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)

We have previously reported an interaction of nitric oxide (NO) and cyclic 3',5'-guanosine monophosphate (cGMP) in erythropoietin (Epo) production. Further studies have been carried out to clarify the role of NO in the hypoxic regulation of Epo production in Epo producing human hepatocellular carcinoma (Hep3B) cells, which produce Epo in response to physiological stimuli. Our reverse transcriptase-polymerase chain reaction (RT-PCR) technique revealed the expression of iNOS mRNA in Hep3B cells after incubation under hypoxic (1% O2) conditions for 6 hr. Hypoxia also significantly increased medium levels of nitrite in Hep3B cells. In order to investigate the role of NO in Epo production in Hep3B cells under normoxic (20% O2) conditions, we have studied the effects of interferon-gamma (IFN-gamma) on Epo production. IFN-gamma is known to induce iNOS and enhance the production of NO. IFN-gamma produced significant increases in medium levels of Epo and nitrite. IFN-gamma also significantly increased cGMP levels in Hep3B cells. Furthermore, NG-nitro-L-arginine methyl ester (L-NAME), an NO synthase inhibitor, significantly decreased IFN-gamma induced elevations in medium levels of Epo and nitrite as well as cGMP levels in Hep3B cells. These results provide further support for an important role of the NO/cGMP system in hypoxic regulation of Epo production in Hep3B cells.
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PMID:Inducible nitric oxide synthase expression and erythropoietin production in human hepatocellular carcinoma cells. 912 39

PACE4 is a processing protease which processes the precursor protein to the mature protein. Currently, four PACE4 isoforms have been reported [Tsuji, A. et al. (1994) Biochem. Biophys. Res. Commun. 200, 943 950]. In this study, we have cloned cDNA encoding a novel isoform, PACE4E, by screening the human brain cerebellum cDNA library and reverse transcriptase polymerase chain reaction analysis of total RNA from human hepatoma HepG2 cells. The PACE4E cDNA encoded an amino acid sequence of 975 residues. The sequence from the amino terminus to Arg900 of PACE4E was identical to the corresponding sequence of PACE4A, but the carboxy terminal sequence (75 residues) was unique and contained a hydrophobic cluster (Leu952-Gly968). PACE4E cDNA was transiently transfected in COS-1 cells, and the expressed proteins were a 112-kDa precursor form and a 105-kDa mature form. They were secreted into the culture medium, but their secretion was retarded compared with that of PACE4A. The expression of a mutant of PACE4E truncated up to the hydrophobic cluster from the carboxy terminus resulted in a remarkable increase in secretion level, suggesting that PACE4E tends to be retained intracellularly due to interaction with the membrane through the hydrophobic cluster. On the contrary, the transient expression experiment of PACE4C showed that only 68-kDa protein (precursor form) was detected in the cell and not secreted into the medium. In addition, coexpression experiment revealed that PACE4E was able to process the precursor form of von Willebrand factor to the mature form, but PACE4C did not process it.
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PMID:A novel human PACE4 isoform, PACE4E is an active processing protease containing a hydrophobic cluster at the carboxy terminus. 919 37

The recently identified cell adhesion regulator (CAR) modulates the process of integrin-mediated cell adhesion. The CAR gene is located on 16q, a locus at which high levels of allelic losses have been demonstrated in advanced human hepatocellular carcinoma (HCC). We studied the possible involvement of the CAR gene in the progression of HCC. With this aim, we determined the expression of CAR mRNA in 30 cases of HCC. Matching pair samples of tumor and adjacent nontumoral liver were analyzed by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). The results were compared with the clinicopathological features of the patients. Every nontumoral liver tissue sample analyzed, expressed CAR mRNA. All tumor samples showed amounts of expression that were equal or lower, compared with those found in their matching controls. Thus, in 16 out of 30 cases (53.3%), CAR mRNA expression in tumor was diminished to less than one tenth of that observed in nontumoral tissue. This group of patients exhibited higher amounts of alpha-fetoprotein, and comprised tumors with poor histological differentiation (Edmondson-Steinert's grades III-IV), higher rates of intrahepatic metastasis and recurrence within the first postoperative year (p < 0.05, respectively). Tumors exhibiting low levels of CAR mRNA were also found to be diagnosed at more advanced TNM stages (p < 0.01). We conclude that downregulation of CAR mRNA expression may play an essential role in the progression of HCC.
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PMID:Association of reduced cell adhesion regulator messenger RNA expression with tumor progression in human hepatocellular carcinoma. 922

