247 results on '"Hampton, Mark B."'
Search Results
202. Induction of apoptosis by phenethyl isothiocyanate in cells overexpressing Bcl-XL
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Cuddihy, Sarah L., Brown, Kristin K., Thomson, Susan J., and Hampton, Mark B.
- Subjects
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APOPTOSIS , *CELL death , *STYRENE , *PHYTOCHEMICALS - Abstract
Abstract: Isothiocyanates are a class of phytochemicals able to induce apoptosis in numerous cells including Jurkat T-lymphoma cells overexpressing the oncoprotein Bcl-2. To test if isothiocyanates are also effective against other anti-apoptotic members of the Bcl-2 family we generated Jurkat cells stably overexpressing Bcl-XL. Phenethyl isothiocyanate (PEITC) was cytotoxic to these cells, with an LD50 ranging from 9 to 18μM depending on the level of Bcl-XL expression. Apoptosis induction in response to PEITC was confirmed by caspase activation and phosphatidylserine exposure. Isothiocyanates specifically target cysteine residues, therefore we tested the hypothesis that PEITC directly impairs Bcl-2 and Bcl-XL activity by interacting with their conserved cysteine residues. Jurkat cells overexpressing double cysteine mutants of Bcl-2 were generated, but they remained sensitive to PEITC. We conclude that PEITC antagonizes the action of anti-apoptotic Bcl-2 family members via an indirect mechanism. [Copyright &y& Elsevier]
- Published
- 2008
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203. The thioredoxin reductase inhibitor auranofin triggers apoptosis through a Bax/Bak-dependent process that involves peroxiredoxin 3 oxidation
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Cox, Andrew G., Brown, Kristin K., Arner, Elias S.J., and Hampton, Mark B.
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CELL death , *CYCLOOXYGENASE 2 inhibitors , *CELECOXIB , *APOPTOSIS - Abstract
Abstract: Thioredoxin reductase (TrxR) is a key selenoprotein antioxidant enzyme and a potential target for anti-cancer drugs. One potent inhibitor of TrxR is the gold (I) compound auranofin, which can trigger mitochondrial-dependent apoptosis pathways. The exact mechanism of apoptosis induction by auranofin is not yet clear, but there are indications that mitochondrial oxidative stress is a central event. We assessed the redox state of the peroxiredoxins (Prxs) in Jurkat T-lymphoma cells treated with auranofin, and found that mitochondrial Prx3 was considerably more sensitive to oxidation than the cytosolic Prx1 and 2, indicating selective mitochondrial stress. Prx3 oxidation was detected at apoptotic doses of auranofin in several cell types, and occurred before other mitochondrial events including cytochrome c release and mitochondrial depolarisation. Auranofin was also able to sensitise U937 cells to TNF-α-mediated apoptosis. Auranofin-induced apoptosis was effectively blocked by the overexpression of Bcl-2, and Bax/Bak deficient mouse embryonic fibroblasts were also resistant to apoptosis, indicating a central role for the pro-apoptotic proteins of this family in auranofin-triggered apoptosis. Auranofin exposure inhibited the proliferation of apoptosis-resistant cells, and at higher doses of auranofin could cause cell death through necrosis. We conclude that auranofin induces apoptosis in cells through a Bax/Bak-dependent mechanism associated with selective disruption of mitochondrial redox homeostasis in conjunction with oxidation of Prx3. [Copyright &y& Elsevier]
- Published
- 2008
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204. Mitochondrial peroxiredoxin 3 is rapidly oxidized in cells treated with isothiocyanates
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Brown, Kristin K., Eriksson, Sofi E., Arnér, Elias S.J., and Hampton, Mark B.
- Subjects
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PHYTOCHEMICALS , *CYSTEINE proteinases , *T cells , *OXIDATION-reduction reaction - Abstract
Abstract: Isothiocyanates are phytochemicals with anti-cancer properties that include the ability to trigger apoptosis. A substantial body of evidence suggests that reaction of the electrophilic isothiocyanate moiety with cysteine residues in cellular proteins and glutathione accounts for their biological activity. In this study we investigated the effect of several different isothiocyanates on the redox states of the cysteine-dependent peroxiredoxins (Prx) in Jurkat T lymphoma cells, and compared this to known effects on the selenoprotein thioredoxin reductase, glutathione reductase and intracellular GSH levels. Interestingly, oxidation of mitochondrial Prx3 could be detected as early as 5 min after exposure of cells to phenethyl isothiocyanate, with complete oxidation occurring at doses that only had small inhibitory effects on total cellular thioredoxin reductase and glutathione reductase activities. Peroxiredoxin oxidation was specific to the mitochondrial isoform with cytoplasmic Prx1 and Prx2 maintained in their reduced forms at all analyzed time points and concentrations of isothiocyanate. Phenethyl isothiocyanate could react with purified Prx3 directly, but it did not oxidize Prx3 or promote its oxidation by hydrogen peroxide. A selection of aromatic and alkyl isothiocyanates were tested and while all lowered cellular GSH levels, only the isothiocyanates that caused Prx3 oxidation were able to trigger cell death. We propose that pro-apoptotic isothiocyanates selectively disrupt mitochondrial redox homeostasis, as indicated by Prx3 oxidation, and that this contributes to their pro-apoptotic activity. [Copyright &y& Elsevier]
- Published
- 2008
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205. Oxidation of mitochondrial peroxiredoxin 3 during the initiation of receptor-mediated apoptosis
- Author
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Cox, Andrew G., Pullar, Juliet M., Hughes, Gillian, Ledgerwood, Elizabeth C., and Hampton, Mark B.
- Subjects
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CELL death , *APOPTOSIS , *GEL electrophoresis , *OXIDATION - Abstract
Abstract: It is hypothesized that activation of death receptors disrupts the redox homeostasis of cells and that this contributes to the induction of apoptosis. The redox status of the peroxiredoxins, which are extremely sensitive to increases in H2O2 and disruption of the thioredoxin system, were monitored in Jurkat T lymphoma cells undergoing Fas-mediated apoptosis. The only detectable change during the early stages of apoptosis was oxidation of mitochondrial peroxiredoxin 3. Increased H2O2 triggers peroxiredoxin overoxidation to a sulphinic acid; however during apoptosis peroxiredoxin 3 was captured as a disulfide, suggesting impairment of the thioredoxin system responsible for maintaining peroxiredoxin 3 in its reduced form. Peroxiredoxin 3 oxidation was an early event, occurring within the same timeframe as increased mitochondrial oxidant production, caspase activation and cytochrome c release. It preceded other major apoptotic events including mitochondrial permeability transition and phosphatidylserine exposure, and glutathione depletion, global thiol protein oxidation and protein carbonylation. Peroxiredoxin 3 oxidation was also observed in U937 cells stimulated with TNF-α. We hypothesize that the selective oxidation of peroxiredoxin 3 leads to an increase in mitochondrial H2O2 and that this may influence the progression of apoptosis. [Copyright &y& Elsevier]
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- 2008
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206. Widespread genomic de novo DNA methylation occurs following CD8 + T cell activation and proliferation.
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Seddon AR, Damiano OM, Hampton MB, and Stevens AJ
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- Humans, CpG Islands, Epigenesis, Genetic, ADAM10 Protein genetics, ADAM10 Protein metabolism, Membrane Proteins genetics, DNA Methylation, CD8-Positive T-Lymphocytes metabolism, CD8-Positive T-Lymphocytes immunology, Lymphocyte Activation, Cell Proliferation
- Abstract
This research investigates the intricate dynamics of DNA methylation in the hours following CD8+ T cell activation, during a critical yet understudied temporal window. DNA methylation is an epigenetic modification central to regulation of gene expression and directing immune responses. Our investigation spanned 96-h post-activation and unveils a nuanced tapestry of global and site-specific methylation changes. We identified 15,626 significant differentially methylated CpGs spread across the genome, with the most significant changes occurring within the genes ADAM10 , ICA1 , and LAPTM5 . While many changes had modest effect sizes, approximately 120 CpGs exhibited a log
2 FC above 1.5, with cell activation and proliferation pathways the most affected. Relatively few of the differentially methylated CpGs occurred along adjacent gene regions. The exceptions were seven differentially methylated gene regions, with the Human T cell Receptor Alpha Joining Genes demonstrating consistent methylation change over a 3kb window. We also investigated whether an inflammatory environment could alter DNA methylation during activation, with proliferating cells exposed to the oxidant glycine chloramine. No substantial differential methylation was observed in this context. The temporal perspective of early activation adds depth to the evolving field of epigenetic immunology, offering insights with implications for therapeutic innovation and expanding our understanding of epigenetic modulation in immune function.- Published
- 2024
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207. Melanoma redox biology and the emergence of drug resistance.
