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1.
Environ Pollut ; 255(Pt 2): 113329, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31600704

RESUMO

Transcription factors including pregnane X receptor (Pxr) and nuclear factor-erythroid 2-related factor-2 (Nrf2) are important modulators of Adenosine triphosphate-binding cassette (ABC) transporters in mammalian cells. However, whether such modulation is conserved in zebrafish embryos remains largely unknown. In this manuscript, pxr- and nrf2-deficient models were constructed with CRISPR/Cas9 system, to evaluate the individual function of Pxr and Nrf2 in the regulation of ABC transporters and detoxification of heavy metal ions like Cd2+ and Ag+. As a result, both Cd2+ and Ag+ conferred extensive interactions with ABC transporters in wild type (WT) embryos: their accumulation and toxicity were affected by the activity of ABC transporters, and they significantly induced the mRNA expressions of ABC transporters. These induction effects were reduced by the mutation of pxr and nrf2, but elevations in the basal expression of ABC transporters compensated for the loss of their inducibility. This could be an explanation for remaining transporter function in both mutant models as well as the unaltered toxicity of metal ions in pxr-deficient embryos. However, mutation of nrf2 disrupted the production of glutathione (GSH), resulting in the enhanced toxicity of Cd2+/Ag+ in zebrafish embryos. In addition, elevated expressions of other transcription factors like aryl hydrocarbon receptor (ahr) 1b, peroxisome proliferator-activated receptor (ppar)-ß, and nrf2 were found in pxr-deficient models without any treatment, while enhanced induction of ahr1b, ppar-ß and pxr could only be seen in nrf2-deficient embryos after the treatment of metal ions, indicating different compensation phenomena for the absence of transcription factors. After all, pxr-deficient and nrf2-deficient zebrafish embryos are useful tools in the functional investigation of Pxr and Nrf2 in the early life stages of aquatic organisms. However, the compensatory mechanisms should be taken into consideration when interpreting the results and need in-depth investigations.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Metais Pesados/toxicidade , Fator 2 Relacionado a NF-E2/genética , Receptor de Pregnano X/genética , Peixe-Zebra/embriologia , Animais , Glutationa/metabolismo , Inativação Metabólica , Receptores de Hidrocarboneto Arílico/metabolismo , Receptores de Esteroides/genética , Fatores de Transcrição/metabolismo , Proteínas de Peixe-Zebra/metabolismo
2.
Nat Cell Biol ; 21(10): 1206-1218, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31548609

RESUMO

Cholesterol activates the master growth regulator, mTORC1 kinase, by promoting its recruitment to the surface of lysosomes by the Rag guanosine triphosphatases (GTPases). The mechanisms that regulate lysosomal cholesterol content to enable mTORC1 signalling are unknown. Here, we show that oxysterol binding protein (OSBP) and its anchors at the endoplasmic reticulum (ER), VAPA and VAPB, deliver cholesterol across ER-lysosome contacts to activate mTORC1. In cells lacking OSBP, but not other VAP-interacting cholesterol carriers, the recruitment of mTORC1 by the Rag GTPases is inhibited owing to impaired transport of cholesterol to lysosomes. By contrast, OSBP-mediated cholesterol trafficking drives constitutive mTORC1 activation in a disease model caused by the loss of the lysosomal cholesterol transporter, Niemann-Pick C1 (NPC1). Chemical and genetic inactivation of OSBP suppresses aberrant mTORC1 signalling and restores autophagic function in cellular models of Niemann-Pick type C (NPC). Thus, ER-lysosome contacts are signalling hubs that enable cholesterol sensing by mTORC1, and targeting the sterol-transfer activity of these signalling hubs could be beneficial in patients with NPC.


Assuntos
Colesterol/metabolismo , Retículo Endoplasmático/metabolismo , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Doenças de Niemann-Pick/metabolismo , Receptores de Esteroides/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células HEK293 , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Receptores de Esteroides/genética , Transdução de Sinais , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/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 , 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 J Radiat Biol ; 95(12): 1696-1707, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31498019

