NADH Oxidase Activity Research Articles

Lambda Red-Mediated Mutagenesis and Efficient Large Scale Affinity Purification of the Escherichia coli NADH:Ubiquinone Oxidoreductase (Complex I)

The proton-pumping NADH:ubiquinone oxidoreductase, the respiratory complex I, couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. The Escherichia coli complex I consists of 13 different subunits named NuoA-N (from NADH:ubiquinone oxidoreductase), that are coded by the genes of the nuo-operon. Genetic manipulation of the operon is difficult due to its enormous size. The enzymatic activity of variants is obscured by an alternative NADH dehydrogenase, and purification of the variants is hampered by their instability. To overcome these problems the entire E. coli nuo-operon was cloned and placed under control of the l-arabinose inducible promoter ParaBAD. The exposed N-terminus of subunit NuoF was chosen for engineering the complex with a hexahistidine-tag by lambda-Red-mediated recombineering. Overproduction of the complex from this construct in a strain which is devoid of any membrane-bound NADH dehydrogenase led to the assembly of a catalytically active complex causing the entire NADH oxidase activity of the cytoplasmic membranes. After solubilization with dodecyl maltoside the engineered complex binds to a Ni2+-iminodiacetic acid matrix allowing the purification of approximately 11 mg of complex I from 25 g of cells. The preparation is pure and monodisperse and comprises all known subunits and cofactors. It contains more lipids than earlier preparations due to the gentle and fast purification procedure. After reconstitution in proteoliposomes it couples the electron transfer with proton translocation in an inhibitor sensitive manner, thus meeting all prerequisites for structural and functional studies.

Dehydromonocrotaline inhibits mitochondrial complex I. A potential mechanism accounting for hepatotoxicity of monocrotaline

Monocrotaline is a pyrrolizidine alkaloid present in plants of the Crotalaria species, which causes cytotoxicity and genotoxicity, including hepatotoxicity in animals and humans. It is metabolized by cytochrome P-450 in the liver to the alkylating agent dehydromonocrotaline. We evaluated the effects of monocrotaline and its metabolite on respiration, membrane potential and ATP levels in isolated rat liver mitochondria, and on respiratory chain complex I NADH oxidase activity in submitochondrial particles. Dehydromonocrotaline, but not the parent compound, showed a concentration-dependent inhibition of glutamate/malate-supported state 3 respiration (respiratory chain complex I), but did not affect succinate-supported respiration (complex II). Only dehydromonocrotaline dissipated mitochondrial membrane potential, depleted ATP, and inhibited complex I NADH oxidase activity (IC 50=62.06 μM) through a non-competitive type of inhibition ( K I=8.1 μM). Therefore, dehydromonocrotaline is an inhibitor of the activity of respiratory chain complex I NADH oxidase, an action potentially accounting for the well-documented monocrotaline's hepatotoxicity to animals and humans. The mechanism probably involves change of the complex I conformation resulting from modification of cysteine thiol groups by the metabolite.

Combating Nitrate Tolerance

Discovered and patented as an explosive by Alfred Nobel in the 1860s, nitroglycerin has been formulated for use in the treatment of symptomatic CAD for over 140 years. In fact, later in life, Nobel himself was prescribed the medication for angina, but refused to take it because of the associated side effect of headache. See page 1729 With glycerol trinitrate (GTN) as the prototype, nitrates represent one of the safest and most rapidly effective pharmacological means to reduce acute symptoms of myocardial ischemia attributable to obstructive coronary disease. This has led, over the years, to the development of long-acting oral and topical preparations. However, efficacy with chronic administration is more difficult to achieve because of the development of therapeutic resistance, generally occurring a few days after initiating treatment. This phenomenon known as nitrate tolerance has been the stimulus for intense investigation of the metabolic fate of nitroglycerin with the idea that modulation of its biotransformation could improve efficacy of chronic treatment. The mechanism of GTN-induced dilation is complex and was not identified until more than 100 years after its discovery. GTN is not a direct vasodilator, rather it must be converted to dinitrate products for vasoactivity. Biotransformation to the active metabolite nitric oxide (NO) occurs in parallel with the formation of glycerol-1,2-dinitrate and involves a dithiol-dependent process.1 It was not until recently that the principal enzyme responsible for biotransformation of GTN was identified. Chen et al1 showed that mitochondrial aldehyde dehydrogenase (ALDH-2) metabolizes GTN to glycerol-1,2-dinitrate and nitrite. This was confirmed by Sydow et al2 using mitochondrial-deficient cultured endothelial cells, although a cytosolic source of ALDH-2 has also been suggested.3 The mitochondrial enzyme converts nanomolar concentrations of GTN to active nitrodilator metabolites in vivo and in vitro, as shown by direct measurements coupled with the use …

