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1.
Viral Immunol ; 31(9): 639-645, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30222506

RESUMO

Nod-like receptor protein 3 (NLRP3), absent in melanoma 2 (AIM2), and interferon gamma inducible protein 16 (IFI16) are innate immune sensors for intracellular microbes, which can be activated by various dangerous signals and subsequently lead to caspase-1 (CASP1) activation and the maturation cleavage of effector molecules pro-IL-1ß and pro-IL-18. Their roles in immunopathology of acute and chronic hepatitis B virus (HBV) infection are still unclear. In this study, we first investigated the activation of NLRP3, AIM2, and IFI16 inflammasomes in peripheral blood mononuclear cells (PBMCs) from patients infected with acute hepatitis B (AHB) and chronic hepatitis B (CHB) by quantitative real-time PCR and enzyme-linked immunosorbent assay. We next analyzed the impact of hepatitis B e antigen (HBeAg) on activation of AIM2 and IFI16 inflammasomes in PBMCs of CHB patients stimulated in vitro with AIM2 and IFI16 agonist ligands, poly (dA:dT) and VACA-70mer, respectively. The results showed that the mRNA expression levels of AIM2, IFI16, and CASP1 in PBMCs from AHB and CHB patients were both upregulated. Furthermore, the mRNA levels of AIM2 and IFI16 in CHB patients were significantly positively correlated with serum HBV loads. However, only in patients with AHB there was elevation of serum IL-1ß and IL-18. There was no activation of NLRP3, AIM2, and IFI16 inflammasomes in CHB patients. Stimulation of PBMCs of CHB patients in vitro with poly (dA:dT) and VACA-70mer induced the activation of AIM2 and IFI16 inflammasomes, respectively. This ligand-induced activation was suppressed by HBeAg. Our results suggest that there exists activation of the AIM2 and IFI16 inflammasomes, but not the NLRP3 inflammasome, in AHB, and the activation of the AIM2 and IFI16 inflammasomes can be inhibited by HBeAg in CHB, which may contribute to HBV-induced immunotolerance.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Hepatite B Crônica/metabolismo , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Doença Aguda , Adulto , DNA Viral/sangue , Proteínas de Ligação a DNA/agonistas , Proteínas de Ligação a DNA/genética , Feminino , Antígenos E da Hepatite B/metabolismo , Hepatite B Crônica/sangue , Hepatite B Crônica/imunologia , Humanos , Imunidade Inata/imunologia , Interleucina-18/sangue , Interleucina-1beta/sangue , Leucócitos Mononucleares/imunologia , Masculino , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteínas Nucleares/agonistas , Proteínas Nucleares/genética , Fosfoproteínas/agonistas , Fosfoproteínas/genética , Polidesoxirribonucleotídeos/antagonistas & inibidores , Polidesoxirribonucleotídeos/farmacologia
2.
Biol Pharm Bull ; 41(9): 1401-1405, 2018 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-29984732

RESUMO

The present study aims to investigate the roles of steroidogenic acute regulatory protein (StAR) in Yangjing Capsule (YC) induced anti-apoptotic effects on Leydig cells and the related mechanism. Leydig tumor cells (MLTC-1) were cultured and treated with YC, and immunofluorescence assay was performed to examine the expression of StAR; furthermore, luciferase reporter assay was conducted to evaluate the impact of YC on StAR promoter; next, MLTC-1 cells were treated with StAR small interfering RNA (siRNA), and flow cytometry was carried out to examine the effect of StAR siRNA on the apoptosis of the cells; furthermore, quantitative (q)RT-PCR and Western blot methods was used to determine the expression of StAR and apoptosis related molecules Bcl-2, Bax and Caspase-3 on both mRNA and protein levels in different groups; finally the secretion of testosterone in different groups was examined by radioimmunoassay. We observed that the YC can increase the expression of StAR in a dose-dependent manner, and YC can activate the promoter of StAR; moreover, transfection of StAR siRNA can block YC induced anti-apoptotic effects and increased production of testosterone. In conclusion, our results suggested that YC might suppress the apoptosis of MLTC-1 cells and enhance the production of testosterone through regulating the expression of StAR.


Assuntos
Apoptose/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Células Intersticiais do Testículo/efeitos dos fármacos , Células Intersticiais do Testículo/metabolismo , Fosfoproteínas/biossíntese , Testosterona/biossíntese , Animais , Apoptose/fisiologia , Cápsulas , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Expressão Gênica , Masculino , Camundongos , Fosfoproteínas/agonistas
3.
J Exp Med ; 215(2): 699-718, 2018 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-29339449

