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1.
Am J Physiol Heart Circ Physiol ; 311(1): H44-53, 2016 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-27199128

RESUMEN

Cardiomyocytes derived from human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs) are increasingly used for in vitro assays and represent an interesting opportunity to increase the data throughput for drug development. In this work, we describe a 96-well recording of synchronous electrical activities from spontaneously beating hiPSC-derived cardiomyocyte monolayers. The signal was obtained with a fast-imaging plate reader using a submillisecond-responding membrane potential recording assay, FluoVolt, based on a newly derived voltage-sensitive fluorescent dye. In our conditions, the toxicity of the dye was moderate and compatible with episodic recordings for >3 h. We show that the waveforms recorded from a whole well or from a single cell-sized zone are equivalent and make available critical functional parameters that are usually accessible only with gold standard techniques like intracellular microelectrode recording. This approach allows accurate identification of the electrophysiological effects of reference drugs on the different phases of the cardiac action potential as follows: fast depolarization (lidocaine), early repolarization (nifedipine, Bay K8644, and veratridine), late repolarization (dofetilide), and diastolic slow depolarization (ivabradine). Furthermore, the data generated with the FluoVolt dye can be pertinently complemented with a calcium-sensitive dye for deeper characterization of the pharmacological responses. In a semiautomated plate reader, the two probes used simultaneously in 96-well plates provide an easy and powerful multiparametric assay to rapidly and precisely evaluate the cardiotropic profile of compounds for drug discovery or cardiac safety.


Asunto(s)
Potenciales de Acción/efectos de los fármacos , Señalización del Calcio/efectos de los fármacos , Colorantes Fluorescentes/metabolismo , Ensayos Analíticos de Alto Rendimiento , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Automatización de Laboratorios , Línea Celular , Relación Dosis-Respuesta a Droga , Colorantes Fluorescentes/toxicidad , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Microscopía Fluorescente , Miocitos Cardíacos/metabolismo , Procesamiento de Señales Asistido por Computador , Factores de Tiempo
2.
J Neurosci ; 31(47): 16928-40, 2011 Nov 23.
Artículo en Inglés | MEDLINE | ID: mdl-22114263

RESUMEN

"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.


Asunto(s)
Eliminación de Gen , N-Metil-3,4-metilenodioxianfetamina/antagonistas & inhibidores , N-Metil-3,4-metilenodioxianfetamina/farmacología , Receptores Acoplados a Proteínas G/deficiencia , Receptores Acoplados a Proteínas G/fisiología , Animales , Dopamina/fisiología , Relación Dosis-Respuesta a Droga , Células HeLa , Humanos , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Distribución Aleatoria , Receptores Acoplados a Proteínas G/genética , Serotonina/fisiología
3.
Stem Cells Int ; 2019: 2945435, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31236114

RESUMEN

The discovery of novel drugs for neurodegenerative diseases has been a real challenge over the last decades. The development of patient- and/or disease-specific in vitro models represents a powerful strategy for the development and validation of lead candidates in preclinical settings. The implementation of a reliable platform modeling dopaminergic neurons will be an asset in the study of dopamine-associated pathologies such as Parkinson's disease. Disease models based on cell reprogramming strategies, using either human-induced pluripotent stem cells or transcription factor-mediated transdifferentiation, are among the most investigated strategies. However, multipotent adult stem cells remain of high interest to devise direct conversion protocols and establish in vitro models that could bypass certain limitations associated with reprogramming strategies. Here, we report the development of a six-step chemically defined protocol that drives the transdifferentiation of human nasal olfactory stem cells into dopaminergic neurons. Morphological changes were progressively accompanied by modifications matching transcript and protein dopaminergic signatures such as LIM homeobox transcription factor 1 alpha (LMX1A), LMX1B, and tyrosine hydroxylase (TH) expression, within 42 days of differentiation. Phenotypic changes were confirmed by the production of dopamine from differentiated neurons. This new strategy paves the way to develop more disease-relevant models by establishing reprogramming-free patient-specific dopaminergic cell models for drug screening and/or target validation for neurodegenerative diseases.