RESUMO

Purpose: Hyperthermia (HT), a clinical treatment involving delivery of heat to tumors, has been used in combination with traditional chemotherapy and radiotherapy to enhance their effects. However, the molecular mechanism underlying the high efficacy of combination therapy is not clear. This study was conducted to identify the molecular mechanism underlying the sensitization of lung cancer to radiotherapy by HT.Materials and methods: Nuclear receptor subfamily 4, group A, member 3 (NR4A3) and Krüppel-like factor 11 (KLF11) expression in non-small-cell lung cancer cells was confirmed by performing real-time quantitative reverse transcription-polymerase chain reaction. Tumor cell proliferation and apoptosis were assessed via a colony-forming assay and Annexin V/propidium iodide staining.Results and conclusions: Expression profile analysis revealed elevated levels of NR4A3 and KLF11 in A549 lung cancer cells after treatment with HT combined with radiation. We also confirmed that NR4A3 and KLF11 induced apoptosis and inhibited cell proliferation by elevating intracellular reactive oxygen species levels. Knockdown of NR4A3 or KLF11 using siRNA led to decreased effects of radiohyperthermia. Finally, the effect of these two factors on lung cancer progression was evaluated by in vivo xenograft studies. Taken together, the results suggest that NR4A3 and KLF11 are critical for increasing the efficacy of radiotherapy in combination with HT.


Assuntos
Proteínas Reguladoras de Apoptose/genética , Proteínas de Ligação a DNA/genética , Progressão da Doença , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Hipertermia Induzida , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/radioterapia , Receptores de Esteroides/genética , Receptores dos Hormônios Tireóideos/genética , Proteínas Repressoras/genética , Células A549 , Animais , Apoptose/efeitos da radiação , Proliferação de Células/efeitos da radiação , Transformação Celular Neoplásica , Terapia Combinada , Humanos , Neoplasias Pulmonares/genética , Masculino , Camundongos
5.
Pestic Biochem Physiol ; 159: 85-90, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31400788

RESUMO

RNA interference (RNAi) is a potentially useful pest control method because of its high specificity. Silencing the expression of important RNAi target genes of pests will block important biological processes and reduce pest damage. Ecdysone is a unique arthropod hormone and the ecdysone receptor (EcR) is a key factor in molting pathway. We investigated the possibility that dsRNA targeting of the EcR of Tetranychus cinnabarinus (TcEcR) could effectively block development from larvae to adults. The mRNA level of TcEcR was highest in the larva stage, and 73.1% of the mites failed to survive the larva stage when TcEcR expression was silenced. Only 11.7% of T. cinnabarinus ingesting dsRNA successfully developed into adults, while 86.7% in the control succeeded in molting across each stage. RNAi significantly increased the developmental intervals of T. cinnabarinus. Under the effects of dsRNA, development times for the larva and first nymph doubled. Phenotype of body size change and death were observed during the development of T. cinnabarinus ingesting dsRNA. These findings suggest that RNAi is a potential means for the control of T. cinnabarinus. Genes in hormone pathways such as EcR are possible RNAi targets.


Assuntos
Larva/metabolismo , Interferência de RNA/fisiologia , Receptores de Esteroides/metabolismo , Tetranychidae/metabolismo , Animais , Tamanho Corporal , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , RNA de Cadeia Dupla/genética , Receptores de Esteroides/genética , Tetranychidae/crescimento & desenvolvimento
6.
Reprod Biol ; 19(2): 210-217, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31262644

RESUMO

Clinical outcomes of fresh embryo transfer in non-hCG triggered in vitro maturation (IVM) cycles are inferior compared to vitrified-warmed embryo transfer. This is a prospective observational pilot study in a consecutive cohort of 31 polycystic ovary syndrome (PCOS) patients and 37 normo-ovulatory egg donors who underwent IVM without fresh embryo transfer between July 2009 and June 2014. All subjects received 150 IU of highly purified menotropin (HP-hMG) daily for three days. On cycle day 6, all patients started transdermal oestradiol (E2) at a daily dose of 9 mg. There was no human chorionic gonadotropin (hCG) trigger before oocyte retrieval (OR). Vaginal micronized progesterone was commenced on the evening after OR, at a daily dose of 600 mg. Additional luteal phase support (LPS) was administered as follows: Group A: no additional LPS; Group B: 1500 IU of hCG administered 4 h after OR and Group C: 5000 IU of hCG administered 4 h after OR + an additional injection of 5000 IU of hCG 1 day before endometrial biopsy. Endometrial biopsy for histology and immunohistochemistry (IHC) was performed on day 5 or 6 after OR. Instead of being downregulated, both PR-B and ERα in endometrial glands and stroma were moderately to strongly expressed in all three protocols, suggesting that the mid-luteal histological signature of endometrial receptivity is deficient in a non-hCG-triggered IVM cycle. Poor clinical outcomes after fresh embryo transfer following IVM are probably related to inappropriate endometrial development which may be linked to the short follicular phase of IVM cycles.


