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1.
Free Radic Biol Med ; 188: 434-446, 2022 08 01.
Article in English | MEDLINE | ID: mdl-35718301

ABSTRACT

Attachment of cargo molecules to lipophilic triphenylphosphonium (TPP+) cations is a widely applied strategy for mitochondrial targeting. We previously demonstrated that the vitamin E-derived antioxidant Trolox increases the levels of active mitochondrial complex I (CI), the first complex of the electron transport chain (ETC), in primary human skin fibroblasts (PHSFs) of Leigh Syndrome (LS) patients with isolated CI deficiency. Primed by this finding, we here studied the cellular effects of mitochondria-targeted Trolox (MitoE10), mitochondria-targeted ubiquinone (MitoQ10) and their mitochondria-targeting moiety decylTPP (C10-TPP+). Chronic treatment (96 h) with these molecules of PHSFs from a healthy subject and an LS patient with isolated CI deficiency (NDUFS7-V122M mutation) did not greatly affect cell number. Unexpectedly, this treatment reduced CI levels/activity, lowered the amount of ETC supercomplexes, inhibited mitochondrial oxygen consumption, increased extracellular acidification, altered mitochondrial morphology and stimulated hydroethidine oxidation. We conclude that the mitochondria-targeting decylTPP moiety is responsible for the observed effects and advocate that every study employing alkylTPP-mediated mitochondrial targeting should routinely include control experiments with the corresponding alkylTPP moiety.


Subject(s)
Electron Transport Complex I , Mitochondria , Electron Transport , Electron Transport Complex I/deficiency , Electron Transport Complex I/metabolism , Fibroblasts/metabolism , Humans , Mitochondria/metabolism , Mitochondrial Diseases
3.
Biochim Biophys Acta Bioenerg ; 1861(8): 148213, 2020 08 01.
Article in English | MEDLINE | ID: mdl-32335026

ABSTRACT

Mutations in NDUFS4, which encodes an accessory subunit of mitochondrial oxidative phosphorylation (OXPHOS) complex I (CI), induce Leigh syndrome (LS). LS is a poorly understood pediatric disorder featuring brain-specific anomalies and early death. To study the LS pathomechanism, we here compared OXPHOS proteomes between various Ndufs4-/- mouse tissues. Ndufs4-/- animals displayed significantly lower CI subunit levels in brain/diaphragm relative to other tissues (liver/heart/kidney/skeletal muscle), whereas other OXPHOS subunit levels were not reduced. Absence of NDUFS4 induced near complete absence of the NDUFA12 accessory subunit, a 50% reduction in other CI subunit levels, and an increase in specific CI assembly factors. Among the latter, NDUFAF2 was most highly increased. Regarding NDUFS4, NDUFA12 and NDUFAF2, identical results were obtained in Ndufs4-/- mouse embryonic fibroblasts (MEFs) and NDUFS4-mutated LS patient cells. Ndufs4-/- MEFs contained active CI in situ but blue-native-PAGE highlighted that NDUFAF2 attached to an inactive CI subcomplex (CI-830) and inactive assemblies of higher MW. In NDUFA12-mutated LS patient cells, NDUFA12 absence did not reduce NDUFS4 levels but triggered NDUFAF2 association to active CI. BN-PAGE revealed no such association in LS patient fibroblasts with mutations in other CI subunit-encoding genes where NDUFAF2 was attached to CI-830 (NDUFS1, NDUFV1 mutation) or not detected (NDUFS7 mutation). Supported by enzymological and CI in silico structural analysis, we conclude that absence of NDUFS4 induces near complete absence of NDUFA12 but not vice versa, and that NDUFAF2 stabilizes active CI in Ndufs4-/- mice and LS patient cells, perhaps in concert with mitochondrial inner membrane lipids.


Subject(s)
Electron Transport Complex I/deficiency , Electron Transport Complex I/genetics , Gene Deletion , Leigh Disease/genetics , Mitochondrial Proteins/metabolism , Molecular Chaperones/metabolism , NADPH Dehydrogenase/metabolism , Animals , Fibroblasts/metabolism , Gene Knockout Techniques , Humans , Leigh Disease/metabolism , Mice , Oxidative Phosphorylation , Protein Stability
4.
Clin Genet ; 97(4): 556-566, 2020 04.
Article in English | MEDLINE | ID: mdl-31957011

ABSTRACT

NGLY1 encodes the enzyme N-glycanase that is involved in the degradation of glycoproteins as part of the endoplasmatic reticulum-associated degradation pathway. Variants in this gene have been described to cause a multisystem disease characterized by neuromotor impairment, neuropathy, intellectual disability, and dysmorphic features. Here, we describe four patients with pathogenic variants in NGLY1. As the clinical features and laboratory results of the patients suggested a multisystem mitochondrial disease, a muscle biopsy had been performed. Biochemical analysis in muscle showed a strongly reduced ATP production rate in all patients, while individual OXPHOS enzyme activities varied from normal to reduced. No causative variants in any mitochondrial disease genes were found using mtDNA analysis and whole exome sequencing. In all four patients, variants in NGLY1 were identified, including two unreported variants (c.849T>G (p.(Cys283Trp)) and c.1067A>G (p.(Glu356Gly)). Western blot analysis of N-glycanase in muscle and fibroblasts showed a complete absence of N-glycanase. One patient showed a decreased basal and maximal oxygen consumption rates in fibroblasts. Mitochondrial morphofunction fibroblast analysis showed patient specific differences when compared to control cell lines. In conclusion, variants in NGLY1 affect mitochondrial energy metabolism which in turn might contribute to the clinical disease course.


