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Nutrients Jun 2018Evidence suggests a relevant role for liver and mitochondrial dysfunction in allergic disease. However, the role of hepatic mitochondrial function in food allergy is...
BACKGROUND
Evidence suggests a relevant role for liver and mitochondrial dysfunction in allergic disease. However, the role of hepatic mitochondrial function in food allergy is largely unknown. We aimed to investigate hepatic mitochondrial dysfunction in a murine model of peanut allergy.
METHODS
Three-week-old C3H/HeOuJ mice were sensitized by the oral route with peanut-extract (PNT). We investigated: 1. the occurrence of effective sensitization to PNT by analysing acute allergic skin response, anaphylactic symptoms score, body temperature, serum mucosal mast cell protease-1 (mMCP-1) and anti-PNT immunoglobulin E (IgE) levels; 2. hepatic involvement by analysing interleukin (IL)-4, IL-5, IL-13, IL-10 and IFN-γ mRNA expression; 3. hepatic mitochondrial oxidation rates and efficiency by polarography, and hydrogen peroxide (H₂O₂) yield, aconitase and superoxide dysmutase activities by spectrophotometry.
RESULTS
Sensitization to PNT was demonstrated by acute allergic skin response, anaphylactic symptoms score, body temperature decrease, serum mMCP-1 and anti-peanut IgE levels. Liver involvement was demonstrated by a significant increase of hepatic Th2 cytokines (IL-4, IL-5 and IL-13) mRNA expression. Mitochondrial dysfunction was demonstrated by lower state 3 respiration rate in the presence of succinate, decreased fatty acid oxidation in the presence of palmitoyl-carnitine, increased yield of ROS proven by the inactivation of aconitase enzyme and higher H₂O₂ mitochondrial release.
CONCLUSIONS
We provide evidence of hepatic mitochondrial dysfunction in a murine model of peanut allergy. These data could open the way to the identification of new mitochondrial targets for innovative preventive and therapeutic strategies against food allergy.
Topics: Allergens; Animals; Arachis; Disease Models, Animal; Energy Metabolism; Female; Immunoglobulin E; Interleukin-13; Interleukin-4; Interleukin-5; Liver; Mice, Inbred C3H; Mitochondria, Liver; Oxidation-Reduction; Oxidative Stress; Peanut Hypersensitivity; Plant Proteins; RNA, Messenger; Th2 Cells; Up-Regulation
PubMed: 29890625
DOI: 10.3390/nu10060744 -
Environmental Science and Pollution... Aug 2018Due to the importance of pollution monitoring in marine ecosystems and lack of a coherent and systematic investigation of heavy metal ions along the southern shores of...
Due to the importance of pollution monitoring in marine ecosystems and lack of a coherent and systematic investigation of heavy metal ions along the southern shores of the Caspian Sea, in the present study, the amount of these metals and As ions in coastal waters along its 780-km-long coast in Iran have been studied. Heavy metals (cobalt, nickel, copper, zinc, cadmium, mercury, lead) and a poisonous metalloid (arsenic) were selected in 59 sampling stations and determined using differential pulse polarography method. The multivariate statistical tools were applied to describe and interpret the experimental data. The overall mean concentrations of studied metals (in microgram per liter; μg L) in the samples were found in the order Zn (10.9) > Ni (7.4) > Cu (5.5) > Pb (1.9) > Hg (1.4) > As (1.3) > Co (1.1) > Cd (0.2). The results when compared with reported international standards confirmed that the sampled waters do contain some of these elements above the suggested maximum permissible limits. Hg and Cu were detected in 54.2 and 72.9% of the samples, almost all above the permissible limits. Ni, Zn, Pb, and Co were detected in 100, 96.6, 93.2, and 88.1%, respectively, while 8.5, 22.0, 3.4, and 1.7% were above the permissible limits. Cd and As were present in 61 and 93% of the samples, and their concentrations were higher than the rate presented by Russian System of Management Chemicals (RSMC). In addition, spatial distribution of heavy metal concentrations showed that Gorgan Bay is an ecosystem serving as a filter, trapping natural and anthropogenic materials that are brought from industrial, commercial, and urbanized areas. The multivariate data analysis reveals that Caspian Sea is contaminated by both anthropogenic as well as pedo-geochemical sources.
Topics: Arsenic; Environmental Monitoring; Iran; Metals, Heavy; Oceans and Seas; Seawater; Water Pollutants, Chemical
PubMed: 29876847
DOI: 10.1007/s11356-018-2455-7 -
Methods in Molecular Biology (Clifton,... 2018Interest in the investigation of mitochondrial dysfunction has seen a resurgence over recent years due to the implication of such dysfunction in both drug-induced...
