A novel mutation (E333D) in the thyroid hormone beta receptor causing resistance to thyroid hormone syndrome.

Resistance to thyroid hormone (RTH) is an inherited syndrome characterized by elevated serum thyroid hormones (TH), failure to suppress pituitary thyroid stimulating hormone (TSH) secretion, and variable peripheral tissue responsiveness to TH. The disorder is associated with diverse mutations in the thyroid hormone beta receptor (TRbeta). Here, we report a novel natural RTH mutation (E333D) located in the large carboxy-terminal ligand binding domain of TRbeta. The mutation was identified in a 22-year-old French woman coming to medical attention because of an increasing overweight. Biochemical tests showed elevated free thyroxine (T4: 20.8 pg/ml (normal, 8.5-18)) and triiodothyronine (T3: 5.7 pg/ml (normal, 1.4-4)) in the serum, together with an inappropriately nonsuppressed TSH level of 4.7 mU/ml (normal, 0.4-4). Her father and her brother's serum tests also showed biochemical abnormalities consistent with RTH. Direct sequencing of the TRbeta gene revealed a heterozygous transition 1284A>C in exon 9 resulting in substitution of glutamic acid 333 by aspartic acid residue (E333D). Further functional analyses of the novel TRbeta mutant were conducted. We found that the E333D mutation neither significantly affected the affinity of the receptor for T3 nor modified heterodimer formation with retinoid X receptor (RXR) when bound to DNA. However, in transient transfection assays, the E333D TRbeta mutant exhibited impaired transcriptional regulation on two distinct positively regulated thyroid response elements (F2- and DR4-TREs) as well as on the negatively regulated human TSHalpha promoter. Moreover, a dominant inhibition of the wild-type TRbeta counterpart transactivation function was observed on both a positive (F2-TRE) and a negative (TSHalpha) promoter. These results strongly suggest that the E333D TRbeta mutation is responsible for the RTH phenotype in the proposita's family.

Identification of second lipolysis activating protein from scorpion Buthus occitanus tunetanus.

Besides the previously described LVP1, a second protein, LVP2, inducing a lipolytic response in adipose cells, was purified from scorpion Buthus occitanus tunetanus venom. It represented 2% of crude venom proteins, with pHi = 6 and molecular mass of 16889 Da. The reduction and the alkylation of LVP2 revealed an heterodimeric structure. Isolated alpha and beta chains of LVP2 have a molecular weight (MW) of 8822 Da and 8902, respectively. This protein was not toxic to mice and stimulated lipolysis on freshly dissociated rat adipocytes in a dose-dependent manner with EC50 = 2 +/- 0.75 microg/ml. LVP2 subunits did not display any lipolytic activity. As previously described for venom and LVP1, beta adrenergic receptor (beta AR) antagonists interfere with LVP2 activity. Furthermore, it is shown that LVP2 competes with [3H] CGP 12177 (beta1/beta2 AR antagonist) for binding to adipocyte plasma membrane with an IC50 of about 10(-7)M. Thus, these results bring original information on the existence of proteins that are present in scorpion venoms and can exert a distinct biological activity on adipocyte lipolysis through a beta-type adreno-receptor pathway.

Low-level arsenite activates the transcription of genes involved in adipose differentiation.

In this study we analyzed gene expression in 3T3-F442A pre-adipocyte cells that differentiate in the presence of micro-molar arsenate concentration. Two concentrations of arsenite (As2O3, 0.25 micromol/L and 0.5 micromol/L) were applied for three days with and without insulin (170 nmol/L) and gene expressions were evaluated by quantitative RT-PCR. The genes included genes of oxidative-stress responses: heme-oxygenase-1 (HO1) and the hypoxia inducible factor 1a (HIF1alpha), genes of cell-cycle: c-jun and Kruppel like factor 5 (KLF5), and genes that play important roles in adipose determination: a peroxisome proliferator-activated receptor (PPARgamma) and a CCAAT/ enhancer binding protein (C/EBPalpha). Arsenite induced the expression of HO1, HIF1alpha, KLF5, PPARgamma and C/EBPalpha. These results suggest that under condition of oxidative stress arsenite induces genes that are required for adipose differentiation.

A novel thyroid hormone receptor-beta mutation, not anticipated to occur in resistance to thyroid hormone, causes variable phenotypes.

