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Digitonin concentration is determinant for mitochondrial supercomplexes analysis by BlueNative page.Biochimica Et Biophysica Acta.... Jan 2021The BlueNative page (BNGE) gel has been the reference technique for studying the electron transport chain organization since it was established 20 years ago. Although...
The BlueNative page (BNGE) gel has been the reference technique for studying the electron transport chain organization since it was established 20 years ago. Although the migration of supercomplexes has been demonstrated being real, there are still several concerns about its ability to reveal genuine interactions between respiratory complexes. Moreover, the use of different solubilization conditions generates conflicting interpretations. Here, we thoroughly compare the impact of different digitonin concentrations on the liquid dispersions' physical properties and correlate with the respiratory complexes' migration pattern and supercomplexes. Our results demonstrate that digitonin concentration generates liquid dispersions with specific size and variability critical to distinguish between a real association of complexes from being trapped in the same micelle.
Topics: Animals; Digitonin; Electron Transport Complex I; Mice; Mitochondria, Heart; Mitochondria, Liver; Mitochondrial Proteins; Native Polyacrylamide Gel Electrophoresis
PubMed: 33129827
DOI: 10.1016/j.bbabio.2020.148332 -
Molecules (Basel, Switzerland) Jul 2022The rhizomes of are commonly consumed as food and also used as medicine. However, the metabolic profile of has not been fully revealed yet. Recently, we developed a...
The rhizomes of are commonly consumed as food and also used as medicine. However, the metabolic profile of has not been fully revealed yet. Recently, we developed a novel evergreen species of P. sibiricum. The objectives of this study were to compare the metabolic profiles of two types of , i.e., the newly developed evergreen type (Gtype) and a wide-type (Wtype), by using UHPLC-Q-Orbitrap-MS-based untargeted metabolomics approach. A total of 263 and 258 compounds in the positive and negative modes of the mass spectra were tentatively identified. Distinctively different metabolomic profiles of these two types of were also revealed by principal component analysis (PCA) and principal coordinates analysis (PCoA). Furthermore, by using partial least squares discriminant analysis (PLS-DA) modeling, it was found that, as compared with Wtype, Gtype samples had significantly higher content of oxyberberine, proliferin, alpinetin, and grandisin. On the other hand, 15 compounds, including herniarin, kaempferol 7-neohesperidoside, benzyl beta-primeveroside, vanillic acid, biochanin A, neoschaftoside, benzyl gentiobioside, cornuside, hydroxytyrosol-glucuronide, apigenin-pentosyl-glucoside, obacunone, 13-alpha-(21)-epoxyeurycomanone, vulgarin, digitonin, and 3-formylindole, were discovered to have higher abundance in Wtype samples. These distinguishing metabolites suggest the different beneficial health potentials and flavor attributes of the two types of rhizomes.
Topics: Chromatography, High Pressure Liquid; Mass Spectrometry; Metabolomics; Polygonatum; Rhizome
PubMed: 35897876
DOI: 10.3390/molecules27154685 -
Scientific Reports Aug 2020Valosin-containing protein (VCP)/p97/Cdc48 is an AAA + ATPase associated with many ubiquitin-dependent cellular pathways that are central to protein quality control....
Valosin-containing protein (VCP)/p97/Cdc48 is an AAA + ATPase associated with many ubiquitin-dependent cellular pathways that are central to protein quality control. VCP binds various cofactors, which determine pathway selectivity and substrate processing. Here, we used co-immunoprecipitation and mass spectrometry studies coupled to in silico analyses to identify the Leishmania infantum VCP (LiVCP) interactome and to predict molecular interactions between LiVCP and its major cofactors. Our data support a largely conserved VCP protein network in Leishmania including known but also novel interaction partners. Network proteomics analysis confirmed LiVCP-cofactor interactions and provided novel insights into cofactor-specific partners and the diversity of LiVCP complexes, including the well-characterized VCP-UFD1-NPL4 complex. Gene Ontology analysis coupled with digitonin fractionation and immunofluorescence studies support cofactor subcellular compartmentalization with either cytoplasmic or organellar or vacuolar localization. Furthermore, in silico models based on 3D homology modeling and protein-protein docking indicated that the conserved binding modules of LiVCP cofactors, except for NPL4, interact with specific binding sites in the hexameric LiVCP protein, similarly to their eukaryotic orthologs. Altogether, these results allowed us to build the first VCP protein interaction network in parasitic protozoa through the identification of known and novel interacting partners potentially associated with distinct VCP complexes.
