scholarly journals Monitoring cytosolic H2O2 fluctuations arising from altered plasma membrane gradients or from mitochondrial activity

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Mercè Carmona ◽  
Laura de Cubas ◽  
Eric Bautista ◽  
Marta Moral-Blanch ◽  
Iria Medraño-Fernández ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Steady State ◽  
Fission Yeast ◽  
Electron Transport Chain ◽  
Metabolic Reprogramming ◽  
Mitochondrial Activity ◽  
Transport Chain ◽  
Steady State Levels ◽  
Low Glucose ◽  
Living Organisms

Abstract Genetically encoded probes monitoring H2O2 fluctuations in living organisms are key to decipher redox signaling events. Here we use a new probe, roGFP2-Tpx1.C169S, to monitor pre-toxic fluctuations of peroxides in fission yeast, where the concentrations linked to signaling or to toxicity have been established. This probe is able to detect nanomolar fluctuations of intracellular H2O2 caused by extracellular peroxides; expression of human aquaporin 8 channels H2O2 entry into fission yeast decreasing membrane gradients. The probe also detects H2O2 bursts from mitochondria after addition of electron transport chain inhibitors, the extent of probe oxidation being proportional to the mitochondrial activity. The oxidation of this probe is an indicator of steady-state levels of H2O2 in different genetic backgrounds. Metabolic reprogramming during growth in low-glucose media causes probe reduction due to the activation of antioxidant cascades. We demonstrate how peroxiredoxin-based probes can be used to monitor physiological H2O2 fluctuations.

Low glucose and high pyruvate reduce the production of 2-oxoaldehydes, improving mitochondrial efficiency, redox regulation and stallion sperm function

2021 ◽  
Author(s):  
J M Ortiz-Rodríguez ◽  
F E Martín-Cano ◽  
G Gaitskell-Phillips ◽  
A Silva ◽  
C Ortega-Ferrusola ◽  
...  
Keyword(s):  
Redox Regulation ◽  
Krebs Cycle ◽  
Tca Cycle ◽  
Mitochondrial Activity ◽  
Atp Production ◽  
Transport Chain ◽  
Low Glucose ◽  
Mitochondrial Efficiency ◽  
Metabolic Strategies ◽  
Go Terms

Abstract Energy metabolism in spermatozoa is complex and involves the metabolism of carbohydrate fatty acids and amino acids. The ATP produced in the electron transport chain (ETC) in the mitochondria appears to be crucial for both sperm motility and maintaining viability, while glycolytic enzymes in the flagella may contribute to ATP production to sustain motility and velocity. Stallion spermatozoa seemingly use diverse metabolic strategies, and in this regard, a study of the metabolic proteome showed that gene ontology (GO) terms and Reactome pathways related to pyruvate metabolism and the Krebs cycle were predominant. Following this, the hypothesis that low glucose concentrations can provide sufficient support for motility and velocity, and thus glucose concentration can be significantly reduced in the medium, was tested. Aliquots of stallion semen in four different media were stored for 48 h at 18°C; a commercial extender containing 67 mM glucose was used as a control. Stallion spermatozoa stored in media with low glucose (1 mM) and high pyruvate (10 mM) (LG-HP) sustained better motility and velocities than those stored in the commercial extender formulated with very high glucose (61.7 ± 1.2% in INRA 96 vs 76.2 ± 1.0% in LG-HP media after 48 h of incubation at 18°C P < 0.0001). Moreover, mitochondrial activity was superior in LG-HP extenders (24.1 ± 1.8% in INRA 96 vs 51.1 ± 0.7% in LG-HP of spermatozoa with active mitochondria after 48 h of storage at 18°C P < 0.0001). Low glucose concentrations may permit more efficient sperm metabolism and redox regulation when substrates for an efficient TCA cycle are provided. The improvement seen using low glucose extenders is due to reductions in the levels of glyoxal and methylglyoxal, 2-oxoaldehydes formed during glycolysis; these compounds are potent electrophiles able to react with proteins, lipids and DNA, causing sperm damage.

