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1.
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
3.
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
4.
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
5.
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
6.
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
7.
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
9.
J Biomol NMR ; 71(4): 203-211, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30121871

RESUMO

NMR studies of human integral membrane proteins provide unique opportunities to probe structure and dynamics at specific locations and on multiple timescales, often with significant implications for disease mechanism and drug development. Since membrane proteins such as G protein-coupled receptors (GPCRs) are highly dynamic and regulated by ligands or other perturbations, NMR methods are potentially well suited to answer basic functional questions (such as addressing the biophysical basis of ligand efficacy) as well as guiding applications (such as novel ligand design). However, such studies on eukaryotic membrane proteins have often been limited by the inability to incorporate optimal isotopic labels for NMR methods developed for large protein/lipid complexes, including methyl TROSY. We review the different expression systems for production of isotopically labeled membrane proteins and highlight the use of the yeast Pichia pastoris to achieve perdeuteration and 13C methyl probe incorporation within isoleucine sidechains. We further illustrate the use of this method for labeling of several biomedically significant GPCRs.


Assuntos
Marcação por Isótopo/métodos , Proteínas de Membrana/análise , Ressonância Magnética Nuclear Biomolecular/métodos , Pichia/química , Animais , Isótopos de Carbono , Deutério , Humanos , Receptores Acoplados a Proteínas-G/análise
10.
Int J Mol Sci ; 19(4)2018 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-29671777

RESUMO

Recently, there have been a number of developments in the fields of calcium and nuclear signaling that point to new avenues for a more effective diagnosis and treatment of prostate cancer. An example is the discovery of new classes of molecules involved in calcium-regulated nuclear import and nuclear calcium signaling, from the G protein-coupled receptor (GPCR) and myosin families. This review surveys the new state of the calcium and nuclear signaling fields with the aim of identifying the unifying themes that hold out promise in the context of the problems presented by prostate cancer. Genomic perturbations, kinase cascades, developmental pathways, and channels and transporters are covered, with an emphasis on nuclear transport and functions. Special attention is paid to the molecular mechanisms behind prostate cancer progression to the malignant forms and the unfavorable response to anti-androgen treatment. The survey leads to some new hypotheses that connect heretofore disparate results and may present a translational interest.


Assuntos
Cálcio/metabolismo , Núcleo Celular/patologia , Miosinas/metabolismo , Próstata/patologia , Neoplasias da Próstata/patologia , Receptores Acoplados a Proteínas-G/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Cálcio/análise , Sinalização do Cálcio , Núcleo Celular/metabolismo , Progressão da Doença , Humanos , Masculino , Miosinas/análise , Próstata/metabolismo , Neoplasias da Próstata/metabolismo , Proteínas Quinases/análise , Proteínas Quinases/metabolismo , Receptores Acoplados a Proteínas-G/análise , Transdução de Sinais
11.
Diagn Pathol ; 13(1): 22, 2018 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-29606134

RESUMO

BACKGROUND: The G protein-coupled bile acid receptor (TGR5) is a cell surface receptor which induces the production of intracellular cAMP and promotes epithelial-mesenchymal transition in gastric cancer cell lines. TGR5 is found in a wide variety of tissues including the kidney. However, the patterns of TGR5 expression have not been well characterized in physiologic kidney or renal neoplasms. We explore the expression of TGR5 in benign renal tissue and renal neoplasms and assess its utility as a diagnostic marker. METHODS: Sixty-one renal cortical neoplasms from 2000 to 2014 were retrieved. TGR5 protein expression was examined by immunohistochemistry. TGR5 mRNA was also measured by real-time PCR. RESULTS: In normal renal tissue, TGR5 was strongly positive in collecting ducts, distal convoluted tubules and thin loop of Henle. Proximal convoluted tubules showed absent or focal weak staining. In clear cell renal cell carcinomas (RCCs), 25 of 27 cases (92%) were negative for TGR5 (p < 0.001). TGR5 mRNA was also significantly decreased in clear cell RCCs, suggesting that decreased TGR5 protein expression may be attributable to the downregulation of TGR5 mRNA in these tumors. All 11 papillary RCCs expressed TGR5 with 45% (5/11) exhibiting moderate to strong staining. All chromophobe RCCs and oncocytomas were positive for TGR5 with weak to moderate staining. TGR5 mRNA expression in these tumors was similar to normal kidney. All urothelial carcinomas of the renal pelvis strongly expressed TGR5 including a poorly differentiated urothelial carcinoma with sarcomatoid features. CONCLUSION: TGR5 is strongly expressed in collecting ducts, distal convoluted tubules and thin loop of Henle. TGR5 protein and mRNA expression were notably decreased in clear cell RCCs and may be helpful in differentiating these tumors from other RCCs.


