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1.
Nucleic Acids Res ; 48(7): 3476-3495, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32052053

RESUMO

The MLR COMPASS complex monomethylates H3K4 that serves to epigenetically mark transcriptional enhancers to drive proper gene expression during animal development. Chromatin enrichment analyses of the Drosophila MLR complex reveals dynamic association with promoters and enhancers in embryos with late stage enrichments biased toward both active and poised enhancers. RNAi depletion of the Cmi (also known as Lpt) subunit that contains the chromatin binding PHD finger domains attenuates enhancer functions, but unexpectedly results in inappropriate enhancer activation during stages when hormone responsive enhancers are poised, revealing critical epigenetic roles involved in both the activation and repression of enhancers depending on developmental context. Cmi is necessary for robust H3K4 monomethylation and H3K27 acetylation that mark active enhancers, but not for the chromatin binding of Trr, the MLR methyltransferase. Our data reveal two likely major regulatory modes of MLR function, contributions to enhancer commissioning in early embryogenesis and bookmarking enhancers to enable rapid transcriptional re-activation at subsequent developmental stages.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Elementos Facilitadores Genéticos , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Coativadores de Receptor Nuclear/metabolismo , Animais , Linhagem Celular , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Ecdisona/farmacologia , Histona-Lisina N-Metiltransferase/metabolismo , Coativadores de Receptor Nuclear/fisiologia , Regiões Promotoras Genéticas , Ativação Transcricional
2.
Nat Commun ; 10(1): 5634, 2019 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-31822677

RESUMO

The blood-feeding behavior of Anopheles females delivers essential nutrients for egg development and drives parasite transmission between humans. Plasmodium growth is adapted to the vector reproductive cycle, but how changes in the reproductive cycle impact parasite development remains unclear. Here, we show that the bloodmeal-induced miR-276-5p fine-tunes the expression of branched-chain amino acid transferase to terminate the reproductive cycle. Silencing of miR-276 prolongs high rates of amino acid (AA) catabolism and increases female fertility, suggesting that timely termination of AA catabolism restricts mosquito investment into reproduction. Prolongation of AA catabolism in P. falciparum-infected females also compromises the development of the transmissible sporozoite forms. Our results suggest that Plasmodium sporogony exploits the surplus mosquito resources available after reproductive investment and demonstrate the crucial role of the mosquito AA metabolism in within-vector parasite proliferation and malaria transmission.


Assuntos
Anopheles/fisiologia , MicroRNAs/metabolismo , Plasmodium falciparum/crescimento & desenvolvimento , Aminoácidos/metabolismo , Animais , Anopheles/efeitos dos fármacos , Sequência de Bases , Ecdisona/farmacologia , Corpo Adiposo/metabolismo , Feminino , Inativação Gênica , MicroRNAs/genética , Modelos Biológicos , Reprodução/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Esteroides/metabolismo , Transaminases/metabolismo
3.
Chemosphere ; 237: 124551, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31549662

RESUMO

To characterize the potential endocrine-disrupting chemicals (EDCs) in the environment that interact with the crustacean ecdysone receptor (EcR), we established a method involving in silico modeling/molecular docking and in vitro reporter gene assay. Cherry shrimp (Neocaridina davidi) EcR (NdEcR) and retinoid X receptor (NdRxR) were identified and cloned for use in this method. A theoretical 3D model of NdEcR ligand-binding domain (LBD) was built in silico based on sequence homology with the established X-ray structure of insect EcR. The interaction of the NdEcR LBD with ecdysteroids, diacylhydrazine (DAH) pesticides, and other potential EDCs was evaluated using molecular docking programs. The results revealed that the ligand-binding pocket in the NdEcR LBD was flexible and adaptive for accommodating ligands of different shapes. The agonistic and antagonistic activities of the candidate compounds were further assessed by in vitro reporter gene assay using human cell lines transiently transfected with NdEcR and NdRxR expression plasmids and a reporter plasmid containing synthesized ecdysone response element. The assay was validated by the dose-dependent responses of EcR-mediated gene transcription after treating the transfected cell lines with ecdysteroids, 20-hydroxyecdysone, and ponasterone A. Examination of the candidate compounds using the reporter gene assay revealed restricted functional specificity to ecdysteroids and DAHs. Three of the tested DAH pesticides originally targeting the insect EcR were found to be weak agonists and strong antagonists of NdEcR. These results suggest that DAHs are potential EDCs for crustaceans that disrupt their ecdysteroid signals by functioning as EcR agonists or antagonists.


