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Signal transducer and activator of transcription 3 (STAT3) is a transcription factor with roles in inflammation and tumorigenicity. A fraction of STAT3 localizes in mitochondria, where it augments tumorigenesis via regulation of mitochondrial functions, including modulation of respiration and redox status. We show a novel mechanism for mitochondrial STAT3 regulation of redox homeostasis in triple‐negative breast cancer cells. Loss of STAT3 diminished complex I dehydrogenase activity and impaired NAD+ regeneration, leading to impaired expression of glutathione biosynthetic genes and other antioxidant genes. Expressing mitochondrially restricted STAT3 or replenishment of the cellular NAD pool restored antioxidant gene expression, as did complementation of the NADH dehydrogenase activity by expression of the STAT3‐independent yeast dehydrogenase, NDI1. These NAD‐regulated processes contributed to malignant phenotypes by promoting clonal cell growth and migration. Proximity interaction and protein pull‐down assays identified three components of complex I that associated with mitochondrial STAT3, providing a potential mechanistic basis for how mitochondrial STAT3 affects complex I activity. Our data document a novel mechanism through which mitochondrial STAT3 indirectly controls antioxidant gene regulation through a retrograde NAD+ signal that is modulated by complex I dehydrogenase activity., STAT3 interacts with respiratory complex I in mitochondria, leading to enhanced NADH dehydrogenase activity, facilitating efficient regeneration of NAD+ during respiration. NAD+ acts as a retrograde signal linking mitochondrial metabolism to changes in nuclear gene expression, leading to induction of antioxidant genes that contribute to the maintenance of redox balance and malignant cell growth, survival, and migration.

Complex I (NADH dehydrogenase) is the first enzyme in the respiratory chain. It catalyses the electron transfer from NADH to ubiquinone that is associated with proton pumping out of the matrix. In this study, we characterized NADH dehydrogenase activity in seven monoxenous trypanosomatid species: Blechomonas ayalai, Herpetomonas tarakana, Kentomonas sorsogonicus, Leptomonas seymouri, Novymonas esmeraldas, Sergeia podlipaevi and Wallacemonas raviniae. We also investigated the subunit composition of the complex I in dixenous Phytomonas serpens, in which its presence and activity have been previously documented. In addition to P. serpens, the complex I is functionally active in N. esmeraldas and S. podlipaevi. We also identified 24–32 subunits of the complex I in individual species by using mass spectrometry. Among them, for the first time, we recognized several proteins of the mitochondrial DNA origin.

Rationale: Reactive oxygen species (ROS) burst from mitochondrial complex I is considered the critical cause of ischemia/reperfusion (I/R) injury. Ginsenoside Rb1 has been reported to protect the heart against I/R injury; however, the underlying mechanism remains unclear. This work aimed to investigate if ginsenoside Rb1 attenuates cardiac I/R injury by inhibiting ROS production from mitochondrial complex I. Methods: In in vivo experiments, mice were given ginsenoside Rb1 and then subjected to I/R injury. Mitochondrial ROS levels in the heart were determined using the mitochondrial-targeted probe MitoB. Mitochondrial proteins were used for TMT-based quantitative proteomic analysis. In in vitro experiments, adult mouse cardiomyocytes were pretreated with ginsenoside Rb1 and then subjected to hypoxia and reoxygenation insult. Mitochondrial ROS, NADH dehydrogenase activity, and conformational changes of mitochondrial complex I were analyzed. Results: Ginsenoside Rb1 decreased mitochondrial ROS production, reduced myocardial infarct size, preserved cardiac function, and limited cardiac fibrosis. Proteomic analysis showed that subunits of NADH dehydrogenase in mitochondrial complex I might be the effector proteins regulated by ginsenoside Rb1. Ginsenoside Rb1 inhibited complex I- but not complex II- or IV-dependent O2 consumption and enzyme activity. The inhibitory effects of ginsenoside Rb1 on mitochondrial I-dependent respiration and reperfusion-induced ROS production were rescued by bypassing complex I using yeast NADH dehydrogenase. Molecular docking and surface plasmon resonance experiments indicated that ginsenoside Rb1 reduced NADH dehydrogenase activity, probably via binding to the ND3 subunit to trap mitochondrial complex I in a deactive form upon reperfusion. Conclusion: Inhibition of mitochondrial complex I-mediated ROS burst elucidated the probable underlying mechanism of ginsenoside Rb1 in alleviating cardiac I/R injury.

AIMS Effects of a proteasome inhibitor, MG-132, on the riboflavin production in Ashbya gossypii were investigated to elucidate the relationship of the riboflavin production with flavoprotein homeostasis. METHODS AND RESULTS The addition of MG-132 to the liquid medium reduced the specific riboflavin production by 79% in A. gossypii at 25 μM after 24 h. The addition of the inhibitor also caused the accumulation of reactive oxygen species and ubiquitinated proteins. These results indicated that MG-132 works in A. gossypii without any genetic engineering and reduces riboflavin production. In the presence of 25 μM MG-132, specific NADH dehydrogenase activity was increased by 1.4-fold compared to DMSO, but specific succinate dehydrogenase (SDH) activity was decreased to 52% compared to DMSO. Additionally, the amount of AgSdh1p (ACR052Wp) was also reduced. Specific riboflavin production was reduced to 22% when 20 mM malonate, a SDH inhibitor, was added to the culture medium. The riboflavin production in heterozygous AgSDH1 gene-disrupted mutant (AgSDH1-/+ ) was reduced to 63% compared to that in wild type. CONCLUSIONS MG-132 suppresses the riboflavin production and SDH activity in A. gossypii. SDH is one of the flavoproteins involved in the riboflavin production in A. gossypii. SIGNIFICANCE AND IMPACT OF THE STUDY This study shows that MG-132 has a negative influence on the riboflavin production and SDH activity in A. gossypii and leads to the elucidation of the connection of the riboflavin production with flavoproteins.

The highly abundant voltage-dependent anion-selective channel (VDAC) allows transit of metabolites across the mitochondrial outer membrane. Previous studies in Neurospora crassa showed that the LoPo strain, expressing 50% of normal VDAC levels, is indistinguishable from wild-type (WT). In contrast, the absence of VDAC (ΔPor-1), or the expression of an N-terminally truncated variant VDAC (ΔN2-12porin), is associated with deficiencies in cytochromes b and aa3 of complexes III and IV and concomitantly increased alternative oxidase (AOX) activity. These observations led us to investigate complex I and complex II activities in these strains, and to explore their mitochondrial bioenergetics. The current study reveals that the total NADH dehydrogenase activity is similar in mitochondria from WT, LoPo, ΔPor-1 and ΔN2-12porin strains; however, in ΔPor-1 most of this activity is the product of rotenone-insensitive alternative NADH dehydrogenases. Unexpectedly, LoPo mitochondria have increased complex II activity. In all mitochondrial types analyzed, oxygen consumption is higher in the presence of the complex II substrate succinate, than with the NADH-linked (complex I) substrates glutamate and malate. When driven by a combination of complex I and II substrates, membrane potentials (Δψ) and oxygen consumption rates (OCR) under non-phosphorylating conditions are similar in all mitochondria. However, as expected, the induction of state 3 (phosphorylating) conditions in ΔPor-1 mitochondria is associated with smaller but significant increases in OCR and smaller decreases in Δψ than those seen in wild-type mitochondria. High ROS production, particularly in the presence of rotenone, was observed under non-phosphorylating conditions in the ΔPor-1 mitochondria. Thus, the absence of VDAC is associated with increased ROS production, in spite of AOX activity and wild-type OCR in ΔPor-1 mitochondria.

