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1.
Einstein (Sao Paulo) ; 18: eAO4560, 2020.
Artigo em Inglês, Português | MEDLINE | ID: mdl-32321078

RESUMO

OBJECTIVE: To investigate if ICI 182,780 (fulvestrant), a selective estrogen receptor alpha/beta (ERα/ERß) antagonist, and G-1, a selective G-protein-coupled receptor (GPER) agonist, can potentially induce autophagy in breast cancer cell lines MCF-7 and SKBr3, and how G-1 affects cell viability. METHODS: Cell viability in MCF-7 and SKBr3 cells was assessed by the MTT assay. To investigate the autophagy flux, MCF-7 cells were transfected with GFP-LC3, a marker of autophagosomes, and analyzed by real-time fluorescence microscopy. MCF-7 and SKBr3 cells were incubated with acridine orange for staining of acidic vesicular organelles and analyzed by flow cytometry as an indicator of autophagy. RESULTS: Regarding cell viability in MCF-7 cells, ICI 182,780 and rapamycin, after 48 hours, led to decreased cell proliferation whereas G-1 did not change viability over the same period. The data showed that neither ICI 182,780 nor G-1 led to increased GFP-LC3 puncta in MCF-7 cells over the 4-hour observation period. The cytometry assay showed that ICI 182,780 led to a higher number of acidic vesicular organelles in MCF-7 cells. G-1, in turn, did not have this effect in any of the cell lines. In contrast, ICI 182,780 and G-1 did not decrease cell viability of SKBr3 cells or induce formation of acidic vesicular organelles, which corresponds to the final step of the autophagy process in this cell line. CONCLUSION: The effect of ICI 182,780 on increasing acidic vesicular organelles in estrogen receptor-positive breast cancer cells appears to be associated with its inhibitory effect on estrogen receptors, and GPER does notseem to be involved. Understanding these mechanisms may guide further investigations of these receptors' involvement in cellular processes of breast cancer resistance.


Assuntos
Autofagia/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Antagonistas do Receptor de Estrogênio/farmacologia , Fulvestranto/farmacologia , Receptores Acoplados a Proteínas-G/agonistas , Análise de Variância , Western Blotting , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Receptor alfa de Estrogênio/antagonistas & inibidores , Receptor beta de Estrogênio/antagonistas & inibidores , Feminino , Citometria de Fluxo/métodos , Humanos , Células MCF-7 , Receptores Acoplados a Proteínas-G/análise , Reprodutibilidade dos Testes , Sirolimo/farmacologia , Fatores de Tempo , Transfecção/métodos
2.
J Am Soc Nephrol ; 31(2): 257-278, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31932472

RESUMO

BACKGROUND: Mononuclear phagocytes (MPs), including macrophages, monocytes, and dendritic cells (DCs), are phagocytic cells with important roles in immunity. The developmental origin of kidney DCs has been highly debated because of the large phenotypic overlap between macrophages and DCs in this tissue. METHODS: We used fate mapping, RNA sequencing, flow cytometry, confocal microscopy, and histo-cytometry to assess the origin and phenotypic and functional properties of renal DCs in healthy kidney and of DCs after cisplatin and ischemia reperfusion-induced kidney injury. RESULTS: Adult kidney contains at least four subsets of MPs with prominent Clec9a-expression history indicating a DC origin. We demonstrate that these populations are phenotypically, functionally, and transcriptionally distinct from each other. We also show these kidney MPs exhibit unique age-dependent developmental heterogeneity. Kidneys from newborn mice contain a prominent population of embryonic-derived MHCIInegF4/80hiCD11blow macrophages that express T cell Ig and mucin domain containing 4 (TIM-4) and MER receptor tyrosine kinase (MERTK). These macrophages are replaced within a few weeks after birth by phenotypically similar cells that express MHCII but lack TIM-4 and MERTK. MHCII+F4/80hi cells exhibit prominent Clec9a-expression history in adulthood but not early life, indicating additional age-dependent developmental heterogeneity. In AKI, MHCIInegF4/80hi cells reappear in adult kidneys as a result of MHCII downregulation by resident MHCII+F4/80hi cells, possibly in response to prostaglandin E2 (PGE2). RNA sequencing further suggests MHCII+F4/80hi cells help coordinate the recruitment of inflammatory cells during renal injury. CONCLUSIONS: Distinct developmental programs contribute to renal DC and macrophage populations throughout life, which could have important implications for therapies targeting these cells.


