The effects of antioxidants and hypohalous acid scavengers on neutrophil activation by hypochlorous acid-modified low-density lipoproteins

Hypochlorous acid-modified human blood low density lipoprotein (LDL–HOCl) was shown to stimulate neutrophils and to increase the luminol- (lm-CL) or lucigenin-enhanced chemiluminescence (lc-CL) of neutrophils. Antioxidants and HOCl scavengers (glutathione, taurine, cysteine, methionine, ceruloplasmin, and human serum albumin (HSA)) were tested for effects on lm-CL, lc-CL, H₂O₂ production, and degranulation of azurophilic granules of neutrophils. All agents used in increasing concentrations were found to decrease lm-CL produced by neutrophils upon stimulation with LDL–HOCl or subsequent treatment with the activator phorbol 12-myristate 13-acetate (PMA). The agents exerted a far lower, if any, effect on lc-CL and the H₂O₂ production by neutrophils in the same conditions. In the majority of cases, a decline in neutrophil chemiluminescence in the presence of the agents was not related to their effect on neutrophil degranulation, but was most likely due to their direct interactions with reactive halogen (RHS) or oxygen (ROS) species generated upon neutrophil activation or to myeloperoxidase (MPO) inhibition. Antioxidants and HOCl scavengers present in the human body were assumed to decelerate the development of oxidative or halogenative stress and thereby prevent neutrophil activation.     

Element fluxes in watershed-lake ecosystems recovering from acidification: Plešné Lake,the Bohemian Forest, 2001–2005

Fluxes of major ions and nutrients were measured in the watershed-lake ecosystem of a strongly acidified lake, Ple?né jezero (Ple?né Lake), in the Czech Republic in hydrological years from 2001 through 2005. The lake is situated in a Norway spruce forest and has a steep watershed between elevations of 1090 and 1378 m. The average water input and output from the ecosystem was 1372 mm and 1157 mm (37 L km^?2 s^?1), respectively, and the water residence time averaged 306 days. Despite ecosystem recovery from acidification occurring since the late 1980s, the Ple?né watershed was an average net source of 25 mmol SO ₄ ^2? m^?2 yr^?1. Nitrogen saturation of the watershed caused low retention of the deposited inorganic N (< 44% on average) before 2004. Then, the watershed became a net source of 28–32 mmol m^?2 yr^?1 of inorganic N in the form of NO ₃ ^? due to climatic effects (a dry summer in 2003 and a cold winter in 2004) and forest dieback caused by a bark beetle attack in 2004. Nitrogen transformations and SO ₄ ^2? release were the dominant terrestrial sources of H⁺ (72 and 49 mmol m^?2 yr^?1, respectively) and the watershed was a net source of 24 mmol H⁺ m^?2 yr^?1. Ionic composition of surface inlets showed seasonal variations, with the most pronounced changes in NO ₃ ^? , ionic Al (Al_i), and DOC concentrations, while the composition of subsurface inlets was more stable. The in-lake biogeochemical processes reduced on average 59% of the incoming H⁺ (251 mmol H⁺ m^?2 yr^?1 on a lake-area basis). NO ₃ ^? assimilation and denitrification, photochemical and microbial decomposition of allochthonous organic acids, and SO ₄ ^2? reduction in the sediments were the most important aquatic H⁺ consuming processes (358, 121, and 59 mmol H⁺ m^?2 yr^?1, respectively), while hydrolysis of Al_i was the dominant in-lake H⁺ generating process (233 mmol H⁺ m^?2 yr^?1). Photochemical liberation from organic complexes was an additional in-lake source of Al_i. The net in-lake retention or removal of total phosphorus, total nitrogen, and silica were on average 50%, 27%, and 23%, respectively. The lake was a net source of NH ₄ ⁺ due to a cease in nitrification (pH < 5) and from NH ₄ ⁺ production by dissimilation exceeding its removal by assimilation.     

