PubMed Health⌕ Search

Biomedical subjects

P Santisteban

Publications and source records attributed to P Santisteban.

At least 19 recordsLinked to original sources
PubMed1992

Expression of thyroglobulin gene in maternal and fetal thyroid in rats.

The relationship between the changes in thyroglobulin (Tg) mRNA and Tg proteins during thyroid development in the fetus and in maternal thyroid glands during gestation and lactation is studied. While the appearance of Tg mRNA (fetal day 15) showed good temporal correlation with that of 12S Tg, no 19S Tg could be detected until 3 days later. The 12S Tg was the predominant protein on days 18 and 19 of gestation in the fetus, while 19S Tg was the predominant protein on fetal days 21-22 and during the postnatal period in the offspring; by the 20th postnatal day, the 19S Tg content per gland was 4 times the amount of 12S (155 vs. 37 micrograms/gland; P less than 0.001). The 19S iodine content in the fetus was the same as that in 12S up to the 21st day of gestation, except for lower values on day 18. From fetal day 22 and through the postnatal period, the iodine content in 19S was 1.6-5.9 times greater than that in 12S. Therefore, the ratio of atoms of iodine per mol Tg during the experimental period changed from 0.75 to 19.5 for 19S and from 0.72 to 7.2 for 12S. The levels of all of the iodoamino acids were low on fetal days 17-19, after which they increased at different rates for each protein. The greatest increase in monoiodotyrosine and T3 corresponded to 12S, while diiodotyrosine and especially T4 showed a greater increase in 19S than in 12S Tg; 20 days after birth, the T4 content in 19S was about 3 times greater than that in 12S Tg. The soluble thyroid proteins from pregnant, lactating, and nonpregnant female controls contained a main protein, 19S, and a smaller amount of 27S. Both 19S Tg and 19S iodine contents were already lower than those in nonpregnant rats at 14 days of pregnancy, and the levels continued to decrease during the experimental period. In contrast, the 27S Tg and 27S iodine levels remained constant and similar to nonpregnant values. Surprisingly, a decrease in the level of Tg mRNA was observed during pregnancy and lactation. We have no explanation for the dramatic decrease in Tg mRNA during the last days of pregnancy. Further studies should help to elucidate the mechanism responsible for the changes in Tg gene expression in the thyroids of pregnant and lactating rats.

Animals↗
PubMed1992

Insulin and insulin-like growth factor I regulate a thyroid-specific nuclear protein that binds to the thyroglobulin promoter.

The mechanism responsible for the stimulation of thyroglobulin (Tg) gene expression by insulin and insulin-like growth factor I (IGF-I) in rat thyroid FRTL-5 cells has been investigated. Both insulin and IGF-I stimulate transcription from the Tg promoter in a transient transfection assay demonstrating that the promoter used contains the DNA signals necessary for insulin and IGF-I regulation. Promoter mutations that interfere with the binding of thyroid transcription factor 1 (TTF-1), TTF-2, and the ubiquitous transcription factor abolish the insulin/IGF-I response, indicating that the three factors may be involved in the observed transcriptional control. Protein-DNA binding studies did not reveal any effect of insulin/IGF-I on the ubiquitous transcription factor and the TTF-1 binding capacity. Instead, TTF-2 is absent in nuclear extracts from cells depleted of serum and insulin. Addition of insulin or IGF-I restores the TTF-2 concentration to normal levels and requires ongoing protein synthesis. The insulin effect was maximal at 24 h and at a concentration of 1 microgram/ml. The same effect was observed with a 10-fold lower concentration of IGF-I. These results suggest that insulin (probably through the IGF-I receptor) and IGF-I modulate the levels of TTF-2, which results in an increased expression of the Tg gene.

Animals↗
PubMed1991

Expression of the growth hormone gene and the pituitary-specific transcription factor GHF-1 in diabetic rats.

Diabetes in the rat is associated with poor growth and decreased GH in the pituitary. In this study we have examined whether this reduction reflects an impairment of GH gene expression. Diabetes was induced by the administration of streptozotocin (7 mg/100 g BW), and 18 days later, GH content, GH mRNA, and GH transcription rate were determined. GH mRNA levels were reduced by more than 80% in the pituitaries of diabetic rats, which had a similarly reduced GH content. The differences observed in transcription fully account for the changes in mRNA concentration, since the transcription rate of the gene was also reduced by a factor of 10 in the diabetic pituitaries. Insulin therapy (3 U/15 days) partially restored these parameters. The expression of the specific transcription factor GHF-1/Pit-1 in diabetic rats was also analyzed. Both GHF-1 mRNA levels and the binding of nuclear proteins to an oligodeoxynucleotide conforming to the GHF-1 proximal binding site in the promoter of the GH gene were normal in the diabetic pituitaries, thus excluding the possibility that decreased availability of this factor could be responsible for the decreased GH transcription. Since diabetes produced an approximately 3-fold reduction of circulating T3, the potential role of thyroid hormones on GH gene expression was also evaluated in thyroidectomized and thyroidectomized diabetic rats. Thyroidectomy decreased GH and GH mRNA to less than 5% of the values found in intact animals, and a single saturating injection of T3 (250 micrograms/100 g BW) resulted in a 8- to 10-fold induction of GH mRNA after 6 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Familial goiter in bongo antelope (Tragelaphus eurycerus).