NADH oxidase activity of rat and human liver xanthine oxidoreductase: potential role in superoxide production

To characterise the NADH oxidase activity of both xanthine dehydrogenase (XD) and xanthine oxidase (XO) forms of rat liver xanthine oxidoreductase (XOR) and to evaluate the potential role of this mammalian enzyme as an O2*- source, kinetics and electron paramagnetic resonance (EPR) spectroscopic studies were performed. A steady-state kinetics study of XD showed that it catalyses NADH oxidation, leading to the formation of one O2*- molecule and half a H(2)O(2) molecule per NADH molecule, at rates 3 times those observed for XO (29.2 +/- 1.6 and 9.38 +/- 0.31 min(-1), respectively). EPR spectra of NADH-reduced XD and XO were qualitatively similar, but they were quantitatively quite different. While NADH efficiently reduced XD, only a great excess of NADH reduced XO. In agreement with reductive titration data, the XD specificity constant for NADH (8.73 +/- 1.36 microM(-1) min(-1)) was found to be higher than that of the XO specificity constant (1.07 +/- 0.09 microM(-1) min(-1)). It was confirmed that, for the reducing substrate xanthine, rat liver XD is also a better O2*- source than XO. These data show that the dehydrogenase form of liver XOR is, thus, intrinsically more efficient at generating O2*- than the oxidase form, independently of the reducing substrate. Most importantly, for comparative purposes, human liver XO activity towards NADH oxidation was also studied, and the kinetics parameters obtained were found to be very similar to those of the XO form of rat liver XOR, foreseeing potential applications of rat liver XOR as a model of the human liver enzyme.

Inhibition of Cyanide-Insensitive Respiration in Klebsiella oxytoca SYSU-011 by 8-Hydroxyquinolone

The inhibition of the cyanide (KCN)-insensitive respiration of Klebsiella oxytoca SYSU-011 by 8-hydroxyquinoline (8-HQ) was determined. Results showed that the profile of the rate of oxygen uptake of normal-grown and 8-HQ-grown K. oxytoca SYSU-011 was biphasic and similar, suggesting that 8-HQ did not inhibit the respiration of normal-grown K. oxytoca SYSU-011. A different biphasic KCN inhibition profile of respiration was observed for KCN-grown cells treated with and without 8-HQ. No decrease in respiration rate of KCN-grown cells and a 40% decrease in respiration rate of KCN-grown cells treated with 8-HQ were observed when KCN concentration was 10(-1) mM. Comparing differences of the profiles of oxygen uptake in KCN-grown cells with and without 8-HQ addition indicated that 8-HQ inhibited expression of the KCN-insensitive pathway carried out by nonheme oxidase. Greater inhibition of NADH oxidase activity by 2-n-heptyl-4-hydroxyquinoline-N-oxide from the cell membrane of the KCN-grown cells treated with 8-HQ, and more H2O2 production from these cells with than without 8-HQ, suggest that the function of the cyanide-insensitive pathway can stabilize the respiration of the cyanide-grown cells to prevent the production of H2O2.

