Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.21.1 (DNase)
 7,655 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A cell-free system prepared from the estrogen-primed chick oviduct was developed and used to study the uptake of cytoplasmic progesterone-receptor complex by isolated nuclei. The receptor and purified nuclei were shown to be stable at 25 degrees, but not at 37 degrees. Thus, nuclear incubations were routinely performed at 25 degrees. Such incubations revealed greater nuclear uptake of the cytoplasmic hormone-receptor complex as compared to control incubations performed at 0 degrees. The uptake process showed a quantitative preference for oviduct nuclei. No net uptake occurred during 0 degrees incubations when the nuclei were preincubated in the absence of cytoplasmic components at 25 degrees. In contrast, the temperature requirement was partially removed by preincubation of the hormone-receptor complex at 25 degrees prior to incubation with nuclei at 0 degrees. Nuclear uptake was not accompanied by measurable alterations in the sedimentation properties of the progesterone receptor. The activation and nuclear uptake of receptor was clearly dependent upon prior binding of steroid hormone to the receptor indicating that the active nuclear form of the receptor could not be generated in the absence of the hormone. Receptor precipitation with ammonium sulfate also partially removed the temperature requirement for nuclear binding. In contrast to temperature activation, ammonium sulfate precipitation activated the receptor in the absence of hormone. It thus seemed likely that temperature and salt activation of receptor occurred via different mechanisms. Although we were able to destroy up to 60% of the nuclear DNA content by treatment with DNase prior to nuclear incubation, some 80 to 85% of the receptor-binding capacity was still present in the treated nuclei. Thus, chick progesterone receptors apparently bind to a relatively DNase-resistant portion of the oviduct genome. The properties of this system indicate its value for further investigation into the initial events of progesterone action in the chick oviduct.
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PMID:Progesterone-binding components of chick oviduct. VIII. Receptor activation and hormone-dependent binding to purified nuclei. 16 39

Several classes of specific progesterone receptor (PR) nuclear binding sites (acceptor sites) have previously been identified in avian oviduct chromatin on the basis of different binding affinities. Recently, two classes of acceptor proteins (AP) that are associated with these binding sites in the avian oviduct have been identified. These APs were termed receptor binding factors (RBF-1 and -2), and one (RBF-1) has been purified [Schuchard et al. (1991) Biochemistry 30, 4535-4542]. The RBF-1 is associated with the highest affinity class of sites in the intact chromatin, and the RBF-2 is associated with the second highest affinity class of sites. The PR binding sites and their associated RBF-2 protein remain with the residual chromatin fraction following extraction by 4 M Gdn-HCl. This Gdn-HCl-treated chromatin has been termed nucleoacidic protein (NAP). This paper describes the 200-fold enrichment of the native RBF-2 class of PR acceptor sites beginning with the DNase I digestion of NAP to obtain DNase-resistant fragment (NAPf) containing approximately 150 bp of DNA. The PR binding sites are further enriched by high-performance or fast protein liquid chromatography and chromatofocusing. Anti-RBF-1/RBF-2 protein antibodies identify antigens that coelute with the PR binding activity. Hybridization analysis of the DNAf from the enriched NAPf demonstrates sequence homologies with the nuclear matrix DNA as well as with genomic sequences of the rapid steroid responding nuclear protooncogenes c-myc and c-jun. However, comparative analyses of the whole genomic DNA with the nuclear matrix DNA indicate that the RBF-2 (NAPf) is largely nonnuclear matrix.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Enrichment of a second class of native acceptor sites for the avian oviduct progesterone receptor as intact chromatin fragments. 189 51

The transcription of the progesterone receptor gene is induced by estrogens and decreased by progestins. Studies were performed to define the regions of the gene and the molecular mechanisms involved. No hormonal regulation could be observed using 5' flanking regions of the gene up to -2762 in front of a heterologous gene. Estrogen and progestin regulation could be observed only when using fragments of the gene extending down to +788. Progressive deletions from the 5' and 3' ends, site-directed mutagenesis and DNase protection experiments with purified estrogen receptor suggested that the biologically active estrogen responsive element (ERE) is present at +698/+723, overlapping the initiation of translation. An oligonucleotide was synthesized bearing this ERE and shown to impart estrogen inducibility to a heterologous gene. Its regulation by anti-estrogens corresponded to that of the in situ progesterone receptor gene since tamoxifen was a partial agonist whereas ICI 164384 was a full antagonist. This ERE also mediated down-regulation by progestins in the presence of the progesterone receptor, even though it has no progesterone receptor binding ability. DNase footprinting showed that this effect was not due to a decrease of estrogen receptor affinity for the ERE in the presence of progesterone receptor. Finally, use of deletion mutants of the progesterone receptor showed that the steroid binding and the DNA binding domains were necessary for down-regulation whereas deletions of various parts of the N-terminal domain were without effect.
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PMID:Characterization of the hormone responsive element involved in the regulation of the progesterone receptor gene. 205 Jan 23

