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:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Detection of rejection after small intestine transplantation (SIT) is difficult, relying largely on histopathology. The purpose of this study was to determine if the intragraft expression of messenger RNA (mRNA) for interleukin-2 receptor (IL-2R), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF) correlated with rejection in a unidirectional, heterotopic rat SIT model. Graft samples were obtained on postoperative day (POD) 3, 5, 7, 8, 9, 10, 12, and 14. After staining, formalin-fixed samples were blindly evaluated for rejection. Reverse transcriptase polymerase chain reaction (rtPCR) using primers specific for beta-actin, IL-2R, IL-6, and TNF was performed on liquid nitrogen-frozen samples. Semiquantitation was accomplished using radionuclide incorporation and beta-scintillation counting. Intestinal histopathology in all isografts (ISO) and POD 3 allografts (ALLO) was normal. Rejection progressed in ALLO from mild on POD 5 to severe by POD 8. rtPCR analysis revealed constitutive expression of IL-2R mRNA in both ISO and ALLO. TNF and IL-6 demonstrated significant increases in mRNA expression in ALLO compared to ISO beginning on POD 5. In summary, intragraft expression of IL-2R mRNA demonstrated late up-regulation in ALLO which did not correlate with rejection. TNF and IL-6 mRNA expression predicted rat SIT rejection. rtPCR analysis of TNF and IL-6 may serve as a useful diagnostic adjunct for rat SIT rejection.
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PMID:Intragraft expression of messenger RNA for interleukin-6 and tumor necrosis factor-alpha is a predictor of rat small intestine transplant rejection. 804 Nov 28

The development of riboprobe expression cassettes for phosphorimager-based quantitation of steady-state transcripts for three different genes using solution hybridization, RNase protection assays is described. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and beta-actin genes are widely used as reporter genes to estimate the amount and integrity of RNA as well as for comparing gene expression among different tissues. To directly compare expression of these two genes in lymphoid tissue and liver, cDNA fragments of beta-actin and GAPDH from both mice and rats were generated by RT-PCR and cloned together into pGEM1 under control of the T7 RNA polymerase promoter. Antisense transcripts from this fusion construct protected the appropriate-sized fragments of beta-actin (115 nt) and GAPDH (214 nt) in RNA isolated from rat spleen, thymus and liver. Expression of GAPDH transcripts was less variable across tissues because this mRNA was only two-fold lower in liver as compared to either thymus or spleen, whereas expression of beta-actin transcripts was eight-fold lower in liver than in these tissues. Two other riboprobe expression cassettes (IGF-I/actin) were constructed by ligating a cDNA fragment of mouse or rat beta-actin that would protect 115 nt to either a mouse or rat IGF-I genomic DNA fragment containing 182 bp of exon 4. These mouse and rat IGF-I/actin riboprobes were used to conclusively demonstrate that rat CSF-1-derived bone marrow macrophages, mouse elicited peritoneal macrophages and the murine PU5-1R macrophage cell line synthesize abundant transcripts for both IGF-I and beta-actin. However, the mouse M1 progenitor myeloid cell line does not express RNA for IGF-I, as demonstrated by the absence of protected transcripts for IGF-I in the presence of abundant protected transcripts for beta-actin. Phosphorimager scanning of the gels revealed that macrophages of both mice and rats express IGF-I transcripts at a level of 60-100% of those found in liver. These data show that a single riboprobe can be developed to generate multigene antisense RNAs that can then be used to quantitatively compare IGF-I transcripts in macrophages and other tissues to an internal standard, with GAPDH transcripts being less variable among tissues than those for beta-actin. This approach should be broadly applicable for measuring a variety of markers of cellular activation.
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PMID:Riboprobe expression cassettes for measuring IGF-I, beta-actin and glyceraldehyde 3-phosphate dehydrogenase transcripts. 830 98

