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:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A receptor tyrosine kinase (RTK), TIE (tyrosine kinase that contains immunoglobulin-like loops and epidermal growth factor [EGF] homology domains), is expressed in vascular endothelial and hematopoietic cells. We generated monoclonal antibodies (MoAbs) against the extracellular domain of TIE and a polyclonal antibody against the TIE carboxyterminus and used them to analyze expression of TIE in hematopoietic cells. Western blotting detected two forms of TIE protein with a molecular mass of 135 and 130 kD in hematopoietic and endothelial cells. Northern blotting analysis revealed that TIE was expressed preferentially in undifferentiated cell lines, especially when megakaryocytic, but not erythroid differentiation was induced. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed that TIE was predominantly expressed in the human hematopoietic progenitor fraction, CD34+ cells. Fluorescence-activated cell sorting (FACS) showed that 42% of CD34+ and 17% of KIT-positive (KIT+) cells were TIE-positive (TIE+). The majority (81%) of the primitive hematopoietic stem cells, CD34+CD38- cells, were TIE+. Assays of progenitor cells and long-term culture-initiating cells (LTC-IC) showed that the TIE+ fraction contained more primitive cells than the TIE- fraction. Some TIE+ cells were in the CD34- fraction, which were CD19+ and CD20+ (B cells). These findings indicate that TIE has a unique spectrum of expression in primitive hematopoietic stem cells and B cells. Although its ligand has not been identified, TIE and its ligand may establish a novel regulatory pathway not only in early hematopoiesis, but also in the differentiation and/or proliferation of B cells.
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PMID:Predominant expression of a receptor tyrosine kinase, TIE, in hematopoietic stem cells and B cells. 854 81

We have studied the actions of the proteinase-activated-receptor-2 (PAR2)-activating polypeptide, SLIGRL-NH2 (SLI-NH2), in rat aorta and in gastric longitudinal muscle preparations. In the phenylephrine-precontracted aorta preparation, SLI-NH2 caused an endothelium-dependent relaxation that mimicked the action of low concentrations (0.5 U/mL) of trypsin and that was blocked by the nitric oxide synthase inhibitor N omega-nitro-L-arginine methyl ester. In endothelium-free aorta ring preparation, SLI-NH2 caused neither a relaxation nor a contraction. In the gastric longitudinal muscle preparation, SLI-NH2 caused a transient contraction that mimicked the action of trypsin (0.5 U/mL) and that was sensitive to inhibitors of cyclooxygenase (indomethacin) and tyrosine kinase (genistein). Further, using a reverse-transcriptase - polymerase chain reaction (RT-PCR) approach we detected, in both assay tissues, mRNA for the rat PAR2 receptor, and we ascertained, using a cloned receptor cDNA obtained from a rat intestinal cDNA library, that the putative N-terminal activating peptide sequence of the rat PAR2 receptor (SLIGRL) is identical with the one previously cloned from murine tissue. We concluded that, like the thrombin receptor, the PAR2 receptor may play a pathophysiologic role in the regulation of vascular and gastric smooth muscle contractility.
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PMID:Detection of functional receptors for the proteinase-activated-receptor-2-activating polypeptide, SLIGRL-NH2, in rat vascular and gastric smooth muscle. 856 91

In neutrophils, binding and phagocytosis facilitate subsequent intracellular killing of microorganisms. Activity of Na+/H+ exchangers (NHEs) participates in these events, especially in regulation of intracellular pH (pHi) by compensating for the H+ load generated by the respiratory burst. Despite the importance of these functions, comparatively little is known regarding the nature and regulation of NHE(s) in neutrophils. The purpose of this study was to identify which NHE(s) are expressed in neutrophils and to elucidate the mechanisms regulating their activity during phagocytosis. Exposure of cells to the phagocytic stimulus opsonized zymosan (OpZ) induced a transient cytosolic acidification followed by a prolonged alkalinization. The latter was inhibited in Na+-free medium and by amiloride analogues and therefore was due to activation of Na+/H+ exchange. Reverse transcriptase PCR and cDNA sequencing demonstrated that mRNA for the NHE-1 but not for NHE-2, 3, or 4 isoforms of the exchanger was expressed. Immunoblotting of purified plasma membranes with isoform-specific antibodies confirmed the presence of NHE-1 protein in neutrophils. Since phagocytosis involves Fcgamma (FcgammaR) and complement receptors such as CR3 (a beta2 integrin) which are linked to pathways involving alterations in intracellular [Ca2+]i and tyrosine phosphorylation, we studied these pathways in relation to activation of NHE-1. Cross-linking of surface bound antibodies (mAb) directed against FcgammaRs (FcgammaRII > FcgammaRIII) but not beta2 integrins induced an amiloride-sensitive cytosolic alkalinization. However, anti-beta2 integrin mAb diminished OpZ-induced alkalinization suggesting that NHE-1 activation involved cooperation between integrins and FcgammaRs. The tyrosine kinase inhibitors genistein and herbimycin blocked cytosolic alkalinization after OpZ or FcgammaR cross-linking suggesting that tyrosine phosphorylation was involved in NHE-I activation. An increase in [Ca2+]i was not required for NHE-1 activation because neither removal of extracellular Ca2+ nor buffering of changes in [Ca2+]i inhibited alkalinization after OpZ or Fc-gammaR cross-linking. In summary, Fc-gammaRs and beta2 integrins cooperate in activation of NHE-1 in neutrophils during phagocytosis by a signaling pathway involving tyrosine phosphorylation.
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PMID:Na+/H+ exchange activity during phagocytosis in human neutrophils: role of Fcgamma receptors and tyrosine kinases. 860 83

