Molecular basis of androgen insensitivity.
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Amino Acid Sequence↗
Biomedical subjects
T R Brown
Publications and source records attributed to T R Brown.
Endocrine evaluation of adults with mild hypospadias.
The goal of our study was to determine the frequency of endocrine abnormalities in sexually mature men with mild hypospadias. For this purpose we evaluated 16 men 22 to 52 years old with mild hypospadias. All patients had a urethral meatus distal to the penoscrotal junction. Serum concentrations of luteinizing hormone, follicle-stimulating hormone and testosterone were measured in all subjects. Dihydrotestosterone and testosterone precursors also were determined in men who had abnormally elevated levels of luteinizing hormone. The level of serum luteinizing hormone in patients was significantly higher than that of normal men, whereas the levels of serum follicle-stimulating hormone and testosterone were similar to those of normal subjects. Of the 16 men with mild hypospadias 3 had abnormally elevated levels of gonadotropins: 2 had primary gonadal dysfunction (elevated luteinizing hormone and follicle-stimulating hormone with low or normal testosterone) and 1 had hormonal evidence of partial androgen insensitivity (elevated luteinizing hormone, normal follicle-stimulating hormone with high normal testosterone and dihydrotestosterone). These patients are rather unique, since they did not have other compromising factors to account for the gonadal failure. In summary, our data demonstrated the presence of endocrine dysfunction in men with mild hypospadias.
Adult↗
Amiodarone-induced changes in lipid metabolism.
Hypothyroidism is a major cause of secondary hypercholesterolemia. Amiodarone treatment alters both the levels of serum lipids and thyroid hormones. We investigated whether the amiodarone-induced changes in lipid metabolism are related to the changes in thyroid hormone levels. Eighteen patients received amiodarone (31 +/- 3 g cumulative dose) for six weeks. Serum triglyceride, total-cholesterol, high density lipoprotein-cholesterol and its subfractions, apolipoproteins B and AI, and plasma post-heparin lipoprotein lipase and hepatic triglyceride lipase activities were determined. Amiodarone treatment caused significant increases in serum total-cholesterol (baseline 4.4 +/- 0.21 (SE), 6 weeks 5.12 +/- 0.26 mmol/l, P less than 0.01), in low density lipoprotein cholesterol (baseline 2.61 +/- 0.26, 6 weeks 3.36 +/- 0.21 mmol/l, P less than 0.05) and in apolipoprotein B (baseline 1.95 +/- 0.15, 6 weeks 2.26 +/- 0.13 mmol/l, P less than 0.01) concentrations. Serum high density lipoprotein and its subfractions, or apolipoprotein AI levels did not change. Plasma post-heparin lipoprotein lipase activity increased (baseline 137 +/- 21, 6 weeks 168 +/- 21 U/ml, P less than 0.01) while hepatic triglyceride lipase did not change. Amiodarone also caused an increase in serum thyroxine (baseline 110 +/- 8, 6 weeks 136 +/- 6 mmol/l, P less than 0.05), although values remained in euthyroid range. In summary, amiodarone therapy increased the concentrations of atherogenic lipoproteins in the serum similar to that seen in hypothyroidism. On the other hand the effect of amiodarone on lipoprotein lipase was opposite to that seen in hypothyroidism. Therefore, amiodarone-induced changes in lipid metabolism cannot be explained solely on the basis of the changes in circulating thyroid hormone levels.
Aged↗
Chemical shift imaging of human brain: axial, sagittal, and coronal P-31 metabolite images.
Multivoxel magnetic resonance (MR) spectroscopy and novel data analysis techniques were developed to obtain high-quality phosphorus-31 metabolite images from the human brain and to overlay each metabolite distribution directly onto corresponding hydrogen-1 MR images. The P-31 MR spectroscopic data were acquired by means of three-dimensional chemical shift imaging (phase encoding in three spatial dimensions) on a 1.5-T clinical instrument equipped with a specially designed quadrature P-31 birdcage coil constructed in the authors' laboratory. Axial, sagittal, and coronal metabolite images based on the area for any one of five peak regions (phosphodiester; phosphocreatine; gamma, alpha, and beta adenosine triphosphate) were generated from 8 X 8 X 8 or 12 X 12 X 8 CSI arrays with voxel sizes of 27 cm3 and 12 cm3, respectively. The positions of these images were aligned with anatomic features by means of the voxel-shifting capability of the Fourier transform. Direct overlays of these metabolite images on corresponding proton images demonstrated excellent correlation with anatomy, factors indicating the utility of this technique for viewing P-31 metabolite levels in all areas of the brain simultaneously.
