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Peroxidative xenobiotic oxidation by partially purified peroxidase and lipoxygenase from human fetal tissues at 10 weeks of gestation.

1. Present study reports the ability of partially purified peroxidase and lipoxygenase from human fetal tissues at 10 weeks of gestation to oxidize selected xenobiotics in vitro. 2. Peroxidase was found to oxidize four different chemicals in the presence of H2O2. Sodium azide and potassium cyanide inhibited peroxidase activity towards guaiacol in a concentration-dependent manner. 3. The dioxygenase and co-oxidase activities of lipoxygenase towards linoleic acid and four model xenobiotics, respectively, were observed. Both the catalytic activities of lipoxygenase were significantly inhibited by < 1.0 microM nordihydroguaiaretic acid. 4. These findings suggest that peroxidase and lipoxygenase may be important pathways for peroxidative xenobiotic oxidation in human fetal tissues.

Female↗

The section-Golgi impregnation procedure. 1. Description of the method and its combination with histochemistry after intracellular iontophoresis or retrograde transport of horseradish peroxidase.

A method is described for impregnating neurons by the Golgi procedure in sections of brain tissue that are of a thickness 80-100 microns, so allowing a variety of histochemical procedures to be carried out prior to Golgi-impregnation. The method has been applied to the retina of the adult primate and to brain sections incubated to reveal horseradish peroxidase activity with substrates that give electron-dense reaction products. Sections containing neurons that have been filled with horseradish peroxidase by intracellular iontophoresis following electrophysiological characterization, or containing neurons retrogradely labelled by horseradish peroxidase, can be impregnated by this Golgi procedure. Subsequent gold-toning of the Golgi-impregnated neurons makes it possible to study, in the electron-microscope, both Golgi-impregnated and peroxidase-containing neurons and their afferent and efferent synaptic contacts within the same section. The procedure also makes it possible to repeat Golgi impregnation on sections already Golgi-impregnated and gold-toned, or to repeat the Golgi impregnation if the first impregnation is not satisfactory. The procedure is illustrated by examples from the monkey retina, cat visual cortex and rat neostriatum.

Animals↗

Nucleus tegmenti pedunculopontinus: efferent connections with special reference to the basal ganglia, studied in the rat by anterograde and retrograde transport of horseradish peroxidase.

The efferent connections of the brain stem nucleus tegmenti pedunculopontinus were studied in the rat using the techniques of anterograde and retrograde transport of the enzyme horseradish peroxidase, laying particular emphasis on that part of pedunculopontinus which receives direct descending projections from the basal ganglia and related nuclei. In a preliminary series of experiments horseradish peroxidase was injected into either the entopeduncular nucleus or the subthalamic nucleus and, following anterograde transport of enzyme, terminal labelling was identified in nucleus tegmenti pedunculopontinus, surrounding the brachium conjunctivum in the caudal mesencephalon. In a subsequent series of experiments, horseradish peroxidase was injected into that region of nucleus tegmenti pedunculopontinus which receives entopeduncular and subthalamic efferents and its efferent projections were studied by anterograde transport of the enzyme. The results indicate that nucleus tegmenti pedunculopontinus gives rise to widely distributed efferent projections which terminate rostrally in mesencephalic, diencephalic and telencephalic structures and caudally in the pontine tegmentum. In the mesencephalon, terminal labelling was found in the pars compacta of the ipsilateral substantia nigra and sometimes in the adjoining ventral tegmental area. Labelling was also found in the ipsilateral half of the periaqueductal grey. In the diencephalon terminal labelling occurred bilaterally in the subthalamic nucleus and ipsilaterally in the intralaminar nuclei of the thalamus. Further rostrally, terminal labelling was particularly evident in the ipsilateral pallidal complex, especially in the caudal two-thirds of the entopeduncular nucleus and the ventral half of the caudal third of the globus pallidus. Caudal to pedunculopontine injection sites dense labelling was observed in the reticular formation of the pontine tegmentum. In a final series of experiments, confirmation of apparent pedunculopontine efferent projections was sought using the retrograde transport of horseradish peroxidase. Enzyme was injected into sites possibly receiving pedunculopontine efferents and the peribrachial area of the brain stem was examined for retrograde cell labelling. In this way, pedunculopontine projections were confirmed to the globus pallidus, entopeduncular nucleus, subthalamic nucleus, substantia nigra, parafascicular nucleus and pontine reticular formation. Injections into the globus pallidus or subthalamic nucleus gave rise to retrograde cell labelling bilaterally in pedunculopontinus. In addition, retrograde transport studies alone demonstrated projections from pedunculopontinus to the cerebral cortex and to the spinal cord. It is concluded that the nucleus tegmenti pedunculopontinus has reciprocal relationships with parts of the basal ganglia and some functionally related nuclei (in particular, the pallidal complex, subthalamic nucleus and substantia nigra).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