Subject(s)
Epilepsies, Myoclonic/genetics , Intellectual Disability/genetics , Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase/genetics , Polyneuropathies/genetics , Child , Child, Preschool , Congenital Disorders of Glycosylation/diagnostic imaging , Congenital Disorders of Glycosylation/genetics , Congenital Disorders of Glycosylation/metabolism , Congenital Disorders of Glycosylation/pathology , Epilepsies, Myoclonic/diagnostic imaging , Epilepsies, Myoclonic/pathology , Female , Humans , Intellectual Disability/diagnostic imaging , Intellectual Disability/pathology , Male , Mitochondria/genetics , Mitochondria/pathology , Mutation/genetics , Polyneuropathies/diagnostic imaging , Polyneuropathies/pathology
6.
Cell Death Dis ; 8(3): e2716, 2017 03 30.
Article in English | MEDLINE | ID: mdl-28358377

ABSTRACT

Inhibition of complex I (CI) of the mitochondrial respiratory chain by BAY 87-2243 ('BAY') triggers death of BRAFV600E melanoma cell lines and inhibits in vivo tumor growth. Here we studied the mechanism by which this inhibition induces melanoma cell death. BAY treatment depolarized the mitochondrial membrane potential (Δψ), increased cellular ROS levels, stimulated lipid peroxidation and reduced glutathione levels. These effects were paralleled by increased opening of the mitochondrial permeability transition pore (mPTP) and stimulation of autophagosome formation and mitophagy. BAY-induced cell death was not due to glucose shortage and inhibited by the antioxidant α-tocopherol and the mPTP inhibitor cyclosporin A. Tumor necrosis factor receptor-associated protein 1 (TRAP1) overexpression in BAY-treated cells lowered ROS levels and inhibited mPTP opening and cell death, whereas the latter was potentiated by TRAP1 knockdown. Knockdown of autophagy-related 5 (ATG5) inhibited the BAY-stimulated autophagosome formation, cellular ROS increase and cell death. Knockdown of phosphatase and tensin homolog-induced putative kinase 1 (PINK1) inhibited the BAY-induced Δψ depolarization, mitophagy stimulation, ROS increase and cell death. Dynamin-related protein 1 (Drp1) knockdown induced mitochondrial filamentation and inhibited BAY-induced cell death. The latter was insensitive to the pancaspase inhibitor z-VAD-FMK, but reduced by necroptosis inhibitors (necrostatin-1, necrostatin-1s)) and knockdown of key necroptosis proteins (receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and mixed lineage kinase domain-like (MLKL)). BAY-induced cell death was also reduced by the ferroptosis inhibitor ferrostatin-1 and overexpression of the ferroptosis-inhibiting protein glutathione peroxidase 4 (GPX4). This overexpression also inhibited the BAY-induced ROS increase and lipid peroxidation. Conversely, GPX4 knockdown potentiated BAY-induced cell death. We propose a chain of events in which: (i) CI inhibition induces mPTP opening and Δψ depolarization, that (ii) stimulate autophagosome formation, mitophagy and an associated ROS increase, leading to (iii) activation of combined necroptotic/ferroptotic cell death.


Subject(s)
Electron Transport Complex I/metabolism , Melanoma/enzymology , Mitophagy , Reactive Oxygen Species/metabolism , Autophagy-Related Protein 5/genetics , Autophagy-Related Protein 5/metabolism , Cell Line, Tumor , Dynamins , Electron Transport Complex I/antagonists & inhibitors , Electron Transport Complex I/genetics , GTP Phosphohydrolases/genetics , GTP Phosphohydrolases/metabolism , HSP90 Heat-Shock Proteins/genetics , HSP90 Heat-Shock Proteins/metabolism , Humans , Melanoma/drug therapy , Melanoma/genetics , Melanoma/pathology , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Mitochondrial Membrane Transport Proteins/genetics , Mitochondrial Membrane Transport Proteins/metabolism , Mitochondrial Permeability Transition Pore , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Oxadiazoles/pharmacology , Protein Kinases/genetics , Protein Kinases/metabolism , Pyrazoles/pharmacology
7.
Sci Rep ; 5: 8035, 2015 Jan 26.
Article in English | MEDLINE | ID: mdl-25620325