HQNO-sensitive NADH:Quinone Oxidoreductase of Bacillus cereus KCTC 3674

The enzymatic properties of NADH:quinone oxidoreductase were examined in Triton X-100 extracts of Bacillus cereus membranes by using the artificial electron acceptors ubiquinone-1 and menadione. Membranes were prepared from B. cereus KCTC 3674 grown aerobically on a complex medium and oxidized with NADH exclusively, whereas deamino-NADH was determined to be poorly oxidized. The NADH oxidase activity was lost completely by solubilization of the membranes with Triton X-100. However, by using the artificial electron acceptors ubiquinone-1 and menadione, NADH oxidation could be observed. The activities of NADH:ubiquinone-1 and NADH:menadione oxidoreductase were enhanced approximately 8-fold and 4-fold, respectively, from the Triton X-100 extracted membranes. The maximum activity of FAD-dependent NADH:ubiquinone-1 oxidoreductase was obtained at about pH 6.0 in the presence of 0.1M NaCl, while the maximum activity of FAD-dependent NADH:menadione oxidoreductase was obtained at about pH 8.0 in the presence of 0.1 M NaCl. The activities of the NADH:ubiquinone-1 and NADH:menadione oxidoreductase were very resistant to such respiratory chain inhibitors as rotenone, capsaicin, and AgNO(3), whereas these activities were sensitive to 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO). Based on these results, we suggest that the aerobic respiratory chain-linked NADH oxidase system of B. cereus KCTC 3674 possesses an HQNO-sensitive NADH:quinone oxidoreductase that lacks an energy coupling site containing FAD as a cofactor.

Early myocardial dysfunction in streptozotocin-induced diabetic mice: a study using in vivo magnetic resonance imaging (MRI)

BackgroundDiabetes is associated with a cardiomyopathy that is independent of coronary artery disease or hypertension. In the present study we used in vivo magnetic resonance imaging (MRI) and echocardiographic techniques to examine and characterize early changes in myocardial function in a mouse model of type 1 diabetes.MethodsDiabetes was induced in 8-week old C57BL/6 mice with two intraperitoneal injections of streptozotocin. The blood glucose levels were maintained at 19–25 mmol/l using intermittent low dosages of long acting insulin glargine. MRI and echocardiography were performed at 4 weeks of diabetes (age of 12 weeks) in diabetic mice and age-matched controls.ResultsAfter 4 weeks of hyperglycemia one marker of mitochondrial function, NADH oxidase activity, was decreased to 50% of control animals. MRI studies of diabetic mice at 4 weeks demonstrated significant deficits in myocardial morphology and functionality including: a decreased left ventricular (LV) wall thickness, an increased LV end-systolic diameter and volume, a diminished LV ejection fraction and cardiac output, a decreased LV circumferential shortening, and decreased LV peak ejection and filling rates. M-mode echocardiographic and Doppler flow studies of diabetic mice at 4 weeks showed a decreased wall thickening and increased E/A ratio, supporting both systolic and diastolic dysfunction.ConclusionOur study demonstrates that MRI interrogation can identify the onset of diabetic cardiomyopathy in mice with its impaired functional capacity and altered morphology. The MRI technique will lend itself to repetitive study of early changes in cardiac function in small animal models of diabetic cardiomyopathy.

Tissue kallikrein infusion prevents cardiomyocyte apoptosis, inflammation and ventricular remodeling after myocardial infarction

We investigated the effect of tissue kallikrein infusion on cardiac protection at acute and sub-acute phases after myocardial infarction (MI). Immediately after MI, rats were infused with purified tissue kallikrein, with or without icatibant (a kinin B2 receptor antagonist). Intramyocardial injection of kallikrein reduced myocardial infarct size and inhibited cardiomyocyte apoptosis at 1 day after MI associated with increased nitric oxide levels, Akt and glycogen synthase kinase-3β phosphorylation and decreased caspase-3 activation. Kallikrein infusion for 7 days improved cardiac function, normalized left ventricular wall thickness and decreased monocyte/macrophage infiltration in the infarct heart. Kallikrein treatment reduced NADH oxidase expression and activity, superoxide formation and malondialdehyde levels, and reduced MAPK and Iκ-Bα phosphorylation, NF-κB activation and MCP-1 and VCAM-1 expression. Kallikrein's effects were all blocked by icatibant. These results indicate that kallikrein through kinin B2 receptor activation prevents apoptosis, inflammation and ventricular remodeling by increased nitric oxide formation and suppression of oxidative stress-mediated signaling pathways.

