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S Filetti

Publications and source records attributed to S Filetti.

At least 37 records · Page 2Linked to original sources
PubMed1987

Molecular cloning of the complementary deoxyribonucleic acid for human thyroid peroxidase.

Five overlapping cDNA clones representing the entire mRNA for human thyroid peroxidase (TPO) have been isolated from a human Graves' thyroid cDNA library. The cDNA sequence has been determined. Human TPO cDNA contains 3060 bases from the start of transcription to the beginning of the poly (A) tail at the 3'-end. The derived amino acid sequence of human TPO consists of 933 amino acids with a mol wt of 102,937. The derived amino acid sequence contains five potential glycosylation sites (Asn-X-Ser/Thr), a probable transmembrane signal peptide sequence at the amino terminus, and a hydrophobic putative membrane-spanning region beginning 85 amino acid residues from the carboxyl terminal end. Comparison of the human TPO amino acid sequence to that of pig TPO shows strong homology extending from the amino terminus to within 44 amino acid residues of the carboxyl-terminus.

Amino Acid Sequence↗
PubMed1986

Thyroid autoregulation: effect of iodine on glucose transport in cultured thyroid cells.

The nonmetabolizable glucose analogs, [3H]2-deoxy-D-glucose and [3H]O-methyl-D-glucose, were used to determine whether iodide influences glucose transport in porcine cells in primary culture. Incubation with iodide (3 h) decreased basal glucose transport with a half-maximum at NaI 3 X 10(-5) M and maximum at 10(-4) M. Iodide (10(-6) M to 10(-4) M) also abolished the stimulatory effect of TSH (1 mU/ml) on glucose transport. The iodide effect on [3H]2-deoxy-D-glucose transport had the following characteristics: 1) it was abolished 24 h after incubation in iodide-free medium; 2) it was prevented by methimazole (3 mM), and correlated with newly formed organic iodine, 3) and it affected the maximum velocity (Vmax) of glucose transport, reducing it from 25.1 to 14.4 and 12.0 nmol/(min mg protein) at 10(-5) M and 10(-4) M NaI, without affecting the Michaelis-Menten constant (Km) (6mM). Iodide-treated cells had a reduced specific binding of [3H]cytochalasin B (38% and 47% with respect to control cells at 10(-5) M and 10(-4) M NaI). These data suggest that iodide treatment reduces the functional carriers mediating glucose transport in the thyroid.

3-O-Methylglucose↗
PubMed1984

On the mechanism of 'escape' from desensitization of the cyclic AMP response to TSH in cultured human thyroid cells.

Studies were conducted to examine the mechanism by which 'escape' from TSH desensitization of the cyclic AMP response to TSH (Endocrinology 109, 1156, 1981) occurs in confluent cultured thyroid cells. At confluence, cell replication and DNA synthesis are suppressed. An attempt was therefore made to reproduce escape in sparse thyroid cell monolayers using inhibitors of DNA synthesis. The concurrent presence of TSH and mitomycin C (5 micrograms/ml) did not influence the induction of desensitization to TSH after 6 h of stimulation, but cAMP levels then rebounded by 24 h; that is, mitomycin C reproduced escape in sparse cells. Hydroxyurea (10 mM) did not reproduce escape in sparse cells. Adenylate cyclase activity was unaltered in plasma membranes prepared from sparse thyroid cells treated with mitomycin C for 24 h. These data suggest that 'escape' from TSH desensitization is related to events occurring during the cell cycle associated with DNA synthesis, and is caused by an alteration in adenylate cyclase substrate or co-factor availability rather than in enzyme activity itself.

1-Methyl-3-isobutylxanthine↗
PubMed1984

Autoregulation by iodine of thyroid protein synthesis: influence of iodine on amino acid transport in cultured thyroid cells.

