Superoxide Anion-generating System Research Articles

Identification of potent antioxidant bioactive peptides from the soluble proteins of chicken egg yolk

Background: Eggs are an excellent nutrient-dense food containing proteins, fats, carbohydrates, minerals, and vitamins. While many proteins are present in egg yolk, there arefew studies on their health benefits. Objective: The aim of this study was to explore the antioxidant peptides derived from the soluble protein fractions of egg yolk. Lipids and lipoproteins were removed with activated carbon and centrifugation after water dilution at acidic pH. Materials and Methods: Compared with 3.6 grams of protein in egg white, egg yolk contains 2.7 grams of protein in a single large egg. The egg yolk soluble protein (EYsP) was digested with pepsin for 2h at pH 3.0 (P-EYsP), followed by digestion with trypsin (P-EYsP-T), α-chymotrypsin (P-EYsP-α), or both enzymes (P-EYsP-T/α). The most active digest was fractionated using sephacryl S-100 gel filtration, followed by reversed phase-HPLC of the most active fraction. The antioxidant activities were evaluated using a superoxide anion-generating system of xanthine oxidase, DPPH-scavenging assay, and yeast cells as an oxidative-stress tolerance cellular model. Results: The intact proteins (EYsP) showed antioxidant activity, but pepsin hydrolysate (P-EYsP) exhibited greater superoxide-scavenging activities than EYsP, while P-EYsP-T, P-EYsP-α and P-EYsP-T/α lacked activities. The P-EYsP and its subsequent proteases (P-EYsP-T, P-EYsP-α and P-EYsP-T/α) exhibited significant DPPH reduction, but P-EYsP-T, P-EYsP-α and P-EYsP-T/α exhibited the strongest DPPH reduction activities. MALDI-TOF-MS analysis revealed five major antioxidant peptides, two derived from yolk glycoprotein 40 (859 Da, 883Da), two from lipovitelline (901Da, 945 Da), and one from livetin (1089 Da). The peptides exhibited potent superoxide anion as well as DPPH scavenging activities and markedly enhanced the tolerance of yeast cells against peroxide-induced oxidative stress.Conclusion: The results show that these bioactive peptides hold a fascinating opportunity for their potential as nutraceuticals in prevention and combating oxidative stress-associated diseases.Keywords: Egg yolk proteins; bioactive peptides; antioxidant; superoxide-scavenging; DPPH-reduction; yeast tolerance

Antioxidant and pro-oxidant mechanisms of (+) catechin in microsomal CYP2E1-dependent oxidative stress

The objectives of this work were to evaluate the effects of catechin on cytochrome P450 2E1 (CYP2E1)-dependent oxidative stress. Microsomes co-expressing human CYP2E1 with NADPH cytochrome P450 reductase and cytochrome b5 were incubated with NADPH and DTPA at pH 7.0. Superoxide anion generation was specifically detected by spin-trapping with DEPMPO. Generation of the DEPMPO-OOH adduct was not observed in the absence of CYP2E1 and in the presence of superoxide dismutase (SOD) or catechin, while catalase was ineffective. Reactive oxygen species generation was detected with 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CPH) by the EPR-detection of its oxidation product, 3-carboxy-proxyl radical (CP●). CP● generation was not observed in the absence of CYP2E1 and in the presence of SOD, while catalase was ineffective. In contrast, catechin increased CPH oxidation, an effect that was not observed in the absence of CYP2E1 or in the presence of SOD (but not catalase), and was not associated with an increase in oxygen consumption. Catechin also increased the non-specific oxidation of the probes CPH and hydroethidine by the superoxide anion-generating system xanthine plus xanthine oxidase. Catechin oxidized CPH in the presence of horseradish peroxidase plus hydrogen peroxide, a catechin radical-generating system. In conclusion, catechin exhibits both antioxidant (superoxide-scavenging) and pro-oxidant effects under CYP2E1-dependent oxidative stress.