Assuntos
Gonadotropina Coriônica/farmacologia , Endométrio/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Síndrome do Ovário Policístico/metabolismo , Receptores de Esteroides/metabolismo , Adulto , Gonadotropina Coriônica/administração & dosagem , Estudos de Coortes , Estradiol/administração & dosagem , Estradiol/farmacologia , Feminino , Humanos , Técnicas de Maturação in Vitro de Oócitos , Projetos Piloto , Progesterona/administração & dosagem , Progesterona/farmacologia , Estudos Prospectivos , Receptores de Esteroides/genética , Adulto Jovem
7.
Insect Biochem Mol Biol ; 112: 103184, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31295549

RESUMO

The rate of carbohydrate metabolism is tightly coordinated with developmental transitions in Drosophila, and fluctuates depending on the requirements of a particular developmental stage. These successive metabolic switches result from changes in the expression levels of genes encoding glycolytic, tricarboxylic acid cycle (TCA), and oxidative phosphorylation enzymes. In this report, we describe a repressive action of ecdysone signaling on the expression of glycolytic genes and enzymes of glycogen metabolism in Drosophila development. The basis of this effect is an interaction between the ecdysone receptor (EcR) and the estrogen-related receptor (ERR), a specific regulator of the Drosophila glycolysis. We found an overlapping DNA-binding pattern for the EcR and ERR in the Drosophila S2 cells. EcR was detected at a subset of the ERR target genes responsible for carbohydrate metabolism. The 20-hydroxyecdysone treatment of both the Drosophila larvae and the S2 cells decreased transcriptional levels of ERR targets. We propose a joint action mode for both the EcR and ERR, for at least a subset of the glycolytic genes. We find that both receptors bind to the same regulatory regions and may form or be part of a joint transcriptional regulatory complex in the Drosophila S2 cells.


Assuntos
Metabolismo dos Carboidratos/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Receptores Estrogênicos/metabolismo , Receptores de Esteroides/metabolismo , Animais , Linhagem Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Ecdisterona/farmacologia , Regulação da Expressão Gênica no Desenvolvimento , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Estrogênicos/genética , Receptores de Esteroides/genética
8.
Mol Med Rep ; 20(2): 1025-1038, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31173207

RESUMO

Hepatocellular carcinoma (HCC) accounts for ~85% of primary liver cancer cases and is a leading cause of mortality worldwide. Effective early diagnosis is difficult for HCC; however, effective biomarkers may be beneficial for diagnosis. In the current study, serum samples, and HCC and adjacent tissue samples were obtained from patients with HCC for the detection of biomarkers using 2­D gel electrophoresis (2­DE) and matrix­assisted laser desorption/ionization­time of flight (TOF)/TOF mass spectrometry. The crude serum samples did not need to be prepared for removal of high abundance proteins. The mRNA expression levels of HCC­associated proteins were detected in tissues using reverse transcription­quantitative PCR. Statistical analysis and database matching were used to identify the differentially expressed proteins detected in the serum and tissue groups. Immunohistochemistry (IHC) was performed to detect the expression of significant proteins in HCC and adjacent tissues. The results revealed ~800 protein spots on a 2­DE gel that were detected in serum samples, and 1,200 spots were identified in the tissue samples. The protein and mRNA expression levels of oxysterol binding protein­like 11 (OSBPL11) in HCC serum and tissue samples were consistent. Pathway analysis demonstrated that members of the apolipoprotein family, particularly apolipoprotein E (APOE), and RAS family members were closely associated in HCC, either directly or via ferratin heavy polypeptide 1. IHC results demonstrated that the APOE protein serves an important role in liver cancer development. The lysis buffer used in the current study was effective for serum protein separation in 2­DE sample preparation. In addition, the present study revealed that downregulated OSBPL11 may be a potential indicator for HCC, and the apolipoprotein family, particularly APOE, and the RAS family may cooperatively serve an important role.


Assuntos
Apolipoproteínas E/genética , Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/genética , Receptores de Esteroides/genética , Idoso , Apolipoproteínas E/sangue , Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/patologia , Estudos de Casos e Controles , Detecção Precoce de Câncer , Eletroforese em Gel Bidimensional , Feminino , Ferritinas/genética , Ferritinas/metabolismo , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Oxirredutases/genética , Oxirredutases/metabolismo , Receptores de Esteroides/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Análise de Sobrevida , Proteínas ras/genética , Proteínas ras/metabolismo
9.
Med Oncol ; 36(8): 66, 2019 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-31183633

