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)

The reverse transcription polymerase chain reaction (RT-PCR) is one of the most useful molecular biology methods in opening the way to understanding of the mechanisms of atherosclerosis on the gene structure and/or expression level. We optimized this technique for assaying expression of the monocyte chemotactic protein type 1 (MCP-1) gene in rabbit aorta with respect to the temperature profile, yield to cycle number, interference of genomic DNA with the RNA matrix, and repeatability. Variability of expression of the constitutive GAPDH gene was also examined. The study was done in 18 New Zealand rabbits allocated to two groups and fed a standard chow for 2 (S1) or 3 (S2) months. The experiment ended with removal of part of the ascending rabbit aorta, from which RNA was isolated. The optimal temperature for binding of specific primers to the MCP-1 and GAPDH genes was 63 degrees C, and the optimal number of cycles for PCR amplification was 22 for MCP-1 and 26 for GAPDH. The GAPDH amplicon size was 465 base pairs in the presence or absence of reverse transcriptase showing contamination of the RNA matrix with genomic DNA. Repeatability of the RT-PCR method was 8.7%, and variability of expression of the GAPDH gene was 7.7%. Thus, RT-PCR adjusted for contaminating genomic DNA provides a reliable way of assaying expression of the MCP-1 gene in rabbit aorta.
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PMID:Optimized RT-PCR method for assaying expression of monocyte chemotactic protein type 1 (MCP-1) in rabbit aorta. 1678 99

Phospholipase A(2) (PLA(2)) is a superfamily of enzymes that may play a major role in airways inflammation. We investigated the effect of interferon-gamma (IFN-gamma) on the gene expression of 19 different PLA(2) types in human monocyte-derived macrophages and nasal epithelial cells (RPMI 2650). The cells were stimulated with IFN-gamma for different lengths of time (up to 48 h), and the mRNA levels of the different PLA(2) types were determined by reverse transcriptase-PCR (RT-PCR) and normalized to those of the house-keeping gene, GAPDH. It appeared that IFN-gamma clearly increased the expression of secretory PLA(2) IID (but not IIA) in macrophages, while both PLA(2) IID and IIA were upregulated in RPMI 2650 cells. Moreover, after 18 h, the mRNA levels of cytosolic PLA(2) IVA were 2-3 times higher in IFN-gamma-stimulated macrophages than controls, while there was no such effect of IFN-gamma in RPMI 2650 cells. Lipopolysaccharide (LPS) augmented the increased gene expression of PLA(2) IVA but decreased both the basal and the IFN-gamma-induced PLA(2) IID mRNA expression in macrophages (but not in RPMI 2650 cells). The NF-kappaB inhibitor Pyrrolidine dithiocarbamate (PDTC) and the phoshatidylinositol 3-kinase (PI3K) inhibitor wortmannin were employed to get an insight into the mechanism behind these observations. Incubation of macrophages with PDTC had no effect on the LPS impairment of PLA(2) IID gene expression, but inhibited the LPS mediated activation of PLA(2) IVA. No significant effect was noted of PDTC on IFN-gamma stimulation, while PI3K had no effect at all on any of the stimuli used. Furthermore, LPS (but not IFN-gamma) increased the mRNA levels of the nuclear factor (NF)-kappaB inhibitors alpha and xi in macrophages, but not in RPMI 2650 cells. These findings indicate that (a) the gene expression of secretory types PLA(2) IID and IIA in response to IFN-gamma is much dependent on cell type, and (b) the regulation of PLA(2) type IID in human macrophages is clearly different from that of PLA(2) type IVA. (c) PLA(2) IVA is probably under control of both NF-kappaB and IFN-gamma-responsive elements (GRE) or IFN-gamma-activating sites (GAS). The possibility that PLA(2) IID is involved in cytokine-mediated inflammation in the nasal mucosa is inferred, as is the potential role of PLA(2) IID in the host defense against LPS-containing bacteria.
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PMID:Interferon gamma-induced gene expression of the novel secretory phospholipase A2 type IID in human monocyte-derived macrophages is inhibited by lipopolysaccharide. 1689 54

