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The HprSR constitutes the bacterial two-component regulatory system engaged by Escherichia coli to reduce the damaging effects of reactive chlorine and oxygen species present in its cytosol. Hypochlorous acid (HOCl) has been shown to be the molecule capable of activating of the HprSR system. HOCl is produced upon pathogen invasion by phagocytic cells of the human innate immune system, particularly neutrophils, to take advantage of its powerful antimicrobial attributes. Therefore, comprehensive studies concerning bacterial sensing and regulatory HprSR system are indispensable in understanding and effectively eliminating pathogens. Here we present the first crystal structure, solved at 1.7 Å resolution, of the HprS cytoplasmic domains arranged as a homodimer. In both protomers, the catalytic ATP-binding domain contains a non-hydrolysable ATP analog coordinated by a magnesium ion. This structure allowed us to provide a detailed characterization of kinase-substrate interaction. Furthermore, the structural data are supported by biophysical studies of kinase interaction with cognate response regulator HprR and substrate ATP. The kinase activity is also assessed in the presence or absence of HprR. [Display omitted] • The first crystal structure of cytoplasmic domains of HprS was determined. • Based on structural analysis, HprS belongs to Type 1 family of histidine kinases. • The HprS structure contained substrate analog, ATPγS, in its catalytic domains. • HprS and its cognate response regulator form stable complex. • The HprS-HprR complex formation has an impact on kinase activity. [ABSTRACT FROM AUTHOR]

[Display omitted] • A novel coumarin-based fluorescent probe (namely CN 2 -CF 3 -S) for HOCl was constructed. • CN 2 -CF 3 -S featured two dimethylamino groups located at its 6- and 7-position. • Simultaneous visualization of lipid droplets and detection of HOCl was achieved with CN 2 -CF 3 -S. • Monitoring the fluctuation of HOCl on skin of the psoriatic mice with CN 2 -CF 3 -S was also achieved. Psoriasis is an autoimmune inflammation-related disease accompanied by a variety of complications. Reactive oxygen species (ROS) are modulators of inflammation, and their excessive production caused by oxidative/anti-oxidative imbalance has been observed in psoriatic patients. Hypochlorous acid (HOCl) is a ROS produced by myeloperoxidase (MPO) from chloride ions (Cl-) and hydrogen peroxide (H 2 O 2). Endogenous HOCl has recently been studied as a potential biomarker of psoriasis underlying the necessity for the development of efficient analytical tools for its detection and real time monitoring. Herein, we designed a novel highly sensitive and selective coumarin-based fluorescent probe for HOCl detection, named CN 2 -CF 3 -S. The probe itself featured negligible fluorescence because of the heavy atom effect of the thiocarbonyl group. However, upon responding to HOCl a conversion to sulfur-free derivative CN 2 -CF 3 -O occurs, resulting in a dramatical fluorescent enhancement setting the detection limit for HOCl of 3.2 nM. The HOCl-recognition mechanism could be ascribed to the HOCl-triggered oxidative desulfurization process that agrees with liquid chromatography-mass spectrometry (LC-MS) analysis and density functional theory (DFT) computations. The probe's design incorporated a synergistic combination of two structural features for efficient lipid droplets (LDs)-targeting imaging. An efficient push–pull system reinforced by the presence of two strongly electron-donating dimethylamino groups equipped CN 2 -CF 3 -O with pronounced solvatochromism realized through the enhanced blue-shifted emission in non-polar media. Meanwhile, the presence of trifluormethylphenyl moiety at the acceptor side led to an increased lipophilicity. The CN 2 -CF 3 -S probe has been successfully utilized to track the endogenous HOCl in cells and on skin of the psoriatic mice in a wash-free manner. As a result, we have demonstrated that the concentration of HOCl in skin might correlate positively with the degree of inflammation in psoriasis. Thus, CN 2 -CF 3 -S constitutes the first example of LDs-imaging fluorescent probe for detecting the psoriasis-linked HOCl, offering a convenient tool for further in-depth investigation of psoriasis pathophysiology. [ABSTRACT FROM AUTHOR]

Hypochlorous acid (HOCl) is commonly utilized in various daily applications. As a toxic reactive oxygen species (ROS) that generated from the industrial and pharmaceutical aspects, it plays a critical role in the mass cycle of environmental system and various biological processes. Understanding the complicated roles of HOCl in environment and biosystems requires the development of precise and efficient detection methods. Thus, in this work, a novel fluorescent probe, MQ-ClO , has been designed to detect hypochlorous acid by utilizing hypochlorous acid-triggered oxidative intramolecular cyclization. This probe exhibits rapid and sensitive response, with a detection limit as low as 35 nM. More importantly, the probe is capable of functioning under highly acidic or basic conditions, exhibiting a wide pH range adaptability from pH 2–11. Furthermore, MQ-ClO has been effectively used to detect HOCl in real water samples. Besides, the low toxicity of MQ-ClO enables its practical application in monitoring endogenous/exogenous HOCl levels in living cells as well as zebrafish. • The first HOCl sensing fluorescence probe a wide pH range adaptability from pH 2–11. • Rapid, highly sensitive and highly selective detections in solutions. • Probe can be used for fluorescent imaging of HOCl in biosystems and environmental waters. [ABSTRACT FROM AUTHOR]

Since the onset of the SARS-CoV-2 pandemic in early 2020, there has been a notable rise in sodium hypochlorite disinfectants. Sodium hypochlorite undergoes hydrolysis to generate hypochlorous acid for virus eradication. This chlorine-based disinfectant is widely utilized for public disinfection due to its effectiveness. Although sodium hypochlorite disinfection is convenient, its excessive and indiscriminate use can harm the water environment and pose a risk to human health. Hypochlorous acid, a reactive oxygen species, plays a crucial role in the troposphere, stratospheric chemistry, and oxidizing capacity. Additionally, hypochlorous acid is vital as a reactive oxygen species in biological systems, and its irregular metabolism and level is associated with several illnesses. Thus, it is crucial to identify hypochlorous acid to comprehend its environmental and biological functions precisely. Here, we constructed a new fluorescent probe, utilizing the twisted intramolecular charge transfer mechanism to quickly and accurately detect hypochlorous acid in environmental water and biosystems. The probe showed a notable increase in fluorescence when exposed to hypochlorous acid, demonstrating its excellent selectivity, fast response time (less than 10 seconds), a large Stokes shift (∼ 102 nm), and a low detection limit of 15.5 nM. [Display omitted] • Simple probe synthesis and detecting setup. • Rapid, highly sensitive and highly selective detections in solutions. • Probe can be used for fluorescent imaging of HOCl in living cells and zebrafish. [ABSTRACT FROM AUTHOR]

Abstract Oxidative stress is a major hallmark of cardiac ischemia/reperfusion (I/R) injury, which is in part due to the release of the enzyme myeloperoxidase (MPO) from activated infiltrating leukocytes, and the subsequent production of the oxidants hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). Although exposure of various cell types to either oxidant is known to cause cellular dysfunction within a variety of pathological settings, the precise role of HOCl and HOSCN in the initiation of tissue damage evident following cardiac I/R injury remains unclear. In this study, we have employed the use of the cardiac myoblast cell line H9c2 as a model for cardiac myocytes and demonstrate that exposure to either oxidant elicits a dose-dependent increase in cytosolic calcium accumulation, depletion of the cellular thiol pool, reduction of glutathione (GSH) levels and loss of mitochondrial inner trans -membrane potential, concomitant with increased necrotic cell death. H9c2 cell recovery from the initial oxidant exposure involves the initiation of cell survival signalling pathways centred around Nrf2-antioxidant response element (ARE) and activator protein 1 (AP-1) activation, with cell survival accompanied by restoration of mitochondrial function following exposure to HOSCN, but not HOCl. These data highlight the cellular responses elicited by HOCl and HOSCN in cardiac myocytes furthering our understanding of the pathogenesis of oxidant injury following cardiac I/R injury. [ABSTRACT FROM AUTHOR]

