Biomedical subjects

Grigory G Borisenko

Publications and source records attributed to Grigory G Borisenko.

11 recordsLinked to original sources

PubMed2006

Oxidation and cytotoxicity of 6-OHDA are mediated by reactive intermediates of COX-2 overexpressed in PC12 cells.

Parkinson's disease is characterized by a progressive loss of dopaminergic neurons, likely associated with dysregulation of oxidation of catechols, such as dopamine (DA) and 6-hydroxydopamine (6-OHDA), and resulting in oxidative stress. The involvement of cyclooxygenase-2 (COX-2) in pathogenesis of Parkinson's disease has been suggested. However, specific COX-2 triggered mechanisms participating in catalysis of DA oxidation and enhanced catechol-induced cytotoxicity remain poorly characterized. Here, we demonstrate that in a model biochemical system, recombinant heme-reconstituted COX-2 induced oxidation of 6-OHDA in the course of its peroxidase (H(2)O(2)-dependent) and cyclooxygenase (arachidonic acid (AA)-dependent) catalytic half-cycles. Similarly, COX-2 was able to stimulate 6-OHDA oxidation during its peroxidase- and cyclooxygenase half-cycles and caused oxidative stress in homogenates of PC12 cells stably overexpressing the enzyme (but not in mock-transfected cells). In addition, the increased levels of COX-2 were associated with enhanced cytotoxicity of 6-OHDA in stably transfected PC12 cells. Finally, co-oxidation of 6-OHDA by COX-2 triggered production of superoxide radicals critical for both propagation of 6-OHDA oxidation and induction of oxidative stress in COX-2 overexpressing cells. Thus, we conclude that both peroxidase and cyclooxygenase half-cycles of COX-2-catalyzed reactions are essential for COX-2-dependent activation of 6-OHDA oxidation, oxygen radical production, oxidative stress, and cytotoxicity.

Animals↗

PubMed2005

Enhanced oxidative stress in iNOS-deficient mice after traumatic brain injury: support for a neuroprotective role of iNOS.

Studies in experimental traumatic brain injury (TBI) suggest both deleterious and protective effects of inducible nitric oxide synthase (iNOS). Early after injury, iNOS may be detrimental via formation of peroxynitrite and iNOS inhibitors are protective. In contrast, we reported impaired long-term functional outcome after TBI in iNOS knockout (ko) versus wild-type (wt) mice. To elucidate potential neuroprotective and neurotoxic mechanisms for iNOS, we studied nitric oxide formation by electron paramagnetic resonance (EPR) spectroscopy using diethyldithiocarbamate-iron (DETC-Fe) as a spin trap and markers of nitrosative (S-nitrosothiol (RSNO, Fluorescent assay); nitrotyrosine (3NT, ELISA)) and oxidative stress (ascorbate, HPLC) at 72 h after controlled cortical impact (CCI) in iNOS ko and wt and in uninjured iNOS ko and wt mice. 3NT immunostaining with macrophage and myeloperoxidase (MPO) dual labeling was also assessed in brain sections. Brain DETC-Fe-NO low-temperature EPR signal intensity was approximately 2-fold greater in wt versus iNOS ko at 72 h after CCI. Ascorbate levels decreased in injured hemisphere in wt and iNOS ko versus uninjured -this decrease was more pronounced in iNOS ko. In wt mice, RSNO and 3NT levels were increased after CCI versus uninjured (50% and 400%, respectively, P < 0.05). RSNO levels were not increased in iNOS ko after CCI. Nitrotyrosine levels increased after CCI in wt and ko versus respective uninjured -this increase was more pronounced in wt (2.34 +/- 0.95 versus 1.27 +/- 0.49 pmol/mg protein, P < 0.05). Increased 3NT immunoreactivity was detected in wt versus iNOS ko at 72 h after CCI, and colocalized with macrophage marker and MPO. Our data support a role for iNOS-derived NO as an endogenous antioxidant after CCI. iNOS also contributes protein nitrosylation and nitration. Colocalization of 3NT with macrophages and MPO suggests generation of nitrating agents by macrophages and/or phagocytosis of nitrated proteins.