Ustilago maydis is an aerobic basidiomycete that depends on oxidative phosphorylation for its ATP supply, pointing to the mitochondrion as a key player in its energy metabolism. Mitochondrial respiratory complexes I, III2, and IV occur in supramolecular structures named respirasome. In this work, we characterized the subunit composition and the kinetics of NADH:Q oxidoreductase activity of the digitonine-solubilized respirasome (1600 kDa) and the free-complex I (990 kDa). In the presence of 2,6-dimethoxy-1,4-benzoquinone (DBQ) and cytochrome c, both the respirasome NADH:O2 and the NADH:DBQ oxidoreductase activities were inhibited by rotenone, antimycin A or cyanide. A value of 2.4 for the NADH oxidized/oxygen reduced ratio was determined for the respirasome activity, while ROS production was less than 0.001% of the oxygen consumption rate. Analysis of the NADH:DBQ oxidoreductase activity showed that respirasome was 3-times more active and showed higher affinity than free-complex I. The results suggest that the contacts between complexes I, III2 and IV in the respirasome increase the catalytic efficiency of complex I and regulate its activity to prevent ROS production.

The cytoplasmic fumarate reductase of Klebsiella pneumoniae (FRD) is a monomeric protein which contains three prosthetic groups: noncovalently bound FMN and FAD plus a covalently bound FMN. In the present work, NADH is revealed to be an inherent electron donor for this enzyme. We found that the fumarate reductase activity of FRD significantly exceeds its NADH dehydrogenase activity. During the catalysis of NADH:fumarate oxidoreductase reaction, FRD turnover is limited by a very low rate (∼10/s) of electron transfer between the noncovalently and covalently bound FMN moieties. Induction of FRD synthesis in K. pneumoniae cells was observed only under anaerobic conditions in the presence of fumarate or malate. Enzymes with the FRD-like domain architecture are widely distributed among various bacteria and apparently comprise a new type of water-soluble NADH:fumarate oxidoreductases.

Bacteroides species are abundant in the human intestine and provide numerous beneficial properties to their hosts. The ability of Bacteroides species to convert host and dietary glycans and polysaccharides to energy is paramount to their success in the human gut. We know a great deal about the molecules that these bacteria extract from the human gut but much less about how they convert those molecules into energy. Here, we show that B. fragilis has a complex respiratory pathway with two different enzymes that transfer electrons from NADH to quinone and a third enzyme complex that may use an electron donor other than NADH. Although fermentation has generally been believed to be the main mechanism of energy generation in Bacteroides, we found that a mutant lacking one of the NADH:quinone oxidoreductases was unable to compete with the wild type in the mammalian gut, revealing the importance of respiration to these abundant gut symbionts., In bacteria, the respiratory pathways that drive molecular transport and ATP synthesis include a variety of enzyme complexes that utilize different electron donors and acceptors. This property allows them to vary the efficiency of energy conservation and to generate different types of electrochemical gradients (H+ or Na+). We know little about the respiratory pathways in Bacteroides species, which are abundant in the human gut, and whether they have a simple or a branched pathway. Here, we combined genetics, enzyme activity measurements, and mammalian gut colonization assays to better understand the first committed step in respiration, the transfer of electrons from NADH to quinone. We found that a model gut Bacteroides species, Bacteroides fragilis, has all three types of putative NADH dehydrogenases that typically transfer electrons from the highly reducing molecule NADH to quinone. Analyses of NADH oxidation and quinone reduction in wild-type and deletion mutants showed that two of these enzymes, Na+-pumping NADH:quinone oxidoreductase (NQR) and NADH dehydrogenase II (NDH2), have NADH dehydrogenase activity, whereas H+-pumping NADH:ubiquinone oxidoreductase (NUO) does not. Under anaerobic conditions, NQR contributes more than 65% of the NADH:quinone oxidoreductase activity. When grown in rich medium, none of the single deletion mutants had a significant growth defect; however, the double Δnqr Δndh2 mutant, which lacked almost all NADH:quinone oxidoreductase activity, had a significantly increased doubling time. Despite unaltered in vitro growth, the single nqr deletion mutant was unable to competitively colonize the gnotobiotic mouse gut, confirming the importance of NQR to respiration in B. fragilis and the overall importance of respiration to this abundant gut symbiont.

The emergence of drug-resistant fungal pathogens is becoming increasingly serious due to overuse of antifungals. Antimicrobial peptides have potent activity against a broad spectrum of pathogens, including fungi, and are considered a potential new class of antifungals. In this study, we examined the activities of the newly designed peptides P-113Du and P-113Tri, together with their parental peptide P-113, against the human fungal pathogen Candida albicans. The results showed that these peptides inhibit mitochondrial complex I, specifically NADH dehydrogenase, of the electron transport chain. Moreover, P-113Du and P-113Tri also block alternative NADH dehydrogenases. Currently, most inhibitors of the mitochondrial complex I are small molecules or artificially-designed antibodies. Here, we demonstrated novel functions of antimicrobial peptides in inhibiting the mitochondrial complex I of C. albicans, providing insight in the development of new antifungal agents.

Type II NADH:quinone oxidoreductases (NDH-2s) are membrane proteins involved in respiratory chains and responsible for the maintenance of NADH/NAD(+) balance in cells. NDH-2s are the only enzymes with NADH dehydrogenase activity present in the respiratory chain of many pathogens, and thus, they were proposed as suitable targets for antimicrobial therapies. In addition, NDH-2s were also considered key players for the treatment of complex I-related neurodegenerative disorders. In this work, we explored substrate-protein interaction in NDH-2 from Escherichia coli (EcNDH-2) combining surface-enhanced infrared absorption spectroscopic studies with electrochemical experiments, fluorescence spectroscopy assays, and quantum chemical calculations. Because of the specific stabilization of substrate complexes of EcNDH-2 immobilized on electrodes, it was possible to demonstrate the presence of two distinct substrate binding sites for NADH and the quinone and to identify a bound semiprotonated quinol as a catalytic intermediate.

The energy-generating membrane protein NADH dehydrogenase (NDH-2), a proposed antibacterial drug target (see “Inhibitors of type II NADH:menaquinone oxidoreductase represent a class of antitubercular drugs” Weinstein et al. 2005 [1]), was screened for modulators of activity in either detergent-solublised or lipid reconstituted (proteolipsome) form. Here we present an annotated list of compounds identified in a small-scale screen against NDH-2. The dataset contains information regarding the libraries screened, the identities of hit compounds and the physicochemical properties governing solubility and permeability. The implications of these data for future antibiotic discovery are discussed in our associated report, “Comparison of lipid and detergent enzyme environments for identifying inhibitors of membrane-bound energy-transducing proteins” [2].