4.
Arch Biochem Biophys ; 477(1): 12-9, 2008 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-18502195

RESUMEN

Melatonin is a neurohormone implicated in both biorhythm synchronization and neuroprotection from oxidative stress. Its functions are mediated by two G-protein-coupled-receptors (MT1 and MT2) and MT3, which corresponds to quinone oxidoreductase 2 (QR2). To determine the binding site of QR2 for melatonin, point mutations of residues crucial for the enzymatic activity of hQR2 were performed. The substitution of the hydrophobic residues Phe126, Ile128 and Phe178 by tyrosines at the active site significantly increased enzymatic activity and decreased the affinity of a structural analog of melatonin, the 2[(125)I]iodo-MCANAT. The mutation of residues implicated in zinc chelating (His(173); His(177)) had no effect on radioligand binding. Destabilisation of the cofactor FAD by mutation N18E showed that 2[(125)I]iodo-MCANAT binding was closely linked to the conformational integrity of human QR2. Surprisingly, the mutations C222F and N161A, which are distant from the determined binding site of the ligand, increased the affinity of 2[(125)I]iodo-MCANAT for hQR2. What seems to better explain the binding variations among the mutants are the activity recorded with BNAH and coenzyme Q1. Various hypotheses are discussed based on the various parameters used in the study: nature of the substrates and co-substrates and nature of the amino acid changes. This study, which constitutes the first structural analysis of hQR2, should enable to better understand the biological role of melatonin on this enzyme and particularly, the discrepancies between the pharmacologies of the melatonin binding site (MT3) and the QR2 catalytic activity.


Asunto(s)
Melatonina/metabolismo , Quinona Reductasas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Western Blotting , Células CHO , Catálisis , Cricetinae , Cricetulus , Humanos , Datos de Secuencia Molecular , Mutagénesis , Quinona Reductasas/química , Quinona Reductasas/genética , Homología de Secuencia de Aminoácido
5.
Br J Pharmacol ; 175(16): 3281-3297, 2018 08.
Artículo en Inglés | MEDLINE | ID: mdl-28898928

RESUMEN

BACKGROUND AND PURPOSE: Recent crystal structures of GPCRs have emphasized the previously unappreciated role of the second extracellular (E2) loop in ligand binding and gating and receptor activation. Here, we have assessed the role of the E2 loop in the activation of the melatonin MT1 receptor and in the inactivation of the closely related orphan receptor GPR50. EXPERIMENTAL APPROACH: Chimeric MT1 -GPR50 receptors were generated and functionally analysed in terms of 2-[125 I]iodomelatonin binding, Gi /cAMP signalling and ß-arrestin2 recruitment. We also used computational molecular dynamics (MD) simulations. KEY RESULTS: MD simulations of 300 ns revealed (i) the tight hairpin structure of the E2 loop of the MT1 receptor (ii) the most suitable features for melatonin binding in MT1 receptors and (iii) major predicted rearrangements upon MT1 receptor activation, stabilizing interaction networks between Phe179 or Gln181 in the E2 loop and transmembrane helixes 5 and 6. Functional assays confirmed these predictions, because reciprocal replacement of MT1 and GPR50 residues/domains led to the predicted loss- and gain-of-melatonin action of MT1 receptors and GPR50 respectively. CONCLUSIONS AND IMPLICATIONS: Our work demonstrated the crucial role of the E2 loop for MT1 receptor and GPR50 function by proposing a model in which the E2 loop is important in stabilizing active MT1 receptor conformations and by showing how evolutionary processes appear to have selected for modifications in the E2 loop in order to make GPR50 unresponsive to melatonin. LINKED ARTICLES: This article is part of a themed section on Recent Developments in Research of Melatonin and its Potential Therapeutic Applications. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.16/issuetoc.


Asunto(s)
Receptor de Melatonina MT1/química , Receptor de Melatonina MT1/metabolismo , Células HEK293 , Humanos , Melatonina/metabolismo , Modelos Moleculares , Proteínas del Tejido Nervioso/metabolismo , Estructura Secundaria de Proteína , Receptores Acoplados a Proteínas G/metabolismo
6.
FEBS Lett ; 581(18): 3572-8, 2007 Jul 24.
Artículo en Inglés | MEDLINE | ID: mdl-17628547

