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:1.14.14.3 (luciferase)
38,195 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

MicroRNA-1 (miR-1) is preferentially expressed in cardiac muscles, and the expression has been demonstrated to be involved in cardiac development and cardiovascular diseases. Here we report that miR-1 is closely related with ischemia/reperfusion injury in a rat model. The level of miR-1 is inversely correlated with Bcl-2 protein expression in cardiomyocytes of the I/R rat model. In vitro, the level of miR-1 was dramatically increased in response to H(2)O(2). Overexpression of miR-1 facilitated H(2)O(2)-induced apoptosis in cardiomyocytes. Inhibition of miR-1 by antisense inhibitory oligonucleotides caused marked resistance to H(2)O(2). Through bioinformatics, we identified the potential target sites for miR-1 on the 3' UTR of Bcl-2. miR-1 significantly reduced the expression of Bcl-2 in the levels of mRNA and protein. The post-transcriptional repression of Bcl-2 was further confirmed by luciferase reporter experiments. These data demonstrated that miR-1 plays an important role in the regulation of cardiomyocyte apoptosis, which is involved in post-transcriptional repression of Bcl-2.
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PMID:MicroRNA-1 regulates cardiomyocyte apoptosis by targeting Bcl-2. 1950 41

Ischemia is a common underlying factor in a number of pathologic conditions ranging from cardiac dysfunction to delayed wound healing. Previous efforts have shown the resulting hypoxia activates the hypoxia inducible factor, a transcription factor with signaling effects through an intranuclear hypoxia response element (HRE). We hypothesized that ischemic conditions should activate these hypoxic signaling pathways in a measurable manner. We tested our hypothesis using variations of an established rabbit ear ischemic wound model and an HRE-luciferase-reporter gene construct. This plasmid construct was transfected into the ears of young, female New Zealand White rabbits, harvested at day 7 and processed to yield a reactive solution. Luminometry was used to quantify luciferase expression in each solution as a marker for HRE activation in each wound. Quantitative readings of hypoxic signaling as measured by luminescence yielded profound and statistically significant differences between the various ischemic models. Our results suggest that the biologic systems for hypoxic signaling can be used to detect local ischemia. HRE-luciferase transfection is an effective tool for quantifying the degree of tissue hypoxia. The caudal ischemic rabbit ear model showed significantly higher levels of hypoxia. Use of a validated model that produces sufficient tissue levels of hypoxia is recommended for meaningful study of ischemic wound healing.
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PMID:Quantifying tissue level ischemia: hypoxia response element-luciferase transfection in a rabbit ear model. 1961 11

Expression and activity of the germinal center kinase [corrected] SLK are increased during kidney development and recovery from renal ischemia-reperfusion injury. SLK promotes apoptosis, in part, via pathway(s) involving apoptosis signal-regulating kinase-1 and p38 mitogen-activated protein kinase. This study addresses the role of p53 as a potential effector of SLK. p53 transactivation was measured after transient transfection of a luciferase reporter plasmid that contains a p53 cis-acting enhancer element. Overexpression of SLK in COS-1 cells and cotransfection of SLK and p53-wild type (wt) cDNAs in glomerular epithelial cells (GECs) stimulated p53 transactivational activity, as measured by a p53 response element-driven luciferase reporter. In GECs, chemical anoxia followed by glucose reexposure (in vitro ischemia-reperfusion) increased p53 reporter activity, and this increase was amplified by overexpression of SLK. Expression of SLK induced p53 phosphorylation on serine (S)-33 and S315. In GECs, cotransfection of SLK with p53-wt, p53-S33A, p53-S315A, or p53-S33A+S315A mutants showed that only the double mutation abolished the SLK-induced increase in p53 reporter activity. SLK-induced stimulation of p53 reporter activity was attenuated by inhibition of JNK. Overexpression of SLK amplified apoptosis induced by subjecting cells to in vitro ischemia-reperfusion injury, while ectopic expression of a dominant negative SLK mutant attenuated the ischemia-reperfusion-induced apoptosis. The p53 transactivation inhibitor pifithrin-alpha significantly attenuated the amount of apoptosis after ischemia-reperfusion and SLK overexpression. Thus SLK induces p53 phosphorylation and transactivation, which enhances apoptosis after in vitro ischemia-reperfusion injury.
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PMID:The Ste20-like kinase SLK promotes p53 transactivation and apoptosis. 1964 Aug 99

