The photoprotein pholasin as a luminescence substrate for detection of superoxide anion radicals and myeloperoxidase activity in stimulated neutrophils

Pholasin, the photoprotein of the common piddock Pholas dactylus, emits an intense luminescence upon oxidation. The contribution of superoxide anion radicals and myeloperoxidase (MPO) to Pholasin luminescence in stimulated neutrophils was investigated. Data on Pholasin luminescence were compared with results of superoxide anion radical generation detected by the cytochrome c test as well as with the release of elastase and MPO. In N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulated neutrophils, most of the luminescence is caused by superoxide anion radicals, whereas MPO shows only a small effect as shown by coincubation with superoxide dismutase (SOD) as well as potassium cyanide (KCN), an inhibitor of MPO. However, both, O₂^- and MPO contribute to light emission in fMLP/cytochalasin B and phorbol myristoyl acetate (PMA) stimulated cells. Thus, the kinetics of O₂^- generation and MPO release can be very well detected by Pholasin luminescence in stimulated neutrophils.

Degranulation of azurophilic granules was assessed using an ELISA test kit for released MPO or detection of elastase activity with MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide in the supernatant of stimulated cells. Both approaches revealed concurrently similar results concerning the amount and kinetics of enzyme release with data of Pholasin luminescence. Both, cytochrome c measurements and Pholasin luminescence indicate that fMLP/cytochalasin B and PMA stimulated neutrophils produce more O₂^- than fMLP stimulated cells. Thus, Pholasin luminescence can be used to detect, sensitively and specifically, O₂^- production and MPO release from stimulated neutrophils.     

Effects of cyclophosphamide on the immune system and disease resistance of Asian catfish Clarias batrachus

Cyclophosphamide (CYP), a multifunctional alkylating agent is known as a potent immunosuppressor in endotherms. Here, an experiment was conducted in an ectothermic Asian catfish species, Clarias batrachus to investigate its effect on non-specific and specific immunity as well as disease resistance against a common bacterial pathogen challenge. CYP was intraperitoneally injected for 3 consecutive days at a rate of 200 mgkg(-1) body weight. After 72 h post-injection, control and CYP-treated fish were screened for superoxide production through nitroblue tetrazolium (NBT) assay, myeloperoxidase (MPO) activity, packed cell volume (PCV) and total protein, lysozyme, alternative complement activity (ACH50) and natural haemagglutinin titre as a measure of non-specific immunity level as well as disease resistance against Aeromonas hydrophila challenge to vaccinated and unvaccinated fish. To study the effect on specific immunity, CYP was injected thrice at an interval of 7 days after bacterin injection and serum antibody titre was measured by bacterial agglutination titre assay. The results showed a significant (P<0.05) decrease in NBT and MPO activities, and percent survival against A. hydrophila challenge (both in unvaccinated and vaccinated fish) in CYP-treated fish, when compared to control fish. The above results support the immunosuppressive action of CYP in freshwater catfish, C. batrachus. The neutrophil activities, as measured through superoxide production and myeloperoxidase levels, might be important contributors during A. hydrophila infection and that treatment with CYP reduces phagocytic killing power and inhibits resistance against aeromoniasis.     

The effect of Euglena viridis on immune response of rohu, Labeo rohita (Ham.)

The study evaluated the effect of dietary doses of Euglena viridis on the immune response and disease resistance of Labeo rohita fingerlings against infection with the bacterial pathogen Aeromonas hydrophila. L. rohita fingerlings were fed with diet containing 0 (Control), 0.1 g, 0.5 g, 1.0 g Euglena powder kg⁻¹ dry diet for 90 days. Biochemical (serum total protein, albumin, globulin, albumin:globulin ratio), haematological (WBC, RBC, haemoglobin content) and immunological (superoxide anion production, lysozyme, serum bactericidal activity) parameters of fish were examined after 30, 60 and 90 days of feeding. Fish were challenged with A. hydrophila 90 days post-feeding and mortalities were recorded over 10 days post-infection. The results demonstrate that fish fed with Euglena showed increased levels of superoxide anion production, lysozyme, serum bactericidal activity, serum protein and albumin (P < 0.05) compared with the control group. Following challenge with A. hydrophila less survivability was observed in the control group (56.65%) than the group fed the experimental diets. The group fed 0.5 g Euglena kg⁻¹ dry diet showed the highest percentage survival (75%). These results indicate that Euglena stimulates the immunity and makes L. rohita more resistant to A. hydrophila infection.     

