RESUMO
BACKGROUND: In cancer patients, circulating cell-free DNA (ccfDNA) can contain tumor-derived DNA (ctDNA), which enables noninvasive diagnosis, real-time monitoring, and treatment susceptibility testing. However, ctDNA fractions are highly variable, which challenges downstream applications. Therefore, established preanalytical work flows in combination with cost-efficient and reproducible reference materials for ccfDNA analyses are crucial for analytical validity and subsequently for clinical decision-making. METHODS: We describe the efforts of the Innovative Medicines Initiative consortium CANCER-ID (http://www.cancer-id.eu) for comparing different technologies for ccfDNA purification, quantification, and characterization in a multicenter setting. To this end, in-house generated mononucleosomal DNA (mnDNA) from lung cancer cell lines carrying known TP53 mutations was spiked in pools of plasma from healthy donors generated from 2 different blood collection tubes (BCTs). ccfDNA extraction was performed at 15 partner sites according to their respective routine practice. Downstream analysis of ccfDNA with respect to recovery, integrity, and mutation analysis was performed centralized at 4 different sites. RESULTS: We demonstrate suitability of mnDNA as a surrogate for ccfDNA as a process quality control from nucleic acid extraction to mutation detection. Although automated extraction protocols and quantitative PCR-based quantification methods yielded the most consistent and precise results, some kits preferentially recovered spiked mnDNA over endogenous ccfDNA. Mutated TP53 fragments derived from mnDNA were consistently detected using both next-generation sequencing-based deep sequencing and droplet digital PCR independently of BCT. CONCLUSIONS: This comprehensive multicenter comparison of ccfDNA preanalytical and analytical work flows is an important contribution to establishing evidence-based guidelines for clinically feasible (pre)analytical work flows.
Assuntos
Ácidos Nucleicos Livres/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Coleta de Amostras Sanguíneas , Linhagem Celular Tumoral , Ácidos Nucleicos Livres/química , Ácidos Nucleicos Livres/normas , DNA Tumoral Circulante/sangue , Análise Mutacional de DNA , Sequenciamento de Nucleotídeos em Larga Escala/normas , Humanos , Neoplasias/genética , Neoplasias/patologia , Nucleossomos/genética , Polimorfismo de Nucleotídeo Único , Fase Pré-Analítica , Reação em Cadeia da Polimerase em Tempo Real/normas , Padrões de Referência , Proteína Supressora de Tumor p53/genéticaRESUMO
Translational research is a continuum between clinical and basic research where the patient is the center of the research process. It brings clinical research to a starting point for the drug discovery process, permitting the generation of a more robust pathophysiological hypothesis essential for a better selection of drug targets and candidate optimization. It also establishes the basis of early proof for clinical concept studies, preferably in phase I, for which biomarkers and surrogate endpoints can often be used. Systems biology is a prerequisite approach to translational research where technologies and expertise are integrated and articulated to support efficient and productive realization of this concept. The first component of systems biology relies on omics-based technologies and integrates the changes in variables, such as genes, proteins and metabolites, into networks that are responsible for an organism's normal and diseased state. The second component of systems biology is in the domain of computational methods, where simulation and modeling create hypotheses of signaling pathways, transcription networks, physiological processes or even cell- or organism-based models. The simulations aim to show the origin of perturbations of the system that lead to pathological states and what treatment could be achieved to ameliorate or normalize the system. This review discusses how translational research and systems biology together could improve global understanding of drug targets, suggest new targets and approaches for therapeutics, and provide a deeper understanding of drug effects. Taken together, these types of analyses can lead to new therapeutic options while improving the safety and efficacy of new and existing medications.
Assuntos
Descoberta de Drogas/métodos , Biologia de Sistemas/métodos , Animais , Humanos , Pesquisa Translacional BiomédicaRESUMO
Background: The Mixed Lymphocyte Reaction (MLR) consists in the allogeneic co-culture of monocytes derived dendritic cells (MoDCs) with T cells from another donor. This in vitro assay is largely used for the assessment of immunotherapy compounds. Nevertheless, the phenotypic changes associated with lymphocyte responsiveness under MLR have never been thoroughly evaluated. Methods: Here, we used multiplex cytokine and chemokine assays, multiparametric flow cytometry and single cell RNA sequencing to deeply characterize T cells activation and function in the context of CD4+- and CD8+-specific MLR kinetics. Results: We showed that CD4+ and CD8+ T cells in MLR share common classical markers of response such as polyfunctionality, increased proliferation and CD25 expression but differ in their kinetics and amplitude of activation as well as their patterns of cytokines secretion and immune checkpoints expression. The analysis of immunoreactive Ki-67+CD25+ T cells identified PBK, LRR1 and MYO1G as new potential markers of MLR response. Using cell-cell communication network inference and pathway analysis on single cell RNA sequencing data, we also highlighted key components of the immunological synapse occurring between T cells and the stimulatory MoDCs together with downstream signaling pathways involved in CD4+ and CD8+ T cells activation. Conclusion: These results provide a deep understanding of the kinetics of the MLR assay for CD4+ or CD8+ T cells and may allow to better characterize compounds impacting MLR and eventually identify new strategies for immunotherapy in cancer.
