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Methods in Molecular Biology (Clifton,... 2023The protozoan parasite, Trypanosoma brucei, offers a simple system to study the growth and duplication of the Golgi. Cell lines stably expressing a photoactivatable GFP...
The protozoan parasite, Trypanosoma brucei, offers a simple system to study the growth and duplication of the Golgi. Cell lines stably expressing a photoactivatable GFP attached to an endogenous Golgi protein are permeabilized using digitonin. Photoactivation followed by imaging can then be used to follow the formation of the new Golgi.
Topics: Animals; Trypanosoma brucei brucei; Golgi Apparatus; Digitonin; Parasites; Protozoan Proteins
PubMed: 36512209
DOI: 10.1007/978-1-0716-2639-9_5 -
Biochimica Et Biophysica Acta.... Apr 2023The FF-ATP synthase uses the energy stored in the electrochemical proton gradient to synthesize ATP. This complex is found in the inner mitochondrial membrane as a...
Deletion of the ATP20 gene in Ustilago maydis produces an unstable dimer of FF-ATP synthase associated with a decrease in mitochondrial ATP synthesis and a high HO production.
The FF-ATP synthase uses the energy stored in the electrochemical proton gradient to synthesize ATP. This complex is found in the inner mitochondrial membrane as a monomer and dimer. The dimer shows higher ATPase activity than the monomer and is essential for cristae folding. The monomer-monomer interface is constituted by subunits a, i/j, e, g, and k. The role of the subunit g in a strict respiratory organism is unknown. A gene knockout was generated in Ustilago maydis to study the role of subunit g on mitochondrial metabolism and cristae architecture. Deletion of the ATP20 gene, encoding the g subunit, did not affect cell growth or glucose consumption, but biomass production was lower in the mutant strain (gΔ strain). Ultrastructure observations showed that mitochondrial size and cristae shape were similar in wild-type and gΔ strains. The mitochondrial membrane potential in both strains had a similar magnitude, but oxygen consumption was higher in the WT strain. ATP synthesis was 20 % lower in the gΔ strain. Additionally, the mutant strain expressed the alternative oxidase in the early stages of growth (exponential phase), probably as a response to ROS stress. Dimer from mutant strain was unstable to digitonin solubilization, avoiding its isolation and kinetic characterization. The isolated monomeric state activated by n-dodecyl-β-D-maltopyranoside showed similar kinetic constants to the monomer from the WT strain. A decrease in mitochondrial ATP synthesis and the presence of the AOX during the exponential growth phase suggests that deletion of the g gene induces ROS stress.
Topics: Hydrogen Peroxide; Mitochondrial Proton-Translocating ATPases; Reactive Oxygen Species; Adenosine Triphosphate
PubMed: 36509127
DOI: 10.1016/j.bbabio.2022.148950 -
Toxicology and Applied Pharmacology Nov 2022Mitochondrial toxicity is one of the causes for drug-induced liver injury, and the classification of phenotypes or mitochondrial toxicity are highly required though...
AIM
Mitochondrial toxicity is one of the causes for drug-induced liver injury, and the classification of phenotypes or mitochondrial toxicity are highly required though there are no molecular-profiling approaches for classifying mitochondrial toxicity. Therefore, the aim of this study was to classify the mechanisms of mitochondrial toxicity by metabolic profiling in vitro and bioinformatics.
MAIN METHODS
We applied an established gas chromatography tandem mass spectrometry-based metabolomics to human hepatoma grade 2 (HepG2) cells that were exposed to mitochondrial toxicants, whose mechanisms are different, such as rotenone (0.1 μM), carbonyl cyanide-3-chlorophenylhydrazone (CCCP, 0.5 μM), nefazodone (20 μM), perhexiline (6.25 μM), or digitonin (positive cytotoxic substance, 4 μM). These concentrations were determined by the Mitochondrial ToxGlo Assay. Galactose medium was used for suppressing the Warburg effect in HepG2 cells, and the metabolome analysis successfully identified 125 metabolites in HepG2 cells. Multivariate, metabolic pathway and network analyses were performed by the R software.
KEY FINDINGS
Metabolic profiling enabled the classifying the mitochondrial toxicity mechanisms of RCC inhibition and uncoupling. The metabolic profiles of respiratory chain complex (RCC) inhibitors (rotenone and nefazodone) and an uncoupler (CCCP) were fully differentiated from those of other compounds. The metabolic pathway analysis revealed that the RCC inhibitors and the uncoupler mainly disrupted TCA-cycle and related metabolic pathways. In addition, the correlation-based network analysis revealed that succinic acid, β-alanine, and glutamic acid were potential metabolic indicators for RCC inhibition and uncoupling.
SIGNIFICANCE
Our results provided new insights into classifying mechanisms of mitochondrial toxicity by in vitro metabolomics.