RESUMO

Nuclear receptor subfamily 4, group A, member 3 (NR4A3) is a member of the NR4A subgroup of orphan nuclear receptors, implicated in the regulation of diverse biological functions, including metabolism, angiogenesis, inflammation, cell proliferation, and apoptosis. Although many reports have suggested the involvement of NR4A3 in the development and/or progression of tumors, its role varies among tumor types. Previously, we reported that DNA hypomethylation at NR4A3 exon 3 is associated with lower survival rate of neuroblastoma (NB) patients. As hypomethylation of this region results in reduced expression of NR4A3, our observations suggested that NR4A3 functions as a tumor suppressor in NB. However, the exact mechanisms underlying its functions have not been clarified. In the present study, we analyzed public databases and showed that reduced NR4A3 expression was associated with shorter survival period of NB in two out of three datasets. An in vitro study revealed that forced expression of NR4A3 in human NB-derived cell line NB1 resulted in elongation of neurites along with overexpression of GAP43, one of the differentiation markers of NB. On the other hand, siRNA-mediated knockdown of NR4A3 suppressed the expression level of GAP43. Interestingly, the forced expression of NR4A3 induced only the GAP43 but not the other molecules involved in NB cell differentiation, such as MYCN, TRKA, and PHOX2B. These results indicated that NR4A3 directly activates the expression of GAP43 and induces differentiated phenotypes of NB cells, without affecting the upstream signals regulating GAP43 expression and NB differentiation.


Assuntos
Proteínas de Ligação a DNA/biossíntese , Neuroblastoma/metabolismo , Receptores de Esteroides/biossíntese , Receptores dos Hormônios Tireóideos/biossíntese , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Progressão da Doença , Proteína GAP-43/biossíntese , Técnicas de Silenciamento de Genes , Humanos , Neuritos/metabolismo , Neuritos/patologia , Neuroblastoma/genética , Neuroblastoma/patologia , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Receptores de Esteroides/genética , Receptores dos Hormônios Tireóideos/genética , Regulação para Cima
10.
Reprod Biol Endocrinol ; 17(1): 48, 2019 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-31226998

RESUMO

BACKGROUND: Puberty in male Atlantic salmon in aquaculture can start as early as after the first winter in seawater, stunts growth and entails welfare problems due to the maturation-associated loss of osmoregulation capacity in seawater. A better understanding of the regulation of puberty is the basis for developing improved cultivation approaches that avoid these problems. Our aim here was to identify morphological and molecular markers signaling the initiation of, and potential involvement in, testis maturation. METHODS: In the first experiment, we monitored for the first time in large Atlantic salmon males several reproductive parameters during 17 months including the first reproductive cycle. Since testicular growth accelerated after the Winter solstice, we focused in the second experiment on the 5 months following the winter solstice, exposing fish from February 1 onwards to the natural photoperiod (NL) or to continuous additional light (LL). RESULTS: In the first experiment, testis weight, plasma androgens and pituitary gonadotropin transcript levels increased with the appearance of type B spermatogonia in the testis, but testicular transcript levels for gonadotropin or androgen receptors did not change while being clearly detectable. In the second experiment, all males kept under NL had been recruited into puberty until June. However, recruitment into puberty was blocked in ~ 40% of the males exposed to LL. The first morphological sign of recruitment was an increased proliferation activity of single spermatogonia and Sertoli cells. Irrespective of the photoperiod, this early sign of testis maturation was accompanied by elevated pituitary gnrhr4 and fshb and testicular igf3 transcript levels as well as increased plasma androgen levels. The transition into puberty occurred again with stable testicular gonadotropin and androgen receptor transcript levels. CONCLUSIONS: The sensitivity to reproductive hormones is already established before puberty starts and up-regulation of testicular hormone receptor expression is not required to facilitate entry into puberty. The increased availability of receptor ligands, on the other hand, may result from an up-regulation of pituitary Gnrh receptor expression, eventually activating testicular growth factor and sex steroid release and driving germ and Sertoli cell proliferation and differentiation.


Assuntos
Hormônios Esteroides Gonadais/metabolismo , Receptores de Esteroides/metabolismo , Salmo salar/metabolismo , Maturidade Sexual , Testículo/metabolismo , Animais , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Regulação da Expressão Gênica/efeitos da radiação , Masculino , Fotoperíodo , Hipófise/metabolismo , Receptores do FSH/genética , Receptores do FSH/metabolismo , Receptores de Esteroides/genética , Reprodução/genética , Reprodução/fisiologia , Salmo salar/genética , Estações do Ano , Água do Mar
11.
Int. microbiol ; 22(2): 169-179, jun. 2019. graf, tab
Artigo em Inglês | IBECS | ID: ibc-184824

