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Biomedical subjects

S Refetoff

Publications and source records attributed to S Refetoff.

At least 19 recordsLinked to original sources
PubMed1996

Functional characteristics of a variant thyrotropin receptor.

A claim has been made that a variant of the human thyrotropin receptor in which Pro52 is replaced by Thr ([Thr52]thyrotropin receptor) is associated with autoimmune thyroid diseases and displays increased responsiveness to thyrotropin. We have analysed the functional characteristics of this variant receptor. Equivalent numbers of of the wild type and of the variant thyrotropin receptor, measured both by 125I-thyrotropin binding and by flow cytofluorimetry, were transiently expressed in COS-7 cells. Under these conditions, the two receptors showed the same degree of constitutive activity for the cAMP pathway, the same affinity for bovine thyrotropin, and a virtually identical responsiveness to bovine thyrotropin for activation of both the cAMP and inositol-phosphate regulatory pathways. Our results show that the [Thr52]thyrotropin receptor variant of the human thyrotropin receptor, which is present in the 12% of the population, does not affect receptor function and represents most likely a simple polymorphism.

Alleles↗
PubMed1996

Expression of thyroid peroxidase in EBV-transformed B cell lines using adenovirus.

Thyroid peroxidase (TPO) is thought to be one of the pathological antigens in Hashimoto's thyroiditis. Epstein-Barr virus-transformed B cell lines (EBVL) can be used for antigen-presenting cells and target cells of cytotoxic T cells. To develop a model for endogenous TPO presentation in EBVL, we constructed a recombinant adenovirus carrying the TPO gene driven by the cytomegalovirus promoter (Ad5-TPO). Enzymatically active human TPO could be expressed in COS cells using Ad5-TPO. the peroxidase activity of the membrane extract from Ad5-TPO-infected COS cells was approximately 6 times higher than that from stably transfected TPO expressing CHO cells. TPO protein expression in the EBVL was analyzed by Western blotting technique. A band at approximately 110 kDa characteristic of hTPO was detected in EBVL infected with Ad5-TPO. hTPO expression in EBVL induced with adenovirus should facilitate understanding of T cell immunity to TPO in patients with autoimmune thyroid diseases.

Adenoviridae↗
PubMed1996

A new family with hyperthyroxinemia caused by transthyretin Val109 misdiagnosed as thyrotoxicosis and resistance to thyroid hormone--a clinical research center study.

Serum transthyretin (TTR) is a protein of liver origin that under normal conditions transports approximately 20% T4. Missense mutations of the TTR gene produce familial amyloidotic polyneuropathy and rarely, euthyroid hyperthyroxinemia (EHT). Of the 3 TTR variants so far identified with increased affinity for T4, Ser6, Thr109, and Met119, only TTR-Thr109 has high enough affinity for T4 to produce consistent hyperthyroxinemia in the heterozygous individuals. Because the mutation GCC-->ACC in codon 109 results in the loss of one Bso FI site in exon 4 of the TTR gene, the use of this enzyme was suggested to screen for TR-Thr109 in subjects with EHT. We investigated a family with dominantly inherited EHT, in which two of eight affected members received ablative thyroid treatment for presumed thyrotoxicosis, and one was misdiagnosed as having resistance to thyroid hormone. All affected individuals had serum reverse T3 concentrations above normal and average T4 50% above the mean of unaffected relatives. Total T3 and TSH levels were within the normal range. Although loss of the Bso FI site in one allele of the two TTR suggested the presence of Thr109, gene sequencing revealed a different mutation in the same codon (GCC-->GTC) producing TTR-Val109. T4-binding to TTR-Val109 was compared to that of the normal TTR-Ala109 and the formerly identified variant TTR-Thr109. Association constants were 1.3, 9.5, and 13.6 X 10(7) M-1 for TTR-Ala109, Val109, and Thr109, respectively. Thus, for equally expressed mutant and normal allele and 20% of serum T4 bound to TTR, the calculated mean serum T4 concentration of heterozygotes for TTR-Val109 should be 50% above the normal mean; the observed value being 55%. These results are in agreement with the observations based on the crystallographic structure of TTR-Thr109 indicating that the extra atoms in Val as in Thr, which are absent in the Ala of the wild type TTR, widen the ligand binding site.

