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Methods in Molecular Biology (Clifton,... 2022The Seahorse Extracellular Flux Analyzer enables the high-throughput characterization of oxidative phosphorylation capacity based on the electron transport chain...
The Seahorse Extracellular Flux Analyzer enables the high-throughput characterization of oxidative phosphorylation capacity based on the electron transport chain organization and regulation with relatively small amount of material. This development over the traditional polarographic Clark-type electrode approaches make it possible to analyze the respiratory features of mitochondria isolated from tissue samples of particular animal models. Here we provide a description of an optimized approach to carry out multi-well measurement of O consumption, with the Agilent Seahorse XFe96 analyzer on mouse brain and muscles to determine the tissue-specific oxidative phosphorylation properties. Protocols include the preparation of the tissue samples, isolation of mitochondria, and analysis of their function; in particular, the preparation and optimization of the reagents and samples.
Topics: Animals; Electron Transport; Mice; Mitochondria; Oxidative Phosphorylation; Oxygen Consumption; Polarography; Smegmamorpha
PubMed: 35771439
DOI: 10.1007/978-1-0716-2309-1_8 -
Journal of Visualized Experiments : JoVE Jun 2022The mitochondrial permeability transition pore (mPTP) is a voltage-gated, nonselective, inner mitochondrial membrane (IMM) mega-channel important in health and disease....
The mitochondrial permeability transition pore (mPTP) is a voltage-gated, nonselective, inner mitochondrial membrane (IMM) mega-channel important in health and disease. The mPTP mediates leakage of protons across the IMM during low-conductance opening and is specifically inhibited by cyclosporine A (CsA). Coenzyme Q (CoQ) is a regulator of the mPTP, and tissue-specific differences have been found in CoQ content and open probability of the mPTP in forebrain and heart mitochondria in a newborn mouse model of fragile X syndrome (FXS, Fmr1 knockout). We developed a technique to determine the voltage threshold for mPTP opening in this mutant strain, exploiting the role of the mPTP as a proton leak channel. To do so, oxygen consumption and membrane potential (ΔΨ) were simultaneously measured in isolated mitochondria using polarography and a tetraphenylphosphonium (TPP) ion-selective electrode during leak respiration. The threshold for mPTP opening was determined by the onset of CsA-mediated inhibition of proton leak at specific membrane potentials. Using this approach, differences in voltage gating of the mPTP were precisely defined in the context of CoQ excess. This novel technique will permit future investigation for enhancing the understanding of physiological and pathological regulation of low-conductance opening of the mPTP.
Topics: Animals; Mice; Calcium; Cyclosporine; Fragile X Mental Retardation Protein; Mitochondria, Heart; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Probability; Protons; Reactive Oxygen Species; Ubiquinone
PubMed: 35723461
DOI: 10.3791/63646 -
Food Chemistry Nov 2022In this study, enhancement of electrochemical performance of electrochemically reduced graphene oxide (ERGO) on a screen-printed carbon electrode (SPCE) (ERGO/SPCE)...
In this study, enhancement of electrochemical performance of electrochemically reduced graphene oxide (ERGO) on a screen-printed carbon electrode (SPCE) (ERGO/SPCE) coupled with ion-pairing (cetyltrimethylammonium bromide, CTAB) for the determination of iodide in table salt has been described. The electrode modification of ERGO/SPCE was conducted using cyclic voltammetric (CV) scanning in the potential range of 1.3-0.4 V for 50 cycles after the drop-casting of graphene oxide (GO) suspension on the SPCE. It was found that the electro-active surface area of ERGO/SPCE was increased by 1.5-fold compared to the bare SPCE. ERGO/SPCE sensor displays linearity towards iodide in the concentration range from 0.020 to 1.0 mg/L (sensitivity = 5.40 µA(mg/L), R = 0.9906) with the limit of detection (LOD) and limit of quantitation (LOQ) of 0.070 mg/L and 0.21 mg/L, respectively. The comparison between polarography and ERGO/SPCE sensor was in good agreement.
Topics: Carbon; Electrochemical Techniques; Electrodes; Graphite; Iodides; Salts; Sodium Chloride, Dietary
PubMed: 35667176
DOI: 10.1016/j.foodchem.2022.133382 -
Medicina (Kaunas, Lithuania) Mar 2022Background and Objectives: Due to the nutritional and behavioral patterns of children, their teeth can be a good indicator of heavy metal uptake from over the years. To...