Alternative oxidase (Aox) is a non-energy-conserving respiratory oxidase found in certain eukaryotes and bacteria, whose role in physiology is not entirely clear. Using the genetically tractable bacterium Vibrio fischeri as a model organism, I have identified a role for Aox to reduce levels of stress in cells exposed to oxygen and nitric oxide (NO). In V. fischeri lacking the NO-detoxifying enzyme flavohemoglobin (Hmp), deletion of aox in cells grown in the presence of oxygen and NO results in alterations to the transcriptome that include increases in transcripts mapping to stress-related genes. Using fluorescence-based reporters, I identified corresponding increases in intracellular reactive oxygen species and decreases in membrane integrity in cells lacking aox Under these growth conditions, activity of Aox is linked to a decrease in NADH levels, indicating coupling of Aox activity with NADH dehydrogenase activity. Taken together, these results suggest that Aox functions to indirectly limit production of ferrous iron and damaging hydroxyl radicals, effectively reducing cellular stress during NO exposure.IMPORTANCE Unlike typical respiratory oxidases, alternative oxidase (Aox) does not directly contribute to energy conservation, and its activity would presumably reduce the efficiency of respiration and associated ATP production. Aox has been identified in certain bacteria, a majority of which are marine associated. The presence of Aox in these bacteria poses the interesting question of how Aox function benefits bacterial growth and survival in the ocean. Using the genetically tractable marine bacterium Vibrio fischeri, I have identified a role for Aox in reduction of stress under conditions where electron flux through the aerobic respiratory pathway is inhibited. These results suggest that Aox activity could positively impact longer-term bacterial fitness and survival under stressful environmental conditions.

YajL is the closest prokaryotic homologue of Parkinson's disease-associated DJ-1, a protein of undefined function involved in the oxidative stress response. We reported recently that YajL and DJ-1 protect cells against oxidative stress-induced protein aggregation by acting as covalent chaperones for the thiol proteome, including the NuoG subunit of NADH dehydrogenase 1, and that NADH dehydrogenase 1 activity is negligible in the yajL mutant. We report here that this mutant compensates for low NADH dehydrogenase activity by utilizing NADH-independent alternative dehydrogenases, including pyruvate oxidase PoxB and D-amino acid dehydrogenase DadA, and mixed acid aerobic fermentations characterized by acetate, lactate, succinate and ethanol excretion. The yajL mutant has a low adenylate energy charge favouring glycolytic flux, and a high NADH/NAD ratio favouring fermentations over pyruvate dehydrogenase and the Krebs cycle. DNA array analysis showed upregulation of genes involved in glycolytic and pentose phosphate pathways and alternative respiratory pathways. Moreover, the yajL mutant preferentially catabolized pyruvate-forming amino acids over Krebs cycle-related amino acids, and thus the yajL mutant utilizes pyruvate-centred respiro-fermentative metabolism to compensate for the NADH dehydrogenase 1 defect and constitutes an interesting model for studying eukaryotic respiratory complex I deficiencies, especially those associated with Alzheimer's and Parkinson's diseases.

Mitochondria of Saccharomyces cerevisiae lack the respiratory complex I, but contain three rotenone-insensitive NADH dehydrogenases distributed on both the external (Nde1 and Nde2) and internal (Ndi1) surfaces of the inner mitochondrial membrane. These enzymes catalyse the transfer of electrons from NADH to ubiquinone without the translocation of protons across the membrane. Due to the high resolution of the Blue Native PAGE (BN-PAGE) technique combined with digitonin solubilization, several bands with NADH dehydrogenase activity were observed on the gel. The use of specific S. cerevisiae single and double mutants of the external alternative elements (ΔNDE1, ΔNDE2, ΔNDE1/ΔNDE2) showed that the high and low molecular weight complexes contained the Ndi1. Some of the Ndi1 associations took place with complexes III and IV, suggesting the formation of respirasome-like structures. Complex II interacted with other proteins to form a high molecular weight supercomplex with a molecular mass around 600 kDa. We also found that the majority of the Ndi1 was in a dimeric form, which is in agreement with the recently reported three-dimensional structure of the protein.

Escherichia coli exposed to tellurite shows augmented membrane lipid peroxidation and ROS content. Also, reduced thiols, protein carbonylation, [Fe-S] center dismantling, and accumulation of key metabolites occur in these bacteria. In spite of this, not much is known about tellurite effects on the E. coli electron transport chain (ETC). In this work, tellurite-mediated damage to the E. coli ETC's NADH dehydrogenases and terminal oxidases was assessed. Mutant lacking ETC components showed delayed growth, decreased oxygen consumption and increased ROS in the presence of the toxicant. Membranes from tellurite-exposed E. coli exhibited decreased oxygen consumption and dNADH/NADH dehydrogenase activity, showing an impairment of NDH-I but not of NDH-II activity. Regarding terminal oxidases, only the bo oxidase complex was affected by tellurite. When assaying NDH-I and NDH-II activity in the presence of superoxide, the NDH-I complex was preferentially damaged. The activity was partly restored in the presence of reducing agents, sulfide and Fe(2+) under anaerobic conditions, suggesting that damage affects NDH-I [4Fe-4S] centers. Finally, augmented membrane protein oxidation along with reduced oxidase activity was observed in the presence of the toxicant. Also, the increased expression of genes encoding alternative terminal oxidases probably reflects a cell's change towards anaerobic respiration when facing tellurite.

Background Enterobacter aerogenes is a facultative anaerobe and is one of the most widely studied bacterial strains because of its ability to use a variety of substrates, to produce hydrogen at a high rate, and its high growth rate during dark fermentation. However, the rate of hydrogen production has not been optimized. In this present study, three strategies to improve hydrogen production in E. aerogenes, namely the disruption of nuoCDE, overexpression of the small RNA RyhB and of NadE to regulate global anaerobic metabolism, and the redistribution of metabolic flux. The goal of this study was to clarify the effect of nuoCDE, RyhB, and NadE on hydrogen production and how the perturbation of NADH influences the yield of hydrogen gas from E. aerogenes. Results NADH dehydrogenase activity was impaired by knocking out nuoCD or nuoCDE in E. aerogenes IAM1183 using the CRISPR-Cas9 system to explore the consequent effect on hydrogen production. The hydrogen yields from IAM1183-CD(∆nuoC/∆nuoD) and IAM1183-CDE (∆nuoC/∆nuoD/∆nuoE) increased, respectively, by 24.5 and 45.6% in batch culture (100 mL serum bottles). The hydrogen produced via the NADH pathway increased significantly in IAM1183-CDE, suggesting that nuoE plays an important role in regulating NADH concentration in E. aerogenes. Batch-cultivating experiments showed that by the overexpression of NadE (N), the hydrogen yields of IAM1183/N, IAM1183-CD/N, and IAM1183-CDE/N increased 1.06-, 1.35-, and 1.55-folds, respectively, compared with IAM1183. Particularly worth mentioning is that the strain IAM118-CDE/N reached 2.28 mol in H2 yield, per mole of glucose consumed. IAN1183/R, IAM1183-CD/R, and IAM1183-CDE/R showed increasing H2 yields in batch culture. Metabolic flux analysis indicated that increased expression of RyhB led to a significant shift in metabolic patterns. We further investigated IAM1183-CDE/N, which had the best hydrogen-producing traits, as a potential candidate for industry applications using a 5-L fermenter; hydrogen production reached up to 1.95 times greater than that measured for IAM1183. Conclusions Knockout of nuoCD or nuoCDE and the overexpression of nadE in E. aerogenes resulted in a redistribution of metabolic flux and improved the hydrogen yield. Overexpression of RyhB had an significant change on the hydrogen production via NADH pathway. A combination of strategies would be a novel approach for developing a more economic and efficient bioprocess for hydrogen production in E. aerogenes. Finally, the latest CRISPR-Cas9 technology was successful for editing genes in E. aerogenes to develop our engineered strain for hydrogen production. Electronic supplementary material The online version of this article (doi:10.1186/s13068-017-0938-2) contains supplementary material, which is available to authorized users.