Effect of Allium sativum on the immunity and survival of Labeo rohita infected with Aeromonas hydrophila

The aim of this study was to evaluate dietary dosages of garlic on the immune response and disease resistance against infections due to the opportunistic pathogen Aeromonas hydrophila in Labeo rohita fingerlings. Garlic, Allium sativum, was incorporated into the diets (0%, 0.1%, 0.5% and 1.0%) of rohu, L. rohita fingerlings (10 ± 2 g). Every 20 days, different biochemical (serum total protein, albumin, globulin, albumin : globulin ratio, blood glucose), haematological (WBC, RBC and haemoglobin content) and immunological (superoxide anion production, lysozyme activity and serum bactericidal activity) parameters were evaluated. Superoxide anion production, lysozyme, serum bactericidal, serum protein and albumin were enhanced in garlic treated groups compared with the control group. After 60 days, fish were challenged with A. hydrophila and mortality (%) was recorded up to day 10 post‐challenge. Survival decreased in control group (57%) up to day 10 after infection. However, this was increased in the garlic treatment group, i.e. 85% survivability in the 1 g garlic kg^?1 (B group) and 5 g garlic kg^?1 (C group), and 71% survivability in the 10 g garlic kg^?1 (D group), respectively. These results indicate that Allium sativum stimulates the immunity and makes L. rohita more resistant to infection by A. hydrophila.     

Comparison of oxidative stress induced by ciprofloxacin and pyoverdin in bacteria and in leukocytes to evaluate toxicity

Oxidative stress induced by ciprofloxacin and pyoverdin, a leukotoxic pigment, was studied by comparing their effect in bacteria and leukocytes. Chemiluminescence (CL) assays with lucigenin or luminol were adapted to measure the stimuli of superoxide anion (O₂^?) and other reactive species of oxygen (ROS) in bacteria. Ciprofloxacin principally induced the production of O₂^? in the three species studied: Staphylococcus aureus, Enterococcus faecalis and Escherichia coli. Lucigenin CL assay showed high oxidative stress in S. aureus due to its low superoxide dismutase (SOD) activity, whereas E. coli exhibited important SOD activity, responsible for little production of O₂^? in absence or presence of ciprofloxacin. Reduction of nitroblue of tetrazolium (NBT) was applied. This assay indicated that there was higher oxidative stress in S. aureus and E. faecalis than in E. coli. The comparison of oxidative stress generated in bacteria and leukocytes was used to check the selective toxicity of ciprofloxacin in comparison with pyoverdin. Ciprofloxacin did not generate significant stimuli of O₂^? in neutrophils, while pyoverdin duplicated the production of O₂^?. CL and NBT were useful to study the leukotoxicity of ciprofloxacin. Oxidative stress caused by the antibiotic and the leukotoxic pigment was similar in bacteria. Copyright © 2003 John Wiley & Sons, Ltd.     

Different effects of concanavalin A and its succinylated derivative on superoxide release in peritoneal macrophages

The effects of tetravalent concanavalin A and its succinylated derivative on the intracellular production of superoxide anion (O₂^?) and its release into cell exterior of peritoneal macrophages were observed. Both tetravalent concanavalin A and its succinylated derivative induced marked enhancement of intracellular reduction of nitroblue tetrazolium, which could be inhibited by α-methyl-D-glucoside. The extent of activation of nitroblue tetrazolium reduction induced by both types of the lectin paralleled the activation ratio of oxygen consumption.There was littele difference in the extent of intracellular O₂^? production induced by two types of the lectin. Nitroblue tetrazolium reduction was not affected significantly by pretreatment with colchicine, rotenone or malonate, inhibitors of the cytoskeletal system and of the electron transport system. In contrast, tetravalent concanavalin A induced a higher rate of superoxide release than did succinylated divalent concanavalin A, which lacks the cross-linking activity of surface glycoproteins.These results indicate that superoxide production following oxygen consumption and superoxide release into cell exterior are controlled independently by a separate membrane mechanism and that superoxide production system is not essentially dependent on the involvement of the cytoskeletal system.     

