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)

Bacterial lipopolysaccharide (LPS, endotoxin) is a ubiquitous component of dust and air pollution and is suspected to contribute after inhalation to an activation of eosinophils in bronchial tissues of asthmatic patients, provoking inflammatory and allergic processes. We were therefore interested in the interaction of eosinophil granulocytes with LPS and have examined the activation of and uptake to human peripheral blood eosinophils by LPS. Eosinophils were stimulated by LPS and the endotoxic component lipid A and the release of tumor necrosis factor alpha (TNF-alpha) and of the eosinophil-specific granule protein eosinophil cationic protein (ECP) was estimated. The results show induction of TNF-alpha and ECP-release by LPS and lipid A in a dose-dependent manner. Anti-CD14 monoclonal antibody (moAb) (clone MEM-18) and the synthetic lipid A partial structure 406 blocked the release of TNF-alpha and ECP by LPS-stimulated eosinophils. Studies with radioactively labeled LPS showed dose-dependent uptake of (3)H-LPS to eosinophils. The (3)H-LPS uptake was found to be specific because preincubation with unlabeled LPS, compound 406 and also anti-CD14 antibodies inhibited uptake of (3)H-LPS to eosinophil granulocytes. By flow cytometry using anti-CD14 moAb and by reverse transcriptase-polymerase chain reaction (RT-PCR) technique, CD14 expression was detectable. Furthermore, messenger RNA (mRNA) expression of Toll-like receptors (TLR) 2 and TLR 4 was detected, indicating the presence of these CD14 coreceptors. The results indicate that eosinophils can take up LPS and can be stimulated by LPS in a CD14-dependent manner. Hence, in addition to allergens, eosinophils interact with endotoxin, a process that possibly exacerbates ongoing inflammatory and allergic processes.
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PMID:The interaction of human peripheral blood eosinophils with bacterial lipopolysaccharide is CD14 dependent. 1113 66

Specific immune cell activation is a hallmark of infections and autoimmune disorders. Quantification of proliferative cell responses by (3)H-thymidine incorporation is a slow process and describes only one type of cellular reaction. We here investigated early immunological responses of purified human peripheral blood mononuclear cells to the direct stimulus alpha CD3 and antigen specific stimulation (human myelin basic protein (hMBP), tetanus toxoid, and influenza vaccine) and compared them to polyclonal LPS stimulation. Cytokine mRNA levels were quantified using real-time quantitative reverse transcriptase polymerase chain reaction (RT PCR) 4 h, 16 h, and 48 h after activation. Proliferation was measured 96 h after initiation of the cultures. Antigen specific responses were detected as early as 4 h after stimulation and followed different kinetics depending on the mode of activation. We demonstrated significant correlations of cytokine mRNA and protein expression for TNF alpha, IL10, and IFN gamma. Expression of IL2 mRNA at 16 h was correlated with proliferation indices at 96 h whereas IL4 mRNA levels were negatively correlated. Early cytokine mRNA expression in stimulated immune cells provides important functional data and is a powerful tool with which to study immunological reactions.
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PMID:Characterization of early immunological responses in primary cultures of differentially activated human peripheral mononuclear cells. 1115 May 44

Exercise can increase plasma inflammatory cytokine concentrations in humans, but tissue responses are not well studied. We examined plasma concentrations and tissue expression of TNFalpha, IL-1beta, and IL-6 following treadmill running in mice. C57B1/6 mice were randomly assigned to: non-exercise control (CON), sacrifice at 0 or 1.5 h after 60 min running (MOD0, MOD 1.5), sacrifice at 0, 1.5, or 3 h after fatiguing running (approximately 3 h) (EX0, EX1.5, EX3), or lipopolysaccharide (25 microg) with no exercise (LPS). Lung, liver, muscle, and brain mRNA expression was analyzed (n = 4-6/group) using reverse transcriptase-rapid polymerase chain reaction (RT-RPCR). Plasma cytokine concentrations were determined (n =4-10/group) by ELISA. Plasma IL-6 was higher in EX1.5, and lung TNFalpha mRNA was higher in EX1.5 and EX3 compared to CON (P < 0.05). No significant increases in plasma cytokine concentrations or tissue cytokine expression were found in other EX groups. LPS significantly increased these cytokine measures in tissues and plasma, with the exception of plasma IL-1beta which was undetectable. The source of the plasma IL-6 following exercise does not appear to be lung, liver, muscle, or brain tissue, and remains to be determined. These data also suggest that tissue level cytokine expression may not necessarily lead to increased plasma cytokine concentrations.
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PMID:Tissue expression and plasma concentrations of TNFalpha, IL-1beta, and IL-6 following treadmill exercise in mice. 1141 67

