Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.49 (reverse transcriptase)
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Aquaporins (AQPs) are a family of water channel proteins that assist in maintenance of the cellular osmotic environment and whole body fluid balance. Specialized organ-specific AQPs are important in physiologic and pathologic processes but little is known about AQPs in the human heart. AQP1 has been identified in rodent heart. We investigated the presence and localization of AQP1 in human heart and skeletal muscle using immunohistochemistry and confocal microscopy, western blot and reverse transcriptase-polymerase chain reaction. There was abundant AQP1 present in both cardiac and skeletal muscle. Immunohistochemistry revealed co-localization of AQP1 with vinculin, a t-tubule marker, and caveolin-3. No novel sequences bearing an NPA box motif common to other AQPs were identified in human heart using degenerative PCR analysis. We conclude that AQP1 is present in the human heart. AQP1 co-localizes with t-tubular and caveolar proteins. Cardiac AQPs may have a role during osmotic stresses including ischemia/reperfusion and cardiopulmonary bypass.
J Mol Cell Cardiol 2004 May
PMID:Expression of aquaporin 1 in human cardiac and skeletal muscle. 1513 60

Glutamate is the only amino acid extracted by healthy myocardium in net amounts, with uptake further increased during hypoxic or ischemic conditions. Glutamate supplementation provides cardioprotection from hypoxic and reperfusion injury through several metabolic pathways that depend upon adequate transport of glutamate into the mitochondria. Glutamate transport across the inner mitochondrial membrane is a key component of the malate/aspartate shuttle. Glutamate transport in the brain has been well characterized since the discovery of the excitatory amino acid transporter (EAAT) family. We hypothesize that a protein similar to EAAT1 found in brain may function as a glutamate transporter in cardiac mitochondria. Rat heart total RNA was screened by reverse transcriptase-polymerase chain reaction with an array of primer pairs derived from the rat brain EAAT1 cDNA sequence, yielding a 3786-bp cDNA comprising a 1638-bp open reading frame identical to rat brain EAAT1 with flanking 5'- and 3'-untranslated regions. Northern blot analysis confirmed a 4-kb mRNA product in rat heart and brain, with greater abundance in brain. A protein of the predicted approximate 60-kD size was recognized in myocardial lysates by an anti-EAAT1 polyclonal antibody produced against an amino-terminal peptide from human EAAT1. The protein enriched in rat heart mitochondria by immunoblot, co-localized with the mitochondrial protein cytochrome c by immunohistochemistry, and further localized to the inner mitochondrial membrane upon digitonin fractionation of the mitochondria. In myocytes overexpressing EAAT1, activity of the malate/aspartate shuttle increased by 33% compared to non-transfected cells (P = 0.004). These data indicate that EAAT1 is expressed in myocardial mitochondria, and functions in the malate/aspartate shuttle, suggesting a role for EAAT1 in myocardial glutamate metabolism.
J Mol Cell Cardiol 2004 Jul
PMID:Localization and function of the brain excitatory amino acid transporter type 1 in cardiac mitochondria. 1524 29

Gene therapy is emerging as a realistic addition to the therapeutic arsenal in heart failure, but the search for suitable vectors for cardiac transfection is still ongoing. In this study, we explore the applicability of recombinant Semliki Forest virus (SFV) in heart failure. SFV was intracoronarily delivered 2 weeks after induction of myocardial infarction in the rat model for heart failure. Duration of SFV expression was determined, and tissue distribution was studied by histochemical, biochemical, and reverse transcriptase-polymerase chain reaction (RT-PCR) analyses. Expression of SFV-mediated transfection in the heart reached its maximum after 48-72 h and subsided within a week. Intracoronary administration of SFV efficiently transfected the non-infarcted cardiac wall, resulting in high levels of beta-galactosidase (beta-gal) activity (1337 +/- 537 IU/mg) and lacZ RNA in the hearts of all rats, whereas brain, kidney, liver, lung, spleen, and testis were lacZ negative. In conclusion, intracoronarily delivered SFV has a favourable distribution pattern, showing expression of the transgene restricted to the heart.
J Mol Cell Cardiol 2004 Jul
PMID:Semliki Forest virus is an efficient and selective vector for gene delivery in infarcted rat heart. 1524 44

