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PubMed2004

Evaluation of the rat bladder-derived relaxant factor by coaxial bioassay system.

The release of bladder-derived relaxant factor in a coaxial bioassay system and the effects of reactive oxygen species were studied. After precontraction with phenylephrine (10(-6)-3x10(-6)) or 50 mM K+, acetylcholine (10(-8)-10(-3) M) induced relaxation in rat anococcygeus muscle mounted within rat bladder in a tissue bath. This relaxation was not altered by the removal of the urothelium or incubation with tetrodotoxin (10(-6) M). However, bupivacaine (10(-4) M) and lidocaine (3 x 10(-4) M) inhibited this response after raising the pH of the nutrient solution to 7.8, and oxybuprocaine (10(-4) M) exerted inhibitory effect at both physiological pH (7.4) and at pH 7.8. Exposure to electrolysis-generated reactive oxygen species or incubation with hydrogen peroxide and pyrogallol did not alter the acetylcholine response. Present results indicate that the bladder-derived relaxant factor does not behave like endothelium-derived hyperpolarizing factor, but its release may be associated with tetrodotoxin-resistant Na+ channels, which are probably in the neurons of the bladder rather than in the urothelium or detrusor muscle. Furthermore, reactive oxygen species do not interact with this relaxing factor, the exact nature and the physiological importance of which, however, remains to be established.

Acetylcholine↗
PubMed2006

Application of spectral beta-correction method and partial least squares for simultaneous determination of V(IV) and V(V) in surfactant media.

Simultaneous determination of V(IV) and V(V) was performed by application of partial least squares when the calibration matrix was obtained using beta-correction spectra. Two reaction between V(IV) and V(V) and Pyrogallol Red as a ligand in presence of cethyltrimethylammoniumbromide (CTAB) media has been investigated and applied to the simultaneous spectrophotometric determination of these species. The parameters controlling behavior of the system were investigated and optimum conditions selected. Determinations were made over the concentration range 0.6-4.50 microg ml-1 of V(IV) and 0.3-5.50 microg ml-1 of V(V). Applying this method to simultaneous determination of these metal ions in several real samples with total relative standard error less than 5% validated the proposed method.

Least-Squares Analysis↗
PubMed2003

Superoxide dismutases from the oyster parasite Perkinsus marinus: purification, biochemical characterization, and development of a plate microassay for activity.

We have isolated and biochemically characterized superoxide dismutase (SOD) activity in cell extracts of clonally cultured Perkinsus marinus, a facultative intracellular parasite of the Eastern oyster, Crassostrea virginica. In order to assess the SOD activity throughout the purification, we developed and optimized a 96-well-plate microassay based on the inhibition of pyrogallol oxidation. The assay was also adapted to identify SOD activity type (Cu/Zn-, Mn-, or FeSOD), even in mixtures of more than one type of SOD. All SOD activity detected in the cell extracts was of the FeSOD type. Most of the SOD activity in P. marinus trophozoites resides in a major component of subunit molecular weight 24 kDa. The protein was purified by affinity chromatography on an anti-SOD antibody-Sepharose column. Amino-terminal peptide sequence of the affinity-purified protein corresponds to the predicted product of the PmSOD1 gene and indicates that amino-terminal processing has taken place. The results are discussed in the context of processing of mitochondrially targeted SODs.

Amino Acid Sequence↗
PubMed2000

The development of the emetic reflex in the house musk shrew, Suncus murinus.

The emetic (retching and vomiting) reflex is an important component of the body's defence system against accidentally ingested toxins and emesis is also a common symptom of disease and a side-effect of a number of pharmacological therapies. The development of the reflex has been the subject of few systematic studies. The aim of this study was to characterise the development of the emetic reflex in Suncus murinus (the house musk shrew) using emetic stimuli acting via three different afferent pathways: motion via the vestibular system, pyrogallol via abdominal vagal afferents and resiniferatoxin (a capsaicin analog) via the brainstem. The emetic reflex was not present to any stimulus prior to postnatal day 10 but the onset of the response to motion lagged behind that to the other stimuli in not being present until postnatal day 15. Body weight was not a determinant of the presence of the reflex. It is proposed that the delayed presence of the emetic reflex in Suncus makes it an ideal species in which to investigate factors regulating its development.

