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PubMed1974

The Use of an Enzyme Electrode in the Analysis of Indole-3-acetic Acid Oxidase Activity in Avena.

A flexible analytical system which allows for the continuous potentiometric monitoring of the disappearance of an electrochemical species, ferrocyanide, by the peroxidase enzyme is described. The ability of peroxidase to mediate the oxidation of indole-3-acetic acid is followed by observing the competition of indole-3-acetic acid with ferrocyanide for the peroxidase enzyme. This is accomplished by examining potentiometrically the decrease in the rate of ferrocyanide oxidation with increasing indole-3-acetic acid concentration. Homogenates of Avena sativa coleoptiles are investigates for both peroxidase and indole-3-acetic acid oxidase activity. Observations are made with respect to H(2)O(2) and ferrocyanide in the presence and absence of indole-3-acetic acid and naphthalene acetic acid and several interpretations of the reaction kinetics are postulated. Solutions previously assayed for indole-3-acetic acid oxidase activity, when dialyzed and reassayed for peroxidase activity, demonstrated an unimpaired ability to oxidize ferrocyanide peroxidatively, suggesting interpretations of the bisubstrate situation which differ slightly from interpretations given in the literature.

Journal Article↗
PubMed1980

Damage to Candida albicans hyphae and pseudohyphae by the myeloperoxidase system and oxidative products of neutrophil metabolism in vitro.

In previous studies, we noted that Candida hyphae and pseudohyphae could be damaged and probably killed by neutrophils, primarily by oxygen-dependent nonphagocytic mechanisms. In extending these studies, amount of damage to hyphae again was measured by inhibition of [(14)C]cytosine uptake. Neutrophils from only one of four patients with chronic granulomatous disease damaged hyphae at all, and neutrophils from this single patient damaged hyphae far less efficiently than simultaneously tested neutrophils from normal control subjects. Neutrophils from neither of two subjects with hereditary myeloperoxidase deficiency damaged the hyphae. This confirmed the importance of oxidative mechanisms in general and myeloperoxidase-mediated systems in particular in damaging Candida hyphae. Several potentially fungicidal oxidative intermediates are produced by metabolic pathways of normal neutrophils, but their relative toxicity for Candida hyphae was previously unknown. To help determine this, cell-free in vitro systems were used to generate these potentially microbicidal products. Myeloperoxidase with hydrogen peroxide, iodide, and chloride resulted in 91.2% damage to hyphal inocula in 11 experiments. There was less damage when either chloride or iodide was omitted, and no damage when myeloperoxidase was omitted or inactivated by heating. Azide, cyanide, and catalase (but not heated catalase) inhibited the damage. Systems for generation of hydrogen peroxide could replace reagent hydrogen peroxide in the myeloperoxidase system. These included glucose oxidase, in the presence of glucose, and xanthine oxidase, in the presence of either hypoxanthine or acetaldehyde. In the presence of myeloperoxidase and a halide, the toxicity of the xanthine oxidase system was not inhibited by superoxide dismutase and, under some conditions, was marginally increased by this enzyme. This suggested that superoxide radical did not damage hyphae directly but served primarily as an intermediate in the production of hydrogen peroxide. The possible damage to hyphae by singlet oxygen was examined using photoactivation of rose bengal. This dye damaged hyphae in the presence of light and oxygen. The effect was almost completely inhibited by putative quenchers of singlet oxygen: histidine, tryptophan, and 1,4-diazobicyclo[2.2.2]octane. These agents also inhibited damage to hyphae by myeloperoxidase, halide, and either hydrogen peroxide or a peroxide source (xanthine oxidase plus acetaldehyde). Myeloperoxidase-mediated damage to hyphae was also inhibited by dimethyl sulfoxide, an antioxidant and scavenger of the hydroxyl radical. These data support the involvement of oxidative mechanisms and the myeloperoxidase-H(2)O(2)-halide system, in particular in damaging hyphae in vitro and perhaps in vivo as well.

Candida albicans↗
PubMed1996

The protective role of glutathione peroxidase in apoptosis induced by reactive oxygen species.