The antifungal activities of chitosan and oligochitosan have been used to control postharvest decay of the fruits. The effect of chitosan and oligochitosan on mycelium growth, spore germination, and mitochondrial function of Rhizopus stolonifer was evaluated in order to establish a connection between fungus development and the main organelle in charge to provide energy to the cell. The mycelium growth of R. stolonifer was significantly reduced on minimum media amended with chitosan or oligochitosan. The highest antifungal indexes were obtained on media containing chitosan or oligochitosan at 2.0 mg ml(-1). Microscopic observation showed that chitosan and oligochitosan affected the spore germination and hyphae morphology. Both polymers increased oxygen consumption of R. stolonifer. Respiratory activity was restored with NADH in permeabilized treated and untreated cells, and was inhibited with rotenone and flavones. Complex III and IV were inhibited by antimycin A and cyanide, respectively, in treated and untreated cells. Chitosan and oligochitosan increased NADH dehydrogenase activity in isolated mitochondria. However, there were not changes in the cytochrome c oxidase and ATPase activities by effect of these polymers. These results suggest that both chitosan and oligochitosan affect the development of R. stolonifer and might be implicated in the mitochondrial dysfunction.

Allelochemical L-DOPA (l-3,4-dihydroxyphenylalanine) inhibits growth of several plant species. However, its mode of action is not well clarified in plants. The present studies were conducted to explore the action mechanism of L-DOPA in cucumber roots. The results revealed that L-DOPA suppressed the root growth of cucumber and induced quinoprotein and melanin formation in the roots. Moreover, L-DOPA not only decreased mitochondrial viability and NADH dehydrogenase (complex I) activity but also increased quinoprotein formation in vitro in isolated mitochondria from cucumber roots. Strong correlations were observed between quinoprotein formation and root growth inhibition, quinoprotein formation and NADH dehydrogenase activity, after L-DOPA treatment. The results suggest that quinoprotein formation and mitochondrial impairment might be involved in growth-inhibition mechanism of L-DOPA in cucumber roots.

Despite advances in clinical therapy, metastasis remains the leading cause of death in breast cancer patients. Mutations in mitochondrial DNA, including those affecting complex I and oxidative phosphorylation, are found in breast tumors and could facilitate metastasis. This study identifies mitochondrial complex I as critical for defining an aggressive phenotype in breast cancer cells. Specific enhancement of mitochondrial complex I activity inhibited tumor growth and metastasis through regulation of the tumor cell NAD+/NADH redox balance, mTORC1 activity, and autophagy. Conversely, nonlethal reduction of NAD+ levels by interfering with nicotinamide phosphoribosyltransferase expression rendered tumor cells more aggressive and increased metastasis. The results translate into a new therapeutic strategy: enhancement of the NAD+/NADH balance through treatment with NAD+ precursors inhibited metastasis in xenograft models, increased animal survival, and strongly interfered with oncogene-driven breast cancer progression in the MMTV-PyMT mouse model. Thus, aberration in mitochondrial complex I NADH dehydrogenase activity can profoundly enhance the aggressiveness of human breast cancer cells, while therapeutic normalization of the NAD+/NADH balance can inhibit metastasis and prevent disease progression.

The first-attempt study employed a proteomics strategy for the identification of abundant proteins from a bioelectricity generation and dye decolorization bacterium Proteus hauseri ZMd44. By using the degenerated primers designed based on the peptide sequences from tandem mass spectroscopy and the whole genomics annotation of the closely associated strain, Proteus penneri ATCC 35198, the genes were successfully obtained for two full-length genes of 543 bp (laccase) and 1,086 bp (Omp F, porin) encoding to 181 amino acids and 362 amino acids, respectively. It explored laccase and NADH dehydrogenase involvement in the oxidation-reduction reaction as well, as porin played an important role in providing channels for related proteins in the accomplishment of electron transportation in P. hauseri. Detailed enzymatic assays indicated that laccase activity of 542.2 U/DCW could be stimulated by 2.5 mM copper induction in LB medium (ca. 293-fold to those without copper induction). Among intracellular proteins, NADH dehydrogenase activity of 257.2 U/mg via mediator riboflavin was in parallel with the decolorizing capability of azo dye Rb160 that only took place in LB medium. From the evaluation of kinetic parameters (Vmax and Km were 0.272 U/min and 0.393 mM with ABTS, 0.046 U/min and 43.8 μM with NADH), it is better to decipher the decolorization mechanism of ZMd44 indicating that laccase and NADH dehydrogenase played the most crucial role for azo dye decolorization.

Chlorogenic acid (CGA), an abundant polyphenol compound in plants, exhibits anti-oxidant effects. The protective effect of CGA in the rat intestine with endotoxin infusion was evaluated. CGA administration ameliorated endotoxin-induced intestinal injury, and decreased the ratio of lactulose/mannitol, the ileum pathological grade, the myeloperoxidase activity in the ileum, and the malondialdehyde content in the ileum and in ileum mitochondria. The small intestine weight, activities of alkaline phosphatase and superoxide dismutase in the ileum, and β-nicotinamide adenine dinucleotide reduce form (NADH) dehydrogenase and succinate dehydrogenase activities in ileum mitochondria were increased. Intestinal permeability was positively correlated with intestinal mitochondrial injury indicated as the level of malondialdehyde in ileum mitochondria, and negatively correlated with NADH dehydrogenase activity. Dietary administration of CGA protected against increased intestinal permeability caused by endotoxin infusion. The protective effect of CGA was probably associated with a decrease in mitochondrial lipid peroxidation levels and an increase in NADH dehydrogenase activity.

We previously isolated respiratory-deficient mutant (RDM) strains of Zymomonas mobilis , which exhibited greater growth and enhanced ethanol production under aerobic conditions. These RDM strains also acquired thermotolerance. Morphologically, the cells of all RDM strains were shorter compared to the wild-type strain. We investigated the respiratory chains of these RDM strains and found that some RDM strains lost NADH dehydrogenase activity, whereas others exhibited reduced cytochrome bd -type ubiquinol oxidase or ubiquinol peroxidase activities. Complementation experiments restored the wild-type phenotype. Some RDM strains seem to have certain mutations other than the corresponding respiratory chain components. RDM strains with deficient NADH dehydrogenase activity displayed the greatest amount of aerobic growth, enhanced ethanol production, and thermotolerance. Nucleotide sequence analysis revealed that all NADH dehydrogenase-deficient strains were mutated within the ndh gene, which includes insertion, deletion, or frameshift. These results suggested that the loss of NADH dehydrogenase activity permits the acquisition of higher aerobic growth, enhanced ethanol production, and thermotolerance in this industrially important strain.