[Display omitted] • A novel fluorescent probe WZ-HOCl for detecting HOCl was designed. • WZ-HOCl could quantitatively detect HOCl with high sensitivity. • WZ-HOCl can distinguish normal hepatocytes and hepatoma cells by detecting HOCl. • WZ-HOCl was successfully used for imaging HOCl in inflammatory and liver disease models. Hypochlorous acid (HOCl) is a common reactive oxygen species (ROS) associated with the development of liver, tumor, inflammatory, and other diseases. In this work, the turn-on fluorescent probe named (WZ-HOCl) with a naphthalimide structure was designed and synthesized to detect endogenous HOCl in disease models. WZ-HOCl can achieve a fast response to HOCl with good linearity in the range of 0–45 μM (LOD = 147 nM). The application of WZ-HOCl in bioimaging was investigated by constructing a series of cellular disease models, and the results showed that WZ-HOCl could sensitively detect endogenous HOCl in inflammatory and liver disease models. It can also be used to differentiate between hepatocytes and hepatoma cells. WZ-HOCl will provide new methods and ideas for fluorescent probes in detecting drug-induced liver injury, alcoholic and non-alcoholic steatohepatitis, and some inflammation-related diseases. [ABSTRACT FROM AUTHOR]

Abstract A new cyclometalated iridium(III) complex-based fluorescence probe (IrCN) for hypochlorite (ClO−) has been synthesized and characterized. The probe displayed nonfluorescent around 577 nm, while a 54-fold enhancement in fluorescence emission intensity was observed after the addition of ClO− due to the removal of C=N isomerization effect. Such "turn-on" fluorescence probe worked excellently in wide pH range (5–12) with short response time (<20 s) and the detection limit was as low as 0.11 μM. In addition, IrCN exhibited high selectivity towards ClO− even in the presence of other competing species. Furthermore, IrCN was successfully integrated in fluorescent test strips for real-time detection of ClO−. Graphical abstract Image 1 Highlights • A new and simple fluorescence probe based on cyclometalated iridium(III) complex was designed and synthesized. • The probe displayed a fast response towards ClO− (<20 s) with a low detection limit of 0.11 μM. • The probe showed high selectivity toward ClO− among other reactive oxygen species. • The probe was successfully applied for visual detection by fluorescent test strips. [ABSTRACT FROM AUTHOR]

Abstract An excellent fluorescent probe with "turn on" phenomenon for sensitive monitoring hypochlorite (ClO−) was prepared using the mild condensation reaction between coumarin and hydroxylamine (NH 2 OH). The probe possessed potent selectivity to ClO− with obvious color changes from yellow to light yellow and green to blue fluorescence emission, which could be noticed by the naked eye. Moreover, the probe has been succeeded in imaging ClO− in living A549 cells and thus has the potential prospect in the visual detection of intracellular ClO−. Graphical abstract Image 1 Highlights • Observable colorimetric and fluorometric changes. • Fast response time. • Highly selective and sensitive response to ClO−. • Strong contrast imaging in living cells. [ABSTRACT FROM AUTHOR]

Abstract Total residual oxidants (TRO) in treated ballast water can produce various disinfection by-products (DBPs) depending on local conditions, such as salinity and organic matter content in water. Because TRO and DBPs are known to be harmful to aquatic organisms and humans, ecotoxicity tests have been proposed for screening the residual toxicity before discharging treated ballast water. In the present study, we aimed to address the decay rates and toxicity changes of TRO under various conditions in salinity, initial TRO concentrations, and residence time of TRO. In addition, the toxicological sensitivities of bioluminescent bacteria Vibrio fischeri and a commonly-used microalgae Skeletonema costatum relative to the residual toxicity of TRO and six selected DBPs were determined. Decay rate of TRO concentration increased as a function of salinity and was affected by the initial concentrations of TRO. Unexpectedly, significant bioluminescence inhibition was observed for hypochlorite-treated water at < 0.1 mg L−1 TRO (expressed as Cl 2), which is a lower concentration than the maximum allowable discharge concentration (MADC) to marine waters established by the International Maritime Organization (IMO). The ecotoxicological thresholds of no observed effective concentration and median effect concentration for all tested DBPs were about 3–10 times lower for V. fischeri than for S. costatum. The results indicate that bioluminescent microbes possess an ecologically-relevant sensitivity to both TRO and DBPs in ballast water. In general, bioassay using V. fischeri was potentially more effective than microalgae for screening the total toxicity of TRO and DBPs in treated ballast water, especially given that ballast water usually contains a highly variable and complex mixture of toxicants. Graphical abstract fx1 Highlights • Decay rates of TRO concentrations in water increased as a function of salinity. • Significant toxicity was detected in the microbe (V. fischeri) at < 0.1 mg L−1 of TRO. • Within 24 h of exposure, microbial toxicity increased in hypochlorite-treated water. • V. fischeri responded more sensitive to DBPs contamination than did for S. costatum. [ABSTRACT FROM AUTHOR]

When neutrophils engulf bacteria, myeloperoxidase converts hydrogen peroxide to hypochlorous acid, which is toxic to all micro-organisms. It has been suggested that some pathogens have virulence factors that target myeloperoxidase to dampen the oxidative reactions of neutrophils. These virulence factors include staphyloxanthin, the golden pigment of Staphylococcus aureus, and enterobactin – a siderophore released by gram-negative bacteria. We investigated the potential of staphyloxanthin and enterobactin to shield bacteria from hypochlorous acid and related chloramines. Clinical strains of S. aureus with high levels of staphyloxanthin and related carotenoids were in general more resistant to low doses of hypochlorous acid than non-pigmented bacteria. But some non-pigmented strains were also resistant to the oxidant. Doses of reactive chlorine species that killed bacteria also bleached their carotenoids. Hypochlorous acid, NH 2 Cl, and NHCl 2 bleached purified staphyloxanthin. When S. aureus were phagocytosed by neutrophils there was no discernible loss of staphyloxanthin. These data suggest that staphyloxanthin is capable of protecting bacteria from low doses of reactive chlorine species formed inside phagosomes. Enterobactin was not an inhibitor of myeloperoxidase. We conclude that staphyloxanthin may protect some bacterial strains against oxidative killing by neutrophils, but enterobactin will not inhibit the production of hypochlorous acid. [ABSTRACT FROM AUTHOR]

Atherosclerosis is characterised by the infiltration of macrophages at sites of inflammation within the vessel wall and the release of myeloperoxidase (MPO), which forms hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN). HOCl is a damaging oxidant implicated in the development of atherosclerosis. Preferential formation of HOSCN occurs under conditions where thiocyanate ions are elevated, as is the case in smokers. HOSCN reacts selectively with thiols, which can result in more enzyme inactivation and damage than HOCl at susceptible sites, which may contribute to atherosclerosis in smokers. In this study, we show that exposure of macrophages to HOSCN results in a time- and dose-dependent increase in the mRNA expression and release of pro-inflammatory cytokines and chemokines, including monocyte chemotactic protein 1, tumour necrosis factor alpha, and interleukins 6, 8 and 1β. At high oxidant concentrations (>200 μM), a significant loss of cellular thiols and increased cell death is observed. HOSCN-induced cytokine/chemokine expression and cell death were decreased on pharmacological inhibition of nuclear factor kappa B. These data highlight a pathway by which HOSCN could promote inflammation and the development of atherosclerosis, in the presence of supra-physiological levels of the precursor thiocyanate, which are achievable by cigarette smoking. [ABSTRACT FROM AUTHOR]

The study starts with a detailed analysis of all the high-resolution rovibrational transitions measured for the minor hypochlorous acid isotopologue H16O37Cl, employing the Marvel (Measured Active Rotational-Vibrational Energy Levels) procedure. The survey considers 16 sources, the wavenumber coverage of the transitions extends from the microwave to the visible region, corresponding to 13 vibrational parent states. Experimental transitions data from older (1994, 1996, and 2000) sources, not published explicitly in the original papers, are presented in here for the first time; the newly reported transitions represent about one third of all the transitions assigned for H16O37Cl. Then a minor update to the transitions and levels of H16O35Cl published in J. Mol. Spectrosc. 384 (2022) 111561 is provided. Proper labelling and internal consistency of the transitions and the empirical rovibrational energies of H16O35Cl and H16O37Cl is enforced utilizing effective Hamiltonian (EH) and first-principles variational nuclear-motion computation results. An iteratively updated line list, based on empirical and EH rovibrational energy levels and first-principles intensities, is used to assign a significant number of new transitions in the 2 ν 2 band (2300–2800 cm−1) for both isotopologues. At the end, for H16O35Cl/H16O37Cl, from the total of 20 349/10 266 experimentally measured transitions considered, 20 119/10 124 could be validated, allowing the derivation of 5760/3933 empirical rovibrational energy levels with well-defined uncertainties. Improved parameters for an effective Hamiltonian corresponding to the 2 ν 2 state are given. A comparison with data in the canonical spectroscopic line-by-line database HITRAN is also provided. • For H16O35Cl/H16O37Cl, a survey of their high-resolution rovibrational spectra provided a total of 20 349/10 266 experimentally measured transitions. • 20 119/10 124 of them could be validated through the MARVEL procedure, allowing the derivation of 5760/3933 empirical rovibrational energy levels with well-defined uncertainties. • The first bending overtone band of HOCl is reanalyzed, yielding a significant number of newly assigned transitions and improved parameters for an effective Hamiltonian. • The validated transitions and the empirical energies are compared to those in the HITRAN datasets. [ABSTRACT FROM AUTHOR]