RESUMEN

Autotaxin is a member of the phosphodiesterase family of enzymes, (NPP2). It is an important secreted protein found in conditioned medium from adipocytes. It also has a putative role in the metastatic process. Based on these observation, further validation of this potential target was necessary, apart from the classical biochemical ones. The construction of a knock out mouse strain for ATX was started. In this paper, we report the generation of a mouse line displaying an inactivated ATX gene product. The KO line was designed in order to generate a functional inactivation of the protein. In this respect, the threonine residue T210 was replaced by an alanine (T210A) leading to a catalytically inactive enzyme. If the experimental work was straight forward, we disappointedly discovered at the final stage that the breeding of heterozygous animals, ATX -/+, led to the generation of a Mendelian repartition of wild-type and heterozygous, but no homozygous were found, strongly suggesting that the ATX deletion is lethal at an early stage of the development. This was confirmed by statistical analysis. Although other reported the same lethality for attempted ATX-/- mice generation [van Meeteren, L.A., Ruurs, P., Stortelers, C., Bouwman, P., van Rooijen, M.A., Pradère, J.P., Pettit, T.R., Wakelam, M.J.O., Saulnier-Blache, J.S., Mummery, C.L., Moolenar, W.H. and Jonkers, J. (2006) Autotaxin, a secreted lysophospholipase D, is essential for blood vessel formation during development, Mol. Cell. Biol. 26, 5015-5022; Tanaka, M., Okudaira, S., Kishi, Y., Ohkawa, R., Isei, S., Ota, M., Noji, S., Yatomi, Y., Aoki, J., and Arai, H. (2006) Autotaxin stabilizes blood vessels and is required for embryonic vasculature by producing lysophosphatidic acid, J. Biol. Chem. 281, 25822-25830], they used more drastic multiple exon deletions in the ATX gene, while we chose a single point mutation. To our knowledge, the present work is the first showing such a lethality in any gene after a point mutation in an enzyme catalytic site.


Asunto(s)
Aminoácidos/genética , Genes Letales/genética , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Mutación/genética , Fosfodiesterasa I/genética , Fosfodiesterasa I/metabolismo , Pirofosfatasas/genética , Pirofosfatasas/metabolismo , Aminoácidos/metabolismo , Animales , Células Cultivadas , Chlorocebus aethiops , Genotipo , Ratones , Ratones Transgénicos , Proteínas Mutantes/genética , Hidrolasas Diéster Fosfóricas
7.
Biochimie ; 89(10): 1264-75, 2007 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-17714848

RESUMEN

NRH:quinone oxidoreductase 2 (QR2) is a long forgotten oxidoreductive enzyme that metabolizes quinones and binds melatonin. We used the potency of the RNA interference (RNAi)-mediated gene silencing to build a cellular model in which the role of QR2 could be studied. Because standard approaches were poorly successful, we successively used: (1) two chemically synthesized fluorescent small interfering RNA (siRNA) duplexes designed and tested for their gene silencing capacity leading to a maximal 40% QR2 gene silencing 48h post-transfection; (2) double transfection and cell-sorting of high fluorescent siRNA-transfected HT22 cells further enhancing QR2 RNAi silencing to 88%; (3) stable QR2 knock-down HT22 cell lines established with H1and U6 promoter driven QR2 short hairpin RNA (shRNA) encoding vectors, resulting in a 71-80% reduction of QR2 enzymatic activity in both QR2 shRNA HT22 cells. Finally, as a first step in the study of this cellular model, we observed a 42-48% reduction of menadione/BNAH-mediated toxicity in QR2 shRNA cells compared to the wild-type HT22 cells. Although becoming widespread and in some cases effective, siRNA-mediated cellular knock-down proves in the present work to be of marginal efficiency. Much development is required for this technique to be of general application.


Asunto(s)
NAD(P)H Deshidrogenasa (Quinona)/genética , Interferencia de ARN , Animales , Línea Celular , Citometría de Flujo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Cinética , Ratones , Ratones Noqueados , Microscopía Fluorescente , NAD(P)H Deshidrogenasa (Quinona)/metabolismo , ARN Interferente Pequeño/genética , Transfección
8.
Biochem J ; 388(Pt 1): 205-15, 2005 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-15636586