Assuntos
Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Teste de Cultura Mista de Linfócitos , Citometria de Fluxo , Análise de Sequência de RNARESUMO
"Ecstasy" [3,4-methylenedioxymetamphetamine (MDMA)] is of considerable interest in light of its prosocial properties and risks associated with widespread recreational use. Recently, it was found to bind trace amine-1 receptors (TA(1)Rs), which modulate dopaminergic transmission. Accordingly, using mice genetically deprived of TA(1)R (TA(1)-KO), we explored their significance to the actions of MDMA, which robustly activated human adenylyl cyclase-coupled TA(1)R transfected into HeLa cells. In wild-type (WT) mice, MDMA elicited a time-, dose-, and ambient temperature-dependent hypothermia and hyperthermia, whereas TA(1)-KO mice displayed hyperthermia only. MDMA-induced increases in dialysate levels of dopamine (DA) in dorsal striatum were amplified in TA(1)-KO mice, despite identical levels of MDMA itself. A similar facilitation of the influence of MDMA upon dopaminergic transmission was acquired in frontal cortex and nucleus accumbens, and induction of locomotion by MDMA was haloperidol-reversibly potentiated in TA(1)-KO versus WT mice. Conversely, genetic deletion of TA(1)R did not affect increases in DA levels evoked by para-chloroamphetamine (PCA), which was inactive at hTA(1) sites. The TA(1)R agonist o-phenyl-3-iodotyramine (o-PIT) blunted the DA-releasing actions of PCA both in vivo (dialysis) and in vitro (synaptosomes) in WT but not TA(1)-KO animals. MDMA-elicited increases in dialysis levels of serotonin (5-HT) were likewise greater in TA(1)-KO versus WT mice, and 5-HT-releasing actions of PCA were blunted in vivo and in vitro by o-PIT in WT mice only. In conclusion, TA(1)Rs exert an inhibitory influence on both dopaminergic and serotonergic transmission, and MDMA auto-inhibits its neurochemical and functional actions by recruitment of TA(1)R. These observations have important implications for the effects of MDMA in humans.
Assuntos
Deleção de Genes , N-Metil-3,4-Metilenodioxianfetamina/antagonistas & inibidores , N-Metil-3,4-Metilenodioxianfetamina/farmacologia , Receptores Acoplados a Proteínas G/deficiência , Receptores Acoplados a Proteínas G/fisiologia , Animais , Dopamina/fisiologia , Relação Dose-Resposta a Droga , Células HeLa , Humanos , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Distribuição Aleatória , Receptores Acoplados a Proteínas G/genética , Serotonina/fisiologiaRESUMO
Sustained overactivation of RhoA is a common component for the pathogenesis of several cardiovascular disorders, including hypertension. Although activity of Rho proteins depends on Rho exchange factors (Rho-GEFs), the identity of Rho-GEFs expressed in vascular smooth muscle cells (VSMC) and participating in the control of Rho protein activity and Rho-dependent functions remains unknown. To address this question, we analyzed by quantitative RT-PCR the expression profile of 28 RhoA-GEFs in arteries of normotensive (saline-treated) and hypertensive (ANG II-treated) rats. Sixteen RhoA-GEFs were downregulated in mesenteric arteries of hypertensive rats, among which nine are also downregulated in cultured VSMC stimulated by ANG II (100 nM, 48 h), suggesting a direct effect of ANG II. Inhibition of type 1 ANG II receptors (losartan, 1 µM) or Rho kinase (fasudil, 10 µM) prevented ANG II-induced RhoA-GEF downregulation. Functionally, ANG II-induced downregulation of RhoA-GEFs is associated with decreased Rho kinase activation in response to endothelin-1, norepinephrine, and U-46619. This work thus identifies a group of RhoA-GEFs that controls RhoA and RhoA-dependent functions in VSMC, and a negative feedback of RhoA/Rho kinase activity on the expression of these RhoA-GEFs that may play an adaptative role to limit RhoA/Rho kinase activation.
