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American Journal of Physiology.... Aug 2019High energy expenditure is reported in cystic fibrosis (CF) animal models and patients. Alterations in skeletal muscle oxidative capacity, fuel utilization, and the...
High energy expenditure is reported in cystic fibrosis (CF) animal models and patients. Alterations in skeletal muscle oxidative capacity, fuel utilization, and the creatine kinase-phosphocreatine system suggest mitochondrial dysfunction. Studies were performed on congenic C57BL/6J and F508del () mice. Indirect calorimetry was used to measure gas exchange to evaluate aerobic capacity during treadmill exercise. The bioenergetic function of skeletal muscle subsarcolemmal (SSM) and interfibrillar mitochondria (IFM) was evaluated using an integrated approach combining measurement of the rate of oxidative phosphorylation by polarography and of electron transport chain activities by spectrophotometry. CF mice have reduced maximal aerobic capacity. In SSM of these mice, oxidative phosphorylation was impaired in the presence of complex I, II, III, and IV substrates except when glutamate was used as substrate. This impairment appeared to be caused by a defect in complex V activity, whereas the oxidative system of the electron transport chain was unchanged. In IFM, oxidative phosphorylation and electron transport chain activities were preserved, whereas complex V activity was reduced, in CF. Furthermore, creatine kinase activity was reduced in both SSM and IFM of CF skeletal muscle. The decreased complex V activity in SSM resulted in reduced oxidative phosphorylation, which could explain the reduced skeletal muscle response to exercise in CF mice. The decrease in mitochondrial creatine kinase activity also contributed to this poor exercise response.
Topics: Animals; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Energy Metabolism; Female; Male; Mice; Mice, Inbred C57BL; Mice, Inbred CFTR; Mice, Transgenic; Mitochondria, Muscle; Muscle, Skeletal; Oxidative Phosphorylation; Oxidative Stress; Physical Conditioning, Animal; Sequence Deletion
PubMed: 31211618
DOI: 10.1152/ajpendo.00064.2019 -
Natural Product Research May 2021This study was focused on screening of the total antioxidant activity of the selected extracts of the mushroom and quinic acid, one of their antidiabetic ingredients,...
Polarography as a technique of choice for the evaluation of total antioxidant activity: The case study of selected extracts and quinic acid, their antidiabetic ingredient.
This study was focused on screening of the total antioxidant activity of the selected extracts of the mushroom and quinic acid, one of their antidiabetic ingredients, by an uncommon electrochemical assay. Indeed, direct current (DC) polarographic HydroxoPerhydroxo Mercury(II) Complex (HPMC) assay based on decrease of anodic limiting current originating from HPMC formation in alkaline solutions of hydrogen peroxide at potential of mercury dissolution, observed upon gradual addition of antioxidants, was applied herein for the estimation of the natural products' antioxidativity. Quinic acid was found to exhibit most promising antioxidant potential (4.0 ± 0.2%µL) being ≈ 2-fold more active than the screened extract samples. Actually, such a finding puts some light on the antioxidativity of cyclic polyols, well understimated class of organic compounds, compared to aromatic (poly)phenolics. As a low cost, easy-to handle and accurate this polarographic assay may be thoroughly recommended for much broader use. [Formula: see text].
Topics: Antioxidants; Biological Products; Coprinus; Hypoglycemic Agents; Mycelium; Polarography; Quinic Acid
PubMed: 31192702
DOI: 10.1080/14786419.2019.1628753 -
Natural Product Research Jan 2021This study focuses on the impact of low and high temperature regimes on the chemical composition and antioxidativity of low sugar berry fruits jellies. High quality...
This study focuses on the impact of low and high temperature regimes on the chemical composition and antioxidativity of low sugar berry fruits jellies. High quality fruits (strawberry, raspberry and blackberry) were collected from Western Serbia region, quite well recognised both nationally and internationally due to an extremely well developed practice in growing berry fruits. The obtained results have clearly indicated the importance of low temperature regime for enriched contents of both total phenolics and anthocyanins followed by an enhanced antioxidativity. L. Čačak Thornless cultivar, the only autochthonous berry fruit variety screened herein, may be firmly recommended as a raw material for industrial production of low sugar blackberry jellies with exceptional characteristics. This innovative procedure of preparing berry fruit jellies have encompassed the application of low temperature regime (55 °C), lower content of sugar (40%), seeds separation from jellies followed by no use of pectin throughout the whole process.