RESUMO

Oxysterol-binding protein is an important non-vesicular trafficking protein involved in the transportation of lipids in eukaryotic cells. Oxysterol-binding protein is identified as oxysterol-binding protein-related proteins (ORPs) in mammals and oxysterol-binding protein homologue (Osh) in yeast. Research has described the function and structure of oxysterol-binding protein in mammals and yeast, but little information about the protein's structure and function in filamentous fungi has been reported. This article focuses on recent advances in the research of Osh proteins in yeast and filamentous fungi, such as Aspergillus oryzae, Aspergillus nidulans, and Candida albicans. Furthermore, we point out some problems in the field, summarizing the membrane contact sites (MCS) of Osh proteins in yeast, and consider the future of Osh protein development


No disponible


Assuntos
Fungos/genética , Receptores de Esteroides/genética , Leveduras/genética , Proteínas de Transporte/genética , Proteínas Fúngicas/genética , Fungos/metabolismo , Receptores de Esteroides/metabolismo , Leveduras/metabolismo , Proteínas de Transporte/metabolismo , Proteínas Fúngicas/metabolismo , Fungos/química , Metabolismo dos Lipídeos , Domínios Proteicos , Receptores de Esteroides/química , Leveduras/química
12.
Environ Sci Pollut Res Int ; 26(21): 21535-21545, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31127518

RESUMO

Glyphosate-based herbicides (GBH) are the most used herbicides worldwide and are considered as endocrine-disrupting compounds (EDC) for non-target organisms. However, effects of GBH on their endocrine systems remain poorly understood. Thus, the aim of this study was to assess the effects of low concentrations of Roundup WG® on growth and reproduction process molecules in both males and females of the decapod crustacean Macrobrachium potiuna, by the relative transcript expression levels of the ecdysteroid receptor (EcR), the molt-inhibiting hormone (MIH), and the vitellogenin (Vg) genes. Prawns were exposed to three concentrations of GBH (0.0065, 0.065, and 0.28 mg L-1) for 7 and 14 days. The results revealed that only in males the three genes transcript levels were influenced by the GBH concentration, time of exposure, and the interaction between the concentrations and time of exposure, suggesting that males were more sensitive to GBH than females. For males, after 7 days of exposure at 0.065 mg L-1, EcR and MIH were over-expressed, while the Vg expression was only over-expressed after 14 days. The present study highlighted that GBH impacted endocrine systems of M. potiuna. Moreover, EcR and MIH gene expressions could be promising EDC biomarkers of exposure in crustaceans. These results also indicate that GBH concentrations, considered secure by regulatory agencies, should be reviewed to minimize the effects on non-target organisms. Potential effects of glyphosate-based herbicides on the endocrine system of decapods Macrobrachium sp.


Assuntos
Disruptores Endócrinos/toxicidade , Glicina/análogos & derivados , Herbicidas/toxicidade , Palaemonidae/fisiologia , Animais , Sistema Endócrino , Feminino , Glicina/toxicidade , Hormônios de Invertebrado , Masculino , Palaemonidae/genética , Receptores de Esteroides/genética
13.
Parasit Vectors ; 12(1): 235, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-31092286

RESUMO

BACKGROUND: Ticks are blood-sucking arthropods that can transmit diseases to humans and animals. These arthropods are the second most important vectors of pathogens. MicroRNAs are a class of conserved small noncoding RNAs that play regulatory roles in gene expression at the post-transcriptional level. Molting is an important biological process in arthropods. Research on the molting process is important for understanding tick physiology and control. METHODS: Dual-luciferase reporter assays were used to assess the role of miRNA let-7 in ecdysteroid receptor (ECR) biology. The expression levels of ECR and let-7 were measured by real-time qPCR before and after tick molting. To explore the function of let-7 and ECR, we performed overexpression and knocking down of let-7 and RNAi of ECR in tick nymphs. The biological function of let-7 in molting was explored by injecting nymphs, ten days after engorgement, with let-7 agomir for overexpression and let-7 antagomir for knocking down. The rate of molting was then determined. ECR dsRNA was injected into ticks to evaluate the function of ECR by gene silencing. The expression of ECR and let-7 was measured using RT-qPCR. All data were analyzed using GraphPad Prism v.6. RESULTS: The results of the luciferase assay using a eukaryotic expression system revealed that ECR was a natural target of let-7. Let-7 overexpressed by agomir affected the rate of molting (P < 0.01) and the period of molting (P < 0.01). Let-7 antagomir for knockdown affected the period of molting (P < 0.01), but there was no effect on the rate of molting (P = 0.27). ECR dsRNA gene silencing significantly affected the rate of molting (P < 0.05). CONCLUSIONS: This study demonstrated that let-7 can regulate the expression of ECR and that let-7 can affect molting in ticks. Our results help to understand the regulation of let-7 by 20-hydroxyecdysone (20E) and will provide a reference for functional analysis studies of microRNAs in ticks.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs/genética , Muda/genética , Receptores de Esteroides/genética , Carrapatos/genética , Animais , Ecdisterona/genética , Ninfa/genética , Ninfa/fisiologia , Interferência de RNA , Reação em Cadeia da Polimerase em Tempo Real , Carrapatos/fisiologia
14.
Gen Comp Endocrinol ; 280: 54-61, 2019 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-30980804