RESUMO

The Hippo pathway plays a vital role in tissue homeostasis and tumorigenesis. The transcription factor IRF3 is essential for innate antiviral immunity. In this study, we discovered IRF3 as an agonist of Yes-associated protein (YAP). The expression of IRF3 is positively correlated with that of YAP and its target genes in gastric cancer; the expression of both IRF3 and YAP is up-regulated and prognosticates patient survival. IRF3 interacts with both YAP and TEAD4 in the nucleus to enhance their interaction, promoting nuclear translocation and activation of YAP. IRF3 and YAP-TEAD4 are associated genome-wide to cobind and coregulate many target genes of the Hippo pathway. Overexpression of active IRF3 increased, but depletion of IRF3 reduced, the occupancy of YAP on the target genes. Knockdown or pharmacological targeting of IRF3 by Amlexanox, a drug used clinically for antiinflammatory treatment, inhibits gastric tumor growth in a YAP-dependent manner. Collectively, our study identifies IRF3 as a positive regulator for YAP, highlighting a new therapeutic target against YAP-driven cancers.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/agonistas , Fator Regulador 3 de Interferon/metabolismo , Terapia de Alvo Molecular , Fosfoproteínas/agonistas , Neoplasias Gástricas/tratamento farmacológico , Aminopiridinas/química , Aminopiridinas/farmacologia , Animais , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Feminino , Estudo de Associação Genômica Ampla , Células HEK293 , Humanos , Fator Regulador 3 de Interferon/genética , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Proteínas Musculares/metabolismo , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Neoplasias Gástricas/patologia , Fatores de Transcrição/metabolismo , Vírus/metabolismo
4.
J Nutr Biochem ; 47: 21-28, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28501702

RESUMO

Polycystic ovary syndrome (PCOS) is a complex endocrinopathy that is characterized by anovulation, hyperandrogenism and polycystic ovary. However, there is a lack of effective treatment for PCOS at present because the pathologic cause of PCOS has not been elucidated. Although it has been known that brown adipose tissue transplantation ameliorates PCOS by activating endogenous BAT, BAT transplantation is not applicable in clinic. Therefore, BAT activation with natural compound could be an effective treatment strategy for PCOS patients. Here, we found that 3 weeks of rutin (a novel compound for BAT activation) treatment increased BAT activation, thereby it improved thermogenesis and systemic insulin sensitivity in dehydroepiandrosterone (DHEA)-induced PCOS rat. In addition, the expression levels of ovarian steroidogenic enzymes such as P450C17, aromatase, 3ß-HSD, 17ß-HSD and STAR were up-regulated in rutin-treated PCOS rat. Furthermore, acyclicity and the serum level of luteinizing hormone were normalized, and a large number of mature ovulated follicle with a reduction of cystic formation were observed in PCOS rat after rutin treatment. Finally, rutin treatment surprisingly improved fertility and birth defect in PCOS rat. Collectively, our results indicate that rutin treatment significantly improves systemic insulin resistance and ovarian malfunction in PCOS, and our findings in this study provide a novel therapeutic option for the treatment of PCOS by activating BAT with rutin.


Assuntos
Tecido Adiposo Marrom/metabolismo , Modelos Animais de Doenças , Resistência à Insulina , Ovário/fisiopatologia , Síndrome do Ovário Policístico/dietoterapia , Rutina/uso terapêutico , Termogênese , Tecido Adiposo Marrom/patologia , Animais , Anovulação/etiologia , Anovulação/prevenção & controle , Anti-Inflamatórios não Esteroides/uso terapêutico , Fármacos Antiobesidade/uso terapêutico , Biomarcadores/sangue , Biomarcadores/metabolismo , Anormalidades Congênitas/etiologia , Anormalidades Congênitas/prevenção & controle , Desidroepiandrosterona , Indução Enzimática , Feminino , Infertilidade Feminina/etiologia , Infertilidade Feminina/prevenção & controle , Hormônio Luteinizante/antagonistas & inibidores , Hormônio Luteinizante/sangue , Ovário/metabolismo , Ovário/patologia , Fosfoproteínas/agonistas , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Síndrome do Ovário Policístico/metabolismo , Síndrome do Ovário Policístico/patologia , Síndrome do Ovário Policístico/fisiopatologia , Ratos Sprague-Dawley , Termografia , Imagem Corporal Total
5.
Biol Trace Elem Res ; 180(2): 233-238, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28349382

RESUMO

Aflatoxins have been considered as one of the major risk factors of male infertility, and aflatoxin B1 (AFB1) is the most highly toxic and prevalent member of the aflatoxins family. Selenium (Se), an essential nutritional trace mineral for normal testicular development and male fertility, has received extensive intensive on protective effects of male reproductive system due to its potential antioxidant and activating testosterone synthesis. To investigate the protective effect of Se on AFB1-induced testicular toxicity, the mice were orally administered with AFB1 (0.75 mg/kg) and Se (0.2 mg/kg or 0.4 mg/kg) for 45 days. We found that that Se elevated testes index, sperm functional parameters (concentration, malformation, and motility), and the level of serum testosterone in AFB1-exposed mice. Moreover, our results showed that Se attenuated the AFB1-induced oxidative stress and the reduction of testicular testosterone synthesis enzyme protein expression such as steroidogenic acute regulatory protein (StAR), P450 side-chain cleavage (P450scc), and 17ß-hydroxysteroid dehydrogenase (17ß-HSD) in AFB1-exposed mice. These results demonstrated that Se conferred protection against AFB1-induced testicular toxicity and can be attributed to its antioxidant and increased testosterone level by stimulating protein expression of StAR and testosterone synthetic enzymes.


