Inherited disturbances of the mitochondrial energy generating system represent a heterogeneous group of disorders associated with a broad spectrum of metabolic abnormalities and clinical symptoms. We used the polarographic and spectrophotometric method for detection of mitochondrial disorders, because these two techniques provide a different insight into mitochondrial function. In six patients suspected of mitochondrial disease we found defects of complex I (two patients), complex III (one patient), complex IV (two patients) and a combination of defect of complex III and IV (one patient). Citrate synthase activity, used as the reference enzyme, was not changed. A comparison of the two methods showed several differences in evaluation of mitochondrial enzymes activity due to the fact that both methods used different conditions for enzyme activity measurements. In contrast to oxygen consumption measurements, where the function of the whole-integrated respiratory chain is characterized, spectrophotometric measurements characterize activities of isolated complexes in disintegrated membranes. However, it may be concluded from our experiments that both methods provide useful and complementary data about mitochondrial energetic functions. Whereas spectrophotometric data are suitable for evaluation of maximal enzyme activities of mitochondrial enzyme complexes, polarographic data provide better information about enzyme activities in cells with mitochondrial defects under in situ conditions., L. Wenchich, Z. Drahota, T. Honzík, H. Hansíková, M. Tesařová, J. Zeman, J. Houštěk., and Obsahuje bibliografii
Remote ischemic preconditioning (RIP)-induced protection of myocardial energetics was well documented on the level of tissue, but data concerning the involvement of mitochondria were missing. We aimed at the identification of changes in membrane properties and respiratory functions induced in rat heart mitochondria by RIP. Experiments were performed on 46 male Wistar rats divided into control and RIP-treated groups of 21 animals each. Blood flow in the occluded area was recorded by MRI angiography in four animals. RIP protocol comprised of three successive 5-min occlusions each followed by 5-min reperfusions of descending branches of the right hind limb femoral artery. The efficacy of RIP was evaluated as the extent of RIP-induced protection against damage to the functions of mitochondria isolated by differential centrifugation after 30-min global ischemia followed by 40-min reperfusion of the hearts in Langendorff mode. Assessments: mitochondrial membrane fluidity with a fluorescent probe DPH, CoQ9 and CoQ10 with HPLC, mitochondrial respiration with the Oxygraph-2k (Oroboros). Results revealed that RIP was affecting the mitochondria. The immediate protection conferred by RIP involves beneficial and prognostically significant effects: a total elimination of ischemia/reperfusion-induced depression of mitochondrial membrane fluidity and a trend for better preservation of mitochondrial state 3 respiration., M. Ferko, I. Kancirová, M. Jašová, S. Čarnická, M. Muráriková, I. Waczulíková, Z. Sumbalová, J. Kucharská, O. uličná, T. Ravingerová, A. Ziegelhöffer., and Obsahuje bibliografii
Our previous preliminary results pointed to possible seasonal variations in Mg2+-ATPase activity of rat heart mitochondria (MIT). It is not too surprising since seasonal differences were already reported in myocardial function, metabolism and ultrastructure of the intact as well as hemodynamically overloaded rabbit hearts and also in other tissues. The present study is aimed to elucidate whether seasonal differences observed in rat heart MIT Mg2+-ATPase activity will be accompanied with changes in membrane fluidity and in the content of conjugated dienes (CD) in the lipid bilayers of MIT membranes as well as whether the above seasonal differences will also be present in the diabetic heart. Our results revealed that values of Mg2+-ATPase activity in the winter/spring-period (W/S-P) exceeded significantly (p<0.05-0.001) those in the summer/autumn-period (S/A-P). Similar trend was also observed in hearts of animals with acute (8 days) streptozotocin diabetes. With the exception of values of CD in the S/A-P, all values of Mg2+-ATPase activities, membrane fluidity and CD concentrations in diabetic hearts exceeded those observed in the healthy hearts. Our results indicate that seasonal differences may play a decisive role in the evaluation of properties and function of rat heart MIT., J. Mujkošová, M. Ferko, P. Humeník, I. Waczulíková, A. Ziegelhöffer., and Obsahuje bibliografii a bibliografické odkazy
