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)

With the ongoing progress in human genome projects, many genes are discovered whose function and/or expression pattern are not known. Most of these genes are expressed in relatively low abundance compared to housekeeping genes such as elongation factor-1alpha and beta-actin. Gene expression is studied by Northern blot assays or by semiquantitative PCR methods. Another method is the visualization of transcripts in tissue or cell cultures by fluorescence in situ hybridization (FISH). However, for low-abundance RNA detection, this method is hampered by its limited detection sensitivity and by the interference of background signals with specific hybridization signals. Background signals are introduced by nonspecific hybridization of probe sequences or nonspecific binding of antibodies used for visualization. To eliminate background signals derived from both sources and to benefit from the peroxidase-driven tyramide signal amplification (TSA), we directly conjugated horseradish peroxidase (HRP) to oligodeoxynucleotides (ODNs) and used these probes to study in the bladder cancer cell line 5637 the expression of various cytokine genes which, according to Northern hybridization and reverse transcriptase-polymerase chain reaction (RT-PCR) assays, are expressed at levels up to 10,000-fold less than abundantly expressed housekeeping genes. The results show that reduction of probe complexity and the limited use of immunocytochemical detection layers strongly reduces noise signals derived from nonspecific binding of nucleic acid probe and antibodies. The use of the HRP-ODNs in combination with TSA allowed detection of low-abundance cytokine mRNAs by FISH.
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PMID:Sensitive mRNA detection by fluorescence in situ hybridization using horseradish peroxidase-labeled oligodeoxynucleotides and tyramide signal amplification. 977 24

We report an RNA targeting strategy, which selectively degrades bcr/abl mRNA in chronic myelogenous leukemia (CML) cells. A 2', 5'-tetraadenylate activator (2-5A) of RNase L was chemically linked to oligonucleotide antisense directed against either the fusion site or against the translation start sequence in bcr/abl mRNA. Selective degradation of the targeted RNA sequences was demonstrated in assays with purified RNase L and decreases of p210(bcr/abl) kinase activity levels were obtained in the CML cell line, K562. Furthermore, the 2-5A-antisense chimeras suppressed growth of K562, while having substantially reduced effects on the promyelocytic leukemia cell line, HL60. Findings were extended to primary CML cells isolated from bone marrow of patients. The 2-5A-antisense treatments both suppressed proliferation of the leukemia cells and selectively depleted levels of bcr/abl mRNA without affecting levels of beta-actin mRNA, determined by reverse transcriptase-polymerase chain reaction (RT-PCR). The specificity of this approach was further shown with control oligonucleotides, such as chimeras containing an inactive dimeric form of 2-5A, antisense lacking 2-5A, or chimeras with altered sequences including several mismatched nucleotides. The control oligonucleotides had either reduced or no effect on CML cell growth and bcr/abl mRNA levels. These findings show that CML cell growth can be selectively suppressed by targeting bcr/abl mRNA with 2-5A-antisense for decay by RNase L and suggest that these compounds should be further explored for their potential as ex vivo purging agents of autologous hematopoietic stem cell transplants from CML patients.
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PMID:2',5'-Oligoadenylate-antisense chimeras cause RNase L to selectively degrade bcr/abl mRNA in chronic myelogenous leukemia cells. 983 40