Interest in the investigation of mitochondrial dysfunction has seen a resurgence over recent years due to the implication of such dysfunction in both drug-induced toxicity and a variety of disease states. Here we describe a methodology to assist in such investigations whereby the oxygen consumption of isolated mitochondria is assessed in a high-throughput fashion using a phosphorescent oxygen-sensitive probe , standard microtiter plates, and plate reader detection. The protocols provided describe the required isolation procedures, initial assay optimization, and subsequent compound screening. Typical data is also provided illustrating the expected activity levels as well as recommended plate maps and data analysis approaches.
Topics: Animals; Biological Assay; Cell Respiration; High-Throughput Screening Assays; Liver; Mitochondria; Myocytes, Cardiac; Oxygen; Oxygen Consumption; Polarography; Rats; Rats, Sprague-Dawley; Toxicity Tests
PubMed: 29850994
DOI: 10.1007/978-1-4939-7831-1_4 -
Methods in Molecular Biology (Clifton,... 2018Protocols for High-Resolution FluoRespirometry of intact cells, permeabilized cells, permeabilized muscle fibers, isolated mitochondria, and tissue homogenates offer...
Protocols for High-Resolution FluoRespirometry of intact cells, permeabilized cells, permeabilized muscle fibers, isolated mitochondria, and tissue homogenates offer sensitive diagnostic tests of integrated mitochondrial function using standard cell culture techniques, small needle biopsies of muscle, and mitochondrial preparation methods. Multiple substrate-uncoupler-inhibitor titration (SUIT) protocols for analysis of oxidative phosphorylation (OXPHOS) improve our understanding of mitochondrial respiratory control and the pathophysiology of mitochondrial diseases. Respiratory states are defined in functional terms to account for the network of metabolic interactions in complex SUIT protocols with stepwise modulation of coupling control and electron transfer pathway states. A regulated degree of intrinsic uncoupling is a hallmark of oxidative phosphorylation, whereas pathological and toxicological dyscoupling is evaluated as a mitochondrial defect. The noncoupled state of maximum respiration is experimentally induced by titration of established uncouplers (CCCP, FCCP, DNP) to collapse the protonmotive force across the mitochondrial inner membrane and measure the electron transfer (ET) capacity (open-circuit operation of respiration). Intrinsic uncoupling and dyscoupling are evaluated as the flux control ratio between non-phosphorylating LEAK respiration (electron flow coupled to proton pumping to compensate for proton leaks) and ET capacity. If OXPHOS capacity (maximally ADP-stimulated O flux) is less than ET capacity, the phosphorylation pathway contributes to flux control. Physiological substrate combinations supporting the NADH and succinate pathway are required to reconstitute tricarboxylic acid cycle function. This supports maximum ET and OXPHOS capacities, due to the additive effect of multiple electron supply pathways converging at the Q-junction. ET pathways with electron entry separately through NADH (pyruvate and malate or glutamate and malate) or succinate (succinate and rotenone) restrict ET capacity and artificially enhance flux control upstream of the Q-cycle, providing diagnostic information on specific ET-pathway branches. O concentration is maintained above air saturation in protocols with permeabilized muscle fibers to avoid experimental O limitation of respiration. Standardized two-point calibration of the polarographic oxygen sensor (static sensor calibration), calibration of the sensor response time (dynamic sensor calibration), and evaluation of instrumental background O flux (systemic flux compensation) provide the unique experimental basis for high accuracy of quantitative results and quality control in High-Resolution FluoRespirometry.
Topics: Animals; Biopsy; Biopsy, Needle; Calibration; Cell Culture Techniques; Cell Membrane Permeability; Cell Respiration; Electron Transport; Fluorometry; HEK293 Cells; Humans; Mice; Mitochondria, Muscle; Muscle Fibers, Skeletal; Oxidative Phosphorylation; Oxygen Consumption; Polarography
PubMed: 29850993
DOI: 10.1007/978-1-4939-7831-1_3 -
Methods in Molecular Biology (Clifton,... 2018In many studies, the evaluation of mitochondrial function is critical to understand how disease conditions or xenobiotics alter mitochondrial function. One of the...
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.
