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)

Multiplex differential display (MDD), a modification of differential display reverse transcriptase polymerase chain reaction (DD-PCR), was used to identify cocaine-dependent regulation of previously known and unknown gene products. Direct comparison of the MDD amplification profiles of duplicate, total RNA samples from the caudate putamen (CPu) of either vehicle or cocaine treated Sprague-Dawley rats indicated that the relative induction of a 240 bp (8G247) product, likely to represent c-fos mRNA, closely paralleled changes in c-fos mRNA as measured by Northern blot analysis. MDD and Northern blot analysis also revealed substantial repression of another PCR product (8G226) at 1 h and 1 day after repeated administration of cocaine. At 2 days after cocaine exposure, the level of 8G226 had returned to control levels. The DNA sequence of 8G226 exhibited near identity with a mouse zinc-finger protein (PZf) and is thus likely to represent a transcriptional regulator. Interestingly, the repression of 8G226 immediately after cocaine treatment is in direct contrast to the cocaine-dependent increase in expression documented for NGFI-A, another zinc-finger protein which also functions as a transcriptional regulator. Detailed characterization of the prolonged reduction in the expression of 8G226 may lead to the identification of additional regulatory pathways that produce changes in cellular response after repeated cocaine exposure.
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PMID:Multiplex differential display identifies a novel zinc-finger protein repressed during withdrawal from cocaine. 938 92

Fetal and neonatal lymphocytes are relatively resistant to activation and cytokine production when stimulated either via their T-cell antigen receptors or lectins. The molecular mechanism(s) responsible for this phenomenon have not been clearly elucidated. We have hypothesized that such defects in fetal/neonatal T-cell activation may be due to lack of expression of the transcriptional regulatory elements required for T-cell activation. We used reverse transcriptase-polymerase chain reaction to examine both fetal and term neonatal cord bloods for mRNA expression of three transcription factors implicated in T-cell activation: c-jun, c-fos, and NF kappa B (p50 subunit). We demonstrate that mRNAs for all three of these regulatory factors are expressed in fetal blood cells by the 27th week of gestation and in term cord bloods. Activation of term infant cord blood mononuclear cells with anti-CD3 monoclonal antibodies resulted in up-regulation of both c-jun and c-fos mRNAs within 15 min of stimulation. However, secretion of IL-2 by anti-CD3-stimulated cord blood mononuclear cells was still blunted compared with control cells from adults. We conclude that fetal nucleated blood cells constitutively express important genes for cytokine regulation and are able to increase intracellular accumulation of the mRNAs for these factors in response to anti-CD3 stimulation. Thus, qualitative differences in the capacity to regulate these factors could not be shown in fetal blood cells. Quantitative experiments comparing binding of these transcription factors to the IL-2 promoter are currently under investigation.
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PMID:Constitutive expression c-fos, c-jun, and NF kappa B mRNA is in nucleated fetal blood cells and up-regulation of c-fos and c-jun with anti-CD3 stimulation. 942 92

A reverse transcriptase followed by a polymerase chain-reaction (RT-PCR) assay was developed for the simultaneous detection and quantitation of proto-oncogene (c-fos and c-myc) mRNAs using an internal standard mRNA glyceraldehyde-6-phosphate dehydrogenase (GAPD). Total cellular RNA was reverse transcribed and PCR amplified with oligonucleotide primers specific to GAPD and either c-fos or c-myc genes. In contrast to Northern blot analysis, the RT-PCR assay is rapid and sensitive enough to quantitate specific proto-oncogene levels from as little as 12-25 ng of total cellular RNA. The reliability of the assay was tested by measuring c-fos and c-myc expression in C3H 10T1/2 mouse embryo fibroblast cells under two different growth states: (a) quiescent cell entry into the proliferative cycle, and (b) plateau phase. Furthermore, the assay was used in measuring variations in c-fos or c-myc expression in HA-1 hamster cells following exposure to the cellular stressing agent, nitric oxide. In serum-stimulated cells, the RT-PCR measurements of transient increase in c-fos (16-fold at 30 min) and c-myc (10-fold at 1 h) mRNA levels were comparable to previously reported results in the literature using a Northern blotting assay. In addition, a two- to fivefold increase in c-fos mRNA levels was observed in plateau phase cells when compared to log phase growth. Furthermore, a transient increase in c-fos mRNA levels (threefold at 2 h) was also observed following cells' exposure to the stressing agent nitric oxide. These results suggest that the multiplex RT-PCR assay represents a significant improvement over current methods to quantitate specific cellular mRNAs under different growth conditions or following environmental insults.
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PMID:A polymerase chain reaction assay for simultaneous detection and quantitation of proto-oncogene and GAPD mRNAs in different cell growth rates. 945 18

