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)

N-Acetylation is a major pathway in the metabolism of hydrazine and arylamine drugs, and has been associated with carcinogen bioactivation. Monomorphic hamster liver N-acetyltransferase (NAT1) cDNA was cloned from hamster liver cells by reverse transcriptase-coupled polymerase chain reaction. The determined nucleotide sequence was identical to that reported for NAT1. The NAT1 coding region was subcloned into the pG1 yeast expression vector, but cell extracts provided only transient acetyltransferase activity. In addition, cDNA was subcloned into the expression vectors pFLAG-ATS and pFLAG-MAC. The latter vectors encoded a tac promoter and appended a low-molecular weight (1 kDa) hydrophilic FLAG marker peptide to the amino terminus of NAT1. Unexpectedly, periplasmic export of FLAGATS-NAT1 by the ompA signal peptide of pFLAG-ATS proved to be detrimental to enzyme activity. High acetyltransferase activity, however, was obtained when the fusion protein was expressed in the cytosol. Enzyme purified to homogeneity by immunoaffinity chromatography exhibited substrate specificity comparable to that of the hamster-derived protein.
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PMID:Hamster monomorphic arylamine N-acetyltransferase: expression in Escherichia coli and purification. 775 38

Using both tumor specimen and cultured tumor cells, we have studied the differentiation of a pineocytoma by light and electron microscopy (EM) and immunohistochemical demonstration of glial, neuronal and neuroendocrine markers. Only interstitial cells were labeled with anti-glial fibrillary acidic protein and anti-S100 protein antibodies. Synaptophysin, neurofilaments and tau labeling was found in cells forming the pineocytomatous rosettes. Some cells also bound the anti-tryptophan hydroxylase antibody (TPOH), but no staining was seen after application of anti-chromogranin A or S-antigen antibodies. EM provided evidence for neurosensory differentiation demonstrating the presence of vesicle-crowned rodlets, cilia (9+0) and fibrous filaments. In culture, tumor cells proliferated slowly and showed positive immunolabeling for vimentin and TPOH. Expression of mRNA coding for TPOH, serotonin N-acetyltransferase, hydroxyindole-O-methyl-transferase and c-myc was found in the tumor using reverse transcriptase-polymerase chain reaction. These results demonstrate neuronal differentiation of this pineocytoma and suggest that the neoplastic pineal cells are capable of synthesizing serotonin and melatonin.
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PMID:Immunohistochemical, ultrastructural, biochemical and in vitro studies of a pineocytoma. 960 Jun

In our previous studies, the opioid receptors located on pinealocytes have been identified and characterized, and these receptors have been found to play a stimulatory role in melatonin synthesis by activating the rate limiting enzyme, N-acetyltransferase (NAT). In the present study, by using reverse transcriptase polymerase chain reaction (RT-PCR) followed by nested-PCR, segments of delta and mu opioid receptors have been amplified from mRNA of rat pineal gland and cerebral cortex. In addition, segments of delta and mu opioid receptors have also been amplified from mRNA of human pineal gland. Furthermore, G(alphai/o)- and G(beta)-protein-coupled receptor mRNAs have been amplified and identified from rat pineal gland. The regulatory effects of morphine on G(alphai/o) and G(beta) mRNA levels have been semiquantitatively analyzed. Acute morphine administration caused significant increase in G(alphai/o), and G(beta), mRNA levels in rat pineal gland, but not in other brain regions. Further studies are needed in order to elaborate the mechanisms of these opioid receptors in regulating G-protein expression in pineal gland.
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PMID:Gene expressions of opioid receptors and G-proteins in pineal glands. 1047 1

Somatostatin is a potent antiproliferative signal in both tumoral and normal mammalian cells, and altered somatostatin receptor (sst) expression is associated with carcinogenesis in human tissues. In this study, two normal and three tumoral human pineal glands were analyzed using the reverse transcriptase-polymerase chain reaction (RT-PCR) for the presence of mRNA coding for the five different somatostatin receptors (sst1-sst5). Pineal parenchymal tumor (PPT) differentiation was confirmed by immunohistochemical detection of neuroendocrine markers (synaptophysin, neurofilaments, and chromogranin A). The presence of mRNA coding for c-myc, a proto-oncogene, and for tryptophan hydroxylase (TPOH), serotonin N-acetyltransferase (NAT), and hydroxyindole-O-methyltransferase (HIOMT), enzymes of the melatonin pathway, was also analyzed by RT-PCR. Only the tumoral tissues contained c-myc mRNA. All five tissues contained TPOH, NAT, and HIOMT mRNA, the levels of HIOMT mRNA being lower in PPT than in the normal pineal gland, suggesting that PPT retain the ability to synthesize melatonin. All tissues contained sst1, sst2, and sst3 transcripts, but not sst4, while small amounts of sst5 mRNA were only found in normal pineal glands. Real-time PCR, performed only with the most abundant subtpe sst2, evidenced an about sixfold higher level in in normal pineal glands. These results demonstrate the presence of somatostatin receptors in the human pineal gland, as described in other species, and point to a differential expression of the sst2 and sst5 subtypes associated with carcinogenesis.
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PMID:Differential somatostatin receptor subtype expression in human normal pineal gland and pineal parenchymal tumors. 1270 86