Vascular peroxidase 1 (VPO1) has been proved to be associated with vascular endothelial cell apoptosis by producing reactive oxygen species. However, the contribution of VPO1 to the development of vascular remodeling (VR) remains to be fully characterized. We explored the role of VPO1 in VR in spontaneously hypertensive rats (SHRs) and the underlying mechanism of losartan in inhibiting VR. Compared to Wistar-Kyoto (WKY) rats, the SHR showed remodeling of their vascular walls. The level of VPO1 and the hydrogen peroxide (H 2 O 2 ) concentration were increased in the SHRs. However, the SHRs pretreated with losartan showed significant inhibition of blood pressure and VR and decreased levels of VPO1 and H 2 O 2 compared to the non-treated SHRs. Angiotensin II significantly increased the expressions of MMP-2, MMP-9 and the concentrations of H 2 O 2 and hypochlorous acid (HOCl) in vascular smooth muscle cells (VSMCs). However, only the H 2 O 2 level increased in VSMCs when transfected with VPO1 shRNA. These results support a critical but previously unrecognized role of VPO1 in VR and suggest that therapies to reduce VPO1 may be novel approaches for VR. [ABSTRACT FROM AUTHOR]

Unmetabolized pharmaceuticals often enter the water treatment plants and exposed to various treatment processes. Among these water treatment processes, disinfection is a process which involves the application of chemical oxidation to remove pathogen. Untreated pharmaceuticals from primary and secondary treatment have the potential to be exposed to the chemical oxidation process during disinfection. This study investigated the kinetics and mechanism of the degradation of sotalol during chlorination process. Chlorination with hypochlorous acid (HOCl) as main reactive oxidant has been known as one of the most commonly used disinfection methods. The second order rate constant for the reaction between sotalol and free available chlorine (FAC) was found to decrease from 60.1 to 39.1 M −1 min −1 when the pH was increased from 6 to 8. This result was mainly attributed by the decreased of HOCl concentration with increasing pH. In the real water samples, the presence of the higher amount of organic content was found to reduce the efficiency of chlorination in the removal of sotalol. This result showed that sotalol competes with natural organic matter to react with HOCl during chlorination. After 24 h of FAC exposure, sotalol was found to produce three stable transformation by-products. These by-products are mainly chlorinated compounds. According to the acute and chronic toxicity calculated using ECOSAR computer program, the transformation by-products are more harmful than sotalol. [ABSTRACT FROM AUTHOR]

Myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are enzymes present in neutrophil and eosinophil leukocytes, respectively. Here, we present the development of a sensitive and specific assay for determination of the halogenating enzymatic activity of MPO and EPO based on the electrophilic attack of HOCl and HOBr on aromatic ring of dansylglycine (DG). We found that the intrinsic fluorescence of DG was promptly depleted by the action of these acids. In the presence of the enzymes, the fluorescence bleaching was dependent of chloride (Cl − ) and bromide (Br − ), which makes the assay able to distinguish the halogenating from the peroxidase activity. A linear correlation was obtained between the hydrogen peroxide (H 2 O 2 ) concentration and the fluorescent decay. Similarly, the enzyme activity was measured by keeping constant H 2 O 2 . The method was applied for studding MPO/EPO specific inhibitors as 5-fluortryptamine (reversible inhibitor) and 4-hydroxybenzhydrazide (irreversible inhibitor). Differently of the taurine chloramine/3,3′,5,5'-tetramethylbenzidine assay, which is among the most used technique, the dansylglycine assay was able to differentiate these inhibitors based on their kinetic behavior. In conclusion, this assay can differentiate the peroxidase and halogenating activity of MPO and EPO. Moreover, the method is adequate for real-time measurement of the production of HOCl and HOBr. [ABSTRACT FROM AUTHOR]

After acute myocardial infarction (AMI), neutrophils are recruited to the affected myocardium. Hypochlorous acid (HOCl) produced by neutrophil myeloperoxidase (MPO) damages cardiomyocytes and potentially expands the primary infarct. Rat cardiomyocyte-like cells were incubated with isolated human neutrophils treated with chemical activators in the absence or presence of nitroxide 4-methoxy-Tempo (MetT; 25 μM) for 4, 6 or 24 h; studies with reagent HOCl served as positive control. Treating cardiomyocytes with activated neutrophils or reagent HOCl resulted in a marked increase in protein tyrosine chlorination and a decline in protein tyrosine phosphatase (PTP) activity. On balance our data also supported an increase in phosphorylation of MAPK p38 and ERK1/2 suggestive of an intracellular hyperphosphorylation status and this was accompanied by decreases in cell viability, as judged by assessing caspases-3/7 activity. For cells exposed to activated neutrophils receptor-mediated uptake of transferrin decreased although total matrix metalloproteinase (MMP) activity was unaffected. Addition of MetT ameliorated protein tyrosine chlorination, decreased MAPK activity and restored receptor-mediated transferrin uptake and PTP activity in cardiomyocytes. Overall, adverse effects of neutrophil-derived HOCl on cultured cardiomyocytes were ameliorated by MetT suggesting that nitroxides may be beneficial to inflammatory pathologies, where neutrophil recruitment/activation is a prominent and early feature. [ABSTRACT FROM AUTHOR]

Myeloperoxidase (MPO) is the most toxic enzyme found in the azurophilic granules of neutrophils. MPO utilizes H 2 O 2 to generate hypochlorous acid (HClO) and other reactive moieties, which kill pathogens during infections. In contrast, in the setting of sterile inflammation, MPO and MPO-derived oxidants are thought to be pathogenic, promoting inflammation and causing tissue damage. In contrast, evidence also exists that MPO can limit the extent of immune responses. Elevated MPO levels and activity are observed in a number of autoimmune diseases including in the central nervous system (CNS) of multiple sclerosis (MS) and the joints of rheumatoid arthritis (RA) patients. A pathogenic role for MPO in driving autoimmune inflammation was demonstrated using mouse models. Mechanisms whereby MPO is thought to contribute to disease pathogenesis include tuning of adaptive immune responses and/or the induction of vascular permeability. [ABSTRACT FROM AUTHOR]

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-derived reactive oxygen species (ROS) such as superoxide and hydrogen peroxide (H 2 O 2 ), have emerged as important molecules in the pathogenesis of diabetic endothelial dysfunction. Additionally, neutrophils-derived myeloperoxidase (MPO) and MPO-catalyzed hypochlorous acid (HOCl) play important roles in the vascular injury. However, it is unknown whether MPO can use vascular-derived ROS to induce diabetic endothelial dysfunction. In the present study, we demonstrated that NADPH oxidase was the main source of ROS formation in high glucose-cultured human umbilical vein endothelial cells (HUVECs), and played a critical role in high glucose-induced endothelial dysfunction such as cell apoptosis, loss of cell viability and reduction of nitric oxide (NO). However, the addition of MPO could amplify the high glucose-induced endothelial dysfunction which was inhibited by the presence of apocynin (NADPH oxidase inhibitor), catalase (H 2 O 2 scavenger), or methionine (HOCl scavenger), demonstrating the contribution of NADPH oxidase-H 2 O 2 -MPO-HOCl pathway in the MPO/high glucose-induced vascular injury. In high glucose-incubated rat aortas, MPO also exacerbated the NADPH oxidase-induced impairment of endothelium-dependent relaxation. Consistent with these in vitro data, in diabetic rat aortas, both MPO expresion and NADPH oxidase activity were increased while the endothelial function was simultaneously impaired. The results suggested that vascular-bound MPO could amplify high glucose-induced vascular injury in diabetes. MPO-NADPH oxidase-HOCl may represent an important pathogenic pathway in diabetic vascular diseases. [ABSTRACT FROM AUTHOR]