RESUMO

The relationship between stress and immunosuppression was investigated in peripheral blood leucocytes (PBL) in rainbow trout, with reference to corticosteroid receptor (CR) expression and responses to cortisol- and/or lipopolysaccharide (LPS)-administration. Confinement stress in shallow water resulted in a sustained elevation of plasma cortisol, whereas lysozyme and immunoglobin levels were suppressed. Significant increases in mRNA levels of caspase-6 and insulin-like growth factor (IGF)-I were observed in PBL isolated from stressed fish. Confinement stress also suppressed proinflammatory cytokine, interleukin (IL)-1ß, expression in PBL. There were decreasing tendencies for the mRNA levels of CRs in PBL of stressed fish. In-vitro treatment of cortisol and LPS on isolated PBL from unstressed trout increased both IL-1 ß and CR mRNA expression. However, in PBL from stressed fish, cortisol and LPS treatment increased IL-1 ß but not CR mRNA levels. Proliferative activities estimated as in-vitro incorporation of bromodeoxyuridine (BrdU) were decreased by cortisol in PBL from the unstressed and stressed fish groups; however, LPS-stimulated proliferation was observed only in the unstressed fish. Ratios of apoptotic PBL quantified as cell fragmentation using an automated cell counter were increased by cortisol in both groups; however, LPS-stimulated apoptosis was observed only in the stressed fish. Our study reveals cortisol has immune-suppressive effects in stressed fish, irrespective of CR down-regulation and desensitization. The complexity of immune-endocrine interaction is shown by the stress-induced attenuation of LPS effects.


Assuntos
Regulação para Baixo/genética , Leucócitos/metabolismo , Oncorhynchus mykiss/genética , Oncorhynchus mykiss/fisiologia , Receptores de Esteroides/genética , Estresse Fisiológico/genética , Animais , Citocinas/genética , DNA Complementar/genética , Regulação para Baixo/efeitos dos fármacos , Hidrocortisona/sangue , Lipopolissacarídeos/farmacologia , Muramidase/sangue , Oncorhynchus mykiss/sangue , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Esteroides/metabolismo
15.
Endocrinology ; 160(5): 1275-1288, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30958537

RESUMO

In mammals, the grainyhead-like transcription factor (GRHL) family is composed of three nuclear proteins that are responsible for driving epithelial cell fate: GRHL1, GRHL2, and GRHL3. GRHL2 is important in maintaining proper tubulogenesis during development and in suppressing the epithelial-to-mesenchymal transition. Within the last decade, evidence indicates both tumor-suppressive and oncogenic roles for GRHL2 in various types of cancers. Recent studies suggest that GRHL2 may be especially important in hormone-dependent cancers, as correlative relationships exist between GRHL2 and various steroid receptors, such as the androgen and estrogen receptors. Acting as a pioneer factor and coactivator, GRHL2 may directly affect steroid receptor transcriptional activity. This review will highlight recent discoveries of GRHL2 activity in cancer and in maintaining the epithelial state, while also exploring recent literature on the role of GRHL2 in hormone-dependent cancers and epigenetics.


Assuntos
Proteínas de Ligação a DNA/genética , Epigênese Genética/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , Receptores de Esteroides/genética , Fatores de Transcrição/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células/genética , Proteínas de Ligação a DNA/metabolismo , Transição Epitelial-Mesenquimal/genética , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Receptores de Esteroides/metabolismo , Fatores de Transcrição/metabolismo
16.
PLoS One ; 14(3): e0214768, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30925160