N-Acetyltransferases (NATs) plays an important role in the first step of arylamine compounds metabolism. Polymorphic NAT is coded for rapid or slow acetylatoion phenotypes, which are recognized to affect cancer risk related to environmental exposure. Aloe-emodin has been shown to exit anticancer activity. The purpose of this study is to examine whether or not aloe-emodin could affect arylamine N-acetyltransferase (NAT) activity and gene expression (NAT mRNA) and DNA-2-aminofluorene (DNA-AF) adduct formation in mouse leukemia cells (L 1210). By using high performance liquid chromatography, N-acetylation and non-N-acetylation of AF were determined and quantitated. By using reverse transcriptase-polymerase chain reaction (RT-PCR) and PCR, NAT mRNA was determined and quantitated. Aloe-emodin displayed a dose-dependent inhibition to cytosolic NAT activity and intact mice leukemia cells. Time-course experiments indicated that N-acetylation of AF measured from intact mice leukemia cells were inhibited by aloe-emodin for up to 24h. Using standard steady-state kinetic analysis, it was demonstrated that aloe-emodin was a possible uncompetitive inhibitor to NAT activity in cytosols. The DNA-AF adduct formation in mouse leukemia cells were inhibited by aloe-emodin. The NAT1 mRNA in mouse leukemia cells were also inhibited by aloe-emodin. This report is the first demonstration which showed aloe-emodin affect mice leukemia cells NAT activity, gene expression (NAT1 mRNA) and DNA-AF on adduct formation.
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PMID:Aloe-emodin inhibited N-acetylation and DNA adduct of 2-aminofluorene and arylamine N-acetyltransferase gene expression in mouse leukemia L 1210 cells. 1280 42

Previous studies have identified and characterized D1- and D2-dopamine receptors in bovine pineal glands. The data indicate that the density of D1-dopamine receptors (974 fmol/mg protein) far exceed that of D2-dopamine receptors (37 fmol/mg protein). The objective of this study was to identify the mRNAs for both D1- and D2-dopamine receptors and to elucidate the status of dopamine and its possible involvement in the pineal function, particularly on melatonin synthesis. The expression of these dopamine receptor subtypes were determined by using a reverse transcriptase-polymerase chain reaction technique with specific pairs of primers to amplify D1- and D2-dopamine receptor mRNAs. Amplification of RNAs from bovine striatum (positive control) and bovine pineal gland resulted in products of the predicted lengths of 231 bp for D1- and 333 bp for D2-dopamine receptors. The results indicate that both D1- and D2-dopamine receptor mRNAs are present in the bovine pineal gland. The role of dopamine receptors was investigated by studying the effects of selective D1- and D2-dopamine agonists and antagonists on the N-acetyltransferase (NAT) activity of cultured bovine pinealocytes. The data showed that SKF-38393, a selective D1-agonist, enhanced NAT activity, and increased melatonin level, and the stimulatory effect was blocked by SCH-23390, a D1-selective antagonist, whereas quinpirole, a selective D2-agonist, inhibited NAT basal activity and decreased the melatonin basal level. Furthermore the inhibitory effect was blocked by D2-selective antagonists, spiperone, haloperidol, and domperidone. The present results indicate that the pineal dopamine receptors have a distinct effect on pineal function. The precise mechanism whereby activation of dopamine receptors altered the NAT activity and melatonin level needs to be further delineated.
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PMID:Effects of D1- and D2-dopamine receptor activation on melatonin synthesis in bovine pinealocytes. 1293

In humans, a polymorphic gene encodes the drug-metabolizing enzyme NAT1 (arylamine N-acetyltransferase Type 1), which is widely expressed throughout the body. While the protein-coding region of NAT1 is contained within a single exon, examination of the human EST (expressed sequence tag) database at the NCBI revealed the presence of nine separate exons, eight of which were located in the 5' non-coding region of NAT1. Differential splicing produced at least eight unique mRNA isoforms that could be grouped according to the location of the first exon, which suggested that NAT1 expression occurs from three alternative promoters. Using RT (reverse transcriptase)-PCR, we identified one major transcript in various epithelial cells derived from different tissues. In contrast, multiple transcripts were observed in blood-derived cell lines (CEM, THP-1 and Jurkat), with a novel variant, not identified in the EST database, found in CEM cells only. The major splice variant increased gene expression 9-11-fold in a luciferase reporter assay, while the other isoforms were similar or slightly greater than the control. We examined the upstream region of the most active splice variant in a promoter-reporter assay, and isolated a 257 bp sequence that produced maximal promoter activity. This sequence lacked a TATA box, but contained a consensus Sp1 site and a CAAT box, as well as several other putative transcription-factor-binding sites. Cell-specific expression of the different NAT1 transcripts may contribute to the variation in NAT1 activity in vivo.
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PMID:Genomic organization of human arylamine N-acetyltransferase Type I reveals alternative promoters that generate different 5'-UTR splice variants with altered translational activities. 1548 85

