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

We have recently purified a novel pituitary polypeptide, designated 7B2. Subsequently, we developed a sensitive and specific radioimmunoassay (RIA) for this novel polypeptide. Our aim in the present study was to investigate the release of 7B2 from rat pituitary induced by various hypothalamic factors [luteinizing hormone-releasing factor (LH-RH), corticotropin-releasing factor (CRF), and growth hormone-releasing factor (GRF)]. The anterior pituitaries were removed from rats and immediately dispersed enzymatically (a mixture of collagenase/dispase/deoxyribonuclease/chicken serum) and plated on collagen-coated multiwell plates in culture medium containing 10% fetal bovine serum. After 2 days of attachment period, the medium was replaced with fresh medium every 24 h. The primary cell culture was incubated with various concentrations of LH-RH, CRF or GRF. Subsequently, the concentrations of IR-7B2, IR-LH, IR-FSH, and IR-ACTH released into the medium were quantified by specific RIA. LH-RH, at a concentration as low as 7.5 ng/ml (6 X 10(9) M: dose range 7.5-60 ng/ml) stimulated the release of IR-7B2, IR-LH, and IR-FSH, by 2- to 3-fold, 17- to 18-fold, and 3-fold, respectively, over basal levels. No significant increase of IR-7B2 was observed when stimulated by CRF or GRF at doses as high as 100 ng/ml. In addition, K+ (50 mM) stimulated the release of all the peptides measured. In conclusion, our studies suggest that the novel peptide 7B2 is under LH-RH control and indirectly confirm the immunohistochemical results of its cellular co-localization in FSH and LH cells.
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PMID:Specific release of a novel pituitary polypeptide, 7B2, from rat anterior pituitary cells in vitro by luteinizing hormone-releasing hormone. 310 Sep 76

In an earlier study on the long-term effects of the pituitary hormone corticotropin (adrenocorticotropin, ACTH), we reported that ACTH promotes the loss of 20-25% of the total actin in isolated rat adrenocortical cells. In a continuation of this work, we report here the purification to near homogeneity of rat adrenal actin. We demonstrate that the protein purified is adrenal actin, and use this actin in the DNAase immunoprecipitation assay of Snabes et al. to confirm our earlier observation of the effect of ACTH on adrenocortical actin. Finally, we describe several changes made in the original protocol of this immunoassay which we believe significantly improve the accuracy of the assay in estimating actin contents in tissue and cell samples.
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PMID:Purification of rat adrenocortical actin and its use in an immunoprecipitation assay to quantitate cellular actin. 352 78

Erythrocyte ghosts were loaded with pancreatic DNase I and fused with Y-1 adrenal tumor cells to test the possibility that this enzyme might inhibit the steroidogenic responses of the cells to ACTH and cyclic AMP. Fusion of erythrocyte ghosts loaded with DNase I, but not those containing albumin, ovalbumin, boiled DNase I, or DNase I with excess G-actin, inhibited the increase in production of 20 alpha-dihydroprogesterone produced by ACTH and dibutyryl cyclic AMP; inhibition was concentration-dependent with 50% inhibition by 3 X 10(7) molecules of DNase I per cell. It was found that inhibition by DNase I was exerted at the step in the steroidogenic pathway at which cholesterol is transported to mitochondria where steroidogenesis begins. This was shown by measuring transport of cholesterol into the inner mitochondrial membrane, by measuring the production of pregnenolone by isolated mitochondria and by demonstrating that DNase I was without effect on the conversion of pregnenolone to 20 alpha-dihydroprogesterone (an end-product of steroid synthesis). The actin content of Y-1 cells was measured by two methods based upon inhibition of DNase I and by SDS gels following centrifugation. The cells were found to contain 2-3 X 10(7) molecules of actin per cell of which two-thirds is present as G-actin. Since DNase I is known to bind to G-actin to give a one to one complex, these and other findings suggest that at least some of the G-actin in the cells may be necessary for the steroidogenic responses to ACTH and cyclic AMP.
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PMID:Role of actin in the responses of adrenal cells to ACTH and cyclic AMP: inhibition by DNase I. 609 Apr 70

Corticotropin (ACTH) induces a characteristic retraction of rat adrenocortical cells in culture. Densitometric analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels of cell lysates showed that ACTH induced a 20% decrease in actin content. These results were fully confirmed by quantitation of actin by the DNase 1 inhibition assay. The decrease in actin is hormone-specific, concentration-dependent, and correlates temporally with the morphological change induced by ACTH. The change in actin content and the reorganization of microfilament ultrastructure may together mediate hormone regulation of cell shape at the cytoskeletal level.
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PMID:Regulation of actin in rat adrenocortical cells by corticotropin. 625 51

