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M Sar

Publications and source records attributed to M Sar.

At least 37 records · Page 2Linked to original sources
PubMed1996

Coordinate modulation of glucocorticoid receptor and glutaminase gene expression in LLC-PK1-F+ cells.

The effect of glucocorticoid receptor on glutaminase gene expression and related glutamine metabolism was studied in proximal tubule-like LCC-PK1-F+ cells. These cells express functional glucocorticoid receptors as demonstrated by immunoreactivity with antiglucocorticoid receptor antibody, specific ligand binding, and a 14-fold increase in chloramphenicol acetyltransferase (CAT) reporter gene activity after exposure to dexamethasone (10(-6)M). Dexamethasone exposure for 18 h increased glutaminase mRNA and activity by 32 and 42%, respectively (both P< 0.05, paired t-test), associated with a small (9%) but significant increase in glutamine utilization (P<0.05). In an effort to elicit a greater response, endogenous glucocorticoid receptors were supplemented by transfecting cells with a plasmid, pMAMGR, expressing the rat glucocorticoid receptor gene. Transfected cells expressed a 39-fold increase in CAT activity with dexamethasone treatment, confirming a higher level of functional receptors, but glutaminase mRNA and activity were now decreased by 34 and 32%, respectively, associated with a 15% fall in glutamine utilization after 18-h exposure to dexamethasone. This biphasic response in glutaminase gene expression was mirrored by glucocorticoid receptor mRNA that increased 41% after dexamethasone in LLC-PK(1)-F+ cells, but decreased 63% after transfection (both P<0.05). These findings are consonant with glucocorticoid receptor gene modulation of glutaminase gene expression and glutamine utilization.

Alanine↗
PubMed1996

Androgen and glucocorticoid receptors in the stroma and epithelium of prostatic hyperplasia and carcinoma.

Differences in stromal and epithelial cell staining for androgen and glucocorticoid receptors (ARs and GRs) were investigated in 20 patients with clinically localized prostatic carcinoma treated by radical prostatectomy. Sections of benign prostatic hyperplasia and prostatic carcinoma from each patient were stained with antibodies to AR and GR using an avidin-biotin peroxidase technique. The specificity of the GR immunoreactivity was established in benign prostatic hyperplasia and prostatic carcinoma by immunohistochemistry using the GR antibody absorbed with synthetic peptide and Western blotting. Nuclear staining intensity and percentage of nuclei stained were summed to obtain AR and GR immunostaining scores. AR staining of prostatic carcinoma epithelial [103 +/- 58 (SD)] and stromal (126 +/- 48) nuclei was less than in benign prostatic hyperplasia (142 +/- 47 and 169 +/- 56; paired Student's t tests, P = 0.02 and P = 0.01); however, no difference in staining intensity occurred between stroma and epithelium in either tissue type. GR stained intensely in stromal cells from benign prostatic hyperplasia (189 +/- 50) and prostatic carcinoma (163 +/- 60). However, prostatic carcinoma epithelial cells (34 +/- 57) had low levels of glucocorticoid receptor staining (P < 10(-7)), and benign prostatic hyperplasia epithelium (74 +/- 51) was intermediate. In most patients, GR could not be detected in nuclei of prostatic carcinoma epithelial cells but was undiminished in stromal cell nuclei. There was no relationship by multivariate regression analysis between AR or GR staining and age, serum prostate-specific antigen, Gleason grade, or pathological stage. In comparison with AR, the greater variability of GR staining in epithelium versus stroma of prostatic carcinoma warrants further study of GR, particularly in the area of stromal-epithelial interaction.

Aged↗
PubMed1995

Androgen receptor antagonist versus agonist activities of the fungicide vinclozolin relative to hydroxyflutamide.

