Clark-type Electrode Research Articles

Real-time Monitoring of Cerebral Blood Flow and Cerebral Oxygenation During Rapid Ventricular Pacing in Neurovascular Surgery: A Pilot Study

Rapid ventricular pacing (RVP) can be used to produce short periods of flow arrest during dissection or rupture of a cerebral aneurysm but carries the risk of inducing cerebral ischemia. This study evaluates the intraoperative effect of RVP on local cerebral blood flow (CBF) and cerebral oxygenation during neurovascular surgery. Five patients undergoing elective cerebrovascular surgery were included in a single-center prospective study. RVP was applied in pacing periods of 40 seconds with 30% and 100% FiO2. Regional cerebral oxygenation was monitored using a Foresight near-infrared spectroscopy sensor. A Clark-type electrode and a thermal diffusion microprobe located in the white matter were used to monitor brain tissue pO2 and CBF, respectively. CBF response to RVP closely followed the blood pressure pattern and resulted in a low-flow state. Unlike CBF, brain tissue pO2 and regional cerebral oxygenation showed a delayed response to RVP, decreasing beyond the pacing period and slowly recovering after RVP cessation. We found a correlation between brain tissue pO2 and regional cerebral oxygenation. Increasing the inspired oxygen concentration had a positive impact on absolute regional cerebral oxygenation and brain tissue pO2 values, but the pattern resulting from applying RVP remained unaltered. RVP reduces CBF and cerebral oxygenation. Brain tissue pO2 and regional cerebral oxygenation are correlated but unlike CBF respond to RVP in a delayed manner. Further research is required to evaluate the impact of longer RVP bursts on brain oxygenation.

Ischemia time impacts on respiratory chain functions and Ca2+-handling of cardiac subsarcolemmal mitochondria subjected to ischemia reperfusion injury

BackgroundMitochondrial impairment can result from myocardial ischemia reperfusion injury (IR). Despite cardioplegic arrest, IR-associated cardiodepression is a major problem in heart surgery. We determined the effect of increasing ischemia time on the respiratory chain (RC) function, the inner membrane polarization and Ca2+ homeostasis of rat cardiac subsarcolemmal mitochondria (SSM).MethodsWistar rat hearts were divided into 4 groups of stop-flow induced warm global IR using a pressure-controlled Langendorff system: 0, 15, 30 and 40 min of ischemia with 30 min of reperfusion, respectively. Myocardial contractility was determined from left ventricular pressure records (dP/dt, dPmax) with an intraventricular balloon. Following reperfusion, SSM were isolated and analyzed regarding electron transport chain (ETC) coupling by polarography (Clark-Type electrode), membrane polarization (JC1 fluorescence) and Ca2+-handling in terms of Ca2+-induced swelling and Ca2+-uptake/release (Calcium Green-5 N® fluorescence).ResultsLV contractility and systolic pressure during reperfusion were impaired by increasing ischemic times. Ischemia reduced ETC oxygen consumption in IR40/30 compared to IR0/30 at complex I-V (8.1 ± 1.2 vs. 18.2 ± 2.0 nmol/min) and II-IV/V (16.4 ± 2.6/14.8 ± 2.3 vs. 2.3 ± 0.6 nmol/min) in state 3 respiration (p < 0.01). Relative membrane potential revealed a distinct hyperpolarization in IR30/30 and IR40/30 (171.5 ± 17.4% and 170.9 ± 13.5%) compared to IR0/30 (p < 0.01), wearing off swiftly after CCCP-induced uncoupling. Excess mitochondrial permeability transition pore (mPTP)-gated Ca2+-induced swelling was recorded in all groups and was most pronounced in IR40/30. Pyruvate addition for mPTP blocking strongly reduced SSM swelling in IR40/30 (relative AUC, ± pyruvate; IR0/30: 1.00 vs. 0.61, IR15/30: 1.68 vs. 1.00, IR30/30: 1.42 vs. 0.75, IR40/30: 1.97 vs. 0.85; p < 0.01). Ca2+-uptake remained unaffected by previous IR. Though Ca2+-release was delayed for ≥30 min of ischemia (p < 0.01), Ca2+ retention was highest in IR15/30 (RFU; IR0/30: 6.3 ± 3.6, IR 15/30 42.9 ± 5.0, IR30/30 15.9 ± 3.8, IR40/30 11.5 ± 6.6; p ≤ 0.01 for IR15/30 against all other groups).ConclusionsIschemia prolongation in IR injury gradually impaired SSM in terms of respiratory chain function and Ca2+-homeostasis. Membrane hyperpolarization appears to be responsible for impaired Ca2+-cycling and ETC function. Ischemia time should be considered an important factor influencing IR experimental data on subsarcolemmal mitochondria. Periods of warm global ischemia should be minimized during cardiac surgery to avoid excessive damage to SSMs.

