Salivary Antigen-5/CAP Family Members Are Cu2+-dependent Antioxidant Enzymes That Scavenge O2⨪ and Inhibit Collagen-induced Platelet Aggregation and Neutrophil Oxidative Burst
Teresa C. F. AssumpçãoDongying MaAlexandra SchwarzKarine ReiterJaime M. SantanaJohn F. AndersenJosé M. C. RibeiroGlenn NardoneLee L. YuIvo M.B. Francischetti
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The function of the antigen-5/CAP family of proteins found in the salivary gland of bloodsucking animals has remained elusive for decades. Antigen-5 members from the hematophagous insects Dipetalogaster maxima (DMAV) and Triatoma infestans (TIAV) were expressed and discovered to attenuate platelet aggregation, ATP secretion, and thromboxane A2 generation by low doses of collagen (<1 μg/ml) but no other agonists. DMAV did not interact with collagen, glycoprotein VI, or integrin α2β1. This inhibitory profile resembles the effects of antioxidants Cu,Zn-superoxide dismutase (Cu,Zn-SOD) in platelet function. Accordingly, DMAV was found to inhibit cytochrome c reduction by O2⨪ generated by the xanthine/xanthine oxidase, implying that it exhibits antioxidant activity. Moreover, our results demonstrate that DMAV blunts the luminescence signal of O2⨪ generated by phorbol 12-myristate 13-acetate-stimulated neutrophils. Mechanistically, inductively coupled plasma mass spectrometry and fluorescence spectroscopy revealed that DMAV, like Cu,Zn-SOD, interacts with Cu2+, which provides redox potential for catalytic removal of O2⨪. Notably, surface plasmon resonance experiments (BIAcore) determined that DMAV binds sulfated glycosaminoglycans (e.g. heparin, KD ∼100 nmol/liter), as reported for extracellular SOD. Finally, fractions of the salivary gland of D. maxima with native DMAV contain Cu2+ and display metal-dependent antioxidant properties. Antigen-5/CAP emerges as novel family of Cu2+-dependent antioxidant enzymes that inhibit neutrophil oxidative burst and negatively modulate platelet aggregation by a unique salivary mechanism. The function of the antigen-5/CAP family of proteins found in the salivary gland of bloodsucking animals has remained elusive for decades. Antigen-5 members from the hematophagous insects Dipetalogaster maxima (DMAV) and Triatoma infestans (TIAV) were expressed and discovered to attenuate platelet aggregation, ATP secretion, and thromboxane A2 generation by low doses of collagen (<1 μg/ml) but no other agonists. DMAV did not interact with collagen, glycoprotein VI, or integrin α2β1. This inhibitory profile resembles the effects of antioxidants Cu,Zn-superoxide dismutase (Cu,Zn-SOD) in platelet function. Accordingly, DMAV was found to inhibit cytochrome c reduction by O2⨪ generated by the xanthine/xanthine oxidase, implying that it exhibits antioxidant activity. Moreover, our results demonstrate that DMAV blunts the luminescence signal of O2⨪ generated by phorbol 12-myristate 13-acetate-stimulated neutrophils. Mechanistically, inductively coupled plasma mass spectrometry and fluorescence spectroscopy revealed that DMAV, like Cu,Zn-SOD, interacts with Cu2+, which provides redox potential for catalytic removal of O2⨪. Notably, surface plasmon resonance experiments (BIAcore) determined that DMAV binds sulfated glycosaminoglycans (e.g. heparin, KD ∼100 nmol/liter), as reported for extracellular SOD. Finally, fractions of the salivary gland of D. maxima with native DMAV contain Cu2+ and display metal-dependent antioxidant properties. Antigen-5/CAP emerges as novel family of Cu2+-dependent antioxidant enzymes that inhibit neutrophil oxidative burst and negatively modulate platelet aggregation by a unique salivary mechanism.Exposure of midlog Bacteroides fragils (VPI 2393) to 2% O2-98% N2 caused a three- to fivefold increase in superoxide dismutase specific activity within the cells. The increase in specific activity was completed within 90 min after exposure to oxygen and was dependent upon protein synthesis. Cells containing the higher superoxide dismutase level were more resistant to the effects of 5 atm of oxygen tension than were cells containing the lower level of superoxide dismutase but were equally resistant to 5 atm of nitrogen tension. Similar results were observed upon comparing viability experiments with B. fragilis and B. vulgatus. Superoxide dismutase activity in sonic extracts of B. fragilis was rapidly inactivated by exposure to 5 mM H2O2 and was inhibited by 1 mM NaN3 but not 5 mM NaCN. The inhibition pattern is identical to the pattern demonstrated for the purified iron-containing enzyme from Escherichia coli B and suggests that the superoxide dismutase in B. fragilis is an iron enzyme.