Regulation of morphological and functional properties of astrocytes by hydrogen peroxide

Effects of hydrogen peroxide on morphological characteristics, proliferation index, and menadione-dependent lucigenin-enhanced chemiluminescence of C6 glioma cells were studied. It was established that H₂O₂ at 5 × 10^?7?1 × 10^?8 M concentrations acted as a regulator of morphological and functional properties of astrocytes, inducing their reactivation, which is manifested as cell body hypertrophy and an increase of proliferative activity and menadione-induced production of superoxide anion radicals (O ₂ ^?? ). Cytodestructive action of hydrogen peroxide at a concentration higher than 1 × 10^?6 M on C6 glioma cells shows itself as a decrease of their proliferation index and the ability to generate O ₂ ^?? under the effect of menadione. Use of lipopolysaccharide B as a functional stimulator has shown that H₂O₂ modifies signaling pathways leading to an increase of mitotic activity of C6 glioma cells and decreases the yield of lucigenin-dependent chemiluminescence of astrocytes under the action of menadione to the level of control values.     

Copper ions and hydrogen peroxide form hypochlorite from NaCl thereby mimicking myeloperoxidase

Sea urchins have elaborated multiple defenses to assure monospermic fertilization. In this work, we have concentrated on a study of the mechanism(s) by which hydrogen peroxide (H₂O₂) prevents polyspermy in Arbacia punctulata. We found that it is not H₂O₂ but probably hypochlorous acid/hypochlorite (HOCl/OCl^?) derived from H₂O₂ that is toxic to the supernumerary sperm. The spermicidal activity of H₂O₂ is potentiated by at least one order of magnitude by cupric ions (Cu²⁺). This increased toxicity is not due to the formation of hydroxyl radicals (·OH) because ·OH scavengers did not counteract the activity of Cu²⁺. More-over, substitution of Cu²⁺ by ferrous ions (Fe²⁺), which are known to cause formation of ·OH from H₂O₂, had no effect on fertilization even at 10²?10³ times higher concentrations. In contrast, 3-amino-1,2,4-triazole (AT), an HOCl/OCl^? scavenger, totally reversed the toxic effects of Cu²⁺. Furthermore, we found that HOCl/OCl^? is generated in solutions of H₂O₂ and Cu²⁺ in the presence of 0.5 M NaCl and that its accumulation is abolished by AT. Thus it is possible that the antifertility properties of copper are due to its ability to mediate formation of HOCl/OCl^?. HOCl/OCl^? generated by Cu²⁺ from H₂O₂ and Cl^?, a low concentration of exogenously added HOCl/OCl^?, or increased concentrations of H₂O₂ has similar inhibitory effects on the fertilization process in sea urchins. Therefore, we suggest that polyspermy is prevented by the action of a myeloperoxidase that affects the formation of HOCl/OCl^? from the Cl^? present in sea water through reaction with H₂O₂ generated by the newly fertilized egg.     

Active oxygen species in pea seedlings during the interactions with symbiotic and pathogenic microorganisms

The level of active oxygen species (AOS)—superoxide anion radical (O ₂ ^·? ) and hydrogen peroxide (H₂O₂)—in pea (Pisum sativum L.) cultivar Marat seedlings was studied upon their inoculation with symbiotic (Rhizobium leguminosarum bv. viceae strain CIAM 1026) and pathogenic (Pseudomonas syringae pv. pisi Sackett) microorganisms. Different patterns of the changes in AOS in pea seedlings during the interactions with the symbiont and the phytopathogen were recorded. It is assumed that O ₂ ^·? and H₂O₂ are involved in the defense and regulatory mechanisms of the host plant.     

Relationship between leaf reddening,ROS and antioxidants in <Emphasis Type="Italic">Buxus microphylla</Emphasis> during overwintering