Assuntos
Retroalimentação Fisiológica/fisiologia , Fatores de Troca do Nucleotídeo Guanina/biossíntese , Hipertensão/fisiopatologia , Músculo Liso Vascular/metabolismo , Quinases Associadas a rho/metabolismo , Angiotensina II/metabolismo , Angiotensina II/toxicidade , Animais , Artérias/metabolismo , Western Blotting , Perfilação da Expressão Gênica , Hipertensão/induzido quimicamente , Masculino , Músculo Liso Vascular/fisiopatologia , RNA Interferente Pequeno , Ratos , Ratos Endogâmicos WKY , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/fisiologia , TransfecçãoRESUMO
The present study describes the effect of (S)-2,3-dihydro-[3,4]cyclopentano-1,2,4-benzothiadiazine-1,1-dioxide (S18986), a positive allosteric modulator of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors, on (S)-AMPA-mediated increases in brain-derived neurotrophic factor (BDNF) mRNA and protein expression in rat primary cortical neuronal cultures. (S)-AMPA (0.01-300 microM) induced a concentration-dependent increase in BDNF mRNA and protein expression (EC(50)=7 microM) with maximal increases (50-fold) compared to untreated cultures observed between 5 and 12 h, whereas for cellular protein levels, maximal expression was detected at 24 h. S18986 alone (< or =300 microM) failed to increase basal BDNF expression. However, S18986 (300 microM) in the presence of increasing concentrations of (S)-AMPA maximally enhanced AMPA-induced expression of BDNF mRNA and protein levels (3-5-fold). S18986 (100-300 microM) potentiated BDNF mRNA induced by 3 microM (S)-AMPA (2-3-fold). Under similar conditions, the AMPA allosteric modulator cyclothiazide induced a potent stimulation of (S)-AMPA-mediated BDNF expression (40-fold; EC(50)=18 microM), whereas IDRA-21 was inactive. Kinetic studies indicated that S18986 (300 microM) in the presence of 3 microM (S)-AMPA was capable of enhancing BDNF mRNA levels for up to 25 h, compared to 3 microM (S)-AMPA alone. On the other hand, S18986 only partially enhanced kainate-mediated expression of BDNF mRNA, but failed to significantly enhance N-methyl-D-aspartate-stimulated BDNF expression levels. In support of these observations, the competitive AMPA receptor antagonist NBQX (1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide) but not the selective NMDA-receptor antagonist, (+)-MK-801 [(5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine], abrogated S18986-induced effects on BDNF expression. S18986-mediated enhancement of (S)-AMPA-evoked BDNF protein expression was markedly attenuated in Ca(2+)-free culture conditions. Furthermore, from a series of kinase inhibitors only the Calmodulin-Kinase II/IV inhibitor (KN-62, 25 microM) significantly inhibited (-85%, P<0.001) AMPA+S18986 stimulated expression of BDNF mRNA. The present study supports the observations that AMPA receptor allosteric modulators can enhance the expression of BDNF mRNA and protein expression via the AMPA receptor in cultured primary neurones. Consequently, the long-term elevation of endogenous BDNF expression by pharmacological intervention with this class of compounds represents a potentially promising therapeutic approach for behavioural disorders implicating cognitive deficits.
Assuntos
Benzotiadiazinas/farmacologia , Fator Neurotrófico Derivado do Encéfalo/efeitos dos fármacos , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , RNA Mensageiro/efeitos dos fármacos , Receptores de AMPA/efeitos dos fármacos , Regulação Alostérica/efeitos dos fármacos , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Células Cultivadas , Córtex Cerebral/citologia , Relação Dose-Resposta a Droga , Sistemas de Liberação de Medicamentos , Transtornos Mentais/tratamento farmacológico , Plasticidade Neuronal/efeitos dos fármacos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Reação em Cadeia da Polimerase , Inibidores de Proteínas Quinases , RNA Mensageiro/metabolismo , Ratos , Receptores de AMPA/metabolismoRESUMO
Neuropeptide Y (NPY) has several receptors; one of them, the neuropeptide Y5 receptor (NPY5) seems involved in feeding behavior in mammals. Although this particular receptor has been extensively studied in the literature, the difficulties encountered to obtain a stable cell line expressing this recombinant receptor have impaired the development of tools necessary to establish its molecular pharmacology. We thus established a method for the functional study of new ligands. It is based upon the cotransfection in human melatonin receptor 1 (MT1)-overexpressing HEK293 cells of three plasmids encoding melanocortin receptor (MC5), neuropeptide Y5 receptor (NPY5) and a cyclic AMP response element-controlled luciferase. Once challenged with alphaMSH, the MC5 receptor activates the cyclic AMP response, through the coupling protein subunit G(s). In contrast, NPY5 agonists, through the NPY5 receptor which is negatively coupled to the same pathway, counteract the alphaMSH-mediated effect on cyclic AMP level. Using appropriate controls, this method can pinpoint compounds with antagonistic activity. Simple and straightforward, this system permits reproducible measurements of agonist or antagonist effects in the presence of neuropeptide Y, the natural agonist. This method has the advantage over already existing methods and beyond its apparent complexity, to enhance the cyclic AMP concentration at a 'physiological' level, by opposition to a forskolin-induced adenylate cyclase activation. Finally, to further validate this assay, we showed results from (1) a series of natural peptidic agonists that permitted the standardization and (2) a series of potent nonpeptidic antagonists (affinity >10(-9) M) that form a new class of active NPY5 receptor antagonists.