PubMed: 36462684
DOI: 10.1016/j.taap.2022.116316 -
Biochimie Apr 2023Accumulation of D-2-hydroxyglutaric acid (D-2-HG) is the biochemical hallmark of D-2-hydroxyglutaric aciduria type I and, particularly, of D-2-hydroxyglutaric aciduria...
Accumulation of D-2-hydroxyglutaric acid (D-2-HG) is the biochemical hallmark of D-2-hydroxyglutaric aciduria type I and, particularly, of D-2-hydroxyglutaric aciduria type II (D2HGA2). D2HGA2 is a metabolic inherited disease caused by gain-of-function mutations in the gene isocitrate dehydrogenase 2. It is clinically characterized by neurological abnormalities and a severe cardiomyopathy whose pathogenesis is still poorly established. The present work investigated the potential cardiotoxicity D-2-HG, by studying its in vitro effects on a large spectrum of bioenergetics parameters in heart of young rats and in cultivated H9c2 cardiac myoblasts. D-2-HG impaired cellular respiration in purified mitochondrial preparations and crude homogenates from heart of young rats, as well as in digitonin-permeabilized H9c2 cells. ATP production and the activities of cytochrome c oxidase (complex IV), alpha-ketoglutarate dehydrogenase, citrate synthase and creatine kinase were also inhibited by D-2-HG, whereas the activities of complexes I, II and II-III of the respiratory chain, glutamate, succinate and malate dehydrogenases were not altered. We also found that this organic acid compromised mitochondrial Ca retention capacity in heart mitochondrial preparations and H9c2 myoblasts. Finally, D-2-HG reduced the viability of H9c2 cardiac myoblasts, as determined by the MTT test and by propidium iodide incorporation. Noteworthy, L-2-hydroxyglutaric acid did not change some of these measurements (complex IV and creatine kinase activities) in heart preparations, indicating a selective inhibitory effect of the enantiomer D. In conclusion, it is presumed that D-2-HG-disrupts mitochondrial bioenergetics and Ca retention capacity, which may be involved in the cardiomyopathy commonly observed in D2HGA2.
Topics: Rats; Animals; Calcium; Cell Survival; Energy Metabolism; Creatine Kinase; Cardiomyopathies
PubMed: 36372308
DOI: 10.1016/j.biochi.2022.11.004 -
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 -
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 -
Computational and Structural... 2022Synthetic lethality (SL) is an emerging therapeutic paradigm in cancer. We introduced a different approach to prioritize SL gene pairs through literature mining and...
Synthetic lethality (SL) is an emerging therapeutic paradigm in cancer. We introduced a different approach to prioritize SL gene pairs through literature mining and -mutant high-throughput screening (HTS) data. We matched essential genes from text-mining and mutant genes from the COSMIC and CCLE HTS datasets to build a prediction model of SL gene pairs. CCLE gene expression data were used to enrich the essential-mutant SL gene pairs using Spearman's correlation coefficient and literature mining. In total, 223 essential trigger terms were extracted and ranked. The threshold of the essential gene score ( ) was set to 10. We identified 586 genes essential for the SL prediction model of colon cancer. Seven essential -mutant SL gene pairs were identified in our model, including -/----/ and - gene pairs. Using -mutant HTS data validation, we identified two potential SL gene pairs, including the (essential gene)- (mutant gene) pair and - pair in the DLD-1 colon cancer cell line (Spearman's correlation values = 0.004786 and 0.00249, respectively). Based on further annotations by PubChem, we observed that digitonin targeted the complex comprising , especially in -mutated HCT116 cancer cells. Moreover, we experimentally demonstrated that exhibited selective vulnerability in -mutant colorectal cancer. We used literature mining and HTS data to identify candidates for SL targets for mutant colon cancer.
PubMed: 36212540
DOI: 10.1016/j.csbj.2022.09.025 -
Drug Metabolism and Disposition: the... Dec 2022As a multitissue organ, the eye possesses unique anatomy and physiology, including differential expression of drug-metabolizing enzymes. Several hydrolytic enzymes that...