Glutathione s-transferase as a marker of oxidative stress in human ejaculated spermatozoa from patients with pathospermia

It is believed that the most common causes of male infertility are impairment of spermatogenesis and sperm functions. Glutathione S-transferases (EC 2.5.1.18) play an important role in sperm physiology, specifically in antioxidant protection against oxidative damage. The catalase decomposition of lipid hydro-peroxides forms as a result of oxidative stress. We used a model of superoxide anion-generating system Fe3+/ascorbate or H2O2-induced stress to study the activity of glutathione s transferase in human ejaculated spermatozoa from patients with pathospermia and products of lipid peroxidation (TBARS) as a marker of oxidative stress. In the present study, dose dependent increase in the level of lipid peroxidation was observed for treatment with Fe3+/ascorbate or H2O2. The TBARS level was higher for sperm cells incubated with superoxide anion-generating system Fe3+/ascorbate than for H2O2. GSTs activity increased in spermatozoa treated with increasing concentration of superoxide anion-generating system Fe3+/ascorbate and H2O. We found that both Fe3+/ascorbate and H2O2 displayed similar inhibitory effects on sperm GSTs activity, however H2O2 at low concentrations activated enzyme activity only in normozoospermic samples, which can be explained as a defence response to oxidative stress. The time course of incubation with 100 μM H2O2 showed a sharp decrease in the enzyme activity during the first 5 min of incubation for both normozoospermic and pathozoospermic men. Preincubation of spermatozoa with GSH completely prevented the ROS-induced inhibition on GSTs only in normozoospermic samples. On the other hand, in pathospermic samples protectory effect of GSH was observed only against non-radical (H2O) radical, but not against radical (superoxide anion-generating system Fe3+/ascorbate) species. The results of our study showed higher oxygen-free radical production, evidenced by increased TBARS level in spermatozoa obtained from infertile men than normozoospermic men. The inhibitory effect of the radical (superoxide anion-generating system Fe3+/ascorbate) species on sperm GSTs activity and products of lipid peroxidation in sperm cells of fertile and infertile men were more expressed compared to non-radical (H2O) species. Our results indicate that estimation of sperm GSTs enzyme assays can be used as a bioindicator for impaired male fertility. The obtained results argue for a biological role of sperm GSTs in susceptibility of spermatozoa to oxidative damage and maintaining sperm antioxidant status.

Synthesis, characterization and potent superoxide dismutase-like activity of novel bis(pyrazole)-2,2'-bipyridyl mixed ligand copper(II) complexes.

Eleven new complexes of Cu(II) chloride and nitrate with bis(pyrazol-1-yl)propane and bis[2-(pyrazol-1-yl)ethyl]ether ligands were prepared and characterized by spectral and electrochemical methods. X-Ray crystal structure determination of bis[2-(3,5-dimethylpyrazol-1-yl)ethyl]etherdinitratocopper revealed a hepta-coordinated structure with the bis(pyrazole) ligand coordinated in a tridentate NNO-fashion and both of the nitrate ions in a bidentate fashion. Reaction of Cu(II) nitrate complexes with 2,2'-bipyridyl led to the displacement of one of the nitrate ions into the outer sphere and the formation of mixed-ligand complexes. Mixed-ligand bipyridyl Cu(II) complexes demonstrated the highest superoxide dismutase (SOD)-like activity in a chemical superoxide anion-generating system, with IC(50) values in the low micromolar range. Density functional theory calculations showed that introduction of a bipypidyl ligand into the complexes dramatically lowered the lowest unoccupied molecular orbital (LUMO) energy level, which explains the increased SOD-like activity of these complexes compared to non-bipy species. These bipy complexes were also effective scavengers of reactive oxygen species generated by phagocytes (human neutrophils and murine bone marrow leukocytes) ex vivo. Thus, these bipy mixed-ligand complexes represent a promising class of SOD mimetics for future development.