Assuntos
Genes Reporter , Luciferases/genética , Receptores de Neuropeptídeo Y/antagonistas & inibidores , Bioensaio , Humanos , Ligantes , Neuropeptídeo Y/farmacologia , Peptídeos/química , Peptídeos/metabolismo , Peptídeos/farmacologia , Receptores de Neuropeptídeo Y/genética , Receptores de Neuropeptídeo Y/metabolismo , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Retinoic acid receptor-related orphan receptor alpha (RORalpha) is a member of the nuclear receptor superfamily. Using RT-PCR, RORalpha mRNA was identified in human aortic smooth muscle cells (hASMC), endothelial cells (EC), as well as in human mammary arteries and atherosclerotic plaques. We found a predominant expression of RORalpha1 in hASMC, and RORalpha4 in EC. RORalpha2 and RORalpha3 were not detected. In arteries, RORalpha4 was predominant compared with RORalpha1. In atherosclerotic plaques, RORalpha expression was significantly decreased. In hASMC stimulated with cytokines, RORalpha expression was increased by 2.5-fold. RORalpha mRNA was also significantly increased (approximately 2-fold) in hASMC and EC cultured under hypoxia.
Assuntos
Endotélio Vascular/metabolismo , Músculo Liso Vascular/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Transativadores/metabolismo , Aorta , Arteriosclerose/genética , Arteriosclerose/metabolismo , Estenose das Carótidas/genética , Estenose das Carótidas/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hipóxia/genética , Hipóxia/metabolismo , Interleucina-1/farmacologia , Lipopolissacarídeos/farmacologia , Artéria Torácica Interna/citologia , Artéria Torácica Interna/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares , Oxigênio/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transativadores/genética , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
1. Endothelin-1 (ET-1) and tumor necrosis factor alpha (TNFalpha) by their action on adipocytes have been independently linked to the pathogenesis of insulino-resistance. In isolated adipocytes, TNFalpha induces the expression of the inducible nitric oxide synthase (iNOS). The purpose of the present work was, in the 3T3-F442A adipocyte cell line, to characterise TNFalpha-induced iNOS expression and to determine whether or not ET-1 could influence TNFalpha-induced iNOS expression and NO production. 2. In differentiated 3T3-F442A, treatment with TNFalpha (20 ng ml(-1)) induced the expression of a functional iNOS as demonstrated by nitrite assay, Western blot, reverse transcription-polymerase chain reaction and Northern blot analysis. TNFalpha-induced iNOS expression requires nuclear factor kappaB activation, but does not necessitate the activation of the PI-3 kinase/Akt and P38-MAP kinase pathways. 3. ET-1, but not ET-3, inhibited the TNFalpha-induced expression of iNOS protein and mRNA as well as nitrite production. The effects of ET-1 were blocked by a specific ETA (BQ123, pA(2) 7.4) but not by a specific ETB receptor antagonist (BQ788). 3T3-F442A adipocytes express the mRNAs for prepro-ET-1 and the ET-A receptor subtype, but not for the ET-B subtype. 4. The inhibitory effect of ET-1 was not affected by bisindolylmaleimide, SB 203580 or indomethacin, inhibitors of protein kinase C, p38-MAP kinase and cyclooxygenase, respectively, and was not associated with cAMP production. However, the effect of ET-1 was partially reversed by wortmannin, suggesting the involvement of PI3 kinase in the transduction signal of ET-1. 5. Differentiated 3T3-F442A adipocytes did not release ET-1 with or without exposure to TNFalpha, although the mRNA for preproET-1 was detected in both pre- and differentiated adipocytes. 6. Thus, these results confirm that adipocytes are a target for circulating ET-1 and demonstrate that the activation of the ETA receptor subtype can prevent TNFalpha-induced iNOS expression.
Assuntos
Adipócitos/enzimologia , Endotelina-1/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/biossíntese , Fator de Necrose Tumoral alfa/farmacologia , Células 3T3 , Adipócitos/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Camundongos , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo II , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/biossíntese , RNA Mensageiro/genéticaRESUMO
Melatonin has a key role in the circadian rhythm relay to periphery organs. Melatonin exerts its multiple roles mainly through two seven transmembrane domain, G-coupled receptors, namely MT1 or MT2 receptors. A pharmacological characterization of these human cloned melatonin hMT1 and hMT2 receptors stably expressed in HEK-293 or CHO cells is presented using a 2-[125I]-iodo-melatonin binding assay and a [35S]-GTPgammaS functional assay. Both reference compounds and new chemically diverse ligands were evaluated. Binding affinities at each receptor were found to be comparable on either HEK-293 or CHO cell membranes. Novel non-selective or selective hMT1 and hMT2 ligands are described. The [35S]-GTPgammaS functional assay was used to define the functional activity of these compounds which included partial, full agonist and/or antagonist activity. None of the compounds acted as an inverse agonist. We report new types of selective antagonists, such as S 25567 and S 26131 for MT1 and S 24601 for MT2. These studies brought other new molecular tools such as the selective MT1 agonist, S 24268, as well as the non-selective antagonist, S 22153. Finally, we also discovered S 25150, the most potent melatonin receptor agonist, so far reported in the literature.