Topics: Animals; Cell Culture Techniques; Cell Membrane Permeability; Cell Respiration; Detergents; Electrodes; Electron Transport Chain Complex Proteins; Electroporation; Humans; Lymphocytes; Membranes, Artificial; Mitochondria; Muscle Cells; Neutrophils; Oxidative Phosphorylation; Oxygen; Oxygen Consumption; Polarography; Rats; Rats, Wistar
PubMed: 29850992
DOI: 10.1007/978-1-4939-7831-1_2 -
Analytical Biochemistry Jul 2018In modern biomedical science, a descriptive study is no longer the major focus of many fields. More researchers are now seeking approaches that will help them obtain... (Review)
Review
In modern biomedical science, a descriptive study is no longer the major focus of many fields. More researchers are now seeking approaches that will help them obtain maximum information from a single sample or model, which will allow them to make more detailed conclusions than previously about mechanisms that underlie certain phenomena. Clearly, simultaneous measurement of multiple parameters will provide more useful information compared to that which can be assessed through parallel studies with multiple single-parameter measurements. Mitochondria are actively involved in the regulation of a number of biochemical processes that are vitally important for normal cell functioning. Dysregulation of cell metabolism occurs under multiple pathological conditions. While changes in mitochondrial and cellular functioning are related to each other, understanding of the details of most mechanisms underlying these relationships are still unknown. It would be appropriate to have an instrument that will help to uncover sequences of events and temporal links among the parameters that involve functional mitochondrial and cellular integration. The current review is focused on the analysis of these technological limitations, and, based on the combined approach, provides hypothetical suggestion on how possibly to create such an instrument.
Topics: Calcium; Electrochemical Techniques; Electrodes; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Swelling; Reactive Oxygen Species
PubMed: 29486206
DOI: 10.1016/j.ab.2018.02.020 -
Methods in Molecular Biology (Clifton,... 2018Mitochondrial oxidative phosphorylation is central for generating ATP and maintaining energy homeostasis in most eukaryotic cells. The ex vivo measurement of...
Mitochondrial oxidative phosphorylation is central for generating ATP and maintaining energy homeostasis in most eukaryotic cells. The ex vivo measurement of mitochondrial oxygen consumption rates in intact cells or isolated organelles is a valuable approach to assess mitochondrial bioenergetics in various experimental conditions. In this chapter, we describe several step-by-step protocols for measuring mitochondrial respiration in intact cells, permeabilized cells (in situ mitochondria), and isolated organelles using both Clark-type polarographic oxygen electrode devices and the newly developed oxygen-sensing fluorophore-based Seahorse technology.
Topics: Animals; Cell Respiration; Cells, Cultured; Electrodes; Hepatocytes; Liver; Mice; Mitochondria; Oxidative Phosphorylation; Oxygen; Oxygen Consumption; Polarography; Primary Cell Culture
PubMed: 29480482
DOI: 10.1007/978-1-4939-7598-3_18 -
Journal of Inorganic Biochemistry Mar 2018Previous studies generally agree that in the blood serum vanadium is transported mainly by human serum transferrin (hTF). In this work through the combined use of...
Previous studies generally agree that in the blood serum vanadium is transported mainly by human serum transferrin (hTF). In this work through the combined use of electrochemical techniques, matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry and small-angle X-ray scattering (SAXS) data it is confirmed that both V and V bind to apo-hTF and holo-hTF. The electrochemical behavior of solutions containing vanadate(V) solutions at pH=7.0, analyzed by using two different voltammetric techniques, with different time windows, at a mercury electrode, Differential Pulse Polarography (DPP) and Cyclic Voltammetry (CV), is consistent with a stepwise reduction of V→V and V→V. Globally the voltammetric data are consistent with the formation of 2:1 complexes in the case of the system V-apo-hTF and both 1:1 and 2:1 complexes in the case of V-holo-hTF; the corresponding conditional formation constants were estimated. MALDI-TOF mass spectrometric data carried out with samples of VOSO and apo-hTF and of NHVO with both apo-hTF and holo-hTF with V:hTF ratios of 3:1 are consistent with the binding of vanadium to the proteins. Additionally the SAXS data suggest that both VOSO and NaVO can effectively interact with human apo-transferrin, but for holo-hTF no clear evidence was obtained supporting the existence or the absence of protein-ligand interactions. This latter data suggest that the conformation of holo-hTF does not change in the presence of either VOSO or NHVO. Therefore, it is anticipated that V or V bound to holo-hTF may be efficiently up-taken by the cells through receptor-mediated endocytosis of hTF.
Topics: Electrochemical Techniques; Endocytosis; Humans; Molecular Conformation; Protein Binding; Scattering, Small Angle; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Transferrin; Vanadium; X-Ray Diffraction
PubMed: 29355752
DOI: 10.1016/j.jinorgbio.2017.12.012 -
Oncotarget Oct 2017Components of the mitochondrial electron transport chain have recently gained much interest as potential therapeutic targets. Since mitochondria are essential for the...