Regulation of elicitin-induced ethylene production in suspension-cultured tobacco BY-2 cells

INF1 elicitin, a proteinaceous elicitor produced by Phytophthora infestans, induces a hypersensitive response in tobacco BY-2 cells. In response to elicitin, tobacco cells produce both reactive oxygen species (ROS) and ethylene (ET). To investigate the regulation of elicitin-induced ET production, we pharmacologically analyzed the effects of several chemicals on ET production. Inhibitors of ROS generation or ROS chelators efficiently inhibited ET production, whereas simultaneous treatment of a superoxide anion-generating system with salicylhydroxamic acid recovered ET production. In an in vitro experiment, superoxide anion was necessary and sufficient for conversion of 1-aminocyclopropane-1-carboxylate (ACC) to ET because ET was produced from ACC solely in the presence of the superoxide-generating chemical KO2. ET production was also inhibited by lipoxygenase (LOX) inhibitors, indicating a possible involvement of LOX-mediated generation of superoxide anion and ET production itself. Furthermore, elicitin-induced ET production was completely inhibited by the protein synthesis inhibitor cycloheximide but recovered after exogenous application of ACC, indicating that de novo protein synthesis is required for ACC accumulation, leading to ET production. We also investigated the effects of several phytohormones on elicitor-induced ET production and discuss their role in the defense response.

Quenching of Intracellular ROS Generation as a Mechanism for Oleate-Induced Reduction of Endothelial Activation and Early Atherogenesis

We previously showed that the exposure of vascular endothelium to oleate results in reduced endothelial activation. We now investigate possible mechanisms for this effect in relation to generation of reactive oxygen species (ROS). We stimulated several types of endothelial cells with cytokines or lipopolysaccharide, with or without preincubation with 10-100 mumol/L oleate. Oleate preincubation reduced VCAM-1 expression in all cell types, as well as macrophage-colony stimulating factor release. We simultaneously measured the concentration of intracellular glutathione (GSH), the activity of GSH-related antioxidant enzymes and the production of intracellular ROS. Stimulation of endothelial cells caused a decrease of GSH and an increase in intracellular ROS. The addition of oleate before stimulation, prevented the depletion of GSH and partially prevented stimuli-induced increase of intracellular ROS. This occurred without any change in the activity of GSH-related antioxidant enzymes, superoxide dismutase and catalase. Furthermore, in a cell-free superoxide anion-generating system, oleate quenched the generation of ROS. These results indicate that oleate may exert direct vascular atheroprotective effects by inhibiting endothelial activation through a quenching of stimuli-induced increase in ROS.

Ascorbate-induced oxidative inactivation of Zn2+-glycerophosphocholine cholinephosphodiesterase.

Zn2+-glycerophosphocholine cholinephosphodiesterase, responsible for the conversion of glycerophosphocholine into glycerol and phosphocholine, was inactivated during incubation with ascorbic acid at 38 degrees C. The inclusion of copper ions or Fe2+ accelerated the ascorbate-induced inactivation, with Cu2+ or Cu+ being much more effective than Fe2+, suggestive of ascorbate-mediated oxidation. Dehydroascorbic acid had no effect on the phosphodiesterase, but H2O2 inactivated the enzyme in a concentration-dependent manner. Also, the enzyme was inactivated partially by a superoxide anion-generating system but not an HOCl generator. In support of involvement of H2O2 in the ascorbate action, catalase and superoxide dismutase expressed a complete and a partial protection, respectively. However, hydroxy radical scavengers such as mannitol, benzoate, or dimethyl sulfoxide were incapable of preventing the ascorbate action, excluding the participation of extraneous .OH. Although p-nitrophenylphosphocholine exhibited a modest protection against the ascorbate action, a remarkable protection was expressed by amino acids, especially by histidine. In addition, imidazole, an electron donor, showed a partial protection. Separately, when Cu2+-induced inactivation of the phosphodiesterase was compared with the ascorbate-mediated one, the protection and pH studies indicate that the mechanism for the ascorbate action is different from that for the Cu2+ action. Here, it is proposed that Zn2+-glycerophosphocholine cholinephosphodiesterase is one of brain membrane proteins susceptible to oxidative inactivation.