The effect of previous transection on quantitative estimates of the preganglionic neurones projecting in the cervical sympathetic trunk of the guinea-pig and the cat made by retrograde labelling of damaged axons by horseradish peroxidase.

The numbers of sympathetic preganglionic neurones in the upper thoracic spinal cord retrogradely labelled after application of horseradish peroxidase to the severed axons of the cervical sympathetic trunk have been determined in guinea-pigs and in cats. Neuronal counts were made on both sides of spinal segments C8 to T10 in control animals and in others in which the enzyme was applied 5-8 days after the left cervical sympathetic trunk had been transected. Labelling was not significantly different between sides of unoperated animals, but after previous cervical trunk section labelling was always less, by from 5 to 100% of that on the control side. The results were not significantly different if the application of horseradish peroxidase was made close to the original lesion or 1 cm more proximal along the nerve trunk. Analysis of the dimensions of labelled cells suggested that the deficit in labelling was greatest for small preganglionic neurones. These findings do not support an earlier report that retrograde labelling from regenerating axons is enhanced at this stage after axotomy, and the possible reasons for the discrepancy in results are discussed. It is suggested that labelling of cell bodies may not be restricted if intact axon sprouts are exposed to horseradish peroxidase. Reduced labelling from axons at the time of a second transection might, at least in part, be due to axon atrophy, emphasizing limitations to labelling when horseradish peroxidase is applied to severed axons of fine diameter. The high numbers and reproducibility of our control data enabled estimates to be made of the segmental distribution of the cell bodies or origin of the axons of the cervical sympathetic trunk in both species.

Animals↗

Topographical organization of the projections from the cerebral cortex to the head of the caudate nucleus. A horseradish peroxidase study in the cat.

The topographical organization of the projections from the cerebral cortex to the head of the caudate nucleus was studied in the cat using the horseradish peroxidase method. Various amounts of horseradish peroxidase were injected into several sites of the head portion of the caudate nucleus at about the frontal level where its cross section was widest. Injections of small amounts of horseradish peroxidase retrogradely labeled neurons in rather limited cortical areas bilaterally, showing the localized organization of the projections. Neurons in the lateral portions of the ventral bank of the cruciate sulcus and in the dorsal bank (areas 4 gamma and 4 delta) were labeled after horseradish peroxidase injections into the dorsolateral part of the head of the caudate nucleus. Neurons in the intermediate portions of the ventral bank (areas 6 a delta and 6 infra fundum) were strongly labeled after dorsolateral or ventrointermediate injections, and neurons in the medial portion (area 6a beta), after dorsomedial, dorsointermediate, ventrointermediate or central injections. These findings indicate that areas 4 gamma and 4 delta project to the dorsolateral part of the caudate nucleus, areas 6a delta and 6 infra fundum to the lateral half, and area 6a beta to a more medial portion. Other findings revealed that the gyrus proreus projects to the medial part of the caudate nucleus and the anterior cingulate gyrus to the dorsal region.

Afferent Pathways↗

Afferent and efferent connections of brainstem locomotor regions: study by means of horseradish peroxidase transport technique.