Overexpression of Apoptosis-Inducing Factor (AIF) and Pro-Survival Function in AML.

Apoptosis inducing factor (AIF) is a bifunctional flavoprotein with NADH oxidase activity with a vital function in bioenergetics and redox metabolism while also inducing apoptosis in cancer cells. The gene is localized on chromosome human X (q25–26). AIF is synthesized in the cytosol as a 67KDa precursor molecule and then imported into the intermembrane space of mitochondria. In response to pro-apoptotic stimuli, the change of mitochondrial membrane potential (MMP) results in release of mature AIF (57KDa) to the cytoplasm and translocation into the nuclei where AIF can induce caspase-independent large scale DNA fragmentation (~ 50 Kbp) and chromatin condensation. The mechanism of induction of cell death by AIF remains poorly understood. We have previously reported that AIF induced cell death is both caspase-dependent and independent and related to mitochondrial membrane depolarization in leukemic cells treated with chemotherapeutic agents. In this study, we investigatethe expression level and function of AIF and its relationship to Bcl-2 and Bcl-2 family members in leukemia cells; andthe expression levels of AIF in AML patient's samples.To study the function of AIF, we introduced AIF si-RNA into K562 CML cell line using lentiviral vectors and established permanent AIF knockdown cell lines. Western blot analysis showed that the expression levels of XIAP, MCL-1, Bcl-xl and suvivin were increased while the AIF expression level was reduced. Cell growth was diminished two-fold (siAIF-clone 2, cell doubling time ~52-hr) to four-fold (siAIF-clone 1, cell doubling time ~99-hr) in AIF knockdown cells compared to the parental cells (24-hr). This suggests that AIF is supporting cell growth.Next, AIF knockdown and scrambled siRNA control cells were treated with Ara-C (10μM) or STI571 (Gleeevec) (2 μM). The frequency of apoptotic cells was determined by flow cytometry (table).24hr48hrControlAra-CSTI571ControlAra-CSTI-571Control17.0430.5630.2320.2148.4876.12siAIF-13.093.3617.037.510.1244.92siAIF-26.776.647.7311.9510.7341.85AIF-siRNA in K562 cells showed increased sensitivity to Ara-C and STI-571 (Gleevec). The results support again a pro-survival function of AIF in K562 cells.To examine the expression levels of AIF in primary AML patients, 148 samples (newly diagnosed n=32, relapsed n=27, refractory n=18, CR n=5, and normal-donors n=31) were analyzed by western blot and 40 samples of newly diagnosed AML were analyzed by RT-PCR. Results show that in comparison with normal CD34+ cells, AIF mRNA levels in newly diagnosed AML patients were significantly higher (95.0 copies AIF per hundred copies ABL1 in AML, 57.4 copies AIF per hundred copies in NBM CD34+, p = 0.02). The AIF/actin protein ratios in AML (0.43±0.05) was increased compared with normal BM-CD34+ (0.12±0.02) (p=0.01). Comparison of AIF expression levels in normal CD34+ cells vs. AML with good (0.5±0.05), bad (0.34±0.05), and intermediate (0.39±0.06) cytogenetics respectively, were significantly different (p<0.002). The transcription levels of AIF also showed significant differences (p<0.04) in patients with different cytogenetics abnormalities.Taken together, we conclude thatAIF transcript and protein levels are significantly higher in AML compared to normal CD34 cells.AIF in K562 cells functions as pro-survival factor for drug sensitivity.These results question the previously assumed predominant pro-apoptotic function of AIF.