Selenium-dependent glutathione peroxidase (GPx) plays a protective role in oxidative stress-induced apoptosis. In this study, we demonstrated that MDBK cells, a bovine renal epithelial cell line, exhibited internucleosomal DNA fragmentation characteristic of apoptotic cell death under selenium-deficient conditions with lower doses of hydrogen peroxide (H2O2) than under selenium-supplemented ones. This was due to a decreased amount of GPx in the cells under selenium-deficient conditions, because other antioxidative enzyme activities were not affected by the selenium supplementation. Cumene hydroperoxide also induced DNA fragmentation in selenium-deficient cells but no ladder formation was observed. Flow cytometric analysis showed that selenium-deficient cells were less capable of scavenging intracellular peroxides after exposure to exogenous H2O2 than selenium-supplemented ones. In contrast, there was no difference in viability between selenium-supplemented and non-supplemented cells in cell survival after exposure to menadione, which activates the electron transport system and increases intracellular superoxide radicals. Clofibrate, a peroxisomal proliferator and an inducer of catalase (CAT), partially protected both Se-deficient and Se-supplemented cells from exogenous H202. We concluded that selenium-deficient cells were more easily brought to apoptotic cell death by peroxides, but not by superoxide radicals, than selenium-supplemented ones and that CAT could compensate for the depletion of GPx to a certain degree by scavenging H2O2.

Animals↗
PubMed2003

Simultaneous determination of L- and D-methotrexate using a sequential injection analysis/amperometric biosensors system.

A sequential injection analysis (SIA) is proposed for the simultaneous determination of L- and D-methotrexate (Mtx) using amperometric biosensors as detectors. A SIA system is proposed due to the highest precision and accuracy and lower consumption of sample and buffer. The amperometric biosensors used as detectors in SIA system were based on L-amino acid oxidase (L-AAOD) or/and L-glutamate oxidase (L-Glox) and horseradish peroxidase (HRP) for the assay of L-Mtx and D-amino acid oxidase (D-AAOD) and HRP for the assay of D-Mtx were selected. The linear concentration ranges are of pmol/l to nmol/l magnitude order, with very low limits of detection. The SIA/biosensors system can be used reliably on-line in synthesis process control, for the simultaneous assay of L- and D-Mtx with a frequency of 34 samples per hour.

Amino Acid Oxidoreductases↗
PubMed1991

Light and electron microscopical visualization of anterogradely labelled corticospinal growth cones using a new combination of HRP staining techniques.

Up until now, the ultrastructural visualization of growth cones of developing long fibre tracts could only be achieved by horseradish peroxidase (HRP) application 'en route', resulting in axonal damage, which in turn may affect growth cone morphology. Besides, this technique results in labelling of passing fibres, thus hampering the identification of axon origin as well as the interpretation of growth cone configuration. In the present investigation a new combination of HRP staining and intensification techniques is presented which makes it possible to visualize anterogradely labelled corticospinal growth cones over long distances in developing rat spinal cord at the light as well as the electron microscopical level. HRP was applied to the originating cells of the corticospinal tract, located in the sensorimotor cortex, and after 24 h was visualized using a procedure which essentially consists of 3 subsequent steps: first a tetramethylbenzidine (TMB)/ammoniumheptamolybdate (AHM) reaction; second diaminobenzidine (DAB)/nickel (Ni) stabilization and finally glucose oxidase intensification. As was verified at the EM level, the staining procedure here described reveals a complete intense black staining of HRP-labelled growth cones of outgrowing corticospinal axons. Therefore, the method described here guarantees a correct analysis of growth cone morphology at the light microscopical and the ultrastructural level. The present procedure is especially valuable in studying the development of long central nervous fibre systems.

Animals↗
PubMed1985

The role of oxygen-derived free radicals in ischemia-induced increases in canine skeletal muscle vascular permeability.