The proton pumping bo oxidase from Vitreoscilla

The cytochrome bo3 quinol oxidase from Vitreoscilla (vbo3) catalyses oxidation of ubiquinol and reduction of O2 to H2O. Data from earlier studies suggested that the free energy released in this reaction is used to pump sodium ions instead of protons across a membrane. Here, we have studied the functional properties of heterologously expressed vbo3 with a variety of methods. (i) Following oxygen consumption with a Clark-type electrode, we did not observe a measurable effect of Na+ on the oxidase activity of purified vbo3 solubilized in detergent or reconstituted in liposomes. (ii) Using fluorescent dyes, we find that vbo3 does not pump Na+ ions, but H+ across the membrane, and that H+-pumping is not influenced by the presence of Na+. (iii) Using an oxygen pulse method, it was found that 2 H+/e− are ejected from proteoliposomes, in agreement with the values found for the H+-pumping bo3 oxidase of Escherichia coli (ecbo3). This coincides with the interpretation that 1 H+/e− is pumped across the membrane and 1 H+/e− is released during quinol oxidation. (iv) When the electron transfer kinetics of vbo3 upon reaction with oxygen were followed in single turnover experiments, a similar sequence of reaction steps was observed as reported for the E. coli enzyme and none of these reactions was notably affected by the presence of Na+. Overall the data show that vbo3 is a proton pumping terminal oxidase, behaving similarly to the Escherichia coli bo3 quinol oxidase.

The Mitochondria-Targeted Antioxidant MitoQ Modulates Mitochondrial Function and Endoplasmic Reticulum Stress in Pancreatic β Cells Exposed to Hyperglycaemia.

Mitochondria-targeted antioxidants such as mitoquinone (MitoQ) have demonstrated protective effects against oxidative damage in several diseases. The increase in reactive oxygen species (ROS) production during glucose metabolism in β cells can be exacerbated under hyperglycaemic conditions such as type 2 diabetes (T2D), thus contributing to β cell function impairment. In the present work, we aimed to evaluate the effect of MitoQ on insulin secretion, oxidative stress, endoplasmic reticulum (ER) stress and nuclear factor kappa B (NFκB) signalling in a pancreatic β cell line under normoglycaemic (NG, 11.1 mM glucose), hyperglycaemic (HG, 25 mM glucose) and lipidic (palmitic acid (PA), 0.5mM) conditions. We incubated the pancreatic β cell line INS-1E with or without MitoQ (0.5µM) under NG, HG and PA conditions. We then assessed the following parameters: glucose-induced insulin secretion, O₂ consumption (with a Clark-type electrode); mitochondrial function, oxidative stress parameters and calcium levels (by fluorescence microscopy); ER stress markers and NFκB-p65 protein levels (by western blotting). MitoQ increased insulin secretion and prevented the enhancement of ROS production and O₂ consumption and decrease in GSH levels that are characteristic under HG conditions. MitoQ also reduced protein levels of ER stress markers (GRP78 and P-eIF2α) and the proinflammatory nuclear transcription factor NFκB-p65, both of which increased under HG. MitoQ did not significantly alter ER stress markers under lipidic conditions. Our findings suggest that treatment with MitoQ modulates mitochondrial function, which in turn ameliorates endoplasmic reticulum stress and NFκB activation, thereby representing potential benefits for pancreatic β cell function.

