JoVE Visualize What is visualize?
Stop Reading. Start Watching.
Advanced Search
Stop Reading. Start Watching.
Regular Search
Find video protocols related to scientific articles indexed in Pubmed.
Structural re-arrangement and peroxidase activation of cytochrome c by anionic analogues of vitamin E: tocopherol-succinate and tocopherol-phosphate.
J. Biol. Chem.
PUBLISHED: 10-02-2014
Show Abstract
Hide Abstract
Cytochrome c (cyt c) is a multi-functional hemoprotein in the mitochondrial intermembrane space whereby its participation in electron shuttling between respiratory complexes III and IV is alternative to its role in apoptosis as a peroxidase activated by interaction with cardiolipin (CL), and resulting in selective CL peroxidation. The switch from electron transfer to peroxidase function requires partial unfolding of the protein upon binding of CL, whose specific features combine negative charges of the two phosphate groups with four hydrophobic fatty acid residues. Assuming that other endogenous small molecule ligands with a hydrophobic chain and a negatively charged functionality may activate cyt c into a peroxidase, we investigated two hydrophobic anionic analogs of vitamin E - lower case Greek alpha-tocopherol succinate (lower case Greek alpha-TOS) and lower case Greek alpha-tocopherol phosphate (lower case Greek alpha-TOP) - as potential inducers of peroxidase activity of cyt c. NMR studies and computational modeling indicate that they interact with cyt c at similar sites previously proposed for CL. Absorption spectroscopy showed that both analogues effectively disrupt the Fe-S(Met80) bond associated with unfolding of cyt c. We found that lower case Greek alpha-TOS and lower case Greek alpha-TOP stimulate peroxidase activity of cyt c. Enhanced peroxidase activity was also observed in isolated rat liver mitochondria incubated with lower case Greek alpha-TOS and tBOOH. A mitochondria-targeted derivative of TOS, triphenylphosphonium-TOS (MitoVES), was more efficient in inducing H2O2-dependent apoptosis in mouse embryonic cyt c+/+ cells than in cyt c-/- cells. Essential for the execution of apoptotic program peroxidase activation of cyt c by lower case Greek alpha-TOS may contribute to its known anti-cancer pharmacological activity.
Related JoVE Video
Inactivation of the ferroptosis regulator Gpx4 triggers acute renal failure in mice.
Nat. Cell Biol.
PUBLISHED: 08-13-2014
Show Abstract
Hide Abstract
Ferroptosis is a non-apoptotic form of cell death induced by small molecules in specific tumour types, and in engineered cells overexpressing oncogenic RAS. Yet, its relevance in non-transformed cells and tissues is unexplored and remains enigmatic. Here, we provide direct genetic evidence that the knockout of glutathione peroxidase 4 (Gpx4) causes cell death in a pathologically relevant form of ferroptosis. Using inducible Gpx4(-/-) mice, we elucidate an essential role for the glutathione/Gpx4 axis in preventing lipid-oxidation-induced acute renal failure and associated death. We furthermore systematically evaluated a library of small molecules for possible ferroptosis inhibitors, leading to the discovery of a potent spiroquinoxalinamine derivative called Liproxstatin-1, which is able to suppress ferroptosis in cells, in Gpx4(-/-) mice, and in a pre-clinical model of ischaemia/reperfusion-induced hepatic damage. In sum, we demonstrate that ferroptosis is a pervasive and dynamic form of cell death, which, when impeded, promises substantial cytoprotection.
Related JoVE Video
Molecular speciation and dynamics of oxidized triacylglycerols in lipid droplets: Mass spectrometry and coarse-grained simulations.
Free Radic. Biol. Med.
PUBLISHED: 08-10-2014
Show Abstract
Hide Abstract
Lipid droplets (LDs) are ubiquitous and physiologically active organelles regulating storage and mobilization of lipids in response to metabolic demands. Among the constituent LD neutral lipids, such as triacylglycerols, cholesterol esters, and free fatty acids, oxidizable polyunsaturated molecular species may be quite abundant, yet the structural and functional roles of their oxidation products have not been studied. Our previous work documented the presence of these peroxidized species in LDs. Assuming that hydrophilic oxygen-containing functionalities may markedly change the hydrophobic/hydrophilic molecular balance, here we utilized computational modeling to test the hypothesis that lipid peroxidation causes redistribution of lipids between the highly hydrophobic core and the polar surface (phospho)lipid monolayer-the area enriched with integrated enzymatic machinery. Using quantitative liquid chromatography/mass spectrometry, we characterized molecular speciation of oxTAGs in LDs of dendritic cells in cancer and hypoxic trophoblasts cells as two cellular models associated with dyslipidemia. Among the many types of oxidized lipids identified, we found that oxidatively truncated forms and hydroxyl derivatives of TAGs were the prevailing oxidized lipid species in LDs in both cell types. Using coarse-grained molecular dynamics (CG-MD) simulations we established that lipid oxidation changed their partitioning whereby oxidized lipids migrated into the outer monolayer of the LD, where they can affect essential metabolic pathways and undergo conversions, possibly leading to the formation of oxygenated lipid mediators.
Related JoVE Video
Copper chelation selectively kills colon cancer cells through redox cycling and generation of reactive oxygen species.
BMC Cancer
PUBLISHED: 07-15-2014
Show Abstract
Hide Abstract
Metals including iron, copper and zinc are essential for physiological processes yet can be toxic at high concentrations. However the role of these metals in the progression of cancer is not well defined. Here we study the anti-tumor activity of the metal chelator, TPEN, and define its mechanism of action.
Related JoVE Video
Abnormalities in the male reproductive system after exposure to diesel and biodiesel blend.
Environ. Mol. Mutagen.
PUBLISHED: 06-30-2014
Show Abstract
Hide Abstract
Altering the fuel source from petroleum-based ultralow sulfur diesel to biodiesel and its blends is considered by many to be a sustainable choice for controlling exposures to particulate material. As the exhaust of biodiesel/diesel blends is composed of a combination of combustion products of polycyclic aromatic hydrocarbons and fatty acid methyl esters, we hypothesize that 50% biodiesel/diesel blend (BD50) exposure could induce harmful outcomes because of its ability to trigger oxidative damage. Here, adverse effects were compared in murine male reproductive organs after pharyngeal aspiration with particles generated by engine fueled with BD50 or neat petroleum diesel (D100). When compared with D100, exposure to BD50 significantly altered sperm integrity, including concentration, motility, and morphological abnormalities, as well as increasing testosterone levels in testes during the time course postexposure. Serum level of luteinizing hormone was significantly depleted only after BD50 exposure. Moreover, we observed that exposure to BD50 significantly increased sperm DNA fragmentation and the upregulation of inflammatory cytokines in the serum and testes on Day 7 postexposure when compared with D100. Histological evaluation of testes sections from BD50 exposure indicated more noticeable interstitial edema, degenerating spermatocytes, and dystrophic seminiferous tubules with arrested spermatogenesis. Significant differences in the level of oxidative stress assessed by accumulation of lipid peroxidation products and depletion of glutathione were detected on exposure to respirable BD50 and D100. Taken together, these results indicate that exposure of mice to inhalable BD50 caused more pronounced adverse effects on male reproductive function than diesel. Environ. Mol. Mutagen., 2014. © 2014 Wiley Periodicals, Inc.
Related JoVE Video
Mitochondrial NM23-H4/NDPK-D: a bifunctional nanoswitch for bioenergetics and lipid signaling.
Naunyn Schmiedebergs Arch. Pharmacol.
PUBLISHED: 06-09-2014
Show Abstract
Hide Abstract
A novel paradigm for the function of the mitochondrial nucleoside diphosphate kinase NM23-H4/NDPK-D is proposed: acting as a bifunctional nanoswitch in bioenergetics and cardiolipin (CL) trafficking and signaling. Similar to some other mitochondrial proteins like cytochrome c or AIF, NM23-H4 seems to have dual functions in bioenergetics and apoptotic signaling. In its bioenergetic phosphotransfer mode, the kinase reversibly phosphorylates NDPs into NTPs, driven by mitochondrially generated ATP. Among others, this reaction can locally supply GTP to mitochondrial GTPases as shown for the dynamin-like GTPase OPA1, found in a complex together with NM23-H4. Further, NM23-H4 is functionally coupled to adenylate translocase (ANT) of the mitochondrial inner membrane (MIM), so generated ADP can stimulate respiration to rapidly regenerate ATP. The lipid transfer mode of NM23-H4 can support, dependent on the presence of CL, the transfer of anionic lipids between membranes in vitro and the sorting of CL from its mitochondrial sites of synthesis (MIM) to the mitochondrial outer membrane (MOM) in vivo. Such (partial) collapse of MIM/MOM CL asymmetry results in CL externalization on the mitochondrial surface, where CL can serve as pro-apoptotic or pro-mitophagic "eat me"-signal. The functional state of NM23-H4 depends on its degree of CL-membrane interaction. In vitro assays have shown that only NM23-H4 that fully cross-links two membranes is lipid transfer competent, but at the same time phosphotransfer (kinase) inactive. Thus, the two functions of NM23-H4 seem to be mutually exclusive. This novel mitochondrial regulatory circuit has potential for the development of interventions in various human pathologies.
Related JoVE Video
Lung macrophages "digest" carbon nanotubes using a superoxide/peroxynitrite oxidative pathway.
ACS Nano
PUBLISHED: 06-04-2014
Show Abstract
Hide Abstract
In contrast to short-lived neutrophils, macrophages display persistent presence in the lung of animals after pulmonary exposure to carbon nanotubes. While effective in the clearance of bacterial pathogens and injured host cells, the ability of macrophages to "digest" carbonaceous nanoparticles has not been documented. Here, we used chemical, biochemical, and cell and animal models and demonstrated oxidative biodegradation of oxidatively functionalized single-walled carbon nanotubes via superoxide/NO* ? peroxynitrite-driven oxidative pathways of activated macrophages facilitating clearance of nanoparticles from the lung.
Related JoVE Video
Graphene oxide attenuates Th2-type immune responses, but augments airway remodeling and hyperresponsiveness in a murine model of asthma.
ACS Nano
PUBLISHED: 05-23-2014
Show Abstract
Hide Abstract
Several lines of evidence indicate that exposure to nanoparticles (NPs) is able to modify airway immune responses, thus facilitating the development of respiratory diseases. Graphene oxide (GO) is a promising carbonaceous nanomaterial with unique physicochemical properties, envisioned for a multitude of medical and industrial applications. In this paper, we determined how exposure to GO modulates the allergic pulmonary response. Using a murine model of ovalbumin (OVA)-induced asthma, we revealed that GO, given at the sensitization stage, augmented airway hyperresponsiveness and airway remodeling in the form of goblet cell hyperplasia and smooth muscle hypertrophy. At the same time, the levels of the cytokines IL-4, IL-5, and IL-13 were reduced in broncho-alveolar lavage (BAL) fluid in GO-exposed mice. Exposure to GO during sensitization with OVA decreased eosinophil accumulation and increased recruitment of macrophages in BAL fluid. In line with the cytokine profiles, sensitization with OVA in the presence of GO stimulated the production of OVA-specific IgG2a and down-regulated the levels of IgE and IgG1. Moreover, exposure to GO increased the macrophage production of the mammalian chitinases, CHI3L1 and AMCase, whose expression is associated with asthma. Finally, molecular modeling has suggested that GO may directly interact with chitinase, affecting AMCase activity, which has been directly proven in our studies. Thus, these data show that GO exposure attenuates Th2 immune response in a model of OVA-induced asthma, but leads to potentiation of airway remodeling and hyperresponsiveness, with the induction of mammalian chitinases.
Related JoVE Video
Deciphering of mitochondrial cardiolipin oxidative signaling in cerebral ischemia-reperfusion.
