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:2.7.10.1 (ERK)
95,504 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cultured macrophages exhibit spreading in response to external stimuli. It is relevant to in vivo morphologic changes of macrophages during extravasation, migration, and differentiation. The present study was performed to elucidate molecular mechanisms that regulate spreading of macrophages. Redox is a crucial factor that modulates a wide range of cell function. We found that macrophages undergo spreading in response to oxidant stress caused by hydrogen peroxide or an oxidant generating agent menadione. To identify signaling pathways involved, a role of mitogen-activated protein (MAP) kinases was investigated. Western blot analysis showed that treatment of macrophages with menadione rapidly induced phosphorylation of extracellular signal-regulated kinases (ERK1, ERK2) and p38 MAP kinase, but not c-Jun N-terminal kinase (JNK). Pharmacologic inhibition of either ERK or p38 activation blunted the macrophage spreading. Similarly, transfection with dominant-negative mutants of ERKs or a mutant p38 significantly suppressed the oxidant-triggered spreading. ERKs and p38 are known to activate serum response element (SRE) via phosphorylation of the ternary complex factor Elk-1. To further identify downstream events, we focused on a role of SRE. Stimulation of macrophages with menadione induced activation of SRE. Intervention in the SRE activation by a dominant-negative mutant of Elk-1 inhibited the menadione-induced spreading. These results suggest that oxygen radical metabolites, the well-known mediators for tissue injury, incite spreading of macrophages via the MAP kinase-SRE signaling pathways.
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PMID:Oxidant stress incites spreading of macrophages via extracellular signal-regulated kinases and p38 mitogen-activated protein kinase. 975 78

The KIT protein is a receptor tyrosine kinase of the platelet derived growth factor (PDGF) receptor family which regulates haematopoiesis, melanogenesis and gut and germ cell development. KIT regulates these diverse processes, at least in part, by inhibiting apoptosis. We have previously found that KIT can suppress p53-mediated apoptosis. The mechanism by which KIT suppresses apoptosis is, however, uncharacterized. Neither is it clear how p53 induces apoptosis. In this report we find that p53-dependent apoptosis proceeds through a pathway involving depolarization of the mitochondrial electropotential gradient (delta(psi)m) and the generation of reactive oxygen species (ROS). KIT activation suppresses p53-induced apoptosis in the mouse DP16 Friend erythroleukemia cell line by preventing delta(psi)m depolarization and ROS generation. Thus, the KIT kinase prevents apoptosis by regulating mitochondrial function and cellular redox state.
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PMID:Inhibition of p53-dependent apoptosis by the KIT tyrosine kinase: regulation of mitochondrial permeability transition and reactive oxygen species generation. 979 94

The aim of this study was to evaluate the ex vivo expansion of normal CD34+ cells in gas-permeable polypropylene bags suitable for clinical use. Cells were cultured for 14 days in serum-free medium supplemented with SCF, IL3, IL6, FLT3-1, G-CSF + MGDF or Epo. The bags supported the expansion of hematopoietic cells in a similar manner to small scale well or flask systems, allowing mean expansions of up to 2193-fold for total nucleated cells, 140-fold for CFU-GM and 66-fold for LTC-IC. Increasing the initial cell concentration from 5 x 10(3) to 1 x 10(5)CD34+ cells/ml induced the production of granulocytic cells with terminal differentiation while simultaneously decreasing the overall extent of expansion of the white blood cells produced. We tested the phagocytic activity and oxidative metabolism of the white blood cells produced. The percentage of phagocytic cells was 39+/-0.5% in expanded cultures derived from fractions initiated at 5 x 10(3), 10(4) or 10(5) cells/ml and 45+/-6% in cultured cells obtained from starting fractions containing 5 x 10(4) cells/ml, as compared to 58+/-4% in normal controls. A study of the potential for oxygen-dependent microbe killing showed that the expanded cells produced H2O2, although in lesser quantities than control cells. We subsequently investigated the possibility of freezing expanded cells. Total cell recovery after thawing was 45+/-4%, while recoveries of progenitors and stem cells ranged from 65 to 90%, without any influence of the initial cell concentration. This new approach could be of major interest for clinical practice, as it would allow evaluation of the quality of a graft prior to its infusion and employs experimental conditions which meet the criteria for potential clinical use.
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PMID:Cell culture bags allow a large extent of ex vivo expansion of LTC-IC and functional mature cells which can subsequently be frozen: interest for a large-scale clinical applications. 981 1