Using reverse transcriptase-PCR and Northern analysis, we have shown that the H4IIE cell line, derived from the Reuber H35 rat hepatoma, contains significant amounts of transcripts for the CaCh3 (neuroendocrine) and CaCh1 (skeletal muscle) L-type voltage-operated calcium channel alpha 1-subunits. Two of the CaCh3 transcripts have a 45 bp deletion in the IVS4 membrane-spanning region which is the result of a mutation in genomic DNA. The deduced amino acid sequences of the PCR-derived clones of CaCh3 indicate that the mutation causes the loss of 15 amino acids from the IVS4 region, including three of the six positively charged residues, which are thought to be part of the voltage-sensing mechanism of voltage-operated Ca2+ channels. Quantitative-PCR and Northern analysis indicate that one of the novel CaCh3 transcripts is present in sufficient amounts to imply it could play a functional role in Ca2+ inflow. RT-PCR analysis of hepatocytes isolated from rat liver detected transcripts of CaCh3 (without the IVS4 mutation) and CaCh2, but at considerably lower levels than observed for the isoforms in the H4IIE cell line. Transcripts of CaCh1 and CaCh2 were also detected at low levels in Jurkat T lymphocytes. Fluorimetric studies with the Ca(2+)-sensitive probe, Fluo-3, have shown that H4IIE cells exhibit receptor-activated and store-activated (thapsigarin-induced), but not depolarisation (extracellular KCl)-induced Ca2+ inflow. The mutant transcripts are unlikely to produce Ca2+ channels that are opened by membrane depolarisation. The idea that they may be opened by other mechanisms is briefly discussed.
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PMID:Novel variants of voltage-operated calcium channel alpha 1-subunit transcripts in a rat liver-derived cell line: deletion in the IVS4 voltage sensing region. 923 51

The cytokeratin 18 related molecules of human hepatocellular carcinoma have been previously recognized through a series of biochemical and immunological approaches. It is suggested that these molecules undergo modulation from human hepatocyte cytokeratin 18. To prove whether these molecules are produced by modulation or protein degradation, we checked the cytokeratin profile of human hepatoma cell line PLC/PRF/5 with the methods used before. These results revealed that the PLC cells have the same cytokeratin 18 related molecules as human hepatocellular carcinoma tissue. The gene expression of the cytokeratin 18 in non-tumor liver tissues, hepatocellular carcinoma and PLC/PRF/5 cells were investigated. First, the mRNAs of non-tumor liver tissues, hepatocellular carcinoma tissues and PLC/PRF/5 cells were collected by the acid guanidinium thiocyanate phenol chloroform method. After transcription into cDNA by reverse transcriptase polymerase chain reaction, the cDNAs of each specimen were amplified by PCR and then digested by SmaI and BamHI restriction enzymes. The digested cDNA fragments were electrophoresed in agarose gel and the base pairs were found to be the same in length between neoplastic and non-neoplastic hepatocytes.
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PMID:The alteration of cytokeratin 18 molecule and its mRNA expression during tumor transformation in hepatoma. 926 84

Hepatocellular carcinoma (HCC) is one of the most common and rapidly fatal malignancies worldwide. Treatment options are severely limited by the frequent presence of metastases. If hepatocyte-specific mRNAs are detected in the circulation, it is possible to infer the presence of circulating, presumably malignant, liver cells. If these can be quantified, it is possible to predict the likelihood of haematogenous metastasis. In this investigation, we have attempted to gain an index of the mass of circulating HCC cells (with reference to the number of hepatoblastoma cells) by measuring the amounts of PCR products for albumin (alb) mRNA and alpha-fetoprotein (afp) mRNA by reverse transcriptase polymerase chain reaction (RT-PCR) and Southern blot analysis. For calibration, total RNA from 1-10(6) HepG2 cells was mixed with total RNA from 10(6) normal peripheral mononuclear cells. A linear relationship was demonstrated between the amount of alb- or afp PCR product and the level of HepG2 total RNA spiked. The assay is sensitive down to a detection level of one HepG2 cell. Alb mRNA was detected in 50% of 18 normal subjects and afp mRNA in only two normal subjects. The alb mRNA cut-off level for the normal was exceeded by seven normal subjects and 34 out of 64 HCC patients, and that for afp mRNA was exceeded by six HCC patients but none of the normal subjects. The level of alb mRNA detected was not linearly proportional to the amount of afp mRNA detected in peripheral blood of the same patients, suggesting heterogeneous expression of alb and afp genes in different circulating tumour cells. In addition, no significant linear association between the levels of afp mRNA and serum AFP was observed. Semiquantification of both mRNA markers for HCC cell detection may prove useful in prediction of metastases.
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PMID:Semiquantification of circulating hepatocellular carcinoma cells by reverse transcriptase polymerase chain reaction. 930 62

Serum amyloid A apolipoproteins (apoSAA) appear to compromise the ability of high density lipoprotein to protect against atherosclerosis and it is of interest to determine whether aortic smooth muscle cells can contribute to local pools of apoSAA in the presence of cytokines that are known to stimulate acute phase apoSAA (A-apoSAA) synthesis in the liver. In this study, the regulation of A-apoSAA synthesis was monitored in cultured neonatal rabbit aortic smooth muscle cells. Constitutive apoSAA3 gene expression was minimal, and only detectable by amplification of the mRNA by reverse transcriptase-polymerase chain reaction. ApoSAA3 gene expression and protein synthesis were stimulated by IL-1 alpha; as little as 0.01 ng/ml of IL-1 alpha stimulated an increase in steady state levels of apoSAA3 mRNA. Interestingly, IL-6 (which is required in addition to IL-1 alpha for the optimal synthesis of A-apoSAA by human hepatoma cells) had little if any effect on apoSAA3 synthesis by the smooth muscle cells. In a time course, it was shown that the stimulation of apoSAA3 mRNA levels was apparent by 1-2 h after the addition of cytokine, and that levels remained elevated in the presence of the cytokine for at least 48 h. Immunoprecipitation using an antiserum directed against apoSAA3 revealed that IL-1 alpha stimulated the synthesis and secretion of apoSAA3 protein in a manner that was consistent with apoSAA3 mRNA expression. The implications of these findings in atherogenesis are discussed.
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PMID:Regulation of extrahepatic apolipoprotein serum amyloid A (ApoSAA) gene expression by interleukin-1 alpha alone: synthesis and secretion of ApoSAA by cultured aortic smooth muscle cells. 931 18