Animals↗

PubMed2005

Quantitative method of measuring phosphatidylserine externalization during apoptosis using electron paramagnetic resonance spectroscopy and annexin-conjugated iron.

We present here the application of a novel assay that measures the absolute amount of phosphatidylserine (PS) externalized on the surface of cells. Although the assay is 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.

Annexins↗

PubMed2004

Oxidative lipidomics of apoptosis: redox catalytic interactions of cytochrome c with cardiolipin and phosphatidylserine.

The primary life-supporting function of cytochrome c (cyt c) is control of cellular energetic metabolism as a mobile shuttle in the electron transport chain of mitochondria. Recently, cyt c's equally important life-terminating function as a trigger and regulator of apoptosis was identified. This dreadful role is realized through the relocalization of mitochondrial cyt c to the cytoplasm where it interacts with Apaf-1 in forming apoptosomes and mediating caspase-9 activation. Although the presence of heme moiety of cyt c is essential for the latter function, cyt c's redox catalytic features are not required. Lately, two other essential functions of cyt c in apoptosis, that may rely heavily on its redox activity have been suggested. Both functions are directed toward oxidation of two negatively charged phospholipids, cardiolipin (CL) in the mitochondria and phosphatidylserine (PS) in the plasma membrane. In both cases, oxidized phospholipids seem to be essential for the transduction of two distinctive apoptotic signals: one is participation of oxidized CL in the formation of the mitochondrial permeability transition pore that facilitates release of cyt c into the cytosol and the other is the contribution of oxidized PS to the externalization and recognition of PS (and possibly oxidized PS) on the cell surface by specialized receptors of phagocytes. In this review, we present a new concept that cyt c actuates both of these oxidative roles through a uniform mechanism: its specific interactions with each of these phospholipids result in the conversion and activation of cyt c, transforming it from an innocuous electron transporter into a calamitous peroxidase capable of oxidizing the activating phospholipids. We also show that this new concept is compatible with a leading role for reactive oxygen species in the execution of the apoptotic program, with cyt c as the main executioner.

Animals↗

PubMed2004

Nitroxides scavenge myeloperoxidase-catalyzed thiyl radicals in model systems and in cells.

Nitroxide radicals possess important antioxidant activity in live tissues because of their ability to scavenge reactive radicals. Despite the fact that, in cells, damaging free radicals are primarily quenched by glutathione (GSH) with subsequent formation of harmful glutathionyl radical (GS(*)), interactions of nitroxide radicals with GS(*) and thiols have not been studied in detail. In addition, intracellular metabolic pathways leading to the formation of secondary amines from nitroxides are unknown. Here we report that GS(*) radicals react efficiently and irreversibly with nitroxides to produce secondary amines. We developed a sensitive method for the detection of GS(*) based on their specific interaction with Ac-Tempo, a nonfluorescent conjugate of fluorogenic acridine with paramagnetic nitroxide Tempo, and used it to characterize interactions between nitroxide and thiyl radicals generated through phenoxyl radical recycling by peroxidase. During reaction of Ac-Tempo with GS(*), Tempo EPR signals decayed and acridine fluorescence concurrently increased. DMPO and PBN, spin traps for GS(*), inhibited this interaction. Using combined HPLC and mass spectrometry, we determined that 90% of the Ac-Tempo was converted into fluorescent acridine (Ac)-piperidine; GSH was primarily oxidized into sulfonic acid. In myeloperoxidase-rich HL-60 cells, Ac-piperidine fluorescence was observed upon stimulation of GS(*) generation by H(2)O(2) and phenol. Development of fluorescence was prevented by preincubation of cells with the thiol-blocking reagent N-ethylmaleimide as well as with peroxidase inhibitiors. Furthermore, Ac-Tempo preserved intracellular GSH and protected cells from phenol/GS(*) toxicity, suggesting a new mechanism for the free-radical scavenging activity of nitroxides in live cells.