scholarly journals The function of ubiquinone in Escherichia coli

1970 ◽  
Vol 117 (3) ◽  
pp. 551-562 ◽  
Author(s):  
G. B. Cox ◽  
N. A. Newton ◽  
F. Gibson ◽  
A. M. Snoswell ◽  
J. A. Hamilton
Keyword(s):  
Escherichia Coli ◽  
Electron Spin Resonance ◽  
Steady State ◽  
Electron Transport ◽  
Electron Spin ◽  
Electron Transport Chain ◽  
Electron Spin Resonance Signal ◽  
Resonance Signal ◽  
Transport Chain ◽  
Spin Resonance

1. The function of ubiquinone in Escherichia coli was studied by using whole cells and membrane preparations of normal E. coli and of a mutant lacking ubiquinone. 2. The mutant lacking ubiquinone, strain AN59 (Ubi−), when grown under aerobic conditions, gave an anaerobic type of growth yield and produced large quantities of lactic acid, indicating that ubiquinone plays a vital role in electron transport. 3. NADH and lactate oxidase activities in membranes from strain AN59 (Ubi−) were greatly impaired and activity was restored by the addition of ubiquinone (Q-1). 4. Comparison of the percentage reduction of flavin, cytochrome b1 and cytochrome a2 in the aerobic steady state in membranes from the normal strain (AN62) and strain AN59 (Ubi−) and the effect of respiratory inhibitors on these percentages in membranes from strain AN62 suggest that ubiquinone functions at more than one site in the electron-transport chain. 5. Membranes from strain AN62, in the absence of substrate, showed an electron-spin-resonance signal attributed to ubisemiquinone. The amount of reduced ubiquinone (50%) found after rapid solvent extraction is consistent with the existence of ubiquinone in membranes as a stabilized ubisemiquinone. 6. The effects of piericidin A on membranes from strain AN62 suggest that this inhibitor acts at the ubiquinone sites: thus inhibition of electron transport is reversed by ubiquinone (Q-1); the aerobic steady-state oxidation–reduction levels of flavins and cytochrome b1 in the presence of the inhibitor are raised to values approximating those found in the membranes of strain AN59 (Ubi−); the inhibitor rapidly eliminates the electron-spin-resonance signal attributed to ubisemiquinone and allows slow oxidation of endogenous ubiquinol in the absence of substrate and prevents reduction of ubiquinone in the presence of substrate. It is concluded that piericidin A separates ubiquinone from the remainder of the electron-transport chain. 7. A scheme is proposed in which ubisemiquinone, complexed to an electron carrier, functions in at least two positions in the electron-transport sequence.

Prolactin increases mRNA encoding Na(+)-TC cotransport polypeptide and hepatic Na(+)-TC cotransport

1995 ◽  
Vol 268 (1) ◽  
pp. G11-G17 ◽  
Author(s):  
Y. Liu ◽  
T. Ganguly ◽  
J. F. Hyde ◽  
M. Vore
Keyword(s):  
Plasma Membrane ◽  
Steady State ◽  
Membrane Vesicles ◽  
Constant Infusion ◽  
Maximal Velocity ◽  
Plasma Membrane Vesicles ◽  
Ovx Rats ◽  
Basolateral Plasma Membrane ◽  
Steady State Levels ◽  
Inhibitor Of Protein Synthesis