Studies were conducted to determine whether the inhibitory effect of iodine on thyroid protein synthesis could be explained by a reduction in intracellular amino acid transport. The nonmetabolizable amino acid [14C]cycloleucine was used as a probe for the L system of neutral amino acid transport in dog thyroid cells during the initial 24 h of primary culture. Uptake of cycloleucine was linear for up to 30 min. At all time points, cycloleucine transport was reduced in cells preincubated for 3 h in NaI (10(-4) M). Inhibition (in a typical experiment) by NaI of cycloleucine transport (10 min) was 19%, 42%, and 69% at 10(-6), 10(-5), and 10(-4) M iodide, respectively. Methimazole (3 mM) together with iodide abolished the inhibitory effect of iodide on cycloleucine transport, implying the necessity of iodide organification. Methimazole itself did not significantly alter cycloleucine transport. T3, T4, MIT, and DIT similarly did not inhibit cycloleucine transport. NaI did not inhibit cycloleucine transport in cells lacking a mechanism for iodide organification. Double reciprocal plots of cycloleucine influx at different substrate concentrations indicated that NaI decreases the maximum velocity of cycloleucine transport (2.1 vs. 4.0 nmol min-1 mg protein-1) without affecting the Km (1 mM). In contrast to influx, iodine did not affect cycloleucine efflux. The inhibitory action of iodine on cycloleucine transport was reversible after removal of extracellular iodide, with full recovery occurring within 24 h. Iodine similarly inhibited the cellular uptake of [14C] alpha-methylaminoisobutyric acid as well as [14C] alpha-aminoisobutyric acid in the presence of 30 mM methyl-alpha-aminoisobutyric acid, that is of specific probes for the A and ASC transport systems of neutral amino acids, respectively. These data indicate that autoregulation by iodine of thyroid protein synthesis occurs, at least in part, by regulation of the maximum velocity of neutral amino acid uptake via the A, ASC, and L transport systems.

Amino Acids↗
PubMed1984

Clinical experience with a human thyroid cell bioassay for thyroid-stimulating immunoglobulin.

A sensitive, specific, and practical bioassay for thyroid-stimulating immunoglobulin (TSI) is now available for clinical use. Fifty-seven of 61 patients with untreated hyperthyroid Graves' disease were TSI positive (sensitivity, 93%). TSI was undetectable in all normal subjects and in patients with Hashimoto's thyroiditis (without concurrent Graves' ophthalmopathy), nontoxic goiter, and toxic nodular goiter (specificity, 100%). The prevalence of TSI in serum declined after therapy, particularly during methimazole or propylthiouracil treatment. TSI correlated well with relapse or remission after antithyroid drug therapy. All 12 patients who were TSI negative at the time of discontinuing antithyroid drug therapy remained in remission (average follow-up of 10 months). TSI values in Graves' disease correlated better with thyroid dysfunction than with ophthalmopathy. Prenatal TSI activity tended to be higher in mothers of infants who developed neonatal Graves' disease than in at-risk mothers who delivered normal infants. However, there was considerable overlap between the two groups.

Biological Assay↗
PubMed1983

Differential effect of protein synthesis inhibition on TSH desensitization at different stages of primary thyroid cell culture.

In contrast to our previous experience with cultured thyroid cells, cycloheximide, actinomycin D and nicotinamide did not prevent TSH-induced desensitization in dog thyroid cells in primary culture for only one day. With continued duration of culture prevention of TSH desensitization by these agents did emerge, but asynchronously. Thus on the second day of primary culture, while cycloheximide and actinomycin D prevented TSH desensitization, nicotinamide remained ineffective. On the third day of primary culture all three agents blocked TSH desensitization. Examination of precursor incorporation into newly synthesized DNA, RNA and protein revealed a temporal association between the appearance of susceptibility to inhibition of TSH desensitization and an increase in DNA and protein synthesis. These data provide an explanation for the discrepant reports regarding the effect of protein synthesis inhibitors on TSH desensitization.

Animals↗
PubMed1983

Evidence that organic iodine attenuates the adenosine 3',5'-monophosphate response to thyrotropin stimulation in thyroid tissue by an action at or near the adenylate cyclase catalytic unit.

Studies were conducted to define more clearly the site in the thyroid adenylate cyclase complex at which iodine exerts its inhibitory effect on activation of this enzyme by TSH. Iodine- and TSH-induced desensitization were additive. Dissociation was observed between the rates of recovery from TSH- and iodine-induced desensitization. Cycloheximide (10(-4) M) prevented recovery from the inhibitory effect of iodine on thyroid adenylate cyclase activation. Preincubation of freshly isolated dog thyroid follicles in 10(-4) M iodide decreased the subsequent cAMP response to cholera toxin (0.5 micrograms/ml) stimulation. This effect of iodide was prevented by 3 mM methimazole. Thyroid adenylate cyclase regulatory protein (Ns) activity was assessed by the ability of detergent extracts of thyroid plasma membranes to reconstitute adenylate cyclase responsiveness to isoproterenol in N-deficient S49 cyc- plasma membranes. Thyroid Ns activities were similar in control and iodide-pretreated thyroid cells. The inhibitory effect of iodine on TSH activation of thyroid cAMP generation was additive to that of inhibition via the alpha 2- adrenergic pathway and also additive to inhibition by 2',5'-dideoxyadenosine (an adenosine P-site agonist). Preincubation of freshly dispersed dog thyroid cells in 10(-4) M NaI reduced the cAMP response to stimulation by 100 microM forskolin. These data provide evidence that in iodine-induced TSH desensitization in the thyroid; 1) TSH receptor function is normal, 2) the regulatory protein (Ns) in the adenylate cyclase stimulatory pathway is functionally unaltered, 3) iodine does not exert its effect via the regulatory protein (Ni) in the pathway that inhibits adenylate cyclase activation, 4) iodine does not act via the adenosine P-site inhibitory pathway, 5) the action of iodine is at or near the adenylate cyclase catalytic unit, and 6) new protein synthesis is necessary for recovery from iodine desensitization.

