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      Apparent Km of mitochondria for oxygen computed from Vmax measured in permeabilized muscle fibers is lower in water enriched in oxygen by electrolysis than injection

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          It has been suggested that oxygen (O 2) diffusion could be favored in water enriched in O 2 by a new electrolytic process because of O 2 trapping in water superstructures (clathrates), which could reduce the local pressure/content relationships for O 2 and facilitate O 2 diffusion along PO 2 gradients.

          Materials and methods

          Mitochondrial respiration was compared in situ in saponin-skinned fibers isolated from the soleus muscles of Wistar rats, in solution enriched in O 2 by injection or the electrolytic process 1) at an O 2 concentration decreasing from 240 µmol/L to 10 µmol/L (132 mmHg to 5 mmHg), with glutamate–malate or N, N, N′, N′-tetramethyl- p-phenylenediamine dihydrochloride (TMPD)–ascorbate (with antimycin A) as substrates; and 2) at increasing adenosine diphosphate (ADP) concentration with glutamate–malate as substrate.


          As expected, maximal respiration decreased with O 2 concentration and, when compared to glutamate–malate, the apparent Km O 2 of mitochondria for O 2 was significantly lower with TMPD–ascorbate with both waters. However, when compared to the water enriched in O 2 by injection, the Km O 2 was significantly lower with both electron donors in water enriched in O 2 by electrolysis. This was not associated with any increase in the sensitivity of mitochondria to ADP; no significant difference was observed for the Km ADP between the two waters.


          In this experiment, a higher affinity of the mitochondria for O 2 was observed in water enriched in O 2 by electrolysis than by injection. This observation is consistent with the hypothesis that O 2 diffusion can be facilitated in water enriched in O 2 by the electrolytic process.

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          Most cited references 25

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          Suppression of nuclear factor-κB activation and inflammation in microglia by physically modified saline.

          Chronic inflammation involving activated microglia and astroglia is becoming a hallmark of many human diseases, including neurodegenerative disorders. Although NF-κB is a multifunctional transcription factor, it is an important target for controlling inflammation as the transcription of many proinflammatory molecules depends on the activation of NF-κB. Here, we have undertaken a novel approach to attenuate NF-κB activation and associated inflammation in activated glial cells. RNS60 is a 0.9% saline solution containing charge-stabilized nanostructures that are generated by subjecting normal saline to Taylor-Couette-Poiseuille (TCP) flow under elevated oxygen pressure. RNS60, but not normal saline, RNS10.3 (TCP-modified saline without excess oxygen), and PNS60 (saline containing excess oxygen without TCP modification) were found to inhibit the production of nitric oxide (NO) and the expression of inducible NO synthase in activated microglia. Similarly, RNS60 also inhibited the expression of inducible NO synthase in activated astroglia. Inhibition of NF-κB activation by RNS60 suggests that RNS60 exerts its anti-inflammatory effect through the inhibition of NF-κB. Interestingly, RNS60 induced the activation of type IA phosphatidylinositol (PI) 3-kinase and Akt and rapidly up-regulated IκBα, a specific endogenous inhibitor of NF-κB. Inhibition of PI 3-kinase and Akt by either chemical inhibitors or dominant-negative mutants abrogated the RNS60-mediated up-regulation of IκBα. Furthermore, we demonstrate that RNS60 induced the activation of cAMP-response element-binding protein (CREB) via the PI 3-kinase-Akt pathway and that RNS60 up-regulated IκBα via CREB. These results describe a novel anti-inflammatory property of RNS60 via type IA PI 3-kinase-Akt-CREB-mediated up-regulation of IκBα, which may be of therapeutic benefit in neurodegenerative disorders.
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            Permeabilized cell and skinned fiber techniques in studies of mitochondrial function in vivo.

            In this chapter we describe in details the permeabilized cell and skinned fiber techniques and their applications for studies of mitochondrial function in vivo. The experience of more than 10 years of research in four countries is summarized. The use of saponin in very low concentration (50-100 microg/ml) for permeabilisation of the sarcolemma leaves all intracellular structures, including mitochondria, completely intact. The intactness of mitochondrial function in these skinned muscle fibers is demonstrated in this work by multiple methods, such as NADH and flavoprotein fluorescence studies, fluorescence imaging, confocal immunofluorescence microscopy and respiratory analysis. Permeabilized cell and skinned fiber techniques have several very significant advantages for studies of mitochondrial function, in comparison with the traditional methods of use of isolated mitochondria: (1) very small tissue samples are required; (2) all cellular population of mitochondria can be investigated; (3) most important, however, is that mitochondria are studied in their natural surrounding. The results of research by using this method show the existence of several new phenomenon--tissue dependence of the mechanism of regulation of mitochondrial respiration, and activation of respiration by selective proteolysis. These phenomena are explained by interaction of mitochondria with other cellular structures in vivo. The details of experimental studies with use of these techniques and problems of kinetic analysis of the results are discussed. Examples of large-scale clinical application of these methods are given.
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              Mitochondrial respiratory parameters in cardiac tissue: a novel method of assessment by using saponin-skinned fibers.

              Respiratory parameters of cardiac mitochondria were determined in the bundles of cardiac fibers skinned by using saponin that specifically removed sarcolemma, but left intracellular structures intact. In the assay medium which simulated the ion composition of cardiac cytoplasm maximal value of state 3 oxygen consumption per mol cytochromes aa3 was close to that value for isolated mitochondria. Ischemia and isopreterenol treatment were found to affect respiratory parameters of mitochondria in saponin-skinned fibers, among them creatine-stimulated respiration decreased most significantly, (3-4)-times under these conditions. The method described can be easily applied for determination of the mitochondrial respiratory parameters in small (5-10 mg) biopsy samples from human heart.

                Author and article information

                Drug Des Devel Ther
                Drug Des Devel Ther
                Drug Design, Development and Therapy
                Drug Design, Development and Therapy
                Dove Medical Press
                13 July 2015
                : 9
                : 3589-3597
                [1 ]Physiology Department, Faculty of Medicine and EA3072, Université de Strasbourg, Strasbourg, France
                [2 ]Clermont Université, Université Blaise Pascal, EA 3533, Laboratoire des Adaptations Métaboliques à l’Exercice en Conditions Physiologiques et Pathologiques, Clermont-Ferrand, France
                [3 ]Danone Research, Centre Daniel Carasso, Palaiseau, France
                [4 ]Department of Anesthesia and Critical Care and EA3072, Hôpital de Hautepierre, Université de Strasbourg, France
                [5 ]Kinesiology Department, Université de Montréal, Montréal, QC, Canada
                [6 ]Department of Sport Medicine and Functional Explorations and INRA UMR 1019, Faculty of Medicine, Université d’Auvergne, Clermont-Ferrand, France
                Author notes
                Correspondence: Ruddy Richard, Service de Médecine du Sport et des Explorations Fonctionnelles, CHU G, Montpied, 58 rue Montalembert, 63003 Clermont-Ferrand Cedex 1, France, Email richard.ruddy@ 123456gmail.com
                © 2015 Zoll et al. This work is published by Dove Medical Press Limited, and licensed under Creative Commons Attribution – Non Commercial (unported, v3.0) License

                The full terms of the License are available at http://creativecommons.org/licenses/by-nc/3.0/. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed.

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