Cloning and expression of the chicken type 2 iodothyronine 5'-deiodinase.
(9/847)
The type 2 iodothyronine deiodinase (D2) is critical for the intracellular production of 3,5,3'-triiodothyronine from thyroxine. The D2 mRNA of higher vertebrates is over 6 kilobases (kb), and no complete cDNA clones have been reported. Using 5'- and 3'-rapid amplification of cDNA ends and two cDNA libraries, we have cloned the 6094-base pair full-length chicken D2 cDNA. The deduced protein is approximately 31 kDa and contains two in-frame UGA codons presumably encoding selenocysteine. One of these is in the highly conserved active catalytic center; the other is near the carboxyl terminus. Unusual features of the cDNA include a selenocysteine insertion sequence element approximately 4.8 kb 3' to the UGA codon in the active center and three short open reading frames in the 5'-untranslated region. The Km of D2 is approximately 1.0 nM for thyroxine, and the reaction is insensitive to inhibition by 6-n-propylthiouracil. Chicken D2 is expressed as a single transcript of approximately 6 kb in different brain regions and in the thyroid and lung. Hypothyroidism increases D2 mRNA in the telencephalon. Unlike in mammals, D2 mRNA and activity are expressed in the liver of the chicken, suggesting a role for D2 in the generation of plasma 3,5,3'-triiodothyronine in this species. (+info)
The interaction between the forkhead thyroid transcription factor TTF-2 and the constitutive factor CTF/NF-1 is required for efficient hormonal regulation of the thyroperoxidase gene transcription.
(10/847)
The forkhead thyroid-specific transcription factor TTF-2 is the main mediator of thyrotropin and insulin regulation of thyroperoxidase (TPO) gene expression. This function depends on multimerization and specific orientation of its DNA-binding site, suggesting that TTF-2 is part of a complex interaction network within the TPO promoter. This was confirmed by transfection experiments and by protein-DNA interaction studies, which demonstrated that CTF/NF1 proteins bind 10 base pairs upstream of the TTF-2-binding site to enhance its action in hormone-induced expression of the TPO gene. GST pull-down assays showed that TTF-2 physically interacts with CTF/NF1 proteins. In addition, we demonstrate that increasing the distance between both transcription factors binding sites by base pair insertion results in loss of promoter activity and in a drastic decrease on the ability of the promoter to respond to the hormones. CTF/NF1 is a family of transcription factors that contributes to constitutive and cell-type specific gene expression. Originally identified as factors implicated in the replication of adenovirus, this group of proteins (CTF/NF1-A, -B, -C, and -X) is now known to be involved in the regulation of several genes. In contrast to other reports regarding the involvement of these proteins in inducible gene expression, we show here that members of this family of transcription factors are regulated by hormones. With the use of specific CTF/NF1 DNA probes and antibodies we demonstrate that CTF/NF1-C is a thyrotropin-, cAMP-, and insulin-inducible protein. Thus CTF/NF1 proteins do not only mediate hormone-induced gene expression cooperating with TTF-2, but are themselves hormonally regulated. All these findings are clearly of important value in understanding the mechanisms governing the transcription regulation of RNA polymerase II promoters, which often contain binding sites for multiple transcription factors. (+info)
Primary biliary cirrhosis associated with painless thyroiditis.
(11/847)
A case of anti-mitochondrial antibody (AMA)-negative primary biliary cirrhosis (PBC) associated with painless thyroiditis is reported in a 47-year-old woman who diagnosed as PBC based on her elevated serum gamma-glutamyl transpeptidase and immunoglobulin M levels, as well as histological findings of destroyed bile ducts surrounded by mononuclear infiltrates in the biopsied liver. She was negative for AMA and had a depressed level of thyroid-stimulating hormone accompanied by increased free thyrosine, thyroxine and triiodothyronine levels and low titers of anti-microsomal and anti-thyroid peroxidase antibodies. Her thyroid disorder corresponded with painless thyroiditis. An association between PBC and hyperthyroidism is rare. Furthermore, an association between AMA-negative PBC and hyperthyroidism due to painless thyroiditis has not previously been reported. (+info)
Urinary iodine and thyroid antibodies in Okinawa, Yamagata, Hyogo, and Nagano, Japan: the differences in iodine intake do not affect thyroid antibody positivity.