Role of mitophagy in hydrogen-induced reduction of LPS-caused mitochondrial injury to macrophages of mice

Objective To evaluate the role of mitophagy in hydrogen-induced reduction of lipopolysaccharide(LPS)-caused mitochondrial injury to macrophages of mice. Methods Macrophage line RAW264.7 cells of mice were routinely cultured and divided into 4 groups(n=6 each)using a random number table method: control group(Con group), LPS group, LPS plus hydrogen group(LPS+ H2 group) and LPS plus hydrogen plus mitophagy inhibitor 3-methyladenine(3-MA) group(LPS+ H2+ 3-MA group). Cells were incubated for 6 h with LPS at the concentration of 1 μg/ml in LPS group.Cells were incubated for 6 h with LPS 1 μg/ml and hydrogen-rich medium 0.6 mmol/L.Cells were incubated for 1 h with 2 mmol/L 3-MA and then incubated for 6 h with LPS 1 μg/ml and hydrogen-rich medium 0.6 mmol/L in LPS+ H2+ 3-MA group.Mitochondrial respiratory control ratio(RCR)was measured using a Clark-type electrode. Mitochondrial membrane potential(MMP)was determined by JC-1 staining.Autophagosomes were counted with a transmission electron microscope.The expression of PTEN-induced putative kinase 1(PINK1), E3 ubiquitin ligase(Parkin), microtubule-associated protein 1 light chain 3 Ⅱ(LC3 Ⅱ)and Beclin-1 was determined by Western blot. Results Compared with Con group, RCR and MMP were significantly decreased, the expression of PINK1, Parkin, LC3Ⅱ and Beclin-1 was up-regulated, and the number of autophagosomes was increased in LPS group(P<0.05). Compared with LPS group, RCR and MMP were significantly increased, the expression of PINK1, Parkin, LC3Ⅱ and Beclin-1 was up-regulated, and the number of autophagosomes was increased in LPS+ H2 group(P<0.05). Compared with LPS+ H2 group, RCR and MMP were significantly decreased, the expression of PINK1, Parkin, LC3Ⅱ and Beclin-1 was down-regulated, and the number of autophagosomes was decreased in LPS+ H2+ 3-MA group(P<0.05). Conclusion Enhanced mitophagy is involved in hydrogen-induced reduction of LPS-caused mitochondirial injury to macrophages of mice. Key words: Hydrogen; Endotoxins; Macrophages; Mitochondria; Autophagy

Time-related changes in hepatic and colonic mitochondrial oxygen consumption after abdominal infection in rats

BackgroundEvidence suggests that early adaptive responses of hepatic mitochondria occur in experimentally induced sepsis. Little is known about both colonic mitochondrial function during abdominal infection and long-term changes in mitochondrial function under inflammatory conditions. We hypothesize that hepatic and colonic mitochondrial oxygen consumption changes time-dependently after sterile laparotomy and in the course of abdominal infection. The aim of the present study was to investigate the hepatic and colonic mitochondrial respiration after sterile laparotomy and abdominal infection over up to 96 h.MethodsAfter approval of the local Animal Care and Use Committee, 95 Wistar rats were randomized into 8 groups (n = 11–12): 1–4 sham (laparotomy only) and 5–8 colon ascendens stent peritonitis (CASP). Healthy, unoperated animals served as controls (n = 9). The mitochondrial respiration in colon and liver homogenates was assessed 24, 48, 72, and 96 h after surgery. Mitochondrial oxygen consumption was determined using a Clark-type electrode. State 2 (oxygen consumption in the presence of the substrates for complexes I and II) and state 3 respiration (ADP dependent) were assessed. The respiratory control ratio (RCR state 3/state 2) and ADP/O ratio (ADP added/oxygen consumed) were calculated for both complexes. Data are presented as means ± SD, two-way ANOVA followed by Tukey’s post hoc test.ResultsHepatic RCR was initially (after 24 h) elevated in both operated groups; after 48 h only, the septic group was elevated compared to controls. In CASP groups, the hepatic ADP/O ratio for complex I was elevated after 24 h (vs. controls) and after 48 h (vs. sham) but declined after 72 h (vs. controls). The ADP/O ratio for complex II stayed unchanged over the time period until 96 h.The colonic RCR and ADP/O did not change over time after sham or CASP operation.ConclusionHepatic, but not colonic, mitochondrial respiration is increased in the initial phase (until 48 h) and normalizes in the longer course of time (until 96 h) of abdominal infection.