The effect of vitamin B6 deficiency on the activity of RNA polymerase and expression of several mRNAs in rat liver was investigated. The activities of RNA polymerase I and II in the liver of vitamin B6-deficient rats were found to be higher than the control rats by 30%. The expression of several mRNAs, including mRNAs for beta-actin and glyceraldehyde-3-phosphate dehydrogenase, and the content of poly(A)+ RNA were also increased in vitamin deficiency. These observations suggest that vitamin B6 influences gene expression in the liver, at least in part, by modulating the activity of RNA polymerase.
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PMID:Vitamin B6 deficiency causes activation of RNA polymerase and general enhancement of gene expression in rat liver. 840 98

Reverse transcriptase polymerase chain reaction (RT-PCR) was used to show that beta-actin RNA levels can be detected in total RNA isolations from as few as two cochleae. In functionally mature chicken cochleae, a low homeostatic level of beta-actin message should be expressed in order to synthesize enough actin to maintain the stereocilia, cuticular plate, junctional complexes of hair cells and the cytoskeletal components of the supporting cells. The RT-PCR product obtained has been characterized by size, restriction digest analysis, and DNA sequencing analysis. These procedures have confirmed that the product is amplified from a chicken beta-actin mRNA target. Subsequently, semi-quantitative RT-PCR techniques were used to demonstrate an upregulation of beta-actin mRNA transcription levels in the cells of the basilar papilla during regeneration following damage from acoustic overstimulation. These studies suggest that RT-PCR can be utilized for analysis of limited quantities of tissue such as that found in the chicken cochlea and indicate promise for further qualitative and quantitative studies on the molecular mechanisms of hair cell transduction and regeneration.
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PMID:Detection of beta-actin mRNA by RT-PCR in normal and regenerating chicken cochleae. 856 47

To evaluate possible functional differences between basic fibroblast growth factor (FGF) 2 isoforms we analyzed the effects of the 18-kDa FGF-2 which mainly localizes in the cytosol and that of the nuclear-targeted 22.5-kDa form on FGF receptors (FGFR) expression. These peptides were expressed at low amounts through a retroviral-infection system. Point mutated FGF-2 cDNAs under the control of the beta-actin promoter were used to infect a pancreatic cell line (AR4 2J) which does not produce FGF-2. Saturation and competition binding studies with 125I-FGF-2 revealed a 3-fold increase in both high and low affinity receptors in cells expressing the 22.5-kDa form and a 2-fold increase only in the high affinity receptors in cells producing the 18-kDa form. Kd values and molecular weights of the high affinity receptors were unaffected. Increasing cell densities or cell treatment with exogenous FGF-2 resulted in FGFR down-regulation as in control cells. Neutralizing anti-FGF-2 antibodies and suramin did not affect receptor density in control and in cells producing the 22.5-kDa form but further increased by 60 and 80%, respectively, the receptor level in cells synthesizing the 18-kDa form. These data suggest the involvement of the intracellular stored FGF-2 in FGFR up-regulation. Although all cells expressed FGFR-1, -2, and -3 mRNA only the FGFR-1 transcript was found increased, 6-fold in 22.5-kDa expressing cells and 3-fold in cell producing the shortest secreted isoform. The increase in FGFR-1 mRNA levels in the 22.5-kDa expressing cells was due to enhanced stability of the transcript. Confocal microscopy detected the presence of FGFR-1 at the cell surface whereas secretory isoforms of the receptor were not observed. Reverse transcriptase-polymerase chain reaction did not reveal significant differences in the expression of FGFR-1 variants. In the 22.5-kDa expressing cells exogenous FGF-2 evoked a stronger translocation of the calcium-phospholipid-dependent PKC. These results indicate that the transfected FGF-2 isoforms up-regulated FGFR-1 mRNA and protein. The 22.5-kDa form acted by increasing FGFR-1 mRNA stability enhancing cell responses to exogenous FGF-2.
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PMID:Differential regulation of fibroblast growth factor (FGF) receptor-1 mRNA and protein by two molecular forms of basic FGF. Modulation of FGFR-1 mRNA stability. 862 30

