Bone morphogenetic protein-2 (BMP-2) and transforming growth factor-β1 (TGF-β1) alter connexin 43 phosphorylation in MC3T3-E1 Cells

BACKGROUND: Bone morphogenetic proteins (BMPs) and transforming growth factor-βs (TGF-βs) are important regulators of bone repair and regeneration. BMP-2 and TGF-β1 have been shown to inhibit gap junctional intercellular communication (GJIC) in MC3T3-E1 cells. Connexin 43 (Cx43) has been shown to mediate GJIC in osteoblasts and it is the predominant gap junctional protein expressed in these murine osteoblast-like cells. We examined the expression, phosphorylation, and subcellular localization of Cx43 after treatment with BMP-2 or TGF-β1 to investigate a possible mechanism for the inhibition of GJIC. RESULTS: Northern blot analysis revealed no detectable change in the expression of Cx43 mRNA. Western blot analysis demonstrated no significant change in the expression of total Cx43 protein. However, significantly higher ratios of unphosphorylated vs. phosphorylated forms of Cx43 were detected after BMP-2 or TGF-β1 treatment. Immunofluorescence and cell protein fractionation revealed no detectable change in the localization of Cx43 between the cytosol and plasma membrane. CONCLUSIONS: BMP-2 and TGF-β1 do not alter expression of Cx43 at the mRNA or protein level. BMP-2 and TGF-β1 may inhibit GJIC by decreasing the phosphorylated form of Cx43 in MC3T3-E1 cells

Cell Cycle Phase-Specific Surface Expression of Nerve Growth Factor Receptors TrkA and p75NTR

[EN]Expression of the nerve growth factor (NGF) receptors TrkA and p75NTR was found to vary at the surface of PC12 cells in a cell cycle phase-specific manner. This was evidenced by using flow cytometric and microscopic analysis of cell populations labeled with antibodies to the extracellular domains of both receptors. Differential expression of these receptors also was evidenced by biotinylation of surface proteins and Western analysis, using antibodies specific for the extracellular domains of TrkA and p75NTR. TrkA is expressed most strongly at the cell surface in M and early G1 phases, whereas p75NTR is expressed mainly in late G1, S, and G2 phases. This expression reflects the molecular and cellular responses to NGF in specific phases of the cell cycle; in the G1 phase NGF elicits both the anti-mitogenic effect, i.e., inhibition of the G1 to S transition, and the differentiation response whereas a survival effect is provoked elsewhere in the cell cycle. A model is proposed relating these responses to the surface expression of the two receptors. These observations open the way for novel approaches to the investigation of the mechanism of NGF signal transduction

EXACT2: the semantics of biomedical protocols

© 2014 Soldatova et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.This article has been made available through the Brunel Open Access Publishing Fund.Background: The reliability and reproducibility of experimental procedures is a cornerstone of scientific practice. There is a pressing technological need for the better representation of biomedical protocols to enable other agents (human or machine) to better reproduce results. A framework that ensures that all information required for the replication of experimental protocols is essential to achieve reproducibility. Methods: We have developed the ontology EXACT2 (EXperimental ACTions) that is designed to capture the full semantics of biomedical protocols required for their reproducibility. To construct EXACT2 we manually inspected hundreds of published and commercial biomedical protocols from several areas of biomedicine. After establishing a clear pattern for extracting the required information we utilized text-mining tools to translate the protocols into a machine amenable format. We have verified the utility of EXACT2 through the successful processing of previously ‘unseen’ (not used for the construction of EXACT2) protocols. Results: The paper reports on a fundamentally new version EXACT2 that supports the semantically-defined representation of biomedical protocols. The ability of EXACT2 to capture the semantics of biomedical procedures was verified through a text mining use case. In this EXACT2 is used as a reference model for text mining tools to identify terms pertinent to experimental actions, and their properties, in biomedical protocols expressed in natural language. An EXACT2-based framework for the translation of biomedical protocols to a machine amenable format is proposed. Conclusions: The EXACT2 ontology is sufficient to record, in a machine processable form, the essential information about biomedical protocols. EXACT2 defines explicit semantics of experimental actions, and can be used by various computer applications. It can serve as a reference model for for the translation of biomedical protocols in natural language into a semantically-defined format.This work has been partially funded by the Brunel University BRIEF award and a grant from Occams Resources

