Aims: Glucose-stimulated insulin secretion (GSIS) in pancreatic β cells was expected to enhance mitochondrial superoxide formation. Hence, we elucidated relevant redox equilibria. Results: Unexpectedly, INS-1E cells at transitions from 3 (11 mM; pancreatic islets from 5 mM) to 25 mM glucose decreased matrix superoxide release rates (MitoSOX Red monitoring validated by MitoB) and H2O2 (mitoHyPer, subtracting mitoSypHer emission). Novel double-channel fluorescence lifetime imaging, approximating free mitochondrial matrix NADHF, indicated its ∼20% decrease. Matrix NAD+F increased on GSIS, indicated by the FAD-emission lifetime decrease, reflecting higher quenching of FAD by NAD+F. The participation of pyruvate/malate and pyruvate/citrate redox shuttles, elevating cytosolic NADPHF (iNAP1 fluorescence monitoring) at the expense of matrix NADHF, was indicated, using citrate (2-oxoglutarate) carrier inhibitors and cytosolic malic enzyme silencing: All changes vanished on these manipulations. 13C-incorporation from 13C-L-glutamine into 13C-citrate reflected the pyruvate/isocitrate shuttle. Matrix NADPHF (iNAP3 monitored) decreased. With decreasing glucose, the suppressor of Complex III site Q electron leak (S3QEL) suppressor caused a higher Complex I IF site contribution, but a lower superoxide fraction ascribed to the Complex III site IIIQo. Thus, the diminished matrix NADHF/NAD+F decreased Complex I flavin site IF superoxide formation on GSIS. Innovation: Mutually validated methods showed decreasing superoxide release into the mitochondrial matrix in pancreatic β cells on GSIS, due to the decreasing matrix NADHF/NAD+F (NADPHF/NADP+F) at increasing cytosolic NADPHF levels. The developed innovative methods enable real-time NADH/NAD+ and NADPH/NADP+ monitoring in any distinct cell compartment. Conclusion: The export of reducing equivalents from mitochondria adjusts lower mitochondrial superoxide production on GSIS, but it does not prevent oxidative stress in pancreatic β cells.

• Klíčová slova
• Complex I, NADH/NAD+ ratio, fluorescence lifetime imaging, glucose-stimulated insulin secretion, mitochondrial superoxide generation, pancreatic β cells,
• MeSH
• adenosintrifosfát metabolismus   MeSH
• beta-buňky metabolismus   MeSH
• buněčné dýchání MeSH
• chromatografie kapalinová MeSH
• energetický metabolismus MeSH
• flavinadenindinukleotid metabolismus   MeSH
• glukosa metabolismus   MeSH
• hmotnostní spektrometrie MeSH
• krysa rodu Rattus MeSH
• kyselina citronová metabolismus   MeSH
• membránový potenciál mitochondrií MeSH
• metabolické sítě a dráhy MeSH
• metabolomika metody   MeSH
• mitochondrie metabolismus   MeSH
• NAD metabolismus   MeSH
• peroxid vodíku metabolismus   MeSH
• sekrece inzulinu * MeSH
• signální transdukce MeSH
• superoxidy metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosintrifosfát MeSH
• flavinadenindinukleotid MeSH
• glukosa MeSH
• kyselina citronová MeSH
• NAD MeSH
• peroxid vodíku MeSH
• superoxidy MeSH

Malate dehydrogenase (decarboxylating) from Tritrichomonas foetus hydrogenosomes was purified close to homogeneity by a combination of differential centrifugation, zwitterionic detergent solubilization, Red-Sepharose chromatography and anion-exchange chromatography. The enzyme with apparent subunit size of 59 kDa and native molecular mass of 308 kDa utilized NAD+ preferentially to NADP+ as a cofactor and required Mn2+ or Mg2+ for its activity. Affinity curves for malate and coenzymes were hyperbolic. Km for malate was 100 microM and 458 microM in the presence of NAD+ and NADP+, respectively. Km for NAD+ and for NADP+ in the presence of malate was 18 microM and 207 microM, respectively. The enzyme is proposed to be a tetramer with a possible physiological role in the maintenance of an appropriate NAD+/NADH ratio in hydrogenosomes.

