501 results on '"Hampton, Mark B."'
Search Results
152. Regulation of Apoptosis by Vitamin C
- Author
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Vissers, Margret C.M., primary, Lee, Wai-Gin, additional, and Hampton, Mark B., additional
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- 2001
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153. Hypochlorous acid causes caspase activation and apoptosis or growth arrest in human endothelial cells
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VISSERS, Margret C.M., primary, PULLAR, Juliet M., additional, and HAMPTON, Mark B., additional
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- 1999
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154. Methods for quantifying phagocytosis and bacterial killing by human neutrophils
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Hampton, Mark B., primary and Winterbourn, Christine C., additional
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- 1999
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155. Involvement of Caspases in Neutrophil Apoptosis: Regulation by Reactive Oxygen Species
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Fadeel, Bengt, primary, Åhlin, Anders, additional, Henter, Jan-Inge, additional, Orrenius, Sten, additional, and Hampton, Mark B., additional
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- 1998
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156. Redox regulation of apoptotic cell death in the immune system
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Hampton, Mark B, primary and Orrenius, Sten, additional
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- 1998
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157. Inside the Neutrophil Phagosome: Oxidants, Myeloperoxidase, and Bacterial Killing
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Hampton, Mark B., primary, Kettle, Anthony J., additional, and Winterbourn, Christine C., additional
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- 1998
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158. Corrigendum to: Cytochrome c release and caspase activation in hydrogen peroxide- and tributyltin-induced apoptosis (FEBS 20394)
- Author
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Stridh, Hélène, primary, Kimland, Monica, additional, Jones, Dean P., additional, Orrenius, Sten, additional, and Hampton, Mark B., additional
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- 1998
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- View/download PDF
159. Activation of NF-κB in human neutrophils during phagocytosis of bacteria independently of oxidant generation
- Author
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Vollebregt, Marieke, primary, Hampton, Mark B., additional, and Winterbourn, Christine C., additional
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- 1998
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160. Cytochrome c release and caspase activation in hydrogen peroxide‐ and tributyltin‐induced apoptosis
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Stridh, Hélène, primary, Kimland, Monica, additional, Jones, Dean P., additional, Orrenius, Sten, additional, and Hampton, Mark B., additional
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- 1998
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161. Redox regulation of apoptotic cell death
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Hampton, Mark B., primary and Orrenius, Sten, additional
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- 1998
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162. Dual regulation of caspase activity by hydrogen peroxide: implications for apoptosis
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Hampton, Mark B, primary and Orrenius, Sten, additional
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- 1997
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163. Mitochondria as the focus of apoptosis research
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Orrenius, Sten, primary, Burgess, David H, additional, Hampton, Mark B, additional, and Zhivotovsky, Boris, additional
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- 1997
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164. Involvement of extracellular calcium in phosphatidylserine exposure during apoptosis
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Hampton, Mark B., primary, Vanags, Daina M., additional, Isabella Pörn-Ares, M., additional, and Orrenius, Sten, additional
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- 1996
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165. Modification of neutrophil oxidant production with diphenyleneiodonium and its effect on bacterial killing
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Hampton, Mark B., primary and Winterbourn, Christine C., additional
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- 1995
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166. A single assay for measuring the rates of phagocytosis and bacterial killing by neutrophils
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Hampton, Mark B, primary, Vissers, Margret C M, additional, and Winterbourn, Christine C, additional
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- 1994
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167. Hyperoxidized peroxiredoxin 2 interacts with the protein disulfideisomerase ERp46.
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PACE, Paul E., PESKIN, Alexander V., HAN, Min-Hi, HAMPTON, Mark B., and WINTERBOURN, Christine C.
- Subjects
PEROXIREDOXINS ,OXIDATION ,CYSTEINE ,OXIDATION-reduction reaction ,ENDOTHELIAL cells ,INTRAMOLECULAR forces ,GAYLUSSACIA - Abstract
Prx (peroxiredoxin) 2 protects cells from deleterious oxidative damage. It catalyses the breakdown of hydroperoxides through a highly reactive cysteine residue and has been linked to chaperone activity that promotes cell survival under conditions of oxidative stress. It may also be involved in redox signalling by binding to other proteins. In the present study we have searched for binding partners of Prx2 in H2O2-treated Jurkat and human umbilical vein endothelial cells and discovered that the hyperoxidized form selectively co-precipitated with the protein disulfide-isomerase ERp46. Mutant analyses revealed that loss of the peroxidative cysteine residue of Prx2 also facilitated complex formation with ERp46, even without H2O2 treatment, whereas the resolving cysteine residue of Prx2 was indispensible for the interaction to occur. The complex involved a stable non-covalent interaction that was disassociated by the reduction of intramolecular disulfides in ERp46, or by disruption of the decameric structure of hyperoxidized Prx2. This is the first example of a protein interaction dependent on the hyperoxidized status of a Prx. [ABSTRACT FROM AUTHOR]
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- 2013
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168. Neutrophil NET Formation with Microbial Stimuli Requires Late Stage NADPH Oxidase Activity.
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Parker, Heather A., Jones, Harry M., Kaldor, Christopher D., Hampton, Mark B., and Winterbourn, Christine C.
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NADPH oxidase ,NEUTROPHILS ,CYTOPLASMIC granules ,CHROMATIN ,PSEUDOMONAS aeruginosa ,PROTEIN kinase C ,CHONDROITIN sulfate proteoglycan - Abstract
Neutrophils respond to a range of stimuli by releasing extracellular traps (NETs), a mesh consisting of chromatin plus granule and cytoplasmic proteins. We have investigated NET release in response to phorbol myristate acetate (PMA), Pseudomonas aeruginosa (PAO1), Staphylococcus aureus and Candida albicans, and the involvement of NADPH oxidase (NOX2) and myeloperoxidase (MPO) activities. An oxidative mechanism was involved with each stimulus, and the NOX2 inhibitor diphenylene iodonium (DPI) gave almost total inhibition. Notably, DPI added up to 60–90 min after stimulation still gave significant inhibition of subsequent NET formation. As most of the NOX2 activity had already occurred by that time, this indicates a requirement for late-stage low-level oxidant production. Inhibition of histone citrullination did not suppress NET formation, indicating that this was not the essential oxidant-dependent step. With PMA and P. aeruginosa PAO1, MPO activity played an important role in the induction of NETs and MPO inhibitors added up to 30–90 min after stimulation suppressed NET formation. NET formation with S. aureus and C. albicans was insensitive to MPO inhibition. Thus, MPO products are important with some stimuli but not others. Our results extend earlier observations with PMA and show that induction of NETs by microbial stimuli requires late stage oxidant production. Others have shown that NET formation involves NOX2-dependent elastase release from granules. As this is an early event, we conclude from our results that there is more than one oxidant-dependent step. [ABSTRACT FROM AUTHOR]
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- 2021
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169. Redox Regulation of the Caspases during Apoptosisa.