In human heart transplantation limited myocardial ischemia duration remains one of the most restricting factors. A new approach towards prolongation of this duration is the combination of cardioplegic arrest and continuous Coronary Oxygen Persufflation (COP) with gaseous oxygen. This technique, which is based on former experiments, was applied in pig hearts which we transplanted orthotopically after a hypothermic preservation time of 14 hours. For cardioplegic arrest we used either Euro-Flush glutathion solution (EFG; n=5), University of Wisconsin solution (UW; n=5), modified Bretschneider HTK cardioplegic solution (mHTK; n=6). In preliminary experiments all three solutions had shown equal cardioprotective qualities. Hearts of the mHTK group were submitted to continuous COP during storage (mHTK+COP). After 14 hours of preservation and orthotopic transplantation the mHTK+COP hearts showed significantly improved cardiac functional recovery compared to hearts preserved by simple cold storage techniques. Hemodynamics measured after 3 hours reperfusion were significantly better in the mHTK+COP group compared to EFG and UW: dp/dtmax in % of baseline+/-standard deviation (SD): 85+/-22, 65+/-26, 36+/-15, CO in % of baseline: 68+/-13, 35+/-8, 39+/-8. Postoperative preload recruitable stroke work in the mHTK+COP hearts was: 51.4+/-23.1 mmHg compared to preoperative: 57.3+/-17.2. ATP of left-ventricular myocardium in the mHTK+COP group: 14.7+2.1 micromol/g dry weight was significantly higher compared to EFG: 10.3+/-4.5 and UW: 5.9+/-3.2. CK-MB in percent of CK in all groups showed no increase during postoperative reperfusion. This study suggests that COP may present an effective complement to cold storage techniques currently used in heart transplantation. Prior to clinical application further investigations regarding long-term survival and endothelial function are required.
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PMID:Coronary oxygen persufflation for long-term myocardial protection. 982 85

Early placental development occurs in an environment of relative hypoxia. Hypoxia promotes angiogenesis and up-regulates vascular endothelial growth factor (VEGF) expression while it down-regulates placenta growth factor (PIGF) that possess 53% homology with VEGF. Morphological studies show poor placental vascular development and an increase in the mitotic index of cytotrophoblasts in intrauterine growth restriction (IUGR). We hypothesized that the reported relatively high oxygen level in the intervillous space in contact with IUGR placental villi will limit angiogenesis by changes in VEGF and PIGF expression and function. Western immunoblot analysis demonstrates a diametric expression of PIGF and VEGF proteins throughout pregnancy with PIGF levels increasing and VEGF levels decreasing, consistent with placental oxygenation. In IUGR placentae, the ratio of PIGF/GAPDH mRNA was increased by 2.3-fold (p < 0.03) and PIGF protein levels were also increased, (p < 0.05) as compared with gestationally-matched normal placentae. PIGF mRNA and protein were localized to the trophoblast bilayer and villous mesenchyme of the human placenta throughout gestation. In vitro studies demonstrated that increasing oxygen tension (hyperoxia) up-regulated PIGF protein in term placental villous explants, whereas hypoxic culture of a term trophoblast choriocarcinoma cell line (BeWo) down-regulated PIGF mRNA and protein and VEGFR-1 (Flt-1) autophosphorylation. The addition of PIGF-1 to a spontaneously transformed first trimester cytotrophoblast cell line stimulated DNA synthesis while PIGF-2 had little effect. VEGF and PIGF exert their biological actions by means of a common receptor VEGFR-1. In the first trimester trophoblast cells, PIGF-1 increased the association of phosphorylated extracellular signal-related kinase (ERK) with VEGFR-1 immunoprecipitates while both PIGF-1 and PIGF-2 also potentiated endogenous VEGF mediated association of phosphorylated extracellular related kinase (ERK) with VEGFR-2 (KDR). More importantly, the addition of PIGF-1 had little effect while PIGF-2 inhibited cell growth in cultured endothelial cells derived from human umbilical vein. Nitric oxide (NO) is reported to promote angiogenesis and PIGF-2 inhibited the basal release of NO from the first trimester trophoblast. The tissue expression and functional studies support the hypothesis of "placental hyperoxia" in early-onset IUGR because hypoxia down-regulates trophoblast PIGF levels, PIGF expression is increased in IUGR, and PIGF-2 inhibits endothelial cell growth. Taken together, these changes provide a cellular explanation for the observed poor angiogenesis in the pathogenesis of IUGR and show that the two PIGF isoforms may modulate trophoblast and endothelial cell function differently, possibly through potentiation of VEGF mediated activation of VEGF-2.
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PMID:Hypoxia down-regulates placenta growth factor, whereas fetal growth restriction up-regulates placenta growth factor expression: molecular evidence for "placental hyperoxia" in intrauterine growth restriction. 1006 4