Adolescent↗
PubMed1995

Molecular and structural characterization of the heat-resistant thyroxine-binding globulin-Chicago.

Thyroxine-binding globulin (TBG) is the main transport protein for thyroxine (T4) in blood. It shares considerable sequence homology with alpha 1-antitrypsin (AT) and other members of the serine proteinase inhibitor (serpin) superfamily of proteins. The crystallographic structure of AT has been determined and was found to represent the archetype of the serpins. This model has been used for structure-function correlations of TBG. Sequence analysis of the heat-resistant variant TBG-Chicago (TBG-CH) revealed a substitution of the normal tyrosine 309 with phenylalanine. For further analysis, vectors containing the coding regions of normal TBG (TBG-N) and TBG-CH were constructed, transcribed in vitro, and expressed in Xenopus oocytes. Both TBGs were secreted into the culture medium and could not be distinguished by gel electrophoresis. Scatchard analysis of T4 binding to TBG-N and -CH revealed no significant differences in binding affinity. The rate of heat denaturation of TBGs was determined by measurement of residual T4 binding capacity after incubation at 60 degrees C for various periods of time. The half-life values of denaturation of TBG-N and -CH were 7 and 132 min, respectively. The tyrosine 309 to phenylalanine substitution of TBG-CH involves a highly conserved phenylalanine residue of the serpins. The respective phenylalanine 312 of AT ties the alpha-helix hI1 to the molecule, thus stabilizing the tertiary structure. A substitution with tyrosine would disrupt this interaction. Accordingly, stabilization of the TBG molecule by replacement of tyrosine with phenylalanine in position 309 causes the increased heat stability of TBG-CH.

Amino Acid Sequence↗
PubMed1995

Molecular cloning of an orphan G-protein-coupled receptor that constitutively activates adenylate cyclase.

A human gene encoding an orphan G-protein-coupled receptor named ACCA (adenylate cyclase constitutive activator) was isolated from a genomic library using as a probe a DNA fragment obtained by low-stringency PCR. Human ACCA (hACCA) is a protein of 330 amino acids that exhibits all the structural hallmarks of the main family of G-protein-coupled receptors. Expression of hACCA resulted in a dramatic stimulation of adenylate cyclase, similar in amplitude to that obtained with other Gs-coupled receptors fully activated by their respective ligands. This stimulation was obtained in a large variety of stable cell lines derived from various organs, and originating from different mammalian species. hACCA was found to be the human homologue of a recently reported mouse orphan receptor (GPCR21). The mouse ACCA (mACCA) was therefore recloned by PCR, and expression of mACCA in Cos-7 cells demonstrated that the mouse receptor behaved similarly as a constitutive activator of adenylate cyclase. It is not known presently whether the stimulation of adenylate cyclase is the result of a true constitutive activity of the receptor or, alternatively, is the consequence of a permanent stimulation by a ubiquitous ligand. The tissue distribution of mACCA was determined by RNase protection assay. Abundant transcripts were found in the brain, whereas lower amounts were detected in testis, ovary and eye. Various hypotheses concerning the constitutive activity of ACCA and their potential biological significance are discussed.

Adenylyl Cyclases↗
PubMed1995

Sleep deprivation in the rat: XIX. Effects of thyroxine administration.