Bacterial endotoxin (lipopolysaccharide; LPS) augments the hepatotoxicity of a number of xenobiotics including allyl alcohol. The mechanism for this effect is known to involve the inflammatory response elicited by LPS. Upregulation of cyclooxygenase-2 (COX-2) and production of eicosanoids are important aspects of inflammation, therefore studies were undertaken to investigate the role of COX-2 in LPS-induced enhancement of liver injury from allyl alcohol. Rats were pretreated (iv) with a noninjurious dose of LPS or sterile saline vehicle and 2 h later were treated (ip) with a noninjurious dose of allyl alcohol or saline vehicle. COX-2 mRNA was determined by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR), and liver injury was assessed from activities in serum of alanine and aspartate aminotransferases (ALT and AST, respectively) and from histology. Liver injury was observed only in rats cotreated with LPS and allyl alcohol. Serum ALT activity was increased by 4 h after administration of LPS and continued to increase through 8 h. COX-2 mRNA was detectable at low levels in livers from rats receiving only the vehicles at any time up to 8 h. Expression of COX-2 mRNA was increased by 30 min after administration of LPS and remained elevated through 6 h. Allyl alcohol treatment alone caused an increase in COX-2 mRNA at 4 h (2 h after allyl alcohol) that lasted less than 2 h. In livers from rats cotreated with LPS and allyl alcohol, levels of COX-2 mRNA were greater than levels seen with either LPS or allyl alcohol alone. The increased expression of COX-2 mRNA was accompanied by an increase in the concentration of prostaglandin (PG) D(2) in plasma. Plasma PGD(2) concentration was increased to a greater extent in rats treated with LPS plus allyl alcohol compared to allyl alcohol or LPS alone. Pretreatment with the COX-2 selective inhibitor, NS-398, abolished the increase in plasma PGD(2) and reduced the increase in ALT and AST activities observed in rats cotreated with LPS and allyl alcohol. NS-398 did not affect liver injury from allyl alcohol alone administered at a larger, hepatotoxic dose. In addition, ibuprofen, a nonselective inhibitor of cyclooxygenases, did not protect against liver injury from LPS plus allyl alcohol. In isolated hepatocytes PGD(2), but not PGE(2), reduced the concentration of allyl alcohol required to cause half-maximal cytotoxicity. These results suggest that products of COX-2 play a role in the augmentation of allyl alcohol-induced liver injury by LPS.
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PMID:Involvement of cyclooxygenase-2 in the potentiation of allyl alcohol-induced liver injury by bacterial lipopolysaccharide. 1144 26

Polymorphonuclear neutrophils (PMNs) are only regarded as being involved in the cleavage of exogenous big endothelin-1 (ET-1) to the active peptide. The aims of the present study were to investigate whether PMNs may themselves express mRNA for prepro-ET-1 (pp-ET-1, a long precursor of 212 amino acids) and to determine the capacity of several PMN stimulants to modulate mRNA expression and the release of ET-1 in culture medium. PMNs, isolated from seven healthy adult volunteers, were stimulated with lipopolysaccharide (LPS, 0.25-10 microg/ml), or LPS (1 microg/ml) + phorbol myristate acetate (PMA, 10 ng/ml) or N-formyl-L-methionyl-L-leucyl-L-phenylalanine (f-MLP, 10(-5) M) or tumour necrosis factor-alpha (TNF-alpha, 50 IU/ml). They were found to express pp-ET-1 mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Enzyme immunoassay (EIA) revealed low levels of ET-1 in the culture supernatants of PMNs stimulated for 3 h with LPS (10 microg/ml) and with LPS + PMA. Control unstimulated PMNs did not express pp-ET-1 mRNA. The local production of ET-1 by PMNs in vivo has significant implications in inflammatory diseases.
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PMID:Gene expression of endothelin-1 (ET-1) and release of mature peptide by activated human neutrophils. 1144 23