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HAMPTON, MARK B., FADEEL, BENGT, and ORRENIUS, STEN
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- 1998
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170. Uptake of Helicobacter pyloriOuter Membrane Vesicles by Gastric Epithelial Cells
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Parker, Heather, Chitcholtan, Kenny, Hampton, Mark B., and Keenan, Jacqueline I.
- Abstract
ABSTRACTHelicobacter pyloribacteria colonize the human stomach where they stimulate a persistent inflammatory response. H. pyloriis considered noninvasive; however, lipopolysaccharide (LPS)-enriched outer membrane vesicles (OMV), continuously shed from the surface of this bacterium, are observed within gastric epithelial cells. The mechanism of vesicle uptake is poorly understood, and this study was undertaken to examine the roles of bacterial VacA cytotoxin and LPS in OMV binding and cholesterol and clathrin-mediated endocytosis in vesicle uptake by gastric epithelial cells. OMV association was examined using a fluorescent membrane dye to label OMV, and a comparison was made between the associations of vesicles from a VacA+strain and OMV from a VacA−isogenic mutant strain. Within 20 min, essentially all associated OMV were intracellular, and vesicle binding appeared to be facilitated by the presence of VacA cytotoxin. Uptake of vesicles from the VacA+strain was inhibited by H. pyloriLPS (58% inhibition with 50 μg/ml LPS), while uptake of OMV from the VacA−mutant strain was less affected (25% inhibition with 50 μg/ml LPS). Vesicle uptake did not require cholesterol. However, uptake of OMV from the VacA−mutant strain was inhibited by a reduction in clathrin-mediated endocytosis (42% with 15 μg/ml chlorpromazine), while uptake of OMV from the VacA+strain was less affected (25% inhibition with 15 μg/ml chlorpromazine). We conclude that VacA toxin enhances the association of H. pyloriOMV with cells and that the presence of the toxin may allow vesicles to exploit more than one pathway of internalization.
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- 2010
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171. Peroxiredoxin 2 functions as a noncatalytic scavenger of low-level hydrogen peroxide in the erythrocyte
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Low, Felicia M., Hampton, Mark B., Peskin, Alexander V., and Winterbourn, Christine C.
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Peroxiredoxin 2 (Prx2), a thiol-dependent peroxidase, is the third most abundant protein in the erythrocyte, and its absence in knock-out mice gives rise to hemolytic anemia. We have found that in human erythrocytes, Prx2 was extremely sensitive to oxidation by H2O2, as dimerization was observed after exposure of 5 × 106 cells/mL to 0.5 μM H2O2. In contrast to Prx2 in Jurkat T lymphocytes, Prx2 was resistant to overoxidation (oxidation of the cysteine thiol to a sulfinic/sulfonic acid) in erythrocytes. Reduction of dimerized Prx2 in the erythrocyte occurred very slowly, with reversal occurring gradually over a 20-minute period. Very low thioredoxin reductase activity was detected in hemolysates. We postulate that this limits the rate of Prx2 regeneration, and this inefficiency in recycling prevents the overoxidation of Prx2. We also found that Prx2 was oxidized by endogenously generated H2O2, which was mainly derived from hemoglobin autoxidation. Our results demonstrate that in the erythrocyte Prx2 is extremely efficient at scavenging H2O2 noncatalytically. Although it does not act as a classical antioxidant enzyme, its high concentration and substrate sensitivity enable it to handle low H2O2 concentrations efficiently. These unique redox properties may account for its nonredundant role in erythrocyte defense against oxidative stress.
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- 2007
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172. Detection of apoptosis by caspase‐3 activation in tracheal aspirate neutrophils from premature infants: relationship with NF‐κB activation
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Cheah, Fook‐Choe, Hampton, Mark B., Darlow, Brian A., Winterbourn, Christine C., and Vissers, Margret C. M.
- Abstract
In premature infants, inflammatory conditions in the lungs may result in the development of chronic lung disease. As neutrophil apoptosis is important for the resolution of inflammation and prevention of tissue injury, we set out to determine the extent of neutrophil apoptosis in tracheal aspirate samples from premature infants. Activation of the transcription factor nuclear factor (NF)‐κB, which causes a delay in neutrophil apoptosis, was also investigated. We obtained 68 tracheal aspirate samples from 27 infants with median gestation and birthweight of 26 weeks and 860 g, respectively. Apoptosis was assessed by immunofluorescent detection of the active form of caspase‐3, this assay being validated with peripheral blood neutrophils. Activation of NF‐κB was monitored by the nuclear translocation of the p65 subunit, detected by immunofluorescence. Cleaved caspase‐3 was detected in 11 of the 68 samples, and a median of 40% of the neutrophils showed activated caspase‐3 (range 3–92%). A majority of the samples did not show evidence of apoptosis. Caspase activation was seen in cells with multilobed nuclear morphology, suggesting that early apoptosis was detectable. There was no significant difference in respiratory outcomes between infants with or without neutrophil apoptosis. Seventeen of the 68 samples (25%) had evidence of activated NF‐κB, and a median of 20% (range 6–41%) of neutrophils showed activation. In all but one tracheal aspirate sample, there was a mutually exclusive relationship between activated caspase‐3 and NF‐κB activation, which supports in vitro observations that NF‐κB activation delays neutrophil apoptosis.
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- 2005
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173. Helicobacter pyloriOuter Membrane Vesicles Modulate Proliferation and Interleukin-8 Production by Gastric Epithelial Cells
- Author
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Ismail, Salim, Hampton, Mark B., and Keenan, Jacqueline I.
- Abstract
ABSTRACTHelicobacter pyloriinfection, which is always associated with gastritis, can progress to ulceration or malignancy. The diversity in clinical outcomes is partly attributed to the expression of virulence factors and adhesins by H. pylori. However, H. pylorimay not have to adhere to the epithelium to cause gastritis. We hypothesize that outer membrane vesicles (OMV), which are constantly shed from the surface of H. pylori, play a role as independent activators of host cell responses. In this study, we found that low doses of OMV from cagPAI+toxigenic and cagPAI−nontoxigenic strains increased proliferation of AGS gastric epithelial cells. At higher doses, we detected growth arrest, increased toxicity, and interleukin-8 (IL-8) production. The only strain differences detected were vacuolation with the toxigenic strain and higher levels of IL-8 production with OMV from the cagPAI−nontoxigenic strain. In summary, we suggest that constitutively shed OMV play a role in promoting the low-grade gastritis associated with H. pyloriinfection.
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- 2003
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174. Redox signalling and regulation of the blood-brain barrier.
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van Leeuwen, Eveline, Hampton, Mark B., and Smyth, Leon C.D.