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Featherston T, Paumann-Page M, and Hampton MB
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- Humans, Animals, Skin Neoplasms drug therapy, Skin Neoplasms pathology, Skin Neoplasms metabolism, Skin Neoplasms genetics, Antineoplastic Agents pharmacology, Antineoplastic Agents therapeutic use, Antioxidants pharmacology, Drug Resistance, Neoplasm, Melanoma drug therapy, Melanoma pathology, Melanoma metabolism, Melanoma genetics, Oxidation-Reduction, Oxidative Stress
- Abstract
Melanoma is the deadliest form of skin cancer, with the loss of approximately 60,000 lives world-wide each year. Despite the development of targeted therapeutics, including compounds that have selectivity for mutant oncoproteins expressed only in cancer cells, many patients are either unresponsive to initial therapy or their tumors acquire resistance. This results in five-year survival rates of below 25%. New strategies that either kill drug-resistant melanoma cells or prevent their emergence would be extremely valuable. Melanoma, like other cancers, has long been described as being under increased oxidative stress, resulting in an increased reliance on antioxidant defense systems. Changes in redox homeostasis are most apparent during metastasis and during the metabolic reprogramming associated with the development of treatment resistance. This review discusses oxidative stress in melanoma, with a particular focus on targeting antioxidant pathways to limit the emergence of drug resistant cells., (Copyright © 2024. Published by Elsevier Inc.)
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- 2024
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208. Oxidation of caspase-8 by hypothiocyanous acid enables TNF-mediated necroptosis.
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Bozonet SM, Magon NJ, Schwartfeger AJ, Konigstorfer A, Heath SG, Vissers MCM, Morris VK, Göbl C, Murphy JM, Salvesen GS, and Hampton MB
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- Animals, Mice, Inflammation metabolism, Oxidation-Reduction drug effects, Fibroblasts drug effects, Fibroblasts enzymology, Fibroblasts metabolism, Peroxidase, Lactoperoxidase, Catalytic Domain, Caspase 8 chemistry, Caspase 8 metabolism, Necroptosis drug effects, Oxidants metabolism, Oxidants pharmacology, Tumor Necrosis Factors metabolism
- 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., Competing Interests: Conflict of interest J. M. M. contributes to the development of necroptosis inhibitors in collaboration with Anaxis Pharma Pty Ltd. G.S.S. consults for Genentech Inc. All other authors declare they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2023
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209. MerA functions as a hypothiocyanous acid reductase and defense mechanism in Staphylococcus aureus.
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Shearer HL, Loi VV, Weiland P, Bange G, Altegoer F, Hampton MB, Antelmann H, and Dickerhof N
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- Humans, Disulfides, Oxidants, Oxidoreductases metabolism, Staphylococcus aureus enzymology, Staphylococcus aureus metabolism
- 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 Cys
43 -Cys48 intramolecular disulfide, and reduced MerACys43 S (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., (© 2023 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)- Published
- 2023
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210. Inside the phagosome: A bacterial perspective.
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Hampton MB and Dickerhof N
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- Humans, Bacteria, Phagocytosis, Phagosomes microbiology, Neutrophils microbiology
- Abstract
The neutrophil phagosome is one of the most hostile environments that bacteria must face and overcome if they are to succeed as pathogens. Targeting bacterial defense mechanisms should lead to new therapies that assist neutrophils to kill pathogens, but this has not yet come to fruition. One of the limiting factors in this effort has been our incomplete knowledge of the complex biochemistry that occurs within the rapidly changing environment of the phagosome. The same compartmentalization that protects host tissue also limits our ability to measure events within the phagosome. In this review, we highlight the limitations in our knowledge, and how the contribution of bacteria to the phagosomal environment is often ignored. There appears to be significant heterogeneity among phagosomes, and it is important to determine whether survivors have more efficient defenses or whether they are ingested into less threatening environments than other bacteria. As part of these efforts, we discuss how monitoring or recovering bacteria from phagosomes can provide insight into the conditions they have faced. We also encourage the use of unbiased screening approaches to identify bacterial genes that are essential for survival inside neutrophil phagosomes., (© 2023 The Authors. Immunological Reviews published by John Wiley & Sons Ltd.)
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- 2023
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211. Site-specific decreases in DNA methylation in replicating cells following exposure to oxidative stress.
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Seddon AR, Das AB, Hampton MB, and Stevens AJ
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- Oxidation-Reduction, Oligonucleotide Array Sequence Analysis, Oxidative Stress genetics, CpG Islands genetics, Epigenesis, Genetic, DNA Methylation genetics, Transcription Factors
- Abstract
Oxidative stress is a common feature of inflammation-driven cancers, and it promotes genomic instability and aggressive tumour phenotypes. It is known that oxidative stress transiently modulates gene expression through the oxidation of transcription factors and associated regulatory proteins. Neutrophils are our most abundant white blood cells and accumulate at sites of infection and inflammation. Activated neutrophils produce hypochlorous acid and chloramines, which can disrupt DNA methylation by oxidizing methionine. The goal of the current study was to determine whether chloramine exposure results in sequence-specific modifications in DNA methylation that enable long-term alterations in transcriptional output. Proliferating Jurkat T-lymphoma cells were exposed to sublethal doses of glycine chloramine and differential methylation patterns were compared using Illumina EPIC 850 K bead chip arrays. There was a substantial genome-wide decrease in methylation 4 h after exposure that correlated with altered RNA expression for 24 and 48 h, indicating sustained impacts on exposed cells. A large proportion of the most significant differentially methylated CpG sites were situated towards chromosomal ends, suggesting that these regions are most susceptible to inhibition of maintenance DNA methylation. This may contribute to epigenetic instability of chromosomal ends in rapidly dividing cells, with potential implications for the regulation of telomere length and cellular longevity., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)
- Published
- 2023
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212. Faecal Myeloperoxidase as a Biomarker of Endoscopic Activity in Inflammatory Bowel Disease.
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Swaminathan A, Borichevsky GM, Edwards TS, Hirschfeld E, Mules TC, Frampton CMA, Day AS, Hampton MB, Kettle AJ, and Gearry RB
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- Female, Humans, Biomarkers metabolism, C-Reactive Protein metabolism, Chronic Disease, Endoscopy, Gastrointestinal, Feces chemistry, Leukocyte L1 Antigen Complex metabolism, Peroxidase metabolism, Severity of Illness Index, Male, Colitis, Ulcerative diagnosis, Colitis, Ulcerative metabolism, Crohn Disease diagnosis, Crohn Disease metabolism, Inflammatory Bowel Diseases diagnosis, Inflammatory Bowel Diseases metabolism
- Abstract
Background and Aims: Inflammatory bowel disease [IBD], consisting of Crohn's disease [CD] and ulcerative colitis [UC], is a relapsing-remitting illness. Treat-to-target IBD management strategies require monitoring of gastrointestinal inflammation. This study aimed to investigate faecal myeloperoxidase [fMPO], a neutrophil granule enzyme, as a biomarker of IBD activity., Methods: Prospectively recruited participants with IBD, undergoing ileocolonoscopy for disease assessment, provided biological samples and completed symptom questionnaires prior to endoscopy. fMPO, C-reactive protein [CRP], and faecal calprotectin [fCal] were compared with validated endoscopic indices [simple endoscopic score for CD and UC endoscopic index of severity]. Receiver operating characteristic [ROC] curves assessed the performance of fMPO, CRP, and fCal in predicting endoscopic disease activity. Baseline biomarkers were used to predict a composite endpoint of complicated disease at 12 months [need for escalation of biologic/immunomodulator due to relapse, steroid use, IBD-related hospitalisation, and surgery]., Results: A total of 172 participants were recruited [91 female, 100 with CD]. fMPO was significantly correlated with endoscopic activity in both CD [r = 0.53, p < 0.01] and UC [r = 0.63, p < 0.01], and with fCal in all patients with IBD [r = 0.82, p < 0.01]. fMPO was effective in predicting moderate-to-severely active CD [AUROC 0.86, p < 0.01] and UC [AUROC 0.92, p < 0.01]. Individuals with a baseline fMPO > 26 µg/g were significantly more likely to reach the composite outcome at 12 months (hazard ratio [HR] 3.71, 95% confidence interval [CI] 2.07-6.64, p < 0.01)., Conclusions: Faecal myeloperoxidase is an accurate biomarker of endoscopic activity in IBD and predicted a more complicated IBD course during follow-up., (© The Author(s) 2022. Published by Oxford University Press on behalf of European Crohn’s and Colitis Organisation.)
- Published
- 2022
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213. Oxidation of bacillithiol during killing of Staphylococcus aureus USA300 inside neutrophil phagosomes.