Topics: Computer Simulation; Leishmania infantum; Multiprotein Complexes; Protein Structure, Quaternary; Protozoan Proteins; Valosin Containing Protein
PubMed: 32753747
DOI: 10.1038/s41598-020-70010-4 -
Pathogens (Basel, Switzerland) Oct 2022In this study, we demonstrate that epimastigotes previously grown in LIT medium supplemented with 20 mM galactose and exposed to sub-lethal concentrations of hydrogen...
In this study, we demonstrate that epimastigotes previously grown in LIT medium supplemented with 20 mM galactose and exposed to sub-lethal concentrations of hydrogen peroxide (100 μM) showed two-fold and five-fold viability when compared to epimastigotes grown in LIT medium supplemented with two different glucose concentrations (20 mM and 1.5 mM), respectively. Similar results were obtained when exposing epimastigotes from all treatments to methylene blue 30 μM. Additionally, through differential centrifugation and the selective permeabilization of cellular membranes with digitonin, we found that phosphoglucomutase activity (a key enzyme in galactose metabolism) occurs predominantly within the cytosolic compartment. Furthermore, after partially permeabilizing epimastigotes with digitonin (0.025 mg × mg of protein), intact glycosomes treated with 20 mM galactose released a higher hexose phosphate concentration to the cytosol in the form of glucose-1-phosphate, when compared to intact glycosomes treated with 20 mM glucose, which predominantly released glucose-6-phosphate. These results shine a light on 's galactose metabolism and its interplay with mechanisms that enable resistance to oxidative stress.
PubMed: 36297231
DOI: 10.3390/pathogens11101174 -
Journal of Pharmaceutical Sciences Jan 2021The fractionation of enough membrane protein from limited samples is challenging for MS-based quantitative targeted absolute proteomics (QTAP) of drug metabolizing...
Differential Detergent Fractionation of Membrane Protein From Small Samples of Hepatocytes and Liver Tissue for Quantitative Proteomic Analysis of Drug Metabolizing Enzymes and Transporters.
The fractionation of enough membrane protein from limited samples is challenging for MS-based quantitative targeted absolute proteomics (QTAP) of drug metabolizing enzymes (DMEs) and transporters. This study evaluated differential detergent fractionation (DDF) of membrane protein from progressively smaller numbers of primary mouse hepatocytes (5 million down to 50,000 cells) and limited liver tissue (25-50 mg) in quantifying select DMEs and transporters by QTAP. Two non-ionic detergents, digitonin and Triton-X-100, were applied in sequence to permeabilize cells and extract membrane proteins. Comparison was made with a membrane protein extraction kit and with homogenization in hypotonic buffer and subsequent differential centrifugation (DC). DDF produced linear membrane protein yields with increasing hepatocyte numbers and better permeabilization evidenced by the higher ratio of cytosolic to membrane protein yields. DDF produced 5-times more membrane protein from liver tissue than DC. The concentration of DMEs and transporters remained consistent in the fractions prepared by DDF from progressively smaller numbers of hepatocytes, but declined in kit fractions. In liver tissue, the concentrations were comparatively higher in DDF versus kit and DC. In conclusion, sequential digitonin and Triton-X-100 fractionation of membrane protein from limited samples is efficient, reproducible and cost-effective for QTAP of DMEs and transporters.
Topics: Animals; Detergents; Hepatocytes; Liver; Membrane Proteins; Mice; Pharmaceutical Preparations; Proteomics
PubMed: 33148403
DOI: 10.1016/j.xphs.2020.10.037 -
Chemical Research in Toxicology Nov 2022Nominal concentrations () in cell culture media are routinely used to define concentration-effect relationships in the toxicology. The actual concentration in the...