RESUMEN

The catabolism of melatonin, whether naturally occurring or ingested, takes place via two pathways: approximately 70% can be accounted for by conjugation (sulpho- and glucurono-conjugation), and approximately 30% by oxidation. It is commonly thought that the interferon-induced enzyme indoleamine 2,3-dioxygenase (EC 1.13.11.42), which oxidizes tryptophan, is also responsible for the oxidation of 5-hydroxytryptamine (serotonin) and its derivative, melatonin. Using the recombinant enzyme expressed in Escherichia coli, we show in the present work that indoleamine 2,3-dioxygenase indeed cleaves tryptophan; however, under the same conditions, it is incapable of cleaving the two other indoleamines. By contrast, myeloperoxidase (EC 1.11.1.7) is capable of cleaving the indole moiety of melatonin. However, when using the peroxidase conditions of assay -- with H2O2 as co-substrate -- indoleamine 2,3-dioxygenase is able to cleave melatonin into its main metabolite, a kynurenine derivative. The present work establishes that the oxidative metabolism of melatonin is due, in the presence of H2O2, to the activities of both myeloperoxidase and indoleamine 2,3-dioxygenase (with lower potency), since both enzymes have Km values for melatonin in the micromolar range. Under these conditions, several indolic compounds can be cleaved by both enzymes, such as tryptamine and 5-hydroxytryptamine. Furthermore, melatonin metabolism results in a kynurenine derivative, the pharmacological action of which remains to be studied, and could amplify the mechanisms of action of melatonin.


Asunto(s)
Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Melatonina/metabolismo , Peroxidasa/metabolismo , Triptófano/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenasa/antagonistas & inhibidores , Indolamina-Pirrol 2,3,-Dioxigenasa/química , Melatonina/química , Modelos Químicos , Estructura Molecular , Oxidación-Reducción , Peroxidasa/química , Especificidad por Sustrato , Triptófano/química
9.
EBioMedicine ; 9: 293-305, 2016 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-27333044

RESUMEN

Autism spectrum disorders affect millions of individuals worldwide, but their heterogeneity complicates therapeutic intervention that is essentially symptomatic. A versatile yet relevant model to rationally screen among hundreds of therapeutic options would help improving clinical practice. Here we investigated whether neurons differentiated from pluripotent stem cells can provide such a tool using SHANK3 haploinsufficiency as a proof of principle. A library of compounds was screened for potential to increase SHANK3 mRNA content in neurons differentiated from control human embryonic stem cells. Using induced pluripotent stem cell technology, active compounds were then evaluated for efficacy in correcting dysfunctional networks of neurons differentiated from individuals with deleterious point mutations of SHANK3. Among 202 compounds tested, lithium and valproic acid showed the best efficacy at corrected SHANK3 haploinsufficiency associated phenotypes in cellulo. Lithium pharmacotherapy was subsequently provided to one patient and, after one year, an encouraging decrease in autism severity was observed. This demonstrated that pluripotent stem cell-derived neurons provide a novel cellular paradigm exploitable in the search for specific disease-modifying treatments.


Asunto(s)
Trastorno del Espectro Autista/patología , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Células Madre Pluripotentes/citología , Trastorno del Espectro Autista/tratamiento farmacológico , Trastorno del Espectro Autista/metabolismo , Diferenciación Celular , Células Cultivadas , Haploinsuficiencia/efectos de los fármacos , Células Madre Embrionarias Humanas , Humanos , Litio/farmacología , Litio/uso terapéutico , Masculino , Proteínas del Tejido Nervioso/metabolismo , Plasticidad Neuronal/efectos de los fármacos , Neuronas/citología , Fenotipo , Células Madre Pluripotentes/metabolismo , ARN Mensajero/metabolismo , Índice de Severidad de la Enfermedad , Transcriptoma/efectos de los fármacos , Ácido Valproico/farmacología
10.
Trends Pharmacol Sci ; 25(9): 449-55, 2004 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-15559245

RESUMEN

Alpha1-adrenoceptors are G-protein-coupled receptors that bind catecholamines. Sixteen distinct human alpha1A-adrenoceptor isoforms have been identified from human tissues, including five full-length and 11 truncated versions. An updated scheme for the identification of alpha1A-adrenoceptor splice variants is proposed. Given the established roles of alpha1-adrenoceptors in benign prostatic hyperplasia, myocardial hypertrophy and other cardiovascular disorders, elucidation of the biological significance of the signaling diversity and potential pharmacological roles of alpha1A-adrenoceptor splice variants are important areas of future research.