RESUMO

The family of oxysterol binding protein (OSBP) and OSBP-related proteins (ORPs) mediate sterol and phospholipid transfer and signaling at membrane contact sites (MCS). The activity of OSBP at MCS is regulated by phosphorylation, but whether this applies to ORPs is unknown. Here we report the functional characterization of a unique proline/serine-rich phosphorylation motif (S762SPSSPSS769) in the lipid binding OSBP-related domain of full-length ORP4L and a truncated variant ORP4S. Phosphorylation was confirmed by mass spectrometry and [32P]PO4 incorporation, and in silico and in vitro assays using purified ORP4L identified putative proline-directed kinases that phosphorylate the site. The functional significance of the phospho-site was assessed by mutating serine 762, S763, S766 and S768 to aspartate or alanine to produce phosphomimetic (S4D) and phosphorylation-deficient (S4A) mutants, respectively. Solution binding of 25-hydroxycholesterol and cholesterol by recombinant ORP4L-S4D and -S4A was similar to wild-type but ORP4L-S4D more effectively extracted cholesterol from liposomes. ORP4L homo-dimerization was unaffected by phosphorylation but gel filtration of ORP4L-S4D indicated that the native conformation was affected. Confocal microscopy revealed that ORP4L-S4D also strongly associated with bundled vimentin filaments, a feature shared with ORP4S which lacks the PH and dimerization domains. We conclude that phosphorylation of a unique serine/proline motif in the ORD induces a conformation change in ORP4L that enhances interaction with vimentin and cholesterol extraction from membranes.


Assuntos
Colesterol/metabolismo , Prolina , Receptores de Esteroides/química , Receptores de Esteroides/metabolismo , Serina , Vimentina/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Linhagem Celular , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Fosforilação , Ligação Proteica , Domínios Proteicos , Receptores de Esteroides/genética
17.
Insect Mol Biol ; 28(5): 676-688, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-30834617

RESUMO

A heterodimer of ultraspiracle (USP) and ecdysone receptor (EcR) mediates 20-hydroxyecdysone (20E) signalling cascade to regulate insect moulting and metamorphosis. However, at least two questions remain to be addressed in terms of the molecular importance of USP in insect species. First, is USP involved in both regulation of ecdysteroidogenesis and mediation of 20E signalling in non-drosophilid insects, as in Drosophila melanogaster? Second, does USP play any role in larval metamorphosis except as the partner of heterodimeric receptor to activate the downstream 20E signalling genes? In this paper, we found that RNA interference (RNAi) of LdUSP in the final (fourth) instar larvae reduced the messenger RNA levels of four ecdysteroidogenesis genes (Ldspo, Ldphm, Lddib and Ldsad) and 20E titre, and repressed the expression of five 20E signal genes (EcRA, HR3, HR4, E74 and E75) in Leptinotarsa decemlineata. The LdUSP RNAi larvae remained as prepupae, with developing antennae, legs and discs of forewings and hindwings. Dietary supplement with 20E restored the expression of the five 20E signal genes, but only partially alleviated the decreased pupation rate in LdUSP RNAi beetles. Knockdown of LdUSP at the penultimate (third) instar larvae did not affect third-fourth instar moulting. However, silencing LdUSP caused similar but less severe impairments on pupation. Accordingly, we propose that USP is undoubtedly necessary for ecdysteroidogenesis, for mediation of 20E signalling and for initiation of metamorphosis in L. decemlineata.


Assuntos
Besouros/crescimento & desenvolvimento , Besouros/genética , Ecdisterona/metabolismo , Receptores de Esteroides/genética , Animais , Besouros/metabolismo , Ecdisterona/farmacologia , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Metamorfose Biológica/genética , Muda/genética , Interferência de RNA , Receptores de Esteroides/metabolismo , Transdução de Sinais
18.
Dev Cell ; 49(2): 220-234.e8, 2019 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-30905771

RESUMO

Lipid transfer proteins (LTPs) acting at membrane contact sites (MCS) between the ER and other organelles contain domains involved in heterotypic (e.g., ER to Golgi) membrane tethering as well as domains involved in lipid transfer. Here, we show that a long ≈90 aa intrinsically unfolded sequence at the N terminus of oxysterol-binding protein (OSBP) controls OSBP orientation and dynamics at MCS. This Gly-Pro-Ala-rich sequence, whose hydrodynamic radius is twice as that of folded domains, prevents the two PH domains of the OSBP dimer from homotypically tethering two Golgi-like membranes and considerably facilitates OSBP in-plane diffusion and recycling at MCS. Although quite distant in sequence, the N terminus of OSBP-related protein-4 (ORP4) has similar effects. We propose that N-terminal sequences of low complexity in ORPs form an entropic barrier that restrains protein orientation, limits protein density, and facilitates protein mobility in the narrow and crowded MCS environment.


