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PubMed2004

The oxidative burst at fertilization is dependent upon activation of the dual oxidase Udx1.

The sea urchin egg is a quiescent cell...until fertilization, when the egg is activated. The classic respiratory burst at fertilization is the result of prodigious hydrogen peroxide production, but the mechanism for this synthesis is not known. Here we quantitate the kinetics of hydrogen peroxide synthesis at a single-cell level using an imaging photon detector, showing that 60 nM hydrogen peroxide accumulates within the perivitelline space of each zygote. We find that the NADPH oxidation activity is enriched at the cell surface and is sensitive to a pharmacological inhibitor of NADPH oxidase enzymes. Finally, we show that a sea urchin dual oxidase homolog, Udx1, is responsible for generating the hydrogen peroxide necessary for the physical block to polyspermy. Phylogenetic analysis of the enzymatic modules in Udx1 suggests a potentially conserved role for the dual oxidase family in hydrogen peroxide production and regulation during fertilization.

Animals↗
PubMed2004

Deficient phospholipase C activity in blood polimorphonuclear neutrophils from patients with liver cirrhosis.

BACKGROUND/AIMS: Circulating neutrophils from cirrhotic patients have a reduced capacity to generate superoxide anion (O(2)(-)), which might contribute to frequent bacterial infections in these patients. We studied the signal transduction pathways involved in the generation of O(2)(-) in neutrophils from 98 cirrhotic patients and 46 healthy controls. METHODS: We measured O(2)(-) production in neutrophils induced by fMLP, opsonized zymosan, TNF alpha, NaF, AlF(4)(-), A23187 and phorbol myristate acetate. Furthermore, we measured phospholipase C activity in neutrophils from healthy controls and end-stage cirrhotic patients. RESULTS: O(2)(-) production was decreased in neutrophils from patients in response to fMLP, opsonized zymosan and TNF alpha. Likewise, response of these cells to G-protein stimulation by fluorides was also decreased. These reduced responses correlated significantly with the degree of liver dysfunction. On the contrary, neutrophils from patients responded normally to A23187 and phorbol esters stimulation indicating that Ca(2+)- and PKC-dependent pathways are intact in these cells. Finally, phospholipase C activity was markedly reduced in neutrophils from end-stage liver cirrhosis. CONCLUSIONS: These data confirm that O(2)(-) generation by neutrophils is decreased in patients with cirrhosis, particularly in those with more severe liver dysfunction, and suggest that this defect involves phosphatidylinositol specific phospholipase C activity.

Adult↗
PubMed2006

Reactive oxygen metabolites and anti-oxidative defenses in aspirin-induced gastric damage in rats: Gastroprotection by Vitamin E.

OBJECTIVE: It has been proposed that neutrophil infiltration and oxygen radicals may be the important prime events that lead to mucosal injury induced by aspirin. Vitamin E acts as a potent antioxidant, and is capable of scavenging free radicals. The aim of this study was to evaluate the oxygen metabolites and anti-oxidative defenses in acute gastric damage induced by aspirin and to find the effects of Vitamin E. METHODS: Ninety-six Wistar rats were divided into four groups of 24 rats each as follows: (1) the control group; (2) the ASA group that received 300mg/kg of ASA; (3) the Vitamin E plus ASA group and (4) the Vitamin E group that received Vitamin E (75 units) alone. At 3, 6, 9 and 24h after the drug administration, six rats were randomly selected from each group and gastric mucosal injury, prostaglandin E2, and the activities of myeloperoxidase, xanthine-oxidase, superoxide dismutase, glutathione peroxidase as well as glutathione level were measured and compared between the groups. RESULTS: Oral administration of ASA caused acute gastric erosions and an increase in myeloperoxidase activity. It also decreased prostaglandin E2, superoxide dismutase activity, glutathione peroxidase activity and glutathione level. Concomitant administration of Vitamin E and ASA restored all the changes toward the control levels. CONCLUSION: Free radicals and suppression of anti-oxidizing enzymes play important roles in gastric damage induced by aspirin. Increased myeloperoxidase activity suggests that activated neutrophils may be a major source of free radicals. Vitamin E protects against ASA-induced damage due to its anti-oxidizing activity.