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BLOOD-brain barrier , *OXIDATION-reduction reaction , *NEUROLOGICAL disorders , *CELL physiology , *OXIDATIVE stress , *BLOOD flow - Abstract
• Oxidative stress and blood-brain barrier disruption are both linked to neurological disease. • Redox signalling occurs under normal circumstances at the blood-brain barrier. • Neurological diseases cause increased oxidative stress in blood-brain barrier cells, disrupting normal signalling pathways. • Targeting redox signalling in blood-brain barrier cells may prevent vascular disruption and slow the progression of neurological disease. Neurological disorders are associated with increased oxidative stress. Reactive oxidants damage tissue and promote cell death, but it is apparent that oxidants can have more subtle effects on cell function through the modulation of redox-sensitive signalling pathways. Cells of the blood-brain barrier regulate the brain microenvironment but become dysfunctional during neurological disease. The blood-brain barrier is maintained by many cell types, and is modulated by redox-sensitive pathways, ranging from the cytoskeletal elements responsible for establishing a barrier, to growth factor and cytokine signalling pathways that influence neurovascular cells. During neurological disease, blood-brain barrier cells are exposed to exogenously generated oxidants from immune cells, as well as increasing endogenously oxidant production. These oxidants impair the function of the blood-brain barrier, leading to increased leakage and reduced blood flow. Reducing the impact of oxidants on the function of blood-brain barrier cells may provide new strategies for delaying the progression of neurological disease. [ABSTRACT FROM AUTHOR]
- Published
- 2020
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175. Cytochrome crelease and caspase activation in hydrogen peroxide‐ and tributyltin‐induced apoptosis
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Stridh, Hélène, Kimland, Monica, Jones, Dean P., Orrenius, Sten, and Hampton, Mark B.
- Abstract
The ability of H2O2and tributyltin (TBT) to trigger pro‐caspase activation via export of cytochrome cfrom mitochondria to the cytoplasm was investigated. Treatment of Jurkat T lymphocytes with H2O2resulted in the appearance of cytochrome cin the cytosol within 2 h. This was at least 1 h before caspase activation was observed. TBT caused cytochrome crelease already after 5 min, followed by caspase activation within 1 h. Measurement of mitochondrial membrane potential (ΔΨm) showed that both H2O2and TBT dissipated ΔΨm, but with different time courses. TBT caused a concomitant loss of ΔΨmand release of cytochrome c, whereas cytochrome crelease and caspase activation preceded any apparent ΔΨmloss in H2O2‐treated cells. Thus, our results suggest that different mechanisms are involved in triggering cytochrome crelease with these apoptosis‐inducing agents.
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- 1998
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176. Glutathione utilization protects Streptococcus pneumoniae against lactoperoxidase-derived hypothiocyanous acid.
- Author
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Shearer, Heather L., Paton, James C., Hampton, Mark B., and Dickerhof, Nina
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STREPTOCOCCUS pneumoniae , *GLUTATHIONE peroxidase , *ATP-binding cassette transporters , *COMMUNITY-acquired pneumonia , *MOLECULAR weights , *LACTOPEROXIDASE , *GLUTATHIONE - Abstract
Streptococcus pneumoniae is the leading cause of community-acquired pneumonia, resulting in more than one million deaths each year worldwide. This pathogen generates large amounts of hydrogen peroxide (H 2 O 2), which will be converted to hypothiocyanous acid (HOSCN) by lactoperoxidase (LPO) in the human respiratory tract. S. pneumoniae has been shown to be more resistant to HOSCN than some bacteria, and sensitizing S. pneumoniae to HOSCN may be a novel treatment strategy for combating this deadly pathogen. In this study we investigated the role of the low molecular weight thiol glutathione in HOSCN resistance. S. pneumoniae does not synthesize glutathione but imports it from the environment via an ABC transporter. Upon treatment of S. pneumoniae with HOSCN, bacterial glutathione was reversibly oxidized in a time- and dose-dependent manner, and intracellular proteins became glutathionylated. Bacterial death was observed when the reduced glutathione pool dropped below 20%. A S. pneumoniae mutant unable to import glutathione (Δ gshT) was more readily killed by exogenous HOSCN. Furthermore, bacterial growth in the presence of LPO converting bacterial H 2 O 2 to HOSCN was significantly impeded in mutants that were unable to import glutathione, or mutants unable to recycle oxidized glutathione (Δ gor). This research highlights the importance of glutathione in protecting S. pneumoniae from HOSCN. Limiting glutathione utilization by S. pneumoniae may be a way to limit colonization and pathogenicity. [Display omitted] • Streptococcus pneumoniae is relatively resistant to lactoperoxidase-derived HOSCN. • S. pneumoniae imports its main LMW thiol GSH from the environment. • GSH is oxidized in HOSCN-treated S. pneumoniae, mirrored by bacterial death. • S. pneumoniae mutants unable to import GSH are more susceptible to killing by HOSCN. • GSH transport and recycling is key for S. pneumoniae growth in the presence of LPO. [ABSTRACT FROM AUTHOR]
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- 2022
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177. Comparing automated cell imaging with conventional methods of measuring cell proliferation and viability.
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Featherston, Therese, Helem, Shaya, Smyth, Leon C. D., Hampton, Mark B., and Paumann-Page, Martina
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AbstractThe ability to assess cell proliferation and viability is essential for assessing new drug treatments, particularly in cancer drug discovery. This study describes a new method that uses a plate reader digital microscopy cell imaging and analysis system to assess cell proliferation and viability. This imaging system utilizes high throughput fluorescence microscopy with two fluorescent probes: cell membrane-impermeable SYTOX green and nuclear binding Hoechst-33342. Here we compare this technology to other known viability assays, namely: propidium iodide (PI)-based flow cytometry, and sulforhodamine B (SRB) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) based plate reader assays. These methods were assessed based on their effectiveness in detecting the cell numbers of two adherent cell lines and one suspension cell line. Automated cell imaging was most accurate at measuring cell number in both adherent and suspension cell lines. The PI-based flow cytometry method was more difficult to use with adherent cells, while the SRB and MTT assays had difficulties when monitoring cells in suspension. Despite these challenges, it was possible to obtain similar results when quantifying the effect of cytotoxic compounds. This study demonstrates that the digital microscopy automated cell imaging system is an effective method for assessing cell proliferation and the cytotoxic effect of compounds on both adherent and suspension cell lines. [ABSTRACT FROM AUTHOR]
- Published
- 2024
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178. Peroxiredoxins in Colorectal Cancer: Predictive Biomarkers of Radiation Response and Therapeutic Targets to Increase Radiation Sensitivity?
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Fischer, Jesse, Eglinton, Tim W., Frizelle, Frank A., and Hampton, Mark B.