1. We report the first experimental evidence of a neuronal C5a receptor (nC5aR) in human cells of neuronal origin. Expression of nC5aR mRNA was demonstrated by the reverse transcriptase-polymerase chain reaction (RT-PCR) in TGW human neuroblastoma cells. 2. Expression of a functional C5aR was supported by the finding that C5a evoked a transient increase in the intracellular calcium level as measured by flow cytometry (FACS). 3. To analyse the function of the nC5aR, an antisense peptide fragment of the C5aR was used. Previous data showed that a C5aR fragment (a peptide termed PR226) has C5aR agonist and antagonist effects in U-937 cells depending on the concentration of the peptide. We found that a multiple antigenic peptide (MAP) form of the same peptide (termed PR226-MAP) induced rapid elevation of nuclear c-fos immunoreactivity and resulted in DNA fragmentation, a characteristic sign of apoptosis, in TGW cells. 4. Early electrophysiological events characteristic of apoptosis were also detected: intermittent calcium current pulses were recorded within 1-2 min of peptide administration. C5a pretreatment delayed the onset of this calcium influx. 5. We also demonstrated that the apoptotic pathway is linked to nC5aR via pertussis toxin-sensitive G-proteins. 6. Although the function of C5a and its receptor on neurons is unknown, these results suggest that an abnormal activation of this signal transduction pathway can result in apoptosis and, subsequently, in neurodegeneration.
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PMID:A neuronal C5a receptor and an associated apoptotic signal transduction pathway. 950 29

Atherosclerosis, like several other vascular diseases, exhibits structural and functional abnormalities resulting partially from an exaggerated proliferation of vascular smooth-muscle cells (VSMCs). Ca2+ channel blockers, such as amlodipine, have been suggested to retard or even prevent the progression of atherosclerosis. To determine the mechanisms involved in these effects, we investigated the influence of amlodipine on VSMC proliferation by using rat aortic VSMCs in culture. Amlodipine (0.1-10 microM) inhibited serum-, basic fibroblast growth factor (bFGF)-, and thrombin-induced VSMC proliferation and DNA synthesis in a concentration-dependent manner, as demonstrated by cell count and bromodeoxyuridine (BrdU)-incorporation measurements, respectively. Delayed addition of amlodipine after VSMC stimulation showed that the drug exerted its effect early in G1 phase of the cell cycle. This observation was confirmed by the finding that amlodipine did not influence DNA synthesis in VSMCs arrested to the G1/S boundary by hydroxyurea treatment. Consistent with its effects on VSMC growth/proliferation, amlodipine also decreased c-myc, c-fos, and c-jun protooncogene expression induced by serum, thrombin, or bFGF within 1 h after cell activation, as assessed by semiquantitative reverse transcriptase (RT)-polymerase chain reaction (PCR) analysis. The calcium channel agonist Bay K 8644, which counteracted the inhibition by nifedipine of bFGF-, thrombin- or serum-induced DNA synthesis, was ineffective to antagonize the inhibitory effect of amlodipine. The aforementioned effects of amlodipine were of similar amplitude, irrespective of the growth-enhancing agent used. This strongly indicates that amlodipine acts downstream of receptor activation to exert its antiproliferative action, probably early in the G1 phase of the cell cycle. Moreover, the lack of antagonistic effect between amlodipine and Bay K 8644 suggests that, in addition to its L-type Ca2+ channel inhibitory effect, amlodipine inhibits other intracellular signaling pathways. Such an interference of amlodipine with mitogenic signaling pathways might contribute to confer a blood vessel-protecting potential on amlodipine.
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PMID:Amlodipine inhibition of serum-, thrombin-, or fibroblast growth factor-induced vascular smooth-muscle cell proliferation. 959 80