Age Factors↗
PubMed1998

Purification and characterization of ascorbate peroxidase in Chlorella vulgaris.

Chlorella vulgaris contained only one isoform of ascorbate peroxidase (AsAP) as the hydrogen peroxide (H2O2)-scavenging system except for catalase at a specific activity of 3.3 +/- 0.2 units/mg protein. The activity of glutathione peroxidase was not detected in the extracts from cells grown in the absence and presence of sodium selenite. We detected the activity of monodehydroascorbate reductase involved in the regeneration of ascorbate, but we failed to detect the dehydroascorbate reductase activity. AsAP has been purified to electrophoretic homogeneity from Chlorella cells. The enzyme was a monomer with a molecular mass of 32 kDa using gel filtration and SDS-polyacrylamide gel electrophoresis. The enzyme showed higher specificity with ascorbate than with pyrogallol. The K(m) values of the enzyme for ascorbate and H2O2 were 111 +/- 8.9 and 20 +/- 2.5 microM, respectively. When the enzyme was diluted with the ascorbate-deleted medium, the half inactivation time was approximately 15 min. The absorption spectra of the purified enzyme and the inhibition by cyanide and azide showed that it is a hemoprotein. The enzyme was markedly inhibited by 0.2 mM p-chloromercuribenzoate. The enzyme cross-reacted by immunoblotting with the monoclonal antibody raised against Euglena cytosolic AsAP. The amino acid sequences in the N-terminal region of Chlorella AsAP showed no significant similarity to any other AsAPs from higher plants and algae.

Amino Acid Sequence↗
PubMed1998

Determination of gallic acid and its metabolites in human plasma and urine by high-performance liquid chromatography.

Gallic acid occurs naturally in plants and has been found to be pharmacologically active as antioxidant, antimutagenic and anticarcinogenic agent. In this work, the metabolism of gallic acid in the human body was investigated. Two methods were developed for the identification and determination of gallic acid and its phenolic metabolites in human plasma and urine by reversed-phase high-performance liquid chromatography using UV detection and involving isocratic elution. One of these methods enables the simultaneous separation and determination of gallic acid (GA), 4-O-methylgallic acid (4OMGA), pyrogallol (PY), 2-O-methylpyrogallol (2OMPY) and resorcinol (RE) in biological fluids. This method is of interest because it allows the separation of a large number of phenolic compounds by isocratic elution using a solution of 4.4 x 10(-3) M phosphoric acid in water as mobile phase. The analysis time for this method, however, is not optimal (57 min). After oral administration of 50 mg GA, 4OMGA rapidly appeared in the plasma and urine besides unchanged GA. Other phenolic compounds, PY, 2OMPY and RE, were not detected. The second method was developed to determine GA and 4OMGA with a short analysis time (25 min).

Chromatography, High Pressure Liquid↗
PubMed2000

The mechanism of nitric oxide and/or superoxide cytotoxicity in endothelial cells.

We examined the mechanism of nitric oxide (NO) and/or superoxide (O2-)-induced cytotoxicity and the importance of thiols in endothelial cells by treating the cells with superoxide dismutase (SOD), catalase (CAT) and hemoglobin (Hb). Pyrogallol, a O2 generator and precursor of hydrogen peroxide (H2O2), had potent cytotoxic effects on the endothelial cells, but this effect was completely abolished by SOD/CAT. Hb, a NO scavenger, protected the endothelial cells from sodium nitroprusside-induced cytotoxicity. The cytotoxic effect of 3-morpholinosydnonimine (SIN-1), which is thought to form peroxynitrite (ONOO-) as a simultaneous O2- and NO generator, was completely blocked by SOD/CAT or Hb. On the other hand, pretreatment of endothelial cells with diethylmaleate, a glutathione depleter, aggravated the cytotoxicity induced by SIN-1, which was prevented by addition of exogenous glutathione and/or SOD/CAT. These data suggest that the cytotoxicity induced by NO, O2- and ONOO- can be blocked by glutathione, and that this is an important cellular protective mechanism against these reactive oxygen species.