To provide a theoretical basis for revealing the mechanism of winter leaf reddening in evergreen species, the relationships between winter leaf reddening, reactive oxygen species (ROS) and the antioxidant system of Buxus microphylla ‘Wintergreen’ were studied. The pigment changes, ROS production, lipid peroxidation and antioxidants activities of sun leaves during the reddening and regreening processes were investigated, using green shade leaves as controls. The carotenoids in the sun leaves increased linearly with reddening but decreased with the regreening. There was no significant difference in either the superoxide anions (O ₂ ^?· ) or malondialdehyde (MDA) changes between the sun and shade leaves, and their O ₂ ^?· contents were positively correlated with MDA. In contrast to the shade leaves, the sun leaves showed a trend in which the hydrogen peroxide (H₂O₂) changes were closely related to the reddening process and positively correlated with carotenoids content but not with the MDA content. A similar trend was observed for catalase (CAT) and ascorbate peroxidase (APX) activity between the sun and shade leaves, but superoxide dismutase (SOD) and peroxidase (POD) activity and the ascorbate (AsA) content differed between these two ecotypes. Furthermore, the sun leaves had higher CAT activity and AsA content than the corresponding shade leaves. These results suggested that H₂O₂ might play an important role in the winter reddening of sun leaves by promoting the accumulation of carotenoids. In addition, SOD, POD and AsA probably play a photoprotective role in winter-red sun leaves, while the changes in O ₂ ^?· , CAT and APX were independent of winter leaf reddening and were more likely responses to stress caused by low temperatures.     

Adiponectin and colon cancer: evidence for inhibitory effects on viability and migration of human colorectal cell lines

Exogenous hydrogen peroxide (H₂O₂) induces oxidative stress and apoptosis in cancer cells. This study evaluated the antiapoptotic effects of pan-caspase and caspase-3, -8, or -9 inhibitors on H₂O₂-treated Calu-6 and A549 lung cancer cells in relation to reactive oxygen species (ROS) and glutathione (GSH). Treatment with 50–500 μM H₂O₂ inhibited the growth of Calu-6 and A549 cells at 24 h and induced apoptosis in these cells. All the tested caspase inhibitors significantly prevented cell death in H₂O₂-treated lung cancer cells. H₂O₂ increased intracellular ROS levels, including that of O ₂ ^·? , at 1 and 24 h. It also increased the activity of catalase but decreased the activity of SOD. In addition, H₂O₂ triggered GSH deletion in Calu-6 and A549 cells at 24 h. It reduced GSH levels in Calu-6 cells at 1 h but increased them at 24 h. Caspase inhibitors decreased O ₂ ^·? levels in H₂O₂-treated Calu-6 cells at 1 h and these inhibitors decreased ROS levels, including that of O ₂ ^·? , in H₂O₂-treated A549 cells at 24 h. Caspase inhibitors partially attenuated GSH depletion in H₂O₂-treated A549 cells and increased GSH levels in these cells at 24 h. However, the inhibitors did not affect GSH deletion and levels in Calu-6 cells at 24 h. In conclusion, H₂O₂ induced caspase-dependent apoptosis in Calu-6 and A549 cells, which was accompanied by increases in ROS and GSH depletion. The antiapoptotic effects of caspase inhibitors were somewhat related to the suppression of H₂O₂-induced oxidative stress and GSH depletion.     

Oxygen Reduction Reaction Affected by Sulfate-Reducing Bacteria: Different Roles of Bacterial Cells and Metabolites

Sulfate-reducing bacteria (SRB) were found to be capable of tolerating a certain amount of oxygen (O₂), but how they affect oxygen reduction reaction (ORR) has not been clear. The present work investigated the impact of SRB on ORR in 3.5 wt% sodium chloride solution with the cyclic voltammetry method. The addition of SRB culture solution hampered both the reduction of O₂ to superoxide (O ₂ ^·? ) and hydrogen peroxide (H₂O₂) to water (H₂O), and the influence of SRB metabolites was much larger than that of bacterial cells. Sulfide and extracellular polymeric substances (EPS), typical inorganic and organic metabolic products, had great impact on ORR. Sulfide played an important role in the decrease of cathodic current for H₂O₂ reduction due to its hydrolysis and chemical reaction activity with H₂O₂. EPS were sticky, easy to adsorb on the electrode surface and abundant in functional groups, which hindered the transformation of O₂ into O ₂ ^·? and favored the reduction of H₂O₂ to H₂O.     