Assuntos
Células Dendríticas/imunologia , Rim/imunologia , Macrófagos/imunologia , Nefrite/imunologia , Lesão Renal Aguda/imunologia , Fatores Etários , Animais , Antígeno CD11b/análise , Receptor 1 de Quimiocina CX3C/análise , Proteínas de Ligação ao Cálcio/análise , Cisplatino/farmacologia , Antígenos de Histocompatibilidade Classe II/análise , Rim/efeitos dos fármacos , Rim/metabolismo , Lectinas Tipo C/análise , Camundongos , Camundongos Endogâmicos C57BL , Receptores Acoplados a Proteínas-G/análise , Receptores Imunológicos/análise
3.
Adv Exp Med Biol ; 1131: 27-72, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646506

RESUMO

Ca2+, Na+ and K+- permeable ion channels as well as GPCRs linked to Ca2+ release are important drug targets. Accordingly, high-throughput fluorescence plate reader assays have contributed substantially to drug discovery efforts and pharmacological characterization of these receptors and ion channels. This chapter describes some of the basic properties of the fluorescent dyes facilitating these assay approaches as well as general methods for establishment and optimisation of fluorescence assays for ion channels and Gq-coupled GPCRs.


Assuntos
Bioensaio , Canais Iônicos , Receptores Acoplados a Proteínas-G , Animais , Bioensaio/tendências , Descoberta de Drogas , Corantes Fluorescentes/metabolismo , Humanos , Canais Iônicos/análise , Receptores Acoplados a Proteínas-G/análise
4.
BMC Cancer ; 19(1): 898, 2019 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-31500591

RESUMO

BACKGROUND: Although the leucine zipper-EF-hand-containing transmembrane protein 1 (LETM1) is one of the mitochondrial inner membrane proteins that is involved in cancer prognosis in various tumors, LETM1 as a biomarker for prognostic evaluation of non-small cell lung carcinoma (NSCLC) has not been well studied. METHODS: To address this issue, we used 75 cases NSCLC, 20 cases adjacent normal lung tissues and NSCLC cell lines. We performed immunohistochemistry staining and western blot analysis as well as immunofluorescence imaging. RESULTS: Our studies show that expression of LETM1 is significantly correlated with the lymph node metastasis (p = 0.003) and the clinical stage (p = 0.005) of NSCLC. The Kaplan-Meier survival analysis revealed that NSCLC patients with positive expression of LETM1 exhibits a shorter overall survival (OS) rate (p = 0.005). The univariate and multivariate Cox regression analysis indicated that LETM1 is a independent poor prognostic marker of NSCLC. In addition, the LETM1 expression is correlated with cancer stemness-related gene LGR5 (p < 0.001) and HIF1α expression (p < 0.001), but not with others. Moreover, LETM1 expression was associated with the expression of cyclin D1 (p = 0.003), p27 (p = 0.001), pPI3K(p85) (p = 0.025), and pAkt-Thr308 (p = 0.004). Further, our studies show in LETM1-positive NSCLC tissues the microvessel density was significantly higher than in the negative ones (p = 0.024). CONCLUSION: These results indicate that LETM1 is a potential prognostic biomarker of NSCLC.


Assuntos
Proteínas de Ligação ao Cálcio/análise , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas de Membrana/análise , Idoso , Biomarcadores Tumorais/análise , Proteínas de Ligação ao Cálcio/genética , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/análise , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Prognóstico , Receptores Acoplados a Proteínas-G/análise , Receptores Acoplados a Proteínas-G/genética
5.
Int J Mol Sci ; 20(16)2019 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-31404983

RESUMO

Hematopoietic stem cell transplantation (HSCT) remains the only curative treatment for several malignant and non-malignant diseases at the cost of serious treatment-related toxicities (TRTs). Recent research on extending the benefits of HSCT to more patients and indications has focused on limiting TRTs and improving immunological effects following proper mobilization and engraftment. Increasing numbers of studies report associations between HSCT outcomes and the expression or the manipulation of G protein-coupled receptors (GPCRs). This large family of cell surface receptors is involved in various human diseases. With ever-better knowledge of their crystal structures and signaling dynamics, GPCRs are already the targets for one third of the current therapeutic arsenal. The present paper assesses the current status of animal and human research on GPCRs in the context of selected HSCT outcomes via a systematized survey and analysis of the literature.