Previous studies indicate that vascular permeability is increased in skeletal muscle subjected to 4 hours of inflow occlusion. However, the mechanism(s) underlying the increase in permeability are unknown. The aim of this study was to assess the role of oxygen-derived free radicals and histamine as putative mediators of the increased permeability in skeletal muscle subjected to 4 hours of inflow occlusion. The osmotic reflection coefficient for total plasma proteins and isogravimetric capillary pressure were estimated in canine gracilis muscle for the following conditions: control, ischemia, and ischemia plus pretreatment with allopurinol (a xanthine oxidase inhibitor), catalase (a peroxidase that reduces hydrogen peroxide to water and molecular oxygen), superoxide dismutase (a superoxide anion scavenger), dimethyl sulfoxide (a hydroxyl radical scavenger), diphenhydramine (a histamine H1-receptor blocker), or cimetidine (a histamine H2-receptor blocker). Ischemia, followed by reperfusion, significantly reduced the reflection coefficient from 0.94 +/- 0.02 to 0.64 +/- 0.02 and isogravimetric capillary pressure from 13.8 +/- 1.0 mm Hg to 6.9 +/- 0.4 mmHg, indicating a dramatic increase in microvascular permeability. Prior treatment with diphenhydramine or cimetidine did not significantly alter the permeability increase induced by ischemia. However, pretreatment with allopurinol, catalase, superoxide dismutase, or dimethylsulfoxide did significantly attenuate the increase in vascular permeability. The results of this study indicate that oxygen radicals are primarily responsible for the increased vascular permeability produced by ischemia-reperfusion, that the hydroxyl radical may represent the primary damaging radical, and that xanthine oxidase may represent the primary source of oxygen-derived free radicals in ischemic skeletal muscle.

Allopurinol↗
PubMed2003

Optimization of a culture medium for ligninolytic enzyme production and synthetic dye decolorization using response surface methodology.

A Box-Wilson central composite design was applied to optimize copper, veratryl alcohol and l-asparagine concentrations for Trametes trogii (BAFC 212) ligninolytic enzyme production in submerged fermentation. Decolorization of different dyes (xylidine, malachite green, and anthraquinone blue) by the ligninolytic fluids from the cultures was compared. The addition of copper stimulated laccase and glyoxal oxidase production, but this response was influenced by the medium N-concentration, with improvement higher at low N-levels. The medium that supported the highest ligninolytic production (22.75 U/ml laccase, 0.34 U/ml manganese peroxidase, and 0.20 U/ml glyoxal oxidase) also showed the greatest ability to decolorize the dyes. Only glyoxal oxidase activity limited biodecoloration efficiency, suggesting the involvement of peroxidases in the process. The addition of 1-hydroxybenzotriazole (a known laccase mediator) to the ligninolytic fluids increased both their range and rate of decolorization. The cell-free supernatant did not decolorize xylidine, poly R-478, azure B, and malachite green as efficiently as the whole broth, but results were similar in the case of indigo carmine and remazol brilliant blue R. This indicates that the mycelial biomass may supply other intracellular or mycelial-bound enzymes, or factors necessary for the catalytic cycle of the enzymes. It also implies that this fungus implements different strategies to degrade dyes with diverse chemical structures.

Alcohol Oxidoreductases↗
PubMed2006

Human Thyroid Oxidases genes promoter activity in thyrocytes does not appear to be functionally dependent on Thyroid Transcription Factor-1 or Pax8.

Thyroid Oxidases (ThOX/DUOX) genes encode proteins that are thought to play a crucial role in the biosynthesis of thyroid hormone by providing the oxidizing agent required to allow the organification of iodine. The expression of these genes is not restricted to the thyroid, but the corresponding mRNAs are found in the thyrocyte more abundantly than in several other cell types. It raises the question whether the same transcription factors, namely Thyroid Transcription Factor-1 (TTF-1) and Pax8, that control the expression of other genes involved in the differentiated thyroid function, also regulate ThOX/DUOX gene transcription in the thyrocyte. We set up a functional co-transfection assay in which fusion proteins composed of the DNA-binding domain of either TTF-1 or Pax8 fused to the repressive domain of the drosophila engrailed protein were used to competitively counteract the activity of endogenous TTF-1 or Pax8 factor in the differentiated thyroid cell line PCCl3. Contrary to the Thyroglobulin or Thyroid Peroxidase promoter, the known regulatory elements of the human ThOX/DUOX genes displayed no reduction in transcriptional activity when either TTF-1 or Pax8 competitor was produced in the cell, indicating that the presently characterized control elements of human ThOX/DUOX genes are not responsive to these thyroid-specific transcription factors.

