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)

The mechanisms of estrogen and progesterone in human cutaneous pigmentation are largely unknown. The molecular identification of estrogen receptor (ER) and progesterone receptor (PR) in the human melanocytes is of great importance to understand the mechanisms. We performed immunocytochemistry analysis and demonstrated that ER and PR were expressed in the cytoplasms and nuclei of human melanocytes. Reverse transcriptase-polymerase chain reaction (RT-PCR) and sequence analysis confirmed the expression of ER and PR at the transcriptional level. Despite of the presence of ER and PR, the physiological and pregnant levels of estrogen and progesterone showed inconsistent effects on the proliferation and tyrosinase activity of cultured human melanocytes. These results suggest that human melanocytes express ER and PR, which have a donor-specific action in human pigmentation. Further studies are needed to elucidate the induction mechanism and functions of these receptors, and the role of estrogen and progesterone in melanocytes.
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PMID:Donor specific response of estrogen and progesterone on cultured human melanocytes. 1185 May 90

A complementary DNA (cDNA) encoding the eggshell zona radiata protein (RBTZR: AF407574) has been cloned from the liver of estradiol-17beta (E(2))-treated rainbow trout (Oncorhynchus mykiss) by reverse-transcriptase polymerase chain reaction (RT-PCR). A set of degenerate primers homologous to the highly conserved cysteine-rich region of the zona radiata protein gene from salmon, winter flounder, medaka and carp were used for the initial RT-PCR. The resulting PCR product was cloned, sequenced and identified as the Zrp gene fragment based on amino acid sequence similarities. Based on the Zrp sequence from the initial PCR, a pair of gene-sequence primers was designed for 3'- and 5'- random amplification of cDNA ends (RACE). Cloning and sequencing of RACE products showed a 1349-bp Zrp gene encoding a 403-amino acid protein with a theoretical molecular mass of approximately 45 kDa. Alignment of the deduced amino acid sequence reveals that RbtZR is similar to piscine and mammalian zona pellucida proteins. The RbtZR gene, together with the estrogen receptor (ER) and vitellogenin (Vtg) genes, was further characterized and comparatively studied for transcriptional and translational expression in xenoestrogen- (nonylphenol, NP) and E(2)-treated juvenile rainbow trout in a time-course experiment. Northern and slot blot analysis showed that the RbtZR mRNA was expressed, in parallel with the ER and Vtg mRNA, in both NP- and E(2)-treated juvenile rainbow trout. Indirect enzyme-linked immunosorbent assay (ELISA) using monoclonal antibody raised against Atlantic salmon Zrp indicated the translational expression of RbtZR protein in blood plasma samples from NP- and E(2)-treated juvenile trout. The differential time-dependent transcriptional and translational expression and use of Zrp, ER and Vtg as sensitive biomarkers in environmental monitoring of endocrine disrupters in fish is discussed.
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PMID:Molecular cloning of rainbow trout (Oncorhynchus mykiss) eggshell zona radiata protein complementary DNA: mRNA expression in 17beta-estradiol- and nonylphenol-treated fish. 1203 56

Raloxifene, a selective estrogen receptor (ER) modulator, is a mixed estrogen agonist/antagonist that has been shown to prevent osteoporosis and breast cancer in women. Because the prostate contains high levels of ER-beta, the present study investigated the effect of raloxifene in three well-characterized, androgen-independent human prostate cancer cell lines: (a) PC3; (b) PC3M; and (c) DU145. Reverse transcriptase-PCR and Western blot analysis for ER-alpha and ER-beta demonstrated that all three cell lines express ER-beta, whereas only PC3 and PC3M cells were positive for ER-alpha. After the treatment with raloxifene, a dramatic increase in cell death was observed in a dose-dependent manner in the three prostate cancer cell lines (10(-9) to 10(-6) M range). Because the three prostate cancer cell lines demonstrated similar morphological changes after the raloxifene treatment, PC3 (ER-alpha/ER-beta+) and DU145 (ER-beta+ only) cells were selected to further characterize the raloxifene-induced cell death. Using the nucleus-specific stain 4',6-diamidino-2-phenylindole, nuclear fragmentation was observed in a time-dependent manner in both cell lines after exposure to 10(-6) M raloxifene. Using the terminal deoxynucleotidyl transferase-mediated nick end labeling apoptotic assay, it was demonstrated that the nuclear fragmentation was caused by apoptosis. To investigate the possibility that caspase activation is involved in raloxifene-induced apoptosis, cells were treated with the pan-caspase inhibitor ZVAD. The results demonstrated that the dramatic change in cellular morphology after treatment with raloxifene was no longer observed when cells were pretreated with ZVAD. Immunoblot demonstrated activation of caspases 8 and 9 in PC3 and DU145 cells, respectively. Taken together, these results demonstrate that the mixed estrogen agonist/antagonist, raloxifene, induces apoptosis in androgen-independent human prostate cancer cell lines.
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PMID:Raloxifene, a mixed estrogen agonist/antagonist, induces apoptosis in androgen-independent human prostate cancer cell lines. 1223 8