Topics: Anthocyanins; Antioxidants; Food-Processing Industry; Fragaria; Fruit; Gels; Phenols; Rubus; Serbia; Sugars; Temperature
PubMed: 31140309
DOI: 10.1080/14786419.2019.1622109 -
Journal of Cardiothoracic Surgery May 2019Mitochondrial impairment can result from myocardial ischemia reperfusion injury (IR). Despite cardioplegic arrest, IR-associated cardiodepression is a major problem in...
BACKGROUND
Mitochondrial 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 Ca homeostasis of rat cardiac subsarcolemmal mitochondria (SSM).
METHODS
Wistar 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 Ca-handling in terms of Ca-induced swelling and Ca-uptake/release (Calcium Green-5 N® fluorescence).
RESULTS
LV 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 Ca-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). Ca-uptake remained unaffected by previous IR. Though Ca-release was delayed for ≥30 min of ischemia (p < 0.01), Ca 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).
CONCLUSIONS
Ischemia prolongation in IR injury gradually impaired SSM in terms of respiratory chain function and Ca-homeostasis. Membrane hyperpolarization appears to be responsible for impaired Ca-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.
Topics: Animals; Calcium; Cardiac Surgical Procedures; Cations; Disease Models, Animal; Electron Transport; Heart Arrest, Induced; Intracellular Membranes; Isolated Heart Preparation; Male; Mitochondria, Heart; Myocardial Reperfusion Injury; Myocardium; Myocytes, Cardiac; Oxygen Consumption; Rats; Rats, Wistar; Recovery of Function; Sarcolemma; Time Factors; Warm Ischemia
PubMed: 31088484
DOI: 10.1186/s13019-019-0911-1 -
Biochimica Et Biophysica Acta. General... Aug 20194-hydroxyphenylacetic acid (HO-PAA) is produced by intestinal microbiota from L-tyrosine. High concentrations in human fecal water have been associated with...
BACKGROUND
4-hydroxyphenylacetic acid (HO-PAA) is produced by intestinal microbiota from L-tyrosine. High concentrations in human fecal water have been associated with cytotoxicity, urging us to test HO-PAA's effects on human colonocytes. We compared these effects with those of phenylacetic acid (PAA), phenol and acetaldehyde, also issued from amino acids fermentation.
METHODS
HT-29 Glc human colonocytes were exposed for 24 h to metabolites at concentrations between 350 and 1000 μM for HO-PAA and PAA, 250-1500 μM for phenol and 25-500 μM for acetaldehyde. We evaluated metabolites'cytotoxicity with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide and DNA quantification assays, reactive oxygen species (ROS) production with H2DCF-DA, and DNA damage with the comet assay. We measured cell oxygen consumption and mitochondrial complexes activity by polarography.
RESULTS
Although HO-PAA displayed no cytotoxic effect on colonocytes, it decreased mitochondrial complex I activity and oxygen consumption. This was paralleled by an increase in ROS production and DNA alteration. Cells pretreatment with N-acetylcysteine, a ROS scavenger, decreased genotoxic effects of HO-PAA, indicating implication of oxidative stress in HO-PAA's genotoxicity. PAA and phenol did not reproduce these effects, but were cytotoxic towards colonocytes. Last, acetaldehyde displayed no effect in terms of cytotoxicity and mitochondrial metabolic activity, but increased DNA damage.
CONCLUSIONS
Several bacterial metabolites produced from amino acids displayed deleterious effects on human colonocytes, in terms of genotoxicity (HO-PAA and acetaldehyde) or cytotoxicity (PAA and phenol).