- Author
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Ashby LV, Springer R, Loi VV, Antelmann H, Hampton MB, Kettle AJ, and Dickerhof N
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- Cysteine analogs & derivatives, Cysteine metabolism, Glucosamine analogs & derivatives, Humans, Hypochlorous Acid metabolism, Hypochlorous Acid pharmacology, Oxidants metabolism, Oxidation-Reduction, Peroxidase metabolism, Phagosomes metabolism, Neutrophils metabolism, Staphylococcus aureus metabolism
- Abstract
Targeting immune evasion tactics of pathogenic bacteria may hold the key to treating recalcitrant bacterial infections. Staphylococcus aureus produces bacillithiol (BSH), its major low-molecular-weight thiol, which is thought to protect this opportunistic human pathogen against the bombardment of oxidants inside neutrophil phagosomes. Here, we show that BSH was oxidized when human neutrophils phagocytosed S. aureus, but provided limited protection to the bacteria. We used mass spectrometry to measure the oxidation of BSH upon exposure of S. aureus USA300 to either a bolus of hypochlorous acid (HOCl) or a flux generated by the neutrophil enzyme myeloperoxidase. Oxidation of BSH and loss of bacterial viability were strongly correlated (r = 0.99, p < 0.001). BSH was fully oxidized after exposure of S. aureus to lethal doses of HOCl. However, there was no relationship between the initial BSH levels and the dose of HOCl required for bacterial killing. In contrast to the HOCl systems, only 50% of total BSH was oxidized when neutrophils killed the majority of phagocytosed bacteria. Oxidation of BSH was decreased upon inhibition of myeloperoxidase, implicating HOCl in phagosomal BSH oxidation. A BSH-deficient S. aureus USA300 mutant was slightly more susceptible to treatment with either HOCl or ammonia chloramine, or to killing within neutrophil phagosomes. Collectively, our data show that myeloperoxidase-derived oxidants react with S. aureus inside neutrophil phagosomes, leading to partial BSH oxidation, and contribute to bacterial killing. However, BSH offers only limited protection against the neutrophil's multifaceted killing mechanisms., (© 2022 The Authors. Journal of Leukocyte Biology published by Wiley Periodicals LLC on behalf of Society for Leukocyte Biology.)
- Published
- 2022
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214. Insights into H 2 O 2 -induced signaling in Jurkat cells from analysis of gene expression.
- Author
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Taylor MF, Black MA, Hampton MB, and Ledgerwood EC
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- Animals, Humans, Jurkat Cells, Oxidants pharmacology, Oxidative Stress, Gene Expression, Mammals, Hydrogen Peroxide pharmacology, Signal Transduction
- Abstract
Hydrogen peroxide (H
2 O2 ) is a ubiquitous oxidant produced in a regulated manner by various enzymes in mammalian cells. H2 O2 reversibly oxidizes thiol groups of cysteine residues to mediate intracellular signaling. While examples of H2 O2- dependent signaling have been reported, the exact molecular mechanism(s) of signaling and the pathways affected are not well understood. Here, the transcriptomic response of Jurkat T cells to H2 O2 was investigated to determine global effects on gene expression. With a low H2 O2 concentration (10 µM) that did not induce an oxidative stress response or cell death, extensive changes in gene expression occurred after 4 h (6803 differentially expressed genes). Of the genes with a greater then 2-fold change in expression, 85% were upregulated suggesting that in a physiological setting H2 O2 predominantly activates gene expression. Pathway analysis identified gene expression signatures associated with FOXO and NTRK signaling. These signatures were associated with an overlapping set of transcriptional regulators. Overall, our results provide a snapshot of gene expression changes in response to H2 O2, which, along with further studies, will lead to new insights into the specific pathways that are activated in response to endogenous production of H2 O2 , and the molecular mechanisms of H2 O2 signaling.- Published
- 2022
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215. A newly identified flavoprotein disulfide reductase Har protects Streptococcus pneumoniae against hypothiocyanous acid.
- Author
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Shearer HL, Pace PE, Paton JC, Hampton MB, and Dickerhof N
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- Animals, Disulfides, Heme, Humans, Hydrogen Peroxide pharmacology, Lactoperoxidase, Mammals metabolism, NAD, Oxidants metabolism, Streptococcus pneumoniae genetics, Streptococcus pneumoniae metabolism, Thioredoxin-Disulfide Reductase genetics, Thioredoxin-Disulfide Reductase metabolism, Anti-Infective Agents, Thiocyanates metabolism, Thiocyanates pharmacology
- 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., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2022
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216. Neutrophil-vascular interactions drive myeloperoxidase accumulation in the brain in Alzheimer's disease.
- Author
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Smyth LCD, Murray HC, Hill M, van Leeuwen E, Highet B, Magon NJ, Osanlouy M, Mathiesen SN, Mockett B, Singh-Bains MK, Morris VK, Clarkson AN, Curtis MA, Abraham WC, Hughes SM, Faull RLM, Kettle AJ, Dragunow M, and Hampton MB
- Subjects
- Animals, Brain metabolism, Humans, Mice, Neutrophils metabolism, Peroxidase metabolism, Alzheimer Disease metabolism, Extracellular Traps metabolism
- 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., (© 2022. The Author(s).)
- Published
- 2022
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217. Hypothiocyanous Acid Disrupts the Barrier Function of Brain Endothelial Cells.
- Author
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van Leeuwen E, Hampton MB, and Smyth LCD
- Abstract
Inflammation is a common feature of neurological diseases. During neuroinflammation, neutrophils are recruited to the brain vasculature, where myeloperoxidase can produce hypochlorous acid and the less well-studied oxidant hypothiocyanous acid (HOSCN). In this study, we exposed primary brain endothelial cells (BECs) to HOSCN and observed a rapid loss of transendothelial electrical resistance (TEER) at sublethal concentrations. Decreased barrier function was associated with a loss of tight junctions at cellular contacts and a concomitant loss of dynamic microtubules. Both tight junction and cytoskeletal disruptions were visible within 30 min of exposure, whereas significant loss of TEER took more than 1 h. The removal of the HOSCN after 30 min prevented subsequent barrier dysfunction. These results indicate that BECs are sensitive to HOSCN, resulting in the eventual loss of barrier function. We hypothesise that this mechanism may be relevant in neutrophil transmigration, with HOSCN facilitating blood-brain barrier opening at the sites of egress. Furthermore, this mechanism may be a way through which neutrophils, residing in the vasculature, can influence neuroinflammation in diseases.
- Published
- 2022
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218. Resistance of Streptococcus pneumoniae to Hypothiocyanous Acid Generated by Host Peroxidases.
- Author
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Shearer HL, Kaldor CD, Hua H, Kettle AJ, Parker HA, and Hampton MB
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- Humans, Hydrogen Peroxide, Hypochlorous Acid metabolism, Lactoperoxidase, Peroxidases, Proteins, Thiocyanates, Peroxidase metabolism, Streptococcus pneumoniae metabolism
- Abstract
Streptococcus pneumoniae is a serious human respiratory pathogen. It generates hydrogen peroxide (H
2 O2 ) as part of its normal metabolism, yet it lacks enzymes that remove this oxidant. Here we show that lactoperoxidase and myeloperoxidase, two host enzymes present in the respiratory tract, convert bacterial H2 O2 into HOSCN that S. pneumoniae can resist. We found that incubation of S. pneumoniae with myeloperoxidase in chloride-rich buffer killed the bacteria due to formation of toxic hypochlorous acid (HOCl). However, the addition of physiological concentrations of thiocyanate protected the bacteria. Similarly, S. pneumoniae remained viable in the presence of lactoperoxidase and thiocyanate even though the majority of bacterial H2 O2 was converted to hypothiocyanous acid (HOSCN). S. pneumoniae and Pseudomonas aeruginosa, another respiratory pathogen, were similarly sensitive to H2 O2 and HOCl. In contrast, S. pneumoniae tolerated much higher doses of HOSCN than P. aeruginosa. When associated with neutrophil extracellular traps (NETs), S. pneumoniae continued to generate H2 O2 , which was converted to HOCl by myeloperoxidase (MPO) present on NETs. However, there was no loss in bacterial viability because HOCl was scavenged by the NET proteins. We conclude that at sites of infection, bacteria will be protected from HOCl by thiocyanate and extracellular proteins including those associated with NETs. Resistance to HOSCN may give S. pneumoniae a survival advantage over other pathogenic bacteria. Understanding the mechanisms by which S. pneumoniae protects itself from HOSCN may reveal novel strategies for limiting the colonization and pathogenicity of this deadly pathogen.- Published
- 2022
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219. Glutathione utilization protects Streptococcus pneumoniae against lactoperoxidase-derived hypothiocyanous acid.
- Author
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Shearer HL, Paton JC, Hampton MB, and Dickerhof N
- Subjects
- Hydrogen Peroxide, Glutathione metabolism, Lactoperoxidase, Streptococcus pneumoniae drug effects, Streptococcus pneumoniae metabolism, Thiocyanates
- 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 O2 ), 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 (ΔgshT) was more readily killed by exogenous HOSCN. Furthermore, bacterial growth in the presence of LPO converting bacterial H2 O2 to HOSCN was significantly impeded in mutants that were unable to import glutathione, or mutants unable to recycle oxidized glutathione (Δgor). 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., (Copyright © 2021 Elsevier Inc. All rights reserved.)- Published
- 2022
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220. Antimicrobial Activity of Neutrophils Against Mycobacteria.
