-PCR can be applied to the analysis of multiple RNA species in small numbers of hematopoietic progenitor cells and show that LD RT-PCR allows the identification and frequency determination of rare cells which are undetectable by flow cytometry.
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PMID:Surface antigen expression on CD34+ cord blood cells: comparative analysis by flow cytometry and limiting dilution (LD) RT-PCR of chymopapain-treated or untreated cells. 887 54

Transgenic rats expressing the rat c-myc gene under the control of the human metallothionein II A promoter were produced. We found that the female transgenic rats were fertile, but that the male transgenic rats were sterile. Atrophy of the seminiferous tubules and depletion of sperm were observed in the sterile male testes. The expression of differential stage-specific mRNAs, including those of the c-kit receptor proto-oncogene, meiotic heat-shock protein 70 gene, acrosin gene, and transition protein 1 gene, was analyzed by the reverse transcriptase-polymerase chain reaction during spermatogenesis. The results suggested that spermatogenesis in these sterile rats were arrested at the prophase of meiosis in the primary spermatocytes. We found that apoptotic DNA fragmentation occurred in primary spermatocytes of the sterile transgenic rats. These results suggest that overexpression of the c-myc gene induces apoptosis at the prophase meiosis of the primary spermatocytes thereby causing male sterility in the c-myc transgenic rats.
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PMID:Overexpression of c-myc induces apoptosis at the prophase of meiosis of rat primary spermatocytes. 895 77

To clarify whether the expression of the WT1 gene in leukemic cells is aberrant or merely reflects that in normal counterparts, the expression levels of the WT1 gene were quantitated for normal hematopoietic progenitor cells. Bone marrow (BM) and umbilical cord blood (CB) cells were fluorescence-activated cell sorting (FACS)-sorted into CD34+ and CD34- cell populations, and the CD34+ cells into nine subsets (CD34+ CD33-, CD34+ CD33+, CD34+ CD38-, CD34+ CD38+, CD34+ HLA-DR-, CD34+ HLA-DR+, CD34+ c-kit(high), CD34+ c-kit(low), and CD34+ c-kit-) according to the expression levels of CD34, CD33, CD38, HLA-DR, and c-kit. Moreover, acute myeloid leukemic cells were also FACS-sorted into four populations (CD34+ CD33-, CD34+ CD33+, CD34- CD33+, and CD34- CD33-). FACS-sorted normal hematopoietic progenitor and leukemic cells and FACS-unsorted leukemic cells were examined for the WT1 expression by quantitative reverse transcriptase-polymerase chain reaction. The WT1 expression in the CD34+ and CD34- cell populations and in the nine CD34+ subsets of BM and CB was at either very low (1.0 to 2.4 x 10(-2)) or undetectable (< 10(-2)) levels (the WT1 expression level of K562 cells was defined as 1.0), whereas the average levels of WT1 expression in FACS-sorted and -unsorted leukemic cells were 2.4 to 9.3 x 10(-1). Thus, the WT1 expression levels in normal hematopoietic progenitor cells were at least 10 times less than those in leukemic cells. Therefore, we could not find any normal counterparts of BM or CB that expressed the WT1 at levels comparable with those in leukemic cells. These results indicate an aberrant overexpression of the WT1 gene in leukemic cells and imply the involvement of this gene in human leukemogenesis.
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PMID:Aberrant overexpression of the Wilms tumor gene (WT1) in human leukemia. 902 64


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