Resistance to thyroid hormones (RTH) is a syndrome characterized by a variable tissue hyposensitivity to thyroid hormones and is linked to mutations in the thyroid hormone receptor-beta (TRbeta) gene. We report here for the first time in vivo the mutation R429W (CCG-->TCG) located in the exon 10. The artificial mutant obtained in vitro displayed a normal T(3)-binding affinity and transactivation function. Therefore, it was thought to produce little, if any, clinical effect and to escape to clinical detection. The present report is at least in part discordant with this prediction since the propositus and his grandmother had an authentic hyperthyroidism with high FT(4) plasma level in the presence of inappropriate TSH. On the other hand, spontaneous variations of clinical features and - interestingly - of plasma FT(4) concentrations with time in the propositus, and the phenotype observed in his mother who never complained with thyrotoxic symptoms, confirmed the in vitro binding and functional predictions. The most intriguing is the clinical course of the grandmother as she first presented with predominant pituitary RTH and a diffuse goiter and finally with a toxic multinodular goiter with normal T(3) and T(4) plasma concentrations and suppressed TSH. In conclusion, we report a novel mutation in the gene encoding the thyroid hormone receptor responsible for predominant pituitary RTH already described in vitro but not in vivo. The fluctuant phenotype of the propositus suggests that other factors modulate the degree of tissue resistance that is under genetic control. Toxic multinodular goiter, possibly due to chronic TSH stimulation during RTH, in addition to the phenotype variability, increases the difficulty to diagnose this thyroid disorder.

[Identification of eight new mutations in the c-erbAB gene of patients with resistance to thyroid hormone]. / Identification de huit nouvelles mutations dans le gène c-erbAB chez des patients atteints de résistance aux hormones thyroïdiennes.

Resistance to thyroid hormone (RTH) is a rare genetic disorder, usually associated with different mutations in the c-erbAB gene that encodes the beta type receptor of thyroid hormone (TRB). It is characterized by elevated serum thyroid hormone and inappropriate TSH secretion. The numerous mutations so far detected are clustered in three hot spot areas in the ligand binding domain of TRB. In the context of a national survey we have detected 16 different mutations in the c-erbAB gene, in 22 families presenting with RTH. Eight of these mutations had not been described previously. Two are located in an area not known to harbor naturally occurring mutations. This observation could lead to define a fourth cluster of mutations in the c-erbAB gene.

Resistance to thyroid hormone in a family with no TRbeta gene anomaly: pathogenic hypotheses.

UNLABELLED: Syndromes of resistance to thyroid hormone (RTH) are almost always linked to a defective triiodothyronine-receptor B gene (TRB). Only six families with RTH exhibiting a normal TRB gene have been reported so far. We report another and discuss possible mechanisms. PATIENTS AND METHODS: We studied a kindred expressing a typical RTH phenotype. DNA was amplified and the TRB gene was sequenced. Linkage analysis assessed linkage between the TRB gene and RTH phenotype. RESULTS: Direct sequencing of the TRB gene failed to identify any anomaly in the coding exons. Linkage analysis demonstrated that the RTH phenotype was not linked to the TRB gene in this family. CONCLUSION: TRB1 and TRB2 genes were not defective in this family. Multiple mechanisms might account for this situation at the pre-receptor, receptor and post-receptor levels. The most likely hypothesis is the involvement of an abnormal nuclear cofactor serving a specific function in the regulation of thyroid hormone action.

Transient expression of c-erbAbeta1 messenger ribonucleic acid and beta1 thyroid hormone receptor early in adipogenesis of Ob 17 cells.

