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)

Thyroid-associated ophthalmopathy (TAO) has a major effect on the two compartments of the retro-orbital (RO) space, leading to enlargement of the extraocular muscles and other RO tissues. T lymphocyte infiltration of RO tissue is a characteristic feature of TAO and there is current interest in whether these T cells are specifically and selectively reactive to RO tissue itself. We recently established 18 T cell lines (TCL) from RO adipose/connective tissue of six patients with severe TAO by using IL-2, anti-CD3 antibodies and irradiated autologous peripheral blood mononuclear cells (PBMC) to maintain the growth of T cells reactive to autologous RO tissue protein fractions. Here we report on the phenotype characteristics and cytokine gene expression profiles of these orbital TCL and on their immunoreactivity to the organ-specific thyroid antigens thyrotropin receptor (TSH-R), thyroidal peroxidase (TPO) and thyroglobulin (TG). Flow cytometry revealed that 10 TCL were predominantly of CD4+ phenotype, three being mostly CD8+ and five neither CD4+ nor CD8+. Analysis with reverse transcriptase-polymerase chain reaction (RT-PCR) of cytokine gene expression revealed both Th1- and Th2-like products in all TCL: IL-2 product (in 17 TCL), interferon-gamma (IFN-gamma) (n = 10), tumour necrosis factor-beta (TNF-beta) (n = 15), IL-4 (n = 12), IL-5 (n = 17), IL-6 (n = 13), TNF-alpha (n = 12) and IL-10 (n = 4). Reactivity to thyroid antigens was observed only in two TCL, the other 16 being uniformly unreactive. Although 10 out of 18 RO tissue-reactive TCL were predominantly CD4+ there were no significant relationships between TCL phenotype, cytokine gene profile, magnitude of reactivity to RO tissue protein or the (rare) occurrence of thyroid reactivity. The findings of both Th1- and Th2-like cytokine gene expression in all RO tissue-reactive TCL support the concept that TAO is a tissue-specific autoimmune disease, distinct immunologically from the thyroid, and involving both T cell and B cell autoimmune mechanisms in disease pathogenesis.
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PMID:Analysis of orbital T cells in thyroid-associated ophthalmopathy. 964 11

Currently, fine-needle aspiration cytology is a valuable tool in the routine diagnosis of suspicious thyroid nodules. We present a very sensitive method for the molecular analysis of the expression of several genes important for normal thyroid function in parallel to the cytological diagnosis. We adapted reverse transcriptase polymerase chain reaction (RT-PCR) to amplify thyroid-typical mRNAs in samples of thyroid carcinoma cells as small as those obtained by fine-needle aspiration biopsy (FNAB), ie, 100-1000 cells, and applied this procedure to four routinely taken FNABs. Gene products such as thyroglobulin (Tg), thyroid-stimulating hormone-receptor (TSHr), sodium/iodide-symporter (NIS), type I iodothyronine-5'-deiodinase (DI), and type II iodothyronine-5'-deiodinase (DII) were analyzed. To establish RT-PCR protocols, serial dilutions of follicular thyroid carcinoma cells, FTC-133, which express these genes at low levels, were initially used for RNA isolation. Successful RNA isolation and reverse transcription were checked by the amplification of beta-actin mRNA. We detected the mRNAs coding for Tg in as little as 10 cells, for NIS in 100 cells, and for TSHr, DI, and DII in 10,000 cells. After preparing cytological smears of four routinely taken FNABs, all above-mentioned thyroid-typical mRNAs were observed by using the material remaining in the needle for RNA isolation followed by RT-PCR. This method offers the possibility of obtaining two different types of information from the same routinely taken thyroid FNAB: the cytological diagnosis and the expression pattern of several diagnostically relevant genes. Therefore, a more specific diagnosis could be rendered in the preoperative state, and may lead to more specific therapy.
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PMID:Reverse transcriptase-polymerase chain reaction analysis of thyrocyte-relevant genes in fine-needle aspiration biopsies of the human thyroid. 984 10

To study the potential roles of cytokines in development and resolution of granulomatous experimental autoimmune thyroiditis (EAT), the kinetics of in vivo expression of cytokine genes in thyroid infiltrates was analysed using reverse transcriptase-PCR (RT-PCR). Both Th1 (IL-2 and IFN-gamma) and Th2 (IL-4 and IL-10) cytokines as well as TGF-betaTNF-alphaIL-12 and IL-1beta were detected in thyroids during both the initial phase and peak of granulomatous EAT. Maximal expression of cytokine genes generally occurred 11-14 days after cell transfer, prior to maximal EAT severity, which occurred 19-21 days after cell transfer. The relative ratios of Th1:Th2 cytokines and mouse thyroglobulin-(MTg)-specific IgG1 and IgG2a autoantibody levels were similar during both the initial phase and peak of EAT. Depletion of CD8(+) T cells did not decrease the severity of EAT but delayed resolution of lesions. Cytokine gene expression in thyroids was not decreased by anti-CD8 treatment. Together, these data indicate that both Th1 and Th2 cytokines produced by CD4(+) T cells are involved in induction and development of granulomatous EAT, and CD8-dependent resolution of granulomatous EAT is apparently not mediated by these cytokines.
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PMID:The kinetics of cytokine gene expression in the thyroids of mice developing granulomatous experimental autoimmune thyroiditis. 987 80