Assuntos
Mobilização de Células-Tronco Hematopoéticas/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Receptores Acoplados a Proteínas-G/análise , Animais , Células-Tronco Hematopoéticas/citologia , Humanos , Resultado do Tratamento
7.
J Agric Food Chem ; 67(27): 7694-7705, 2019 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-31250637

RESUMO

Liver plays a central role in modulating blood glucose level. Our most recent findings suggested that supplementation with microbiota metabolite sodium butyrate (NaB) could ameliorate progression of type 2 diabetes mellitus (T2DM) and decrease blood HbA1c in db/db mice. To further investigate the role of butyrate in homeostasis of blood glucose and glycogen metabolism, we carried out the present study. In db/db mice, we found significant hypertrophy and steatosis in hepatic lobules accompanied by reduced glycogen storage, and expression of GPR43 was significantly decreased by 59.38 ± 3.33%; NaB administration significantly increased NaB receptor G-protein coupled receptor 43 (GPR43) level and increased glycogen storage in both mice and HepG2 cells. Glucose transporter 2 (GLUT2) and sodium-glucose cotransporter 1 (SGLT1) on cell membrane were upregulated by NaB. The activation of intracellular signaling Protein kinase B (PKB), also known as AKT, was inhibited while glycogen synthase kinase 3 (GSK3) was activated by NaB in both in vivo and in vitro studies. The present study demonstrated that microbiota metabolite NaB possessed beneficial effects on preserving blood glucose homeostasis by promoting glycogen metabolism in liver cells, and the GPR43-AKT-GSK3 signaling pathway should contribute to this effect.


Assuntos
Ácido Butírico/administração & dosagem , Diabetes Mellitus Tipo 2/metabolismo , Glicogênio Hepático/metabolismo , Animais , Glicemia/análise , Ácido Butírico/metabolismo , Imunofluorescência , Microbioma Gastrointestinal/fisiologia , Transportador de Glucose Tipo 2/análise , Hemoglobina A Glicada/análise , Quinase 3 da Glicogênio Sintase/metabolismo , Células Hep G2 , Homeostase/efeitos dos fármacos , Humanos , Fígado/química , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Acoplados a Proteínas-G/análise , Transdução de Sinais/efeitos dos fármacos , Transportador 1 de Glucose-Sódio/análise
8.
Inflammopharmacology ; 27(6): 1309-1318, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31168686

RESUMO

SB-706375 is a selective receptor antagonist of human urotensin-II (hU-II), which can block the aorta contraction induced by hU-II in rats. The effect of SB-706375 on myocardial ischaemia-reperfusion (I/R) injury is unclear. The major objective of this study was to investigate whether SB-706375 has a protective effect on myocardial I/R injury in rats and explore its possible mechanisms. Isolated hearts of Adult Sprague-Dawley were perfused in a Langendorff apparatus, and haemodynamic parameters, lactate dehydrogenase (LDH), creatine phosphokinase-MB (CK-MB), cardiac troponin I (cTnI), RhoA, and the protein expressions of U-II receptor (UTR), receptor-interacting protein 3 (RIP3), Rho-associated coiled-coil-containing protein kinase 1 (ROCK1) and Rho-associated coiled-coil-containing protein kinase 2 (ROCK2) were assessed. We found that SB-706375 (1 × 10-6 and 1 × 10-5 mol/L) significantly inhibited the changes of haemodynamic parameters and reduced LDH and CK-MB activities and also cTnI level in the coronary effluents in the heart subjected to myocardial I/R injury. Further experiments studies showed that SB-706375 obviously prevented myocardial I/R increased RhoA activity and UTR, RIP3, ROCK1, and ROCK2 protein expressions. ROCK inhibition abolished the improving effect of SB-706375 on myocardial I/R-induced haemodynamic change in the isolated perfused rat heart. These findings suggested that SB-706375 provides cardio-protection against I/R injury in isolated rats by blocking UTR-RhoA/ROCK-RIP3 pathway.