ABSTRACT

In primary fibroblasts from Leigh Syndrome (LS) patients, isolated mitochondrial complex I deficiency is associated with increased reactive oxygen species levels and mitochondrial morpho-functional changes. Empirical evidence suggests these aberrations constitute linked therapeutic targets for small chemical molecules. However, the latter generally induce multiple subtle effects, meaning that in vitro potency analysis or single-parameter high-throughput cell screening are of limited use to identify these molecules. We combine automated image quantification and artificial intelligence to discriminate between primary fibroblasts of a healthy individual and a LS patient based upon their mitochondrial morpho-functional phenotype. We then evaluate the effects of newly developed Trolox variants in LS patient cells. This revealed that Trolox ornithylamide hydrochloride best counterbalanced mitochondrial morpho-functional aberrations, effectively scavenged ROS and increased the maximal activity of mitochondrial complexes I, IV and citrate synthase. Our results suggest that Trolox-derived antioxidants are promising candidates in therapy development for human mitochondrial disorders.


Subject(s)
Electron Transport Complex I/deficiency , Leigh Disease/genetics , Machine Learning , Mitochondrial Diseases/genetics , Chromans/administration & dosage , Citrate (si)-Synthase/metabolism , Electron Transport Complex I/genetics , Electron Transport Complex I/metabolism , Fibroblasts/drug effects , Fibroblasts/metabolism , Fibroblasts/pathology , Humans , Leigh Disease/drug therapy , Leigh Disease/pathology , Membrane Potential, Mitochondrial/drug effects , Mitochondria/drug effects , Mitochondria/metabolism , Mitochondria/pathology , Mitochondrial Diseases/drug therapy , Mitochondrial Diseases/metabolism , Mitochondrial Diseases/pathology , Oxidative Phosphorylation/drug effects , Reactive Oxygen Species/metabolism
8.
Antioxid Redox Signal ; 17(12): 1657-69, 2012 Dec 15.
Article in English | MEDLINE | ID: mdl-22559215

ABSTRACT

AIMS: Cell regulation by signaling reactive oxygen species (sROS) is often incorrectly studied through extracellular oxidant addition. Here, we used the membrane-permeable antioxidant Trolox to examine the role of sROS in mitochondrial morphology, oxidative phosphorylation (OXPHOS), and cytosolic calcium (Ca(2+)) handling in healthy human skin fibroblasts. RESULTS AND INNOVATION: Trolox treatment reduced the levels of 5-(and-6)-chloromethyl-2',7'-dichlorodihydro-fluorescein (CM-H(2)DCF) oxidizing ROS, lowered cellular lipid peroxidation, and induced a less oxidized mitochondrial thiol redox state. This was paralleled by increased glutathione- and mitofusin-dependent mitochondrial filamentation, increased expression of fully assembled mitochondrial complex I, elevated activity of citrate synthase and OXPHOS enzymes, and a higher cellular O(2) consumption. In contrast, Trolox did not alter hydroethidium oxidation, cytosolic thiol redox state, mitochondrial NAD(P)H levels, or mitochondrial membrane potential. Whole genome expression profiling revealed that Trolox did not trigger significant changes in gene expression, suggesting that Trolox acts downstream of this process. Cytosolic Ca(2+) transients, induced by the hormone bradykinin, were of a higher amplitude and decayed faster in Trolox-treated cells. These effects were dose-dependently antagonized by hydrogen peroxide. CONCLUSIONS: Our findings suggest that Trolox-sensitive sROS are upstream regulators of mitochondrial mitofusin levels, morphology, and function in healthy human skin fibroblasts. This information not only facilitates the interpretation of antioxidant effects in cell models (of oxidative-stress), but also contributes to a better understanding of ROS-related human pathologies, including mitochondrial disorders.


Subject(s)
Calcium/metabolism , Chromans/pharmacology , Cytosol/metabolism , Mitochondria/metabolism , Reactive Oxygen Species/metabolism , Animals , Blotting, Western , CHO Cells , Cells, Cultured , Cricetinae , HeLa Cells , Humans , Lipid Peroxidation/drug effects , Oxidative Phosphorylation/drug effects
9.
Biochim Biophys Acta ; 1817(10): 1925-36, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22430089