Induction of the iron-containing superoxide dismutase in Entamoeba histolytica by a superoxide anion-generating system or by iron chelation

The regulation of superoxide dismutase (SOD) expression was studied in 4 Entamoeba histolytica isolates. In comparison to anaerobic conditions, cultivation of the amoebae in the presence of superoxide radical anions or a ferrous iron chelator revealed substantial increase of SOD expression. Under the different culture conditions, all SOD activity could be exclusively attributed to an iron-containing type (FeSOD). Northern blot analysis revealed that FeSOD expression was regulated on the transcriptional level. Within the 5’-flanking region of the amoebic FeSOD gene, a 19-bp fragment was found with 68% sequence identity to the consensus motif of the binding site for the ferric uptake regulation gene product of Escherichia coli. Electrophoretic mobility shift assays with this 19-bp fragment and with amoebic nuclear extracts revealed specific DNA/protein complex formation. The results indicate that the regulation of E. histolytica FeSOD expression is similar to that of the manganese-containing SOD (MnSOD) of E. coli.

On the pleiotropy of indoleamine actions in a dinoflagellate, Gonyaulax polyedra: comparison of 5-methoxytryptamine with 5- and 6-fluorotryptamines

In Gonyaulax polyedra, 5-methoxytryptamine (5-MT), 5-fluorotryptamine (5-FT) and 6-fluorotryptamine (6-FT) are similarly potent stimulators of bioluminescence. When given in long-days (light/dark cycle of 16:8 hr), 5-methoxytryptamine is a strong inducer of cyst formation, leading to complete encystment of the entire cell population down to concentrations of 5 × 10 −6 M, whereas 5- and 6-fluorotryptamines only elicit incomplete encystment, especially with regard to the capability of the individual cells to form cyst walls (5-FT: about one half of cells; 6-FT: maximally 1 5 ); the fluorotryptamines lead, however, to an immobilization of cells and retractions of the protoplast from the theca (6-FT ⩾ 5 × 10 −6 M; 5-FT ⩾ 10 −5 M). Gonyaulax extracts catalyse photo-oxidatin of the three indoleamines: 5-MT leads to an accumulation of the homologous 5-methoylated kynuramine, whereas only minor amounts of fluorinated kynuramines were obtained. In a superoxide anion-generating system, 5-MT and 5-FT are predominantly oxidized to substituted kynuramines; 6-FT undergoes the homologous reaction, but is further converted into a non-fluorescent product.

Depression of cardiac sarcolemmal phospholipase D activity by oxidant-induced thiol modification.

Myocardial phospholipase D (PLD) is primarily localized at the sarcolemmal level and selectively hydrolyzes phosphatidylcholine to form phosphatidic acid as part of the signal transduction mechanisms for regulating Ca2+ movements in the heart. Since the myocardial cell damage induced by oxidative stress is associated with abnormalities in Ca2+ homeostasis and thiol status, we examined the thiol group dependence and the effects of oxidant species on this enzyme. Sarcolemmal membranes isolated from rat heart were exposed to several types of thiol group modifiers. Alkylation with N-ethylmaleimide or methyl methanethiosulfonate, mercaptide formation with p-chloromercuriphenylsulfonic acid, and thiol-disulfide exchange with 5,5'-dithio-bis(2-nitrobenzoate) depressed sarcolemmal PLD activity; in all cases the depression was prevented by dithiothreitol. At different concentrations of N-ethylmaleimide the PLD depression correlated well (r = 0.98) with the decrease in total thiol group content of the membrane. The enzyme activity was not affected by xanthine-xanthine oxidase, a superoxide anion-generating system, but was depressed by hydrogen peroxide (H2O2) in a concentration-dependent manner. This inhibitory effect was prevented by catalase as well as by dithiothreitol, but not by D-mannitol. The effect of a hydroxyl radical-generating system (Fenton reaction) could not be assessed because of an interfering direct inhibition by Fe2+. Dithiothreitol was also able to restore PLD activity in H2O2-pretreated membranes and to prevent a severe deactivation of the enzyme by hypochlorous acid (HOCI). Protection by glutathione and inhibition by its oxidized form were also observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased sensitivity of Candida albicans cells accumulating 14 alpha-methylated sterols to active oxygen: possible relevance to in vivo efficacies of azole antifungal agents.