Assessing changes in tissue oxygenation by near-infrared spectroscopy following brachial plexus block for arteriovenous fistula surgery: A prospective observational pilot study.

Near-infrared spectroscopy (NIRS) can be used to measure tissue oxygen saturation (StO2) in different sites and in a wide range of clinical scenarios. Peripheral regional anaesthesia induces vascular changes causing increased arterial blood flow and venodilatation, but its effect on StO2 is still under debate. This is especially so for patients undergoing arteriovenous fistula surgery, wherein latest data suggest an improved outcome under brachial plexus block (BPB) compared with local anaesthesia, but no data are available. The aim of this study was to investigate changes in StO2 following BPB prior to arteriovenous fistula surgery using NIRS. A prospective observational study. A secondary teaching hospital from August 2016 to March 2017. Fifteen patients undergoing arteriovenous fistula surgery. Ultrasound-guided BPB in 15 patients undergoing arteriovenous fistula surgery. StO2 at baseline and compared with baseline and the contralateral arm following BPB measured using NIRS of the thenar eminence (NIRSth). Baseline values of StO2 assessed by NIRSth were 42.6 ± 7.7% in the arteriovenous fistula arm and 42.7 ± 9.7% in the contralateral arm. There was no significant difference between the two. Five minutes after BPB, there was a significant increase in StO2 of the blocked arm, compared with the control arm expressed as difference of absolute values (7.1 ± 9.7%). At 60 min, an absolute difference of 21.0 ± 13.5% was reached. The absolute increase in StO2 of the blocked arm compared with baseline reached significance after 5 min (8.8 ± 4.6%) and increased up to 23.2 ± 8.2% after 60 min. NIRSth indicates that BPB significantly increases StO2 of the arteriovenous fistula arm in patients undergoing haemodialysis. Clinicaltrials.gov: NCT03044496.

Regulation of the oxidative balance with coenzyme Q10 sensitizes human glioblastoma cells to radiation and temozolomide

ObjectivesTo investigate how the modulation of the oxidative balance affects cytotoxic therapies in glioblastoma, in vitro. Material and methodsHuman glioblastoma U251 and T98 cells and normal astrocytes C8D1A were loaded with coenzyme Q10 (CoQ). Mitochondrial superoxide ion (O2−) and H2O2 were measured by fluorescence microscopy. OXPHOS performance was assessed in U251 cells with an oxytherm Clark-type electrode. Radio- and chemotherapy cytotoxicity was assessed by immunostaining of γH2AX (24 h), annexin V and nuclei morphology, at short (72 h) and long (15 d) time. Hif-1α, SOD1, SOD2 and NQO1 were determined by immunolabeling. Catalase activity was measured by classic enzymatic assay. Glutathione levels and total antioxidant capacity were quantified using commercial kits. ResultsCoQ did not affect oxygen consumption but reduced the level of O2− and H2O2 while shifted to a pro-oxidant cell status mainly due to a decrease in catalase activity and SOD2 level. Hif-1α was dampened, echoed by a decrease lactate and several key metabolites involved in glutathione synthesis. CoQ-treated cells were twofold more sensitive than control to radiation-induced DNA damage and apoptosis in short and long-term clonogenic assays, potentiating TMZ-induced cytotoxicity, without affecting non-transformed astrocytes. ConclusionsCoQ acts as sensitizer for cytotoxic therapies, disarming GBM cells, but not normal astrocytes, against further pro-oxidant injuries, being potentially useful in clinical practice for this fatal pathology.

Differential temperature effects on dissipation of excess light energy and energy partitioning in lut2 mutant of Arabidopsis thaliana under photoinhibitory conditions.