We have shown that prolactin (Prl) increases the transhepatic transport of taurocholate (TC) in postpartum rats and following treatment of ovariectomized (Ovx) rats with ovine Prl (oPrl). The present studies were designed to determine if treatment of Ovx rats with oPrl (100, 300, or 600 micrograms/day, 7 days iv) 1) increases Na(+)-TC cotransport in basolateral plasma membrane vesicles (bLPM), 2) induces a corresponding increase in the steady-state levels of Na(+)-TC cotransport polypeptide (Ntcp mRNA), and 3) if the oPrl-mediated increase in Na(+)-TC cotransport activity is blocked by cycloheximide, an inhibitor of protein synthesis. oPrl (300 micrograms/day) induced a twofold increase in the maximal velocity for Na(+)-TC cotransport in both hepatocytes and bLPM vesicles with little change in the Michaelis constant. Infusion of oPrl at a dose of 100, 300, or 600 micrograms/day increased steady-state Ntcp mRNA four-, ten-, and twofold, respectively. Finally, cycloheximide blocked the oPrl-mediated increase in Na(+)-TC cotransport but did not affect basal activity. These data support the hypothesis that an increase in Ntcp mRNA followed by increased synthesis and incorporation of Ntcp in the plasma membrane is responsible for the oPrl-mediated increase in Na(+)-TC cotransport in the basolateral plasma membrane domain of the hepatocyte.

scholarly journals Targeting the complex I and III of mitochondrial electron transport chain as a potentially viable option in liver cancer management

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Qin Yang ◽  
Ling Wang ◽  
Jiaye Liu ◽  
Wanlu Cao ◽  
Qiuwei Pan ◽  
...  
Keyword(s):  
Electron Transport ◽  
Liver Cancer ◽  
Therapeutic Target ◽  
Complex I ◽  
Electron Transport Chain ◽  
Metabolic Reprogramming ◽  
Complex Iii ◽  
Mitochondrial Electron Transport Chain ◽  
Potential Therapeutic Target ◽  
Transport Chain

AbstractLiver cancer is one of the most common and lethal types of oncological disease in the world, with limited treatment options. New treatment modalities are desperately needed, but their development is hampered by a lack of insight into the underlying molecular mechanisms of disease. It is clear that metabolic reprogramming in mitochondrial function is intimately linked to the liver cancer process, prompting the possibility to explore mitochondrial biochemistry as a potential therapeutic target. Here we report that depletion of mitochondrial DNA, pharmacologic inhibition of mitochondrial electron transport chain (mETC) complex I/complex III, or genetic of mETC complex I restricts cancer cell growth and clonogenicity in various preclinical models of liver cancer, including cell lines, mouse liver organoids, and murine xenografts. The restriction is linked to the production of reactive oxygen species, apoptosis induction and reduced ATP generation. As a result, our findings suggest that the mETC compartment of mitochondria could be a potential therapeutic target in liver cancer.

Proton Translocations in Isolated Spinach Chloroplasts after Single-turnover Actinic Flashes

1979 ◽  
Vol 6 (3) ◽  
pp. 289
Author(s):  
A.B Hope ◽  
A Morland
Keyword(s):  
Steady State ◽  
Electron Transport ◽  
Electron Transport Chain ◽  
Neutral Red ◽  
Plastoquinone Pool ◽  
Single Turnover ◽  
Transport Chain ◽  
Initial Reduction ◽  
Proton Uptake ◽  
Cresol Red

Proton movements were monitored with cresol red and neutral red. Absorbance changes of these dyes following actinic flashes were measured both for the steady state and for sequences of 6-9 flashes given to separate dark-adapted suspensions of chloroplasts. The cresol red changes corresponded to a steady-state proton uptake (�H*+ss) of 0.0015-0.0017 mol H+/mol Chl per flash (ferricyanide as electron acceptor) or 0.0031 mol H+/mol Chl per flash (methyl viologen). Assuming 600 Chl per electron transport chain, these uptakes corresponded to H+/e- = 1 or 2 respectively. In flash sequences with ferricyanide, a minimum of about 0.6 �H*+ss was noted after flash No. 3. Controls, and considerations of the partitioning of unprotonated neutral red into the thylakoid membrane phase, strongly indicated that neutral red was responding to an acidification of the intrathylakoid aqueous phase. The steady-state signal was interpreted as 2 H+ deposited per electron transport chain per flash (H+/e- = 2), one proton from the oxidation of water and one from the oxidation of plastoquinone. Patterns of proton deposition following sequences of flashes and their change upon adding dibromothymoquinone suggested that (a) protons are produced one at a time in the advance of the 'S-states' (Z*n+ � Z(n+1)+ ); and (b) the precursor to the plastoquinone pool, B, is a special plastoquinone, requiring 1 e- and 1 H+ for its initial reduction but passing on to the plastoquinone pool 2 e- upon the next flash. The proton deposition on flash No. 1 corresponded to a small proportion of reduced B in dark-adapted chloroplasts, as previously postulated.