Assuntos
Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miocárdio/patologia , Pirrolidinas/farmacologia , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores , Receptores Acoplados a Proteínas-G/antagonistas & inibidores , Sulfonamidas/farmacologia , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Quinases Associadas a rho/antagonistas & inibidores , Animais , Feminino , Masculino , Necrose , Ratos , Ratos Sprague-Dawley , Proteína Serina-Treonina Quinases de Interação com Receptores/fisiologia , Receptores Acoplados a Proteínas-G/análise , Transdução de Sinais/fisiologia , Proteínas rho de Ligação ao GTP/fisiologia , Quinases Associadas a rho/fisiologia
9.
Histochem Cell Biol ; 152(2): 155-166, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31111198

RESUMO

Trace amine-associated receptors are G protein-coupled receptors of which TAAR1 is the most well-studied. Recently, Vattai et al. (J Cancer Res Clin Oncol 143:1637-1647 https://doi.org/10.1007/s00432-017-2420-8 , 2017) reported that expression of TAAR1 may be a marker of breast cancer (BC) survival, with a positive correlation also suggested between TAAR1 expression and HER2 positivity. Neither a role for TAAR1 in breast tissue, nor in cancer, had previously been suspected. We, therefore, sought to provide independent validation and to further examine these putative relationships. First, a bioinformatic analysis on 58 total samples including normal breast tissue, BC-related cell lines, and tumour samples representing different BC sub-types found no clear correlation between TAAR1 mRNA levels and any BC subtype, including HER2 + . We next confirmed the bioinformatics data correlated to protein expression using a well validated anti-human TAAR1 antibody. TAAR1 mRNA levels correlated with the relative intensity of immunofluorescence staining in six BC cell lines (MCF-7, T47D, MDA-MB-231, SKBR3, MDA-MB-468, BT-474), but not in the MCF-10A immortalized mammary gland line, which had high mRNA but low protein levels. As expected, TAAR1 protein was intracellular in all cell lines. Surprisingly MCF-7, SKBR3, and MDA-MB-468 showed pronounced nuclear localization. The relative protein expression in MCF-7, MDA-MB-231, and MCF-10A lines was further confirmed by semi-quantitative flow cytometry. Finally, we demonstrate that the commercially available anti-TAAR1 antibody has poor selectivity, which likely explains the lack of correlation with the previous study. Therefore, while we clearly demonstrate variable expression and sub-cellular localization of TAAR1 across BC cell lines, we find no evidence for association with BC subtype.


Assuntos
Neoplasias da Mama/genética , Receptores Acoplados a Proteínas-G/análise , Receptores Acoplados a Proteínas-G/genética , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/metabolismo , Linhagem Celular , Biologia Computacional , Feminino , Citometria de Fluxo , Imunofluorescência , Humanos , RNA Mensageiro/análise , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo
10.
J Sci Food Agric ; 99(11): 4952-4962, 2019 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-30953347

RESUMO

BACKGROUND: Long-term artificial sweetener consumption has been reported to induce glucose intolerance, and the intestinal microbiota seems as an important target. While the impacts of artificial sweeteners on energy balance remain controversial, this work aimed to evaluate the protective effects in mice of a low digestible carbohydrate (LDC) diet on plasma glucose, plasma fasting insulin, sweet taste receptors, glucose transporters, and absorption of carbohydrates, together with consumption of acesulfame potassium (AK) or saccharin (SAC). RESULTS: Artificial sweetener was administered to mice for 12 weeks to induce glucose metabolism disorders; mice were treated with an LDC diet for the final 6 weeks. The experimental groups were treated with an LDC diet that had the same energy as the normal-diet group. Prolonged administration of artificial sweeteners led to metabolic dysfunction, characterized by significantly increased plasma glucose, insulin resistance, sweet taste receptors, glucose transporters, and absorption of carbohydrates. Treatment with an LDC diet positively modulated these altered parameters, suggesting overall beneficial effects of an LDC diet on detrimental changes associated with artificial sweeteners. CONCLUSIONS: Reducing digestible carbohydrates in the diet can significantly reduce the absorption of carbohydrates and improve glucose metabolism disorders caused by dietary factors. These effects may be due to the fact that reducing the amount of digestible carbohydrates in the feed can reduce the number of intestinal sweet receptors induced by exposure to artificial sweeteners. © 2019 Society of Chemical Industry.


