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)

Only small numbers of cells from solid tumours are needed for haematogenous metastasis. Detection is difficult because existing techniques are not sensitive enough. We have used reverse transcriptase to make complementary DNA from peripheral blood messenger RNA, and the polymerase chain reaction (PCR) to amplify cDNA specific for a gene actively transcribed only in the tumour tissue type. We prepared cDNA from peripheral blood of seven patients with malignant melanoma, four patients with other metastatic cancers, and four healthy subjects, as well as from several melanoma-derived cell lines. PCR was used to amplify the gene for tyrosinase, a tissue-specific gene in melanocytes. Since normal melanocytes are not thought to circulate in peripheral blood, detection of tyrosinase transcription in peripheral blood should indicate the presence of circulating cancer cells. The method was highly sensitive and could detect a single melanoma cell from a cell line in 2 ml normal blood. Blood samples from four of the seven patients with malignant melanoma gave positive results, whereas all eight control subjects gave negative results. This method does not depend on the characterisation of cancer-specific genetic abnormalities and can be applied to any cancer for which tissue-specific genes can be identified, including epithelial cancers. It could prove useful in the diagnosis of primary or metastatic cancers, in assessing prognosis, and in detecting residual disease after treatment.
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PMID:Detection of melanoma cells in peripheral blood by means of reverse transcriptase and polymerase chain reaction. 171 20

Several derivatives of levodopa have been shown to be potent inhibitors of the sulfhydryl enzyme, RNA dependent DNA polymerase, reverse transcriptase (RT). In the presence of the polyphenol oxidase, tyrosinase, the inhibitory values were between 10(-6) M and 10(-5) M. Structure-activity studies revealed that active oxidation or reduction was necessary for this potent inhibitory response. Spectrophotometric analysis showed that the presence of both the quinone and quinol was required for maximum inhibitory activity. These data suggest that the common intermediate of oxidation of quinols or reduction of quinones (i.e., semiquinone) is the active species. The use of tyrosinase provides a convenient model for the detection of the actual inhibitory interaction of a free-radical (semiquinone) with a biologically important macromolecule, reverse transcriptase.
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PMID:Inhibition of reverse transcriptase by tyrosinase generated quinones related to levodopa and dopamine. 617 37

The relationship between the process of neural induction and the control of tyrosinase gene expression in the cells that derive from the neural crest of amphibians has been examined at the molecular level. [3H] Tyrosinase cDNA was utilized as a probe to measure the levels of tyrosinase RNA transcripts present in Rana pipiens embryos from the time of fertilization through Stage 25 of cleavage (operculum complete, 240 h) and to correlate these levels with those published for the rate of tyrosinase protein synthesis. R. pipiens [3H]tyrosinase cDNA was synthesized from a purified tyrosinase mRNA template using avian myeloblastosis virus reverse transcriptase and was enriched for full length copies by preparative polyacrylamide gel electrophoresis. This cDNA product was estimated to represent greater than 90% by length of tyrosinase mRNA and hybridized to tyrosinase mRNA to greater than 97% within two orders of magnitude of R0t values. The extent of hybridization of [3H]tyrosinase cDNA to total embryonic RNA throughout early development paralleled the rate of synthesis of tyrosinase protein. Tyrosinase RNA transcripts were first detected at Stage 12-13 (0.0032% of total RNA) and rose to maximal levels by Stage 19 (0.011%). This represents a 50-fold increase from preinduction levels. These results are consistent with a model which predicts that one of the early events in the development of neural crest derivatives is the transcriptionally dependent accumulation of functionally mature tyrosinase mRNA.
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PMID:Control of tyrosinase gene expression and its relationship to neural crest induction in Rana pipiens. II. Measurement of tyrosinase mRNA accumulation during early embryogenesis using a specific cDNA probe. 641 41

We applied an enzyme-linked immunospot (ELISPOT) assay for the detection and quantification of blood-derived CD8+ T cells recognizing peptide antigens presented by HLA-A2.1. CD8+ T lymphocytes were isolated from peripheral blood and were stimulated for 40 h with peptide-loaded A2.1-positive 0.174 x CEM.T2 cells. Tumor necrosis factor alpha (TNF-alpha) secreted by single T cells in response to antigen contact was trapped on nitrocellulose membranes precoated with anti-TNF-alpha antibodies and was then immunochemically visualized as spots. With this assay, up to 25% of cloned cytolytic T lymphocytes (CTL) were detected during the test period that recognized defined melanoma antigens in association with HLA-A2.1. CD8+ lymphocytes responsive to a known immunogenic HLA-A2.1-binding peptide from reverse transcriptase of the human immunodeficiency virus (HIV) were only detectable in HIV-infected patients, but not in anti-HIV-negative donors. T cells reacting with a peptide derived from a mutated cyclin-dependent kinase 4 (CDK4-R24C) were exclusively detected among CD8+ lymphocytes isolated from blood of the patient, whose melanoma had previously been found to carry the CDK4-R24C allele. T cells responding to HLA-A2.1-associated peptides of normal melanocyte differentiation antigens tyrosinase and Melan-A/MART-1 were found at low frequencies in almost all donors tested, which might reflect a natural autoimmunity to these antigens. However, in a melanoma patient we found a few days after surgery of melanoma metastases high frequencies of T cells against Melan-A/MART-1 and tyrosinase peptides (up to 38 per 10(5) CD8+ T cells), which gradually decreased during the following months. In an HIV-infected patient with progressive disease we observed a loss of T cells reactive with the HIV reverse transcriptase peptide. These observations provide evidence that peptide-dependent TNF-alpha spot formation in vitro resulted from previous antigen exposure in vivo. Therefore, the TNF-alpha ELISPOT assay might be useful in monitoring antigen-specific T lymphocyte responses during the natural course of diseases as well as during therapeutic interventions aiming at the induction of protective T cell immunity. In addition, it might help to identify immunodominant T cell epitopes.
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PMID:Detection and quantification of blood-derived CD8+ T lymphocytes secreting tumor necrosis factor alpha in response to HLA-A2.1-binding melanoma and viral peptide antigens. 866 32