Assuntos
Crustáceos/efeitos dos fármacos , Ecdisteroides/farmacologia , Praguicidas/toxicidade , Receptores de Esteroides/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Simulação por Computador , Crustáceos/metabolismo , Decápodes/genética , Ecdisona/metabolismo , Ecdisona/farmacologia , Ecdisteroides/toxicidade , Ecdisterona/análogos & derivados , Ecdisterona/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Humanos , Simulação de Acoplamento Molecular , Praguicidas/química , Praguicidas/metabolismo , Filogenia , Receptores de Esteroides/agonistas , Receptores de Esteroides/antagonistas & inibidores , Receptores de Esteroides/genética , Receptores X Retinoide/química , Receptores X Retinoide/genética , Receptores X Retinoide/metabolismo
4.
Int Immunopharmacol ; 73: 405-413, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31152978

RESUMO

The search for new biologically active compounds is a topic of current research because of their ubiquitous availability and low toxicity. Plants of the Silene genus contain secondary metabolites known as phytoecdysteroids that reportedly have various biological activities. α-Ecdysone is a phytoecdysteroid with biological activity that has not been thoroughly investigated to date. Therefore, we investigated the immunomodulatory and anti-inflammatory effects of α-ecdysone on LPS-treated RAW264.7macrophage cells and in a zebrafish model. To explore these activities, RAW264.7 cells were pretreated with α-ecdysone (0.1-10 µM) for 24 h and then with LPS to induce inflammation. We assayed membrane fluidity, lysosomal enzyme activity, and superoxide generation to determine the immunomodulatory activity. Using ELISA, we examined the levels of the pro-inflammatory cytokines prostaglandin (PGE2) and interleukin-1ß (IL-1ß), as well as the protein expression of cyclooxygenase-2 (COX-2), tumor necrosis factor-alpha (TNF-α) and heme­oxygenase-1 (HO-1) by immunoblotting. We also investigated the subcellular localization of the nuclear transcription factor (NF-κB) subunits and expression of the mitogen-activated protein kinase (MAPK) pathway. We found that α-ecdysone is a potent immunostimulator that enhances membrane fluidity and lysosomal enzyme activity and generates superoxide anions. Simultaneously, α-ecdysone inhibited nitric oxide levels and suppressed the levels of pro-inflammatory mediators and cytokines. Furthermore, α-ecdysone increased HO-1 and nuclear factor erythroid 2-related factor (Nrf2) production, mitigated NF-κB subunit proteins in the nucleus and decreased MAPKs and Akt activation. These results suggest that α-ecdysone is a good immunostimulator with anti-inflammatory effects that occur via inhibition of pro-inflammatory mediators and cytokines through stimulation of HO-1 and Nrf-2 production.


Assuntos
Anti-Inflamatórios/farmacologia , Ecdisona/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , Animais , Heme Oxigenase-1/metabolismo , Larva , Lipopolissacarídeos/farmacologia , Proteínas de Membrana/metabolismo , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Células RAW 264.7 , Transdução de Sinais/efeitos dos fármacos , Coluna Vertebral/anormalidades , Peixe-Zebra
5.
J Asian Nat Prod Res ; 21(3): 217-226, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30739491

RESUMO

Two new azafluoranthene alkaloids (1 and 2), and a new phytoecdysone (3), were isolated from the stems of Cyclea barbata Miers, together with six known compounds (4-9). Their structures were elucidated by spectroscopic data analysis and comparison with published data. This is the first report of azafluoranthene alkaloids (1 and 2) and phytoecdysones (3, 8, and 9) from Cyclea genus. In in vitro bioassay, four isolates (3, 5, 6, and 9) showed moderate hepatoprotective activity against N-acetyl-p-aminophenol (APAP)-induced toxicity in HepG2 cells.


Assuntos
Alcaloides/química , Antineoplásicos Fitogênicos/química , Cyclea/química , Ecdisona/química , Fitosteróis/química , Alcaloides/isolamento & purificação , Alcaloides/farmacologia , Antineoplásicos Fitogênicos/isolamento & purificação , Antineoplásicos Fitogênicos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Ecdisona/farmacologia , Células Hep G2 , Humanos , Modelos Moleculares , Estrutura Molecular , Fitosteróis/farmacologia
6.
Gen Comp Endocrinol ; 278: 68-78, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-30243885

RESUMO

Ecdysone, diapause hormone and a diapause hormone analog are all capable of breaking pupal diapause and prompting initiation of adult development in the cotton earworm, Helicoverpa zea. In this study we asked whether these three chemically-distinct diapause terminators elicit the same effect on expression of a collection of microRNAs and transcripts encoding components of the ecdysone signaling pathway. Injection of all three endocrine agents resulted in downregulation of one miRNA, miR-277-3p, a miRNA previously linked to the insulin/FOXO signaling pathway, and all three agents promoted upregulation of spook, a member of the ecdysone biosynthesis pathway, and iswi, an ecdysone-responsive transcript. Other miRNA and mRNA responses varied depending on the agent used to terminate diapause, thus suggesting that different endocrine pathways and mechanisms can lead to the same final developmental response.