• MeSH
• biologické modely MeSH
• malátdehydrogenasa izolace a purifikace   metabolismus   MeSH
• maláty metabolismus   MeSH
• mangan metabolismus   MeSH
• NAD metabolismus   MeSH
• NADP metabolismus   MeSH
• organely enzymologie   MeSH
• Tritrichomonas foetus enzymologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• malátdehydrogenasa MeSH
• malate dehydrogenase (decarboxylating) MeSH Prohlížeč
• maláty MeSH
• mangan MeSH
• NAD MeSH
• NADP MeSH

Recent evidence suggests that energy metabolism contributes to molecular mechanisms controlling stem cell identity. For example, human embryonic stem cells (hESCs) receive their metabolic energy mostly via glycolysis rather than mitochondrial oxidative phosphorylation. This suggests a connection of metabolic homeostasis to stemness. Nicotinamide adenine dinucleotide (NAD) is an important cellular redox carrier and a cofactor for various metabolic pathways, including glycolysis. Therefore, accurate determination of NAD cellular levels and dynamics is of growing importance for understanding the physiology of stem cells. Conventional analytic methods for the determination of metabolite levels rely on linear calibration curves. However, in actual practice many two-enzyme cycling assays, such as the assay systems used in this work, display prominently nonlinear behavior. Here we present a diaphorase/lactate dehydrogenase NAD cycling assay optimized for hESCs, together with a mechanism-based, nonlinear regression models for the determination of NAD(+), NADH, and total NAD. We also present experimental data on metabolic homeostasis of hESC under various physiological conditions. We show that NAD(+)/NADH ratio varies considerably with time in culture after routine change of medium, while the total NAD content undergoes relatively minor changes. In addition, we show that the NAD(+)/NADH ratio, as well as the total NAD levels, vary between stem cells and their differentiated counterparts. Importantly, the NAD(+)/NADH ratio was found to be substantially higher in hESC-derived fibroblasts versus hESCs. Overall, our nonlinear mathematical model is applicable to other enzymatic amplification systems.

• MeSH
• buněčné extrakty MeSH
• elektroforéza kapilární MeSH
• embryonální kmenové buňky metabolismus   MeSH
• kalibrace MeSH
• lidé MeSH
• NAD metabolismus   MeSH
• nelineární dynamika * MeSH
• oxaziny metabolismus   MeSH
• regresní analýza MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• buněčné extrakty MeSH
• NAD MeSH
• oxaziny MeSH
• resorufin MeSH Prohlížeč

The homodimeric flavoprotein FerB of Paracoccus denitrificans catalyzed the reduction of chromate with NADH as electron donor. When present, oxygen was reduced concomitantly with chromate. The recombinant enzyme had a maximum activity at pH 5.0. The stoichiometric ratio of NADH oxidized to chromate reduced was found to be 1.53 ± 0.09 (O(2) absent) or > 2 (O(2) present), the apparent K (M) value for chromate amounted to 70 ± 10 μM with the maximum rate of 2.9 ± 0.3 μmol NADH s(-1) (mg protein)(-1). Diode-array spectrophotometry and experiments with one-electron acceptors provided evidence for oxygen consumption being due to a flavin semiquinone, formed transiently during the interaction of FerB with chromate. At the whole-cell level, a ferB mutant strain displayed only slightly diminished rate of chromate reduction when compared to the wild-type parental strain. Anaerobically grown cells were more active than cells grown aerobically. The activity could be partly inhibited by antimycin, suggesting an involvement of the respiratory chain. Chromate concentrations above ten micromolars transiently slowed or halted culture growth, with the effect being more pronounced for the mutant strain. It appears, therefore, that, rather than directly reducing chromate, FerB confers a protection of cells against the oxidative stress accompanying chromate reduction. With a strain carrying the chromosomally integrated ferB promoter-lacZ fusion, it was shown that the ferB gene is not inducible by chromate.

• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• chromany metabolismus   MeSH
• flavinadenindinukleotid analogy a deriváty   metabolismus   MeSH
• flavoproteiny genetika   metabolismus   MeSH
• FMN-reduktasa genetika   metabolismus   MeSH
• koncentrace vodíkových iontů MeSH
• NAD metabolismus   MeSH
• oxidace-redukce MeSH
• oxidační stres MeSH
• oxidoreduktasy genetika   metabolismus   MeSH
• Paracoccus denitrificans enzymologie   genetika   MeSH
• spotřeba kyslíku MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• chromany MeSH
• chromate reductase MeSH Prohlížeč
• ferric citrate iron reductase MeSH Prohlížeč
• flavin semiquinone MeSH Prohlížeč
• flavinadenindinukleotid MeSH
• flavoproteiny MeSH
• FMN-reduktasa MeSH
• NAD MeSH
• oxidoreduktasy MeSH