The sensitivity of Candida albicans cells to killing by hydrogen peroxide was found to increase markedly when they were grown in the presence of sub-growth-inhibitory concentrations of the azole drug clotrimazole (CTZ). A superoxide anion-generating system consisting of xanthine and xanthine oxidase also killed such CTZ-treated cells more efficiently than control cells, but this seemed to be accounted for by hydrogen peroxide secondarily formed from superoxide anion as judged by the effect of catalase and superoxide dismutase. The increased sensitivity to hydrogen peroxide was considered to be attributable to the inhibition of 14 alpha-demethylation of ergosterol biosynthesis by CTZ, since a 14 alpha-demethylation-deficient mutant of C. albicans exhibited a similar phenotype. It is suggested that the in vivo efficacy of azole antifungal agents against C. albicans infection is at least partially due to the sensitization of the fungal cells to the oxygen-dependent microbicidal system of the phagocyte.

Evidence for the involvement of distinct signal transduction pathways in the regulation of constitutive and interferon gamma-dependent gene expression of NADPH oxidase components (gp91-phox, p47-phox, and p22- phox) and high-affinity receptor for IgG (Fc gamma R-I) in human polymorphonuclear leukocytes

We recently showed that mRNA levels coding the high-affinity Fc gamma receptor for IgG (Fc gamma R-I, CD64) and two of the components of the phagocytic superoxide anion-generating system--the heavy-chain subunit of cytochrome b558 (gp91-phox) and the 47-Kd cytosolic factor (p47- phox)--are modulated by interferon gamma (IFN-gamma). In this study, we examined whether dexamethasone (DEX) affects gp91-phox and p47-phox mRNA expression of human polymorphonuclear leukocytes (PMN), treated or not with IFN-gamma. We also investigated whether staurosporine, a general inhibitor of protein kinases, influences gp91-phox, p47-phox, and Fc gamma R-I gene expression in PMN treated with or without IFN- gamma. We found that (1) gp91-phox mRNA steady-state levels, expressed in control or IFN-gamma-treated PMN, were significantly inhibited, in a dose-dependent fashion, by both DEX and staurosporine; (2) p47-phox mRNA steady-state levels, expressed in control or IFN-gamma-treated PMN, were not influenced by DEX, but were markedly depressed by staurosporine; (3) no changes of spectrophotometric cytochrome b558 were found in PMN treated for up to 20 hours with the inhibitors, regardless of the presence of IFN-gamma; (4) both DEX and staurosporine dose-dependently inhibited IFN-gamma-induced Fc gamma R-I mRNA and protein expression; and (5) stability of gp91-phox and Fc gamma R-I messages in IFN-gamma-treated PMN was not altered by the presence of DEX. Our results demonstrate that gp91-phox, p47-phox, and Fc gamma R-I gene expression of PMN is governed by specific and independent biochemical pathways. Moreover, IFN-gamma activates different signal transduction pathways to modulate mRNA expression of gp91-phox, p47- phox, and Fc gamma R-I.