Animals↗
PubMed2000

Enzymatic degradation of cichoric acid in Echinacea purpurea preparations.

Cichoric acid (2R,3R-O-dicaffeoyltartaric acid) (1) is highly susceptible to enzymatic degradation during the preparation of Echinacea purpurea products. Degradation of 1 and other caffeic acid derivatives can be inhibited by antioxidants added to the extraction solvent or in buffered protein extracts saturated with nitrogen. Inhibitor studies conducted with protein extracts prepared from dried overground parts of E. purpurea revealed that polyphenol oxidases (PPO) but not peroxidases are responsible for the oxidative degradation of exogenous and endogenous caffeic acid derivatives. With a view to stabilizing aqueous extracts with respect to their content of 1, the effects of ascorbic acid and ethanol were tested. Compound 1 was not stable under conditions where oxidative processes could almost be excluded. It was found that an esterase hydrolyzing the ester bonds between tartaric acid and caffeic acid is still active under PPO inhibitory conditions. Finally, addition of 40% ethanol and 50 mM ascorbic acid to aqueous extracts of "Echinaceae purpureae herba" resulted in a constant amount of cichoric acid over four weeks.

Caffeic Acids↗
PubMed1980

New type of antibody-enzyme conjugate which specifically kills Candida albicans.

A new type of antibody-enzyme conjugate was made, and its possible application to Candida infection was studied. Both lactoperoxidase and xanthine oxidase were conjugated to specific antibody against Candida albicans. In vitro microbiocidal activity of the new antibody-enzyme conjugate, when incubated together with xanthine and minute amount of halides, showed a remarkable level of candidacidal ability. When the new antibody-enzyme conjugate was given to Candida-infected mice, followed by injecting xanthine and a minute amount of halides, about 50% of these heavily infected mice survived, whereas all control nontreated mice died. These data suggest that the further eleboration of this new antibody-enzyme conjugate might lead us to improve our therapeutic methods of clinical medicine.

Animals↗
PubMed1987

Structure and properties of enzyme graft copolymers: effects of using dissolved agarose on horseradish peroxidase immobilization.

The immobilization of horseradish peroxidase (HRP) onto dissolved agarose by a photochemically initiated graft copolymerization reaction, carried out at room temperature, was studied. Enzyme immobilization parameters such as the catalyst (FeCl3) and the enzyme concentration were considered. Using hexhydro-1,3,5-triacryloyl-s-triazine (HTsT) as vinyl monomer, the agarose/HTsT ratio was the main reaction parameter controlling the copolymer characteristics. By increasing the polymer content of the sample better stability properties were obtained. For the samples with agarose/HTsT ratios of 20/40 and 40/20 (S 20-40, S 40-20) the residual activities after 240 min at 60 degrees C were respectively 47 and 18%. The residual activity in continuous working was 33% for S 40-20 (after 20 h) and 64% for S 20-40 (after 70 h). For both the synthesized copolymers no limitation to substrate diffusion was found but the flexibility of immobilized enzyme decreased with the increase of polymer content as indicated by the Km values that were 0.90 X 10(-4) mol/liter for the sample S 40-20, and 1.50 X 10(-4) mol/liter for the sample S 20-40. Other enzymes (glucose oxidase, alpha-chymotrypsin, and lipoxidase), besides HRP, were immobilized with good yields, showing the wide applicability of the proposed methodology for the preparation of a solid biocatalyst which can be conveniently stored in water suspension or as lyophilized material.

Chymotrypsin↗
PubMed1986

Enols of aldehydes in the peroxidase/oxidase-promoted generation of excited triplet species.

General (acid and base) or specific (fluoride ion) catalysis generates the enol of isobutanal and propanal from the corresponding trimethylsilyl enol ethers. The enols are directly rapidly oxidized by peroxidase (acting as an oxidase) to triplet acetone or triplet acetaldehyde, respectively, and formic acid. Due to the faster rate of reaction and the absence of quenching by excess aldehyde, the excited carbonyl emits more strongly than when the aldehyde itself is the substrate. With both enols the emission is pure phosphorescence. Both triplet acetone and triplet acetaldehyde are generated within the enzyme, as shown by the different quenching by D- and L-tryptophan, and are somewhat protected from oxygen quenching, as attested by the very fact that phosphorescence is observed. The use of enol precursors as substrates opens wide possibilities for photochemical investigations in the absence of light over a much broader range of experimental conditions.