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Abstract Background The small intestine is highly susceptible to surgical stress even at remote locations. An earlier study using a rat model indicated that oxidative stress plays an important role in this process. The enzyme xanthine oxidase is an important source of free radicals in the small intestine. The role of this enzyme in intestinal damage after surgical stress was examined. Methods Rats pretreated with xanthine oxidase inhibitors were subjected to surgical stress by opening the abdomen and handling the intestine, as done during laparotomy. Enterocytes at various stages of differentiation were isolated and the protection offered by xanthine oxidase inhibitors against damage due to surgical stress was determined and compared with normal controls. Protection against ultrastructural changes to the mucosa, as well as mitochondrial function was examined. Results Surgical stress affected both the villus as well as crypt cells, causing increased superoxide generation, accompanied by increased activity of xanthine oxidase. Xanthine oxidase inhibitors ameliorated the increased superoxide generation, and protected against mitochondrial damage and ultrastructural changes in the intestine. Conclusion Surgical stress affects both the villus and crypt cell populations in the small intestine. The enzyme xanthine oxidase maybe an important mediator of surgical stress in the intestine.
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Thirteen phenolic compounds were tested for their inhibitory effects on xanthine oxidase. The enzyme xanthine oxidase catalyses the oxidation of hypoxanthine to xanthine and of xanthine to uric acid, which has lambda max of 295 nm, forming the basis for a spectrophotometric assay of the activity of xanthine oxidase. The results showed that purpurogallin and silymarin group displayed the inhibitory effects on xanthine oxidase (IC50 = 2.96 +/- 0.12 and 27.58 +/- 3.48 microM, respectively). Their apparent inhibition constants (Ki) were 1.16 and 5.85 microM, and induced uncompetitive and mixed type (non-competitive-uncompetitive) inhibitions respectively, with respect to the substrate xanthine.
Hypoxanthine
Xanthine
Uncompetitive inhibitor
Allopurinol
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Xanthine dehydrogenase
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Allopurinol
Xanthine
Xanthine dehydrogenase
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Xanthine oxidase inhibitor
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New Xanthine oxidase inhibitors are in development and these need to be evaluated reliably before being introduced into clinical investigations. The presence of valid, easy and reliable in vivo bioassy to evaluate the Xanthine oxidase inhibition is of utmost importance to achieve steady paces in drug discovery attempts to treat gout disease. The objective of the current study was to develop and validate a bioassay procedure that can be employed for in vivo evaluation of drugs investigated as xanthine oxidase inhibitors. Exploiting rats as an animal model to carry out the bioassay is proposed to establish a novel approach to screen and evaluate the xanthine oxidase inhibitory activity of potential agents and/or plant extracts. Allopurinol was used as a reference treatment to inhibit Xanthine oxidase while the degree of enzyme inhibition was indirectly measured by determining the blood levels of 6mercaptopurine (6-MP) as a surrogate chemical marker. In this model, serum obtained from: untreated rats, rats treated with 6-MP alone and rats treated with 6-MP and different doses of allopurinol were analyzed, for levels of 6-mercaptopurine. An elevated plasma 6-mercaptopurine level in the allopurinol treated rats as compared to untreated rats was an indication of an in vivo inhibition of the enzyme Xanthine oxidase.