- Subjects
PEROXIREDOXINS ,COLON cancer ,BIOLOGICAL tags ,RADIATION ,SENSITIVITY analysis - Abstract
Colorectal cancer (CRC) is the third most common cancer in the Western world, with one-third of cases located in the rectum. Preoperative radiotherapy is the standard of care for many patients with rectal cancer but has a highly variable response rate. The ability to predict response would be of great clinical utility. The response of cells to ionizing radiation is known to involve immediate damage to biomolecules and more sustained disruption of redox homeostasis leading to cell death. The peroxiredoxins are an important group of thiol-dependent antioxidants involved in protecting cells from oxidative stress and regulating signaling pathways involved in cellular responses to oxidative stress. All six human peroxiredoxins have shown increased expression in CRC and may be associated with clinicopathological features and tumor response to ionizing radiation. Peroxiredoxins can act as markers of oxidative stress in various biological systems but they have not been investigated in this capacity in CRC. As such, there is currently insufficient evidence to support the role of peroxiredoxins as clinical biomarkers, but it is an area worthy of investigation. Future research should focus on the in vivo response of rectal cancer to radiotherapy and the redox status of peroxiredoxins in rectal cancer cells, in order to predict response to radiotherapy. The peroxiredoxin system is also a potential therapeutic target for CRC. [ABSTRACT FROM AUTHOR]
- Published
- 2018
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179. Evaluating the bactericidal action of hypochlorous acid in culture media.
- Author
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Ashby, Louisa V., Springer, Reuben, Hampton, Mark B., Kettle, Anthony J., and Winterbourn, Christine C.
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HYPOCHLORITES , *BACTERICIDAL action , *CELL suspensions , *CELL culture , *OXIDIZING agents , *CULTURE - Abstract
The bactericidal activity of the physiological oxidant hypochlorous acid (HOCl) is commonly studied in a variety of laboratory media. Reactive with numerous targets, HOCl will rapidly lose its toxicity via reduction or be converted to chloramines and other less toxic species. The objective of this study was to test the influence of various media, temperature and reaction time on the toxicity of HOCl. After incubating bacteria in media dosed with reagent HOCl, the bactericidal outcome was measured by colony forming ability. In parallel, we determined the HOCl and chloramine content after dosing media alone. Our results showed that more reagent HOCl was required to kill bacteria in culture media than in aqueous buffer, and this corresponded to the lower concentration of reactive chlorine species achieved in the media. RPMI and MOPS minimal medium retained significant oxidising equivalents after HOCl-dosing, but more nutrient-rich media such as MEM, DMEM, LB and TSB, had higher scavenging capacity. Other factors that lowered the bactericidal strength of HOCl were longer lag-times and raised temperature when pre-dosing media, and insufficient incubation time of cells with the HOCl-treated media. In summary, we demonstrate that the choice of media as well as procedural details within experiments crucially impact the cellular toxicity of HOCl. These factors influence the nature and concentration of oxidants generated, and therefore are critical in affecting cellular responses. Image 1 • Reagent HOCl reacts with the culture media of cell suspensions. • The bactericidal effect of HOCl depends on the scavenging capability of media. • HOCl will generate different reactive species depending on media constituents. • Variations in temperature and time also affect the bactericidal effect of oxidants. [ABSTRACT FROM AUTHOR]
- Published
- 2020
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180. Redox biology: Signaling via a peroxiredoxin sensor.
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Winterbourn, Christine C and Hampton, Mark B
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HYDROGEN peroxide , *TRANSCRIPTION factors , *THIOLS , *DISULFIDES , *OXIDATION of proteins - Abstract
The author discusses hydrogen peroxide that regulates cell signaling pathways through oxidation of specific thiol proteins, and reflects on a study which described a relay system involving peroxiredoxin 2 (Prx2) as a peroxide sensor that oxidizes the mammalian transcription factor STAT3 via a mixed disulfide intermediate. It also discusses two possible molecular mechanisms can be responsible for the transfer of oxidizing equivalents between the thiols of Prx2 and a target such as STAT3.
- Published
- 2015
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181. Corrigendum to: Cytochrome c release and caspase activation in hydrogen peroxide- and tributyltin-induced apoptosis (FEBS 20394) [FEBS Letters 429 (1998) 351–355]1PII: S0014-5793(98)00630-91
- Author
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Stridh, Hélène, Kimland, Monica, Jones, Dean P., Orrenius, Sten, and Hampton, Mark B.
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182. Oxidation of caspase-8 by hypothiocyanous acid enables TNF-mediated necroptosis.
- Author
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Bozonet, Stephanie M., Magon, Nicholas J., Schwartfeger, Abigail J., Konigstorfer, Andreas, Heath, Sarah G., Vissers, Margreet C. M., Morris, Vanessa K., Göbl, Christoph, Murphy, James M., Salvesen, Guy S., and Hampton, Mark B.
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CASPASES , *RECEPTOR-interacting proteins , *TUMOR necrosis factors , *PROTEIN kinases , *CASPASE inhibitors - Abstract
Necroptosis is a form of regulated cell death triggered by various host and pathogen-derived molecules during infection and inflammation. The essential step leading to necroptosis is phosphorylation of the mixed lineage kinase domain-like protein by receptor-interacting protein kinase 3. Caspase-8 cleaves receptor-interacting protein kinases to block necroptosis, so synthetic caspase inhibitors are required to study this process in experimental models. However, it is unclear how caspase-8 activity is regulated in a physiological setting. The active site cysteine of caspases is sensitive to oxidative inactivation, so we hypothesized that oxidants generated at sites of inflammation can inhibit caspase-8 and promote necroptosis. Here, we discovered that hypothiocyanous acid (HOSCN), an oxidant generated in vivo by heme peroxidases including myeloperoxidase and lactoperoxidase, is a potent caspase-8 inhibitor. We found HOSCN was able to promote necroptosis in mouse fibroblasts treated with tumor necrosis factor. We also demonstrate purified caspase-8 was inactivated by low concentrations of HOSCN, with the predominant product being a disulfide-linked dimer between Cys360 and Cys409 of the large and small catalytic subunits. We show oxidation still occurred in the presence of reducing agents, and reduction of the dimer was slow, consistent with HOSCN being a powerful physiological caspase inhibitor. While the initial oxidation product is a dimer, further modification also occurred in cells treated with HOSCN, leading to higher molecular weight caspase-8 species. Taken together, these findings indicate major disruption of caspase-8 function and suggest a novel mechanism for the promotion of necroptosis at sites of inflammation. [ABSTRACT FROM AUTHOR]
- Published
- 2023
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183. MerA functions as a hypothiocyanous acid reductase and defense mechanism in Staphylococcus aureus.
