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)

Human thyroglobulin mRNA was isolated from Graves' goitres by size selection of total poly(A)-rich RNA in a sucrose gradient. It sedimented at 33 S, as in other mammalian species, and showed a single component of approximately 8500 bases by gel electrophoresis. cDNA was synthesized from the 33-S RNA by using reverse transcriptase in the presence of human placenta ribonuclease inhibitor and in conditions allowing the formation of long transcripts. The latter was made double-stranded using reverse transcriptase and blunt-ended with nuclease S1. After tailing with dCTP and terminal transferase, the double-stranded cDNA was annealed to pBR322 DNA that had been cleaved at the endonuclease PstI site and tailed with dGTP. The resulting plasmids were used to transform Escherichia coli C600 cells and four cloned recombinants were selected. Each plasmid DNA was shown to contain a sequence complementary to human thyroglobulin mRNA by hybridization with a labeled 33-S mRNA, visualization of cDNA . mRNA hybrids by electron microscopy and filter hybridization selection of mRNA directing the synthesis of immunologically related thyroglobulin peptides in the reticulocyte lysate. The four inserted DNA sequences were 1400 - 1800 base pairs long, two of them showing an homologous sequence of 1100 base pairs. Together, the four cloned DNA fragments represented 63% of the 8500 bases of human thyroglobulin mRNA.
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PMID:Cloning of four DNA fragments complementary to human thyroglobulin messenger RNA. 617 25

A recent report has identified a new autoantigen called D1 that appears to be associated with Graves' ophthalmopathy and is expressed in the thyroid and eye muscle. To better characterize the tissue specificity and disease relevance of this antigen, we evaluated the expression of D1 RNA in various human tissues using a reverse transcriptase polymerase chain reaction assay. These studies indicate a wide tissue distribution of the messenger RNA for this antigen, including the thyroid, eye muscle, parathyroid, spleen, skeletal muscle, and uterus. There were variations in the relative amounts of specific message for D1 in the different tissues, with the uterus, thyroid, and eye muscle having the greatest amount of product per microgram of total RNA. A maltose binding protein-D1 fusion protein was expressed in Escherichia coli, purified, and used to assess serologic reactivity to D1 by Western blot. Autoantibodies to this antigen were noted in 19 of 24 (78%) of Hashimoto's disease patients, 26 of 41 (63%) of Graves' disease patients, and in 9 of 17 (53%) of normal controls. Sixty percent of Graves' disease patients with clinical ophthalmopathy had antibodies to D1, as did 63% of Graves' patients without signs or symptoms of clinical ophthalmopathy. There was no correlation between reactivity to D1 and either clinical measures of hyperthyroidism or antibody titers to thyroid peroxidase or thyroglobulin. The presence of autoantibodies to this antigen in patients with Hashimoto's disease, in Graves' disease patients without ophthalmopathy and in normal controls indicate that serologic recognition of this antigen is not restricted to patients with ophthalmopathy. In addition, the expression of messenger RNA for this antigen in multiple types of cells questions the tissue specificity of this autoantigen.
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PMID:Tissue specificity and serologic reactivity of an autoantigen associated with autoimmune thyroid disease. 834 48