ABSTRACT

Human mitochondrial complex I (CI) deficiency is associated with progressive neurological disorders. To better understand the CI pathomechanism, we here studied how deletion of the CI gene NDUFS4 affects cell metabolism. To this end we compared immortalized mouse embryonic fibroblasts (MEFs) derived from wildtype (wt) and whole-body NDUFS4 knockout (KO) mice. Mitochondria from KO cells lacked the NDUFS4 protein and mitoplasts displayed virtually no CI activity, moderately reduced CII, CIII and CIV activities and normal citrate synthase and CV (F(o)F(1)-ATPase) activity. Native electrophoresis of KO cell mitochondrial fractions revealed two distinct CI subcomplexes of ~830kDa (enzymatically inactive) and ~200kDa (active). The level of fully-assembled CII-CV was not affected by NDUFS4 gene deletion. KO cells exhibited a moderately reduced maximal and routine O(2) consumption, which was fully inhibited by acute application of the CI inhibitor rotenone. The aberrant CI assembly and reduced O(2) consumption in KO cells were fully normalized by NDUFS4 gene complementation. Cellular [NAD(+)]/[NADH] ratio, lactate production and mitochondrial tetramethyl rhodamine methyl ester (TMRM) accumulation were slightly increased in KO cells. In contrast, NDUFS4 gene deletion did not detectably alter [NADP(+)]/[NADPH] ratio, cellular glucose consumption, the protein levels of hexokinases (I and II) and phosphorylated pyruvate dehydrogenase (P-PDH), total cellular adenosine triphosphate (ATP) level, free cytosolic [ATP], cell growth rate, and reactive oxygen species (ROS) levels. We conclude that the NDUFS4 subunit is of key importance in CI stabilization and that, due to the metabolic properties of the immortalized MEFs, NDUFS4 gene deletion has only modest effects at the live cell level. This article is part of a special issue entitled: 17th European Bioenergetics Conference (EBEC 2012).


Subject(s)
Electron Transport Complex I/metabolism , Embryo, Mammalian/enzymology , Fibroblasts/enzymology , Mitochondria/enzymology , Mitochondrial Proteins/metabolism , Adenosine Triphosphate/genetics , Adenosine Triphosphate/metabolism , Animals , Cell Line, Transformed , Electron Transport Complex I/genetics , Embryo, Mammalian/cytology , Enzyme Stability/physiology , Fibroblasts/cytology , Gene Deletion , Humans , Lactic Acid/metabolism , Mice , Mice, Knockout , Mitochondria/genetics , Mitochondria/metabolism , Mitochondrial Proteins/genetics , NAD/genetics , NAD/metabolism , NADP/genetics , NADP/metabolism , Phosphorylation/physiology , Proton-Translocating ATPases/genetics , Proton-Translocating ATPases/metabolism , Pyruvate Dehydrogenase Complex/genetics , Pyruvate Dehydrogenase Complex/metabolism
10.
Biochim Biophys Acta ; 1822(2): 168-75, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22036843

ABSTRACT

In this study, we investigated the pathogenicity of a homozygous Asp446Asn mutation in the NDUFS2 gene of a patient with a mitochondrial respiratory chain complex I deficiency. The clinical, biochemical, and genetic features of the NDUFS2 patient were compared with those of 4 patients with previously identified NDUFS2 mutations. All 5 patients presented with Leigh syndrome. In addition, 3 out of 5 showed hypertrophic cardiomyopathy. Complex I amounts in the patient carrying the Asp446Asn mutation were normal, while the complex I activity was strongly reduced, showing that the NDUFS2 mutation affects complex I enzymatic function. By contrast, the 4 other NDUFS2 patients showed both a reduced amount and activity of complex I. The enzymatic defect in fibroblasts of the patient carrying the Asp446Asn mutation was rescued by transduction of wild type NDUFS2. A 3-D model of the catalytic core of complex I showed that the mutated amino acid residue resides near the coenzyme Q binding pocket. However, the K(M) of complex I for coenzyme Q analogs of the Asp446Asn mutated complex I was similar to the K(M) observed in other complex I defects and in controls. We propose that the mutation interferes with the reduction of coenzyme Q or with the coupling of coenzyme Q reduction with the conformational changes involved in proton pumping of complex I.


Subject(s)
Electron Transport Complex I/genetics , Leigh Disease/genetics , Mitochondria/enzymology , Mutation , NADH Dehydrogenase/genetics , Amino Acid Sequence , Animals , Cardiomyopathy, Hypertrophic/genetics , Cardiomyopathy, Hypertrophic/metabolism , Catalysis , Electron Transport Complex I/metabolism , Female , Fibroblasts/metabolism , Homozygote , Humans , Infant , Infant, Newborn , Leigh Disease/enzymology , Leigh Disease/metabolism , Membrane Potential, Mitochondrial/genetics , Membrane Potential, Mitochondrial/physiology , Mitochondria/metabolism , Mitochondrial Membranes/enzymology , Mitochondrial Membranes/metabolism , Models, Molecular , Molecular Sequence Data , NADH Dehydrogenase/metabolism , Protein Conformation , Transduction, Genetic/methods , Ubiquinone/metabolism
11.
Clin Nutr ; 28(1): 59-64, 2009 Feb.
Article in English | MEDLINE | ID: mdl-18952326