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Parker HA, Forrester L, Kaldor CD, Dickerhof N, and Hampton MB
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- Animals, Biomarkers, Cytotoxicity, Immunologic, Disease Susceptibility immunology, Extracellular Traps genetics, Extracellular Traps immunology, Host-Pathogen Interactions genetics, Humans, Immunity, Innate, Mycobacterium Infections diagnosis, Mycobacterium Infections metabolism, Neutrophil Activation genetics, Neutrophil Activation immunology, Neutrophils microbiology, Neutrophils pathology, Oxidants metabolism, Oxidative Stress, Phagocytosis genetics, Phagocytosis immunology, Phagosomes metabolism, Host-Pathogen Interactions immunology, Mycobacterium immunology, Mycobacterium Infections immunology, Mycobacterium Infections microbiology, Neutrophils immunology, Neutrophils metabolism
- Abstract
The mycobacterium genus contains a broad range of species, including the human pathogens M. tuberculosis and M. leprae . These bacteria are best known for their residence inside host cells. Neutrophils are frequently observed at sites of mycobacterial infection, but their role in clearance is not well understood. In this review, we discuss how neutrophils attempt to control mycobacterial infections, either through the ingestion of bacteria into intracellular phagosomes, or the release of neutrophil extracellular traps (NETs). Despite their powerful antimicrobial activity, including the production of reactive oxidants such as hypochlorous acid, neutrophils appear ineffective in killing pathogenic mycobacteria. We explore mycobacterial resistance mechanisms, and how thwarting neutrophil action exacerbates disease pathology. A better understanding of how mycobacteria protect themselves from neutrophils will aid the development of novel strategies that facilitate bacterial clearance and limit host tissue damage., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Parker, Forrester, Kaldor, Dickerhof and Hampton.)
- Published
- 2021
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221. Macrophage migration inhibitory factor inhibits neutrophil apoptosis by inducing cytokine release from mononuclear cells.
- Author
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Schindler L, Zwissler L, Krammer C, Hendgen-Cotta U, Rassaf T, Hampton MB, Dickerhof N, and Bernhagen J
- Subjects
- Cytokines immunology, Humans, Inflammation immunology, Receptors, Interleukin-8B immunology, Apoptosis immunology, Intramolecular Oxidoreductases immunology, Leukocytes, Mononuclear immunology, Macrophage Migration-Inhibitory Factors immunology, Neutrophils immunology
- Abstract
The chemokine-like inflammatory cytokine macrophage migration inhibitory factor (MIF) is a pivotal driver of acute and chronic inflammatory conditions, cardiovascular disease, autoimmunity, and cancer. MIF modulates the early inflammatory response through various mechanisms, including regulation of neutrophil recruitment and fate, but the mechanisms and the role of the more recently described MIF homolog MIF-2 (D-dopachrome tautomerase; D-DT) are incompletely understood. Here, we show that both MIF and MIF-2/D-DT inhibit neutrophil apoptosis. This is not a direct effect, but involves the activation of mononuclear cells, which secrete CXCL8 and other prosurvival mediators to promote neutrophil survival. Individually, CXCL8 and MIF (or MIF-2) did not significantly inhibit neutrophil apoptosis, but in combination they elicited a synergistic response, promoting neutrophil survival even in the absence of mononuclear cells. The use of receptor-specific inhibitors provided evidence for a causal role of the noncognate MIF receptor CXCR2 expressed on both monocytes and neutrophils in MIF-mediated neutrophil survival. We suggest that the ability to inhibit neutrophil apoptosis contributes to the proinflammatory role ascribed to MIF, and propose that blocking the interaction between MIF and CXCR2 could be an important anti-inflammatory strategy in the early inflammatory response., (© 2021 The Authors. Journal of Leukocyte Biology published by Wiley Periodicals LLC on behalf of Society for Leukocyte Biology.)
- Published
- 2021
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222. Ascorbate Inhibits Proliferation and Promotes Myeloid Differentiation in TP53 -Mutant Leukemia.
- Author
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Smith-Díaz CC, Magon NJ, McKenzie JL, Hampton MB, Vissers MCM, and Das AB
- Abstract
Loss-of-function mutations in the DNA demethylase TET2 are associated with the dysregulation of hematopoietic stem cell differentiation and arise in approximately 10% of de novo acute myeloid leukemia (AML). TET2 mutations coexist with other mutations in AML, including TP53 mutations, which can indicate a particularly poor prognosis. Ascorbate can function as an epigenetic therapeutic in pathological contexts involving heterozygous TET2 mutations by restoring TET2 activity. How this response is affected when myeloid leukemia cells harbor mutations in both TET2 and TP53 is unknown. Therefore, we examined the effects of ascorbate on the SKM-1 AML cell line that has mutated TET2 and TP53 . Sustained treatment with ascorbate inhibited proliferation and promoted the differentiation of these cells. Furthermore, ascorbate treatment significantly increased 5-hydroxymethylcytosine, suggesting increased TET activity as the likely mechanism. We also investigated whether ascorbate affected the cytotoxicity of Prima-1
Met , a drug that reactivates some p53 mutants and is currently in clinical trials for AML. We found that the addition of ascorbate had a minimal effect on Prima-1Met -induced cytotoxicity, with small increases or decreases in cytotoxicity being observed depending on the timing of treatment. Collectively, these data suggest that ascorbate could exert a beneficial anti-proliferative effect on AML cells harboring both TET2 and TP53 mutations whilst not interfering with targeted cytotoxic therapies such as Prima-1Met ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Smith-Díaz, Magon, McKenzie, Hampton, Vissers and Das.)- Published
- 2021
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223. Peroxiredoxin 2 oxidation reveals hydrogen peroxide generation within erythrocytes during high-dose vitamin C administration.
- Author
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Pearson AG, Pullar JM, Cook J, Spencer ES, Vissers MC, Carr AC, and Hampton MB
- Subjects
- Ascorbic Acid, Erythrocytes metabolism, Homeodomain Proteins, Humans, Oxidation-Reduction, Hydrogen Peroxide, Peroxiredoxins metabolism
- Abstract
Intravenous infusion of high dose (>10 g) vitamin C (IVC) is a common alternative cancer therapy. IVC results in millimolar levels of circulating ascorbate, which is proposed to generate cytotoxic quantities of H
2 O2 in the presence of transition metal ions. In this study we report on the in vitro and in vivo effects of millimolar ascorbate on erythrocytes. Addition of ascorbate to whole blood increased erythrocyte intracellular ascorbate approximately 35-fold. Within 10 min of ascorbate addition, we detected increased oxidation of erythrocyte peroxiredoxin 2 (Prx2), a major thiol antioxidant protein and a sensitive marker of H2 O2 production. Up to 50% of Prx2 was present in the oxidised form after 60 min. The presence of extracellular catalase, removal of plasma or the addition of a metal chelator did not prevent ascorbate-induced Prx2 oxidation, suggesting that the H2 O2 responsible for Prx2 oxidation was generated within the erythrocyte. Ascorbate is known to increase the rate of haemoglobin autoxidation and H2 O2 production. Through spectral monitoring of oxidised haemoglobin we estimated a generation rate of 15 μM H2 O2 /min inside erythrocytes. We also investigated changes in erythrocyte ascorbate concentration and Prx2 oxidation following IVC infusion in a cohort of patients with cancer. Plasma ascorbate levels ranged from 7.8 to 35 mM immediately post infusion, while erythrocyte ascorbate levels reached 1.5-3.4 mM 4 h after beginning infusion. Transient oxidation of erythrocyte Prx2 was observed. We conclude that erythrocytes accumulate ascorbate during IVC infusion, providing a significant reservoir of ascorbate, and this ascorbate increases H2 O2 generation within the cells. The consequence of increased erythrocyte Prx2 oxidation warrants further investigation., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)- Published
- 2021
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224. Macrophage migration inhibitory factor (MIF) enhances hypochlorous acid production in phagocytic neutrophils.
- Author
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Schindler L, Smyth LCD, Bernhagen J, Hampton MB, and Dickerhof N
- Subjects
- Humans, Hypochlorous Acid, Intramolecular Oxidoreductases, Neutrophils, Phagocytosis, Extracellular Traps, Macrophage Migration-Inhibitory Factors
- Abstract
Background: Macrophage migration inhibitory factor (MIF) is an important immuno-regulatory cytokine and is elevated in inflammatory conditions. Neutrophils are the first immune cells to migrate to sites of infection and inflammation, where they generate, among other mediators, the potent oxidant hypochlorous acid (HOCl). Here, we investigated the impact of MIF on HOCl production in neutrophils in response to phagocytic stimuli., Methods: Production of HOCl during phagocytosis of zymosan was determined using the specific fluorescent probe R19-S in combination with flow cytometry and live cell microscopy. The rate of phagocytosis was monitored using fluorescently-labeled zymosan. Alternatively, HOCl production was assessed during phagocytosis of Pseudomonas aeruginosa by measuring the oxidation of bacterial glutathione to the HOCl-specific product glutathione sulfonamide. Formation of neutrophil extracellular traps (NETs), an oxidant-dependent process, was quantified using a SYTOX Green plate assay., Results: Exposure of human neutrophils to MIF doubled the proportion of neutrophils producing HOCl during early stages of zymosan phagocytosis, and the concentration of HOCl produced was greater. During phagocytosis of P. aeruginosa, a greater fraction of bacterial glutathione was oxidized to glutathione sulfonamide in MIF-treated compared to control neutrophils. The ability of MIF to increase neutrophil HOCl production was independent of the rate of phagocytosis and could be blocked by the MIF inhibitor 4-IPP. Neutrophils pre-treated with MIF produced more NETs than control cells in response to PMA., Conclusion: Our results suggest a role for MIF in potentiating HOCl production in neutrophils in response to phagocytic stimuli. We propose that this newly discovered activity of MIF contributes to its role in mediating the inflammatory response and enhances host defence., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)
- Published
- 2021
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225. Evaluating the bactericidal action of hypochlorous acid in culture media.