Reactive oxygen and nitrogen species’ effect on <Emphasis Type="Italic">lux</Emphasis>-biosensors based on <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Salmonella typhimurium</Emphasis>

The effect of reactive oxygen and nitrogen species on lux-biosensors based on the Escherichia coli K12 MG1655 and Salmonella typhimurium LT2 host strains was investigated. The bioactivity of exogenous free radicals to the constitutively luminescent E. coli strain with plasmid pXen7 decreased in the order H₂O₂ > OCl^– > NO^? > RОO^? > ONOO^–> O₂^?- while the bioluminescence of S. typhimurium strain transformed with this plasmid decreased in the order NO^? > H₂O₂ > ONOO^– > RОO^? > OCl^– > O₂^?- The cross-reactivity of induced lux-biosensors to reactive oxygen and nitrogen species, the threshold sensitivity and the luminescence amplitude dependences from the plasmid specificity and the host strain were indicated. The biosensors with plasmid pSoxS′::lux possessed a wider range of sensitivity, including H₂O₂ and OCl^–, along with O₂^?- and NO^?. Among the used reactive oxygen and nitrogen species, H₂O₂ showed the highest induction activity concerning to the plasmids pKatG′::lux, pSoxS′::lux and pRecA′::lux. The inducible lux-biosensors based on S. typhimurium host strain possessed a higher sensitivity to the reactive oxygen and nitrogen species in comparison with the E. coli lux-biosensors.     

Reaction of 3′,5′-di-O-acetyl-2′-deoxyguansoine with hypobromous acid

Hypobromous acid (HOBr) is formed by eosinophil peroxidase and myeloperoxidase in the presence of H₂O₂, Cl^?, and Br^? in the host defense system of humans, protecting against invading bacteria. However, the formed HOBr may cause damage to DNA and its components in the host. When a guanine nucleoside (3′,5′-di-O-acetyl-2′-deoxyguansoine) was treated with HOBr at pH 7.4, spiroiminodihydantoin, guanidinohydantoin/iminoallantoin, dehydro-iminoallantoin, diimino-imidazole, amino-imidazolone, and diamino-oxazolone nucleosides were generated in addition to an 8-bromoguanine nucleoside. The major products were spiroiminodihydantoin under neutral conditions and guanidinohydantoin/iminoallantoin under mildly acidic conditions. All the products were formed in the reaction with HOCl in the presence of Br^?. These products were also produced by eosinophil peroxidase or myeloperoxidase in the presence of H₂O₂, Cl^?, and Br^?. The results suggest that the products other than 8-bromoguanine may also have importance for mutagenesis by the reaction of HOBr with guanine residues in nucleotides and DNA.     

Effect of cadmium on the distribution of hydroxyl radical, superoxide and hydrogen peroxide in barley root tip

In the present study, we investigated the alteration of reactive oxygen species production along the longitudinal axis of barley root tips during Cd treatment. In unstressed barley root tips, H₂O₂ production decreased from the root apex towards the differentiation zone where again, a slight increase was observed towards the more mature region of root. An opposite pattern was observed for O ₂ ^?? and OH^? generation. The amount of both O ₂ ^?? and OH^? was highest in the elongation zone, decreased in the root apex and at the differentiation zone of root, then increased again towards the more mature region of root. An elevated Cd-induced O ₂ ^?? production started in the elongation zone and increased further along the differentiation zone of barley root tip. In contrast, Cd-induced H₂O₂ production was localised to the root elongation zone and to the beginning of the differentiation zone. In contrast to Cd-induced H₂O₂ and O ₂ ^?? production, Cd reduced OH^? production along the whole barley root tip. Our results suggest that not only an increase but also the spatial distribution of reactive oxygen species production is involved in the Cd-induced stress response of barley root tip.     