We determined the expression of Th-2 type cytokines, interleukin-4 (IL-4) and IL-5, and of the Th-1 type cytokine, interferon-gamma (IFN-gamma), in the Brown-Norway rat. Rats were intraperitoneally sensitized with ovalbumin and 21 days later were either exposed to ovalbumin or saline aerosol. The value -log PC300 (PC300 = concentration of acetylcholine needed to increase baseline lung resistance by 300%) was 2.49 +/- 0.15 in sensitized, exposed rats, was higher than in sensitized, saline-exposed or naive rats (1.54 +/- 0.27 and 1.63 +/- 0.06 respectively, P < 0.05). There was a significant increase in eosinophils in bronchoalveolar lavage fluid and in airway submucosal airway tissues in the sensitized exposed group. Reverse-transcriptase polymerase chain reaction was performed on total lung RNA using primers for IL-4, IL-5, IFN-gamma and beta-actin. IL-4 and IL-5 mRNA levels in control and sensitized saline-exposed rats were not detectable, but increased levels were found in sensitized and ovalbumin-exposed rats with levels of 0.25 +/- 0.01 and 0.98 +/- 0.02% of beta-actin mRNA as assessed by densitometric measurements. Expression of IFN-gamma mRNA was significantly reduced in sensitized and ovalbumin-exposed rats. As in asthmatic airways, there is an increased expression of Th-2 cytokines, IL-4 and IL-5, together with a reduction in the Th-1 cytokine, IFN-gamma, thus supporting a role for Th-2 cytokines in allergic eosinophilic inflammation.
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PMID:Expression of Th-2 cytokines interleukin-4 and -5 and of Th-1 cytokine interferon-gamma in ovalbumin-exposed sensitized Brown-Norway rats. 869 Apr 57

We measured, by employing a quantitative reverse-transcriptase PCR procedure, the relative (to beta-actin) levels of amyloid precursor protein APP751 and APP770 mRNA isoforms in lymphocytes obtained from 64 cognitively intact subjects ranging in ages from 20 to 91 years and in 19 patients with sporadic Alzheimer's disease. A positive correlation was observed between the relative lymphocyte APP751 mRNA levels and subject age for the cognitively intact cohort. No difference in lymphocyte APP751 mRNA levels was observed between Alzheimer's disease patients and their age-matched controls (> 55 years of age). However, the ratio of lymphocyte APP751:APP770 mRNA levels was significantly lower in Alzheimer's disease subjects compared to the > 55-year-old cohort. This decreased ratio is most likely due to an average 31% increase in the lymphocyte APP770 isoform in Alzheimer's disease patients compared to 12% in the > 55-year-old cognitively intact group. Marked individual differences in amount of APP mRNA isoforms were encountered among all the subject groups and in the < or = 55-year-old cohort, a 10-fold variation in individual APP751 mRNA levels was observed. The relevance of these findings in lymphocytes to the pathogenesis of Alzheimer's disease is discussed.
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PMID:Changes in expression of lymphocyte amyloid precursor protein mRNA isoforms in normal aging and Alzheimer's disease. 871 62

