Regional physiological adaptation of the central nervous system deiodinases to iodine deficiency.

The goal of the present investigation was to analyze the types 2 (D2) and 3 (D3) iodothyronine deiodinases in various structures within the central nervous system (CNS) in response to iodine deficiency. After 5-6 wk of low-iodine diet (LID) or LID + 2 microg potassium iodide/ml (LID + KI; control), rats' brains were processed for in situ hybridization histochemistry for D2 and D3 mRNA or dissected, frozen in liquid nitrogen, and processed for D2 and D3 activities. LID did not affect weight gain or serum triiodothyronine, but plasma thyroxine (T4) was undetectable. In the LID + KI animals, D3 activities were highest in the cerebral cortex (CO) and hippocampus (HI), followed by the olfactory bulb and was lowest in cerebellum (CE). Iodine deficiency decreased D3 mRNA expression in all CNS regions, and these changes were accompanied by three- to eightfold decreases in D3 activity. In control animals, D2 activity in the medial basal hypothalamus (MBH) was similar to that in pituitary gland. Of the CNS D2-expressing regions analyzed, the two most responsive to iodine deficiency were the CO and HI, in which an approximately 20-fold increase in D2 activity occurred. Other regions, i.e., CE, lateral hypothalamus, MBH, and pituitary gland, showed smaller increases. The distribution of and changes in D2 mRNA were similar to those of D2 activity. Our results indicate that decreases in the expression of D3 and increases in D2 are an integral peripheral component of the physiological response of the CNS to iodine deficiency.

The human, but not rat, dio2 gene is stimulated by thyroid transcription factor-1 (TTF-1).

Types 1 and 2 iodothyronine deiodinases (D1 and D2) catalyze the production of T(3) from T(4). D2 mRNA is abundant in the human thyroid but very low in adult rat thyroid, whereas D1 activity is high in both. To understand the molecular regulation of these genes in thyroid cells, the effect of thyroid transcription factor 1 (TTF-1) and the paired domain-containing protein 8 (Pax-8) on the transcriptional activity of the deiodinase promoters were studied. Both the approximately 6.5-kb hdio2 sequence and its most 3' 633 bp were activated 10-fold by transiently expressed TTF-1 in COS-7 cells, but the hdio1 was unaffected. Surprisingly, the response of the rdio2 gene to TTF-1 was only 3-fold despite the 73% identity with the proximal 633-bp region of hdio2 including complete conservation of a functional cAMP response element at -90. Neither human nor rat dio2 nor human dio1 was induced by Pax-8. The binding affinity of four putative TTF-1 binding sites in hdio2 were compared by a semiquantitative gel retardation assay using in vitro expressed TTF-1 homeodomain protein. Only two sites, D and C1 (both of which are absent in rdio2), had significant affinity. Functional analyses showed that both sites are required for the full response to TTF-1. These results can explain the differential expression of dio2 in thyroid and potentially other tissues in humans and rats.

Characterization of the 5'-flanking and 5'-untranslated regions of the cyclic adenosine 3',5'-monophosphate-responsive human type 2 iodothyronine deiodinase gene.

The type 2 iodothyronine deiodinase (D2) catalyzes T4 activation. In humans, unlike rodents, it is widely expressed, and its action probably contributes to both intracellular and plasma T3 pools. We have isolated the 6.5-kb 5'-flanking region (FR) and the previously uncloned 553 nucleotides (nt) of the 5'-untranslated region (UTR) of hdio2. The 5'-UTR is complex, with three transcription start sites (TSS) (708, 31, and approximately 24 nt 5' to the ATG), an alternatively spliced approximately 300-nt intron in the 5'-UTR, and three short open reading frames 5' to the initiator ATG. The previously reported approximately 7.5-kb D2 messenger RNA (mRNA) is actually an approximately 7-kb doublet that is present in thyroid, pituitary, cardiac and skeletal muscle, and possibly brain, but with only the longer transcript in placenta. A canonical cAMP response element-binding protein-binding site is present at about 90 bp 5' to the most 5'-TSS. It accounts for the robust response of the 6.8-kb hdio2 5'-FR to protein kinase A. Forskolin increases D2 mRNA in human thyroid cells, which may explain the high D2 mRNA in Graves' thyroid and thyroid adenomas. The hdio2 gene structure and Northern blot results suggest that D2 expression is tightly controlled and tissue specific.

Cloning and expression of the chicken type 2 iodothyronine 5'-deiodinase.

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.

Regional expression of the type 3 iodothyronine deiodinase messenger ribonucleic acid in the rat central nervous system and its regulation by thyroid hormone.