Background and Objectives: Due to the nutritional and behavioral patterns of children, their teeth can be a good indicator of heavy metal uptake from over the years. To determine the amount of Zn, Cu, Cd, and Pb accumulated in the body, primary teeth of children in Zanjan, Iran, were examined with a polarography device. Materials and Methods: Samples were collected from dentistry clinics of Zanjan, Iran, and were prepared for acid digestion, and then were analyzed by a polarography device for determining the concentration of lead, copper, zinc, and cadmium. Results: Data were analyzed by a t-independent test to compare different groups (p < 0.05). Based on the results obtained from this study, the mean concentrations of zinc, lead, copper, and cadmium were 245, 7.66, 5.33, and 0.0879 µg/g, respectively, which shows that the amount of each of the four elements was more than the amounts that have been reported for different countries. The results showed no significant difference between age, tooth type, and jaw groups. Conclusions: We conclude that primary teeth are an important biological indicator to evaluate the concentration of heavy elements in the human body. The high concentrations of these elements in the primary teeth analyzed in this study could be attributed to the high concentrations of these elements in the environment of Zanjan.
Topics: Cadmium; Child; Dentistry; Humans; Metals, Heavy; Polarography; Zinc
PubMed: 35334624
DOI: 10.3390/medicina58030448 -
NeuroImage Feb 2022Resting-state functional MRI (rsfMRI) provides a view of human brain organization based on correlation patterns of blood oxygen level dependent (BOLD) signals recorded...
Resting-state functional MRI (rsfMRI) provides a view of human brain organization based on correlation patterns of blood oxygen level dependent (BOLD) signals recorded across the whole brain. The neural basis of resting-state BOLD fluctuations and their correlation remains poorly understood. We simultaneously recorded oxygen level, spikes, and local field potential (LFP) at multiple sites in awake, resting monkeys. Following a spike, the average local oxygen and LFP voltage responses each resemble a task-driven BOLD response, with LFP preceding oxygen by 0.5 s. Between sites, features of the long-range correlation patterns of oxygen, LFP, and spikes are similar to features seen in rsfMRI. Most of the variance shared between sites lies in the infraslow frequency band (0.01-0.1 Hz) and in the infraslow envelope of higher-frequency bands (e.g. gamma LFP). While gamma LFP and infraslow LFP are both strong correlates of local oxygen, infraslow LFP explains significantly more of the variance shared between correlated oxygen signals than any other electrophysiological signal. Together these findings are consistent with a causal relationship between infraslow LFP and long-range oxygen correlations in the resting state.
Topics: Animals; Brain; Brain Mapping; Electrophysiological Phenomena; Image Processing, Computer-Assisted; Magnetic Resonance Imaging; Oxygen; Primates; Rest
PubMed: 34923136
DOI: 10.1016/j.neuroimage.2021.118728 -
Frontiers in Nutrition 2021Fatty acid overload, either of the saturated palmitic acid (PA) or the unsaturated oleic acid (OA), causes triglyceride accumulation into specialized organelles termed...
Fatty acid overload, either of the saturated palmitic acid (PA) or the unsaturated oleic acid (OA), causes triglyceride accumulation into specialized organelles termed lipid droplets (LD). However, only PA overload leads to liver damage mediated by mitochondrial dysfunction. Whether these divergent outcomes stem from differential effects of PA and OA on LD and mitochondria joint dynamics remains to be uncovered. Here, we contrast how both fatty acids impact the morphology and interaction between both organelles and mitochondrial bioenergetics in HepG2 cells. Using confocal microscopy, we showed that short-term (2-24 h) OA overload promotes more and bigger LD accumulation than PA. Oxygen polarography indicated that both treatments stimulated mitochondrial respiration; however, OA favored an overall build-up of the mitochondrial potential, and PA evoked mitochondrial fragmentation, concomitant with an ATP-oriented metabolism. Even though PA-induced a lesser increase in LD-mitochondria proximity than OA, those LD associated with highly active mitochondria suggest that they interact mainly to fuel fatty acid oxidation and ATP synthesis (that is, metabolically "active" LD). On the contrary, OA overload seemingly stimulated LD-mitochondria interaction mainly for LD growth (thus metabolically "passive" LDs). In sum, these differences point out that OA readily accumulates in LD, likely reducing their toxicity, while PA preferably stimulates mitochondrial oxidative metabolism, which may contribute to liver damage progression.
PubMed: 34869541
DOI: 10.3389/fnut.2021.775382 -
International Journal of Molecular... Aug 2021The effect of weightlessness on gametogenesis and the functional state of female germ cells are still poorly understood. We studied the ovaries of , the full development...