Evidence for the Involvement of Distinct Signal Transduction Pathways in the Regulation of Constitutive and Interferon γ-Dependent Gene Expression of NADPH Oxidase Components (gp91-phox, p47-phox, and p22-phox) and High-Affinity Receptor for IgG (FcγR-I) in Human Polymorphonuclear Leukocytes

AbstractWe recently showed that mRNA levels coding the high-affinity Fcγ receptor for IgG (FcγR-I, CD64) and two of the components of the phagocytic superoxide anion-generating system—the heavy-chain subunit of cytochrome b558 (gp91-phox) and the 47-Kd cytosolic factor (p47-phox)—are modulated by interferon gamma (IFN-γ). In this study, we examined whether dexamethasone (DEX) affects gp91-phox and p47-phox mRNA expression of human polymorphonuclear leukocytes (PMN), treated or not with IFN-γ. We also investigated whether staurosporine, a general inhibitor of protein kinases, influences gp91-phox, p47-phox, and FcγR-I gene expression in PMN treated with or without IFN-γ. We found that (1) gp91-phox mRNA steady-state levels, expressed in control or IFN-γ-treated PMN, were significantly inhibited, in a dose-dependent fashion, by both DEX and staurosporine; (2) p47-phox mRNA steady-state levels, expressed in control or IFN-γ-treated PMN, were not influenced by DEX, but were markedly depressed by staurosporine; (3) no changes of spectrophotometric cytochrome b558 were found in PMN treated for up to 20 hours with the inhibitors, regardless of the presence of IFN-γ; (4) both DEX and staurosporine dose-dependently inhibited IFN-γ-induced FcγR-I mRNA and protein expression; and (5) stability of gp91-phox and FcγR-I messages in IFN-γ-treated PMN was not altered by the presence of DEX. Our results demonstrate that gp91-phox, p47-phox, and FcγR-I gene expression of PMN is governed by specific and independent biochemical pathways. Moreover, IFN-γ activates different signal transduction pathways to modulate mRNA expression of gp91-phox, p47-phox, and FcγR-I. © 1992 by The American Society of Hematology. 0006-4971/92/7903-0021$3.00/0

Molecular basis of interferon-gamma and lipopolysaccharide enhancement of phagocyte respiratory burst capability. Studies on the gene expression of several NADPH oxidase components

In this study, we analyzed the expression of genes encoding for components of the phagocyte superoxide anion-generating system in human phagocytes treated with interferon-gamma (IFN-gamma) or lipopolysaccharide (LPS). Human neutrophils express high levels of the 47-kDa cytosolic factor (p47-phox), which are down-regulated after treatment with IFN-gamma, but not with LPS. On the contrary, the steady-state levels of the heavy chain subunit of cytochrome b558 (gp91-phox) were increased by IFN-gamma and LPS in human monocyte-derived macrophages and neutrophils in a time- and dose-dependent fashion, whereas cytochrome b558 light chain subunit (p22-phox) mRNA was not influenced by either agent. Studies on post-transcriptional regulation at the level of mRNA stability indicate that, in neutrophils, IFN-gamma has no influence on gp91-phox and p47-phox mRNA half-lives. The content of the two cytochrome b558 subunits was quantified by enzyme-linked immunosorbent assay, which revealed that, in neutrophils, gp91-phox levels doubled after 4 h of treatment with IFN-gamma or LPS. Monocyte/macrophage maturation was associated with a gradual decrease in gp91-phox mRNA and protein levels, which were both restored by treatment with IFN-gamma for 24-48 h. These results suggest that induction of the gp91-phox gene and protein product by IFN-gamma or LPS is an important requirement in the mechanism of the enhancement of neutrophil and macrophage oxidative metabolism.