Novel Role of Kallistatin in Protection Against Myocardial Ischemia–Reperfusion Injury by Preventing Apoptosis and Inflammation

Kallistatin is a serine proteinase inhibitor that has been shown to reduce joint swelling and to inhibit inflammation in a rat model of arthritis. In this study, we investigated the effect and mechanisms of kallistatin on cardiac function after myocardial ischemia-reperfusion (I/R) injury. The human kallistatin gene in an adenoviral vector was delivered locally into rat heart 4 days before 30-min ischemia followed by 24-hr reperfusion. Kallistatin gene transfer significantly reduced myocardial infarct size and left ventricle end-diastolic pressure and improved cardiac contractility. Kallistatin significantly reduced I/R-induced cardiomyocyte apoptosis as identified by TUNEL and Hoechst staining, DNA laddering, cell viability, and caspase-3 activity in ischemic myocardium and in primary cultured cardiomyocytes. Kallistatin also reduced intramyocardial monocyte/macrophage and neutrophil accumulation in conjunction with decreased expression of monocyte chemoattractant protein-1, tumor necrosis factor-alpha, and intercellular adhesion molecule-1. Kallistatin delivery promoted cardiac endothelial nitric oxide synthase activation and increased nitric oxide (NO) formation, but inhibited NADH oxidase activity, p22phox expression, and superoxide production. Moreover, kallistatin reduced the phosphorylation of apoptosis signal-regulating kinase-1 and mitogen-activated protein kinases (MAPKs), but increased Akt and glycogen synthase kinase-3beta phosphorylation. The effects of kallistatin on cardiac function, oxidative stress, and these signal transduction events were all blocked by Nomega-nitro-L-argi-nine methyl ester. These results indicate a novel role of kallistatin in cardiac protection after I/R injury through increased NO formation and Akt-glycogen synthase kinase-3beta signaling and suppression of oxidative stress and MAPK activation.

Cadmium-induced microsomal membrane-bound peroxidases mediated hydrogen peroxide production in barley roots

The effect of cadmium on microsomal membrane-bound peroxidases and their involvement in hydrogen peroxide production was studied in barley roots. One anionic and two cationic peroxidases were detected, which were strongly activated by Cd treatment. Positive correlation was found between root growth inhibition and increased peroxidase, NADH oxidase activity and H2O2 generation in root microsomal membrane fraction of Cd-treated barley roots.

The Group B Streptococcus NADH oxidase Nox‐2 is involved in fatty acid biosynthesis during aerobic growth and contributes to virulence

Numerous Streptococcaceae produce an H2O-forming NADH oxidase, Nox-2, which has been generally implicated in aerobic survival. We examined the roles of Nox-2 in Group B Streptococcus (GBS), a leading agent of neonatal infections. While nox2 inactivation caused an aerobic growth arrest, no improvement was seen by addition of antioxidants to cultures, suggesting that this defect was not due to accumulation of toxic oxygen species. Using several approaches, we show that the observed inability of the nox2 mutant to grow aerobically is mainly due to an underlying defect in fatty acid (FA) biosynthesis: (i) the nox2 aerobic growth defect is fully and rapidly complemented by adding oleic acid to culture medium, and (ii) direct assimilation of this unsaturated FA in both wild type (WT) and nox2 GBS membranes is demonstrated and correlated with mutant growth rescue. We propose that NAD+ depletion in the nox2 mutant results in reduced acetyl-CoA production, which perturbs FA biosynthesis and hence blocks growth in aerobiosis. The nox2 aerobic growth defect was also complemented when GBS respiration metabolism was activated by exogenous haem and menaquinone. The membrane NADH oxidase activity generated by the functional respiratory chain thus compensates the cytoplasmic NADH oxidase deficiency. The nox2 mutant was attenuated for virulence, as assessed in lung, intraperitoneal and intravenous murine infection models. As the nox2 defect seems only to affect aerobic growth of GBS, its reduced virulence supports the suggestion that aerobic conditions and NADH oxidase activities are relevant to the GBS infection process.