The high-light-induced alterations in photosynthetic performance of photosystem II (PSII) and photosystem I (PSI) as well as effectiveness of dissipation of excessive absorbed light during illumination for different periods of time at room (22°C) and low (8-10°C) temperature of leaves of Arabidopsis thaliana, wt and lut2, were followed with the aim of unraveling the role of lutein in the process of photoinhibition. Photosynthetic parameters of PSII and PSI were determined on whole leaves by PAM fluorometer and oxygen evolving activity-by a Clark-type electrode. In thylakoid membranes, isolated from non-illuminated and illuminated for 4.5h leaves of wt and lut2 the photochemical activity of PSII and PSI and energy interaction between the main pigment-protein complexeswas determined. Results indicate that in non-illuminated leaves of lut2 the maximum rate of oxygen evolution and energy utilization in PSII is lower, excitation pressure of PSII is higher and cyclic electron transport around PSI is faster than in wt leaves. Under high-light illumination, lut2 leaves are more sensitive in respect to PSII performance and the extent of increase of excitation pressure of PSII, ΦNO, and cyclic electron transport around PSI are higher than in wt leaves, especially when illumination is performed at low temperature. Significant part of the excessive light energy is dissipated via mechanism, not dependent on ∆pH and to functioning of xanthophyll cycle in LHCII, operating more intensively in lut2 leaves.

Improvement of the rice photosynthetic apparatus defence under cadmium stress modulated by salicylic acid supply to roots

The present study was conducted to investigate the effect of exogenous salicylic acid (SA) added to the nutrient solution on the growth parameters and the functions of the photosynthetic apparatus of rice plants under cadmium (Cd) stress. Our investigations have shown that 10 µM SA has an optimal effect in rice plants grown hydroponically. Pulse amplitude modulated chlorophyll fluorescence, low-temperature chlorophyll fluorescence, oxygen evolution (measured with Clark-type and Joliot-type electrodes) and P700 photo-oxidation measurements were carried out to assess the effect of SA on the activity of the photosynthetic apparatus. The levels of three important parameters associated with oxidative stress (hydrogen peroxide, lipid peroxidation and proline content) were measured. The application of low concentration of SA significantly decreased the levels of hydrogen peroxide, lipid peroxidation and proline under Cd stress. The results revealed that low concentration of SA, applied in plants exposed to 150 µM CdCl2, significantly improves plant growth, photochemical activities of both photosystems, the electron flow from QA to plastoquinone, energetic distribution between pigment-protein complexes and the kinetic parameters of oxygen-evolving reactions. This study suggests that exogenous application of 10 µM SA through the rooting medium has a protective effect against Cd toxicity in rice plants. The possible molecular mechanisms involved in the defence effect of SA on the function of photosynthetic apparatus are discussed.

Evaluation of Respiration with Clark-Type Electrode in Isolated Mitochondria and Permeabilized Animal Cells.

In many studies, the evaluation of mitochondrial function is critical to understand how disease conditions or xenobiotics alter mitochondrial function. One of the classic end points that can be assessed is oxygen consumption, which can be performed under controlled yet artificial conditions. Oxygen is the terminal acceptor in the mitochondrial respiratory chain, namely, at an enzyme called cytochrome oxidase, which produces water in the process and pumps protons from the matrix to the intermembrane space. Several techniques are available to measure oxygen consumption, including polarography with oxygen electrodes or fluorescent/luminescent probes. The present chapter will deal with the determination of mitochondrial oxygen consumption by means of the Clark-type electrode, which has been widely used in the literature and still remains to be a reliable technique. We focus our technical description in the measurement of oxygen consumption by isolated mitochondrial fractions and by permeabilized cells.

A protocol for quantifying hydrogen evolution by dye-sensitized molecular photocathodes and its implementation for evaluating a new covalent architecture based on an optimized dye-catalyst dyad.

A protocol that combines gas chromatography and a high-sensitivity micro Clark-type electrode is described to quantify hydrogen production across gas and solution phases for systems operating at very low currents such as dye-sensitized H2-evolving photocathodes. Data indicate that a significant fraction of H2 remains in aqueous solution even after several hours of experiments. Using this protocol, re-evaluation of a dye-sensitized H2-evolving photocathode based on a dye-catalyst dyad showed a reproducible 66% increase of the faradaic efficiency compared with previously reported headspace GC measurements [Kaeffer et al., J. Am. Chem. Soc., 2016, 138, 12308-12311]. This dyad was based on an organic push-pull dye where donor and acceptor are separated by one thiophene group. Insertion of a second thiophene group between the donor and acceptor led to a more efficient system with 30% improved faradaic efficiency for H2 evolution.