We performed high-sensitivity flow cytometry and Western blotting to study the expression of the low-affinity NGF receptor (p75NGFR) and of the transmembrane tyrosine kinase (Trk) family of high-affinity receptors for the different neurotrophic factors on Epstein-Barr virus (EBV)-transformed human B lymphocytes. Reverse transcriptase (RT) polymerase chain reaction (PCR) with single and multiple sets of primers (multiplex RT-PCR) was used to survey the repertoire of neurotrophin receptor transcripts in this cell line. We demonstrated that transformed B cells express detectable levels of Trk b and its mRNA. Conversely, negative results were obtained for p75NGFR, Trk a, and Trk c. Exposure of EBV-transformed B lymphocytes to brain-derived neurotrophic factor (BDNF) triggered the phosphorylation of Trk b, as demonstrated by Western blots of cell lysates probed with monoclonal antibody against phosphotyrosine.
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PMID:Expression of high- and low-affinity neurotrophin receptors on human transformed B lymphocytes. 863 56

The c-abl proto-oncogene encodes a nuclear tyrosine kinase that can phosphorylate the mammalian RNA polymerase II (RNAP II) on its C-terminal repeated domain (CTD) in vitro. Phosphorylation of the CTD has previously been shown to require the tyrosine kinase and the SH2 domain of Abl. We show here that a CTD-interacting domain (CTD-ID) at the C-terminal region of c-Abl is also required. Deletion of the CTD-ID causes the Km 0.4 microM to increase by 2 orders of magnitude. Direct binding of the CTD-ID to CTD and to RNAP II could be demonstrated in vitro. Phosphorylation of a recombinant glutathione S-transferase-CTD by c-Abl was observed in cotransfected COS cells. Mutant Abl proteins lacking the CTD-ID, while capable of autophosphorylation, neither phosphorylated nor associated with the glutathione S-transferase-CTD in vivo. Transient overexpression of c-Abl also led to a four- to fivefold increase in the phosphotyrosine content of the RNAP II large subunit. Moreover, the large subunit of RNAP II could be coprecipitated with c-Abl. Tyrosine phosphorylation of the coprecipitated RNAP II was again dependent on the presence of the CTD-ID in Abl. Finally, the ability of c-Abl to phosphorylate and associate with RNAP II could be correlated with the enhancement of transcription by c-Abl in transient cotransfection assays. Taken together, these observations demonstrate that c-Abl can function as a CTD kinase in vitro as well as in vivo.
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PMID:Identification of a binding site in c-Ab1 tyrosine kinase for the C-terminal repeated domain of RNA polymerase II. 866 51

The breakpoints of the translocation t(2;5)(p23;q35) associated with Ki-1-positive anaplastic large cell lymphoma (Ki-1 ALCL) have recently been cloned. They involve a novel tyrosine kinase gene, ALK, at 2p23 and the nucleophosmin gene, NPM, at 5q35. Reverse transcriptase-polymerase chain reaction (RT-PCR) with NPM and ALK primers detects a consistent fusion product in Ki-1 ALCL cases that have the translocation. In the course of a survey of 15 cases of Ki-1 ALCL, we identified a single case with a slightly smaller NPM-ALK RT-PCR product, among 12 cases positive for this fusion RNA. Sequencing of this novel NPM-ALK RT-PCR product showed an in-frame junction of NPM to ALK, 30 bases distal to the usual ALK junction site, but at the usual NPM Junction site. The predicted chimeric protein in this case is thus shorter by 10 amino acids, but the putative ALK catalytic domain remains intact. PCR with ALK primers bracketing the novel fusion point, performed on either cDNA or genomic DNA, yielded the same product, confirming that this novel ALK fusion point was located within an exon. Hybridization analysis of the genomic junction fragment isolated by long-range DNA PCR suggested that the ALK genomic breakpoint was also exonic. Cloning and sequencing of the genomic breakpoint confirmed that the break occurred within the 5' portion of the ALK exon participating in the fusion junction, 28 bases 3' to the normal ALK exon boundary, resulting in the use of a cryptic splice acceptor site two bases distal to the breakpoint. This case demonstrates that, in translocations resulting in chimeric transcripts, genomic breakpoints may rarely lie within an exon, provided that the reading frame is maintained and no domains presumed critical to tumorigenesis are deleted.
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PMID:Molecular variant of the NPM-ALK rearrangement of Ki-1 lymphoma involving a cryptic ALK splice site. 872 82

