Variable transcriptional activity and ligand binding of mutant beta 1 3,5,3'-triiodothyronine receptors from four families with generalized resistance to thyroid hormone.

Mutations in the gene encoding the human beta 1 T3 receptor (hTR beta 1) have been associated with generalized resistance to thyroid hormone (GRTH). We measured the T3-binding affinity and transcriptional regulatory capacity of the mutant hTR beta 1 from four unrelated kindreds with GRTH. These mutations are contained in different functional regions of the ligand-binding domain. The T3 affinity of the mutant receptors correlated well with the degree of impairment of their trans-activating function in a transient cotransfection system in HeLa cells; two mutant receptors with undetectable ligand affinity showed no transcriptional activity, whereas the two other mutants characterized by a 2- and 5-fold reduction in T3 affinity required 5- and 15-fold higher T3 concentrations for half-maximal activity in the cotransfection assay, respectively. All of the mutant hTR beta 1s were able to inhibit the function of transfected normal hTR beta 1 and endogenous retinoic acid receptor in activating a palindromic positive T3 response element (TRE). In the partially functional mutants this dominant negative effect could be completely reversed by increased T3 concentrations. The dominant negative potency did not depend on the type of TRE used; mutant hTR beta 1s were able to inhibit normal receptor function to the same degree on a dimer-permissive palindromic TRE as on a nondimer-permissive inverted repeat of two identical half-sites separated by five spacer bases. However, the dominant negative potency was dependent on the absolute amount of receptor expression vector transfected. The expression of normal and mutant hTR beta 1 was assessed by immunocytochemistry. The hTR beta 1 protein levels in HeLa cells paralleled the amount of transfected expression vector. Moreover, all the mutant receptors were properly expressed in the nuclei of the transfected cells. These data suggest that different mutations in the ligand-binding domain of the human hTR beta 1 result in a variable degree of functional impairment, which may partially explain the phenotypic differences between kindreds with GRTH. Our findings suggest that competition for binding to the TRE and possibly the binding of limiting accessory factors may be more important in mediating the dominant negative effect than the formation of normal/mutant T3 receptor dimers.

Growth hormone (GH) responses to the hexapeptide GH-releasing peptide and GH-releasing hormone (GHRH) in the cynomolgus macaque: evidence for non-GHRH-mediated responses.

GH-releasing peptide (GHRP; His-D-Trp-Ala-Trp-D-Phe-Lys-NH2), a hexapeptide derived from enkephalin, has been shown to have GH-releasing activity in man and several animal species. To characterize the GHRP dose-response curve and compare it with that of GH-releasing hormone [GHRH-(1-44)NH2], six unanesthetized young adult cynomolgus macaques were tested with a range of iv doses of GHRP or GHRH in random order. Animals were fitted with vests and tethers. Blood samples were obtained before and at 15-min intervals after the administration of drugs. Doses ranged from 0.03-3 mg/kg for GHRP and from 1-30 micrograms/kg for GHRH. The dose-response curves for the two peptides were not parallel. GHRP had lower potency, but evoked a much higher peak GH response than GHRH (greater than 55 vs. 12 micrograms/L). Because one of the proposed mechanisms of action of GHRP is the inhibition of somatostatin (SS), we tested the effects of propranolol, which inhibits SS, on the GH responses to GHRH and GHRP. Propranolol was given at a dose of 14 micrograms/kg, iv, 10 min before the injection of saline, GHRH (10 micrograms/kg), or GHRP (1 mg/kg). GH responses to propranolol alone did not differ from those to placebo (peak, 6 +/- 2 vs. 8 +/- 2 micrograms/L). However, propranolol pretreatment doubled the GH responses to both GHRH and GHRP compared with those to GHRH or GHRP alone 28 +/- 5 micrograms/L vs. 14 +/- 5 (P less than 0.05) and 54 +/- 2 vs. 25 +/- 6 micrograms/L (P less than 0.001), respectively]. These results show that GHRP causes a potent dose-dependent release of GH in this primate species. Since GHRP can produce a greater maximal GH response than GHRH, mechanisms other than release of endogenous GHRH must be involved.

An arginine to histidine mutation in codon 315 of the c-erbA beta thyroid hormone receptor in a kindred with generalized resistance to thyroid hormones results in a receptor with significant 3,5,3'-triiodothyronine binding activity.

Generalized resistance to thyroid hormones results from diverse mutations in the T3-binding domain of the c-erbA beta thyroid hormone receptor, and different kindreds have variable phenotypes. However, the T3-binding affinities of these mutant receptors studied in vitro have all been severely reduced compared to wild type. We report here a new kindred, CL, with a mutation further upstream than previously reported, a guanine to adenine base substitution at nucleotide 1244 in codon 315 changing an arginine to histidine. This base substitution was the only one found in codons 90-456 of genomic sequence and was formally shown to be a mutation by screening 51 random individuals. The kindred CL receptor complementary DNA was recreated, and the mutant receptor synthesized with rabbit reticulocyte lysate had a T3-binding affinity of 2.4 +/- 0.9 x 10(10) M-1 compared to the wild-type human placental receptor affinity of 5.2 +/- 1.6 x 10(10) M-1. Affected members of this kindred appeared clinically to have a relatively mild degree of resistance with mean total thyroxine of only 192 +/- 24 nmol/L and inappropriately normal TSH levels. Kindred CL is an example of mild generalized resistance to thyroid hormones correlated with a mutation in the beta-receptor that resulted in only a modest deficiency in T3-binding activity.