Components of the mitochondrial electron transport chain have recently gained much interest as potential therapeutic targets. Since mitochondria are essential for the supply of energy that is required for both angiogenic and tumourigenic activity, targeting the mitochondria represents a promising potential therapeutic approach for treating cancer. Here we investigate the established anti-angiogenesis drugs combretastatin A4, thalidomide, OGT 2115 and tranilast that we hypothesise are able to exert a direct anti-cancer effect in the absence of vasculature by targeting the mitochondria. Drug cytotoxicity was measured using the MTT assay. Mitochondrial function was measured in intact isolated mitochondria using polarography, fluorimetry and enzymatic assays to measure mitochondrial oxygen consumption, membrane potential and complex I-IV activities respectively. Combretastatin A4, OGT 2115 and tranilast were both shown to decrease mitochondrial oxygen consumption. OGT 2115 and tranilast decreased mitochondrial membrane potential and reduced complex I activity while combretastatin A4 and thalidomide did not. OGT 2115 inhibited mitochondrial complex II-III activity while combretastatin A4, thalidomide and tranilast did not. Combretastatin A4, thalidomide and OGT 2115 induced bi-phasic concentration-dependent increases and decreases in mitochondrial complex IV activity while tranilast had no evident effect. These data demonstrate that combretastatin A4, thalidomide, OGT 2115 and tranilast are all mitochondrial modulators. OGT 2115 and tranilast are both mitochondrial inhibitors capable of eliciting concentration-dependent reductions in cell viability by decreasing mitochondrial membrane potential and oxygen consumption.
PubMed: 29179466
DOI: 10.18632/oncotarget.20858 -
Journal of Inherited Metabolic Disease Mar 2018Propionic acidemia (PA) is a classical inborn error of metabolism with high morbidity that results from the inability of the propionyl-CoA carboxylase (PCC) enzyme to...
UNLABELLED
Propionic acidemia (PA) is a classical inborn error of metabolism with high morbidity that results from the inability of the propionyl-CoA carboxylase (PCC) enzyme to convert propionyl-CoA to methylmalonyl-CoA. PA is inherited in an autosomal recessive fashion due to functional loss of both alleles of either PCCA or PCCB. These genes are highly conserved across evolutionarily diverse species and share extensive similarity with pcca-1 and pccb-1 in the nematode, Caenorhabditis elegans. Here, we report the global metabolic effects of deletion in a single PCC gene, either pcca-1 or pccb-1, in C. elegans. Animal lifespan was significantly reduced relative to wild-type worms in both mutant strains, although to a greater degree in pcca-1. Mitochondrial oxidative phosphorylation (OXPHOS) capacity and efficiency as determined by direct polarography of isolated mitochondria were also significantly reduced in both mutant strains. While in vivo quantitation of mitochondrial physiology was normal in pccb-1 mutants, pcca-1 deletion mutants had significantly increased mitochondrial matrix oxidant burden as well as significantly decreased mitochondrial membrane potential and mitochondrial content. Whole worm steady-state free amino acid profiling by UPLC revealed reduced levels in both mutant strains of the glutathione precursor cysteine, possibly suggestive of increased oxidative stress. Intermediary metabolic flux analysis by GC/MS with 1,6-C-glucose further showed both PCC deletion strains had decreased accumulation of a distal tricarboxylic acid (TCA) cycle metabolic intermediate (+1 malate), isotopic enrichment in a proximal TCA cycle intermediate (+1 citrate), and increased +1 lactate accumulation. GC/MS analysis further revealed accumulation in the PCC mutants of a small amount of 3-hydroxypropionate, which appeared to be metabolized in C. elegans to oxalate through a unique metabolic pathway. Collectively, these detailed metabolic investigations in translational PA model animals with genetic-based PCC deficiency reveal their significantly dysregulated energy metabolism at multiple levels, including reduced mitochondrial OXPHOS capacity, increased oxidative stress, and inhibition of distal TCA cycle flux, culminating in reduced animal lifespan. These findings demonstrate that the pathophysiology of PA extends well beyond what has classically been understood as a single PCC enzyme deficiency with toxic precursor accumulation, and suggest that therapeutically targeting the globally disrupted energy metabolism may offer novel treatment opportunities for PA.
SUMMARY
Two C. elegans model animals of propionic acidemia with single-gene pcca-1 or pccb-1 deletions have reduced lifespan with significantly reduced mitochondrial energy metabolism and increased oxidative stress, reflecting the disease's broader pathophysiology beyond a single enzyme deficiency with toxic precursor accumulation.
Topics: Animals; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Disease Models, Animal; Energy Metabolism; Gene Deletion; Genetic Predisposition to Disease; Longevity; Membrane Potential, Mitochondrial; Methylmalonyl-CoA Decarboxylase; Mitochondria; Oxidative Stress; Phenotype; Propionic Acidemia
PubMed: 29159707
DOI: 10.1007/s10545-017-0111-x