Assuntos
Biomarcadores Tumorais/análise , Neoplasias Renais/patologia , Receptores Acoplados a Proteínas-G/biossíntese , Humanos , Rim/metabolismo , Receptores Acoplados a Proteínas-G/análise
12.
Proc Natl Acad Sci U S A ; 115(11): E2653-E2662, 2018 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-29487210

RESUMO

There is currently an unmet need for versatile techniques to monitor the assembly and dynamics of ternary complexes in live cells. Here we describe bioluminescence resonance energy transfer with fluorescence enhancement by combined transfer (BRETFect), a high-throughput technique that enables robust spectrometric detection of ternary protein complexes based on increased energy transfer from a luciferase to a fluorescent acceptor in the presence of a fluorescent intermediate. Its unique donor-intermediate-acceptor relay system is designed so that the acceptor can receive energy either directly from the donor or indirectly via the intermediate in a combined transfer, taking advantage of the entire luciferase emission spectrum. BRETFect was used to study the ligand-dependent cofactor interaction properties of the estrogen receptors ERα and ERß, which form homo- or heterodimers whose distinctive regulatory properties are difficult to dissect using traditional methods. BRETFect uncovered the relative capacities of hetero- vs. homodimers to recruit receptor-specific cofactors and regulatory proteins, and to interact with common cofactors in the presence of receptor-specific ligands. BRETFect was also used to follow the assembly of ternary complexes between the V2R vasopressin receptor and two different intracellular effectors, illustrating its use for dissection of ternary protein-protein interactions engaged by G protein-coupled receptors. Our results indicate that BRETFect represents a powerful and versatile technique to monitor the dynamics of ternary interactions within multimeric complexes in live cells.


Assuntos
Técnicas Citológicas/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Fatores de Complexo Ternário/metabolismo , Células HEK293 , Humanos , Proteínas Luminescentes , Receptores Citoplasmáticos e Nucleares/análise , Receptores Citoplasmáticos e Nucleares/química , Receptores Acoplados a Proteínas-G/análise , Receptores Acoplados a Proteínas-G/química , Fatores de Complexo Ternário/análise , Fatores de Complexo Ternário/química
13.
Arch Oral Biol ; 85: 84-97, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29035722

RESUMO

OBJECTIVE: Intradental sensory receptors trigger painful sensations and unperceived mechanosensitivity, but the receptor bases for those functions are only partly defined. We present new evidence here concerning complex endings of myelinated axons in rat molars. DESIGN: We sectioned mature rat jaws in sagittal and transverse planes to analyze neural immunoreactivity (IR) for parvalbumin, peripherin, neurofilament protein, neurotrophin receptors, synaptophysin, calcitonin gene-related peptide (CGRP), or mas-related g-protein-receptor-d (Mrgprd). RESULTS: We found two complex sensory systems in mature rat molar dentin that labeled with neurofilament protein-IR, plus either parvalbumin-IR or peripherin-IR. The parvalbumin-IR system made extensively branched, beaded endings focused into dentin throughout each pulp horn. The peripherin-IR system primarily made unbeaded, fork-shaped dentinal endings scattered throughout crown including cervical regions. Both of these systems differed from neuropeptide CGRP-IR. In molar pulp we found peripherin- and parvalbumin-IR layered endings, either near special horizontal plexus arrays or in small coiled endings near tangled plexus, each with specific foci for specific pulp horns. Parvalbumin-IR nerve fibers had Aß axons (5-7µm diameter), while peripherin-IR axons were thinner Aδ size (2-5µm). Mechano-nociceptive Mrgprd-IR was only found in peripherin-IR axons. CONCLUSIONS: Complex somatosensory receptors in rat molars include two types of dentinal endings that both differ from CGRP-IR endings, and at least two newly defined types of pulpal endings. The PV-IR neurons with their widely branched, synaptophysin-rich, intradentinal beaded endings are good candidates for endodontic non-nociceptive, low threshold, unperceived mechanoreceptors. The complex molar dentinal and pulpal sensory systems were not found in rat incisors.


