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Item Activation of APE/Ref-1 redox activity is mediated by reactive oxygen species and PKC phosphorylation(2001-05) Hsieh, Marlene M.; Hegde, Vijay; Kelley, Mark R.; Deutsch, Walter A.Reactive oxygen species (ROS) arise through normal cellular aerobic respiration, and, in combination with external sources such as ionizing radiation, cigarette tar and smoke, and particulate matter generated by combustion, can have a profound negative effect on cellular macromolecules such as DNA that may lead to a number of human pathological disorders including accelerated aging and cancer. A major end product of ROS damage to DNA is the formation of apurinic/apyrimidinic (AP) sites, which without removal are known to halt mRNA and DNA synthesis, or act as non-coding lesions resulting in the increased generation of DNA mutations. In human cells, the major enzyme in correcting the deleterious effects of AP sites in DNA is through the participation of AP endonuclease (APE), which initiates the removal of baseless sites in DNA through the catalytic scission of the phosphodiester bond 5′ and adjacent to an AP site. Interestingly, APE also possesses an activity (Ref-1) that controls the redox status of a number of transcription factors including Fos and Jun. The means by which APE/Ref-1 is directed to carry out such disparate roles are unknown. The presence of a number of phosphorylation sites scattered throughout both functional domains of APE/Ref-1 however offered one possible mechanism that we reasoned could play a role in dictating how this protein responds to different stimuli. Here we show that the in vitro redox activity of APE/Ref-1 is stimulated by PKC phosphorylation. Furthermore, when human cells were exposed to the PKC activator phorbol 12-myristate 13-acetate, an increase in redox activity was observed that corresponded to an increase in the phosphorylation status of APE/Ref-1. Importantly, human cells exposed to the oxidizing agent hypochlorite, followed by methyl methanesulfanate, responded with an increase in redox activity by APE/Ref-1 that also involved an increase in PKC activity and a corresponding increase in the phosphorylation of APE/Ref-1. These results suggest that the ability of APE/Ref-1 to perform its in vivo redox function is correlated to its susceptibility to PKC phosphorylation that notably occurs in response to DNA damaging agents.Item Apurinic/Apyrimidinic Endonuclease/Redox Factor-1 (APE1/Ref-1) redox function negatively regulates NRF2(2015-01) Fishel, Melissa L.; Wu, Xue; Devlin, Cecilia M.; Logsdon, Derek P.; Jiang, Yanlin; Luo, Meihua; He, Ying; Yu, Zhangsheng; Tong, Yan; Lipking, Kelsey P.; Maitra, Anirban; Rajeshkumar, N. V.; Scandura, Glenda; Kelley, Mark R.; Ivan, Mircea; Department of Pediatrics, Indiana University School of MedicineApurinic/apyrimidinic endonuclease/redox factor-1 (APE1/Ref-1) (henceforth referred to as Ref-1) is a multifunctional protein that in addition to its base excision DNA repair activity exerts redox control of multiple transcription factors, including nuclear factor κ-light chain enhancer of activated B cells (NF-κB), STAT3, activator protein-1 (AP-1), hypoxia-inducible factor-1 (HIF-1), and tumor protein 53 (p53). In recent years, Ref-1 has emerged as a promising therapeutic target in cancer, particularly in pancreatic ductal carcinoma. Although a significant amount of research has centered on Ref-1, no wide-ranging approach had been performed on the effects of Ref-1 inhibition and transcription factor activity perturbation. Starting with a broader approach, we identified a previously unsuspected effect on the nuclear factor erythroid-related factor 2 (NRF2), a critical regulator of cellular defenses against oxidative stress. Based on genetic and small molecule inhibitor-based methodologies, we demonstrated that repression of Ref-1 potently activates NRF2 and its downstream targets in a dose-dependent fashion, and that the redox, rather than the DNA repair function of Ref-1 is critical for this effect. Intriguingly, our results also indicate that this pathway does not involve reactive oxygen species. The link between Ref-1 and NRF2 appears to be present in all cells tested in vitro, noncancerous and cancerous, including patient-derived tumor samples. In particular, we focused on understanding the implications of the novel interaction between these two pathways in primary pancreatic ductal adenocarcinoma tumor cells and provide the first evidence that this mechanism has implications for overcoming the resistance against experimental drugs targeting Ref-1 activity, with clear translational implications.Item Dietary Polyphenols in Cancer Chemoprevention: Implications in Pancreatic Cancer(MDPI, 2020-07-23) Thyagarajan, Anita; Forino, Andrew S.; Konger, Raymond L.; Sahu, Ravi P.; Pathology and Laboratory Medicine, School of MedicineNaturally occurring dietary agents present in a wide variety of plant products, are rich sources of phytochemicals