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L Lamas

Publications and source records attributed to L Lamas.

At least 19 recordsLinked to original sources
PubMed1997

Identification and quantitation of iodotyrosines and iodothyronines in proteins using high-performance liquid chromatography by photodiode-array ultraviolet-visible detection.

We describe a new method for the separation, identification and quantitation of iodotyrosines and iodothyronines [3-monoiodo-L-tyrosine (MIT), 3,5-diiodo-L-tyrosine (DIT), L-thyronine (T0), 3,5-diiodo-L-thyronine (T2), 3,5,3'-triiodo-L-thyronine (T3), reverse 3,3',5'-triiodo-L-thyronine (rT3) and 3,3',5,5'-tetraiodo-L-thyronine (T4)]. Reversed-phase high-performance liquid chromatography (RP-HPLC) was performed on a Nucleosil C8 column with photodiode-array UV-Vis detection. A clearly defined elution profile was obtained of each iodoamino acid (iodotyrosines and iodothyronines) using a linear gradient from 20 to 80% phase B (90% acetonitrile, 10% water, 0.1% TFA), phase A (water, 0.1% TFA, pH 2.0) eluted over 40 min. Iodoamino acid composition was determined, taking into account retention times and spectral characteristics. Thyroid protein samples were digested enzymatically and the complex mixture of IAA was then injected onto the RP-HPLC system. A photodiode-array detector with a dynamic range in the UV-Vis region was used in the HPLC system to monitor the absorbance at different wavelengths continuously, collecting data which were compared with standard samples. Each IAA was quantitated using linear calibration curves obtained at 280 nm. This method allowed identification and quantitation of iodoamino acids from diverse sources in the range 2-500 ng, avoiding the need to radiolabel samples. The technique was tested with in vitro iodinated and non-iodinated human thyroglobulin and the recoveries ranged from 84 to 91%.

Animals↗
PubMed1995

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.

Base Sequence↗
PubMed1992

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.

Amino Acid Sequence↗
PubMed1989

Consensus sequences for early iodination and hormonogenesis in human thyroglobulin.

Thyroglobulin from a human goiter, containing four atoms of iodine/molecule (660,000 daltons), was iodinated with Na 125I and KI in vitro to achieve a net addition of either 2 or 7.8 atoms of iodine/molecule. After fractionation by high performance liquid chromatography, iodinated tryptic peptides from S-cyanoethylated 125I-thyroglobulin were purified, sequenced, characterized by [125I]iodoamino acid distribution, and localized within thyroglobulins primary structure based upon the published cDNA sequence, (Malthiery, Y., and Lissitsky, S. (1987) Eur. J. Biochem. 165, 491-498). The addition of 2 atoms of iodine/molecule of thyroglobulin produced iodotyrosyls at five principal sites, with no 125I-hormone formation. The addition of 7.8 atoms iodinated the same sites more heavily, produced iodotyrosyls at 10 additional sites, and formed iodothyronines at 5 sites. After addition of 2 atoms of iodine, tyrosyl 24 and 11% of thyroglobulins 125I, while tyrosyl 2572 had 24%, but with 7.8 added atoms of iodine, tyrosyl 24 had more of the thyroglobulins [125I]iodothyronine (36 versus 26%). Since tyrosyls 149, 866, and 1466 were iodinated early but did not form the inner rings of iodothyronines, they are attractive candidates for donors of outer iodothyronyl rings. The sequences around the iodotyrosyls fall into three consensus groups, as follows: 1) Glu/Asp-Tyr, associated with synthesis of thyroxine (residues 24, 2572, and 1309), or iodotyrosine (residues 2586 and 991); 2) Ser/Thr-Tyr-Ser, associated with synthesis of iodothyronine (residue 2765) and iodotyrosine (1466 and 883); and 3) Glu-X-Tyr, 7 of the remaining 8 iodotyrosyls occur in this sequence, and we found iodine incorporation at each place this sequence appears in the thyroglobulin molecule. Iodine has been found at homologues of most of these sites in thyroglobulins of other species. We conclude that the primary structure of thyroglobulin, and particularly these consensus sequences, have a major role in the formation of thyroid hormones and their iodinated precursors.

Amino Acid Sequence↗
PubMed1989

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.

Animals↗
PubMed1988

Improvement in HPLC fractionation of thyroxine-containing thyroglobulin tryptic peptides by prior Accell ion-exchange column chromatography.

The isolation and purification of the peptides containing the hormonogenic tyrosyl residues in thyroglobulin is of great interest to the understanding of structure-function relationships in this iodoprotein. This is usually performed in reduced alkylated selectively hydrolyzed thyroglobulin by subsequent HPLC fractionation. However, the main difficulty encountered when trying to isolate these peptides is their disproportion with respect to the total number of possible peptides (14 vs a total of 508). Several HPLC runs with different mobile phases are necessary and each run is accompanied with significant losses of the peptides to be purified. In an attempt to improve the separation of the T4-containing peptides in thyroglobulin tryptic digests from the much more abundant iodotyrosine-containing ones, which are present as contaminants, we have used a very simple and fast step prior to the HPLC fractionation as it is a self-packed ion-exchange column chromatography. This preliminary step results in an improvement in the separation of these peptides and leads to a relative enrichment of the hormonogenic peptides falling in different zones of the HPLC chromatogram, which facilitates their subsequent separation and purification by HPLC.