Assuntos
Aflatoxina B1/antagonistas & inibidores , Suplementos Nutricionais , Infertilidade Masculina/prevenção & controle , Estresse Oxidativo , Substâncias Protetoras/uso terapêutico , Selênio/uso terapêutico , Testículo/efeitos dos fármacos , 17-Hidroxiesteroide Desidrogenases/antagonistas & inibidores , 17-Hidroxiesteroide Desidrogenases/química , 17-Hidroxiesteroide Desidrogenases/metabolismo , Aflatoxina B1/toxicidade , Animais , Animais não Endogâmicos , Antioxidantes/uso terapêutico , Biomarcadores/sangue , Biomarcadores/metabolismo , Carcinógenos Ambientais/química , Carcinógenos Ambientais/toxicidade , Enzima de Clivagem da Cadeia Lateral do Colesterol/antagonistas & inibidores , Enzima de Clivagem da Cadeia Lateral do Colesterol/química , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Contaminação de Alimentos , Doenças Transmitidas por Alimentos/etiologia , Doenças Transmitidas por Alimentos/prevenção & controle , Infertilidade Masculina/sangue , Infertilidade Masculina/induzido quimicamente , Infertilidade Masculina/metabolismo , Masculino , Camundongos , Estresse Oxidativo/efeitos dos fármacos , Fosfoproteínas/agonistas , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/metabolismo , Substâncias Protetoras/administração & dosagem , Selênio/administração & dosagem , Análise do Sêmen , Selenito de Sódio/administração & dosagem , Testículo/metabolismo , Testosterona/biossíntese , Testosterona/sangue
6.
J Steroid Biochem Mol Biol ; 171: 66-74, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28242260

RESUMO

Steroid hormones, estrogen and androgen, control transcription in various reproductive and non-reproductive tissues. Both hormones are known to be important for control of sperm release from the seminiferous epithelium (spermiation), a process characterized by extensive remodeling of actin filaments and endocytosis. Earlier studies with an estrogen (E2)-induced rat model of spermiation failure revealed genes involved in actin remodeling (Arpc1b and Evl) and endocytosis (Picalm, Eea1, and Stx5a) to be differentially regulated. Further, among these genes, Arpc1b and Evl were found to be estrogen-responsive whereas Eea1 and Stx5a were androgen-responsive and Picalm was responsive to both hormones in seminiferous tubule cultures. Yet, the mechanism by which these genes are regulated by estrogen and androgen in the testis was unclear. Here, we report the presence of a functional estrogen response element (ERE) upstream of Arpc1b and Evl genes and androgen response element (ARE) upstream of Picalm, Eea1, and Stx5a genes. Chromatin immunoprecipitation in control versus E2-treated testes revealed significant changes in estrogen receptor beta (ERß) recruitment along with coregulators to the EREs upstream of Arpc1b and Evl genes and androgen receptor (AR) at AREs upstream of Picalm, Eea1, and Stx5a genes. Enrichment patterns of these EREs/AREs with coregulators, activating and repressing histone modifications along with RNA polymerase II recruitment, correlated with the observed expression patterns of these genes upon E2 treatment. Taken together, our results reveal direct targets of estrogen and androgen in the testes and provide insights into transcriptional control of sperm release by the two steroid hormones.


Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/agonistas , Receptor beta de Estrogênio/agonistas , Estrogênios/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Proteínas dos Microfilamentos/agonistas , Fosfoproteínas/agonistas , Elementos de Resposta/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Androgênios/metabolismo , Animais , Células Cultivadas , Imunoprecipitação da Cromatina , Estradiol/administração & dosagem , Receptor beta de Estrogênio/metabolismo , Estrogênios/administração & dosagem , Injeções Subcutâneas , Masculino , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Proteínas Monoméricas de Montagem de Clatrina/agonistas , Proteínas Monoméricas de Montagem de Clatrina/genética , Proteínas Monoméricas de Montagem de Clatrina/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas Qa-SNARE/agonistas , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , Ratos Sprague-Dawley , Receptores Androgênicos/química , Receptores Androgênicos/metabolismo , Células de Sertoli/citologia , Células de Sertoli/efeitos dos fármacos , Células de Sertoli/metabolismo , Espermatogênese/efeitos dos fármacos , Espermatozoides/citologia , Espermatozoides/metabolismo , Proteínas de Transporte Vesicular/agonistas , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
7.
Nature ; 541(7638): 541-545, 2017 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-28068668

RESUMO

Cell fate perturbations underlie many human diseases, including breast cancer. Unfortunately, the mechanisms by which breast cell fate are regulated are largely unknown. The mammary gland epithelium consists of differentiated luminal epithelial and basal myoepithelial cells, as well as undifferentiated stem cells and more restricted progenitors. Breast cancer originates from this epithelium, but the molecular mechanisms that underlie breast epithelial hierarchy remain ill-defined. Here, we use a high-content confocal image-based short hairpin RNA screen to identify tumour suppressors that regulate breast cell fate in primary human breast epithelial cells. We show that ablation of the large tumour suppressor kinases (LATS) 1 and 2 (refs 5, 6), which are part of the Hippo pathway, promotes the luminal phenotype and increases the number of bipotent and luminal progenitors, the proposed cells-of-origin of most human breast cancers. Mechanistically, we have identified a direct interaction between Hippo and oestrogen receptor-α (ERα) signalling. In the presence of LATS, ERα was targeted for ubiquitination and Ddb1-cullin4-associated-factor 1 (DCAF1)-dependent proteasomal degradation. Absence of LATS stabilized ERα and the Hippo effectors YAP and TAZ (hereafter YAP/TAZ), which together control breast cell fate through intrinsic and paracrine mechanisms. Our findings reveal a non-canonical (that is, YAP/TAZ-independent) effect of LATS in the regulation of human breast cell fate.