There has been much controversy about the presence of TNF-alpha within thyroid tissue. We therefore conducted a study to determine if TNF-alpha mRNA is present in thyroid tissue and thyroid-derived cells. Semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was employed with a heterologous competitor fragment. Significantly lower levels of TNF-alpha mRNA were found in the autonomous nodules from patients with thyroid autonomy (TA; n = 4; 5.7 +/- 1.3 arbitrary units (AU) (mean +/- s.e.m.); P < 0.03) and in normal thyroid tissue (n = 2, 7.0 +/- 3.1 AU) compared with tissue from patients with Graves' disease (GD; n = 13; 27.9 +/- 10.3 AU), non-toxic multinodular goitre (NTG; n = 5; 20.9 +/- 5.8 AU) and perinodular tissue from TA patients (20.3 +/- 4.0 AU). Higher levels were detected in tissues from patients with Hashimoto's thyroiditis (HT; n = 2; 51.3 +/- 10.3 AU). Cultures of pure thyroid-derived fibroblasts (46 +/- 18 AU thyrocytes (33 +/- 8 AU), and the anaplastic thyroid carcinoma cell lines 8505 C (39 +/- 11 AU), SW 1736 (214 +/- 16 AU) and C643 (3 +/- 1 AU) showed significantly lower TNF-alpha mRNA levels than thyroid-derived lymphocytes (1650 +/- 32 AU). TNF-alpha was detected in the supernatants of unstimulated lymphocytes (22.1 +/- 1.1 pg/ml) and SW 1736 cells (3.5 +/- 0.9 pg/ml), but not in unstimulated fibroblasts and thyrocytes. Using an intracellular labelling technique in flow cytometry, the immunophenotype of stimulated TNF-alpha-positive lymphocytes was determined as predominantly CD3+CD45RO+. Our results suggest that TNF-alpha is present in the thyroid tissue of different thyroid disorders. Thyroid-derived lymphocytes are potential TNF-alpha producers and may thus locally influence thyroid function.
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PMID:Expression of tumour necrosis factor-alpha (TNF-alpha) mRNA and protein in pathological thyroid tissue and carcinoma cell lines. 869 23

We have developed a competitive reverse transcriptase-polymerase chain reaction (RT-PCR) assay for the quantitation of 1D mRNA, which encodes a 64-kDa protein associated with thyroid-associated ophthalmopathy, in preparations of total RNA from a variety of human tissues. This competitive RT-PCR assay is based on coamplification of competing 1D cDNA internal control template (pGEM-1D') and target cDNA template. 1D-specific mRNA was quantified in 10 human tissues. The level of 1D transcript expression in these tissues decreased in the following order: thyroid (5 pg/mg total RNA) > eye muscle (3.2 pg/mg) > skeletal muscle (2.4 pg/mg) > ovary (2 pg/mg) > cerebellum (0.4 pg/mg), kidney (0.33 pg/mg), pancreas (0.27 pg/mg), spleen (0.22 pg/mg), and thymus (0.19 pg/microgram) > retina (0.016 pg/mg). Graves' disease may be a multisystem autoimmune disorder of the connective tissue and skeletal muscle (and thyroid) that is mainly localized in the orbit and skin. One mechanism for this localization may be increased expression of target autoantigens, of which the 64-kDa protein 1D is a candidate, in the involved tissues. The higher expression of the 1D molecule in thyroid and eye supports this hypothesis.
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PMID:Tissue distribution and quantitation of a gene expressing a 64-kDa antigen associated with thyroid-associated ophthalmopathy. 881 Oct 43

Healthy humans have CD4+ T cells specific for self-components. Since autoreactive T cells in autoimmune patients may use a limited number of TCR V-region genes, we investigated here whether this also occurs for the potentially autoreactive CD4+ cells present in healthy persons. We studied CD4+ cells specific for human TSH receptor (TSHr) sequences, that are present with high frequency in healthy subjects and, as expected, in Graves' disease (GD) patients. We used short-term CD4+ cell lines propagated from four GD patients and five healthy subjects by cycles of stimulation with a pool of overlapping synthetic peptides corresponding to the putative extracellular parts of the TSHr sequence. The lines recognized the pool of TSHr peptides specifically and vigorously. Their epitope repertoire had been characterized previously: each line recognized one or a few TSHr peptides, different for each subject. We determined their TCR Vbeta usage by a semi-quantitative reverse transcriptase PCR assay, using primers specific for each known human Vbeta region family, in conjunction with a constant region primer. Six lines preferentially used one Vbeta family (42-94%), different for each line. In all lines, three or less Vbeta families accounted for approximately 60% or more of the Vbeta usage. Different Vbeta regions were used by each subject. There was no obvious difference between the Vbeta usage of the lines from GD patients and healthy controls. These results suggest that a limited pool of potentially autoreactive T cells survives clonal deletion. The pathogenic CD4+ cells involved in autoimmune diseases are likely recruited from that pool, since they have similar characteristics of epitope and TCR repertoire as the CD4+ cells specific for the same autoantigen in healthy subjects.
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PMID:TCR vbeta usage of TSH receptor-specific CD4+ T cells in Graves' disease patients and healthy humans. 937 76