- Author
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Shearer, Heather L., Loi, Vu V., Weiland, Paul, Bange, Gert, Altegoer, Florian, Hampton, Mark B., Antelmann, Haike, and Dickerhof, Nina
- Subjects
- *
CRYSTAL structure , *ACIDS , *DISULFIDES , *OXIDIZING agents - Abstract
The major pathogen Staphylococcus aureus has to cope with host‐derived oxidative stress to cause infections in humans. Here, we report that S. aureus tolerates high concentrations of hypothiocyanous acid (HOSCN), a key antimicrobial oxidant produced in the respiratory tract. We discovered that the flavoprotein disulfide reductase (FDR) MerA protects S. aureus from this oxidant by functioning as a HOSCN reductase, with its deletion sensitizing bacteria to HOSCN. Crystal structures of homodimeric MerA (2.4 Å) with a Cys43–Cys48 intramolecular disulfide, and reduced MerACys43S (1.6 Å) showed the FAD cofactor close to the active site, supporting that MerA functions as a group I FDR. MerA is controlled by the redox‐sensitive repressor HypR, which we show to be oxidized to intermolecular disulfides under HOSCN stress, resulting in its inactivation and derepression of merA transcription to promote HOSCN tolerance. Our study highlights the HOSCN tolerance of S. aureus and characterizes the structure and function of MerA as a major HOSCN defense mechanism. Crippling the capacity to respond to HOSCN may be a novel strategy for treating S. aureus infections. [ABSTRACT FROM AUTHOR]
- Published
- 2023
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184. N-terminal Proline of Macrophage Migration Inhibitory Factor as a Biological Target of Isothiocyanates
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Brown, Kristin K and Hampton, Mark B
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- 2010
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185. A newly identified flavoprotein disulfide reductase Har protects Streptococcus pneumoniae against hypothiocyanous acid.
- Author
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Shearer, Heather L., Pace, Paul E., Paton, James C., Hampton, Mark B., and Dickerhof, Nina
- Subjects
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STREPTOCOCCUS pneumoniae , *HYDROGEN peroxide , *LACTOPEROXIDASE , *THIOREDOXIN , *BACTERIAL growth , *PEROXIDASE , *FLAVOPROTEINS - Abstract
Hypothiocyanous acid (HOSCN) is an antimicrobial oxidant produced from hydrogen peroxide and thiocyanate anions by heme peroxidases in secretory fluids such as in the human respiratory tract. Some respiratory tract pathogens display tolerance to this oxidant, which suggests that there might be therapeutic value in targeting HOSCN defense mechanisms. However, surprisingly little is known about how bacteria protect themselves from HOSCN. We hypothesized that tolerant pathogens have a flavoprotein disulfide reductase that uses NAD(P)H to directly reduce HOSCN, similar to thioredoxin reductase in mammalian cells. Here, we report the discovery of a previously uncharacterized flavoprotein disulfide reductase with HOSCN reductase activity, which we term Har (hypothiocyanous acid reductase), in Streptococcus pneumoniae, a bacterium previously found to be tolerant of HOSCN. S. pneumoniae generates large amounts of hydrogen peroxide that can be converted to HOSCN in the respiratory tract. Using deletion mutants, we demonstrate that the HOSCN reductase is dispensable for growth of S. pneumoniae in the presence of lactoperoxidase and thiocyanate. However, bacterial growth in the HOSCN-generating system was completely crippled when deletion of HOSCN reductase activity was combined with disruption of GSH import or recycling. Our findings identify a new bacterial HOSCN reductase and demonstrate a role for this protein in combination with GSH utilization to protect S. pneumoniae from HOSCN. [ABSTRACT FROM AUTHOR]
- Published
- 2022
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186. Corrigendum to: Cytochrome crelease and caspase activation in hydrogen peroxide‐ and tributyltin‐induced apoptosis (FEBS 20394)
- Author
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Stridh, Hélène, Kimland, Monica, Jones, Dean P., Orrenius, Sten, and Hampton, Mark B.
- Published
- 1998
- Full Text
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187. Neutrophil-vascular interactions drive myeloperoxidase accumulation in the brain in Alzheimer's disease.
- Author
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Smyth, Leon C. D., Murray, Helen C., Hill, Madison, van Leeuwen, Eve, Highet, Blake, Magon, Nicholas J., Osanlouy, Mahyar, Mathiesen, Sophie N., Mockett, Bruce, Singh-Bains, Malvindar K., Morris, Vanessa K., Clarkson, Andrew N., Curtis, Maurice A., Abraham, Wickliffe C., Hughes, Stephanie M., Faull, Richard L. M., Kettle, Anthony J., Dragunow, Mike, and Hampton, Mark B.
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ALZHEIMER'S disease , *MYELOPEROXIDASE , *AMYLOID beta-protein precursor - Abstract
Introduction: Neutrophil accumulation is a well-established feature of Alzheimer's disease (AD) and has been linked to cognitive impairment by modulating disease-relevant neuroinflammatory and vascular pathways. Neutrophils express high levels of the oxidant-generating enzyme myeloperoxidase (MPO), however there has been controversy regarding the cellular source and localisation of MPO in the AD brain. Materials and methods: We used immunostaining and immunoassays to quantify the accumulation of neutrophils in human AD tissue microarrays and in the brains of APP/PS1 mice. We also used multiplexed immunolabelling to define the presence of NETs in AD. Results: There was an increase in neutrophils in AD brains as well as in the murine APP/PS1 model of AD. Indeed, MPO expression was almost exclusively confined to S100A8-positive neutrophils in both human AD and murine APP/PS1 brains. The vascular localisation of neutrophils in both human AD and mouse models of AD was striking and driven by enhanced neutrophil adhesion to small vessels. We also observed rare infiltrating neutrophils and deposits of MPO around plaques. Citrullinated histone H3, a marker of neutrophil extracellular traps (NETs), was also detected in human AD cases at these sites, indicating the presence of extracellular MPO in the vasculature. Finally, there was a reduction in the endothelial glycocalyx in AD that may be responsible for non-productive neutrophil adhesion to the vasculature. Conclusion: Our report indicates that vascular changes may drive neutrophil adhesion and NETosis, and that neutrophil-derived MPO may lead to vascular oxidative stress and be a relevant therapeutic target in AD. [ABSTRACT FROM AUTHOR]
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- 2022
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188. Redox proteomics of thiol proteins in mouse heart during ischemia/reperfusion using ICAT reagents and mass spectrometry
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Kumar, Vikas, Kleffmann, Torsten, Hampton, Mark B., Cannell, Mark B., and Winterbourn, Christine C.
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PROTEOMICS , *THIOLS , *OXYGEN in the body , *MASS spectrometry , *REPERFUSION injury , *N-ethylmaleimide , *LIPID metabolism - Abstract
Abstract: There is strong evidence for the involvement of reactive oxygen species in ischemia/reperfusion injury. Although oxidation of individual thiol proteins has been reported, more extensive redox proteomics of hearts subjected to ischemia/reperfusion has not been performed. We have carried out an exploratory study using mass spectrometry with isotope-coded affinity tags (ICAT) aimed at identifying reversible oxidative changes to protein thiols in Langendorff perfused isolated mouse hearts subjected to 20min ischemia with or without aerobic reperfusion for 5 or 30min. Reduced thiols were blocked by adding N-ethylmaleimide during protein extraction, then reversibly oxidized thiols in extracts of control perfused and treated hearts were reduced and labeled with the light and heavy ICAT reagents, respectively. Protein extracts were mixed in equal amounts and relative proportions of the isotope-labeled peaks were used to quantify oxidative changes between the control and the treated groups. Approximately 300 peptides with ICAT signatures were reliably identified in each sample, with 181 peptides from 118 proteins common to all treatments. A proportion showed elevated ICAT ratios, consistent with reversible thiol oxidation. This was most evident after early reperfusion, with apparent reversal after longer reperfusion. In comparison, there was gradual accumulation of protein carbonyls and loss of GSH with longer reperfusion. Many of the thiol changes were in mitochondrial proteins, including components of electron transport complexes and enzymes involved in lipid metabolism. The results are consistent with mitochondria being a major site of oxidant generation during early cardiac reperfusion and mitochondrial thiol proteins being targets for oxidation. [Copyright &y& Elsevier]
- Published
- 2013
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189. Reactions of Superoxide with Myeloperoxidase.