A Transverse Tubule NADPH Oxidase Activity Stimulates Calcium Release from Isolated Triads via Ryanodine Receptor Type 1 S -Glutathionylation

We report here the presence of an NADPH oxidase (NOX) activity both in intact and in isolated transverse tubules and in triads isolated from mammalian skeletal muscle, as established by immunochemical, enzymatic, and pharmacological criteria. Immunohistochemical determinations with NOX antibodies showed that the gp91(phox) membrane subunit and the cytoplasmic regulatory p47(phox) subunit co-localized in transverse tubules of adult mice fibers with the alpha1s subunit of dihydropyridine receptors. Western blot analysis revealed that isolated triads contained the integral membrane subunits gp91(phox) and p22(phox), which were markedly enriched in isolated transverse tubules but absent from junctional sarcoplasmic reticulum vesicles. Isolated triads and transverse tubules, but not junctional sarcoplasmic reticulum, also contained varying amounts of the cytoplasmic NOX regulatory subunits p47(phox) and p67(phox). NADPH or NADH elicited superoxide anion and hydrogen peroxide generation by isolated triads; both activities were inhibited by NOX inhibitors but not by rotenone. NADH diminished the total thiol content of triads by one-third; catalase or apocynin, a NOX inhibitor, prevented this effect. NADPH enhanced the activity of ryanodine receptor type 1 (RyR1) in triads, measured through [3H]ryanodine binding and calcium release kinetics, and increased significantly RyR1 S-glutathionylation over basal levels. Preincubation with reducing agents or NOX inhibitors abolished the enhancement of RyR1 activity produced by NADPH and prevented NADPH-induced RyR1 S-glutathionylation. We propose that reactive oxygen species generated by the transverse tubule NOX activate via redox modification the neighboring RyR1 Ca2+ release channels. Possible implications of this putative mechanism for skeletal muscle function are discussed.

Response of the Microaerophilic Bifidobacterium Species, B. boum and B. thermophilum , to Oxygen

We investigated the effects of O2 on Bifidobacterium species using liquid shaking cultures under various O2 concentrations. Although most of the Bifidobacterium species we selected showed O2 sensitivity, two species, B. boum and B. thermophilum, demonstrated microaerophilic profiles. The growth of B. bifidum and B. longum was inhibited under high-O2 conditions accompanied by the accumulation of H2O2 in the medium, and growth was restored by adding catalase to the medium. B. boum and B. thermophilum grew well even under 20% O2 conditions without H2O2 accumulation, and growth was stimulated compared to anoxic growth. H2O-forming NADH oxidase activities were detected dominantly in cell extracts of B. boum and B. thermophilum under acidic reaction conditions (pH 5.0 to 6.0).

Mode of Inhibitory Action of Δlac-Acetogenins, a New Class of Inhibitors of Bovine Heart Mitochondrial Complex I

We have revealed that Deltalac-acetogenins, a new class of inhibitors of bovine heart mitochondrial complex I (NADH-ubiquinone oxidoreductase), act differently from ordinary inhibitors such as rotenone and piericidin A [Ichimaru et al. (2005) Biochemistry 44, 816-825]. Since a detailed study of these unique inhibitors might provide new insight into the terminal electron transfer step of the enzyme, we further characterized their inhibitory action using the most potent Deltalac-acetogenin derivative (compound 1). Unlike ordinary complex I inhibitors, 1 had a dose-response curve for inhibition of the reduction of exogenous short-chain ubiquinones that was difficult to explain with a simple bimolecular association model. The inhibitory effect of 1 on ubiquinol-NAD(+) oxidoreductase activity (reverse electron transfer) was much weaker than that on NADH oxidase activity (forward electron transfer), indicating a direction-specific effect. These results suggest that the binding site of 1 is not identical to that of ubiquinone and the binding of 1 to the enzyme secondarily (or indirectly) disturbs the redox reaction of ubiquinone. Using endogenous and exogenous ubiquinone as an electron acceptor of complex I, we investigated the effect of 1 in combination with different ordinary inhibitors on the superoxide production from the enzyme. The results indicated that the level of superoxide production induced by 1 is significantly lower than that induced by ordinary inhibitors probably because of fewer electron leaks from the ubisemiquinone radical to molecular oxygen and that the site of inhibition by 1 is downstream of that by ordinary inhibitors. The unique inhibitory action of hydrophobic Deltalac-acetogenins may be closely associated with the dynamic function of the membrane domain of complex I.

