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)

Clara cell 10-kD (cc10-kD) protein has been suggested to be the human counterpart of rabbit uteroglobin (UG). Like UG, this protein is also a potent inhibitor of phospholipase A2 (PLA2) and a substrate of transglutaminase. Although it has been established that UG gene expression in the rabbit endometrium is stimulated by progesterone, the expression of cc10-kD gene in the human endometrium is not clearly understood. The present study was undertaken to determine whether the cc10-kD gene is expressed in the human endometrium and whether its level of expression changes in relation to the ovarian menstrual cycle. Using reverse transcriptase polymerase chain reaction (RT-PCR) and immunofluorescence, we demonstrate that the cc10-kD gene is expressed in different stages of the menstrual cycle and that the highest level of expression is reached during the luteal phase. These results suggest that like rabbit UG, cc10-kD gene expression in the human endometrium may be stimulated by progesterone. Since cc10-kD is a potent inhibitor of PLA2 activity, this protein may play an important physiological role in regulating eicosanoid levels in the human uterus.
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PMID:Expression of Clara cell 10-kD gene in the human endometrium and its relationship to ovarian menstrual cycle. 751 78

Inflammation has been considered to be related to carcinogenesis. Previously, we demonstrated that 1-hydroxyanthraquinone (1-HA), a naturally occurring carcinogen, induced severe inflammation such as ulcerative colitis in colonic mucosa. We also showed that indomethacin inhibited the tumorigenicity of 1-HA. In this study, we examined the expressions of major enzymes in arachidonic acid cascade related to inflammation in the colon mucosa of rats treated with 1-HA. After the treatment of 1% 1-HA diet, colon lesions were observed and RNA was extracted from mucosa and neoplasms. The mRNA expressions of group II phospholipase A2, cyclooxygenase-2 and 5-lipoxygenase, were examined by using a reverse transcriptase polymerase chain reaction. The expressions of phospholipase A2 and cyclooxygenase were significantly increased in non-neoplastic mucosa in rats treated with 1-HA compared with those in control rats. The expressions in the neoplasms induced by 1-HA were also increased. Phospholipase A2, especially, was much higher in the neoplasms than in non-neoplastic mucosa. However, the expression of 5-lipoxygenase showed no change in the non-neoplastic mucosa and neoplasms of rats treated with 1-HA, compared with that in control rats. These findings suggest that the inflammation induced by 1-HA may be related to the metabolites through a cyclooxygenase pathway, which indicates a prostaglandin synthesis, but not through a lipoxygenase pathway, which indicates a leukotriene synthesis in arachidonic acid cascade.
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PMID:The mRNA overexpression of inflammatory enzymes, phospholipase A2 and cyclooxygenase, in the large bowel mucosa and neoplasms of F344 rats treated with naturally occurring carcinogen, 1-hydroxyanthraquinone. 758 82

Here we show that secretory phospholipase A2 (sPLA2) that is immunochemically indistinguishable from type II sPLA2 is (i) stored in neuroendocrine cells, (ii) released in response to neurotransmitters or depolarization, and (iii) involved in the regulation of catecholamine secretion by these cells. Rat brain synaptic vesicle fractions contained PLA2 activity, which was neutralized completely by an antibody raised against rat type II sPLA2. sPLA2 immunoreactive with anti-(type II sPLA2) antibody was released from synaptosomes in response to depolarization evoked by a high concentration of potassium in the presence of Ca2+. Rat pheochromocytoma PC12 cells, which differentiated into adherent cells similar to sympathetic neurons in response to nerve growth factor, were used for the detailed analysis of the dynamics and function of sPLA2 in neuronal cells. Antibody against rat type II sPLA2 precipitated approximately 80% of the PLA2 activity in PC12 cell lysates. Transcript for type II sPLA2 was detected in PC12 cells by reverse transcriptase-PCR. When neuronally differentiated PC12 cells were stimulated with carbamylcholine or potassium, sPLA2 was released into the medium and reached a maximal approximately 40% release by 15 min. Inhibitors specific to type II sPLA2 suppressed catecholamine secretion by PC12 cells which had been activated by carbamylcholine. Furthermore, treatment of PC12 cells with exogenous type II sPLA2 alone elicited catecholamine secretion. These observations indicate that sPLA2 released from neuronal cells may regulate the degranulation process leading to release of neurotransmitters and are compatible with our earlier finding that this enzyme is involved in the degranulation of rat mast cells.
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PMID:Release of secretory phospholipase A2 from rat neuronal cells and its possible function in the regulation of catecholamine secretion. 880 65

