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Docobo-Perez F.,University of Seville | Docobo-Perez F.,A+ Network | Docobo-Perez F.,Campus Hospital Universitario Virgen Del Rocio | Nordmann P.,University Paris - Sud | And 9 more authors.
International Journal of Antimicrobial Agents | Year: 2012

New Delhi metallo-β-lactamase-1 (NDM-1)-producing Enterobacteriaceae have emerged as a global threat. The aim of this study was to assess the efficacies of colistin and tigecycline in an experimental model of pneumonia caused by NDM-1-producing Escherichia coli and Klebsiella pneumoniae. The susceptibilities of K. pneumoniae NDM, E. coli NDM and K. pneumoniae ATCC 29665 were determined using the broth microdilution technique. The pharmacokinetics of colistin and tigecycline in an experimental model of pneumonia were performed using immunocompetent C57BL/6 mice. Mice were treated with colistin (60 mg/kg/day) or tigecycline (10 mg/kg/day). Mortality, bacteraemia and lung bacterial concentrations were recorded. The strains were susceptible to colistin and tigecycline. The ratio of area under the concentration-time curve/minimum inhibitory concentration (AUC/MIC) for colistin was 158.5 (all three strains) and that for tigecycline was 18.5 (K. pneumoniae NDM) and 37 (K. pneumoniae ATCC 29665 and E. coli NDM). In vivo, colistin decreased bacterial lung concentrations of K. pneumoniae NDM and K. pneumoniae ATCC 29665 by 1.16 log colony-forming units (CFU)/g and 2.23 log CFU/g, respectively, compared with controls (not significant). Tigecycline reduced K. pneumoniae NDM and K. pneumoniae ATCC 29665 load by 2.67 log CFU/g and 4.62 log CFU/g (P < 0.05). Colistin and tigecycline decreased lung concentrations of E. coli NDM by 2.27 log CFU/g and 4.15 log CFU/g (P < 0.05), respectively, compared with controls, and was more active than colistin (P < 0.05). In conclusion, these results suggest that colistin is inappropriate for treating pneumonia due to NDM-1-producing K. pneumoniae and its efficacy was suboptimal against NDM-1-producing E. coli. A high tigecycline dose was efficacious for treating experimental pneumonia due to NDM-1-producing E. coli and K. pneumoniae. © 2011 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. Source

Fernandez-Tenorio M.,Hospital Universitario Virgen Del Rocio | Porras-Gonzalez C.,Hospital Universitario Virgen Del Rocio | Castellano A.,Hospital Universitario Virgen Del Rocio | Del Valle-Rodriguez A.,New York University | And 3 more authors.
Circulation Research | Year: 2011

Background: Sustained vascular smooth muscle contraction is mediated by extracellular Ca2+ influx through L-type voltage-gated Ca 2+ channels (VGCC) and RhoA/Rho-associated kinase (ROCK)-dependent Ca2+ sensitization of the contractile machinery. VGCC activation can also trigger an ion-independent metabotropic pathway that involves G-protein/phospholipase C activation, inositol 1,4,5-trisphosphate synthesis, and Ca2+ release from the sarcoplasmic reticulum (calcium channel-induced Ca release). We have studied the functional role of calcium channel-induced Ca release and the inter-relations between Ca2+ channel and RhoA/ROCK activation. Methods and results: We have used normal and genetically modified animals to study single myocyte electrophysiology and fluorimetry as well as cytosolic Ca2+ and diameter in intact arteries. These analyses were complemented with measurement of tension and RhoA activity in normal and reversibly permeabilized arterial rings. We have found that, unexpectedly, L-type Ca channel activation and subsequent metabotropic Ca release from sarcoplasmic reticulum participate in depolarization-evoked RhoA/ROCK activity and sustained arterial contraction. We show that these phenomena do not depend on the change in the membrane potential itself, or the mere release of Ca from the sarcoplasmic reticulum, but they require the simultaneous activation of VGCC and the downstream metabotropic pathway with concomitant Ca2+ release. During protracted depolarizations, refilling of the stores by a residual extracellular Ca2+ influx through VGCC helps maintaining RhoA activity and sustained arterial contraction. Conclusions: These findings reveal that calcium channel-induced Ca release has a major role in tonic vascular smooth muscle contractility because it links membrane depolarization and Ca channel activation with metabotropic Ca release and sensitization (RhoA/ROCK stimulation). © 2011 American Heart Association, Inc. Source

Basilico F.,Max Planck Institute of Molecular Physiology | Basilico F.,Italian National Cancer Institute | Maffini S.,Max Planck Institute of Molecular Physiology | Weir J.R.,Max Planck Institute of Molecular Physiology | And 17 more authors.
eLife | Year: 2014

Kinetochores, multi-subunit complexes that assemble at the interface with centromeres, bind spindle microtubules to ensure faithful delivery of chromosomes during cell division. The configuration and function of the kinetochore-centromere interface is poorly understood. We report that a protein at this interface, CENP-M, is structurally and evolutionarily related to small GTPases but is incapable of GTP-binding and conformational switching. We show that CENP-M is crucially required for the assembly and stability of a tetramer also comprising CENP-I, CENP-H, and CENP-K, the HIKM complex, which we extensively characterize through a combination of structural, biochemical, and cell biological approaches. A point mutant affecting the CENP-M/CENP-I interaction hampers kinetochore assembly and chromosome alignment and prevents kinetochore recruitment of the CENP-T/W complex, questioning a role of CENP-T/W as founder of an independent axis of kinetochore assembly. Our studies identify a single pathway having CENP-C as founder, and CENP-H/I/K/M and CENP-T/W as CENP-C-dependent followers. Source

Fernandez-Aguera M.C.,University of Seville | Fernandez-Aguera M.C.,Campus Hospital Universitario Virgen Del Rocio | Gao L.,University of Seville | Gao L.,Campus Hospital Universitario Virgen Del Rocio | And 13 more authors.
Cell Metabolism | Year: 2015

O2 sensing is essential for mammalian homeostasis. Peripheral chemoreceptors such as the carotid body (CB) contain cells with O2-sensitive K+ channels, which are inhibited by hypoxia to trigger fast adaptive cardiorespiratory reflexes. How variations of O2 tension (PO2) are detected and the mechanisms whereby these changes are conveyed to membrane ion channels have remained elusive. We have studied acute O2 sensing in conditional knockout mice lacking mitochondrial complex I (MCI) genes. We inactivated Ndufs2, which encodes a protein that participates in ubiquinone binding. Ndufs2-null mice lose the hyperventilatory response to hypoxia, although they respond to hypercapnia. Ndufs2-deficient CB cells have normal functions and ATP content but are insensitive to changes in PO2. Our data suggest that chemoreceptor cells have a specialized succinate-dependent metabolism that induces an MCI state during hypoxia, characterized by the production of reactive oxygen species and accumulation of reduced pyridine nucleotides, which signal neighboring K+ channels. © 2015 Elsevier Inc. Source

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