Adult↗
Antioxidants delay the onset of thyroiditis in obese strain chickens.
Dietary iodine has been shown to be important in the induction of thyroiditis in susceptible chicken strains although the underlying mechanism remains unknown. Iodine may exert its effects through the formation of reactive oxidative radicals which would cause thyroidal injury and initiate infiltration. We have tested this hypothesis by examining the ability of butylated hydroxyanisole (BHA), ethoxyquin, and other antioxidants to prevent thyroiditis in Obese strain (OS) chickens, a strain that develops severe disease by 4 weeks of age. BHA, when administered from hatching until death at 5 weeks of age, reduced thyroidal infiltration and serum levels of antibodies binding thyroglobulin, T3, T4. Similar effects were observed with the antioxidant ethoxyquin. Weaker antioxidants such as vitamins C and E and beta-carotene had only slight or negligible effects on these parameters. BHA reduced thyroiditis in OS chicks killed at 3 and 5 weeks of age, but not at 8 weeks. When BHA treatment was initiated after the development of severe disease, it did not reduce thyroglobulin antibody levels. To determine the mechanism by which BHA reduces thyroiditis, studies were performed to assess the effect of BHA on thyroid function and on the immune responses to exogenous antigens. BHA had no effect on thyroid function in normal strain chickens since thyroidal radioiodine uptake and organification and serum T3 and T4 levels were unaffected. BHA did not alter immune responses to exogenous antigens such as sheep red blood cells or Brucella abortus in OS chickens. In summary, potent antioxidant drugs delayed the onset of thyroiditis when treatment was initiated before the onset of disease, suggesting that reactive oxygen intermediates are involved in the early stages of pathogenesis. However, the site of action remains unknown since they had no detectable effects on thyroid function or general immune responses.
Animals↗
Functional characterization of naturally occurring mutant androgen receptors from subjects with complete androgen insensitivity.
Mutations in the androgen receptor (AR) are thought to cause complete androgen insensitivity (CAIS) in 46,XY human subjects who have a female phenotype despite normal adult male concentrations of plasma testosterone. Assays of AR binding in cultured skin fibroblasts from subjects with CAIS show either an apparent absence of AR (AR-) or normal levels of AR (AR+) binding. In several subjects with CAIS, AR-, no gross AR mutation was detected by Southern blot analyses of genomic DNA and normal sized 10 kilobase mRNA was present on Northern blots of poly(A+) RNA from cultured genital skin fibroblasts. We have used the polymerase chain reaction to amplify individual exons within the human AR gene of subjects with CAIS and have identified point mutations in three subjects. In one AR- subject (R774C), amino acid 774 was changed from arginine (CGC) to cysteine (TGC), in another AR- subject (R831Q), arginine (CGA) was changed to glutamine (CAA) at position 831, and in an AR+ subject (V866M) a methionine (ATG) was substituted for valine (GTG) at position 866. Transfection of wild type and mutant AR cDNA clones into COS cells results in detection of AR protein by immunoblotting. AR ligand binding activity is absent in cells transfected with AR mutants R774C and R831Q, but present with AR mutant V866M. Androgen binding in cells transfected with AR mutant V866M has a 6-fold lower apparent binding affinity than that of wild-type AR. Transcriptional activation of the MMTV-CAT reporter gene was androgen dependent and specific and nearly maximal at physiological concentrations (10(-10) M) of androgen when wild-type AR was transfected into cells, whereas neither AR mutants R774C nor R831Q were able to stimulate CAT activity even at 10(-8) M androgen. AR mutant V866M was able to stimulate CAT activity but the androgen dose dependency was shifted toward pharmacological concentrations of steroid that exceed in vivo levels. The molecular basis of CAIS in humans exhibits genetic heterogeneity. Our study shows that some cases of CAIS are explained by an inability to form a functional AR-steroid complex and hence, the AR is unable to activate transcription of genes essential for male sex differentiation during fetal development.