Assuntos
Clonagem Molecular/métodos , Melatonina/análogos & derivados , Melatonina/metabolismo , Receptor MT1 de Melatonina/metabolismo , Receptor MT2 de Melatonina/metabolismo , Animais , Células CHO , Linhagem Celular , Cricetinae , Relação Dose-Resposta a Droga , Humanos , Ligantes , Melatonina/química , Ligação Proteica/fisiologia , Receptor MT1 de Melatonina/agonistas , Receptor MT1 de Melatonina/antagonistas & inibidores , Receptor MT1 de Melatonina/genética , Receptor MT2 de Melatonina/agonistas , Receptor MT2 de Melatonina/antagonistas & inibidores , Receptor MT2 de Melatonina/genéticaRESUMO
BACKGROUND: The sterol regulatory element-binding protein (SREBP) 1c contributes to the transcriptional coordination of cholesterol, fatty acid, and carbohydrate metabolisms. Alterations in these processes accelerate the progression of hepatic steatosis and insulin resistance during aging and obesity. METHODS: Using an ex vivo chromatin immunoprecipitation coupled to microarray (ChIP-on-chip) technique combined with genome-wide gene expression analysis, we analyzed the transcriptomic adaptations mediated by Srebp-1c binding to gene promoters in the liver of mice fed with a low-fat diet or a high-fat diet (HFD) for either 1 or 12 months. RESULTS: Aging had a higher transcriptional impact than HFD and modified the expression of genes involved in fatty acid oxidation and oxidative stress. HFD was associated with a marked induction of genes involved in lipid and cholesterol metabolism. The prolonged high-fat feeding together with the aging effects stimulates inflammatory pathways. ChIP-on-chip applied to aging and HFD analyses revealed that the binding of SREBP-1c to a series of promoters accompanied a paralleled modification of gene expression. Therefore, SREBP-1c could play a role in aging and high-fat feeding through the regulation of genes involved in lipid metabolism and inflammatory response. CONCLUSIONS: This study represents an original ex vivo experiment to elucidate the molecular events involved in metabolic disorders.
Assuntos
Envelhecimento/genética , Gorduras na Dieta/administração & dosagem , Fígado/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo Real , Transcrição Gênica , TranscriptomaRESUMO
Studies of 5-HT-glutamate interactions suggest that activation of brain 5-HT(2A) receptors leads to an AMPA receptor-mediated induction of the immediate early (activity-dependent) gene, Arc (Arg3.1). In this respect, noradrenaline-glutamate interactions are poorly characterised. Here we investigated the influence on regional brain Arc gene expression of selective blockade of α(2)-adrenoceptors in rats. Several complementary techniques were used: qPCR (mRNA, discrete tissue punches), in situ hybridisation (mRNA, sections) and immunocytochemistry. The α(2)-adrenoceptor antagonist, RX 821002, dose-dependently and time-dependently (maximal effect 2 h) increased Arc mRNA levels as demonstrated both by qPCR and in situ hybridisation. The α(2)-adrenoceptor antagonist, atipamezole, also increased Arc mRNA in in situ hybridisation studies. Changes in Arc mRNA after RX 821002 were of similar magnitude in punches and intact tissue sections and region-specific, with effects being most pronounced in parietal cortex and caudate putamen, less robust in frontal cortex, and not detectable in hippocampal sub-regions. Both qPCR and in situ hybridisation studies demonstrated that RX 821002-induced Arc mRNA was blocked by the AMPA antagonist, GYKI 52466. Pretreatment with the NMDA antagonist MK 801 also prevented RX 821002-induced Arc mRNA, as did the mGluR5 antagonist MPEP, whilst the mGluR2/3 antagonist, LY341495, had no effect. Finally, immunocytochemical studies showed that RX 821002 increased Arc-immunoreactivity in cells in close apposition to α(2)-adrenoceptor-positive processes. Thus, employing three complementary techniques, these observations demonstrate that blockade of α(2)-adrenoceptors triggers brain expression of the immediate early gene, Arc, and that this effect involves the recruitment of AMPA, NMDA and mGluR5 but not mGluR2/3 glutamatergic receptors.