cDNA expression arrays were used to identify mRNA expression markers for cardiac myxoma. The RNA profile analysis suggests that cardiac myxoma should be considered as a stand-alone tissue rather than a pathological modification of particular normal tissue. The analysis reveals a set of genes which are highly and steadily expressed in cardiac myxomas and can serve as an mRNA expression markers of the tumour. Marker status of selected genes was confirmed by reverse transcriptase polymerase chain reaction analysis. Genes MIA (melanoma inhibitory activity) and PLA2G2A (phospholipase A2, group IIA) show the highest specificity as cardiac myxoma markers, since they have more than 10-fold higher RNA level in cardiac myxomas than in any one of 15 normal tissues tested. Among markers of myxoma at least three are participants of phospholipid metabolism: ANXA3, PLA2G2A, and phospholipid transfer protein. Tissue inhibitor of metalloproteinase 1 and secretory leucocyte protease inhibitor are inhibitors of proteases degrading extracellular matrix proteins and participating in cell proliferation regulation. MIA, SPP1, fibromodulin are modulators or participants of the interaction between extracellular matrix proteins and their cell surface receptors. SOX9 is a transcription factor required for chondrocyte differentiation. Calretenin (CALB2) is an intracellular calcium-binding protein with poorly understood function.
J Mol Cell Cardiol 2004 Sep
PMID:Gene expression analysis to identify mRNA markers of cardiac myxoma. 1535 Aug 45

Functional ATP-sensitive potassium (K(ATP)) channels can be reconstituted by expression of various combinations of different pore-forming subunits (Kir6.1 and Kir6.2) and sulfonylurea receptor (SUR) subunits. Using dominant negative and gene knockout approaches, Kir6.2 subunits have been identified as required pore-forming components of plasmalemmal K(ATP) channels in ventricular myocytes. Previous data obtained in heterologous expression systems suggest that Kir6.1 and Kir6.2 subunits are capable of forming a functional heteromultimeric channel complex. However, until now the existence of such heteromultimeric Kir6.1/Kir6.2 complexes has not been demonstrated for native K(ATP) channels. The primary aim of this study was to identify the molecular composition of native K(ATP) channels in primary human coronary artery endothelial cells (HCAEC) and smooth muscle cells (HCASMC) from human origin. We specifically investigated the potential that heteromultimeric Kir6.1/Kir6.2 channels exist in these cells. Using reverse transcriptase-polymerase chain reaction, we detected the expression of Kir6.1, Kir6.2, and SUR2B in both cell types. Western blotting and immunoprecipitation assays demonstrated the presence of Kir6.1 protein in both HCAEC and HCASMC; however, Kir6.2 protein was only expressed in HCAEC. Interaction between Kir6.1 and Kir6.2 subunits was demonstrated by reciprocal co-immunoprecipitation of these two subunits in HCAEC. Furthermore, Kir6.1 and Kir6.2 were detected in the immunoprecipitate when using an anti-SUR2 antibody. Confocal microscopy imaging demonstrated Kir6.1 and Kir6.2 subunits to co-localize at the cell surface membrane in HCAEC. In conclusion, our data characterize the molecular composition of primary human coronary smooth muscle and endothelial cells. We demonstrate that human coronary endothelial K(ATP) channels consist of a heteromultimeric complex of Kir6.1, Kir6.2, and SUR2B subunits.
J Mol Cell Cardiol 2004 Oct
PMID:K ATP channels of primary human coronary artery endothelial cells consist of a heteromultimeric complex of Kir6.1, Kir6.2, and SUR2B subunits. 1538 Jun 76