J. Cereb. Blood Flow Metab.
PUBLISHED: 05-16-2014
Show Abstract
Hide Abstract
It is believed that biosynthesis of lipid mediators in the central nervous system after cerebral ischemia-reperfusion starts with phospholipid hydrolysis by calcium-dependent phospholipases and is followed by oxygenation of released fatty acids (FAs). Here, we report an alternative pathway whereby cereberal ischemia-reperfusion triggered oxygenation of a mitochondria-specific phospholipid, cardiolipin (CL), is followed by its hydrolysis to yield monolyso-CLs and oxygenated derivatives of fatty (linoleic) acids. We used a model of global cerebral ischemia-reperfusion characterized by 9?minutes of asphyxia leading to asystole followed by cardiopulmonary resuscitation in postnatal day 17 rats. Global ischemia and cardiopulmonary resuscitation resulted in: (1) selective oxidation and hydrolysis of CLs, (2) accumulation of lyso-CLs and oxygenated free FAs, (3) activation of caspase 3/7 in the brain, and (4) motor and cognitive dysfunction. On the basis of these findings, we used a mitochondria targeted nitroxide electron scavenger, which prevented CL oxidation and subsequent hydrolysis, attenuated caspase activation, and improved neurocognitive outcome when administered after cardiac arrest. These data show that calcium-independent CL oxidation and subsequent hydrolysis represent a previously unidentified pathogenic mechanism of brain injury incurred by ischemia-reperfusion and a clinically relevant therapeutic target.Journal of Cerebral Blood Flow & Metabolism advance online publication, 19 November 2014; doi:10.1038/jcbfm.2014.204.
Related JoVE Video
A mitochondrial pathway for biosynthesis of lipid mediators.
Nat Chem
PUBLISHED: 03-16-2014
Show Abstract
Hide Abstract
The central role of mitochondria in metabolic pathways and in cell-death mechanisms requires sophisticated signalling systems. Essential in this signalling process is an array of lipid mediators derived from polyunsaturated fatty acids. However, the molecular machinery for the production of oxygenated polyunsaturated fatty acids is localized in the cytosol and their biosynthesis has not been identified in mitochondria. Here we report that a range of diversified polyunsaturated molecular species derived from a mitochondria-specific phospholipid, cardiolipin (CL), is oxidized by the intermembrane-space haemoprotein, cytochrome c. We show that a number of oxygenated CL species undergo phospholipase A2-catalysed hydrolysis and thus generate multiple oxygenated fatty acids, including well-known lipid mediators. This represents a new biosynthetic pathway for lipid mediators. We demonstrate that this pathway, which includes the oxidation of polyunsaturated CLs and accumulation of their hydrolysis products (oxygenated linoleic, arachidonic acids and monolysocardiolipins), is activated in vivo after acute tissue injury.
Related JoVE Video
Quantitative method of measuring phosphatidylserine externalization during apoptosis using electron paramagnetic resonance (EPR) spectroscopy and annexin-conjugated iron.
Methods Mol. Biol.
PUBLISHED: 03-14-2014
Show Abstract
Hide Abstract
We present here the application of a novel assay that measures the absolute amount of PS externalized on the surface of cells. While based on the same annexin binding principle as the fluorescent flow cytometry assay, we use paramagnetic iron as the ultimate reporter molecule, establishing a linear relationship between signal amplitude and amount of PS on the cell surface, allowing a quantitative assay of PS externalization over a wide dynamic range. The application of this technique, alone and in concert with the PS oxidation method presented in the previous chapter, will greatly aid in studying the mechanistic connection between lipid peroxidation and translocation events during apoptosis.
Related JoVE Video
Quantification of selective phosphatidylserine oxidation during apoptosis.
Methods Mol. Biol.
PUBLISHED: 03-14-2014
Show Abstract
Hide Abstract
Membrane phospholipids are gaining increasing attention as important mediators in a variety of signal transduction processes. Oxidation and changes in membrane topography of lipids are likely important elements in the regulation of phospholipid-dependent signaling. Phosphatidylserine (PS), in particular, is implicated in the regulation of macrophage-dependent clearance of apoptotic cell "corpses" in a pathway likely mediated by selective oxidation and translocation of PS in the plasma membrane. Here we describe our highly sensitive and specific assay to measure differential lipid peroxidation in individual phospholipid classes in live cells using metabolic integration of the fluorescent oxidation-sensitive fatty acid analog, cis- parinaric acid and resolution of specific phospholipids by high-pressure liquid chromatography. These experimental approaches can provide insight into the roles and mechanisms of PS oxidation in the identification and clearance of apoptotic cells.
Related JoVE Video
TNFR1/phox interaction and TNFR1 mitochondrial translocation Thwart silica-induced pulmonary fibrosis.
J. Immunol.
PUBLISHED: 03-12-2014
Show Abstract
Hide Abstract
Macrophages play a fundamental role in innate immunity and the pathogenesis of silicosis. Phagocytosis of silica particles is associated with the generation of reactive oxygen species (ROS), secretion of cytokines, such as TNF, and cell death that contribute to silica-induced lung disease. In macrophages, ROS production is executed primarily by activation of the NADPH oxidase (Phox) and by generation of mitochondrial ROS (mtROS); however, the relative contribution is unclear, and the effects on macrophage function and fate are unknown. In this study, we used primary human and mouse macrophages (C57BL/6, BALB/c, and p47(phox-/-)) and macrophage cell lines (RAW 264.7 and IC21) to investigate the contribution of Phox and mtROS to silica-induced lung injury. We demonstrate that reduced p47(phox) expression in IC21 macrophages is linked to enhanced mtROS generation, cardiolipin oxidation, and accumulation of cardiolipin hydrolysis products, culminating in cell death. mtROS production is also observed in p47(phox-/-) macrophages, and p47(phox-/-) mice exhibit increased inflammation and fibrosis in the lung following silica exposure. Silica induces interaction between TNFR1 and Phox in RAW 264.7 macrophages. Moreover, TNFR1 expression in mitochondria decreased mtROS production and increased RAW 264.7 macrophage survival to silica. These results identify TNFR1/Phox interaction as a key event in the pathogenesis of silicosis that prevents mtROS formation and reduces macrophage apoptosis.
Related JoVE Video
Long-chain acyl-CoA dehydrogenase deficiency as a cause of pulmonary surfactant dysfunction.
J. Biol. Chem.
PUBLISHED: 03-03-2014
Show Abstract
Hide Abstract
Long-chain acyl-CoA dehydrogenase (LCAD) is a mitochondrial fatty acid oxidation enzyme whose expression in humans is low or absent in organs known to utilize fatty acids for energy such as heart, muscle, and liver. This study demonstrates localization of LCAD to human alveolar type II pneumocytes, which synthesize and secrete pulmonary surfactant. The physiological role of LCAD and the fatty acid oxidation pathway in lung was subsequently studied using LCAD knock-out mice. Lung fatty acid oxidation was reduced in LCAD(-/-) mice. LCAD(-/-) mice demonstrated reduced pulmonary compliance, but histological examination of lung tissue revealed no obvious signs of inflammation or pathology. The changes in lung mechanics were found to be due to pulmonary surfactant dysfunction. Large aggregate surfactant isolated from LCAD(-/-) mouse lavage fluid had significantly reduced phospholipid content as well as alterations in the acyl chain composition of phosphatidylcholine and phosphatidylglycerol. LCAD(-/-) surfactant demonstrated functional abnormalities when subjected to dynamic compression-expansion cycling on a constrained drop surfactometer. Serum albumin, which has been shown to degrade and inactivate pulmonary surfactant, was significantly increased in LCAD(-/-) lavage fluid, suggesting increased epithelial permeability. Finally, we identified two cases of sudden unexplained infant death where no lung LCAD antigen was detectable. Both infants were homozygous for an amino acid changing polymorphism (K333Q). These findings for the first time identify the fatty acid oxidation pathway and LCAD in particular as factors contributing to the pathophysiology of pulmonary disease.
Related JoVE Video
E3 ligase subunit Fbxo15 and PINK1 kinase regulate cardiolipin synthase 1 stability and mitochondrial function in pneumonia.
Cell Rep
PUBLISHED: 02-28-2014
Show Abstract
Hide Abstract
Acute lung injury (ALI) is linked to mitochondrial injury, resulting in impaired cellular oxygen utilization; however, it is unknown how these events are linked on the molecular level. Cardiolipin, a mitochondrial-specific lipid, is generated by cardiolipin synthase (CLS1). Here, we show that S. aureus activates a ubiquitin E3 ligase component, Fbxo15, that is sufficient to mediate proteasomal degradation of CLS1 in epithelia, resulting in decreased cardiolipin availability and disrupted mitochondrial function. CLS1 is destabilized by the phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1), which binds CLS1 to phosphorylate and regulates CLS1 disposal. Like Fbxo15, PINK1 interacts with and regulates levels of CLS1 through a mechanism dependent upon Thr219. S. aureus infection upregulates this Fbxo15-PINK1 pathway to impair mitochondrial integrity, and Pink1 knockout mice are less prone to S. aureus-induced ALI. Thus, ALI-associated disruption of cellular bioenergetics involves bioeffectors that utilize a phosphodegron to elicit ubiquitin-mediated disposal of a key mitochondrial enzyme.
Related JoVE Video
Designing inhibitors of cytochrome c/cardiolipin peroxidase complexes: mitochondria-targeted imidazole-substituted fatty acids.
Free Radic. Biol. Med.
PUBLISHED: 02-27-2014
Show Abstract
Hide Abstract
Mitochondria have emerged as the major regulatory platform responsible for the coordination of numerous metabolic reactions as well as cell death processes, whereby the execution of intrinsic apoptosis includes the production of reactive oxygen species fueling oxidation of cardiolipin (CL) catalyzed by cytochrome (Cyt) c. As this oxidation occurs within the peroxidase complex of Cyt c with CL, the latter represents a promising target for the discovery and design of drugs with antiapoptotic mechanisms of action. In this work, we designed and synthesized a new group of mitochondria-targeted imidazole-substituted analogs of stearic acid TPP-n-ISAs with various positions of the attached imidazole group on the fatty acid (n = 6, 8, 10, 13, and 14). By using a combination of absorption spectroscopy and EPR protocols (continuous wave electron paramagnetic resonance and electron spin echo envelope modulation) we demonstrated that TPP-n-ISAs indeed were able to potently suppress CL-induced structural rearrangements in Cyt c, paving the way to its peroxidase competence. TPP-n-ISA analogs preserved the low-spin hexa-coordinated heme-iron state in Cyt c/CL complexes whereby TPP-6-ISA displayed a significantly more effective preservation pattern than TPP-14-ISA. Elucidation of these intermolecular stabilization mechanisms of Cyt c identified TPP-6-ISA as an effective inhibitor of the peroxidase function of Cyt c/CL complexes with a significant antiapoptotic potential realized in mouse embryonic cells exposed to ionizing irradiation. These experimental findings were detailed and supported by all-atom molecular dynamics simulations. Based on the experimental data and computation predictions, we identified TPP-6-ISA as a candidate drug with optimized antiapoptotic potency.
Related JoVE Video
Oxidized lipids block antigen cross-presentation by dendritic cells in cancer.