Chronic total sleep deprivation (TSD) in the rat produces an initial elevation and then declining body temperatures, increasing metabolic rate and eventual death. Because TSD rats will engage in warming behavior, one hypothesis is that the metabolic increase is an unsuccessful attempt at warming to combat a lethal hypothermia. However, TSD rats also undergo weight loss and progressive deterioration of skin and fur, suggesting TSD-induced pathological catabolic activity, possibly secondary to increased metabolic rate, that could be lethal. To evaluate these alternatives, the metabolic rate of rats was increased by thyroxine (T4) treatment while subjecting them to TSD. Compared to TSD rats not given T4, they had higher metabolic rates, higher body temperatures and reduced warming behavior, but their survival period was 37% shorter. Thus, it is unlikely that hypothermia is the cause of death in TSD rats. Weight and appearance declined more rapidly in T4-treated rats, but at the same proportions of survival time, skin pathology and decline in appearance were less evident in T4-treated rats than in TSD rats not given T4. Thus, there is some doubt whether a general pathological catabolic process is the cause of death. It is also possible that a specific morbid process normally reversed by sleep was accelerated by T4 administration.

Animals↗
PubMed1995

Effects of aging on glucose regulation during wakefulness and sleep.

Glucose intolerance, reduced sleep efficiency, and disturbed circadian rhythmicity occur in aging. In normal young subjects, glucose regulation is modulated by sleep and circadian rhythmicity. To examine age-related alterations in the temporal pattern of glucose tolerance and insulin secretion, eight modestly overweight healthy older men, eight weight-matched young men, and six young lean men were studied during constant glucose infusion for 53 h. Levels of glucose, insulin, C-peptide, and growth hormone (GH) were measured every 20 min. Rates of insulin and GH secretion were calculated by deconvolution. In older volunteers, sleep ws shallow and more fragmented than in young subjects but was nevertheless associated with robust glucose elevations. However, postsleep increases of insulin secretion were markedly dampened. During wakefulness, the normal morning-to-evening increase in glucose was preserved in the elderly, but insulin secretion failed to increase proportionately. Thus decreased glucose tolerance in aging is associated with insulin resistance and also with a relative insensitivity of the beta-cell to the modulation of glucose regulation by sleep and circadian rhythmicity.

Adult↗
PubMed1995

Studies on the repression of basal transcription (silencing) by artificial and natural human thyroid hormone receptor-beta mutants.

Thyroid hormone receptors (TRs) are ligand-dependent transcription factors that regulate target gene transcription. Interestingly, in the absence of ligand, TRs also can repress basal transcription of positively regulated target genes, suggesting that unliganded TR may have a distinct role in gene regulation. In this paper, DNA binding, truncation, and natural human TR beta mutants were used in cotransfection and electrophoretic mobility shift assays to study various aspects of TR-mediated basal repression. Presently, little is known about the role(s) of natural human TR beta mutants on basal repression. These results show that: 1) TR binding to DNA likely is required for basal repression; 2) the amino-terminal region of TR is not required for basal repression; 3) TR homodimer binding is not absolutely required for basal repression, as TR mutants that selectively form TR-retinoid X receptor heterodimers can mediate basal repression; and 4) TR mutants with poor T3-binding affinity likely have constitutive basal repression, even in the presence of ligand. These findings provide new insight on the mechanism of basal repression by unliganded TRs.

Base Sequence↗
PubMed1995

Linkage of familial dysalbuminemic hyperthyroxinemia to the albumin gene in a large Amish kindred.

Familial dysalbuminemic hyperthyroxinemia (FDH) is the most common cause of inherited euthyroid hyperthyroxinemia in Caucasians. Transmitted as an autosomal dominant trait, it is always associated with high serum total T4 (TT4) and more rarely with elevated total T3 (TT3) and/or rT3 (TrT3) concentrations. Free T4, by dialysis, and TSH levels are normal, suggesting the presence of a T4-binding protein abnormality. The abnormal serum T4 carrier shares some physical and immunological properties with albumin, suggesting that it may be albumin itself. Here we show linkage between FDH and the albumin gene in a large Amish family of Swiss descent, using as markers a SacI polymorphism in the coding sequence of the albumin gene and the group-specific component (Gc) gene, located less than 1 centimorgan from the albumin gene. Blood samples were obtained from 160 members of this kindred, and 22 had FDH identified by the pattern of T4 binding to serum proteins separated by isoelectric focusing. Serum TT4 values were above the normal range in all subjects expressing the FDH phenotype, and TrT3 levels were above the normal range in only one half. TT4 concentrations correlated positively with TrT3 and TT3. All TT3 values were, however, within the normal range. Free T4 and TSH levels were normal, confirming the euthyroid state in these subjects. FDH was associated with the albumin SacI(+)/Gc 1S haplotype, yielding a LOD (logarithm of the odds ratio) score of 5.53, with a recombination frequency of 0. These data provide strong support that a variant albumin is the cause of FDH in this kindred.