Animals↗
PubMed1995

Subchronic application of humic acids and associated compounds provokes histological changes of goitre in the rat.

1. Different preparations of humic acids, a pyrogallol oxydation product (PYROP) and resorcinol were administered to male and female rats with the drinking water for 8 to 14 weeks. 2. By histometrical evaluation of the thyroid glands, an increase of the mean epithelial cell height of follicular epithelial cells was established in the 0.1% humic acid groups, 0.1% PYROP group and the 0.004% resorcinol groups. Females seemed to be more sensitive than males. Additionally, in some experiments, a decrease of the mean follicular diameter was found, which was the result of newly formed little follicles. The changes must be considered as a precursor of goitre. 3. In one of the experiments, in which circulating thyroid hormones were investigated, the serum thyroxine level in the male 0.1% humic acid group and the free thyroxine index in the female 0.1% humic group were diminished as compared to the control groups.

Animals↗
PubMed2003

Method for analysis of tannic acid and its metabolites in biological samples: application to tannic acid metabolism in the rat.

A new analytical method for measuring tannic acid (TA) using tannase was developed and applied to the investigation of TA metabolism in the rat following oral administration at a dose of 1.0 g/kg. The proposed method for TA determination was based on the enzymatic hydrolysis of TA to gallic acid (GA) and subsequent determination by HPLC. TA metabolites were determined by HPLC. 4-O-Methylgallic acid (4-OMGA), pyrogallol (PY), and resorcinol (RE) were detected in serum. TA was excreted into urine as GA (0.01%), 4-OMGA (0.10%), PY (0.24%), and RE (2.06%) and into feces as TA (62.74%), GA (0.19%), PY (0.02%), and RE (0.76%) within 54 h after oral administration. It was suggested that >60% of TA remained unchanged but that some was hydrolyzed to GA by tannase in the intestine and further metabolized to 4-OMGA, PY, and RE.

Animals↗
PubMed2002

Screening of ubiquitous plant constituents for COX-2 inhibition with a scintillation proximity based assay.

A rapid semi-homogeneous cyclooxygenase-2 (COX-2) enzymatic assay using scintillation proximity assay (SPA) technology was developed, and 49 ubiquitous plant secondary metabolites were screened for inhibition of COX-2-catalyzed prostaglandin E(2) (PGE(2)) biosynthesis. Assay conditions were optimized with respect to reaction time, amount of antibody, radiolabeled PGE(2), and SPA beads, and the kinetic parameter, K(m), was estimated. The assay was validated with two natural triterpenoids, ursolic and oleanolic acid, known to inhibit COX-2, as well as with four synthetic COX inhibitors, NS-398, rofecoxib, indomethacin, and aspirin. Plant metabolites of different biosynthetic origin representing several substance classes, including alkaloids, anthraquinones, flavonoids, phenylpropanes, steroids, and terpenes, were screened for inhibition of COX-2-catalyzed PGE(2) production. Of these 49 plant metabolites, eugenol, pyrogallol, and cinnamaldehyde (with IC(50) values of 129, 144, and 245 microM, respectively) were found to inhibit COX-2. This study showed that a COX-2-catalyzed PGE(2) assay using SPA is suitable for screening natural compounds with respect to COX-2 inhibition.

Acrolein↗
PubMed1999

Comparison of the redox forms of nitrogen monoxide with the nitrergic transmitter in the rat anococcygeus muscle.