J. Immunol.
PUBLISHED: 02-19-2014
Show Abstract
Hide Abstract
Cross-presentation is one of the main features of dendritic cells (DCs), which is critically important for the development of spontaneous and therapy-inducible antitumor immune responses. Patients, at early stages of cancer, have normal presence of DCs. However, the difficulties in the development of antitumor responses in patients with low tumor burden raised the question of the mechanisms of DC dysfunction. In this study, we found that, in differentiated DCs, tumor-derived factors blocked the cross-presentation of exogenous Ags without inhibiting the Ag presentation of endogenous protein or peptides. This effect was caused by intracellular accumulation of different types of oxidized neutral lipids: triglycerides, cholesterol esters, and fatty acids. In contrast, the accumulation of nonoxidized lipids did not affect cross-presentation. Oxidized lipids blocked cross-presentation by reducing the expression of peptide-MHC class I complexes on the cell surface. Thus, this study suggests the novel role of oxidized lipids in the regulation of cross-presentation.
Related JoVE Video
LC3 binds externalized cardiolipin on injured mitochondria to signal mitophagy in neurons: Implications for Parkinson disease.
Autophagy
PUBLISHED: 11-26-2013
Show Abstract
Hide Abstract
Mitophagy, or the selective clearance of mitochondria by autophagy, plays a key role in mitochondrial quality control. Due to their postmitotic nature and metabolic dependence on mitochondria, either insufficient or unchecked mitophagy is detrimental to neurons. To better understand signals that regulate this process, we treated primary rat cortical neurons with the electron transport chain complex I inhibitor rotenone to elicit mitophagy. The lipidomic profiles of mitochondria from control or injured neurons were analyzed by mass spectrometry, revealing a significant redistribution of cardiolipin (CL) from the inner mitochondrial membrane to the outer mitochondrial surface. Direct liposome-binding studies, computational modeling, and site-directed mutagenesis indicate that microtubule-associated protein 1 light chain 3 (MAP1LC3/LC3), a defining protein of autophagic membranes, binds to CL. Preventing this interaction inhibits rotenone-induced mitochondrial delivery to autophagosomes and lysosomes and attenuates mitochondrial loss as assessed by western blot. The CL-LC3 interaction is also important for mitophagy induced by other stimuli including 6-hydroxydopamine, another chemical model of Parkinson disease. Given that a conserved LC3 phosphorylation site is adjacent to key residues involved in CL binding, signaling pathways could potentially modulate this interaction to fine-tune the mitochondrial recycling response.
Related JoVE Video
Long-Term Effects of Carbon Containing Engineered Nanomaterials and Asbestos in the Lung: One Year Post Exposure Comparisons.
Am. J. Physiol. Lung Cell Mol. Physiol.
PUBLISHED: 11-08-2013
Show Abstract
Hide Abstract
The hallmark geometric feature of single-walled carbon nanotubes (SWCNT) and carbon nanofibers (CNF) - high length to width ratio - makes them similar to a hazardous agent - asbestos. Very limited data are available concerning long-term effects of pulmonary exposure to SWCNT or CNF. Here we compared inflammatory, fibrogenic and genotoxic effects of CNF, SWCNT or asbestos in mice one year after pharyngeal aspiration. In addition, we compared pulmonary responses to SWCNT by bolus dosing through pharyngeal aspiration and inhalation 5h/day for 4 days, to evaluate the effect of dose rate. The aspiration studies showed that, these particles can be visualized in the lung at one year post-exposure, while some translocate to lymphatics. All these particles induced chronic bronchopneumonia and lymphadenitis, accompanied by pulmonary fibrosis. CNF and asbestos were found to promote the greatest degree of inflammation, followed by SWCNT, while SWCNT were the most fibrogenic of these three particles. Further, SWCNT induced cytogenetic alterations seen as micronuclei formation and nuclear protrusions in vivo. Importantly, inhalation exposure to SWCNT showed significantly greater inflammatory, fibrotic and genotoxic effects than bolus pharyngeal aspiration. Finally, SWCNT and CNF, but not asbestos exposures, increased the incidence of K-ras oncogene mutations in the lung. No increased lung tumor incidence occurred after 1 year post exposure to SWCNT, CNF and asbestos. Overall, our data suggest that long-term pulmonary toxicity of SWCNT, CNF and asbestos - is defined not only by their chemical composition but also by the specific surface area and type of exposure.
Related JoVE Video
The hydrogen-peroxide-induced radical behaviour in human cytochrome c-phospholipid complexes: implications for the enhanced pro-apoptotic activity of the G41S mutant.
Biochem. J.
PUBLISHED: 10-09-2013
Show Abstract
Hide Abstract
We have investigated whether the pro-apoptotic properties of the G41S mutant of human cytochrome c can be explained by a higher than wild-type peroxidase activity triggered by phospholipid binding. A key complex in mitochondrial apoptosis involves cytochrome c and the phospholipid cardiolipin. In this complex cytochrome c has its native axial Met80 ligand dissociated from the haem-iron, considerably augmenting the peroxidase capability of the haem group upon H2O2 binding. By EPR spectroscopy we reveal that the magnitude of changes in the paramagnetic haem states, as well as the yield of protein-bound free radical, is dependent on the phospholipid used and is considerably greater in the G41S mutant. A high-resolution X-ray crystal structure of human cytochrome c was determined and, in combination with the radical EPR signal analysis, two tyrosine residues, Tyr46 and Tyr48, have been rationalized to be putative radical sites. Subsequent single and double tyrosine-to-phenylalanine mutations revealed that the EPR signal of the radical, found to be similar in all variants, including G41S and wild-type, originates not from a single tyrosine residue, but is instead a superimposition of multiple EPR signals from different radical sites. We propose a mechanism of multiple radical formations in the cytochrome c-phospholipid complexes under H2O2 treatment, consistent with the stabilization of the radical in the G41S mutant, which elicits a greater peroxidase activity from cytochrome c and thus has implications in mitochondrial apoptosis.
Related JoVE Video
Characterization of cardiolipins and their oxidation products by LC-MS analysis.
Chem. Phys. Lipids
PUBLISHED: 09-19-2013
Show Abstract
Hide Abstract
Cardiolipins, a class of mitochondria-specific lipid molecules, is one of the most unusual and ancient phospholipids found in essentially all living species. Typical of mammalian cells is the presence of vulnerable to oxidation polyunsaturated fatty acid resides in CL molecules. The overall role and involvement of cardiolipin oxidation (CLox) products in major intracellular signaling as well as extracellular inflammatory and immune responses have been established. However, identification of individual peroxidized molecular species in the context of their ability to induce specific biological responses has not been yet achieved. This is due, at least in part, to technological difficulties in detection, identification, structural characterization and quantitation of CLox associated with their very low abundance and exquisite diversification. This dictates the need for the development of new methodologies for reliable, sensitive and selective analysis of both CLox. LC-MS-based oxidative lipidomics with high mass accuracy instrumentation as well as new software packages are promising in achieving the goals of expedited and reliable analysis of cardiolipin oxygenated species in biosamples.
Related JoVE Video
Cardiolipin asymmetry, oxidation and signaling.
Chem. Phys. Lipids
PUBLISHED: 09-19-2013
Show Abstract
Hide Abstract
Cardiolipins (CLs) are ancient and unusual dimeric phospholipids localized in the plasma membrane of bacteria and in the inner mitochondrial membrane of eukaryotes. In mitochondria, two types of asymmetries - trans-membrane and molecular - are essential determinants of CL functions. In this review, we describe CL-based signaling mitochondrial pathways realized via modulation of trans-membrane asymmetry and leading to externalization and peroxidation of CLs in mitophagy and apoptosis, respectively. We discuss possible mechanisms of CL translocations from the inner leaflet of the inner to the outer leaflet of the outer mitochondrial membranes. We present redox reaction mechanisms of cytochrome c-catalyzed CL peroxidation as a required stage in the execution of apoptosis. We also emphasize the significance of CL-related metabolic pathways as new targets for drug discovery. Finally, a remarkable diversity of polyunsaturated CL species and their oxidation products have evolved in eukaryotes vs. prokaryotes. This diversity - associated with CL molecular asymmetry - is presented as the basis for mitochondrial communications language.
Related JoVE Video
Cardiolipin externalization to the outer mitochondrial membrane acts as an elimination signal for mitophagy in neuronal cells.
Nat. Cell Biol.
PUBLISHED: 08-07-2013
Show Abstract
Hide Abstract
Recognition of injured mitochondria for degradation by macroautophagy is essential for cellular health, but the mechanisms remain poorly understood. Cardiolipin is an inner mitochondrial membrane phospholipid. We found that rotenone, staurosporine, 6-hydroxydopamine and other pro-mitophagy stimuli caused externalization of cardiolipin to the mitochondrial surface in primary cortical neurons and SH-SY5Y cells. RNAi knockdown of cardiolipin synthase or of phospholipid scramblase-3, which transports cardiolipin to the outer mitochondrial membrane, decreased the delivery of mitochondria to autophagosomes. Furthermore, we found that the autophagy protein microtubule-associated-protein-1 light chain 3 (LC3), which mediates both autophagosome formation and cargo recognition, contains cardiolipin-binding sites important for the engulfment of mitochondria by the autophagic system. Mutation of LC3 residues predicted as cardiolipin-interaction sites by computational modelling inhibited its participation in mitophagy. These data indicate that redistribution of cardiolipin serves as an eat-me signal for the elimination of damaged mitochondria from neuronal cells.
Related JoVE Video
Evaluation of potential ionizing irradiation protectors and mitigators using clonogenic survival of human umbilical cord blood hematopoietic progenitor cells.
Exp. Hematol.
PUBLISHED: 08-01-2013
Show Abstract
Hide Abstract
We evaluated the use of colony formation (colony-forming unit-granulocyte macrophage [CFU-GM], burst-forming unit erythroid [BFU-E], and colony-forming unit-granulocyte-erythroid-megakaryocyte-monocytes [CFU-GEMM]) by human umbilical cord blood (CB) hematopoietic progenitor cells for testing novel small molecule ionizing irradiation protectors and mitigators. The following compounds were added before (protection) or after (mitigation) ionizing irradiation: GS-nitroxides (JP4-039 and XJB-5-131), the bifunctional sulfoxide MMS-350, the phosphoinositol-3-kinase inhibitor LY29400, triphenylphosphonium-imidazole fatty acid, the nitric oxide synthase inhibitor (MCF-201-89), the p53/mdm2/mdm4 inhibitor (BEB55), methoxamine, isoproterenol, propranolol, and the adenosine triphosphate-sensitive potassium channel blocker (glyburide). The drugs XJB-5-131, JP4-039, and MMS-350 were radiation protectors for CFU-GM. JP4-039 was also a radiation protector for CFU-GEMM. The drugs XJB-5-131, JP4-039, and MMS-350 were radiation mitigators for BFU-E, MMS-350 and JP4-039 were mitigators for CFU-GM, and MMS350 was a mitigator for CFU-GEMM. In contrast, other drugs were effective in murine assays; TTP-IOA, LY294002, MCF201-89, BEB55, propranolol, isoproterenol, methoxamine, and glyburide but showed no significant protection or mitigation in human CB assays. These data support the testing of new candidate clinical radiation protectors and mitigators using human CB clonogenic assays early in the drug discovery process, thus reducing the need for animal experiments.
Related JoVE Video
Oxidative stress and dermal toxicity of iron oxide nanoparticles in vitro.
Cell Biochem. Biophys.
