Hexokinase inhibits flux of fluorescently labeled ATP through mitochondrial outer membrane porin

AbstractMitochondrial function requires maintaining metabolite fluxes across the mitochondrial outer membrane, which is mediated primarily by the voltage dependent anion channel (VDAC). We applied fluorescence correlation spectroscopy (FCS) to study regulation of the VDAC functional state by monitoring distribution of fluorescently labeled ATP (BODIPY-FL-ATP) in isolated intact rat liver and heart mitochondria. Addition of mitochondria to BODIPY-FL-ATP solution resulted in accumulation of the fluorescent probe in these organelles. The addition of hexokinase II (HKII) isolated from rat heart led to a decrease in the BODIPY-FL-ATP accumulation, while a 15-residue peptide corresponding to the N-terminal domain of hexokinase did not produce this effect. Therefore, the hexokinase-induced inhibition of the ATP flow mediated by VDAC was revealed in isolated mitochondria

Diazepam inhibits cell respiration and induces fragmentation of mitochondrial reticulum

AbstractDiazepam (70–150 μg/ml) significantly inhibits oxygen consumption by pig kidney embryo cells and causes the cellular ATP level to fall. The maximum inhibitory effect develops after 1.5–2.5 h of diazepam treatment. In isolated mitochondria diazepam inhibits respiration in state 2 and 3u with glutamate and in state 3u with succinate. Ethylrhodamine staining and electron microscopic study reveal fragmentation of mitochondria in living cells

Interrelations of mitochondrial fragmentation and cell death under ischemia/reoxygenation and UV-irradiation: Protective effects of SkQ1, lithium ions and insulin

AbstractMitochondria-targeted antioxidant 10-(6-plastoquinonyl)decyltriphenyl-phosphonium (SkQ1) as well as insulin and the inhibitor of glycogen-synthase kinase, Li+ are shown to (i) protect renal tubular cells from an apoptotic death and (ii) diminish mitochondrial fission (the thread-grain transition) induced by ischemia/reoxygenation. However, SkQ1 and LiCl protected the mitochondrial reticulum of skin fibroblasts from ultraviolet-induced fission but were ineffective in preventing a further cell death. This means that mitochondrial fission is not essential for apoptotic cascade progression

ОСВОБОЖДЕНИЕ ОТ УГОЛОВНОЙ ОТВЕТСТВЕННОСТИ ПРИ ЭКОНОМИЧЕСКИХ ПРЕСТ УПЛЕНИЯХ

The institution of exemption from criminal liability for crimes committed in the sphere of the economic activity is subjected to the critical analysis. Its position and the extent of its viability under the current law are examined; the author focuses on its inconsistency, confusion and technical imperfection. According to the author, the greater the damage caused by an economic crime, the greater the benefit received by the state if the business owner makes a decision to meet the statutory terms and conditions of exemption from criminal liability.Taking into account the current policy of humanization and special indulgence to entrepreneurs who have committed crimes, and having assessed his own benefit, the entrepreneur can choose punishment rather than exemption from criminal liability because of the greater rigidity of the latter. As a result, the relative impunity increases the attractiveness of the economic sphere for organized crime groups.В статье автор подвергает критическому анализу институт освобождения от уголовной ответственности по делам о преступлениях в сфере экономической деятельности. Рассматриваются его состояние и степень необходимости в рамках законодательства в действующей редакции, при этом автор акцентирует внимание на ее непоследовательности, запутанности и техническом несовершенстве. По мнению автора, чем больший вред причинен экономическим преступлением, тем бо́льшую выгоду получает государство, если предприниматель примет решение о выполнении предусмотренных законом условий освобождения от уголовной ответственности. Учитывая современную политику гуманизации и особого снисхождения к предпринимателям, совершившим преступления, оценив свою собственную выгоду, предприниматель может выбрать наказание, а не освобождение от уголовной ответственности, по причине болей жесткости последнего. В итоге относительная безнаказанность повысит привлекательность экономической сферы для сформировавшихся организованных преступных группировок

The role of mitochondria in oxidative and nitrosative stress during ischemia/reperfusion in the rat kidney