The mechanism of antiandrogenic activity of vinclozolin (3-(3,5-dichlorophenyl)-5-methyl-5-vinyloxazolidine-2,4-dione), a dicarboximide fungicide under investigation for its potential adverse effects on human male reproduction, was investigated using recombinant human androgen receptor (AR). The two primary metabolites of vinclozolin in plants and mammals are M1 (2-[[3,5-dichlorophenyl)-carbamoyl]oxy]-2-methyl-3-butenoic acid) and M2 (3',5'-dichloro-2-hydroxy-2-methylbut-3-enanilide). Both metabolites, in a dose-dependent manner, target AR to the nucleus and inhibit androgen-induced transactivation mediated by the mouse mammary tumor virus promoter. M2 is a 50-fold more potent inhibitor than M1 and only 2-fold less than hydroxyflutamide. In the presence of dihydrotestosterone (50 nM), M2 (0.2-10 microM) inhibits androgen-induced AR binding to androgen response element DNA. In the absence of dihydrotestosterone, concentrations of 10 microM M2 or hydroxyflutamide promote AR binding to androgen response element DNA and activation of transcription. Agonist activities of M2 and hydroxyflutamide occur at 10-fold lower concentrations with the mutant AR (Thr877 to Ala) endogenous to LNCaP human prostate cancer cells. The results indicate that androgen antagonists can act as agonists, depending on ligand binding affinity, concentration, and the presence of competing natural ligands.

Androgen Receptor Antagonists↗
PubMed1995

Immunocytochemical analysis of hormone mediated nuclear translocation of wild type and mutant glucocorticoid receptors.

We have analyzed structural and functional features of the human glucocorticoid receptor (hGR) for their effects on receptor subcellular distribution. COS 1 cells transiently transfected with wild type and mutant hGR cDNAs were assessed immunocytochemically using well-characterized antipeptide antibodies to the hGR. The effect of administration of steroid hormones (and the antiglucocorticoid RU486) on receptor localization was evaluated. Unliganded wild type receptors expressed in COS 1 cells were predominately cytoplasmic. Addition of glucocorticoids or the glucocorticoid receptor antagonist, RU486, resulted in complete translocation of these receptors into the nucleus whereas non-glucocorticoid steroids or dibutyryl cAMP were not effective in promoting nuclear translocation. Thus, nuclear translocation was specific for steroids capable of high affinity binding to the hGR. To elucidate the potential role of receptor domains in receptor localization, COS 1 cells transiently transfected with various receptor cDNA mutants were analyzed in a similar manner. Translocation of an hGR deletion mutant lacking the majority of the amino terminus (deletion of amino acids 77-262) was identical to the wild type receptor despite the absence of a transactivation domain. Receptors in which the DNA binding domain was either partially or totally deleted showed an impaired capacity to undergo hormone-inducible nuclear translocation. Deletion of the hinge region of the hGR (which also contains part of the nuclear localization signal, NL1) resulted in receptor localization in the cytoplasm. Mutants in the ligand binding domain exhibited two localization phenotypes, exclusively nuclear or cytoplasmic. Receptor mutants truncated after amino acid 550 were found in the nucleus in the presence and absence of hormone consistent with the existence of nuclear localization inhibitory sequences in the ligand binding domain of the receptor. However, a linker insertion mutant (at amino acid 582) which results in a receptor deficient in ligand binding did not undergo nuclear translocation indicating that nuclear localization inhibitory sequences were intact in this mutant. The role of receptor phosphorylation on hormone induced nuclear translocation was also examined. Mouse glucocorticoid receptors which contained mutations of certain hormone inducible phosphorylation sites exhibited translocation properties similar to wild type mGR indicating that these phosphorylation sites on the receptor do not play a major role in hormone inducible nuclear translocation.

Animals↗
PubMed1995

Steroid hormone receptors in astrocytic neoplasms.

The presence of specific steroid hormone-binding receptors has been correlated with the clinical response to hormonal therapy in a number of different neoplasias, including breast and prostate cancer. In this article, we investigated the expression of the androgen, estrogen, glucocorticoid, and progesterone receptor messenger ribonucleic acid (mRNA) and protein in a number of astrocytic neoplasms of various histological grades. Androgen and glucocorticoid receptor mRNA were detected in all astrocytic neoplasms examined, regardless of histological subtype. In contrast, progesterone receptor mRNA was observed more frequently in high-grade tumors than in low-grade tumors. Estrogen receptor mRNA was undetectable in all astrocytic tumors examined. These studies suggest a possible adjunct clinical use of hormonal therapy for the treatment of astrocytomas. Specific antagonists and agonists may allow the modulation of the growth of these tumors. Development of this body of knowledge may lead to the development of better treatment for these aggressive tumors.

