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)

In order to gain further understanding of the physiology of basic fibroblast growth factor (FGF-beta) in the mammalian reproductive tract, the expression of FGF-beta mRNA in the rat and porcine ovary has been examined by in situ hybridization and quantitative reverse transcriptase-polymerase chain reaction techniques during different stages of the estrus cycle. The results confirm that an increase in FGF-beta mRNA levels occurs over the course of the estrus cycle. No FGF-beta gene expression was detected during diestrus, the non-hormonal phase of the cycle, or at the early proestrus stage of the cycle. During late proestrus and estrus, FGF-beta mRNA was predominantly localized to granulosa cells of the dominant follicles, and to a lesser extent, to secondary antral follicles not committed to ovulation. These cells also expressed FGF-beta mRNA during this phase of follicular development, albeit in low abundance. During metestrus, after ovulation, in the newly formed corpora lutea FGF-beta mRNA levels were maximal, however on entering the next cycle commencing at diestrus, no FGF-beta mRNA was observed in the degenerating corpora lutea. These results indicate that expression of the FGF-beta gene is differentially regulated during the estrus cycle. The biological significance of this expression and the potential role of FGF-beta in local intra-ovarian regulation of the repetitive cycles of follicular differentiation, proliferation and maturation associated with ovarian revascularization are discussed.
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PMID:Studies on basic fibroblast growth factor (FGF-beta) gene expression in the rat and pig ovary using in situ hybridization and quantitative reverse transcriptase--polymerase chain reaction techniques. 137 33

We have previously shown that basic fibroblast growth factor (bFGF) stimulates megakaryocytopoiesis and granulopoiesis in vitro and that normal haematopoietic cells and several leukaemic cell lines express FGF receptors. In this paper, we demonstrate by reverse transcriptase-mediated polymerase chain reaction (RT-PCR) that bFGF mRNA is expressed in two leukaemia cell lines with megakaryocytic features (Meg-01 and K562), in two lymphocytic cell lines (Hut 78 and CA) and in normal human peripheral blood mononuclear cells. In addition, the conditioned media of Meg-01, but not Dami, contained a potent fibroblast-stimulating activity which could be neutralized by bFGF antibodies. Furthermore, bFGF antibody significantly inhibited the autocrine growth of Meg-01 cells in vitro. However, we could not detect cell-associated 18 kDa bFGF or HMW bFGF by immunofluorescence, immunoprecipitation or Western blotting. These data indicate that bFGF is expressed by certain haematopoietic cells and support further a role of this FGF prototype in haematopoiesis.
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PMID:Constitutive and selective expression of basic fibroblast growth factor in human leukaemia cell lines. 754 90

Heparan sulfate proteoglycans are thought to be obligatory for receptor binding and subsequent mitogenic activity of basic fibroblast growth factor (FGF-2). In a previous study (Nurcombe V., Ford, M. D., Wildschut, J., Bartlett, P. F. (1993) Science 260, 103-106) we have shown that primary cultures of mouse neuroepithelial cells and a cell line derived from then, 2.3D, secrete a heparan sulfate proteoglycan with a high affinity for FGF-2. In this study, a combination of affinity chromatography and gel chromatography was used to further isolate heparan sulfate side chains with high affinity for FGF-2. These active chains had an average molecular weight of 18,000-20,000. In order to determine whether heparan sulfate chains with specificity for FGF-2 also displayed selectivity for the different FGF receptors, peptides designed to the heparin-binding region of the receptors were used in competitive inhibition studies. The structure of the predicted heparin-binding domain of the FGF receptor 1 was modeled on the basis of its presumed secondary and tertiary structure homology with immunoglobulin loops. These results suggested that many of the basic residues within the second immunoglobulin loop of the FGF receptor 1 form a basic domain in the molecule and therefore form part of a heparin-binding site. Peptides homologous to this region of FGF receptor 1 were shown to inhibit mitogenesis in 2.3D cells, while those to FGF receptor types 2, 3, and 4 did not. A reverse transcriptase-polymerase chain reaction assay designed to detect expression of the four FGF receptors types demonstrated that FGF receptors 1 and 3 were present on the 2.3D cell line but that receptors 2 and 4 were not. These findings indicate that unique heparan sulfate domains interact with specific cell-surface receptors to direct cellular responses.
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PMID:Heparan sulfates mediate the binding of basic fibroblast growth factor to a specific receptor on neural precursor cells. 755 20