In this review, some of the recent developments in probes and assay techniques specific for superoxide (O 2 – ) and hydrogen peroxide (H 2 O 2 ) are discussed. Over the last decade, significant progress has been made in O 2 – and H 2 O 2 detection due to syntheses of new redox probes, better understanding of their chemistry, and development of specific and sensitive assays. For superoxide detection, hydroethidine (HE) is the most suitable probe, as the product, 2-hydroxyethidium, is specific for O 2 – . In addition, HE-derived dimeric products are specific for one-electron oxidants. As red-fluorescent ethidium is always formed from HE intracellularly, chromatographic techniques are required for detecting 2-hydroxyethidium. HE analogs, Mito-SOX and hydropropidine, exhibit the same reaction chemistry with O 2 – and one-electron oxidants. Thus, mitochondrial superoxide can be unequivocally detected using HPLC-based methods and not by fluorescence microscopy. Aromatic boronate-based probes react quantitatively with H 2 O 2 , forming a phenolic product. However, peroxynitrite and hypochlorite react more rapidly with boronates, forming the same product. Using ROS-specific probes and HPLC assays, it is possible to screen chemical libraries to discover specific inhibitors of NADPH oxidases. We hope that rigorous detection of O 2 – and H 2 O 2 in different cellular compartments will improve our understanding of their role in redox signaling. [ABSTRACT FROM AUTHOR]

Myocardial inflammation following acute myocardial infarct (AMI) is associated with risk of congestive heart failure. Pro-inflammatory neutrophils were recruited to the damaged myocardium 24 h after permanent coronary ligation in rats to induce AMI as judged by the presence of immune-positive myeloperoxidase (MPO) in the tissues; MPO generates the oxidant hypochlorous acid (HOCl). Neutrophils were absent in hearts from Control (untreated) and surgical Sham. Similarly, rats exposed to 1 h coronary ligation (Ischemia) showed no neutrophil infiltrate. Concomitantly, MPO activity increased in left ventricular (LV) homogenates prepared from the AMI group and this was inhibited by paracetamol and the nitroxide TEMPO. The same LV-homogenates showed increased 3-chlorotyrosine/tyrosine ratios (biomarker for MPO-activity). Combined 2D gel/Western blot indicated cardiac myoglobin (Mb) was modified after AMI. Subsequent MALDI-TOF and LC-MS/MS analysis of isolated protein spots revealed increased Mb oxidation in hearts from the AMI group relative to Control, Sham and Ischemia groups. Peptide mass mapping revealed oxidation of Met9 and Met132 to the corresponding sulfoxides yet Cys67 remained unmodified. Therefore, neutrophil-generated HOCl can oxidize cardiac Mb after AMI and this may impact on its function within the affected myocardium: oxidized Mb maybe a useful marker of myocardial inflammation. [ABSTRACT FROM AUTHOR]

General markers of HOCl induced changes in food proteins were evaluated and compared with the specific indicator 3-chlorotyrosine using dairy proteins at various oxidant/protein ratios and at different pHs. Protein aggregation was more pronounced at alkaline pH and already observed at a ratio of 0.3 mmol HOCl/g protein. Tryptophan, methionine, tyrosine and lysine in whey proteins showed more degradation at pH 8.0, whereas methionine and histidine in caseins were more vulnerable for degradation at pH 5.8. Total thiol content was strongly decreased, up to 75% at 4.8 mmol HOCl/g whey protein with more degradation at acidic pH while in caseins it remained constant. The available lysine content notably decreased upon HOCl treatment and was more pronounced at pH 8.0. The levels of 3-chlorotyrosine increased as function of the oxidant/protein ratio and reached a maximum at 2.8 mmol HOCl/g whey and casein proteins. The 3-chlorotyrosine concentration was observed the least at pH 8.0, while the increase in protein carbonyls depended only on the HOCl/protein ratio, but not on the pH. It is concluded that 3-chlorotyrosine provides a more accurate assessment of the impact of HOCl damage on proteins in foods. [ABSTRACT FROM AUTHOR]

Myeloperoxidase (MPO)-derived product hypochlorous acid (HOCl) is able to induce cellular senescence and MPO is also expressed in endothelial cells besides the well-recognized immune cells. This study aims to clarify the association of endothelium-derived MPO with endothelial senescence in hyperlipidemia. The rats were fed with high-fat diet for 8 weeks to establish a hyperlipidemic model, which showed an increase in plasma lipids, endothelium-derived MPO expression, endothelial senescence and endothelial dysfunction concomitant with a reduction in glycogen synthase kinase 3 beta (GSK-3β) activity and phosphorylated β-catenin (p-β-catenin) level as well as an increase in β-catenin and p53 levels within the endothelium. Next, human umbilical vein endothelial cells (HUVECs) were incubated with oxidized low density lipoprotein (ox-LDL, 100 μg/ml) for 24 h to establish a senescent cell model in vitro. Consistent with the finding in vivo, ox-LDL-induced MPO expression and HUVECs senescence, accompanied by a decrease in GSK-3β activity and p-β-catenin level as well as an increase in HOCl content, β-catenin and p53 levels; these phenomena were attenuated by MPO inhibitor. Replacement of ox-LDL with HOCl could also induce HUVECs senescence and activate the β-catenin/p53 pathway. Based on these observations, we conclude that endothelium-derived MPO is upregulated in hyperlipidemic rats, which may contribute to the accelerated vascular endothelial senescence through a mechanism involving the β-catenin/p53 pathway. [ABSTRACT FROM AUTHOR]

Heme b containing chlorite dismutase (Cld) catalyses the conversion of chlorite to chloride and dioxygen which includes an unusual O O bond formation. This review summarizes our knowledge about the interaction of chlorite with heme enzymes and introduces the biological role, phylogeny and structure of functional chlorite dismutases with differences in overall structure and subunit architecture. The paper sums up the available experimental and computational studies on chlorite degradation by water soluble porphyrin complexes as well as a model based on the active site of Cld. Finally, it reports the available biochemical and biophysical data of Clds from different organisms which allow the presentation of a general reaction mechanism. It includes binding of chlorite to ferric Cld followed by subsequent heterolytic O Cl bond cleavage leading to the formation of Compound I and hypochlorite, which finally recombine for production of chloride and O 2 . The role of the Cld-typical distal arginine in catalysis is discussed together with the pH dependence of the reaction and the role of transiently produced hypochlorite in irreversible inactivation of the enzyme. [ABSTRACT FROM AUTHOR]

Atherosclerosis is characterised by the accumulation of lipids within macrophages in the artery wall. Low-density lipoprotein (LDL) is the source of this lipid, owing to the uptake of oxidised LDL by scavenger receptors. Myeloperoxidase (MPO) released by leukocytes during inflammation produces oxidants that are implicated in atherosclerosis. Modification of LDL by the MPO oxidant hypochlorous acid (HOCl), results in extensive lipid accumulation by macrophages. However, the reactivity of the other major MPO oxidant, hypothiocyanous acid (HOSCN) with LDL is poorly characterised, which is significant given that thiocyanate is the favoured substrate for MPO. In this study, we comprehensively compare the reactivity of HOCl and HOSCN with LDL, and show key differences in the profile of oxidative damage observed. HOSCN selectively modifies Cys residues on apolipoprotein B100, and oxidises cholesteryl esters resulting in formation of lipid hydroperoxides, 9-hydroxy-10,12-octadecadienoic acid (9-HODE) and F 2 -isoprostanes. The modification of LDL by HOSCN results macrophage lipid accumulation, though generally to a lesser extent than HOCl-modified LDL. This suggests that a change in the ratio of HOSCN:HOCl formation by MPO from variations in plasma thiocyanate levels, will influence the nature of LDL oxidation in vivo , and has implications for the progression of atherosclerosis. [ABSTRACT FROM AUTHOR]