Assuntos
Antagonistas de Receptores Adrenérgicos alfa 2/farmacologia , Química Encefálica/efeitos dos fármacos , Proteínas do Citoesqueleto/biossíntese , Proteínas do Citoesqueleto/genética , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Receptores de Glutamato/efeitos dos fármacos , Animais , Antagonistas de Aminoácidos Excitatórios/farmacologia , Idazoxano/análogos & derivados , Idazoxano/farmacologia , Processamento de Imagem Assistida por Computador , Imidazóis/farmacologia , Imuno-Histoquímica , Hibridização In Situ , Masculino , Reação em Cadeia da Polimerase , RNA/biossíntese , RNA/genética , Ratos , Ratos Sprague-Dawley , Receptor de Glutamato Metabotrópico 5 , Receptores de AMPA/antagonistas & inibidores , Receptores de Glutamato Metabotrópico/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Serotonina/fisiologiaRESUMO
To improve our understanding of the molecular events underlying the effects of positive allosteric modulators of the alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (S)-AMPA-type glutamate receptors, gene expression profiles of primary cortical culture were measured by Agilent-Microarray technique under (S)-AMPA (1µM) stimulation for 0.5, 6, 24 and 48h in the presence or absence of S70340 (30µM), an allosteric potentiator of AMPA receptors. (S)-AMPA and S70340 treatment alone have little effect on gene expression whereas as early as 6h, their combination induced a large number of genes known to decrease apoptosis and mediate cell survival. Pathway analyses of (S)-AMPA+S70340 treatment-mediated gene expression from 6 to 48h further suggested the activation of cellular functions including neuron differentiation and neurite outgrowth. A proportion of genes implicated in these functions encode proteins involved in environmental cues and are expressed in growth cones, such as extracellular matrix component proteins and filopodia microfilament-associated proteins. Time course analysis of mRNA expression combined with in silico promoter analysis revealed an enrichment in the cAMP response element (CRE) among co-regulated genes. This study demonstrated that S70340-mediated AMPA potentialisation activated genes and functional processes involved in neuroprotective and cognitive effects and describes putative new functional biomarkers.
Assuntos
Córtex Cerebral/fisiologia , Perfilação da Expressão Gênica/métodos , Estudo de Associação Genômica Ampla/métodos , Receptores de AMPA/agonistas , Receptores de AMPA/fisiologia , Animais , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Redes Reguladoras de Genes/genética , Ratos , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/análogos & derivados , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/farmacologiaRESUMO
Rosiglitazone (RSG), developed for the treatment of type 2 diabetes mellitus, is known to have potent effects on carbohydrate and lipid metabolism leading to the improvement of insulin sensitivity in target tissues. To further assess the capacity of RSG to normalize gene expression in insulin-sensitive tissues, we compared groups of 18-day-treated db/db mice with increasing oral doses of RSG (10, 30, and 100 mg/kg/d) with untreated non-diabetic littermates (db/+). For this aim, transcriptional changes were measured in liver, inguinal adipose tissue (IAT) and soleus muscle using microarrays and real-time PCR. In parallel, targeted metabolomic assessment of lipids (triglycerides (TGs) and free fatty acids (FFAs)) in plasma and tissues was performed by UPLC-MS methods. Multivariate analyses revealed a relationship between the differential gene expressions in liver and liver trioleate content and between blood glucose levels and a combination of differentially expressed genes measured in liver, IAT, and muscle. In summary, we have integrated gene expression and targeted metabolomic data to present a comprehensive overview of RSG-induced changes in a diabetes mouse model and improved the molecular understanding of how RSG ameliorates diabetes through its effect on the major insulin-sensitive tissues.
RESUMO
In order to interpret the effects of melatonin ligands in rats, we need to determine their activity at the receptor subtype level in the corresponding species. Thus, the rat melatonin rMT(1) receptor was cloned using DNA fragments for exon 1 and 2 amplified from rat genomic DNA followed by screening of a rat genomic library for the full length exon sequences. The rat rMT(2) receptor subtype was cloned in a similar manner with the exception of exon 1 which was identified by screening a rat genomic library with exon 1 of the human hMT(2) receptor. The coding region of these receptors translates proteins of 353 and 364 amino acids, respectively, for rMT(1) and rMT(2). A 55% homology was observed between both rat isoforms. The entire contiguous rat MT(1) and MT(2) receptor coding sequences were cloned, stably expressed in CHO cells and characterized in binding assay using 2-[(125)I]-Iodomelatonin. The dissociation constants (K(d)) for rMT(1) and rMT(2) were 42 and 130 pM, respectively. Chemically diverse compounds previously characterized at human MT(1) and MT(2) receptors were evaluated at rMT(1) and rMT(2) receptors, for their binding affinity and functionality in [(35)S]-GTPgammaS binding assay. Some, but not all, compounds shared a similar binding affinity and functionality at both rat and human corresponding subtypes. A different pharmacological profile of the MT(1) subtype has also been observed previously between human and ovine species. These in vitro results obtained with the rat melatonin receptors are thus of importance to understand the physiological roles of each subtype in animal models.