The 1st step in the action of a steroid hormone involves entering a target cell where it is recognized and bound by a soluble macromolecule called a cytosol receptor (Re) specific for that hormone. The receptor hormone complex (ReS) is translocated to the cell's nucleus (RnS) where it binds to a large number of sites on chromatin. The binding of RnS to acceptor sites is thought to make gene sites available for transcription by RNA polymerase which subsequently results in elevated cellular RNA and protein synthesis. The 3-H steroid exchange assay based on the temperature dependence of the rate of steroid dissociation can be used to differentiate occupied and unoccupied receptors. The method has been used to examine the relationship between nuclear binding of the Rn estradiol complex and the stimulation of growth processes in the rat uterus. A single injection of .2 mcg/100 g body weight of estradiol causes the nuclear accumulation and retention of approximately 10-20% of the total number of uterine Re sites. Nuclear occupancy for 6 or more hours appears to be a requirement for stimulation of late uterotrophic events such as DNA synthesis, sustained stimulation of RNA polymerase activities, and cellular hypertrophy and hyperplasia. Estriol, a short-acting estrogen, has been classified as a weak estrogen, but it acts as such only when administered in a single injection, in which case it stimulates all early uterotrophic events but does not stimulate significant uterine growth. When estriol is present in a continuous fashion it is a highly effective estrogen. Longterm nuclear retention of the RnE complexes is necessary for uterine hypertrophy and hyperplasia. RnE complexes appear to interact with the genome to open gene sites for transcription; continued transcriptional activity is required for full uterine growth. Nonsteroidal estrogen antagonists have estrogen properties in some cells but act as estrogen antagonists in others. Progesterone appears to modify or redirect estrogen action by modulating estrogen receptor levels. Progesterone may act by reducing the level of available cytoplasmic estrogen receptors and hence decreasing the likelihood of receptor binding or by interfering with the nuclear retention of the RnE complex and decreasing the ability of these complexes to stimulate transcriptional events.
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PMID:Steroid hormone receptors and mechanism of action. 1229 12

The biological actions of estrogens are mediated via two distinct intranuclear estrogen receptor (ER) proteins, ERalpha and ERbeta. We have used an in vitro chromatin assembly and transcription system to compare the transcriptional activities of the two ERs in the context of chromatin, the physiological template for transcription by RNA polymerase II. We find that under conditions where many biochemical activities of the receptors are similar (e.g. ligand binding, chromatin binding, chromatin remodeling and co-activator recruitment), liganded ERalpha is a much more potent transcriptional activator than ERbeta with chromatin templates, but not with naked DNA. This difference is attributable to the N-terminal A/B region of ERalpha, which contains a transferable activation function that facilitates transcription specifically with chromatin templates. Interestingly, chromatin selectively restricts ligand-dependent transcriptional activation by ERbeta under some conditions (e.g. with a closed chromatin architecture), while allowing it under other conditions (e.g. with an open chromatin architecture). Collectively, our results define an important role for chromatin in determining signaling outcomes mediated by distinct subtypes of signal-transducing transcriptional activator proteins.
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PMID:Chromatin exposes intrinsic differences in the transcriptional activities of estrogen receptors alpha and beta. 1255 60

