Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human sebaceous glands (SG) and hair follicles (HF) are target structures in the skin for androgen action. They contain steroid enzymes, capable of transforming weak androgens into the target-tissue-active androgens testosterone (T) and dihydrotestosterone (DHT), which bind to the androgen receptor (AR) to regulate cellular transcription. The AR from HF and SG from human scalp tissue has been purified greater than 86,000 times by phenyl-sepharose, DEAE-sephacel, gel filtration chromatography, and ultrafiltration. Sucrose density gradient analysis and non-denaturing gradient polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS)-PAGE revealed two molecular species of AR, an active form called monomer, capable of binding DHT with great specificity (4S, m = 62,000 Da, Kd = 0.6 nM, Bmax 8260 fmol/micrograms protein), and the other, an inactive form of the monomer called tetramer (10.8S, m = 252,000 Da, Kd = 2.9 nM). The two species are interconvertible, and after purification each appeared as a single band on SDS-PAGE. The conversion of the monomer to the tetramer AR form is influenced by reduced and oxidized glutathione, and possibly by an endogenous disulfide converting factor (DCF). Furthermore, biochemical events in the androgenic signal transduction sequence were shown to be stimulated by androgens via the AR. These include the total nuclear AR content, chromatin binding of AR complexes, and stimulation of RNA polymerase II, thus influencing gene expression, which is important in understanding regulation of HF growth and SG proliferation.
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PMID:Purification of androgen receptors in human sebocytes and hair. 158 31

Controlled digestion of rat ventral prostate nuclei by careful adjustment of conditions of temperature, divalent cation concentration, ionic strength and micrococcal nuclease:DNA ratios yielded oligonucleosome fractions corresponding to less than 10% of the total genome which contain the majority of RNA polymerase B activity and androgen-receptor complexes of the nucleus. These parameters were affected acutely by androgen withdrawal and administration: furthermore, such manipulations affected the susceptibility to micrococcal nuclease release of prostate binding protein gene sequences. This transcriptionally-active androgen-influenced fraction was considered ideal for studies of interaction of chromatin components with androgen receptor protein. Androgen receptor was purified approximately 20 000-fold from rat prostate cytosol. The purified protein retained its ability to stimulate RNA polymerase B activity in prostate nuclei and chromatin fractions, and its properties of binding to chromatin and to DNA. However, although purified receptor protein showed tissue-specific binding to prostate chromatin and enhanced binding to fractions released by low nuclease digestion, no such specificity was indicated by binding to total DNA, DNA from specific fractions or cloned prostatic binding protein cDNA.
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PMID:Interaction of androgen receptors with chromatin and DNA. 653 19

This study was designed to characterize mouse kidney ornithine decarboxylase (ODC) activity as an androgenic end point and to use ODC activity to detect an androgenic effect of antiandrogens. Enzyme activity was not affected by freezing the whole kidney or the 15,000 X g supernatant for up to 7 days. ODC activity in female mice had a diurnal variation that peaked at midday. This diurnal variation did not affect the androgenic response of ODC. Enzyme activity was lower in females than in males and, in both sexes, could be induced further to similar levels with testosterone treatment. A single dose of crystalline testosterone induced a marked increase in activity, which peaked sharply, up to 100-fold above baseline, 12-17 h after treatment. Enzyme activity could be maintained with continued treatment for at least 28 days and reached levels up to 1,000-fold above baseline. The response was specific for androgens and required a functional androgen receptor. Other hormones had permissive effects. The early androgen-stimulated response (less than 24 h) was partially diminished by hypophysectomy. Propylthiouracil reduced both early and chronic responses. Genetic factors were also involved. The testosterone-stimulated response of C57BL/6J mice was consistently approximately half that of DBA/2J mice. Using this very specific and sensitive increase in ODC activity as an end point, we did not detect an androgenic response to treatment with the antiandrogens, cyproterone acetate (6-chloro - 17 alpha - acetoxyl - 1,2 alpha - methylene - 4,6- pregnadiene- 3,20-dione) and flutamide (4'-nitro-3'-trifluoromethylisobutyranilide), despite an increase in RNA polymerase activity. The functionality of the polymerase activity induced by antiandrogens thus remains in question. These data suggest that mouse renal ODC activity can be a useful tool for future study of androgen action at the physiological and molecular level.
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PMID:Androgen and progestin stimulation of ornithine decarboxylase activity in the mouse kidney. 668 54