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Kettle, Anthony J., Anderson, Robert F., Hampton, Mark B., and Winterbourn, Christine C.
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SUPEROXIDES , *PEROXIDASE , *MICROORGANISMS , *RADIATION chemistry , *ORGANIC compounds , *HYDROGEN peroxide - Abstract
When neutrophils ingest bacteria, they discharge superoxide and myeloperoxidase into phagosomes. Both are essential for killing of the phagocytosed micro-organisms. It is generally accepted that superoxide is a precursor of hydrogen peroxide which myeloperoxidase uses to oxidize chloride to hypochlorous acid. Previously, we demonstrated that superoxide modulates the chlorination activity of myeloperoxidase by reacting with its ferric and compound II redox states. In this investigation we used pulse radiolysis to determine kinetic parameters of superoxide reacting with redox forms of myeloperoxidase and used these data in a steady-state kinetic analysis. We provide evidence that superoxide reacts with compound I and compound III. Our estimates of the rate constants for the reaction of superoxide with compound I, compound II, and compound III are 5 × 106 M-1 s-1, 5.5 ± 0.4 × 106 M-1 s-1, and 1.3 ± 0.2 × 105 M-1 s-1, respectively. These reactions define new activities for myeloperoxidase. It will act as a superoxide dismutase when superoxide reacts consecutively with ferric myeloperoxidase and compound III. It will also act as a superoxidase by using hydrogen peroxide to oxidize superoxide via compound I and compound II. The favorable kinetics of these reactions indicate that, within the confines of a phagosome, superoxide will react with myeloperoxidase and affect the reactions it will catalyze. These interactions of superoxide and myeloperoxidase will have a major influence on the way neutrophils use oxygen to kill bacteria. Consequently, superoxide should be viewed as a cosubstrate that myeloperoxidase uses to elicit bacterial killing. [ABSTRACT FROM AUTHOR]
- Published
- 2007
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190. NADPH oxidase involvement in the pathology of Helicobacter pylori infection
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Keenan, Jacqueline I., Peterson, Richard A., and Hampton, Mark B.
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OXIDASES , *HELICOBACTER pylori , *INFECTION , *PATHOLOGY - Abstract
Abstract: Neutrophil oxidants are hypothesized to damage the gastric mucosa and promote carcinogenesis in people infected with Helicobacter pylori. To investigate this process we used wild-type and chronic granulomatous disease (CGD) mice with a targeted disruption of the gp91phox subunit of the NADPH oxidase. The mice were innoculated with a mouse-adapted strain of H. pylori and changes in gastric pathology were examined 12 and 30 weeks after infection. Glandular atrophy, a precursor lesion in the development of intestinal-type gastric carcinoma, and epithelial cell proliferation were both dramatically increased in the gastric body of CGD animals within 12 weeks. This correlated strongly with increased numbers of neutrophils in the mucosa (Pearson coefficient 0.97, P < 0.001). H. pylori is a noninvasive bacterium, and there was no increase in bacterial numbers in the CGD animals. Closer examination of the gastric tissue indicated the presence of degenerate neutrophils associated with atrophy. We hypothesize that the release of granule constituents from these neutrophils contributes to tissue damage. This is exacerbated by the absence of a functional NADPH oxidase, and is consistent with this enzyme complex having an important role in dampening the inflammatory response. [Copyright &y& Elsevier]
- Published
- 2005
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191. Hairpin-bisulfite sequencing of cells exposed to decitabine documents the process of DNA demethylation.
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Mayyas, Issam M, Weeks, Robert J, Day, Robert C, Magrath, Helena E, O'Connor, Karina M, Kardailsky, Olga, Hore, Timothy A, Hampton, Mark B, and Morison, Ian M
- Published
- 2021
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192. Regulation of the epigenetic landscape by immune cell oxidants.
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Das, Andrew B., Seddon, Annika R., O'Connor, Karina M., and Hampton, Mark B.
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EPIGENETICS , *OXIDIZING agents , *EPIGENOMICS , *DNA methylation , *REDUCTION potential , *OXIDATIVE stress , *MOLECULAR pathology - Abstract
Excessive production of microbicidal oxidants by neutrophils can damage host tissue. The short-term response of cells to oxidative stress is well understood, but the mechanisms behind long-term consequences require further clarification. Epigenetic pathways mediate cellular adaptation, and are therefore a potential target of oxidative stress. Indeed, there is evidence that many proteins and metabolites involved in epigenetic pathways are redox sensitive. In this review we provide an overview of the epigenetic landscape and discuss the potential for redox regulation. Using this information, we highlight specific examples where neutrophil oxidants react with epigenetic pathway components. We also use published data from redox proteomics to map out known intersections between oxidative stress and epigenetics that may signpost helpful directions for future investigation. Finally, we discuss the role neutrophils play in adaptive pathologies with a focus on tumour initiation and progression. We hope this information will stimulate further discourse on the emerging field of redox epigenomics. [Display omitted] • Neutrophils participate in tumour initiation and progression. • Dysregulation of epigenetic pathways is seen in these adaptive pathologies. • Many components of epigenetic pathways are redox-sensitive. • Neutrophil oxidants react with key enzymes and decrease cofactor availability. • Sublethal oxidant exposure modifies DNA methylation in replicating cells. [ABSTRACT FROM AUTHOR]
- Published
- 2021
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193. Mycobacterium smegmatis Resists the Bactericidal Activity of Hypochlorous Acid Produced in Neutrophil Phagosomes.
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Parker, Heather A., Dickerhof, Nina, Forrester, Lorna, Ryburn, Heath, Smyth, Leon, Messens, Joris, Aung, Htin L., Cook, Gregory M., Kettle, Anthony J., and Hampton, Mark B.