Four related genes encode four different secretory phospholipase A2 (sPLA2) enzymes in mammals, namely the well described Group I and IIA enzymes and the more recently described Groups IIC and V. A large body of research has putatively demonstrated that the Group IIA sPLA2 is involved in diverse pathologic processes, such as rheumatoid arthritis, septic shock, intestinal neoplasia, and epidermal hyperplasia, as well as in cellular signaling by regulating the formation of arachidonate-derived lipid messengers. However, we demonstrate herein the involvement of another sPLA2, i.e. the Group V sPLA2, in arachidonic acid release and prostaglandin production in the mouse macrophage-like cell line P388D1. Abundant message for Group V sPLA2 was detected in both resting and activated cells. In contrast, Group IIA sPLA2 message was undetectable as analyzed by Northern blot and reverse transcriptase-polymerase chain reaction. Moreover, blockage of Group V sPLA2 gene expression by antisense RNA oligonucleotides resulted in inhibition of prostaglandin E2 production as well as reduction of the amount of sPLA2 protein at the cellular surface. Collectively, these results uncover Group V sPLA2 as a novel effector involved in arachidonic acid-mediated signal transduction.
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PMID:Novel group V phospholipase A2 involved in arachidonic acid mobilization in murine P388D1 macrophages. 894 2

Secretory nonpancreatic type II phospholipase A2 (snpPLA2) hydrolyzes fatty acids at the sn-2 position in phospholipids releasing free fatty acids (FFAs) and lysophospholipids. These products may act as intracellular second messengers or can be further metabolized into proinflammatory lipid mediators. The presence of snpPLA2 in extracellular fluids and serum during inflammation has suggested a role of the enzyme in this process. However, the presence of snpPLA2 in a variety of normal tissues suggests that snpPLA2 may also have physiological functions. Atherosclerosis appears to have an inflammatory component. Here we report on the snpPLA2 localization in normal and atherosclerotic lesions and on the properties of the isolated enzyme. A strong snpPLA2 immunoreactivity was observed in the arterial media that was colocalized with alpha-actin-positive vascular smooth muscle cells (SMCs) in both normal and atherosclerotic vessels. In aortic atherosclerotic lesions, snpPLA2 was observed colocalized with CD68-positive macrophages and HHF-35-positive SMCs and extracellularly in the lipid core. snpPLA2 was isolated from human normal arteries and from aorta with lesions. The enzyme was isolated by acid extraction of normal arterial tissues followed by immunoaffinity chromatography. The purified snpPLA2 had an expected molecular weight of 14 kD by polyacrylamide gel electrophoresis and appeared as a single band in immunoblotting. The enzymatic activity was followed by measuring release of fatty acids from phospholipid liposomes or LDL as substrates. The enzymatic activity was inhibited with two specific inhibitors for human snpPLA2: (1) monoclonal antibody 187 and (2) LY311727, a synthetic selective inhibitor. The mRNA for snpPLA2 was detected with reverse transcriptase polymerase chain reaction. These results indicate that snpPLA2 is present in human arteries and that it is able to hydrolyze phospholipids in LDL. The results support the hypothesis that snpPLA2 can release proinflammatory lipids at places of LDL deposition in the arterial wall.
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PMID:Localization of nonpancreatic secretory phospholipase A2 in normal and atherosclerotic arteries. Activity of the isolated enzyme on low-density lipoproteins. 908 85

Prostaglandin D2 (PGD2) synthesis in activated mast cells occurs in two phases, an early phase that is dependent on prostaglandin synthase 1 and a delayed phase that is dependent on activation-induced prostaglandin synthase 2 gene expression. Early phase PGD2 synthesis in activated mast cells also requires the activity of a secretory phospholipase A2 (PLA2). It has been thought that the secretory PLA2 expressed in mast cells is group IIa PLA2, encoded by the Pla2 g2a gene. However, activated bone marrow-derived mast cells prepared from Pla2 g2a+/+ mice and mast cells prepared from mice with a mutation in the Pla2 g2a gene both demonstrate early phase PGD2 synthesis. Moreover, mast cells from both murine strains secrete PLA2 activity following activation. Northern and reverse transcriptase/polymerase chain reaction analyses demonstrate that mast cells from Pla2 g2a+/+ and Pla2 g2a-/- mice do not express group IIa PLA2 message. Instead, Northern and reverse transcriptase/polymerase chain reaction analyses demonstrate that both Pla2 g2a+/+ and Pla2 g2a-/- mast cells express mRNA for group V PLA2, encoded by the Pla2gV gene. An antisense oligonucleotide directed against group V PLA2 is also able to inhibit both the early phase of PGD2 production and the secretion of PLA2 activity by activated mast cells. Our data suggest that (i) group IIa PLA2 does not play a significant role in murine mast cell prostaglandin synthesis, (ii) group V PLA2 mediates early mast cell PGD2 production and transcellular PGE2 production in murine mast cells, and (iii) much of the data, based on studies with chemical inhibitors and antibodies, suggesting that group IIa PLA2 is responsible for arachidonic acid mobilization needs to be reevaluated.
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PMID:Analysis of the secretory phospholipase A2 that mediates prostaglandin production in mast cells. 915 7

Rat lecithin:cholesterol acyltransferase (LCAT) cDNA was obtained by reverse transcriptase/polymerase chain reaction amplification of rat liver total RNA. A consensus sequence was derived from four independent clones from two strains of rats. In vitro expression of rat LCAT cDNA in COS cells resulted in secreted enzyme protein with the same fatty acyl specificity for phospholipase A2 activity and cholesterol esterification as rat plasma LCAT, but different from that of recombinant or human plasma LCAT.
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PMID:Cloning and in vitro expression of rat lecithin:cholesterol acyltransferase. 921 4