Reactive oxygen in the tumor promotion stage of skin carcinogenesis

Exposure of isolated SENCAR mouse epidermal cells to the tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA) in vitro resulted in the production of oxidant species detected as chemiluminescence. This oxidant response can be inhibited by superoxide dismutase and copper complexes but not catalase or scavengers of hydroxyl radical or singlet oxygen, suggesting that the oxidant is superoxide anion. Inhibitors of various parts of the arachidonate cascade affect the TPA-induced oxidant response in a manner that corresponds to their effects on in vivo tumor promotion experiments. Agents that inhibit lipoxygenase activity, i.e. nordihydroguaiaretic acid, benoxaprofen, but not agents that are cyclooxygenase inhibitors, i.e. indomethacin, are effective in suppressing the oxidant response to TPA. Phospholipase C but not phospholipase A2 or D produced an oxidant response kinetically similar to that elicited by TPA. The inhibitors of TPA-induced oxidants inhibited the phospholipase C response to the same extent, suggesting that TPA and phospholipase C may produce an oxidant species through a common mechanism, via phospholipid turnover-protein kinase C activation. The relevance of oxidant production to the tumor promotion process is suggested by the ability of exogenous xanthine/xanthine oxidase, a superoxide anion-generating system, to induce ornithine decarboxylase, a characteristic of TPA-treated cells. In addition, oxidant production is significantly lower in cells from the TPA-promotion resistant C57BL/6J mouse. These studies provide further support for a role for reactive oxygens in the tumor promotion process.

Monoclonal antibodies to a particulate superoxide-forming system stimulate a respiratory burst in intact guinea pig neutrophils.

Monoclonal rat antibodies were produced against a subcellular preparation of phorbol 12-myristate 13-acetate (PMA)-stimulated guinea pig neutrophils that retains NADPH-oxidase activity. Two antibodies, 1A10.4 and IG4, were isolated that bind to a surface antigen restricted to guinea pig neutrophils from bone marrow and peritoneal exudate and to macrophages and that trigger a respiratory burst in neutrophils in the presence of cytochalasin B. Intact antibody 1A10.4, subclass IgG2c, can trigger superoxide anion release directly; F(ab')2 fragments of 1A10.4 and intact IG4 require further cross-linking by F(ab')2 fragments of anti-rat immunoglobulin antibody. Both antibodies recognize the same antigen, a proteolipid of apparent molecular mass 10 kDa. Immunoprecipitation of solubilized oxidase activity with 1A10.4 brings down this activity as part of a macromolecular complex. Surface expression of the antigen is increased on treatment of cells with both PMA and cytochalasin B. 1A10.4 also triggers release of the granule enzyme beta-glucuronidase. Triggering of a respiratory burst by the antibodies appears distinct from the PMA and fMet-Leu-Phe signalling systems. These studies indicate that the antigen defined by antibodies 1A10.4 and IG4 becomes associated with the superoxide anion-generating system of neutrophils but may play a more general role in signal transduction in phagocytic cells.

Hemoglobin. A biologic fenton reagent.

Iron and iron compounds may facilitate hydroxyl-radical generation from activated oxygen species. Earlier work on the generation of this radical has been focused on simple, low-molecular-weight iron compounds. We hypothesized that free hemoglobin, like other iron-rich substances, might also mediate hydroxyl-radical generation. We find: 1) In the presence of a superoxide anion-generating system (hypoxanthine and xanthine oxidase), hemoglobin promotes hydroxyl-radical formation in a dose-dependent fashion. 2) This generation of hydroxyl radical is greatly decreased by prior oxidation of the hemoglobin, equilibration of hemoglobin with carbon monoxide, or addition of catalase, while added superoxide dismutase has little effect. Therefore, hydroxyl radical probably arises primarily via reaction between the ferrous heme iron and H2O2. 3) In further support of this, hydroxyl radical forms as readily upon the addition of H2O2 to hemoglobin. 4) Hemoglobin also increases hypoxanthine/xanthine oxidase-driven peroxidation of poly-unsaturated fatty acids such as arachidonic acid and human red cell membrane lipids. 5) The addition of sufficient haptoglobin (the plasma hemoglobin-binding protein) suppresses both hemoglobin-driven hydroxyl radical and malondialdehyde generation. Thus, free hemoglobin may be biologically hazardous, in part because it acts as a "Fenton" reagent, having the potential to catalyze hydroxyl-radical generation in areas of inflammation. Haptoglobin, which binds hemoglobin very tightly, blocks this through a presently unknown mechanism. An important physiologic function of haptoglobin may be prevention of such hemoglobin-mediated oxidation.