Amino Acid Sequence↗
Thyroid dysfunction in adults over age 55 years. A study in an urban US community.
The prevalence of thyroid dysfunction was determined in a healthy urban population over the age of 55 years. A highly sensitive serum thyrotropin assay was used initially to screen 968 subjects. Elevated values (greater than 6 mU/L) were found in 7.3%, while suppressed values (less than 0.1 mU/L) were present in 2.5% subjects. Protirelin stimulation testing demonstrated exaggerated responses in 95% of the subjects with elevated thyrotropin levels and subnormal responses in 81% of the subjects with suppressed thyrotropin levels. Thyroid dysfunction, as defined by abnormalities of both serum thyrotropin level and protirelin response, was calculated to be present in 8.9% of the population. The prevalence was greater in whites (vs blacks), in women, and in subjects older than 75 years as compared with the 55- to 64-year age group. Hypothyroidism was calculated to be present in 6.9% subjects. Despite an increased prevalence of thyroid autoantibodies in these subjects, only 8.5% of them had subnormal serum thyroxine concentrations. Hyperthyroidism was calculated to be present in 2.0% of the population, two thirds of whom were taking thyroid hormone preparations. These results suggest a significant prevalence of thyroid dysfunction in the elderly, with important sex and racial differences.
Aged↗
Use of time clocks for employees in health-care institutions.
The reasons for time clock use in health-care institutions, the categories of workers required to use a time clock and other timekeeping methods, and the incidence of time-clock-related conflicts were studied. A questionnaire was mailed to a random sample of 565 hospitals in October 1989. Usable responses were received from 340 (60.0%) of the institutions. Reasons given for time clock use included payroll tabulation, overtime calculation, and ensuring fair payment. Thirty-four institutions (10.0%) required all employees to clock in, and 179 (52.6%) required some employees to do so. A written time card completed by the employee was the method used most frequently if a time clock was not used. Clinical pharmacists were required to clock in at 51 institutions (15.0%), staff pharmacists at 62 (18.2%), and pharmacy technicians at 144 (42.9%). Clinical nurse specialists and registered nurses clocked in at 88 (25.9%) and 169 (49.7%) hospitals, respectively, and licensed practical nurses and nurse's aides each clocked in at 176 hospitals (51.8%). Less than 6% of the hospitals required salaried pharmacists or nurses to use a time clock. Of the respondents, 152 (44.7%) reported that they were not aware of any conflicts or that no conflicts had been experienced. Inconvenience and inaccurate clocking were the most common sources of conflict cited. Most hospitals use time clocks for nonsalaried employees for bookkeeping purposes; dissatisfaction with this method of tracking hours worked does not appear to be widespread.
Labor Unions↗
Identification of D-sorbitol 3-phosphate in the normal and diabetic mammalian lens.
The 31P NMR spectra of mammalian lenses show, in addition to previously identified compounds such as ATP, Pi, glycerophosphorylcholine, and alpha-glycerophosphate, an unusual phosphorylated metabolite resonating at 6.5 ppm. The concentration of this compound increases manyfold in the lenses of diabetic rats concomitant with activation of the aldose reductase pathway. We have purified this material and have identified it as D-sorbitol 3-phosphate. It appears likely that this unusual metabolite of sorbitol may reflect or be part of the metabolic aberration of diabetes in the lens.
Animals↗
Bicarbonate abolishes intracellular alkalinization in mitogen-stimulated 3T3 cells.