GENERAL SIGNIFICANCE
This study helps understanding the consequences of intestinal microbiota's metabolic activity on the host since amino acids fermentation can lead to the formation of compounds toxic towards colonic epithelial cells.
Topics: Amino Acids; Bacteria; Colon; DNA; HT29 Cells; Humans; In Vitro Techniques; Oxidative Stress; Oxygen Consumption
PubMed: 31034912
DOI: 10.1016/j.bbagen.2019.04.018 -
Biochimica Et Biophysica Acta.... Jul 2019Information on binding and rearrangement of pivotal water molecules could support understanding of light-driven water oxidation at the catalytic MnCaO cluster of...
Information on binding and rearrangement of pivotal water molecules could support understanding of light-driven water oxidation at the catalytic MnCaO cluster of photosystem II (PSII). To address this point, the binding of ammonia (NH)-a possible substrate-water analogue-has been investigated and discussed in the context of putative reaction mechanisms. By time-resolved detection of O formation after light-flash excitation, we discriminate three NH/NH binding sites jointly characterized by a K value around 25 mM (of NH), but differing in their influence on the O-formation step. At 100 mM NHCl (pH 7.5), we observe (1) a PSII fraction with complete inhibition of O-formation, (2) fast O-formation with a time constant of 1.7 ms at 20 °C (Fast-PSII), and (3) slow O-formation with a time constant of 36 ms at 20 °C (Slow-PSII). For the Fast-PSII, we determine an activation enthalpy of 223 ± 11 meV. Activation enthalpy and entropy of the Fast-PSII are essentially identical to the corresponding figures in the absence NH/NH binding. For the Slow-PSII, the activation enthalpy is 323 ± 11 meV and thus significantly increased, whereas the activation entropy remains essentially unchanged. We conclude: (1) The fully-inhibitory binding site could relate to bound NH replacing one of the two substrate-water molecules. (2) The Fast-PSII may relate to NH/NH binding in the S-state of PSII followed by unbinding before onset of the OO bond formation step, but also more intricate mechanisms are not excluded. (3) In the Slow-PSII, NH/NH binding increases the energetic barrier of the OO bond formation step significantly.
Topics: Ammonia; Models, Molecular; Oxidation-Reduction; Oxygen; Photosystem II Protein Complex; Spinacia oleracea; Water
PubMed: 31034801
DOI: 10.1016/j.bbabio.2019.04.005 -
Analytical and Bioanalytical Chemistry May 2019The origin of the method of standard additions (SAM) and of the use of internal standard (IS) in instrumental chemical analysis and their spread into other areas has...
The origin of the method of standard additions (SAM) and of the use of internal standard (IS) in instrumental chemical analysis and their spread into other areas has been reviewed. Recorded applications of IS range from flame spectroscopy in 1877 through multiple techniques to current use in NMR and standard additions with isotopically labelled internal standards in hyphenated techniques. For SAM, applications stemmed from polarography in 1937 and spread to most instrumental methods. Some misconceptions on priorities are corrected.
PubMed: 30941480
DOI: 10.1007/s00216-019-01754-w -
Structural Dynamics (Melville, N.Y.) Mar 2019Time-resolved crystallography is a powerful technique to elucidate molecular mechanisms at both spatial (angstroms) and temporal (picoseconds to seconds) resolutions. We...
Time-resolved crystallography is a powerful technique to elucidate molecular mechanisms at both spatial (angstroms) and temporal (picoseconds to seconds) resolutions. We recently discovered an unusually slow reaction at room temperature that occurs on the order of days: the reverse oxidative decay of the chemically labile (6S)-5,6,7,8-tetrahydrofolate in complex with its producing enzyme dihydrofolate reductase. Here, we report the critical analysis of a representative dataset at an intermediate reaction time point. A quinonoid-like intermediate state lying between tetrahydrofolate and dihydrofolate features a near coplanar geometry of the bicyclic pterin moiety, and a tetrahedral C6 geometry is proposed based on the apparent mFo-DFc omit electron densities of the ligand. The presence of this intermediate is strongly supported by Bayesian difference refinement. Isomorphous Fo-Fo difference map and multi-state refinement analyses suggest the presence of end-state ligand populations as well, although the putative intermediate state is likely the most populated. A similar quinonoid intermediate previously proposed to transiently exist during the oxidation of tetrahydrofolate was confirmed by polarography and UV-vis spectroscopy to be relatively stable in the oxidation of its close analog tetrahydropterin. We postulate that the constraints on the ligand imposed by the interactions with the protein environment might be the origin of the slow reaction observed by time-resolved crystallography.