Intracellular calcium ion concentration ([Ca2+]i) is a key regulator of ischemia-reperfusion injury (IRI) and ischemic preconditioning (IPC), with myocardial sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) acting as the major regulatory protein in the control of [Ca2+]i. In this study, we examined the effect of the SERCA2a promoter in IRI and IPC. The SERCA2a promoter fragment was acquired using PCR and molecular cloning technology, and was inserted into a luciferase reporter gene vector to construct reporter gene plasmids. Primary cultured neonatal rat cardiomyocytes were treated by analog ischemic treatment to produce in vitro models of IRI and IPC. Using an in vitro gene transfer assay, SERCA2a transcriptional activity was measured under conditions of IRI and IPC. Results from this study showed that (1) short duration analog ischemia treatment (analog IPC) results in a significant decrease in transcriptional activity, (2) analog IRI caused a dramatic depression in SERCA2a gene expression in transcription initiation levels, and (3) analog IPC attenuated IRI-induced transcriptional depression, even though IPC itself significantly depressed transcriptional activity. In conclusion, the SERCA2a promoter plays a significant role in IRI and IPC, with IPC able to precondition the SERCA2a promoter against the deleterious effects of IRI-induced injury.
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PMID:The role of the rat sarcoplasmic reticulum Ca2+-ATPase promoter in myocardial ischemia-preconditioning. 1971 49

MicroRNAs (miRs) participate in most cellular functions by posttranscriptional regulation of gene expression albeit with little information regarding their role in ischemic preconditioning (IP) of stem cells. We report that IP of bone marrow-derived mesenchymal stem cells (MSCs) with two cycles of 30-min ischemia/reoxygenation (I/R) supported their survival under subsequent longer exposure to anoxia and following engraftment in the infarcted heart. IP significantly reduced apoptosis in MSCs through activation of Akt (Ser(473)) and ERK1/2 (Thr(202)/Tyr(204)) and nuclear translocation of hypoxia-inducible factor-1alpha (HIF-1alpha). We observed concomitant induction of miR-210 in the preconditioned MSCs ((PC)MSCs). Inhibition of HIF-1alpha or of miR-210 abrogated the cytoprotective effects of preconditioning. Extrapolation of these data to in vivo studies in a rat model of acute myocardial infarction predominantly improved stem cell survival after engraftment with a role for miR-210. Notably, multiple I/R cycles more effectively regulated the miR-210 and hence promoted MSC survival compared with single-cycle hypoxia of an equal duration. Real time PCR array for rat apoptotic genes, computational target gene analyses, and luciferase reporter assay identified FLICE-associated huge protein (FLASH)/caspase-8-associated protein-2 (Casp8ap2) in (PC)MSCs as the target gene of miR-210. Induction of FLASH/CASP8AP2 in miR-210 knocked-down (PC)MSCs resulted in increased cell apoptosis. Taken together, these data demonstrated that cytoprotection afforded by IP was regulated by miR-210 induction via FLASH/Casp8ap2 suppression. These results highlighted that IP by multiple short episodes of I/R is a novel strategy to promote stem cell survival.
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PMID:Ischemic preconditioning augments survival of stem cells via miR-210 expression by targeting caspase-8-associated protein 2. 1972 Nov 36