(12/847)
Excess iodine intake may affect the development of Hashimoto's thyroiditis. Kelp consumption is very high in Okinawa. We expected a high prevalence of Hashimoto's thyroiditis in Okinawa. We studied urinary iodine excretion and the positivities of anti-thyroglobulin antibodies (TGAb) and anti-thyroid peroxidase antibodies (TPOAb) in the residents of Nishihara in Okinawa, Yamagata in Yamagata, Kobe in Hyogo, and Hotaka in Nagano, Japan. TGAb and/or TPOAb were positive in 142 (13.7%) of 1039 subjects in Nishihara, in 16 (16.0%) of 100 subjects in Yamagata, in 31 (13.4%) of 232 subjects in Kobe, and in 35 (13.9%) of 252 subjects in Hotaka; TGAb and/or TPOAb positivity was about the same in these 4 areas. One tenth of the subjects with positive TGAb and/or TPOAb had hypothyroidism; the frequencies of hypothyroidism in those with positive TGAb and/or TPOAb were about the same in Nishihara, Yamagata, Kobe, and Hotaka. The iodine concentration in samples of morning urine correlated well with the 24-h urine iodine excretion. The urinary iodine excretion was 1.5 mg/day in Nishihara. There were no differences between Nishihara and Yamagata in the urinary iodine concentration, but the urinary iodine concentrations in Kobe and Hotaka were less than those in Nishihara or Yamagata. The amounts of iodine excretion in Kobe and Hotaka were moderate, and less than those in Nishihara or Yamagata. The amounts of iodine intake in Kobe and Hotaka were less than those in Nishihara or Yamagata, but TGAb and/or TPOAb positivity was about the same in Nishihara, Yamagata, Kobe, and Hotaka. The differences in dietary iodine intake do not affect TGAb and/or TPOAb positivity. (+info)
Rarity of autoantibodies to a major autoantigen, thyroid peroxidase, that interact with denatured antigen or with epitopes outside the immunodominant region.
(13/847)
The nature of the autoantibody repertoire to the dominant autoantigen in human autoimmune thyroid disease is controversial. There is evidence that autoantibodies to thyroid peroxidase (TPO) interact with overlapping conformational epitopes in an immunodominant region and binding to denatured (DN) protein is decreased. Contrary data demonstrate TPO autoantibody reactivity with DN-TPO or polypeptide fragments. However, none of the TPO-specific, human monoclonal autoantibodies isolated to date preferentially recognize denatured autoantigen. We therefore searched an immunoglobulin gene phage display library for human autoantibodies that bind TPO denatured by reduction and alkylation (DN-TPO). Thyroid-infiltrating B cells from a typical TPO autoantibody-positive patient were the source of mRNA for library construction. Surprisingly, the library enriched after panning on DN-TPO, as well as a panel of individual clones, preferentially bound native (N)-TPO. Of 13 clones selected using DN-TPO or N-TPO, 12 clones recognized the TPO immunodominant region. Moreover, regardless of selection with N-TPO or DN-TPO, their heavy and light chains were encoded by similar VDJ and Vkappa combinations. One clone (DN4), isolated using DN-TPO, did not interact with the TPO immunodominant region and its H chain derives from a different VH gene. Although DN4 binds specifically to TPO, its affinity is low, unlike the high affinities of other human TPO autoantibodies. In conclusion, human monoclonal autoantibodies that preferentially recognize denatured TPO could not be isolated from an immunoglobulin gene library despite selection with denatured protein. Our findings demonstrate the bias of the human B cell repertoire towards recognition of an immunodominant region on the conformationally intact form of a major thyroid autoantigen. (+info)
Presence of a soluble inhibitor of thyroid iodination in primary cultures of thyroid cells.