Acridines↗

PubMed2004

Glutathione propagates oxidative stress triggered by myeloperoxidase in HL-60 cells. Evidence for glutathionyl radical-induced peroxidation of phospholipids and cytotoxicity.

Glutathione acts as a universal scavenger of free radicals at the expense of the formation of the glutathionyl radicals (GS*). Here we demonstrated that GS* radicals specifically interact with a reporter molecule, paramagnetic and non-fluorescent 4-((9-acridinecarbonyl)-amino)-2,2,6,6-tetramethylpiperidine-1-oxyl (Ac-Tempo), and convert it into a non-paramagnetic fluorescent product, identified as 4-((9-acridinecarbonyl)amino)-2,2,6,6-tetramethylpiperidine (Ac-piperidine). Horseradish peroxidase-, myeloperoxidase-, and cyclooxygenasecatalyzed oxidation of phenol in the presence of H2O2 and GSH caused the generation of phenoxyl radicals and GS* radicals, of which only the latter reacted with Ac-Tempo. Oxidation of several other phenolic compounds (e.g. etoposide and tyrosine) was accompanied by the formation of GS* radicals along with a characteristic fluorescence response from Ac-Tempo. In myeloperoxidase-rich HL-60 cells treated with H2O2 and phenol, fluorescence microscopic imaging of Ac-Tempo revealed the production of GS* radicals. A thiol-blocking reagent, N-ethylmaleimide, as well as myeloperoxidase inhibitors (succinyl acetone and azide), blocked formation of fluorescent acridine-piperidine. H2O2/phenolinduced peroxidation of major classes of phospholipids in HL-60 cells was completely inhibited by Ac-Tempo, indicating that GS* radicals were responsible for phospholipid peroxidation. Thus, GSH, commonly viewed as a universal free radical scavenger and major intracellular antioxidant, acts as a pro-oxidant during myeloperoxidase-catalyzed metabolism of phenol in HL-60 cells.

Cell Survival↗

PubMed2004

Ascorbate as a "redox sensor" and protector against irradiation-induced oxidative stress in 32D CL 3 hematopoietic cells and subclones overexpressing human manganese superoxide dismutase.

PURPOSE: To determine whether increased expression of manganese superoxide dismutase (MnSOD) protects cells from irradiation by preventing the production of reactive oxygen species (ROS), a new approach to detecting free radical intermediates using ascorbate as an endogenous spin trap was used. MATERIALS AND METHODS: Cells from the 32D cl 3 hematopoietic cell line or a subclone overexpressing MnSOD (2C6) were incubated with dehydroascorbate for 30 min and irradiated to doses from 0 to 50 Gy. Radical intermediates reacting with spin traps or ascorbate were measured by electron spin resonance spectroscopy. Results were compared to irradiation-induced changes in thiol levels, irradiation survival curves, and accumulation of 8-OHdG as a measurement of DNA oxidative damage. RESULTS: Manganese superoxide dismutase-overexpressing 2C6 cells maintained higher levels of ascorbate (5.4 +/- 0.5 and 2.6 +/- 0.5 nmol/10(6) cells, respectively) and thiols (14.0 +/- 0.1 and 11.1 +/- 0.2 nmol/10(6) cells) compared to 32D cl 3 parent cells. Cells overexpressing MnSOD produced lower levels of ROS than did the parental 32D cl 3 cells, as evidenced by lower expenditure of ascorbate and GSH after irradiation. Increased ascorbate levels protected both 32D cl 3 and 2C6 cells from irradiation killing, as demonstrated by an increased shoulder on survival curves and decreased DNA 8-OHdG accumulation. CONCLUSIONS: Manganese superoxide dismutase overexpression protects 2C6 cells from irradiation damage by scavenging ROS that readily interact with major endogenous antioxidants--ascorbate and GSH--in nontransfected hematopoietic 32D cl 3 cells.

Animals↗

PubMed2004

Cytochrome c release is required for phosphatidylserine peroxidation during Fas-triggered apoptosis in lung epithelial A549 cells.