Congenital defects in thyroglobulin (Tg) synthesis in animals have proven to be useful models for the study of Tg synthesis and regulation. Defects in Tg synthesis have been well described in Afrikander cattle, Australia Merino sheep, and goats in The Netherlands. This report describes a study of goiter in a nondomesticated bovine species, bongo antelope (Tragelaphus eurycerus), an African bovid. Three animals housed at the National Zoological Park, Washington, D.C. were studied; two had visible goiters, and a third bongo had microscopic evidence of goiter. Tg extracted from thyroid glands or thyroid colloid from these animals had a high mol wt component that was greater than 220K daltons and differed in apparent mol wt from 19S Tg from domestic cattle. Thyroid extracts also had thyroid albumin; albumin was more than half the total protein in colloid extract. The animals with goiter were euthyroid according to their circulating levels of thyroid hormones.

Animals↗
PubMed1989

Retinoic acid regulates growth hormone gene expression.

Vitamin A is required for normal growth and development, and retinoic acid (RA) may be the active metabolite in this process. Recent evidence indicates that RA acts through binding to a nuclear receptor which belongs to the steroid/thyroid hormone receptor superfamily. The receptors seem to associate with hormone-response elements in the target genes resulting in the activation (or inhibition) of transcription. Although no interaction of RA-receptor complex with specific DNA sequences has yet been reported, the homology of the different receptors suggests their mechanisms of action are similar. We therefore examined whether the effects of RA on growth could be related to changes in the expression of the growth hormone gene which is known to be transcriptionally regulated by both thyroid and glucocorticoid hormones. Our results show that RA controls growth hormone production in pituitary GH1 cells and that its effect is synergistic with that caused by these hormones.

Dose-Response Relationship, Drug↗
PubMed1989

Cloning and characterization of a cDNA that encodes a 70-kDa novel human thyroid autoantigen.

cDNA clones were isolated by screening a human thyroid carcinoma lambda gt11 library with immunoglobulins purified from serum of a patient with autoimmune Graves' disease. One clone (ML8) containing a 1.25-kilobase (kb) insert hybridized with a single 2.0-kb poly(A+) mRNA in human thyroid and lymphocytes but not in human brain, liver, kidney, or muscle. In addition, this probe also hybridized with a single 2.0-kb poly(A+) mRNA from a rat thyroid cell line (FRTL-5). An apparently full length 2,074-base pair (bp) human cDNA was obtained and sequenced. The nucleotide sequence of the 2,074-bp cDNA includes a 5'-noncoding sequence of 17 bp, a 1827-bp open reading frame, and a 222-bp 3'-noncoding sequence. The canonical polyadenylation signal AATAAA is present 18 bp upstream of the poly(A) tail. This cDNA encodes a 69,812-dalton protein with two potential N-linked glycosylation sites and at least one potential membrane spanning domain. Immunoprecipitation of the in vitro translated protein by sera from several patients with Graves' disease argues that the 69,812-dalton protein is an autoantigen.

Amino Acid Sequence↗
PubMed1989

Receptors of the thyroid: the thyrotropin receptor is only the first violinist of a symphony orchestra.

A basic reason for undertaking these studies was to further our knowledge of the structure and function of the TSH receptor as well as its interaction with other receptors on thyroid cells. The multiplicity of observations suggests the approach is bearing fruit, does not provide a simple answer, and can have pitfalls. We hope they may also contribute to understanding the structure and function of autoantigens in Graves' disease and glycoprotein hormone receptors in general. The authors are grateful to their collaborators in the National Dental Institute, particularly Drs. Bellur Prabhakar, Edward Oates, and Abner Notkins, in the National Cancer Institute, Drs. W. O. McBride and M. Lerman for their contributions to the cloning studies.

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↗
PubMed1989

Sequence and chromosomal assignment of a novel cDNA identified by immunoscreening of a thyroid expression library: similarity to a family of mitochondrial solute carrier proteins.