In the murine Ob 17 preadipocyte cell line, the thyroid hormone T3 is an adipogenic factor necessary at an early stage for differentiation into adipocyte. We demonstrate here that this T3 dependence may involve a transient expression (at both the messenger RNA and the protein levels) of c-ErbA beta-type receptors (T3R), although a large body of T3R remained the product of the c-erbAalpha gene, as previously described. c-ErbAbeta1 (and not beta2) expression emerged significantly at growth arrest, peaked 2 days later, and almost disappeared in maturing adipocytes. This expression is related to the presence of T3, as total deprivation of culture medium from T3 prevented it, and the addition of 1.5 nM T3 to preconfluent cultures was able to restore it. When cells were cultured in the presence of T3 and thus were able to differentiate, the c-erbAbeta peak was accompanied by sequential rapid increases in CAAT/enhancer-binding protein-delta(C/EBPdelta), peroxisome proliferator-activated-gamma receptor (PPARgamma), and C/EBPalpha gene expressions. On the contrary, under thyroid hormone-deprived culture conditions that result in nondifferentiation of the preadipocytes, c-erbAbeta1, PPARgamma, and the large C/EBPalpha expressions were blunted, and a moderate early increase in c-erbAalpha1 transcripts was sustained for a longer period. Addition of T3 to T3-deprived preconfluent cells restored PPARgamma and C/EBPalpha expressions. Taken together, the results highlight the important role of T3 in the adipogenesis of Ob 17 cells through the involvement of both beta1 and alpha1 T3R subtypes.

A novel resistance to thyroid hormone associated with a new mutation (T329N) in the thyroid hormone receptor beta gene.

Resistance to thyroid hormone (RTH) is a syndrome of elevated serum thyroxine, inappropriately "normal" serum thyrotropin (TSH) and reduced thyroid hormone responsiveness associated with point mutations in the thyroid hormone receptor-beta (TRbeta) gene. We describe a novel point mutation resulting in a cytosine for adenine substitution at nucleotide 1271 (exon 9) that results in the substitution of threonine for asparagine (T329N). This mutation was identified in a 30-year-old woman who was investigated for recurrent spontaneous abortions and was found to have RTH. Dextrothyroxine (D-T4) therapy was instituted. At 8 mg per day 2 pregnancies followed with the delivery of a healthy boy and an RTH-affected girl another miscarriage occurred on D-T4 treatment at 6 mg per day. The T329N mutation, which was also identified in the daughter, markedly reduces the affinity of TRbeta for triiodothyronine (T3). Formation of T329N mutant TR homodimers and heterodimers with RXRalpha on thyroid hormone response element F2 (TRE F2) was not affected, but the ability of T3 to interrupt T329N mutant TRbeta homodimerization was markedly reduced. The T329N mutant TRbeta was transcriptionally inactive in transient expression assays. In cotransfection assays with wild-type TRbeta1, the mutant TRbeta1 functioned in a dominant negative manner. The results suggest that the T329N mutation in the T3-binding domain of TRbeta is responsible for RTH in the proposita's family.

Detection of a new de novo mutation at codon 251 of exon 8 of thyroid hormone receptor beta gene in an Italian kindred with resistance to thyroid hormone.

Resistance to thyroid hormone (RTH) is almost invariably associated with mutations of the thyroid hormone (TH) receptor beta (hTR beta) gene and is inherited as an autosomal dominant disease. Mutations of hTR beta identified in patients affected by RTH cluster generally at two spots of the ligand binding domain. We investigated whether an Italian kindred with RTH had a mutation in the thyroid hormone (TH) receptor beta gene. Blood samples were obtained from the available family members for biochemical and genetic analyses. Thyroid function tests in basal conditions, and in the case of the propositus also following incremental doses of T3, were performed. Exon 4 to 10 of hTR beta gene were amplified using the polymerase chain reaction (PCR) and the mutation was identified by direct sequence analysis. The affinity constant of this mutated receptor for T3 was measured by in vitro transcription-translation and was then compared with that of wild type. We identified a heterozygous G to A transition at nucleotide 1037 of exon 8 at codon 251, resulting in a glycine (G) to glutamic acid (E) substitution (G251E) in the patient affected by RTH and in his affected offspring, but not in the normal family members. This novel mutation represents a de novo mutation since both parents of the index case were unaffected and did not have this genomic mutation. When expressed in vitro, the mutant protein (G251E) showed a marked decrease of the affinity for T3, suggesting an impaired ligand-dependent transactivation activity of this mutant receptor. In vivo studies with incremental doses of L-T3 demonstrated a reduced sensitivity to TH in the index case, in particular at the pituitary level where the thyrotrophs' activity was not completely inhibited even by 200 micrograms/day of L-T3. G251E mutation represents the fourth mutation described up to now in exon 8 of hTR beta among the subjects affected by RTH. A third cluster of mutations of the c-erbA beta gene located proximally with respect to the other two so far described begins to emerge in RTH patients.