Assuntos
Dieta com Restrição de Carboidratos , Carboidratos da Dieta/farmacocinética , Duodeno/metabolismo , Transtornos do Metabolismo de Glucose/induzido quimicamente , Edulcorantes/efeitos adversos , Animais , Glicemia/análise , Carboidratos da Dieta/administração & dosagem , Digestão , Duodeno/química , Microbioma Gastrointestinal/efeitos dos fármacos , Intolerância à Glucose/induzido quimicamente , Transtornos do Metabolismo de Glucose/metabolismo , Insulina/sangue , Resistência à Insulina , Absorção Intestinal/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Receptores Acoplados a Proteínas-G/análise , Receptores Acoplados a Proteínas-G/efeitos dos fármacos , Paladar/efeitos dos fármacos , Ganho de Peso
11.
Med Sci Monit Basic Res ; 25: 76-87, 2019 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-30842391

RESUMO

BACKGROUND The aim of this study was to determine if components of the endocannabinoid system are modulated in uterine leiomyomas (fibroids). Components studied included cannabinoid receptors 1 (CB1) and 2 (CB2); the G protein-coupled receptor GPR55; transient potential vanilloid receptor 1 (TRPV1) and the endocannabinoid modulating enzymes N-acylphosphatidylethanolamine-specific phospholipase D (NAPE-PLD) and fatty acid amide hydrolase (FAAH), and their N-acylethanolamine (NAE) ligands: N-arachidonylethanolamine (AEA), N-oleoylethanolamine (OEA), and N-palmityolethanaolamine (PEA). MATERIAL AND METHODS Transcript levels of CB1, CB2, TRPV1, GPR55, NAPE-PLD, and FAAH were measured using RT-PCR and correlated with the tissue levels of the 3 NAEs in myometrial tissues. The tissues studied were: 1) fibroids, 2) myometrium adjacent/juxtaposed to the fibroid lesions, and 3) normal myometrium. Thirty-seven samples were processed for NAE measurements and 28 samples were used for RT-PCR analyses. RESULTS FAAH expression was significantly lower in fibroids, resulting in a NAPE-PLD: FAAH ratio that favors higher AEA levels in pre-menopausal tissues, whilst PEA levels were significantly lower, particularly in post-menopausal women, suggesting PEA protects against fibroid pathogenesis. The CB1: CB2 ratio was lower in fibroids, suggesting that loss of CB1 expression affects the fibroid cell phenotype. Significant correlations between reduced FAAH, CB1, and GPR55 expression and PEA in fibroids indicate that the loss of these endocannabinoid system components are biomarkers of leiomyomata. CONCLUSIONS Loss of expression of CB1, FAAH, GPR55, and PEA production are linked to the pathogenesis of uterine fibroids and further understanding of this might eventually lead to better disease indicators or the development of therapeutic potentials that might eventually be used in the management of uterine fibroids.


Assuntos
Endocanabinoides/metabolismo , Leiomioma/metabolismo , Leiomioma/fisiopatologia , Adulto , Idoso , Amidoidrolases/análise , Biópsia , Etanolaminas/metabolismo , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Pessoa de Meia-Idade , Ácidos Oleicos/metabolismo , Fosfolipase D/análise , Receptor CB1 de Canabinoide/análise , Receptor CB2 de Canabinoide/análise , Receptores Acoplados a Proteínas-G/análise , Canais de Cátion TRPV/análise , Útero/fisiopatologia
12.
Curr Opin Cell Biol ; 57: 115-122, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30849632

RESUMO

G protein-coupled receptors (GPCRs), belonging to the largest class of membrane proteins, play a prominent role in many (patho)physiological processes and are, therefore, important drug targets. Although most often targeted by small molecules, these receptors have become interesting targets for antibodies and antibody fragments, especially camelid-derived heavy chain-only antibodies and fragments thereof (nanobodies). The small size and molecular structure of nanobodies allow GPCR-binding and modulation, from both the intracellular and extracellular sides. These molecular features make nanobodies attractive tools to study, modulate, and exploit GPCRs. Besides modulating GPCR activity as monovalent or multivalent constructs, nanobodies can also be functionalized for imaging and therapy. Moreover, GPCR-binding nanobodies have been instrumental in obtaining crystal structures of GPCRs, facilitating structure-based drug discovery. Here, we describe the current status and future perspectives of nanobodies targeting GPCRs intra and extracellularly.


Assuntos
Receptores Acoplados a Proteínas-G/análise , Receptores Acoplados a Proteínas-G/metabolismo , Anticorpos de Domínio Único/metabolismo , Animais , Descoberta de Drogas , Humanos , Transdução de Sinais , Anticorpos de Domínio Único/química
13.
Biochem Biophys Res Commun ; 511(2): 280-286, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30782484

