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:3.1.27.1 (RNase)
16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cardiopulmonary bypass is a planned support technique that results in a period of myocardial ischemia and reperfusion. In addition, it is associated with an inflammatory response likely involving endothelial cell activation. In previous studies, we showed that E-selectin and intercellular adhesion molecule-1 (ICAM-1) messenger ribonucleic acid (mRNA) are increased in human myocardium after cardiopulmonary bypass. We have now examined the expression of P-selectin mRNA by ribonuclease protection in paired atrial biopsy specimens from 12 patients before and after cardiopulmonary bypass. By means of immunocytochemistry, we have also examined the endothelial cell surface expression of P-selectin protein, as well as that of E-selectin and ICAM-1 in three additional patients. Patient ages ranged from 1 day to 8.5 years (median 12 months), and cardiopulmonary bypass times ranged from 46 to 196 minutes (median 144 minutes). By ribonuclease protection, there was marked variability in the expression of P-selectin in biopsy specimens before bypass. However, when compared with prebypass levels, P-selectin mRNA decreased modestly in 10 of 12 patients after bypass (median decrease 1.5-fold, p = 0.016). As seen with immunocytochemistry, P-selectin protein was distributed diffusely through the vascular bed on large vessels and small vessels before bypass but was virtually absent on capillaries in specimens taken after bypass. E-selectin, which was absent in prebypass biopsy specimens, was induced in one of the three specimens after bypass, but no change in ICAM-1 protein expression above baseline was noted. We also find that cultured human endothelial cells treated with tumor necrosis factor-alpha in doses which induce ICAM-1 mRNA simultaneously decrease their expression of P-selectin mRNA as compared with untreated cells. These observations suggest that endothelial P-selectin is transcriptionally downregulated after cardiopulmonary bypass at times when E-selectin and ICAM-1 are induced. Furthermore, we find that E-selectin and ICAM-1 are expressed at times and at sites where P-selectin is absent. Although it is possible that P-selectin may have been induced and lost at early times before reperfusion, these data suggest that endothelial P-selectin plays a limited role in the inflammatory response that ensues after cardiopulmonary bypass.
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PMID:P-selectin expression in myocardium of children undergoing cardiopulmonary bypass. 747 58

Protein disulfide isomerase (PDI) is an enzyme that catalyzes the formation as well as the isomerization of disulfide bonds. In this study, antibodies against PDI were used to show PDI antigen on the platelet surface by indirect immunofluorescence microscopy and by flow cytometry. The platelets were not activated, as evidenced by the absence of staining by an antibody against P-selectin. Permeabilized platelets showed little cytosolic PDI by indirect immunofluorescence microscopy, suggesting that the majority of platelet PDI is localized to the platelet surface. PDI activity against "scrambled" RNase was shown with intact platelets. The activity was inhibited by inhibitors of PDI and by an antibody against PDI. Other blood cells showed little PDI. Platelet surface PDI may play a role in the various physiological and pathophysiologic processes in which platelets are involved.
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PMID:Localization of protein disulfide isomerase to the external surface of the platelet plasma membrane. 766 65

P-selectin, an adhesion receptor for leukocytes, is synthesized selectively by megakaryocytes and endothelial cells. We have cloned the 5'-flanking region of the human P-selectin gene and conducted a preliminary analysis of its features. As determined by primer extension, RNase protection, and anchored polymerase chain reaction cloning, there were multiple transcriptional initiation sites from -95 to -25 nucleotides relative to the start of protein-coding sequence. Transfection of bovine aortic endothelial cells with serially truncated segments of the 5'-flanking region linked to luciferase indicated that the sequence from -249 to -13 was sufficient to promote high level gene expression. Deletions to -197, -147, and -128 gradually reduced expression to basal levels, and further deletion to -100 abolished expression. The sequence from -309 to -13 supported only basal luciferase expression in COS-7, 293, or HeLa cells. Putative regulatory elements in the short 5'-flanking sequence included a CACCC sequence, two inverted repeats similar to binding sites for the ETS and NF-kappa B/rel families, a GATA motif, and a sequence related to the GT-IIC element of the SV40 enhancer. The GATA element was functional, as it bound recombinant GATA-2, and mutations in the core sequence impaired both nuclear protein binding and gene expression. These data suggest that the P-selectin gene is regulated by a combination of cis elements and their cognate transcription factors.
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PMID:Characterization of the promoter for the human P-selectin gene. 769 74