Bile salt-dependent lipase transcripts in human fetal tissues.

In human fetal pancreas, we identified two cDNA transcripts of the bile salt-dependent lipase (BSDL) using reverse transcription followed by polymerase chain reaction (RT-PCR). The sequence of four overlapping segments obtained by RT-PCR matched the sequence of the 2.2 kb cDNA cloned from human adult pancreas (Reue et al. (1991) J. Lipid Res. 32, 267-276). A second RT-PCR product of approx. 1.1 kb was evidenced, the sequence of which corresponds to that of the BSDL-pseudogene transcript (Nilsson et al. (1993) Genomics, 17, 416-422). The short transcript is present in all tissues examined whereas the former one (2.2 kb) is either poorly (in liver and kidney) or not at all expressed in adult tissues, excepted in the pancreas. On the other hand, the 2.2 kb transcript specific of the BSDL gene was detected in all fetal tissues examined as early as the 6th week of gestation. Results also suggested that the fetal pancreas contains more 2.2 kb transcript than its adult counterpart. Therewith, BSDL was immuno-precipitated from fetal liver. The role of BSDL-gene expression during the fetal life is discussed.

Expression of a 46 kDa protein in human pancreatic tumors and its possible relationship with the bile salt-dependent lipase.

Bile salt-dependent lipase (BSDL), an enzyme normally found in human pancreatic secretions is a 100 kDa glycoprotein. A BSDL-specific 477 bp cDNA probe was prepared by performing polymerase chain reaction experiments. This cDNA was used to probe mRNAs extracted from human pancreatic tissue and tumoral cell lines. Two mRNAs were detected in normal human pancreas at 2.2 and 1.3 to 1.5 kb. In human pancreatic tumoral cells, mRNAs encoding for the BSDL were detected using in situ hybridization, and proteins with an M(r) of 46,000 to 48,000 were translated into an in vitro system using mRNAs extracted from these cells. Using an immunoprecipitation procedure, we observed here that the specific BSDL polyclonal antibodies recognized three proteins of 100 +/- 5 (p100), 46 +/- 2 (p46) and 22.7 +/- 1.2 (p23) kDa, respectively in the soluble extracts of normal adult human pancreas. The p100 protein was probably the glycosylated product resulting from the translation of the 2.2 kb transcript. The p46 protein, which electrophoresed as a doublet was the main component immunoprecipitated from extract of a differentiated human pancreatic adenocarcinoma as well as from the extracts of two pancreatic cell lines, BxPC-3 and SOJ-6. In addition, the p46 immunoform of the BSDL was detected in cell-free medium from SOJ-6 cell line and its expression was found to be correlated with the secretion of an esterolytic activity on 4-nitrophenyl caproate, whereas the BxPC-3 cell line neither secreted the p46 nor showed any esterolytic activity on this substrate. The p46 may be either a short variant of BSDL resulting from the translation of the 1.3 to 1.5 kb transcript or a protein structurally related to the enzyme. The p46 doublet immunoform was detected in the human pancreatic secretion.

Regulation by thyrotropin of acyl-CoA: cholesterol acyltransferase in cultured thyroid cells.

ACAT activity measured in microsomes of thyroid cells cultured for 4 days in the presence of TSH (1 mU/ml) was two or three times lower than that of the control cells cultured for the same period in the absence of TSH. The pool of cellular cholesterol which served as the ACAT substrate, was not exchangeable with exogenous cholesterol provided in the form of liposomes. However, the incubation of microsomes with liposomes made it possible to increase the cholesterol content in the microsomes by 35%, and this resulted in the activation of ACAT activity. Nevertheless, maximum activity measured after activation in the microsomes of the control cells remained higher than that of the microsomes of cells cultured in the presence of TSH. These findings would suggest that TSH acted by diminishing the cellular content in ACAT enzyme, as well as modifying the distribution of cholesterol in the intracellular membranes.

Chronic regulation by thyrotropin of arachidonic acid incorporation in cholesteryl esters of cultured thyroid cells.

During short term incubations, radioactive arachidonic acid and palmitic acid were incorporated in the cholesteryl ester fraction of the lipids of cultured thyroid cells. Three times more arachidonic than palmitic acid was incorporated and the incorporation of both was dependent upon the culture conditions: the presence of 1 mU/ml thyrotropin in the culture medium during four days almost completely inhibited the subsequent incorporation of the two fatty acids in the cholesteryl ester fraction whereas the total cholesterol and cholesteryl ester content of the cells was not affected.