Chemical composition of the Tatra Mountain lakes: Recovery from acidification

Ninety-one lakes distributed along the Tatra Mountains (most of lakes > 1 ha and 65% of lakes > 0.01 ha) were sampled and analysed for ionic and nutrient composition in September 2004 (15 years after reduction in acid deposition). Eighty-one lakes were in alpine zone and ten lakes in Norway spruce forest. The results were compared to similar lake surveys from 1994 (the beginning of water recovery from acidification) and 1984 (maximum acidification). Atmospheric deposition of SO ₄ ^2? and inorganic N decreased 57% and 35%, respectively, in this region from the late 1980s to 2000. Lake water concentrations of SO ₄ ^2? and NO ₃ ^? have decreased both by ～50% on average (to 23 and 19 μmol L^?1, respectively, in 2004) since 1984. While the decrease in SO ₄ ^2? concentrations was stable throughout 1984–2004, most of the NO ₃ ^? decrease occurred from 1994 to 2004. The declines in SO ₄ ^2? and NO ₃ ^? concentrations depended on catchment coverage with vegetation, being most rapid for SO ₄ ^2? in forest lakes and for NO ₃ ^? in rocky lakes. Concentrations of the sum of base cations (dominated by Ca²⁺) significantly decreased between 1984 and 2004, with the highest change in rocky lakes. Most of this decline occurred between 1994 and 2004. Acid neutralising capacity (ANC) did not change in the 1984–1994 period, but increased on average by 29 μmol L^?1 between 1994 and 2004, with the highest change in rocky lakes. Over the last decade, the proportion of lakes with ANC > 150 μmol L^?1 increased from 15% to 21% and that of ANC < 20 μmol L^?1 decreased from 37% to 20%. The highest decline in H⁺ and Al concentrations occurred in the most acid lakes. On a regional basis, no significant change was observed for total phosphorus, total organic nitrogen, and dissolved organic carbon (DOC) in the 1994–2004 period. However, these parameters increased in forest lakes, which exhibited an increasing trend in DOC concentrations, inversely related (P < 0.001) to their decreasing ionic strength (30% on average in 1994–2004).     

Magnificant role of intracellular reactive oxygen species production and its scavenging encompasses downstream processes

Environmental stresses are often associated with production of certain deleterious chemical entities called reactive oxygen species (ROS), which include hydrogen peroxide (H₂O₂), superoxide radical (O₂^?), hydroxyl radical (OH^?). In plants, ROS are formed by the inevitable leakage of electrons onto O₂ from the electron transport activities of chloroplasts, mitochondria, peroxisomes, vacuole and plasma membranes or as a byproduct of various metabolic pathways. Plants have their own antioxidant defense mechanisms to encounter ROS that is of enzymic and non-enzymic nature. Coordinated activities of these antioxidants regulate ROS detoxification and reduces oxidative load in plants. Though ROS are always regarded to impart negative impact on plants, some reports consider them to be important in regulating key cellular functions; however, such reports in plant are limited. On the other hand, specific ROS function as signaling molecules and activate signal transduction processes in response to various stresses is a matter of investigation.     

Element fluxes in watershed-lake ecosystems recovering from acidification: Čertovo Lake,the Bohemian Forest, 2001–2005

Fluxes of major ions and nutrients were measured in the watershed-lake ecosystem of a strongly acidified lake, ?ertovo jezero (?ertovo Lake), in the 2001 through 2005 hydrological years. Water balance was estimated from precipitation and throughfall amounts, and measured outflow from the lake. The average water input into and outflow from the watershed-lake ecosystem was 1461 mm and 1271 mm (40 L km^?2 s^?1), respectively, and the water residence time in the lake averaged 662 days. The ecosystem has been recovering from acidification since the late 1980s. Still, however, ?ertovo watershed was an average net source of 23 mmol m^?2 yr^?1 of SO ₄ ^2? . Nitrogen saturation of the watershed caused low retention of the deposited inorganic N (23% on average). After a dry summer in 2003 and a cold winter in 2004, the watershed became a net source of inorganic N (19 mmol m^?2 yr^?1). Nitrogen transformations and SO ₄ ^2? release were the dominant terrestrial sources of H⁺ (81 and 47 mmol m^?2 yr^?1, respectively) and the watershed was a net source of 42 mmol H⁺ m^?2 yr^?1. Ionic composition of tributaries showed seasonal variations with the most pronounced changes in NO ₃ ^? , base cations, DOC, and ionic Al (Al_i) concentrations. The in-lake biogeochemical processes reduced the incoming H⁺ by ～50% (i.e., neutralized on average 222 mmol H⁺ m^?2 yr^?1, on a lake-area basis). Denitrification, SO ₄ ^2? reduction, and photochemical and microbial decomposition of allochthonous organic matter were the most important in-lake H⁺ consuming processes (215, 85, and 122 mmol H⁺ m^?2 yr^?1, respectively), while hydrolysis of Al_i was the dominant H⁺ generating process (96 mmol H⁺ m^?2 yr^?1) in ?ertovo Lake. Photochemical liberation from organic complexes was an additional in-lake source of Al_i. The net in-lake retention or removal of nutrients (carbon, phosphorus, nitrogen, and silica) varied between 18% and 34% of their inputs.     