The very low-density lipoprotein (VLDL) receptor is a member of the low-density lipoprotein (LDL) receptor gene family with distinct tissue distribution and function. VLDL receptors are also expressed in vascular smooth muscle cells (VSMCs) and have been shown to be upregulated in atherosclerotic lesions. In the present study, we examined the effects of interleukin-1beta (IL-1beta) on the uptake of betaVLDL and its receptor expression in rat VSMCs. IL-1beta downregulated expression of the VLDL receptor in a time and dose-dependent manner as shown by Western blotting, Northern blotting, and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Treatment with IL-1beta significantly reduced the uptake of beta-VLDL but not LDL in VSMCs. Use of specific pharmacologic inhibitors indicated that the tyrosine kinase inhibitors, herbimycin A and geldanamycin, completely reversed IL-1beta-induced downregulation of the VLDL receptor expression. Another tyrosine kinase inhibitor, genistein, the protein kinase C inhibitors, GF109203X and H7, the mitogen-activated protein (MAP) kinase inhibitors (MEK inhibitor PD098059 for [MEK] and SB203580 for p38-MAP kinase), and the protein kinase A inhibitor, KT5270 all had no effect on receptor expression. In addition, the c-Src specific inhibitor PP2 or adenoviral-mediated gene transfer of kinase inactive (KI)-c-Src failed to reverse IL-1beta-induced downregulation of VLDL receptor expression. These results indicate that IL-1beta attenuates uptake of VLDL through downregulation of its receptor in VSMCs, and that this downregulation is mediated through a benzoquinone ansamycin-dependent but c-Src-independent pathway.
J Mol Cell Cardiol 2005 Apr
PMID:Interleukin-1beta attenuates beta-very low-density lipoprotein uptake and its receptor expression in vascular smooth muscle cells. 1580 40

Endogenous oxidative stress is a likely cause of cardiac myocyte death in vivo. We examined the early (0-2 h) changes in the proteome of isolated cardiac myocytes from neonatal rats exposed to H2O2 (0.1 mM), focussing on proteins with apparent molecular masses of between 20 and 30 kDa. Proteins were separated by two-dimensional gel electrophoresis (2DGE), located by silver-staining and identified by mass spectrometry. Incorporation of [35S]methionine or 32Pi was also studied. For selected proteins, transcript abundance was examined by reverse transcriptase-polymerase chain reaction. Of the 38 protein spots in the region, 23 were identified. Two families showed changes in 2DGE migration or abundance with H2O2 treatment: the peroxiredoxins and two small heat shock protein (Hsp) family members: heat shock 27 kDa protein 1 (Hsp25) and alphaB-crystallin. Peroxiredoxins shifted to lower pI values and this was probably attributable to 'over-oxidation' of active site Cys-residues. Hsp25 also shifted to lower pI values but this was attributable to phosphorylation. alphaB-crystallin migration was unchanged but its abundance decreased. Transcripts encoding peroxiredoxins 2 and 5 increased significantly. In addition, 10 further proteins were identified. For two (glutathione S-transferase pi, translationally-controlled tumour protein), we could not find any previous references indicating their occurrence in cardiac myocytes. We conclude that exposure of cardiac myocytes to oxidative stress causes post-translational modification in two protein families involved in cytoprotection. These changes may be potentially useful diagnostically. In the short term, oxidative stress causes few detectable changes in global protein abundance as assessed by silver-staining.
J Mol Cell Cardiol 2006 Jan
PMID:Effects of oxidative stress on the cardiac myocyte proteome: modifications to peroxiredoxins and small heat shock proteins. 1632 8

A method is described for an aptamer-based affinity assay using a combination of two nonconventional techniques, temperature gradient focusing (TGF) and field-amplified continuous sample injection TGF (FACSI-TGF), with fluorescence detection. Human immunodeficiency virus reverse transcriptase (HIVRT) is used as the protein target for the assay. The TGF and FACSI-TGF assays are compared to similar results obtained with conventional CE. A range of starting aptamer concentrations are used to determine the optimal LOD for human immunodeficiency virus reverse transcriptase (HIVRT) using each approach. The results indicate that the LODs for HIVRT obtained with TGF and FACSI-TGF are comparable to or even lower than the LODs obtained with conventional CE in spite of the inferior detector used for the TGF and FACSI-TGF assays (arc lamp and low-cost CCD for TGF versus LIF with PMT for CE). It is hypothesized that this is due to the greater reproducibility of the TGF and FACSI-TGF techniques since they do not employ a defined sample injection. The lowest LOD achieved with the new aptamer assay approach is more than an order of magnitude lower than that reported for a similar CE-based aptamer assay for the same target.
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PMID:Development of aptamer-based affinity assays using temperature gradient focusing: minimization of the limit of detection. 1864 83