Cytosolic androgen receptors from neocortex, hypothalamus and anterior pituitary and ventral prostate glands were analysed by miniature isoelectric focusing and sucrose density-gradient centrifugation before and after precipitation of [3H]dihydrotestosterone (DHT)-bound complexes with ammonium sulphate. In the hypothalamus and neocortex (NH4)2SO4 precipitation appeared to cause heterodisperse peaks, and in the case of the ventral prostate there was a clear shift to a more basic isoelectric point. After sucrose density-gradient centrifugation all cytosols sedimented as large aggregates which appeared to dissociate into subunits in 0.4 M-KCl gradients. The functional significance of these altered forms was tested by nuclear uptake studies of cytosolic [3H]DHT-bound complexes, which could only be detected in brain and pituitary nuclei after prior precipitation with (NH4)2SO4, which also significantly increased extraction of ventral prostate [3H]DHT-bound complexes from the nucleus. The nuclei apparently responded to the (NH4)2SO4-precipitated and redissolved complexes by increased RNA polymerase activity. These results are consistent with the possibility that the neural androgen receptor is altered before interaction with the genome, and this alteration may be necessary for the action of the hormone to be expressed.
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PMID:Analysis of activated androgen receptors in rat brain and anterior pituitary and ventral prostate glands: nuclear binding and RNA polymerase activity. 674 98

Androgens stringently regulate the concentrations of the polyamines, spermine and spermidine, in rat ventral prostate. The intracellular distribution of the polyamines is also dependent on the androgenic milieu; a small proportion of polyamines is associated tightly with the nucleus. Soluble nucleolar chromatin contains RNA polymerase A and protein kinase activities, both of which are regulated by androgens and markedly stimulated by polyamines. It remains to be established whether the polyamines modulate these enzyme moieties directly, or whether the phosphorylation of other nucleolar proteins plays a crucial role in the androgenic regulation of rRNA synthesis. Using a protocol centered on affinity chromatography, the purification of the androgen receptor protein to a state approaching homogeneity has been accomplished. The purified receptor stimulates the RNA polymerase A and protein kinase activities associated with prostate nucleolar chromatin. The significance of these findings in the light of correct concepts of steroid hormone action is discussed.
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PMID:Enhanced transcription of rRNA genes by purified androgen receptor complexes in vitro. 688 53

The DNA sequence of the genes for the androgen receptor (AR) and TATA-binding protein (TBP), like many other genes encoding transcription factors, contains a series of tandem CAG repeats. Here we explore the capacity of complementary peptide nucleic acids (PNAs) to invade the CAG triplets of the AR and TBP genes in human prostatic cancer cells and show that the PNAs readily entered the nuclei of lysolecithin-permeabilized cells and effectively inhibited sense transcription of unique AR and TBP DNA sequences downstream of the site of PNA.DNA hybridization, but not upstream of that site. These PNAs had little or no effect on transcription of the c-myc gene, which lacks a CAG triplet domain. Conversely, a PNA complementary to a unique sequence of the c-myc gene did not inhibit transcription of the AR or TBP genes but did inhibit c-myc transcription. Comparisons of PNA effects on sense and antisense transcription of the AR, TBP, and c-myc genes confirm that progression of the RNA polymerase complex beyond the site of PNA.DNA hybridization is impaired in both directions. Suppression of the AR gene results in refolding of a transcriptionally active nucleosome containing a unique 17-mer AR DNA sequence.
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PMID:Invasion of the CAG triplet repeats by a complementary peptide nucleic acid inhibits transcription of the androgen receptor and TATA-binding protein genes and correlates with refolding of an active nucleosome containing a unique AR gene sequence. 866 37

Mutations of the androgen receptor (AR) gene and protein are associated with complete androgen insensitivity syndromes (CAIS) in individuals with XY genotypes causing them to develop as phenotypic females. Splice site mutations of the AR gene are very rare and in this report we describe the consequences of a novel G --> A mutation at the exon 7/intron 7 splice junction of the AR gene that resulted in CAIS in two siblings. Reverse transcriptase-polymerase chain reaction (RT-PCR) of the AR transcript in patient's fibroblasts was performed and sequencing of the product showed omission of exon 7, with exon 6 being spliced directly to exon 8. This resulted in a shift of the reading frame and the introduction of a premature stop codon 10 amino acids into exon 8. Immunoblot analyses showed that the resultant AR protein was partially deleted in its C-terminal region and was approximately 1.5 kDa smaller than the wild type. This truncated AR was non-functional as it was unable to bind its physiological ligand (dihydrotestosterone) in androgen-binding assays. This is the first documentation of a point mutation in the AR gene which causes exon skipping and proves that the mutation is the cause of CAIS in our two subjects.
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PMID:A novel splice site mutation in the androgen receptor gene results in exon skipping and a non-functional truncated protein. 929 79