An increase in intracellular pH (pHi) following mitogenic stimulation has been reported in a variety of mammalian cells (W. Moolenaar, Annu. Rev. Physiol., 48:363-376, 1986; E. Rozengurt, Science, 234:161-166, 1986). This increase is currently believed to constitute a "permissive" signal in the process of cell activation (A.E. Lagarde and J.M. Pouyssegur, Cancer Biochem. Biophys. 9:1-14, 1986). Since the majority of studies of this phenomenon have been conducted in the nonphysiological milieu of bicarbonate-free solutions, we have undertaken a study of the effects of bicarbonate and CO2 on mitogen-induced intracellular alkalinization in NIH 3T3 cells. Using nuclear magnetic resonance (NMR) spectroscopy and novel 31P NMR pH indicators (2-amino-phosphono-carboxylic acids) we found that mitogen induces an increase in pHi of 0.16 units only in cells bathed in medium containing low concentrations of bicarbonate (less than 1 mM) and not in cells bathed in medium containing physiological levels of bicarbonate (10-30 mM). In addition to abolishing the mitogen-induced alkalinization, bicarbonate stabilizes pHi at 7.25 units as the external pH (pHe) is varied from 7.0 to 7.6. In contrast, in a bicarbonate-free medium pHi increases from 6.9 to 7.3 over the same range of external pHs. At a constant external pH, increasing the bicarbonate/CO2 concentration results in an increase in pHi from 6.9 in bicarbonate-free solution to 7.25 in a bicarbonate-buffered medium. This relationship is hyperbolic with half-maximal effect occurring at a concentration of 0.4 mM bicarbonate at pH 7.05 and 37 degrees C. Our results suggest that the observations of mitogen-induced alkalinization may be due to the use of nonphysiological bicarbonate-free media. Since this increase in pHi is not observed in physiological media where bicarbonate concentrations are usually greater than 20 mM, we conclude that an increase in pHi is not an obligatory or usual part of the cellular response to growth factors in vivo.
Animals↗
In vivo T1 values of phosphorus metabolites in human liver and muscle determined at 1.5 T by chemical shift imaging.
A procedure for obtaining T1 values for phosphorous metabolites in localized regions of human subjects, using a standard 1.5 T MR imager, is described. 31P spectra and T1 values localized to the liver, and to abdominal and calf muscle of healthy volunteers were obtained by means of a multi-slice spectroscopy technique, consisting of a chemical shift imaging (CSI) sequence with a B1-insensitive excitation and one dimension of phase encoding, used in conjunction with a surface coil. An examination consisting of proton imaging, shimming and collection of 31P progressive saturation spectroscopic data for T1 determination required 1 h to perform. Shimming on the signal from the body region detected by the surface coil gave spectra of excellent spectral resolution. Quantification of all peaks in the localized 31P spectra was carried out with the PIQABLE algorithm, and T1 values were calculated for inorganic phosphate (Pi), the phosphodiester region, and the ATP alpha-, beta- and gamma peaks of liver, and for calf muscle Pi, phosphocreatine (PCr), and the three ATP peaks. The precisions of the measurement and of the entire process for obtaining and quantifying localized spectra by one-dimensional CSI were determined, and the accuracy of T1 values obtained by this means was verified. The temporal variation in T1 values obtained in a series of examinations of a single normal subject was also assessed. The consistency of the T1 values obtained in this study with in vivo T1 values obtained by other techniques is a stringent test of accuracy of localized spectra obtained with CSI.
Adenosine Triphosphate↗
Influence of cell culture conditions on aromatase activity in human genital skin fibroblasts.