Receptor for advanced glycation end products (RAGE) and connective tissue growth factor (CTGF) play a key role in diabetic myocardial fibrosis, and peroxisome proliferator-activated receptor-gamma (PPAR-gamma) activation has been reported to reduce RAGE and CTGF expression. This study investigated the effects of the PPAR-gamma agonist, rosiglitazone, on myocardial expression of RAGE and CTGF, extent of cardiac fibrosis, and left ventricular (LV) diastolic function in type 2 diabetic (T2D) rats. Twenty-week-old T2D rats were randomized to treatment with either 20 weeks of rosiglitazone (20 mg/kg) or saline (n = 10 in each group). Serial echocardiographic examinations were performed just before randomization (20 weeks) and at study completion (40 weeks). Fibrosis extent and RAGE and CTGF expression were assessed in previously imaged hearts by picrosirius red staining, and by real-time reverse transcriptase-coupled polymerase chain reaction (RT-PCR) and immunoblotting, respectively. Results of the latter assessments were further validated by immunohistochemical staining. Rosiglitazone treatment significantly improved E/A ratio in serial echocardiography assessment, and reduced LV collagen volume fraction as demonstrated by picrosirius red staining. Real-time RT-PCR and immunoblots of myocardial tissue from rosiglitazone-treated rats revealed reduced RAGE and CTGF mRNA and protein signals compared to those of saline-treated T2D rats, which were consistent with reduced proportions of myocardial RAGE and CTGF staining in the hearts of T2D rats. PPAR-gamma agonist therapy reduces cardiac fibrosis and improves LV diastolic dysfunction as assessed by serial echocardiographic imaging. Suppression of RAGE and CTGF expression in the diabetic myocardium appears to contribute to the antifibrotic effect of rosiglitazone. These results support the potential of PPAR-gamma agonists as antifibrotic agents in diabetic cardiomyopathy.
Basic Res Cardiol 2010 May
PMID:Peroxisome proliferator-activated receptor-gamma activation attenuates cardiac fibrosis in type 2 diabetic rats: the effect of rosiglitazone on myocardial expression of receptor for advanced glycation end products and of connective tissue growth factor. 1990 20

The 10-23 DNA enzyme (10-23 DNAzyme), a single-stranded DNA (ssDNA) molecule, can efficiently and specifically cleave almost any target RNA molecules. Therefore, it is regarded as one of the promising tools in gene therapy. However, there are still some obstacles, such as low efficiency of cellular uptake and instability in vivo, in its application. Taking advantage of the mechanism of Moloney mouse leukemia virus (MMLV) reverse transcriptase (RT), we investigate the construction of a novel ssDNA expression vector in this study. In order to improve the expression efficiency, the mmlv-rt gene and ODN-PMT (an oligodeoxynucleotide including other essential sequences for generating ssDNA) were cloned into a single plasmid under the control of 2 separated promoters. The ability of the vector to generate specific 10-23 DNAzyme in mammalian cell was tested by constructing a tryptophan-aspartate-containing coat protein (taco) gene-specific 10-23 DNAzyme expression plasmid. The potential of the expressed 10-23 DNAzyme to suppress TACO expression was also investigated. Our results indicated that this vector generates desired 10-23 DNAzyme in mammalian cells. The expressed 10-23 DNAzyme targeting taco gene can reduce TACO expression both at mRNA level (by 78.26%) and at protein level (by 75.30%).
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PMID:The 10-23 DNA enzyme generated by a novel expression vector mediate inhibition of taco expression in macrophage. 2005 15


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