Hepatic enzyme induction is often evoked as the cause for a variety of effects in animal studies, e.g. hepatic hypertrophy and secondary thyroid neoplasms in rodents. In clinical practice, enzyme induction often enhances drug clearance and may result in reduced efficacy. For example, carbamazepine or rifampin treatment induces P450 3A in humans, and as a result, dramatically reduces the efficacy of midazolam or cyclosporine. Due to species differences in substrate specificity and the regulation of various drug-metabolizing enzymes, assessing enzyme induction in human tissues is a desirable goal. Since induction often occurs as a result of increased synthesis of mRNA coding for a particular enzyme, induction may be quantified by measuring specific mRNA levels. We describe an approach to quantifying mRNA levels using reverse transcriptase-polymerase chain reaction (RT-PCR). This approach makes use of either radiolabeled PCR primers or fluorimetric quantification of product and does not require the synthesis of a competitor RNA or DNA molecule. Thus, Cyp2B1/2 mRNA can be shown to be induced 17-fold in the H4IIE rat hepatoma cell line. Likewise, Cyp3A and Cyp2A6 mRNAs can be shown to be induced in primary human hepatocytes cultured on collagen-coated plates and treated with rifampin for 72 h. By contrast, mRNA levels for Cyp1A1 and Cyp2E1 were not increased by rifampin treatment. This report demonstrates the potential of this approach for examining a number of mRNAs from drug-treated cultured cells, as a means of assessing metabolic enzyme induction.
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PMID:Quantitative reverse transcriptase/PCR assay for the measurement of induction in cultured hepatocytes. 940 49

Telomerase is a specialized type of reverse transcriptase which catalyzes the synthesis and extension of telomeric DNA (for review, see ref.1). This enzyme is highly active in most cancer cells, but is inactive in most somatic cells. This striking observation led to the suggestion that telomerase might be important for the continued growth or progression of cancer cells. However, little is known about the molecular mechanism of telomerase activation in cancer cells. Human telomerase reverse transcriptase (hTRT) has recently been identified as a putative human telomerase catalytic subunit. We transfected the gene encoding hTRT into telomerase-negative human normal fibroblast cells and demonstrated that expression of wild-type hTRT induces telomerase activity, whereas hTRT mutants containing mutations in regions conserved among other reverse transcriptases did not. Hepatocellular carcinoma (20 samples) and non-cancerous liver tissues (19 samples) were examined for telomerase activity and expression of hTRT, the human telomerase RNA component (hTR; encoded by TERC) and the human telomerase-associated protein (hTLP1; encoded by TEP1). A significant correlation between hTRT expression and telomerase activity was observed. These results indicate that the hTRT protein is the catalytic subunit of human telomerase, and that it plays a key role in the activation of telomerase in cancer cells.
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PMID:Telomerase activation by hTRT in human normal fibroblasts and hepatocellular carcinomas. 942 3

Hepatitis B virus (HBV) and hepatitis C virus (HCV) are known to be associated with hepatocellular carcinoma (HCC). In this study, we investigated the prevalence of the newly described hepatitis G virus (HGV) in patients with HCC. The sera of 85 patients (66 male, 19 female, 61 +/- 11 years) with HCC were studied for the presence of HGV RNA by reverse transcriptase-polymerase chain reaction. Seventeen (20%) of 85 patients with HCC, 10 (16%) of 61 patients with chronic hepatitis B without HCC and 14 (20%) of 68 patients with chronic hepatitis C without HCC were infected with HGV, a significantly higher proportion when compared with two (2%) of 85 healthy controls (P < 0.01). When grouped according to the underlying cause of liver disease, HCC patients with HBV infection (33%), HCV infection (21%), alcoholic liver disease (17%), or cryptogenic cirrhosis (15%) had similar serum levels of HGV RNA. Four of the 17 (24%) HGV-positive patients with HCC were coinfected with HBV and six (35%) with HCV; thus, 59% of HGV-positive patients with HCC were coinfected with other hepatotropic viruses. Seven (41%) HGV-positive patients were infected with HGV only. Patients with HGV infection were more likely to have a history of blood transfusion than patients without HGV infection (P = 0.024). Hence, the prevalence of HGV is significantly higher in patients with HCC in comparison with the healthy population.
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PMID:Prevalence of hepatitis G virus in patients with hepatocellular carcinoma. 943 Mar 61


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