Acetone↗
PubMed2002

Spleen and liver pigmented macrophages of Rana esculenta L. A new melanogenic system?

The present study reports the results of a morpho-functional analysis of spleen pigmented cells from Rana esculenta L. and comparison with liver melanin-synthesizing cells, belonging to the macrophage cell lineage. Cytological and cytochemical analyses show that parenchymal pigmented cells of the spleen, like those of the liver, are positive to peroxidase and lipase reactions and have phagocytic properties. The observation of premelanosomes in various stages of differentiation, together with the demonstration of dopa oxidase activity in the melanosome proteins, indicate that spleen pigmented macrophages have endogenous melanogenic ability as do liver pigmented macrophages. Attempts to demonstrate tyrosinehydroxylase activity in melanosome protein extracts from frog spleen and liver, using the same protocol as for mammalian tyrosinases, gave negative results. As regards the dopa oxidase activity revealed, some of its properties differ from the typical behaviour observed for tyrosinases from different sources. Peroxidase activity is shown in spleen and liver melanosome proteins with p-phenylenediamine-pyrocatechol (PPD-PC), and not with typical peroxidase substrates. Suitable inhibition tests revealed that dopa oxidase and peroxidase activities might be supported by two different proteins. Liver melanosome extracts display a very strong laccase (dimethoxyphenoloxidase) activity but spleen extracts do not. Differences observed in the enzymatic properties of the spleen and liver melanosomes suggest that pigmented macrophages may undergo tissue-specific differentiation. These preliminary data show that the melanin pathway of pigmented macrophages is different from that of melanocytes and may pave the way to identification of a new melanogenic pathway in vertebrates.

Animals↗
PubMed1986

Kinetics of the oxidation of reduced nicotinamide adenine dinucleotide by horseradish peroxidase compounds I and II.

The transient state kinetics of the oxidation of reduced nicotinamide adenine dinucleotide (NADH) by horseradish peroxidase compound I and II (HRP-I and HRP-II) was investigated as a function of pH at 25.0 degrees C in aqueous solutions of ionic strength 0.11 using both a stopped-flow apparatus and a conventional spectrophotometer. In agreement with studies using many other substrates, the pH dependence of the HRP-I-NADH reaction can be explained in terms of a single ionization of pKa = 4.7 +/- 0.5 at the active site of HRP-I. Contrary to studies with other substrates, the pH dependence of the HRP-II-NADH reaction can be interpreted in terms of a single ionization with pKa of 4.2 +/- 1.4 at the active site of HRP-II. An apparent reversibility of the HRP-II-NADH reaction was observed. Over the pH range of 4-10 the rate constant for the reaction of HRP-I with NADH varied from 2.6 X 10(5) to 5.6 X 10(2) M-1 s-1 and of HRP-II with NADH varied from 4.4 X 10(4) to 4.1 M-1 s-1. These rate constants must be taken into consideration to explain quantitatively the oxidase reaction of horseradish peroxidase with NADH.

Horseradish Peroxidase↗
PubMedDate not supplied

[Biochemical indicators of oxygen poisoning].

Content of extra erythrocyte hemoglobin (EEH), total peroxidase activity (TPA), tseruloplasmin oxidase activity (TOA) and chemiluminescence intensity have been investigated in plasma. The dose-dependent changes of EEN, TPA, TOA are shown in experiments on the animals under hyperbaric oxygen (HBO) pressure (0.2, 0.3, 0.5, 0.7 MPa from 30 min. to 120 min.) and EEH, TPA--under hyperbaric air (1.1 MPa; 4 hours). All these parameters have been studied in patients treated with HBO for the assessment of HBO efficacy in the clinical investigations. These biochemical parameters may be used as informative tests of the functional state of the organism under HBO and hyperbaric air.

Adult↗
PubMed1989

Ultrastructural and functional evidence for the survival of corticogeniculate neurons in kainic acid-lesioned lateral geniculate nucleus.