Type 3 iodothyronine deiodinase (D3) is a selenoenzyme that inactivates thyroid hormone. It is necessary for T3 homeostasis in the central nervous system. D3 activity has been identified in many regions of the brain and parallels thyroid status, but the level at which it is regulated and its specific cellular locations are not known. We evaluated the effect of thyroid status on the expression of the D3 gene within the central nervous system using in situ hybridization histochemistry. D3 messenger RNA (mRNA) was identified throughout, but with high focal expression in the hippocampal pyramidal neurons, granule cells of the dentate nucleus, and layers II-VI of the cerebral cortex. In every region, D3 mRNA abundance was correlated with thyroid status. Four different D3 transcripts were identified by Northern analyses, with evidence for region-specific processing, and D3 mRNA increased 4- to 50-fold from the euthyroid to the hyperthyroid state. D3 mRNA was not detectable in hypothyroid brain. In the central nervous system, the D3 gene is highly T3 responsive, and its focal localization within the hippocampus and cerebral cortex suggests an important role for T3 homeostasis in memory and cognitive functions.

Expression of the Brazil nut methionine-rich protein and mutants with increased methionine in transgenic potato.

A cDNA encoding the methionine-rich (19 mol% Met) protein in Brazil nut was placed under the regulation of CaMV 35S promoter and nopaline synthase terminator and introduced into the potato cultivar Russet Burbank via Agrobacterium-mediated transformation. To further enhance the Met content in the transgenic plants, chimeric genes containing four mutant constructs, BoxIa (with 5 additional Met), BoxIIa (2 additional Met), BoxIaIIa (7 additional Met), and BoxIIa2 (7 additional Met), were also generated by sequence modifications of the cDNA and transferred into potato. Analysis of the microtubers and leaves of the transgenic potato plants revealed, in general, with the exception of the BoxIIa2, the presence of mRNA transcripts of the expected size and the correctly processed Met-rich 9 kDa subunit polypeptides. The expression levels in the leaves among the various constructs and individual transgenic plants varied between <0.01% and 0.2% of total protein. The corresponding expression in the tubers was usually 2- to 4-fold lower than in leaves. In the case of BoxIIa2, which contains two tandem repeats of the BoxIIa mutant sequence, a larger (10.5-11 kDa) polypeptide was detected. These findings demonstrated that it is feasible to exploit the variable region of the Brazil Nut 2S protein for enhanced Met contents and perhaps for other desirable properties.

Study on the binding of cerium(III) ion and spermine to TRNA(Phe).

Fluorescence of Ce(III) aqua ion at pH 6.0 is found to be in good linear relationship with its concentration, and the intensity is strong enough to be employed to determine its concentration. TRNA(Phe) evidently quench this fluorescence. Fluorescence titration experiments were performed to Ce(III)-tRNA(Phe) system, the binding number and association constant was estimated with a Scatchard plot. Two classes of binding sites with association constant of 5.2 x 10(7) and 4.4 x 10(6) M, respectively, were found. Addition of spermine slightly decrease binding number and association constant of Ce(III) ion.

Regional distribution of type 2 thyroxine deiodinase messenger ribonucleic acid in rat hypothalamus and pituitary and its regulation by thyroid hormone.

To identify the specific locations of type 2 deiodinase (D2) messenger RNA (mRNA) in the hypothalamus and pituitary gland and determine its regulation by thyroid hormone, we performed in situ hybridization histochemistry, Northern analysis, and quantitative RT-PCR in euthyroid, hypothyroid, and hyperthyroid rats. By in situ hybridization histochemistry, silver grains were concentrated over ependymal cells lining the floor and infralateral walls of the third ventricle extending from the rostral tip of the median eminence (ME) to the infundibular recess, surrounding blood vessels in the arcuate nucleus (ARC), and in the ME adjacent to the portal vessels and overlying the tuberoinfundibular sulci. Silver grains also accumulated over distinct cells in the midportion of the anterior pituitary. In hypothyroid animals, an increase in signal intensity was observed in the caudal hypothalamus, and a marked increase in the number of positive cells occurred in the anterior pituitary. Microdissection of the hypothalamus for Northern and PCR analysis established the authenticity of D2 mRNA in the caudal hypothalamus, and confirmed that the majority of D2 mRNA is concentrated in this region. The distribution of D2 mRNA suggests its expression in specialized ependymal cells, termed tanycytes, originating from the third ventricle. Thus, the tanycyte is the source of the high D2 activity previously found in the ARC-ME region of the hypothalamus. The results indicate that tanycytes may have a previously unrecognized integral role in feedback regulation of TSH secretion by T4.