Acute renal metabolic effect of metformin assessed with hyperpolarised MRI in rats.

Metformin inhibits hepatic mitochondrial glycerol phosphate dehydrogenase, thereby increasing cytosolic lactate and suppressing gluconeogenesis flux in the liver. This inhibition alters cytosolic and mitochondrial reduction-oxidation (redox) potential, which has been reported to protect organ function in several disease states including diabetes. In this study, we investigated the acute metabolic and functional changes induced by metformin in the kidneys of both healthy and insulinopenic Wistar rats used as a model of diabetes. Diabetes was induced by intravenous injection of streptozotocin, and kidney metabolism in healthy and diabetic animals was investigated 4weeks thereafter using hyperpolarised 13C-MRI, Clark-type electrodes and biochemical analysis. Metformin increased renal blood flow, but did not change total kidney oxygen consumption. In healthy rat kidneys, metformin increased [1-13C]lactate production and reduced mitochondrial [1-13C]pyruvate oxidation (decreased the 13C-bicarbonate/[1-13C]pyruvate ratio) within 30min of administration. Corresponding alterations to indices of mitochondrial, cytosolic and whole-cell redox potential were observed. Pyruvate oxidation was maintained in the diabetic rats, suggesting that the diabetic state abrogates metabolic reprogramming caused by metformin. This study demonstrates that metformin-induced acute metabolic alterations in healthy kidneys favoured anaerobic metabolism at the expense of aerobic metabolism. The results suggest that metformin directly alters the renal redox state, with elevated renal cytosolic redox states as well as decreased mitochondrial redox state. These findings suggest redox biology as a novel target to eliminate the renal complications associated with metformin treatment in individuals with impaired renal function.

Effect of hydrogen on expression of Drp1 in myocardial mitochondria of septic mice

Objective To evaluate the effect of hydrogen on the expression of Drp1 in the myocardial mitochondria of septic mice. Methods Fifty-four pathogen-free healthy male ICR mice, aged 6 weeks, weighing 20-25 g, were divided into 3 groups(n=18 each)using a random number table: sham operation group(S group), sepsis group(Sep group)and hydrogen group(H2 group). Sepsis was produced by cecal ligation and puncture in anesthetized mice.The mice inhaled 2% hydrogen for 1 h starting from 1 and 6 h after operation in group H2.The mice were sacrificed at 1 day after operation, and hearts were removed for microscopic examination of the pathological changes which were scored and for determination of apoptosis in cardiomyocytes.Apoptosis rate was calculated.The myocardial mitochondria were isolated for measurement of respiratory control ratio(RCR)(using a Clark-type electrode), ATP content(by using a bioluminescence technique)and Drp1 expression(by Western blot). Results Compared with group S, the pathological score and apoptosis rate were significantly increased, the RCR and ATP content in mitochondria were decreased, and the expression of Drp1 was up-regulated in Sep and H2 groups(P<0.05). Compared with group Sep, the pathological score and apoptosis rate were significantly decreased, the RCR and ATP content in mitochondria were increased, and the expression of Drp1 was down-regulated in group H2(P<0.05). Conclusion The mechanism by which hydrogen mitigates myocardial injury is related to down-regulating expression of Drp1 in the myocardial mitochondria and improving mitochondrial function in septic mice. Key words: Hydrogen; Sepsis; Myocardial injury; Mitochondrial proteins

Nucleoside reverse transcriptase inhibitors induced hepatocellular mitochondrial DNA lesions and compensatory enhancement of mitochondrial function and DNA repair