Production of reactive oxygen species in decoupled, Ca2+-depleted PSII and their use in assigning a function to chloride on both sides of PSII

Extraction of Ca²⁺ from the oxygen-evolving complex of photosystem II (PSII) in the absence of a chelator inhibits O₂ evolution without significant inhibition of the light-dependent reduction of the exogenous electron acceptor, 2,6-dichlorophenolindophenol (DCPIP) on the reducing side of PSII. The phenomenon is known as “the decoupling effect” (Semin et al. Photosynth Res 98:235–249, 2008). Extraction of Cl^? from Ca²⁺-depleted membranes (PSII[–Ca]) suppresses the reduction of DCPIP. In the current study we investigated the nature of the oxidized substrate and the nature of the product(s) of the substrate oxidation. After elimination of all other possible donors, water was identified as the substrate. Generation of reactive oxygen species HO, H₂O₂, and O ₂ ^·? , as possible products of water oxidation in PSII(–Ca) membranes was examined. During the investigation of O ₂ ^·? production in PSII(–Ca) samples, we found that (i) O ₂ ^·? is formed on the acceptor side of PSII due to the reduction of O₂; (ii) depletion of Cl^? does not inhibit water oxidation, but (iii) Cl^? depletion does decrease the efficiency of the reduction of exogenous electron acceptors. In the absence of Cl^? under aerobic conditions, electron transport is diverted from reducing exogenous acceptors to reducing O₂, thereby increasing the rate of O ₂ ^·? generation. From these observations we conclude that the product of water oxidation is H₂O₂ and that Cl^? anions are not involved in the oxidation of water to H₂O₂ in decoupled PSII(–Ca) membranes. These results also indicate that Cl^? anions are not directly involved in water oxidation by the Mn cluster in the native PSII membranes, but possibly provide access for H₂O molecules to the Mn₄CaO₅ cluster and/or facilitate the release of H⁺ ions into the lumenal space.     

Oxidative processes at initial stages of interaction of nodule bacteria (<Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis>) and pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.): A review

A possible physiological mechanism of legume-Rhizobium symbiosis, consisting in regulation of the intensity of oxidative processes by the macrosymbiont in response to infection with Rhizobium, was analyzed using our own and published data. The results used in the analysis included data on the content of reactive oxygen species (O ₂ ^·? and H₂O₂), activity of antioxidant enzymes (superoxide dismutase, catalase, and peroxidase), and intensity of lipid peroxidation proceeding with the involvement of lipophilic phenolic compounds of the microsymbiont.     

Microbial sulfate-reducing activities in anoxic sediment from Marine Lake Grevelingen: screening of electron donors and acceptors

Sulfate-reducing bacteria in marine sediments mainly utilize sulfate as a terminal electron acceptor with different organic compounds as electron donors. This study investigated microbial sulfate-reducing activity of coastal sediment from Marine Lake Grevelingen (MLG), the Netherlands using different electron donors and electron acceptors. All four electron donors (ethanol, lactate, acetate and methane) showed sulfate-reducing activity with sulfate as electron acceptor, suggesting the presence of an active sulfate-reducing bacterial population in the sediment, even at dissolved sulfide concentrations exceeding 12 mM. Ethanol showed the highest sulfate reduction rate of 55 µmol g _VSS ^?1 day^?1 compared to lactate (32 µmol g _VSS ^?1 day^?1), acetate (26 µmol g _VSS ^?1 day^?1) and methane (4.7 µmol g _VSS ^?1 day^?1). Sulfide production using thiosulfate and elemental sulfur as electron acceptors and methane as the electron donor was observed, however, mainly by disproportionation rather than by anaerobic oxidation of methane coupled to sulfate reduction. This study showed that the MLG sediment is capable of performing sulfate reduction by using diverse electron donors, including the gaseous and cheap electron donor methane.     