Currently, fine-needle aspiration cytology is a valuable tool in the routine diagnosis of suspicious thyroid nodules. We present a very sensitive method for the molecular analysis of the expression of several genes important for normal thyroid function in parallel to the cytological diagnosis. We adapted reverse transcriptase polymerase chain reaction (RT-PCR) to amplify thyroid-typical mRNAs in samples of thyroid carcinoma cells as small as those obtained by fine-needle aspiration biopsy (FNAB), ie, 100-1000 cells, and applied this procedure to four routinely taken FNABs. Gene products such as thyroglobulin (Tg), thyroid-stimulating hormone-receptor (TSHr), sodium/iodide-symporter (NIS), type I iodothyronine-5'-deiodinase (DI), and type II iodothyronine-5'-deiodinase (DII) were analyzed. To establish RT-PCR protocols, serial dilutions of follicular thyroid carcinoma cells, FTC-133, which express these genes at low levels, were initially used for RNA isolation. Successful RNA isolation and reverse transcription were checked by the amplification of beta-actin mRNA. We detected the mRNAs coding for Tg in as little as 10 cells, for NIS in 100 cells, and for TSHr, DI, and DII in 10,000 cells. After preparing cytological smears of four routinely taken FNABs, all above-mentioned thyroid-typical mRNAs were observed by using the material remaining in the needle for RNA isolation followed by RT-PCR. This method offers the possibility of obtaining two different types of information from the same routinely taken thyroid FNAB: the cytological diagnosis and the expression pattern of several diagnostically relevant genes. Therefore, a more specific diagnosis could be rendered in the preoperative state, and may lead to more specific therapy.
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PMID:Reverse transcriptase-polymerase chain reaction analysis of thyrocyte-relevant genes in fine-needle aspiration biopsies of the human thyroid. 984 10

We describe a synthetic 769-bp DNA internal standard, GABARQuant 1, for measuring mRNAs of 13 GABA(A) receptor subunits by reverse transcriptase-polymerase chain reaction (RT-PCR). When it is transcribed into cRNA, added in known amounts to target mRNAs in extracts from rat or mouse tissue. competitively reverse transcribed into cDNA, and amplified by the polymerase chain reaction (PCR), the relative intensities of the amplified, stained target and standard DNA bands enable measurement of small amounts of mRNAs for GABA(A) receptor subunits alpha1-6, beta1-3, gamma1-3 and delta and the three cellular markers beta-actin, light neurofilament protein, and glutamine synthetase. For the subunits, most standard products (263-504 bp) differ in size from target products (398-564 bp) by 10-20%. Primer pairs span at least one intron, to prevent interference by genomic DNA, and at least one rat versus mouse restriction fragment length polymorphism (RFLP), to enable rat products to be distinguished from mouse products.
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PMID:A synthetic standard for competitive RT-PCR quantitation of 13 GABA receptor type A subunit mRNAs in rats and mice. 987 45

Alzheimer's disease (AD) has been hypothesized to be associated with oxidative stress. In this study, the expression of key oxidative stress-handling genes was studied in hippocampus, inferior parietal lobule, and cerebellum of 10 AD subjects and 10 control subjects using reverse transcriptase-polymerase chain reaction (RT-PCR). The content of Mn-, Cu,Zn-superoxide dismutases (Mn- and Cu,Zn-SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and glutathione reductase (GSSG-R) mRNAs, and the "marker genes" (beta-actin and cyclophilin) mRNAs was determined. This study suggests that gene responses to oxidative stress can be significantly modulated by the general decrease of transcription in the AD brain. To determine if the particular oxidative stress handling gene transcription was induced or suppressed in AD, the "oxidative stress-handling gene/beta-actin" ratios were quantified and compared with control values in all brain regions studied. The Mn-SOD mRNA/beta-actin mRNA ratio was unchanged in all regions of the AD brain studied, but an increase of the Cu,Zn-SOD mRNA/beta-actin mRNA ratio was observed in the AD inferior parietal lobule. The levels of peroxidation handling (CAT, GSHPx, and GSSG-R) mRNAs normalized to beta-actin mRNA level were elevated in hippocampus and inferior parietal lobule, but not in cerebellum of AD patients, which may reflect the protective gene response to the increased peroxidation in the brain regions showing severe AD pathology. The results of this study suggest that region-specific differences of the magnitude of ROS-mediated injury rather than primary deficits of oxidative stress handling gene transcription are likely to contribute to the variable intensity of neurodegeneration in different areas of AD brain.
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PMID:The expression of key oxidative stress-handling genes in different brain regions in Alzheimer's disease. 1009 42