Oxidation of phosphatidylserine (PtdSer) has been shown to play a pivotal role in signaling during cell apoptosis and subsequent recognition of apoptotic cells by phagocytes. However, the redox catalytic mechanisms involved in selective PtdSer oxidation during apoptosis remain poorly understood. Here we employed anti-Fas antibody CH-11-treated A549 cells as a physiologically relevant model to investigate the involvement of PtdSer oxidation and its potential mechanism during apoptosis. We demonstrated that ligation of CH-11 with its cognate receptor initiated execution of apoptotic program in interferon gamma-pretreated A549 cells as evidenced by activation of caspase and DNA fragmentation. A significant increase of cytochrome c (cyt c) content in the cytosol as early as 2 h after CH-11 exposure was detected indicating that Fas-induced apoptosis in A549 cells proceeds via extrinsic type II pathway and includes mitochondrial signaling. PtdSer was selectively oxidized 3 h after anti-Fas triggering while two more abundant phospholipids--phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn)--and the major intracellular antioxidant, glutathione, remained nonoxidized. A pan-caspase inhibitor, z-VAD, fully blocked cyt c release and oxidation of PtdSer in Fas-treated A549 cells. On the other hand, z-DQMD, a caspase-3 inhibitor, completely inhibited caspase-3 activity but did not fully block caspase-8 activation and release of cyt c. Importantly, z-DQMD failed to protect PtdSer from oxidation. In addition, in a model system, we demonstrated that peroxidase activity of cyt c was greatly enhanced in the presence of dioleoylphosphatidylserine containing liposomes by monitoring oxidation of 2',7'-dichlorodihydrofluorescein to 2',7'-dichlorofluorescein. We further showed that peroxidase activity of cyt c catalyzed oxidation of 1-palmitoyl-2-arachidonoyl-3-glycero-phosphoserine using a newly developed HPLC assay. MS analysis of 1-palmitoyl-2-arachidonoyl-3-glycero-phosphoserine revealed that in addition to its mono- and dihydroperoxides, several different PtdSer oxidation products can be formed. Overall, we concluded that cyt c acts as a catalyst of PtdSer oxidation during Fas-triggered A549 cell apoptosis.

Antibodies↗

PubMed2004

Arachidonic acid-induced carbon-centered radicals and phospholipid peroxidation in cyclo-oxygenase-2-transfected PC12 cells.

Cyclo-oxygenase-2 (COX-2) is believed to induce neuronal oxidative stress via production of radicals. While oxygen radicals are not directly involved in COX-2-catalytic cycle, superoxide anion radicals have been repeatedly reported to play a critical role in COX-2-associated oxidative stress. To resolve the controversy, we characterized production of free radicals in PC12 cells in which COX-2 expression was manipulated either genetically or by direct protein transfection and compared them with those generated by a recombinant COX-2 in a cell-free system. Using spin-traps alpha-(4-pyridyl-1-oxide)-N-t-butylnitrone, 5,5-dimethyl-1-pyrroline-N-oxide and 4-((9-acridinecarbonyl) amino)-2,2,6,6- tetramethylpiperidine-1-oxyl (Ac-Tempo), we observed arachidonic acid (AA)-dependent production of carbon-centered radicals by heme-reconstituted recombinant COX-2. No oxygen radicals or thiyl radicals have been detected. COX-2 also catalyzed AA-dependent one-electron co-oxidation of ascorbate to ascorbate radicals. Next, we used two different approaches of COX-2 expression in cells, PCXII cells which express isopropyl-1-thio-beta-D-galactopyranoside inducible COX-2, and PC12 cells transfected with COX-2 using a protein delivery reagent, Chariot. In both models, COX-2-dependent AA-induced generation of carbon-centered radicals was documented using spin-traps and Ac-Tempo. No oxygen radical formation was detected in COX-2-transfected cells by either spin-traps or fluorogenic probe, dihydroethidium. In the presence of ascorbate, AA-induced COX-2-dependent ascorbate radicals were detected. AA caused a significant and selective oxidation of one of the major phospholipids, phosphatidylserine (PS). PS was not a direct substrate for COX-2 but was co-oxidized in the presence of AA. The radical generation and PS oxidation were inhibited by COX-2 inhibitors, niflumic acid, nimesulide, or NS-398. Thus, COX-2 generated carbon-centered radicals but not oxygen radicals or thiyl radicals are responsible for oxidative stress in AA-challenged PC12 cells overexpressing COX-2.

