Spontaneously remitting insulin autoimmune syndrome in a patient taking alpha-lipoic acid.

Insulin autoimmune syndrome (IAS), or Hirata disease, is a rare hypoglycaemic disorder caused by the presence of high titer of insulin autoantibodies (IAA) in patients without previous exposure to exogenous insulin. Even though its pathogenesis is not fully understood, striking evidences link IAS to previous exposure to sulphydryl-containing medications, like alpha-lipoic acid, a widely used nutritional supplement. Although challenging, a careful differential diagnosis from other causes of hyperinsulinaemic hypoglycaemia (such as insulinoma) is mandatory, since these conditions require different therapeutic approaches. In the present study, we report a 35-year-old woman originally from Sri Lanka who was referred to our University Hospital on suspicion of occult insulinoma. Her medical history was positive for endometriosis, treated with estroprogestins and alpha-lipoic acid. The latter supplement was begun 2 weeks before the first hypoglycaemic episode. Our tests confirmed the presence of hypoglycaemia associated with high insulin and C-peptide concentrations. When insulin concentrations were compared using different assays, the results were significantly different. Moreover, insulin values significantly decreased after precipitation with polyethylene glycol. An assay for IAA proved positive (530 U/mL). A genetic analysis revealed the presence of HLA-DRB1*04,15, an immunogenetic determinant associated with IAS. On the basis of clinical data we avoided a first-line approach with immunosuppressive treatments, and the patient was advised to modify her diet, with the introduction of frequent low-caloric meals. During follow-up evaluations, glucose levels (registered trough a flash glucose monitoring system) resulted progressively more stable. IAA titer progressively decreased, being undetectable by the fifteenth month, thus indicating the remission of the IAS. Learning points: Insulin autoimmune syndrome (IAS) is a rare cause of hyperinsulinaemic hypoglycaemia, whose prevalence is higher in East Asian populations due to the higher prevalence of specific immunogenetic determinants. Nevertheless, an increasing number of IAS cases is being reported worldwide, due to the wide diffusion of medications such as alpha-lipoic acid. Differential diagnosis of IAS from other causes of hyperinsulinemic hypoglycaemia is challenging. Even though many tests can be suggestive of IAS, the gold standard remains the detection of IAAs, despite that dedicated commercial kits are not widely available. The therapeutic approach to IAS is problematic. As a matter of fact IAS is often a self-remitting disease, but sometimes needs aggressive immunosuppression. The benefits and risks of any therapeutic choice should be carefully weighted and tailored on the single patient.

Clinical aspects and therapeutic outcome in thyrotropin-secreting pituitary adenomas: a single center experience.

BACKGROUND AND AIM: The management of pituitary adenomas secreting TSH has evolved considerably over the last decades.We report the clinical features, management, and outcome of a large monocentric series. MATERIAL AND METHODS: A monocentric retrospective cohort of 26 patients admitted to our Department of Endocrinology between 1983 and 2007, followed for a period up to 204 months. The diagnosis of TSH-secreting adenoma was based on clinical and biochemical findings of central hyperthyroidism. Evaluation of basal and dynamic pituitary function, magnetic resonance imaging or computerized tomography scan were performed in all patients. Twenty-two patients, of whom 15 pre-treated by somatostatin analogs (SSA), underwent trans-sphenoidal surgery and were regularly re-evaluated. RESULTS: The number of cases increased over the years. Age at diagnosis, micro- to macroadenoma ratio, and mean estimated latency between first symptoms and diagnosis did not appreciably change over time. Latency was significantly shorter in macroadenomas. Following surgery, 55% of patients obtained remission (success rate of 40 and 67% in macro- and microadenomas, respectively). SSA pre-treatment led to an apparent although not statistically- significant increase in success rate in micro- but not in macroadenomas. CONCLUSIONS: In a monocentric group of 26 TSH-secreting adenomas the high ratio between micro- and macroadenomas remained stable over time with a significantly shorter diagnosis latency in macroadenomas. A more precocious recognition of the tumors and possibly the use of presurgical SSA allowed a high remission rate. A varied combination of neurosurgery, SSA, radiotherapy, and thyroid ablation led to the control of the disease in all the patients studied.

A special case of bilateral ovarian metastases in a woman with papillary carcinoma of the thyroid.