Reoxygenation following ischemia causes tissue oxidative stress. We studied the role of oxidative stress caused by kidney ischemia/reperfusion (I/R) on the mitochondria of renal tissue slices. I/R caused the mitochondria to be swollen, fragmented, and have lower membrane potential. The mitochondria generated more reactive oxygen species (ROS) and nitric oxide (NO) in situ as measu red by fluorescence of ROS- and NO-sensitive probes. Infusion of lithium ion, an inhibitor of glycogen kinase synthase-3, caused phosphorylation of its Ser-9 and restored the membrane potential and decreased ROS production of the mitochondrial fraction. Ischemic kidney and hypoxic rat preconditioning improved mitochondrial membrane potential and lowered ROS production caused by subsequent I/R similar to lithium ion infusion. Preconditioning normalized NO production in mitochondria as well. The drop in the mitochondrial membrane potential was prevented by NO synthase inhibition, demonstrating a strong contribution of NO to changes in mitochondrial energy metabolism during the I/R transition. Mitochondria in the I/R-stressed kidney contained less cytochrome c and more pro-apoptotic Bax, consistent with apoptotic degradation

Combined therapeutic benefit of mitochondria-targeted antioxidant, MitoQ10, and angiotensin receptor blocker, losartan, on cardiovascular function

<b>Objective:</b><p></p> Mitochondria-derived reactive oxygen species (ROS) play important roles in the development of cardiovascular disease highlighting the need for novel targeted therapies. This study assessed the potential therapeutic benefit of combining the mitochondria-specific antioxidant, MitoQ<sub>10</sub>, with the low-dose angiotensin receptor blocker (ARB), losartan, on attenuation of hypertension and left ventricular hypertrophy. In parallel, we investigated the impact of MitoQ<sub>10</sub> on cardiac hypertrophy in a neonatal cardiomyocyte cell line.<p></p> <b>Methods and results:</b><p></p> Eight-week-old male stroke-prone spontaneously hypertensive rats (SHRSPs, <i>n</i> = 8–11) were treated with low-dose losartan (2.5 mg/kg per day); MitoQ<sub>10</sub> (500 μmol/l); a combination of MitoQ<sub>10</sub> and losartan (M + L); or vehicle for 8 weeks. Systolic pressure and pulse pressure were significantly lower in M + L rats (167.1 ± 2.9 mmHg; 50.2 ± 2.05 mmHg) than in untreated SHRSP (206.6 ± 9 mmHg, P < 0.001; 63.7 ± 2.7 mmHg, P = 0.001) and demonstrated greater improvement than MitoQ10 or low-dose losartan alone, as measured by radiotelemetry. Left ventricular mass index was significantly reduced from 22.8 ± 0.74 to 20.1 ± 0.61 mg/mm in the combination group (P < 0.05). Picrosirius red staining showed significantly reduced cardiac fibrosis in M + L rats (0.82 ± 0.22 A.U.) compared with control (5.94 ± 1.35 A.U., P < 0.01). In H9c2 neonatal rat cardiomyocytes, MitoQ<sub>10</sub> significantly inhibited angiotensin II mediated hypertrophy in a dose-dependent manner (500 nmol/l MitoQ<sub>10</sub> 153.7 ± 3.1 microns vs. angiotensin II 200.1 ± 3.6 microns, P <0.001).<p></p> <b>Conclusion:</b><p></p> Combining MitoQ<sub>10</sub> and low-dose losartan provides additive therapeutic benefit, significantly attenuating development of hypertension and reducing left ventricular hypertrophy. In addition, MitoQ<sub>10</sub> mediates a direct antihypertrophic effect on rat cardiomyocytes <i>in vitro</i>. MitoQ<sub>10</sub> has potential as a novel therapeutic intervention in conjunction with current antihypertensive drugs.<p></p&gt

The involvement of the aspartate triad of the active center in all catalytic activities of multisubunit RNA polymerase

Three conserved aspartate residues in the largest subunit of multisubunit RNA polymerases (RNAPs) coordinate two Mg(2+) ions involved in the catalysis of phosphodiester bond synthesis. A structural model based on the stereochemistry of nucleotidyl transfer reaction as well as recent crystallographic data predict that these Mg(2+) ions should also be involved in the reverse reaction of pyrophosphorolysis as well as in the endo- and exonucleolytic cleavage of the nascent RNA. Here, we check these predictions by constructing point substitutions of each of the three Asp residues in the β′ subunit of Escherichia coli RNAP and testing the mutant enzymes' functions. Using artificially assembled elongation complexes, we demonstrate that substitutions of any of the three aspartates dramatically reduce all known RNAP catalytic activities, supporting the model's predictions that same amino acids participate in all RNAP catalytic reactions. We demonstrate that though substitutions in the DFDGD motif decrease Mg(2+) binding to free RNAP below detection limits, the apparent affinity to Mg(2+) in transcription complexes formed by the mutant and wild-type RNAPs is similar, suggesting that NTP substrates and/or nucleic acids actively contribute to the retention of active center Mg(2+)