Assuntos
Mama/citologia , Mama/enzimologia , Diferenciação Celular , Linhagem da Célula , Receptor alfa de Estrogênio/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/agonistas , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Mama/patologia , Proteínas de Transporte/metabolismo , Células Cultivadas , Receptor alfa de Estrogênio/agonistas , Feminino , Genes Supressores de Tumor , Humanos , Fosfoproteínas/agonistas , Fosfoproteínas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Serina-Treonina Quinases/deficiência , Proteólise , Transdução de Sinais , Fatores de Transcrição , Proteínas Supressoras de Tumor/deficiência , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases
8.
FEBS Lett ; 589(18): 2401-8, 2015 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-26226422

RESUMO

Many in vitro data have shown that the efficacy of several opioid drugs is correlated with differential mu-opioid (MOP) receptor phosphorylation. Label-free semiquantitative on-line nanoflow liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) analyses were performed to compare the endogenous MOP receptor phosphorylation patterns of mice administered with morphine, etonitazene and fentanyl. The analysis identified S363, T370 and S375 as phosphorylated residues in the carboxy-terminus. Only T370 and S375 were regulated by agonists, with a higher propensity to promote double phosphorylation for high efficacy agonists. Our study provides confirmation that differential agonist-driven multi-site phosphorylation of MOP receptor occurs in vivo and validate the use of MS to study endogenous GPCR phosphorylation.


Assuntos
Encéfalo/metabolismo , Fosfoproteínas/metabolismo , Proteômica , Receptores Opioides mu/metabolismo , Sequência de Aminoácidos , Analgésicos Opioides/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Espectrometria de Massas , Camundongos , Dados de Sequência Molecular , Fosfoproteínas/agonistas , Fosfoproteínas/química , Fosforilação/efeitos dos fármacos , Receptores Opioides mu/agonistas , Receptores Opioides mu/química
9.
J Biochem ; 158(5): 413-23, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25979969

RESUMO

Transcriptional co-activator with PSD-95/Dlg-A/ZO-1 (PDZ)-binding motif (TAZ) regulates in cell proliferation and differentiation. In mesenchymal stem cells it promotes osteogenesis and myogenesis, and suppresses adipogenesis. TAZ activators are expected to prevent osteoporosis, obesity and muscle atrophy. TAZ activation induces epithelial-mesenchymal transition, confers stemness to cancer cells and leads to poor clinical prognosis in cancer patients. In this point of view, TAZ inhibitors should contribute to cancer therapy. Thus, TAZ attracts attention as a two-faced drug target. We screened for TAZ modulators by using human lung cancer A549 cells expressing the fluorescent reporter. Through this assay, we obtained TAZ activator candidates. We unexpectedly found that ethacridine, a widely used antiseptic and abortifacient, enhances the interaction of TAZ and protein phosphatases and increases unphosphorylated and nuclear TAZ. Ethacridine inhibits adipogenesis in mesenchymal C3H10T1/2 cells through the activation of TAZ. This finding suggests that ethacridine is a bona fide TAZ activator and supports that our assay is useful to discover TAZ activators.


Assuntos
Adipogenia/efeitos dos fármacos , Fármacos Antiobesidade/farmacologia , Etacridina/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/agonistas , Células-Tronco Mesenquimais/efeitos dos fármacos , Proteína Fosfatase 1/metabolismo , Proteína Fosfatase 2/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/agonistas , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Genes Reporter/efeitos dos fármacos , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Fosfoproteínas/agonistas , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Regiões Promotoras Genéticas/efeitos dos fármacos , Proteína Fosfatase 1/química , Proteína Fosfatase 1/genética , Proteína Fosfatase 2/química , Proteína Fosfatase 2/genética , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
10.
Food Chem Toxicol ; 59: 303-10, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23811533

RESUMO

Microcystins (MCs) are a family of cyclic heptapeptides that are produced by blooming algae Microcystis. MCs have been implicated in the development of liver cancer, necrosis and even intrahepatic bleeding. Effective prophylactic approaches and complete removal of MCs are urgently needed. Accumulating evidence suggests that microcystin-LR (MC-LR)-induced damage is accompanied by oxidative stress. Supplementation of Se can enhance resistance to oxidative stress. Therefore, in the present study, we investigated the protective effects of κ-Selenocarrageenan (Se-Car), a kind of organic Se compound, in Balb/c mice exposed to MC-LR. Our results proved that Se-Car could significantly ameliorate the hepatic damage induced by MC-LR, including serum markers of liver dysfunction, oxidative damages and histological alterations. Furthermore, Se-Car could significantly alleviate the up-regulation of the molecular targets indicating mitochondrial dysfunction and endoplasmic reticulum stress induced by MC-LR. In conclusion, Se-Car showed clear protection against toxicity induced by MC-LR. Thus, Se-Car could be useful as a new category of anti-MC-LR toxicity reagent.