Afferent and efferent connections of the hypothalamic and mesencephalic locomotor regions and also the bulbar locomotor strip were studied in cat using retrograde (horseradish peroxidase) transport technique. To study the sources of afferent projections, the enzyme microinjections were performed exactly into the same brain sites eliciting treadmill locomotion by means of electrical stimulation. When studying efferent projections horseradish peroxidase labeled neurons were revealed within locomotor regions after enzyme microinjections into different brain structures. Experimental data have shown that the hypothalamic and mesencephalic locomotor regions have mutual afferent and efferent projections with numerous brain areas including interconnections. Apart from the entopeduncular nucleus, the great number of different sensory nuclei are noted: among the sources of afferent projections are the nucleus tractus spinalis nervi trigemini, nucleus cuneatus, nucleus tractus solitarius and vestibular nuclei. In addition, after horseradish peroxidase injection into the mesencephalic locomotor region labeled neurons were found in the cochlear nuclei. Direct descending neuronal projections of the hypothalamic and mesencephalic locomotor regions are distributed mainly in the ipsilateral brainstem. Only a few of them reach the lumbar spinal cord. The most marked efferent projections of given regions are those to the brainstem reticular formation. After horseradish peroxidase injection into a functionally identified bulbar locomotor strip, labeled neurons were revealed in different stem regions mainly caudal to the enzyme injection site. The existence of a locomotor strip as an independent structural formation is called into question. When studying the locomotor region connections, the structural heterogeneity of these regions is revealed. Transitory fibers of ascending tracts are presumably within their limits side by side with neurons. The role of these fibers in stepping initiation by electrical stimulation of locomotor regions remains uncertain.

Animals↗

Different rates of horseradish peroxidase transport in the optic nerve of neonatal and adult rats.

In order to evaluate the effects of age upon fast transport kinetics, we studied the velocity of horseradish peroxidase transport along the optic pathway of neonatal and adult rats. Rate of horseradish peroxidase movement was assessed by following the displacement of horseradish peroxidase activity in the optic nerve with time after injection. We estimated a rate about 100 mm/day for bidirectional transport of horseradish peroxidase in the optic nerve of neonatal rats, while a two-fold higher rate was observed for anterograde transport in adult animals. Developmental regulation of fast transport rate may ensure relative constancy of the time required to connect the cell body with axon terminals.

Animals↗

Structure and chromosomal localization of the gene encoding barley seed peroxidase BP 2A.

A clone, lambda Prx6.1, coding for a barley seed peroxidase (BP; EC 1.11.1.7), was isolated from a genomic library using a cDNA coding for the barley seed peroxidase, BP 1, as a probe. The nucleotide sequence coded for a BP showing 73% amino acid (aa) sequence identity with BP 1 and less than 50% similarity with other sequenced plant peroxidases. The aa composition is 92% identical to that determined for BP 2 purified from mature barley grains, and therefore the gene product is named BP 2A. The alignment suggests that the coding region is interrupted by a 76-bp intron having the consensuses GT and AG, at the 5' and 3' ends, respectively. Alignment with BP 1 suggests that BP 2A has a leader peptide of 36 aa and the mature protein is 319 aa. Alanine and leucine account for 50% of the residues of the leader peptide. Of the codons used 90% have a C or G in the third position. The promoter shows a putative abscisic acid-response element, 5'-GTACGTGTC, 115 bp upstream from the start codon. The BP 2A-encoding gene was RFLP-mapped on barley chromosome 3, and we suggest for this peroxidase locus the name Prx6.

Amino Acid Sequence↗

Whole-cell bacterial peroxidase test with isoproterenol as the hydrogen donor.

The beta-adrenergic compound isoproterenol was used as oxidizable reagent in a whole-cell assay for the detection of bacterial peroxidase activities. Isoproterenol has been shown to constitute a useful reagent for detecting peroxidase activities in enzymatic tests, utilizing standard purified enzymes, and in the microbiological application proposed. The procedure developed is simple and rapid to perform. In contrast to currently used whole-cell tests for bacterial peroxidases, the assay described here does not need preliminary permeabilization; moreover, the compound utilized does not have related toxicological problems. Therefore, the isoproterenol assay may represent a low-cost safe additional peroxidase test in clinical bacteriology.

Bacteria↗

Susceptibility of glutathione peroxidase to proteolysis after oxidative alteration by peroxides and hydroxyl radicals.

Glutathione peroxidase is a key enzyme in the antioxidant system of the cells. This enzyme has been shown to be irreversibly inactivated by H2O2, tert-butyl hydroperoxide (tert-BHP) and hydroxyl radicals when incubated without GSH. We observed that in our experimental conditions glutathione peroxidase was not degraded by trypsin or chymotrypsin while degraded by pronase, papaïn, pepsin, and lysosomal proteases. Hydroxyl radicals and superoxide anions but not H2O2 or tert-BHP could also fragment the enzyme on their own. A former incubation with H2O2, tert-BHP, or hydroxyl radicals also increased the proteolytic susceptibility of glutathione peroxidase. Like superoxide dismutase (SOD) and other oxidatively denatured proteins, glutathione peroxidase inactivated by peroxides or free radicals seems to be degraded preferentially by proteases. As hydroxyl radicals can fragment the enzyme by themselves, the increased proteolytic susceptibility afterwards is easily understood while the increased susceptibility induced by H2O2 and tert-BHP seems to be more specific.