Summary Pentatricopeptide repeat (PPR) proteins are involved in the modification of organelle transcripts. In this study, we investigated the molecular function in rice of the mitochondrial PPR-encoding gene MITOCHONDRIAL PPR25 (MPR25), which belongs to the E subgroup of the PPR family. A Tos17 knockout mutant of MPR25 exhibited growth retardation and pale-green leaves with reduced chlorophyll content during the early stages of plant development. The photosynthetic rate in the mpr25 mutant was significantly decreased, especially under strong light conditions, although the respiration rate did not differ from that of wild-type plants. MPR25 was preferentially expressed in leaves. FLAG-tagged MPR25 accumulated in mitochondria but not in chloroplasts. Direct sequencing revealed that the mpr25 mutant fails to edit a C–U RNA editing site at nucleotide 1580 of nad5, which encodes a subunit of complex I (NADH dehydrogenase) of the respiratory chain in mitochondria. RNA editing of this site is responsible for a change in amino acid from serine to leucine. Recombinant MPR25 directly interacted with the proximal region of the editing site of nad5 transcripts. However, the NADH dehydrogenase activity of complex I was not affected in the mutant. By contrast, genes encoding alternative NADH dehydrogenases and alternative oxidase were up-regulated. The mpr25 mutant may therefore provide new information on the coordinated interaction between mitochondria and chloroplasts.

Cytoplasmic male sterility (CMS) is an important trait in rice (Oryza sativa L.) breeding because it provides a source for producing hybrid seeds. In rice CMS lines, ATPases involved in the oxidative phosphorylation complexes are believed to be dysfunctional due to the expression of rice CMS-related gene orf79. In the present study, a new type of CMS line named CMS-ZA (ZidaoA) was used. We found an orf79 homologous gene (named orfZ79) in three different rice lines (a CMS line, a maintainer line, and a hybrid). However, no detectable expression products of orfZ79 were found in the three lines. We evaluated the ATPase and NADH dehydrogenase activities of the three lines using in-gel catalytic assays. Our results show that the sterile line has intact ATPase activity, while NADH DHase activity is clearly decreased. To investigate NADH dehydrogenase deficiency, we measured NADH DHase activity in etiolated seedlings and green seedlings from the ZidaoA CMS sterile line and its corresponding maintainer line. We note that the NADH DHase activity of the sterile line was more deficient in green seedlings than that in etiolated seedlings. Our results show a possible role of NADH DHase deficiency to cause rice CMS.

Mutations in mitochondrial oxidative phosphorylation complex I are associated with multiple pathologies, and complex I has been proposed as a crucial regulator of animal longevity. In yeast, the single-subunit NADH dehydrogenase Ndi1 serves as a non-proton-translocating alternative enzyme that replaces complex I, bringing about the reoxidation of intramitochondrial NADH. We have created transgenic strains of Drosophila that express yeast NDI1 ubiquitously. Mitochondrial extracts from NDI1 -expressing flies displayed a rotenone-insensitive NADH dehydrogenase activity, and functionality of the enzyme in vivo was confirmed by the rescue of lethality resulting from RNAi knockdown of complex I. NDI1 expression increased median, mean, and maximum lifespan independently of dietary restriction, and with no change in sirtuin activity. NDI1 expression mitigated the aging associated decline in respiratory capacity and the accompanying increase in mitochondrial reactive oxygen species production, and resulted in decreased accumulation of markers of oxidative damage in aged flies. Our results support a central role of mitochondrial oxidative phosphorylation complex I in influencing longevity via oxidative stress, independently of pathways connected to nutrition and growth signaling.

Acetogenic bacteria grow by the oxidation of various substrates coupled to the reduction of carbon dioxide (acetogenesis) or other electron acceptors but the mechanisms of energy conservation are still enigmatic. Here, we report the presence of a rnf gene cluster rnfCDGEAB in Acetobacterium woodii that is speculated to encode a novel, energy-conserving ferredoxin:NAD(+)-oxidoreductase complex composed of at least six different subunits. Transcriptional analysis revealed that the genes constitute an operon. RnfC and RnfG were heterologously produced and antibodies were generated. Western blot analyses demonstrated that these subunits were produced and are associated with the cytoplasmic membrane. The subunits were present in cells respiring with either carbon dioxide or caffeate. A preparation with NADH dehydrogenase activity was obtained from detergent solubilized membranes that contained RnfC and RnfG.

The Na + -translocating NADH:quinone oxidoreductase (Na + -NQR) from Vibrio cholerae is a membrane-bound, respiratory Na + pump. Its NqrF subunit contains one FAD and a [2Fe–2S] cluster and catalyzes the initial oxidation of NADH. A soluble variant of NqrF lacking its hydrophobic, N-terminal helix (NqrF′) was produced in V. cholerae wild type and nqr deletion strain. Under identical conditions of growth and induction, the yield of NqrF′ increased by 30% in the presence of the Na + -NQR. FAD-containing NqrF′ species with or without the FeS cluster were observed, indicating that assembly of the FeS center, but not insertion of the flavin cofactor, was limited during overproduction in V. cholerae . A comparison of these distinct NqrF′ species with regard to specific NADH dehydrogenase activity, pH dependence of activity and thermal inactivation showed that NqrF′ lacking the [2Fe–2S] cluster was less stable, partially unfolded, and therefore prone to proteolytic degradation in V. cholerae . We conclude that the overall yield of NqrF′ critically depends on the amount of fully assembled, FeS-containing NqrF′ in the V. cholerae host cells. The Na + -NQR is proposed to increase the stability of NqrF′ by stimulating the maturation of FeS centers.

To study effects of mitochondrial complex I (CI, NADH:ubiquinone oxidoreductase) deficiency, we inactivated the Ndufs4 gene, which encodes an 18 kDa subunit of the 45-protein CI complex. Although small, Ndufs4 knockout (KO) mice appeared healthy until approximately 5 weeks of age, when ataxic signs began, progressing to death at approximately 7 weeks. KO mice manifested encephalomyopathy including a retarded growth rate, lethargy, loss of motor skill, blindness, and elevated serum lactate. CI activity in submitochondrial particles from KO mice was undetectable by spectrophotometric assays. However, CI-driven oxygen consumption by intact tissue was about half that of controls. Native gel electrophoresis revealed reduced levels of intact CI. These data suggest that CI fails to assemble properly or is unstable without NDUFS4. KO muscle has normal morphology but low NADH dehydrogenase activity and subsarcolemmal aggregates of mitochondria. Nonetheless, total oxygen consumption and muscle ATP and phosphocreatine concentrations measured in vivo were within normal parameters.

NADH dehydrogenase activity was characterized in the mitochondrial lysates of Phytomonas serpens, a trypanosomatid flagellate parasitizing plants. Two different high molecular weight NADH dehydrogenases were characterized by native PAGE and detected by direct in-gel activity staining. The association of NADH dehydrogenase activities with two distinct multisubunit complexes was revealed in the second dimension performed under denaturing conditions. One subunit present in both complexes cross-reacted with the antibody against the 39 kDa subunit of bovine complex I. Out of several subunits analyzed by MS, one contained a domain characteristic for the LYR family subunit of the NADH:ubiquinone oxidoreductases. Spectrophotometric measurement of the NADH:ubiquinone 10 and NADH:ferricyanide dehydrogenase activities revealed their different sensitivities to rotenone, piericidin, and diphenyl iodonium.