Base Sequence↗
PubMed1995

Do clinical manifestations of resistance to thyroid hormone correlate with the functional alteration of the corresponding mutant thyroid hormone-beta receptors?

Resistance to thyroid hormone (RTH), a syndrome characterized by variable tissue hyposensitivity to thyroid hormone, is linked to mutations in the thyroid hormone receptor-beta (TR beta) gene. The purpose of this study was to determine whether the clinical phenotypes of RTH can be translated in terms of functional impairment of the corresponding mutant TR beta. Data from 124 subjects with RTH representing 18 different mutant TR beta s, showed that serum free T4 levels correlated with the degree of T3-binding impairment of the corresponding TR beta in 12 of these mutant TR beta s (group I), but not in the remaining 6 (group II). In subjects from both groups studied in detail by the administration of incremental doses of T3, the degree of thyrotroph resistance to T3 correlated with the magnitude of endogenous free T4 elevation at baseline, but did not parallel the resistance of peripheral tissues. In transfection studies, all group I mutant TR beta s inhibited positive transactivation by the wild type TR beta s to a similar degree in the presence of 1 nmol/L T3, whereas group II mutant TR beta s exerted a weaker inhibition that was not related to their T3-dependent trans-activation when tested alone. Similar results were obtained with negatively regulated reporter genes. It is concluded that the clinical severity of RTH, determined by thyrotroph resistance, can be predicted from the degree of T3 binding impairment and dominant negative potency of mutant TR beta s, but the degree of peripheral tissue resistance and related clinical manifestations is limited by putative genetic or environmental factors that modulate the effect of thyroid hormone.

Adolescent↗
PubMed1995

Thyroid function tests and characterization of thyroxine-binding globulin in the carbohydrate-deficient glycoprotein syndrome type I.

Carbohydrate-deficient glycoprotein (CDG) syndrome is a newly recognized hereditary disorder that presents with psychomotor retardation, cerebellar ataxia, peripheral sensorimotor neuropathy, and, variably, skeletal abnormalities, lipodystrophy, and retinitis pigmentosa. These abnormalities appear to be produced by a defect that causes reduced carbohydrate content in glycoproteins. We studied seven patients with CDG type I belonging to five unrelated families. The concentration of serum TBG, a glycoprotein of hepatic origin, was measured by RIA and T4 saturation and was found to be below the normal range in three of the seven patients and normal in four of them. More than half of the total serum TBG had reduced sialic acid content and localized on isoelectric focusing (IEF) as two prominent bands cathodal to the three major bands of normal TBG. The latter two bands are responsible for the characteristic IEF pattern or CDG syndrome. TBG in patients with CDG had immunoreactivity indistinguishable from that of normal TBG and had normal affinity for T4, T3, and rT3. Serum total T4, T3, and rT3 were below the normal range in seven, five, and seven patients, respectively. The free T4 index was also below normal in four patients, but the free T4 concentration, measured by equilibrium dialysis at low dilution, and serum TSH were in the midnormal range. The serum total T4 and rT3 levels were disproportionately reduced relative to the serum TBG concentration and compared to the concentrations of these iodothyronines in matched subjects with inherited partial TBG deficiency. Chronic illness cannot explain these changes, because, contrary to patients with nonthyroidal illness, those with CDG had significantly higher serum total T3/T4 and lower rT3/T4 ratios. It is concluded that IEF of TBG is a rapid and simple method for the diagnosis of CDG type I and that the abnormal pattern can be detected as early as 5 days postpartum. Patients with CDG are chemically euthyroid, and it is postulated that the reduction in serum iodothyronine concentrations beyond that explained on the basis of low TBG levels may be due to the interference with binding to TBG by an unidentified substance.