Assuntos
Diapausa/genética , Ecdisona/farmacologia , MicroRNAs/metabolismo , Mariposas/crescimento & desenvolvimento , Mariposas/genética , Neuropeptídeos/farmacologia , Zea mays/parasitologia , Animais , Diapausa/efeitos dos fármacos , Ecdisona/biossíntese , Ecdisterona/farmacologia , MicroRNAs/genética , Mariposas/efeitos dos fármacos , Pupa/efeitos dos fármacos , Pupa/crescimento & desenvolvimento , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
7.
Cell Physiol Biochem ; 49(4): 1633-1645, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30227391

RESUMO

BACKGROUND/AIMS: Ecdysteroids are steroidal insect molting hormones that also exist in herbs. Ecdysteroid-containing adaptogens have been popularly used to improve well-being and by bodybuilders for muscle growth. However, the use of ecdysone in mammals is also associated with kidney growth and enlargement, indications of disturbed kidney homeostasis. The underlying pathogenic mechanism remains to be clarified. METHODS: Virtual screening tools were employed to identify compounds that are homologous to ecdysone and to predict putative ecdysone-interacting proteins. The kidney effect of ecdysone was examined in vitro and in vivo and compared with that of aldosterone. Cellular apoptosis was estimated by terminal deoxynucleotidyl transferase dUTP nick end labeling. Cell motility was assessed by scratch-wound cell migration assay. Blood urea nitrogen was measured to evaluate renal function. Western immunblot analysis was employed to determine the expression profile of interested proteins. RESULTS: Computational molecular structure analysis revealed that ecdysone is highly homologous to aldosterone. Moreover, virtual screening based on compound-protein interaction profiles identified the Mineralocorticoid Receptor (MR) to potentially interact with ecdysone. Accordingly, to assess potential biological functions of ecdysone in mammals, ecdysone was applied to mineralocorticoid-sensitive inner medullar collecting duct cells. Ecdysone induced mesenchymal accumulation of extracellular matrix and epithelial dedifferentiation characterized by de novo expression of α-smooth muscle actin. In addition, ecdysone elicited cellular apoptosis and retarded cell motility, akin to the effect of aldosterone. In vivo, daily treatment of mice with ecdysone increased cell apoptosis in the kidney, impaired renal function and elicited early signs of renal fibrogenesis, marked by deposition of collagen and fibronectin in tubulointerstitium, reminiscent of the action of aldosterone. The MR signaling pathway is likely responsible for the cellular and pathobiological effects of ecdysone, as evidenced by strong ecdysone-induced MR nuclear translocation in renal tubular cells both in vitro and in vivo, while blockade of MR by concomitant spironolactone treatment largely abolished the detrimental effects of ecdysone. CONCLUSION: Our findings suggest that ecdysone induces mineralocorticoid-dependent activities that impair renal function and elicit renal injury.


Assuntos
Apoptose/efeitos dos fármacos , Ecdisona/farmacologia , Mineralocorticoides/farmacologia , Insuficiência Renal Crônica/patologia , Aldosterona/farmacologia , Animais , Nitrogênio da Ureia Sanguínea , Desdiferenciação Celular , Linhagem Celular , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/ultraestrutura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Mineralocorticoides/metabolismo , Insuficiência Renal Crônica/metabolismo , Transdução de Sinais/efeitos dos fármacos
8.
Sci Rep ; 8(1): 3793, 2018 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-29491422

RESUMO

Taking advantage of a large transcriptomic dataset recently obtained in the sentinel crustacean amphipod Gammarus fossarum, we developed an approach based on sequence similarity and phylogenetic reconstruction to identify key players involved in the endocrine regulation of G. fossarum. Our work identified three genes of interest: the nuclear receptors RXR and E75, and the regulator broad-complex (BR). Their involvement in the regulation of molting and reproduction, along with their sensitivity to chemical contamination were experimentally assessed by studying gene expression during the female reproductive cycle, and after laboratory exposure to model endocrine disrupting compounds (EDCs): pyriproxyfen, tebufenozide and piperonyl butoxide. RXR expression suggested a role of this gene in ecdysis and post-molting processes. E75 presented two expression peaks that suggested a role in vitellogenesis, and molting. BR expression showed no variation during molting/reproductive cycle. After exposure to the three EDCs, a strong inhibition of the inter-molt E75 peak was observed with tebufenozide, and an induction of RXR after exposure to pyriproxyfen and piperonyl butoxide. These results confirm the implication of RXR and E75 in hormonal regulation of female reproductive cycles in G. fossarum and their sensitivity towards EDCs opens the possibility of using them as specific endocrine disruption biomarkers.