Restricted expression of oncofetal fibronectin mRNA in the tissues of thyroid papillary and anaplastic carcinoma has recently been shown by both Northern blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR). Oncofetal fibronectin mRNA can be a target of gene diagnosis and targeted gene therapy, provided it is expressed in all cancer cells in the tissues. To investigate this criterion in thyroid cancer tissues, we measured their expression of oncofetal fibronectin mRNA using in situ hybridization. An abundant expression of oncofetal fibronectin mRNA was found in all the observed cancer cells of six papillary carcinomas and an anaplastic carcinoma, but not in the tissues of normal thyroid, Graves' disease, adenomatous goitre, follicular adenoma, follicular carcinoma or medullary carcinoma. This result encourages us to establish gene diagnosis of thyroid papillary and anaplastic carcinomas by detecting oncofetal fibronectin mRNA in biopsies.
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PMID:Restricted expression of oncofetal fibronectin mRNA in thyroid papillary and anaplastic carcinoma: an in situ hybridization study. 968 97

To evaluate the regulatory mechanism of human Type 1 iodothyronine deiodinase (D1) gene expression, we measured the D1 mRNA levels in peripheral blood mononuclear cells (PBMC) in normal control subjects and in patients with Graves' disease. We used competitive reverse transcriptase-polymerase chain reaction with the deleted complimentary RNA of D1 as the standard for quantification. The D1 mRNA levels in PBMC were increased significantly in patients with Graves' disease compared with that in normal controls. There was a significant (p < 0.01) positive correlation (r=0.698) between D1 mRNA level and serum T3 concentration. When PBMC from the normal volunteers were cultured with various doses of T3, the quantity of D1 mRNA increased significantly in a dose-dependent manner. These findings indicate that PBMC D1 mRNA is actually up-regulated by T3 in vivo, and we postulate that a vicious spiral of increasing T3 and D1 is responsible for the exacerbation of thyrotoxicosis in hyperthyroid Graves' disease.
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PMID:Quantitative measurements for type 1 deiodinase messenger ribonucleic acid in human peripheral blood mononuclear cells: mechanism of the preferential increase of T3 in hyperthyroid Graves' disease. 978 99

We addressed the role of soluble Fas (sFas), which suppresses Fas-mediated apoptosis, in the pathogenesis of Graves' disease (GD). The serum concentration of sFas was measured by enzyme-linked immunosorbent assay and the expression of sFas mRNA in thyroid tissues by reverse transcriptase-polymerase chain reaction. The serum concentration of sFas was significantly increased in untreated GD (mean+/-SD: 1.57+/-0.48 ng/mL) compared to age-matched control subjects (0.77+/-0.46 ng/mL). The serum sFas level tended to decrease after the medication of antithyroid drugs for 6 to 8 weeks and was significantly decreased in patients who were euthyroid for more than 3 years (0.98+/-0.23 ng/mL), compared to that in untreated GD. The concentration of serum sFas was significantly correlated with anti-thyrotropin (TSH) receptor antibody titers, but not with the other clinical parameters (free triiodothyronine [FT3], free thyroxine [FT4], TSH, antithyroglobulin antibody titer, antimicrosomal antibody titer, or 123I uptake). The sFas mRNA was detected in thyroid tissue, cultured thyrocytes, and intrathyroidal lymphocytes. sFas was detected in supernatant of cultured thyrocytes from patients with GD. Its production by thyrocytes was induced by culture with interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha). The present study confirms serum sFas increases in GD and provides evidence of local production of sFas by thyrocytes and its regulation by cytokines. These data suggest that sFas may play a role in the pathogenesis of GD.
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PMID:Increased serum soluble Fas in patients with Graves' disease. 1031 38