We have measured the amount of fibroblast growth factor 1 (FGF-1) mRNA and protein in primary breast cancers and non-malignant breast tissue and have found greatly reduced levels in breast cancer compared with non-malignant tissue. A total of 116 breast cancers and 37 biopsies taken from non-malignant breast were compared for FGF-1 mRNA expression using reverse transcriptase-polymerase chain reaction (RT-PCR) and significantly lower levels were found in the cancer tissues (P < 0.001). These findings were confirmed at the protein level where four out of five breast cancers contained no detectable FGF-1 and a fifth cancer had a low level of FGF-1 compared with three samples from reduction mammoplasties. Similar results were obtained from breast cell lines in which 80% of cancer cell lines had very low levels of FGF-1, whereas all non-malignant breast cell lines contained higher levels of FGF-1. Immunohistochemical analysis indicated that FGF-1 was present in the luminal epithelial cells of the non-malignant breast but was absent from cancer cells. The decreased levels of FGF-1 in breast cancer may indicate that stimulation of cancer cells is resulting in down-regulation of FGF-1 expression or may implicate FGF-1 as a differentiation factor rather than a growth factor at its physiological concentration in the breast.
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PMID:Expression of fibroblast growth factor 1 is lower in breast cancer than in the normal human breast. 851 54

Previous studies have indicated that growth factors such as epidermal growth factor, transforming growth factor alpha, and fibroblast growth factor 1 (FGF-1) have important regulatory functions in murine urothelial wound healing and tumorigenesis. Immunocytochemical analyses suggest that these factors are also involved in human urothelium. Yet, little is known about the functional effects of these growth factors on human urothelial cells. We established organoid-like primary cultures of normal human urothelium on porous membranes. Direct functional effects of growth factors were examined on confluent cultures reflecting intact urothelium. Immunocytochemistry was performed with a panel of specific antibodies against growth factors and their receptors on both cultures and the corresponding tissue sections. Lacking the appropriate antibodies, we performed reverse transcriptase PCR to detect FGF receptor mRNA in cultures and dissected tissue. The proliferation was stimulated by transforming growth factor alpha, FGF-1, and weakly by FGF-7, but not by FGF-2. TGF beta 1 inhibited proliferation. In contrast to mouse urothelium, none of the growth factors showed an effect on differentiation. The functional data correlate with the urothelial expression of epidermal growth factor receptors, TGF beta receptor types I and II, the (low) protein expression of FGF receptor 1, and the presence of FGF-7 receptor (FGF receptor 2 (IIIb)) mRNA. The organotypic nature of the cultures permits the study of growth factor interactions between urothelial cells. The data indicate that FGF-1, transforming growth factor alpha, and TGF beta 1 contribute differently to the maintenance of human urothelium.
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PMID:Functions of fibroblast and transforming growth factors in primary organoid-like cultures of normal human urothelium. 876 15