PubMed: 30868089
DOI: 10.1063/1.5086436 -
Die Pharmazie Jan 2019The electrochemical behavior of the echinocandin antifungals anidulafungin (AF) and micafungin (MF) has been investigated by differential pulse polarography (DPP). The...
The electrochemical behavior of the echinocandin antifungals anidulafungin (AF) and micafungin (MF) has been investigated by differential pulse polarography (DPP). The measurements were carried out in a supporting electrolyte solution consisting of Britton-Robinson buffer and methanol at various substance concentrations and pH values. An amperometric cell with a three electrode system consisting of a dropping mercury electrode (DME) as working electrode, an auxiliary platinum electrode and an Ag/AgCl reference electrode was used in all experiments. AF was electrochemically reduced at potentials between -1.3 and -1.5 V. MF showed a first reduction peak (a) between -1.0 and -1.4 V and a second peak (b) between -1.5 and -1.8 V. A strong pH-dependence was observed, with optimal results at pH 2.0-3.0 for the AF peak, pH 2.0 for the MF peak (a) and pH 5.0 for the MF peak (b). A linear correlation between the concentration and the peak current has been demonstrated for all reduction peaks. MF peak (a) showed a similar behavior to the AF peak regarding shape, peak current and pH-dependence. Therefore, it can be assumed that both reductions are based on the same mechanism, a two-step reduction of the N-acyl group.
Topics: Anidulafungin; Antifungal Agents; Electrochemistry; Electrodes; Hydrogen-Ion Concentration; Mercury; Micafungin; Polarography
PubMed: 30782245
DOI: 10.1691/ph.2019.8680 -
Physiologia Plantarum May 2019High solar flux is known to diminish photosynthetic growth rates, reducing biomass productivity and lowering disease tolerance. Photosystem II (PSII) of plants is...
High solar flux is known to diminish photosynthetic growth rates, reducing biomass productivity and lowering disease tolerance. Photosystem II (PSII) of plants is susceptible to photodamage (also known as photoinactivation) in strong light, resulting in severe loss of water oxidation capacity and destruction of the water-oxidizing complex (WOC). The repair of damaged PSIIs comes at a high energy cost and requires de novo biosynthesis of damaged PSII subunits, reassembly of the WOC inorganic cofactors and membrane remodeling. Employing membrane-inlet mass spectrometry and O -polarography under flashing light conditions, we demonstrate that newly synthesized PSII complexes are far more susceptible to photodamage than are mature PSII complexes. We examined these 'PSII birth defects' in barley seedlings and plastids (etiochloroplasts and chloroplasts) isolated at various times during de-etiolation as chloroplast development begins and matures in synchronization with thylakoid membrane biogenesis and grana membrane formation. We show that the degree of PSII photodamage decreases simultaneously with biogenesis of the PSII turnover efficiency measured by O -polarography, and with grana membrane stacking, as determined by electron microscopy. Our data from fluorescence, Q -inhibitor binding, and thermoluminescence studies indicate that the decline of the high-light susceptibility of PSII to photodamage is coincident with appearance of electron transfer capability Q → Q during de-etiolation. This rate depends in turn on the downstream clearing of electrons upon buildup of the complete linear electron transfer chain and the formation of stacked grana membranes capable of longer-range energy transfer.
Topics: Chloroplasts; Mass Spectrometry; Microscopy, Electron; Organelle Biogenesis; Photosynthesis; Photosystem II Protein Complex
PubMed: 30693529
DOI: 10.1111/ppl.12932