Myeloperoxidase (MPO) is expressed by myeloid cells for the purpose of catalyzing the formation of hypochlorous acid, from chloride ions and reaction with a hydrogen peroxide-charged heme covalently bound to the enzyme. Most peroxidase enzymes both plant and mammalian are inhibited by benzoic acid hydrazide (BAH)-containing compounds, but the mechanism underlying MPO inhibition by BAH compounds is largely unknown. Recently, we reported MPO inhibition by BAH and 4-(trifluoromethyl)-BAH was due to hydrolysis of the ester bond between MPO heavy chain glutamate 242 ( HC Glu 242 ) residue and the heme pyrrole A ring, freeing the heme linked light chain MPO subunit from the larger remaining heavy chain portion. Here we probed the structure and function relationship behind this ester bond cleavage using a panel of BAH analogs to gain insight into the constraints imposed by the MPO active site and channel leading to the buried protoporphyrin IX ring. In addition, we show evidence that destruction of the heme ring does not occur by tracking the heme prosthetic group and provide evidence that the mechanism of hydrolysis follows a potential attack of the HC Glu 242 carbonyl leading to a rearrangement causing the release of the vinyl-sulfonium linkage between HC Met 243 and the pyrrole A ring. [ABSTRACT FROM AUTHOR]

OmpD is the major Salmonella enterica serovar Typhimurium ( S. Typhimurium) porin and mediates hydrogen peroxide (H 2 O 2 ) influx. The results described herein extend this finding to hypochlorous acid (HOCl), another reactive oxygen species that is also part of the oxidative burst generated by the phagosome. S. Typhimurium cells lacking OmpD show decreased HOCl influx, and OmpD-reconstituted proteoliposomes show an increase in the uptake of the toxic compound. To understand this physiologically relevant process, we investigated the role of key OmpD residues in H 2 O 2 and NaOCl transport. Using a theoretical approach, residue K16 was defined as a major contributor to the channel electrostatic properties, and E111 was shown to directly participate in the size-exclusion limit of the channel. Together, we provide theoretical, genetic, and biochemical evidence that OmpD mediates H 2 O 2 and NaOCl uptake, and that key residues of the channel are implicated in this process. [ABSTRACT FROM AUTHOR]

Ceruloplasmin (Cp) is a copper-containing ferroxidase with potent antioxidant activity. Cp is expressed by hepatocytes and activated macrophages and has been known as physiologic inhibitor of myeloperoxidase (MPO). Enzymatic activity of MPO produces anti-microbial agents and strong prooxidants such as hypochlorous acid and has a potential to damage host tissue at the sites of inflammation and infection. Thus Cp–MPO interaction and inhibition of MPO has previously been suggested as an important control mechanism of excessive MPO activity. Our aim in this study was to identify minimal Cp domain or peptide that interacts with MPO. We first confirmed Cp–MPO interaction by ELISA and surface plasmon resonance (SPR). SPR analysis of the interaction yielded 30 nM affinity between Cp and MPO. We then designed and synthesized 87 overlapping peptides spanning the entire amino acid sequence of Cp. Each of the peptides was tested whether it binds to MPO by direct binding ELISA. Two of the 87 peptides, P18 and P76 strongly interacted with MPO. Amino acid sequence analysis of identified peptides revealed high sequence and structural homology between them. Further structural analysis of Cp’s crystal structure by PyMOL software unfolded that both peptides represent surface-exposed sites of Cp and face nearly the same direction. To confirm our finding we raised anti-P18 antisera in rabbit and demonstrated that this antisera disrupts Cp–MPO binding and rescues MPO activity. Collectively, our results confirm Cp–MPO interaction and identify two nearly identical sites on Cp that specifically bind MPO. We propose that inhibition of MPO by Cp requires two nearly identical sites on Cp to bind homodimeric MPO simultaneously and at an angle of at least 120 degrees, which, in turn, exerts tension on MPO and results in conformational change. [ABSTRACT FROM AUTHOR]

Abstract: Soybean proteins are largely used in processed food products. Controlled soybean protein oxidation may improve its physico-chemical properties but it can lead to severe protein degradation as well. The indirect lipid induced and direct hypochlorous acid induced soybean protein oxidation were investigated and compared. Protein oxidation led to severe changes on protein level as evidenced by formation of protein bound carbonyls and changes in the electrophoretic pattern. During hypochlorous acid induced oxidation, significant losses of histidine, arginine, tyrosine, methionine and tryptophan residues were observed with severe aggregation. Lipid induced oxidation, however, led to less severe modifications on amino acid level. Lysine and histidine residues were the preferred targets being probably modified via interaction with secondary oxidation products while modification of methionine, tryptophan and tyrosine residues took place only under the most severe oxidizing conditions. The most dramatic changes upon oxidation of proteins were the aggregation mainly due to the interaction of secondary oxidation products with nucleophilic amino acids either via Michael addition or through formation of Schiff bases. [Copyright &y& Elsevier]

Abstract: Nanosized titanium dioxide (TiO2) is a common component of sunscreen preparations and cosmetics as it reflects UV and visible light in accordance to Rayleigh’s law. However, in aqueous environments, TiO2 is an efficient photocatalyst, producing superoxide and hydroxyl (HO ) radicals, which are highly damaging to biomolecules. We investigated the role of TiO2 in promoting the peroxidation of linoleic acid (LA) alone and in the presence of hypochlorous acid (HOCl). TiO2 significantly enhanced peroxidation of LA, which was further enhanced in the presence of HOCl. This latter finding involved the formation of singlet molecular oxygen in a Russell-type mechanism appearing to involve preformed lipid hydroperoxides (LOOH). In addition to lipid peroxidation, HOCl also mediated formation of 18:1 monochlorohydrins, which in the presence of TiO2 appeared to decompose to kinetic products which supplemented peroxidation of linoleic acid. We present a theoretical mechanism which fits the available experimental data and may partially explain the dichotomy associated with HOCls role in lipid modification. [Copyright &y& Elsevier]

Abstract: Generation of hypochlorous acid (HOCl), an important microbicidal agent, is considered to be the main function of myeloperoxidase (MPO), an enzyme present in phagocytes. High amounts of MPO are present in neutrophil azurophilic granules, which are mobilized into the phagolysosome vacuole during phagocytosis. MPO is also present in monocytes and macrophages, although to a lesser degree than in neutrophils. In the present study, we investigated the distribution of MPO in murine peritoneal cells using flow cytometry, confocal microscopy (CM) and transmission electron microscopy (TEM). MPO was observed in macrophages, and surprisingly, we detected MPO in B lymphocytes, specifically in B1-a. MPO was present in cytoplasmic granules, vesicles, mitochondria and the nucleus of murine peritoneal cells. Together, these findings suggest that, in addition to its known microbicidal activity, MPO has a myriad of other unanticipated cellular functions. [Copyright &y& Elsevier]

Abstract: Neutrophil oxidants, including the myeloperoxidase products, HOCl and chloramines, have been linked to endothelial dysfunction in inflammatory diseases such as atherosclerosis. As they react preferentially with sulfur centers, thiol proteins are likely to be cellular targets. Our objectives were to establish whether there is selective protein oxidation in vascular endothelial cells treated with HOCl or chloramines, and to identify sensitive proteins. Cells were treated with HOCl, glycine chloramine and monochloramine, reversibly oxidized cysteines were labeled and separated by 1D or 2D SDS–PAGE, and proteins were characterized by mass spectrometry. Selective protein oxidation was observed, with chloramines and HOCl causing more changes than H2O2. Cyclophilin A was one of the most sensitive targets, particularly with glycine chloramine. Cyclophilin A was also oxidized in Jurkat T cells where its identity was confirmed using a knockout cell line. The product was a mixed disulfide with glutathione, with glutathionylation at Cys-161. Glyceraldehyde-3-phosphate dehydrogenase, peroxiredoxins and cofilin were also highly sensitive to HOCl/chloramines. Cyclophilins are becoming recognized as redox regulatory proteins, and glutathionylation is an important mechanism for redox regulation. Cells lacking Cyclophilin A showed more glutathionylation of other proteins than wild-type cells, suggesting that cyclophilin-regulated deglutathionylation could contribute to redox changes in HOCl/chloramine-exposed cells. [Copyright &y& Elsevier]