Assuntos
Antitoxinas/uso terapêutico , Toxinas Bacterianas/antagonistas & inibidores , Carragenina/uso terapêutico , Insuficiência Hepática/prevenção & controle , Fígado/efeitos dos fármacos , Toxinas Marinhas/antagonistas & inibidores , Microcistinas/antagonistas & inibidores , Compostos Organosselênicos/uso terapêutico , Animais , Toxinas Bacterianas/toxicidade , Biomarcadores/sangue , Proteínas de Transporte/agonistas , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Fatores de Iniciação em Eucariotos , Insuficiência Hepática/induzido quimicamente , Insuficiência Hepática/metabolismo , Insuficiência Hepática/patologia , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Fígado/fisiopatologia , Masculino , Toxinas Marinhas/toxicidade , Camundongos , Camundongos Endogâmicos BALB C , Microcistinas/toxicidade , Microcystis/metabolismo , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Hepáticas/patologia , Estresse Oxidativo/efeitos dos fármacos , Fosfoproteínas/agonistas , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Distribuição Aleatória , Transdução de Sinais/efeitos dos fármacos , Análise de Sobrevida
11.
FEBS J ; 280(16): 3920-7, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23773620

RESUMO

Adiponectin is an adipocyte hormone that is predominantly secreted by adipocytes, and has important roles in glucose and lipid homeostasis. Recent studies have shown that adiponectin is also involved in the regulation of many endocrine organs, such as the ovary, adrenal gland, and pituitary. However, its biological role in male testes is largely unexplored. The present findings demonstrate the presence of adeponectin receptors (adiponectin receptor 1 and adiponectin receptor 2) in TM3 cells derived from mouse Leydig cells. Proinflammatory cytokine treatment significantly downregulated mRNA and protein levels of adiponectin receptor 1 and adiponectin receptor 2. However, adiponectin pretreatment successfully inhibited the signaling pathway mediated by proinflammatory cytokines. At the molecular level, we provide compelling evidence that adeponectin achieves this by suppressing nuclear factor-κB activation through promotion of AMP-activated protein kinase phosphorylation. Thus, our data clearly indicate that adiponectin plays a protective role in Leydig cells through its anti-inflammatory actions.


Assuntos
Adiponectina/metabolismo , Citocinas/antagonistas & inibidores , Regulação para Baixo , Células Intersticiais do Testículo/metabolismo , NF-kappa B/antagonistas & inibidores , Receptores de Adiponectina/metabolismo , Transdução de Sinais , 3-Hidroxiesteroide Desidrogenases/antagonistas & inibidores , 3-Hidroxiesteroide Desidrogenases/química , 3-Hidroxiesteroide Desidrogenases/genética , 3-Hidroxiesteroide Desidrogenases/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Linhagem Celular , Ciclo-Oxigenase 2/química , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Citocinas/metabolismo , Inativação Gênica , Células Intersticiais do Testículo/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/química , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Fosfoproteínas/agonistas , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Receptores de Adiponectina/antagonistas & inibidores , Receptores de Adiponectina/genética , Regulação para Cima
12.
Mol Cell Endocrinol ; 376(1-2): 85-92, 2013 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-23791847

RESUMO

Calcium, in combination with vitamin D, is an effective treatment for osteoporosis. Since bone mineralisation occurs concurrently with osteoblast to osteocyte transition, we hypothesised that calcium would stimulate this process. The effect of calcium (1.8-11.8mM) was tested on human primary osteoblast (NHBC) differentiation in vitro. Cultures were assayed for cell-associated mineral and gene expression associated with osteoblast differentiation and mineralisation. Treatment with calcium resulted in a striking dose- and time-dependent increase in cell-associated mineralisation. Calcium appeared to promote osteoblast to osteocyte differentiation, as indicated by increased expression of osteocalcin (OCN), E11, dentin matrix protein 1 (DMP1) and SOST mRNA. The expression of the osteoclast inhibitor, osteoprotegerin, was dramatically enhanced by calcium. Calcium also increased the ratio of PHEX mRNA expression relative to that of MEPE, suggesting a mechanism for the pro-anabolic effect. Consistent with this, calcium-dependent mineralisation was reversed in the presence of MEPE-ASARM peptides. This study suggests that calcium promotes osteoblast to osteocyte transition and concurrent matrix mineralisation, at least in part through the PHEX-MEPE axis.