ABSTRACT

BACKGROUND & AIMS: Lipid-induced immune modulation might contribute to the increased infection rate that is observed in patients using parenteral nutrition. We previously showed that emulsions containing medium-chain triglycerides (LCT/MCTs or pure MCTs), but not pure long-chain triglycerides (LCTs), impair neutrophil functions, modulate cell-signaling and induce neutrophil activation in vitro. It has recently been shown that medium-chain fatty acids are ligands for GPR84, a pertussis toxin (PT)-sensitive G-protein-coupled receptor (GPCR). This finding urged us to investigate whether MCT-induced neutrophil activation is mediated by PT-sensitive GPCRs. METHODS: Neutrophils isolated from blood of healthy volunteers were pre-incubated with PT (0.5-1 microg/mL, 1.5 h) and analyzed for the effect of this pre-incubation on LCT/MCT (2.5 mmol/L)-dependent modulation of serum-treated zymosan (STZ)-induced intracellular Ca(2+) mobilization and on LCT/MCT (5 mmol/L)-induced expression of cell surface adhesion (CD11b) and degranulation (CD66b) markers and oxygen radical (ROS) production. RESULTS: PT did not inhibit the effects of LCT/MCT on the STZ-induced increase in cytosolic free Ca(2+) concentration. LCT/MCT increased ROS production to 146% of unstimulated cells. However, pre-incubation with PT did not inhibit the LCT/MCT-induced ROS production. Furthermore, the LCT/MCT-induced increase in CD11b and CD66b expression (196% and 235% of unstimulated cells, respectively) was not inhibited by pre-incubation with PT. CONCLUSION: LCT/MCT-induced neutrophil activation does not involve the action of a PT-sensitive G-protein-coupled receptor.


Subject(s)
Neutrophil Activation/drug effects , Parenteral Nutrition , Receptors, Cell Surface/metabolism , Receptors, G-Protein-Coupled/metabolism , Triglycerides/pharmacology , CD11b Antigen/metabolism , Calcium/metabolism , Calcium Signaling/drug effects , Cell Adhesion/drug effects , Cell Degranulation/drug effects , Cells, Cultured , Fat Emulsions, Intravenous/chemistry , Humans , Neutrophil Activation/immunology , Neutrophils/drug effects , Neutrophils/immunology , Parenteral Nutrition/adverse effects , Reactive Oxygen Species/metabolism , Triglycerides/chemistry , Zymosan/pharmacology
12.
Biochim Biophys Acta ; 1777(7-8): 853-9, 2008.
Article in English | MEDLINE | ID: mdl-18435906

ABSTRACT

Deficiency of mitochondrial NADH:ubiquinone oxidoreductase (complex I), is associated with a variety of clinical phenotypes such as Leigh syndrome, encephalomyopathy and cardiomyopathy. Circumstantial evidence suggests that increased reactive oxygen species (ROS) levels contribute to the pathogenesis of these disorders. Here we assessed the effect of the water-soluble vitamin E derivative Trolox on ROS levels, and the amount and activity of complex I in fibroblasts of six children with isolated complex I deficiency caused by a mutation in the NDUFS1, NDUFS2, NDUFS7, NDUFS8 or NDUFV1 gene. Patient cells displayed increased ROS levels and a variable decrease in complex I activity and amount. For control cells, the ratio between activity and amount was 1 whereas for the patients this ratio was below 1, indicating a defect in intrinsic catalytic activity of complex I in the latter cells. Trolox treatment dramatically reduced ROS levels in both control and patient cells, which was paralleled by a substantial increase in the amount of complex I. Although the ratio between the increase in activity and amount of complex I was exactly proportional in control cells it varied between 0.1 and 0.8 for the patients. Our findings suggest that the expression of complex I is regulated by ROS. Furthermore, they provide evidence that both the amount and intrinsic activity of complex I are decreased in inherited complex I deficiency. The finding that Trolox treatment increased the amount of complex I might aid the future development of antioxidant treatment strategies for patients. However, such treatment may only be beneficial to patients with a relatively small reduction in intrinsic catalytic defect of the complex.


Subject(s)
Chromans/pharmacology , Electron Transport Complex I/deficiency , Electron Transport Complex I/genetics , Electron Transport Complex I/drug effects , Fibroblasts/enzymology , Genetic Diseases, Inborn/enzymology , Genetic Diseases, Inborn/genetics , Humans , Kinetics , Mitochondria/enzymology , Mutation , Oxidative Phosphorylation , Phenotype , Protein Subunits/genetics , Skin/enzymology
13.
Am J Clin Nutr ; 87(3): 539-47, 2008 Mar.
Article in English | MEDLINE | ID: mdl-18326590