Because the measurement of aromatase activity in cultured human genital skin fibroblasts has been proposed as a means of studying estrogen production in men, we investigated the influence of culture conditions on aromatase activity. Genital skin fibroblasts were seeded onto culture plates at a density of 1 X 10(6) cells/plate and aromatase activity was determined over a 1-mo. period. Enzyme activity rose slowly over the first 14 d but then rose rapidly to a 10-fold higher plateau by Day 28. The rise in aromatase activity was similar whether activity was normalized for protein or for DNA content. When cells were seeded at the usual density of 1 X 10(6) or at 0.25 X 10(6) cells/plate, aromatase activity was consistently lower during the first 2 wk in cells plated at lower density, but thereafter the levels of enzyme activity in the two groups converged. In cells plated at the lower density, the lower activity observed in the first 2 wk was associated with a lower Vmax. Preincubation of cells plated at one density with conditioned medium from cells plated at the other density did not change the relative levels of activity in the two groups. By contrast, dihydrotestosterone (DHT) receptor binding and 5 alpha-reductase activity were similar at all time points, despite differences in plating density. In additional experiments, the culture medium was replaced daily rather than every 3rd d, and aromatase activity was assayed on Day 7. In cells fed daily, DNA and protein content were twice that of cells fed every 3rd d. By contrast, aromatase activity declined to 30% of that in the latter group. DHT and dexamethasone receptor binding and 5 alpha-reductase activity were similar in the two groups. In summary, factors such as plating density, culture density, and frequency of media replacement dramatically influence aromatase activity in cultured human genital skin fibroblasts. Therefore, the interpretation of aromatase activity data obtained from cultured cells in relation to physiologic or pathologic states should be viewed with appropriate caution.
Adult↗
Comparison of the effects of amiodarone and ipodate on the rat heart.
Chronic treatment of rats with amiodarone has been shown to produce hypothyroid-like effects such as a reduction in body and heart weight and increased synthesis of the low ATPase V3-cardiac isomyosin (Bagchi, Brown, Schneider and Banerjee 1987). In this report, we have tested the hypothesis that amiodarone causes these effects through the inhibition of intracellular production of triiodothyronine (T3) from thyroxine (T4) by comparing the effects of amiodarone with those of ipodate, a potent inhibitor of T4 to T3 conversion. Separate groups of rats were given dietary ipodate and amiodarone respectively for six weeks. Both agents increased serum T4 and T4/T3 ratios, a finding consistent with the inhibition of peripheral T4 to T3 conversion. However, ipodate failed to produce hypothyroid-like effects on body weight, heart weight and isomyosin transitions similar to those found in the amiodarone group. These data indicate that the hypothyroid-like effects of amiodarone on the rat heart are not due to the inhibition of intracellular generation of T3 from T4.
Amiodarone↗
Sequence of the intron/exon junctions of the coding region of the human androgen receptor gene and identification of a point mutation in a family with complete androgen insensitivity.
Androgens act through a receptor protein (AR) to mediate sex differentiation and development of the male phenotype. We have isolated the eight exons in the amino acid coding region of the AR gene from a human X chromosome library. Nucleotide sequences of the AR gene intron/exon boundaries were determined for use in designing synthetic oligonucleotide primers to bracket coding exons for amplification by the polymerase chain reaction. Genomic DNA was amplified from 46,XY phenotypic female siblings with complete androgen insensitivity syndrome. AR binding affinity for dihydrotestosterone in the affected siblings was lower than in normal males, but the binding capacity was normal. Sequence analysis of amplified exons demonstrated within the AR steroid-binding domain (exon G) a single guanine to adenine mutation, resulting in replacement of valine with methionine at amino acid residue 866. As expected, the carrier mother had both normal and mutant AR genes. Thus, a single point mutation in the steroid-binding domain of the AR gene correlated with the expression of an AR protein ineffective in stimulating male sexual development.
Alleles↗
Androgen receptors in boys with isolated bilateral cryptorchidism.
A study was conducted in boys with bilateral cryptorchidism, who were to undergo surgical orchiopexy, to determine if target-organ androgen insensitivity might play a role in the failure of the testes to descend into the scrotum. Nine boys older than 1 year in whom bilateral undescended testes was the only genitourinary abnormality were evaluated. Each subject was administered a six-week course of human chorionic gonadotropin (hCG), 3000 U/m2 of body surface area, intramuscularly injected daily for five days and then twice per week. Basal and hCG-stimulated levels of testosterone were normal. However, hCG administration failed to induce testicular descent in all cases. At the time of surgery, scrotal skin and testicular biopsy specimens were obtained for propagation of cells in tissue culture. Androgen receptor levels and binding affinity were normal for the androgen-specific ligands dihydrotestosterone and metribolone in both skin fibroblasts and testicular cells. In addition, 5 alpha-reductase activity was normal in scrotal skin fibroblasts. Nine boys with bilateral cryptorchidism and normal testicular androgen biosynthesis had normal androgen receptor-binding activity and 5 alpha-reductase activity in cultured scrotal skin fibroblasts and testicular cells. Therefore, bilateral maldescent of the testes in these boys with cryptorchidism was not due to androgen insensitivity.