Adolescent↗
PubMed1995

Androgen receptor expression in meningiomas.

The predominance of meningiomas in females, the accelerated growth of these tumors during the luteal phase of the menstrual cycle and during pregnancy, and the association between meningiomas and breast cancer have led to a number of studies examining the potential role of steroids in the growth of meningiomas. The possibility that androgens play a role in meningioma proliferation has been suggested by a small number of investigators. The aim of this study was to examine the expression of androgen receptor messenger ribonucleic acid (mRNA) and correlate it using immunochemistry with the nuclear localization of androgen receptor in a large number of meningiomas. Thirty-nine meningiomas were examined by Northern blot analysis for the presence of measurable amounts of androgen receptor mRNA and eight of these were analyzed by immunohistochemistry for receptor protein. Sixty-seven percent of the meningiomas expressed androgen receptor mRNA. There was a marked predominance of women among the patients whose tumors expressed androgen receptor; 69% were women and 31% were men. The immunohistochemical data correlated with Northern blot analysis of mRNA. The staining was predominantly nuclear, suggesting that the androgen receptor resides in a location that can activate gene expression.

Adult↗
PubMed1994

A ligand-dependent bipartite nuclear targeting signal in the human androgen receptor. Requirement for the DNA-binding domain and modulation by NH2-terminal and carboxyl-terminal sequences.

The amino acid sequence requirements for androgen-dependent androgen receptor nuclear import were determined by immunostaining transiently expressed full-length wild type and mutant human androgen receptors (AR) in monkey kidney COS cells and measuring transcriptional activity by cotransfection with a luciferase reporter vector in monkey kidney CV1 cells. Mutagenesis studies revealed a bipartite nuclear targeting sequence in the DNA binding and hinge regions at amino acids 617-633, consisting of two clusters of basic amino acids separated by 10 amino acids, (sequence: see text). In a series of deletion mutants, AR NH2-terminal fragments (residues 1-639 through 1-723) displayed constitutive nuclear import, and transcriptional activity was similar to that of the ligand-activated full-length wild type AR. In contrast, nuclear import and transcriptional activation were inhibited by sequence extensions into the steroid-binding domain (1-771). Constitutive nuclear import was regained in part by NH2-terminal deletions of full-length AR. Expression of AR/pyruvate kinase chimeras defined a sequence required for pre-dominant nuclear localization as residues 580-661, comprised of the second zinc finger region of the DNA-binding domain, the 17-amino-acid putative targeting sequence, and 28 residues of flanking carboxyl-terminal sequence. These studies suggest that the bipartite nuclear targeting sequence of AR includes flanking sequence and is modulated by interactions between the NH2-and carboxyl-terminal regions.

Amino Acid Sequence↗
PubMed1994

Extended junctional sarcoplasmic reticulum of avian cardiac muscle contains functional ryanodine receptors.

The ryanodine receptor (RYR)/Ca2+ release channel of avian cardiac muscle was localized by immunocytochemical techniques and biochemically characterized using isolated membrane and receptor protein fractions. Monoclonal antibody C3-33 raised against the canine cardiac RYR bound to the junctional sarcoplasmic reticulum of pigeon and finch hearts, both at peripheral couplings and at extended junctional sarcoplasmic reticulum (EJSR). Immunoblots of sarcoplasmic reticulum vesicles from pigeon and finch hearts showed this antibody recognized a single high molecular weight protein, which co-migrated with the canine M(r) 565,000 RYR/Ca2+ release channel polypeptide. The pigeon heart RYR bound [3H]ryanodine with high affinity in a Ca(2+)-dependent manner, comparable to the canine cardiac RYR. Purification of the pigeon RYR yielded a 30 S protein complex, which bound the maximum calculated amount of [3H]ryanodine ((440 +/- 60) pmol/mg protein), assuming one high affinity site/tetrameric 30 S RYR comprised of M(r) 565,000 polypeptides. Autoradiography of isolated finch cardiac myocytes indicated [3H]ryanodine binding throughout the cells. These results suggest that avian heart contains a single population of RYRs, and thereby support the hypothesis that avian EJSR contains functional calcium release channels which, because of the absence of transverse tubules, can be located micrometers away from the surface membrane in avian heart.