The Bcl-2 family has been shown to be vital regulators of programmed cell death in numerous systems. To investigate the role of such proteins in the regulation of apoptosis of eosinophils, the expression of Bcl-2 and homologues Bcl-xL (death antagonists), Bax, and Bcl-xS (death agonists) were examined by immunoblot, flow cytometry, and reverse transcriptase-polymerase chain reaction analysis. Potential modulation of apoptosis-associated molecules during spontaneous apoptosis and in the presence of interleukin (IL)-5 was also investigated. Peripheral blood eosinophils were found to express constitutively Bax and Bcl-x, but Bcl-2 was absent. Analysis of mRNA revealed that the bcl-xL isoform predominated, although bcl-xS was also detectable. Spontaneous apoptosis due to culturing in the absence of cytokines for 24 h did not result in modulation of any of the Bcl-2 homologues examined. Culturing eosinophils in the presence of 100 pg/ml IL-5 for 24 h significantly reduced apoptosis (P < 0.01) to 10.7 +/- 2.6% compared with 46.8 +/- 7.4% in the absence of IL-5, and induced Bcl-2 mRNA and protein expression, with no detectable change in Bax, Bcl-x, or beta-actin as a control. This investigation indicates a specific profile of apoptotic molecules in eosinophils distinct from that of neutrophils, and indicates that survival-enhancing IL-5 modulates the expression of Bcl-2 in vitro.
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PMID:Expression of Bcl-2 and its homologues in human eosinophils. Modulation by interleukin-5. 1010 Oct 4

Calpain, a Ca2+-activated cysteine protease, has been implicated in apoptosis of immune cells. Since central nervous system (CNS) is abundant in calpain, the possible involvement of calpain in apoptosis of CNS cells needs to be investigated. We studied calpain expression in rat C6 glioma cells exposed to reactive hydroxyl radical (.OH) [formed via the Fenton reaction (Fe2++H2O2+H+-->Fe3++H2O+.OH)], interferon-gamma (IFN-gamma), and calcium ionophore (A23187). Cell death, cell cycle, calpain expression, and calpain activity were examined. Diverse stimuli induced apoptosis in C6 cells morphologically (chromatin condensation as detected by light microscopy) and biochemically [DNA fragmentation as detected by TdT-mediated dUTP Nick-End Labeling (TUNEL) assay]. Oxidative stress arrested a population of C6 cells at the G2/M phase of cell cycle. The levels of mRNA expression of six genes were analyzed by the reverse transcriptase-polymerase chain reaction (RT-PCR). Diverse stimuli did not alter beta-actin (internal control) expression, but increased calpain expression, and the upregulated bax (pro-apoptotic)/bcl-2 (anti-apoptotic) ratio. There was no significant increase in expression of calpastatin (endogenous calpain inhibitor). Western blot analysis showed an increase in calpain content and degradation of myelin-associated glycoprotein (MAG), a calpain substrate. Pretreatment of C6 cells with calpeptin (a cell-permeable calpain inhibitor) blocked calpain overexpression, MAG degradation, and DNA fragmentation. We conclude that calpain overexpression due to.OH stress, IFN-gamma stimulation, or Ca2+ influx is involved in C6 cell death, which is attenuated by a calpain-specific inhibitor.
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PMID:Diverse stimuli induce calpain overexpression and apoptosis in C6 glioma cells. 1035 May 26