In this study, the effect of 4-hydroxynonenal (HNE), a peroxidation product of omega-6-poly-unsaturated fatty acids, on the expression of the c-fos proto-oncogene and growth factor-induced proliferation of HeLa carcinoma cells in vitro was investigated. The Fos protein forms the heterodimer AP-1 with the Jun protein and regulates the cell cycle by inducing cyclin D1. Agents that are able to induce c-fos include serum, platelet-derived growth factor (PDGF), and epidermal growth factor (EGF), all of which were used in this study. The proliferation rate was determined by cell counting (viable and dead cells according to trypan blue exclusion) and the BrdU assay. The c-fos mRNA level was monitored by the reverse transcriptase/polymerase chain reaction. In the absence of HNE, serum-deprived cells responded to serum stimulation with a more than 10-fold increase of the c-fos mRNA level as well as with an increased rate of DNA synthesis and cell multiplication. Both EGF and PDGF (applied in combination with insulin) were able to substitute for FCS and induced rapid growth of the tumor cells preincubated in serum-deprived medium. In the absence of growth factors a negative correlation between the HNE concentration (range: 1-250 microM) and the c-fos mRNA level was observed. We suppose that HNE interferes in this case with the basal activity of the c-fos promoter. EGF, when applied after the HNE treatment, induced rapid growth of the tumor cells preincubated in serum-free medium, if HNE was used in a physiological concentration (1 microM). No difference was observed compared to the HNE-free control. c-fos mRNA level was nearly unchanged. In contrast, a cytotoxic concentration of the aldehyde (100 microM) caused a complete inhibition of proliferation, although a twofold increase of the c-fos mRNA level immediately after the aldehyde treatment was observed. A similar effect of HNE in cytotoxic concentration on c-fos expression was observed when cells were grown in presence of PDGF instead of EGF. Hence, in both cases HNE possibly interferes with the signal transduction pathway, which is initiated by external growth factors. The increased c-fos expression might be part of an abortive attempt to overcome the stressful condition raised by a cytotoxic concentration of HNE.
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PMID:4-Hydroxynonenal modifies the effects of serum growth factors on the expression of the c-fos proto-oncogene and the proliferation of HeLa carcinoma cells. 965 20

The expression pattern of c-fos, c-jun, c-kit and stem cell factor (SCF) has been investigated in developing human placenta using the highly sensitive technique of in situ reverse transcriptase-polymerase chain reaction (RT-PCR). Specific transcripts of all genes under study were observed in first-trimester placenta sections. c-fos, c-jun, c-kit and SCF transcripts were localized in cells of the villous stroma; fos, jun and kit-specific mRNAs were also found in endothelial cells; fos, kit and SCF mRNAs were detected in villous trophoblast cells. In mid-trimester and term placenta specimens only SCF transcripts were observed, restricted to trophoblast cells. The lack of c-fos transcripts in placenta from the second and third trimesters is a finding that contrasts with data from the literature obtained using extractive techniques. Parallel immunocytochemistry of placenta specimens from the three pregnancy stages under study revealed the fos protein only in first-trimester placenta, in agreement with the in situ RT-PCR findings. We conclude that the in situ RT-PCR technique is most suitable for gene expression studies because of its high level of sensitivity in correctly assigning the signal to specific cell types in complex tissues.
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PMID:Localization of fos, jun, kit and SCF mRNA in human placenta throughout gestation using in situ RT-PCR. 1008 77

Hepatocytes entrapped in collagen gel and cultured in serum-free conditions survived longer than cells cultured on plastic (5 days vs. 3 weeks), showed fewer signs of early cell senescence (no increase in c-fos oncoprotein expression), and maintained the expression of differentiated hepatic metabolic functions over a longer period of time. Cells cultured in collagen gels retained their ability to respond to hormones. The insulin-stimulated glycogen synthesis rate remained fairly constant during 18 days in culture (between 5.4 +/- 0.37 and 9 +/- 2.7 nmol glucose/h/microg DNA). Collagen-cultured hepatocytes recovered glycogen stores to levels similar to those found in liver, or in hepatocytes isolated from fed rats. Urea synthesis from ammonia remained stable for more than 2 weeks (average value, 23 +/- 4 nmol urea/h/microg DNA). The rate of albumin synthesis in collagen-entrapped cells was maintained above the day-1 level during 18 days in culture. Cells showed high levels of glutathione (GSH) (1,278 +/- 152 pmol/microg DNA). Biotransformation activities CYP4501A1, CYP4502A2, CYP4502B1, and CYP4503A1 remained fairly stable in collagen-cultured hepatocytes. CYP4502E1 and CYP4502C11 decreased but were still measurable after 18 days. After 4 days in culture, GST activity returned to levels observed in isolated hepatocytes. In contrast with plastic cultures, cells responded to CYP450 inducers (methylcholanthrene for CYP4501A1, CYP4501A2, and glutathione-transferase, and ethanol for CYP4502E1) for more than 2 weeks. CYP4501A1, CYP4501A2, and glutathione-transferase A2 (GST A2) induction was preceded by an increase in specific mRNA, while the effects on CYP4502E1 seemed to be at a posttranslational level. Analysis of the expression of relevant hepatic genes by reverse Northern and semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) revealed that culturing hepatocytes in collagen gels results in a sustained higher expression of key liver transcription factor genes DBP, C/EBP-alpha and -beta, and HNF-1 and -4, as well as specific liver enzyme genes (phosphoenol pyryvate carboxykinase, and carbamoylphosphate-synthetase I).
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PMID:Long-term expression of differentiated functions in hepatocytes cultured in three-dimensional collagen matrix. 1009 8

Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a naturally occurring compound shown to inhibit carcinogen-induced preneoplastic lesion formation in mouse mammary organ culture and tumorigenesis in the two-stage mouse skin model. Cancer chemopreventive potential was also suggested in various assays reflective of the three major stages of carcinogenesis. Anti-initiation activity was indicated by its antioxidant and antimutagenic effects, inhibition of the hydroperoxidase function of cyclooxygenase (COX), and induction of phase II drug-metabolizing enzymes. Antipromotion activity was indicated by antiinflammatory effects, inhibition of production of arachidonic acid metabolites catalyzed by either COX-1 or COX-2, and chemical carcinogen-induced neoplastic transformation of mouse embryo fibroblasts. Antiprogression activity was demonstrated by its ability to induce human promyelocytic leukemia (HL-60) cell differentiation. Moreover, pretreatment of mouse skin with resveratrol significantly counteracted 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced oxidative stress, as evidenced by numerous biochemical responses. Resveratrol reduced the generation of hydrogen peroxide, and normalized levels of myeloperoxidase and oxidized-glutathione reductase activities. It also restored glutathione levels and superoxide dismutase activity. As judged by the reverse transcriptase-polymerase chain reaction, resveratrol selectively inhibited TPA-induced expression of c-fos and transforming growth factor-beta 1 (TGF-beta 1), but did not affect other TPA-induced gene products including COX-1, COX-2, c-myc, c-jun, and tumor necrosis factor-alpha. These data indicate that resveratrol may interfere with reactive oxidant pathways and/or modulate the expression of c-fos and TGF-beta 1 to inhibit tumorigenesis in mouse skin. As reported herein, in addition to the activities described above, resveratrol inhibited the de novo formation of inducible nitric oxide synthase (iNOS) in mouse macrophages stimulated with lipopolysaccharide. This finding suggests an additional mechanism by which resveratrol may function as a cancer chemopreventive agent.
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PMID:Cancer chemopreventive activity of resveratrol. 1037 Aug 67

Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a natural product shown to inhibit carcinogen-induced pre-neoplastic lesions in mouse mammary organ culture and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted mouse skin tumors. Application of TPA to mouse skin induces oxidative stress, as evidenced by numerous biochemical responses, including significant generation of H2O2 and enhanced levels of myeloperoxidase and oxidized glutathione reductase activities and decreases in glutathione levels and superoxide dismutase activity. TPA treatment also elevates the expression of cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), c-myc, c-fos, c-jun, transforming growth factor-beta1 (TGF-beta1) and tumor necrosis factor-alpha (TNF-alpha). As currently reported, pre-treatment of mouse skin with resveratrol negated several of these TPA-induced effects in a dose-dependent manner. H2O2 and glutathione levels were restored to control levels, as were myeloperoxidase, oxidized glutathione reductase and superoxide dismutase activities. As judged by reverse transcriptase-polymerase chain reaction (RT-PCR), TPA-induced increases in the expression of c-fos and TGF-beta1 were selectively inhibited. These data suggest that resveratrol inhibits tumorigenesis in mouse skin through interference with pathways of reactive oxidants and possibly by modulating the expression of c-fos and TGF-beta1.
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PMID:Effects of resveratrol on 12-O-tetradecanoylphorbol-13-acetate-induced oxidative events and gene expression in mouse skin. 1038 Nov 33


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