Unique antioxidant effects of herbal leaf tea and stem tea from Moringa oleifera L. especially on superoxide anion radical generation systems

ABSTRACT

This study aimed to investigate the unique antioxidative effects of Japanese moringa products, herbal leaf tea and stem tea, using established free radical assays, focusing on superoxide anion (O₂^?) radical generation systems. Hot-water extracts from moringa teas resulted in different but lower scavenging activities than Trolox in four synthetic free radical models. Interestingly, these extracts further showed higher O₂^? radical scavenging effects than Trolox in the phenazine methosulfate-NADH-nitroblue tetrazolium and xanthine oxidase assay systems. Incubating human neutrophils in the presence of these tea extracts rather than Trolox effectively suppressed cellular O₂^? radical generation. Among the eight known phenolic constituents of moringa leaves, caffeic acid and chlorogenic acid may be responsible for the O₂^–specific radical scavenging capacity stronger than that of Trolox. These results suggest that moringa herbal teas are a good source of natural antioxidants for preventing O₂^? radical-mediated disorders.

Abbreviations: O₂^?: superoxide anion; ROS: reactive oxygen species; H₂O₂: hydrogen peroxide; XOD: xanthine oxidase; DPPH: 1,1-diphenyl-2-picrylhydrazyl; ABTS⁺: 2,2′-azinobis(2-ethylbenzothiazoline-6-sulfonic acid) cation; CPZ⁺: chlorpromazine cation; PMS: phenazine methosulfate; NBT: nitroblue tetrazolium; PMA: phorbol 12-myristate 13-acetate     

Superoxide Anion Production by Human Neutrophils Activated by Trichomonas vaginalis

Neutrophils are the predominant inflammatory cells found in vaginal discharges of patients infected with Trichomonas vaginalis. In this study, we examined superoxide anion (O₂^.-) production by neutrophils activated by T. vaginalis. Human neutrophils produced superoxide anions when stimulated with either a lysate of T. vaginalis, its membrane component (MC), or excretory-secretory product (ESP). To assess the role of trichomonad protease in production of superoxide anions by neutrophils, T. vaginalis lysate, ESP, and MC were each pretreated with a protease inhibitor cocktail before incubation with neutrophils. Superoxide anion production was significantly decreased by this treatment. Trichomonad growth was inhibited by preincubation with supernatants of neutrophils incubated for 3 hr with T. vaginalis lysate. Furthermore, myeloperoxidase (MPO) production by neutrophils was stimulated by live trichomonads. These results indicate that the production of superoxide anions and MPO by neutrophils stimulated with T. vaginalis may be a part of defense mechanisms of neutrophils in trichomoniasis.     

Negative air ions as a source of superoxide

The physico-chemical characteristics and possible formation mechanisms of negative air ions are considered. It was found that the products of oxygen and nitrogen negative ionization reduce ferricytochromec and nitroblue tetrazolium, and that these reactions were inhibited by superoxide dismutase. The interaction of negatively ionized oxygen with water led to hydrogen peroxide accumulation, which was inhibited by tetranitromethane or catalase. Nitrogen ionization under these conditions caused the formation of the hydrated electron e _aq ^— and the superoxide anion O ₂ ^— . The data obtained indicate that the biological activity of negative air ions may be dependent on superoxide. The generation of reactive oxygen ions in the gas phase and also at a gas/water interface is described. A scheme for superoxide production under oxygen and nitrogen ionization is proposed.     