A critical role for Kalirin in NGF signaling through TrkA

Kalirin is a multidomain guanine nucleotide exchange factor (GEF) that activates Rho proteins, inducing cytoskeletal rearrangement in neurons. Although much is known about the effects of Kalirin on Rho GTPases and neuronal morphology, little is known about the association of Kalirin with the receptor/signaling systems that affect neuronal morphology. Our experiments demonstrate that Kalirin binds to and colocalizes with the TrkA neurotrophin receptor in neurons. In PC12 cells, inhibition of Kalirin expression using antisense RNA decreased nerve growth factor (NGF)-induced TrkA autophosphorylation and process extension. Kalirin overexpression potentiated neurotrophin-stimulated TrkA autophosphorylation and neurite outgrowth in PC12 cells at a low concentration of NGF. Furthermore, elevated Kalirin expression resulted in catalytic activation of TrkA, as demonstrated by in vitro kinase assays and increased NGF-stimulated cellular activation of Rac, Mek, and CREB. Domain mapping demonstrated that the N-terminal Kalirin pleckstrin homology domain mediates the interaction with TrkA. The effects of Kalirin on TrkA provide a molecular basis for the requirement of Kalirin in process extension from PC12 cells and for previously observed effects on axonal extension and dendritic maintenance. The interaction of TrkA with the pleckstrin homology domain of Kalirin may be one example of a general mechanism whereby receptor/Rho GEF pairings play an important role in receptor tyrosine kinase activation and signal transduction

The Toronto prehospital hypertonic resuscitation-head injury and multi organ dysfunction trial (TOPHR HIT) - Methods and data collection tools

<p>Abstract</p> <p>Background</p> <p>Clinical trials evaluating the use of hypertonic saline in the treatment of hypovolemia and head trauma suggest no survival superiority over normal saline; however subgroup analyses suggest there may be a reduction in the inflammatory response and multiorgan failure which may lead to better survival and enhanced neurocognitive function. We describe a feasibility study of randomizing head injured patients to hypertonic saline and dextran vs. normal saline administration in the out of hospital setting.</p> <p>Methods/Design</p> <p>This feasibility study employs a randomized, placebo-controlled design evaluating normal saline compared with a single dose of 250 ml of 7.5% hypertonic saline in 6% dextran 70 in the management of traumatic brain injuries. The primary feasibility endpoints of the trial were: 1) baseline survival rates for the treatment and control group to aid in the design of a definitive multicentre trial, 2) randomization compliance rate, 3) ease of protocol implementation in the out-of-hospital setting, and 4) adverse event rate of HSD infusion.</p> <p>The secondary objectives include measuring the effect of HSD in modulating the immuno-inflammatory response to severe head injury and its effect on modulating the release of neuro-biomarkers into serum; evaluating the role of serum neuro-biomarkers in predicting patient outcome and clinical response to HSD intervention; evaluating effects of HSD on brain atrophy post-injury and neurocognitive and neuropsychological outcomes.</p> <p>Discussion</p> <p>We anticipate three aspects of the trial will present challenges to trial success; ethical demands associated with a waiver of consent trial, challenging follow up and comprehensive accurate timely data collection of patient identifiers and clinical or laboratory values. In addition all the data collection tools had to be derived de novo as none existed in the literature.</p> <p>Trial registration number</p> <p>NCT00878631</p

Activation directe de la calcineurine A endogène (impact biologique d aptamères peptidiques sélectifs)