Hypochlorous Acid Generated by Neutrophils Inactivates ADAMTS13: AN OXIDATIVE MECHANISM FOR REGULATING ADAMTS13 PROTEOLYTIC ACTIVITY DURING INFLAMMATION*

ADAMTS13 is a plasma metalloproteinase that cleaves large multimeric forms of von Willebrand factor (VWF) to smaller, less adhesive forms. ADAMTS13 activity is reduced in systemic inflammatory syndromes, but the cause is unknown. Here, we examined whether neutrophil-derived oxidants can regulate ADAMTS13 activity. We exposed ADAMTS13 to hypochlorous acid (HOCl), produced by a myeloperoxidase-H₂O₂-Cl⁻ system, and determined its residual proteolytic activity using both a VWF A2 peptide substrate and multimeric plasma VWF. Treatment with 25 nm myeloperoxidase plus 50 μm H₂O₂ reduced ADAMTS13 activity by >85%. Using mass spectrometry, we demonstrated that Met²⁴⁹, Met³³¹, and Met⁴⁹⁶ in important functional domains of ADAMTS13 were oxidized to methionine sulfoxide in an HOCl concentration-dependent manner. The loss of enzyme activity correlated with the extent of oxidation of these residues. These Met residues were also oxidized in ADAMTS13 exposed to activated human neutrophils, accompanied by reduced enzyme activity. ADAMTS13 treated with either neutrophil elastase or plasmin was inhibited to a lesser extent, especially in the presence of plasma. These observations suggest that oxidation could be an important mechanism for ADAMTS13 inactivation during inflammation and contribute to the prothrombotic tendency associated with inflammation.     

Superoxide production pathways in aortas of diabetic rats: beneficial effects of insulin therapy and endurance training

Superoxide (O ₂ ^·? ) overproduction, by decreasing the nitric oxide (^·NO) bioavailability, contributes to vascular complications in type 1 diabetes. In this disease, the vascular O ₂ ^·? can be produced by the NADPH oxidase (NOX), nitric oxide synthase (NOS), and xanthine oxidase (XO). This study aimed to determine the contribution of each enzymatic pathway in hyperglycemia-induced O ₂ ^·? overproduction, and the effects of an endurance training program and insulin therapy, associated or not, on the O ₂ ^·? production (amount and related enzymes) in diabetic rats. Forty male Wistar rats were divided into diabetic (D), diabetic treated with insulin (D-Ins), diabetic trained (D-Tr), or diabetic insulin-treated and trained (D-Ins + Tr) groups. An additional healthy group was used as control. Insulin therapy (Glargine Lantus, Sanofi) and endurance training (treadmill run: 60 min/day, 25 m/min, 5 days/week) started 1 week after diabetes induction by streptozotocin (45 mg/kg), and lasted for 8 weeks. At the end of the protocol, the O ₂ ^·? production in aorta rings was evaluated by histochemical analyses (DHE staining). Each production pathway was studied by inhibiting NOX (apocynin), NOS (L-Name), or XO (allopurinol) before DHE staining. Diabetic rats exhibited hyperglycemia-induced O ₂ ^·? overproduction, resulting from NOX, NOS, and XO activation. Insulin therapy and endurance training, associated or not, decreased efficiently and similarly the O ₂ ^·? overproduction. Insulin therapy reduced the hyperglycemia and decreased the three enzymatic pathways implicated in the O ₂ ^·? production. Endurance training decreased directly the NOS and XO activity. While both therapeutic strategies activated different pathways, their association did not reduce the O ₂ ^·? overproduction more significantly.     

The effect of total inorganic nitrogen and the balance between its ionic forms on adventitious bud formation and callus growth of ‘Węgierka Zwykła’ plum (<Emphasis Type="Italic">Prunus domestica</Emphasis> L.)

The effect of different ammonium NH ₄ ⁺ and nitrate NO ₃ ^? ratios (4:1, 2:1, 1:1, 1:2, 1:4, 1:6) on organogenesis of ‘W?gierka Zwyk?a’ leaf explants cultivated on media with nitrogen levels equalling full- or half-MS was investigated. On media with total nitrogen equal to ½ MS, explant regeneration increased significantly and was highest on media with 1:2 or 1:4 NH ₄ ⁺ :NO ₃ ^? ratio. An excess of ammonium versus nitrate ions had a negative effect on both regeneration and biomass. Addition of potassium to the medium increased the fresh weight of explants and the number of adventitious buds.