Assuntos
Dentina/inervação , Mecanorreceptores/fisiologia , Dente Molar/inervação , Nociceptores/fisiologia , Animais , Axônios , Biomarcadores/análise , Peptídeo Relacionado com Gene de Calcitonina/análise , Imuno-Histoquímica , Masculino , Proteínas do Tecido Nervoso/análise , Proteínas de Neurofilamentos/análise , Parvalbuminas/análise , Periferinas/análise , Ratos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas-G/análise , Sinaptofisina/análise
14.
Biochem Biophys Res Commun ; 495(1): 131-135, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29080746

RESUMO

It has been reported that a functional fat-taste receptor, GPR120, is present in chicken oral tissues, and that chickens can detect fat taste in a behavioral test. However, although triglycerides need to be digested to free fatty acids to be recognized by fat-taste receptors such as GPR120, it remains unknown whether lipase activities exist in chicken oral tissues. To examine this question, we first cloned another fat-taste receptor candidate gene, CD36, from the chicken palate. Then, using RT-PCR, we determined that GPR120 and CD36 were broadly expressed in chicken oral and gastrointestinal tissues. Also by RT-PCR, we confirmed that several lipase genes were expressed in both oral and gastrointestinal tissues. Finally, we analyzed the lipase activities of oral tissues by using a fluorogenic triglyceride analog as a lipase substrate. We found there are functional lipases in oral tissues as well as in the stomach and pancreas. These results suggested that chickens have a basic fat-taste reception system that incorporates a triglycerides/oral-lipases/free fatty acids/GPR120 axis and CD36 axis.


Assuntos
Antígenos CD36/metabolismo , Galinhas/fisiologia , Gorduras na Dieta/metabolismo , Lipase/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Paladar , Sequência de Aminoácidos , Animais , Sequência de Bases , Antígenos CD36/análise , Antígenos CD36/genética , Galinhas/genética , Clonagem Molecular , Ácidos Graxos não Esterificados/metabolismo , Expressão Gênica , Lipase/análise , Lipase/genética , Palato/metabolismo , Receptores Acoplados a Proteínas-G/análise , Receptores Acoplados a Proteínas-G/genética , Papilas Gustativas/fisiologia , Percepção Gustatória , Triglicerídeos/metabolismo
15.
Elife ; 62017 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-29022879

RESUMO

The human distal limbs have a high spatial acuity for noxious stimuli but a low density of pain-sensing neurites. To elucidate mechanisms underlying regional differences in processing nociception, we sparsely traced non-peptidergic nociceptors across the body using a newly generated MrgprdCreERT2 mouse line. We found that mouse plantar paw skin is also innervated by a low density of Mrgprd+ nociceptors, while individual arbors in different locations are comparable in size. Surprisingly, the central arbors of plantar paw and trunk innervating nociceptors have distinct morphologies in the spinal cord. This regional difference is well correlated with a heightened signal transmission for plantar paw circuits, as revealed by both spinal cord slice recordings and behavior assays. Taken together, our results elucidate a novel somatotopic functional organization of the mammalian pain system and suggest that regional central arbor structure could facilitate the "enlarged representation" of plantar paw regions in the CNS.