P-selectin translocation to the surface of endothelial cells is increased after exposure to the nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME), resulting in increased endothelial adhesiveness. L-NAME (3 mM) was added to human cultured iliac vein endothelial cells for 1, 2, 4, and 6 h, and P-selectin mRNA expression was quantified by a ribonuclease protection assay. In parallel experiments, the NO donor, SPM-5185 (10 microM), was added to human iliac venous endothelial cells, and P-selectin mRNA expression quantified. P-selectin protein synthesis was quantified by Western blot analysis. L-NAME caused increased expression of P-selection RNA at 2-4 h, whereas D-NAME, the stereoisomer lacking NO synthase-inhibitory activity, had no effect. The stimulatory effect of L-NAME was reversed by addition of 3 mM L-arginine. SPM-5185 decreased P-selectin mRNA over the same time period (P < 0.02). The increased P-selectin mRNA expression induced by L-NAME was paralleled by an increase in P-selectin protein synthesis. The effects of SPM-5185 and L-arginine were also paralleled by decreases in P-selectin protein synthesis and in decreased adherence of human neutrophils to human iliac venous endothelial cells. The peak effect of inhibition of NO synthesis or addition of exogenous NO occurred at 2-4 h. These results suggest a regulatory effect of NO on endothelial P-selectin expression that modulates early leukocyte-endothelial cell interactions to preserve vascular homeostasis.
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PMID:Regulation of P-selectin expression in human endothelial cells by nitric oxide. 927 91

Murine traumatic shock is associated with increased adherence of neutrophils to the vascular endothelium resulting in neutrophil infiltration and tissue damage. We examined the effects of trauma on the expression of the adhesion molecule P-selectin in several vital organs (i.e., heart, lungs, liver, kidneys, and small intestine) 2 h after induction of Noble-Collip drum trauma in anesthetized rats. Total RNA was extracted from these organs and P-selectin mRNA was quantified by RNase protection assays. P-selectin mRNA was significantly increased over control nontraumatized, anesthetized rats in all vital organs (P<0.05 or less), with the largest increase occurring in the lung (P<0.01). Immunohistochemical analysis showed increased expression of P-selectin protein in postcapillary venules of all vital organs after trauma. To further investigate the possible mechanisms of increased P-selectin mRNA transcription promoter activity during trauma, we quantified binding of proteins from nuclear extracts to the kappaB site (-218GGGGGTGACCC[-207]) of the P-selectin gene by electrophoretic mobility shift assay. We confirmed the results of NF-kappaB binding by demonstrating increases in p50 and p52, as well as decreases in IkappaB in cytoplasmic and nuclear extracts from the lungs of trauma rats by Western blotting. Increased activity of the transcription factor, nuclear factor kappaB (NF-kappaB), occurred in all vital organs of the trauma rats compared to sham-operated controls. Our findings suggest that severe trauma results in up-regulation of P-selectin at the transcriptional level, which is partly controlled by an NF-kappaB-responsive element in the region of the P-selectin promoter. This increased activation of NF-kappB binding may contribute to the widespread increases in P-selectin expression observed in several vital organs 2 h after trauma, which in turn may play a key role in the pathogenesis of traumatic shock.
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PMID:P-selectin is up-regulated in vital organs during murine traumatic shock. 940 46