RESUMO

In the mammalian taste system, the taste receptor type 2 (T2R) family mediates bitter taste, and the taste receptor type 1 (T1R) family mediates sweet and umami tastes (the heterodimer of T1R2/T1R3 forms the sweet taste receptor, and the heterodimer of T1R1/T1R3 forms the umami taste receptor). In the chicken genome, bitter (T2R1, T2R2, and T2R7) and umami (T1R1 and T1R3) taste receptor genes have been found. However, the localization of these taste receptors in the taste buds of chickens has not been elucidated. In the present study, we demonstrated that the bitter taste receptor T2R7 and the umami taste receptor subunit T1R1 were expressed specifically in the taste buds of chickens labeled by Vimentin, a molecular marker for chicken taste buds. We analyzed the distributions of T2R7 and T1R1 on the oral epithelial sheets of chickens and among 3 different oral tissues of chickens: the palate, the base of the oral cavity, and the posterior tongue. We found that the distribution patterns and numbers were similar between taste bud clusters expressing these receptors and those expressing Vimentin. These results indicated broad distributions of T2R7 and T1R1 in the gustatory tissues of the chicken oral cavity. In addition, 3D-reconstructed images clearly revealed that high levels of T2R7 and T1R1 were expressed in Vimentin-negative taste bud cells. Taken together, the present results indicated the presence of bitter and umami sensing systems in the taste buds of chickens, and broad distribution of T2R7 and T1R1 in the chicken oral cavity.


Assuntos
Proteínas Aviárias/análise , Galinhas/anatomia & histologia , Receptores Acoplados a Proteínas-G/análise , Papilas Gustativas/ultraestrutura , Vimentina/análise , Animais , Galinhas/fisiologia , Paladar , Papilas Gustativas/química , Papilas Gustativas/citologia , Percepção Gustatória
14.
ACS Chem Neurosci ; 10(4): 1884-1891, 2019 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-30726666

RESUMO

Neuropeptides are chemical messengers that act to regulate a number of physiological processes, including feeding, reward, pain, and memory, among others. PEN is one of the most abundant hypothalamic neuropeptides; however, until recently, its target receptor remained unknown. In this Review, we summarize recent developments in research focusing on PEN and its receptor GPR83. We describe the studies leading to the deorphanization of GPR83 as the receptor for PEN. We also describe the signaling mediated by the PEN-GPR83 system, as well as the physiological roles in which PEN-GPR83 has been implicated. As studies have suggested a role for the PEN-GPR83 system in food intake and body weight regulation, as well as in drug addiction and reward disorders, a thorough understanding of this novel neuropeptide-receptor system will help identify novel therapeutic targets to treat pathophysiological conditions involving PEN-GPR83.


Assuntos
Hipotálamo/metabolismo , Neuropeptídeos/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Recompensa , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Animais , Comportamento Aditivo/genética , Comportamento Aditivo/metabolismo , Humanos , Hipotálamo/química , Neuropeptídeos/análise , Neuropeptídeos/genética , Receptores Acoplados a Proteínas-G/análise , Receptores Acoplados a Proteínas-G/genética
15.
Biosci Rep ; 39(2)2019 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-30760632

RESUMO

G protein-coupled receptor 30 (GPR30), or G protein-coupled estrogen receptor (GPER), is a G protein-coupled receptor (GPCR) that is currently attracting considerable attention in breast cancer and cardiometabolic regulation. The receptor was reported to be a novel membrane estrogen receptor mediating rapid non-genomic responses. However, questions remain about both the cognate ligand and the subcellular localization of receptor activity. Here, we used human embryonic kidney (HEK) 293 (HEK293) cells ectopically expressing N-terminally FLAG-tagged human GPR30 and three unique antibodies (Ab) specifically targetting the receptor N-terminal domain (N-domain) to investigate the role of N-glycosylation in receptor maturation and activity, the latter assayed by constitutive receptor-stimulated extracellular-regulated protein kinase (ERK) 1/2 (ERK1/2) activity. GPR30 expression was complex with receptor species spanning from approximately 40 kDa to higher molecular masses and localized in the endoplasmatic reticulum (ER), the plasma membrane (PM), and endocytic vesicles. The receptor contains three conserved asparagines, Asn25, Asn32, and Asn44, in consensus N-glycosylation motifs, all in the N-domain, and PNGase F treatment showed that at least one of them is N-glycosylated. Mutating Asn44 to isoleucine inactivated the receptor, yielding a unique receptor species at approximately 20 kDa that was recognized by Ab only in a denatured state. On the other hand, mutating Asn25 or Asn32 either individually or in combination, or truncating successively N-domain residues 1-42, had no significant effect either on receptor structure, maturation, or activity. Thus, Asn44 in the GPR30 N-domain is required for receptor structure and activity, whereas N-domain residues 1-42, including specifically Asn25 and Asn32, do not play any major structural or functional role(s).