Assuntos
Receptor MT1 de Melatonina/genética , Receptor MT1 de Melatonina/metabolismo , Receptor MT2 de Melatonina/genética , Receptor MT2 de Melatonina/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Clonagem Molecular , Cricetinae , Cricetulus , Éxons/genética , Humanos , Melatonina/análogos & derivados , Melatonina/metabolismo , Dados de Sequência Molecular , Ratos , Receptor MT1 de Melatonina/agonistas , Receptor MT1 de Melatonina/antagonistas & inibidores , Receptor MT2 de Melatonina/agonistas , Receptor MT2 de Melatonina/antagonistas & inibidores , Análise de Sequência de DNARESUMO
Autotaxin is a type II ectonucleotide pyrophosphate phosphodiesterase enzyme. It has been recently discovered that it also has a lysophospholipase D activity. This enzyme probably provides most of the extracellular lysophosphatidic acid from lysophosphatidylcholine. The cloning and tissue distribution of the three isoforms (imaginatively called alpha, beta, and gamma) from human and mouse are reported in this study, as well as their tissue distribution by PCR in the human and mouse. The fate of the alpha isoform from human was also studied after purification and using mass spectrometry. Indeed, this particular isoform expresses the intron 12 in which a cleavage site is present, leading to a rapid catabolism of the isoform. For the human isoform gamma and the total autotaxin mRNA expression, quantitative PCR is presented in 21 tissues. The isoforms were expressed in two different hosts, insect cells and Chinese hamster ovary cells, and were highly purified. The characteristics of the six purified isoforms (pH and temperature dependence, K(m) and V(max) values, and their dependence on metal ions) are presented in this study. Their sensitivity to a small molecule inhibitor, hypericin, is also shown. Finally, the specificity of the isoforms toward a large family of lysophosphatidylcholines is reported. This study is the first complete description of the reported autotaxin isoforms.
Assuntos
Regulação Enzimológica da Expressão Gênica/fisiologia , Lisofosfatidilcolinas/metabolismo , Lisofosfolipídeos/metabolismo , Complexos Multienzimáticos/biossíntese , Complexos Multienzimáticos/genética , Fosfodiesterase I/biossíntese , Fosfodiesterase I/genética , Pirofosfatases/biossíntese , Pirofosfatases/genética , Animais , Antracenos , Sequência de Bases , Células CHO , Células COS , Chlorocebus aethiops , Cricetinae , Cricetulus , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/biossíntese , Isoenzimas/genética , Lisofosfatidilcolinas/genética , Lisofosfolipídeos/genética , Camundongos , Dados de Sequência Molecular , Complexos Multienzimáticos/antagonistas & inibidores , Especificidade de Órgãos/efeitos dos fármacos , Especificidade de Órgãos/fisiologia , Perileno/análogos & derivados , Perileno/farmacologia , Fosfodiesterase I/antagonistas & inibidores , Diester Fosfórico Hidrolases/biossíntese , Diester Fosfórico Hidrolases/genética , Pirofosfatases/antagonistas & inibidores , Especificidade por Substrato/efeitos dos fármacos , Especificidade por Substrato/fisiologiaRESUMO
Neuropeptide Y (NPY) is present in the hypothalamus, where it is believed to play a key role in the control of food intake. Evidence for this assertion has come from studies demonstrating that acute administration of NPY into the hypothalamus or into the brain ventricles leads to increased food intake. In the case of chronic administration, the hyperphagic effects of NPY are prolonged leading to the development of an obese state. NPY levels in the hypothalamus are temporally correlated with food intake and are markedly elevated in response to energy depletion. However, attempts to demonstrate an important role for NPY in the control of food intake using NPY knockout mice, NPY antisense oligodeoxynucleotides and anti-NPY antibodies has produced equivocal results. Despite this many pharmaceutical companies have moved ahead with the search for agonists and antagonists of NPY receptor subtypes as anti-obesity agents. Antagonists of the NPY Y(1) and NPY Y(5) receptor subtype initially looked promising since analogs of NPY with high selectivity for these receptors strongly stimulated food intake. However, attempts to inhibit the signaling of NPY through the NPY Y(1) and NPY Y(5) receptors has produced equivocal effects on food intake. Recent observations that the gut derived peptide PYY(3-36) suppresses appetite by stimulating both peripherally and centrally located NPY Y(2) receptors remain controversial in animals but the effects look promising in human studies. Whether this will be the long awaited therapy based on manipulation of NPY receptors will await further studies of long term efficacy and more importantly a favorable side effect profile.