Adenylyl Cyclases↗
PubMed1982

Studies on the cyclic AMP response to thyroid stimulating immunoglobulin (TSI) and thyrotropin (TSH) in human thyroid cell monolayers.

Studies were conducted on the cultured human thyroid cell bioassay for thyroid stimulating immunoglobulin (TSI) and thyrotropin (TSH). In confirmation of the data of Kasagi, et al.,15 incubation of human thyroid cells in Hank's balanced salt solution deficient in NaCl increased the sensitivity of the c-AMP response to bovine TSH by approximately one order of magnitude. Half-maximal stimulation was attained at approximately 0.1 mU bTSH/ml. The effect of NaCl in the medium was greater with stimulation by TSI greater than hTSH greater than bTSH. In contract to incubations in NaCl(+)medium, with NaCl(-) medium most (70%-80%) of the c-AMP produced was released into the medium; this proportion remaining relatively constant over a wide range of bTSH and hTSH concentrations. At TSI concentrations higher than 3mg/ml efflux of c-AMP into the medium was significantly diminished. Stimulation by cholera toxin and prostaglandin E of thyroid cell c-AMP generation was not enhanced in NaCl(-) medium, in contrast to stimulation by TSH and TSI. The presence of 10(-4)m cycloheximide in NaCl(+) medium enhanced the c-AMP response to physiological concentrations of TSH. As with NaCl(-) medium, cycloheximide increased the sensitivity but not the maximum response of the c-AMP response to TSH. However no additivity was observed with NaCl(-) medium and cycloheximide. Human thyroid cells obtained from patients with Graves' disease are relatively insensitive to TSI stimulation. In NaCl(-) medium, however, the sensitivity of these cells to TSI stimulation is sufficient to enable them to be utilized in the TSI assay. The present state of the TSI assay is discussed.

Alprostadil↗
PubMed1982

High performance gel permeation chromatography of bovine thyrotropin (TSH): effect of column stability and mobile phase variation.

Because of previously observed variability in the elution patterns of radiolabeled bovine TSH (bTSH) and bTSH bioactivity (in a particular bTSH preparation) on gel permeation high performance liquid chromatography (GPC), studies were conducted to examine the effect of different conditions on the elution of this material. Continued use of a Waters' I-125 column at pH 7.2 demonstrated progressive retardation in the elution of bioactivity and radioactivity in this highly-purified bTSH preparation, with progressive but incomplete separation of these two functions. With decreasing mobile phase pH the elution positions of TSH bioactivity and radioactivity advanced to a coincident peak near to the void volume (pH 4). Addition to the mobile phase of the ion pair reagents tetrabutyl-ammonium phosphate (PIC-A) and pentane sulfonic acid (PIC B5) produced marked alterations in the elution positions of TSH radioactivity and bioactivity, with separation of these functions in the presence of the former reagent. These data indicate the present limitations of GPC for the purification of TSH.

Animals↗
PubMed1982

Evidence for species specificity in the interaction between thyrotropin and thyroid-stimulating immunoglobulin and their receptor in thyroid tissue.

The cAMP response in cultured human and dog thyroid cells was used to examine the relationship between human TSH, nonprimate TSH, and thyroid-stimulating immunoglobulin (TSI) bioactivity in human and nonhuman thyroid tissue. The bovine TSH (bTSH) to human TSH potency ratio was approximately 6-fold greater in dog than in human thyroid cells. Relative bioactivity of bTSH and TSI aslo differed in these cell types. Thus, four TSI samples produced approximately 6-fold greater stimulation relative to bTSH in human thyroid than in dog thyroid cells. It is discussed why these data suggest that the TSH receptor as well as TSH and TSI display species specificity as defined by the classical concept of this term.

Animals↗
PubMed1982

Role of thyrotrophin-releasing hormone in the development of pituitary-thyroid axis in four anencephalic infants.