Phospholipase A2 has been considered to play a role in physiological membrane turnover in cardiac tissue and in the degradation of membrane lipids under pathophysiological conditions, such as ischemia and reperfusion. We report the cloning of a cDNA encoding a member of the Ca2+-dependent, low molecular mass phospholipase A2 (PLA2) present in rat heart. The cDNA predicts a mature protein of 146 amino acid residues including a 21 amino acid sequence at the N-terminal end, which has the features characteristic of eukaryotic secretory signal peptides. The deduced amino acid sequence constitutes an enzyme of the group II class of PLA2s, and resembles PLA2s from other mammalian sources. A Northern blot analysis performed to determine the tissue distribution showed that rat ileum contains the largest amount of the PLA2 transcript among the tissues examined, a weaker signal was present in heart, spleen and soleus muscle, and no signal could be detected in EDL muscle, stomach, liver, kidney, brain and lung. Northern blot analysis and reverse transcriptase-polymerase chain reaction (RT-PCR) techniques indicate the presence of this enzyme in neonatal and adult rat cardiomyocytes and in a cultured rat cardiac fibroblast-like cell line, but not in rat cardiac-derived endothelial cell lines. Transcription levels of rat heart group II PLA2 in isolated neonatal rat cardiomyocytes were found to increase after stimulating the cells with tumor necrosis factor-alpha (TNF-alpha) or the alpha1-adrenergic agonist phenylephrine.
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PMID:Cloning and cellular distribution of a group II phospholipase A2 expressed in the heart. 928 42

Angiotensin II (Ang II) elicits an Ang II type 2 (AT2) receptor-mediated increase in delayed-rectifier K+ current (IK) in neurons cultured from newborn rat hypothalamus and brainstem. This effect involves a pertussis toxin (PTX)-sensitive Gi protein and is abolished by inhibition of serine and threonine phosphatase 2A (PP-2A). Here, we determined that Ang II stimulates [3H]arachidonic acid (AA) release from cultured neurons via AT2 receptors. This effect of Ang II was blocked by inhibition of phospholipase A2 (PLA2) and by PTX. Because AA and its metabolites are powerful modulators of neuronal K+ currents, we investigated the involvement of PLA2 and AA in the AT2 receptor-mediated stimulation of IK by Ang II. Single-cell reverse transcriptase (RT)-PCR analyses revealed the presence of PLA2 mRNA in neurons that responded to Ang II with an increase in IK. The stimulation of neuronal IK by Ang II was attenuated by selective inhibitors of PLA2 and was mimicked by application of AA to neurons. Inhibition of lipoxygenase (LO) enzymes significantly reduced both Ang II- and AA-stimulated IK, and the 12-LO metabolite of AA 12S-hydroxyeicosatetraenoic acid (12S-HETE) stimulated IK. These data indicate the involvement of a PLA2, AA, and LO metabolite intracellular pathway in the AT2 receptor-mediated stimulation of neuronal IK by Ang II. Furthermore, the demonstration that inhibition of PP-2A abolished the stimulatory effects of Ang II, AA, and 12S-HETE on neuronal IK but did not alter Ang II-stimulated [3H]-AA release suggests that PP-2A is a distal event in this pathway.
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PMID:Angiotensin II type 2 receptor stimulation of neuronal delayed-rectifier potassium current involves phospholipase A2 and arachidonic acid. 942 10

In the present study a protocol of in situ reverse transcriptase-nested polymerase chain reaction (in situ RT-nested PCR) was examined based on the following modifications. (i) To exclude false positive signals caused by "DNA repair mechanisms" and "endogenous priming", a two-step PCR was applied after reverse transcription. The first step was performed in the presence of extrinsic primers and unlabeled nucleotides with a maximum of PCR cycles possible without destroying the cell morphology. The second step consisted of only one annealing/elongation reaction, the target sequence marked by addition of digoxigenin-labeled nucleotides and intrinsic primers. (ii) In order to prevent amplifications of genomic DNA nested primer pairs were applied crossing intron sequences. (iii) To minimize the diffusion of PCR products in cells, the extrinsic primers were extended with complementary 5(prime, variant)-tails. This approach results in the generation of high molecular weight concatamers during PCR cycles. By applying this protocol, immunostainings specific for phospholipase A2 of type IIA mRNA were exclusively detectable in the cytoplasm of HepG2 hepatoma cells, which were used as a model system, whereas the nuclei were unstained. Multiple control experiments yielded completely negative results. These data suggest that the in situ RT-nested PCR, which in comparison to the method of in situ RT-PCR-in situ-hybridisation is simpler and less time-consuming, can be used as an alternative approach to identify intracellular nucleic acids.
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PMID:In situ reverse transcriptase-nested polymerase chain reaction to identify intracellular nucleic acids without the necessity of DNAse pretreatment and hybridisation. 1145 34


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