Tissue engineering of articular cartilage usually requires the isolation and culture of chondrocytes. Previous studies have suggested that enzymatic isolation may alter the metabolic activity and growth rate of chondrocytes. This study examined the effects of 4 common isolation protocols on chondrocyte gene expression, morphology, and total cell yield immediately following the digest (t = 0) and after 2 culture periods (24 h and 1 week). Cartilage explants were digested using 1 of 4 protocols: (1) 6-h collagenase digest, (2) 22-h collagenase digest, (3) 45-min trypsin digest followed by a 3-h collagenase digest, or (4) 1.5-h pronase digest followed by a 3-h collagenase digest. Gene expression levels for glyceraldehyde-3-phosphate dehydrogenase, type I collagen, type II collagen, aggrecan, superficial zone protein, matrix metalloproteinase- 1, and tissue inhibitor of metalloproteinase-1 were measured at t = 0 h, 24 h, and 1 week using quantitative reverse transcriptase-polymerase chain reaction. In this study, cell yield was greatest for the 22-h collagenase and pronase-collagenase digests. However, the data indicate that a 6-h collagenase digest has the fewest gene expression changes compared to native cells. For tissue engineering, data from this study suggest that when cell yield is critical, a 22-h collagenase digest is preferable, but when obtaining cells closest to native chondrocytes is more desired, the 6-h collagenase digest is more beneficial.
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PMID:The effects of isolation on chondrocyte gene expression. 1699 90

A subtracted cDNA library was used to identify specific genes that increase in post-mortem muscle of rainbow trout (Oncorhynchus mykiss) during on-ice storage. Of the 200 cDNAs analyzed, 82 had significant homologies to previously identified genes from salmonids and other species such as homologues of troponin I, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and so on, whereas 40 had no significant homologies and were designated as unknown. Comparison of gene expression profiles by dot blot hybridization confirmed an increase or induction of mRNA in the muscle after 3 h of on-ice storage compared to that at 0 h after death. Real-time reverse transcriptase-Polymerase Chain Reaction analysis showed that troponin I and GAPDH mRNAs were increased by 24 h and, in particular, that the change in troponin I mRNA was greater than that of GAPDH mRNA. These results suggest that the increased mRNAs in rainbow trout muscle occurred by transcription immediately after death.
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PMID:Post-mortem changes in gene expression of the muscle tissue of rainbow trout, Oncorhynchus mykiss. 1714 27

Endothelium-derived nitric oxide (NO) plays an important role in the prevention of platelet aggregation and adhesion to the vascular wall. Endothelial nitric oxide synthase (eNOS) and L-arginine/NO pathway are both present in human platelets. Platelet-derived NO inhibits excessive activation and aggregation of platelets. However, the expression level of the eNOS gene in human platelets has yet to be elucidated. The current study investigates the individual expression level of platelet eNOS mRNA using the real-time reverse transcriptase-polymerase chain reaction (RT-PCR) detection method. eNOS mRNA expression was examined in platelets isolated from 50 subjects: 11 male smokers, 15 male nonsmokers, and 24 female non-smokers. After extraction of platelet total RNA, eNOS (target) and GAPDH (internal control) mRNA expression levels were quantitated using real-time RT-PCR. The expression levels of eNOS mRNA (relative copy numbers) were significantly lower in male smokers (59+/-17) than in male nonsmokers (195+/-71, P < .03), and higher in female nonsmokers (285+/-60) than in the male nonsmokers (195+/-71, P < .03). By multiple linear regression analysis, cigarette smoking (P = .008) and diabetes mellitus (P = .047) were found to be significantly negative predictors, and antioxidant (vitamin E) treatment (P = .01) was a significantly positive predictor of platelet eNOS mRNA expression. Age, other medications, and other risk factors for coronary artery disease were not significant. Using this method, eNOS mRNA abundance in human platelets was detected and quantitated in real-time. The intraplatelet eNOS mRNA expression levels were significantly decreased in cigarette smokers. Low platelet NO synthesis in smokers may result in the augmentation of platelet aggregation and thrombus formation, developing into acute coronary syndromes.
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PMID:The effects of long-term smoking on endothelial nitric oxide synthase mRNA expression in human platelets as detected with real-time quantitative RT-PCR. 1716 95