Anencephaly provides a unique model for studying endocrine functions in absence of hypothalamic influence. We previously reported that in anencephalic newborns both pituitary TSH-secreting cells and the thyroid were normal and were able to function if adequately stimulated. In order to verify if the normal development of the pituitary-thyroid axis in these infants depends on TRH of extrahypothalamic origin, we measured endogenous TRH levels in the clear fluid of a cyst of the cerebro-vasculosa in 4 anencephalic newborns. In these cysts were also injected 200 micrograms of synthetic TRH and evaluated TSH response in peripheral blood samples. Endogenous TRH was detectable in the cysts of the cerebro-vasculosa in 3 of the 4 infants. In all 4 cases serum TSH sharply increased after TRH administration. Our data suggest that the normal development of the pituitary-thyroid axis in anencephalic infants either requires no TRH or depends on extrahypothalamic TRH. In this latter case TRH produced by other areas of the central nervous system might be secreted into the cysts of the cerebro-vasculosa, actively transported to the hypophyseal vessels, and might thus reach the pituitary to stimulate TSH-cells growth and function.

Anencephaly↗
PubMed1981

Prevention by nicotinamide of desensitization to thyrotropin stimulation in cultured human thyroid cells.

The presence of 50 mM nicotinamide together with 100 milliunits/ml of TSH in the incubation medium prevented the decline in human thyroid cell cAMP from maximum, stimulated levels (15-30 min) that occurs when the cells are exposed to TSH alone. Nicotinamide in the absence of TSH did not increase thyroid cell cAMP content. TSH desensitization, and its prevention by nicotinamide, occurred in the presence or absence of 3-isobutyl-methylxanthine. 1-Methyl nicotinamide and N'-methyl nicotinamide similarly prevented TSH desensitization. Recovery from TSH desensitization was prolonged and incomplete after 72 h. The presence of 50 mM nicotinamide hastened recovery from desensitization. Desensitization of the cAMP response to 10(6) M prostaglandin E1 and 1 mM adenosine was unaffected by nicotinamide. Other inhibitors of poly(ADP-ribose) polymerase activity, 5-bromouridine, 5-bromo-2'-deoxyuridine, and thymidine (all at 50 mM) completely or partially prevented TSH desensitization. Pyridoxine (50 mM) similarly prevented this phenomenon. As with dog thyroid cells, 10(-4) M cycloheximide blocked TSH desensitization. The combination of 10(-4) M cycloheximide and 50 mM nicotinamide had a synergistic effect in augmenting the thyroid cell cAMP response to TSH stimulation.

Cells, Cultured↗
PubMed1981

Hormonal stimulation of eucaryotic cell ADP-ribosylation.

The effect of thyrotropin (TSH) on the ADP-ribosylation of endogenous thyroid cell acceptor proteins was examined. Cells were "permeabilized" at 4 degrees C in hypotonic medium and then exposed to [(32)P]- or [(3)H-adenine]NAD(+). The net incorporation of labeled ADP-ribose was measured by trichloroacetic acid precipitation. TSH (100 mU/ml) enhanced ADP-ribosylation with a maximum effect after 30-60 min in the majority of experiments. TSH stimulation was observed even when the incubation contained 1,000-fold more exogenous NAD(+) than the amount of NAD(+) contributed by the permeabilized cells, indicating an effect on enzymatic activity rather than an alteration in NAD(+) pool size or specific activity. No incorporation of radioactivity from labeled NAD(+) was observed in cells not rendered permeable to NAD(+) by hypotonic shock. TSH did not increase the rate of disappearance of trichloroacetic-precipitable radioactivity and did not contain intrinsic NAD(+) glycohydrolase activity. Alkali and snake venom phosphodiesterase, but not ribonuclease or deoxyribonuclease digestion of trichloroacetic acid precipitable thyroid cell radioactivity, revealed primarily 5'-AMP, consistent with an effect of TSH on mono-ADP ribosylation. Nicotinamide and thymidine (50 mM) inhibited both basal and TSH-stimulated ADP-ribosylation of thyroid cell protein. Dibutyryl cyclic (c)AMP (0.1 mM) inhibited endogenous ADP-ribosylation by approximately 35% but had no effect at lower concentrations. 0.5 mM isobutylmethylxanthine inhibited this reaction by approximately 60%. We suggest that TSH enhances thyroid cell ADP-ribosylation by a mechanism independent of cAMP as a second messenger, and that ADP-ribosylation plays a role in the expression of TSH.

Adenosine Diphosphate Ribose↗
PubMed1981

Insulin receptor down-regulation, prevention at a post-receptor site.

Nicotinamide (50mM) prevented insulin-mediated down-regulation of insulin receptors in IM-9 lymphoblastoid cells. Half-maximum effectiveness was between 10 and 33mM. Nicotinamide did not influence insulin binding to the cells, cell viability, insulin degradation or protein synthesis. A variety of inhibitors of ADP-ribosylation reactions besides nicotinamide, most of them pyridine analogues, similarly prevented insulin-induced receptor loss. Spermine decreased the number of insulin receptors in IM-9 cells, but this effect was not inhibited by nicotinamide.

Cells, Cultured↗