Sustained mammary gland-directed, ponasterone A-inducible expression in transgenic mice.
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The ability to regulate temporal- and spatial-specific expression of target genes in transgenic mice will facilitate analysis of gene function and enable the generation of murine models of human diseases. The genetic analysis of mammary gland tumorigenesis requires the development of mammary gland-specific transgenics, which are tightly regulated throughout the adult mammary epithelium. Analysis of genes implicated in mammary gland tumorigenesis has been hampered by mosaic transgene expression and the findings that homozygous deletion of several candidate genes (cyclin D1, Stat5A, prolactin receptor) abrogates normal mammary gland development. We describe the development of transgenic mouse lines in which sustained transgene expression was inducibly regulated, both specifically and homogeneously, in the adult mammary gland epithelium. Transgenes encoding RXRalpha and a chimeric ecdysone receptor under control of a modified MMTV-LTR, which targets mammary gland expression, were used. These transgenic 'receptor' lines were crossed with transgenic 'enhancer' lines in which the ecdysone/RXR binding site induced ligand-dependent expression of transgenic beta-galactosidase. Pharmacokinetic analysis of a highly bioactive ligand (ponasterone A), identified through screening ecdysteroids from local plants, demonstrated sustained release and transgene expression in vivo. This transgenic model with both tightly regulated and homogeneous transgene expression, which was sustained in vivo using ligands readily extracted from local flora, has broad practical applicability for genetic analysis of mammary gland disease. (+info)
Molecular determinants of differential ligand sensitivities of insect ecdysteroid receptors.
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The functional receptor for insect ecdysteroid hormones is a heterodimer consisting of two nuclear hormone receptors, ecdysteroid receptor (EcR) and the retinoid X receptor homologue Ultraspiracle (USP). Although ecdysone is commonly thought to be a hormone precursor and 20-hydroxyecdysone (20E), the physiologically active steroid, little is known about the relative activity of ecdysteroids in various arthropods. As a step toward characterization of potential differential ligand recognition, we have analyzed the activities of various ecdysteroids using gel mobility shift assays and transfection assays in Schneider-2 (S2) cells. Ecdysone showed little activation of the Drosophila melanogaster receptor complex (DmEcR-USP). In contrast, this steroid functioned as a potent ligand for the mosquito Aedes aegypti receptor complex (AaEcR-USP), significantly enhancing DNA binding and transactivating a reporter gene in S2 cells. The mosquito receptor also displayed higher hormone-independent DNA binding activity than the Drosophila receptor. Subunit-swapping experiments indicated that the EcR protein, not the USP protein, was responsible for ligand specificity. Using domain-swapping techniques, we made a series of Aedes and Drosophila EcR chimeric constructs. Differential ligand responsiveness was mapped near the C terminus of the ligand binding domain, within the identity box previously implicated in the dimerization specificity of nuclear receptors. This region includes helices 9 and 10, as determined by comparison with available crystal structures obtained from other nuclear receptors. Site-directed mutagenesis revealed that Phe529 in Aedes EcR, corresponding to Tyr611 in Drosophila EcR, was most critical for ligand specificity and hormone-independent DNA binding activity. These results demonstrated that ecdysone could function as a bona fide ligand in a species-specific manner. (+info)
Puffing activities and binding of ecdysteroid to polytene chromosomes of Drosophila melanogaster.
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Salivary glands of third instar Drosophila melanogaster larvae were incubated in vitro in the presence of 5 x 10(-6) M 20-hydroxy-ecdysone. Steroid hormone was localized on the polytene chromosomes of the salivary gland by a combination of photoaffinity-labeling and indirect immunofluorescence microscopy. Steroid hormone binding to chromosomal loci and their puffing activity was correlated for the larval/prepupal puffing cycle characterized by puff stages 1-10. In general, there was a good correlation between the sequential and temporal puffing activity induced by 20-hydroxy-ecdysone and the binding of ecdysteroid hormone to these puffs. Ecdysteroid hormone was detected at intermolt, and at early and late puffs with two notable exceptions. Ecdysteroid was not detected at the two well-studied puffs at 23E and at 25AC, the former being an early puff, which is activated in the presence of 20-hydroxy-ecdysone, and the latter being an intermolt puff, which regresses more rapidly in the presence of hormone. Ecdysteroid hormone was present at puffs as long as the respective puff was active. Also, it apparently accumulated at late puff sites after induction. Since ecdysteroid binding to chromosomal loci is temporal as well as sequential during the larval/prepupal puffing cycle, additional factors besides steroid hormone are necessary for sequentially regulating puffing and concomitant gene activity during development from larvae to prepupae. (+info)
A new model for 20-hydroxyecdysone and dibenzoylhydrazine binding: a homology modeling and docking approach.
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The ecdysone receptor (ECR), a nuclear transcription factor controlling insect development, is a novel target for insecticides such as dibenzoylhydrazines with low environmental and toxicological impacts. To understand the high selectivity of such synthetic molecules toward ECR, two homology models of the Chironomus tentans ECR ligand-binding domain (LDB) have been constructed by taking as templates the known LBD crystal structures of the retinoic acid and vitamin D receptors. Docking of 20-hydroxyecdysone (20E) and dibenzoylhydrazines to the receptor suggests a novel superposition of the natural and synthetic molecules; the N-tert-butyl substituent of the dibenzoylhydrazines extends significantly beyond the 20E volume. Our ECR-LBD protein models rationalize how 20E and dibenzoylhydrazines interact with the ligand-binding pocket. The homology model complexes provide new insights that can be exploited in the rational design of new environmentally safe insecticides. (+info)
Deltorphin II-induced rapid desensitization of delta-opioid receptor requires both phosphorylation and internalization of the receptor.