Selective estrogen receptor modulators (SERMs) are steroidal or nonsteroidal compounds that can exhibit either estrogen-like agonistic effects or estrogen-antagonistic effects depending on the target tissue. While SERM actions in the breast, bone, and uterus have been well characterized, their effects in the brain are considerably less well understood. Previous work by our laboratory has demonstrated a beneficial effect of tamoxifen in the reduction of ischemic stroke damage in ovariectomized female rats. The present study utilized neuronal cell culture models to attempt to understand the mechanisms of tamoxifen-mediated neuroprotection. Neither physiologic doses of 17beta-E2 nor clinically therapeutic doses of tamoxifen directly protected GT1-7 neurons or purified cultures of rat cerebrocortical neurons from several forms of cell death. Reverse transcriptase polymerase chain reaction and Western blot analysis revealed that GT1-7 neurons possessed both estrogen receptor-alpha (ERalpha) and ERbeta mRNA and protein, whereas purified embryonic rat cortical neurons only expressed appreciable levels of ERalpha transcript and protein, with little to no expression of ERbeta. In contrast to the lack of protection in the purified neuronal cultures, both 17beta- E2 and tamoxifen significantly protected mixed glial/ neuronal cortical cultures from cell death, suggesting that glia may facilitate 17beta-E2-and tamoxifen-mediated neuroprotection. Furthermore, astrocyte-conditioned media and exogenous transforming growth factor-beta1, a documented astrocyte-derived cytokine, were shown to rescue purified cortical neurons from cell death. Together, these findings support a role for astrocytes in neuroprotection and raise the intriguing possibility that astrocytes may help mediate the neuroprotective effect of 17beta-E2 and tamoxifen.
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PMID:Neuroprotective effects of estrogen and tamoxifen in vitro: a facilitative role for glia? 1277 4

Tamoxifen, a breast cancer therapeutic, is a tissue-selective estrogen receptor modulator (SERM), which acts as an antiestrogen in the mammary tissue and displays estrogenic activity in other tissues such as bone and uterus. In order to understand the mechanisms underlying the antiestrogenic effect of this prototype SERM, we performed an analysis of the cofactors that interact with ER complexed with 4-hydroxytamoxifen (OHT) at natural target genes in a human breast tumor cell line MCF-7. Employing chromatin immunoprecipitation (ChIP), we observed that treatment with OHT rapidly induces the binding of ERalpha to the E-responsive promoter regions of pS2 and c-myc genes. Promoter-bound OHT-complexed ERa coordinately recruited the components of a multiprotein complex containing the corepressor NCoR, histone deacetylase 3 (HDAC3), and a WD40-repeat protein TBL1. Surprisingly, the OHT-complexed ERalpha also recruited a chromatin-remodeling NuRD complex in which histone deacetylase 1 (HDAC1) is associated with several polypeptides including metastasis-associated protein 1/2 (MTA1/2), and SWI2/SNF2-related ATPase Mi2. Kinetic studies revealed that following OHT addition the recruitment of these HDAC complexes to pS2 or the c-myc promoter occurs in a sequential manner; the NCoR-HDAC3 complex is recruited earlier than the NuRD complex. Serial ChIP experiments indicated that the ER-NCoR-HDAC3 and ER-NuRD complexes are distinct, and they do not occupy the target gene promoter simultaneously. We also established a close temporal link between the appearance of the HDAC complexes at the E-responsive regions of pS2 and c-myc promoters, local hypoacetylation of specific lysine residues in N-terminal tails of histones H3 and H4, and disappearance of RNA polymerase II from the target gene loci. Collectively, our studies indicated that transcriptional repression by tamoxifen-bound ER at E-regulated gene promoters involves a dynamic interplay of multiple distinct chromatin-modifying/remodeling complexes.
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PMID:Recruitment of distinct chromatin-modifying complexes by tamoxifen-complexed estrogen receptor at natural target gene promoters in vivo. 1472 73