Sequence analysis of two clones found repressed in melanoma cell lines in earlier studies showed 9F2 to be identical with the TRP-1 gene and 6F5 with TRP-2 containing a long untranslated 3' end. For further investigation of the expression of the tyrosinase gene family in normal and malignant melanocytic cells, a series of melanoma cell lines and of cultured melanocytes were analyzed by Northern blotting and by reverse transcriptase-polymerase chain reaction (RT-PCR). The Northern blots were probed with cDNA fragments specific for TRP-1, TRP-2, and tyrosinase, for nested tyrosinase-PCR the outer primers specified a 284 bp and the nested primers a 207 bp fragment. Investigations on 14 established melanoma cell lines grown in different media compared with seven normal human melanocyte (NHM) cultures revealed that all three pigment genes were expressed in NHM, whereas pigment gene expression was found repressed in nearly all melanoma cell lines and was completely absent in 4 of 14 specimen. In particular, tyrosinase and TRP-2 genes were found always to be expressed together, and TRP-1 mRNA alone was absent in four melanoma cell lines. Negativity of cultured melanoma cells for tyrosinase mRNA was confirmed by nested RT-PCR, and gene deletion was ruled out by genomic Southern blots. The gene expression seemed independent from the type of medium used for cultivation. These findings indicate repressed or lacking expression of pigment genes in melanoma cell lines, most likely due to regulatory mechanisms, and that differences may exist between tyrosinase and TRP-2 on one hand and TRP-1 on the other. Overall, it seemed that RT-PCR for tyrosinase has limited value for identifying melanoma cells in the peripheral blood of melanoma patients; TRP-1, TRP-2, and other, additional markers may be required.
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PMID:Incomplete expression of the tyrosinase gene family (tyrosinase, TRP-1, and TRP-2) in human malignant melanoma cells in vitro. 878 39

Retinal pigment epithelial (RPE) cells, which proliferate and dedifferentiate under several pathological conditions, and cultured RPE cells have been considered a good model for comparison. In this investigation, we compared the properties of RPE cells in proliferative vitreoretinopathy with that of cultured human RPE cells. mRNAs of RPE cells from patients with proliferative vitreoretinopathy and from cultured human RPE cells were extracted, and reverse transcriptase-polymerase chain reaction was performed. We also examined cells that were aspirated from bare RPE surface from a patient with a giant retinal tear. We amplified the interleukin-6 gene in the proliferative membranes and cultured RPE cells. We also amplified the tyrosinase gene in seven of eight proliferative membranes, as well as tyrosinase-related proteins and cellular retinaldehyde-binding protein genes, but not the tyrosinase gene in cultured RPE cells. The cells aspirated from bare RPE surface showed reduced activity for expressing interleukin-6 and tyrosinase genes. The dedifferentiation characteristics of cultured RPE cells were different, in that they were less active than RPE cells in proliferative membranes for expressing the genes of melanogenesis, which are essential for pigment cells. Interleukin-6 and genes that were related to melanogenesis were expressed in the proliferative membranes and may play an important role in the generation of proliferative vitreoretinopathy.
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PMID:The properties of retinal pigment epithelial cells in proliferative vitreoretinopathy compared with cultured retinal pigment epithelial cells. 898 78