Assuntos
Anfípodes/metabolismo , Biomarcadores/metabolismo , Ecdisona/farmacologia , Disruptores Endócrinos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Espécies Sentinelas/metabolismo , Anfípodes/genética , Animais , Perfilação da Expressão Gênica , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores X Retinoide/genética , Receptores X Retinoide/metabolismo , Espécies Sentinelas/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
9.
Biochim Biophys Acta Gene Regul Mech ; 1861(2): 178-189, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29410380

RESUMO

Transcriptional activation is often represented as a "one-step process" that involves the simultaneous recruitment of co-activator proteins, leading to a change in gene status. Using Drosophila developmental ecdysone-dependent genes as a model, we demonstrated that activation of transcription is instead a continuous process that consists of a number of steps at which different phases of transcription (initiation or elongation) are stimulated. Thorough evaluation of the behaviour of multiple transcriptional complexes during the early activation process has shown that the pathways by which activation proceeds for different genes may vary considerably, even in response to the same induction signal. RNA polymerase II recruitment is an important step that is involved in one of the pathways. RNA polymerase II recruitment is accompanied by the recruitment of a significant number of transcriptional coactivators as well as slight changes in the chromatin structure. The second pathway involves the stimulation of transcriptional elongation as its key step. The level of coactivator binding to the promoter shows almost no increase, whereas chromatin modification levels change significantly.


Assuntos
Drosophila melanogaster/genética , Regiões Promotoras Genéticas/genética , Transdução de Sinais/genética , Ativação Transcricional/genética , Animais , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Ecdisona/farmacologia , Modelos Genéticos , Ligação Proteica , Interferência de RNA , RNA Polimerase II/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Tempo , Ativação Transcricional/efeitos dos fármacos , Fatores de Transcrição de p300-CBP/genética , Fatores de Transcrição de p300-CBP/metabolismo
10.
Mol Cell ; 66(1): 63-76.e6, 2017 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-28366641

RESUMO

Nuclear pore complex components (Nups) have been implicated in transcriptional regulation, yet what regulatory steps are controlled by metazoan Nups remains unclear. We identified the presence of multiple Nups at promoters, enhancers, and insulators in the Drosophila genome. In line with this binding, we uncovered a functional role for Nup98 in mediating enhancer-promoter looping at ecdysone-inducible genes. These genes were found to be stably associated with nuclear pores before and after activation. Although changing levels of Nup98 disrupted enhancer-promoter contacts, it did not affect ongoing transcription but instead compromised subsequent transcriptional activation or transcriptional memory. In support of the enhancer-looping role, we found Nup98 to gain and retain physical interactions with architectural proteins upon stimulation with ecdysone. Together, our data identify Nups as a class of architectural proteins for enhancers and supports a model in which animal genomes use the nuclear pore as an organizing scaffold for inducible poised genes.


Assuntos
Cromatina/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Elementos Facilitadores Genéticos , Regiões Promotoras Genéticas , Transcrição Genética , Ativação Transcricional , Animais , Animais Geneticamente Modificados , Sítios de Ligação , Linhagem Celular , Cromatina/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/genética , Ecdisona/farmacologia , Genótipo , Elementos Isolantes , Mutação , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Fenótipo , Ligação Proteica , Interferência de RNA , Transcrição Genética/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Transfecção
11.
Proc Natl Acad Sci U S A ; 114(6): 1419-1423, 2017 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-28115695

RESUMO

The wing polyphenism of pea aphids is a compelling laboratory model with which to study the molecular mechanisms underlying phenotypic plasticity. In this polyphenism, environmental stressors such as high aphid density cause asexual, viviparous adult female aphids to alter the developmental fate of their embryos from wingless to winged morphs. This polyphenism is transgenerational, in that the pea aphid mother experiences the environmental signals, but it is her offspring that are affected. Previous research suggested that the steroid hormone ecdysone may play a role in this polyphenism. Here, we analyzed ecdysone-related gene expression patterns and found that they were consistent with a down-regulation of the ecdysone pathway being involved in the production of winged offspring. We therefore predicted that reduced ecdysone signaling would result in more winged offspring. Experimental injections of ecdysone or its analog resulted in a decreased production of winged offspring. Conversely, interfering with ecdysone signaling using an ecdysone receptor antagonist or knocking down the ecdysone receptor gene with RNAi resulted in an increased production of winged offspring. Our results are therefore consistent with the idea that ecdysone plays a causative role in the regulation of the proportion of winged offspring produced in response to crowding in this polyphenism. Our results also show that an environmentally regulated maternal hormone can mediate phenotype production in the next generation, as well as provide significant insight into the molecular mechanisms underlying the functioning of transgenerational phenotypic plasticity.