Viable and inactivated Porphyromonas gingivalis dose-dependently induced interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) secretion in human umbilical vein endothelial cells (HUVECs). The inactivated P. gingivalis, in comparison with viable bacteria, tended to enhance the production of both chemokines more strongly. The production of MCP-1 protein began increasing immediately after stimulation by P. gingivalis, and there was a nearly linear increase from 0 to 8 h of incubation, whereas IL-8 production showed a linear increase between 4 and 12 h of incubation. The IL-8 and MCP-1 mRNA expressions in HUVECs as determined by reverse transcriptase-polymerase chain reaction (RT-PCR) or Quantikine mRNA colorimetric quantification kits were found to be enhanced by P. gingivalis. Furthermore, the time courses of IL-8 and MCP-1 mRNA expressions were in accordance with those of protein production. Addition of polymyxin B or boiling did not weaken the stimulatory effect of P. gingivalis, which inhibited the effect of Escherichia coli lipopolysaccharide (E. coli LPS) and tumour necrosis factor-alpha (TNF-alpha), respectively. In contrast, the induction of IL-8 and MCP-1 by P. gingivalis was significantly reduced by anti-CD14 antibody. Our results suggest that some heat-stable component of P. gingivalis, including LPS, may be responsible for the induction of IL-8 and MCP-1 in HUVECs by a CD14-dependent mechanism. These effects might be involved in the accumulation and activation of neutrophils and monocytes at an early stage of the periodontal pathogenesis.
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PMID:CD14-mediated induction of interleukin-8 and monocyte chemoattractant protein-1 by a heat-resistant constituent of Porphyromonas gingivalis in endothelial cells. 1241 Jul 98

To profile postmortem degradation of mRNA, total RNA was extracted, at given postmortem intervals, from the brain, lung, heart and liver of rats left at 20 degrees C. In electrophoretic analysis, total RNA was most stable in the brain, moderately stable in the lung and heart, and most unstable in the liver. Northern blot analysis of total RNA extracts from the brain and liver of dead rats with a cDNA probe for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) showed that GAPDH mRNA degraded in a similar fashion to total RNA. Analysis of the postmortem degradation profile of GAPDH mRNA with real-time reverse transcriptase-polymerase chain reaction (real-time RT-PCR) gave results consistent with those above, indicating that real-time RT-PCR is reliable for estimation of the mRNA level in specimens from dead bodies. Real-time RT-PCR analysis showed that degradation rates of three housekeeping genes, GAPDH, beta-actin and hypoxanthine guanine phosphoribosyltransferase, in the brains of dead rats were similar. The degradation rate of interleukin-1beta (IL-1beta) mRNA induced by intravenous injection of LPS to rats was higher than that of GAPDH mRNA in the lung. In real-time RT-PCR analysis using GAPDH mRNA as an internal standard, the detection level of IL-1beta mRNA decreased in the postmortem interval. However, enhanced expression of IL-1beta was detected for at least 3 days postmortem.
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PMID:Degradation profile of mRNA in a dead rat body: basic semi-quantification study. 1247 33

Tissues must quickly recognize injury to respond to the rapid pace of microbial growth. In skin, dermal microvascular endothelial cells must also react to danger signals from the surrounding tissue and immediately participate by initiating the wound repair process. Components of the extracellular matrix such as hyaluronan are rapidly broken down into smaller molecular weight oligosaccharides in a wound, and these can activate a variety of biological processes. This study set out to determine if hyaluronan fragments released following injury can stimulate endothelial cells and what mechanism is responsible for this response. Using genechip microarray analysis, a response to hyaluronan fragments was detected in endothelial cells with the most significant increase observed for the chemokine IL-8. This observation was verified with qualitative reverse transcriptase-PCR and ELISA in human endothelial cell culture, and in a mouse model by observing serum levels of MIP-2 and KC following hyaluronan fragment administration in vivo. Activation was TLR4-dependent, as shown by use of TLR4 blocking antibody and TLR4-deficient mice, but not due to the presence of undetected contaminants as shown by inactivation following digestion with the hyaluronan-degrading enzyme chondroitinase ABC or incubation with the hyaluronan-specific blocking peptide Pep-1. Inactivation of LPS activity failed to diminish the action of hyaluronan fragments. These observations suggest that endogenous components of the extracellular matrix can stimulate endothelia to trigger recognition of injury in the initial stages of the wound defense and repair response.
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PMID:Hyaluronan fragments stimulate endothelial recognition of injury through TLR4. 1476 99