possessing medicinal properties, and thus, have been used in folk medicine for ages to treat various ailments. The beneficial effects of such dietary components are frequently attributed to their anti-inflammatory and antioxidant properties, particularly in regards to their antineoplastic activities. As many tumor types exhibit greater oxidative stress levels that are implicated in favoring autonomous cell growth activation, most chemotherapeutic agents can also enhance tumoral oxidative stress levels in part via generating reactive oxygen species (ROS). While ROS-mediated imbalance of the cellular redox potential can provide novel drug targets, as a consequence, this ROS-mediated excessive damage to cellular functions, including oncogenic mutagenesis, has also been implicated in inducing chemoresistance. This remains one of the major challenges in the treatment and management of human malignancies. Antioxidant-enriched natural compounds offer one of the promising approaches in mitigating some of the underlying mechanisms involved in tumorigenesis and metastasis, and therefore, have been extensively explored in cancer chemoprevention. Among various groups of dietary phytochemicals, polyphenols have been extensively explored for their underlying chemopreventive mechanisms in other cancer models. Thus, the current review highlights the significance and mechanisms of some of the highly studied polyphenolic compounds, with greater emphasis on pancreatic cancer chemoprevention.Item Effects of inhibiting antioxidant pathways on cellular hydrogen sulfide and polysulfide metabolism(Elsevier, 2019-05) Olson, Kenneth R.; Gao, Yan; Medicine, School of MedicineElaborate antioxidant pathways have evolved to minimize the threat of excessive reactive oxygen species (ROS) and to regulate ROS as signaling entities. ROS are chemically and functionally similar to reactive sulfur species (RSS) and both ROS and RSS have been shown to be metabolized by the antioxidant enzymes, superoxide dismutase and catalase. Here we use fluorophores to examine the effects of a variety of inhibitors of antioxidant pathways on metabolism of two important RSS, hydrogen sulfide (H2S with AzMC) and polysulfides (H2Sn, where n = 2–7, with SSP4) in HEK293 cells. Cells were exposed to inhibitors for up to 5 days in normoxia (21% O2) and hypoxia (5% O2), conditions also known to affect ROS production. Decreasing intracellular glutathione (GSH) with l-buthionine-sulfoximine (BSO) or diethyl maleate (DEM) decreased H2S production for 5 days but did not affect H2Sn. The glutathione reductase inhibitor, auranofin, initially decreased H2S and H2Sn but after two days H2Sn increased over controls. Inhibition of peroxiredoxins with conoidin A decreased H2S and increased H2Sn, whereas the glutathione peroxidase inhibitor, tiopronin, increased H2S. Aminoadipic acid, an inhibitor of cystine uptake did not affect either H2S or H2Sn. In buffer, the glutathione reductase and thioredoxin reductase inhibitor, 2-AAPA, the glutathione peroxidase mimetic, ebselen, and tiopronin variously reacted directly with AzMC and SSP4, reacted with H2S and H2S2, or optically interfered with AzMC or SSP4 fluorescence. Collectively these results show that antioxidant inhibitors, generally known for their ability to increase cellular ROS, have various effects on cellular RSS. These findings suggest that the inhibitors may affect cellular sulfur metabolism pathways that are not related to ROS production and in some instances they may directly affect RSS or the methods used to measure them. They also illustrate the importance of carefully evaluating RSS metabolism when biologically or pharmacologically attempting to manipulate ROS.Item Major Collateral Vessels Develop from Pre-existing Small Arteries through RAC2/NOX2 Independent Mechanisms(2009-03-18T18:45:14Z) DiStasi, Matthew Robert; Unthank, JosephThere is no consensus on which vascular segment or what size of vessels is most important in the process of collateral growth, the degree to which these vessels can enlarge, or the mechanisms that mediate collateral vessel expansion and its impairment. Chapter I identifies the major collateral vessels that develop in response to femoral arterial occlusion in the pig, rat, and mouse hindlimbs for comparison to humans. Pre-existent small named arteries enlarged ~2-3-fold to become the major collateral vessels in each species, these major collaterals displayed characteristics similar to large arteries experiencing flow-mediated outward remodeling, and important differences in vascular wall thickness were observed between rodents and pigs. Chapter II utilized Rac2-/- and Nox2-/- mice to investigate the hypothesis that Nox2-NAD(P)H oxidase is required for major collateral growth subsequent to femoral arterial occlusion. Previous studies suggest bone marrow cell (BMC)-derived reactive oxygen species (ROS) produced by the Nox2 subunit of NAD(P)H oxidase plays an important role in neovascularization and recovery of hindlimb perfusion