Glia cell line-derived neurotrophic factor (GDNF), a recently cloned member of the transforming growth factor-beta (TGF-beta) superfamily, has been implicated in the survival, morphological and functional differentiation of midbrain dopaminergic neurons and motoneurons in vitro and in vivo. The factor may thus have utility in the treatment of various human neurodegenerative disorders. Mechanisms regulating expression of GDNF in normal and diseased brain as a possible means to increase the local availability of GDNF are only beginning to be explored. We have established and employed a competitive reverse transcriptase-polymerase chain reaction (RT-PCR) to study and compare levels of expression of GDNF mRNA in several cell types and to investigate its regulation. GDNF expression was clearly evident in primary cultured astrocytes, the glioma B49 and C6 cell, but less pronounced in the Schwannoma RN22 cell lines. Little or no signal could be observed in neuroblastoma cell lines (IMR32, LAN-1) or the pheochromocytoma cell line PC12, emphasizing the glial character of this factor. Using the C6 cell line we found that fibroblast growth factor-2 (FGF-2; bFGF) can increase GDNF mRNA levels, whereas FGF-1, platelet-derived growth factor (PDGF), and vasoactive intestinal polypeptide (VIP) are apparently ineffective. Several other factors (forskolin, kainic acid, triiodothyronine dexamethasone, GDNF, TGF-beta 1, and interleukin-6) appear to have slightly negative effects on GDNF mRNA levels at the concentrations tested. To further explore the relationship between FGF-2 and GDNF, we also addressed the question whether GDNF, like FGF-2, may have an effect on C6 cell proliferation. We conclude that (1) glial and glial tumor cells, rather than neuronal cell lines, express GDNF, (2) that FGF-2 has a prominent inductive effect on GDNF expression and (3) that GDNF stimulates C6 cell proliferation. Finally, these data suggest that neurotrophic actions of FGF-2 in mixed glial-neuronal cell cultures might be mediated in part by GDNF.
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PMID:GDNF mRNA levels are induced by FGF-2 in rat C6 glioblastoma cells. 888 50

In mated sows, the level of placental vascularization has a direct effect on fetal growth and litter birth weight. Vascularization of the endometrium and uterus under the control of various polypeptide growth factors is an important early stage in this process. Basic fibroblast growth factor (FGF-2), a polypeptide distributed throughout the mesodermal and neuroectodermal tissues of many species, is a vascular endothelial cell mitogen in vitro and has been implicated in neovascularization and wound healing in vivo. As part of our studies of the distribution of FGF-2 in uterine tissue and its role in placental development and embryo implantation, the localization and changes in the abundance of porcine FGF-2 mRNA in the uterus of mated and unmated gilts were investigated by in situ hybridization procedures. These procedures were based on the use of [alpha35S]-dATP-labeled oligonucleotide probes and a novel set of digoxigenin-labeled oligonucleotide probes generated by reverse transcriptase-polymerase chain reaction (RT-PCR) methods and anti-sense labeling strategies from the corresponding mRNA templates. With these in situ hybridization procedures, porcine FGF-2 mRNA was localized during the first 30 days of pregnancy to specific tissue areas in the porcine uterus comprising glandular and luminal epithelial cells and stromal cells of both the stratum functionalis and stratum basalis regions of the endometrium, and within the smooth muscle of myometrium and the associated blood vessels. However, no significant increase in the level of FGF-2 mRNA within these tissues was detected during these early stages of pregnancy or during the estrous cycle of unmated gilts. These distribution and abundance patterns are only partially compatible with other recent observations suggesting a possible role for changing levels of the mature polypeptide form of FGF-2 in the reproductive tract of sows during the early stages of pregnancy.
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PMID:Localization of basic fibroblast growth factor mRNA (FGF-2 mRNA) in the uterus of mated and unmated gilts. 891 4

In the present investigation, oligonucleotide primers of high hybridisation stringency have been used in combination with optimised reverse transcriptase-polymerase chain reaction (RT-PCR) methods for the determination of the cDNA sequence corresponding to porcine FGF-2 mRNA present in brain and uterine tissue. Application of these optimised methods have overcome previous limitations associated with the low abundance of the porcine FGF-2 mRNA, and allowed as little as 100 micrograms of tissue to be employed to generate the complete cDNA nucleotide sequences as well as to provide specific template fragments selected for their suitability in subsequent ligation and mutagenesis studies with conventional expression vectors. Comparisons of the cDNA nucleotide and the deduced amino-acid sequence of porcine FGF-2 and the known FGF-2s from other species have indicated nucleotide sequence homologies of 95.5% with the bovine, 94.7% with the human and 88.7% with the rat FGF-2 cDNA whilst amino-acid sequence homologies of 100% with the bovine, 98.7% with the human and 96.8% with the rat FGF-2, respectively, were found. Based on these investigations, application of analogous strategies and methods with low abundance mRNAs related to other members of this family of growth factors, as well as very low abundance mRNAs of other protein growth factor, in the pig should now be readily realised.
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PMID:Application of an optimised reverse transcriptase-polymerase chain reaction method to determine the cDNA nucleotide sequence of porcine basic fibroblast growth factor. 902 66