We have studied by immunocytochemistry and monoclonal antibodies the presence and localization of estrogen receptors, progesterone receptors, and a 24-kD estrogen-regulated heat shock protein in biopsies from breast and endometrial cancer patients. Three different tissue processing protocols were used to colocalize the antigens in the same tissue sections: a) frozen sections, b) formalin fixation with routine paraffin embedding, and c) picric acid-formaldehyde (PAF) fixation with a rapid embedding in paraffin. Frozen sections showed good receptor staining but poor 24-kD protein immunoreactivity, while routine paraffin sections (with or without DNase pretreatment) were inadequate to reveal the nuclear receptor proteins at the same level seen in frozen sections. On the other hand, all three proteins could be detected satisfactorily in PAF-fixed paraffin-embedded tissue. Using this procedure we were able to visualize 24-kD protein and estrogen receptor or progesterone receptor in individual cells in paraffin sections. The study revealed that in all of the estrogen receptor positive breast and endometrial tumor samples, almost 90% of the cells expressing the cytoplasmic 24-kD protein contained estrogen receptor in the cell nucleus. In contrast, 24-kD immunoreactive cells did not express progesterone receptors in almost 40% of the progesterone receptor positive tumor samples.
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PMID:Colocalization of estrogen and progesterone receptors with an estrogen-regulated heat shock protein in paraffin sections of human breast and endometrial cancer tissue. 208 75

Monoclonal antibodies (mAB) against progesterone receptor (PR) and the peroxidase-antiperoxidase (PAP) method to visualize PR in paraffin sections from 68 human breast cancers were used. Ten mAB, which recognize human PR on frozen sections, were tested. Six could detect PR in paraffin sections, with Li 417, LET 456, and LET 126 giving the best results. LET 126 antibody was used for most further studies. The effects of fixation with picric acid-formaldehyde (PAF), buffered-formalin, or with Bouin solution were investigated; all fixation methods allowed PR immunolabeling, although PAF or buffered formalin usually gave the best results. Positive staining was seen in the nucleus of carcinoma cells. Variations in intensity and extent of immunoreactivity were observed in all sections and among different regions of the same specimen. These were probably related to the heterogeneity of the tumor cell population. Results were compared with the PR content in the respective tumor tissues, determined by steroid-binding assay, and with immunocytochemistry on frozen sections. It was shown that there were correlations between the immunocytochemical staining (positive or negative) and the steroid binding assay (80%) and between the immunocytochemical staining on paraffin sections and on frozen sections (78%). Weaker intensity and fewer number of PR-positive cells were found for paraffin-embedded tumors. Estrogen receptors were also detected on adjacent sections from the same paraffin-embedded tissues by use of monoclonal anti-ER antibodies (ERICA-kit[Abbott Laboratories, Chicago, IL]) and DNase pretreatment. In conclusion, this immunocytochemical method for detection of PR and ER on paraffin sections offers an alternative to frozen tissue. It allows histologic and immunocytochemical studies on the same sample and retrospective studies on stored tissue blocks.
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PMID:Immunocytochemical staining of progesterone receptor in paraffin sections of human breast cancers. 267 23

It has been reported that the response of target cells to steroid hormone (SH) stimulation may depend on their position in the cell cycle. The DNA and RNA contents of malignant cells of the endometrium cultured in vitro were measured using flow cytometry (FCM). We also measured estrogen receptor (ER) and progesterone receptor (PR) levels of cells at different positions in the cell cycle. The G1 and S phases of the cell cycle were investigated using cells synchronized by sodium n-butyrate (G1 block), methotrexate (S block), and excess thymidine (S block). For DNA measurements, the cells were stained with propidium iodide following RNase treatment. For RNA measurements (double-stranded RNA) the cells were treated with DNase. We found that S phase synchronization by methotrexate was 136.2% of control (100%). Using the excess thymidine block and release procedure, the S phase fraction was 185.1% of control. G1 phase synchronization by sodium n-butyrate was 134% of control. The estrogen receptor level in G1 phase synchronized cells increased to 5.94 fmol/micrograms DNA in the cytosol and 12.35 fmol/micrograms DNA in the nuclear fraction. These levels represent a sevenfold total increase over that of the control estrogen receptor level. Cells in S phase showed no significant increase in estrogen receptor levels over control cells. Based on this study, the functional increase of the steroid receptor was most significant in the G1 phase.
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PMID:Relationship between changes of the steroid receptor and synchronization in human endometrial adenocarcinoma cells in vitro. 320 23