Journal Article↗
PubMed2006

Flavonoid oxidation in plants: from biochemical properties to physiological functions.

Flavonoids protect plants against various biotic and abiotic stresses, and their occurrence in human diet participates in preventing degenerative diseases. Many of the biological roles of flavonoids are attributed to their potential cytotoxicity and antioxidant abilities. Flavonoid oxidation contributes to these chemical and biological properties and can lead to the formation of brown pigments in plant tissues as well as plant-derived foods and beverages. Flavonoid oxidation in planta is mainly catalyzed by polyphenol oxidases (catechol oxidases and laccases) and peroxidases. These activities are induced during seed and plant development, and by environmental stresses such as pathogen attacks. Their complex mode of action is regulated at several levels, involving transcriptional to post-translational mechanisms together with the differential subcellular compartmentalization of enzymes and substrates.

Flavonoids↗
PubMed1998

Resonance Raman microspectroscopy of myeloperoxidase and cytochrome b558 in human neutrophilic granulocytes.

With (resonance) Raman microscospectroscopy, it is possible to investigate the chemical constitution of a very small volume (0.5 fl) in a living cell. We have measured resonance Raman spectra in the cytoplasm of living normal, myeloperoxidase (MPO)-deficient, and cytochrome b558-deficient neutrophils and in isolated specific and azurophilic granule fractions, using an excitation wavelength of 413.1 nm. Similar experiments were performed after reduction of the redox centers by the addition of sodium dithionite. The specific and azurophilic granules in both redox states appeared to have clearly distinguishable Raman spectra when exciting at a wavelength of 413.1 nm. The azurophilic granules and the cytochrome b558-deficient neutrophils showed Raman spectra similar to that of the isolated MPO. The spectra of the specific granules and the MPO-deficient neutrophils corresponded very well to published cytochrome b558 spectra. The resonance Raman spectrum of the cytoplasmic region of normal neutrophilic granulocytes could be fitted with a combination of the spectra of the specific and azurophilic granules, which shows that the Raman signal of neutrophilic granulocytes mainly originates from MPO and cytochrome b558, at an excitation wavelength of 413.1 nm.

Cytochrome b Group↗
PubMed1988

Definitive characterization of uric acid as an interferent in peroxidase indicator reactions and a proposed mechanism of action.

We have characterized and identified uric acid as an interferent to peroxidase catalyzed reactions where hydrogen peroxide is generated at relatively low concentrations. The implications of these findings are important for those utilizing peroxidase as an indicator reaction where low primary substrate concentrations require their preliminary extraction or chemical modification. We have shown that the elimination of uric acid as an interferent from biologic fluid obviates the necessity for such treatment. In amniotic fluid, our data suggests that uric acid represents the only interference to peroxidase-catalyzed reactions, especially when using p-substituted phenols as proton donors. The removal of uric acid has been shown to eliminate hydrogen peroxide reduction and should allow for an increase in sensitivity and specificity for measurements incorporating a peroxidase-coupled indicator reaction, hence, more effective use of these reaction sequences. To our knowledge, this is the first report of a mechanism to eliminate hydrogen peroxide reduction in amniotic fluid.

Amniotic Fluid↗
PubMed1999

YC-1 potentiates the antiplatelet effect of hydrogen peroxide via sensitization of soluble guanylate cyclase.

In the present study, we showed that 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1), a nitric oxide (NO)-independent activator of soluble guanylate cyclase, could potentiate H2O2-induced inhibition of platelet aggregation and increase of platelet cGMP levels. The synergistic effect of YC-1 and H2O2 on platelet aggregation and increases of cGMP were almost completely prevented by catalase and a selective soluble guanylate cyclase inhibitor (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), or partially attenuated by the hydroxyl radical scavenger mannitol. In contrast, superoxide dismutase failed to influence H2O2/YC-1-induced inhibition of aggregation. Furthermore, YC-1 could enhance the activation of soluble guanylate cyclase caused by FeSO4/H2O2 and, this effect was prevented markedly by mannitol. These results suggest that YC-1 may enhance the antiaggregatory effect of H2O2 via the sensitization of platelet soluble guanylate cyclase. In addition, this phenomenon is, at least in part, dependent on H2O2-derived hydroxyl radical.