Breaking down the wall: Fractionation of mycobacteria

Mycobacterium spp. possess a complex cell envelope that consists of a plasma membrane, a peptidoglycan–arabinogalactan complex which in turn is esterified by mycolic acids that form with other non-bound lipids an asymmetric permeability barrier and an outer layer, also called a capsule in the case of pathogenic species. In order to investigate the functional roles of the cell envelope components, especially those of the major pathogens Mycobacterium tuberculosis and Mycobacterium leprae, it is necessary to fractionate the envelope by breaking the unusual wall that covers these bacteria. To this aim we first compared the efficiency of high pressure (cell disrupter/French press) with those of pathogen-compatible breakage methods such as sonication, bead beater and lysozyme treatment using the non-pathogenic Mycobacterium smegmatis. When the distribution of various specific markers of the cell envelope compartments, which include mycolic acids, arabinose, NADH oxidase activity, cell wall and cytosolic proteins, were determined sonication combined with lysozyme treatment was found to be the best option. The protocol of subcellular fractionation was then validated for pathogenic species by applying the method to Mycobacterium bovis BCG cells, an attenuated strain of the M. tuberculosis complex.

Modulation by homocysteine of the iberiotoxin-sensitive, Ca 2+-activated K + channels of porcine coronary artery smooth muscle cells

We evaluated the acute effect of homocysteine on the iberiotoxin-sensitive, Ca 2+-activated K + (BK Ca) channels of the porcine coronary artery smooth muscle cells. NS 1619 (1 to 30 μM) caused a concentration-dependent enhancement of the BK Ca amplitude (recorded using the whole-cell, membrane-rupture configuration) only with an elevated [Ca 2+] i of ∼ 444 nM, but not with [Ca 2+] i of ∼ 100 nM. Homocysteine (30 μM) caused a small inhibition (∼ 16%) of the BK Ca amplitude ([Ca 2+] i = ∼ 444 nM), and a greater inhibition (∼ 77%) was observed with 100 μM NADH present in the pipette solution. The inhibition persisted after washing. With NADPH (100 μM), a smaller magnitude of inhibition (∼ 34%) of the BK Ca amplitude was recorded. The NS 1619-mediated enhancement of the BK Ca amplitude (with elevated [Ca 2+] i plus NADH in the pipette) was attenuated by homocysteine. The homocysteine-mediated inhibition of the BK Ca amplitude was suppressed by Tiron (10 mM) or diphenylene iodonium (30 nM), applied alone, but not by superoxide dismutase (500 U/ml) and catalase (500 U/ml). Generation of superoxide ( O 2 −) of the smooth muscle cells (with NADH presence), measured using the lucigenin-enhanced chemiluminescence, was markedly increased by angiotensin II (100 nM) and homocysteine (30 μM). The chemiluminescence signal was sensitive to apocynin (300 μM) or Tiron, applied alone, but not to superoxide dismutase and catalase. In conclusion, our results demonstrate that acute homocysteine application inhibits the iberiotoxin-sensitive BK Ca channels (with elevated [Ca 2+] i and NADH present) which is probably caused by the NADH oxidase activation and the concomitant generation of intracellular superoxide.