- Subjects
- *
MYCOBACTERIUM smegmatis , *PHAGOSOMES , *HYPOCHLORITES , *MYCOBACTERIAL diseases , *NEUTROPHILS - Abstract
Neutrophils are often the major leukocyte at sites of mycobacterial infection, yet little is known about their ability to kill mycobacteria. In this study we have investigated whether the potent antibacterial oxidant hypochlorous acid (HOCl) contributes to killing of Mycobacterium smegmatis when this bacterium is phagocytosed by human neutrophils. We found that M. smegmatis were ingested by neutrophils into intracellular phagosomes but were killed slowly. We measured a t1/2 of 30 min for the survival of M. smegmatis inside neutrophils, which is 5 times longer than that reported for Staphylococcus aureus and 15 times longer than Escherichia coli. Live-cell imaging indicated that neutrophils generated HOCl in phagosomes containing M. smegmatis; however, inhibition of HOCl production did not alter the rate of bacterial killing. Also, the doses of HOCl that are likely to be produced inside phagosomes failed to kill isolated bacteria. Lethal doses of reagent HOCl caused oxidation of mycothiol, the main low-m.w. thiol in this bacterium. In contrast, phagocytosed M. smegmatis maintained their original level of reduced mycothiol. Collectively, these findings suggest that M. smegmatis can cope with the HOCl that is produced inside neutrophil phagosomes. A mycothioldeficient mutant was killed by neutrophils at the same rate as wild-type bacteria, indicating that mycothiol itself is not the main driver of M. smegmatis resistance. Understanding how M. smegmatis avoids killing by phagosomal HOCl could provide new opportunities to sensitize pathogenic mycobacteria to destruction by the innate immune system. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
194. Genome-wide impact of hydrogen peroxide on maintenance DNA methylation in replicating cells.
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Seddon, Annika R., Liau, Yusmiati, Pace, Paul E., Miller, Allison L., Das, Andrew B., Kennedy, Martin A., Hampton, Mark B., and Stevens, Aaron J.
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DNA methylation , *HYDROGEN peroxide , *DNA methyltransferases , *EPIGENOMICS , *CELL division , *OXIDATIVE stress - Abstract
Background: Environmental factors, such as oxidative stress, have the potential to modify the epigenetic landscape of cells. We have previously shown that DNA methyltransferase (DNMT) activity can be inhibited by sublethal doses of hydrogen peroxide (H2O2). However, site-specific changes in DNA methylation and the reversibility of any changes have not been explored. Using bead chip array technology, differential methylation was assessed in Jurkat T-lymphoma cells following exposure to H2O2. Results: Sublethal H2O2 exposure was associated with an initial genome-wide decrease in DNA methylation in replicating cells, which was largely corrected 72 h later. However, some alterations were conserved through subsequent cycles of cell division. Significant changes to the variability of DNA methylation were also observed both globally and at the site-specific level. Conclusions: This research indicates that increased exposure to H2O2 can result in long-term alterations to DNA methylation patterns, providing a mechanism for environmental factors to have prolonged impact on gene expression. [ABSTRACT FROM AUTHOR]
- Published
- 2021
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- View/download PDF
195. Inhibition of DNA methylation in proliferating human lymphoma cells by immune cell oxidants.
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O'Connor, Karina M., Das, Andrew B., Winterbourn, Christine C., and Hampton, Mark B.
- Subjects
- *
DNA methylation , *DNA methyltransferases , *OXIDIZING agents , *T cells , *CELLS , *INHIBITION (Chemistry) - Abstract
Excessive generation of oxidants by immune cells results in acute tissue damage. One mechanism by which oxidant exposure could have long-term effects is modulation of epigenetic pathways. We hypothesized that methylation of newly synthesized DNA in proliferating cells can be altered by oxidants that target DNA methyltransferase activity or deplete its substrate, the methyl donor SAM. To this end, we investigated the effect of two oxidants produced by neutrophils, H2O2 and glycine chloramine, on maintenance DNA methylation in Jurkat T lymphoma cells. Using cell synchronization and MS-based analysis, we measured heavy deoxycytidine isotope incorporation into newly synthesized DNA and observed that a sublethal bolus of glycine chloramine, but not H2O2, significantly inhibited DNA methylation. Both oxidants inhibited DNA methyltransferase 1 activity, but only chloramine depleted SAM, suggesting that removal of substrate was the most effective means of inhibiting DNA methylation. These results indicate that immune cell- derived oxidants generated during inflammation have the potential to affect the epigenome of neighboring cells. [ABSTRACT FROM AUTHOR]
- Published
- 2020
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196. Quantifying mitochondrial respiration in human lymphocytes and monocytes challenged with hydrogen peroxide.
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Pearson, Andree G., Zawari, Masuma, Pearson, John F., and Hampton, Mark B.
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AEROBIC capacity , *HYDROGEN peroxide , *RESPIRATION , *LYMPHOCYTES , *MONOCYTES , *BLOOD cells - Abstract
Improved technology for the bioenergetic profiling of human blood cells enables population-based screening for alterations in mitochondrial respiration. Mitochondria are sensitive to oxidative stress, and the aim of this study was to quantify mitochondrial respiration in freshly isolated lymphocytes and monocytes challenged with a bolus of H2O2. Mitochondrial reserve capacity, calculated as the difference between basal oxygen consumption and maximal activity after uncoupling of the electron transport chain, was the most sensitive to H2O2. Treatment of lymphocytes with 20 μM H2O2 reduced the reserve capacity by approximately 50%, while monocyte reserve capacity was five times more resistant. Healthy donors of a similar age were tested to determine the variation between individuals, and within the same individuals tested on several different occasions. Lymphocytes obtained from a population of people aged 70–80 years showed a similar inhibition upon challenge with H2O2 as those aged 18–25 years, indicating no decline in resilience with age. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
197. Structure-function analyses of alkylhydroperoxidase D from Streptococcus pneumoniae reveal an unusual three-cysteine active site architecture.
- Author
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Yanxiang Meng, Sheen, Campbell R., Magon, Nicholas J., Hampton, Mark B., and Dobson, Renwick C. J.
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STREPTOCOCCUS pneumoniae , *QUATERNARY structure , *HYDROGEN peroxide , *MYCOBACTERIUM tuberculosis , *ARCHITECTURE , *CRYSTAL structure , *CHARGE exchange - Abstract
During aerobic growth, the Gram-positive facultative anaerobe and opportunistic human pathogen Streptococcus pneumoniae generates large amounts of hydrogen peroxide that can accumulate to millimolar concentrations. The mechanism by which this catalase-negative bacterium can withstand endogenous hydrogen peroxide is incompletely understood. The enzyme alkylhydroperoxidase D (AhpD) has been shown to contribute to pneumococcal virulence and oxidative stress responses in vivo. We demonstrate here that SpAhpD exhibits weak thiol-dependent peroxidase activity and, unlike the previously reported Mycobacterium tuberculosis AhpC/D system, SpAhpD does not mediate electron transfer to SpAhpC. A 2.3-Å resolution crystal structure revealed several unusual structural features, including a three-cysteine active site architecture that is buried in a deep pocket, in contrast to the two-cysteine active site found in other AhpD enzymes. All single-cysteine SpAhpD variants remained partially active, and LC-MS/MS analyses revealed that the third cysteine, Cys-163, formed disulfide bonds with either of two cysteines in the canonical Cys-78-X-X-Cys-81 motif. We observed that SpAhpD formed a dimeric quaternary structure both in the crystal and in solution, and that the highly conserved Asn-76 of the AhpD core motif is important for SpAhpD folding. In summary, SpAhpD is a weak peroxidase and does not transfer electrons to AhpC, and therefore does not fit existing models of bacterial AhpD antioxidant defense mechanisms. We propose that it is unlikely that SpAhpD removes peroxides either directly or via AhpC, and that SpAhpD cysteine oxidation may act as a redox switch or mediate electron transfer with other thiol proteins. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
198. Identification of Streptococcus pneumoniae genes associated with hypothiocyanous acid tolerance through genome-wide screening.