- Author
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Ashby LV, Springer R, Hampton MB, Kettle AJ, and Winterbourn CC
- Subjects
- Bacteria, Chlorine, Culture Media, Oxidants pharmacology, Chloramines pharmacology, Hypochlorous Acid
- 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., (Copyright © 2020 Elsevier Inc. All rights reserved.)
- Published
- 2020
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226. Redox signalling and regulation of the blood-brain barrier.
- Author
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van Leeuwen E, Hampton MB, and Smyth LCD
- Subjects
- Animals, Astrocytes metabolism, Astrocytes pathology, Blood-Brain Barrier enzymology, Blood-Brain Barrier metabolism, Cell Death drug effects, Endothelial Cells enzymology, Endothelial Cells metabolism, Humans, Inflammation enzymology, Inflammation immunology, Microglia enzymology, Microglia metabolism, Nervous System Diseases enzymology, Nervous System Diseases physiopathology, Neutrophils enzymology, Neutrophils metabolism, Oxidation-Reduction drug effects, Oxidative Stress drug effects, Pericytes enzymology, Pericytes metabolism, Pericytes pathology, Signal Transduction genetics, Blood-Brain Barrier cytology, Inflammation metabolism, Nervous System Diseases metabolism, Oxidative Stress physiology
- Abstract
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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)
- Published
- 2020
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227. Structure-function analyses of alkylhydroperoxidase D from Streptococcus pneumoniae reveal an unusual three-cysteine active site architecture.
- Author
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Meng Y, Sheen CR, Magon NJ, Hampton MB, and Dobson RCJ
- Subjects
- Amino Acid Motifs, Amino Acid Sequence, Bacterial Proteins chemistry, Bacterial Proteins genetics, Biocatalysis, Catalytic Domain, Chromatography, High Pressure Liquid, Crystallography, X-Ray, Cysteine chemistry, Cysteine metabolism, Dimerization, Disulfides chemistry, Dithiothreitol chemistry, Mutagenesis, Site-Directed, Peroxidases chemistry, Peroxidases genetics, Protein Structure, Quaternary, Sequence Alignment, Tandem Mass Spectrometry, Bacterial Proteins metabolism, Peroxidases metabolism, Streptococcus pneumoniae enzymology
- 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 Sp AhpD exhibits weak thiol-dependent peroxidase activity and, unlike the previously reported Mycobacterium tuberculosis AhpC/D system, Sp AhpD does not mediate electron transfer to Sp AhpC. 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 Sp AhpD 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 Sp AhpD 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 Sp AhpD folding. In summary, Sp AhpD 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 Sp AhpD removes peroxides either directly or via AhpC, and that Sp AhpD cysteine oxidation may act as a redox switch or mediate electron transfer with other thiol proteins., (© 2020 Meng et al.)
- Published
- 2020
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228. Analysis of Neutrophil Bactericidal Activity.
- Author
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Magon NJ, Parker HA, Ashby LV, Springer RJ, and Hampton MB
- Subjects
- Algorithms, Cell Survival, Defense Mechanisms, Humans, Models, Theoretical, Neutrophils metabolism, Phagosomes metabolism, Staphylococcus aureus immunology, Host-Pathogen Interactions immunology, Neutrophils immunology, Neutrophils microbiology, Phagocytosis
- Abstract
This chapter describes three methods for measuring the bactericidal activity of neutrophils. All utilize colony counting techniques to quantify viable bacteria. A simple "one-step" protocol provides a composite measure of phagocytosis and killing, while a "two-step" protocol that separates extracellular and intracellular bacteria allows calculation of rate constants for both of these processes. We also present a method for selectively monitoring the long-term survival of bacteria within the phagosome. This may have application in identifying resistant strains and searching for compounds that sensitize pathogens to destruction.
- Published
- 2020
- Full Text
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229. Prolonged exposure to hypoxia induces an autophagy-like cell survival program in human neutrophils.
- Author
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Talla U, Bozonet SM, Parker HA, Hampton MB, and Vissers MCM
- Subjects
- Biomarkers, Caspases metabolism, Cell Hypoxia, Cell Survival, Humans, Neutrophils ultrastructure, Peroxidase metabolism, Phagocytosis immunology, Respiratory Burst immunology, Autophagy, Hypoxia metabolism, Neutrophils immunology, Neutrophils metabolism
- Abstract
Neutrophils contribute to low oxygen availability at inflammatory sites through the generation of reactive oxidants. They are also functionally affected by hypoxia, which delays neutrophil apoptosis. However, the eventual fate of neutrophils in hypoxic conditions is unknown and this is important for their effective clearance and the resolution of inflammation. We have monitored the survival and function of normal human neutrophils exposed to hypoxia over a 48 h period. Apoptosis was delayed, and the cells remained intact even at 48 h. However, hypoxia promoted significant changes in neutrophil morphology with the appearance of many new cytoplasmic vesicles, often containing cell material, within 5 hours of exposure to low O
2 . This coincided with an increase in LC3B-II expression, indicative of autophagosome formation and an autophagy-like process. In hypoxic conditions, neutrophils preferentially lost myeloperoxidase, a marker of azurophil granules. Short-term (2 h) hypoxic exposure resulted in sustained potential to generate superoxide when O2 was restored, but the capacity for oxidant production was lost with longer periods of hypoxia. Phagocytic ability was unchanged by hypoxia, and bacterial killing by neutrophils in both normoxic and hypoxic conditions was substantially diminished after 24 hours. However, pre-exposure to hypoxia resulted in an enhanced ability to kill bacteria by oxidant-independent mechanisms. Our data provide the first evidence for hypoxia as a driver of neutrophil autophagy that can influence the function and ultimate fate of these cells, including their eventual clearance and the resolution of inflammation., (©2019 Society for Leukocyte Biology.)- Published
- 2019
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230. Post-translational regulation of macrophage migration inhibitory factor: Basis for functional fine-tuning.
- Author
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Schindler L, Dickerhof N, Hampton MB, and Bernhagen J
- Subjects
- Amino Acid Sequence genetics, Animals, Cysteine metabolism, Humans, Macrophage Migration-Inhibitory Factors metabolism, Protein Conformation, Antigens, Differentiation, B-Lymphocyte genetics, Histocompatibility Antigens Class II genetics, Macrophage Migration-Inhibitory Factors genetics, Oxidation-Reduction, Protein Processing, Post-Translational genetics
- Abstract
Macrophage migration inhibitory factor (MIF) is a chemokine-like protein and an important mediator in the inflammatory response. Unlike most other pro-inflammatory cytokines, a number of cell types constitutively express MIF and secretion occurs from preformed stores. MIF is an evolutionarily conserved protein that shows a remarkable functional diversity, including specific binding to surface CD74 and chemokine receptors and the presence of two intrinsic tautomerase and oxidoreductase activities. Several studies have shown that MIF is subject to post-translational modification, particularly redox-dependent modification of the catalytic proline and cysteine residues. In this review, we summarize and discuss MIF post-translational modifications and their effects on the biological properties of this protein. We propose that the redox-sensitive residues in MIF will be modified at sites of inflammation and that this will add further depth to the functional diversity of this intriguing cytokine., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)
- Published
- 2018
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231. Thioredoxin reductase 1 and NADPH directly protect protein tyrosine phosphatase 1B from inactivation during H 2 O 2 exposure.