Assuntos
Cálcio/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Osteócitos/efeitos dos fármacos , RNA Mensageiro/genética , Biomarcadores/metabolismo , Proteínas Morfogenéticas Ósseas/agonistas , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Cálcio/metabolismo , Diferenciação Celular , Relação Dose-Resposta a Droga , Proteínas da Matriz Extracelular/agonistas , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Feminino , Marcadores Genéticos/genética , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteocalcina/agonistas , Osteocalcina/genética , Osteocalcina/metabolismo , Osteócitos/citologia , Osteócitos/metabolismo , Osteoprotegerina/agonistas , Osteoprotegerina/genética , Osteoprotegerina/metabolismo , Endopeptidase Neutra Reguladora de Fosfato PHEX/genética , Endopeptidase Neutra Reguladora de Fosfato PHEX/metabolismo , Fosfoproteínas/agonistas , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Cultura Primária de Células , RNA Mensageiro/metabolismo , Transdução de Sinais
13.
Platelets ; 23(8): 617-25, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22273509

RESUMO

The main responses of P2Y(1) ligation are platelet shape change and transient aggregation while P2Y(12) ligation amplifies P2Y(1)-induced aggregation and accelerates aggregation, secretion and thromboxane A(2) production induced by other agonist-receptor complexes. We searched for new targets of P2Y signalling using micro-arrays with 144 peptides representing known phosphosites of protein tyrosine kinases. ADP induced phosphorylation of peptides representing surface receptors, second messenger enzymes and cytoskeletal proteins. Strong phosphorylation was found in peptides representing Ephrin-receptor family members. Blockade of P2Y(1/12) inhibited phosphorylation of EphA4- and EphB1-peptides on micro-arrays. The EphA2/4 inhibitor 2,5-dimethylpyrrolyl benzoic acid derivative interfered with P2Y(1/12)-induced EphA4 phosphorylation, left P2Y(1)-induced aggregation unchanged but inhibited with P2Y(12)-induced secretion, second phase aggregation and thrombus formation on collagen at 1600 s(-1). These results show that platelet EphA4 is an important intermediate in P2Y(12)-induced granule secretion.


Assuntos
Plaquetas/enzimologia , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptor EphA4/agonistas , Receptores Purinérgicos P2Y12/metabolismo , Vesículas Secretórias/enzimologia , Difosfato de Adenosina/farmacologia , Plaquetas/citologia , Plaquetas/efeitos dos fármacos , Efrina-A4/agonistas , Efrina-A4/metabolismo , Humanos , Ligantes , Fosfoproteínas/agonistas , Fosfoproteínas/antagonistas & inibidores , Fosforilação , Ativação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Análise Serial de Proteínas , Antagonistas do Receptor Purinérgico P2/farmacologia , Receptor Cross-Talk , Receptor EphA4/metabolismo , Receptores Purinérgicos P2Y1/metabolismo , Vesículas Secretórias/efeitos dos fármacos , Transdução de Sinais
14.
PLoS One ; 3(11): e3776, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-19020663

RESUMO

Hematopoietic stem cells (HSCs) are probably the best-studied adult tissue-restricted stem cells. Although methods for flow cytometric detection of phosphoproteins in hematopoeitic progenitors and mature cells are available, analogous protocols for HSC are lacking. We present a robust method to study intracellular signaling in immunophenotypically-defined murine HSC/progenitor cell (HPC)-enriched populations. Using this method, we uncover differences in the response dynamics of several phosphoproteins representative of the Ras/MAP-Kinase(K), PI3K, mTOR and Jak/STAT pathways in HSC/HPCs stimulated by Scf, Thpo, as well as several other important HSC/HPC agonists.


Assuntos
Citometria de Fluxo/métodos , Células-Tronco Hematopoéticas/citologia , Fosfoproteínas/química , Células-Tronco/citologia , Animais , Antígenos CD34/biossíntese , Separação Celular , Humanos , Imunofenotipagem , Cinética , Ligantes , Camundongos , Fosfoproteínas/agonistas , Fosforilação , Fatores de Tempo
15.
Am J Physiol Cell Physiol ; 290(3): C892-9, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16267107

RESUMO

Phosphatase holoenzyme inhibitor (PHI)-1 is one of the newest members of the family of protein phosphatase inhibitor proteins. In isolated enzyme systems, several kinases, including PKC and rho kinase (ROCK), have been shown to phosphorylate PHI-1. However, it is largely unknown whether PHI-1 is phosphorylated in response to agonist stimulation in intact cells. We investigated this question in primary cultured rat aortic vascular smooth muscle cells (VSMCs). Using two-dimensional polyacrylamide gel electrophoresis and immunoblot, we found that there are two major PHI-1 spots under resting conditions: a minor spot with an acidic isoelectric point (pI) and a major spot with a more alkaline pI. Interestingly, U-46619, a G protein-coupled receptor agonist, caused a significant increase in the acidic spot, suggesting that it may represent a phosphorylated form of PHI-1. This was confirmed by phosphatase treatment and by a specific phospho-PHI-1 antibody. Furthermore, we found that angiotensin II, thrombin, and U-46619 increased phosphorylated PHI-1 from 9% of total PHI-1 in resting cells to 18%, 18%, and 30%, respectively. We also found that inhibition of ROCK by Y-27632 or H-1152 selectively diminished U-46619-induced CPI-17 phosphorylation, whereas it did not affect PHI-1 phosphorylation. Activation of ROCK by expressing V14RhoA selectively induced CPI-17 phosphorylation without affecting PHI-1 phosphorylation. In contrast, inhibition of PKC by GF-109203X or by PKC downregulation selectively diminished U-46619-induced PHI-1 phosphorylation without significantly affecting U-46619-induced CPI-17 phosphorylation. Activating PKC by PMA induced PHI-1 phosphorylation. Together, our results show for the first time that agonist induces PHI-1 phosphorylation in VSMCs and divergent kinase signaling couples agonist stimulation to PHI-1 and CPI-17 phosphorylation.


