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F. Monaco, C. Santolamazza, I. De Ros, and A. Andreoli

Abstract.

The effect of 6-propyl-2-thiouracil (PTU), and 1-methyl-2-mercaptoimidazole (MMI) on thyroglobulin (Tg) biosynthesis has been studied in vivo and in vitro. In vivo experiments were performed in rats treated for 20 days with PTU or MMI. analyzing soluble and particulate, cold and 125I-labelled, Tg. Thyroglobulin biosynthesis was also investigated by in vitro experiments, incubating thyroid tissue with labelled amino acid and carbohydrate in the presence of antithyroid compounds.

It has been found that in vivo antithyroid agents decrease the amount of soluble Tg and increase the proportion of particulate Tg. Tg from treated animals is poorly iodinated being mainly represented by its 12S subunit.

In vitro studies demonstrate that PTU and MMI inhibit Tg biosynthesis which is impaired in the polypeptide synthesis as wellas in carbohydrate chains addition.

Thus the inhibition of the hormonogenetic processes induced by antithyroid treatment leading to a depressed iodinating activity also appears to be related to a significant impairment of the production of the Tg molecule, the specific iodine acceptor.

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M Centanni, G Canettieri, N Viceconti, R Sibilla, A Bei, and M Andreoli

OBJECTIVE: We have studied the effect of tryptophan on cellular [(125)I]tri-iodothyronine (T3) uptake by mouse thymocytes. MATERIALS AND METHODS: Mouse thymocytes (20 x 10(6 )cells/ml) were suspended in Krebs-Ringer solution buffered by Tris-HCl and incubation (23 degrees C at pH7.45+/-0.6), in the presence or absence of 1mM tryptophan, was started by adding 25 pM [(125)I]T3. At the end of incubation, samples were cooled in ice, centrifuged over a 30% sucrose cushion and the cell-associated radioactivity was measured in the pellet. RESULTS: Tryptophan reduced both the total and the saturable fraction of [(125)I]T3 uptake by 44% (P=0.0009) and 60% (P=0.0006) respectively, following 1 min of incubation. This effect was specific and dose-dependent, being maximal at 5mM concentration (-82%). In contrast, the pre-exposure of cells to tryptophan for up to 2h had no effect on the subsequent uptake of [(125)I]T3, in the absence of tryptophan. The effect of D-tryptophan on saturable T3 uptake was not different from that obtained using the L-stereoisomer. Tryptophan reduced the V(max) of the initial rate of saturable [(125)I]T3 uptake by two-thirds without affecting the apparent K(m) (2.2 nM) of the process, thus indicating the non-competitive nature of the inhibition. In sodium-free medium the saturable [(125)I]T3 uptake was reduced by 43%. The inhibitory effect of tryptophan on [(125)I]T3 uptake was exerted in both the presence and the absence of sodium. In fact, the inhibitory effect of tryptophan on T3 transport was greater and significantly different (P=0.0046) from that obtained by sodium depletion alone. CONCLUSIONS: Tryptophan interferes with both the sodium-dependent and -independent components of [(125)I]T3 uptake by a dose-dependent, non-competitive mechanism which operates in cis-modality at the plasma membrane level of mouse thymocytes.

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FS Celi, G Canettieri, D Mentuccia, L Proietti-Pannunzi, A Fumarola, R Sibilla, V Predazzi, M Ferraro, M Andreoli, and M Centanni

OBJECTIVE: The selenoenzyme type 2 iodothyronine 5' deiodinase (DII) catalyzes the conversion of thyroxine into its active form tri-iodothyronine (T3), modulating thyroid hormone homeostasis in a local, tissue-specific manner. The amphibian, rodent and human cDNAs encoding this enzyme have been recently cloned and expressed. At present, little information regarding the genomic structure of mammalian DII is available. DESIGN AND METHODS: The complete structure, including intron-exon junctions, of the human DII (hDII) gene was obtained by long PCR and rapid amplification of cDNA ends (RACE). Chromosomal assignment of the hDII gene was performed by fluorescence in situ hybridization using a highly specific probe. RESULTS AND CONCLUSIONS: Our data demonstrated that hDII is a single copy gene located on chromosome 14, position 14q24.3. The gene spans over 15 kb, and the 7 kb transcript is encoded by three exons of 149 bp, 273 bp and 6.6 kb separated respectively by two 274 bp and 7.4 kb introns. A restriction map of the hDII gene is also reported. These data will help in further studies of the role of DII in the maintenance of peripheral thyroid hormone homeostasis.