A cell-free system was used to characterize the binding reaction between the progesterone receptor and nuclear acceptor sites prepared from rat placenta. Two forms of receptor-acceptor complex were examined. One was extracted from nuclei by exposure to 0.6 M KCl; the other type was resistant to salt extraction. Kinetic analysis indicated that the binding reactions were saturable (3-4 pmol binding sites/mg DNA) and of high affinity (Kd = 3-6 nM). Acceptor binding was specific for placental nuclei and did not occur with nuclei prepared from spleen or with denatured nuclei from placenta. Acceptor sites were further characterized by their sensitivity to RNase, DNase I, and protease. RNase treatment had no influence on receptor-acceptor binding. However, DNase I reduced the number of KCl-resistant acceptor sites by 41%, but only a 19% reduction occurred in KCl-extractable acceptor sites (P less than 0.05). Protease removed 34% and 48% of the KCl-resistant and -extractable acceptor sites, respectively, and combined treatment with DNase and protease eliminated 76% of acceptor-binding activity. The endogenous inhibitor previously described from rat placental cytosol blocked acceptor-binding sites in a concentration-dependent manner, a decrease of 1.15 pmol sites/mg inhibitor protein for resistant sites and 0.76 pmol/mg inhibitor protein for extractable sites. However, receptor-acceptor binding was not altered by treating nuclei with actinomycin D or chloroquine. Mercurial reagents reduced receptor-acceptor interaction by 80% and 94% in KCl-resistant and -extractable sites, respectively, whereas sulfhydryl alkylating agents reduced binding 35% and 76%. Pyridoxal phosphate destroyed 88-93% of acceptor binding. The results of these studies suggest that the progesterone receptor acceptor sites are composed of a complex of chromatin protein and DNA in rat placenta. Furthermore, the binding reaction requires the participation of sulfhydryl and terminal amino groups.
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PMID:Nuclear acceptor sites for progesterone-receptor complexes in rat placenta. 329 40

The steroid receptor interactions in vitro with specific acceptor sites composed of acceptor protein-DNA complexes fulfill many of the criteria of a physiologically significant binding system. Chromatin acceptor sites for many steroid receptors (especially for the progesterone and estrogen receptor) are specific since they are saturable and competitive with unlabelled receptors, have high affinity for the receptor, distinguish between functional and nonfunctional receptors and demonstrate target tissue specificity. Pure DNA as acceptor sites does not display many of these properties. Therefore, it is clear that certain chromatin proteins provide the necessary specificity for the acceptor sites for the steroid receptors. For the progesterone receptor in the chick oviduct, these nuclear sites appear to contain specific chromosomal proteins as well as specific DNA sequences. The substitution of other chromosomal proteins or the genomic DNAs from evolutionarily distant organisms results in a loss of the specific nuclear binding. The nuclear acceptor sites appear to be resistant to the DNase activity which is not characteristic of transcriptionally active domains of the genome. Further studies using the ovalbumin gene sequences from genomic clones also indicate that none of the sequences within this domain and the 3-k flanking regions appear to contain the specific acceptor sequences. These observations have led to development of a model suggesting that the steroid receptors bind to acceptor sites distant from the structural genes the steroids ultimately regulate. Neighboring these acceptor sites are regulatory genes which code for regulatory substances which in turn (as secondary messengers) regulate at great distances the expression of the structural gene. This model might better fit the sex steroids which require 1-2 h to measurably alter gene transcription, as opposed to the glucocorticoids which more rapidly alter gene expression.
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PMID:Nuclear acceptor sites for sex steroid hormone receptors in chromatin. 369 76

Monoclonal antibodies which block the binding of the avian oviduct progesterone receptor (PR) to nuclear acceptor sites have been prepared. We have previously shown that such acceptor sites consist of complexes of specific nonhistone proteins and DNA. The antigen was a reconstituted, enriched nuclear site for avian oviduct PR formed by reannealing a partially purified acceptor protein to pure hen DNA to reconstitute native-like acceptor sites. These reconstituted acceptor sites were then partially digested with deoxyribonuclease I and injected into BALB/c mice. The spleen cells were fused with NS-1 mouse myeloma cells. Hybridomas were then grown and tested for the ability of their culture media to 1) inhibit PR binding to avian oviduct nucleoacidic protein (NAP) which contains the native-like acceptor sites, but 2) to not inhibit PR binding to pure hen DNA. Three hybridoma clones produced ascites fluids which inhibited PR binding to intact oviduct chromatin and NAP but not to pure hen DNA. Control ascites fluids, prepared against other protein antigens, showed no inhibition of PR binding. The three positive ascites fluids contained low concentrations of immunoglobulin (0.3-0.5 mg/ml). The antibodies did not affect the stability of the receptor and did not interact with PR when analyzed by sucrose density gradient sedimentation. Direct binding of the antibodies to the NAP is shown by an enzyme-linked immunosorbent assay. The monoclonal antibodies display a partial species specificity with regard to the source of NAP in that PR binding to hen oviduct NAP was inhibited by greater than 90%, while PR binding to human uterine NAP was inhibited less than 40% by the antibodies. Further characterization of these candidate antiacceptor site monoclonal antibodies with regard to tissue, species, and steroid receptor specificities are underway.
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PMID:Monoclonal antibodies recognizing the nuclear binding sites of the avian oviduct progesterone receptor. 369 12

Immunocytochemical study in paraffin sections of human endometrium showed that the receptor contents for both oestrogen and progesterone receptors were lower than in the frozen sections although the staining patterns were similar in these two section types. Pretreating the specimens with proteolytic enzymes like trypsin, DNase and pronase improved the oestrogen receptor staining but a better result with progesterone receptor staining was obtained when no enzymatic pretreatment was applied to the sections.
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PMID:Oestrogen and progesterone receptors in normal human endometrium: comparison of immunocytochemical analysis on frozen and paraffin sections with or without enzymatic pretreatment. 763 70


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