Previously suggested antioxidant mechanisms for mitochondria-targeted plastoquinone SkQ1 included: i) ion-pairing of cationic SkQ1+ with free fatty acid anions resulting in uncoupling; ii) SkQ1H2 ability to interact with lipoperoxyl radical; iii) interference with electron flow at the inner ubiquinone (Q) binding site of Complex III (Qi), involving the reduction of SkQ1 to SkQ1H2 by ubiquinol. We elucidated SkQ1 antioxidant properties by confocal fluorescence semi-quantification of mitochondrial superoxide (Jm) and cytosolic H2O2 (Jc) release rates in HepG2 cells. Only in glycolytic cells, SkQ1 prevented the rotenone-induced enhancement of Jm and Jc but not basal releases without rotenone. The effect ceased in glutaminolytic aglycemic cells, in which the redox parameter NAD(P)H/FAD increased after rotenone in contrast to its decrease in glycolytic cells. Autofluorescence decay indicated decreased NADPH/NADH ratios with rotenone in both metabolic modes. SkQ1 did not increase cell respiration and diminished Jm established high by antimycin or myxothiazol but not by stigmatellin. The revealed SkQ1 antioxidant modes reflect its reduction to SkQ1H2 at Complex I IQ or Complex III Qi site. Both reductions diminish electron diversions to oxygen thus attenuating superoxide formation. Resulting SkQ1H2 oxidizes back to SkQ1at the second (flavin) Complex I site, previously indicated for MitoQ10. Regeneration proceeds only at lower NAD(P)H/FAD in glycolytic cells. In contrast, cyclic SkQ1 reduction/SkQ1H2 oxidation does not substantiate antioxidant activity in intact cells in the absence of oxidative stress (neither pro-oxidant activity, representing a great advantage). A targeted delivery to oxidative-stressed tissues is suggested for the effective antioxidant therapy based on SkQ1.

• Klíčová slova
• HepG2 cells, Mitochondria-targeted antioxidant SkQ1, Mitochondrial Complex I superoxide formation, Mitochondrial Complex III superoxide formation, NAD(P)H fluorescence lifetime imaging microscopy,
• MeSH
• antimycin A analogy a deriváty   farmakologie   MeSH
• antioxidancia farmakologie   MeSH
• buňky Hep G2 MeSH
• flavinadenindinukleotid metabolismus   MeSH
• glykolýza MeSH
• lidé MeSH
• methakryláty farmakologie   MeSH
• mitochondrie účinky léků   metabolismus   MeSH
• NAD metabolismus   MeSH
• oxidace-redukce MeSH
• oxidační stres MeSH
• oxidativní fosforylace * MeSH
• plastochinon analogy a deriváty   farmakologie   MeSH
• polyeny farmakologie   MeSH
• respirační komplex I metabolismus   MeSH
• respirační komplex III metabolismus   MeSH
• rotenon farmakologie   MeSH
• superoxidy metabolismus   MeSH
• thiazoly farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 10-(6'-plastoquinonyl)decyltriphenylphosphonium MeSH Prohlížeč
• antimycin A MeSH
• antimycin MeSH Prohlížeč
• antioxidancia MeSH
• flavinadenindinukleotid MeSH
• methakryláty MeSH
• myxothiazol MeSH Prohlížeč
• NAD MeSH
• plastochinon MeSH
• polyeny MeSH
• respirační komplex I MeSH
• respirační komplex III MeSH
• rotenon MeSH
• stigmatellin MeSH Prohlížeč
• superoxidy MeSH
• thiazoly MeSH

Long-term peritoneal dialysis is associated with alterations in peritoneal function, like the development of high small solute transfer rates and impaired ultrafiltration. Also, morphologic changes can develop, the most prominent being loss of mesothelium, vasculopathy, and interstitial fibrosis. Current research suggests peritoneal inflammation as the driving force for these alterations. In this review, the available evidence for inflammation is examined and a new hypothesis is put forward consisting of high glucose-induced pseudohypoxia. Hypoxia of cells is characterized by a high (oxidized-reduced nicotinamide dinucleotide ratio) NADH-NAD+ ratio in their cytosol. Pseudohypoxia is similar but occurs when excessive amounts of glucose are metabolized, as is the case for peritoneal interstitial cells in peritoneal dialysis. The glucose-induced high NADH-NAD+ ratio upregulates the hypoxia-inducible factor-1 gene, which stimulates not only the glucose transporter-1 gene but also many profibrotic genes like TGFβ, vascular endothelial growth factor, plasminogen activator inhibitor-1, and connective tissue growth factor, all known to be involved in the development of peritoneal fibrosis. This review discusses the causes and consequences of pseudohypoxia in peritoneal dialysis and the available options for treatment and prevention. Reducing peritoneal exposure to the excessively high dialysate glucose load is the cornerstone to avoid the pseudohypoxia-induced alterations. This can partly be done by the use of icodextrin or by combinations of low molecular mass osmotic agents, all in a low dose. The addition of alanyl-glutamine to the dialysis solution needs further clinical investigation.