Papillary thyroid carcinoma is a slow growing tumor with low metastatic potential. The most frequent sites of distant metastases are lung and bone; less frequent sites are brain, liver, kidney, and skin. Ovarian metastases from papillary thyroid carcinoma are exceptional. We describe a case of bilateral ovarian metastases from a papillary thyroid carcinoma associated with autoimmune thyroiditis in a 38-year-old woman who underwent thyroidectomy and cervical lymph-node dissection 7 years before, followed by 948 mCi of 131I. A primary ovarian cancer could be excluded by the typical pathological aspects of a papillary thyroid carcinoma in a context of an aggressive form of thyroid cancer. On the other hand, the clinical history and the absence of normal thyroid epithelium and teratomatous components could exclude a papillary thyroid carcinoma arising in struma ovarii. This is a singular case of papillary thyroid carcinoma metastasizing to the ovary, combined with an autoimmune thyroiditis.

Androgen receptor polymorphism (CAG repeats) and androgenicity.

Objective Polymorphism of the androgen receptor (AR) has been related to various pathophysiological conditions, such as osteoporosis and infertility. The objectives of this study were to evaluate the frequency of distribution in a normal Italian population and to assess CAG repeats (CAGr) in other conditions, such as hypoandrogenism, potentially influenced by AR polymorphism. Patients and measurements CAGr polymorphism was determined in a group of 91 healthy normoandrogenized subjects, 29 hypoandrogenized patients (hypoplasia of prostate and seminal vesicles, reduced beard or body hair, etc.) and 29 infertile patients by direct sequencing. Results The mean (+/- SD) number of CAG repeats [(CAGr)n] was 21.5 (+/- 1.7) in the control group, 21.4 (+/- 2.0) in the infertile patients and 24.0 (+/- 2.9) in the hypoandrogenic males. The difference was statistically significant between this last group and the other two (P < 0.0001), while there was no difference between normal controls and infertile patients. The frequency distribution showed a shift towards higher CAG length in hypoandrogenized patients compared to controls and infertile patients. If we used a cut-off point of 24.9 (2 SD above the mean), the percentage of patients with 25 or more CAGr repeats was 38% among hypoandrogenized patients, 7% among infertile patients and 5% among the control group. In hypoandrogenized subjects (CAGr)n correlated slightly with testis and prostate volume. The number of CAG repeats was not associated with any of the hormonal parameters, including testosterone, evaluated in the three groups. Conclusions Our normal population, representing subjects from Central Italy, is superimposable on other European populations with regard to (CAGr)n distribution. Hypoandrogenic males have a shift in the frequency distribution towards longer (CAGr)n. Infertile patients are not statistically different from the control group. These findings suggest that, given the same amount of circulating testosterone, as in our hypoandrogenized and control group, the final net androgenic phenotypical effect is due to AR polymorphism.

Effects of Perennial Peanut (Arachis glabrata) Ground Cover on Nematode Communities in Citrus.

The effects of perennial peanut (Arachis glabrata) ground cover on the nematode community in a citrus orchard were examined. Samples were taken from two different ground cover treatments (perennial peanut or bare ground) at each of three distances from the tree trunk. Richness, measured as total numbers of nematode genera per sample, and total numbers of nematodes were greatest in the perennial peanut treatment (P < 0.05). Abundance of many genera of bacterivores, fungivores, and omnivores were increased by the perennial peanut ground cover. Total numbers of plant parasites were greater in perennial peanut treatments on three of the five sampling dates (P < 0.05), mainly due to trends in numbers of Mesocriconema. Distance from a tree trunk and the interaction of ground cover treatments and proximity to a tree trunk were most influential for Belonolaimus and Hoplolaimus. Although differences among treatments were observed for nematode genera and trophic groups, ecological indices were not consistently sensitive to treatments. Among several ecological indices evaluated, richness was most often affected by ground cover treatment.

Thyroid hormone receptors in neonatal, prepubertal, and adult rat testis.