Background Previous study showed that mitochondrial ND6 (mitND6) gene missense mutation resulted in NADH dehydrogenase deficiency and was associated with tumor metastasis in several mouse tumor cell lines. In the present study, we investigated the possible role of mitND6 gene nonsense and missense mutations in the metastasis of human lung adenocarcinoma. Methods The presence of mitND6 gene mutations was screened by DNA sequencing of tumor tissues from 87 primary lung adenocarcinoma patients and the correlation of the mutations with the clinical features was analyzed. In addition, we constructed cytoplasmic hybrid cells with denucleared primary lung adenocarcinoma cell as the mitochondria donor and mitochondria depleted lung adenocarcinoma A549 cell as the nuclear donor. Using these cells, we studied the effects of mitND6 gene nonsense and missense mutations on cell migration and invasion through wounding healing and matrigel-coated transwell assay. The effects of mitND6 gene mutations on NADH dehydrogenase activity and ROS production were analyzed by spectrophotometry and flow cytometry. Results mitND6 gene nonsense and missense mutations were detected in 11 of 87 lung adenocarcinoma specimens and was correlated with the clinical features including age, pathological grade, tumor stage, lymph node metastasis and survival rate. Moreover, A549 cell containing mitND6 gene nonsense and missense mutation exhibited significantly lower activity of NADH dehydrogenase, higher level of ROS, higher capacity of cell migration and invasion, and higher pAKT and pERK1/ERK2 expression level than cells with the wild type mitND6 gene. In addition, NADH dehydrogenase inhibitor rotenone was found to significantly promote the migration and invasion of A549 cells. Conclusions Our data suggest that mitND6 gene nonsense and missense mutation might promote cell migration and invasion in lung adenocarcinoma, probably by NADH dehydrogenase deficiency induced over-production of ROS.

Mitochondrial NADH-dehydrogenase, succinate dehydrogenase and cytochrome oxidase activities of peripheral blood leukocytes of rats with the grafted Guerin's carcinoma were studied in the dynamics of oncogenesis under the conditions of the preliminary low-level irradiation. Tumor growth was accompanied by a decrease in NADH-dehydrogenase activity, an increase of succinate dehydrogenase activity. Cytochrome oxidase activity of leucocytes remained at the control level up to the terminal stages of tumor growth. Preliminary low-level irradiation of the tumor bearing animals caused a tendency to the decrease of enzymatic activities studied. This tendency was observed from the initial stages of oncogenesis.Issledovana NADN-degidrogenaznaia, suktsinatdegidrogenaznaia i tsitokhromoksidaznaia aktivnost' mitokhondriĭ leĭkotsitov perifericheskoĭ krovi krys s transplantirovannoĭ kartsinomoĭ Gerena v dinamike onkogeneza v usloviiakh predvaritel'nogo oblucheniia malymi dozami radiatsii. Pokazano, chto v dinamike rosta kartsinomy Gerena nabliudaetsia tormozhenie NADH-degidrogenaznoĭ aktivnosti na fone aktivatsii suktsinatdegidrogenazy c sokhraneniem na terminal'nykh étapakh rosta opukholi tsitokhromoksidaznoĭ aktivnosti na urovne znacheniĭ kontrolia. V usloviiakh predvaritel'nogo nizkodozovogo oblucheniia opukholenositeleĭ nabliudaetsia tendentsiia k tormozheniiu issleduemykh fermentativnykh aktivnosteĭ, nachinaia uzhe s nachal'nykh étapov onkogeneza.

International audience; Complex I (CI) deficiency is the most common respiratory chain defect representing more than 30% of mitochondrial diseases. CI is an L-shaped multi-subunit complex with a peripheral arm protruding into the mitochondrial matrix and a membrane arm. CI sequentially assembled into main assembly intermediates: the P (pumping), Q (Quinone) and N (NADH dehydrogenase) modules. In this study, we analyzed 11 fibroblast cell lines derived from patients with inherited CI deficiency resulting from mutations in the nuclear or mitochondrial DNA and impacting these different modules. In patient cells carrying a mutation located in the matrix arm of CI, blue native-polyacrylamide gel electrophoresis (BN-PAGE) revealed a significant reduction of fully assembled CI enzyme and an accumulation of intermediates of the N module. In these cell lines with an assembly defect, NADH dehydrogenase activity was partly functional, even though CI was not fully assembled. We further demonstrated that this functional N module was responsible for ROS production through the reduced flavin mononucleotide. Due to the assembly defect, the FMN site was not re-oxidized leading to a significant oxidative stress in cell lines with an assembly defect. These findings not only highlight the relationship between CI assembly and oxidative stress, but also show the suitability of BN-PAGE analysis in evaluating the consequences of CI dysfunction. Moreover, these data suggest that the use of antioxidants may be particularly relevant for patients displaying a CI assembly defect.

In standard laboratory strains of the obligate aerobic yeast Yarrowia lipolytica, respiratory chain complex I (proton-translocating NADH : ubiquinone oxidoreductase) is an essential enzyme, since alternative NADH dehydrogenase activity is located exclusively at the external face of the mitochondrial inner membrane. Deletions and other loss-of-function mutations in genes for nuclear coded subunits of complex I can be obtained only when an internal version of the latter enzyme, termed NDH2i, is introduced. In contrast to recent findings with Neurospora crassa, external alternative NADH dehydrogenase activity is dispensable in complex I deletion strains of Y. lipolytica. We used regulable promoters to create strains which express internal alternative NADH dehydrogenase in a substrate-dependent manner. The ability to switch between complex I-dependent and -independent mode of growth simply by changing the carbon source is an important prerequisite for screens for both loss-of-function and inhibitor resistance mutation. The isocitrate lyase promoter (pICL1), in combination with a NDH2i allele that results in reduced expression and activity, was most promising. In the presence of complex I inhibitors, this construct allowed growth on acetate, but not on glucose minimal media. A somewhat higher background was observed with the acyl-CoA oxidase 2 (pPOX2) promoter on glucose minimal media. Copyright © 2006 John Wiley & Sons, Ltd.

Propofol is a widely used intravenous anesthetic agent. Our previous study showed that a therapeutic concentration of propofol can modulate macrophage functions. Mitochondria play critical roles in the maintenance of macrophage activities. This study attempted to evaluate further the effects of mitochondria on the propofol-induced suppression of macrophage functions using mouse macrophage-like Raw 264.7 cells as the experimental model. Macrophages were exposed to a clinically relevant concentration of propofol for 1, 6, and 24 h. Analysis by the Trypan blue exclusion method revealed that propofol was not cytotoxic to macrophages. Exposure of macrophages to propofol did not affect mitochondrial NADH dehydrogenase activity of complex I. However, analysis of flow cytometry showed that propofol significantly decreased the mitochondrial membrane potential of macrophages. Cellular levels of ATP in macrophages were significantly reduced after propofol administration. In parallel with the dysfunction of mitochondria, the chemotactic analysis showed that exposure to propofol significantly inhibited the migration of macrophages. This study shows that a therapeutic concentration of propofol can specifically reduce the mitochondrial membrane potential, but there is no such effect on complex I NADH dehydrogenase activity. Modulation of the mitochondrial membrane potential may decrease the biosynthesis of cellular ATP and thus reduce the chemotactic activity of macrophages. This study provides in vitro data to validate mitochondrial dysfunction as a possible critical cause for propofol-induced immunosuppression of macrophage functions.