[Analysis on the health status of residents from Diseases Surveillance Points in Gansu Province].

We have surveyed a population size of 6633315 from Diseases Surveillance Points (DSP) system in Gansu province for the last eleven years. The annual birth rate was 18.20% with an annual standard mortality rate 545.80/10(5). The annual standard mortality for male and female were 607.53/10(5) and 483.29/10(5) respectively. The major causes of death were Respiratory system diseases, Cardiovascular diseases, Neoplasms, Injuries, Digestive system diseases, Pediatric diseases, Infectious diseases in sequence. In eleven years, there seemed to be a rising trend in the mortalities of following diseases as: Cerebrovascular diseases, Ischemic heart diseases, Rheumatic fever and heart disease, Lung Cancer, Liver Cancer, Cancer of the Esophagus, Intestinal cancer, Cervical cancer, Injury, Congenital abnomalities, to different degrees. However, an obvious descending trend on the morbidity and mortality of infectious diseases was moticed. The average life expectancy was 71.05 years in DSP, with male 69.57 years, and female 72.72 years. Diseases with higher PYLL were Injuries, Neoplasms, Respiratory system diseases and the like. Data suggested not only the prevention andcontrol of infectious diseases, but also the surveillance of injuries and the prevention and control of chronic diseases should be strengthened.

Effects of inhibition of norepinephrine synthesis on spontaneous and growth hormone-releasing hormone-induced GH secretion in cynomolgus macaques: evidence for increased hypothalamic somatostatin tone.

To define the role of noradrenergic regulation of growth hormone (GH) secretion in a primate species, spontaneous and GH-releasing hormone (GHRH) stimulated GH secretion was studied in 6 chronically catheterized adult male cynomolgus monkeys before and after inhibition of norepinephrine synthesis. Blood samples were obtained at 15-min intervals during an 8-hour period to characterize the pattern of GH secretion, and the GH response to GHRH, 10 micrograms/kg i.v., was determined. These measurements were repeated 2 weeks later, 2 h after the intravenous administration of 12.5 mg/kg of the dopamine-beta-hydroxylase inhibitor diethyldithiocarbamate (DDTC), which has been shown to be an effective norepinephrine synthesis inhibitor in the rat. Spontaneous and stimulated GH secretory patterns before and after DDTC administration were compared. Both the frequency and the amplitude of spontaneous GH pulses were markedly reduced by DDTC [3.8 +/- 0.4 before vs. 1.8 +/- 0.4 peaks/8 h after DDTC (p less than 0.005) and 5.5 +/- 0.7 vs. 2.0 +/- 0.6 ng/ml (p less than 0.01)]. Areas under the curve were also reduced by DDTC treatment [10.8 +/- 1.0 vs. 5.7 +/- 1.1 ng.h/ml (p less than 0.01)], and DDTC administration diminished the peak GH responses to GHRH [12 +/- 6 vs. 4 +/- 2 ng/ml (p less than 0.05)]. These results are consistent with the belief that DDTC is a potent inhibitor of spontaneous and GHRH-induced GH secretion. The action of DDTC could be mediated by a reduction in GHRH due to reduced norepinephrine synthesis, by an increase in somatostatin release through a dopaminergic stimulus, or by a direct dopaminergic effect on somatotrophs.

Generalized thyroid hormone resistance: identification of an arginine to cystine mutation in codon 315 of the c-erb A beta thyroid hormone receptor.

The present report studies a large kindred (WR) with generalized thyroid hormone resistance that has varying degrees of neuropsychological dysfunction, hyperactivity, poor attention span, decreased IQ and/or abnormalities in spatial perception. In this kindred, there has been found tight linkage of the syndrome with the c-erb A beta gene. The present study was performed to identify the presence of a possible gene mutation as a cause for this syndrome. DNA from peripheral leukocytes was isolated from 15 unaffected and 8 affected individuals from the kindred. Primers encompassing exons 9 (nucleotides 1171-1429) and 10 (nucleotides 1430-1698) were synthesized and used in PCR reactions to amplify these exons. Direct sequencing revealed a consistent substitution in each affected subject, but in none of the unaffected individuals, of a C to T change in one allele from nucleotide 1243, resulting in an arg to cys change in codon 315. The mutant and wild-type human beta 1 receptors were prepared and their translated proteins were analyzed for T3 binding. The WR T3 receptor from affected patients had reduced T3 binding affinity, with values approximately 2.5 x 10(10) M-1 compared to about 5 x 10(10) M-1 in normals. In summary, we have: i) identified a consistent and reproducible mutation of a C to T change in nucleotide 1243 in each of the affected but in none of the unaffected individuals of a large well characterized kindred with generalized thyroid hormone resistance; and ii) noted that the WR allele causes an approximate 50% decrease in the T3 binding affinity.(ABSTRACT TRUNCATED AT 250 WORDS)