- Author
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Dagnell M, Pace PE, Cheng Q, Frijhoff J, Östman A, Arnér ESJ, Hampton MB, and Winterbourn CC
- Subjects
- Animals, Auranofin pharmacology, Catalytic Domain, Cells, Cultured, Dimerization, Embryo, Mammalian cytology, Homeodomain Proteins chemistry, Homeodomain Proteins genetics, Homeodomain Proteins metabolism, Humans, Hydrogen Peroxide pharmacology, Mice, Oxidants pharmacology, Oxidation-Reduction, Peptide Fragments chemistry, Peptide Fragments genetics, Peptide Fragments metabolism, Protein Interaction Domains and Motifs, Protein Tyrosine Phosphatase, Non-Receptor Type 1 antagonists & inhibitors, Protein Tyrosine Phosphatase, Non-Receptor Type 1 genetics, Rats, Receptor-Like Protein Tyrosine Phosphatases, Class 3 chemistry, Receptor-Like Protein Tyrosine Phosphatases, Class 3 genetics, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins metabolism, Selenocysteine chemistry, Selenocysteine metabolism, Thioredoxin Reductase 1 antagonists & inhibitors, Thioredoxin Reductase 1 chemistry, Thioredoxin Reductase 1 genetics, Thioredoxins chemistry, Thioredoxins genetics, Thioredoxins metabolism, NADP metabolism, Protein Tyrosine Phosphatase, Non-Receptor Type 1 metabolism, Receptor-Like Protein Tyrosine Phosphatases, Class 3 metabolism, Thioredoxin Reductase 1 metabolism
- Abstract
Regulation of growth factor signaling involves reversible inactivation of protein tyrosine phosphatases (PTPs) through the oxidation and reduction of their active site cysteine. However, there is limited mechanistic understanding of these redox events and their co-ordination in the presence of cellular antioxidant networks. Here we investigated interactions between PTP1B and the peroxiredoxin 2 (Prx2)/thioredoxin 1 (Trx1)/thioredoxin reductase 1 (TrxR1) network. We found that Prx2 becomes oxidized in PDGF-treated fibroblasts, but only when TrxR1 has first been inhibited. Using purified proteins, we also found that PTP1B is relatively insensitive to inactivation by H
2 O2 but found no evidence for a relay mechanism in which Prx2 or Trx1 facilitates PTP1B oxidation. Instead, these proteins prevented PTP1B inactivation by H2 O2 Intriguingly, we discovered that TrxR1/NADPH directly protects PTP1B from inactivation when present during the H2 O2 exposure. This protection was dependent on the concentration of TrxR1 and independent of Trx1 and Prx2. The protection was blocked by auranofin and required an intact selenocysteine residue in TrxR1. This activity likely involves reduction of the sulfenic acid intermediate form of PTP1B by TrxR1 and is therefore distinct from the previously described reactivation of end-point oxidized PTP1B, which requires both Trx1 and TrxR1. The ability of TrxR1 to directly reduce an oxidized phosphatase is a novel activity that can help explain previously observed increases in PTP1B oxidation and PDGF receptor phosphorylation in TrxR1 knockout cells. The activity of TrxR1 is therefore of potential relevance for understanding the mechanisms of redox regulation of growth factor signaling pathways., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)- Published
- 2017
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232. The marine cytotoxin portimine is a potent and selective inducer of apoptosis.
- Author
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Cuddihy SL, Drake S, Harwood DT, Selwood AI, McNabb PS, and Hampton MB
- Subjects
- Animals, Apoptosis Regulatory Proteins genetics, Apoptosis Regulatory Proteins metabolism, Cell Line, Cytotoxins chemistry, Fibroblasts cytology, Fibroblasts drug effects, Fibroblasts metabolism, Humans, Imines chemistry, Jurkat Cells, Marine Toxins chemistry, Mice, Molecular Structure, Apoptosis drug effects, Cytotoxins toxicity, Imines toxicity, Marine Toxins toxicity
- Abstract
Portimine is a recently discovered member of a class of marine micro-algal toxins called cyclic imines. In dramatic contrast to related compounds in this toxin class, portimine has very low acute toxicity to mice but is highly cytotoxic to cultured cells. In this study we show that portimine kills human Jurkat T-lymphoma cells and mouse embryonic fibroblasts (MEFs), with LC
50 values of 6 and 2.5 nM respectively. Treated cells displayed rapid caspase activation and phosphatidylserine exposure, indicative of apoptotic cell death. Jurkat cells overexpressing the anti-apoptotic protein Bcl-2 or Bax/Bak knockout MEFs were completely protected from portimine. This protection was apparent even at high concentrations of portimine, with no evidence of necrotic cell death, indicating that portimine is a selective chemical inducer of apoptosis. Treatment of the Bcl-2-overexpressing cells with both portimine and the Bcl-2 inhibitor ABT-737 proved a powerful combination, causing >90 % death. We conclude that portimine is one of the most potent naturally derived inducers of apoptosis to be discovered, and it displays strong selectivity for the induction of apoptotic pathways.- Published
- 2016
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233. Accumulation of oxidized peroxiredoxin 2 in red blood cells and its prevention.
- Author
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Bayer SB, Hampton MB, and Winterbourn CC
- Subjects
- Acetylcysteine pharmacology, Adenine pharmacology, Adolescent, Adult, Aged, Antioxidants pharmacology, Buffers, Electrophoresis, Polyacrylamide Gel, Erythrocytes drug effects, Female, Glucose pharmacology, Glutathione blood, Humans, Hydrogen Peroxide pharmacology, Inosine pharmacology, Male, Middle Aged, Oxidation-Reduction, Phosphates pharmacology, Pyruvic Acid pharmacology, Solutions pharmacology, Sulfhydryl Compounds blood, Thioctic Acid analogs & derivatives, Thioctic Acid pharmacology, Young Adult, Blood Preservation, Erythrocytes chemistry, Peroxiredoxins blood
- Abstract
Background: The thiol protein peroxiredoxin 2 (Prx2) is a major red blood cell (RBC) antioxidant that breaks down hydroperoxides and in the process is converted to an oxidized disulfide. Our objective was to determine whether Prx2 becomes oxidized during storage of RBCs, to understand the underlying mechanism, and to find ways of preventing the accumulation of the oxidized form., Study Design and Methods: RBCs were stored for up to 6 weeks under simulated blood banking conditions and Prx2 oxidation was monitored by nonreducing gel electrophoresis. The ability of the cells to reverse Prx2 oxidation after storage and to respond to added hydrogen peroxide was also evaluated., Results: Prx2 remained predominantly reduced during the first 3 weeks of storage, and then the oxidized form accumulated progressively. In contrast to fresh cells, oxidation was not reversed by incubation with glucose. Storage of RBCs in a high-pH, low-chloride, and high-phosphate/bicarbonate buffer (EAS-76v6) largely prevented accumulation of oxidized Prx for at least 6 weeks, and dihydrolipoic acid (DHLA), but not Rejuvesol, N-acetylcysteine, or α-lipoic acid, was able to reverse or protect against Prx2 oxidation. Additional, Prx2 oxidation occurred when hydrogen peroxide was added. However, this was reversible, suggesting that the reductive capacity was compromised in some but not in all cells., Conclusion: Prx2 remains mostly reduced in a high-pH storage solution with buffering capacity. Addition of DHLA to stored RBCs might be advantageous. Prx2 redox status could be used as a biomarker for the quality of stored RBCs., (© 2015 AABB.)
- Published
- 2015
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234. Analysis of neutrophil bactericidal activity.
- Author
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Parker HA, Magon NJ, Green JN, Hampton MB, and Winterbourn CC
- Subjects
- Colony-Forming Units Assay, Fluorometry methods, Humans, Staphylococcus aureus immunology, Bacteria immunology, Neutrophils immunology, Phagocytosis immunology
- Abstract
This chapter describes two methods for measuring the bactericidal activity of neutrophils. These are a new simple fluorescence-based assay, which quantifies bactericidal activity by measuring changes in bacterial fluorescence associated with a loss of membrane potential over time, and a more traditional colony counting protocol. Two variations of these techniques are presented: a "one-step" protocol providing a composite measure of phagocytosis and killing, and a "two-step" protocol that allows calculation of separate rate constants for both of these processes.
- Published
- 2014
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235. Macrophage migration inhibitory factor gene polymorphisms in inflammatory bowel disease: an association study in New Zealand Caucasians and meta-analysis.
- Author
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Falvey JD, Bentley RW, Merriman TR, Hampton MB, Barclay ML, Gearry RB, and Roberts RL
- Subjects
- Adult, Case-Control Studies, Colitis, Ulcerative ethnology, Colitis, Ulcerative immunology, Crohn Disease ethnology, Crohn Disease immunology, Female, Gene Frequency, Genetic Association Studies, Genetic Predisposition to Disease, Haplotypes, Humans, Male, Middle Aged, New Zealand epidemiology, Odds Ratio, Phenotype, Promoter Regions, Genetic, Risk Factors, Young Adult, Asian People genetics, Colitis, Ulcerative genetics, Crohn Disease genetics, Intramolecular Oxidoreductases genetics, Macrophage Migration-Inhibitory Factors genetics, Polymorphism, Single Nucleotide, White People genetics
- Abstract
Aim: To investigate the association of macrophage migration inhibitory factor (MIF) promoter polymorphisms with inflammatory bowel disease (IBD) risk., Methods: One thousand and six New Zealand Caucasian cases and 540 Caucasian controls were genotyped for the MIF SNP -173G > C (rs755622) and the repeat polymorphism CATT₅₋₈ (rs5844572) using a pre-designed TaqMan SNP assay and capillary electrophoresis, respectively. Data were analysed for single site and haplotype association with IBD risk and phenotype. Meta-analysis was employed, to assess cumulative evidence of association of MIF -173G > C with IBD. All published genotype data for MIF -173G > C in IBD were identified using PubMed and subsequently searching the references of all PubMed-identified studies. Imputed genotypes for MIF -173G > C were generated from the Wellcome Trust Case Control Consortium (and National Institute of Diabetes and Digestive and Kidney Diseases). Separate meta-analyses were performed on Caucasian Crohn's disease (CD) (3863 patients, 6031 controls), Caucasian ulcerative colitis (UC) (1260 patients, 1987 controls), and East Asian UC (416 patients and 789 controls) datasets using the Mantel-Haenszel method. The New Zealand dataset had 93% power, and the meta-analyses had 100% power to detect an effect size of OR = 1.40 at α = 0.05, respectively., Results: In our New Zealand dataset, single-site analysis found no evidence of association of MIF polymorphisms with overall risk of CD, UC, and IBD or disease phenotype (all P values > 0.05). Haplotype analysis found the CATT₅/-173C haplotype occurred at a higher frequency in New Zealand controls compared to IBD patients (0.6 vs 0.01; P = 0.03, OR = 0.22; 95%CI: 0.05-0.99), but this association did not survive bonferroni correction. Meta-analysis of our New Zealand MIF -173G > C data with data from seven additional Caucasian datasets using a random effects model found no association of MIF polymorphisms with CD, UC, or overall IBD. Similarly, meta-analysis of all published MIF -173G > C data from East Asian datasets (416 UC patients, 789 controls) found no association of this promoter polymorphism with UC., Conclusion: We found no evidence of association of MIF promoter polymorphisms with IBD.