Asunto(s)
Receptores Adrenérgicos alfa 1/genética , Animales , Mapeo Cromosómico , Humanos , Isoformas de Proteínas/clasificación , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Receptores Adrenérgicos alfa 1/clasificación , Receptores Adrenérgicos alfa 1/fisiología , Transducción de Señal , Terminología como Asunto
11.
Biochem Pharmacol ; 71(1-2): 74-88, 2005 Dec 19.
Artículo en Inglés | MEDLINE | ID: mdl-16293234

RESUMEN

Melatonin acts through a series of molecular targets: the G-protein coupled receptors, MT1 and MT2, and a third binding site, MT3, recently identified as the enzyme NRH:quinone oxydoreductase 2 (QR2). The relationship between the multiple physiological functions of melatonin and this enzyme remains unclear. Because of the relationship of QR2 with the redox status of cells, these studies could bring the first tools for a molecular rationale of the antioxidant effects of melatonin. In the present paper, we used a QR2-stably expressing cell line and hamster kidneys to compare the 2-[125I]-iodomelatonin and 2-[125I]-iodo-5-methoxycarbonylamino-N-acetyltryptamine binding data, and to characterize the MT3 binding site. We designed and tested compounds from two distinct chemicals series in a displacement assay of the two MT3 ligands, 2-[125I]-iodomelatonin and 2-[125I]-iodo-5-methoxycarbonylamino-N-acetyltryptamine from their cloned target. We also tested their ability to inhibit QR2 catalytic activity. These compounds were separated into two classes: those that bind within the catalytic site (and being inhibitors) and those that bind outside it (and therefore not being inhibitors). Compounds range from potent ligands (K(i) = 1 nM) to potent inhibitors (14 nM), and include one compound [NMDPEF: N-[2-(2-methoxy-6H-dipyrido[2,3-a:3,2-e]pyrrolizin-11-yl)ethyl]-2-furamide] active on both parameters in the low nanomolar range. To dissect the physio-pathological pathways in which QR2, MT3 and melatonin meet, one needs more compounds binding to MT3 and/or inhibitors of QR2 enzymatic activity. The compounds described in the present paper are new tools for such a task.


Asunto(s)
Melatonina/metabolismo , Quinona Reductasas/metabolismo , Animales , Sitios de Unión , Unión Competitiva , Células CHO , Cricetinae
12.
Br J Pharmacol ; 139(5): 935-44, 2003 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-12839867

RESUMEN

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.


Asunto(s)
Adipocitos/enzimología , Endotelina-1/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/biosíntesis , Factor de Necrosis Tumoral alfa/farmacología , Células 3T3 , Adipocitos/efectos de los fármacos , Animales , Relación Dosis-Respuesta a Droga , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Ratones , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo II , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/biosíntesis , ARN Mensajero/genética
13.
Protein Sci ; 21(9): 1323-33, 2012 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-22760822

RESUMEN

The human thioredoxin (TRX)-interacting protein is found in multiple subcellular compartments and plays a major role in redox homeostasis, particularly in the context of metabolism (e.g., lipidemia and glycemia) and apoptosis. A molecular approach to the protein's modus operandi is still needed because some aspects of the TRX-interacting protein-mediated regulation of TRX are not clearly understood. To this end, His-tagged TRX-interacting proteins were over-expressed in Escherichia coli. Because the protein is expressed mainly in inclusion bodies, it was denatured in high concentrations of guanidium hydrochloride, centrifuged, and purified by Ni-NTA affinity chromatography. His-TRX-interacting protein was then refolded by dialysis and its restructuring monitored by circular dichroism spectrometry. This preparation resulted in the formation of a covalent complex with recombinant human TRX, demonstrating that association occurs without the intervention of other partner proteins. Multiple cysteine-to-serine mutants of TRX-interacting protein were produced and purified. These mutations were efficient in limiting the formation of disulfide-linked homo-oligomers in an oxidizing environment. The mutants were also used to gain functional insight into the formation of the TRX-interacting protein-TRX complexes. These complexes were able to form in the absence of internal disulfide bridges. A mutant with all but one cysteine changed to serine (Cys ²47) also showed an enhanced capacity to form complexes with TRX demonstrating, in a pure molecular system, that this particular cysteine is likely responsible for the disulfide bridge between TRX-interacting protein and TRX.