PUBLISHED: 06-15-2013
Show Abstract
Hide Abstract
A number of commercially available metal/metal oxide nanoparticles (NPs) such as superparamagnetic iron oxide (SPION) are utilized by the medical field for a wide variety of applications. These NPs may able to induce dermal toxicity via their physical nature and reactive surface properties. We hypothesize that SPION may be toxic to skin via the ability of particles to be internalized and thereby initiate oxidative stress, inducing redox-sensitive transcription factors affecting/leading to inflammation. Due to the skins susceptibility to UV radiation, it is also of importance to address the combined effect of UVB and NPs co-exposure. To test this hypothesis, the effects of dextran-coated SPION of different sizes (15-50 nm) and manufacturers (MicroMod, Rostock-Warnemunde, Germany and KTH-Royal Institute of Technology, Stockholm, Sweden) were evaluated in two cell lines: normal human epidermal keratinocytes (HEK) and murine epidermal cells (JB6 P(+)). HEK cells exposed to 20 nm (KTH and MicroMod) had a decrease in viability, while the 15 and 50 nm particles were not cytotoxic. HEK cells were also capable of internalizing the KTH particles (15 and 20 nm) but not the MicroMod SPION (20 and 50 nm). IL-8 and IL-6 were also elevated in HEK cells following exposure to SPION. Exposure of JB6 P(+) cells to all SPIONs evaluated resulted in activation of AP-1. Exposure to SPION alone was not sufficient to induce NF-?B activation; however, co-exposure with UVB resulted in significant NF-?B induction in cells exposed to 15 and 20 nm KTH SPION and 50 nm MicroMod particles. Pre-exposure of JB6 P(+) cells to UVB followed by NPs induced a significant depletion of glutathione, release of cytokines, and cell damage as assessed by release of lactate dehydrogenase. Altogether, these data indicate that co-exposure to UVB and SPIONs was associated with induction of oxidative stress and release of inflammatory mediators. These results verify the need to thoroughly evaluate the adverse effects of UVB when evaluating dermal toxicity of engineered NPs on skin.
Related JoVE Video
Screening of biochemical and molecular mechanisms of secondary injury and repair in the brain after experimental blast-induced traumatic brain injury in rats.
J. Neurotrauma
PUBLISHED: 06-05-2013
Show Abstract
Hide Abstract
Abstract Explosive blast-induced traumatic brain injury (TBI) is the signature insult in modern combat casualty care and has been linked to post-traumatic stress disorder, memory loss, and chronic traumatic encephalopathy. In this article we report on blast-induced mild TBI (mTBI) characterized by fiber-tract degeneration and axonal injury revealed by cupric silver staining in adult male rats after head-only exposure to 35 psi in a helium-driven shock tube with head restraint. We now explore pathways of secondary injury and repair using biochemical/molecular strategies. Injury produced ?25% mortality from apnea. Shams received identical anesthesia exposure. Rats were sacrificed at 2 or 24 h, and brain was sampled in the hippocampus and prefrontal cortex. Hippocampal samples were used to assess gene array (RatRef-12 Expression BeadChip; Illumina, Inc., San Diego, CA) and oxidative stress (OS; ascorbate, glutathione, low-molecular-weight thiols [LMWT], protein thiols, and 4-hydroxynonenal [HNE]). Cortical samples were used to assess neuroinflammation (cytokines, chemokines, and growth factors; Luminex Corporation, Austin, TX) and purines (adenosine triphosphate [ATP], adenosine diphosphate, adenosine, inosine, 2-AMP [adenosine monophosphate], and 5-AMP). Gene array revealed marked increases in astrocyte and neuroinflammatory markers at 24 h (glial fibrillary acidic protein, vimentin, and complement component 1) with expression patterns bioinformatically consistent with those noted in Alzheimers disease and long-term potentiation. Ascorbate, LMWT, and protein thiols were reduced at 2 and 24 h; by 24 h, HNE was increased. At 2 h, multiple cytokines and chemokines (interleukin [IL]-1?, IL-6, IL-10, and macrophage inflammatory protein 1 alpha [MIP-1?]) were increased; by 24 h, only MIP-1? remained elevated. ATP was not depleted, and adenosine correlated with 2-cyclic AMP (cAMP), and not 5-cAMP. Our data reveal (1) gene-array alterations similar to disorders of memory processing and a marked astrocyte response, (2) OS, (3) neuroinflammation with a sustained chemokine response, and (4) adenosine production despite lack of energy failure-possibly resulting from metabolism of 2-3-cAMP. A robust biochemical/molecular response occurs after blast-induced mTBI, with the body protected from blast and the head constrained to limit motion.
Related JoVE Video
Dual acute proinflammatory and antifibrotic pulmonary effects of short palate, lung, and nasal epithelium clone-1 after exposure to carbon nanotubes.
Am. J. Respir. Cell Mol. Biol.
PUBLISHED: 06-01-2013
Show Abstract
Hide Abstract
Carbon nanotubes (CNTs; allotropes of carbon with a cylindrical nanostructure) have emerged as one of the most commonly used types of nanomaterials, with numerous applications in industry and biomedicine. However, the inhalation of CNTs has been shown to elicit pulmonary toxicity, accompanied by a robust inflammatory response with an early-onset fibrotic phase. Epithelial host-defense proteins represent an important component of the pulmonary innate immune response to foreign inhalants such as particles and bacteria. The short palate, lung, and nasal epithelium clone-1 (SPLUNC1) protein, a member of the bactericidal/permeability-increasing-fold (BPIF)-containing protein family, is a 25-kD secretory protein that is expressed in nasal, oropharyngeal, and lung epithelia, and has been shown to have multiple functions, including antimicrobial and chemotactic activities, as well as surfactant properties. This study sought to assess the importance of SPLUNC1-mediated pulmonary responses in airway epithelial secretions, and to explore the biological relevance of SPLUNC1 to inhaled particles in a single-walled carbon nanotube (SWCNT) model. Using Scgb1a1-hSPLUNC1 transgenic mice, we observed that SPLUNC1 significantly modified host inflammatory responses by increasing leukocyte recruitment and enhancing phagocytic activity. Furthermore, we found that transgenic mice were more susceptible to SWCNT exposure at the acute phase, but showed resistance against lung fibrogenesis through pathological changes in the long term. The binding of SPLUNC1 also attenuated SWCNT-induced TNF-? secretion by RAW 264.7 macrophages. Taken together, our data indicate that SPLUNC1 is an important component of mucosal innate immune defense against pulmonary inhaled particles.
Related JoVE Video
Effect of antioxidants on enzyme-catalysed biodegradation of carbon nanotubes.
J Mater Chem B Mater Biol Med
PUBLISHED: 04-30-2013
Show Abstract
Hide Abstract
The growing applications of carbon nanotubes (CNTs) inevitably increase the risk of exposure to this potentially toxic nanomaterial. In an attempt to address this issue, research has been implemented to study the biodegradation of CNTs. In particular, myeloperoxidase (MPO), an enzyme expressed by inflammatory cells of animals including humans, catalyse the degradation of oxidized carbon nanomaterials. While reactive intermediates generated by MPO efficiently degrade oxidized single-walled carbon nanotubes (o-SWCNTs); the exact mechanism of enzyme-catalysed biodegradation remains ambiguous. In this work, we tried to explain enzymatic oxidation in terms of redox potentials by employing competitive substrates for MPO such as chloride, which is oxidized by MPO to form a strong oxidant (hypochlorite), and antioxidants that have lower redox potentials than CNTs. Employing transmission electron microscopy, Raman spectroscopy, and vis-NIR absorption spectroscopy, we demonstrate that the addition of antioxidants, L-ascorbic acid and L-glutathione, with or without chloride significantly mitigates MPO-catalysed biodegradation of o-SWCNTs. This study focuses on a fundamental understanding of the mechanisms of enzymatic biodegradation of CNTs and the impact of antioxidants on these pathways.
Related JoVE Video
Biodiesel versus diesel exposure: enhanced pulmonary inflammation, oxidative stress, and differential morphological changes in the mouse lung.
Toxicol. Appl. Pharmacol.
PUBLISHED: 04-22-2013
Show Abstract
Hide Abstract
The use of biodiesel (BD) or its blends with petroleum diesel (D) is considered to be a viable approach to reduce occupational and environmental exposures to particulate matter (PM). Due to its lower particulate mass emissions compared to D, use of BD is thought to alleviate adverse health effects. Considering BD fuel is mainly composed of unsaturated fatty acids, we hypothesize that BD exhaust particles could induce pronounced adverse outcomes, due to their ability to readily oxidize. The main objective of this study was to compare the effects of particles generated by engine fueled with neat BD and neat petroleum-based D. Biomarkers of tissue damage and inflammation were significantly elevated in lungs of mice exposed to BD particulates. Additionally, BD particulates caused a significant accumulation of oxidatively modified proteins and an increase in 4-hydroxynonenal. The up-regulation of inflammatory cytokines/chemokines/growth factors was higher in lungs upon BD particulate exposure. Histological evaluation of lung sections indicated presence of lymphocytic infiltrate and impaired clearance with prolonged retention of BD particulate in pigment laden macrophages. Taken together, these results clearly indicate that BD exhaust particles could exert more toxic effects compared to D.
Related JoVE Video
Biodegradation of single-walled carbon nanotubes by eosinophil peroxidase.
Small
PUBLISHED: 02-27-2013
Show Abstract
Hide Abstract
Eosinophil peroxidase (EPO) is one of the major oxidant-producing enzymes during inflammatory states in the human lung. The degradation of single-walled carbon nanotubes (SWCNTs) upon incubation with human EPO and H?O? is reported. Biodegradation of SWCNTs is higher in the presence of NaBr, but neither EPO alone nor H?O? alone caused the degradation of nanotubes. Molecular modeling reveals two binding sites for SWCNTs on EPO, one located at the proximal side (same side as the catalytic site) and the other on the distal side of EPO. The oxidized groups on SWCNTs in both cases are stabilized by electrostatic interactions with positively charged residues. Biodegradation of SWCNTs can also be executed in an ex vivo culture system using primary murine eosinophils stimulated to undergo degranulation. Biodegradation is proven by a range of methods including transmission electron microscopy, UV-visible-NIR spectroscopy, Raman spectroscopy, and confocal Raman imaging. Thus, human EPO (in vitro) and ex vivo activated eosinophils mediate biodegradation of SWCNTs: an observation that is relevant to pulmonary responses to these materials.
Related JoVE Video
Peroxidase-mediated biodegradation of carbon nanotubes in vitro and in vivo.
Adv. Drug Deliv. Rev.
PUBLISHED: 01-29-2013
Show Abstract
Hide Abstract
As a result of their unique electronic, optical, and mechanical properties, carbon nanotubes (CNTs) have been implemented in therapeutic and imaging applications. In an idealized situation, CNTs would be disposed of after they transport their theranostic payloads. Biodegradation represents an attractive pathway for the elimination of CNT carriers post-delivery and may be integral in catalyzing the release of the cargo from the delivery vehicle. Accordingly, recent research efforts have focused on peroxidase-driven biodegradation of CNTs. In this review, we not only summarize recent efforts to biodegrade CNTs in the test tube, in vitro, and in vivo, but also attempt to explore the fundamental parameters underlying degradation. Encouraged by the in vivo results obtained to date, we envision a future, where carbon-based nano-containers, which are specifically designed to target organs/cells, deliver their cargo, and biodegrade via peroxidase-driven mechanism, will represent an attractive therapeutic delivery option in nanomedicine.
Related JoVE Video
Molecular modeling in structural nano-toxicology: Interactions of nano-particles with nano-machinery of cells.
Adv. Drug Deliv. Rev.
PUBLISHED: 01-29-2013
Show Abstract
Hide Abstract
Over the past two decades, nanotechnology has emerged as a key player in various disciplines of science and technology. Some of the most exciting applications are in the field of biomedicine - for theranostics (for combined diagnostic and therapeutic purposes) as well as for exploration of biological systems. A detailed understanding of the molecular interactions between nanoparticles and biological nano-machinery - macromolecules, membranes, and intracellular organelles - is crucial for obtaining adequate information on mechanisms of action of nanomaterials as well as a perspective on the long term effects of these materials and their possible toxicological outcomes. This review focuses on the use of structure-based computational molecular modeling as a tool to understand and to predict the interactions between nanomaterials and nano-biosystems. We review major approaches and provide examples of computational analysis of the structural principles behind such interactions. A rationale on how nanoparticles of different sizes, shape, structure and chemical properties can affect the organization and functions of nano-machinery of cells is also presented.