Marrow stromal cells mediate the effect of 1alpha,25-dihydroxyvitamin D3 on formation of osteoclast-like cells from undifferentiated hematopoetic precursors in bone marrow. Induction by the vitamin D hormone of multinucleated, calcitonin receptor- and tartrate-resistant acid phosphatase-positive cells in primary mouse bone marrow culture can be modulated by other members of the steroid/thyroid hormone family, such as triiodothyronine, which has a positive effect, as well as 17beta-estradiol and 5alpha-dihydrotestosterone, which both act as inhibitors of osteoclastogenesis. In an attempt to relate these effects of the steroid/thyroid hormones to the presence of their respective nuclear receptors, we studied expression of the vitamin D receptor (VDR), estrogen receptor (ER)-alpha and -beta, thyroid hormone receptor (TR)-alpha and -beta, and androgen receptor (AR) in total bone marrow as well as primary marrow stromal cell cultures. By using reverse-transcriptase-polymerase chain reaction, in both cases amplification products were obtained, which were identified by multiple restriction fragment length analysis as transcripts from mRNA specific for the ligand-binding domains of the VDR, ER-alpha, ER-beta, TR-alpha, TR-beta, and AR. Specific immunostaining by indirect peroxidase labeling revealed that among the various cell types present in bone marrow, the steroid/ thyroid hormone receptors are abundant particularly in marrow stromal cells. In another series of experiments, we extended our survey on receptor expression also to stromal/osteoblastic cell lines. At the mRNA level, the complete repertoire of steroid/thyroid hormone receptors was present in preadipocytic ST2 cells as well as in osteoblastic MC3T3-E1 cells. By immunocytochemical staining of the latter, it became apparent that single cells exhibit wide variations in intensity of specific signals for all the receptors investigated, so that, notably in contrast to primary stromal cells and ST2 cells, MC3T3-E1 display a mosaic pattern of receptor protein expression.
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PMID:Expression of the vitamin D receptor, of estrogen and thyroid hormone receptor alpha- and beta-isoforms, and of the androgen receptor in cultures of native mouse bone marrow and of stromal/osteoblastic cells. 1032 6

The androgen receptor (AR), like other steroid receptors, modulates the activity of the general transcription machinery on the core promoter to exert its function as a regulator. Co-immunoprecipitation of prostate cancer LNCaP cell extract using protein A-Sepharose coupled with anti-AR antibody indicates that the AR interacts with the general transcription factor TFIIH in a physiological condition. Co-transfection of cdk activating kinase (CAK), the kinase moiety of TFIIH, enhanced AR-mediated transcription in a ligand-dependent manner in human prostate cancer PC-3 and LNCaP cells, and in a ligand-independent manner in human prostate cancer DU145 cells. Detailed interaction studies further revealed that the AR NH(2)-terminal domain interacting with CAK was essential for the CAK-induced AR transactivation. Together, our data suggest that the AR may interact with TFIIH for efficient communication with the general transcription factors/RNA polymerase II on the core promoter.
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PMID:From androgen receptor to the general transcription factor TFIIH. Identification of cdk activating kinase (CAK) as an androgen receptor NH(2)-terminal associated coactivator. 1073 72

Gonadal steroids are potent modulators of gonadotropin releasing hormone (GnRH) secretion, and androgen binding sites and 5alpha-reductase activity have been found in the immortalized GnRH secreting cell line GT1-1, suggesting the existence of a direct androgenic control of GnRH dynamics. Two isoforms of the 5alpha-reductase have been cloned with very different biochemical/functional properties: 5alpha-reductase type 1 (widely distributed in the body) and 5alpha-reductase type 2 (confined in androgen target structures). We have analysed whether, in GT1-1, androgen binding sites are linked to "classical" androgen receptor, and which 5alpha-reductase isoform is active. Reverse transcriptase-polymerase chain reaction analysis showed that the mRNAs coding for androgen receptor and for the two 5alpha-reductase isoforms are all expressed in GT1-1 cells. However, the 5alpha-reductase enzymatic reaction showed a peak of activity at a narrow pH around 5.5, the optimum for the 5alpha-reductase type 2. The affinity for testosterone, of the enzyme present in GT1-1 cells, was very similar to that observed for the recombinant type 2 isozyme expressed in yeasts. The data indicate that GT1-1 cells (i) express a "classical" androgen receptor and (ii) contain the 5alpha-reductase type 2 isoform, a specific marker of androgen-responsiveness.
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PMID:5Alpha-reductase type 2 and androgen receptor expression in gonadotropin releasing hormone GT1-1 cells. 1126 23


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