Asunto(s)
Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Disulfuros/metabolismo , Tiorredoxinas/metabolismo , Sustitución de Aminoácidos , Animales , Proteínas Portadoras/química , Proteínas Portadoras/aislamiento & purificación , Línea Celular , Clonación Molecular , Cisteína/química , Cisteína/genética , Cisteína/metabolismo , Escherichia coli/genética , Humanos , Oxidación-Reducción , Replegamiento Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo
14.
Eur J Pharmacol ; 677(1-3): 15-21, 2012 Feb 29.
Artículo en Inglés | MEDLINE | ID: mdl-22202844

RESUMEN

The main melatonin receptors are two G-protein coupled receptors named MT(1) and MT(2). Having described the molecular pharmacology of the human versions of these receptors, we turned to two of the three species most useful in studying melatonin physiology: rat and sheep (a diurnal species used to understand the relationship between circadian rhythm and depression). We also employed previously used compounds to describe the mouse melatonin receptors; despite the early cloning of mouse receptors, few molecular pharmacology studies on these receptors exist. To our surprise, we detected no major differences between the data obtained from mice and those from other species.


Asunto(s)
Receptor de Melatonina MT1/química , Receptor de Melatonina MT1/metabolismo , Receptor de Melatonina MT2/química , Receptor de Melatonina MT2/metabolismo , Secuencia de Aminoácidos , Animales , Células CHO , Cricetinae , Cricetulus , Genómica , Humanos , Ratones , Datos de Secuencia Molecular , Transporte de Proteínas , Ratas , Alineación de Secuencia , Ovinos , Especificidad de la Especie
15.
PLoS One ; 6(2): e17237, 2011 Feb 24.
Artículo en Inglés | MEDLINE | ID: mdl-21390316

RESUMEN

c-Yes, a member of the Src tyrosine kinase family, is found highly activated in colon carcinoma but its importance relative to c-Src has remained unclear. Here we show that, in HT29 colon carcinoma cells, silencing of c-Yes, but not of c-Src, selectively leads to an increase of cell clustering associated with a localisation of ß-catenin at cell membranes and a reduction of expression of ß-catenin target genes. c-Yes silencing induced an increase in apoptosis, inhibition of growth in soft-agar and in mouse xenografts, inhibition of cell migration and loss of the capacity to generate liver metastases in mice. Re-introduction of c-Yes, but not c -Src, restores transforming properties of c-Yes depleted cells. Moreover, we found that c-Yes kinase activity is required for its role in ß-catenin localisation and growth in soft agar, whereas kinase activity is dispensable for its role in cell migration. We conclude that c-Yes regulates specific oncogenic signalling pathways important for colon cancer progression that is not shared with c-Src.


Asunto(s)
Carcinoma/patología , Transformación Celular Neoplásica/genética , Neoplasias del Colon/patología , Proteínas Proto-Oncogénicas c-yes/fisiología , Animales , Carcinoma/genética , Línea Celular Tumoral , Neoplasias del Colon/genética , Progresión de la Enfermedad , Femenino , Técnicas de Silenciamiento del Gen , Células HCT116 , Células HT29 , Humanos , Ratones , Ratones Desnudos , Ratones SCID , Especificidad de Órganos/genética , Proteínas Proto-Oncogénicas c-yes/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-yes/genética , Proteínas Proto-Oncogénicas pp60(c-src)/genética , Proteínas Proto-Oncogénicas pp60(c-src)/fisiología , Transducción de Señal/genética , Transducción de Señal/fisiología , Trasplante Heterólogo , Familia-src Quinasas/antagonistas & inhibidores , Familia-src Quinasas/genética , Familia-src Quinasas/fisiología
16.
Chem Biol Interact ; 186(2): 103-9, 2010 Jul 30.
Artículo en Inglés | MEDLINE | ID: mdl-20399199