The effect of weightlessness on gametogenesis and the functional state of female germ cells are still poorly understood. We studied the ovaries of , the full development cycle of which (from zygote to sexually mature adults) passed under simulated microgravity by a random positioning machine. The rate of cellular respiration was studied by polarography as a parameter reflecting the functional state of mitochondria. The content of cytoskeletal proteins and histones was determined using Western blotting. The relative content of mRNA was determined using qRT-PCR. The results obtained indicated an increase in the rate of cellular respiration under simulated microgravity conditions during the full cycle of gametogenesis in due to complex I of the respiratory chain. In addition, an increase in the contents of actin cytoskeleton components was observed against the background of an increase in the mRNA content of the cytoskeleton's encoding genes. Moreover, we observed an increase in the relative content of histone H3 acetylated at Lys9 and Lys27, which may explain the increase in the expression of cytoskeletal genes. In conclusion, the formation of an adaptive pattern of functioning of the ovaries that developed under simulated microgravity includes structural and functional changes and epigenetic regulation.
Topics: Animals; Cell Respiration; Cytoskeleton; Drosophila melanogaster; Electron Transport Complex I; Female; Histones; Oogenesis; Ovary; Ovum; Transcriptome; Weightlessness Simulation
PubMed: 34502148
DOI: 10.3390/ijms22179234 -
International Journal of Molecular... Aug 2021Olivacine and ellipticine are model anticancer drugs acting as topoisomerase II inhibitors. Here, we present investigations performed on four olivacine derivatives in...
Olivacine and ellipticine are model anticancer drugs acting as topoisomerase II inhibitors. Here, we present investigations performed on four olivacine derivatives in light of their antitumor activity. The aim of this study was to identify the best antitumor compound among the four tested olivacine derivatives. The study was performed using CCRF/CEM and MCF-7 cell lines. Comet assay, polarography, inhibition of topoisomerase II activity, histone acetylation, and molecular docking studies were performed. Each tested compound displayed interaction with DNA and topoisomerase II, but did not cause histone acetylation. Compound (9-methoxy-5,6-dimethyl-1-({[1-hydroxy-2-(hydroxymethyl)butan-2-yl]amino}methyl)-6-pyrido[4,3-]carbazole) was found to be the best candidate as an anticancer drug because it had the highest affinity for topoisomerase II and caused the least genotoxic damage in cells.
Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Survival; DNA; DNA Topoisomerases, Type II; Ellipticines; Humans; Molecular Docking Simulation; Neoplasms; Topoisomerase II Inhibitors; Tumor Cells, Cultured
PubMed: 34445198
DOI: 10.3390/ijms22168492 -
International Journal of Analytical... 2021This review article represents the collection and discussion of various analytical methods available in the literature for the determination of allopurinol (ALLP) in... (Review)
Review
This review article represents the collection and discussion of various analytical methods available in the literature for the determination of allopurinol (ALLP) in pharmaceutical and biological samples consisting of HPLC, UV-visible method, near-IR spectroscopy, spectrofluorometry, capillary electrophoresis, polarography, voltammetry, and hyphenated techniques such as LC-MS, LC-MS/MS, UPLC-MS/MS, and GC-MS. The anticipated review provides details about the comparative utilization of various analytical techniques for the determination of ALLP. The present review article can be effectively explored to conduct future analytical investigation for the estimation of ALLP.
PubMed: 34194505
DOI: 10.1155/2021/5558651 -
Chinese Medicine Jun 2021Musk, the dried secretion from the musk sac gland which is located between the navel and genitals of mature male musk deer, is utilized as oriental medicine in east... (Review)
Review
Musk, the dried secretion from the musk sac gland which is located between the navel and genitals of mature male musk deer, is utilized as oriental medicine in east Asia. It has been utilized to treat conditions such as stroke, coma, neurasthenia, convulsions, and heart diseases in China since ancient times. This paper aims to provide a comprehensive overview of musk in zoology, chemical composition, pharmacology, clinical applications, and quality control according to the up-to-date literature. Studies found that musk mainly contains macrocyclic ketones, pyridine, steroids, fatty acids, amino acids, peptides, and proteins, whilst the main active ingredient is muscone. Modern pharmacological studies have proven that musk possesses potent anti-inflammatory effects, neuroprotective effects, anti-cancer effects, antioxidant effects, etc. Moreover, muscone, the main active ingredient, possesses anti-inflammatory, neuroprotective, antioxidant, and other pharmacological effects. In the quality control of musk, muscone is usually the main detection indicator, and the common analytical method is GC, and researchers have established novel and convenient methods such as HPLC-RI, RP-UPLC-ELSD, and Single-Sweep Polarography. In addition, quality evaluation methods based on steroids and the bioactivity of musk have been established. As for the identification of musk, due to various objective factors such as the availability of synthetic Muscone, it is not sufficient to rely on muscone alone as an identification index. To date, some novel technologies have also been introduced into the identification of musk, such as the electronic nose and DNA barcoding technology. In future research, more in vivo experiments and clinical studies are encouraged to fully explain the pharmacological effects and toxicity of musk, and more comprehensive methods are needed to evaluate and control the quality of musk.
PubMed: 34147113
DOI: 10.1186/s13020-021-00457-8