Astrocytes are essential cells for maintaining brain integrity. We have recently shown that the transcription factor C/EBP homologous protein (CHOP), associated with endoplasmic reticulum (ER) stress, plays a key role in the astrocyte death induced by ischemia. Meanwhile, mediators of apoptosis downstream of CHOP in the ER stress-dependent pathway remain to be elucidated. Our aim in this work was to determine whether caspase-11, able to activate apoptotic and proinflammatory pathways, is implicated in ER stress-dependent astrocyte death in ischemic conditions. According to our results, caspase-11 is up-regulated in primary astrocyte cultures following either oxygen and glucose deprivation (OGD) or treatment with the ER-stress inducers thapsigargin and tunicamycin. Moreover, these same stimuli increased caspase-11 mRNA levels and luciferase activity driven by a caspase-11 promoter, indicating that caspase-11 is regulated at the transcriptional level. Our data also illustrate the involvement of ER stress-associated CHOP in caspase-11 regulation, insofar as CHOP overexpression by means of an adenoviral vector caused a significant raise in caspase-11. In turn, caspase-11 suppression with siRNA rescued astrocytes from OGD- and ER stress-induced death, supporting the idea that caspase-11 is responsible for the deleterious effects of ischemia on astrocytes. Finally, inhibition of caspase-1 and caspase-3 significantly reduced astrocyte death, which indicates that these proteases act as death effectors of caspase-11. In conclusion, our work contributes to clarifying the pathways leading to astrocyte death in response to ischemia by defining caspase-11 as a key mediator of the ER stress response acting downstream of CHOP.
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PMID:Caspase-11 mediates ischemia-induced astrocyte death: involvement of endoplasmic reticulum stress and C/EBP homologous protein. 1989 Sep 20

Mu-opioid receptor expression increases during neurogenesis, regulates the survival of maturing neurons and is implicated in ischemia-induced neuronal death. The repressor element 1 silencing transcription factor (REST), a regulator of a subset of genes in differentiating and post-mitotic neurons, is involved in its transcriptional repression. Extracellular signaling molecules and mechanisms that control the human mu-opioid receptor (hMOR) gene transcription are not clearly understood. We examined the role of protein kinase C (PKC) on hMOR transcription in a model of neuronal cells and in the context of the potential influence of REST. In native SH-SY5Y neuroblastoma cells, PKC activation with phorbol 12-myristate 13-acetate (PMA, 16 nM, 24h) down-regulated hMOR transcription and concomitantly elevated the REST binding activity to repressor element 1 of the hMOR promoter. In contrast, PMA activated hMOR gene transcription when REST expression was knocked down by an antisense strategy or by retinoic acid-induced cell differentiation. PMA acts through a PKC-dependent pathway requiring downstream MAP kinases and the transcription factor AP-1. In a series of hMOR-luciferase promoter/reporter constructs transfected into SH-SY5Y cells and PC12 cells, PMA up-regulated hMOR transcription in PC12 cells lacking REST, and in SH-SY5Y cells either transfected with constructs deficient in the REST DNA binding element or when REST was down-regulated in retinoic acid-differentiated cells. These findings help explain how hMOR transcription is regulated and may clarify its contribution to epigenetic modifications and reprogramming of differentiated neuronal cells exposed to PKC-activating agents.
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PMID:Transcription factor REST negatively influences the protein kinase C-dependent up-regulation of human mu-opioid receptor gene transcription. 1991 83