Assuntos
Afídeos/efeitos dos fármacos , Ecdisona/farmacologia , Morfogênese/efeitos dos fármacos , Asas de Animais/efeitos dos fármacos , Animais , Afídeos/embriologia , Afídeos/genética , Aglomeração , Ecdisona/metabolismo , Ecdisterona/farmacologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Morfogênese/genética , Ervilhas/parasitologia , Fenótipo , Interferência de RNA , Receptores de Esteroides/antagonistas & inibidores , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Transdução de Sinais , Triterpenos/farmacologia , Asas de Animais/embriologia , Asas de Animais/metabolismo
12.
G3 (Bethesda) ; 6(8): 2629-42, 2016 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-27226164

RESUMO

Multiple aspects of Drosophila oogenesis, including germline stem cell activity, germ cell differentiation, and follicle survival, are regulated by the steroid hormone ecdysone. While the transcriptional targets of ecdysone signaling during development have been studied extensively, targets in the ovary remain largely unknown. Early studies of salivary gland polytene chromosomes led to a model in which ecdysone stimulates a hierarchical transcriptional cascade, wherein a core group of ecdysone-sensitive transcription factors induce tissue-specific responses by activating secondary branches of transcriptional targets. More recently, genome-wide approaches have identified hundreds of putative ecdysone-responsive targets. Determining whether these putative targets represent bona fide targets in vivo, however, requires that they be tested via traditional mutant analysis in a cell-type specific fashion. To investigate the molecular mechanisms whereby ecdysone signaling regulates oogenesis, we used genetic mosaic analysis to screen putative ecdysone-responsive genes for novel roles in the control of the earliest steps of oogenesis. We identified a cohort of genes required for stem cell maintenance, stem and progenitor cell proliferation, and follicle encapsulation, growth, and survival. These genes encode transcription factors, chromatin modulators, and factors required for RNA transport, stability, and ribosome biogenesis, suggesting that ecdysone might control a wide range of molecular processes during oogenesis. Our results suggest that, although ecdysone target genes are known to have cell type-specific roles, many ecdysone response genes that control larval or pupal cell types at developmental transitions are used reiteratively in the adult ovary. These results provide novel insights into the molecular mechanisms by which ecdysone signaling controls oogenesis, laying new ground for future studies.


Assuntos
Drosophila melanogaster/genética , Ecdisona/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Oogênese/genética , Ovário/fisiologia , Animais , Animais Geneticamente Modificados , Linhagem da Célula , Sobrevivência Celular/genética , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Ecdisona/farmacologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Técnicas Genéticas , Ribonucleoproteínas Nucleares Heterogêneas/genética , Larva/efeitos dos fármacos , Larva/genética , Mosaicismo , Mutação , Oogênese/efeitos dos fármacos , Ovário/citologia , Pupa/genética , Receptores de Esteroides/genética , Células-Tronco/fisiologia
13.
Bioorg Med Chem Lett ; 26(9): 2344-8, 2016 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-26988299

RESUMO

Bioassay-guided fractionation of an ethanolic extract of Zoanthus spp. collected in Taiwan has resulted in the isolation of one new ecdysone, zoanthone A (1), along with thirteen known compounds (2-14). The structures of these compounds were determined by spectroscopic methods, especially 2D NMR analyses. The in vitro antiviral activities of all isolated ecdysones (1-14) against dengue virus type 2 (DENV-2) were evaluated using DENV infectious system. New compound (1) exhibited potent antiviral activity (EC50=19.61 ± 2.46 µM) with a selectivity index (CC50/EC50) value of 36.7. The structure-activity relationships of isolated ecdysones against DENV-2 were concluded. Molecular docking information of 3 and NS5 polymerase was performed either.


Assuntos
Antozoários/química , Antivirais/farmacologia , Vírus da Dengue/efeitos dos fármacos , Ecdisona/farmacologia , Animais , Antivirais/química , Ecdisona/química , Relação Estrutura-Atividade
14.
G3 (Bethesda) ; 6(3): 683-94, 2016 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-26772746

RESUMO

Steroid hormones induce cascades of gene activation and repression with transformative effects on cell fate . Steroid transduction plays a major role in the development and physiology of nearly all metazoan species, and in the progression of the most common forms of cancer. Despite the paramount importance of steroids in developmental and translational biology, a complete map of transcriptional response has not been developed for any hormone . In the case of 20-hydroxyecdysone (ecdysone) in Drosophila melanogaster, these trajectories range from apoptosis to immortalization. We mapped the ecdysone transduction network in a cohort of 41 cell lines, the largest such atlas yet assembled. We found that the early transcriptional response mirrors the distinctiveness of physiological origins: genes respond in restricted patterns, conditional on the expression levels of dozens of transcription factors. Only a small cohort of genes is constitutively modulated independent of initial cell state. Ecdysone-responsive genes tend to organize into directional same-stranded units, with consecutive genes induced from the same strand. Here, we identify half of the ecdysone receptor heterodimer as the primary rate-limiting step in the response, and find that initial receptor isoform levels modulate the activated cohort of target transcription factors. This atlas of steroid response reveals organizing principles of gene regulation by a model type II nuclear receptor and lays the foundation for comprehensive and predictive understanding of the ecdysone transduction network in the fruit fly.