Related JoVE Video
Effect of hyperoxia on resuscitation of experimental combined traumatic brain injury and hemorrhagic shock in mice.
Anesthesiology
PUBLISHED: 01-10-2013
Show Abstract
Hide Abstract
Hypotension and hypoxemia worsen traumatic brain injury outcomes. Hyperoxic resuscitation is controversial. The authors proposed that hyperoxia would improve hemodynamics and neuronal survival by augmenting oxygen delivery despite increased oxidative stress and neuroinflammation in experimental combined controlled cortical impact plus hemorrhagic shock in mice.
Related JoVE Video
Strategies for discovery of small molecule radiation protectors and radiation mitigators.
Front Oncol
PUBLISHED: 12-01-2011
Show Abstract
Hide Abstract
Mitochondrial targeted radiation damage protectors (delivered prior to irradiation) and mitigators (delivered after irradiation, but before the appearance of symptoms associated with radiation syndrome) have been a recent focus in drug discovery for (1) normal tissue radiation protection during fractionated radiotherapy, and (2) radiation terrorism counter measures. Several categories of such molecules have been discovered: nitroxide-linked hybrid molecules, including GS-nitroxide, GS-nitric oxide synthase inhibitors, p53/mdm2/mdm4 inhibitors, and pharmaceutical agents including inhibitors of the phosphoinositide-3-kinase pathway and the anti-seizure medicine, carbamazepine. Evaluation of potential new radiation dose modifying molecules to protect normal tissue includes: clonogenic radiation survival curves, assays for apoptosis and DNA repair, and irradiation-induced depletion of antioxidant stores. Studies of organ specific radioprotection and in total body irradiation-induced hematopoietic syndrome in the mouse model for protection/mitigation facilitate rational means by which to move candidate small molecule drugs along the drug discovery pipeline into clinical development.
Related JoVE Video
Mitochondrial injury after mechanical stretch of cortical neurons in vitro: biomarkers of apoptosis and selective peroxidation of anionic phospholipids.
J. Neurotrauma
PUBLISHED: 11-04-2011
Show Abstract
Hide Abstract
Mechanical injury of neurites accompanied by rupture of mitochondrial membranes may lead to immediate nonspecific release and spreading of pro-apoptotic factors and activation of proteases, that is, execution of apoptotic program. In the current work, we studied the time course of the major biomarkers of apoptosis as they are induced by exposure of rat cortical neurons to mechanical stretch. By using transmission electron microscopy, we found that mitochondria in the neurites were damaged early (1?h) after mechanical stretch injury whereas somal mitochondria were significantly more resistant and demonstrated structural damage and degenerative mitochondrial changes at a later time point after stretch (12?h). We also report that the stretch injury caused immediate activation of reactive oxygen species production followed by selective oxidation of a mitochondria-specific phospholipid, cardiolipin, whose individual peroxidized molecular species have been identified and quantified by electrospray ionization mass spectrometry analysis. Most abundant neuronal phospholipids - phosphatidylcholine, phophatidylethanolamine - did not undergo oxidative modification. Simultaneously, a small-scale release of cytochrome c was observed. Notably, caspase activation and phosphatidylserine externalization - two irreversible apoptotic events designating a point of no return - are substantially delayed and do not occur until 6-12?h after the initial impact. The early onset of reactive oxygen species production and cytochrome c release may be relevant to direct stretch-induced damage to mitochondria. The delayed emergence of apoptotic neuronal death after the immediate mechanical damage to mitochondria suggests a possible window of opportunity for targeted therapies.
Related JoVE Video
Mitochondria targeting of non-peroxidizable triphenylphosphonium conjugated oleic acid protects mouse embryonic cells against apoptosis: role of cardiolipin remodeling.
FEBS Lett.
PUBLISHED: 11-01-2011
Show Abstract
Hide Abstract
Peroxidation of cardiolipin in mitochondria is essential for the execution of apoptosis. We suggested that integration of oleic acid into cardiolipin generates non-oxidizable cardiolipin species hence protects cells against apoptosis. We synthesized mitochondria-targeted triphenylphosphonium oleic acid ester. Using lipidomics analysis we found that pretreatment of mouse embryonic cells with triphenylphosphonium oleic acid ester resulted in decreased contents of polyunsaturated cardiolipins and elevation of its species containing oleic acid residues. This caused suppression of apoptosis induced by actinomycin D. Triacsin C, an inhibitor of acyl-CoA synthase, blocked integration of oleic acid into cardiolipin and restored cell sensitivity to apoptosis.
Related JoVE Video
Amelioration of radiation esophagitis by orally administered p53/Mdm2/Mdm4 inhibitor (BEB55) or GS-nitroxide.
In Vivo
PUBLISHED: 10-25-2011
Show Abstract
Hide Abstract
Esophagitis is a significant toxicity of radiation therapy for lung cancer. In this study, reduction of irradiation esophagitis in mice, by orally administered p53/Mdm2/Mdm4 inhibitor, BEB55, or the GS-nitroxide, JP4-039, was evaluated.
Related JoVE Video
Mass-spectrometric characterization of peroxidized and hydrolyzed lipids in plasma and dendritic cells of tumor-bearing animals.
Biochem. Biophys. Res. Commun.
PUBLISHED: 08-12-2011
Show Abstract
Hide Abstract
Dendritic cells are the most potent antigen presenting cells responsible for the development of immune responses in cancer. However, it is known that the function of dendritic cells in tumor-bearing hosts is severely compromised. Our previous studies demonstrated that the defects in dendritic cell function are due, to a large extent, to the accumulation of high amounts of lipids--predominantly triglycerides--in a substantial proportion of dendritic cells in tumor-bearing mice and patients with cancer. The dendritic cells accumulation of lipids is likely associated with their up-regulation of a scavenger receptor A. This receptor is primarily responsible for uptake of modified lipids. Here, by using different versions of liquid chromatography-mass spectrometry, we identified several molecular species of oxygenated lipids in plasma of tumor-bearing animals that may be responsible for their uptake and accumulation by dendritic cells via scavenger receptor A-dependent pathway--the effect that may be associated with the loss of dendritic cells immune surveillance function in cancer.
Related JoVE Video
Global phospholipidomics analysis reveals selective pulmonary peroxidation profiles upon inhalation of single-walled carbon nanotubes.
ACS Nano
PUBLISHED: 08-04-2011
Show Abstract
Hide Abstract
It is commonly believed that nanomaterials cause nonspecific oxidative damage. Our mass spectrometry-based oxidative lipidomics analysis of all major phospholipid classes revealed highly selective patterns of pulmonary peroxidation after inhalation exposure of mice to single-walled carbon nanotubes. No oxidized molecular species were found in the two most abundant phospholipid classes: phosphatidylcholine and phosphatidylethanolamine. Peroxidation products were identified in three relatively minor classes of anionic phospholipids, cardiolipin, phosphatidylserine, and phosphatidylinositol, whereby oxygenation of polyunsaturated fatty acid residues also showed unusual substrate specificity. This nonrandom peroxidation coincided with the accumulation of apoptotic cells in the lung. A similar selective phospholipid peroxidation profile was detected upon incubation of a mixture of total lung lipids with H(2)O(2)/cytochrome c known to catalyze cardiolipin and phosphatidylserine peroxidation in apoptotic cells. The characterized specific phospholipid peroxidation signaling pathways indicate new approaches to the development of mitochondria-targeted regulators of cardiolipin peroxidation to protect against deleterious effects of pro-apoptotic effects of single-walled carbon nanotubes in the lung.
Related JoVE Video
A mitochondria-targeted inhibitor of cytochrome c peroxidase mitigates radiation-induced death.
Nat Commun
PUBLISHED: 08-01-2011
Show Abstract
Hide Abstract
The risk of radionuclide release in terrorist acts or exposure of healthy tissue during radiotherapy demand potent radioprotectants/radiomitigators. Ionizing radiation induces cell death by initiating the selective peroxidation of cardiolipin in mitochondria by the peroxidase activity of its complex with cytochrome c leading to release of haemoprotein into the cytosol and commitment to the apoptotic program. Here we design and synthesize mitochondria-targeted triphenylphosphonium-conjugated imidazole-substituted oleic and stearic acids that blocked peroxidase activity of cytochrome c/cardiolipin complex by specifically binding to its haem-iron. We show that both compounds inhibit pro-apoptotic oxidative events, suppress cyt c release, prevent cell death, and protect mice against lethal doses of irradiation. Significant radioprotective/radiomitigative effects of imidazole-substituted oleic acid are observed after pretreatment of mice from 1?h before through 24?h after the irradiation.
Related JoVE Video
A high-throughput screening assay of ascorbate in brain samples.
J. Neurosci. Methods
PUBLISHED: 07-31-2011
Show Abstract
Hide Abstract
Ascorbate is a vital reductant/free radical scavenger in the CNS, whose content defines - to a large extent - the redox status and the antioxidant reserves. Quick, reliable and specific methods for its measurement in brain samples are highly desirable. We have developed a new high-throughput screening assay for measurements of ascorbate using a fluorescence plate-reader. This assay is based on a direct reaction of ascorbate with a nitroxide radical conjugated with a fluorogenic acridine moiety, 4-((9-acridinecarbonyl)-amino)-2,2,6,6-tetramethylpiperidine-1-oxyl radical (AC-TEMPO), yielding fluorescent hydroxylamine product (AC-TEMPO-H). The reaction was monitored over time using fluorescence and electron spin resonance techniques. The appearance of fluorescent AC-TEMPO-H was linear within the range of 3.75-75?M AscH(-) in the sample (0.5-10?M AscH(-) in the well). Assay was validated with high performance liquid chromatography method. The concentration of ascorbate in murine tissue samples, including brain samples after traumatic brain injury and hemorrhagic shock, was measured.
Related JoVE Video
Are mitochondrial reactive oxygen species required for autophagy?
Biochem. Biophys. Res. Commun.
PUBLISHED: 07-07-2011
Show Abstract
Hide Abstract
Reactive oxygen species (ROS) are said to participate in the autophagy signaling. Supporting evidence is obscured by interference of autophagy and apoptosis, whereby the latter heavily relies on ROS signaling. To dissect autophagy from apoptosis we knocked down expression of cytochrome c, the key component of mitochondria-dependent apoptosis, in HeLa cells using shRNA. In cytochrome c deficient HeLa1.2 cells, electron transport was compromised due to the lack of electron shuttle between mitochondrial respiratory complexes III and IV. A rapid and robust LC3-I/II conversion and mitochondria degradation were observed in HeLa1.2 cells treated with staurosporine (STS). Neither generation of superoxide nor accumulation of H(2)O(2) was detected in STS-treated HeLa1.2 cells. A membrane permeable antioxidant, PEG-SOD, plus catalase exerted no effect on STS-induced LC3-I/II conversion and mitochondria degradation. Further, STS caused autophagy in mitochondria DNA-deficient ?° HeLa1.2 cells in which both electron transport and ROS generation were completely disrupted. Counter to the widespread view, we conclude that mitochondrial ROS are not required for the induction of autophagy.
Related JoVE Video
Direct effects of carbon nanotubes on dendritic cells induce immune suppression upon pulmonary exposure.
ACS Nano
PUBLISHED: 06-15-2011
Show Abstract
Hide Abstract
Pharyngeal aspiration of single-walled carbon nanotubes (SWCNTs) caused inflammation, pulmonary damage, and an altered cytokine network in the lung. Local inflammatory response in vivo was accompanied by modified systemic immunity as documented by decreased proliferation of splenic T cells. Preincubation of nai?ve T cells in vitro with SWCNT-treated dendritic cells reduced proliferation of T cells. Our data suggest that in vivo exposure to SWCNT modifies systemic immunity by modulating dendritic cell function.