Our group has shown that mechanical stimulation increases the stiffness of stem cell-collagen sponge constructs at 14 days in culture and subsequent rabbit patellar tendon repairs at 12 weeks postsurgery. What remains unclear is which genes might be responsible for this increase in stiffness. Therefore, the objective of this study was to determine how a tensile stimulus affects the gene expression of stem cell-collagen sponge constructs used to repair rabbit central patellar tendon defects. Tissue-engineered constructs were created by seeding mesenchymal stem cells (MSCs) from 10 adult rabbits at 0.14 x 10(6) cells/construct in type I collagen sponges. Half of the constructs were mechanically stimulated once every 5 min for 8 h/d to a peak strain of 2.4% for 2 weeks. The other half remained in an incubator without mechanical stimulation for 2 weeks. After 14 days in culture, half of the stimulated and nonstimulated constructs were prepared to determine the expression of collagen type I, collagen type III, decorin, fibronectin, and glyceraldehyde-3-phosphate dehydrogenase genes using real-time quantitative reverse transcriptase polymerase chain reaction. The remaining constructs were mechanically tested to determine their mechanical properties. Two weeks of in vitro mechanical stimulation significantly increased collagen type I and collagen type III gene expression of the stem cell-collagen sponge constructs. Stimulated constructs showed 3 and 4 times greater collagen type I (p = 0.0001) and collagen type III gene expression (p = 0.001) than nonstimulated controls. Stimulated constructs also had 2.5 times the linear stiffness and 4 times the linear modulus of nonstimulated constructs. However, mechanical stimulation did not significantly increase decorin or fibronectin gene expression (p = 0.2) after 14 days in culture. This study shows that mechanical stimulation of cell-sponge constructs produces similar increases in the expression of 2 structural genes, as well as linear stiffness and linear modulus.
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PMID:Mechanical stimulation increases collagen type I and collagen type III gene expression of stem cell-collagen sponge constructs for patellar tendon repair. 1751 15

The quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) is a sensitive and very efficient technique for quantification of gene expression. However, qRT-PCR relies on accurate normalization of gene expression data, as RNA recovery and cDNA synthesis efficiency might vary from sample to sample. In the present study, six putative reference genes were validated for normalization of gene expression in three different tissues and in white blood cells from pigs experimentally infected with the common respiratory pathogen Actinobacillus pleuropneumoniae. Two dedicated validation programs (geNorm and Normfinder) were used to rank the six reference genes from best to worst. qRT-PCR data for the proinflammatory cytokine IL-6 was normalized using the proposed genes from geNorm and Normfinder as well as the commonly used reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). IL-6 expression was quantified in white blood cells, liver, lymph nodes and tonsils from 10 infected pigs and 5 control pigs. After normalization using either geNorm or Normfinder IL-6 was shown to be significantly up-regulated (P<0.05) in all of the tissues from infected animals compared to non-infected control animals with a good agreement of expression differences between the two programs. On the contrary, normalization of IL-6 expression data from blood using GAPDH rendered the difference between infected and non-infected groups non-significant, and resulted in significantly different values compared to geNorm (P=0.01). Based on these results, we recommend to validate putative reference genes before normalization.
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PMID:Validation of putative reference genes for qRT-PCR normalization in tissues and blood from pigs infected with Actinobacillus pleuropneumoniae. 1754 55