Thyroid hormone (TH) is involved in the differentiation and development of rat testis, whereas its role in adult testis function is still undefined. The aim of our work has been to further analyze the presence in the testis of rats of various ages of messenger RNA (mRNA) coding the different TH receptor (TR) subtypes using a sensitive assay, such as reverse transcriptase-polymerase chain reaction (RT-PCR). To rule out the possibility of an "illegitimate transcription," we have analyzed both T3-binding capacity of adult rat testis and the presence in the same organ of TR proteins by immunohistochemistry, using specific antibodies directed against the various TR isoforms. Messenger RNA coding for TR alpha1 and alpha2 isoforms was clearly visible in gels prepared from RT-PCR samples obtained from the testis of rats of all ages, including adults, whereas mRNA for the TR beta1-beta2 was absent. The T3 maximal binding capacity (Cmax) by nuclear extracts of testicular homogenates gradually decreased from birth to adulthood, still remaining significantly detectable in adult testis, and represented approximately 1% of the Cmax observed in the liver. The immunostaining technique revealed an intense nuclear staining along the basement membrane of testicular tubules prepared from rats of all ages and incubated with an antipeptide antibody specific for TR alpha1 (alpha1-403). Staining with an antipeptide antibody specific for TR beta1 (beta-62) was never present. Our data show that mRNAs coding for the functional TR alpha1, and also for the still undefined alpha2, are present in the testis of rats of all ages. T3-binding activity and immunohistochemical studies confirmed that the message is translated into proteins. The transcriptional activity clearly decreased from birth to adulthood, but it still remained significantly present. The presence of a TR alpha1 message indicates that the adult rat testis may be directly responsive to T3 and, therefore, suggests an action of TH on rat testis that is not only developmental, but also metabolic.

Ontogenetic pattern of thyroid hormone receptor expression in the human testis.

We studied the spatiotemporal distribution of thyroid hormone nuclear receptors (TRs) alpha1 and alpha2 and beta messenger RNA (mRNA) levels in normal human testicular tissue during development and in adulthood. Nonpathological specimens from five aborted fetuses (17 and 23 weeks of gestation, three and two cases, respectively) and from four patients undergoing orchiectomy (18 months old and 38-, 42-, and 52-yr-old, respectively) were analyzed by Northern blot, semiquantitative RT-PCR amplification using DNA sequences or specifically designed primers for the TR isoforms, and in situ hybridization. By using PCR amplification, we found that TRalpha1 and TRalpha2 are both expressed at different levels in fetal and adult testis. At all ages TRalpha2 is found at higher levels. Northern analysis showed hybridization signals corresponding to the expression of TRalpha2 and TRalpha in a ratio that increased from 2.6 at 17 weeks of gestation to 12.0 in adulthood. In fact, the expression of TRalpha1 dramatically decreased throughout development, being faintly detectable in the adult testis. Expression of TRbeta was not detected at any age studied. This finding was further confirmed by PCR, which did not amplify TRbeta either in fetal or in adult testis mRNAs. In situ hybridization studies showed the absence of TRbeta and that TRalpha1 and TRalpha2 colocalized in Sertoli cells of prepubertal testis, whereas germ and interstitial cells appeared devoid of TR mRNA signals. From these results it can be concluded that the human testis exclusively expresses TRalpha, which is localized in Sertoli cells, TRbeta being always undetectable. Fetal and prepubertal ages represent the period of maximal expression of TRalpha1 and TRalpha2. The alpha2/alpha1 ratio rises dramatically after development. These results confirm a critical window for the action of thyroid hormone in human testis, in the period of maximal expression of T3 binding isoform TRalpha1, and may account for the macroorchidism without virilization occurring when hyposecretion of thyroid hormones occurs before puberty.

Toxic adenoma and papillary thyroid carcinoma in a patient with Graves' disease.

A case of a very rare association of toxic adenoma and papillary carcinoma with Graves' disease is presented. A 34-year-old woman developed Graves' disease with mild ophthalmopathy. An ultrasound revealed diffuse thyroid enlargement with a hypoechoic pattern and a hypoechoic nodule with regular edges of 1.6 cm in diameter at the lower pole of the left lobe. A thyroid 131I scintiscan showed a diffuse and homogeneous 131I distribution. The 131I uptake (RAIU) was elevated. One year later, while still on a low dose of methimazole, the patient had a recurrence of hyperthyroidism following an iodine load from a contrast agent. A further thyroid ultrasound confirmed the previously described pattern but showed a new hypoechoic nodule of 1.1 cm with irregular edges in the left lobe. A thyroid 131I scintiscan this time demonstrated a hyperactive area localised in the larger nodule and a lower diffuse uptake of the remaining tissue. Because of the worsening of the symptoms of hyperthyroidism, the patient had a left lobectomy. On histological examination, the larger nodule was well encapsulated and showed the characteristics of a hyperfunctioning follicular adenoma. The smaller nodule was a typically unencapsulated papillary carcinoma. Several other microfoci of papillary carcinoma were also found in the adjacent tissue. Completion of thyroidectomy was therefore performed, followed by 131I ablative therapy and thyroxine suppressive treatment. This observation suggests that the chronic abnormal stimulation of the thyroid gland by the thyroid-stimulating antibody (TSAb) may facilitate the neoplastic transformation of the thyrocytes in individuals with a critical genetic background.

