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)

Gap junctions, which serve as intercellular channels providing direct cytoplasmic continuity and ionic current flow between adjacent cells, are constituted by connexin proteins. Using an in vitro model of bicuculline-induced epileptiform activity, we asked whether increased connexin levels occur during epileptiform activity in the intact whole hippocampus, freshly isolated from young (15-day-old) mouse brain. Exposure to bicuculline (10 microM), for 2-10 h, induced persistent changes in electrical activities that included enhanced spontaneous field activity (4 h), an epileptiform response to single electrical stimulation (6 h), and spontaneous epileptiform activity (6 h). These electrophysiological changes were not reversed by up to 60 min perfusion with normal artificial cerebrospinal fluid, but were greatly depressed by the gap junction uncoupler, carbenoxolone (120 microM, 10 min). Data from RNase protection assay and immunoblotting showed that among several detected gap junctions, only connexin 32 was affected. After 2-6 h exposure to bicuculline, the connexin 32 mRNA expression was upregulated to 2-3-fold control (P < 0.01), and its protein level was significantly elevated the following 6 h (P < 0.01), at which time electrophysiologically measured evidence of clearly epileptiform activity was apparent. In addition, the transcription factor, c-fos protein, but not the cAMP response element-binding protein, was also found to be increased at the early stage of bicuculline exposure (2 h) compared to control (P < 0.05).Thus, we have found that exposing the acutely isolated hippocampus to bicuculline, induced increased c-fos protein, followed by increased connexin 32 transcript and protein, and concurrently, persistent epileptiform activity that was depressed by carbenoxolone.
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PMID:Upregulation of gap junction connexin 32 with epileptiform activity in the isolated mouse hippocampus. 1151 26

Antioxidants have been proposed as a promising treatment for restenosis after percutaneous transluminal coronary angioplasty (PTCA), but their mechanism of action remains unclear. Here, we investigated the effect of antioxidants on gene expression in the artery after balloon denudation. We developed a sensitive ribonuclease (RNase) protection assay for the messenger RNA (mRNA) levels of immediate early (IE) genes (c-jun, c-fos and c-myc), as well as platelet-derived growth factor-A (PDGF-A), platelet-derived growth factor-beta receptor, transforming growth factor-beta 1, and vascular endothelial growth factor. New Zealand White rabbits were fed a 0.17% cholesterol diet containing vehicle, BO-653 or probucol, and balloon denudation for iliac arteries was performed. The iliac arteries were then removed at 4 h after the denudation, for IE genes, and 10 days after for growth factors and receptors. Both BO-653 and probucol significantly reduced neointimal thickening, compared with the control. In terms of gene expression, BO-653, but not probucol, significantly inhibited c-myc induction. On the other hand, probucol, but not BO-653, significantly inhibited PDGF-A expression. Neither treatment had any effect on the expression of other genes. These results suggest that antioxidants affect the gene expression of the neointimal response and that both BO-653 and probucol inhibit gene expression in specific manners.
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PMID:Effect of BO-653 and probucol on c-MYC and PDGF-A messenger RNA of the iliac artery after balloon denudation in cholesterol-fed rabbits. 1188 18

TaqMan, a variation of fluorescent PCR, is a powerful tool for gene expression and polymorphism studies. Here we describe the design and evaluation of 27 new TaqMan primer-probe sets for rat genes that play a key role in neural signaling. These newly designed and synthesized probes were tested and then used for quantification of RNA isolated from rat brain. The usual length of common TaqMan probes is 25 bases or less. In these studies we constructed probes with lengths of 25-39 bases to span exon-exon junctions of nucleic acids to avoid the influence of DNA contamination upon the RNA quantification. The specific sequences at these positions required probes of these lengths to optimize hybridization. We found that the relocation of the quencher from the traditional 3' position to an internal one increases the sensitivity of probe up to 30 fold. Substitution of 6-carboxyfluorescein with Alexa Fluor 488 as fluorophore and TAMRA with non-fluorescent quencher dabcyl was also investigated. We also describe the evaluation of part of a newly designed set of 27 TaqMan primer-probes for the measurement of differences in gene expression levels in samples from the caudate putamen region of rat brain after 'binge' paradigm cocaine administration. Cocaine-induced alterations in expression of c-fos and preprodynorphin mRNAs measured by TaqMan were confirmed by ribonuclease protection assay.
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PMID:Optimizing primer--probe design for fluorescent PCR. 1258 47

Chronic (18 h) exposure of cultured hippocampal slices to the type-A GABA receptor blocker, bicuculline methiodide (BMI) 10 micro m increased the levels of connexin 43 (Cx43) and connexin 32 (Cx32) mRNAs, but not connexin 26 and connexin 36, as demonstrated by RNase protection assays. The levels of Cx43 and Cx32 proteins in membrane fractions detected by western blotting were also significantly increased. Immunoblotting indicated that BMI also promoted a significant expression of the transcription protein c-fos. The rate of fluorescence recovery after photobleaching, an index of gap junctional coupling, was also significantly increased, whereas it was blocked by the gap junctional blocker, carbenoxolone (100 micro m). Extracellular recordings in CA1 stratum pyramidale, performed in BMI-free solution, demonstrated that BMI-exposed cultures possessed synaptic responses characteristic of epileptiform discharges: (i) significantly greater frequency of spontaneous epileptiform discharges, (ii) post-synaptic potentials with multiple population spikes, and (iii) significantly longer duration of primary afterdischarges. Carbenoxolone (100 micro m), but not its inactive analog, oleanolic acid (100 micro m), reversibly inhibited spontaneous and evoked epileptiform discharges. The findings of BMI-induced parallel increases in levels of gap junction expression and function, and the increase in epileptiform discharges, which were sensitive to gap junctional blockers, are consistent with the hypothesis that increased gap junctional communication plays an intrinsic role in the epileptogenic process.
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PMID:Epileptiform activity in hippocampal slice cultures exposed chronically to bicuculline: increased gap junctional function and expression. 1285 82