1. A sustained tone was produced in rat isolated anococcygeus muscles with guanethidine and clonidine and relaxant responses were elicited by electrical stimulation of its nitrergic nerves and by the three redox forms of nitrogen monoxide. 2. The nitroxyl anion (NO ) was donated by dissociation of Angeli's salt; the free radical (NO*) was from an aqueous solution of nitric oxide gas; the nitrosonium cation (NO+) was donated by dissociation of nitrosonium tetrafluoroborate. 3. The concentrations producing approximately 50% relaxations of the anococcygeus muscle were 0.3 microM for Angeli's salt (nitroxyl), 0.5 microM for NO* and 100 microM for nitrosonium tetrafluoroborate. Nitrergic nerve stimulation at 1 Hz for 10 s produced equivalent relaxant responses. 4. The superoxide generator pyrogallol (100 microM) had no effect on responses to nitrergic nerve stimulation or Angeli's salt but significantly reduced responses to NO* and nitrosonium tetrafluoroborate. 5. The NO* scavenger carboxy-PTIO (100 microM) had no effect on responses to nitrergic nerve stimulation or Angeli's salt but significantly reduced responses to NO* and nitrosonium tetrafluoroborate. 6. Hydroxocobalamin (30 microM) had no significant effect on responses to the nitrergic transmitter, enhanced the response to Angeli's salt, and significantly reduced responses to NO* and nitrosonium tetrafluoroborate. 7. The findings suggest that the nitroxyl anion donated by Angeli's salt is a better candidate than NO* to serve as the nitrergic transmitter in the rat anococcygeus muscle, although it still does not behave exactly like the transmitter.

Animals↗
PubMed2001

p53 regulates ceramide formation by neutral sphingomyelinase through reactive oxygen species in human glioma cells.

The present study was designed to elucidate the relationship between p53 and ceramide, both of which are involved in apoptotic signaling. Treatment of human glioma cells with etoposide caused apoptosis only in cells expressing functional p53. p53 activation was followed by the formation of reactive oxygen species (ROS), superoxide anion (O2-*) measured by hydroethidium oxidation into ethidium and hydrogen peroxide (H2O2) measured by oxidation of 2',7'-dichlorofluorescin (DCFH) into 2',7'-dichlorofluorescein (DCF), which was accompanied with ceramide generation through the activation of neutral, but not acid, sphingomyelinase. Superoxide dismutase (SOD), a selective antioxidant for O2-*, had no effects on p53 expression but inhibited ceramide generation and apoptotic cell death caused by etoposide. However, catalase, a specific antioxidant for H2O2, only weakly inhibited and sodium formate, a hydroxyl radical (* OH) scavenger, unaffected etoposide-induced apoptosis. Like etoposide-induced cell death, treatment of glioma cells with the O2-*-releasing agent, pyrogallol, induced typical apoptosis and ceramide generation even in the presence of catalase. In contrast, human glioma cells lacking functional p53, either due to mutation or the expression of E6 protein of human papillomavirus, were highly resistant to etoposide and exhibited no significant change in the ceramide level. Moreover, expression of functional p53 protein in glioma cells expressing mutant p53 using a temperature-sensitive human p53(Val138) induced ceramide accumulation by the activation of neutral sphingomyelinase which was dependent on the generation of O2-*. Taken together, these results suggest that p53 may modulate ceramide generation by activation of neutral sphingomyelinase through the formation of O2-*, but not its downstream compounds H2O2 or * OH.

Apoptosis↗
PubMed2006

Peroxynitrite-modified DNA: a better antigen for systemic lupus erythematosus anti-DNA autoantibodies.

Native human DNA was modified by ONOO- (peroxynitrite), generated by the synergistic action of sodium nitroprusside [Na2Fe(CN)5NO], an NO donor, and pyrogallol, a superoxide donor. The modifications were analysed by UV absorption characteristics, fluorescence emission transitions, nuclease S1 digestibility and melting-temperature studies. Modified DNA was found to be highly immunogenic, inducing high-titre immunogen-specific antibodies in experimental animals. A maximum of 85% inhibition of the antibody binding was observed in competition ELISA with immunogen as inhibitor. SLE (systemic lupus erythematosus) anti-DNA autoantibodies recognized modified DNA as a better antigen than the native analogue. Modification of native DNA by ONOO-, forming neo-epitopes on the molecule, may be one of the factors for the induction of the autoimmune response seen in SLE.

Animals↗
PubMed2001

Amine oxidase-like activity of polyphenols. Mechanism and properties.