The identification of stromal cell-derived factor (SDF)-1alpha as a chemoattractant for human progenitor cells suggests that this chemokine and its receptor might represent critical determinants for the homing, retention, and exit of precursor cells from hematopoietic organs. In this study, we investigated the expression profile of CXCR4 receptor and the biological activity of SDF-1alpha during megakaryocytopoiesis. CD34(+) cells from bone marrow and cord blood were purified and induced to differentiate toward the megakaryocyte lineage by a combination of stem-cell factor (SCF) and recombinant human pegylated megakaryocyte growth and development factor (PEG-rhuMGDF). After 6 days of culture, a time where mature and immature megakaryocytes were present, CD41(+) cells were immunopurified and CXCR4mRNA expression was studied. High transcript levels were detected by a RNase protection assay in cultured megakaryocytes derived from cord blood CD34(+) cells as well as in peripheral blood platelets. The transcript levels were about equivalent to that found in activated T cells. By flow cytometry, a large fraction (ranging from 30% to 100%) of CD41(+) cells showed high levels of CXCR4 antigen on their surface, its expression increasing in parallel with the CD41 antigen during megakaryocytic differentiation. CXCR4 protein was also detected on peripheral blood platelets. SDF-1alpha acts on megakaryocytes by inducing intracellular calcium mobilization and actin polymerization. In addition, in in vitro transmigration experiments, a significant proportion of megakaryocytes was observed to respond to this chemokine. This cell migration was inhibited by pertussis toxin, indicating coupling of this signal to heterotrimeric guanine nucleotide binding proteins. Although a close correlation between CD41a and CXCR4 expession was observed, cell surface markers as well as morphological criteria indicate a preferential attraction of immature megakaryocytes (low level of CD41a and CD42a), suggesting that SDF-1alpha is a potent attractant for immature megakaryocytic cells but is less active on fully mature megakaryocytes. This hypothesis was further supported by the observation that SDF-1alpha induced the migration of colony forming unit-megakaryocyte progenitors (CFU-MK) and the expression of activation-dependent P-selectin (CD62P) surface antigen on early megakaryocytes, although no effect was observed on mature megakaryocytes and platelets. These results indicate that CXCR4 is expressed by human megakaryocytes and platelets. Furthermore, based on the lower responses of mature megakaryocytes and platelets to SDF-1alpha as compared with early precursors, these data suggest a role for this chemokine in the maintenance and homing during early stages of megakaryocyte development. Moreover, because megakaryocytes are also reported to express CD4, it becomes important to reevaluate the role of direct infection of these cells by the human immunodeficiency virus (HIV)-1 in HIV-1-related thrombocytopenia.
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PMID:Phenotypic and functional evidence for the expression of CXCR4 receptor during megakaryocytopoiesis. 1002 79

Thrombopoietin (TPO) is a hematopoietic growth factor that regulates megakaryocytopoiesis and platelet production through binding to its receptor, Mpl, encoded by the c-mpl proto-oncogene. Circulating levels of TPO are regulated by receptor-mediated uptake and degradation. To better understand this mode of TPO regulation, we examined whether expression of Mpl was regulated by its ligand. Using RNase protection analysis, we found no differences in the levels of c-mpl transcripts in megakaryocytes (MKs) produced in vitro either in the presence or absence of TPO and in platelets (PLTs) obtained from mice hyperstimulated in vivo by ectopic secretion of TPO. Similarly, Western blot analysis of MKs produced in the presence or absence of TPO showed no difference in Mpl levels. Levels of Mpl, GpIIb, or P-selectin were virtually identical in platelet lysates obtained from normal, TPO knockout and mildly TPO-stimulated mice. In contrast, the expression of Mpl was significantly reduced in PLTs from severely thrombocythemic mice. These results show that TPO does not have a major effect on the transcription or translation of Mpl. However, they do suggest that an excess of circulating TPO can lead to the disappearance of Mpl from PLTs via catabolism.
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PMID:High-level expression of Mpl in platelets and megakaryocytes is independent of thrombopoietin. 1021 80

Classic ischemic preconditioning transiently (30 to 120 minutes) protects the myocardium against subsequent lethal ischemia/reperfusion injury. After dissipation of this acute protection, a second window of protection (SWOP) appears 12 to 24 hours later; this SWOP lasts up to 3 days. Several triggers induce a SWOP, including brief repetitive cycles of coronary artery occlusion, rapid ventricular pacing, stimulation of adenosine A(1) receptors, and administration of wall fragments of Gram-negative bacteria, such as lipopolysaccharide (LPS). The aim of this study was to investigate whether lipoteichoic acid (LTA), a cell wall fragment of Gram-positive bacteria, can induce a SWOP in a rat model of left anterior descending coronary artery (LAD) occlusion (25 minutes) and reperfusion (2 hours). Thus, 166 male Wistar rats were pretreated (2 to 24 hours) with saline, LTA (1 mg/kg IP), or LPS (1 mg/kg IP) and subjected to LAD occlusion/reperfusion. Pretreatment with LTA or LPS for 16 hours led to a substantial, approximately 65%, reduction in infarct size and a reduction in the release of cardiac troponin T into the plasma. The dose of LTA used had no toxic effect (on any of the parameters studied), whereas the same dose of LPS caused a time-dependent activation of the coagulation system and liver injury. By use of RNase protection assays, it was determined that LPS caused a time-dependent induction of tumor necrosis factor-alpha, interleukin-1beta, and manganese superoxide dismutase mRNA content in the heart, whereas LTA failed to induce manganese superoxide dismutase. LPS also caused an upregulation of the expression of intercellular adhesion molecule-1 and P-selectin, whereas LTA downregulated these molecules and attenuated the accumulation of polymorphonuclear granulocytes caused by myocardial ischemia/reperfusion. This study demonstrates for the first time that pretreatment with LTA at 8 to 24 hours before myocardial ischemia significantly reduces (1) infarct size, (2) cardiac troponin T, and (3) the histological signs of tissue injury in rats subjected to LAD occlusion and reperfusion. The mechanism(s) underlying the observed cardioprotective effects of LTA warrants further investigation but is likely to be related to its ability to inhibit the interactions between the coronary vascular endothelium and polymorphonuclear granulocytes. Therefore, LTA represents a novel and promising agent capable of enhancing myocardial tolerance to ischemia/reperfusion injury.
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PMID:Lipoteichoic acid induces delayed protection in the rat heart: A comparison with endotoxin. 1084 67