Related JoVE Video
Assessments of thiyl radicals in biosystems: difficulties and new applications.
Anal. Chem.
PUBLISHED: 06-01-2011
Show Abstract
Hide Abstract
The high reactivity of thiyl radicals (RS(•)), which results in half-lives on the order of microseconds, hinders their analysis in biological systems. This Feature reviews the contemporary approaches to assessment of RS(•) using EPR spin trapping, mass spectrometric, immunological, and HPLC protocols.
Related JoVE Video
Succinobucol induces apoptosis in vascular smooth muscle cells.
Free Radic. Biol. Med.
PUBLISHED: 05-31-2011
Show Abstract
Hide Abstract
Probucol inhibits the proliferation of vascular smooth muscle cells in vitro and in vivo, and the drug reduces intimal hyperplasia and atherosclerosis in animals via induction of heme oxygenase-1 (HO-1). Because the succinyl ester of probucol, succinobucol, recently failed as an antiatherogenic drug in humans, we investigated its effects on smooth muscle cell proliferation. Succinobucol and probucol induced HO-1 and decreased cell proliferation in rat aortic smooth muscle cells. However, whereas inhibition of HO-1 reversed the antiproliferative effects of probucol, this was not observed with succinobucol. Instead, succinobucol but not probucol induced caspase activity and apoptosis, and it increased mitochondrial oxidation of hydroethidine to ethidium, suggestive of the participation of H(2)O(2) and cytochrome c. Also, succinobucol but not probucol converted cytochrome c into a peroxidase in the presence of H(2)O(2), and succinobucol-induced apoptosis was decreased in cells that lacked cytochrome c or a functional mitochondrial complex II. In addition, succinobucol increased apoptosis of vascular smooth muscle cells in vivo after balloon angioplasty-mediated vascular injury. Our results suggest that succinobucol induces apoptosis via a pathway involving mitochondrial complex II, H(2)O(2), and cytochrome c. These unexpected results are discussed in light of the failure of succinobucol as an antiatherogenic drug in humans.
Related JoVE Video
Topography of tyrosine residues and their involvement in peroxidation of polyunsaturated cardiolipin in cytochrome c/cardiolipin peroxidase complexes.
Biochim. Biophys. Acta
PUBLISHED: 04-13-2011
Show Abstract
Hide Abstract
Formation of cytochrome c (cyt c)/cardiolipin (CL) peroxidase complex selective toward peroxidation of polyunsaturated CLs is a pre-requisite for mitochondrial membrane permeabilization. Tyrosine residues - via the generation of tyrosyl radicals (Tyr) - are likely reactive intermediates of the peroxidase cycle leading to CL peroxidation. We used mutants of horse heart cyt c in which each of the four Tyr residues was substituted for Phe and assessed their contribution to the peroxidase catalysis. Tyr67Phe mutation was associated with a partial loss of the oxygenase function of the cyt c/CL complex and the lowest concentration of H(2)O(2)-induced Tyr radicals in electron paramagnetic resonance (EPR) spectra. Our MS experiments directly demonstrated decreased production of CL-hydroperoxides (CL-OOH) by Tyr67Phe mutant. Similarly, oxidation of a phenolic substrate, Amplex Red, was affected to a greater extent in Tyr67Phe than in three other mutants. Tyr67Phe mutant exerted high resistance to H(2)O(2)-induced oligomerization. Measurements of Tyr fluorescence, hetero-nuclear magnetic resonance (NMR) and computer simulations position Tyr67 in close proximity to the porphyrin ring heme iron and one of the two axial heme-iron ligand residues, Met80. Thus, the highly conserved Tyr67 is a likely electron-donor (radical acceptor) in the oxygenase half-reaction of the cyt c/CL peroxidase complex.
Related JoVE Video
A manganese-porphyrin complex decomposes H(2)O(2), inhibits apoptosis, and acts as a radiation mitigator in vivo.
ACS Med Chem Lett
PUBLISHED: 04-08-2011
Show Abstract
Hide Abstract
Ionizing radiation triggers mitochondrial overproduction of H(2)O(2) with concomitant induction of intrinsic apoptosis, whereby clearance of H(2)O(2) upon overexpression of mitochondrial catalase increases radioresistance in vitro and in vivo. As an alternative to gene therapy, we tested the potential of Mn((III))-porphyrin complexes to clear mitochondrial H(2)O(2). We report that triphenyl-[(2E)-2-[4-[(1Z,4Z,9Z,15Z)-10,15,20-tris(4-aminophenyl)-21,23-dihydroporphyrin-5-yl]phenyl]iminoethyl]phosphonium-Mn((III)) compartmentalizes preferentially into mitochondria of mouse embryonic cells, reacts with H(2)O(2), impedes ?-ray-induced mitochondrial apoptosis, and increases the survival of mice exposed to whole body irradiation with ?-rays.
Related JoVE Video
Blast exposure in rats with body shielding is characterized primarily by diffuse axonal injury.
J. Neurotrauma
PUBLISHED: 04-01-2011
Show Abstract
Hide Abstract
Blast-induced traumatic brain injury (TBI) is the signature insult in combat casualty care. Survival with neurological damage from otherwise lethal blast exposures has become possible with body armor use. We characterized the neuropathologic alterations produced by a single blast exposure in rats using a helium-driven shock tube to generate a nominal exposure of 35 pounds per square inch (PSI) (positive phase duration ? 4 msec). Using an IACUC-approved protocol, isoflurane-anesthetized rats were placed in a steel wedge (to shield the body) 7 feet inside the end of the tube. The left side faced the blast wave (with head-only exposure); the wedge apex focused a Mach stem onto the rats head. The insult produced ? 25% mortality (due to impact apnea). Surviving and sham rats were perfusion-fixed at 24 h, 72 h, or 2 weeks post-blast. Neuropathologic evaluations were performed utilizing hematoxylin and eosin, amino cupric silver, and a variety of immunohistochemical stains for amyloid precursor protein (APP), glial fibrillary acidic protein (GFAP), ionized calcium-binding adapter molecule 1 (Iba1), ED1, and rat IgG. Multifocal axonal degeneration, as evidenced by staining with amino cupric silver, was present in all blast-exposed rats at all time points. Deep cerebellar and brainstem white matter tracts were most heavily stained with amino cupric silver, with the morphologic staining patterns suggesting a process of diffuse axonal injury. Silver-stained sections revealed mild multifocal neuronal death at 24 h and 72 h. GFAP, ED1, and Iba1 staining were not prominently increased, although small numbers of reactive microglia were seen within areas of neuronal death. Increased blood-brain barrier permeability (as measured by IgG staining) was seen at 24 h and primarily affected the contralateral cortex. Axonal injury was the most prominent feature during the initial 2 weeks following blast exposure, although degeneration of other neuronal processes was also present. Strikingly, silver staining revealed otherwise undetected abnormalities, and therefore represents a recommended outcome measure in future studies of blast TBI.
Related JoVE Video
Normoxic versus hyperoxic resuscitation in pediatric asphyxial cardiac arrest: effects on oxidative stress.
Crit. Care Med.
PUBLISHED: 03-05-2011
Show Abstract
Hide Abstract
To determine the effects of normoxic vs. hyperoxic resuscitation on oxidative stress in a model of pediatric asphyxial cardiac arrest.
Related JoVE Video
The enzymatic oxidation of graphene oxide.
ACS Nano
PUBLISHED: 02-23-2011
Show Abstract
Hide Abstract
Two-dimensional graphitic carbon is a new material with many emerging applications, and studying its chemical properties is an important goal. Here, we reported a new phenomenon--the enzymatic oxidation of a single layer of graphitic carbon by horseradish peroxidase (HRP). In the presence of low concentrations of hydrogen peroxide (?40 ?M), HRP catalyzed the oxidation of graphene oxide, which resulted in the formation of holes on its basal plane. During the same period of analysis, HRP failed to oxidize chemically reduced graphene oxide (RGO). The enzymatic oxidation was characterized by Raman, ultraviolet-visible, electron paramagnetic resonance, Fourier transform infrared spectroscopy, transmission electron microscopy, atomic force microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and gas chromatography-mass spectrometry. Computational docking studies indicated that HRP was preferentially bound to the basal plane rather than the edge for both graphene oxide and RGO. Owing to the more dynamic nature of HRP on graphene oxide, the heme active site of HRP was in closer proximity to graphene oxide compared to RGO, thereby facilitating the oxidation of the basal plane of graphene oxide. We also studied the electronic properties of the reduced intermediate product, holey reduced graphene oxide (hRGO), using field-effect transistor (FET) measurements. While RGO exhibited a V-shaped transfer characteristic similar to a single layer of graphene that was attributed to its zero band gap, hRGO demonstrated a p-type semiconducting behavior with a positive shift in the Dirac points. This p-type behavior rendered hRGO, which can be conceptualized as interconnected graphene nanoribbons, as a potentially attractive material for FET sensors.
Related JoVE Video
Oxidative lipidomics of ?-radiation-induced lung injury: mass spectrometric characterization of cardiolipin and phosphatidylserine peroxidation.
Radiat. Res.
PUBLISHED: 02-21-2011
Show Abstract
Hide Abstract
Oxidative damage plays a significant role in the pathogenesis of ?-radiation-induced lung injury. Endothelium is a preferred target for early radiation-induced damage and apoptosis. Given the newly discovered role of oxidized phospholipids in apoptotic signaling, we performed oxidative lipidomics analysis of phospholipids in irradiated mouse lungs and cultured mouse lung endothelial cells. C57BL/6NHsd female mice were subjected to total-body irradiation (10 Gy, 15 Gy) and euthanized 24 h thereafter. Mouse lung endothelial cells were analyzed 48 h after ? irradiation (15 Gy). We found that radiation-induced apoptosis in vivo and in vitro was accompanied by non-random oxidation of phospholipids. Cardiolipin and phosphatidylserine were the major oxidized phospholipids, while more abundant phospholipids (phosphatidylcholine, phosphatidylethanolamine) remained non-oxidized. Electrospray ionization mass spectrometry analysis revealed the formation of cardiolipin and phosphatidylserine oxygenated molecular species in the irradiated lung and cells. Analysis of fatty acids after hydrolysis of cardiolipin and phosphatidylserine by phospholipase A(2) revealed the presence of mono-hydroperoxy and/or mono-hydroxy/mono-epoxy, mono-hydroperoxy/mono-oxo molecular species of linoleic acid. We speculate that cyt c-driven oxidations of cardiolipin and phosphatidylserine associated with the execution of apoptosis in pulmonary endothelial cells are important contributors to endothelium dysfunction in ?-radiation-induced lung injury.
Related JoVE Video
The multiple functions of cytochrome c and their regulation in life and death decisions of the mammalian cell: From respiration to apoptosis.
Mitochondrion
PUBLISHED: 01-26-2011
Show Abstract
Hide Abstract
Cytochrome c (Cytc) is essential in mitochondrial electron transport and intrinsic type II apoptosis. Mammalian Cytc also scavenges reactive oxygen species (ROS) under healthy conditions, produces ROS with the co-factor p66(Shc), and oxidizes cardiolipin during apoptosis. The recent finding that Cytc is phosphorylated in vivo underpins a model for the pivotal role of Cytc regulation in making life and death decisions. An apoptotic sequence of events is proposed involving changes in Cytc phosphorylation, increased ROS via increased mitochondrial membrane potentials or the p66(Shc) pathway, and oxidation of cardiolipin by Cytc followed by its release from the mitochondria. Cytc regulation in respiration and cell death is discussed in a human disease context including neurodegenerative and cardiovascular diseases, cancer, and sepsis.