Animals↗

A ready-to-use fluorimetric biosensor for superoxide radical using superoxide dismutase and peroxidase immobilized in sol-gel glasses.

In this work, a highly sensitive fluorescent biosensor for quantitative superoxide radical detection, based on the coupled reaction superoxide dismutase-peroxidase enzymes and the use of the probe Amplex red, is described. Superoxide anion radical was produced via oxidation of xanthine by xanthine oxidase. Dismutation of superoxide was catalyzed by superoxide dismutase, generating hydrogen peroxide, which reacted stoichiometrically with the nonfluorescent Amplex red, in the presence of peroxidase, yielding the red-fluorescent oxidation product resorufin. The coupled superoxide dismutase-peroxidase system was immobilized in a single sol-gel matrix. The enzymatic activity of the encapsulated superoxide dismutase-peroxidase system was nearly identical to that of one of the soluble enzymes, indicating that sol-gel encapsulation preserved the hierarchy of the enzyme's activity. Specificity and reusability of the encapsulated system for up to four cycles were also demonstrated. The fluorescent biosensor was able to detect concentrations of superoxide as low as 20 nM in phospholipid model membranes composed of saturated or unsaturated phospholipids. These facts make this biosensor a simple, reliable, and highly sensitive method with a potential use in biological systems, food, and drinks.

Anions↗

Polyclonal antibodies mediated immobilization of a peroxidase from ammonium sulphate fractionated bitter gourd (Momordica charantia) proteins.

Polyclonal antibody bound Sepharose 4B support has been exploited for the immobilization of bitter gourd peroxidase directly from ammonium sulphate precipitated proteins. Immunoaffinity immobilized bitter gourd peroxidase exhibited high yield of immobilization. IgG-Sepharose 4B bound bitter gourd peroxidase showed a higher stability against heat, chaotropic agents (urea and guanidinium chloride), detergents (cetyl trimethyl ammonium bromide and Surf Excel), proteolytic enzyme (trypsin) and water-miscible organic solvents (propanol, THF and dioxane). The activity of immobilized bitter gourd peroxidase was significantly enhanced in the presence of cetyl trimethyl ammonium bromide and after treatment with trypsin as compared to soluble enzyme.

Ammonium Sulfate↗

Palm tree peroxidase-based biosensor with unique characteristics for hydrogen peroxide monitoring.

Three amperometric enzyme electrodes have been constructed by adsorbing anionic royal palm tree peroxidase (RPTP), anionic sweet potato peroxidase (SPP), or cationic horseradish peroxidase (HRP-C) on spectroscopic graphite electrodes. The resulting H(2)O(2)-sensitive biosensors were characterized both in a flow injection system and in batch mode to evaluate their main bioelectrochemical parameters, such as pH dependency, I(max), K(M)(app), detection limit, linear range, operational and storage stability. The obtained results showed a distinctly different behavior for the plant peroxidase electrodes, demonstrating uniquely superior characteristics of the RPTP-based sensors. The broader linear range observed for the RPTP-based biosensor is explained by a high stability of this enzyme in presence of H(2)O(2). The higher storage and operational stability of RPTP-based biosensor as well as its capability to measure hydrogen peroxide under acidic conditions connect with an extremely high thermal and pH-stability of RPTP.

Arecaceae↗

A proposed molecular basis for the selective resveratrol inhibition of the PGHS-1 peroxidase activity.

Docking results have enabled us to propose how resveratrol could act as a selective PGHS-1 peroxidase site inhibitor. The docking model has predicted a slightly less favorable DeltaG(bind) (-17.9 kcal/mol) of the resveratrol to the PGHS-2 peroxidase site in comparison with its corresponding binding to the PGHS-1 (-20.4 kcal/mol). The formation of hydrogen bonds among the hydroxyl groups of the resveratrol phenyl rings, the backbone of Fe-heme and the carbonyl group of Leu294 inside the PGHS-1 peroxidase site, associated with the absence of His214 in the backbone of PGHS-1, are essential features that are required to maintain the aromatic rings of the natural product parallel to the Fe-heme group and transverse to the peroxidase access channel promoting a large steric hindrance at this site and its consequent selective inhibition.