Nucleoside reverse transcriptase inhibitors (NRTIs) are the backbone of combined antiretroviral therapy (cART) and are widely used in anti-human immunodeficiency virus (HIV) therapy. Long-term administration of NRTIs can result in mitochondrial dysfunction in certain HIV-1–infected patients. However, NRTI–associated liver mitochondrial toxicity is not well known. Herein, the liver autopsy of acquired immune deficiency syndrome (AIDS) patients and the liver tissues of mice with 12 months of NRTI exposure were used to identify NRTI–associated liver toxicity with immunofluorescence, quantitative real-time polymerase chain reaction (qPCR), Amplex red and horseradish peroxidase, and cloning and sequencing. Laser capture microdissection was used to capture hepatocytes from liver tissues. We observed DNA oxidative damage and mitochondrial DNA (mtDNA) loss in the livers of AIDS patients, and cART patients had higher DNA oxidative damage and lower DNA repair function in liver tissues than non-cART patients. We also observed liver oxidative damage, increased DNA repair and mtDNA loss in mice with exposure to four different NRTIs for 12 months, and hepatocytes had no more mtDNA loss than liver tissues. Although NRTIs could induce mitochondrial hydrogen peroxide production, increased mitochondrial oxygen consumption was found with a Clark-type electrode. The captured hepatocytes had greater diversity in their mtDNA D-loop, dehydrogenase subunit1 (ND1) and ND4 than the controls. Long-term NRTI exposure induced single nucleotide variation in hepatocellular mtDNA D-loop, ND1 and ND4. Our findings indicate that NRTIs can induce liver mtDNA lesions, but simultaneously enhance mitochondrial function and mtDNA repair.

Abstract 147: The Valosin-Containing Protein Regulates Mitochondrial Respiration and Membrane Permeability via Inducible Nitric Oxide Synthase in the Mouse Heart

Introduction: Mitochondrial dysfunction plays a critical role in mediating cell death during ischemia/reperfusion (IR). We previously found that overexpressing valosin-containing protein (VCP), an ATPase associate-binding protein, in a transgenic (TG) mouse heart increases the expression of inducible nitric oxide synthase (iNOS) and reduces infarct size during IR. We tested the hypothesis that VCP mediates cardiac protection by increasing mitochondrial respiration and inhibiting the opening of the mitochondrial permeability transition pore (mPTP) via iNOS. Methods and Results: Cardiac mitochondria were isolated from the heart tissues of adult VCP TG and their litter-matched WT mice (N=6/group). VCP TG mice increased the expression of VCP by 3.1 folds and iNOS by 4.5 folds in the mitochondrial fraction vs WT ( p &lt;0.01). Mitochondrial respiration was determined by oxygen consumption rate (OCR) in an airtight chamber with a Clark-type electrode. Compared to WT, mitochondria from VCP TG showed a significant increase in OCR during ADP-dependent State 3 under Complex I stimulation while no significant change was seen in oligomycin induced-State 4, increasing the efficiency of mitochondrial respiration, as measured by the respiratory control ratio (State 3/State 4). VCP TG mice also displayed a significant increase in their maximum respiration capacity versus WT, measured after the addition of the uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone. However, there was no difference between VCP TG and WT on Complex II-dependent respiration rate, suggesting VCP’s predominance on Complex I-stimulated respiration. mPTP opening was induced by Ca 2+ overload in isolated mitochondria and was measured by a swelling assay. Compared to WT mice, VCP TG showed significantly less mitochondrial swelling under the Ca 2+ overload ( p &lt;0.05). To test whether protection by VCP is mediated by iNOS, a bigenic VCP TG /iNOS knock out mouse was generated. Deletion of iNOS from the VCP TG abolishes the effects of VCP on mitochondrial respiration and mPTP opening. Conclusion: VCP prevents the opening of the mPTP and increases mitochondrial respiratory efficiency in an iNOS dependent manner, which may represent a novel mechanism of cardiac protection.

The wheat mutant DELLA-encoding gene (Rht-B1c) affects plant photosynthetic responses to cadmium stress