Abstract: Optimal and efficient killing of ingested microbes by human neutrophils is mediated in large part by the action of hypochlorous acid produced by the myeloperoxidase–H2O2–chloride system in phagosomes. Myeloperoxidase gene transcription is limited to early myeloid precursors in the bone marrow, when myeloperoxidase is synthesized and stored in azurophilic granules for subsequent release from stimulated neutrophils. Promyeloperoxidase, the 90kDa myeloperoxidase precursor synthesized in the endoplasmic reticulum (ER), contains a 125-amino acid pro-region whose function and fate during myeloperoxidase biosynthesis are unknown. Promyeloperoxidase has two fates during myeloperoxidase biosynthesis; the majority undergoes proteolytic processing to generate mature myeloperoxidase, while the remainder is constitutively secreted from the cells in bone marrow. We used a promyelocytic cell line that produces endogenous myeloperoxidase as well as human embryonic kidney cells stably expressing normal and mutant forms of myeloperoxidase to examine proteolytic processing of promyeloperoxidase. We demonstrated that CMK-RVKR, an inhibitor of subtilisin-like proteinases, blocked cleavage of the pro-peptide of promyeloperoxidase in a post-ER compartment. Mutants with alanine substitution of basic residues in the predicted proteinase cleavage site failed to undergo maturation to normal myeloperoxidase subunits and were arrested at the promyeloperoxidase stage. Whereas specific mutants varied as to their stability, secreted promyeloperoxidase from the mutants retained the capacity to generate hypochlorous acid. Taken together, these studies demonstrate proconvertase-dependent cleavage of promyeloperoxidase as an essential step in normal proteolytic processing and granule targeting of myeloperoxidase. Furthermore, although mutations in the proteinase cleavage site reduced intracellular stability of the mutants, the integrity of the heme group was not compromised, as chlorinating activity was retained in the secreted promyeloperoxidase. [Copyright &y& Elsevier]

Abstract: Perspectives for the use of carbon nanotubes in biomedical applications depend largely on their ability to degrade in the body into products that can be easily cleared out. Carboxylated single-walled carbon nanotubes (c-SWCNTs) were shown to be degraded by oxidants generated by peroxidases in the presence of hydrogen peroxide. In the present study we demonstrated that conjugation of poly(ethylene glycol) (PEG) to c-SWCNTs does not interfere with their degradation by peroxidase/H2O2 system or by hypochlorite. Comparison of different heme-containing proteins for their ability to degrade PEG-SWCNTs has led us to conclude that the myeloperoxidase (MPO) product hypochlorous acid (HOCl) is the major oxidant that may be responsible for biodegradation of PEG-SWCNTs in vivo. MPO is secreted mainly by neutrophils upon activation. We hypothesize that SWCNTs may enhance neutrophil activation and therefore stimulate their own biodegradation due to MPO-generated HOCl. PEG-SWCNTs at concentrations similar to those commonly used in in vivo studies were found to activate isolated human neutrophils to produce HOCl. Both PEG-SWCNTs and c-SWCNTs enhanced HOCl generation from isolated neutrophils upon serum-opsonized zymosan stimulation. Both types of nanotubes were also found to activate neutrophils in whole blood samples. Intraperitoneal injection of a low dose of PEG-SWCNTs into mice induced an increase in percentage of circulating neutrophils and activation of neutrophils and macrophages in the peritoneal cavity, suggesting the evolution of an inflammatory response. Activated neutrophils can produce high local concentrations of HOCl, thereby creating the conditions favorable for degradation of the nanotubes. [Copyright &y& Elsevier]

Abstract: Hypochlorous acid (HOCl) is a potent oxidant produced by the enzyme myeloperoxidase, which is released by neutrophils under inflammatory conditions. Although important in the immune system, HOCl can also damage host tissue, which contributes to the development of disease. HOCl reacts readily with free amino groups to form N-chloramines, which also cause damage in vivo, owing to the extracellular release of myeloperoxidase and production of HOCl. HOCl and N-chloramines react readily with cellular thiols, which causes dysfunction via enzyme inactivation and modulation of redox signaling processes. In this study, the ability of HOCl and model N-chloramines produced on histamine and ammonia at inflammatory sites, to oxidize specific thiol-containing proteins in human coronary artery endothelial cells was investigated. Using a proteomics approach with the thiol-specific probe, 5-iodoacetamidofluorescein, we show that several proteins including peptidylprolyl isomerase A (cyclophilin A), protein disulfide isomerase, glyceraldehyde-3-phosphate dehydrogenase and galectin-1 are particularly sensitive to oxidation by HOCl and N-chloramines formed at inflammatory sites. This will contribute to cellular dysfunction and may play a role in inflammatory disease pathogenesis. [Copyright &y& Elsevier]

Abstract: The objectives of the present study were to evaluate the spread of Salmonella Enteritidis to different cutting boards (wood, triclosan-treated plastic, glass, and stainless steel) from contaminated poultry skin (5 log CFU/g) and then to tomatoes and to analyze the effect of different protocols used to clean these surfaces to control contamination. The following procedures were simulated: (1) no cleaning after handling contaminated poultry skin; (2) rinsing in running water; (3) cleaning with dish soap and mechanical scrubbing; and (4) cleaning with dish soap and mechanical scrubbing, followed by disinfection with hypochlorite. The pathogen was recovered from all surfaces following procedure 1, with counts ranging from 1.90 to 2.80 log, as well as from the tomatoes handled on it. Reduced numbers of S. Enteritidis were recovered using the other procedures, both from the surfaces and from the tomatoes. Counts were undetectable after procedure 4. From all surfaces evaluated, wood was the most difficult to clean, and stainless steel was the easiest. The use of hypochlorite as a disinfecting agent helped to reduce cross-contamination. [Copyright &y& Elsevier]

Abstract: Myeloperoxidase is abundantly present in inflammatory diseases where activation of monocytes/macrophages and T-cell-mediated immune response occurs. The potent oxidant hypochlorous acid (HOCl), generated by the myeloperoxidase–H2O2–chloride system of activated phagocytes, converts low-density lipoprotein (LDL) into a proinflammatory lipoprotein particle. Here, we investigated the apoptotic effect of HOCl–LDL, an in vivo occurring LDL modification, on human T-cell lymphoblast-like Jurkat cells. Experiments revealed that HOCl–LDL, depending on the oxidant:lipoprotein molar ratio, induces apoptosis via activation of caspase-3, PARP cleavage and accumulation of reactive oxygen species. The absence of Fas-associated protein with death domain or caspase-8 in mutant cells did not prevent HOCl–LDL induced apoptosis. In contrast, overexpression of the anti-apoptotic Bcl-2 protein protects Jurkat cells against HOCl–LDL-induced apoptosis and prevents accumulation of reactive oxygen species. We conclude that HOCl–LDL-mediated apoptosis in Jurkat cells follows predominantly the intrinsic, mitochondrial pathway. In situ experiments revealed that an antibody raised against HOCl–LDL recognized epitopes that colocalize both with myeloperoxidase and CD3-positive T-cells in human decidual tissue where local stimulation of the immune system occurs. We provide convincing evidence that formation of HOCl-modified (lipo)proteins generated by the myeloperoxidase–H2O2–chloride system contributes to apoptosis in T-cells. [Copyright &y& Elsevier]

Abstract: Modification/chlorination of high-density lipoprotein (HDL) by hypochlorous acid (HOCl), formed by the myeloperoxidase–H2O2–chloride system of activated phagocytes, converts an anti-atherogenic lipoprotein into a pro-inflammatory lipoprotein particle. Chlorinated HDL is present in human lesion material, binds to and is internalized by endothelial cells and impairs expression and activity of endothelial nitric oxide synthase. The present study aimed at clarifying whether exposure of endothelial cells to pro-inflammatory HOCl–HDL impacts on expression of heme oxygenase-1, a potential rescue pathway against endothelial dysfunction. Our findings revealed that HDL modified by HOCl, added as reagent or generated enzymatically, induced phosphorylation of p42/44 mitogen-activated protein kinase, expression of transcription factor early growth response-1 (Egr-1) and enhanced expression of heme oxygenase-1 in human endothelial cells. Upregulation of heme oxygenase-1 could be blocked by an inhibitor upstream of p42/44 mitogen-activated protein kinase and/or knockdown of Egr-1 by RNA-interference. Electrophoretic mobility shift assays demonstrated HOCl–HDL-mediated induction of the Egr-1 DNA binding activity. Immunocytochemical and immunoblotting experiments demonstrated HOCl–HDL-induced translocation of Egr-1 to the nucleus. The present study demonstrates a novel compensatory pathway against adverse effects of HOCl–HDL, providing cytoprotection in a number of pathological conditions including cardiovascular disease. [Copyright &y& Elsevier]