The pupillary membrane (PM) is a transient ocular capillary network, which can serve as a model system in which to study the mechanism of capillary regression. Previous work has shown that there is a tight correlation between the cessation of blood flow in a capillary segment and the appearance of apoptotic capillary cells throughout the segment. This pattern of cell death is referred to as synchronous apoptosis (Lang, R. A., Lustig, M., Francois, F., Sellinger, M. and Plesken, H. (1994) Development 120, 3395-3404; Meeson, A., Palmer, M., Calfon, M. and Lang, R. A. (1996) Development 122, 3929-3938). In the present study, we have investigated whether the cause of synchronous apoptosis might be a segmental deficiency of either oxygen or a survival factor. Labeling with the compound EF5 in a normal PM indicated no segmental hypoxia; this argued that oxygen deprivation was unlikely to be the cause of synchronous apoptosis. When rat plasma was used as a source of survival factors in an in vitro PM explant assay, inhibition of vascular endothelial growth factor (VEGF) all but eliminated the activity of plasma in suppressing apoptosis. This argued that VEGF was an important plasma survival factor. Furthermore, inhibition of VEGF in vivo using fusion proteins of the human Flk-1/KDR receptor resulted in a significantly increased number of capillaries showing synchronous apoptosis. This provides evidence that VEGF is necessary for endothelial cell survival in this system and in addition, that VEGF deprivation mediated by flow cessation is a component of synchronous apoptosis.
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PMID:VEGF deprivation-induced apoptosis is a component of programmed capillary regression. 1006 34

Exposure to high levels of inspired oxygen leads to respiratory failure and death in many animal models. Endothelial cell death is an early finding, before the onset of respiratory failure. Vascular endothelial growth factor (VEGF) is highly expressed in the lungs of adult animals. In the present study, adult Sprague-Dawley rats were exposed to >95% FiO2 for 24 or 48 hours. Northern blot analysis revealed a marked reduction in VEGF mRNA abundance by 24 hours, which decreased to less than 50% of control by 48 hours. In situ hybridization revealed that VEGF was highly expressed in distal airway epithelial cells in controls but disappeared in the oxygen-exposed animals. Immunohistochemistry and Western blot analyses demonstrated that VEGF protein was decreased at 48 hours. TUNEL staining demonstrated the presence of apoptotic cells coincident with the decline in VEGF. Abundance of VEGF receptor mRNAs (Flt-1 and KDR/Flk) decreased in the late time points of the study (48 hours), possibly secondary to the loss of endothelial cells. We speculate that VEGF functions as a survival factor in the normal adult rat lung, and its loss during hyperoxia contributes to the pathophysiology of oxygen-induced lung damage.
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PMID:Exposure to hyperoxia decreases the expression of vascular endothelial growth factor and its receptors in adult rat lungs. 1007 60

While post-transplant pancreatitis is still a frequently occurring complication of whole pancreas transplantation, dysfunction of the endocrine tissue is rarely observed. Given that microcirculatory disorders play a major role in the pathogenesis of pancreatitis, we hypothesized a dissociation of endocrine and exocrine microvascular control in pancreas transplantation (cold ischemia-reperfusion) and studied this dissociation quantitatively, analyzing the pancreatic microcirculation after heterotopic isogeneic pancreaticoduodenal transplantation in rats by means of fluorescence microscopy. Functional capillary density (FCD) of both exocrine and endocrine tissue of pancreatic grafts after 1 h of cold storage in HTK solution did not differ when compared to sham-operated, time-matched controls. Intermittent capillary perfusion, which is absent under sham control conditions and which is proposed to be operative as a compensatory mechanism to counteract malperfusion, was observed in 52% of the exocrine, but in only 8% of the endocrine, tissue studied (p < 0.05). In contrast, cold storage of pancreatic grafts for 6 h in HTK resulted in a complete loss of intermittent capillary perfusion in exocrine tissue and, consequently, marked exocrine perfusion failure (decrease in FCD), while FCD of pancreatic endocrine tissue was preserved without any significant change in the incidence of intermittent capillary perfusion. Thus, our results indicate a higher susceptibility of the exocrine pancreas to cold ischemia/reperfusion events that is associated with significant alterations in nutritive perfusion and, thus, with limitations of the oxygen supply to the tissue. This may lead to inflammatory tissue reactions in the clinical setting of pancreas transplantation.
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PMID:Exocrine, but not endocrine, tissue is susceptible to microvascular ischemia/reperfusion injury following pancreas transplantation in the rat. 1008 Apr 6