Assuntos
Drosophila/genética , Drosophila/metabolismo , Regulação da Expressão Gênica , Hormônios/metabolismo , Transdução de Sinais , Animais , Linhagem Celular , Análise por Conglomerados , Ecdisona/metabolismo , Ecdisona/farmacologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Hormônios/farmacologia , Isoformas de Proteínas , Receptores de Esteroides/metabolismo , Esteroides/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética/efeitos dos fármacos , Transcriptoma
15.
Artigo em Inglês | MEDLINE | ID: mdl-26627129

RESUMO

Three full-length cDNAs encoding lipoprotein homologs were identified in Tigriopus kingsejongensis, a newly identified copepod from Antarctica. Structural and transcriptional analyses revealed homology with two vitellogenin-like proteins, Tik-Vg1 and Tik-Vg2, which were 1855 and 1795 amino acids in length, respectively, along with a third protein, Tik-MEP, which produced a 1517-residue protein with similarity to a melanin engaging protein (MEP) in insects Phylogenetic analysis showed that Vgs in Maxillopods including two Tik-Vgs belong to the arthropod vitellogenin-like clade, which includes clottable proteins (CPs) in decapod crustaceans and vitellogenins in insects. Tik-MEP clustered together with insect MEPs, which appear to have evolved before the apoB-like and arthropod Vg-like clades. Interestingly, no genes orthologous to those found in the apoB clade were identified in Maxillopoda, suggesting that functions of large lipid transfer proteins (LLTPs) in reproduction and lipid metabolism may be different from those in insect and decapod crustaceans. As suggested by phylogenetic analyses, the two Tik-Vgs belonging to the arthropod Vg-like clade appear to play major roles in oocyte maturation, while Vgs belonging to the apoB clade function primarily in the reproduction of decapod crustaceans. Transcriptional analysis of Tik-Vg expression revealed a 24-fold increase in mature and ovigerous females compared with immature female, whereas expression of Tik-MEP remained low through all reproductive stages. Acute temperature changes did not affect the transcription of Tik-Vg genes, whereas Tik-MEP appeared to be affected by temperature change. Among the three hormones thought to be involved in molting and reproduction in arthropods, only farnesoic acid (FA) induced transcription of the two Tik-Vg genes. Regardless of developmental stage and hormone treatment, Tik-Vg1 and Tik-Vg2 exhibited a strong positive correlation in expression, suggesting that expression of these genes may be regulated by the same transcriptional machinery.


Assuntos
Copépodes/genética , DNA Complementar/genética , RNA Mensageiro/genética , Transcrição Genética , Vitelogeninas/genética , Sequência de Aminoácidos , Animais , Regiões Antárticas , Clonagem Molecular , Copépodes/classificação , Copépodes/efeitos dos fármacos , Copépodes/crescimento & desenvolvimento , DNA Complementar/metabolismo , Ecdisona/farmacologia , Ácidos Graxos Insaturados/farmacologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Dados de Sequência Molecular , Filogenia , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Temperatura , Vitelogeninas/metabolismo
16.
Parasit Vectors ; 8: 422, 2015 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-26272689