A rapid and simple assay for the study of thromboxane B2 synthesis by intact human platelets.

Conversion of 1-14C-arachidonic acid (AA) to thromboxane B2 (TXB2) by human platelets was studied by using a new, simple technique. Organic solvent extraction was avoided by spotting aliquots of the reaction mixture directly on thin layer chromatography (TLC) plates. In this way it was possible to study the kinetic parameters of the formation of TXB2. Moreover, the rapidity and simplicity of the assay should be particularly suitable when studying the possible relationships between thromboxane synthesis and aggregation function in human platelets, where a large number of determinations is required.

Modulation by catecholamines and thyrotrophin of cyclic AMP response to beta-adrenergic stimulation by cultured porcine thyroid cells.

Thyroid cell cyclic AMP synthesis is stimulated by beta-adrenergic agonists. We have characterized this sensitivity on cultured porcine thyroid cells and have studied its modulation by chronic treatment with thyrotrophin. The synthesis of cyclic AMP in intact porcine thyroid cells in primary culture was stimulated by the beta-adrenergic agonist, isoproterenol. This stimulation was dose-dependent and was inhibited by the beta-adrenergic antagonists propranolol and alprenolol. The cell responsiveness (i.e. the response elicited by 5 microM-isoproterenol after 5-min stimulation) was increased when the cells were cultured in the absence of thyrotrophin. Thyrotrophin, when present in the culture medium at the onset of culturing, inhibited this increase. A concentration of 100 microunits. thyrotrophin/ml was sufficient to reduce the cyclic AMP response to 15% of its control value. Prostaglandin E2 or dibutyryl cyclic AMP did not mimic the effect of thyrotrophin. The low sensitivity of thyrotrophin-treated cells to beta-adrenergic agonists could be explained by a decreased number of beta-adrenergic receptors. [125I]Iodohydroxybenzyl pindolol specific binding was ten times greater in membrane preparations of control cells than in membranes derived from thyrotrophin-treated cells. The beta-adrenergic sensitivity of cultured thyroid cells was also decreased after long-term treatment by terbutaline. A time- and dose-dependent desensitization was observed.

Polarity of three-dimensional structures derived from isolated hog thyroid cells in primary culture.

When cultured in polystyrene dishes subjected to previous treatment and supplied with a serum-containing medium, hog thyroid cells form monolayers displaying dome-like arrangements after three to four days. Cells involved in formation of "domes" are morphologically polarized; the apical microvilli of these cells point toward the culture medium. When the tissue is cultured in untreated polystyrene dishes, thyroid cells remain in suspension; their aggregates swell progressively and form hollow spheres encompassed by a single layer of cells. The polarity of the cells forming such spheres is inverse in comparison to the condition characteristic of the intact thyroid gland. When culture medium is supplemented with TSH, PGE1, PGE2 or dBC, structures resembling true follicles are formed in both types of cultures. Gelatin, added to suspension cultures, is also capable of promoting follicle formation. Cultured thyroid cells regularly form an epithelial layer as a result of the interaction of cellular processes. However, the polarization of this layer depends on culture conditions. Thus, structures with either a normal follicle-like polarization of their cells or showing an inverted type of polarization can be obtained.

[Polyuridylic sequences and negative strands of Sindbis virus-specific RNAs: study by affinity chromatography on poly (A)-sepharose columns (author's transl)]. / Séquences poly(U) et chaines complémentaires des ARN spécifiques du virus Sindbis: étude par chromatographie d'affinité sur poly (A)-sépharose

The experimental conditions of affinity chromatography on poly (A)-Sepharose columns have been determined. This method makes obvious the existence of polyuridylic acid sequences on the negative strands of Sinbis virus-spedific RNAs. The isolated RNAs are negative and positive strands hybrids. By polyacrylamide gel electrophoresis, it has been shown that the negative strands have the same length as the 26 S interjacent RNA at the 6th hour, and as the 42 S virion RNA at the 9th hour postinfection. The polyadenylic acid sequences of the virion RNA and of the replicative intermediate are therefore probably genetically coded.