Animals↗

PubMed2003

Peroxidation and externalization of phosphatidylserine associated with release of cytochrome c from mitochondria.

Production of reactive oxygen species (ROS) during apoptosis is associated with peroxidation of phospholipids particularly of phosphatidylserine (PS). The mechanism(s) underlying preferential PS oxidation are not well understood. We hypothesized that cytochrome c (cyt c) released from mitochondria into cytosol acts as a catalyst that utilizes ROS generated by disrupted mitochondrial electron transport for PS oxidation. Selectivity of PS oxidation is achieved via specific interactions of positively charged cyt c with negatively charged PS. To test the hypothesis we employed temporary transfection of Jurkat cells with a pro-apoptotic peptide, DP1, a conjugate consisting of a protein transduction domain, PTD-5, and an antimicrobial domain, KLA [(KLAKLAK)2], known to selectively disrupt mitochondria. We report that treatment of Jurkat cells with DP1 yielded rapid and effective release of cyt c from mitochondria and its accumulation in cytosol accompanied by production of H2O2. Remarkably, this resulted in selective peroxidation of PS while more abundant phospholipids such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE) remained nonoxidized. Neither PTD-5 alone nor KLA alone exerted any effect on PS peroxidation. Redox catalytic involvement of cyt c in PS oxidation was further supported by our data demonstrating that: (i) specific interactions of cyt c with PS resulted in the formation of EPR-detectable protein-centered tyrosyl radicals of cyt c upon its interaction with H2O2 in the presence of PS-containing liposomes, and (ii) integration of cyt c into cytochrome c null (Cyt c -/-) cells or HL-60 cells specifically stimulates PS oxidation in the presence of H2O2 or t-BuOOH, respectively. We further demonstrated that DP1 elicited externalization of PS on the surface of Jurkat cells and enhanced their recognition and phagocytosis by J774A.1 macrophages. Our results are compatible with the hypothesis that catalysis of selective PS oxidation during apoptosis by cytosolic cyt c is important for PS-dependent signaling pathways such as PS externalization and recognition by macrophage receptors.

Apoptosis↗

PubMed2003

Macrophage recognition of externalized phosphatidylserine and phagocytosis of apoptotic Jurkat cells--existence of a threshold.

Phosphatidylserine (PS) is predominantly confined to the inner leaflet of plasma membrane in cells, but it is externalized on the cell surface during apoptosis. This externalized PS is required for effective phagocytosis of apoptotic cells by macrophages. Because PS trans-bilayer asymmetry is not absolute in different types of nonapoptotic cells, we hypothesized that the amounts of externalized PS may be critical for macrophage discrimination between apoptotic and nonapoptotic cells. We developed a sensitive electron paramagnetic resonance method to quantify the amounts of externalized PS based on specific binding of paramagnetic annexin V-microbead conjugates with PS on cell surfaces. Using this technique, we found that nonapoptotic Jurkat cells externalize 0.9 pmol of endogenous PS/10(6) Jurkat cells. For cells with different amounts of integrated exogenous PS on their surface, no phagocytic response was observed at PS levels <5 pmol/10(6) Jurkat cells; at higher PS concentrations, phagocytosis increased in a concentration-dependent manner. Apoptosis in Jurkat cells caused externalization of approximately 240 pmol PS/10(6) Jurkat cells; these amounts of externalized PS are manyfold higher than the threshold amounts of PS required for phagocytosis. Thus, macrophages have a sensitivity threshold for PS externalized on the cell surface that provides for reliable recognition and distinction between normal cells with low contents of externalized PS and apoptotic cells with remarkably elevated PS levels.

Animals↗