Assuntos
Proteínas de Transporte/metabolismo , Receptores de Esteroides/metabolismo , Proteínas de Transporte/fisiologia , Linhagem Celular , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Células HeLa , Humanos , Lipídeos/fisiologia , Membranas Mitocondriais/metabolismo , Organelas/metabolismo , Domínios Proteicos/fisiologia , Receptores de Esteroides/genética , Receptores de Esteroides/fisiologia , Esteróis/metabolismo
19.
BMC Med Genet ; 20(1): 43, 2019 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-30894143

RESUMO

BACKGROUND: To investigate the clinical features and the underlying causal gene of a family with hereditary late-onset deafness in Inner Mongolia of China, and to provide evidence for the early genetic screening and diagnosis of this disease. METHODS: Family data were collected to draw a pedigree. Audiological testing and physical examination of the family members were conducted following questionnaire. Genomic DNA was extracted from peripheral blood of 5 family members (3 patients and 2 normal control) and subjected to whole genome sequencing for identifying deafness casual genes. The pathogenic variant in the deafness gene was further confirmed by Sanger sequencing. RESULTS: The family is composed of a total of 6 generations, with 53 traceable individuals. In this family,19 of them were diagnosed with post lingual deafness with the age of onset between 10 and 40 years, displaying delayed and progressive hearing loss. Patients with hearing loss showed bilateral symmetry and mild to severe sensorineural deafness. The pattern of deafness inheritance in this family is autosomal dominant. Whole genome sequencing identified a novel pathogenic frameshift mutation, c.158_159delAA (p.Gln53Arg fs*100) in the gene OSBPL2 (Oxysterol-binding protein-related protein 2, NM_144498.2), which is absent from genomic data of 201 unrelated normal subjects. This pathogenic variant was further validated by Sanger sequencing, and was found to co-segregate in this family. CONCLUSIONS: Whole genome sequencing identified a two-nucleotide deletion in OSBPL2 (c.158_159delAA) as the pathogenic variant for deafness in the family. Our finding expands the mutational spectrum of OSBPL2 and contributes to the pathogenic variant list in genetic counseling for deafness screening.


Assuntos
Mutação da Fase de Leitura , Perda Auditiva/congênito , Perda Auditiva/genética , Receptores de Esteroides/genética , Sequenciamento Completo do Genoma/métodos , Adulto , Idade de Início , Grupo com Ancestrais do Continente Asiático/genética , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Mongólia , Linhagem , Fenótipo
20.
Ecotoxicol Environ Saf ; 174: 675-682, 2019 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-30878007

RESUMO

The endocrine disrupting properties of bisphenol A (BPA) discharged to the environment have been newly identified by the European Chemicals Agency, increasing the need to assess the environmental endocrine disrupting potentials of its alternatives with which it shares close structural features. However, few investigations of the environmental endocrine disrupting functions of BPA analogs have been conducted to date. In this study, the endocrine disrupting effects of a BPA analog of bisphenol P (BPP) were investigated in the nonbiting midge (Chironomus tentans), a model organism in ecotoxicology. An initial ex vivo test using salivary gland cells explanted from the larvae and a subsequent in vivo test using embryos and larvae revealed the upregulatory effects of BPP on ecdysone receptor genes encoding the ecdysone receptor (EcR) and the early responsive gene E74, with a similar temporal pattern of gene activation. Partial life cycle and full life cycle toxicity tests demonstrated BPP altered embryo hatching, larval emergence, and adult sex ratio at concentrations close to the effective concentrations for hormonal genetic endpoints in embryos and larvae after 48 h of exposure. Although embryos appeared to be more sensitive to BPP than the fourth instar larvae, the impact on neither life stage seemed enough to estimate the developmental impairment of the insects. These results demonstrate the ecdysone pathway is a target of BPP, and that long-term exposure could cause apical effects on the development of C. tentans. The endocrine disrupting effects towards aquatic organisms, as well as the high persistence and bioconcentration potential, indicate an urgent need to assess the environmental risks associated with BPP.


Assuntos
Compostos Benzidrílicos/toxicidade , Chironomidae/fisiologia , Disruptores Endócrinos/toxicidade , Poluentes Ambientais/toxicidade , Estágios do Ciclo de Vida/efeitos dos fármacos , Fenóis/toxicidade , Animais , Compostos Benzidrílicos/química , Chironomidae/genética , Chironomidae/crescimento & desenvolvimento , Biomarcadores Ambientais , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Larva/genética , Larva/crescimento & desenvolvimento , Larva/fisiologia , Fenóis/química , Receptores de Esteroides/genética
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