Assuntos
Asparagina/metabolismo , Receptores Estrogênicos/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Sequência de Aminoácidos , Asparagina/análise , Glicosilação , Células HEK293 , Humanos , Proteína Quinase 1 Ativada por Mitógeno/análise , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/análise , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Conformação Proteica , Domínios Proteicos , Receptores Estrogênicos/análise , Receptores Acoplados a Proteínas-G/análise
16.
Laryngoscope ; 129(9): E307-E312, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-30675726

RESUMO

OBJECTIVES/HYPOTHESIS: Taste sensitivity varies greatly among individuals influencing eating behavior and health, consequently the disorders of this sense can affect the quality of life. The ability to perceive the bitter of thiourea compounds, such as phenylthiocarbamide (PTC), has been largely reported as a marker of the general taste sensitivity, food preferences, and health. PTC sensitivity is mediated by the TAS2R38 receptor and its genetic common variants. We study the role of the TAS2R38 receptor in taste disorders with the aim of understanding if these can be genetically determined. STUDY DESIGN: Prospective cohort study. METHODS: Differences in the PTC responsiveness between the patients cohort and healthy controls were assessed. All subjects received standardized tests for smell and taste function and were genotyped for the TAS2R38 gene. RESULTS: PAV/PAV homozygous patients gave high PTC ratings, whereas PAV/AVI genotypes reported lower values, which are similar to those determined in AVI/AVI or rare genotypes. In addition, the patients cohort did not meet the Hardy-Weinberg equilibrium at the TAS2R38 locus, showing a very low frequency of subjects carrying the PAV/AVI diplotype. Independently, in healthy controls who were in equilibrium at the locus, PAV/PAV homozygous and heterozygous rated PTC bitterness higher compared to AVI/AVI or rare genotypes. CONCLUSIONS: Our findings, by showing that an only taster haplotype (PAV) is not sufficient to evoke high responses of TAS2R38 receptor in patients with taste disorders, suggest that the genetic constitution may represent a risk factor for the development of taste disorders. LEVEL OF EVIDENCE: 2c Laryngoscope, 129:E307-E312, 2019.


Assuntos
Receptores Acoplados a Proteínas-G/análise , Distúrbios do Paladar/genética , Paladar/genética , Idoso , Estudos de Casos e Controles , Feminino , Genótipo , Homozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Feniltioureia/análise , Projetos Piloto , Estudos Prospectivos , Saliva/química
17.
Life Sci ; 217: 49-56, 2019 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-30503722

RESUMO

AIMS: GPR18, a G protein-coupled receptor (GPCR), is involved in bacterial clearance and survival in microbial sepsis. In this study, we examine GPR18 expression on polymorphonuclear neutrophils (PMNs) of patients with sepsis and to determine the potential association with disease severity and outcomes. MAIN METHODS: We enrolled 81 patients admitted at the intensive care unit (ICU) with the diagnosis of sepsis. PMNs GPR18 expression was measured by flow cytometry at admission. Sequential Organ Failure Assessment (SOFA) and Acute Physiology and Chronic Health Evaluation (APACHEII) as well as other biomarker were measured at admission. Cox regression analysis was used to determine the influence of PMNs GPR18 expression on 28-day mortality. KEY FINDINGS: Patients with sepsis had a decreased percentage of PMNs bearing GPR18 in comparison with healthy subjects (P < 0.001). Compared with survivors, non-survivors had lower percentage of GPR18-positive PMNs, but higher SOFA, APACHEIIscores, and WBC count. There were inverse correlations between the percentage of GPR18-positive PMNs and APACHEII, SOFA score and C-recreative protein (CRP). Using Kaplan-Meier analysis, high percentage of PMNs expressing GPR18 (≥43.7%) was associated with a preferable 28-day survival (P = 0.004). High percentage of PMNs expressing GPR18 (≥43.7%) was significantly and independently associated with 28-day mortality, with a hazard ratio of 0.36 (P = 0.37). Moreover, LPS-Toll-like receptor (TLR)4 signaling mediated the GPR18 expression on PMNs. SIGNIFICANCE: These results indicate that decreased percentage GPR18-positive PMNs is associated with increased severity and poorer outcome of sepsis.