Assuntos
Proteínas Musculares/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Fosfoproteínas/metabolismo , Proteínas/metabolismo , Transdução de Sinais/fisiologia , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Angiotensina II/farmacologia , Animais , Proteínas Musculares/agonistas , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/efeitos dos fármacos , Fosfoproteínas/agonistas , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas/agonistas , Ratos , Ratos Sprague-Dawley , Trombina/farmacologia
16.
Exp Cell Res ; 298(1): 207-17, 2004 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-15242775

RESUMO

We have previously reported that expression of the constitutively active mutant of Galpha11 or stimulation of m1 muscarinic acetylcholine receptor induced proteolytic activation of Rho-associated kinase (ROCK-I) by caspase and apoptosis in HeLa cells. In this study, we investigate the molecular mechanisms of Galphaq/11-induced apoptosis in m1 muscarinic acetylcholine receptor-expressing HeLa cells. Overexpression of Bcl-2 inhibited carbachol-induced ROCK-I cleavage, indicating a mitochondrial apoptotic pathway. Overexpression of the constitutively active mutant of Akt that delivers an anti-apoptotic survival signal had a similar influence. Insulin, a major survival factor in many cells, strongly increased phosphorylation of Akt, which was completely blocked by carbachol. This latter effect was partially inhibited by treatment with the tyrosine phosphatase inhibitors, orthovanadate and pervanadate. In parallel with these observations, carbachol attenuated insulin-stimulated tyrosine phosphorylation of insulin receptor substrate-1, an effect eliminated by orthovanadate. On the other hand, carbachol induced rapid stimulation of endogenous RhoA, and expression of a constitutively active mutant of RhoA increased ROCK-I cleavage. Orthovanadate and the dominant negative mutant of RhoA partially, and their combination completely, inhibited carbachol-induced ROCK-I cleavage and apoptosis. These results demonstrate that Gq/11 signaling induces apoptosis by reducing insulin-stimulated Akt phosphorylation through tyrosine dephosphorylation and activating RhoA in HeLa cells.


Assuntos
Apoptose/fisiologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais/fisiologia , Proteína rhoA de Ligação ao GTP/metabolismo , Apoptose/efeitos dos fármacos , Carbacol/farmacologia , Agonistas Colinérgicos/farmacologia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Inibidores Enzimáticos/farmacologia , Células HeLa , Humanos , Insulina/metabolismo , Insulina/farmacologia , Proteínas Substratos do Receptor de Insulina , Peptídeos e Proteínas de Sinalização Intracelular , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mutação/genética , Fosfoproteínas/agonistas , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Serina-Treonina Quinases/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-akt , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptor Muscarínico M1/efeitos dos fármacos , Receptor Muscarínico M1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tirosina/metabolismo , Quinases Associadas a rho
17.
Prog Cell Cycle Res ; 5: 375-82, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-14593732

RESUMO

The tumour suppressor activity of p53 plays a major role in limiting abnormal proliferation, and inactivation of the p53 response is becoming increasingly accepted as a hallmark of cancer. In contrast, both p63 and p73, which are close relatives of p53, are rarely mutated in tumour cells. At a theoretical level, therapeutic approaches that reinstate p53 activity, or augment p63 and p73, provide plausible and potentially efficacious routes towards new cancer treatments. Equally important is the clinical need to increase the efficacy of conventional anti-cancer drugs. Incapacitating the p53 response to limit the side effects in healthy cells may be one approach towards increasing the therapeutic window of many current anti-cancer drugs. Nevertheless, while cancer drug discovery focussed on p53 is an exciting and realistic possibility, translating this concept into a clinical setting is likely to be challenging.


Assuntos
Antineoplásicos/efeitos adversos , Divisão Celular/fisiologia , Desenho de Drogas , Proteínas de Membrana , Neoplasias/tratamento farmacológico , Proteína Supressora de Tumor p53/metabolismo , Animais , Divisão Celular/efeitos dos fármacos , Proteínas de Ligação a DNA/agonistas , Proteínas de Ligação a DNA/metabolismo , Genes Supressores de Tumor , Humanos , Estrutura Molecular , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Nucleares/agonistas , Proteínas Nucleares/metabolismo , Fosfoproteínas/agonistas , Fosfoproteínas/metabolismo , Transativadores/agonistas , Transativadores/metabolismo , Fatores de Transcrição , Proteína Tumoral p73 , Proteína Supressora de Tumor p53/agonistas , Proteínas Supressoras de Tumor
18.
Clin Exp Pharmacol Physiol ; 30(9): 643-8, 2003 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12940882