Assuntos
Anatomia Regional , Nociceptores/citologia , Nociceptores/fisiologia , Receptores Acoplados a Proteínas-G/análise , Pele/inervação , Animais , Camundongos , Nociceptividade , Receptores Acoplados a Proteínas-G/genética
16.
Cell ; 170(6): 1149-1163.e12, 2017 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-28886383

RESUMO

The diversity of mesenchymal cell types in the lung that influence epithelial homeostasis and regeneration is poorly defined. We used genetic lineage tracing, single-cell RNA sequencing, and organoid culture approaches to show that Lgr5 and Lgr6, well-known markers of stem cells in epithelial tissues, are markers of mesenchymal cells in the adult lung. Lgr6+ cells comprise a subpopulation of smooth muscle cells surrounding airway epithelia and promote airway differentiation of epithelial progenitors via Wnt-Fgf10 cooperation. Genetic ablation of Lgr6+ cells impairs airway injury repair in vivo. Distinct Lgr5+ cells are located in alveolar compartments and are sufficient to promote alveolar differentiation of epithelial progenitors through Wnt activation. Modulating Wnt activity altered differentiation outcomes specified by mesenchymal cells. This identification of region- and lineage-specific crosstalk between epithelium and their neighboring mesenchymal partners provides new understanding of how different cell types are maintained in the adult lung.


Assuntos
Pulmão/citologia , Mesoderma/citologia , Animais , Homeostase , Pulmão/fisiologia , Camundongos , Organoides/citologia , Alvéolos Pulmonares/citologia , Receptores Acoplados a Proteínas-G/análise , Análise de Sequência de RNA , Análise de Célula Única , Transcrição Genética
17.
Fertil Steril ; 108(5): 858-867.e2, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28923287

RESUMO

OBJECTIVE: To characterize leucine-rich repeat containing G protein-coupled receptor 5-positive (LGR5+) cells from the endometrium of women with endometriosis. DESIGN: Prospective experimental study. SETTING: University hospital/fertility clinic. PATIENT(S): Twenty-seven women with endometriosis who underwent surgery and 12 healthy egg donors, together comprising 39 endometrial samples. INTERVENTION(S): Obtaining of uterine aspirates by using a Cornier Pipelle. MAIN OUTCOMES MEASURE(S): Immunofluorescence in formalin-fixed paraffin-embedded tissue from mice and healthy and pathologic human endometrium using antibodies against LGR5, E-cadherin, and cytokeratin, and epithelial and stromal LGR5+ cells isolated from healthy and pathologic human eutopic endometrium by fluorescence-activated cell sorting and transcriptomic characterization by RNA high sequencing. RESULT(S): Immunofluorescence showed that LGR5+ cells colocalized with epithelial markers in the stroma of the endometrium only in endometriotic patients. The results from RNA high sequencing of LGR5+ cells from epithelium and stroma did not show any statistically significant differences between them. The LGR5+ versus LGR5- cells in pathologic endometrium showed 394 differentially expressed genes. The LGR5+ cells in deep-infiltrating endometriosis expressed inflammatory markers not present in the other types of the disease. CONCLUSION(S): Our results revealed the presence of aberrantly located LGR5+ cells coexpressing epithelial markers in the stromal compartment of women with endometriosis. These cells have a statistically significantly different expression profile in deep-infiltrating endometriosis in comparison with other types of endometriosis, independent of the menstrual cycle phase. Further studies are needed to elucidate their role and influence in reproductive outcomes.


Assuntos
Endometriose/metabolismo , Endométrio/química , Receptores Acoplados a Proteínas-G/análise , Células Estromais/química , Biomarcadores/análise , Estudos de Casos e Controles , Endometriose/genética , Endometriose/patologia , Endométrio/patologia , Feminino , Imunofluorescência , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Estudos Prospectivos , Receptores Acoplados a Proteínas-G/genética , Análise de Sequência de RNA , Células Estromais/patologia
18.
Chem Biol Interact ; 277: 176-184, 2017 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-28947257