Acetonitriles↗
PubMed1986

Evidence, by in vitro enzymatic iodination of thyroglobulin, that the efficiency of coupling is determined by the initial iodide concentration.

The rate of iodination and the extent and the efficiency of coupling, assessed by the T4 residues formed per atom of iodine bound, have been studied by in vitro enzymatic iodination of iodine-poor (0.01% I) human goiter thyroglobulin (Tgb; 1 microM) with varying concentrations of iodide (0.75 X 10(-5)-10(-4) M I-), lactoperoxidase (0.1-5 micrograms/ml), and hydrogen peroxide (H2O2) generated from glucose (1 mg/ml) and glucose oxidase (0.025-2.5 mU/ml). Under the experimental conditions used, variations in the H2O2 concentration affect the rate of iodination, but not the extent of coupling. Variations in the peroxidase concentration do not affect the rate of iodination or the coupling efficiency. Variations in the iodide concentration do not affect the rate of iodination, but do affect the coupling efficiency, which is higher the lower the initial iodide concentration. Both iodination and coupling take place almost simultaneously. They are catalyzed by peroxidase, and iodination is completed earlier the lower the iodide concentration. Therefore, once iodination is complete, coupling alone, with high efficiency, takes place. This suggests that the active peroxidase compounds formed will be mainly directed toward the catalysis of iodination or coupling, depending on the actual iodide concentration present in the incubation medium. Provided that initially nonlimiting noninhibiting H2O2 and iodide concentrations are used and that iodination and coupling are allowed to proceed to completion, the extent of coupling is a function of the degree of iodination of Tgb achieved, and its efficiency is determined not just by the native structure of Tgb, but also by the initial iodide concentration used.

Goiter, Nodular↗
PubMed1986

Intrathyroidal thyroglobulin in streptozotocin-diabetic rats.

In order to elucidate whether or not the 'coupling defect' observed in thyroids from diabetic rats is due to a structural defect of intrathyroidal thyroglobulin (Tg), the sedimentation pattern and the stability of the thyroidal soluble iodoproteins were studied in control (C), food restricted (FR), diabetic (D) and insulin-treated diabetic (D+I) rats fed a low iodine diet either with (NID) or without (LID) iodide supplementation and labelled with 125I: acutely, 24 h prior to sacrifice and chronically, by feeding the corresponding diet labelled for 60 days. Diabetes resulted in a decrease of thyroidal weight, an increase of both thyroidal 127I content and concentration and decreased plasma TSH, irrespective of the diet. Insulin treatment reversed these alterations. Food restriction led to an intermediate situation between C and D. The iodoamino acid distribution in the acutely labelled thyroidal soluble iodoproteins showed a significant increase in the percent of organified 125I found as iodotyrosines (MIT and DIT) and a decrease of that found as iodothyronines (T3 and T4) both in D and FR. However, in the isotopically equilibrated groups, no differences were found except in LID-D where a slight increase in DIT and a decrease in T3 was found as compared to the corresponding control. The sedimentation patterns of both acutely and chronically labelled thyroidal soluble iodoproteins from all experimental groups displayed two peaks. The main one, corresponding to Tg, had a slightly lower sedimentation coefficient than the 19 S internal marker run in parallel, while the second one, relatively small, formed probably by dissociation of the main Tg peak, sedimented more slowly (12- 14 S).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Effects of high doses of iodide on thyroid secretion: evidence for the presence of iodinated membrane tubulin.

The transient inhibitory effect on thyroid secretion produced by high doses of iodide was investigated with respect to changes in the level of in vivo iodination of membrane tubulin. Iodinated tubulin, characterized by gel electrophoresis, immunoprecipitation, and peptide mapping, was shown to be associated with a thyroid membrane fraction, and totally absent from cytoplasmic proteins. The administration of an acute dose of 5 mg KI, although it inhibited thyroid secretion (as shown by an increase in TSH), did not have a significant effect on the level of iodination of membrane tubulin. Thus, the observed inhibition of thyroid secretion is probably unrelated to the level of iodination of tubulin. Although its function is not known at present, iodinated tubulin is probably involved in membrane-related phenomena.

Animals↗
PubMed1984

Cerebral hypothyroidism in rats with adult-onset iodine deficiency.