Allopurinol
Xanthine oxidase inhibitor
Xanthine dehydrogenase
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Superoxide dismutases (EC 1.15.1.1) in vascular plants representing different evolutionary levels were characterized using polyacrylamide gel electrophoresis. The three forms of the enzyme were distinguished from each other based on the following criteria: a) the Cu-Zn enzyme is sensitive to cyanide wherease the Fe and Mn enzymes are not; and b) the Cu-Zn and Fe enzymes are inhibited by H(2)O(2) whereas the Mn enzyme is H(2)O(2)-resistant. Of the 43 plant families investigated, the Fe-containing superoxide dismutase was found in three families: Gingkoaceae, Nymphaceae, and Cruciferae.
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Rats were fed purified diets containing 21 per cent, 12 per cent, or 8 per cent casein with or without added p -dimethylaminoazobenzene. At the end of 1, 2, and 4 months, six rats from each of the six dietary groups were analyzed for xanthine oxidase in the liver, kidney, lung, spleen, intestine, and blood. A method for determining blood xanthine oxidase was described.
By comparison with chow-fed rats, the purified 21 per cent casein diet alone decreased the xanthine oxidase somewhat in the liver, spleen, lung, and intestine, but not in the kidney. The 8 per cent casein diet gave a further marked decrease in the liver enzyme only, and a marked increase in the blood xanthine oxidase.
p -Dimethylaminoazobenzene decreased the xanthine oxidase in kidney and blood, irrespective of diet, and decreased the enzyme in liver synergistically with a low protein intake; the dye had no depressant effect on the xanthine oxidase in lung, spleen, or intestine.
The same xanthine oxidase activity was found in the liver homogenates after dialysis. The activity of added milk xanthine oxidase was increased slightly more in the liver homogenates of dye-free rats than in dye-fed rats. Methylene blue added to the liver homogenate in the aerobic test system increased the xanthine oxidase activity more in the dye-fed rats than in the controls. The effect of methylene blue on the xanthine oxidase activity of the liver homogenate was lost on dialysis, especially with livers from the low protein groups. These results indicated that the livers of dye-fed rats contained a small amount of nondialyzable inhibitor of xanthine oxidase that affected primarily the auto-oxidizable rather than the dehydrogenase portion of the enzyme. The livers also contained less enzyme than the dye-free controls. The effect of the dye in decreasing the endogenous respiration of the liver homogenate was due to a decreased xanthine oxidase activity of the liver.
The low protein diets were characterized by fatty infiltration and hydropic degeneration of the liver. Dye feeding led to bile-duct proliferation and cystadenomas in the 21 per cent casein group and bile-duct carcinomas in the other dietary groups.
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Influence of low-dosage pre-irradiation on xanthine oxidase levels in Guerin's carcinoma was studied with particular emphasis on free oxygen generation and free sulphohydrils in a fraction of proteins characterized by xanthine oxidase activity. Enhanced growth of tumor correlated with that of xanthine oxidase activity involving higher levels of enzymatic O-form due to thiol groups oxidation. Under similar conditions, xanthine oxidase and free oxygen generation levels were higher due to SH-group oxidation than in intact animals. Terminal stages involved dropping xanthine oxidase levels and enzymatic protein degradation.
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Xanthine dehydrogenase
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The antioxidant activity of rosemary antioxidant compared with BHT antioxidant on snake oil and grape seed oil was discussed in this paper,UV / VIS spectra of rosemary antioxidant was investigated in this paper.The results showed that rosemary antioxidant had the best antioxidant activity,when its concentration was between 0.04% to 0.05%.The oxidation resistance decrease with the increase of the amount.When the amount rose to some degree,it turned out to be catalytic oxidation.The rosemary antioxidant had great absorption at the UVA band,which could improve the protection of UVA in Sunscreen cosmetic.
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