Animals↗
PubMed1994

Regulation of the human glucocorticoid receptor by long-term and chronic treatment with glucocorticoid.

HeLa S3 cells that contain endogenous glucocorticoid receptors (GR) were treated with dexamethasone (DEX) for periods of time ranging from 24 h to 2 weeks or chronically over a 2-year period. Regulation of GR protein and mRNA were examined by affinity labeling, Western blotting, and Northern blotting. Relatively short-term treatment of cells with DEX for 24 or 48 h revealed more profound down-regulation of GR protein than of GR mRNA. However, by 2 weeks of DEX treatment, the levels of both receptor protein and mRNA were both maximally down-regulated. Cells that had been chronically DEX treated (for up to 2 years) had no measurable GR protein or mRNA. The down-regulation of receptor protein and RNA that occurred after 2 weeks of DEX treatment is completely reversible upon DEX removal, whereas reversibility did not occur with cells that had been chronically treated with DEX. Furthermore, transfection of a glucocorticoid responsive reporter plasmid into these chronically DEX-treated cells demonstrated that these cells were no longer responsive to steroid treatment. However, cotransfection of a plasmid encoding the human GR into these chronically DEX-treated cells resulted in restored production of GR and responsiveness to hormone, indicating that the defect in these cells occurs only at the receptor level.

Blotting, Northern↗
PubMed1994

Characterization of mutant androgen receptors causing partial androgen insensitivity syndrome.

The androgen insensitivity syndrome (AIS) is an X-linked disorder caused by mutations of the androgen receptor (AR) gene resulting in a spectrum of sex phenotypes that ranges from complete female (complete AIS) to nearly complete male (partial AIS). Using the polymerase chain reaction and denaturing gradient gel electrophoresis, we have analyzed the AR gene in three 46,XY individuals with partial AIS. In one subject whose androgen insensitivity was manifest at birth by clitoromegaly, posterior labial fusion, and a urogenital sinus, androgen-binding affinity in genital skin fibroblasts was similar to that of the control. In this subject, a mutation was identified in exon C encoding the second zinc finger of the androgen receptor. The mutation converted a leucine residue at position 616 to arginine, causing greatly reduced binding of receptor to an androgen-response element DNA sequence. However, the mutant AR retained a low level of transcriptional activity at physiological androgen concentrations in keeping with the subject's phenotype of partial AIS. In the second subject, who also had an ambiguous external genital phenotype, a single base mutation was identified in exon G, converting arginine at position 840 to histidine. Androgen-binding affinity in genital skin fibroblasts of this subject was 7-fold lower than control, and the mutant receptor had reduced transcriptional activity. In the third subject, who had a female phenotype with normal pubic hair reflecting a low degree of androgen responsiveness, the valine residue at position 889 was replaced by methionine. This mutant receptor had apparent normal androgen-binding affinity but reduced androgen-binding capacity when examined by expression of the recreated mutant AR in COS 7 cells. These results demonstrate the clinical, functional, and molecular heterogeneity in the syndrome of partial androgen insensitivity.

Adolescent↗
PubMed1994

Intragenic sequences of the human glucocorticoid receptor complementary DNA mediate hormone-inducible receptor messenger RNA down-regulation through multiple mechanisms.