As a multitissue organ, the eye possesses unique anatomy and physiology, including differential expression of drug-metabolizing enzymes. Several hydrolytic enzymes that play a major role in drug metabolism and bioactivation of prodrugs have been detected in ocular tissues, but data on their quantitative expression is scarce. Also, many ophthalmic drugs are prone to hydrolysis. Metabolic characterization of individual ocular tissues is useful for the drug development process, and therefore, seven individual ocular tissues from human eyes were analyzed for the activity and expression of carboxylesterases (CESs) and arylacetamide deacetylase (AADAC). Generic and selective human esterase substrates 4-nitrophenyl acetate (most esterases), D-luciferin methyl ester (CES1), fluorescein diacetate and procaine (CES2), and phenacetin (AADAC) were applied to determine the enzymes' specific activities. Enzyme kinetics and inhibition studies were performed with isoform-selective inhibitors digitonin (CES1) and verapamil and diltiazem (CES2). Enzyme contents were determined using quantitative targeted proteomics, and CES2 expression was confirmed by western blotting. The expression and activity of human CES1 among ocular tissues varied by >10-fold, with the highest levels found in the retina and iris-ciliary body. In contrast, human CES2 expression appeared lower and more similar between tissues, whereas AADAC could not be detected. Inhibition studies showed that hydrolysis of fluorescein diacetate is also catalyzed by enzymes other than CES2. This study provides, for the first time, quantitative information on the tissue-dependent expression of human ocular esterases, which can be useful for the development of ocular drugs, prodrugs, and in pharmacokinetic modeling of the eye. SIGNIFICANCE STATEMENT: Novel and comprehensive data on the protein expression and activities of carboxylesterases from individual human eye tissues are generated. In combination with previous reports on preclinical species, this study will improve the understanding of interspecies differences in ocular drug metabolism and aid the development of ocular pharmacokinetics models.
Topics: Humans; Carboxylic Ester Hydrolases; Carboxylesterase; Prodrugs; Fluoresceins; Hydrolysis
PubMed: 36195336
DOI: 10.1124/dmd.122.000993 -
International Journal of Pharmaceutics Nov 2022Migraine is a highly prevalent neurological disease affecting circa 1 billion patients worldwide with severe incapacitating symptoms, which significantly diminishes the...
Migraine is a highly prevalent neurological disease affecting circa 1 billion patients worldwide with severe incapacitating symptoms, which significantly diminishes the quality of life. As self-medication practice, oral administration of triptans is the most common option, despite its relatively slow therapeutic onset and low drug bioavailability. To overcome these issues, here we present, to the best of our knowledge, the first study on the possibility of oral transmucosal delivery of one of the safest triptans, namely eletriptan hydrobromide (EB). Based on a comprehensive set of in vitro and ex vivo experiments, we highlight the conditions required for oral transmucosal delivery, potentially giving rise to similar, or even higher, drug plasma concentrations expected from conventional oral administration. With histology and tissue integrity studies, we conclude that EB neither induces morphological changes nor impairs the integrity of the mucosal barrier following 4 h of exposure. On a cellular level, EB is internalized in human oral keratinocytes within the first 5 min without inducing toxicity at the relevant concentrations for transmucosal delivery. Considering that the pK of EB falls within the physiologically range, we systematically investigated the effect of pH on both solubility and transmucosal permeation. When the pH is increased from 6.8 to 10.4, the drug solubility decreases drastically from 14.7 to 0.07 mg/mL. At pH 6.8, EB gave rise to the highest drug flux and total permeated amount across mucosa, while at pH 10.4 EB shows greater permeability coefficient and thus higher ratio of permeated drug versus applied drug. Permeation experiments with model membranes confirmed the pH dependent permeation profile of EB. The distribution of EB in different cellular compartments of keratinocytes is pH dependent. In brief, high drug ionization leads to higher association with the cell membrane, suggesting ionic interactions between EB and the phospholipid head groups. Moreover, we show that the chemical permeation enhancer DMSO can be used to enhance the drug permeation significantly (i.e., 12 to 36-fold increase). Taken together, this study presents important findings on transmucosal delivery of eletriptan via the oral cavity and paves the way for clinical investigations for a fast and safe migraine treatment.
Topics: Humans; Quality of Life; Dimethyl Sulfoxide; Tryptamines; Administration, Oral; Pharmaceutical Preparations; Migraine Disorders; Phospholipids
PubMed: 36155795
DOI: 10.1016/j.ijpharm.2022.122222 -
Bioluminescent test systems based on firefly luciferase for studying stress effects on living cells.Biophysical Reviews Aug 2022The bioluminescent luciferin-luciferase reaction is based on the oxidation of D-luciferin by oxygen in the presence of ATP and magnesium ions, catalyzed by firefly... (Review)
Review
The bioluminescent luciferin-luciferase reaction is based on the oxidation of D-luciferin by oxygen in the presence of ATP and magnesium ions, catalyzed by firefly luciferase. The possibilities of using this reaction to study the influence of external effectors of a physical and chemical nature (temperature exposure, additions of drugs, membrane-active compounds, etc.) on living cells (prokaryotes and eukaryotes) are considered. Examples of the use of test systems based on living cells producing thermostable firefly luciferase for monitoring cellular homeostasis are given. The study of the kinetics of changes in the concentration of ATP and luciferase inside and outside cells made it possible to determine in dynamics the metabolic activity, cytotoxicity, and survival of cells under conditions of cellular stress, to study the processes of ATP synthesis/hydrolysis, and to evaluate the effectiveness of lytic agents in changing the permeability of the cell membrane.
PubMed: 36124280
DOI: 10.1007/s12551-022-00978-y