To evaluate the specificity and applicability to the study of human tumor cells of the reverse transcription (RT) in situ PCR and RT polymerase chain reaction (PCR) in situ hybridization techniques, we examined five melanoma cell lines and five nonmelanoma lines for tyrosinase mRNA using primers specific for tyrosinase. Each procedural step was optimized and minutely controlled, and results from the in situ techniques and solution-phase RT-PCR were compared. All melanoma lines showed a specific pattern of perinuclear cytoplasmic reaction not seen in nonmelanoma lines. There was exact agreement between the results from the RT in situ PCR and RT-PCR in situ hybridization techniques and those from solution-phase RT-PCR. Ribonuclease digestion abolished cytoplasmic staining, as did omission of the reverse transcriptase step. Nuclear staining was seen in melanoma and nonmelanoma lines, apparently as a result of DNA synthesis from repair-replication and mispriming or nonspecific amplification. Neither high concentrations of deoxyribonuclease nor long incubation periods abolished this effect completely. Demonstration of cytoplasmic mRNA by RT in situ PCR and RT-PCR in situ hybridization specifically identifies cells of melanocytic lineage.
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PMID:Demonstration of cytoplasmic tyrosinase mRNA in tissue-cultured cells by reverse transcription (RT) in situ polymerase chain reaction (PCR) and RT PCR in situ hybridization. 902 34

While not all circulating tumor cells survive, one could postulate that patients with circulating tumor cells might be candidates for adjuvant chemotherapy because of the risk of relapse. Recently, reverse transcriptase-polymerase chain reaction (RT-PCR) for the detection of circulating tumor cells has been suggested as a potential technique for staging of cancer. In malignant melanoma, the detection of circulating melanoma cells by tyrosinase RT-PCR correlated with the clinical stage of patients and was an independent prognostic factor for recurrence. A strong association was found between the detection of neuroblastoma cells in circulation by tyrosine hydroxylase RT-PCR with bone marrow micrometastasis. This method may be of use to identify early signs of relapse in disease-free patients. In prostatic cancer, the frequency of positivity for detection of circulating tumor cells in peripheral blood by PSA RT-PCR increased with tumor stages but a significant proportion of patients with metastatic disease were negative. The prognostic significance of the detection of tumor cell in blood in other epithelial tumors is not established and will require further long-term follow-up study.
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PMID:[Molecular diagnostic detection of circulating tumor cells and their prognostic implications]. 905 Nov 25

Dopamine acts, under appropriate conditions, as a selective neurotoxin. This toxicity is attributed to the autoxidation of the neurotransmitter into a reactive quinone that covalently modifies cellular macromolecules (i.e. proteins and nucleic acids). The oxidation of the catecholamine to a quinone is greatly accelerated by the enzyme tyrosinase. There is controversy, however, as to whether or not tyrosinase is expressed in human brain. In the present study, RT-PCR was utilized to demonstrate the presence of tyrosinase mRNA in post-mortem human brain tissues. Using gene-specific amplification primers, specific tyrosinase amplicons were detected following analysis of RNA from substantia nigra of four individuals. Analysis of cerebellar RNA from the same individuals produced no amplification products. Control reactions performed in the absence of reverse transcriptase failed to generate PCR products for any tissue tested. Three amplicons were subjected to direct DNA sequencing and all proved to be identical with tyrosinase sequences, thus obviating the possibility of amplification of a related gene. It is clear, therefore, that the tyrosinase gene is expressed in the human substantia nigra, lending support to previous studies describing tyrosinase-like activity and immunoreactive protein in the brain. This enzyme could be central to dopamine neurotoxicity as well as contribute to the neurodegeneration associated with Parkinson's disease.
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PMID:Tyrosinase mRNA is expressed in human substantia nigra. 910 85

Melanomas tend to become less pigmented in the course of malignant progression. Thus, as proliferation increases, the tumors are decreasingly characterized by the tissue-specific phenotype of normally differentiated melanocytes. To learn whether the decline in melanization is associated with a shift from constitutive to alternative splicing of some pigment gene pre-mRNAs, melanomas were collected from Tyr-SV40E transgenic mice of the standard C57BL/6 strain. The mRNAs of the tyrosinase gene, which has a key role in melanogenesis, were analyzed by quantitative reverse transcriptase-PCR in 34 samples from 16 cutaneous tumors and 9 metastases. The cutaneous tumors included some cases with distinct melanotic and amelanotic zones, which were separately analyzed. All tyrosinase transcripts found in the melanomas were also found in normal skin melanocytes. However, the Delta1b and Delta1d alternatively spliced transcripts, due to deletions within the first exon, were specifically augmented in most of the tumors over their very low levels in skin; the exceptions were some all-amelanotic tumors in which no tyrosinase transcripts were detected. The level of Delta1b rose as high as 11.3% of total tyrosinase mRNAs as compared with 0.6% in skin; Delta1d reached 4.0% as compared with 0. 8% in skin. Expression of these splice variants was highest in the melanotic components of zonal primary tumors, relatively lower in their amelanotic components, and still lower in all-amelanotic primary tumors and amelanotic metastases. The increase in Delta1b and Delta1d transcripts may be predicted to increase the levels of unusual peptides, which could have antigenic potential in the tumors, especially in the relatively early phases of malignancy. Analyses of the alternative transcripts of other pigment genes may identify additional candidate antigens, ultimately enabling melanoma cells in all phases of the disease to be represented as a basis for immune intervention.
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PMID:Selective increase in specific alternative splice variants of tyrosinase in murine melanomas: a projected basis for immunotherapy. 914 37


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