PT and aPTT cannot be interchangeably used with ROTEM(®) CT. Based on the results of ROTEM(®), recommended thresholds for PT and aPTT might overestimate the need for coagulation therapy. A good correlation was found between the fibrinogen level and the FibTEM assay. In addition, ROTEM(®) offered faster turnaround times.
Mitochondria from transgenic mice, expressing enzymatically active mitochondrial creatine kinase in liver, were analyzed for opening of the permeability transition pore in the absence and presence of creatine kinase substrates but with no external adenine nucleotides added. In mitochondria from these transgenic mice, cyclosporin A-inhibited pore opening was delayed by creatine or cyclocreatine but not by -guanidinopropionic acid. This observation correlated with the ability of these substrates to stimulate state 3 respiration in the presence of extramitochondrial ATP. The dependence of transition pore opening on calcium and magnesium concentration was studied in the presence and absence of creatine. If mitochondrial creatine kinase activity decreased (i.e. by omitting magnesium from the medium), protection of permeability transition pore opening by creatine or cyclocreatine was no longer seen. Likewise, when creatine kinase was added externally to liver mitochondria from wild-type mice that do not express mitochondrial creatine kinase in liver, no protective effect on pore opening by creatine and its analog was observed. All these findings indicate that mitochondrial creatine kinase activity located within the intermembrane and intercristae space, in conjunction with its tight functional coupling to oxidative phosphorylation, via the adenine nucleotide translocase, can modulate mitochondrial permeability transition in the presence of creatine. These results are of relevance for the design of creatine analogs for cell protection as potential adjuvant therapeutic tools against neurodegenerative diseases.
Nucleoside diphosphate kinases (NDPK), 3 encoded by NME genes (also called NM23), catalyze the exchange of ␥-phosphate between di-and triphosphonucleosides and participate in the regulation of intracellular nucleotide homeostasis. They mainly utilize ATP formed by oxidative phosphorylation to synthesize the other triphosphonucleosides, in particular GTP (1). Given the poor substrate selectivity of NDPKs, it is assumed that specificity could arise from the presence of different isoforms at different subcellular localizations. Associated in networks with other nucleotide-metabolizing enzymes such as adenylate kinases, creatine kinases, and glycolytic enzymes, NDPKs participate in high energy phosphoryl transfer and signal communication in the cell (2). Up to now nine genes encoding NDPK or NDPK-like proteins have been identified (3, 4), but little is known about their respective role within the cell. The most studied, NDPK-A and -B, encoded by NME1 and NME2 genes, respectively, play a key role in tumor progression and metastasis dissemination (5, 6).NDPK activity has been found associated with different cellular compartments, such as cytosol, nucleus, plasma membrane, and mitochondria. Precise localization in the latter organelles has been a matter of debate. Depending on species and tissue examined, NDPK activity was reported in both the matrix and the intermembrane/cristae space (7), including the so-called contact sites between inner and outer membrane (8 -10). In mammalian liver (rat and rabbit), the NDPK activity was mainly associated with an extra-matrix compartment, probably the intermembrane/cristae space, whereas in heart activity was more abundant in the matrix (11). For mitochondrial NDPK in matrix, many functions have been proposed ranging from nucleotide supply for mitochondrial nucleic acid and protein synthesis to functional interaction with the Krebs * This work was supported by the Germaine de Stael Program for FrancoSwiss collaboration (to U. S. and M.-L. L.), the Agence Nationale de la Recherche (Chaire d'Excellence (to U. S.)), the Marie Curie Intraeuropean Fellowship of the European Community (to M. T.-S.), INSERM, and grants from the Groupement des Entreprises Françaises contre le Cancer and from the Association pour la Recherche contre le Cancer (to M.-L. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The presence of N-methyl-D-aspartate receptor (NMDAR) was previously shown in rat red blood cells (RBCs) and in a UT-7/Epo human myeloid cell line differentiating into erythroid lineage. Here we have characterized the subunit composition of the NMDAR and monitored its function during human erythropoiesis and in circulating RBCs. Expression of the NMDARs subunits was assessed in erythroid progenitors during ex vivo erythropoiesis and in circulating human RBCs using quantitative PCR and flow cytometry. Receptor activity was monitored using a radiolabeled antagonist binding assay, live imaging of Ca 2ϩ uptake, patch clamp, and monitoring of cell volume changes. The receptor tetramers in erythroid precursor cells are composed of the NR1, NR2A, 2C, 2D, NR3A, and 3B subunits of which the glycine-binding NR3A and 3B and glutamate-binding NR2C and 2D subunits prevailed. Functional receptor is required for survival of erythroid precursors. Circulating RBCs retain a low number of the receptor copies that is higher in young cells compared with mature and senescent RBC populations. In circulating RBCs the receptor activity is controlled by plasma glutamate and glycine. Modulation of the NMDAR activity in RBCs by agonists or antagonists is associated with the alterations in whole cell ion currents. Activation of the receptor results in the transient Ca 2ϩ accumulation, cell shrinkage, and alteration in the intracellular pH, which is associated with the change in hemoglobin oxygen affinity. Thus functional NMDARs are present in erythroid precursor cells and in circulating RBCs. These receptors contribute to intracellular Ca 2ϩ homeostasis and modulate oxygen delivery to peripheral tissues.
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