RESUMO

1. Many clinical studies have suggested a relationship between oestrogen and insulin sensitivity. In the present study, HepG2 cells were divided into four groups: (i) control, incubated with 1 nmol/L insulin; (ii) the HI group, which was incubated with 100 nmol/L insulin to induce insulin resistance; (iii) the E2 group, in which control cells were incubated with 1 nmol/L insulin plus 1 nmol/L oestradiol; and (iv) the HI + E2 group, in which insulin-resistant cells were incubated with 100 nmol/L insulin + 1 nmol/L oestradiol. 2. A high concentration of insulin decreased the activity of phosphofructo-1-kinase (PFK), pyruvate dehydrogenase (PDH) and glycogen synthase (GS), as well as decreasing the expression of insulin receptor (IR) and insulin receptor substrate-2 (IRS-2). High insulin had no effect on glucose transport or the expression of insulin receptor-1 (IRS-1). 3. The addition of oestradiol to control cells increased glucose transport, the activity of PFK, PDH and GS and the expression of IRS-1 and IRS-2, but had no effect on the expression of IR. 4. Treatment of insulin-resistant HepG2 cells with oestradiol attenuated HI-induced decreases, except for IR, and the expression of IRS-1 was significantly higher than control, attaining levels seen in group 3. The expression of IRS-2 was significant higher than in insulin-resistant cells, but did not reach control levels. Changes in the activity of PFK, PDH and GS were the same as the changes seen in the expression of IRS-2. 5. These results suggest that high concentrations of insulin induce insulin resistance in HepG2 cells, whereas oestradiol improves glucose metabolism and insulin signal transduction of cells by enhancing the activity of key enzymes involved in glucose metabolism and the expression of IRS-1 and IRS-2.


Assuntos
Estradiol/farmacologia , Glucose/metabolismo , Insulina/fisiologia , Fosfoproteínas/biossíntese , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Proteínas Substratos do Receptor de Insulina , Peptídeos e Proteínas de Sinalização Intracelular , Fosfoproteínas/agonistas , Receptor de Insulina/agonistas , Receptor de Insulina/biossíntese , Transdução de Sinais/fisiologia
19.
J Biol Chem ; 278(25): 22578-85, 2003 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-12697772

RESUMO

Missense mutations of the ligand binding domain of hepatocyte nuclear factor (HNF)-4alpha result in maturity onset diabetes of the young (MODY)-1. We show here that MODY-1 as well as Gln-185 missense mutants of the ligand binding domain of HNF-4alpha fail to transactivate transcription of HNF-4alpha-responsive genes. Defective transactivation by these mutants is accounted for by their reduced binding affinities for fatty acyl agonist ligands of HNF-4alpha. These mutants may be rescued by exogenous fatty acid agonist ligands of HNF-4alpha, yielding transcriptional activities in the wild type range. The effect of added ligands is synergistic with that of transcriptional coactivators of HNF-4alpha. These findings may indicate the means for treating selected MODY-1 subjects with HNF-4alpha agonist nutrients and drugs.


Assuntos
Proteínas de Ligação a DNA , Diabetes Mellitus Tipo 2/genética , Fosfoproteínas/genética , Fatores de Transcrição/genética , Ativação Transcricional , Acil Coenzima A/farmacologia , Substituição de Aminoácidos , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Células COS , Variação Genética , Células HeLa , Fator 4 Nuclear de Hepatócito , Humanos , Cinética , Ligantes , Mutação de Sentido Incorreto , Fosfoproteínas/agonistas , Fosfoproteínas/química , Proteínas Recombinantes/metabolismo , Fatores de Transcrição/agonistas , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Transfecção , Células Tumorais Cultivadas
20.
Nat Neurosci ; 5(5): 415-24, 2002 May.
Artigo em Inglês | MEDLINE | ID: mdl-11976702

RESUMO

GABA (gamma-aminobutyric acid)(B) receptors are heterodimeric G protein-coupled receptors that mediate slow synaptic inhibition in the central nervous system. Here we show that the functional coupling of GABA(B)R1/GABA(B)R2 receptors to inwardly rectifying K(+) channels rapidly desensitizes. This effect is alleviated after direct phosphorylation of a single serine residue (Ser892) in the cytoplasmic tail of GABA(B)R2 by cyclic AMP (cAMP)-dependent protein kinase (PKA). Basal phosphorylation of this residue is evident in rat brain membranes and in cultured neurons. Phosphorylation of Ser892 is modulated positively by pathways that elevate cAMP concentration, such as those involving forskolin and beta-adrenergic receptors. GABA(B) receptor agonists reduce receptor phosphorylation, which is consistent with PKA functioning in the control of GABA(B)-activated currents. Mechanistically, phosphorylation of Ser892 specifically enhances the membrane stability of GABA(B) receptors. We conclude that signaling pathways that activate PKA may have profound effects on GABA(B) receptor-mediated synaptic inhibition. These results also challenge the accepted view that phosphorylation is a universal negative modulator of G protein-coupled receptors.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Receptores de GABA-B/metabolismo , Animais , Encéfalo/metabolismo , Química Encefálica , Células CHO , Células COS , Membrana Celular/química , Membrana Celular/metabolismo , Células Cultivadas , Cricetinae , AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Agonistas GABAérgicos/farmacologia , Agonistas dos Receptores de GABA-B , Humanos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Técnicas de Patch-Clamp , Fosfoproteínas/agonistas , Fosfoproteínas/análise , Fosfoproteínas/metabolismo , Fosforilação , Canais de Potássio/metabolismo , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/análise , Isoformas de Proteínas/metabolismo , Ratos , Receptores de GABA-B/análise , Proteínas Recombinantes/metabolismo , Transdução de Sinais/fisiologia
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