Dye-linked l-proline dehydrogenase catalyzes the oxidation of l-proline in the presence of artificial electron acceptors such as 2, 6-dichloroindophenol and ferricyanide. The enzyme from the hyperthermophilic archaeon Thermococcus profundus was purified and characterized for the first time in archaea by Sakuraba et al. in 2001. In this study, cloning and sequencing analyses of the gene encoding the enzyme and functional analysis of the subunits were performed. The gene formed an operon that consisted of four genes, pdhA, pdhB, pdhF, and pdhX, which are tandemly arranged in the order of pdhA-F-X-B. SDS-PAGE analysis of the purified recombinant enzyme showed four different bands corresponding to alpha (54 kDa), beta (43 kDa), gamma (19 kDa), and delta (8 kDa) subunits encoded by pdhA, pdhB, pdhF, and pdhX, respectively, and the molecular ratio of these subunits was determined to be equal. This indicates that the enzyme consists of a heterotetrameric alphabetagammadelta structure. Functional analysis of each subunit revealed that the beta subunit catalyzed the dye-linked l-proline dehydrogenase reaction by itself and that, unexpectedly, the alpha subunit exhibited dye-linked NADH dehydrogenase activity. This is the first example showing the existence of a bifunctional dye-linked l-proline/NADH dehydrogenase complex. On the basis of genome analysis, similar gene clusters were observed in the genomes of Pyrococcus horikoshii, Pyrococcus abyssi, Pyrococcus furiosus, and Archaeoglobus fulgidus. These results indicate that the dye-linked l-proline dehydrogenase is a novel type of heterotetrameric amino acid dehydrogenase that might be widely distributed in the hyperthermophilic archaeal strain.

Oat ( Avena sativa L. cv. Prevision) plants grown under high light intensity showed a decrease in the quantum yield of photosystem (PS) II, the capacity of photosynthetic electron transport and photochemical quenching, and an increase in non-photochemical quenching as consequence of the photoinhibition of PS II. PS I was more stable than PS II in oat plants exposed to strong light treatments, probably because it plays a photoprotective role though cyclic electron flow. PS I photoinhibition was only observed after 10 photoperiods of high light intensity, as revealed by decreased PS I activity and the degradation of PSI A/B protein. However, PS I was more sensitive to photoinhibition in the presence of antimycin A, which is an inhibitor of one pathway of the cyclic electron transport. Immunoblot analysis using a specific antibody against the NdhH subunit of the thylakoidal NADH dehydrogenase (NADH DH) complex revealed an increase in the amount of this subunit in response to photoinhibitory conditions. Additionally, an increase in NADH dehydrogenase activity in the stroma thylakoids was also observed under these conditions. Treatment with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), which stimulates the cyclic electron flow, decreased the maximal quantum yield of PSII ( Φ PS II) and caused an increase in PSI and NADH dehydrogenase activities of the stroma thylakoids. Treatment with antimycin A under high light intensity also caused an increase in the NADH dehydrogenase activity of the stroma thylakoids. The results are in agreement with an increase of the thylakoidal NADH dehydrogenase complex and a stimulation of the cyclic electron flow involving the thylakoidal complex in response to photoinhibition conditions.

Although isopentenyl diphosphate-dimethylallyl diphosphate isomerase is thought to be essential for archaea because they use the mevalonate pathway, its corresponding activity has not been detected in any archaea. A novel type of the enzyme, which has no sequence similarity to the known, well-studied type of enzymes, was recently reported in some bacterial strains. In this study, we describe the cloning of a gene of a homologue of the novel bacterial isomerase from a thermoacidophilic archaeon Sulfolobus shibatae. The gene was heterologously expressed in Escherichia coli, and the recombinant enzyme was purified and characterized. The thermostable archaeal enzyme is tetrameric, and requires NAD(P)H and Mg2+ for activity, similar to its bacterial homologues. Using its apoenzyme, we were able to confirm that the archaeal enzyme is strictly dependent on FMN. Moreover, we provide evidence to show that the enzyme also has NADH dehydrogenase activity although it catalyzes the isomerase reaction without consuming any detectable amount of NADH.

Keeping a cytosolic redox balance is a prerequisite for living cells in order to maintain a metabolic activity and enable growth. During growth of Saccharomyces cerevisiae, an excess of NADH is generated in the cytosol. Aerobically, it has been shown that the external NADH dehydrogenase, Nde1p and Nde2p, as well as the glycerol-3-phosphate dehydrogenase shuttle, comprising the cytoplasmic glycerol-3-phosphate dehydrogenase, Gpdlp, and the mitochondrial glycerol-3-phosphate dehydrogenase, Gut2p, are the most important mechanisms for mitochondrial oxidation of cytosolic NADH. In this review we summarize the recent results showing (i) the contribution of each of the mechanisms involved in mitochondrial oxidation of the cytosolic NADH, under different physiological situations; (ii) the kinetic and structural properties of these metabolic pathways in order to channel NADH from cytosolic dehydrogenases to the inner mitochondrial membrane and (iii) the organization in supramolecular complexes and, the peculiar ensuing kinetic regulation of some of the enzymes (i.e. Gut2p inhibition by external NADH dehydrogenase activity) leading to a highly integrated functioning of enzymes having a similar physiological function. The cell physiological consequences of such an organized and regulated network are discussed.

Three rotenone-insensitive NADH dehydrogenases are present in the mitochondria of yeast Saccharomyces cerevisiae, which lack complex I. To elucidate the functions of these enzymes, superoxide production was determined in yeast mitochondria. The low levels of hydrogen peroxide (0.10 to 0.18 nmol/min/mg) produced in mitochondria incubated with succinate, malate, or NADH were stimulated 9-fold by antimycin A. Myxothiazol and stigmatellin blocked completely hydrogen peroxide formation with succinate or malate, indicating that the cytochrome bc(1) complex is the source of superoxide; however, these inhibitors only inhibited 46% hydrogen peroxide formation with NADH as substrate. Diphenyliodonium inhibited hydrogen peroxide formation (with NADH as substrate) by 64%. Superoxide formation, determined by EPR and acetylated cytochrome c reduction in mitochondria was stimulated by antimycin A, and partially inhibited by myxothiazol and stigmatellin. Proteinase K digestion of mitoplasts reduced 95% NADH dehydrogenase activity with a similar inhibition of superoxide production. Mild detergent treatment of the proteinase-treated mitoplasts resulted in an increase in NADH dehydrogenase activity due to the oxidation of exogenous NADH by the internal NADH dehydrogenase; however, little increase in superoxide production was observed. These results suggest that the external NADH dehydrogenase is a potential source of superoxide in S. cerevisiae mitochondria.