Related JoVE Video
Two strategies for the development of mitochondrion-targeted small molecule radiation damage mitigators.
Int. J. Radiat. Oncol. Biol. Phys.
PUBLISHED: 01-18-2011
Show Abstract
Hide Abstract
To evaluate the effectiveness of mitigation of acute ionizing radiation damage by mitochondrion-targeted small molecules.
Related JoVE Video
LPS-induced decrease in intracellular labile zinc, [Zn]i, contributes to apoptosis in cultured sheep pulmonary artery endothelial cells.
Am. J. Physiol. Lung Cell Mol. Physiol.
PUBLISHED: 01-14-2011
Show Abstract
Hide Abstract
A role in signal transduction for a vanishingly small labile pool of intracellular zinc ([Zn](i)) has been inferred by the sensitivity of various physiological pathways to zinc chelators such as N,N,N,N-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) and/or associations with changes in nonprotein-bound zinc-sensitive fluorophores. Although we (44) reported that LPS-induced apoptosis in cultured sheep pulmonary artery endothelial cells (SPAEC) was exacerbated by TPEN, 1) we did not detect acute (30 min) changes in [Zn](i), and 2) it is unclear from other reports whether LPS increases or decreases [Zn](i) and whether elevations or decreases in [Zn](i) are associated with cell death and/or apoptosis. In the present study, we used both chemical (FluoZin-3 via live cell epifluorescence microscopy and fluorescence-activated cell sorting) and genetic (luciferase activity of a chimeric reporter encoding zinc-sensitive metal-response element and changes in steady-state mRNA of zinc importer, SLC39A14 or ZIP14) techniques to show that LPS caused a delayed time-dependent (2-4 h) decrease in [Zn](i) in SPAEC. A contributory role of decreases in [Zn](i) in LPS-induced apoptosis (as determined by caspase-3/7 activation, annexin-V binding, and cytochrome c release) in SPAECs was revealed by mimicking the effect of LPS with the zinc chelator, TPEN, and inhibiting LPS- (or TPEN)-induced apoptosis with exogenous zinc. Collectively, these are the first data demonstrating a signaling role for decrease in [Zn](i) in pulmonary endothelial cells and suggest that endogenous levels of labile zinc may affect sensitivity of pulmonary endothelium to the important and complex proapoptotic stimulus of LPS.
Related JoVE Video
Cytoprotective effects of albumin, nitrosated or reduced, in cultured rat pulmonary vascular cells.
Am. J. Physiol. Lung Cell Mol. Physiol.
PUBLISHED: 01-14-2011
Show Abstract
Hide Abstract
S-nitrosoalbumin (SNO-Alb) has been shown to be an efficacious cytoprotective molecule in acute lung injury, as well as ischemia-reperfusion injury in heart and skeletal muscle. Nonetheless, limited information is available on the cellular mechanism of such protection. Accordingly, we investigated the protective effects of SNO-Alb [ and its denitrosated congener, reduced albumin (SH-Alb) ] on tert-butyl hydroperoxide (tBH)-mediated cytotoxicity in cultured rat pulmonary microvascular endothelial cells (RPMEC), as well as hydrogen sulfide (H(2)S)-mediated cytotoxicity in rat pulmonary artery smooth muscle cells (RPASMC). We noted that tBH caused a concentration-dependent necrosis in RPMEC, and pretreatment of RPMEC with SNO-Alb dose-dependently decreased the sensitivity of these cells to tBH. A component of SNO-Alb cytoprotection was sensitive to N(G)-nitro-L-arginine methyl ester and was associated with activation of endothelial nitric oxide synthase (eNOS), phenomena that could be reproduced with pretreatment with SH-Alb. Exogenous H(2)S caused concentration-dependent apoptosis in RPASMC due to activation of ERK1/2 and p38, as well as downregulation of Bcl-2. Pretreatment with SNO-Alb reduced H(2)S-mediated apoptosis in a concentration-dependent manner that was associated with SNO-Alb-mediated inhibition of activation of ERK1/2 and p38. Pretreatment with SNO-Alb reduced toxicity of 1 mM sodium hydrosulfide in an N(G)-nitro-L-arginine methyl ester-sensitive fashion in RPASMC that expressed gp60 and neuronal NOS and was capable of transporting fluorescently labeled SH-Alb. Therefore, SNO-Alb is cytoprotective against models of oxidant-induced necrosis (tBH) and inhibitors of cellular respiration and apoptosis (H(2)S) in both pulmonary endothelium and smooth muscle, respectively, and a component of such protection can be attributed to a SH-Alb-mediated activation of constitutive NOS.
Related JoVE Video
Intraesophageal administration of GS-nitroxide (JP4-039) protects against ionizing irradiation-induced esophagitis.
In Vivo
PUBLISHED: 12-18-2010
Show Abstract
Hide Abstract
this study evaluated esophageal radioprotection by the Gramicidin S (GS) derived-nitroxide, JP4-039, a mitochondrial targeting peptide-isostere covalently-linked to 4-amino-Tempo, delivered in a novel swallowed oil-based (F15) formulation.
Related JoVE Video
Myeloperoxidase-dependent oxidation of etoposide in human myeloid progenitor CD34+ cells.
Mol. Pharmacol.
PUBLISHED: 11-19-2010
Show Abstract
Hide Abstract
Etoposide is a widely used anticancer drug successfully used for the treatment of many types of cancer in children and adults. Its use, however, is associated with an increased risk of development of secondary acute myelogenous leukemia involving the mixed-lineage leukemia (MLL) gene (11q23) translocations. Previous studies demonstrated that the phenoxyl radical of etoposide can be produced by action of myeloperoxidase (MPO), an enzyme found in developing myeloid progenitor cells, the likely origin for myeloid leukemias. We hypothesized, therefore, that one-electron oxidation of etoposide by MPO to its phenoxyl radical is important for converting this anticancer drug to genotoxic and carcinogenic species in human CD34(+) myeloid progenitor cells. In the present study, using electron paramagnetic resonance spectroscopy, we provide conclusive evidence for MPO-dependent formation of etoposide phenoxyl radicals in growth factor-mobilized CD34(+) cells isolated from human umbilical cord blood and demonstrate that MPO-induced oxidation of etoposide is amplified in the presence of phenol. Formation of etoposide radicals resulted in the oxidation of endogenous thiols, thus providing evidence for etoposide-mediated MPO-catalyzed redox cycling that may play a role in enhanced etoposide genotoxicity. In separate studies, etoposide-induced DNA damage and MLL gene rearrangements were demonstrated to be dependent in part on MPO activity in CD34(+) cells. Together, our results are consistent with the idea that MPO-dependent oxidation of etoposide in human hematopoietic CD34(+) cells makes these cells especially prone to the induction of etoposide-related acute myeloid leukemia.
Related JoVE Video
Mass-spectrometry based oxidative lipidomics and lipid imaging: applications in traumatic brain injury.
J. Neurochem.
PUBLISHED: 11-19-2010
Show Abstract
Hide Abstract
Lipids, particularly phospholipids, are fundamental to CNS tissue architecture and function. Endogenous polyunsaturated fatty acid chains of phospholipids possess cis-double bonds each separated by one methylene group. These phospholipids are very susceptible to free-radical attack and oxidative modifications. A combination of analytical methods including different versions of chromatography and mass spectrometry allows detailed information to be obtained on the content and distribution of lipids and their oxidation products thus constituting the newly emerging field of oxidative lipidomics. It is becoming evident that specific oxidative modifications of lipids are critical to a number of cellular functions, disease states and responses to oxidative stresses. Oxidative lipidomics is beginning to provide new mechanistic insights into traumatic brain injury which may have significant translational potential for development of therapies in acute CNS insults. In particular, selective oxidation of a mitochondria-specific phospholipid, cardiolipin, has been associated with the initiation and progression of apoptosis in injured neurons thus indicating new drug discovery targets. Furthermore, imaging mass-spectrometry represents an exciting new opportunity for correlating maps of lipid profiles and their oxidation products with structure and neuropathology. This review is focused on these most recent advancements in the field of lipidomics and oxidative lipidomics based on the applications of mass spectrometry and imaging mass spectrometry as they relate to studies of phospholipids in traumatic brain injury.
Related JoVE Video
Lipid antioxidants: free radical scavenging versus regulation of enzymatic lipid peroxidation.
J Clin Biochem Nutr
PUBLISHED: 09-30-2010
Show Abstract
Hide Abstract
The essentiality of polyunsaturated lipids makes membranes susceptible to peroxidative modifications. One of the most contemporary examples includes selective peroxidation of cardiolipin in mitochondria of cells undergoing apoptosis. Cardiolipin peroxidation products are required for the mitochondrial membrane permeabilization, release of pro-apoptotic factors and completion of the cell death program. Therefore, search for effective inhibitors of cardiolipin peroxidation is critical to discovery and development of anti-apoptotic antioxidants. Mitochondria contain significant amounts of ?-tocopherol, a well known scavenger of reactive free radicals. In the present study, we used an oxidative lipidomics approach to evaluate the effect of ?-tocopherol and its homologues with different lengths of the side-chain such as 2,5,7,8,-tetramethyl-2(4-methylpentyl)-6-chromanol and 2,2,5,7,8-pentamethyl-6-chromanol, on oxidation of tetralinoleoyl cardiolipin induced by cytochrome c in the presence of hydrogen peroxide. Our data indicate that vitamin E homologues inhibit not only accumulation of tetralinoleoyl cardiolipin hydroperoxides but also hydroxy-derivatives of tetralinoleoyl cardiolipin formed in the enzymatic peroxidase half-reaction catalyzed by cytochrome c. This suggests that protective effects of vitamin E homologues against tetralinoleoyl cardiolipin peroxidation catalyzed by cytochrome c/hydrogen peroxide are realized largely due to their effects on the peroxidase activity of cytochrome c towards tetralinoleoyl cardiolipin rather than via their scavenging activity.
Related JoVE Video
Phosphomimetic substitution of cytochrome C tyrosine 48 decreases respiration and binding to cardiolipin and abolishes ability to trigger downstream caspase activation.
Biochemistry
PUBLISHED: 07-01-2010
Show Abstract
Hide Abstract
Mammalian cytochrome c (Cytc) transfers electrons from the bc(1) complex to cytochrome c oxidase (CcO) as part of the mitochondrial electron transport chain, and it also participates in type II apoptosis. Our recent discovery of two tyrosine phosphorylation sites in Cytc, Tyr97 in bovine heart and Tyr48 in bovine liver, indicates that Cytc functions are regulated through cell signaling. To characterize the role of Cytc tyrosine phosphorylation in detail using an independent approach, we here overexpressed and purified a Tyr48Glu mutant Cytc, mimicking the in vivo Tyr48 phosphorylation found in cow liver, along with wild-type and Tyr48Phe variants as controls. The midpoint redox potential of the phosphomimetic mutant was decreased by 45 mV compared to control (192 vs 237 mV). Similar to Tyr48 in vivo phosphorylated Cytc, direct kinetic analysis of the Cytc reaction with isolated CcO revealed decreased V(max) for the Tyr48Glu mutant by 30% compared to wild type or the Tyr48Phe variants. Moreover, the phosphomimetic substitution resulted in major changes of Cytc functions related to apoptosis. The binding affinity of Tyr48Glu Cytc to cardiolipin was decreased by about 30% compared to wild type or the Tyr48Phe variants, and Cytc peroxidase activity of the Tyr48Glu mutant was cardiolipin-inducible only at high cardiolipin concentration, unlike controls. Importantly, the Tyr48Glu Cytc failed to induce any detectable downstream activation of caspase-3. Our data suggest that in vivo Tyr48 phosphorylation might serve as an antiapoptotic switch and highlight the strategic position and role of the conserved Cytc residue Tyr48 in regulating multiple functions of Cytc.