A guanidinium isothiocyanate (GITC) lysis solution was evaluated for its efficacy in preserving nucleic acids for subsequent analysis after prolonged storage at room temperature. Aliquots of thyroid crude cell lysates were stored at 22 degrees C for 8 weeks in the GITC solution. Crude cell lysates stored for 2 weeks in the GITC solution consistently provided adequate RNA and DNA for reverse transcriptase-polymerase chain reaction (RT-PCR) and PCR, respectively, of beta 2-microglobulin, thyroid stimulating hormone receptor (TSHR), and thyroglobulin. Interestingly, the thyroglobulin and TSHR messages from thyroid fine needle aspirate samples were detected by RT-PCR only when stored at room temperature for less than 1 week, whereas the RT-PCR product for beta 2-microglobulin was detectable in 95% of the samples at 3 months. In addition, thyroglobulin DNA was amplified by PCR in nearly all samples stored at 22 degrees C for 2 months. These data suggest that GITC solutions can be used to preserve nucleic acids in most circumstance until transported to laboratories equipped and staffed with appropriate resources.
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PMID:Preservation of nucleic acids for polymerase chain reaction after prolonged storage at room temperature. 1020 68

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

In this work we have extended our initial molecular studies of a consanguineous family with two affected goitrous siblings (H.S.N. and Ac.S.N.) with defective thyroglobulin (Tg) synthesis and secretion because of a homozygotic deletion of a fragment of 138 nucleotides (nt) in the central region of the Tg mRNA, identified previously in H.S.N. In order to identify the intron/exon boundaries and to analyze the regions responsible for pre-mRNA processing corresponding to a 138 nt deletion, we performed a screening of a human genomic library. The intron/exon junction sequences were determined from one positive clone by sequencing both strands of the DNA template. The results showed that the deletion mapped between positions 5549 and 5686 of the Tg mRNA and corresponded to exon 30. The positions of the exon limits differed by three nucleotides from the previously reported data obtained from direct sequencing of the deleted reverse transcriptase-polymerase chain reaction fragment from H.S.N. These variations are because the intron/exon junctions in this region were not available at the time when the deletion was first described. The deletion does not affect the reading frame of the resulting mRNA and is potentially fully translatable into a Tg polypeptide chain that is shortened by 46 residues. The same 138 nt deletion was observed in reverse transcriptase-polymerase chain reaction studies performed in the thyroid tissues from Ac.S.N. Genomic DNA analysis showed that a G to T transversion was observed at position +1 in the donor site of intron 30. Both affected patients (H.S.N. and Ac.S.N.) are homozygous for the mutation whereas the normal sister (At.S.N.) had a normal allele pattern. The functional consequences of the deletion are related to structural changes in the protein molecule that either could modify the normal routing of the translation product through the membrane system of the cell or could impair the coupling reaction. Probably the mutant Tg polypeptide might be functionally active in the production of thyroid hormone, because in the presence of a normal iodine ingestion (approximately 150 microg/day), Ac.S.N. was able to maintain normal serum levels of total triiodothyronine (T3) associated with relatively low serum total thyroxine (T4) with normal somatic development without signs of brain damage.
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PMID:Congenital goiter with hypothyroidism caused by a 5' splice site mutation in the thyroglobulin gene. 1148 98

We have isolated the novel gene SMOC-1 that encodes a secreted modular protein containing an EF-hand calcium-binding domain homologous to that in BM-40. It further consists of two thyroglobulin-like domains, a follistatin-like domain and a novel domain. Recombinant expression in human cells showed that SMOC-1 is a glycoprotein with a calcium-dependent conformation. Results from Northern blots, reverse transcriptase-PCR, and immunoblots revealed a widespread expression in many tissues. Immunofluorescence studies with an antiserum directed against recombinant human SMOC-1 demonstrated a basement membrane localization of the protein and additionally its presence in other extracellular matrices. Immunogold electron microscopy confirmed the localization of SMOC-1 within basement membranes in kidney and skeletal muscle as well as its expression in the zona pellucida surrounding the oocyte.
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PMID:Characterization of SMOC-1, a novel modular calcium-binding protein in basement membranes. 1213 Jun 37