The Reactivity of 4-Thiouridine with Peroxidase and Superoxide Radicals

Rice leaf slices stimulated with blast fungus hyphal component reduced nitroblue tetrazolium in a damped oscillatory profile with relaxing half wavelength in a medium containing glucose, when the respective rate of reduction was plotted against the function of time after the application of blast fungus hyphal component. In the presence of 110μm FAD and glucose, the wave number of the reduction profile increased 4- to 5-fold when compared to that in the absence of exogenous FAD. Exogenous FAD in the increasing concentration of 70 to 110 μm, which was added in the presence of glucose, gave a positive heterotropic-like response upon the reduction of nitroblue tetrazolium with rice leaf slices which were press-injured and stimulated. Exogenous pyrroloquinoline quinone in the increasing concentration of 10^?3 to 10^?1 μm, which was added in the presence of glucose, gave an inhibition upon the reduction. From sediment of the homogenate of stimulated rice leaf slices, the nitroblue tetrazolium reducing redox-enzyme system was solubilized by Triton X-100 and was electrophoretically isolated in a sharp blue band on a polyacrylamide slab gel containing Triton X-100, when the electrophoresed gel was stained by nitroblue tetrazolium or Coomassie brilliant blue. In the solubilized solution, the presence of b-type cytochrome was observed by the oxidation-reduction difference spectrum.     

Assay of superoxide dismutase activity in animal tissues

Convenient assays for superoxide dismutase have necessarily been of the indirect type. It was observed that among the different methods used for the assay of superoxide dismutase in rat liver homogenate, namely the xanthine-xanthine oxidase ferricytochromec, xanthine-xanthine oxidase nitroblue tetrazolium, and pyrogallol autoxidation methods, a modified pyrogallol autoxidation method appeared to be simple, rapid and reproducible. The xanthine-xanthine oxidase ferricytochromec method was applicable only to dialysed crude tissue homogenates. The xanthine-xanthine oxidase nitroblue tetrazolium method, either with sodium carbonate solution, pH 10.2, or potassium phosphate buffer, pH 7·8, was not applicable to rat liver homogenate even after extensive dialysis. Using the modified pyrogallol autoxidation method, data have been obtained for superoxide dismutase activity in different tissues of rat. The effect of age, including neonatal and postnatal development on the activity, as well as activity in normal and cancerous human tissues were also studied. The pyrogallol method has also been used for the assay of iron-containing superoxide dismutase inEscherichia coli and for the identification of superoxide dismutase on polyacrylamide gels after electrophoresis.     

Investigation of Cadmium-Induced Genotoxicity and Oxidative Stress Response in Indian Major Carp,Labeo rohita

We investigated genotoxicity and oxidative stress in the gills of Labeo rohita exposed to 33.6, 67.1, and 100.6 mg L^–1of cadmium chloride at 96 h. Genotoxicity was assessed using single cell gel electrophoresis whereas oxidative stress was monitored through lipid peroxidation induction and antioxidant response parameters, namely reduced glutathione (GSH), glutathione peroxidase, glutathione-S-transferase, superoxide dismutase, and catalase (CAT) activities. Significant (p < .05) effect of both concentration and time of exposure was observed on the extent of DNA damage in treated fish. Similarly, malondialdehyde content, level of GSH, and activities of antioxidant enzymes were significantly elevated in treated groups, except CAT. The increased DNA damage and lipid peroxidation (LPO) content along with fluctuation in antioxidant defense system in fish indicated the interaction of cadmium (Cd) with DNA repair processes and production of reactive oxygen species. Thus, Cd is liable for induction of LPO, alteration of antioxidant defenses, and DNA damage in gills of L. rohita.     