Stimulation of the human neutrophil superoxide anion-generating system with 1- O-hexadecyl/octadecyl-2-acetyl-sn-glyceryl-3-phosphorycholine

1- O-Hexadecyl/octadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC) stimulated a time- and concentration-dependent generation of superoxide anion (O 2 −) by human neutrophils. Maximum production of O 2 − occurred 60 sec subsequent to cell contact with AGEPC. Superoxide generation was reduced significantly if cells were not preincubated with cytochalasin B prior to exprosure to AGEPC (0.01 to 10 μM). A time-dependent desensitization for O 2 − production was demonstrated in neutrophilis which were stimulated with AGEPC prior to contact with cytochalisin B. The rate and amount of O 2 − generated by AGEPC-activated neutrophils were enhanced significantly in the presence of extracellular calcium. However, incubation of neutrophils with ethyleneglycol-bis(β-amino-ethyl ether) N, N′-tetra-acetate (EGTA) in calcium-free medium had no effect on the 2-deoxy- D-glucose (2-DG) AGEPC-induced O 2 − production was suppressed by the glycolytic inhibitor 2-deoxy- D-glucose (2-DG) and the sulfhydryl reagents N-ethylmaleimide (NEM) and iodoacetic acid (1A). Sodium cyanide was inactive. Pretreatment of neutrophils with AGEPC reduced cell responsiveness to subsequent exposure to this stimulus. Desensitization of the O 2 −-generating system activated with AGEPC appears to be stimulus specific for leukotriene B 4 (LTB 4) and N-formyl-methionyl-leucyl-phenylalanine (FMLP) in that these stimuli were capable of inducing O 2 − production by cells pretreated with AGEPC. However, neutrophils pretreated with AGEPC were desensitized to zymosan-activated serum (ZAS).

Superoxide dismutase assay using alkaline dimethylsulfoxide as superoxide anion-generating system

Alkaline dimethylsulfoxide as a superoxide anion-generating system in association with cytochrome c as a superoxide anion-indicating scavenger has been used to develop a new assay for superoxide dismutase. The assay is sensitive (one unit of enzymatic activity is provided by 110 ng of purified copper-containing superoxide dismutase) and highly specific. The nature of this system prevents the usual interferences and its simplicity allows for multiple, rapid measurements of superoxide dismutase activity in biological preparations using either normal or automated procedures.

Effect of traumatic injury on membrane phosphatase activity in cat spinal cord

The enzymatic activities of the spinal cord membrane phosphatases, (Na,K)ATPase, KpNPPase, 5′-nucleotidase, MgATPase, and MgpNPPase, were determined following contusion injury. Within 30 min after injury, the activities of (Na,K)ATPase, KpNPPase, and 5′-nucleotidase were suppressed at the injury site and remained suppressed for 24 h. Considerable loss of (Na,K)ATPase and KpNPPase activity was seen along the longitudinal axis of the spinal cord for the first 4 h after injury, whereas at 24 h after injury, there was almost complete restitution in the activity of those enzymes. No similar changes in activity were observed for the MgATPase, whereas the activity of the MgpNPPase progressively worsened with time at the injury site. The deduce the pathobiochemical process that accounts for the loss of spinal membrane phosphatase activity, spinal cord membranes were incubated in vitro in the presence of a superoxide anion-generating system, as well as in the presence of trypsin and lysolecithin. It was found that superoxide anions inhibited only the activity of transport phosphatase, KpNPPase, whereas trypsin inhibited the activity of both KpNPPase and MgpNPPase. No inhibition of 5′-nucleotidase was observed by superoxide anions; however, the activity of 5′-nucleotidase was enhanced by trypsin, lysolecithin, or both in concert. These results suggest that the pathobiochemical process that accounts for the loss of spinal membrane phosphatase activity that follows injury can be attributed to the concerted effects of free radical attack as well as the activation of proteolytic and lipolytic enzymes. Inactivation of spinal membrane phosphatase can occur by direct attack of these processes on the membrane or by loss of membrane material, e.g., solubilization.