Antioxidants↗
PubMed1999

Redox hydrogel based bienzyme electrode for L-glutamate monitoring.

Amperometric bienzyme electrodes based on coupled L-glutamate oxidase (GlOx) and horseradish peroxidase (HRP) were constructed for the direct monitoring of L-glutamate in a flow injection (FI)-system. The bienzyme electrodes were constructed by coating solid graphite rods with a premixed solution containing GlOx and HRP crosslinked with a redox polymer formed of poly(1-vinylimidazole) complexed with (osmium (4-4'-dimethylbpy)2 Cl)II/III. Poly(ethylene glycol) diglycidyl ether (PEGDGE) was used as the crosslinker and the modified electrodes were inserted as the working electrode in a conventional three electrode flow through amperometric cell operated at -0.05 V versus Ag¿AgCl (0.1 M KCl). The bienzyme electrode was optimized with regard to wire composition, Os-loading of the wires, enzyme ratios, coating procedure, flow rate, effect of poly(ethyleneimine) addition, etc. The optimized electrodes were characterized by a sensitivity of 88.36 +/- 0.14 microA mM(-1) cm(-2), a detection limit of 0.3 microM (calculated as three times the signal-to-noise ratio), a response time of less than 10 s and responded linearly between 0.3 and 250 microM (linear regression coefficient = 0.999) with an operational stability of only 3% sensitivity loss during 8 h of continuous FI operation at a sample throughput of 30 injections h(-1).

Amino Acid Oxidoreductases↗
PubMed2000

Oxidative stress as a prerequisite for aflatoxin production by Aspergillus parasiticus.

The relevance of free radical generation and oxidative stress with regard to aflatoxin production was examined by comparing the oxygen requirement and antioxidant status of a toxigenic strain of Aspergillus parasiticus with that of a nontoxigenic strain at early (trophophase) and late logarithmic (idiophase) growth phases. In comparison to the nontoxigenic strain, wherein the oxygen requirements were relatively unaltered at various growth phases, the toxigenic strain exhibited greater oxygen requirements at trophophase coinciding with onset of aflatoxin production. The activities of antioxidant enzymes such as xanthine oxidase, superoxide dismutase, and glutathione peroxidase and the mycelial contents of thiobarbituric acid-reactive substances as well as of reduced glutathione were all enhanced during the progression of toxigenic strain from trophophase to idiophase. The combined results suggest that aflatoxin production by the toxigenic strain may be a consequence of increased oxidative stress leading to enhanced lipid peroxidation and free radical generation.

Aflatoxins↗
PubMed2003

Graphite-Teflon composite bienzyme amperometric biosensors for monitoring of alcohols.

Composite graphite-Teflon electrodes, in which the enzymes alcohol oxidase (AOD) and horseradish peroxidase (HRP), as well as the mediator ferrocene, are incorporated into the electrode matrix, are reported for the reliable monitoring of alcohols in food and beverages. The bienzyme electrodes are constructed by simple physical inclusion of the enzymes and the mediator in the bulk of graphite-70% Teflon rigid cylindrical pellets. The composite biosensors are robust and reusable because of the renewability of the electrode surface by polishing. Reproducible amperometric responses at 0.00 V were obtained with different electrodes constructed from the same pellet and from different pellets. No significant loss of the enzymes activities was found after at least 3 months of storage at 0 degrees C. The detection limits obtained by amperometry in stirred solutions can be advantageously compared with those achieved with commercial sensors for alcohols. The bienzyme electrodes are suitable to be used under flow-injection conditions, as well as for amperometric detection in HPLC. The bioelectrodes were employed for the determination of ethanol in beers, wines and liquors, using both batch- and flow-injection modes, and for the determination of methanol in wines and liquors by HPLC with amperometric detection. Only a dilution of the beverages was needed as sample treatment in all cases.

Alcoholic Beverages↗
PubMed2003

Chemical modification of biocatalysts.

Although several powerful methods exist for the redesign of enzyme structure and function these are typically limited to the 20 most abundant proteinogenic amino acids. The use of chemical modification overcomes this limitation to allow virtually unlimited alteration of amino acid sidechain structures. If heterogeneous mixtures of enzyme products are to be avoided, however, the required chemistry should be efficient, selective and compatible with aqueous conditions. Recent advances have been made in the modification of proteinases, aminotransferases and redox enzymes.