The isolated bovine adrenocortical cells are prepared aseptically by the use of collagenase and deoxyribonuclease. The isolated cells are suspended in Ham F-10 medium containing 5% fetal calf serum, 10% newborn calf serum, 2.5% horse serum and antibiotics. The seeded cells are cultured at 37 degrees C in a humidified atmosphere of 5% CO2 in air. Steroidogenic activity for ACTH reached the maximum in the 2- to 3-day primary cultured cells; the maximum response to ACTH in these cells is more intense than that in freshly isolated bovine adrenocortical cells. The primary cultured cells have prostaglandin, muscarinic, ATP and beta-adrenergic receptors that are linked to steroidogenesis in addition to ACTH and aldosterone receptors. Thus primary cultured bovine adrenocortical cells are a useful tool to study these receptors and the intracellular events that are associated with the receptors. We also demonstrated that the fura 2 loaded primary cultured monolayer cells on glass cover slips provide us much more information than suspended cells in the study of intracellular Ca2+ mobilization in adrenocortical cells.
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PMID:[Primary culture of bovine adrenocortical cells]. 811 88

DNA elements governing transcription of the ovine cytochrome P-450 side-chain cleavage (CYP11A1) gene were investigated. Three overlapping genomic clones for the ovine CYP11A1 gene were isolated and characterized. The transcriptional start site was located 51 nucleotides upstream from the initiating methionine. Gene transfer experiments were conducted in murine adrenocortical Y1 cells and human choriocarcinoma JEG-3 cells using chloramphenicol acetyltransferase reporter gene constructs containing promoter fragments from -2700 to -177 bp. The results demonstrate that DNA elements sufficient to convey a basal level of expression and cyclic AMP (cAMP) responsiveness lie within 177 bp of the transcriptional start, although the possibility that additional regulatory elements reside outside this 177 bp has not been excluded. The ovine 5' flanking sequence demonstrated 92% homology with the bovine sequence, extending over the entire fragment. In contrast, only four significant regions of conservation between the ovine, murine, rat and human CYP11A1 promoters were found. These regions are positioned within 200 bp upstream of the transcriptional start site. DNase 1 footprinting was performed to identify DNA elements able to bind nuclear proteins. Primary adrenocortical and placental tissues from sheep were used as the source of nuclear extracts to detect DNA-protein interactions relevant to CYP11A1 gene expression in vivo. Five regions of protection were detected in the first -634 bp of the ovine CYP11A1 promoter. Three of these elements corresponded to the regions which are well-conserved between species. The other two elements resembled activating protein-1 (AP-1) and AP-4 sites and overlapping AP-2/Sp1 sites, and are conserved in the bovine gene but not in other species. Nuclear protein extracts from adrenals of sheep with different serum ACTH levels (i.e. ACTH-treated, dexamethasone-treated and untreated sheep) protected similar regions of the ovine CYP11A1 promoter fragment. Similarly, the regions protected did not differ when nuclear protein from JEG-3 cells treated with cAMP was compared with that of untreated JEG-3 cells. These results suggest that induction of CYP11A1 gene transcription by ACTH in the ovine adrenal and by cAMP in JEG-3 cells in culture is not mediated by changes in binding of the proteins that interact directly with these footprinted elements. The elements footprinted by extracts from primary ovine tissue lie within the 177 bp sufficient for cAMP-regulated expression. The correspondence of these elements either to regions conserved between species or to known consensus binding sites suggests that these sequences are cis elements involved in regulating transcription of the ovine CYP11A1 gene in vivo.
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PMID:Molecular cloning and characterization of the cyclic AMP-responsive ovine CYP11A1 (cholesterol side-chain cleavage) gene promoter: DNase 1 protection of conserved consensus elements. 837 14

This study investigated the direct effects of hydrocortisone (HS), corticotropin-releasing factor (CRF), and adrenocorticotropin (ACTH) on basal and gonadotropin-releasing hormone (GnRH)-stimulated secretion of follicle-stimulating hormone (FSH) from dispersed pig pituitary cells in vitro. Pig pituitaries were dispersed into cells with collagenase, DNAase, and hyaluronidase and then cultured in McCoy's 5a medium containing horse serum (10%) and fetal calf serum (2.5%) pretreated with dextran-coated charcoal for 3 days. Cells were preincubated with steroids, CRF, or ACTH before GnRH was added. HS did not affect basal FSH secretion after 72 h of incubation. Treatment of pituitary cells with increasing concentrations (0.001-800 micrograms/ml) of HS for 72 h resulted in a dose-dependent decrease in GnRH-stimulated FSH release. HS pretreatment did not cause a change in cellular FSH content. Increasing duration (6-72 h) of treatment with HS (200 micrograms/ml) led to a time-dependent decrease in GnRH-stimulated FSH release, achieving statistical significance by 12 h. Porcine ACTH had no influence on basal and GnRH-stimulated FSH secretion. CRF decreased GnRH-stimulated FSH secretion in a dose-dependent manner, and the inhibitory effect required preincubation (6-18 h) with CRF. HS inhibited the FSH secretory responses to phospholipase C, melittin, and 8-bromo-cAMP but did not affect the response to 1,2-dioctanoyl-sn-glycerol and ionophore A23187. These results indicate that both cortisol and CRF can act directly on pig pituitary to inhibit FSH responsiveness to GnRH.
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PMID:Actions of corticotropin-releasing factor or cortisol on follicle-stimulating hormone secretion by isolated pig pituitary cells. 839 May 96