Тhe sensitivity to cadmium (Cd) stress of two near-isogenic wheat lines with differences at the Rht-B1 locus, Rht-B1a (tall wild type, encoding DELLA proteins) and Rht-B1c (dwarf mutant, encoding modified DELLA proteins), was investigated. The effects of 100 μM CdCl2 on plant growth, pigment content and functional activity of the photosynthetic apparatus of wheat seedlings grown on a nutrient solution were evaluated through a combination of PAM chlorophyll fluorescence, oxygen evolution, oxidation-reduction kinetics of P700 and 77 K fluorescence. The results showed that the wheat mutant (Rht-B1c) was more tolerant to Cd stress compared to the wild type (Rht-B1a), as evidenced by the lower reductions in plant growth and pigment content, lower inhibition of photosystem I (PSI) and photosystem II (PSII) photochemistry and of the oxygen evolution measured with Clark-type and Joliot-type electrodes. Furthermore, the enhanced Cd tolerance was accompanied by increased Cd accumulation within mutant plant tissues.The molecular mechanisms through which the Rht-B1c mutation improves plant tolerance to Cd stress involve structural alterations in the mutant photosynthetic membranes leading to better protection of the Mn cluster of oxygen-evolving complex and increased capacity for PSI cyclic electron transport, protecting photochemical activity of the photosynthetic apparatus under stress. This study suggests a role for the Rht-B1c-encoded DELLA proteins in protective mechanisms and tolerance of the photosynthetic apparatus in wheat plants exposed to heavy metals stress.

Adipose tissue is influenced by hypoxia of obstructive sleep apnea syndrome independent of obesity

AimsObstructive sleep apnea syndrome (OSAS) is associated with increased cardiovascular risk and diabetes independent of obesity. We investigated whether adipose tissue dysfunction is exacerbated due to increased tissue hypoxia. MethodsAdipose tissue (AT) oxygenation was measured with a Clarke-type electrode (pATO2) in 16 men with OSAS before and after 4 months of continuous positive airway pressure therapy (CPAP) and in BMI-matched controls. Oxygenation was simultaneously monitored in arterial blood by pulse oximetry (SaO2); mixed blood in AT microcirculation by reflectance spectroscopy (SATO2) along with blood flow. Markers of hypoxia, adipo- and angiogenesis, inflammation and fibrosis were analysed in AT and serum. ResultsOSAS subjects were more insulin resistant. Despite lower arterial SaO2 (95.4±1.3% vs. 97.1±1.6%, P=0.013) in subjects with OSAS, there was no difference in the oxygen content of AT microcirculation (61.6±18.4 vs. 72.2±7.0%, P=0.07) or pATO2 (49.2±7.5 vs. 50.4±14.7mmHg, P=0.83) between groups. Resting AT blood flow was higher in OSAS compared to controls (108.5±22.7 vs. 78.9±24.9au, P<0.005) and strongly associated with inflammation markers IL-6 and MCP-1. AT of OSAS subjects showed increased inflammation (TNFA P=0.049) and fibrosis (COL3A1 P=0.02), a trend of higher HIF1A expression (P=0.06) and reduced adipogenesis (PPARG P=0.006). After CPAP, only expression of the lipid deposition marker LPL increased (30%, P=0.047). ConclusionsAdipose tissue of awake OSAS subjects appears no more hypoxic than adipose tissue of BMI-matched controls despite daytime hypoxaemia. Increased adipose tissue blood flow may be explained by an increased inflammatory response. We observe features of adipose dysfunction in subjects with OSAS, which attribute to increased cardiometabolic risk associated with this condition.

Textbook of Limnology

Textbook of Limnology

AgNO3 dependant modulation of glucose mediated respiration kinetics in Escherichia coli at different pH and temperature.

The inhibitory role of AgNO3 on glucose-mediated respiration in Escherichia coli has been investigated as a function of pH and temperature using Clark-type electrode, environmental scanning electron microscopy, and computational tools. In the given concentration of bacterial suspension (1 × 108 CFU/ml), E. coli showed an increasing nonlinear trend of tetra-phasic respiration between 1-133 μM glucose concentration within 20 min. The glucose concentrations above 133 μM did not result any linear increment in respiration but rather showed a partial inhibition at higher glucose concentrations (266-1066 μM). In the presence of glucose, AgNO3 caused a concentration-dependent (47-1960 μM) inhibition of the respiration rate within 4 min of its addition. The respiration rate was the highest at pH 7-8 and then was decreased on either side of this pH range. The inhibitory action of AgNO3 upon bacterial respiration was the highest at 37 °C. The observations of the respiration data were well supported by the altered bacterial morphology as observed in electron microscopic study. Docking study indicated the AgNO3 binding to different amino acids of all respiratory complex enzymes in E. coli and thereby explaining its interference with the respiratory chain. Copyright © 2016 John Wiley & Sons, Ltd.