subsequent to femoral arterial occlusion; but did not investigate collateral growth. The hematopoietic cell restricted protein Rac2 has been shown to bind to and activate Nox2-NAD(P)H oxidase and Rac2-/- and Nox2-/- leukocytes display impaired ROS related functions. The data demonstrated that Rac2 and Nox2 are not essential for major collateral growth, but both are important for the recovery of hindlimb perfusion and preservation of distal tissue morphology. Chapter III investigated BMC and antioxidant therapy in the age-related impairment of collateral growth. Aging, like all cardiovascular disease risk factors is associated with elevated ROS and impaired collateral growth. Studies also suggest BMCs promote collateral growth by secreting paracrine factors but elevated ROS may affect the efficacy of BMCs. The data revealed that neither BMC injection nor antioxidant therapy via apocynin enhanced the process of major collateral artery growth in aged mice.Item Reactive oxygen species' role in endothelial dysfunction by electron paramagnetic resonance(2013-08-29) Wassall, Cynthia D.; Kemple, Marvin D.; Joglekar, Yogesh; Petrache, Horia; Nageswara Rao, B. D.; Durbin, Stephen M.; Decca, RicardoThe endothelium is a single layer of cells lining the arteries and is involved in many physiological reactions which are responsible for vascular tone. Free radicals are important participants in these chemical reactions in the endothelium. Here we quantify free radicals, ex vivo, in biological tissue with continuous wave electron paramagnetic resonance (EPR). In all of the experiments in this thesis, we use a novel EPR spin trapping technique that has been developed for tissue segments. EPR spin trapping is often considered the ‘gold standard’ in reactive oxygen species (ROS) detection because of its sensitivity and non-invasive nature. In all experiments, tissue was placed in physiological saline solution with 190-mM PBN (N-tert-butyl-α-phenylnitrone), 10% by volume dimethyl-sulphoxide (DMSO) for cryopreservation, and incubated in the dark for between 30 minutes up to 2 hours at 37°C while gently being stirred. Tissue and supernatant were then loaded into a syringe and frozen at -80°C until EPR analysis. In our experiments, the EPR spectra were normalized with respect to tissue volume. Conducting experiments at liquid nitrogen temperature leads to some experimental advantages. The freezing of the spin adducts renders them stable over a longer period, which allows ample time to analyze tissue samples for ROS. The dielectric constant of ice is greatly reduced over its liquid counterpart; this property of water enables larger sample volumes to be inserted into the EPR cavity without overloading it and leads to enhanced signal detection. Due to Maxwell-Boltzmann statistics, the population difference goes up as the temperature goes down, so this phenomenon enhances the signal intensity as well. With the ‘gold standard’ assertion in mind, we investigated whether slicing tissue to assay ROS that is commonly used in fluorescence experiments will show more free radical generation than tissue of a similar volume that remains unsliced. Sliced tissue exhibited a 76% increase in ROS generation; this implies that higher ROS concentrations in sliced tissue indicate extraneous ROS generation not associated with the ROS stimulus of interest. We also investigated the role of ROS in chronic flow overload (CFO). Elevation of shear stress that increases production of vascular ROS has not been well investigated. We hypothesize that CFO increases ROS production mediated in part by NADPH oxidase, which leads to endothelial dysfunction. ROS production increased threefold in response to CFO. The endothelium dependent vasorelaxation was compromised in the CFO group. Treatment with apocynin significantly reduced ROS production in the vessel wall, preserved endothelial function, and inhibited expressions of p22/p47phox and NOX2/NOX4. The present data implicate NADPH oxidase produced ROS and eNOS uncoupling in endothelial dysfunction at 1 wk of CFO. In further work, a swine right ventricular hypertrophy (RVH) model induced by pulmonary artery (PA) banding was used to study right coronary artery (RCA) endothelial function and ROS level. Endothelial function was compromised in RCA of RVH as attributed to insufficient endothelial nitric oxide synthase cofactor tetrahydrobiopterin. In conclusion, stretch due to outward remodeling of RCA during RVH (at constant wall shear stress), similar to vessel stretch in hypertension, appears to induce ROS elevation, endothelial dysfunction, and an increase in basal tone. Finally, although hypertension-induced vascular stiffness and dysfunction are well established in patients and animal models, we hypothesize that stretch or distension due to hypertension and outward expansion is the cause of endothelial dysfunction mediated by angiotensin II type 1 (AT1) receptor in coronary arteries. The expression and activation of AT1 receptor and the production