Endopeptidases↗
PubMed2001

Detection of electrochemical enzymatic reactions by surface plasmon resonance measurement.

We describe the surface plasmon resonance (SPR) detection of an enzymatic turnover reaction and the measurement of glucose concentration using a multienzyme layer modified gold electrode. We constructed an osmium redox polymer mediated enzyme sensor on a gold thin-film electrode and monitored electrochemical reaction by SPR measurement. Unlike the usual binding assay with SPR, here we used SPR to detect the redox state of an electron mediator that was the result of the electron-transfer reaction of sequential enzymatic reactions. Therefore, the degree of refractive index change was independent of the dielectric property of the substrate and enzymatic molecular recognition was converted to refractive index change with amplification. For the quantitative evaluation of glucose with this method, we used chronopotentiometry and a linear relation was obtained between the glucose concentration and the rate of refractive index change.

Electrochemistry↗
PubMed2000

Redox hydrogel-based amperometric bienzyme electrodes for fish freshness monitoring

This work presents the design and optimization of amperometric biosensors for the determination of biogenic amines (e.g., histamine, putrescine, cadaverine, tyramine, cystamine, agmatine, spermidine), commonly present in food products, and their application for monitoring of freshness in fish samples. The biosensors were used as the working electrodes of a three-electrode electrochemical cell of wall-jet type, operated at -50 mV vs Ag/AgCl, in a flow injection system. Two different bienzyme electrode designs were considered, one based on the two enzymes [a newly isolated and purified amine oxidase (AO) and horseradish peroxidase (HRP)] simply adsorbed onto graphite electrodes, and one when they were cross-linked to an Os-based redox polymer. The redox hydrogel-based biosensors showed better biosensors characteristics, i.e., sensitivity of 0.194 A M-1 cm-2 for putrescine and 0.073 A M-1 cm-2 for histamine, and detection limits (calculated as three times the signal-to-noise ratio) of 0.17 microM for putrescine and 0.33 microM for histamine. The optimized redox hydrogel-based biosensors were evaluated in terms of stability and selectivity, and were used for the determination of total amine content in fish samples kept for 10 days in different conditions.

Journal Article↗
PubMed1987

Ovothiols, a family of redox-active mercaptohistidine compounds from marine invertebrate eggs.

We have previously reported a novel thiol compound, 1-methyl-N alpha,N alpha-dimethyl-4-mercaptohistidine, or ovothiol, present at high concentration in the eggs of the sea urchin Strongylocentrotus purpuratus [Turner, E., Klevit, R., Hopkins, P. B., & Shapiro, B. M. (1986) J. Biol. Chem. 261, 13056-13063]. Here we report two related compounds, 1-methyl-N alpha-methyl-4-mercaptohistidine, or ovothiol B, from the scallop Chlamys hastata, and 1-methyl-4-mercaptohistidine, or ovothiol A, from the starfish Evasterias troschelii. These two compounds, as well as the S. purpuratus compound now designated ovothiol C, were isolated from eggs or ovarian tissue by S-carboxymethylation with [3H]iodoacetic acid, ion-exchange chromatography and ion-pairing high-pressure liquid chromatography. The structures of S-(carboxymethyl)ovothiols A and B were determined by 1H NMR, and that of ovothiol A was confirmed by comparison with authentic methylhistidine samples after desulfuration with Raney nickel. In the ovary of each species, the predominant methylation form of ovothiol accounts for at least 80% of the total 4-mercaptohistidine. The ovothiol concentration of the ovary far exceeds that of the testis or somatic tissues. The ovothiol C content of unfertilized S. purpuratus eggs is 1.14 mumol/10(6) eggs, equivalent to approximately 4.3 mM average concentration; the glutathione (GSH + GSSG) content is 0.9 mumol/10(6) eggs. In this species, high ovothiol levels persisted for the first 2 weeks of embryonic development. Ovothiol and glutathione account for virtually all of the trichloroacetic acid soluble-SH groups in the egg; these results are compared to several previous studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids, Sulfur↗