Nominal concentrations () in cell culture media are routinely used to define concentration-effect relationships in the toxicology. The actual concentration in the medium () can be affected by adsorption processes, evaporation, or degradation of chemicals. Therefore, we measured the total and free concentration of 12 chemicals, covering a wide range of lipophilicity (log -0.07-6.84), in the culture medium () and cells () after incubation with Balb/c 3T3 cells for up to 48 h. Measured values were compared to predictions using an as yet unpublished mass balance model that combined relevant equations from similar models published by others. The total for all chemicals except tamoxifen (TAM) were similar to the . This was attributed to the cellular uptake of TAM and accumulation into lysosomes. The free (i.e., unbound) for the low/no protein binding chemicals were similar to the , whereas values of all moderately to highly protein-bound chemicals were less than 30% of the . Of the 12 chemicals, the two most hydrophilic chemicals, acetaminophen (APAP) and caffeine (CAF), were the only ones for which the was the same as the . The for all other chemicals tended to increase over time and were all 2- to 274-fold higher than . Measurements of , using a digitonin method to release cytosol, compared well with (using a freeze-thaw method) for four chemicals (CAF, APAP, FLU, and KET), indicating that both methods could be used. The mass balance model predicted the total within 30% of the measured values for 11 chemicals. The free of all 12 chemicals were predicted within 3-fold of the measured values. There was a poorer prediction of values, with a median overprediction of 3- to 4-fold. In conclusion, while the number of chemicals in the study is limited, it demonstrates the large differences between and total and free and , which were also relatively well predicted by the mass balance model.
Topics: Mice; Animals; Acetaminophen; Hydrophobic and Hydrophilic Interactions; Protein Binding; Cell Culture Techniques
PubMed: 36264934
DOI: 10.1021/acs.chemrestox.2c00128 -
Cells Feb 2022Cytotoxicity quantification of nanoparticles is commonly performed by biochemical assays to evaluate their biocompatibility and safety. We explored quantitative phase...
Cytotoxicity quantification of nanoparticles is commonly performed by biochemical assays to evaluate their biocompatibility and safety. We explored quantitative phase imaging (QPI) with digital holographic microscopy (DHM) as a time-resolved in vitro assay to quantify effects caused by three different types of organic nanoparticles in development for medical use. Label-free proliferation quantification of native cell populations facilitates cytotoxicity testing in biomedical nanotechnology. Therefore, DHM quantitative phase images from measurements on nanomaterial and control agent incubated cells were acquired over 24 h, from which the temporal course of the cellular dry mass was calculated within the observed field of view. The impact of LipImage™ 815 lipidots nanoparticles, as well as empty and cabazitaxel-loaded poly(alkyl cyanoacrylate) nanoparticles on the dry mass development of four different cell lines (RAW 264.7, NIH-3T3, NRK-52E, and RLE-6TN), was observed vs. digitonin as cytotoxicity control and cells in culture medium. The acquired QPI data were compared to a colorimetric cell viability assay (WST-8) to explore the use of the DHM assay with standard biochemical analysis methods downstream. Our results show that QPI with DHM is highly suitable to identify harmful or low-toxic nanomaterials. The presented DHM assay can be implemented with commercial microscopes. The capability for imaging of native cells and the compatibility with common 96-well plates allows high-throughput systems and future embedding into existing experimental routines for in vitro cytotoxicity assessment.
Topics: Biological Assay; Cell Line; Holography; Microscopy; Nanoparticles
PubMed: 35203295
DOI: 10.3390/cells11040644 -
Current Research in Microbial Sciences 2022Chagas disease (CD), caused by , occurs in several countries in Latin America and non-endemic countries. Heterogeneity among population has been the Achilles' heel to...