1. The biological activities of the proteinase-activated receptor number 2 (PAR-2)-derived peptides, SLIGRL (PP6) SLIGRL-NH2 (PP6-NH2) and SLIGR-NH2 (PP5-NH2) were measured in mouse and rat gastric longitudinal muscle (LM) tissue and in a rat aortic ring preparation and the actions of the PAR-2-derived peptides were compared with trypsin and with the actions of the thrombin receptor activating peptide, SFLLR-NH2 (TP5-NH2). 2. From a neonatal rat intestinal cDNA library, and from intestinal and kidney-derived cDNA, the coding region of the rat PAR-2 receptor was cloned and sequenced, thereby establishing its close sequence identity with the previously described mouse PAR-2 receptor; and this information, along with a reverse-transcriptase (RT) polymerase chain reaction (PCR) analysis of cDNA derived from gastric and aortic tissue was used to establish the concurrent presence of PAR-2 and thrombin receptor mRNA in both tissues. 3. In the mouse and rat gastric preparations, the PAR-2-derived polypeptides, PP6, PP6-HN2 and PP5-NH2 caused contractile responses that mimicked the contractile actions of low concentrations of trypsin (5 u/ml-1; 10 nM) and that were equivalent to contractions caused by TP5-NH2. 4. The cumulative exposure of the rat LM tissue to PP6-NH2 led to a desensitization of the contractile response to this polypeptide, but not to TP5-NH2 and vice versa, so as to indicate a lack of cross-desensitization between the receptors responsive to the PAR-2 and thrombin receptor-derived peptides. 5. In the rat gastric preparation, the potencies of the PAR-2-activating peptides were lower than the potency of TP5-NH2 (potency order: TP5-NH2 > > PP6-NH2 > or = PP6 > PP5-NH2); PP6 was a partial agonist in this preparation. 6. The contractile actions of PP6 and PP6-NH2 in the rat gastric preparation required the presence of extracellular calcium, were inhibited by nifedipine and were blocked by the cyclo-oxygenase inhibitor, indomethacin and by the tyrosine kinase inhibitor, genistein, but not by the kinase C inhibitor, GF109203X. The contractile responses were not blocked by atropine, chlorpheniramine, phenoxybenzamine, propranolol, ritanserin or tetrodotoxin. 7. In a precontracted rat aortic ring preparation, with an intact endothelium, all of the PAR-2-derived peptides caused a prompt relaxation response that was blocked by the nitric oxide synthase inhibitor, N omega-nitro-L-arginine-methyl ester (L-NAME) but not by D-NAME; in an endothelium-free preparation, which possessed mRNA for both the PAR-2 and thrombin receptors, the PAR-2-activating peptides caused neither a relaxation nor a contraction, in contrast with the contractile action of TP5-NH2. The relaxation response to PP6-NH2 was not blocked by atropine, chlorpheniramine, genistein, indomethacin, propranolol or ritanserin. 8. In the rat aortic preparation, the potencies of PP6, PP6-NH2 and PP5-NH2 were greater than those of the thrombin receptor activating peptide, TP5-NH2 (potency order: PP6-NH2 > or = PP6 > PP5-NH2 > TP5-NH2). 9. In the rat aortic preparation, the relaxant actions of the PAR-2-derived peptides were mimicked by trypsin, at concentrations (0.5-1 u ml-1; 1-2 nM) lower than those that can activate the thrombin receptor. 10. The bioassay data obtained with the PAR-2 peptides and with trypsin, along with the molecular cloning/RT-PCR analysis, point to the presence of functional PAR-2 receptors that can activate distinct responses in the gastric and vascular smooth muscle preparations. These responses were comparable to those resulting from thrombin receptor activation in the same tissues, so as to suggest that the receptor for the PAR-2-activating peptides may play a physiological role as far reaching as the one proposed for the thrombin receptor.
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PMID:Rat proteinase-activated receptor-2 (PAR-2): cDNA sequence and activity of receptor-derived peptides in gastric and vascular tissue. 876 73