ABSTRACT

BACKGROUND: It remains unclear whether immune modulation by lipids contributes to the high risk of infectious complications that is associated with the use of parenteral nutrition. Although mixed long- and medium-chain triacylglycerol (LCT-MCT)-containing emulsions, but not pure LCT emulsions, activate neutrophils and impair crucial leukocyte functions in vitro, in vivo studies have failed to corroborate these findings. OBJECTIVES: The present investigation was conducted to evaluate the effects of LCT and LCT-MCT on immune function in healthy humans and to assess whether the lack of in vivo effects results from sampling errors due to extravascular sequestration of activated neutrophils. DESIGN: Saline, LCT-MCT, and LCT emulsions were administered intravenously for 4.5 h to 12 healthy volunteers in a randomized crossover design. Plasma triacylglycerol concentrations were clamped at a clinically relevant concentration of 3-5 mmol/L. Leukocyte population counts and neutrophil activation were assessed before and after infusion. Leukocyte sequestration was evaluated by monitoring the distribution of Technetium-99m-labeled autologous leukocytes during infusions. RESULTS: Whereas LCT exerted no greater effects than did saline, LCT-MCT significantly decreased lymphocyte counts. However, no evidence for neutrophil activation was found with either lipid. Moreover, the clearance of radiolabeled leukocytes from the liver, spleen, and lungs was not altered by any lipid, which suggested that lipid emulsions do not induce leukocyte sequestration. CONCLUSIONS: Short-term infusion of LCT-MCT (but not LCT) to healthy humans modulates leukocyte population counts but, in clear contrast with the in vitro situation, does not induce neutrophil activation. These disparate findings cannot be explained by MCT-induced leukocyte sequestration.


Subject(s)
Fat Emulsions, Intravenous/pharmacology , Leukocytes/drug effects , Neutrophil Activation/drug effects , Parenteral Nutrition, Total/methods , Triglycerides , Adult , Cross-Over Studies , Fat Emulsions, Intravenous/adverse effects , Female , Humans , Leukocyte Count , Leukocytes/immunology , Leukocytes/physiology , Male , Middle Aged , Neutrophil Activation/physiology , Radiopharmaceuticals/metabolism , Reactive Oxygen Species/metabolism , Time Factors , Triglycerides/blood , Triglycerides/chemistry , Triglycerides/immunology , Triglycerides/pharmacology
14.
J Gen Virol ; 88(Pt 11): 3027-3030, 2007 Nov.
Article in English | MEDLINE | ID: mdl-17947526

ABSTRACT

During enterovirus infection, host cell membranes are rigorously rearranged and modified. One ubiquitously expressed lipid-modifying enzyme that might contribute to these alterations is phospholipase D (PLD). Here, we investigated PLD activity in coxsackievirus-infected cells. We show that PLD activity is not required for efficient coxsackievirus RNA replication. Instead, PLD activity rapidly decreased upon infection and upon ectopic expression of the viral 3A protein, which inhibits the PLD activator ADP-ribosylation factor 1. However, similar decreases were observed during infection with coxsackieviruses carrying defective mutant 3A proteins. Possible causes for the reduction of PLD activity and the biological consequences are discussed.


Subject(s)
Enterovirus/enzymology , Phospholipase D/metabolism , Viral Proteins/metabolism , ADP-Ribosylation Factor 1/antagonists & inhibitors , Animals , Cell Line , Chlorocebus aethiops , Phospholipase D/antagonists & inhibitors , RNA, Viral/biosynthesis , Viral Proteins/antagonists & inhibitors , Virus Replication
15.
Biochim Biophys Acta ; 1772(9): 1041-51, 2007 Sep.
Article in English | MEDLINE | ID: mdl-17600689

ABSTRACT

Isolated complex I deficiency is the most common enzymatic defect of the oxidative phosphorylation (OXPHOS) system, causing a wide range of clinical phenotypes. We reported before that the rates at which reactive oxygen species (ROS)-sensitive dyes are converted into their fluorescent oxidation products are markedly increased in cultured skin fibroblasts of patients with nuclear-inherited isolated complex I deficiency. Using video-imaging microscopy we show here that these cells also display a marked increase in NAD(P)H autofluorescence. Linear regression analysis revealed a negative correlation with the residual complex I activity and a positive correlation with the oxidation rates of the ROS-sensitive dyes 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein and hydroethidine for a cohort of 10 patient cell lines. On the other hand, video-imaging microscopy of cells expressing reduction-oxidation sensitive GFP1 in either the mitochondrial matrix or cytosol showed the absence of any detectable change in thiol redox state. In agreement with this result, neither the glutathione nor the glutathione disulfide content differed significantly between patient and healthy fibroblasts. Finally, video-rate confocal microscopy of cells loaded with C11-BODIPY(581/591) demonstrated that the extent of lipid peroxidation, which is regarded as a measure of oxidative damage, was not altered in patient fibroblasts. Our results indicate that fibroblasts of patients with isolated complex I deficiency maintain their thiol redox state despite marked increases in ROS production.


Subject(s)
Cytosol/metabolism , Electron Transport Complex I/deficiency , Mitochondria/metabolism , Mitochondrial Diseases/genetics , Mitochondrial Diseases/metabolism , Oxidation-Reduction , Sulfhydryl Compounds/metabolism , Adult , Case-Control Studies , Cells, Cultured , Child, Preschool , Electron Transport Complex I/metabolism , Fibroblasts/drug effects , Fibroblasts/metabolism , Glutathione/metabolism , Humans , Infant , Infant, Newborn , Lipid Peroxidation , Rotenone/pharmacology , Uncoupling Agents/pharmacology
16.
Am J Physiol Cell Physiol ; 293(1): C22-9, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17428841