Related JoVE Video
Unusual peroxidase activity of polynitroxylated pegylated hemoglobin: Elimination of H(2)O(2) coupled with intramolecular oxidation of nitroxides.
Biochem. Biophys. Res. Commun.
PUBLISHED: 06-28-2010
Show Abstract
Hide Abstract
Polynitroxylated hemoglobin (Hb(AcTPO)(12)) has been developed as a hemoglobin-based oxygen carrier. While Hb(AcTPO)(12) has been shown to exert beneficial effects in a number of models of oxidative injury, its peroxidase activity has not been characterized thus far. In the blood stream, Hb(AcTPO)(12) undergoes reduction by ascorbate to its hydroxylamine form Hb(AcTPOH)(12). Here we report that Hb(AcTPOH)(12) exhibits peroxidase activity where H(2)O(2) is utilized for intramolecular oxidation of its TPOH residues to TPO. This represents an unusual redox-catalytic mechanism whereby reduction of H(2)O(2) is achieved at the expense of reducing equivalents of ascorbate converted into those of Hb(AcTPOH)(12), a new propensity that cannot be directly associated with ascorbate.
Related JoVE Video
Dynamic regulation of cardiolipin by the lipid pump Atp8b1 determines the severity of lung injury in experimental pneumonia.
Nat. Med.
PUBLISHED: 06-11-2010
Show Abstract
Hide Abstract
Pneumonia remains the leading cause of death from infection in the US, yet fundamentally new conceptual models underlying its pathogenesis have not emerged. We show that humans and mice with bacterial pneumonia have markedly elevated amounts of cardiolipin, a rare, mitochondrial-specific phospholipid, in lung fluid and find that it potently disrupts surfactant function. Intratracheal cardiolipin administration in mice recapitulates the clinical phenotype of pneumonia, including impaired lung mechanics, modulation of cell survival and cytokine networks and lung consolidation. We have identified and characterized the activity of a unique cardiolipin transporter, the P-type ATPase transmembrane lipid pump Atp8b1, a mutant version of which is associated with severe pneumonia in humans and mice. Atp8b1 bound and internalized cardiolipin from extracellular fluid via a basic residue-enriched motif. Administration of a peptide encompassing the cardiolipin binding motif or Atp8b1 gene transfer in mice lessened bacteria-induced lung injury and improved survival. The results unveil a new paradigm whereby Atp8b1 is a cardiolipin importer whose capacity to remove cardiolipin from lung fluid is exceeded during inflammation or when Atp8b1 is defective. This discovery opens the door for new therapeutic strategies directed at modulating the abundance or molecular interactions of cardiolipin in pneumonia.
Related JoVE Video
Lipid accumulation and dendritic cell dysfunction in cancer.
Nat. Med.
PUBLISHED: 05-27-2010
Show Abstract
Hide Abstract
Dendritic cells (DCs), a type of professional antigen-presenting cells, are responsible for initiation and maintenance of immune responses. Here we report that a substantial proportion of DCs in tumor-bearing mice and people with cancer have high amounts of triglycerides as compared with DCs from tumor-free mice and healthy individuals. In our studies, lipid accumulation in DCs was caused by increased uptake of extracellular lipids due to upregulation of scavenger receptor A. DCs with high lipid content were not able to effectively stimulate allogeneic T cells or present tumor-associated antigens. DCs with high and normal lipid levels did not differ in expression of major histocompatibility complex and co-stimulatory molecules. However, lipid-laden DCs had a reduced capacity to process antigens. Pharmacological normalization of lipid abundance in DCs with an inhibitor of acetyl-CoA carboxylase restored the functional activity of DCs and substantially enhanced the effects of cancer vaccines. These findings suggest that immune responses in cancer can be improved by manipulating the lipid levels in DCs.
Related JoVE Video
Protection of normal brain cells from ?-irradiation-induced apoptosis by a mitochondria-targeted triphenyl-phosphonium-nitroxide: a possible utility in glioblastoma therapy.
J. Neurooncol.
PUBLISHED: 04-28-2010
Show Abstract
Hide Abstract
Glioblastoma multiforme is the most frequent and aggressive primary brain tumor. A strong rationale to identify innovative approaches to treat these tumors is required since treatment failures result in local recurrences and median survivals range from 9 to 12 months. Glioma cells are reported to have less mitochondrial content compared to adjacent normal brain cells. Based on this difference, we suggest a new strategy, utilizing protection of normal brain cells by mitochondria-targeted electron scavengers and antioxidants-nitroxides-thus allowing for the escalation of the radiation doses. In this paper, we report that a conjugate of nitroxide with a hydrophobic cation, triphenyl-phosphonium (TPEY-Tempo), significantly protected brain endothelial cells from ?-irradiation-induced apoptosis while radiosensitizing brain tumor cells. Thus, TPEY-Tempo may be a promising adjunct in the treatment of glioblastoma with the potential to not only prolong survival but also to maintain quality of life and reduce treatment toxicity.
Related JoVE Video
Oxidative lipidomics of hyperoxic acute lung injury: mass spectrometric characterization of cardiolipin and phosphatidylserine peroxidation.
Am. J. Physiol. Lung Cell Mol. Physiol.
PUBLISHED: 04-23-2010
Show Abstract
Hide Abstract
Reactive oxygen species have been shown to play a significant role in hyperoxia-induced acute lung injury, in part, by inducing apoptosis of pulmonary endothelium. However, the signaling roles of phospholipid oxidation products in pulmonary endothelial apoptosis have not been studied. Using an oxidative lipidomics approach, we identified individual molecular species of phospholipids involved in the apoptosis-associated peroxidation process in a hyperoxic lung. C57BL/6 mice were killed 72 h after exposure to hyperoxia (100% oxygen). We found that hyperoxia-induced apoptosis (documented by activation of caspase-3 and -7 and histochemical terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling staining of pulmonary endothelium) was accompanied by nonrandom oxidation of pulmonary lipids. Two anionic phospholipids, mitochondria-specific cardiolipin (CL) and extramitochondrial phosphatidylserine (PS), were the two major oxidized phospholipids in hyperoxic lung. Using electrospray ionization mass spectrometry, we identified several oxygenation products in CL and PS. Quantitative assessments revealed a significant decrease of CL and PS molecular species containing C(18:2), C(20:4), C(22:5), and C(22:6) fatty acids. Similarly, exposure of mouse pulmonary endothelial cells (MLEC) to hyperoxia (95% oxygen; 72 h) resulted in activation of caspase-3 and -7 and significantly decreased the content of CL molecular species containing C(18:2) and C(20:4) as well as PS molecular species containing C(22:5) and C(22:6). Oxygenated molecular species were found in the same two anionic phospholipids, CL and PS, in MLEC exposed to hyperoxia. Treatment of MLEC with a mitochondria-targeted radical scavenger, a conjugate of hemi-gramicidin S with nitroxide, XJB-5-131, resulted in significantly lower oxidation of both CL and PS and a decrease in hyperoxia-induced changes in caspase-3 and -7 activation. We speculate that cytochrome c driven oxidation of CL and PS is associated with the signaling role of these oxygenated species participating in the execution of apoptosis and clearance of pulmonary endothelial cells, thus contributing to hyperoxic lung injury.
Related JoVE Video
?-Synuclein levels are elevated in cerebrospinal fluid following traumatic brain injury in infants and children: the effect of therapeutic hypothermia.
Dev. Neurosci.
PUBLISHED: 04-06-2010
Show Abstract
Hide Abstract
?-Synuclein is one of the most abundant proteins in presynaptic terminals. Normal expression of ?-synuclein is essential for neuronal survival and it prevents the initiation of apoptosis in neurons through covalent cross-linking of cytochrome c released from mitochondria. Exocytosis of ?-synuclein occurs with neuronal mitochondrial dysfunction, making its detection in cerebrospinal fluid (CSF) of children after severe traumatic brain injury (TBI) a potentially important marker of injury. Experimental therapeutic hypothermia (TH) improves mitochondrial function and attenuates cell death, and therefore may also affect CSF ?-synuclein concentrations. We assessed ?-synuclein levels in CSF of 47 infants and children with severe TBI using a commercial ELISA for detection of monomeric protein. 23 patients were randomized to TH based on published protocols where cooling (32-33°C) was initiated within 6-24 h, maintained for 48 h, and then followed by slow rewarming. CSF samples were obtained continuously via an intraventricular catheter for 6 days after TBI. Control CSF (n = 9) was sampled from children receiving lumbar puncture for CSF analysis of infection that was proven negative. Associations of initial Glasgow Coma Scale (GCS) score, age, gender, treatment, mechanism of injury and Glasgow Outcome Scale (GOS) score with CSF ?-synuclein were compared by multivariate regression analysis. CSF ?-synuclein levels were elevated in TBI patients compared to controls (p = 0.0093), with a temporal profile showing an early, approximately 5-fold increase on days 1-3 followed by a delayed, >10-fold increase on days 4-6 versus control. ?-Synuclein levels were higher in patients treated with normothermia versus hypothermia (p = 0.0033), in patients aged <4 years versus ?4 years (p < 0.0001), in females versus males (p = 0.0007), in nonaccidental TBI versus accidental TBI victims (p = 0.0003), and in patients with global versus focal injury on computed tomography of the brain (p = 0.046). Comparisons of CSF ?-synuclein levels with initial GCS and GOS scores were not statistically significant. Further studies are needed to evaluate the conformational status of ?-synuclein in CSF, and whether TH affects ?-synuclein aggregation.
Related JoVE Video
The cyclooxygenase site, but not the peroxidase site of cyclooxygenase-2 is required for neurotoxicity in hypoxic and ischemic injury.
J. Neurochem.
PUBLISHED: 03-17-2010
Show Abstract
Hide Abstract
Cyclooxygenase-2 (COX-2) activity has been implicated in the pathogenesis of ischemic injury, but the exact mechanisms responsible for its toxicity remain unclear. Infection of primary neurons with an adenovirus expressing wild type (WT) COX-2 increased the susceptibility of neurons to hypoxia. Infection with an adenoviral vector expressing COX-2 with a mutation at the cyclooxygenase site did not increase susceptibility to hypoxia, whereas over-expression of COX-2 with a mutation in the peroxidase site produced similar susceptibility to hypoxia as WT COX-2. Primary neuronal cultures obtained from transgenic mice bearing a mutation in the COX-2 cylooxygenase site were protected from hypoxia. Mice with a mutation in the cyclooxygenase site had smaller infarctions 24 h after 70 min of middle cerebral artery occlusion than WT control mice. COX-2 activity had no effect on the formation of protein carbonyls. Ascorbate radicals were detected by electron paramagnetic resonance as a product of recombinant COX-2 activity and were blocked by COX-2 inhibitors. Similarly, formation of ascorbate radicals was inhibited in the presence of COX-2 inhibitors and in homogenates obtained from COX-2 null mice. Taken together, these results indicate that the cyclooxygenase activity of COX-2 is necessary to exacerbate neuronal hypoxia/ischemia injury rather than the peroxidase activity of the enzyme.
Related JoVE Video
N-acetylcysteine does not prevent hepatorenal ischaemia-reperfusion injury in patients undergoing orthotopic liver transplantation.
Nephrol. Dial. Transplant.
PUBLISHED: 02-22-2010
Show Abstract
Hide Abstract
Glutathione (GSH) acts as a free radical scavenger that may be helpful in preventing reperfusion injury. N-acetylcysteine (NAC) replenishes GSH stores. The aims of this study were to evaluate the efficacy of NAC in improving liver graft performance and reducing the incidence of post-operative acute kidney injury (AKI).
Related JoVE Video

What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.