The molecular mechanisms by which bone morphogenetic proteins (BMPs) promote skeletal cell differentiation were investigated in the murine mesenchymal stem cell line C3H10T1/2. Both BMP-7 and BMP-2 induced C3H10T1/2 cells to undergo a sequential pattern of chondrogenic followed by osteogenic differentiation that was dependent on both the concentration and the continuous presence of BMP in the growth media. Differentiation was determined by the expression of chondrogenesis and osteogenesis associated matrix genes. Subsequent experiments using BMP-7 demonstrated that withdrawal of BMP from the growth media led to a complete loss of skeletal cell differentiation accompanied by adipogenic differentiation of these cells. Continuous treatment with BMP-7 increased the expression of Sox9, Msx 2, and c-fos during the periods of chondrogenic differentiation after which point their expression decreased. In contrast, Dlx 5 expression was induced by BMP-7 treatment and remained elevated throughout the time-course of skeletal cell differentiation. Runx2/Cbfa1 was not detected by ribonuclease protection assay (RPA) and did not appear to be induced by BMP-7. The sequential nature of differentiation of chondrocytic and osteoblastic cells and the necessity for continuous BMP treatment to maintain skeletal cell differentiation suggests that the maintenance of selective differentiation of the two skeletal cell lineages might be dependent on BMP-7-regulated expression of other morphogenetic factors. An examination of the expression of Wnt, transforming growth factor-beta (TGF-beta), and the hedgehog family of morphogens showed that Wnt 5b, Wnt 11, BMP-4, growth and differentiation factor-1 (GDF-1), Sonic hedgehog (Shh), and Indian hedgehog (Ihh) were endogenously expressed by C3H10T1/2 cells. Wnt 11, BMP-4, and GDF-1 expression were inhibited by BMP-7 treatment in a dose-dependent manner while Wnt 5b and Shh were selectively induced by BMP-7 during the period of chondrogenic differentiation. Ihh expression also showed induction by BMP-7 treatment, however, the period of maximal expression was during the later time-points, corresponding to osteogenic differentiation. An interesting phenomenon was that BMP-7 activity could be further enhanced twofold by growing the cells in a more nutrient-rich media. In summary, the murine mesenchymal stem cell line C3H10T1/2 was induced to follow an endochondral sequence of chondrogenic and osteogenic differentiation dependent on both dose and continual presence of BMP-7 and enhanced by a nutrient-rich media. Our preliminary results suggest that the induction of osteogenesis is dependent on the secondary regulation of factors that control osteogenesis through an autocrine mechanism.
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PMID:BMP treatment of C3H10T1/2 mesenchymal stem cells induces both chondrogenesis and osteogenesis. 1463 86

Exposure to environmental pollutants may severely affect lung growth and development. The present study was designed to test the hypothesis that lung damage caused either by ozone or lipopolysaccharide (LPS) occurs through distinct early responses, which are age dependent in the postnatal lung. C57Bl/6 mice ages 4, 10, and 56 days were exposed to inhalation of LPS with an estimated deposited dose of 26 EU and examined 0.5, 1, or 4 h post inhalation exposure; or to 1 or 2.5 ppm ozone for 4 h or sequential exposures of LPS followed by ozone. Abundance of c-fos, c-jun, interleukin (IL)-1beta, Toll-like receptor (TLR) 2, TLR 4, and tumor necrosis factor (TNF) alpha message levels were measured by RNase protection assay. Exposure to ozone for 4 h induced a c-fos and c-jun response in 4-; 10-; and 56-day-old mice in a dose-dependent manner, was localized to conducting and terminal airways, and also induced TLR 4 message abundance in 10- and 56-day-old mice. Exposure to LPS induced c-fos and c-jun 30 and 60 min postinhalation in 10- and 56-day-old mice only. TLR 2 and 4 message abundance was increased at 10 and 56 days, but was undetectable at 4 days of age, and correlated with proinflamatory message induction. Exposure to LPS followed by ozone increased message abundance of IL-1beta, TNFalpha, TLR 2, TLR 4, and c-jun/c-fos at 10 and 56 days, suggesting that combined exposures that induce cellular stresses can regulate gene expression by activating signaling pathways that operate through both transcription factors activator protein (AP)-1 and nuclear factor (NF)-kappaB. However, only c-jun/c-fos and TNFalpha were elevated in 4-day-old mice after sequential exposures, suggesting that the early activation of the inflammatory response after sequential exposures may occur through a TLR-independent pathway. These results suggest that sequential exposures induce multiple signaling pathways that are age dependent.
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PMID:Postnatal lung development: immediate-early gene responses post ozone and LPS exposure. 1686 5


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