An immunoglobulin G (IgG) preparation of the serum from a patient with active Graves' disease was used to isolate cDNA clones from a lambda gt11 cDNA library of human thyroid follicular carcinoma tissue by immunoscreening. One of these clones, hML-7, is further characterized herein by sequencing, Northern analysis, and chromosomal mapping. The clone reacted with IgG preparations from the sera of 14 of 19 patients with active Graves' disease but not with IgG preparations from 11 normal individuals, three patients with toxic thyroid adenoma, and three with rheumatoid arthritis. The hML-7 cDNA hybridized to a 3.6 kilobase (kb) mRNA transcript in poly(A+) RNA preparations from human thyroid tissue and continuously cultured rat thyroid cells; expression of this transcript in rat FRTL-5 thyroid cells was positively regulated by TSH. The 3.6 kb transcript was less abundant in rat liver (BRL3A) cells or differentiated rat (L6) myoblasts than in cultured rat thyroid cells and was not detectable in mouse L-M fibroblasts, human IM-9 lymphocytes, Chinese hamster ovary cells, or human cervical carcinoma cells. The cDNA from hML-7 was sequenced and compared with the sequence of cross-hybridizing cDNA clones isolated from human Graves' thyroid and rat FRTL-5 thyroid cell lambda gt11 expression libraries. A 1.05 kb open reading frame, which is highly conserved between human and rat, was defined. The predicted amino acid sequence of 348 residues exhibited a strong homology with the mitochondrial ADP/ATP carrier protein (adenine nucleotide translocase; ADP/ATP translocator) and with two other members of the same mitochondrial protein family, the phosphate carrier and the hydrogen ion uncoupling protein. The gene represented by the hML-7 cDNA has been assigned to human chromosome 10.

Amino Acid Sequence↗
PubMed1987

Characterization of phosphate residues on thyroglobulin.

Follicular 19 S thyroglobulin (molecular weight 660,000) from rat, human, and bovine thyroid tissues contains approximately 10-12 mol of phosphate/mol of protein. These phosphate residues can be radiolabeled when rat thyroid hemilobes, FRTL-5 rat thyroid cells, or bovine thyroid slices are incubated in vitro with [32P]phosphate. Thus labeled, the [32P]phosphate residues comigrate with unlabeled 19 S follicular thyroglobulin on sucrose gradients and gel filtration columns; are specifically immunoprecipitated by an antibody preparation to rat or bovine thyroglobulin as appropriate; and co-migrate with authentic 19 S thyroglobulin when subjected to analytic or preparative gel electrophoresis. Tunicamycin prevents approximately 50% of the phosphate from being incorporated into FRTL-5 cell thyroglobulin. Approximately one-half of the phosphate in FRTL-5 cell or bovine thyroglobulin can also be released by enzymatic deglycosylation and can be located in Pronase-digested peptides which contain mannose, are endo-beta-N-acetylglucosaminidase H but not neuraminidase-sensitive, and release a dually labeled oligosaccharide containing mannose and phosphate after endo-beta-N-acetylglucosaminidase H digestion. The remainder of the phosphate is in alkali-sensitive phosphoserine residues (3-4/mol of protein) and phosphotyrosine residues (approximately 2/mol of protein). This is evidenced by electrophoresis of acid hydrolysates of 32P-labeled thyroglobulin and by reactivity with antibodies directed against phosphotyrosine residues. The phosphoserine and phosphotyrosine residues do not appear to be randomly located through the thyroglobulin molecule since approximately 75-85% of the phosphotyrosine and phosphoserine residues were recovered in a approximately 15-kDa tryptic peptide or a approximately 24-kDa cyanogen bromide peptide, each almost devoid of carbohydrate. 31P nuclear magnetic resonance studies of bovine thyroglobulin confirm the presence and heterogeneity of the phosphate residues on thyroglobulin preparations.

Animals↗
PubMed1987

Thyroglobulin gene expression is regulated by insulin and insulin-like growth factor I, as well as thyrotropin, in FRTL-5 thyroid cells.

In FRTL-5 thyroid cells depleted of thyrotropin, insulin, and serum for 3 days, growth and synthesis of thyroglobulin are inhibited. Readdition of insulin or IGF-I in the absence of thyrotropin stimulates thyroglobulin synthesis or thyroglobulin mRNA level by 2-4-fold but has no effect on cell growth. The half-maximal effects of insulin and IGF-I are at 100 and 10 ng/ml, respectively. In both cases, the increased mRNA levels are accompanied by an increase in transcription rate. Maximally effective concentrations of insulin or IGF-I are additive with thyrotropin in increasing thyroglobulin mRNA levels and in increasing transcription; in contrast, the effect of insulin is not additive with IGF-I.

Cell Division↗
PubMed1987

Norepinephrine and thyrotropin stimulation of iodide efflux in FRTL-5 thyroid cells involves metabolites of arachidonic acid and is associated with the iodination of thyroglobulin.