Abstract: Taurine is the most abundant free amino acid in leukocytes and can react with HOBr to produce taurine bromamine (Tau-NHBr). The aim of this study was to assess the ability of Tau-NHBr to oxidize tryptophan, either free or as a residue in albumin. We have demonstrated that Tau-NHBr is a powerful oxidant for tryptophan. Importantly, in comparison to taurine chloramine, HOCl or HOBr, Tau-NHBr exhibits a degree of selectivity for tryptophan. Oxidation of albumin by Tau-NHBr resulted in emission of light, and the quantum yield was more than 10-fold more efficient than that of the other oxidants. The fluorescence band corresponding to oxidized albumin (λ ex 350/λ em 450), which is characteristic of the formation of formylkynurenine, was significantly higher in reactions using Tau-NHBr. Excitation of the fluorescent probe 8-anilino-1-naphthalenesulfonate at 295nm was used to assess the depletion of tryptophan residues in albumin. Results from this experiment further supported a higher efficiency of oxidation of tryptophan residues by Tau-NHBr. Other parameters of protein oxidation, including cysteine depletion and formation of carbonyl groups, were not significantly different between the oxidants tested. In conclusion, these results indicate that Tau-NHBr has a higher affinity for tryptophan residues in proteins. [Copyright &y& Elsevier]

Abstract: The objectives of this study were to investigate the efficacy of two sanitizers, i.e. hypochlorous and peracetic acids, in reducing coliforms and Escherichia coli levels on tomato fruits and lettuce leaves, and to mathematically predict the relationship among the initial bacterial load, type of vegetable/fruit, types and concentration of sanitizer and residual microorganism levels after the sanitizing, by applying artificial neural networks (ANNs). The E. coli and coliforms used in this study were isolated from the two food types, and their cultures were activated in Tryptic Soy Broth (ca. 6–7log10 cfu/ml) before inoculating onto the fruit and vegetable. Both sanitizers reduced the number of the micro-organisms. However, as the hypochlorous acid concentration was increased, the level of viable coliforms and E. coli on the tomato fruits was reduced around 2–3log10 cfu/g (p ≤ 0.05), compared to only about 1log10 cfu/g reduction on lettuce leaves (p ≤ 0.05). Conversely, when the peracetic acid concentration was increased, the coliforms and E. coli levels on tomato fruits were reduced by some 3–4log10 cfu/g (p > 0.05) compared to only about 2log10 cfu/g on lettuce leaves (p > 0.05). The best sum square error from the neural prediction of residual coliforms and E. coli were 0.50 and 0.84, respectively, and the maximum R 2 of residual coliforms and E. coli were 0.85 and 0.72, respectively. Only one hidden layer with three hidden neurons for coliforms and five for E. coli, were required to model this data. [ABSTRACT FROM AUTHOR]

Abstract: HDL-cholesterol levels are inversely correlated to the risk of cardiovascular disease. In recent years the concept that not only the quantity, but also the quality of HDL is related to their atheroprotective function has gained momentum. In fact several studies have showed that HDL can shift their properties from anti-atherogenic to pro-atherogenic upon chemical or enzymatic “modification”. However, not all kind of modifications affect the antiatherogenic properties of HDL. For example, tyrosylation of HDL improves its ability to remove cholesterol from cultured cells and inhibits mice atherosclerotic lesion formation; oxidation of HDL3 with 15-lipoxygenase or with copper ions for short time induce the formation of pre-β-migrating particles that are highly effective as cholesterol acceptors from lipid laden cells. Myeloperoxidase modifies HDL and apoA-I and reduces their ability to promote ABCA1-mediated cholesterol efflux. In the present study we show that modification with low concentration HOCl (a myeloperoxidase product) induces the formation of pre-β-migrating particles, thus improving the function of HDL in the reverse cholesterol transport, without affecting the anti-inflammatory activity. At higher HOCl concentration, pre-β-migrating particles were not detectable and the anti-inflammatory properties of HDL were lost. These findings suggest that during early phases of inflammation, when a low HOCl concentration is generated, changes in HDL occur that increase their ability to remove cholesterol and sparing anti-inflammatory properties; later during acute inflammation, when higher HOCl concentration are present changes in HDL occur that severely decrease their ability to remove cholesterol from macrophages and to protect endothelial cells from pro-inflammatory stimuli. [Copyright &y& Elsevier]

Abstract: We compared the apoptotic mechanism involved in U937 human monocytic cell line in presence of oxidized low-density lipoproteins (oxLDL) obtained after treatment with hypochlorous acid (HOCl) or copper (Cu). Both types of oxLDL induced U937 apoptotic cell death via the mitochondrial pathway. In contrast to HOCl-oxLDL, Cu-oxLDL induced apoptosis via a caspase-independent mechanism, with no activation of pro-caspase-3, but via the release of apoptosis inducing factor (AIF) from mitochondria. The apoptotic program of the monocyte differs depending on the mode of LDL oxidation, based on differences in the oxidatively modified components of the two oxLDL types. [Copyright &y& Elsevier]

Abstract: The prospects for using bacterial DNA as an intrinsic probe for HOCl and secondary oxidants/chlorinating agents associated with it has been evaluated using both in vitro and in vivo studies. Single-strand and double-strand breaks occurred in bare plasmid DNA that had been exposed to high levels of HOCl, although these reactions were very inefficient compared to polynucleotide chain cleavage caused by the OH -generating reagent, peroxynitrite. Plasmid nicking was not increased when intact Escherichia coli were exposed to HOCl; rather, the amount of recoverable plasmid diminished in a dose-dependent manner. At concentration levels of HOCl exceeding lethal doses, genomic bacterial DNA underwent extensive fragmentation and the amount of precipitable DNA–protein complexes increased several-fold. The 5-chlorocytosine content of plasmid and genomic DNA isolated from HOCl-exposed E. coli was also slightly elevated above controls, as measured by mass spectrometry of the deaminated product, 5-chlorouracil. However, the yields were not dose-dependent over the bactericidal concentration range. Genomic DNA recovered from E. coli that had been subjected to phagocytosis by human neutrophils occasionally showed small increases in 5-chlorocytosine content when compared to analogous cellular reactions where myeloperoxidase activity was inhibited by azide ion. Overall, the amount of isolable 5-chlorouracil from the HOCl-exposed bacterial cells was far less than the damage manifested in polynucleotide bond cleavage and cross-linking. [Copyright &y& Elsevier]

Abstract: Inflammatory liver diseases are associated with oxidative stress mediated particularly by neutrophilic granulocytes. At inflammatory loci, hypochlorous acid (HOCl) is generated by myeloperoxidase. HOCl reacts with a large variety of molecules and induces (among other reactions) the formation of lysophosphatidylcholine (LPC) from polyunsaturated phosphatidylcholines (PC). As liver tissue contains huge amounts of polyunsaturated PC species enhanced LPC concentrations are detectable under these conditions. However, human liver contains also major amounts of polyunsaturated phosphatidylethanolamine (PE). It is so far widely unknown, if PE oxidation by HOCl leads to the generation of LPE in a similar way as observed in the case of PC. Using MALDI-TOF mass spectrometry (MS) and 31P NMR spectroscopy, LPC generation from unsaturated PC could be verified in the presence of HOCl. In contrast, unsaturated PE led exclusively to chlorohydrins and other oxidation products but not to LPE. Although these data were obtained with a quite simple model system, it is obvious that LPC is a much more suitable biomarker of oxidative stress than LPE: LPC is more readily generated and also more sensitively detectable by means of mass spectrometry and other spectroscopic methods. Nevertheless, it will also be shown that the nitrile of LPE is also generated. However, this compound is exclusively detectable as negative ion. [Copyright &y& Elsevier]