RESUMO

BACKGROUND: Searching for new effective drugs against human and animal toxoplasmosis we decided to test the anti-Toxoplasma potential of phytoecdysteroids (α-ecdysone and 20-hydroxyecdysone) characterized by the pleiotropic activity on mammalian organisms including the enhancement of host's anti-parasitic defence. This objective was accomplished by the in vitro evaluation of T. gondii growth in phytoecdysteroid-treated immunocompetent cells of selected hosts: humans and two strains of inbred mice with genetically determined different susceptibility to toxoplasmosis. METHODS: Peripheral mononuclear blood cells were isolated from Toxoplasma-positive and Toxoplasma-negative women (N = 43) and men (N = 21). Non-infected mice (C57BL/6, N = 10 and BALB/c, N = 14) and mice (BALB/c, N = 10) challenged intraperitoneally with 5 tissue cysts of the T. gondii DX strain were also used in this study as a source of splenocytes. The effects of phytoecdysteroids on the viability of human PBMC and mouse splenocytes were evaluated using the MTT assay. The influence of phytoecdysteroids on PBMCs, splenocytes and T. gondii proliferation was measured using radioactivity tests (the level of 3[H] uracil incorporation by toxoplasms or 3[H] thymidine by PBMCs and splenocytes), which was confirmed by quantitative Real-Time PCR. Statistical analysis was performed using SigmaStat 3.5 (Systat Software GmbH). The best-fit IC50 curves were plotted using GraphPad Prism 6.0 (GraphPad Software, Inc.). RESULTS: Our results showed that phytoecdysteroids promote the multiplication of Toxoplasma in cultures of human or murine immune cells, in contrast to another apicomplexan parasite, Babesia gibsoni. Additionally, the tested phytoecdysteroids did not stimulate the in vitro secretion of the essential protective cytokines (IFN-γ, IL-2 and IL-10), neither by human nor by murine immune cells involved in an effective intracellular killing of the parasite. CONCLUSIONS: Judging by the effect of phytoecdysteroids on the T. gondii proliferation, demonstrated for the first time in this study, it seems that these compounds should not be taken into consideration as potential medications to treat toxoplasmosis. Phytoecdysteroids included in the food are most likely not harmful for human or animal health but certain nutrients containing ecdysteroids at high concentrations could promote T. gondii proliferation in chronically infected and immunocompromised individuals. In order to assess the real impact of ecdysteroids on the course of natural T. gondii invasion, in vivo research should be undertaken because it cannot be ruled out that the in vivo effect will be different than the in vitro one. However, taking into account the possible stimulating effect of ecdysteroids on some opportunistic parasites (such as Toxoplasma or Strongyloides) further studies are necessary and should focus on the mechanisms of their action, which directly or indirectly enhance the parasite growth. Since ecdysteroids are considered as potential drugs, it is essential to determine their effect on various parasitic pathogens, which may infect the host at the same time, especially in immunocompromised individuals.


Assuntos
Citocinas/metabolismo , Ecdisona/farmacologia , Ecdisterona/farmacologia , Leucócitos Mononucleares/parasitologia , Toxoplasma/crescimento & desenvolvimento , Toxoplasmose/parasitologia , Adulto , Idoso , Animais , Encéfalo/parasitologia , Sobrevivência Celular/efeitos dos fármacos , Citocinas/genética , Relação Dose-Resposta a Droga , Ecdisona/administração & dosagem , Ecdisterona/administração & dosagem , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Baço/citologia , Baço/parasitologia , Adulto Jovem
17.
Mol Immunol ; 66(2): 325-39, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25931442

RESUMO

In insects, humoral response to injury is accomplished by the production of antimicrobial peptides (AMPs) which are secreted in the hemolymph to eliminate the pathogen. Drosophila Malpighian tubules (MTs), however, are unique immune organs that show constitutive expression of AMPs even in unchallenged conditions and the onset of immune response is developmental stage dependent. Earlier reports have shown ecdysone positively regulates immune response after pathogenic challenge however, a robust response requires prior potentiation by the hormone. Here we provide evidence to show that MTs do not require prior potentiation with ecdysone hormone for expression of AMPs and they respond to ecdysone very fast even without immune challenge, although the different AMPs Diptericin, Cecropin, Attacin, Drosocin show differential expression in response to ecdysone. We show that early gene Broad complex (BR-C) could be regulating the IMD pathway by activating Relish and physically interacting with it to activate AMPs expression. BR-C depletion from Malpighian tubules renders the flies susceptible to infection. We also show that in MTs ecdysone signaling is transduced by EcR-B1 and B2. In the absence of ecdysone signaling the IMD pathway associated genes are down regulated and activation and translocation of transcription factor Relish is also affected.


Assuntos
Peptídeos Catiônicos Antimicrobianos/imunologia , Proteínas de Drosophila/imunologia , Drosophila melanogaster/imunologia , Ecdisona/imunologia , Túbulos de Malpighi/imunologia , Fatores de Transcrição/imunologia , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/microbiologia , Ecdisona/farmacologia , Escherichia coli/imunologia , Regulação da Expressão Gênica no Desenvolvimento , Hemolinfa/química , Hemolinfa/imunologia , Imunidade Humoral , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Larva/imunologia , Larva/microbiologia , Túbulos de Malpighi/química , Túbulos de Malpighi/efeitos dos fármacos , Receptores de Esteroides/genética , Receptores de Esteroides/imunologia , Transdução de Sinais , Fatores de Transcrição/genética
18.
Sci Rep ; 5: 8675, 2015 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-25728569