of ROS were up regulated and endothelial function deteriorated in the RCA. The acute inhibition of AT1 receptor and NADPH oxidase partially restored the endothelial function. Stretch or distension activates the AT1 receptor which mediates ROS production; this collectively leads to endothelial dysfunction in coronary arteries.Item Streptococcus agalactiae npx Is Required for Survival in Human Placental Macrophages and Full Virulence in a Model of Ascending Vaginal Infection during Pregnancy(American Society for Microbiology, 2022-11-21) Lu, Jacky; Moore, Rebecca E.; Spice, Sabrina K.; Doster, Ryan S.; Guevara, Miriam A.; Francis, Jamisha D.; Noble, Kristen N.; Rogers, Lisa M.; Talbert, Julie A.; Korir, Michelle L.; Townsend, Steven D.; Aronoff, David M.; Manning, Shannon D.; Gaddy, Jennifer A.; Medicine, School of MedicineStreptococcus agalactiae, also known as group B Streptococcus (GBS), is a Gram-positive encapsulated bacterium that colonizes the gastrointestinal tract of 30 to 50% of humans. GBS causes invasive infection during pregnancy that can lead to chorioamnionitis, funisitis, preterm prelabor rupture of membranes (PPROM), preterm birth, neonatal sepsis, and maternal and fetal demise. Upon infecting the host, GBS encounters sentinel innate immune cells, such as macrophages, within reproductive tissues. Once phagocytosed by macrophages, GBS upregulates the expression of the gene npx, which encodes an NADH peroxidase. GBS mutants with an npx deletion (Δnpx) are exquisitely sensitive to reactive oxygen stress. Furthermore, we have shown that npx is required for GBS survival in both THP-1 and placental macrophages. In an in vivo murine model of ascending GBS vaginal infection during pregnancy, npx is required for invading reproductive tissues and is critical for inducing disease progression, including PPROM and preterm birth. Reproductive tissue cytokine production was also significantly diminished in Δnpx mutant-infected animals compared to that in animals infected with wild-type (WT) GBS. Complementation in trans reversed this phenotype, indicating that npx is critical for GBS survival and the initiation of proinflammatory signaling in the gravid host.Item What Can Cellular Redox, Iron, and Reactive Oxygen Species Suggest About the Mechanisms and Potential Therapy of COVID-19?(Frontiers, 2020-12) Muhoberac, Barry B.; Chemistry and Chemical Biology, School of ScienceAccumulating evidence suggests that there are important contributions to coronavirus disease (COVID-19) from redox imbalance and improperly coordinated iron, which cause cellular oxidative damage and stress. Cells have developed elaborate redox-dependent processes to handle and store iron, and their disfunction leads to several serious diseases. Cellular reductants are important as reactive oxygen species (ROS) scavengers and to power enzymatic repair mechanisms, but they also may help generate toxic ROS. These complicated interrelationships are presented in terms of a cellular redox/iron/ROS triad, including ROS generation both at improperly coordinated iron and enzymatically, ROS interconvertibility, cellular signaling and damage, and reductant and iron chelator concentration-dependent effects. This perspective provides the rational necessary to strongly suggest that COVID-19 disrupts this interdependent triad, producing a substantial contribution to the ROS load, which causes direct ROS-induced protein and phospholipid damage, taxes cellular resources and repair mechanisms, and alters cellular signaling, especially in the more critical acute respiratory distress syndrome (ARDS) phase of the infection. Specific suggestions for therapeutic interventions using reductants and chelators that may help treat COVID-19 are discussed.Item β-aminoisobutyric Acid, L-BAIBA, Is a Muscle-Derived Osteocyte Survival Factor(Cell Press, 2018-02-06) Kitase, Yukiko; Vallejo, Julian A.; Gutheil, William; Vemula, Harika; Jähn, Katharina; Yi, Jianxun; Zhou, Jingsong; Brotto, Marco; Bonewald, Lynda F.; Anatomy and Cell Biology, School of MedicineExercise has beneficial effects on metabolism and on tissues. The exercise-induced muscle factor β-aminoisobutyric acid (BAIBA) plays a critical role in the browning of white fat and in insulin resistance. Here we show another function for BAIBA, that of a bone-protective factor that prevents osteocyte cell death induced by reactive oxygen species (ROS). L-BAIBA was as or more protective than estrogen or N-acetyl cysteine, signaling through the Mas-Related G Protein-Coupled Receptor Type D (MRGPRD) to prevent the breakdown of mitochondria due to ROS. BAIBA supplied in drinking water prevented bone loss and loss of muscle function in the murine hindlimb unloading model, a model of osteocyte apoptosis. The protective effect of BAIBA was lost with age, not due to loss of the muscle capacity to produce BAIBA but likely to reduced Mrgprd expression with aging. This has implications for understanding the attenuated effect of exercise on bone with aging.,