Des approches thérapeutiques visant à la stimulation de la régénération et/ou à l inhibition des processus de dégénérescence neuromusculaire pourraient constituer des stratégies efficaces pour préserver le tonus musculaire des patients et augmenter ainsi leur espérance de vie. L activation de la Calcineurine A (CnA), une phosphatase des sérines et thréonines, contrôle une large gamme de réseaux régulateurs dans le muscle squelettique, notamment en stimulant l expression de gènes spécifiques des fibres musculaires lentes (de type I). La CnA est considérée comme un acteur clé de la réponse hypertrophique et du processus de régénération dans le muscle squelettique. L activation de la CnA est ainsi considérée comme une stratégie potentielle pour stimuler la régénération musculaire dans les cas de myopathie. Nous avons identifié un aptamère peptidique qui active la CNA in vitro et in vivo. Dans un modèle murin d atrophie musculaire induite par dénervation, l aptamère a montré de significatives capacités thérapeutiques. L effet curatif de l aptamère a notamment été observable par une augmentation générale de la surface des muscles traités, mais aussi par un accroissement de la surface individuelle des fibres musculaires.Une augmentation du niveau de NFAT nucléaire dans ces fibres a été observée, en cohérence avec les capacités d activation de la CnA par notre aptamère. Par ailleurs, une autre observation faite dans les muscles traités avec l aptamère a été l augmentation de noyaux centraux, caractéristiques de la présence de nouvelles fibres. Finalement, l identification du site d interaction entre la CnA et notre aptamère, permise par l utilisation de plusieurs formes tronquées de la phosphatase, a offert un aperçu du mécanisme d action de l aptamère à l échelle moléculaire. Dans l ensemble, les études présentées ici ont offert la première démonstration qu une activation directe de la CnA endogène a un impact significatif sur les processus cellulaires, résultant en la stimulation de la régénération musculaire et l amélioration de l état physiopathologique chez les modèles animaux utilisés.Therapeutic approaches leading to the stimulation of regeneration, and/or inhibition of degeneration processes in neuromuscular disorders are believed to offer valid therapeutic strategies that would preserve muscle tone and contribute to the quality of life while lengthening patient life span. Activation of CalcineurinA (CnA), a threonine-serine phosphatase, controls gene regulatory programs in skeletal muscle by stimulating slow muscle fiber (type I) gene expression. This phosphatase has been also identified as a key mediator in the hypertrophic response and in skeletal muscle regeneration. Activation of CnA is, therefore, considered as a potentially interesting means of stimulating muscle regeneration in myopathies. We have identified a peptide aptamer that activates CnA in vitro, in cells and in vivo. In a mouse model for denervation-induced muscle atrophy, CnA-activating peptide aptamers show significant positive impact. This is reflected in larger overall muscle cross-sectional surface area due to an increased number of fibers and larger individual fiber surface area. Insight into the biological mechanism is afforded by observation of increased levels of nuclear NFAT transcription factor in these fibers, in agreement with peptide aptamer-mediated activation of CnA. Furthermore, a significant increase in central nuclei, characteristic of the presence of new fibers, is observed in muscles treated with the peptide aptamers specifically activating CnA. Identification of the specific binding site of the peptide aptamer on CnA was achieved using several truncations of the phosphatase, offering insight into the molecular mechanism of action. Together, these studies offer the first proof that direct activation of endogenous CnA has a measureable impact on cellular responses resulting in stimulation of muscle regeneration and enhancement of pathophysiological state in selected animal models.LYON-ENS Sciences (693872304) / SudocSudocFranceF

Caractérisation d aptamères peptidiques suppresseurs et de leur(s) cible(s) dans le contexte de la mort cellulaire Bax-dependante