Tamoxifen is the primary hormonal therapy for breast cancer and is also used as a breast cancer chemopreventative agent. A major problem with tamoxifen therapy is undesirable endometrial proliferation. To identify proteins associated with the growth stimulatory effects of tamoxifen in an ER-positive model, the present study profiled total cellular and secreted proteins regulated by estradiol and selective estrogen receptor modifiers (SERMs) in the Ishikawa endometrial adenocarcinoma cell line using two-dimensional gel electrophoresis. Following 24 h incubation with 10(-8) M estradiol, 10(-7) M 4-hydroxytamoxifen, or 10(-7) M EM-652 (Acolbifene), nine proteins exhibited significant increase in expression. The proteins identified were heat shock protein 90-alpha, and -beta, heterogeneous nuclear ribonucleoprotein F, RNA polymerase II-mediating protein, cytoskeletal keratin 8, cytoskeletal keratin 18, ubiquitin-conjugating enzyme E2-18 kDa and nucleoside diphosphate kinase B. These protein profiles may serve as novel indices of SERM response and may also provide insight into novel mechanisms of SERM-mediated growth.
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PMID:Selective estrogen receptor modulator regulated proteins in endometrial cancer cells. 1514 34

Although evidences are emerging that dietary isoflavones have beneficial effects in treatment of hyperlipidemia and cardiovascular diseases, the underlying molecular mechanism has not yet been extensively characterized. In this report, we showed that genistein, one of the major isoflavones, increased expression of genes involved in lipid catabolism such as carnitine palmitoyltransferase 1, liver form (CPT1L) in HepG2 cells, when assayed by real-time reverse-transcriptase polymerase chain reactions as well as Western blotting analysis. The increase in mRNA-level of CPT1L after genistein treatment was not changed in the presence of ICI182780, a potent inhibitor of estrogen receptor, suggesting that this effect of genistein was estrogen receptor-independent. Since these genes involved in fatty acid catabolism are considered putative downstream target genes of peroxisome proliferators-activated receptor alpha (PPARalpha), we examined whether expression of PPARalpha was modulated by genistein treatment. Interestingly, genistein induced expression of PPARalpha at both mRNA- and protein-level. Further, genistein activated transcriptional activity of PPARalpha, when determined by reporter gene analysis, suggesting genistein as a potential ligand for PPARalpha. Taken together, this study provides a picture of the regulatory action of genistein, as an activator of PPARalpha in fatty acid catabolism and potential use of genistein as lipid-lowering agent.
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PMID:Genistein enhances expression of genes involved in fatty acid catabolism through activation of PPARalpha. 1519 99

We sought an in vitro primate model for serotonin neurons. Rhesus monkey embryonic stem (ES) cell colonies were isolated and differentiated into embryoid bodies (EBs), then transferred to serum-free medium with 1% insulin-transferrin-selenium for 7 days to induce neural precursor cell (NPC) formation. NPCs were cultured in medium with 1% N-2 neural supplement and human fibroblast growth factor 2 (FGF2, 10 ng/ml) for 7 days to stimulate cell proliferation. Lastly, NPCs were dispersed into single cells and cultured without FGF2 for another 7 days to obtain terminal differentiation. Terminal cells were characterized for neuronal and serotonergic markers. Over 95% of the NPCs were immunopositive for nestin and Musashi1. Terminally differentiated cells appeared in both small and large morphologies. Most (>95%) of the mature cells (both small and large) were immunopositive for neuron-specific nuclear protein (NeuN), synaptophysin, microtubule-associated protein (MAP2C), Tau-1, neurofilament 160 (NF-160), beta-tubulin (TujIII), tryptophan hydroxylase (TPH), serotonin, the serotonin reuptake transporter (SERT), estrogen receptor-beta (ERbeta), and progestin receptor (PR), but not estrogen receptor-alpha (ERalpha). Less than 2-3% of cells were positive for tyrosine hydroxylase (TH). Reverse transcriptase polymerase chain reaction (RT-PCR) detected mRNA transcripts for TPH-1, TPH-2, SERT, 5-HT1A-autoreceptor, ERbeta, and PR in the differentiated population. A low level of expression of ERalpha mRNA was also detected. Quantitative RT-PCR indicated that the relative abundance of TPH-2 mRNA was greater than TPH-1 mRNA. Serotonin as measured by ELISA increased 3-fold in the mature stage compared to the selection and expansion stages. In summary, a remarkably high percentage of cells derived from monkey ES cells exhibited neuronal plus serotonergic markers as well as nuclear steroid receptors similar to primate CNS serotonin neurons, suggesting that these cells may serve as a useful primate model for serotonergic neurons.
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PMID:Serotonin neurons derived from rhesus monkey embryonic stem cells: similarities to CNS serotonin neurons. 1524 35


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