Assuntos
Peso Corporal/fisiologia , Sistemas de Liberação de Medicamentos/métodos , Receptores de Neuropeptídeos/fisiologia , Animais , Peso Corporal/efeitos dos fármacos , Humanos , Receptores de Neuropeptídeos/agonistas , Receptores de Neuropeptídeos/antagonistas & inibidoresRESUMO
Transcriptional mechanisms controlling human UCP3 gene expression in skeletal muscle remain poorly understood. Experiments based on plasmid electrotransfer into tibialis anterior muscle of C57/BL6 male mice were set up in order to functionally analyze the hUCP3 gene promoter. These transfection experiments showed that a 6300 bp region upstream of the transcription initiation site was sufficient to mediate maximal promoter activity. Further analyses with a series of 5(')-deleted constructs demonstrated that the hUCP3 gene minimal promoter was located between nucleotides -284 and -40. Furthermore, an essential region was identified between nucleotides -284 and -224. The analysis of this region revealed a putative response element for PPAR located between nucleotides -281 and -269. Finally, mutations of potential cis-acting elements within the hUCP3 minimal promoter showed the presence of two TATA boxes (-198/-194 and -45/-41) required for constitutive UCP3 gene expression. To our knowledge, this is the first time that molecular characterization of the UCP3 promoter has been achieved using an in vivo experimental model.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/genética , Músculo Esquelético/metabolismo , Músculos/metabolismo , Regiões Promotoras Genéticas , Animais , Células CHO , Células COS , Cricetinae , DNA/metabolismo , Eletroporação , Deleção de Genes , Genes Reporter , Humanos , Canais Iônicos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Mitocondriais , Modelos Genéticos , Mutagênese Sítio-Dirigida , Mutação , Plasmídeos/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Transcrição Gênica , Transfecção , Proteína Desacopladora 3RESUMO
The neuropeptide Y Y5 receptor gene generates two splice variants, referred to here as Y5(L) (long isoform) and Y5(S) (short isoform). Y5(L) mRNA differs from Y5(S) mRNA in its 5' end, generating a putative open reading frame with 30 additional nucleotides upstream of the initiator AUG compared with the Y5(S) mRNA. The purpose of the present work was to investigate the existence of the Y5(L) mRNA. The authenticity of this transcript was confirmed by isolating part of its 5' untranslated region through 5' rapid amplification of cDNA ends and analysing its tissue distribution. To study the initiation of translation on Y5(L) mRNA, we cloned the Y5(L) cDNA and two Y5(L) cDNA mutants lacking the first or the second putative initiation start codon. Transient expression of the three plasmids in COS-7 cells and saturation binding experiments using (125)I-labelled polypeptide YY (PYY) as a ligand showed that initiation of translation on Y5(L) mRNA could start at the first AUG, giving rise to a Y5(L) receptor with an N-terminal 10-amino-acid extension when compared with the Y5(S) receptor. The human Y5(L) and Y5(S) receptor isoforms displayed similar affinity constants (1.3 nM and 1.5 nM respectively). [(125)I]PYY binding to COS-7 cells expressing either the Y5(L) or the Y5(S) isoform was inhibited with the same rank order of potency by a selection of six chemically diverse compounds: PYY>neuropeptide Y>pancreatic polypeptide>CGP71683A>Synaptic 34>Banyu 6. Comparison of the tissue distribution of Y5(L) and Y5(S) mRNAs, as determined by reverse transcription-PCR analysis, indicated that expression of Y5(L) mRNA occurs in a tissue-specific manner. Finally, we have shown that the two AUG triplets contained in the 5' untranslated region of Y5(L) mRNA did not affect receptor expression.
Assuntos
Regiões 5' não Traduzidas , Arginina/análogos & derivados , Receptores de Neuropeptídeo Y/efeitos dos fármacos , Receptores de Neuropeptídeo Y/genética , Processamento Alternativo , Animais , Arginina/farmacologia , Sequência de Bases , Encéfalo/metabolismo , Células COS/efeitos dos fármacos , Clonagem Molecular , Códon de Iniciação , Humanos , Dados de Sequência Molecular , Neuropeptídeo Y/metabolismo , Neuropeptídeo Y/farmacologia , Biossíntese de Proteínas , Isoformas de Proteínas/efeitos dos fármacos , Isoformas de Proteínas/genética , RNA Mensageiro/metabolismo , Receptores de Neuropeptídeo Y/antagonistas & inibidores , Receptores de Neuropeptídeo Y/metabolismo , Transcrição Gênica , Repetições de Trinucleotídeos/genéticaRESUMO
Uncoupling protein-3 (UCP3), which is expressed abundantly in skeletal muscle, is one of the carrier proteins dissipating the transmitochondrial electrochemical gradient as heat and has therefore been implicated in the regulation of energy metabolism. Myoblasts or differentiated muscle cells in vitro expressed little if any UCP3, compared with the levels detected in biopsies of skeletal muscle. In the present report, we sought to investigate UCP3 mRNA expression in human muscle generated by myoblast transplantation in the skeletal muscle of an immunodeficient mouse model. Time course experiments demonstrated that 7-8 weeks following transplantation fully differentiated human muscle fibers were formed. The presence of differentiated human muscle fibers was assessed by quantitative PCR measurement of the human alpha-actin mRNA together with immunohistochemical staining using specific antibodies for spectrin and the slow adult myosin heavy chain. Interestingly, we found that the expression of UCP3 mRNA was dependant on human muscle differentiation and that the UCP3 mRNA level was comparable with that found in human muscle biopsies. Moreover, the human UCP3 (hUCP3) promoter seems to be fully functional, since triiodothyronine treatment of the mice not only stimulated the mouse UCP3 (mUCP3) mRNA expression but also strongly stimulated the hUCP3 mRNA expression in human fibers formed after myoblast transplantation. To our knowledge, this is the first time that primary myoblasts could be induced to express the UCP3 gene at a level comparable of that found in human muscle fibers.