The breakpoints of the translocation t(2;5)(p23;q35) associated with Ki-1-positive anaplastic large-cell lymphoma (Ki-1 ALCL) involve a novel tyrosine kinase gene, ALK, at 2p23 and the nucleophosmin gene, NPM, at 5q35. Reverse transcriptase-polymerase chain reaction (RT-PCR) using NPM and ALK primers detects a consistent fusion product in Ki-1 ALCL cases with the translocation, resulting from genomic breakpoints within the same respective introns of NPM and ALK. To examine the feasibility of long-range DNA PCR with the same exonic NPM and ALK primers for the detection of the genomic NPM-ALK rearrangement, we examined 20 cases of Ki-1 ALCL previously characterized by NPM-ALK RT-PCR. Ten cases were positive for the NPM-ALK fusion RNA and 10 were negative. We first confirmed that both the NPM and ALK normal introns are relatively short, approximately 1 and 2 kb, respectively, suggesting that the largest possible size for the chimeric NPM-ALK intron would be about 3 kb. All 10 cases positive by RT-PCR were also positive by long-range DNA PCR. The DNA PCR products ranged, as expected, from the sizes of the normal introns, between 0.5 and 2.5 kb. All 10 RT-PCR-negative cases were also negative by long-range DNA PCR, and control templates for RT-PCR and long-range DNA PCR were successfully amplified. Thus, we have shown that the introns involved by the NPM-ALK rearrangement seen in some Ki-1 lymphomas are relatively short, making the genomic rearrangement amenable to reliable detection by long-range DNA PCR. Furthermore, the variability observed in the sizes of chimeric introns in evidence against clustering of the genomic breakpoints within these introns.
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PMID:Detection of the NPM-ALK genomic rearrangement of Ki-1 lymphoma and isolation of the involved NPM and ALK introns. 886 27

Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen with potent angiogenic and vascular permeability-inducing properties, both of which may be important for the function of islets of Langerhans. In this study, we have examined the expression of VEGF and its tyrosine kinase receptors (flt and flk-1) in isolated rat islets of Langerhans in vitro. When analyzed by in situ hybridization, islet tissue showed a significant 4.6-fold increase in VEGF mRNA expression over time in culture from 0 to 7 days. Islet tissue exposed to hypoxic/anoxic conditions for a period of 8 hr showed a 3.7-fold increase in VEGF mRNA when analyzed by Northern blot hybridization. Reverse transcriptase-polymerase chain reaction revealed the presence of both flt and flk-1 in freshly isolated islets, and two VEGF isoforms, namely VEGF120 and VEGF164. Three rodent beta-cell lines derived from insulinomas (RINm5F-2A, INS-1, and MIN6) were also found to express VEGF by Northern blot hybridization. However, neither hypoxia/anoxia nor low (0.3 g/L)- or high (3.0 g/L)-glucose culture conditions modulated their expression of VEGF. VEGF derived from RINm5F-2A cells was bioactive in a three-dimensional in vitro model of angiogenesis, which assays for endothelial cell invasion and capillary morphogenesis. These findings demonstrate, first, that devascularization increases VEGF expression in isolated islet tissue, and they point to VEGF as a potentially important endogenous angiogenic stimulus for subsequent revascularization in vivo. Second, our observations raise the possibility that survival of transplanted islets may be improved by increasing VEGF expression before transplantation.
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PMID:Vascular endothelial growth factor is increased in devascularized rat islets of Langerhans in vitro. 903 36

The largest subunit of RNA polymerase II contains a C-terminal repeated domain (CTD) that is the site of phosphorylation by serine (threonine) and tyrosine kinases. Phosphorylation of the CTD is correlated with transcription elongation. A number of different kinases have previously been shown to phosphorylate the CTD; among them is a nuclear tyrosine kinase encoded by the c-abl proto-oncogene. The processive and high stoichiometric phosphorylation of RNA polymerase II by c-Abl requires the tyrosine kinase, the SH2 domain, and a CTD-interacting domain (CTD-ID) in the Abl protein. The physiological tyrosine phosphorylation of RNA polymerase II by c-Abl in DNA damage response has previously been demonstrated. Basal tyrosine phosphorylation of RNA polymerase II, however, is observed in cells derived from abl-deficient mice, indicating the existence of other CTD tyrosine kinases. In this report, we show that the tyrosine kinase encoded by an Abl-related gene (Arg) also phosphorylates the CTD in vitro and in transfected cells. The SH2 and kinase domain of Arg are 95% identical to that of c-Abl. However, these two proteins share only 29% identity in the large C-terminal region. Interestingly, a CTD-ID is also found in the C-terminal region of Arg. Mapping studies and sequence analysis have led to the identification of the CTD-ID that is highly conserved among the divergent C-terminal regions of Abl and Arg. These results indicate that tyrosine phosphorylation of RNA polymerase II CTD could be catalyzed by either c-Abl or Arg kinase.
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PMID:Tyrosine phosphorylation of RNA polymerase II carboxyl-terminal domain by the Abl-related gene product. 922 69


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