Three novel mutations at serine 314 in the thyroid hormone beta receptor differentially impair ligand binding in the syndrome of resistance to thyroid hormone.

The syndrome of resistance to thyroid hormone is associated with diverse mutations in the ligand-binding domain of the thyroid hormone beta receptor, localizing to three clusters around the hormone binding cavity. Here, we report three novel resistance to thyroid hormone mutations (S314C, S314F, and S314Y), due to different nucleotide substitutions in the same codon, occurring in six separate families. Functional characterization of these mutant receptors showed marked differences in their properties. S314F and S314Y receptor mutants exhibited significant transcriptional impairment in keeping with negligible ligand binding and were potent dominant negative inhibitors of wild-type receptor action. In contrast, the S314C mutant bound ligand with reduced affinity, such that its functional impairment and dominant negative activity manifest at low concentrations of thyroid hormone, but are more reversible at higher T3 concentrations. The degree of functional impairment of mutant receptors in vitro may correlate with the magnitude of thyroid dysfunction in vivo. Modelling these mutations using the crystal structure of thyroid hormone receptor beta shows why ligand binding is perturbed and why the phenylalanine/tyrosine mutations are more deleterious than cysteine.

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.

Thyroid hormone receptor (alpha) distribution in hamster and sheep brain: colocalization in gonadotropin-releasing hormone and other identified neurons.

Thyroid hormones appear to play an important role in the seasonal reproductive transitions of a number of mammalian and avian species. These seasonal transitions as well as the effects of thyroid hormones on the reproductive neuroendocrine axis are mediated by the GnRH system. How thyroid hormones affect the GnRH system is unclear. Double label immunocytochemistry was used to examine GnRH- and other neurotransmitter/neuropeptide-containing neurons for thyroid hormone receptor (alphaTHR) colocalization in two seasonal breeders, the golden hamster and the sheep. AlphaTHR was identified in hamster and sheep brain by Western blot analysis. Furthermore, alphaTHR immunoreactivity was widely distributed in brain and was colocalized in identified populations: GnRH neurons (hamster, 28%; sheep, 46%); dopaminergic neurons of the A14 (hypothalamic) and A16 (olfactory bulb) cell groups, but not in the hypothalamic A13 cell group; and neurophysin-immunoreactive neurons of the supraoptic and paraventricular nuclei. The finding of alphaTHR in GnRH and A14 dopamine neurons provides an anatomical substrate for direct thyroid hormone action on the reproductive neuroendocrine system of these two seasonally breeding species. It remains to be determined whether the GnRH gene itself or the gene of another constituent within the same GnRH neuron is responsive to thyroid hormones.

Identification and characterization of a novel de novo mutation (L346V) in the thyroid hormone receptor beta gene in a family with generalized thyroid hormone resistance.

We have investigated an Italian family with generalized resistance to thyroid hormone (RTH), consisting of two individuals with elevated serum thyroid hormones (TH) and a non-suppressed TSH, together with unaffected family members, for a mutation in the thyroid hormone receptor beta gene (hTR beta). We have identified a single nucleotide substitution (1321 CTT to GTT) corresponding to a leucine to valine substitution at codon 346 (L346V) in the predicted protein. The index case and her affected child are heterozygous for the receptor defect, with normal sequence in unaffected family members. Furthermore, both parents of the index case were unaffected, suggesting that the mutation had arisen de novo. When expressed in vitro, the L346V mutant receptor showed a marked reduction in its affinity for tri-iodothyronine (T3), impaired ligand-dependent transactivation and potent dominant negative activity. Its functional impairment could not be alleviated, even at supraphysiological concentrations of T3, suggesting that the mutation might interfere with the intrinsic ligand-dependent transactivation function (AF-2) located in the hormone binding domain of hTR beta. Finally, the presence of the L346V mutation in the son of the propositus, who died from complications associated with congenital heart disease, raises the possibility that RTH might have contributed to the pathogenesis or severity of the latter.

