Browsing by Subject "GREEN FLUORESCENT PROTEIN"

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  • Ludwig, Anastasia; Rivera Baeza, Claudio; Uvarov, Pavel (2017)
    Background: Cation-chloride cotransporters (CCCs) are indispensable for maintaining chloride homeostasis in multiple cell types, but K-Cl cotransporter KCC2 is the only CCC member with an exclusively neuronal expression in mammals. KCC2 is critical for rendering fast hyperpolarizing responses of ionotropic.-aminobutyric acid and glycine receptors in adult neurons, for neuronal migration in the developing central nervous system, and for the formation and maintenance of small dendritic protrusions-dendritic spines. Deficit in KCC2 expression and/or activity is associated with epilepsy and neuropathic pain, and effective strategies are required to search for novel drugs augmenting KCC2 function. Results: We revised current methods to develop a noninvasive optical approach for assessing KCC2 transport activity using a previously characterized genetically encoded chloride sensor. Our protocol directly assesses dynamics of KCC2-mediated chloride efflux and allows measuring genuine KCC2 activity with good spatial and temporal resolution. As a proof of concept, we used this approach to compare transport activities of the two known KCC2 splice isoforms, KCC2a and KCC2b, in mouse neuronal Neuro-2a cells. Conclusions: Our noninvasive optical protocol proved to be efficient for assessment of furosemide-sensitive chloride fluxes. Transport activities of the N-terminal splice isoforms KCC2a and KCC2b obtained by the novel approach matched to those reported previously using standard methods for measuring chloride fluxes.
  • Medina, Igor; Friedel, Perrine; Rivera, Claudio; Kahle, Kristopher T.; Kourdougli, Nazim; Uvarov, Pavel; Pellegrino, Christophe (2014)
  • Leppä, Elli; Linden, Anni-Maija; Vekovischeva, Olga Y.; Swinny, Jerome D.; Rantanen, Ville; Toppila, Esko; Hoeger, Harald; Sieghart, Werner; Wulff, Peer; Wisden, William; Korpi, Esa R. (2011)
  • Stepanenko, Olesya V.; Stepanenko, Olga V.; Kuznetsova, Irina M.; Verkhusha, Vladislav V.; Turoverov, Konstantin K. (2014)
  • Vannini, Nicola; Campos, Vasco; Girotra, Mukul; Trachsel, Vincent; Rojas-Sutterlin, Shanti; Tratwal, Josefine; Ragusa, Simone; Stefanidis, Evangelos; Ryu, Dongryeol; Rainer, Pernille Y.; Nikitin, Gena; Giger, Sonja; Li, Terytty Y.; Semilietof, Aikaterini; Oggier, Aurelien; Yersin, Yannick; Tauzin, Loic; Pirinen, Eija; Cheng, Wan-Chen; Ratajczak, Joanna; Canto, Carles; Ehrbar, Martin; Sizzano, Federico; Petrova, Tatiana V.; Vanhecke, Dominique; Zhang, Lianjun; Romero, Pedro; Nahimana, Aimable; Cherix, Stephane; Duchosal, Michel A.; Ho, Ping-Chih; Deplancke, Bart; Coukos, George; Auwerx, Johan; Lutolf, Matthias P.; Naveiras, Olaia (2019)
    It has been recently shown that increased oxidative phosphorylation, as reflected by increased mitochondrial activity, together with impairment of the mitochondrial stress response, can severely compromise hematopoietic stem cell (HSC) regeneration. Here we show that the NAD(+)-boosting agent nicotinamide riboside (NR) reduces mitochondrial activity within HSCs through increased mitochondrial clearance, leading to increased asymmetric HSC divisions. NR dietary supplementation results in a significantly enlarged pool of progenitors, without concurrent HSC exhaustion, improves survival by 80%, and accelerates blood recovery after murine lethal irradiation and limiting-HSC transplantation. In immune-deficient mice, NR increased the production of human leucocytes from hCD34+ progenitors. Our work demonstrates for the first time a positive effect of NAD(+)-boosting strategies on the most primitive blood stem cells, establishing a link between HSC mitochondrial stress, mitophagy, and stem-cell fate decision, and unveiling the potential of NR to improve recovery of patients suffering from hematological failure including post chemo- and radiotherapy.