Post-traumatic inflammation is connected to monocyte dysfunction characterized by reduced NF-kappaB translocation during the first post-traumatic days. Because the exact dynamic of monocytic NF-kappaB translocation in patients directly after trauma remains unclear, the aim of this pilot study was to measure the intranuclear presence of NF-kappaB in monocytes from patients with multiple injuries initially after the trauma and during the early post-traumatic period and to compare these results with downstream-placed mRNA expression alteration of TNF-alpha, as well as with clinical data. Eleven patients were enrolled with an Injury Severity Score of 16 to 66 points, and blood samples were drawn on admission within 90 min and at 6, 12, 24, 48, and 72 h after trauma. NF-kappaB translocation of monocytic nuclear protein was analyzed by electrophoretic mobility shift assay and was quantified by densitometry as arbitrary units. In addition, monocytes of healthy volunteers were analyzed either native (-, control) or after LPS stimulation (+, control). For determination of downstream mRNA encoding for TNF-alpha, quantitative reverse transcriptase-PCR was performed. For both parameters, the negative control values were set as baseline (=1) and results from positive controls and patients were given as a relative alteration ratio without unit. Initial post-traumatic NF-kappaB translocation was significantly increased in trauma patients on admission (88 +/- 37) and 6 h after trauma (59 +/- 28) compared with the baseline level. In contrast, TNF-alpha mRNA was not increased on admission (1.7 +/- 0.9) and decreased even below baseline after 12 h. The substantial information of our study arises from the analysis of the dynamic of NF-kappaB translocation of monocytes. Enabled by closely matched sequential blood sampling strictly standardized to the traumatic event, an essential increase of monocytic signal transduction and transcription could be elucidated in the very early post-traumatic period, which precedes the down-regulation of the innate immune system.
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PMID:Initial posttraumatic translocation of NF-kappaB and TNF-alpha mRNA expression in peripheral blood monocytes of trauma patients with multiple injuries: a pilot study. 1554 23

P-glycoprotein (PGP) encoded by the Mdr1 gene mediates the excretion of drugs in organs such as the liver and kidney. Inflammation has been shown to suppress the expression and activity of PGP in rodent liver, thus potentially altering the pharmacokinetics of drugs that are substrates of PGP. Here we examined the effect of endotoxin (lipopolysaccharide; LPS)-induced inflammation on the disposition of the PGP substrate doxorubicin (DOX) in the mouse. Male CD-1 mice received 5 mg/kg LPS intraperitoneally. DOX (5 mg/kg) was administered intravenously 24 h after LPS treatment. The time course of DOX levels in plasma, urine, bile, and tissues was analyzed by high performance liquid chromatography. PGP protein and mRNA expression in liver and kidney was measured using Western blots and reverse transcriptase polymerase chain reaction. As compared to controls, LPS-treated mice exhibited a significant decrease (50%) in biliary clearance and 3-fold increased renal clearance of DOX. These changes were associated with strongly reduced PGP protein levels (30% controls, p < 0.05) in the liver and increased PGP levels in the kidney (140% controls, p < 0.05). Hepatic mRNA levels of all Mdr isoforms were reduced in LPS-treated mice, whereas renal Mdr1b levels were increased. In LPS-treated mice, we also measured an increased area under the plasma concentration-time curve and reduced systemic clearance of DOX, as well as a 2- to 5-fold increase in the urinary excretion of the doxorubicin and doxorubicinol aglycones. Our data suggest that endotoxin-induced inflammation in mice causes differential regulation of PGP in liver and kidney, thereby altering the clearance profile of DOX.
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PMID:Impact of endotoxin-induced changes in P-glycoprotein expression on disposition of doxorubicin in mice. 1577 72


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