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Similar to other G protein-coupled receptors, rapid phosphorylation of the delta-opioid receptor in the presence of agonist has been reported. Hence, agonist-induced desensitization of the delta-opioid receptor has been suggested to be via the receptor phosphorylation, arrestin-mediated pathway. However, due to the highly efficient coupling between the delta-opioid receptor and the adenylyl cyclase, the direct correlation between the rates of receptor phosphorylation and receptor desensitization as measured by the adenylyl cyclase activity could not be established. In the current studies, using an ecdysone-inducible expression system to control the delta-opioid receptor levels in HEK293 cells, we could demonstrate that the rate of deltorphin II-induced receptor desensitization is dependent on the receptor level. Only at receptor concentrations +info)
Conserved molecular mechanism for the stage specificity of the mosquito vitellogenic response to ecdysone.
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In the mosquito Aedes aegypti, the adult female becomes competent for a vitellogenic response to ecdysone after previtellogenic development. Here, we show that betaFTZ-F1, the nuclear receptor implicated as a competence factor for stage-specific responses to ecdysone during Drosophila metamorphosis, serves a similar function during mosquito vitellogenesis. AaFTZ-F1 is expressed highly in the mosquito fat body during pre- and postvitellogenic periods when ecdysteroid titers are low. The mosquito AaFTZ-F1 transcript nearly disappears in mid-vitellogenesis when ecdysteroid titers are high. An expression peak of HR3, a nuclear receptor implicated in the activation of betaFTZ-F1 in Drosophila, precedes each rise in mosquito FTZ-F1 expression. In in vitro fat body culture, AaFTZ-F1 expression is inhibited by 20-hydroxyecdysone (20E) and superactivated by its withdrawal. Following in vitro AaFTZ-F1 superactivation, a secondary 20E challenge results in superinduction of the early AaE75 gene and the late target VCP gene. Electrophoretic mobility-shift assays show that the onset of ecdysone-response competence in the mosquito fat body is correlated with the appearance of the functional AaFTZ-F1 protein at the end of the previtellogenic development. These findings suggest that a conserved molecular mechanism for controlling stage specificity is reiteratively used during metamorphic and reproductive responses to ecdysone. (+info)
Ecdysteroid coordinates optic lobe neurogenesis via a nitric oxide signaling pathway.
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Proliferation of neural precursors in the optic lobe of Manduca sexta is controlled by circulating steroids and by local production of nitric oxide (NO). Diaphorase staining, anti-NO synthase (NOS) immunocytochemistry and the NO-indicator, DAF-2, show that cells throughout the optic anlage contain NOS and produce NO. Signaling via NO inhibits proliferation in the anlage. When exposed to low levels of ecdysteroid, NO production is stimulated and proliferation ceases. When steroid levels are increased, NO production begins to decrease within 15 minutes independent of RNA or protein synthesis and cells rapidly resume proliferation. Resumption of proliferation is not due simply to the removal of NO repression though, but also requires an ecdysteroid stimulatory pathway. The consequence of these opposing pathways is a sharpening of the responsiveness to the steroid, thereby facilitating a tight coordination between development of the different elements of the adult visual system. (+info)
Synthesis of a molt-inhibiting hormone of the American crayfish, Procambarus clarkii, and determination of the location of its disulfide linkages.
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A molt-inhibiting hormone (Prc-MIH) of the American crayfish, Procambarus clarkii, a member of the type II CHH family, was chemically synthesized and the location of its three disulfide linkages was determined. Prc-MIH consists of 75 amino acid residues and was synthesized by a thioester method. Two peptide segments, Boc-[Cys(Acm)(7,24,27), Lys(Boc)(19)]-Prc-MIH(1-39)-SCH(2)CH(2)CO-Nle-NH(2) and H-[Cys(Acm)(40,44,53), Lys(Boc)(42,51,67)]-Prc-MIH(40-75)-NH(2), were prepared using peptides obtained via the Boc solid-phase method. Condensation of the building blocks in the presence of silver chloride, 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine, and N, N-diisopropylethylamine, followed by removal of the protecting groups, gave the reduced form of Prc-MIH(1-75)-NH(2). This product was converted to the native form of Prc-MIH (synthetic Prc-MIH) in a buffer which contained cysteine and cystine. The synthetic Prc-MIH showed the same behavior by RP-HPLC and biological activity assays as the natural Prc-MIH. The disulfide bond between Cys7 and Cys44 was determined by isolation of a fragment from an enzymatic digest of the synthetic Prc-MIH by RP-HPLC, followed by mass analysis. The disulfide bonds between Cys24 and Cys40 and between Cys27 and Cys53 were determined by comparing the elution position of an enzymatic digest of the synthetic Prc-MIH with authentic chemically synthesized samples, which contained three types of possible disulfide linkages. (+info)