Dopamine, due to metabolism by monoamine oxidase or autoxidation, can generate toxic products such as hydrogen peroxide, oxygen-derived radicals, semiquinones, and quinones and thus exert its neurotoxic effects. Intracerebroventricular injection of dopamine into rats pretreated with the monoamine oxidase nonselective inhibitor pargyline caused mortality in a dose-dependent manner with LD50 = 90 micrograms. Norepinephrine was less effective with LD50 = 141 micrograms. The iron chelator desferrioxamine completely protected against dopamine-induced mortality. In the absence of pargyline more rats survived, indicating that the products of dopamine enzymatic metabolism are not the main contributors to dopamine-induced toxicity. Biochemical analysis of frontal cortex and striatum from rats that received a lethal dose of dopamine did not show any difference from control rats in lipid and protein peroxidation and glutathione reductase and peroxidase activities. Moreover, dopamine significantly reduced the formation of iron-induced malondialdehyde in vitro, thus suggesting that earlier events in cell damage are involved in dopamine toxicity. Indeed, dopamine inhibited mitochondrial NADH dehydrogenase activity with IC50 = 8 microM, and that of norepinephrine was twice as much (IC50 = 15 microM). Dopamine-induced inhibition of NADH dehydrogenase activity was only partially reversed by desferrioxamine, which had no effect on norepinephrine-induced inhibition. These results suggest that catecholamines can cause toxicity not only by inducing an oxidative stress state but also possibly through direct interaction with the mitochondrial electron transport system. The latter was further supported by the ability of ADP to reverse dopamine-induced inhibition of NADH dehydrogenase activity in a dose-dependent manner.

Most (78%) mitochondrial genomes in the studied mutant strain of Drosophila subobscura have undergone a large-scale deletion (5 kb) in the coding region. This mutation is stable, and is transmitted intact to the offspring. This animal model of major rearrangements of mitochondrial genomes can be used to analyse the involvement of the nuclear genome in the production and maintenance of these rearrangements. Successive backcrosses between mutant strain females and wild-type males yield a biphasic change in heteroplasmy level: (a) a 5% decrease in mutated genomes per generation (from 78 to 55%), until the nuclear genome is virtually replaced by the wild-type genome (seven to eight crosses); and (b) a continuous decrease of 0.5% per generation when the nuclear context is completely wild-type. In parallel with these changes, NADH dehydrogenase activity, which is halved in the mutant strain (five subunits of this complex are affected by the mutation), gradually increases and stabilizes near the wild-type activity. A return to a nuclear context is accompanied by the opposite phenomena: progressive increase in heteroplasmy level and stabilization at the value seen in the wild-type strain and a decrease in the activity of complex I. These results indicate that the nuclear genome plays an important role in the control of heteroplasmy level and probably in the production of rearranged genomes.

The rotenone sensitive NADH:menaquinone oxidoreductase (NDH-I or complex I) from the thermohalophilic bacterium Rhodothermus marinus has been purified and characterized. Three of its subunits react with antibodies against 78, 51, and 21.3c kDa subunits of Neurospora crassa complex I. The optimum conditions for NADH dehydrogenase activity are 50 degrees C and pH 8.1, and the enzyme presents a KM of 9 microM for NADH. The enzyme also displays NADH:quinone oxidoreductase activity with two menaquinone analogs, 1,4-naphtoquinone (NQ) and 2,3-dimethyl-1,4-naphtoquinone (DMN), being the last one rotenone sensitive, indicating the complex integrity as purified. When incorporated in liposomes, a stimulation of the NADH:DMN oxidoreductase activity is observed by dissipation of the membrane potential, upon addition of CCCP. The purified enzyme contains 13.5 +/- 3.5 iron atoms and approximately 3.7 menaquinone per FMN. At least five iron-sulfur centers are observed by EPR spectroscopy: two [2Fe-2S](2+/1+) and three [4Fe-4S](2+/1+) centers. By fluorescence spectroscopy a still unidentified chromophore was detected in R. marinus complex I.

NAD(+) metabolism is an essential regulator of cellular redox reactions, energy pathways, and a substrate provider for NAD(+) consuming enzymes. We recently demonstrated that enhancement of NAD(+)/NADH levels in breast cancer cells with impaired mitochondrial NADH dehydrogenase activity, through augmentation of complex I or by supplementing tumor cell nutrients with NAD(+) precursors, inhibits tumorigenicity and metastasis. To more fully understand how aberrantly low NAD(+) levels promote tumor cell dissemination, we here asked whether inhibition of NAD(+) salvage pathway activity by reduction in nicotinamide phosphoribosyltransferase (NAMPT) expression can impact metastasis and tumor cell adhesive functions. We show that knockdown of NAMPT, the enzyme catalyzing the rate-limiting step of the NAD(+) salvage pathway, enhances metastatic aggressiveness in human breast cancer cells and involves modulation of integrin expression and function. Reduction in NAMPT expression is associated with upregulation of select adhesion receptors, particularly αvβ3 and β1 integrins, and results in increased breast cancer cell attachment to extracellular matrix proteins, a key function in tumor cell dissemination. Interestingly, NAMPT downregulation prompts expression of integrin αvβ3 in a high affinity conformation, known to promote tumor cell adhesive interactions during hematogenous metastasis. NAMPT has been selected as a therapeutic target for cancer therapy based on the essential functions of this enzyme in NAD(+) metabolism, cellular redox, DNA repair and energy pathways. Notably, our results indicate that incomplete inhibition of NAMPT, which impedes NAD(+) metabolism but does not kill a tumor cell can alter its phenotype to be more aggressive and metastatic. This phenomenon could promote cancer recurrence, even if NAMPT inhibition initially reduces tumor growth.

Chloroplast-encoded NDH polypeptides (components of the plastid Ndh complex) and the NADH dehydrogenase activity of the Ndh complex (NADH-DH) increased under photooxidative stress. The possible involvement of H2O2-mediated signaling in the photooxidative induction of chloroplastic ndh genes was thoroughly studied. We have analyzed the changes in the NADH-DH and steady-state levels of NDH-F polypeptide and ndhB andndhF transcripts in barley (Hordeum vulgare cv Hassan) leaves. Subapical leaf segments were incubated in growing light (GL), photooxidative light (PhL), GL and H2O2 (GL + H2O2), or PhL and 50 nm paraquat in the incubation medium. Treatments with H2O2 under GL mimicked the photooxidative stimulus, causing a dose-dependent increase of NADH-DH and NDH-F polypeptide. The kinetic of Ndh complex induction was further studied in leaves pre-incubated with or without the H2O2-scavenger dimethyltiourea. NADH-DH and NDH-F polypeptide rapidly increased up to 16 h in PhL, GL+ H2O2, and, at higher rate, in PhL and paraquat. The observed increases of NADH-DH and NDH-F after 4 h in PhL and GL + H2O2 were not accompanied by significant changes in ndhB and ndhF transcripts. However, at 16-h incubations NADH-DH and NDH-F changes closely correlated with higher ndhB and ndhFtranscript levels. All these effects were prevented by dimethylthiourea. It is proposed that the induction of chloroplasticndh genes under photooxidative stress is mediated by H2O2 through mechanisms that involve a rapid translation of pre-existing transcripts and the increase of thendh transcript levels.