Glucocorticoid receptors (GR) are ligand-dependent transcription factors that play a critical role in the endocrine control of cell growth, differentiation, and death. These steroid receptors are widely recognized to undergo down-regulation after exposure to ligand in cell cultures and animals, including humans. This reduction in cellular receptor levels leads to insensitivity to subsequent hormone administration. The mechanisms controlling homologous down-regulation of the GR are, however, poorly understood. We have previously shown (1) that a transfected human GR (hGR) complementary DNA (cDNA) contains sequences that are sufficient to recapitulate the down-regulation of both hGR messenger RNA (mRNA) and protein seen in vivo. We have now evaluated potential mechanisms involved in the hormonal regulation of the hGR mRNA and, further, have identified an intragenic domain of the hGR cDNA that contains the down-regulatory signal. Glucocorticoid treatment of COS-1 cells expressing a transfected hGR cDNA resulted in down-regulation of the hGR mRNA in the presence of cycloheximide or actinomycin-D, suggesting that a glucocorticoid-inducible protein was not essential for down-regulation. We show that prolonged receptor occupation by ligand leads to increased GR mRNA turnover, and furthermore, that either the agonist dexamethasone or the antagonist RU486 decreased transcription of the hGR cDNA. To resolve which receptor cDNA sequences are critical in down-regulation, a cotransfection strategy was employed in which a series of hGR cDNA deletion mutants was transfected in conjunction with the full-length hGR cDNA. The effects of glucocorticoid on the regulation of receptor mRNAs encoded by the mutant receptor cDNAs were examined. Deletions within the 5' half of the receptor cDNA produced transcripts that were susceptible to glucocorticoid-mediated down-regulation, whereas deletion of sequences located in the 3'-end of the receptor-coding sequence (corresponding to amino acids 550-697) resulted in receptor transcripts that were only minimally down-regulated by glucocorticoid. Together these studies indicate that multiple mechanisms control GR mRNA abundance, and an intragenic element within the ligand-binding domain is critical for this down-regulation.

Animals↗
PubMed1993

Steroid requirement for androgen receptor dimerization and DNA binding. Modulation by intramolecular interactions between the NH2-terminal and steroid-binding domains.

Infection of Spodoptera frugiperda Sf9 insect cells with recombinant human androgen receptor (AR) baculovirus results in expression of a 118-kDa phosphoprotein that displays high affinity androgen binding and androgen-dependent targeting to the nucleus. Using the DNA mobility shift assay, specific in vitro binding of full-length AR to androgen response element DNA (ARE) requires intracellular hormone exposure. The ability of a variety of steroids to induce ARE binding paralleled their transcriptional potential. Certain antihormones, cyproterone acetate and RU486, promote ARE binding, but a pure antiandrogen, hydroxyflutamide, inhibits AR binding to ARE DNA. AR dimerization requires incubation of recombinant baculovirus-infected insect cells with androgen, but only when one or both components of the dimer contain the NH2-terminal domain. Based on the intensities of ARE binding and lack of binding to an ARE half-site, it appears that, unlike the glucocorticoid receptor, AR binds DNA primarily as a dimer. Thus, full-length baculovirus-expressed AR requires intracellular hormone exposure for dimerization and ARE binding to overcome inhibition imposed by the AR NH2-terminal domain. Antihormones with agonist activity promote dimerization and ARE binding, while a pure antiandrogen blocks AR DNA binding. It is concluded that intramolecular interactions between the NH2-terminal and steroid-binding domains are regulated by the specificity of hormone binding and modulate receptor dimerization and DNA binding.

Androgens↗
PubMed1993

Dexamethasone target sites in the central nervous system and their potential relevance to mental illness.

1. The topical distribution of tritiated dexamethasone (DEX), a potent synthetic glucocorticoid of widespread use in the diagnosis and assessment of mental illness, was studied in rat CNS by autoradiography to obtain information on potential target sites for feedback and other centrally mediated effects of glucocorticoids. 2. The cells of the arcuate nucleus of the hypothalamus and the lateral thalamic nuclei displayed the most concentrated nuclear accumulation of silver grains. 3. Significant accumulation, exceeding that found in the hippocampal formation, also occurred in the cells of the ventromedial, periventricular, and paraventricular nuclei of the hypothalamus, the locus ceruleus, the nucleus tractus solitarii, and the area postrema, none of which are targeted by corticosterone, the native glucocorticoid of the rat. 4. Nuclear accumulation of silver grains was prominent in neural and glial cells of the cerebral cortex, the olfactory nucleus, the dorsolateral septum, the amygdala, the subfornical organ, the lateral parabrachial, medial trapezoid, and dorsal reticular nuclei, the nucleus centralis of the raphe, the cerebellum, and vascular tissues. 5. The localization of DEX in hypothalamic and brain-stem nuclei coincided with that of the glucocorticoid receptor, possibly implicating these sites in direct or modulating effects of glucocorticoids in various forms of mental disturbance, including depression, anxiety, panic disorders, and alcohol withdrawal. 6. The extent to which various CNS regions targeted by DEX feature in negative feedback control of adrenocortical secretion remains to be defined, as does the site of impaired feedback disclosed by the dexamethasone suppression test in psychiatric patients.