ABSTRACT

Malfunction of NADH:ubiquinone oxidoreductase or complex I (CI), the first and largest complex of the mitochondrial oxidative phosphorylation system, has been implicated in a wide variety of human disorders. To demonstrate a quantitative relationship between CI amount and activity and mitochondrial shape and cellular reactive oxygen species (ROS) levels, we recently combined native electrophoresis and confocal and video microscopy of dermal fibroblasts of healthy control subjects and children with isolated CI deficiency. Individual mitochondria appeared fragmented and/or less branched in patient fibroblasts with a severely reduced CI amount and activity (class I), whereas patient cells in which these latter parameters were only moderately reduced displayed a normal mitochondrial morphology (class II). Moreover, cellular ROS levels were significantly more increased in class I compared with class II cells. We propose a mechanism in which a mutation-induced decrease in the cellular amount and activity of CI leads to enhanced ROS levels, which, in turn, induce mitochondrial fragmentation when not appropriately counterbalanced by the cell's antioxidant defense systems.


Subject(s)
Electron Transport Complex I/metabolism , Fibroblasts/metabolism , Mitochondria/metabolism , Mitochondrial Diseases/metabolism , Oxidative Phosphorylation , Reactive Oxygen Species/metabolism , Cell Line , Child , Cluster Analysis , Electron Transport Complex I/antagonists & inhibitors , Electron Transport Complex I/deficiency , Electron Transport Complex I/genetics , Electrophoresis , Enzyme Inhibitors/pharmacology , Fibroblasts/drug effects , Fibroblasts/enzymology , Fibroblasts/pathology , Genetic Predisposition to Disease , Humans , Membrane Potential, Mitochondrial , Microscopy, Confocal , Microscopy, Video , Mitochondria/drug effects , Mitochondria/enzymology , Mitochondria/pathology , Mitochondrial Diseases/enzymology , Mitochondrial Diseases/genetics , Mitochondrial Diseases/pathology , Mitochondrial Proteins/metabolism , Mitochondrial Size , Mutation , Oxidative Phosphorylation/drug effects , Reproducibility of Results , Rotenone/pharmacology , Severity of Illness Index , Uncoupling Agents/pharmacology
17.
Biochim Biophys Acta ; 1772(3): 373-81, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17289351

ABSTRACT

Deficiency of NADH:ubiquinone oxidoreductase or complex I (CI) is the most common cause of disorders of the oxidative phosphorylation system in humans. Using life cell imaging and blue-native electrophoresis we quantitatively compared superoxide production and CI amount and activity in cultured skin fibroblasts of 7 healthy control subjects and 21 children with inherited isolated CI deficiency. Thirteen children had a disease causing mutation in one of the nuclear-encoded CI subunits, whereas in the remainder the genetic cause of the disease is not yet established. Superoxide production was significantly increased in all but two of the patient cell lines. An inverse relationship with the amount and residual activity of CI was observed. In agreement with this finding, rotenone, a potent inhibitor of CI activity, dose-dependently increased superoxide production in healthy control cells. Also in this case an inverse relationship with the residual activity of CI was observed. In sharp contrast, however, rotenone did not decrease the amount of CI. The data presented show that superoxide production is increased in inherited CI deficiency and that this increase is primarily a consequence of the reduction in cellular CI activity and not of a further leakage of electrons from mutationally malformed complexes.


Subject(s)
Electron Transport Complex I/deficiency , Metabolism, Inborn Errors/metabolism , Oxidative Phosphorylation , Superoxides/metabolism , Child, Preschool , Electron Transport Complex I/analysis , Electron Transport Complex I/genetics , Female , Fibroblasts/enzymology , Humans , Infant , Infant, Newborn , Male , Metabolism, Inborn Errors/enzymology , Metabolism, Inborn Errors/genetics , Rotenone/administration & dosage , Skin/enzymology , Superoxides/analysis , Uncoupling Agents/administration & dosage
18.
Cytometry A ; 69(12): 1184-92, 2006 Dec 01.
Article in English | MEDLINE | ID: mdl-17066472

ABSTRACT

BACKGROUND: Mitochondrial dysfunction may lead to increased oxidative stress and consequent changes in cell spreading. Here, we describe and validate a novel method for simultaneous quantification of these two parameters. METHODS: Human skin fibroblasts were loaded with 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein (CM-H(2)DCF), and its oxidative conversion into CM-DCF was monitored as a function of time by video-rate confocal microscopy and real-time image averaging. Cell size was determined after binarization of the acquired images. RESULTS: At the lowest practical laser output, CM-DCF formation occurred with zero order kinetics, indicating that [CM-H(2)DCF] was not rate-limiting and that the rate of [CM-DCF] formation (V(CM-DCF)) was a function of the cellular oxidant level. Analysis of fibroblasts of a healthy control subject and a patient with a deficiency of NADH:ubiquinone oxidoreductase, the first complex of the oxidative phosphorylation system, revealed a significant increase in cellular oxidant level in the latter cells that was, however, not accompanied by a change in cell spreading. Conversely, chronic treatment with 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), a derivative of vitamin E, markedly decreased the oxidant level and cell spreading in both control and patient fibroblasts. CONCLUSIONS: We present a reliable method for simultaneous quantification of oxidant levels and cell spreading in living cells.