Identification and Characterization of AIFsh2, a Mitochondrial Apoptosis-inducing Factor (AIF) Isoform with NADH Oxidase Activity

Apoptosis-inducing factor (AIF) is a bifunctional NADH oxidase involved in mitochondrial respiration and caspase-independent apoptosis. Three alternatively spliced mRNA isoforms of AIF have been identified previously: AIF, AIF-exB, and AIFsh. Here, we report the cloning and the biochemical characterization of a new isoform named AIF short 2 (AIFsh2). AIFsh2 transcript includes a previously unknown exon placed between exons 9 and 10 of AIF. The resulting AIFsh2 protein, which localizes in mitochondria, corresponds to the oxidoreductase domain of AIF. In this way, AIFsh2 exhibits similar NADH oxidase activity to AIF and generates reactive oxygen species. Like AIF, AIFsh2 is released from mitochondria to cytosol after an apoptotic insult in a calpain or cathepsin-dependent manner. However, in contrast to AIF, AIFsh2 does not induce nuclear apoptosis. Thus, it seems that the reactive oxygen species produced by the oxidoreductase domain of AIF/AIFsh2 are not important for AIF-dependent nuclear apoptosis. In addition, we demonstrate that the AIFsh2 mRNA is absent in normal brain tissue, whereas it is expressed in neuroblastoma-derived cells, suggesting a different regulation in normal and transformed cells from the brain lineage. Together, our results reveal that AIF yields an original and independent genetic regulation of the two AIF functions. This is an important issue to understand the physiological role of this protein.

Loss of Aif function causes cell death in the mouse embryo, but the temporal progression of patterning is normal

Apoptosis-inducing factor (AIF) is an evolutionarily conserved, ubiquitously expressed flavoprotein with NADH oxidase activity that is normally confined to mitochondria. In mammalian cells, AIF is released from mitochondria in response to apoptotic stimuli and translocates to the nucleus where it is thought to bind DNA and contribute to chromatinolysis and cell death in a caspase-independent manner. Here we describe the consequences of inactivating Aif in the early mouse embryo. Unexpectedly, we found that both the apoptosis-dependent process of cavitation in embryoid bodies and apoptosis associated with embryonic neural tube closure occur in the absence of AIF, indicating that Aif function is not required for apoptotic cell death in early mouse embryos. By embryonic day 9 (E9), loss of Aif function causes abnormal cell death, presumably because of reduced mitochondrial respiratory chain complex I activity. Because of this cell death, Aif null embryos fail to increase significantly in size after E9. Remarkably, patterning processes continue on an essentially normal schedule, such that E10 Aif null embryos with only approximately 1/10 the normal number of cells have the same somite number as their wild-type littermates. These observations show that pattern formation in the mouse can occur independent of embryo size and cell number.

Examination of Lactobacillus plantarum lactate metabolism side effects in relation to the modulation of aeration parameters

The characterization of global aerobic metabolism of Lactobacillus plantarum LP652 under different aeration levels, in order to optimize acetate production kinetics and to suppress H2O2 toxicity. Cultures of L. plantarum were grown on different aeration conditions. After sugar exhaustion and in the presence of oxygen, lactate was converted to acetate, H2O2 and carbon dioxide with concomitant ATP production. Physiological assays were performed at selected intervals in order to assess enzyme activity and vitality of the strain during lactic acid conversion. The maximal aerated condition led to fast lactate-to-acetate conversion kinetics between 8 and 12 h, but H2O2 immediately accumulated, thus affecting cell metabolism. Pyruvate oxidase activity was highly enhanced by oxygen tension and was responsible for H2O2 production after 12 h of culture, whereas lactate oxidase and NADH-dependent lactate dehydrogenase activities were not correlated to metabolite production. Limited NADH oxidase (NOX) and NADH peroxidase (NPR) activities were probably responsible for toxic H2O2 levels in over-aerated cultures. Modulating initial airflow led to the maximal specific activity of NOX and NPR observed after 24 h of culture, thus promoting H2O2 destruction and strain vitality at the end of the process. Optimal aeration conditions were determined to minimize H2O2 concentration level during growth on lactate.