scholarly journals Steady-state kinetic analysis of mitochondrial respiratory enzymes from bovine heart mitochondria

2021 ◽  
Vol 64 (1) ◽  
Author(s):  
Dayoung Kim ◽  
Eun Ko ◽  
Moonsung Choi ◽  
Sooim Shin
Keyword(s):  
Steady State ◽  
Electron Transport ◽  
Kinetic Analysis ◽  
Kinetic Parameters ◽  
Complex I ◽  
Electron Transport Chain ◽  
Complex Iii ◽  
Steady State Kinetic ◽  
Transport Chain ◽  
State Kinetic

AbstractMitochondria is a decisive organelle of cells that produces adenosine triphosphate (ATP) by the process of oxidative phosphorylation of the Krebs cycle and the electron transport chain. The electron transport chain system of mitochondria embodies multiple enzyme supercomplexes including complex I to V which located in the inner membrane. Although the simple enzyme activity of some as-isolated complex has been studied so far, the steady-state kinetic analysis of each complex within the form of mitochondrial supercomplex has not been studied in depth. To this end, kinetic parameters of mitochondrial complex I–IV were determined using steady-kinetic analysis using corresponding substrates of them. Catalytic activity and binding affinity between substrates and enzymes were obtained by fitting the data to the Michaelis–Menten equation. Acquired kinetic parameters represented distinctive values depending on the complexes that can be interpreted by the characteristics of the enzymes including the distinction of substrates or the ratio of the enzyme itself under the supercomplex form. The indirect kcat of the mitochondrial enzymes were varied from 0.0609 to 0.334 s−1 in order of complex III, II, I, and IV and Km of substrates were also diverse from 5.1 μM to 12.14 mM. This is the first attempt to get exact kinetic values that should provide profound information to evaluate the mitochondrial function practically in advance.

scholarly journals Genetic Landscape of Electron Transport Chain Complex I Dependency in Acute Myeloid Leukemia

2019 ◽  
Author(s):  
Irène Baccelli ◽  
Yves Gareau ◽  
Bernhard Lehnertz ◽  
Stéphane Gingras ◽  
Jean-François Spinella ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Electron Transport ◽  
Complex I ◽  
Myeloid Leukemia ◽  
Electron Transport Chain ◽  
Chain Complex ◽  
Mitochondrial Activity ◽  
Selective Toxicity ◽  
Transport Chain ◽  
Acute Myeloid

AbstractInhibition of oxidative phosphorylation (OXPHOS) is a promising therapeutic strategy in Acute Myeloid Leukemia (AML), but patients respond heterogeneously. Through chemically interrogation of 200 sequenced specimens, we identified Mubritinib as a strong in vitro and in vivo anti-leukemic compound, acting through ubiquinone-dependent inhibition of Electron Transport Chain complex I (ETC1). ETC1 targeting showed selective toxicity against a subgroup of chemotherapy-resistant leukemias exhibiting OXPHOS hyperactivity, high expression of mitochondrial activity-related genes, and mutations affecting NPM1, FLT3 and DNMT3A. Altogether, our work thus identifies a novel ETC1 inhibitor with high clinical potential and reveals the landscape of OXPHOS dependency in AML.

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