Subject(s)
Fluoresceins/chemistry , Fluorescent Dyes/chemistry , Microscopy, Confocal/methods , Oxidants/analysis , Oxidative Stress , Antioxidants/pharmacology , Cell Movement , Cell Size , Fibroblasts/chemistry , Fluoresceins/pharmacokinetics , Fluorescent Dyes/pharmacokinetics , Humans , Microscopy, Video/methods , Oxidation-Reduction , Skin/chemistry , Skin/cytology
19.
Am J Physiol Cell Physiol ; 291(2): C308-16, 2006 Aug.
Article in English | MEDLINE | ID: mdl-16554405

ABSTRACT

Previously, we reported that both the bradykinin (Bk)-induced increase in mitochondrial ATP concentration ([ATP]M) and the rate of cytosolic Ca2+ removal are significantly decreased in skin fibroblasts from a patient with an isolated complex I deficiency. Here we demonstrate that the mitochondrial Ca2+ indicator rhod-2 can be used to selectively buffer the Bk-induced increase in mitochondrial Ca2+ concentration ([Ca2+]M) and, consequently, the Ca2+-stimulated increase in [ATP]M, thus allowing studies of how the increase in [ATP]M and the cytosolic Ca2+ removal rate are related. Luminometry of healthy fibroblasts expressing either aequorin or luciferase in the mitochondrial matrix showed that rhod-2 dose dependently decreased the Bk-induced increase in [Ca2+]M and [ATP]M by maximally 80 and 90%, respectively. Digital imaging microscopy of cells coloaded with the cytosolic Ca2+ indicator fura-2 revealed that, in parallel, rhod-2 maximally decreased the cytosolic Ca2+ removal rate by 20%. These findings demonstrate that increased mitochondrial ATP production is required for accelerating cytosolic Ca2+ removal during stimulation with a Ca2+-mobilizing agonist. In contrast, complex I-deficient patient fibroblasts displayed a cytosolic Ca2+ removal rate that was already decreased by 40% compared with healthy fibroblasts. Rhod-2 did not further decrease this rate, indicating the absence of mitochondrial ATP supply to the cytosolic Ca2+ pumps. This work reveals the usefulness of rhodamine-based Ca2+ indicators in examining the role of intramitochondrial Ca2+ in mitochondrial (patho) physiology.


Subject(s)
Adenosine Triphosphate/biosynthesis , Bradykinin/administration & dosage , Calcium Signaling/drug effects , Calcium/metabolism , Electron Transport Complex I/deficiency , Fibroblasts/metabolism , Mitochondria/metabolism , Cells, Cultured , Dose-Response Relationship, Drug , Fibroblasts/drug effects , Humans , Mitochondria/drug effects
20.
Biochim Biophys Acta ; 1762(1): 115-23, 2006 Jan.
Article in English | MEDLINE | ID: mdl-16213125

ABSTRACT

Although a large number of mutations causing malfunction of complex I (NADH:ubiquinone oxidoreductase) of the OXPHOS system is now known, their cell biological consequences remain obscure. We previously showed that the bradykinin (Bk)-induced increase in mitochondrial [ATP] ([ATP](M)) is significantly reduced in primary skin fibroblasts from a patient with an isolated complex I deficiency. The present work addresses the mechanism(s) underlying this impaired response. Luminometry of fibroblasts from 6 healthy subjects and 14 genetically characterized patients expressing mitochondria targeted luciferase revealed that the Bk-induced increase in [ATP](M) was significantly, but to a variable degree, decreased in 10 patients. The same variation was observed for the increases in mitochondrial [Ca(2+)] ([Ca(2+)](M)), measured with mitochondria targeted aequorin, and cytosolic [Ca(2+)] ([Ca(2+)](C)), measured with fura-2, and for the Ca(2+) content of the endoplasmic reticulum (ER), calculated from the increase in [Ca(2+)](C) evoked by thapsigargin, an inhibitor of the ER Ca(2+) ATPase. Regression analysis revealed that the increase in [ATP](M) was directly proportional to the increases in [Ca(2+)](C) and [Ca(2+)](M) and to the ER Ca(2+) content. Our findings provide evidence that a pathological reduction in ER Ca(2+) content is the direct cause of the impaired Bk-induced increase in [ATP](M) in human complex I deficiency.


Subject(s)
Adenosine Triphosphate/agonists , Adenosine Triphosphate/biosynthesis , Calcium Signaling/drug effects , Calcium/metabolism , Electron Transport Complex I/deficiency , Endoplasmic Reticulum/metabolism , Mitochondria/metabolism , Adult , Bradykinin/pharmacology , Cells, Cultured , Child , Child, Preschool , Fibroblasts/pathology , Humans , Infant , Infant, Newborn
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