Abstract: The prevalence of Alicyclobacillus spp. and other spore-forming spoilage organisms in food handling and processing environments presents a sanitation challenge to manufacturers of products such as juices and beverages. The objectives of this study were to determine the efficacy of chlorine dioxide and sodium hypochlorite in killing Alicyclobacillus spores in situ and to evaluate the efficacy of various chlorine dioxide and hypochlorite sanitizing regimes on Alicyclobacillus spp. spores on stainless steel, wood, and rubber conveyor material. Five or two log CFU/ml spore concentrations were left in aqueous solution or inoculated onto stainless steel, rubber, or wood coupons and challenged with sanitizer for varied time intervals. After treatment, the coupons were placed in sterile sample bags, massaged with neutralizing buffer, and enumerated on Ali agar. Surfaces were also examined before and after treatment by scanning electron microscopy to confirm destruction or removal of the spores. For both five and two log CFU/ml spore concentrations, treatments of 50 and 100 ppm of chlorine dioxide and 1000 and 2000 ppm of hypochlorite, respectively, were the most effective. Of the range of chlorine dioxide concentrations and contact time regimes evaluated for all surfaces, the most effective concentration/time regime applied was 100 ppm for 10 min. Reductions ranged from 0 to 4.5 log CFU/coupon. Chlorine dioxide was least effective when applied to wood. Hypochlorite was not efficient at eliminating Alicyclobacillus spores from any of the food contact surfaces at any time and concentration combinations tested. Chlorine dioxide is an alternative treatment to kill spores of Alicyclobacillus spp. in the processing environment. [Copyright &y& Elsevier]

Abstract: The heme protein myeloperoxidase (MPO) functions as a catalyst for lipoprotein oxidation. Hypochlorous acid (HOCl), a potent two-electron oxidant formed by the MPO–H2O2–chloride system of activated phagocytes, modifies antiatherogenic high-density lipoprotein (HDL). The structural heterogeneity and oxidative susceptibility of HDL particle subfractions were probed with HOCl. All distinct five HDL subfraction were modified by HOCl as demonstrated by the consumption of tryptophan residues and free amino groups, cross-linking of apolipoprotein AI, formation of HOCl-modified epitopes, increased electrophoretic mobility and altered content of unsaturated fatty acids in HDL subclasses. Small, dense HDL3 were less susceptible to oxidative modification than large, light HDL2 on a total mass basis at a fixed HOCl:HDL mass ratio of 1:32, but in contrast not on a particle number basis at a fixed HOCl:HDL molar ratio of 97:1. We conclude that structural and physicochemical differences between HDL subclasses do not influence their intrinsic susceptibility to oxidative attack by HOCl. [Copyright &y& Elsevier]

This study presents the isolation of a novel strain of Dehalococcoides mccartyi , NIT01, which can completely dechlorinate up to 4.0 mM of trichloroethene to ethene via 1,2- cis -dichroroethene and vinyl chloride within 25 days. Strain NIT01 dechlorinated chloroethenes (CEs) at a temperature range of 25–32 °C and pH range of 6.5–7.8. The activity of the strain was inhibited by salt at more than 1.3% and inactivated by 1 h exposure to 2.0% air or 0.5 ppm hypochlorous acid. The genome of NIT01 was highly similar to that of the Dehalococcoides strains DCMB5, GT, 11a5, CBDB1, and CG5, and all included identical 16S rRNA genes. Moreover, NIT01 had 19 rdhA genes including NIT01- rdhA7 and rdhA13 , which are almost identical to vcrA and pceA that encode known dehalogenases for tetrachloroethene and vinyl chloride, respectively. We also extracted RdhAs from the membrane fraction of NIT01 using 0.5% n-dodecyl-β- d -maltoside and separated them by anion exchange chromatography to identify those involved in CE dechlorination. LC/MS identification of the LDS-PAGE bands and RdhA activities in the fractions indicated cellular expression of six RdhAs. NIT01-RdhA7 (VcrA) and NIT01-RdhA15 were highly detected and NIT01-RdhA6 was the third-most detected. Among these three RdhAs, NIT01-RdhA15 and NIT01-RdhA6 had no biochemically identified relatives and were suggested to be novel functional dehalogenases for CEs. The expression of multiple dehalogenases may support bacterial tolerance to high concentrations of CEs. • Dehalococcoides mccartyi NIT01 completely dechlorinates up to 4.0 mM TCE. • NIT01 had 19 rdhA genes including vcrA and pceA in the genome. • NIT01 expressed six RdhAs including NIT01-RdhA7 (VcrA). • The multiple RdhAs may support tolerance to high concentrations of chloroethenes. [ABSTRACT FROM AUTHOR]

Abstract: The outbreaks of West Nile virus (WNV), an emerging flavivirus recently implicated in outbreaks of fatal encephalitis, necessitate the development of effective anti-WNV drugs. In this study, it is demonstrated that human serum albumin is transformed into a WNV antiviral substance by hypochlorite (HOCl) modification. The HOCl-modified albumin efficiently neutralized WNV in vitro (EC50 =300 nM) and showed binding to a recombinant protein, representing the domain III of the WNV external envelope E glycoprotein. [Copyright &y& Elsevier]

Abstract: Ten heat-resistant lactococcal phages in M17 broth were inactivated by exposure to a range of different hypochlorite concentrations (2000–5000mgl−1). Deviations from first-order kinetics as sigmoidal shapes were observed in the survival curves of all bacteriophages. Therefore, an empirical model with four parameters was used to describe hypochlorite inactivation. The model provided good fit for all phages; however, it was observed that there was a high correlation between the parameters of this model. That is why, the number of parameters was reduced from four to two with a slight loss of goodness-of-fit and this reduced model produced fits comparable to the original model. Alternative D and z values were also proposed for the model. By comparing the times necessary to reduce the number of phages six- or seven-logs, the most hypochlorite-resistant phages were found as: φpld67 37, φpll47 21 and φpld66 36. Demonstration of this model may also be used for other bacteriophages inactivated by other biocides. [Copyright &y& Elsevier]

The parasite Perkinsus marinus has devastated natural and farmed oyster populations along the Atlantic and Gulf coasts of North America. When viable P. marinus trophozoites are engulfed by oyster hemocytes, the typical accumulation of reactive oxygen species (ROS) normally associated with phagocyte activity is not observed. One hypothesis to explain this is that the parasite rapidly removes ROS. A manifestation of efficient ROS removal should be a high level of resistance to exogenous ROS. We investigated the in vitro susceptibility of P. marinus to ROS as compared to the estuarine bacterium Vibrio splendidus. We find that P. marinus is markedly less susceptible than V. splendidus to superoxide and hydrogen peroxide (H2O2), but equally sensitive to hypochlorite. Viable P. marinus trophozoites degrade H2O2 in vitro, but lack detectable catalase activity. However, extracts contain an ascorbate dependent peroxidase activity that may contribute to H2O2 removal in vitro and in vivo. [Copyright &y& Elsevier]

Hypochlorite or its acid, hypochlorous acid, may exert both beneficial and toxic effects in vivo. In order to understand the role and action of hypochlorite, the formation of active oxygen species and its kinetics were studied in the reactions of hypochlorite with peroxides and amino acids. It was found that tert-butyl hydroperoxide and methyl linoleate hydroperoxide reacted with hypochlorite to give peroxyl and/or alkoxyl radicals with little formation of singlet oxygen in contrast to hydrogen peroxide, which gave singlet oxygen exclusively. Amino acids and ascorbate reacted with hypochlorite much faster than peroxides. Free radical-mediated lipid peroxidation of micelles and membranes in aqueous suspensions was induced by hypochlorite, the chain initiation being the decomposition of hydroperoxides by hypochlorite. It was suppressed efficiently by ebselen which reduced hydroperoxides and by α-tocopherol, which broke chain propagation, but less effectively by hydrophilic antioxidants present in the aqueous phase. Cysteine suppressed the oxidation, but it was poorer antioxidant than α-tocopherol. Ascorbate also exerted moderate antioxidant capacity, but it acted as a synergist with α-tocopherol. Taken together, it was suggested that the primary target of hypochlorite must be sulfhydryl and amino groups in proteins and that the lipid peroxidation may proceed as the secondary reaction, which is induced by radicals generated from sulfenyl chlorides and chloramines. [Copyright &y& Elsevier]