Positive modulation of the a9a10 nicotinic cholinergic receptor by ascorbic acid

Background and Purpose: The activation of α9α10 nicotinic cholinergic receptors (nAChRs) present at the synapse between efferent olivocochlear fibres and cochlear hair cells can prevent acoustic trauma. Hence, pharmacological potentiators of these receptors could be useful therapeutically. In this work, we characterize ascorbic acid as a positive modulator of recombinant α9α10 nAChRs. Experimental Approach: ACh-evoked responses were analysed under two-electrode voltage-clamp recordings in Xenopus laevis oocytes injected with α9 and α10 cRNAs. Key Results: Ascorbic acid potentiated ACh responses in X. laevis oocytes expressing α9α10 (but not α4β2 or α7) nAChRs, in a concentration-dependent manner, with an effective concentration range of 1–30 mM. The compound did not affect the receptor's current–voltage profile nor its apparent affinity for ACh, but it significantly enhanced the maximal evoked currents (percentage of ACh maximal response, 240 ± 20%). This effect was specific for the L form of reduced ascorbic acid. Substitution of the extracellular cysteine residues present in loop C of the ACh binding site did not affect the potentiation. Ascorbic acid turned into a partial agonist of α9α10 nAChRs bearing a point mutation at the pore domain of the channel (TM2 V13′T mutant). A positive allosteric mechanism of action rather than an antioxidant effect of ascorbic acid is proposed. Conclusions and Implications: The present work describes one of the few agents that activates or potentiates α9α10 nAChRs and leads to new avenues for designing drugs with potential therapeutic use in inner ear disorders.Fil: Boffi, Juan Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; ArgentinaFil: Wedemeyer, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; ArgentinaFil: Lipovsek, Maria Marcela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; ArgentinaFil: Katz, Eleonora. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; ArgentinaFil: Calvo, Daniel Juan. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; ArgentinaFil: Elgoyhen, Ana Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Farmacología; Argentin

Differential Contribution of Subunit Interfaces to α 9 α 10 Nicotinic Acetylcholine Receptor Function

Nicotinic acetylcholine receptors can be assembled from either homomeric or heteromeric pentameric subunit combinations. At the interface of the extracellular domains of adjacent subunits lies the acetylcholine binding site, composed of a principal component provided by one subunit and a complementary component of the adjacent subunit. Compared with neuronal nicotinic acetylcholine cholinergic receptors (nAChRs) assembled from α and β subunits, the α9α10 receptor is an atypical member of the family. It is a heteromeric receptor composed only of α subunits. Whereas mammalian α9 subunits can form functional homomeric α9 receptors, α10 subunits do not generate functional channels when expressed heterologously. Hence, it has been proposed that α10 might serve as a structural subunit, much like a β subunit of heteromeric nAChRs, providing only complementary components to the agonist binding site. Here, we have made use of site-directed mutagenesis to examine the contribution of subunit interface domains to α9α10 receptors by a combination of electrophysiological and radioligand binding studies. Characterization of receptors containing Y190T mutations revealed unexpectedly that both α9 and α10 subunits equally contribute to the principal components of the α9α10 nAChR. In addition, we have shown that the introduction of a W55T mutation impairs receptor binding and function in the rat α9 subunit but not in the α10 subunit, indicating that the contribution of α9 and α10 subunits to complementary components of the ligand-binding site is nonequivalent. We conclude that this asymmetry, which is supported by molecular docking studies, results from adaptive amino acid changes acquired only during the evolution of mammalian α10 subunits.Fil: Boffi, Juan Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Marcovich, Irina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Gill Thind, JasKiran K.. University College London; Reino UnidoFil: Corradi, Jeremias. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Collins, Toby. University College London; Reino UnidoFil: Lipovsek, Maria Marcela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Moglie, Marcelo Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Plazas, Paola Viviana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Craig, Patricio Oliver. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Millar, Neil S.. University College London; Reino UnidoFil: Bouzat, Cecilia Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Elgoyhen, Ana Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentin

STAT5 transcriptional activity is impaired by LIF in a mammary epithelial cell line

Gene regulation mediated by STAT factors has been implicated in cellular functions with relevance to a variety of processes. Particularly, STAT5 and STAT3 play a crucial role in mammary epithelium displaying reciprocal activation kinetics during pregnancy, lactation and involution. Here, we show that LIF treatment of mammary epithelial HC11 cells reduces the phosphorylation levels and transcriptional activity of p-STAT5 in correlation with STAT3 phosphorylation. We have also found that STAT5 activity is negatively modulated by this cytokine, both on a gene whose expression is induced, as well as on a promoter repressed by STAT5. Besides, our results show that lactogenic hormones increase LIF effect on gene induction without modifying STAT3 phosphorylation state. Our findings strongly suggest that there is crosstalk between STAT5 and STAT3 pathways that could modulate their ability to regulate gene expression.Fil: Rodriguez Granillo, Agustina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Boffi, Juan Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Barañao, Jose Lino Salvador. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Kordon, Edith Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Pecci, Adali. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Guberman, Alejandra Sonia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentin

Maintenance of murine embryonic stem cells' self-renewal and pluripotency with increase in proliferation rate by a bovine granulosa cell line-conditioned medium

Murine embryonic stem cells (mESCs) are pluripotent cells that can be propagated in an undifferentiated state in continuous culture on a feeder layer or without feeders in the presence of leukemia inhibitory factor (LIF). Although there has been a great advance since their establishment, ESC culture is still complex and expensive. Therefore, finding culture conditions that maintain the self-renewal of ESCs, preventing their differentiation and promoting their proliferation, is still an area of great interest. In this work, we studied the effects of the conditioned medium from a bovine granulosa cell line (BGC-CM) on the maintenance of self-renewal and pluripotency of mESCs. We found that this medium is able to maintain mESCs' self-renewal while preserving its critical properties without LIF addition. mESCs cultured in BGC-CM expressed the stem cell markers Oct4, Sox2, Nanog, SSEA-1, Klf4, Rex1, and ECAT1. Moreover, mESCs cultured in BGC-CM gave rise to embryoid bodies and teratomas that differentiated effectively to diverse cell populations from endoderm, mesoderm, and ectoderm. Further, we found that mESCs cultured in BGC-CM have an increased proliferation rate compared with cells grown in the mESC standard culture medium supplemented with LIF. These findings may provide a powerful tool to culture mESCs for long periods of time with high proliferation rate while preserving its basic characteristics, contributing to the application of these cells to assess potential tissue engineering and cellular therapy applications. © Copyright 2011, Mary Ann Liebert, Inc.Fil: Losino, Noelia Ivana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Luzzani, Carlos Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Solari, Claudia María. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Boffi, Juan Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Louis Tisserand, Mariana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Sevlever, Gustavo. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; ArgentinaFil: Barañao, Jose Lino Salvador. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Guberman, Alejandra Sonia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentin

Deep learning-enhanced light-field imaging with continuous validation

Light field microscopy (LFM) has emerged as a powerful tool for fast volumetric image acquisition in biology, but its effective throughput and widespread use has been hampered by a computationally demanding and artefact-prone image reconstruction process. Here, we present a novel framework consisting of a hybrid light-field light-sheet microscope and deep learning-based volume reconstruction, where single light-sheet acquisitions continuously serve as training data and validation for the convolutional neural network reconstructing the LFM volume. Our network delivers high-quality reconstructions at video-rate throughput and we demonstrate the capabilities of our approach by imaging medaka heart dynamics and zebrafish neural activity

Tracking the Molecular Evolution of Calcium Permeability in a Nicotinic Acetylcholine Receptor

Nicotinic acetylcholine receptors are a family of ligand-gated nonselective cationic channels that participate in fundamental physiological processes at both the central and the peripheral nervous system. The extent of calcium entry through ligand-gated ion channels defines their distinct functions. The α9α10 nicotinic cholinergic receptor, expressed in cochlear hair cells, is a peculiar member of the family as it shows differences in the extent of calcium permeability across species. In particular, mammalian α9α10 receptors are among the ligand-gated ion channels which exhibit the highest calcium selectivity. This acquired differential property provides the unique opportunity of studying how protein function was shaped along evolutionary history, by tracking its evolutionary record and experimentally defining the amino acid changes involved. We have applied a molecular evolution approach of ancestral sequence reconstruction, together with molecular dynamics simulations and an evolutionary-based mutagenesis strategy, in order to trace the molecular events that yielded a high calcium permeable nicotinic α9α10 mammalian receptor. Only three specific amino acid substitutions in the α9 subunit were directly involved. These are located at the extracellular vestibule and at the exit of the channel pore and not at the transmembrane region 2 of the protein as previously thought. Moreover, we show that these three critical substitutions only increase calcium permeability in the context of the mammalian but not the avian receptor, stressing the relevance of overall protein structure on defining functional properties. These results highlight the importance of tracking evolutionarily acquired changes in protein sequence underlying fundamental functional properties of ligand-gated ion channels.Fil: Lipovsek, Maria Marcela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular ; ArgentinaFil: Fierro, Angélica. Pontificia Universidad Católica de Chile; ChileFil: Perez, Edwin G.. Pontificia Universidad Católica de Chile; ChileFil: Boffi, Juan Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular ; ArgentinaFil: Millar, Neil S.. University College London; Estados UnidosFil: Fuchs, Paul A.. University Johns Hopkins; Estados UnidosFil: Katz, Eleonora. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular ; ArgentinaFil: Elgoyhen, Ana Belen. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Farmacología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular ; Argentin