An alternative approach to conventional protein-based body fluid identification is gene expression profiling analysis. In the present work, we report the development of sensitive and robust multiplex quantitative reverse transcriptase-PCR assays for the identification of blood, saliva, semen, and menstrual blood. Each body fluid assay comprises a triplex system that detects transcripts from two body fluid-specific genes and a housekeeping gene GAPDH. The body fluid-specific genes include erythroid delta-aminolevulinate synthase (ALAS2) and beta-spectrin (SPTB) for blood, statherin (STATH) and histatin 3 (HTN3) for saliva, protamine 1 (PRM1) and protamine 2 (PRM2) for semen, and matrix metalloproteinase 7 (MMP7) and matrix metalloproteinase 10 (MMP10) for menstrual blood. Normalization of both body fluid-specific genes to the housekeeping gene by means of appropriate cycle threshold metrics ensures the high specificity of each assay for its cognate body fluid.
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PMID:mRNA profiling for body fluid identification by multiplex quantitative RT-PCR. 1786 68

The use of ribonucleic acid (RNA) extracted from Hepes glutamic acid buffer-mediated organic solvent protection effect (HOPE)-fixed tissues in quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) is fairly novel. We compared qRT-PCR analysis of formalin- and HOPE-fixed, paraffin-embedded lymph node tissues from Mycobacterium bovis-infected cattle by extracting total RNA using a commercial kit (Ambion) and a Trizol method. RNA extracted from HOPE-fixed tissues showed comparable quantities between the commercial kit (82.7-107.9 microg/ml total RNA) and the Trizol method (87-161.1 microg/ml total RNA), displaying a high degree of integrity when analyzed by electrophoresis. RNA extracted from formalin-fixed tissues using the commercial kit produced similar concentrations (80.6-145.7 microg/ml total RNA) in comparison to the HOPE tissue; however, the integrity was compromised. Extraction of RNA from the formalin-fixed tissues using Trizol was unsuccessful. Following qRT-PCR for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), total RNA from HOPE-fixed tissues showed higher levels of target messenger ribonucleic acid (mRNA) (4.05 x 10(-2)pg/100 ng total RNA using the commercial kit and 6.45 x 10(-2)pg/100 ng total RNA using Trizol) in comparison to formalin-fixed tissues (5.69 x 10(-4)pg/100 ng total RNA). This could be attributed to RNA degradation by exposure to formalin fixation. In conclusion, the HOPE fixative proved to be a better source for RNA extraction from cattle lymph nodes and subsequent qRT-PCR.
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PMID:RNA isolation and quantitative PCR from HOPE- and formalin-fixed bovine lymph node tissues. 1798 5

Feline allergic skin disease is thought to be associated with dermal infiltration of Th(2) lymphocytes and synthesis of associated cytokines. In this study, real-time RT-PCR assays were developed to measure feline interleukin (IL)-2, IL-4, IL-5, IL-6, IL-10, IL12 (p35 and p40), IL-18, tumour necrosis factor-alpha (TNF-alpha), transforming growth factor-beta (TGF-beta), interferon-gamma (IFN-gamma) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in the skin of healthy control cats, and in the lesional and non-lesional skin of cats with allergic skin disease. Total RNA was extracted from skin biopsies using the RNeasy Mini Kit with on-column and in-solution DNase digestion steps. cDNA was synthesised using Improm-II reverse transcriptase and random hexamers. Real-time PCR was carried out using an iCycler IQ system (Bio-Rad), and gene-specific primers were designed to span an exon/exon junction of each cytokine gene. Taq-man probes were used to add specificity to the system. Messenger RNA from the housekeeping gene GAPDH was used for normalisation of the cytokine threshold cycle. The eleven cytokine mRNA transcripts quantified were present at varying levels, but there was no apparent difference in expression between normal, non-lesional and lesional skin. TGF-beta represented the most abundant transcript while IL-4, IL-5, IL-6, IL-10, IL-12, IL-18 and TNF-alpha were present at levels approximately 1000-fold less. IL-2 and INF-gamma represented the least abundant templates with no detectable copies in most RNA samples. This quantitative analysis of cytokine mRNA expression in feline skin biopsies has suggested that there is not a simple Th(2) bias in lesional skin of cats with allergic dermatopathies.
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PMID:Quantitative real-time RT-PCR measurement of cytokine mRNA expression in the skin of normal cats and cats with allergic skin disease. 1819 Dec 30


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