Bone marrow-derived mesenchymal stem cells (MSCs) are multipotent and secrete angiogenic factors, which could help patients with occlusive arterial diseases. We hypothesize that MSCs, in comparison to fibroblasts, survive better under hypoxic conditions in vitro and in vivo. MSCs and fibroblasts from L2G mice expressing firefly luciferase and GFP were cultured in normoxic and hypoxic conditions for 24 hours. In vitro cell viability was tested by detecting apoptosis and necrosis. MSCs released higher amounts of VEGF (281.1 +/- 62.6 pg/ml) under hypoxic conditions compared to normoxia (154.9 +/- 52.3 pg/ml, p = NS), but were less tolerant to hypoxia (45 +/- 7.9%) than fibroblasts (28.1 +/- 3.6%, p = NS). A hindlimb ischemia model was created by ligating the femoral artery of 18 FVB mice. After one week, 1 x 106 cells (MSCs, fibroblasts or saline) were injected into the limb muscles of each animal (n = 6 per group). Bioluminescence measurement to assess the viability of luciferase positive cells showed significant proliferation of MSCs on day four compared to fibroblasts (p = 0.001). Three weeks after cell delivery, the capillary to muscle fiber ratio of ischemic areas was analyzed. In the MSC group, vessel density was significantly higher than in the fibroblast or control group (0.5 +/- 0.08 and 0.3 +/- 0.03). Under hypoxia, MSCs produced more VEGF compared to normal conditions and MSC transplantation into murine ischemic limbs led to an increase in vessel density, although MSC survival was limited. This study suggests that MSC transplantation may be an effective and clinically relevant tool in the therapy of occlusive arterial diseases.
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PMID:Angiogenic effects despite limited cell survival of bone marrow-derived mesenchymal stem cells under ischemia. 2037 63

Small molecules inhibiting hypoxia inducible factor (HIF) prolyl hydroxylases (PHDs) are the focus of drug development efforts directed toward the treatment of ischemia and metabolic imbalance. A cell-based reporter produced by fusing HIF-1 alpha oxygen degradable domain (ODD) to luciferase was shown to work as a capture assay monitoring stability of the overexpressed luciferase-labeled HIF PHD substrate under conditions more physiological than in vitro test tubes. High throughput screening identified novel catechol and oxyquinoline pharmacophores with a "branching motif" immediately adjacent to a Fe-binding motif that fits selectively into the HIF PHD active site in in silico models. In accord with their structure-activity relationship in the primary screen, the best "hits" stabilize HIF1 alpha, upregulate known HIF target genes in a human neuronal line, and exert neuroprotective effects in established model of oxidative stress in cortical neurons.
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PMID:Utilization of an in vivo reporter for high throughput identification of branched small molecule regulators of hypoxic adaptation. 2041

Among the identified microRNAs (miRs) thus far, ~50% of mammalian miRs are clustered in the genome and transcribed as polycistronic primary transcripts. However, whether clustered miRs mediate non-redundant and cooperative functions remains poorly understood. In this study, we first identified activation of the promoter of miR-144/451 by GATA-4, a critical transcription factor in the heart. Next, we observed that ectopic expression of miR-144 and -451 individually augmented cardiomyocyte survival, which was further improved by overexpression of miR-144/451, compared to control cells in response to simulated ischemia/reperfusion. In contrast, knockdown of endogenous miR-144 and -451 revealed opposite effects. Using luciferase reporter assay and western blot analysis, we also validated that both miR-144 and miR-451 target CUG triplet repeat-binding protein 2 (CUGBP2), a ubiquitously expressed RNA-binding protein, known to interact with COX-2 3'UTR and inhibit its translation. Accordingly, protein levels of CUGBP2 were greatly reduced and COX-2 activity was markedly increased in miR-144-, miR-451-, and miR-144/451-overexpressing cardiomyocytes, compared to GFP cells. Furthermore, inhibition of COX-2 activity by either NS-398 or DUP-697 partially offset protective effects of the miR-144/451 cluster. Together, these data indicate that both partners of the miR-144/451 cluster confer protection against simulated I/R-induced cardiomyocyte death via targeting CUGBP2-COX-2 pathway, at least in part. Thus, both miR-144 and miR-451 may represent new therapeutic agents for the treatment of ischemic heart disease.
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PMID:Synergistic effects of the GATA-4-mediated miR-144/451 cluster in protection against simulated ischemia/reperfusion-induced cardiomyocyte death. 2070 14


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