RESUMO

The activation of the G protein-coupled estrogen receptor (GPER) by its specific agonist G-1 inhibits prostate cancer and 17ß-estradiol-stimulated breast cancer cell proliferation. Tamoxifen (TAM), which also activates the GPER, decreases melanoma cell proliferation, but its action mechanism remains controversial. Here we investigated the expression and the effects of GPER activation by G-1, TAM and its key metabolite endoxifen (EDX) on melanoma cells. Mouse melanoma K1735-M2 cells expressed GPER and G-1 reduced cell biomass, and the number of viable cells, without increasing cell death. Rather, G-1 decreased cell division by blocking cell cycle progression in G2. Likewise, TAM and EDX exhibited an antiproliferative activity in melanoma cells due to decreased cell division. Both G-1 and the antiestrogens showed a trend to decrease the levels of phosphorylated ERK 1/2 after 1 h treatment, although only EDX, the most potent antiproliferative antiestrogen, induced significant effects. Importantly, the targeting of GPER with siRNA abolished the cytostatic activity of both G-1 and antiestrogens, suggesting that the antitumor actions of antiestrogens in melanoma cells involve GPER activation. Our results unveil a new target for melanoma therapy and identify GPER as a key mediator of antiestrogen antiproliferative effects, which may contribute to select the patients that benefit from an antiestrogen-containing regimen.


Assuntos
Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Ciclopentanos/farmacologia , Melanoma/tratamento farmacológico , Quinolinas/farmacologia , Receptores Acoplados a Proteínas-G/agonistas , Animais , Linhagem Celular Tumoral , Melanoma/metabolismo , Camundongos , Receptores Estrogênicos/análise , Receptores Estrogênicos/metabolismo , Receptores Acoplados a Proteínas-G/análise , Receptores Acoplados a Proteínas-G/metabolismo
19.
Methods Mol Biol ; 1647: 1-18, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28808992

RESUMO

The combination of photoaffinity labeling (PAL) and quantitative chemoproteomics enables the comprehensive, unbiased determination of protein interaction profiles to support target identification of bioactive small molecules. This approach is amenable to cells in culture and compatible with pharmacologically relevant transmembrane target classes like G-protein coupled receptors and ions channels which have been notoriously hard to access by conventional chemoproteomics approaches. Here, we describe a strategy that combines PAL probe titration and competition with excess parental compounds with the goal of enabling the identification of specific interactors as well as assessing the functional relevance of a binding event for the phenotype under investigation.


Assuntos
Marcadores de Fotoafinidade/química , Proteômica/métodos , Bibliotecas de Moléculas Pequenas/análise , Química Click , Condutometria , Desenho de Drogas , Proteínas de Ligação ao GTP/análise , Células HEK293 , Humanos , Espectrometria de Massas , Receptores Acoplados a Proteínas-G/análise
20.
Cell Tissue Res ; 370(2): 267-273, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28766044

RESUMO

Primary familial brain calcification (PFBC) is a neuropsychiatric disorder characterized by bilateral cerebral calcification with diverse neurologic or psychiatric symptoms. Recently, XPR1 variation has accounted for PFBC as another new causative gene. However, little is known about the distribution and basic function of XPR1 and its interaction with the other three pathogenic genes for PFBC (SLC20A2, PDGFRB and PDGFB). The aim of this study was to further clarify the role of XPR1 in PFBC brain pathology. As a result, gene expression profiles showed that XPR1 mRNA was widely expressed throughout the mouse brain. Cerebellum and striatum, most commonly affected in PFBC, contained a higher level of XPR1 protein than other brain regions. Additionally, XPR1 deficiency seriously affected Pi efflux and XPR1 mutations seemed to have an effect through haploinsufficiency mechanism. The immunoprecipitation and immunohistochemical studies demonstrated that XPR1 could interact with PDGFRB and might form a complex on the cell membrane. These results suggested that XPR1 played a fundamental role in the maintenance of cellular phosphate balance in the brain. This provided us with a novel perspective on understanding the pathophysiology of PFBC. The expression networks and interaction with the known pathogenic genes could shed new light on additional candidate genes for PFBC.


Assuntos
Encefalopatias/genética , Encéfalo/metabolismo , Calcinose/genética , Receptores Acoplados a Proteínas-G/genética , Receptores Virais/genética , Transcriptoma , Animais , Encéfalo/patologia , Encefalopatias/metabolismo , Encefalopatias/patologia , Calcinose/metabolismo , Calcinose/patologia , Expressão Gênica , Predisposição Genética para Doença , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Mapas de Interação de Proteínas , RNA Mensageiro/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/análise , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptores Acoplados a Proteínas-G/análise , Receptores Acoplados a Proteínas-G/metabolismo , Receptores Virais/análise , Receptores Virais/metabolismo , Regulação para Cima
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