Long-term intracavernous self-injection with prostaglandin E1 for the treatment of erectile dysfunction.

Intracavernous self-injection with vasoactive drugs has become a widely accepted treatment for erectile dysfunctions (ED). Sixty-eight patients affected by ED have been followed during long-term (at least 1 year) treatment with intracavernous prostaglandin E1. Each patient underwent a penile dynamic ultrasound examination using a high-definition probe (13 MHz) before and during the treatment. The drop-out rate was 8.8%. An improvement in spontaneous erections was reported by 13% of patients. The onset of fibrotic nodules occurred in three (4.4%) patients. In one of the latter cases the occurrence of nodules could be related to the frequency and dose of the drug administered, whereas in the other two cases no such correlation could be hypothesized. These findings draw attention to this possible side-effect of long-term treatment with PGE1.

Three different thyroid hormone receptor isoforms are detected in a pure culture of ovine oligodendrocytes.

Thyroid hormones are important for the normal development of the central nervous system. In humans, the period around the end of the intrauterine life and the first few months of neonatal life is critically dependent on the presence of normal amounts of thyroid hormone. There are significant events occurring during this time; myelination is one. Myelin is synthesized by oligodendrocytes. A panel of site-specific polyclonal antibodies against alpha-1 thyroid hormone receptor (TR), alpha-2 variant TR, and beta-1 TR isoforms has been employed to investigate the presence of TR isoforms in a pure culture of ovine oligodendrocytes by the avidin-biotin peroxidase immunocytochemical method. Strong nuclear staining was obtained with all the anti-TR antibodies; no reaction products were detected in the cytoplasm or cellular processes. By contrast, an anti-myelin basic protein antibody gave strong cytoplasmic and process staining; no nuclear staining was seen. These latter results served to 1) confirm that the cells under study are oligodendrocytes; and 2) prove that the nuclear staining with anti-TR antibodies is specific. Preimmune sera were totally negative. Scatchard analysis of [125I] T3 binding by isolated oligodendrocyte nuclei demonstrated the existence of high-affinity--low-capacity T3 binding sites with a Ka of approximately 6 x 10(-9) M and a maximal binding capacity of approximately 20 fmol/100 micrograms of DNA. Our results demonstrate that differentiated oligodendrocytes express alpha-1 and alpha-2 variant and beta-1 isoforms of TR at the protein level and support the notion of a direct impact of thyroid hormones on oligodendrocytes in their regulation of myelin synthesis.

Antibodies against restricted sequences in human c-ErbA hinge domain recognize differentially natural mammalian alpha- or beta-type triiodothyronine receptors and interfere differently with hormone binding.

In our first report, rabbit antibodies directed to recombinant polypeptides of human alpha-type c-ErbA sequences recognized natural triiodothyronine (T3) receptors (TR) in adipocytes (mouse Ob 17 cell line) but not in liver (mouse, rat). Moreover, some of them, directed to the sequence 150-228, markedly interfered with hormone binding to adipocyte T3 receptors. We now raised antibodies against shorter synthetic peptides within this alpha-type 150-228 c-ErbA sequence, which encompasses part of the hinge (D) domain and N-terminus of the E domain (alpha-150-166 and alpha 172-191) and against a beta-type c-ErbA sequence (beta 204-220 aligned on alpha 150-166, and differing by eight amino acids). Our present antibodies, which bear the expected c-ErbA alpha- or beta-type specificity, immunoprecipitated the TR in nuclear extracts, with a different pattern between tissues: exclusive precipitation by anti-c-ErbA alpha antibodies in Ob 17 adipocytes; large but non-exclusive precipitation by anti-cErbA beta antibodies in rat or mouse liver, which also expresses some alpha-type TR. This pattern of discriminative immunoprecipitation, also obtained in parallel analysis using our previously described antibodies to other c-ErbA alpha or beta sequences (anti-alpha 144-162, anti-alpha 1 403-410 and anti-beta 62-82), roughly verifies results of c-erbA mRNA expression in these tissues. Slight differences appeared in the extent of alpha-type TR recognition by antibodies directed to alpha 172-191, whether TR were liganded or not to T3 before antibody addition. This evokes a different conformation of this region after hormone binding. Most interestingly, these anti-alpha 172-191 antibodies lowered the Ka for T3 and extensively dissociated the adipocyte T3-TR complexes; they interfered poorly with the binding of T3 in liver nuclear extracts. This strongly supports the concept that internal sequences in c-ErbA alpha, more precisely in a restricted C-terminal part of the D domain, are necessary for efficient T3 binding, which also need the C-terminal part of domain E.