Adolescent↗
PubMed1995

High prevalence of thyroxine-binding globulin deficiency among Bedouin infants in southern Israel.

A high prevalence of thyroxine-binding globulin deficiency was found among Bedouin newborns in the Negev area (southern Israel) during a study lasting 2 years. This prevalence is significantly higher than those reported in other populations. Moreover, thyroxine-binding globulin deficiency was found to be more common than congenital hypothyroidism among the Bedouin. The results of thyroxine-binding globulin analysis in four patients may suggest the coexistence of at least two different mutations among the Bedouin population.

Ethnicity↗
PubMed1995

Neurocognitive characteristics of individuals with resistance to thyroid hormone: comparisons with individuals with attention-deficit hyperactivity disorder.

Previous studies have demonstrated an association between resistance to thyroid hormone (RTH) and attention-deficit hyperactivity disorder (ADHD). To determine if the neurocognitive characteristics in individuals with RTH are similar to those observed in ADHD, 12 children with RTH from 7 families were matched to 12 children with ADHD without RTH. Subjects were administered standardized intellectual, developmental, and school achievement tests. Parent and teacher ratings of children's hyperactivity and attention were similar for both groups, as were measures of attention, impulsivity, and verbal IQ. Children with RTH displayed lower nonverbal intelligence (performance IQ = 85) and academic achievement (> 1-2 SD below the mean) when compared with those with ADHD only (performance IQ = 99; achievement within 2 SD). Although children with RTH have behavioral characteristics similar to those with ADHD, their significantly weaker abilities of perceptual-organization and lower school achievement suggest a more severe neurobehavioral impairment than ADHD.

Adolescent↗
PubMed1994

Expression of a thyroid hormone-responsive recombinant gene introduced into adult mice livers by replication-defective adenovirus can be regulated by endogenous thyroid hormone receptor.

We constructed a recombinant adenovirus carrying the firefly luciferase gene driven by the thymidine kinase promoter and controlled by the palindromic thyroid hormone/retinoic acid-responsive element. The same adenovirus vector without the hormone-responsive element was used as control. Regulation of the luciferase gene expression was tested in pituitary-derived GH cells and hepatoma-derived HepG2 cells infected with the recombinant adenoviruses. Administration of triiodothyronine to GH cells and all-trans-retinoic acid to HepG2 cells resulted in 8.0 +/- 0.3-fold and 4.6 +/- 0.5-fold induction of luciferase activity, respectively. No significant increase was observed with the control adenovirus. Hormonal regulation was also examined in adult mice. Mice depleted of thyroid hormone were injected intravenously with the recombinant adenoviruses and given 4 times the replacement dose of triiodothyronine or vehicle only for 4 days. Hormone administration resulted in 4.2-fold increase of luciferase activity in liver homogenates. No significant effect was observed in animals injected with the control adenovirus. This recombinant adenovirus provides a new experimental system in the study of thyroid hormone and retinoic acid action and offers the potential to regulate by physiological means the expression of genes transferred for the purpose of therapy.

Adenoviridae↗
PubMed1994

An identical missense mutation in the albumin gene results in familial dysalbuminemic hyperthyroxinemia in 8 unrelated families.

Familial dysalbuminemic hyperthyroxinemia (FDH) is the most common form of inherited increase of serum thyroxine in Caucasians. It is the result of increased thyroxine-binding to serum proteins and is inherited as a dominant trait. The entire coding region of the albumin gene of a subject with FDH was sequenced. A single nucleotide substitution, G to A transition in codon 218, was found in one of the two alleles, resulting in the replacement of the normal Arg with His. This mutation was found in 9 affected family members but not in 8 unaffected relatives and 18 unrelated normal individuals. The same missense mutation was found in 12 other subjects with FDH belonging to 7 unrelated families. In every individual with FDH, the mutation was associated with the Sac I+ polymorphism in the albumin gene, strongly suggesting a founder effect.

Arginine↗