- Published
- 2013
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236. Redox proteomics of thiol proteins in mouse heart during ischemia/reperfusion using ICAT reagents and mass spectrometry.
- Author
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Kumar V, Kleffmann T, Hampton MB, Cannell MB, and Winterbourn CC
- Subjects
- Animals, Isotope Labeling, Mass Spectrometry, Mice, Mitochondrial Proteins classification, Mitochondrial Proteins isolation & purification, Proteomics, Reactive Oxygen Species metabolism, Sulfhydryl Compounds metabolism, Mitochondrial Proteins metabolism, Myocardium metabolism, Oxidation-Reduction, Reperfusion Injury metabolism
- 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 20 min ischemia with or without aerobic reperfusion for 5 or 30 min. 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 © 2013 Elsevier Inc. All rights reserved.)
- Published
- 2013
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237. Using food to reduce H. pylori-associated inflammation.
- Author
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Keenan JI, Salm N, Wallace AJ, and Hampton MB
- Subjects
- Cell Line, Functional Food, Helicobacter pylori growth & development, Humans, Interleukin-8 metabolism, Tumor Necrosis Factor-alpha pharmacology, Brassica, Fatty Acids, Omega-3, Helicobacter Infections complications, Honey, Inflammation complications
- Abstract
Inflammation is widely recognized as a risk factor for gastric H. pylori-associated disease and disruption of this process provides a potential target for intervention. Using an in vitro system, broccoli sprouts, manuka honey and omega-3 oil, singly and in combination, were screened for their ability to limit H. pylori-associated inflammation. Each food significantly attenuated the release of IL-8 by H. pylori-infected cells, although the magnitude of this effect was variable. Only broccoli sprouts (0.125 mg/mL, w/v) were able to inhibit IL-8 release in response to TNFα, suggesting it acted by a different mechanism to the other two foods. The combination of manuka honey (1.25%, v/v) with omega-3 oil (0.006%, v/v) failed further to reduce IL-8 levels below those observed with honey alone, but the same concentrations of omega-3 oil and manuka honey independently enhanced the antiinflammatory effect of the isothiocyanate-rich broccoli sprouts. The results suggest that in the future certain foods may find increased clinical use as a non-antimicrobial approach for reducing the inflammation that is a major risk factor for H. pylori-associated disease, notably gastric cancer., (Copyright © 2012 John Wiley & Sons, Ltd.)
- Published
- 2012
- Full Text
- View/download PDF
238. Individual and combined effects of foods on Helicobacter pylori growth.
- Author
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Keenan JI, Salm N, Hampton MB, and Wallace AJ
- Subjects
- Brassica chemistry, Honey, Microbial Sensitivity Tests, Functional Food, Helicobacter Infections diet therapy, Helicobacter pylori growth & development
- Abstract
Eradication of H. pylori can reduce the risk of non-cardia gastric cancer developing in infected humans. Thus, the consumption of foods that inhibit the growth of these bacteria may provide an alternative to current therapies that include antibiotics, proton pump inhibitors and/or bismuth salts. This study describes a simple broth dilution assay developed to screen a range of foods for their individual and combined effects on H. pylori growth. It was found that foods with measurable anti-H. pylori activity have an effect greater in combination than the sum of foods tested singly, and that this was most noticeable with a combination of broccoli sprouts and blackcurrant oil. The results suggest that food synergy should be considered in any nutraceutical approach to H. pylori infection., (Copyright (c) 2010 John Wiley & Sons, Ltd.)
- Published
- 2010
- Full Text
- View/download PDF
239. Measuring mitochondrial protein thiol redox state.
- Author
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Requejo R, Chouchani ET, Hurd TR, Menger KE, Hampton MB, and Murphy MP
- Subjects
- Blotting, Western, Glutathione chemistry, Glutathione metabolism, Liver enzymology, Mitochondrial Proteins analysis, Mitochondrial Proteins metabolism, Myocytes, Cardiac enzymology, Oxidation-Reduction, Protein Binding, Signal Transduction, Sulfhydryl Compounds analysis, Sulfhydryl Compounds metabolism, Mitochondrial Proteins chemistry, Sulfhydryl Compounds chemistry
- Abstract
Protein thiols are an important component of mammalian intramitochondrial antioxidant defenses owing to their selective interaction with reactive oxygen and nitrogen species (ROS and RNS). Reversible modifications of protein thiols resulting from these interactions are also an important aspect of redox signal transduction. Therefore, to assess how mitochondria respond to oxidative stress and act as nodes in redox signaling pathways, it is important to measure general changes to protein thiol redox states and also to identify the specific mitochondrial thiol proteins involved. Here we outline some of the approaches that can be used to accomplish these goals and thereby infer the multiple roles of mammalian mitochondrial protein thiols in antioxidant defense and redox signaling., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)
- Published
- 2010
- Full Text
- View/download PDF
240. Measuring the redox state of cellular peroxiredoxins by immunoblotting.
- Author
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Cox AG, Winterbourn CC, and Hampton MB
- Subjects
- Animals, Cells, Cultured, Humans, Immunoblotting trends, Oxidation-Reduction, Time Factors, Immunoblotting methods, Peroxiredoxins chemistry
- Abstract
The peroxiredoxins (Prxs) are a family of thiol peroxidases that scavenge hydroperoxides and peroxynitrite. The abundance and reactivity of these proteins makes them primary targets for cellular H(2)O(2). The catalytic cycle of typical 2-Cys Prxs involves formation of an intermolecular disulfide bond between peroxidatic and resolving cysteines on opposing subunits. Rapid alterations in the ratio of reduced monomer and oxidized dimer have been detected in the cytoplasm and mitochondria of cultured cells exposed to various exogenous and endogenous sources of oxidative stress. Here we describe immunoblot methods to monitor the interconversion of individual 2-Cys Prxs in cultured cells. We also outline an adaptation of this method to measure the extent to which individual 2-Cys Prxs become hyper oxidized in treated cells. Together, these methods enable the redox status of cellular Prxs to be assessed and quantified in a rapid and robust manner., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)
- Published
- 2010
- Full Text
- View/download PDF
241. Proteomic detection of oxidized and reduced thiol proteins in cultured cells.
- Author
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Cuddihy SL, Baty JW, Brown KK, Winterbourn CC, and Hampton MB
- Subjects
- Electrophoresis, Gel, Two-Dimensional methods, Humans, Molecular Structure, Oxidation-Reduction, Cells, Cultured chemistry, Proteins chemistry, Proteome analysis, Sulfhydryl Compounds chemistry
- Abstract
The oxidation and reduction of cysteine residues is emerging as an important post-translational control of protein function. We describe a method for fluorescent labelling of either reduced or oxidized thiols in combination with two-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (2DE) to detect changes in the redox proteome of cultured cells. Reduced thiols are labelled with the fluorescent compound 5-iodoacetamidofluorescein. To monitor oxidized thiols, the reduced thiols are first blocked with N-ethyl-maleimide, then the oxidized thiols reduced with dithiothreitol and labelled with 5-iodoacetamidofluorescein. The method is illustrated by treating Jurkat T-lymphoma cells with hydrogen peroxide and monitoring increased labelling of oxidized thiol proteins. A decrease in labelling can also be detected, and this is attributed to the formation of higher oxidation states of cysteine that are not reduced by dithiothreitol.