Binding Sites↗

Relationship between malondialdehyde level and glutathione peroxidase activity in diabetic rats.

BACKGROUND: This investigation describes the relationship between glutathione peroxidase activities, an antioxidant enzyme, and the oxidative status in diabetic rat blood. METHODS: Malondialdehyde level and glutathione peroxidase activity were measured by spectrophotometric techniques. RESULTS AND CONCLUSIONS: Malondialdehyde content in the diabetic rats group was increased compared to that in the controls [3.08+/-0.32 (mean+/-S.E.) vs. 1.15+/-0.29 mM/g hemoglobin, P>0.01]. Glutathione peroxidase activity in the diabetic rats group was increased compared to that in the control [10.27+/-1.39 (mean+/-S.E.) vs. 3.14+/-0.38 micromol NADPH/min/g hemoglobin, P>0.01]. Our results show a positive correlation between serum glutathione peroxidase and malondialdehyde concentration in diabetic rats.

Animals↗

Two enzymes in one; two yeast peroxiredoxins display oxidative stress-dependent switching from a peroxidase to a molecular chaperone function.

Although a great deal is known biochemically about peroxiredoxins (Prxs), little is known about their real physiological function. We show here that two cytosolic yeast Prxs, cPrxI and II, which display diversity in structure and apparent molecular weights (MW), can act alternatively as peroxidases and molecular chaperones. The peroxidase function predominates in the lower MW forms, whereas the chaperone function predominates in the higher MW complexes. Oxidative stress and heat shock exposure of yeasts causes the protein structures of cPrxI and II to shift from low MW species to high MW complexes. This triggers a peroxidase-to-chaperone functional switch. These in vivo changes are primarily guided by the active peroxidase site residue, Cys(47), which serves as an efficient "H(2)O(2)-sensor" in the cells. The chaperone function of these proteins enhances yeast resistance to heat shock.

Hot Temperature↗

The two manganese peroxidases Pr-MnP2 and Pr-MnP3 of Phlebia radiata, a lignin-degrading basidiomycete, are phylogenetically and structurally divergent.

Two new, at primary sequence and protein structure levels different, manganese peroxidase encoding genes from the white rot basidiomycete Phlebia radiata are described. Both genes are expressed in liquid cultures of P. radiata containing milled alder wood or glucose as carbon source, and high Mn(2+) concentration. The gene Pr-mnp2 contains 7 introns and codes for a 390 amino-acid polypeptide, whereas Pr-mnp3 presents 11 introns and codes for a 362 amino-acid protein. The 3-D molecular models confirm this diversity; the predicted Pr-MnP2 with a long C-terminal extension has the highest structural similarity with the crystal structure of Phanerochaete chrysosporium MnP1, whereas the shorter Pr-MnP3 protein is structurally more related to lignin peroxidases (P. chrysosporium LiPH8/H2). In Pr-MnP3, however, an alanine replaces the exposed tryptophan present in LiP and versatile peroxidases, and both Pr-MnPs include the conserved Mn(2+)-binding amino-acid ligands. This is the first occasion when two enzymes of similar function and origin fall into phylogenetically distinct subfamilies within the expanding dendrogram of the class II fungal secretory heme peroxidases.

Amino Acid Sequence↗

Phenol removal using Brassica juncea hairy roots: role of inherent peroxidase and H(2)O(2).

Removal of phenol, a major pollutant in aqueous effluents was studied using plant hairy root cultures. Among four different species of hairy roots tested, Brassica juncea showed the highest potential for phenol removal. The effect of phenol concentration and reuse in a batch system was studied using B. juncea hairy root cultures. Unlike most of the studies reported earlier, phenol removal by the hairy roots was seen to take place without the need for addition of external hydrogen peroxide (H(2)O(2)). To understand the mechanism of phenol removal, levels of peroxidase and phenol oxidase were monitored in the hairy roots. Peroxidase activity in the roots was enhanced when exposed to phenol, while phenol oxidase remained constant. Since peroxidase has a pre-requisite for H(2)O(2), the levels of H(2)O(2) were monitored for its in situ synthesis. H(2)O(2) levels were seen to increase in the presence of phenol. Thus, a mechanism wherein hairy roots also produce H(2)O(2) besides peroxidase, as a protection strategy of plant against xenobiotic stress is plausible.

Biodegradation, Environmental↗