Evaluation of the ontogeny of thyroid hormone receptor isotypes in rat brain and liver using an immunohistochemical technique.

We performed an immunohistochemical study on rat brain and liver during fetal and neonatal life using rabbit antipeptide polyclonal antibodies able to recognize each thyroid hormone receptor (TR) isoform. The expression of TR alpha-1, alpha-2 and beta-1 proteins from 14 days of gestation to 21 days after birth was evaluated. Frozen tissues from 14 (F14), 17 (F17) and 21 (F21)-day-old fetuses and from 5 (N5), 16 (N16) and 21 (N21)-day old newborn rats were stained with anti-TR antibodies using an avidin-biotin-peroxidase system. The antipeptide antibodies utilized in the present study were characterized previously: alpha-144 antibody recognizes both TR alpha-1 and alpha-2; alpha-2-431 antibody is specific for TR variant alpha-2, and beta-62 antibody specifically reacts with the TR beta-1 isoform. The expression of TR alpha-1 was deduced by comparing the staining obtained with alpha-144 and alpha-2-431 antibodies. We demonstrated that each TR isoform is expressed in rat brain from 14 days of gestation and that the alpha isoform was predominant in the early stage. The three TR isoforms were expressed in both neural cell nuclei and in glial cell nuclei. As far as the liver is concerned, at F14 the expression of TR isoforms was weaker in hepatocytes when, on the contrary, TR alpha was clearly detected in hematopoietic cells. The expression of TRs in hepatocytes becomes evident later. The data that we obtained, although not quantitative, emphasize the presence of each TR isoform in brain and liver from 14 days of fetal rat life.

Region-specific anti-thyroid hormone receptor (TR) antibodies detect changes in TR structure due to ligand-binding and dimerization.

There are multiple factors that potentially can induce structural changes in DNA-bound thyroid hormone receptors (TRs) including protein-protein interactions, ligand-binding to TRs, and the thyroid hormone response element (TRE) sequence. We used a battery of anti-TR antibodies that recognize the amino-terminal, hinge, or carboxy-terminal regions of TRs to study changes in the epitope regions of in vitro translated TRs in electrophoretic mobility shift assays. We found that the carboxy-terminal and hinge region antibodies recognized TR homodimers but not TR/T3-receptor auxiliary protein or TR/retinoid X receptor heterodimers. The amino-terminal antibodies detected conformational changes due to ligand binding. In contrast, each antibody recognized TR complexes bound to TREs containing half-sites arranged in three different orientations. These results suggest that dimerization with nuclear proteins and ligand-binding, rather than the orientation of TRE half-sites, cause changes in several TR subregions.

Antipeptide polyclonal antibodies specifically recognize each human thyroid hormone receptor isoform.

We characterized four antipeptide polyclonal antibodies (abs) able to specifically recognize each thyroid hormone receptor (TR) isoform. The abs immunoprecipitated both the in vitro synthesized receptor and the receptor expressed in E. coli and their specificity was confirmed by competition studies and immunohistochemistry. Ab activity measured by enzyme-linked immunosorbent assay decreased after preabsorption of each ab with the immunizing peptide or the specific receptor protein expressed in E. coli. No specific activity was detectable in enzyme-linked immunosorbent assay, no nuclear staining was observed after affinity column immunoabsorption, and the specific bands obtained in Western blot analysis disappeared after preabsorption with the specific TR isoform expressed in E. coli. By immunohistochemical studies we detected coordinate expression of each receptor isoform in most tissues examined. However, in heart and muscle, the beta-isoform is expressed at a very low level compared to the alpha-isoform in spite of the significant TR beta mRNA levels previously demonstrated by Northern blot analysis. We also demonstrated a different pattern of distribution of alpha- and beta-isoforms in rat testis. In this tissue the TR alpha is significantly expressed in spermatogonia nuclei, but in spermatids the beta-isoform is predominant, and only the TR beta is detectable in mature spermatozoa.