We describe a new, simple and reliable semiautomated strategy for quantifying mRNA from archival specimens by using oligo(dT)25 paramagnetic beads and the reverse-transcriptase polymerase chain reaction (PCR) coupled with quantitative digital image analysis (Q-DIA). To evaluate the experimental conditions, we examined thymidylate synthase (TS) gene expression in mRNA isolated from both flash-frozen and formalin-fixed paraffin-embedded human biopsy samples using biopsy material obtained from 2 patients prior to chemotherapy with 5-fluorouracil. Following the electrophoretic separation of the PCR products through a 20% polyacrylamide gel, quantitation of the perimeters of the silver-nitrate-stained PCR products will be done by Q-DIA using a video frame-grabber board attached to a CCD camera using Image-Pro+ software. Validation of this approach will involve a comparison of the observed gene expression levels to TS protein levels obtained by tissue homogenization assays of TS, tetrahydrofolate, 5,10-methylenetetrahydrofolate, 2'-deoxyuridine-5'-monophosphate and 5-fluoro-2'-deoxyuridylate (FdUMP), by established [3H]FdUMP ligand-binding assays. The novelty of this method is that it offers a low-cost means whereby Q-DIA is performed directly from the gel to rapidly and accurately determine the level of TS gene expression, which is standardized against the beta-actin housekeeping gene. In the protocol described herein, gene expression studies can be done quickly and without the use of radioactive substances in both normal clinical samples shock frozen at the time of surgical excision and in formalin-fixed paraffin-embedded archival samples, which are commonly available in all hospital pathology departments. To demonstrate the utility of this method, mRNA was extracted from both nonpathological and tumor biopsies originating from both types of material from the same patients. TS gene expression in the flash-frozen and archival materials was compared to the level of TS intracellular enzyme activity in the same samples and a correlation of 89 and 80% between the shock-frozen and archival material relative to TS intracellular enzyme activity levels was observed. These findings suggest that routine semiautomated quantitative analysis of rare mRNA transcripts, e.g. TS, from archival material can be applied for retrospective studies.
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PMID:Detection of thymidylate synthase gene expression levels in formalin-fixed paraffin embedded tissue by semiquantitative, nonradioactive reverse transcriptase polymerase chain reaction. 898 25

Cisplatin is an anticancer agent frequently used as an alternative to the nitrosoureas in brain tumor chemotherapy. We describe the use of a technique of quantitative reverse transcription-polymerase chain reaction (RT-PCR) to examine the damage induced in the glutathione S-transferase (GST)-pi gene by cisplatin and the subsequent repair of this damage in cells of the MGR3 human glioblastoma multiforme cell line. The relationship between cisplatin dose and the extent of damage in the GST-pi gene was determined over cisplatin concentrations (0-10 microM) within the clinically achievable range. Total RNA was purified from control and cisplatin-treated cells, and both the full-length GST-pi cDNA and control 200-bp beta-actin cDNA were amplified by RT-PCR. The cDNA reaction products were electrophoresed, Southern hybridized, and quantitated densitometrically. A decrease in GST-pi mRNA representing damage to the GST-pi gene was observed with increasing cisplatin concentrations, up to a maximum of 75% at 10 microM cisplatin. Repair of the GST-pi gene in cells treated with cisplatin, assessed as recovery of transcriptional activity of the gene, was shown to occur even after 48 hr following drug removal. A potent RNA polymerase II inhibitor, alpha-amanitin, was used to show that the GST-pi mRNA quantitated in this RT-PCR assay resulted from de novo RNA transcription of the GST-pi gene with little contribution from preexisting GST-pi transcripts. The results demonstrate that the GST pi gene, which is actively transcribed and often overexpressed in human glioma cells, is a target for cisplatin, but that the damage to the gene is efficiently repaired in these cells. The RT-PCR assay has the potential for use in the detection of DNA damage induced by genotoxic agents in other actively transcribed genes and for assessing the repair of gene-specific DNA lesions in cells.
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PMID:Detection of DNA damage in transcriptionally active genes by RT-PCR and assessment of repair of cisplatin-induced damage in the glutathione S-transferase-pi gene in human glioblastoma cells. 907 88

Thymidylate synthase (TS) provides the only de novo source of thymidylate for DNA synthesis and is a key target for cancer chemotherapeutic agents. We investigated the TS gene expression by semiquantitative reverse-transcriptase polymerase chain reaction in metastatic melanoma and compared the results with those from control tissues. The relative TS/beta-actin level ratios were 0.5, 0.9, 0.3, 0.4, and 0.5 (mean 0.5) in skin, lymph node, thyroid, muscle, and spleen, respectively. In metastatic melanoma samples, the ratios varied from 0.9 to 2.7 (mean 2.0). The differences of expression levels between these two groups of samples were statistically highly significant (p = 0.0000713). A similar statistical significance (p = 0.0002) was observed between patients achieving a complete response and patients who had progressive disease despite immunochemotherapy. There was no clear relationship between a high TS/ beta-actin ratio and the S phase fraction, as all melanomas had a high S phase fraction.
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PMID:Increased thymidylate synthase gene expression in metastatic melanoma. 907 87


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