Les aptamères peptidiques sont des protéines combinatoires capables de moduler spécifiquement une fonction de leur cible. Une sélection fonctionelle d aptamères peptidiques capables d inhiber la mort cellulaire Bax-dependante chez la levure et en cellules mammaifères a été effectuée. Deux aptamères peptidiques ont été sélectionnés (Apta-32 et Apta-34). L objectif de ce travail de thèse a été de caractériser ces deux aptamères peptidiques et leur(s) cible(s) dans le contexte de la mort cellulaire Bax-dependante. La première partie est l étude de l Apta-34 qui cible une protéine (C34) contenant un domaine de mort et ayant des fonctions pro-apoptotiques. Nous avons montré que lors de l induction de l apoptose, C34 est transloquée du noyau (sa localisation principale) au cytoplasme. Dans les mêmes conditions, Apta-34 co-localise avec C34 dans le noyau, empêchant, ou du moins retardant, sa sortie du noyau. De plus nous avons identifié le site de liaison d Apta-34 sur C34, qui est localisé dans les 215 amino acides en N-terminale de la protéine, une région qui contient un site prédictif d export nucléaire. Finalement, nous avons montré que la délétion de l homologue de C34 protège contre la mort induite par hBax en levure. La seconde partie est l étude d Apta-32 qui cible deux paralogues (C32a et b) d une famille de protéine impliquée dans le traffic membranaire dans les voies de l endocytose. Nous avons montré qu Apta-32 se lie à un domaine fonctionnel de C32. Des études in silico de docking ont permis d identifier trois sites distincts de liaison d Apta-32 sur ce domaine. Le site dominant est composé d acides aminés qui partagent des propriétés physico-chimiques communes entre les différents interacteurs d Apta-32 (C32a, C32b et l homologue levure) mais pas avec des homologues qui ne lient pas Apta-32. De plus un screening double hybride d une banque de cDNA levure a permis d identifier des cibles mevure d Apta-32. Finalement, des études préliminaires chez l embryons de drosophile, permettent de suggérer que l expression d Apta-32 peut entraîner un défaut de la phagocytose. Cette étude a permis d identifier des régulateurs de la mort cellulaires impliqués dans deux processus cellulaires distincts.Peptide aptamers are small combinatorial proteins able to specifically modulate a function of their target. A functional selection of peptide aptamers able to inhibit Bax-dependent cell death in yeast and mammalian systems has been performed. Two peptide aptamers have been selected (Apta-32 and Apta-34). The aim of this thesis project was to characterize those two inhibitory peptide aptamers and their targets in order to understand their function in the Bax-dependent cell death. The first part focuses on Apta-34 that targets a Death Domain-containing protein (T34) that has pro-apoptotic functions. We showed that during the induction of apoptosis T34 translocates from nucleus (its major localization site) to the cytoplasm. In the same conditions, Apta-34 co-localizes with T34 in the nucleus, inhibiting or at least delaying its exit from the nucleus. Moreover we identified that Apta-34 binds to the well conserved 215 N-terminal amino acids of T34 that contains a putative Nuclear Export Signal. Finally we showed that the deletion of its homologue prevents hBax-induced cell death in yeast. The second part focuses on Apta-32 that targets two paralogues (T32a and b) of a family of proteins involved in the endocytotic membrane trafficking. We showed that Apta-32 is binding to a functional domain of T32. By in silico docking studies we identified 3 distinct binding sites of Apta-32 on this domain. The dominant binding site is composed by amino acid that share physico-chemical properties between binders of Apta-32 (T32a, T32b and a yeast homologue) but not with homologues that do not bind Apta-32. Moreover we identified yeast targets of Apta-32 by yeast two hybrid yeast cDNA library screening. Finally preliminary observations on drosophila embryos expressing Apta-32 suggest that Apta-32 expression could lead to a defect on phagocytosis. This study leads to the identification of regulators of the cell death acting on two distinct pathways.LYON-ENS Sciences (693872304) / SudocSudocFranceF

Expression et devenir des récepteurs au Nerve Growth Factor (NGF), TrkA et p75NTR

LYON-ENS Sciences (693872304) / SudocSudocFranceF

Modulation de la signalisation du Nerve Growth Factor par les Cavéolines 1 et 2

Le Nerve Growth Factor (NGF) est une neurotrophine qui conditionne la survie et la différenciation de certaines populations de neurones. La transduction du signal du NGF repose sur deux récepteurs transmenbranaires : le récepteur p75NTR, qui fixe toutes les neutrophines et le récepteur tyrosine kinase TrkA, spécifique du NGF. L'étude de la localisation précise de ces récepteurs montre qu'ils sont localisés dans des spécialisations structurales et biochimiques de la membrane plasmique enrichies en cholestérol et en glycosphingolipides, les microdomaines membranaires (rafts et cavéoles). Les cavéolines 1 et 2 sont des constituants des cavéoles, impliquées dans la régulation du trafic membranaire et dans la transduction du signal. Les cavéolines 1 et 2 exercent une modulation différentielle sur la signalisation du NGF. L'expression de cavéoline 1 inhibe l'arrêt de prolifération et la différenciation des cellules PC12 exposées au NGF. A l'inverse, l'expression de cavéoline 2 exerce un effet potentiateur sur la différenciation induite par le NGF et sur la survie cellulaire en présence de NGF. La cavéoline 1 est co-localisée avec TrkA à la surface cellulaire et dans l'endosome. Son effet inhibiteur sur la signalisation du NGF semble reposer d'une part sur une modification des paramètres de trafic cellulaire de TrkA, vraisemblablement en interférant avec la sortie des microdomaines membranaires et avec l'internalisation et, d'autre part, sur l'inhibition intracellulaire des effecteurs de TrkA, se traduisant par l'inhibition de la phosphorylation du facteur de transcription CREB et la non-induction de l'inhibiteur de cycle cellulaire p21wafI/CIPI. Les mécanismes de l'effet potentiateur de la cavéoline 2 sont en revanche moins évidents puisque la cavéoline 2 est restreinte à l'appareil de Golgi et ne se co-localise pas avec TrkA. Son effet potentiateur suggère l'existence d'un mécanisme de régulation négative de TrkA qui serait lui-m^eme inhibé par la cavéoline 2.LYON-ENS Sciences (693872304) / SudocSudocFranceF