• Klíčová slova
• connective tissue growth factor, glucose exposure, inflammation, peritoneal dialysis, peritoneal membrane alterations, plasminogen activator inhibitor-1, pseudohypoxia, vascular endothelial growth factor,
• MeSH
• dialyzační roztoky škodlivé účinky   metabolismus   MeSH
• glukosa škodlivé účinky   metabolismus   MeSH
• hypoxie MeSH
• lidé MeSH
• NAD * metabolismus   MeSH
• peritoneální dialýza * škodlivé účinky   MeSH
• peritoneum metabolismus   MeSH
• vaskulární endoteliální růstový faktor A metabolismus   MeSH
• zánět MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• dialyzační roztoky MeSH
• glukosa MeSH
• NAD * MeSH
• vaskulární endoteliální růstový faktor A MeSH

We investigated iron uptake mechanisms in five marine microalgae from different ecologically important phyla: the diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana, the prasinophyceae Ostreococcus tauri and Micromonas pusilla, and the coccolithophore Emiliania huxleyi. Among these species, only the two diatoms were clearly able to reduce iron, via an inducible (P. tricornutum) or constitutive (T. pseudonana) ferrireductase system displaying characteristics similar to the yeast (Saccharomyces cerevisiae) flavohemoproteins proteins. Iron uptake mechanisms probably involve very different components according to the species, but the species we studied shared common features. Regardless of the presence and/or induction of a ferrireductase system, all the species were able to take up both ferric and ferrous iron, and iron reduction was not a prerequisite for uptake. Iron uptake decreased with increasing the affinity constants of iron-ligand complexes and with increasing ligand-iron ratios. Therefore, at least one step of the iron uptake mechanism involves a thermodynamically controlled process. Another step escapes to simple thermodynamic rules and involves specific and strong binding of ferric as well as ferrous iron at the cell surface before uptake of iron. Binding was paradoxically increased in iron-rich conditions, whereas uptake per se was induced in all species only after prolonged iron deprivation. We sought cell proteins loaded with iron following iron uptake. One such protein in O. tauri may be ferritin, and in P. tricornutum, Isip1 may be involved. We conclude that the species we studied have uptake systems for both ferric and ferrous iron, both involving specific iron binding at the cell surface.

• MeSH
• autoradiografie MeSH
• biologické modely MeSH
• buněčná membrána účinky léků   metabolismus   MeSH
• chelátory železa farmakologie   MeSH
• FMN-reduktasa metabolismus   MeSH
• fylogeneze MeSH
• kinetika MeSH
• ligandy MeSH
• mikrořasy účinky léků   enzymologie   růst a vývoj   metabolismus   MeSH
• oxidace-redukce účinky léků   MeSH
• Saccharomyces cerevisiae účinky léků   metabolismus   MeSH
• transport elektronů účinky léků   MeSH
• vodní organismy růst a vývoj   metabolismus   MeSH
• železo metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• chelátory železa MeSH
• ferric citrate iron reductase MeSH Prohlížeč
• FMN-reduktasa MeSH
• ligandy MeSH
• železo MeSH

FTIR, circular dichroism (CD) and fluorescence spectroscopies were used to characterize conformational changes in horse liver alcohol dehydrogenase (HLADH) and ketoreductase (KRED 117) upon physical and covalent immobilizations on silica particles (functionalized with amino, epoxy and thiol groups) of different sizes. Conformational changes for immobilized enzymes were associated with high and low frequency shifts of the amide I and II bands. CD spectra of native HLADH and KRED 117 characterized with a negative peak at 222nm indicating a α-helical structure. The disappearance of the negative peak in the CD spectra of immobilized enzymes and appearance of a positive peak at 222nm supported these observations. These findings demonstrated unfolding of folded enzymes and exposure of the amino acid residues during denaturation with a red shift in tryptophan fluorescence. The decrease in specific activities (by 60-70% in all cases) for both immobilized enzymes was correlated to those of conformational changes. Silica-attached enzyme-NADH systems were evaluated for enantioselective reduction of 1-(p-methoxyphenyl)-propan-2-one. Conformational changes enhanced the enantioselectivity of immobilized HLADH with a switch in its stereoselectivity. In the case of immobilized KRED 117, kinetic values (V(max) and K(m)) were lower than that of the free enzyme, without enhancing enzyme enantio- and stereoselectivity.