Assuntos
Neutrófilos/patologia , Receptores Acoplados a Proteínas-G/análise , Sepse/diagnóstico , APACHE , Idoso , Biomarcadores/análise , Células Cultivadas , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Prognóstico , Sepse/epidemiologia , Sepse/patologia
19.
Breast Cancer Res Treat ; 174(1): 121-127, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30478785

RESUMO

BACKGROUND: The role of G-protein-coupled estrogen receptor 1 (GPER-1) in the development of tamoxifen resistance in breast cancer is a highly controversial issue. The aim of this study was to determine the expression of GPER-1 in the clinical routine under conditions of endocrine treatment. PATIENTS AND METHODS: GPER-1 expression was analyzed in 442 patients with primary invasive breast cancer. GPER-1 score of > 3 was determined as positive. Expression data were correlated with clinical and pathological characteristics and patient survival. RESULTS: GPER-1 expression was observed in 352 (80.9%) cases, and positively correlated with estrogen and progesterone receptor status (p = 0.0001). GPER-1 positivity was associated with an increased grade of differentiation (p = 0.0001) and with a low level of Ki-67 expression (p = 0.0001). High GPER-1 expression was associated with a decreased level upon systemic treatment (p = 0.011). In the whole cohort, GPER-1 expression was associated with prolonged disease-free survival (DFS). DFS between tamoxifen- and aromatase inhibitor-treated GPER-1-positive patients was similar (p = 0.090). Notably, after matching the analysis for the most important prognostic factors, DFS for tamoxifen-treated GPER-1-positive patients was 69.1%, which is a percentage that is significantly lower compared to DFS for GPER-1-positive patients treated with aromatase inhibitors (92.7%) (p = 0.005). CONCLUSION: GPER-1 expression is a favorable prognostic factor in breast cancer patients. Its predictive role for poor benefit form tamoxifen treatment should be investigated in further studies.


Assuntos
Biomarcadores Tumorais/análise , Neoplasias da Mama/patologia , Receptores Estrogênicos/biossíntese , Receptores Acoplados a Proteínas-G/biossíntese , Moduladores Seletivos de Receptor Estrogênico/uso terapêutico , Tamoxifeno/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/mortalidade , Intervalo Livre de Doença , Feminino , Humanos , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Prognóstico , Receptores Estrogênicos/análise , Receptores Acoplados a Proteínas-G/análise
20.
Iran Biomed J ; 23(2): 107-20, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30501144

RESUMO

Background: Two of the Wnt signaling pathway target genes, tumor necrosis factor receptor family member (TROY) and leucine-rich G-protein coupled receptor (LGR5), are involved in the generation and maintenance of gastrointestinal epithelium. A negative modulatory role has recently been assigned to TROY, in this pathway. Here, we have examined their simultaneous expression in gastric carcinogenesis. Methods: Tumor and paired adjacent tissues of intestinal-type gastric cancer (GC) patients (n = 30) were evaluated for LGR5 and TROY expression by immunohistochemistry. The combination of the percentage of positively¬ stained cells and the intensity of staining was defined as the composite score and compared between groups. The obtained findings were re-evaluated in a mouse model. Results: TROY expression in the tumor tissue was significantly lower than that of the adjacent tissue (2.5 ± 0.9 vs. 3.3 ± 0.9, p = 0.004), which was coincident with higher LGR5 expression (3.6 ± 1.1 vs. 2.7 ± 0.9, p = 0.001). This observation was prominent at stages II/III of GC, leading to a statistically significant mean difference of expression between these two molecules (p = 0.005). In the H. pylori infected-mouse model, this inverse expression was observed in transition from early (8-16 w) to late (26-50 w) time points, post treatment (p = 0.002). Conclusion: Our data demonstrates an inverse trend between TROY down-regulation and LGR5 up-regulation in GC tumors, as well as in response to H. pylori infection in mice. These findings support a potential negative modulatory role for TROY on LGR5 expression.


Assuntos
Regulação Neoplásica da Expressão Gênica , Receptores Acoplados a Proteínas-G/genética , Receptores do Fator de Necrose Tumoral/genética , Neoplasias Gástricas/genética , Idoso , Animais , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/genética , Feminino , Infecções por Helicobacter/genética , Infecções por Helicobacter/metabolismo , Infecções por Helicobacter/patologia , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Receptores Acoplados a Proteínas-G/análise , Receptores Acoplados a Proteínas-G/biossíntese , Receptores do Fator de Necrose Tumoral/análise , Receptores do Fator de Necrose Tumoral/biossíntese , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia
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