- Published
- 2009
- Full Text
- View/download PDF
242. Outer membrane vesicles enhance the carcinogenic potential of Helicobacter pylori.
- Author
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Chitcholtan K, Hampton MB, and Keenan JI
- Subjects
- Bacterial Proteins, Cell Line, Tumor, Cell Membrane metabolism, Cell Membrane virology, Fluorescent Antibody Technique, Glutathione metabolism, Humans, Iron metabolism, Micronuclei, Chromosome-Defective, Oxidative Stress physiology, Protein Carbonylation physiology, Stomach Neoplasms pathology, Transport Vesicles metabolism, Cell Transformation, Neoplastic, Helicobacter pylori pathogenicity, Stomach Neoplasms virology, Transport Vesicles virology
- Abstract
Chronic Helicobacter pylori infection is associated with an increased risk of gastric carcinogenesis. These non-invasive bacteria colonize the gastric mucosa and constitutively shed small outer membrane vesicles (OMV). In this study, we investigated the direct effect of H.pylori OMV on cellular events associated with carcinogenesis. We observed increased micronuclei formation in AGS human gastric epithelial cells treated with OMV isolated from a toxigenic H.pylori strain (60190). This effect was absent in OMV from strain 60190v:1 that has a mutant vacA, indicating VacA-dependent micronuclei formation. VacA induces intracellular vacuolation, and reduced acridine orange staining indicated disruption in the integrity of these vacuoles. This was accompanied by an alteration in iron metabolism and glutathione (GSH) loss, suggesting a role for oxidative stress in genomic damage. Increasing intracellular GSH levels with a GSH ester abrogated the VacA-mediated increase in micronuclei formation. In conclusion, OMV-mediated delivery of VacA to the gastric epithelium may constitute a new mechanism for H.pylori-induced gastric carcinogenesis.
- Published
- 2008
- Full Text
- View/download PDF
243. Thiol chemistry and specificity in redox signaling.
- Author
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Winterbourn CC and Hampton MB
- Subjects
- Animals, Humans, Models, Biological, Oxidants chemistry, Oxidants metabolism, Oxidation-Reduction, Reactive Oxygen Species metabolism, Substrate Specificity, Signal Transduction, Sulfhydryl Compounds chemistry, Sulfhydryl Compounds metabolism
- Abstract
Exposure of cells to sublethal oxidative stress results in the modulation of various signaling pathways. Oxidants can activate and inactivate transcription factors, membrane channels, and metabolic enzymes, and regulate calcium-dependent and phosphorylation signaling pathways. Oxidation and reduction of thiol proteins are thought to be the major mechanisms by which reactive oxidants integrate into cellular signal transduction pathways. This review focuses on mechanisms for sensing and transmitting redox signals, from the perspective of their chemical reactivity with specific oxidants. We discuss substrate preferences for different oxidants and how the kinetics of these reactions determines how each oxidant will react in a cell. This kinetic approach helps to identify initial oxidant-sensitive targets and elucidate mechanisms involved in transmission of redox signals. It indicates that only those proteins with very high reactivity, such as peroxiredoxins, are likely to be direct targets for hydrogen peroxide. Other more modestly reactive thiol proteins such as protein tyrosine phosphatases are more likely to become oxidized by an indirect mechanism. The review also examines oxidative changes observed during receptor-mediated signaling, the strengths and limitations of detection methods for reactive oxidant production, and the evidence for hydrogen peroxide acting as the second messenger. We discuss areas where observations in cell systems can be rationalized with the reactivity of specific oxidants and where further work is needed to understand the mechanisms involved.
- Published
- 2008
- Full Text
- View/download PDF
244. Bcl-2 over-expression promotes genomic instability by inhibiting apoptosis of cells exposed to hydrogen peroxide.
- Author
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Cox AG and Hampton MB
- Subjects
- Apoptosis drug effects, Caspases metabolism, Cell Division drug effects, Cell Survival drug effects, Humans, Jurkat Cells, Lipid Peroxidation drug effects, Lipid Peroxidation physiology, Lymphoma, T-Cell, Lysosomes drug effects, Lysosomes ultrastructure, Necrosis, Oxidative Stress drug effects, Oxidative Stress physiology, Apoptosis physiology, Genomic Instability genetics, Hydrogen Peroxide pharmacology, Proto-Oncogene Proteins c-bcl-2 genetics
- Abstract
The anti-apoptotic oncogene bcl-2 is hypothesized to increase the antioxidant status of cells, thereby protecting them from oxidative stress. In this study, we examined hydrogen peroxide (H2O2)-mediated oxidative stress in Jurkat T lymphoma cells. Over-expression of Bcl-2 did not inhibit cytotoxicity at doses of H2O2 that caused necrosis (>200 microM), but it did block cell death at apoptotic doses (<200 microM). However, these cells exhibited the same initial level of protein and lipid oxidation following exposure to H2O2 as the parental cells, indicating that the anti-apoptotic activity is not associated with general antioxidant properties. Bcl-2 expression was able to protect against secondary protein carbonyl formation, which was linked to lysosome stabilization. Assessment of micronuclei formation in cells over-expressing Bcl-2 showed evidence of increased genomic instability, consistent with the impairment of apoptosis in damaged cells. We conclude that while Bcl-2 can block cytotoxicity associated with apoptosis-inducing levels of oxidative stress, it does not protect the cells from the stress itself. Bcl-2 may promote tumourigenesis by preventing the removal of oxidatively damaged cells.
- Published
- 2007
- Full Text
- View/download PDF
245. Analysis of neutrophil bactericidal activity.
- Author
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Green JN, Winterbourn CC, and Hampton MB
- Subjects
- Algorithms, Animals, Guidelines as Topic, Humans, Neutrophils metabolism, Neutrophils microbiology, Staphylococcus aureus physiology, Statistics as Topic, Blood Bactericidal Activity, Neutrophil Activation physiology, Neutrophils immunology
- Abstract
The primary function of neutrophils is to engulf and destroy invading pathogens. If the bactericidal capacity of neutrophils is defective, an individual may suffer from enhanced susceptibility to potentially fatal microbial infection. To identify such defects, and to investigate the mechanisms used to kill bacteria, the bactericidal activity of neutrophils must be accurately quantified. This chapter provides details of a comprehensive microbiological technique that quantifies neutrophil bactericidal activity by measuring the loss of viability of ingested bacteria over time. Two variations of this technique are presented: a simple "one-step" protocol providing a composite measure of phagocytosis and killing, and a more advanced "two-step" protocol that allows calculation of separate rate constants for both of these processes.
- Published
- 2007
- Full Text
- View/download PDF
246. Detection of apoptosis by caspase-3 activation in tracheal aspirate neutrophils from premature infants: relationship with NF-kappaB activation.
- Author
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Cheah FC, Hampton MB, Darlow BA, Winterbourn CC, and Vissers MC
- Subjects
- Adult, Caspase 3, Humans, Infant, Newborn, NF-kappa B metabolism, Suction, Time Factors, Trachea cytology, Apoptosis immunology, Caspases immunology, NF-kappa B immunology, Neutrophils immunology, Trachea immunology
- 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)-kappaB, 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-kappaB 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-kappaB, 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-kappaB activation, which supports in vitro observations that NF-kappaB activation delays neutrophil apoptosis.
- Published
- 2005
- Full Text
- View/download PDF
247. The chemopreventive agent phenethyl isothiocyanate sensitizes cells to Fas-mediated apoptosis.
- Author
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Pullar JM, Thomson SJ, King MJ, Turnbull CI, Midwinter RG, and Hampton MB
- Subjects
- Aldehyde Dehydrogenase antagonists & inhibitors, Buthionine Sulfoximine pharmacology, Caspase 3, Caspases metabolism, Chemoprevention, Humans, Jurkat Cells, MAP Kinase Kinase 4, Mitogen-Activated Protein Kinase 1 metabolism, Mitogen-Activated Protein Kinase 3, Mitogen-Activated Protein Kinase Kinases metabolism, Mitogen-Activated Protein Kinases metabolism, Proto-Oncogene Proteins c-bcl-2 metabolism, Signal Transduction, Urinary Bladder Neoplasms metabolism, p38 Mitogen-Activated Protein Kinases, Anticarcinogenic Agents pharmacology, Apoptosis drug effects, Drug Resistance, Neoplasm, Isothiocyanates pharmacology, JNK Mitogen-Activated Protein Kinases, Urinary Bladder Neoplasms pathology, fas Receptor metabolism
- Abstract
The chemopreventive properties of the isothiocyanates have been attributed to their ability to inhibit phase I enzymes that activate procarcinogens, induce phase II protective enzymes and trigger apoptosis in transformed cells. In this study we provide evidence for a new mechanism of chemoprevention, wherein sublethal doses of phenethyl isothiocyanate (PEITC) sensitize cells to Fas-mediated apoptosis. The phenomenon was observed in the Fas-resistant T24 bladder carcinoma cell line and in Jurkat T cells overexpressing the anti-apoptotic protein Bcl-2. Caspase-3-like activity was increased up to 20-fold of that observed with either PEITC or anti-Fas antibody alone. While PEITC activated ERK, JNK and p38, inhibitors of these MAP kinases did not block apoptosis. PEITC transiently depleted cellular glutathione, providing a putative mechanism for sensitizing the cells to apoptosis. However, lowering glutathione with buthionine sulfoximine did not mimic the effect of PEITC. Instead, we propose that PEITC promotes apoptosis by directly modifying intracellular thiol proteins. The ability of PEITC to sensitize cells to receptor-mediated apoptosis provides an additional mechanism to explain its chemopreventive properties.
- Published
- 2004
- Full Text
- View/download PDF
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