- Author
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Shearer, Heather L., Pace, Paul E., Smith, Leah M., Fineran, Peter C., Matthews, Allison J., Camilli, Andrew, Dickerhof, Nina, and Hampton, Mark B.
- Abstract
Streptococcus pneumoniae is a commensal bacterium and invasive pathogen that causes millions of deaths worldwide. The pneumococcal vaccine offers limited protection, and the rise of antimicrobial resistance will make treatment increasingly challenging, emphasizing the need for new antipneumococcal strategies. One possibility is to target antioxidant defenses to render S. pneumoniae more susceptible to oxidants produced by the immune system. Human peroxidase enzymes will convert bacterial-derived hydrogen peroxide to hypothiocyanous acid (HOSCN) at sites of colonization and infection. Here, we used saturation transposon mutagenesis and deep sequencing to identify genes that enable S. pneumoniae to tolerate HOSCN. We identified 37 genes associated with S. pneumoniae HOSCN tolerance, including genes involved in metabolism, membrane transport, DNA repair, and oxidant detoxification. Single-gene deletion mutants of the identified antioxidant defense genes sodA, spxB, trxA, and ahpD were generated and their ability to survive HOSCN was assessed. With the exception of ΔahpD, all deletion mutants showed significantly greater sensitivity to HOSCN, validating the result of the genome-wide screen. The activity of hypothiocyanous acid reductase or glutathione reductase, known to be important for S. pneumoniae tolerance of HOSCN, was increased in three of the mutants, highlighting the compensatory potential of antioxidant systems. Double deletion of the gene encoding glutathione reductase and sodA sensitized the bacteria significantly more than single deletion. The HOSCN defense systems identified in this study may be viable targets for novel therapeutics against this deadly pathogen. [ABSTRACT FROM AUTHOR]
- Published
- 2023
- Full Text
- View/download PDF
199. Peroxiredoxin interaction with the cytoskeletal-regulatory protein CRMP2: Investigation of a putative redox relay.
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Pace, Paul E., Peskin, Alexander V., Konigstorfer, Andreas, Jasoni, Christine J., Winterbourn, Christine C., and Hampton, Mark B.
- Subjects
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PEROXIREDOXINS , *OXIDATION-reduction reaction , *CYTOSKELETAL proteins , *CYSTEINE , *MICROTUBULES - Abstract
Abstract Hydrogen peroxide (H 2 O 2) acts as a signaling molecule in cells by oxidising cysteine residues in regulatory proteins such as phosphatases, kinases and transcription factors. It is unclear exactly how many of these proteins are specifically targeted by H 2 O 2 because they appear too unreactive to be directly oxidised. One proposal is that peroxiredoxins (Prxs) initially react with H 2 O 2 and then oxidise adjacent proteins via a thiol relay mechanism. The aim of this study was to identify constitutive interaction partners of Prx2 in Jurkat T-lymphoma cells, in which thiol protein oxidation occurs at low micromolar concentrations of H 2 O 2. Immunoprecipitation and proximity ligation assays identified a physical interaction between collapsin response mediator protein 2 (CRMP2) and cytoplasmic Prx2. CRMP2 regulates microtubule structure during lymphocyte migration and neuronal development. Exposure of Jurkat cells to low micromolar levels of H 2 O 2 caused rapid and reversible oxidation of CRMP2, in parallel with Prx2 oxidation, despite purified recombinant CRMP2 protein reacting slowly with H 2 O 2 (k~1 M−1s−1). Lowering Prx expression should inhibit oxidation of proteins oxidised by a relay mechanism, however knockout of Prx2 had no effect on CRMP2 oxidation. CRMP2 also interacted with Prx1, suggesting redundancy in single knockout cells. Prx 1 and 2 double knockout Jurkat cells were not viable. An interaction between Prx2 and CRMP2 was also detected in other human and rodent cells, including primary neurons. However, low concentrations of H 2 O 2 did not cause CRMP2 oxidation in these cells. This indicates a cell-type specific mechanism for promoting CRMP2 oxidation in Jurkat cells, with insufficient evidence to attribute oxidation to a Prx-dependent redox relay. Highlights • Cytosolic peroxiredoxins constitutively interact with CRMP2 in several different cell types. • Addition of H 2 O 2 to Jurkat T lymphoma cells results in CRMP2 oxidation. • Recombinant CRMP2 has low reactivity with H 2 O 2. • CRMP2 remained sensitive to oxidation in single peroxiredoxin knockout Jurkat cells. • The data suggest a cell-type specific mechanism to increase the sensitivity of CRMP2 to oxidation. Graphical abstract fx1 [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
- View/download PDF
200. Frailty in surgical patients.
- Author
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Richards, Simon J. G., Frizelle, Frank A., Geddes, John A., Eglinton, Tim W., and Hampton, Mark B.
- Subjects
- *
FRAGILITY (Psychology) , *OLDER patients , *HEALTH of older people , *FRAIL elderly , *SURGICAL complications - Abstract
Objective: To describe the current definitions, aetiology, assessment tools and clinical implications of frailty in modern surgical practice.Background: Frailty is a critical issue in modern surgical practice due to its association with adverse health events and poor post-operative outcomes. The global population is rapidly ageing resulting in more older patients presenting for surgery. With this, the number of frail patients presenting for surgery is also increasing. Despite the identification of frailty as a significant predictor of poor health outcomes, there is currently no consensus on how to define, measure and diagnose this important syndrome.Methods: Relevant references were identified through keyword searches of the Cochran, MEDLINE and EMbase databases.Results: Despite the lack of a gold standard operational definition, frailty can be conceptualised as a state of increased vulnerability resulting from a decline in physiological reserve and function across multiple organ systems, such that the ability to withstand stressors is impaired. Multiple studies have shown a strong association between frailty and adverse peri-operative outcomes. Frailty may be assessed using multiple tools; however, the ideal tool for use in a clinical setting has yet to be identified. Despite the association between frailty and adverse outcomes, few interventions have been shown to improve outcomes in these patients.Conclusion: Frailty encompasses a group of individuals at high risk of adverse post-operative outcomes. Further work exploring ways to optimally assess and target interventions towards these patients should be the focus of ongoing research. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
- View/download PDF
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