Animals↗
PubMed1993

Progestagen nuclear binding sites in the male genital tract of the mouse, studied by autoradiography.

Autoradiograms were prepared after injection of 125I progestagen (sp. act. 2200 Ci/mmol) to prepubertal and young adult mice. Nuclear concentration of radioactivity was found in smooth muscle cells at the beginning and the end of the deferent duct and in fibroblasts around the fusion of the deferent duct with the urethra. Nuclear labeling was enhanced in adult animals pretreated with oestradiol-valerate. In prepubertal mice nuclear labeling was more abundant than in adult mice and found in all smooth muscle cells of the deferent duct. No nuclear labeling was observed in other accessory sex organs. Nuclear labeling was prevented by injection of excess of unlabeled R5020. The presence of progestin receptors in smooth muscle cells of the deferent duct suggests a regulatory effect of progestin on contractions in analogy to progestin effects on oviductal and uteral smooth muscle cells. The presence of nuclear progestin receptors in the periurethral region may indicate an involvement of progestins in the etiology and regulation of benign prostate hyperplasia.

Animals↗
PubMed1993

Immunohistochemistry of the androgen receptor in human benign and malignant prostate tissue.

The role of the androgen receptor in the development and progression of prostatic carcinoma has not been defined. The development of androgen receptor antibodies has provided new opportunities for direct immunohistochemical analysis. We compared the androgen receptor staining characteristics of fresh human prostatic carcinoma with benign prostatic hyperplasia (BPH) using an avidin-biotin complex method. Cancer and BPH obtained from the same radical retropubic prostatectomy specimen in 10 prostate cancer patients (68.5 +/- 7.3 years old standard deviation) and BPH from 10 noncancer patients (71.5 +/- 7.7 years old) were incubated with AR52, a rabbit polyclonal antibody against a synthetic androgen receptor peptide. Nuclei within each section were graded for intensity of androgen receptor staining (0-absent, 1-weak, 2-moderate or 3-strong) and the percentage (0 to 100%) of nuclei sampled staining at each of these intensity levels was determined. A total intensity score (0 to 300) was the summation of the products of each intensity score (0 to 3) and their corresponding percentages. Cancer sections (166 +/- 69) stained less intensely and more heterogeneously than BPH in cancer patients (246 +/- 41, Student's t test p < 0.05) and noncancer patients (225 +/- 39, p < 0.05). The decreased intensity and greater heterogeneity of androgen receptor staining in cancer tissue may implicate a quantitative or functional difference in androgen receptor between prostatic carcinoma and BPH.

Aged↗
PubMed1992

Expression of a cardiac Ca(2+)-release channel isoform in mammalian brain.

Mammalian brain possesses ryanodine-sensitive Ca2+ channels, which in muscle cells mediate rapid Ca2+ release from intracellular stores during excitation-contraction coupling. Analysis of bovine brain ryanodine receptor (RyR) channels suggests specific expression of the cardiac-muscle RyR isoform in mammalian brain. Localization using cardiac-muscle RyR-specific antibodies and antisense RNA revealed that brain RyRs were present in dendrites, cell bodies and terminals of rat forebrain, and highly enriched in the hippocampus. Activity of skeletal-muscle RyR channels is coupled to sarcolemmal voltage sensors, in contrast with cardiac-muscle RyR channels, which are known to be Ca(2+)-induced Ca(2+)-release channels. Thus Ca(2+)-induced Ca2+ release from intracellular stores mediated by brain RyR channels may be a major Ca(2+)-signalling pathway in specific regions of mammalian brain, and hence may play a fundamental role in neuronal Ca2+ homoeostasis.

Animals↗