3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
Evaluation of manual methods for integration of in vivo phosphorus NMR spectra.
Eight human subjects measured peak integrals from a set of simulated 31P NMR spectra by triangulation, by extrapolation of tangents from the baseline of computed integrals, and by cursor demarcation of computed integrals using standard commercial software. The test spectra were qualitatively similar to phosphorus spectra routinely acquired from mammalian skeletal muscle. All three of the above methods were found to underestimate the areas of relatively broad peaks (e.g., ATP peaks were underestimated by 20-40%), even when the peak signal/rms-noise ratio was better than 8/1. This bias resulted in major errors in calculated metabolite ratios. Of the three methods, triangulation was the least accurate, and showed the greatest variation between subjects. Cursor demarcation of computed integrals was the most accurate manual method. The same test spectra were also integrated by two fully-automated fitting algorithms. Although not free of bias, these methods were generally superior to the manual methods.
Adult↗
Decreased thyroidal response to thyrotropin in type II diabetes mellitus.
The responses to TRH and bovine TSH (bTSH) were compared in 19 men with uncontrolled type II diabetes mellitus and eight healthy control subjects. Baseline serum TSH, T3 and T4 were similar in both groups and the rise of serum TSH, T3 and T4 following the intravenous (IV) administration of TRH (500 micrograms) was not significantly different. Diabetic subjects showed a blunted response to the subcutaneous (sc) administration of bTSH (5 U) when their maximal serum T3 and T4 values were compared with controls (T4, 9.4 +/- 0.3 v 12.3 +/- 1.1 micrograms/dL, P less than .005; T3, 185 +/- 9 v 233 +/- 17 ng/dL, P less than .025; diabetic v control). When the response to bTSH was examined in seven patients after 4 to 5 days of strict glycemic control, the maximal T3 response was found to increase in six, and the maximal T4 response in five. These data show that the thyroidal secretory response to large doses of TSH is decreased in uncontrolled diabetes mellitus and that strict glycemic control frequently improves the response.
Adult↗
Studies of up-regulation of androgen receptors in genital skin fibroblasts.
Prolonged exposure of genital skin fibroblasts (GSF) to dihydrotestosterone (DHT) increases androgen receptor binding of steroid, a process termed 'up-regulation'. Because the extent of up-regulation appears to be quite variable, we have investigated the optimal conditions and the molecular mechanisms that control this phenomenon in seven strains of normal neonatal GSF. When GSF were incubated for 1-48 h with 3 nM methyltrienolone (R1881), maximal up-regulation was reached by 20 h and remained constant thereafter. With DHT, rapid steroid metabolism required replenishment of DHT for maximum up-regulation. Up-regulation levels following 20 h incubation with DHT (including steroid replenishment) and R1881 were 2.07-fold (range = 1.1-3.3) and 2.35-fold (range = 1.86-3.33), respectively. The greater variability observed with DHT may be related to variable rates of steroid catabolism among cell strains. Half-maximal up-regulation was attained at 0.29 nM R1881, which approximates the Kd. Maximal up-regulation was reached only with continuous exposure to R1881 for 24 h. It was completely inhibited by actinomycin D (0.5 micrograms/ml) or cycloheximide (10 micrograms/ml). Following up-regulation, removal of R1881 for 24 h resulted in a highly variable decline of androgen receptors among cell strains. Maximal up-regulation could be reinduced by exposure to R1881 again for an additional 24 h. During up-regulation, androgen receptor levels in nuclei and nuclear matrix rose with increments comparable to those obtained in whole cells. We conclude that the extent of up-regulation and its rate of decline differ greatly among normal cell strains. Hence, its study in cells of patients with androgen insensitivity may have limited value.(ABSTRACT TRUNCATED AT 250 WORDS)
Cells, Cultured↗