Cyanide catalyzes the oxidation of alpha-hydroxyaldehydes and related compounds: monitored as the reduction of dioxygen, cytochrome c, and nitroblue tetrazolium

Cyanide catalyzed the oxidation of α-hydroxycarbonyls and of related compounds. In the cases of glyceraldehyde 3-phosphate and of dihydroxyacetone phosphate the tautomeric enediol was the obligatory intermediate which reacted with cyanide yielding the active reductant. Cytochrome c, nitroblue tetrazolium, and dioxygen were all reduced by this reductant. In the case of dioxygen the product was the superoxide radical which could then secondarily reduce cytochrome c or nitroblue tetrazolium. In air-equilibrated reaction mixtures, at 25 °C, approximately 35% of cytochrome c reduction and 95% of nitroblue tetrazolium reduction was mediated by superoxide, as judged from susceptibilities to inhibition by superoxide dismutase. Since the oxidations observed were univalent, carbon-centered radicals appear to be necessary intermediates, and their secondary reactions generated a multiplicity of products, seen as smears on thin-layer chromatograms. Free cyanide must be regenerated during these secondary reactions, since cyanide functioned catalytically in the overall process. A partial mechanism has been proposed in explanation of these observations.     

The Effects of Culture Conditions on the Secretion of Human Lysozyme by Saccharomyces cerevisiae A2-1-1A

The effects of initial glucose concentrations on the cell growth, glucose usage, and human lysozyme (HLY) secretion under the ENOl promoter were examined in the Saccharomyces cerevisiae strain A2–1–1A harboring a multicopy plasmid. By increasing the initial glucose from 2 % to 10 %, the HLY secretion increased 7 ~ 8 fold although the cell growth was not affected. By adding a mixture of mineral salts to the basal medium, the HLY secretion was increased about twice due to the continuity of the HLY expression at the stationary phase of cell growth.

The high HLY secretion (5.5 mg per liter, 47-fold higher than the original level) was achieved by the strain A2–1–1A grown in the synthetic basal medium containing 10% initial glucose, and supplemented with mineral salts containing ammonium sulfate, potassium phosphate, potassium chloride, magnesium sulfate, and iron sulfate.     

Lysozyme activities and immunoglobulin concentrations in seminal plasma and spermatozoa of different teleost species and indications on its significance for sperm function

The occurrence of lysozyme and immunoglobulin (Ig) in semen of different teleost species (brown trout—Salmo trutta, perch—Perca fluviatilis, burbot—Lota lota) was studied. In all investigated species lysozyme activities (1.13-1.45 U ml⁻¹) and Ig concentrations (T-Ig: 1.11-1.61 μg ml⁻¹, IgG [measured only in brown trout]: 1.49 μg ml⁻¹) were detected in seminal plasma. Ig was also found in spermatozoa (T-Ig: 0.234-0.357 μg/g protein, IgG: 0.198 μg ml⁻¹) while spermatozoal lysozyme activities were low and fluctuating (0.093-0.164 U/g protein). In Salmo trutta lysozyme activities and immunoglobulin levels were compared between semen samples with high and low sperm motility as motility is an indicator for sperm fertility. Lysozyme activities were higher in seminal plasma of samples with high motility than in those with low motility while seminal plasma and spermatozoal immunoglobulin concentrations (T-Ig, IgG) were increased in samples with low motility in comparison to samples with high motility. Seminal plasma and spermatozoal IgG concentrations and seminal plasma lysozyme activities showed significant correlations with the sperm motility rate and swimming velocity. Moreover, lysozyme improved the viability of spermatozoa in in vitro experiments. Possible physiological meanings of these results are discussed.     

Effects of physical exercise and aging on ascorbic acid and superoxide anion levels in peritoneal macrophages from mice and guinea pigs

The relationship of physical activity and aging, two processes with a high production of oxygen-free radicals to the ascorbate and superoxide anion (O ₂ ^- ) contents of peritoneal macrophages was studied in two animal species: guinea-pig (in which ascorbic acid is a vitamin) and mouse (in which ascorbic acid is not a vitamin). The effects of exhaustive exercise were examined in young and old animals. The results show that macrophages from old animals have a lower ascorbate content than those from young ones, whereas with exercise the ascorbate content increased in both old and young animals. This increase was higher in young than in old animals, and more evident in mice than in guinea-pigs. Aging also resulted in an increase in the O ₂ ^- levels of macrophages. With exercise these levels decreased in young mice but increased in young guinea-pigs. In old animals the exhaustive exercise did not change the O ₂ ^- levels. The results suggest in general a lack of correlation between the intracellular ascorbate and O ₂ ^- levels in relation to both physical exercise and aging.Abbreviations PBS phosphate buffered saline - NBT nitroblue tetrazolium - PEC peritoneal exudate cells - PMN polymorphonuclear     