Follow-up of recurrent differentiated thyroid carcinoma involves the measurement of serum thyroglobulin (Tg). However, Tg autoantibodies are present in a high proportion of thyroid carcinoma patients (up to 25%) and these can interfere with the Tg immunoassays. To overcome this obstacle, investigators have used real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) to measure Tg mRNA in the blood of patients with differentiated thyroid cancer, with varying degrees of success. In the present study, we demonstrate the first reported use of the PAXgene Blood RNA collection tube and extraction kit method for the preparation of RT-PCR-quality RNA with subsequent deployment of the latter in the development of a specific, sensitive, and reproducible Taqman assay for the detection and quantification of thyroglobulin mRNA. Beta-actin mRNA was also assayed and results are expressed as a ratio of Tg to beta-actin mRNA. The intra-assay coefficient of variations (CVs) for Tg and beta-actin mRNA assay were 27.7% and 25.4%, respectively. Inter-assay CVs were 20.8% and 28.8%, respectively, for the two assays. Tg mRNA was detected in all cancer subjects (n = 42) and healthy individuals (n = 20). Tg mRNA was significantly higher in cancer patients than in the healthy subjects (0.00169 +/- 0.00013 vs. 0.00051 +/- 0.00015; P<0.0001). Fourteen cancer patients had detectable levels of serum Tg, and Tg mRNA levels tended to be higher in these than in cancer subjects with undetectable serum Tg (0.00188 +/- 0.00021 vs. 0.00157 +/- 0.000178; P = 0.08). Circulatory Tg mRNA measurement may serve a useful role in the assessment of thyroid cancer.
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PMID:Real-time quantitative PCR measurement of thyroglobulin mRNA in peripheral blood of thyroid cancer patients and healthy subjects. 1525 54

Chemokines represent a group of small, secreted proteins mainly involved in navigating leukocytes towards site of inflammation. Some chemokines have been implicated in the pathogenesis of autoimmune diseases, which are characterized by an ectopic retention of leukocytes within the target organ, ultimately leading to loss of function. To determine the chemokines profile expressed in the thyroid gland upon chronic exposure to interferon-gamma (IFNgamma), we analyzed C57BL6 transgenic mice that aberrantly express IFNgamma under control of the thyroglobulin promoter. We compared by reverse transcriptase PCR the thyroidal expression of 10 chemokines (CCL1 through 5 and CXCL9 through 13) in thyr-IFNgamma transgenics and wild-type littermates. We found that transgenics exclusively expressed CCL4, CXCL9, and CXCL11, and showed increased expression of CCL5 and CXCL10. This chemokine profile was associated with moderate mononuclear cell infiltration of the thyroid stroma that, however, decreased significantly after 2 months of age and did not organize into lymphoid structures. Our findings indicate that the isolated expression of IFNgamma is capable of recruiting mononuclear cells but they do not progress to full lymphoid transformation of the thyroid.
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PMID:Early chemokine expression induced by interferon-gamma in a murine model of Hashimoto's thyroiditis. 1550 31

Usually thyroid cells isolated from tissue obtained by surgery or thyroid cell lines are used to investigate the pathogenesis of autoimmune thyroid diseases. Isolation and cultivation of thyrocytes from fine-needle aspiration biopsy (FNAB) has not yet been published. The aim of this study was to isolate and cultivate thyrocytes from samples of FNAB. FNAB samples were obtained from nine adults and nine children with Hashimoto's thyroiditis (HT). The aspiration material was filtered resulting in small samples of tissue on the surface of the filter membrane. These tissue fragments were digested by collagenase I and dispase II. The yielding cells were cultivated for 3 weeks in Ham's F12 Kaighn's Modification medium in presence of 1 mU/mL bovine thyrotropin (TSH), 10 microg/mL human insulin, 6 microg/mL transferrin, and 10(-8) M hydrocortisone. Finally, isolated thyroid cells were characterized by determination of gene expression of thyrotropin receptor (TSHR), thyroperoxidase (TPO), and thyroglobulin (Tg) using a nested reverse transcriptase-polymerase chain reaction (RT-PCR). Thyroid cells obtained by FNAB can be maintained over a time period of approximately 3 weeks. Depending on the sample size a final number of 1000-14,000 cells was gained per FNAB. In addition, all cells isolated by the described method expressed TPO mRNA. TSHR mRNA was found in 4 samples, whereas 15 samples were Tg mRNA-positive. There were no differences with respect to the expression TSHR and TPO mRNA between samples from adults and children. The isolation and cultivation of thyroid cells obtained by FNAB has been established. In contrast to surgical specimen, this technique provides an easy access to thyrocytes derived from individual patients allowing repeated sampling to investigate the time progression of the chronic disease or the effect of treatment over time.
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PMID:Isolation of thyroid cells obtained by fine-needle aspiration biopsy. 1618 6


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