RESUMO

G-protein-coupled receptors (GPCRs) are involved in animal steroid hormone signaling, but their mechanism is unclear. In this research, we report that a GPCR called ErGPCR-2 controls steroid hormone 20-hydroxyecdysone (20E) signaling in the cell membrane of the lepidopteran insect Helicoverpa armigera. ErGPCR-2 was highly expressed during molting and metamorphosis. 20E, via ErGPCR-2, regulated rapid intracellular calcium increase, protein phosphorylation, gene transcription, and insect metamorphosis. ErGPCR-2 was located in the cell surface and was internalized by 20E induction. GPCR kinase 2 participated in 20E-induced ErGPCR-2 phosphorylation and internalization. The internalized ErGPCR-2 was degraded by proteases to desensitize 20E signaling. ErGPCR-2 knockdown suppressed the entrance of 20E analog [(3)H] ponasterone A ([(3)H]Pon A) into the cells. ErGPCR-2 overexpression or blocking of ErGPCR-2 internalization increased the entrance of [(3)H]Pon A into the cells. However, ErGPCR-2 did not bind to [(3)H]Pon A. Results suggest that ErGPCR-2 transmits steroid hormone 20E signaling and controls 20E entrance into cells in the cell membrane.


Assuntos
Membrana Celular/metabolismo , Hormônios/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Esteroides/metabolismo , Animais , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Ecdisona/farmacologia , Endocitose/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Inativação Gênica/efeitos dos fármacos , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Lepidópteros/efeitos dos fármacos , Lepidópteros/genética , Lepidópteros/crescimento & desenvolvimento , Lepidópteros/metabolismo , Modelos Biológicos , Muda/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Transcrição Genética/efeitos dos fármacos , Trítio/metabolismo , Regulação para Cima/efeitos dos fármacos
19.
Curr Biol ; 25(8): 993-1004, 2015 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-25802149

RESUMO

Disruptions in energy homeostasis severely affect reproduction in many organisms and are linked to several reproductive disorders in humans. As a result, understanding the mechanisms that control nutrient accumulation in the oocyte will provide valuable insights into the links between metabolic disease and reproductive dysfunction. We show that the steroid hormone ecdysone functions in Drosophila to control lipid metabolism and support oocyte production. First, local EcR-mediated signaling induces a stage-specific accumulation of lipids in stage-10 oocytes. EcR induces lipid accumulation by promoting the activation of the lipogenic transcription factor SREBP and by controlling the expression of the low-density lipoprotein (LDL) receptor homolog, LpR2. Second, global signaling via the ecdysone receptor, EcR, establishes a female metabolic state and promotes whole-body triglyceride and glycogen storage at high levels. EcR acts in the CNS to mediate these effects, in part by promoting higher levels of feeding in females. Thus, ecdysone functions at two levels to support reproduction: first by inducing lipid accumulation in the late stages of oocyte development and second by providing a signal that coordinates lipid metabolism in the germline with whole-animal lipid homeostasis. Ecdysone regulation allows females to assess the demands of oogenesis and alter their behavior and metabolic state to support the biosynthetic requirements of oocyte production.


Assuntos
Ecdisona/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Esteroides/farmacologia , Animais , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Comportamento Alimentar/efeitos dos fármacos , Feminino , Glicogênio/metabolismo , Oócitos/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Esteroides/metabolismo , Proteínas de Ligação a Elemento Regulador de Esterol/metabolismo , Triglicerídeos/metabolismo
20.
Int J Mol Sci ; 16(2): 3335-49, 2015 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-25654229

RESUMO

EcR (ecdysone receptor)-mediated ecdysone signaling pathway contributes to regulate the transcription of genes involved in various processes during insect development. In this work, we detected the expression of EcR gene in silkworm ovary-derived BmN4 cells and found that EcR RNAi result in an alteration of cell shape, indicating that EcR may orchestrate cell cycle progression. EcR RNAi and EcR overexpression analysis revealed that in the cultured BmN4 cells, EcR respectively promoted and suppressed the transcription of E2F-1 and CycE, two genes controlling cell cycle progression. Further examination demonstrated that ecdysone application in BmN4 cells not only changed the transcription of these two cell cycle genes like that under EcR overexpression, but also induced cell cycle arrest at G2/M phase. In vivo analysis confirmed that E2F-1 expression was elevated in silk gland of silkworm larvae after ecdysone application, which is same as its response to ecdysone in BmN4 cells. However, ecdysone also promotes CycE transcription in silk gland, and this is converse with the observation in BmN4 cells. These results provide new insights into understanding the roles of EcR-mediated ecdysone signaling in the regulation of cell cycle.


Assuntos
Bombyx/genética , Proteínas de Ciclo Celular/genética , Regulação da Expressão Gênica , Receptores de Esteroides/metabolismo , Transcrição Genética , Animais , Bombyx/efeitos dos fármacos , Bombyx/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular , Relação Dose-Resposta a Droga , Ecdisona/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Interferência de RNA , Receptores de Esteroides/genética
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