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:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

It has previously been demonstrated that the epidermis is a rich source of proinflammatory cytokines and growth factors and that complex interactions between these factors may affect inflammatory responses in skin. To investigate whether IL-10 (cytokine synthesis inhibitory factor) is part of this complex process, RNA was extracted from normal epidermis at various times after application of various chemicals to murine skin and mRNA signals for IL-10 were sought using a quantitative reverse-transcriptase-polymerase chain reaction technique. IL-10 signal strength was normalized to that of beta-actin in each sample. IL-10 mRNA signals were occasionally identified in normal epidermis but were uniformly enhanced 4 h after hapten application, and were maximal after 12 h. Contact allergens induced IL-10 mRNA signals whereas vehicles and irritants did not. Depletion of Langerhans cells, Thy-1+ dendritic epidermal cells, and T lymphocytes demonstrated that keratinocytes were the main source of IL-10 mRNA. IL-10 signals were also detected in mRNA derived from PAM 212 (spontaneously transformed keratinocyte) cells. IL-10 protein could be detected by immunoprecipitation, with an IL-10 mAB, of supernatants obtained 16 h after cultured epidermal cells were coupled with hapten. This study demonstrates that murine keratinocytes are capable of producing IL-10 mRNA and protein, and that signal strength of IL-10 mRNA is enhanced by hapten application.
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PMID:Identification and induction of keratinocyte-derived IL-10. 160 65

Normal C57BL/10SnJ myoblasts were transplanted into the tibialis anterior of C57BL/10SnJ, C57BL/ScSn mdx, or BALB/c mice. These transplantations allowed us to investigate the immune response not only against MHC but also against dystrophin introduced in the dystrophic muscles by such transplantations. Recently, our group reported following myoblast transplantations cellular infiltration of the host muscle by class II MHC cells, macrophages, and lymphocytes expressing CD4 or CD8 and IL-2 receptors. In the present study, activation of these infiltrating lymphocytes was investigated by measuring the expression of granzyme B mRNA. We used reverse-transcriptase polymerase chain reaction to detect granzyme B mRNA at various intervals after myoblast transplantations. To standardize the results, the mRNA were reverse transcribed using an oligo (dt) so that beta-actin mRNA could also be amplified from the same cDNA preparation. Granzyme B mRNA was increased for at least 3 weeks after MHC alloincompatible grafts. The absence of increased granzyme B expression after allocompatible transplantation in mdx mice suggests that dystrophin is not sufficiently immunogenic to induce short term acute rejection. These results indicate that lymphocytes infiltrated in muscles injected with histoincompatible myoblasts are activated and sustain the requirement for an adequate immunosuppression after such transplantations.
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PMID:Increased granzyme B mRNA after alloincompatible myoblast transplantation. 749 74

Thirty-five patients with Philadelphia chromosome (Ph1)-positive chronic myelogenous leukemia (CML) were classified on the basis of the fusion pattern of bcr-abl mRNA determined by the reverse-transcriptase-polymerase chain reaction (RT-PCR) method. Semiquantitative assay of the bcr exon 2/abl exon 2 fused mRNA (b2-a2) and bcr exon 3/abl exon 2 fused mRNA (b3-a2) resulted in 21 patients showing b3-a2 type mRNA, seven showing b2-a2 type and seven showing coexpression. Quantification of the autoradiographic signals of amplified products was estimated using an MCID image analysis system. The relative intensity was defined as the ratio of bcr-abl signal to that of beta-actin. The relationship between the semiquantified bcr-abl mRNA and the platelet/megakaryocyte counts was analyzed. A possible correlation was found between the semiquantified b3-a2 type mRNA and the platelet (p < .05, N = 28) and megakaryocyte (p < .05, N = 13) counts of these patients. This finding suggests the possibility that b3-a2 mRNA may affect the thrombopoietic activity in Ph1-positive CML in a dose-response manner.
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PMID:Possible correlation of b3-a2-type bcr-abl messenger RNA defined by semiquantitative RT-PCR to platelet and megakaryocyte counts in Philadelphia-positive chronic myelogenous leukemia. 752 Jul 86

Hodgkin's disease (HD) and Ki-1 positive anaplastic large cell lymphoma (Ki-1 ALCL) appear pathologically and immunohistochemically related, and a common histogenesis has been postulated in at least some cases. The breakpoints of the t(2;5) (p23;q35) [corrected] translocation, which is reported in about 40% of Ki-1 ALCL, have recently been cloned. They involve a novel tyrosine kinase gene, ALK, at 2p23 and the nucleophosmin gene, NPM, at 5q35. Reverse transcriptase polymerase chain reaction (RT-PCR) using NPM and ALK primers consistently detects a fusion product in Ki-1 ALCL cases with the translocation. To determine if this tumor-specific genetic alteration also occurs in HD, we performed NPM-ALK RT-PCR on RNA samples extracted from 40 lymph node biopsies of HD (25 nodular sclerosis, 11 mixed cellularity, 2 lymphocyte depleted, 2 lymphocyte predominant). Using control samples, the sensitivity of the NPM-ALK RT-PCR assay was shown to be at least 1:10(4). Amplifiable template was confirmed in all samples by RT-PCR using beta-actin primers. None of the 40 cases showed the expected 177-bp RT-PCR product indicative of the translocation. We conclude that the most common primary genetic alteration in Ki-1 ALCL, the t(2;5), is absent or very infrequent in typical cases of HD. These results further support the concept that HD and Ki-1 ALCL are pathogenetically distinct entities.
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PMID:Reverse transcriptase polymerase chain reaction for the Ki-1 anaplastic large cell lymphoma-associated t(2;5) translocation in Hodgkin's disease. 752 17

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

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


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