In this study, self-organizing map (SOM) gene cluster techniques are applied to the analysis of cDNA microarray analysis of gene expression changes occurring in the early stages of genitourinary inflammation. We determined the time course of lipopolysaccharide (LPS)-induced gene expression in experimental cystitis. Mice were euthanized 0.5, 1, 4, and 24 h after LPS instillation into the urinary bladder, and gene expression was determined using four replicate Atlas mouse cDNA expression arrays containing 588 known genes at each time point. SOM gene cluster analysis, performed without preconditions, identified functionally significant gene clusters based on the kinetics of change in gene expression. Genes were classified as follows: 1) expressed at time 0; 2) early genes (peak expression between 0.5 and 1 h); and 3) late genes (peak expression between 4 and 24 h). One gene cluster maintained a constant level of expression during the entire time period studied. In contrast, LPS treatment downregulated the expression of some genes expressed at time 0, in a cluster including transcription factors, protooncogenes, apoptosis-related proteins (cysteine protease), intracellular kinases, and growth factors. Gene upregulation in response to LPS was observed as early as 0.5 h in a cluster including the interleukin-6 (IL-6) receptor, alpha- and beta-nerve growth factor (alpha- and beta-NGF), vascular endothelial growth factor receptor-1 (VEGF R1), C-C chemokine receptor, and P-selectin. Another tight cluster of genes with marked expression at 1 h after LPS and insignificant expression at all other time points studied included the protooncogenes c-Fos, Fos-B, Fra-2, Jun-B, Jun-D, and Egr-1. Almost all interleukin genes were upregulated as early as 1 h after stimulation with LPS. Nuclear factor-kappaB (NF-kappaB) pathway genes collected in a single cluster with a peak expression 4 h after LPS stimulation. In contrast, most of the interleukin receptors and chemokine receptors presented a late peak of expression 24 h after LPS coinciding with the peak of neutrophil infiltration into the bladder wall. Selected cDNA microarray observations were confirmed by RNase protection assay. In conclusion, the cDNA array experimental approach provided a global profile of gene expression changes in bladder tissue after stimulation with LPS. SOM techniques identified functionally significant gene clusters, providing a powerful technical basis for future analysis of mechanisms of bladder inflammation.
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PMID:Time course of LPS-induced gene expression in a mouse model of genitourinary inflammation. 1128 68

Tissue factor (TF) is the primary cellular initiator of the coagulation protease cascade and serves as a cell surface receptor and a specific cofactor for plasma factors VII/VIIa. Because there is evidence that TF is regulated by a P-selectin dependent gene, we examined TF mRNA expression in the lungs during murine traumatic shock in the presence and absence of recombinant soluble P-selectin glycoprotein ligand-1 (rsPSGL.Ig) by using ribonuclease protection assays. Moreover, we studied the level of TF mRNA expression in mice with their P-selectin gene deleted (P-selectin -/-). Our data show that TF mRNA was significantly increased (+143%; P < 0.001) in the lungs 2 h after trauma compared with control rats subjected to sham trauma, which exhibited reduced TF mRNA expression (-34%; P < 0.001) after systemic administration of rsPSGL.Ig. The expression of TF mRNA was also significantly decreased (-29%; P < 0.05) in the lungs of P-selectin -/- mice compared with wild-type control C57B16 mice. The present results provide evidence for a P-selectin-dependent mechanism that enhances TF gene expression in traumatic shock. The major support for this mechanism is that either blockade of P-selectin by rsPSGL.Ig or deletion of the P-selectin gene leads to significant decreases in TF mRNA expression in the lung. These results are consistent with the concept that TF interacting with P-selectin may play a significant role in the pathophysiology of trauma.
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PMID:Pulmonary tissue factor mRNA expression during murine traumatic shock: effect of P-selectin blockade. 1130 34


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