Superresolution microscopy enables to see previously hidden details of cellular structures. However, it requires to use high irradiation intensities that may cause artefacts and photodamage of fragile biological samples. Newly developed confocal technique and Olympus super resolution (OSR), that combines spinning disk confocal with structured illumination microscopy, represents a reliable and fast superresolution avoiding photodamage. We demonstrate an OSR microscope platform, that enables subsecond, multicolour data acquisition. It also provides access to subdiffraction structured illumination imaging. We show that OSR allows live-cell experiments without any noticeable cellular damage. OSR system has an improved lateral (∼∼2) and axial (∼∼3) resolution compared with conventional confocal imaging. Moreover, OSR is compatible with both fixed and live cell imaging. and Superrezoluční mikroskopie umožňuje zobrazovat dříve nepoznané detaily jemných buněčných struktur. Vyžaduje však světelný zdroj vysoké intenzity, který může způsobovat artefakty a foto-poškození citlivých biologických vzorků. Nově vyvinutá konfokální technologie superrezolučního mikroskopu Olympus (OSR) kombinuje rotující disk se strukturovaným mikroskopickým osvětlením a představuje spolehlivé a rychlé superrezoluční zobrazení, které minimalizuje foto-toxicitu. V tomto příspěvku představujeme platformu OSR mikroskopu, která umožňuje subsekundovou vícebarevnou akvizici dat a subdifrační zobrazení ve strukturovaném osvětlení. OSR umožňuje zobrazování živých buněk bez jejich zjevného poškození. Ve srovnání s konvenčním konfokálním zobrazováním má systém OSR vylepšené laterální (~ 2) a axiální (~ 3) rozlišení. OSR je navíc kompatibilní se zobrazováním fixovaných i živých buněk.
Calorie restriction (CR) has been shown to decrease reactive oxygen species (ROS) production an d retard aging in a variety of species. It has been proposed that alterations in membrane saturation are central to these ac tions of CR. As a step towards testing this theory, mice were assigned to 4 dietary groups (control and 3 CR groups) and fed AIN-93G diets at 95 % (control) or 60 % (CR) of ad libitum for 8 months. To manipulate membrane composition, the primary dietary fats for the CR groups were soybean oil (also used in the control diet), fish oil or lard. Skeletal muscle mitochondrial lipid composition, proton leak, and H 2 O 2 production were measured. Phospholipid fatty acid composition in CR mice was altered in a manner that reflected the n-3 and n-6 fatty acid profiles of their respective dietary lipid sources. Dietary lipid composition did not alter proton leak kinetics between the CR groups . However, the capacity of mitochondrial complex III to produce ROS was decreased in the CR lard compared to the other CR groups. The results of this study indicate that dietary lipid composition can influence ROS production in muscle mitochondria of CR mice. It remains to be determined if lard or other dietary oils can maximize the CR- induced decreases in ROS production., Y. Chen ... [et al.]., and Obsahuje bibliografii a bibliografické odkazy
The transformed C6 glial cells in cultures were treated with sodium mercaptoborate (Na2B12H11SH, BSH), a carrier of atomic targets (10B) of thermal neutrons for the neutron capture therapy of brain tumors. As shown by light microscopy, the therapeutic dose of BSH (100 µg/ml) did not alter the gross morphology and growth of the population of cells within a 72 h treatment interval. Electron microscopic analysis of these cells revealed activation of nucleoli and, occasionally, enlarged and bifurcated mitochondria. After 200 µg BSH/ml and 72 h treatment, growth of the cell population was inhibited and ultrastructural changes became more profound. They included condensation of chromatin and its allocation to the nuclear envelope which formed deeper invaginations. Mitochondria further increased in size and were characterized by slim or angular cristae. Moreover, in circumscribed segments of some of the slightly swollen mitochondria their cristae disappeared or were reduced to fine pouch-like structures localized near the continuous outer membrane, suggestive for a non-destructive restructuring of the inner mitochondrial membrane. The smooth pinocytotic vesicles near the plasma membrane, lysosomes and heterogeneous dense bodies were more frequent. The revealed subcellular targets of BSH may initiate the development of pharmacological protocols aimed to further improve the tolerance to BSH by the healthy tissues of patients undergoing BNCT of brain tumors, e.g. by intervention into the oxidative stress triggered likely by the altered mitochondria., V. Mareš, D. Krajčí, V. Lisá., and Obsahuje bibliografii