Rats fed chronically a low iodine diet may have low serum T4 and high circulating TSH, despite normal serum T3. As the brain depends to a great extent on intracellular generation of T3 from T4 for its total and nuclear T3, we have carried out two experiments to determine whether the brain of iodine-deficient rats may become hypothyroid, despite normal serum T3 levels. In both experiments we confirmed previous data, showing that the pituitary and liver of iodine-deficient rats with very low plasma T4 levels are hypothyroid as compared to those of animals receiving the same diet supplemented with KI, though not as markedly as animals which had undetectable circulating levels of both T4 and T3 as a consequence of chronic ingestion of KC1O-4, or of surgical thyroidectomy. We have further found that the nuclear T3 content was decreased in the brain of iodine-deficient rats, as compared with the animals on the iodine-supplemented diet. The nuclear to plasma ratios of labeled T3 showed that the uptake of this hormone into liver and brain nuclei is not decreased in the iodine-deficient rats as compared with those on the iodine-supplemented diet. This finding indicates that the decreased liver and brain nuclear T3 contents of iodine-deficient rats are likely to be a consequence of the marked reduction of their T4 pool, leading to decreased amounts of intracellularly generated T3. The number of spines on shafts of pyramidal neurons from the visual cortex of iodine-deficient rats was lower than that of rats fed the same diet supplemented with KI. Their distributions along the shaft were also not the same. Such changes might well be an index of cerebral hypothyroidism, as they are similar to those found after thyroidectomy of adult rats. It is concluded from the present findings that normal circulating T3 levels may not be sufficient to maintain brain euthyroidism in rats fed a diet iodine deficient enough to result in very low circulating T4 levels.

Animals↗
PubMed1982

Are iodine-deficient rats euthyroid?

Inhabitants of many severe endemic goiter areas have low serum T4 and high circulating TSH, despite normal levels of T3. This situation may be produced experimentally chronically feeding rats a low iodine diet (LID). We fed rats a Remington-type LID and gave them 1% NaClO4 in their drinking water for 2 days. After this, the animals were divided into three groups. One group was fed LID, supplemented with 5 micrograms I/rat.day and was used as the control group. Another group was fed LID alone. The third group was fed LID and given 1% NaClO4 to drink. The latter treatment was used to induce severe hypothyroidism. Animals were killed 1, 2, 3, and 5 weeks after the onset of these treatment schedules. The following measurements were made on some or all groups of animals: body and thyroid weights; thyroidal I content; soluble labeled iodoprotein profile; thyroidal labeled iodoamino acid distribution pattern; plasma T4, T3, and TSH; pituitary GH content; and liver intramitochondrial alpha-glycerophosphate dehydrogenase and cytosolic malic enzyme activities. T4 and T3 concentrations were also measured in liver nuclei of the animals killed 5 weeks after the onset of treatment. As assessed from various indices of thyroid function, the LID rats became iodine deficient, although not as markedly as those given LID and ClO4-, The plasma T4 decreased to undetectable levels, and plasma TSH increased, whereas circulating T3 remained normal throughout in the LID rats. In rats given LID and ClO4-, plasma T4 decreased sooner than in rats given LID alone; plasma T3 levels also became undetectable, and TSH increased more markedly and sooner than in rats given LID alone. As measured at the end of 5 weeks of treatment, pituitary GH content, and liver alpha-glycerophosphate dehydrogenase and malic enzyme activities were lower in rats given LID than in the euthyroid LID- and I--treated controls. They were not, however, as markedly reduced as in the severely hypothyroid LID- and ClO4--treated rats. In spite of normal plasma T3 levels, the concentration of T3 in liver nuclei of the rats given LID alone was significantly lower than that of the LID- and I--treated controls. The results show that the thyrotrophs, somatotrophs, and livers of rats given LID alone are not like those of euthyroid rats despite normal circulating T3 levels. In iodine-deficient rats, there is a discrepancy between the measured indices of thyroid hormone action in the liver and the circulating T3 level, but not between biological activity and liver nuclear T3 concentration. It remains to be seen whether the same is true in the anterior pituitary.

Animals↗
PubMed1981

The effect of varying iodine content on the proteolytic activity of rat thyroid lysosomes.

The possibility that the iodine supply modulates thyroid lysosomal activity was investigated in rats receiving chronic or acute increasing doses of iodide. The lysosomal activity or the various experimental groups was determined by measuring the activity of beta-glycerophosphatase and cathepsin D both in thyroid homogenates and in semipurified thyroid lysosomal preparations, and the degradation of labelled thyroglobulin by the various lysosomal fractions. In the chronic experiments beta-glycerophosphatase and cathepsin D activities increased with the iodide supply of the animals up to 100 micrograms I/rat and decreased slightly thereafter. In the acute experiments the activity of these enzymes increased up to 1000 micrograms I/rat and decreased above 5000 micrograms I/rat. The proteolytic activity of lysosomal fractions from the various experimental groups towards thyroglobulin decreased slightly with increased iodide supply both in chronic and acute experiments. The results suggest that thyroid lysosomal activity may participate in the autoregulation of thyroid secretion by inducing synthesis of new enzymes and modulating thyroglobulin degradation.

Animals↗
PubMed1980

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.

Animals↗