Chagas disease (CD), caused by , occurs in several countries in Latin America and non-endemic countries. Heterogeneity among population has been the Achilles' heel to find a better treatment for CD. In this study, we characterized the biochemical parameters and mitochondrial bioenergetics of epimastigotes differentiated from eight isolates (I1-I8) obtained from Brazilian CD patients. Molecular analysis of parasites DTUs grouped all of them as TcII. The profile of the growth curves in axenic cultures was distinct among them, except for I1 and I3 and I2 and I4. Doubling times, growth rates, cell body length, and resistance to benznidazole were also significantly different among them. All the isolates were more glucose-dependent than other strains adapted to grow in axenic culture. Mitochondrial bioenergetics analysis showed that each isolate behaved differently regarding oxygen consumption rates in non-permeabilized and in digitonin-permeabilized cells in the presence of a complex II-linked substrate. When complex IV-linked respiratory chain substrate was used to provide electrons to the mitochondrial respiratory chain (MRC), similarity among the isolates was higher. Our findings show that TcII epimastigotes derived from patients' trypomastigotes displayed their own characteristics , highlighting the intra-TcII diversity, especially regarding the functionality of mitochondrial respiratory complexes II and IV. Understanding intraspecific biological features help us to move a step further on our comprehension regarding parasite's survival and adaptability offering clues to improve the development of new therapies for CD.
PubMed: 35199071
DOI: 10.1016/j.crmicr.2022.100110 -
Journal of Neurochemistry Apr 2020Acid-sensing ion channel 1a (ASIC1a) is well-known to play a major pathophysiological role during brain ischemia linked to acute acidosis of ~pH 6, whereas its function...
Acid-sensing ion channel 1a (ASIC1a) is well-known to play a major pathophysiological role during brain ischemia linked to acute acidosis of ~pH 6, whereas its function during physiological brain activity, linked to much milder pH changes, is still poorly understood. Here, by performing live cell imaging utilizing Na and Ca sensitive and spatially specific fluorescent dyes, we investigated the role of ASIC1a in cytosolic Na and Ca signals elicited by a mild extracellular drop from pH 7.4 to 7.0 and how these affect mitochondrial Na and Ca signaling or metabolic activity. We show that in mouse primary cortical neurons, this small extracellular pH change triggers cytosolic Na and Ca waves that propagate to mitochondria. Inhibiting ASIC1a with Psalmotoxin 1 or ASIC1a gene knockout blocked not only the cytosolic but also the mitochondrial Na and Ca signals. Moreover, physiological activation of ASIC1a by this pH shift enhances mitochondrial respiration and evokes mitochondrial Na signaling even in digitonin-permeabilized neurons. Altogether our results indicate that ASIC1a is critical in linking physiological extracellular pH stimuli to mitochondrial ion signaling and metabolic activity and thus is an important metabolic sensor.
Topics: Acid Sensing Ion Channels; Animals; Cerebral Cortex; Energy Metabolism; Homeostasis; Hydrogen-Ion Concentration; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitochondria; Neurons; Signal Transduction
PubMed: 31976561
DOI: 10.1111/jnc.14971 -
Journal of Fungi (Basel, Switzerland) Jan 2021Respiratory supercomplexes are found in mitochondria of eukaryotic cells and some bacteria. A hypothetical role of these supercomplexes is electron channeling, which in...
Respiratory supercomplexes are found in mitochondria of eukaryotic cells and some bacteria. A hypothetical role of these supercomplexes is electron channeling, which in principle should increase the respiratory chain efficiency and ATP synthesis. In addition to the four classic respiratory complexes and the ATP synthase, mitochondria contain three type II NADH dehydrogenases (NADH for reduced nicotinamide adenine dinucleotide) and the alternative oxidase. Changes in the composition of the respiratory supercomplexes due to energy requirements have been reported in certain organisms. In this study, we addressed the organization of the mitochondrial respiratory complexes in under diverse energy conditions. Supercomplexes were obtained by solubilization of mitochondria with digitonin and separated by blue native polyacrylamide gel electrophoresis (BN-PAGE). The molecular mass of supercomplexes and their probable stoichiometries were 1200 kDa (I:IV), 1400 kDa (I:III), 1600 kDa (I:III:IV), and 1800 kDa (I:III:IV). Concerning the ATP synthase, approximately half of the protein is present as a dimer and half as a monomer. The distribution of respiratory supercomplexes was the same in all growth conditions. We did not find evidence for the association of complex II and the alternative NADH dehydrogenases with other respiratory complexes.
PubMed: 33440829
DOI: 10.3390/jof7010042