Using a pharmacophore model for ATP-competitive inhibitors interacting with the active site of the EGFR protein tyrosine kinase together with published X-ray crystal data of quercetin (2) in complex with the Hck tyrosine kinase and of deschloroflavopiridol (3b) in complex with CDK2, a putative binding mode of the isoflavone genistein (1) was proposed. Then, based on literature data suggesting that a salicylic acid function, which is represented by the 5-hydroxy-4-keto motif in 1, could serve as a pharmacophore replacement of a pyrimidine ring, superposition of 1 onto the potent EGFR tyrosine kinase inhibitor 4-(3'-chlorophenylamino)-6, 7-dimethoxyquinazoline (4) led to 3'-chloro-5,7-dihydroxyisoflavone (6) as a target structure which in fact was 10 times more potent than 1. The putative binding mode of 6 suggests a sulfur-aromatic interaction of the m-chlorophenyl moiety with Cys 773 in the "sugar pocket" of the EGFR kinase model. Replacement of the oxygen in the chromenone ring of 6 by a nitrogen atom further improved the inhibitory activity against the EGFR kinase. With IC50 values of 38 and 8 nM, respectively, the quinolones 11 and 12 were the most potent compounds of the series. N-Alkylation of 11 did not further improve enzyme inhibitory activity but led to derivatives with cellular activity in the lower micromolar range.
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PMID:Use of a pharmacophore model for the design of EGFR tyrosine kinase inhibitors: isoflavones and 3-phenyl-4(1H)-quinolones. 1009 Jul 85

The paradigm for the response to hypoxia is erythropoietin gene expression; activation of hypoxia-inducible factor-1 (HIF-1) results in erythropoietin production. Previously, we found that oxygen deprivation induced tissue factor, especially in mononuclear phagocytes, by an early growth response (Egr-1)-dependent pathway without involvement of HIF-1 (Yan, S.-F., Zou, Y.-S., Gao, Y., Zhai, C., Mackman, N., Lee, S., Milbrandt, J., Pinsky, D., Kisiel, W., and Stern, D. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8298-8303). Now, we show that cultured monocytes subjected to hypoxia (pO2 approximately 12 torr) displayed increased Egr-1 expression because of de novo biosynthesis, with a approximately 10-fold increased rate of transcription. Transfection of monocytes with Egr-1 promoter-luciferase constructs localized elements responsible for hypoxia-enhanced expression to -424/-65, a region including EBS (ets binding site)-SRE (serum response element)-EBS and SRE-EBS-SRE sites. Further studies with each of these regions ligated to the basal thymidine kinase promoter and luciferase demonstrated that EBS sites in the element spanning -424/-375 were critical for hypoxia-enhanceable gene expression. These data suggested that an activated ets factor, such as Elk-1, in complex with serum response factor, was the likely proximal trigger of Egr-1 transcription. Indeed, hypoxia induced activation of Elk-1, and suppression of Elk-1 blocked up-regulation of Egr-1 transcription. The signaling cascade preceding Elk-1 activation in response to oxygen deprivation was traced to activation of protein kinase C-betaII, Raf, mitogen-activated protein kinase/extracellular signal-regulated protein kinase kinase and mitogen-activated protein kinases. Comparable hypoxia-mediated Egr-1 induction and activation were observed in cultured hepatoma-derived cells deficient in HIF-1beta and wild-type hepatoma cells, indicating that the HIF-1 and Egr-1 pathways are initiated independently in response to oxygen deprivation. We propose that activation of Egr-1 in response to hypoxia induces a different facet of the adaptive response than HIF-1, one component of which causes expression of tissue factor, resulting in fibrin deposition.
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PMID:Hypoxia-associated induction of early growth response-1 gene expression. 1032 6


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