The ability to concentrate iodide, a fundamental property of normally functioning thyroid tissue, is altered in various thyroid diseases. Given the critical role of the Na+/I- symporter (NIS) in controlling iodide access to the thyroid gland, altered expression of NIS may be responsible, at least in part, for an enhanced or diminished capacity to concentrate iodide. In this study, we used Northern blot analysis, a newly established quantitative polymerase chain reaction (PCR) assay and in addition hNIS-directed immunohistochemical analysis to assess the levels of hNIS mRNA and protein expression in various localized and diffuse benign thyroid abnormalities, including Graves' disease (GD), scintigraphically cold solitary benign thyroid nodule (CBTN), nontoxic multinodular goiter (NMNG), solitary autonomously functioning thyroid nodule (AFTN), and mild diffuse iodine deficiency goiter (IDG). In addition, in view of the recent identification of putative binding sites for the transcription factors thyroid transcription factor-1 (TTF-1) and human paired-box-protein-8 (Pax-8) in the human NIS gene promoter, we used reverse transcriptase-polymerase chain reaction (RT-PCR) to assess in these same samples the levels of TTF-1 and Pax-8 gene expression. Northern blot analysis revealed high levels of hNIS gene expression in thyroid specimens derived from patients with GD and AFTN. In contrast, levels of hNIS mRNA expression were moderate in NMNG, low in diffuse IDG, and very low in CBTN. Quantitative RT-PCR analysis of hNIS mRNA transcripts revealed variable but generally low levels of hNIS gene expression in IDG and NMNG, and undetectable or very low levels of hNIS mRNA in all scintigraphically CBTN studied. In contrast, markedly elevated levels of hNIS mRNA transcripts were detected in active GD (up to 17-fold) and AFTN (up to 25-fold). Immunohistochemical analysis revealed abundant hNIS protein expression by thyroid follicular cells in GD, moderate and heterogeneous levels in NMNG, and very low levels in CBTN. hNIS mRNA levels were correlated with TTF-1 and Pax-8 gene expression in GD and, to a lesser degree, in AFTN, NMNG, and IDG, but not in CBTN. In general, hNIS gene expression was more closely correlated with TTF-1 as compared to Pax-8 gene expression. In conclusion, the abundance of hNIS mRNA and protein expression in a broad range of benign thyroid pathologies correlated well with their functional state as assessed by thyroid scintigraphy. In addition to TTF-1 and Pax-8, other transcription factors and enhancer elements may contribute to regulation of NIS gene promoter activity.
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PMID:Analysis of human sodium/iodide symporter, thyroid transcription factor-1, and paired-box-protein-8 gene expression in benign thyroid diseases. 1036 77

The tissue specific origin of thyroglobulin (Tg) in the blood has made this protein very applicable as a diagnostic marker in various thyroid diseases such as differentiated thyroid cancer (DTC), subacute thyroiditis (ST), destructive thyroiditis, Graves' disease (GD) and congenital thyroid diseases, although the mechanism by which Tg is released and the molecular structure in which it appears in the circulation are not fully understood. This review first describes serum Tg measurements using the immuno-radiometric assay kits available in Japan and the problem of this assay in relation to interference by the autoantibody to Tg. Finally, future directions of serum Tg measurement and its clinical application are considered based on the recent evidence that the molecular form of Tg in the circulation has characteristics that differ for specific thyroid diseases (DTC, ST or GD), and that the detection of the Tg molecule by reverse transcriptase polymerase chain reaction (RT-PCR) is possible from using thyroid cells in peripheral circulation and is effective in diagnosis and monitoring of DTC.
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PMID:[Serum thyroglobulin measurement and its clinical significance]. 1048 54


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