After a kainic acid lesion in the dorsal lateral geniculate nucleus of rat, retrograde axonal transport of fluorescent dyes is blocked in corticogeniculate but not in retinogeniculate neurons. This inhibition, however, can be reversed by electrical stimulation in the subcortical white matter (Woodward and Coull, Brain Research 454 (1988) 106-115). These observations suggest that retrograde axonal transport in corticogeniculate neurons is impulse-dependent and that neuronal activity in this pathway is reduced as a consequence of the lesions. To test this we examined retrograde transport of horseradish peroxidase (HRP) and cytochrome oxidase activity in the cortex of lesioned animals. Unilateral kainic acid lesions in the geniculate inhibit the retrograde transport of HRP, but this inhibition is reversed by electrical stimulation of white matter. Moreover, histochemical staining for cytochrome oxidase activity is less intense over visual cortex on the lesioned side, implying that cortical activity in intrinsic and efferent pathways is reduced as a consequence of removal of geniculate afferents. Inasmuch as the retrograde transport of HRP is dependent upon impulse activity in neurons and is thought to be mediated by synaptic vesicle recycling, these results suggest that terminals of corticogeniculate fibers survive the kainic acid lesions in the geniculate and are capable of releasing synaptic vesicles. Ultrastructural examination of lesioned geniculates strongly supports this conclusion and reveals the presence of axon terminal profiles which are filled with small round synaptic vesicles and have membrane specializations reminiscent of synaptic contacts. These terminal profiles are presumed to be of retinal and cortical origin.

Animals↗
PubMed2000

Haemocytes of the clam Tapes philippinarum (Adams & Reeve, 1850): morphofunctional characterisation.

Tapes philippinarum is a bivalve mollusc of the Pacific Ocean, successfully imported for human consumption into the northern Adriatic Sea (Europe). For better knowledge of its considerable adaptive ability in comparison with similar autochthonous species, a morpho-functional characterisation of its haemocytes was carried out with the establishment of short-term cell cultures (60 min at 25 degrees C). Various methods of cytochemical staining identified four cell types in the haemolymph: granulocytes (48.05% +/- 1.43), hyalinocytes (32.18% +/- 0.99), haemoblasts (18.97% +/- 0.63) and serous cells (0.8% +/- 0.19). The granulocytes, possessing cytoplasmic granules with differing dye affinity, included basophils, neutrophils and acidophils. Such granules stained vitally with Neutral Red, and correspond to lysosomes. Hydrolytic and oxidative enzymes were mainly detectable after stimulation in the presence of yeast cells. Both granulocytes and hyalinocytes were positive for alkaline phosphatase, non-specific esterase, peroxidase, and cytochrome C oxidase, whereas only granulocytes were positive for beta-glucuronidase, acid esterase, and arylsulphatase. Both cell types were competent phagocytes towards yeast and plasma had an opsonising effect. Moreover, the respiratory burst accompanied phagocytosis with superoxide anion production, recognisable through cytoplasmic deposits of formazan after treatment with nitro blue tetrazolium. Haemoblasts were small undifferentiated cells which, due to their morphology and positivity to the anti-CD34 antibody, show the typical features of stem cells. Serous cells, probably arising from Keber's gland and belonging to another differentiation pathway, contained non-sulphate acid mucopolysaccharides and play an important role in early defence mechanisms, taking part in the formation of clots.

5'-Nucleotidase↗
PubMed1986

Monoamine oxidase in rat ovary during the estrous cycle. A histochemical study by a new coupled peroxidatic oxidation method.

A new coupled peroxidatic oxidation method for histochemical detection of monoamine oxidase (MAO) was applied to rat ovary. With this new method, fixed tissues could be used, and two forms of MAO could be identified by use of selective inhibitors. MAO activity was observed in the corpora lutea, interstitial gland cells, and blood vessels. In the corpora lutea, no activity was detected during the first estrous cycle, but strong activity was observed in the next two cycles. MAO in blood vessels showed characteristic changes of activity during the estrous cycle. The results suggest that MAO activity might possibly be involved in ovulation and progesterone metabolism in the ovary. Like other organs, rat ovary was found to contain two types of MAO; type A MAO was predominant in the corpora lutea. On the other hand, only one type of MAO, type B, was found in the blood vessels.

Animals↗