Ca2+-dependent and TSH-, norepinephrine (NE)-, and A23187-induced iodide (I-) efflux from FRTL-5 rat thyroid cells is inhibited by quinacrine and trifluoroperazine, agents that inhibit phospholipase A2 activity. Furthermore, I- efflux can be stimulated by an activator of phospholipase A2 activity, melittin. Phospholipase A2 action releases arachidonic acid from phospholipids; arachidonic acid enhances I- efflux in FRTL-5 cells. Inhibitors of arachidonic acid metabolism via the lipoxygenase pathway, 5,8,11,14-eicosatetraynoic acid and nordihydroguaiaretic acid, and via the cytochrome P450-linked epoxygenase pathway, piperonyl butoxide and 2-diethylaminoethyl-2,2-diphenyl valerate, but not an inhibitor of the cyclooxygenase pathway, indomethacin, can inhibit TSH-, NE-, and A23187-induced I- efflux. TSH, NE, and arachidonic acid stimulation of I- efflux in FRTL-5 cells is associated with increased iodination of thyroglobulin, which is blocked by 10 microM 5,8,11,14-eicosatetraynoic acid and 50 microM piperonyl butoxide. The data thus suggest that TSH- and NE-induced I- efflux from FRTL-5 thyroid cells involves lipoxygenase and/or epoxygenase metabolites of arachidonic acid, released from phospholipids upon Ca2+-dependent activation of phospholipase A2. Since this process is associated with the iodination of thyroglobulin, TSH- and NE-induced I- efflux in FRTL-5 cells may represent the transport of I- from the cell into the follicular lumen in vivo.

Animals↗
PubMed1987

TSH receptor structure.

When solubilized, radiolabelled membrane preparations from FRTL-5 rat thyroid cells are applied to TSH affinity columns, two separate peaks of protein can be eluted by high salts/high pH and low pH buffers, respectively. Immunoprecipitation with monoclonal antibodies to the TSH receptor shows that both peaks contain proteins related to the TSH receptor. If extracts were from cells grown without TSH, one peak has a approximately 300 K and the other a approximately 70 K protein the 70 K protein can be derived from the purified 300 K protein in vitro. A 50 and 20 K protein can be derived from the 70 K protein. If extracts are from cells grown with TSH, the peaks contain a multiplicity of additional immuno-precipitable bands of approximately 200, 175, 130, 90, 50, 20 K etc. These bands are shown to result from the ability of TSH to increase the synthesis (3-4-fold) and degradation (2-3-fold) of the 300 and 70 K proteins. The 300/70 K protein fractions are reactive with monoclonal autoimmune thyroid stimulating antibodies and contain a specific disialo ganglioside. The ganglioside migrates near GM2, i.e., like a lower order ganglioside, and contains fucose. In translation experiments, the monoclonal antibodies to the TSH receptor identify a single mRNA component which produces a protein of approximately 220 K. This protein is not present in thyroid cells which have no functional TSH receptor and which cannot be surface labelled with monoclonal antibodies to the TSH receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Monoclonal antibody studies defining the origin and properties of autoantibodies in Graves' disease.

The present report summarizes experiments with monoclonal antibodies to the TSH receptor. The data provide further insight into the TSH receptor structure and into the basis of autoimmune antibodies implicated in the pathogenesis of Graves' disease. They resolve many clinical questions and provide new approaches to enhance our understanding of autoimmune disease. In one new approach, it has been noted that the 11E8 TBIAb can precipitate the phosphorylated beta subunit of the insulin and IGF1 receptor. This cross-reactivity or recognition of determinants adjacent to the TSH receptor may not be random. Insulin, IGF1, alpha 1 adrenergic, and TSH receptors have been linked to a synergistic cascade response system of the thyroid involving growth, thyroglobulin biosynthesis, iodination of thyroglobulin, and thyroid hormone formation. Future studies with the monoclonals may help unravel this cascade system and its regulatory relationships, along with the relationships between autoimmune thyroid disease and autoimmune diseases of other organs.

Antibodies, Anti-Idiotypic↗
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

Iodine suppression of iodide uptake in FRTL-5 thyroid cells.

Exposure of FRTL-5 cells to iodide (I-) in excess of 3 microM suppresses the concentrative uptake of I-. The depression of I- uptake measured at the steady state is due to decrease in the rate of I- influx and not to an effect on I- efflux. Exposure to NaI is associated with decreased T4 secretion and also depressed Na+-dependent amino acid accumulation. The depression in I- and amino acid transports increases proportionately with the duration of exposure and concentration of I- used but is not associated with alterations in FRTL-5 cell cAMP levels. The I- suppression effect is blocked, however, when methimazole is present during the incubation with NaI. In agreement with studies in vivo, I- suppression in FRTL-5 cells appears to depend on an intermediate in the organification process and to be independent of a TSH-induced cAMP-mediated action.

Amino Acids↗