RESUMEN

Quinone reductase 2 is a cytosolic enzyme which catalyses the reduction of quinones, such as menadione and coenzymes Q. Despite a relatively close sequence-based resemblance to NAD(P)H:quinone oxidoreductase 1 (QR1), it has many different features. QR2 is the third melatonin binding site (MT3). It is inhibited in the micromolar range by melatonin, and does not accept conventional phosphorylated nicotinamides as hydride donors. QR2 has a powerful capacity to activate quinones leading to unexpected toxicity situations. In the present paper, we report the characterization of three QR2 modulators: melatonin, resveratrol and S29434. The latter compound inhibits QR2 activity with an IC(50) in the low nanomolar range. The potency of the modulators ranged as follows, from the least to the most potent: melatonin

Asunto(s)
Inhibidores Enzimáticos/farmacología , Piridinas/farmacología , Alcaloides de Pirrolicidina/farmacología , Quinona Reductasas/antagonistas & inhibidores , Animales , Secuencia de Bases , Cartilla de ADN/genética , Inhibidores Enzimáticos/química , Furanos/química , Furanos/farmacología , Humanos , Técnicas In Vitro , Cinética , Melatonina/farmacología , Piridinas/química , Alcaloides de Pirrolicidina/química , Quinona Reductasas/genética , Quinona Reductasas/metabolismo , Receptores de Melatonina/metabolismo , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Resveratrol , Spodoptera , Estilbenos/farmacología , Especificidad por Sustrato
17.
J Biomol Screen ; 15(8): 956-67, 2010 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-20625180

RESUMEN

The development of cell-based assays for high-throughput screening (HTS) approaches often requires the generation of stable transformant cell lines. However, these cell lines are essentially created by random integration of a gene of interest (GOI) with no control over the level and stability of gene expression. The authors developed a targeted integration system in Chinese hamster ovary (CHO) cells, called the cellular genome positioning system (cGPS), based on the stimulation of homologous gene targeting by meganucleases. Five different GOIs were knocked in at the same locus in cGPS CHO-K1 cells. Further characterization revealed that the cGPS CHO-K1 system is more rapid (2-week protocol), efficient (all selected clones expressed the GOI), reproducible (GOI expression level variation of 12%), and stable over time (no change in GOI expression after 23 weeks of culture) than classical random integration. Moreover, in all cGPS CHO-K1 targeted clones, the recombinant protein was biologically active and its properties similar to the endogenous protein. This fast and robust method opens the door for creating large collections of cell lines of better quality and expressing therapeutically relevant GOIs at physiological levels, thereby enhancing the potential scope of HTS.


Asunto(s)
Células/metabolismo , Desoxirribonucleasas/fisiología , Marcación de Gen/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Mutagénesis Sitio-Dirigida/métodos , Animales , Células CHO , Línea Celular , Células/citología , Mapeo Cromosómico/métodos , Cricetinae , Cricetulus , Desoxirribonucleasas/metabolismo , Modelos Biológicos , Factores de Tiempo , Transfección
18.
J Biol Chem ; 283(12): 7776-89, 2008 Mar 21.
Artículo en Inglés | MEDLINE | ID: mdl-18175805

RESUMEN

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.


Asunto(s)
Regulación Enzimológica de la Expresión Génica/fisiología , Lisofosfatidilcolinas/metabolismo , Lisofosfolípidos/metabolismo , Complejos Multienzimáticos/biosíntesis , Complejos Multienzimáticos/genética , Fosfodiesterasa I/biosíntesis , Fosfodiesterasa I/genética , Pirofosfatasas/biosíntesis , Pirofosfatasas/genética , Animales , Antracenos , Secuencia de Bases , Células CHO , Células COS , Chlorocebus aethiops , Cricetinae , Cricetulus , Inhibidores Enzimáticos/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/biosíntesis , Isoenzimas/genética , Lisofosfatidilcolinas/genética , Lisofosfolípidos/genética , Ratones , Datos de Secuencia Molecular , Complejos Multienzimáticos/antagonistas & inhibidores , Especificidad de Órganos/efectos de los fármacos , Especificidad de Órganos/fisiología , Perileno/análogos & derivados , Perileno/farmacología , Fosfodiesterasa I/antagonistas & inhibidores , Hidrolasas Diéster Fosfóricas/biosíntesis , Hidrolasas Diéster Fosfóricas/genética , Pirofosfatasas/antagonistas & inhibidores , Especificidad por Sustrato/efectos de los fármacos , Especificidad por Sustrato/fisiología
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