Polyphenols in several oxidation systems gained amine oxidase-like activity, probably due to the formation of the corresponding quinones. In the presence of Cu(II), o- and p-phenolic compounds exhibited amine oxidase-like activity, whereas only the o-phenolic compounds showed the activity in the presence of 1,1-diphenyl-2-picrylhydrazyl radical. The activity was determined by measuring the conversion of benzylamine to benzaldehyde by HPLC. Moreover, gallic acid, chlorogenic acid, and caffeic acid, which are plant polyphenols, converted the lysine residue of bovine serum albumin to alpha-amino-adipic semialdehyde residue, indicating lysyl oxidase-like activity. We also characterized the activity of pyrocatechol, hydroquinone, and pyrogallol in the presence of Cu(II). The oxidative deamination was accelerated at a higher pH, and required O2 and transition metal ions. Furthermore, EDTA markedly inhibited the reaction but not beta-aminopropionitrile, which is a specific inhibitor of lysyl oxidase. Catalase significantly inhibited the oxidation, implying the participation of hydroxyl radical in the reaction, but superoxide dismutase stimulated the oxidation, probably due to its radical formation activity. We discussed the mechanism of the oxidative deamination by polyphenols and the possible significance of the activity for biological systems.

Amine Oxidase (Copper-Containing)↗
PubMed1975

Breaking of seed dormancy by catalase inhibition.

Germination of some dormant seeds is promoted by solutions of thiourea, sodium nitrite, and hydroxylamine salts. The promotions are accompanied by irreversible inhibition of catalase (EC 1.11.1.6) in extracts from the seeds. The seeds are also promoted in germination by catechol and pyrogallol solutions. These effects are recorded for lettuce (Lactuca sativa L. cv. Grand Rapids) and pigweed (Amaranthus albus L.) seeds. The results indicae that metabolically derived hydrogen peroxide, spared from decomposition by catalase inhibition, oxidizes reduced NADPH required as the oxidant in the pentose pathway of glucose use. The metabolic system for such use of H2O2 involves the enzymes, peroxidase (EC 1.11.1.7) and pyridine nucleotide quinone oxidoreductase (EC 1.6.99.2), which are present in the dormant seed prior to imbibition of water.

Catalase↗
PubMed1986

Mechanism of action of some inhibitors of endothelium-derived relaxing factor.

The mechanism of the inhibitory action of phenidone, 3-amino-1-[m-(trifluoromethyl)phenyl]-2-pyrazoline (BW 755C), dithiothreitol, hydroquinone, and pyrogallol on the vascular relaxation induced by endothelium-derived relaxing factor (EDRF) was investigated. EDRF was released from porcine aortic endothelial cells in culture and bioassayed on a cascade of superfused rabbit aortic strips. These compounds inhibited EDRF-induced relaxation of vascular strips, without affecting the relaxation induced by glyceryl trinitrate, and their inhibitory potency was markedly attenuated (by more than 1 order of magnitude) by the addition of superoxide dismutase (5-15 units/ml) or oxidized cytochrome c (20-40 microM) but not by catalase (30 units/ml) or heat-inactivated superoxide dismutase. These data indicate that the above five inhibitors inactivate EDRF through the formation of superoxide ions, which have recently been shown to destroy EDRF. The inhibition of EDRF by these compounds is therefore attributable to their redox properties rather than to any specific biological action.

4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyraz↗
PubMed1999

Infusion of plasma expanders may lead to unexpected results in urinary protein assays.

Overt proteinuria was detected in the urine of a potential kidney donor, ultimately leading to the refusal of the kidneys for transplantation purposes. Histological examination of the kidneys did not reveal any abnormalities. Searching for substances that could have interfered with the urinary total protein assay, the role of infused, modified gelatin plasma expanders was investigated. We therefore measured the concentration of protein before and after the addition of various artificial plasma expanders to urine. Only when Biuret reagent or Pyrogallol Red dye were used did we find elevated concentrations of protein. Other methods, including the turbidimetric assays, did not detect additional amounts of protein in the spiked urine. We conclude that the infusion of modified gelatin solutions may cause apparent proteinuria. This effect is not observed with starch-based plasma expanders. Clinical chemists and clinicians should be aware of this phenomenon and possibly repeat the analysis with a different technique.

Albumins↗