(14/847)
Monolayer cultures of thyroid cells lose their iodide organification capacity a few days before the disappearance of thyroid peroxidase (TPO) activity. The present studies were performed in order to clarify this point. The above mentioned difference was due to the presence of an inhibitor in the monolayer thyroid cells culture, given that total homogenate prepared from confluent cells caused a significant inhibition of activity of TPO from fresh tissue. The inhibitor was localized in the 105000g supernatant of the homogenate of the cell culture, but not in a similar preparation obtained from fresh thyroid. It is thermostable, dialyzable and has a molecular weight of less than 2 kDa. Addition of the inhibitor at the end of the reaction of tyrosine iodination failed to alter the results. This fact suggests that the compound does not destroy the iodinated product. The presence of the cytosolic inhibitor was observed in monolayer thyroid cell cultures of different species (bovine, porcine, rat and human) but not in free follicles cultures. (+info)
Effect of nicotine on type 2 deiodinase activity in cultured rat glial cells.
(15/847)
Intracellular generation of triiodothyronine (T3) from thyroxine (T4) by type 2 deiodinase (D2) in the mammalian brain, plays a key role in thyroid hormone action. The presence of D2 in rat astrocytes suggests the importance of glial cells in the regulation of intracellular T3 levels in the rat central nervous system (CNS). To analyze further the factors that regulate D2 activity in the CNS, we investigated the effects of nicotine and of mecamylamine, which inhibits the binding of nicotine with nicotinic acetylcholine receptors, on D2 activity in cultured mixed glial cells of the rat brain. We incubated cultured mixed glial cells obtained from neonatal Wistar rats in the presence of 10 mM dithiothreitol, 2 nM [125I] reverse T3 and 1 mM 6-N-propyl-2-thiouracil for 2 h at 37 degrees C, and the released 125I- was counted in a gamma counter. D2 activity of cultured cells was dependent on the temperature and the amount of protein. The basal D2 activity of rat mixed glial cells was 1.9 +/- 0.2 fmol of I- released/mg protein/h (mean +/- SEM). The addition of 10(-11), 2 x 10(-11), 10(-10), and 10(-9) M nicotine significantly increased D2 activity to approximately 2.2-, 2.4, 3.5- and 2.9-fold the basal level, respectively. D2 activity stimulated by 10(-8) M nicotine (2.5-fold) reached a peak after 9 h incubation. The stimulatory effect of nicotine was completely blocked by 10(-6) M mecamylamine. In conclusion, nicotine increases D2 activity probably via nicotinic acetylcholine receptors, and may influence brain function, at least in part, by affecting thyroid hormone metabolism. (+info)
Distinct immunological and biochemical properties of thyroid peroxidase purified from human thyroid glands and recombinant protein produced in insect cells.
(16/847)
The biosynthesis of thyroid hormone from thyroglobulin is catalysed by thyroid peroxidase (TPO), an integral membrane protein. TPO is also a major autoantigen in autoimmune thyroid disease and autoantibodies to TPO are markers for disease activity. Large quantities of purified TPO are essential for elucidating its structure and understanding its role in disease activity. We describe the high yield purification of full-length recombinant human TPO from baculovirus infected insect cells and compare it to purified native TPO from human thyroid glands. In contrast to native human TPO, the human TPO produced in insect cells as a recombinant protein was insoluble and resistant to solubilisation in detergents. Reversible substitution of lysine residues with citraconic anhydride led to increased solubility of the recombinant TPO, allowing high-yield purification by monoclonal antibody chromatography. The purified enzyme preparation was shown to be TPO by its reactivity with monoclonal and polyclonal antibodies by enzyme linked immunosorbent assay and Western blotting. Both the human and recombinant purified TPO preparations also react with sera from patients with autoimmune thyroid disease, although the binding of conformational dependent autoantibodies was considerably lower to the recombinant TPO than to the native TPO. This suggests that the recombinant TPO may differ in some aspects of its tertiary structure. The purified recombinant TPO was devoid of enzyme activity, in contrast to the enzymatically active, purified human TPO preparations. Both preparations contained comparable amounts of haem (R(z)=0.269), but a shift in the Soret band of recombinant TPO (402 nm) from that of natural TPO (409 nm) indicates that the lack of enzymatic activity of the recombinant enzyme may be due to changes in the protein backbone surrounding the haem. Both the purified native and recombinant TPO, under non-denaturing conditions, show evidence of high molecular mass oligomers, although the latter preparation is prone to a greater degree of aggregation. In conclusion, our studies indicate that recombinant TPO generated in insect cells is conformationally distinct from the native TPO, is insoluble and enzymatically inactive, consistent with the difficulties associated with its purification and crystallisation. (+info)