• MeSH
• aldehydreduktasa MeSH
• aldo-keto reduktasy MeSH
• alkoholdehydrogenasa chemie   MeSH
• alkoholoxidoreduktasy chemie   MeSH
• cirkulární dichroismus MeSH
• enzymy imobilizované chemie   MeSH
• fluorescenční spektrometrie MeSH
• játra enzymologie   MeSH
• kinetika MeSH
• koenzymy chemie   MeSH
• koně MeSH
• NAD chemie   MeSH
• nanočástice chemie   ultrastruktura   MeSH
• oxid křemičitý chemie   MeSH
• rozbalení proteinů MeSH
• sekundární struktura proteinů MeSH
• spektroskopie infračervená s Fourierovou transformací MeSH
• stereoizomerie MeSH
• substrátová specifita MeSH
• terciární struktura proteinů MeSH
• tryptofan chemie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aldehydreduktasa MeSH
• aldo-keto reduktasy MeSH
• alkoholdehydrogenasa MeSH
• alkoholoxidoreduktasy MeSH
• enzymy imobilizované MeSH
• koenzymy MeSH
• NAD MeSH
• oxid křemičitý MeSH
• tryptofan MeSH

Addition of hydrogen peroxide (greater than 10 mM) to aerated derepressed cells of S. cerevisiae in the absence of substrate caused a boost of endogenous respiration and both intra- and extracellular acidification, without any significant change in cellular ATP level. Furthermore, a hyperpolarization of the plasma membrane was indicated by an enhanced accumulation of tetraphenylphosphonium in the cells. The extracellular pH attained was as low as 3.5. The acidification could be suspended by the H(+)-ATPase inhibitors diethylstilbestrol and dicyclohexylcarbodiimide and was, in general, associated with an opposite flux of K+. K+ also stimulated the H(+)-ATPase activity in the purified plasma membrane fraction. These results are consistent with the plasma membrane H(+)-ATPase being involved in the H+ extrusion induced by H2O2 in the absence of substrate. Extended exposure of cells to H2O2 led eventually to an arrest of both respiration and ion fluxes that could be again lifted by depolarizing the plasma membrane. Along with differences in the cellular NADH/NAD+ ratio and in the participation of organic acids, this makes the H2O2-induced acidification distinct from that induced by glucose.

• MeSH
• aktivace enzymů MeSH
• buněčná membrána enzymologie   MeSH
• dicyklohexylkarbodiimid farmakologie   MeSH
• diethylstilbestrol farmakologie   MeSH
• draslík metabolismus   MeSH
• karbonylkyanid-m-chlorfenylhydrazon farmakologie   MeSH
• koncentrace vodíkových iontů MeSH
• NAD metabolismus   MeSH
• oniové sloučeniny MeSH
• organofosforové sloučeniny MeSH
• oxidace-redukce MeSH
• peroxid vodíku farmakologie   MeSH
• protonové ATPasy metabolismus   MeSH
• pufry MeSH
• Saccharomyces cerevisiae enzymologie   MeSH
• spotřeba kyslíku MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• dicyklohexylkarbodiimid MeSH
• diethylstilbestrol MeSH
• draslík MeSH
• karbonylkyanid-m-chlorfenylhydrazon MeSH
• NAD MeSH
• oniové sloučeniny MeSH
• organofosforové sloučeniny MeSH
• peroxid vodíku MeSH
• protonové ATPasy MeSH
• pufry MeSH
• tetraphenylphosphonium MeSH Prohlížeč

Malate dehydrogenase, malic enzyme, succinate dehydrogenase, and fumarate reductase activities have been studied in the cytoplasm and mitochondria of Taenia crassiceps larvae. The results show that these larvae contain enzymes for anaerobic acquisition of energy with terminal fumarate reductase, but some facts, as the high ratio of succinate dehydrogenase activity to fumarate reductase activity and the low proportion of fumarate reductase in the whole NADH oxidase activity in mitochondria, suggest that aerobic processes are also involved in the energy acquisition in this parasite.

• MeSH
• aerobióza MeSH
• cytoplazma enzymologie   MeSH
• malátdehydrogenasa metabolismus   MeSH
• mitochondrie enzymologie   MeSH
• oxidoreduktasy působící na CH-CH vazby * MeSH
• oxidoreduktasy metabolismus   MeSH
• sukcinátdehydrogenasa metabolismus   MeSH
• Taenia enzymologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• fumarate reductase (NADH) MeSH Prohlížeč
• malátdehydrogenasa MeSH
• oxidoreduktasy působící na CH-CH vazby * MeSH
• oxidoreduktasy MeSH
• sukcinátdehydrogenasa MeSH