V79 Chinese hamster cells were genetically engineered for the stable co-expression of human cytochrome P450 1A2 and the polymorphic N-acetyltransferase 2 alleles *4, *5B, *6A and *13, in order to generate an in vitro tool for studying the metabolism-dependent toxicity of aromatic amines. N-acetyltransferase 2*4-encoding cDNA was generated by the polymerase chain reaction (PCR) with defined primers from the genomic DNA of a human liver donor homozygous for *4, and served as a template to generate the *5B, *6A and *13 isoforms by site-directed mutagenesis. Human cytochrome P450 (CYP) 1A2-encoding cDNA was generated by the PCR from genomic DNA of the recombinant V79MZh1A2 cell line. All the cDNAs were inserted into a CMV promoter-containing plasmid in conjunction with the selectable marker genes, neomycin and hydromycin. The recombinant expression plasmids were transfected for stable integration into the genomic DNA of the V79 cells. Several cellular clones were obtained and checked for the genomic integration of intact cDNAs with the PCR on the genomic DNA of the recombinant cells. Stable expression was confirmed by the reverse transcriptase PCR (RT-PCR) on RNA preparations. Metabolic function was tested with ethoxyresorufin as a marker substrate for CYP1A2, and 2-aminofluorene and N-sulphametazine for N-acetyltransferase activity, and compared to data obtained from biological samples. 7-Ethoxyresorufin-O-deethylase activities ranged from 0.2 to 4 pmol resorufin/min/mg total protein. The N-acetylation of sulphametazine ranged from 0.07 to 1.7 nmol N-acetyl-sulphametazine/mg total protein/min. Selected clones showing activities in the range of physiological activities were submitted to metabolism dependent mutagenicity studies. In particular, the polymorphism-dependent N-acetylation of 2-aminofluorene and the role of CYP1A2 and N-acetyltransferase in the mutagenicity of 2-aminofluorene, were investigated. Surprisingly, the mutagenicity of 2-aminofluorene is dramatically reduced in V79 cells co-expressing CYP1A2 and N-acetyltransferase, compared to V79 cells expressing CYP1A2 only, pointing to a significant species-dependent difference in the metabolic activation of aromatic amines between rats and humans.
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PMID:Heterologous co-expression of human cytochrome P450 1A2 and polymorphic forms of N-acetyltransferase 2 for studies on aromatic amines in V79 Chinese hamster cells. 1637 32

There are functional inter-relationships between the beta cells of the endocrine pancreas and the pineal gland, where the synchronizing circadian molecule melatonin originates. The aim of this study was to elucidate a putative interaction between insulin and melatonin in diabetic patients and a diabetic rat model. We analyzed glucose, insulin, and melatonin levels of type 2 patients, as well as type 2 diabetic Goto Kakizaki (GK) rats by radioimmunoassay. Expression of pancreatic melatonin and pineal insulin receptors, as well as arylalkylamine-N-acetyltransferase (AANAT), was determined by real-time reverse transcriptase polymerase chain reaction (RT-PCR). The AANAT enzyme activity was measured in pineal homogenates. Diabetic patients showed a decrease in melatonin levels, while in the pancreas of GK rats an upregulation of the melatonin-receptor mRNA was determined. The pancreatic islets of GK rats showed expression of the mRNA for the pancreatic melatonin (MT1) receptor, which had previously been identified in rats and insulinoma (INS1) cells. Besides their presence in animal cells, the MT1-receptor transcript was also detected in human pancreas by RT-PCR. Whereas the rat pancreatic mRNA expression of the MT1-receptor was significantly increased, the activity of the pineal AANAT enzyme was reduced. The latter observation was in accordance with plasma melatonin levels. The insulin-receptor mRNA of the pineal gland was found to be reduced in GK rats. Our observations suggest a functional inter-relationship between melatonin and insulin, and may indicate a reduction of melatonin in the genesis of diabetes.
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PMID:Diabetic Goto Kakizaki rats as well as type 2 diabetic patients show a decreased diurnal serum melatonin level and an increased pancreatic melatonin-receptor status. 1644 50