Tissue-specific inactivation of a gene using the Cre-loxP system has been used as an important tool to define its role in which the inactivation of the gene in every cell type results in an embryonic lethality. The expression of Cre recombinase (Cre) can be regulated by controlling the timing or spatial distribution of Cre expression via tissue-specific promoters, ligand-inducible promoters, and ligand-dependent Cre fusion proteins. The rat insulin promoter (RIP) has been used in this study to drive the expression of Cre, specifically in the beta cells. The Cre coding sequence was ligated with the RIP and the isolated RIP-Cre transgene was microinjected into one cell embryo to establish a transgenic mouse line. Tissue specificity of the rat insulin promoter was demonstrated by reverse transcriptase polymerase chain reaction using total RNA from pancreas and other tissues of the RIP-Cre transgenic mice. In addition, the efficiency and specificity of RIP was further analyzed by crossbreeding the RIP-Cre transgenic mice with reporter mice bearing a beta-actin-loxP-CAT-loxP-lacZ transgene. In these mice, lacZ is expressed only after excision of the floxed-CAT gene by Cre-mediated recombination. Here, we present the data for beta cell-specific expression of lacZ in the bigenic mice, as proof of concept in a mouse model for targeting beta cell-specific gene(s). The RIP-Cre transgenic mice will be used as a potential tool for targeting the excision of beta cell-specific gene(s) to study their role in islet cell physiology.
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PMID:Beta cell-specific ablation of target gene using Cre-loxP system in transgenic mice. 1035 20

Direct reverse transcriptase in situ polymerase chain reaction (RT-in situ PCR) of selected mRNA expression in rat mast cells (MC) and alveolar macrophages (AM) was optimized. Rat peritoneal mast cells (PMC), rat cultured mast cells (RCMC), rat bronchoalveolar lavage cells (BALC) or rat cultured alveolar macrophages (NR8383) were studied for the detection of mRNA for beta-actin, TNF-alpha and/or CD8alpha. Each type of cell has unique optimal conditions for RT-in situ PCR. The following parameters were carefully evaluated for optimization: protease digestion, DNAse digestion, heparinase digestion, RT, PCR cycle number and signal development with chromagen. Heparinase digestion was required for PMC mRNA detection because they contain large amounts of heparin proteoglycan, which is a potent inhibitor of RT and Taq polymerase enzymes. Only a few PCR cycles were needed to produce a cytoplasmic signal for mRNA transcripts in RCMC, whereas other types of cells (PMC, BALC and NR8383) needed at least 20 cycles for mRNA detection. The mRNA signal in PMC was localized to the perinuclear region, whereas mRNA in other cell types (RCMC, BALC and NR8383) were detected throughout the cytoplasm. Furthermore, modified Southern blot analysis for TNF-alpha in RCMC treated with RT-in situ PCR demonstrated the specificity of amplification product. The modified and optimized protocols for this procedure were successfully applied to detect and localize several mRNA transcripts in rat MC and AM. The approach is valuable and can be used to further study selected gene expression in these and other cell types.
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PMID:Reverse transcriptase in situ polymerase chain reaction for gene expression in rat mast cells and macrophages. 1041 Sep 80

Coronary arteriosclerosis is an underlying condition in acute myocardial infarction (AMI), unstable angina pectoris (UAP) and stable angina pectoris (SAP), and is also related to restenosis (RS) following coronary intervention. To investigate the pathogenesis of this condition, a quantitative reverse transcriptase polymerase chain reaction was used to determine relative levels of mRNA for interleukin (IL)-1beta, IL-6, IL-8, transforming growth factor beta (TGF-beta), intercellular adhesion molecule (ICAM)-1, E-selectin and vascular cell adhesion molecule (VCAM)-1 using directional coronary atherectomy (DCA) specimens. Eleven patients with AMI, 7 with UAP, 10 with SAP and 6 with RS following a previous coronary intervention underwent DCA. The mRNA intensity for each molecule was expressed by comparing it with that of beta-actin mRNA. The AMI and UAP patients showed high frequencies of mRNA for IL-1beta, IL-8, TGF-beta, and ICAM-1 together with strong intensities of expression, whereas SAP patients showed decreased mRNA expression for these molecules. Increased IL-6 mRNA expression was observed only in AMI samples. Specimens from RS patients revealed an accumulated expression of proinflammatory cytokines, except for IL-6, as well as of TGF-beta. The study suggests that variation in mRNA expression may reflect the pathophysiology of specific types of coronary artery disease, and remodeling following vascular injury.
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PMID:Expression of cytokine and adhesion molecule mRNA in atherectomy specimens from patients with coronary artery disease. 1047 71


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