Double disruption of the proteinase genes, tppA and pepE, increases the production level of human lysozyme by Aspergillus oryzae

In this study, we investigated the effects of proteinase gene disruption on heterologous protein production by Aspergillus oryzae. The human lysozyme (HLY) was selected for recombinant production as a model for the heterologous protein. A tandem HLY construct fused with α-amylase (AmyB) was expressed by A. oryzae in which the Kex2 cleavage site was inserted at the upstream of HLY. HLY was successfully processed from AmyB and produced in the medium. We performed a systematic disruption analysis of five proteinase genes (pepA, pepE, alpA, tppA, and palB) in the HLY-producing strain with the adeA selectable marker. Comparative analysis indicated that disruption of the tppA gene encoding a tripeptidyl peptidase resulted in the highest increase (36%) in the HLY production. We further deleted the tppA gene in the pepE or palB disruptant with another selectable marker, argB. Consequently, a double disruption of the tppA and pepE genes led to a 63% increase in the HLY production compared to the control strain. This is the first study to report that the double disruption of the tppA and pepE genes improved the production level of a heterologous protein by filamentous fungi. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The phagocytic ability of neutrophils and serum lysozyme activity in experimentally infected carp, Cyprinus carpio L.

The phagocytosis of neutrophils and serum lysozyme activity were investigated in carp experimentally infected with Pseudomonas alcaligenes and Aeromonas punctata. The total leucocyte numbers, relative leucocyte counts, nitroblue tetrazolium (NBT) reduction, NBT test rate, lysozyme activity and lysozyme index were examined on days 7, 14 and 21 after injection. On days 7 and 14 there was a significant increase in total leucocyte numbers and serum lysozyme activity, but a decrease in the NBT test rate and lysozyme index. NBT reduction was unchanged compared with the control group. On day 21 the total leucocyte numbers and lysozyme activity had declined and were less than control values, but there was a significant increase of the NBT test rate, NBT reduction and lysozyme index.     

Inhibition of myeloperoxidase- and neutrophil-mediated oxidant production by tetraethyl and tetramethyl nitroxides

The powerful oxidant HOCl (hypochlorous acid and its corresponding anion, ⁻OCl) generated by the myeloperoxidase (MPO)–H₂O₂–Cl⁻ system of activated leukocytes is strongly associated with multiple human inflammatory diseases; consequently there is considerable interest in inhibition of this enzyme. Nitroxides are established antioxidants of low toxicity that can attenuate oxidation in animal models, with this ascribed to superoxide dismutase or radical-scavenging activities. We have shown (M.D. Rees et al., Biochem. J. 421, 79–86, 2009) that nitroxides, including 4-amino-TEMPO (4-amino-2,2,6,6-tetramethylpiperidin-1-yloxyl radical), are potent inhibitors of HOCl formation by isolated MPO and activated neutrophils, with IC₅₀ values of ~1 and ~6 µM respectively. The utility of tetramethyl-substituted nitroxides is, however, limited by their rapid reduction by biological reductants. The corresponding tetraethyl-substituted nitroxides have, however, been reported to be less susceptible to reduction. In this study we show that the tetraethyl species were reduced less rapidly than the tetramethyl species by both human plasma (89–99% decreased rate of reduction) and activated human neutrophils (62–75% decreased rate). The tetraethyl-substituted nitroxides retained their ability to inhibit HOCl production by MPO and activated neutrophils with IC₅₀ values in the low-micromolar range; in some cases inhibition was enhanced compared to tetramethyl substitution. Nitroxides with rigid structures (fused oxaspiro rings) were, however, inactive. Overall, these data indicate that tetraethyl-substituted nitroxides are potent inhibitors of oxidant formation by MPO, with longer plasma and cellular half-lives compared to the tetramethyl species, potentially allowing lower doses to be employed.