Luis Lamas

Congenital human thyroglobulin defect due to low expression of the thyroid-specific transcription factor TTF-1

TTF-1 and Pax-8 are thyroid-specific transcription factors, from homeo and paired box genes, respectively, that are responsible for thyroid development and for thyroglobulin and thyroperoxidase gene expression. However, TTF-1 and Pax-8 preferentially bind to the thyroglobulin and thyroperoxidase promoters, respectively. Here, we have studied a patient with defective thyroglobulin synthesis. Thyroglobulin mRNA was found at very low levels while the mRNA for thyroperoxidase was found to be more abundant compared with control tissue. The low levels of thyroglobulin mRNA are caused by a transcriptional defect due to the virtual absence of TTF-1 expression as determined by Northern blot analysis, reverse transcriptase-PCR, and electrophoretic mobility shift assays. The level of Pax-8 mRNA was the same in the goiter and in the control thyroid. These results are the first reported evidence of a congenital goiter with a thyroglobulin synthesis defect due to the low expression of the thyroid-specific transcription factor TTF-1. Moreover, these data suggest that TTF-1 and Pax-8 would be differentially regulating thyroglobulin and thyroperoxidase gene transcription.

Identification of peptides containing aromatic amino acids, cysteine, iodotyrosine and iodothyronine by high-performance liquid chromatography with photodiode-array detection

Abstract A general method for the identification of peptides containing tyrosine, tryptophan, phenylalanine and cysteine and a preliminary study of the identification of peptides containing monoiodotyrosine, diiodotyrosine, triiodothyronine and thyroxine by high-performance liquid chromatography photodiode-array ultraviolet—visible detection is reported. The technique was tested with an immunoglobulin light chain and with an in vitro iodinated urinar human complex-forming glycoprotein, heterogeneous in charge (protein HC), and human thyroglobulin (Tg) after enzymatic digestion. The system continuously monitors wavelengths and collects data that can be analysed by comparison with standard spectra via software routines. This procedure saves sample, time and reagents and avoids the use of radioactive reagents.

Efficient thyroid hormone formation by in vitro iodination of a segment of rat thyroglobulin fused to Staphylococcal protein A

A polypeptide of 224 amino acids from the C terminus of rat thyroglobulin fused to Staphylococcal protein A (TgC 224), containing 3 tyrosines which have been shown to be hormonogenic in vivo (Tyr‐2555, ‐2569 and ‐2748), forms thyroid hormones with relatively high efficiency upon in vitro enzymatic iodination using, most likely, the hormonogenic Tyr‐2555 and Tyr‐2569. Acetylcholinesterase, which has sequence and structural homology with the C terminus of the thyroglobulin molecule and bovine serum albumin, used as control proteins, formed thyroid hormones with lower efficiency. These results validate our experimental approach to define the structural requirements for thyroid hormone formation using thyroglobulin fragments.

Re-evaluation of some factors affecting the stability of rat thyroglobulin

The stability of thyroglobulin obtained after in vivo or in vitro labeling of thyroids from rats kept chronically on an iodine-poor diet (about 30 micrograms I/kg) has been studied by sucrose gradient density centrifugation. The role played by factors such as the degree of iodination, the procedure of labeling, the temperature and the ionic strength, has been reevaluated. Under the experimental conditions used in this study, the stability of thyroglobulin from rats on iodine-poor diet decreases with increasing time of the diet. For comparable levels of iodination, in vivo labeling results in a more stable thyroglobulin than in vitro labeling of the glands: The sedimentation coefficient of in vivo labeled poorly iodinated thyroglobulin is slightly lower (17.5--18.5 S) than that of well iodinated thyroglobulin (19.0 S), independent of the temperature and/or ionic strength used, though at low temperature and/or low ionic strength there is some unfolding of thyroglobulin. The results obtained demonstrate that in order to compare data on thyroglobulin stability it is necessary to keep the experimental conditions constant.

Subcellular Localization of Iodinated Thyroid Tubulin

Subcellular fractions enriched in mitochondria, plasma membranes, microsomes and Golgi apparatus were obtained from thyroid glands of rats injected with I125. Autoradiography of SDS-polyacrylamide gels revealed the presence of a number of radiolabelled proteins in each membrane fraction. One polypeptide, with the same electrophoretic mobility as brain tubulin, was found in all fractions except the plasma membranes and was immunoprecipitated with commercial anti-tubulin monoclonal antibodies. Hydrolysis of Asp-Pro linkages of I125 labelled tubulin with formic acid indicated that there were iodination sites in both the carboxy terminal one third and the amino terminal two thirds of the molecule. These results, together with the absence of iodinated tubulin from the cytosolic fraction, are consistent with the idea that a population of thyroid membrane tubulin is iodinated at multiple sites either just before or after insertion into intracellular membranes where it may act as an anchorage point for microtubule-membrane interactions.