Basic fibroblast growth factor (bFGF) and its specific receptors have diverse roles on a variety of cell types, such as the induction of vascular smooth-muscle cell proliferation which contributes to restenosis after coronary balloon angioplasty. bFGF is also known to interact with heparan sulphate proteoglycans present on the cell surface or in the extracellular matrix. In this study, the binding of 125I-bFGF to human aortic smooth-muscle cells was investigated. 125I-bFGF binding to these cells was reversible and saturable. Scatchard analysis revealed the presence of two distinct binding sites: a high-affinity receptor (Kd=38+/-7 pM; 1480+/-220 sites/cell) and a low-affinity non-saturable binding site (Kd=8. 0+/-2.0 nM). Pretreatment of the cells with heparinase resulted in a large reduction of 125I-bFGF binding to its low-affinity receptors, suggesting that they are heparin-like molecules. The specificity of the low- and high-affinity binding sites for bFGF was determined with acidic FGF, platelet-derived growth factor-BB and epidermal growth factor, which did not compete for 125I-bFGF binding. Expression of FGF receptor isoforms analysed by reverse transcriptase-PCR revealed the presence of only the type-1 receptor. Binding to low-affinity binding sites was antagonized by heparin, suramin, protamine sulphate and platelet factor 4. Unexpectedly, these molecules also reduced the binding of 125I-bFGF to its high-affinity sites. Consistent with these results, heparin, suramin, protamine sulphate and platelet factor 4 inhibited bFGF-induced proliferation of human aortic smooth-muscle cells. Heparin abrogated bFGF-induced release of tissue-type plasminogen activator by these cells. These observations suggest that the interaction of bFGF with human aortic smooth-muscle cells is different from that described for other cells such as endothelial cells, in which heparin acts as a potentiating factor of the mitogenic activity of bFGF.
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PMID:Heparin inhibits the binding of basic fibroblast growth factor to cultured human aortic smooth-muscle cells. 930 14

Basic fibroblast growth factor (FGF-2) plays an important role in myocardial growth and development and in particular cardiac myocyte proliferation. FGF-2 exerts its effects by binding to cell surface receptors (FGFR-1) of the tyrosine kinase family. We have detected the presence of both long and short isoforms of FGFR-1 in embryonic and adult mouse heart. In this report, we have examined the ability of long and short FGFR-1 isoforms to signal a mitogenic response. Assessment of RNA from rat myoblast H9c2 cells by reverse transcriptase-polymerase chain reaction and RNA blotting revealed that they were deficient in transcripts corresponding to long and short FGFR-1 species. Hybrid genes containing the cDNAs coding for long and short FGFR-1 isoforms directed by the myosin light chain-2 promoter and simian virus 40 enhancer sequences, were used to transiently transfect H9c2 cells. Total tyrosine phosphorylation was increased 2.0 and 2.6 fold in H9c2 cells transfected with the long and short FGFR-1 isoforms, respectively, compared to 'control' transfected H9c2 cells. This was accompanied by a 2.1 and 2.0 fold increase in DNA synthesis, as measured by tritiated thymidine incorporation, in H9c2 cells expressing the long and short FGFR-1 isoforms, respectively. To assess effects on proliferation, H9c2 cells were stably transfected with the myosin light chain-2/FGFR-1 cDNA genes. The rate of proliferation was increased 1.6 and 3.1 fold in H9c2 cells stably expressing the long and short FGFR-1 isoforms, respectively, compared to 'control' H9c2 cells. In contrast to non transfected H9c2 cells, treatment of H9c2 cells stably expressing long FGFR-1 with FGF-2 for 24 h resulted in a slight increase (1.3 fold, p < 0.02) in cell number. However, a greater response (1.5 fold, p < 0.0005) was observed with H9c2 cells stably expressing short FGFR-1 after treatment with FGF-2. These results suggest that both long and short FGFR-1 isoforms are capable of signalling a mitogenic response.
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PMID:Expression of fibroblast growth factor receptor-1 in rat heart H9c2 myoblasts increases cell proliferation. 940 49


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