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Afferent fiber connections from lower brain stem to hypothalamus studied by the horseradish peroxidase method with special reference to noradrenaline innervation.

Attempts were made to determine the afferent projections to the anterior hypothalamus including the preoptic area from the lower brain stem by means of the horseradish peroxidase method combined with monoamine oxidase staining to identify noradrenaline (NA) neurons. In addition to this technique, a histofluorescence analysis was performed. NA fibers in the medial part of the anterior hypothalamus were mainly supplied by A1 and A2 NA neuron groups, while the lateral part and periventricular zone received NA terminals from both pontine and medulla oblongata NA neuron groups. Furthermore, the present study indicated that there were direct projections to the anterior hypothalamus from non-noradrenergic neurons in the lower brain stem: nuclei raphe dorsalis, centralis superior, cells in the mesencephalic and pontine central gray matter, nuclei parabrachialis lateralis and medialis, cells around fasciculus longitudinalis medialis.

Afferent Pathways↗

Aluminium increases xanthine oxidase activity and disturbs antioxidant status in the rat.

The mechanisms responsible for the neurotoxic effects of Al remain poorly understood. In order to determine whether Al promotes oxidative stress in vivo, we measured the enzymatic activity of xanthine oxidase (XO), superoxide dismutase (SOD), glutathione peroxidase (GPX), glutathione-S-transferase (GST) and glutathione reductase (GR) in four groups of rats after eight days of intraperitoneal administration of variable concentrations of Al (0, 5, 10, and 15 mg/kg body weight, respectively). XO activity was measured in both plasma and liver samples, and the activities of the remaining enzymes were further determined in the brain and red blood cells (RBC). The most significant changes were observed in XO and GPX activities, that were enhanced and depressed, respectively. In both instances, the enzyme activities were correlated with Al concentrations, either positively (XO) or negatively (GPX). Enhancement of XO and inhibition of GPX activity may lead to the accumulation of intermediate toxic compounds such as hydrogen peroxide and hydroxyl radicals, since SOD activity is increased as well. The latter finding must be taken with some caution because previous studies have shown contradictory results in this field. Our data suggest that Al toxicity could be mediated by its action on both pro- and anti-oxidant enzymes. The biological significance of these findings remains to be established.

Aluminum↗

Inducible peroxidases mediate nitration of anopheles midgut cells undergoing apoptosis in response to Plasmodium invasion.

Plasmodium berghei invasion of Anopheles stephensi midgut cells causes severe damage, induces expression of nitric-oxide synthase, and leads to apoptosis. The present study indicates that invasion results in tyrosine nitration, catalyzed as a two-step reaction in which nitric-oxide synthase induction is followed by increased peroxidase activity. Ookinete invasion induced localized expression of peroxidase enzymes, which catalyzed protein nitration in vitro in the presence of nitrite and H(2)O(2). Histochemical stainings revealed that when a parasite migrates laterally and invades more than one cell, the pattern of induced peroxidase activity is similar to that observed for tyrosine nitration. In Anopheles gambiae, ookinete invasion elicited similar responses; it induced expression of 5 of the 16 peroxidase genes predicted by the genome sequence and decreased mRNA levels of one of them. One of these inducible peroxidases has a C-terminal oxidase domain homologous to the catalytic moiety of phagocyte NADPH oxidase and could provide high local levels of superoxide anion (O(2)), that when dismutated would generate the local increase in H(2)O(2) required for nitration. Chemically induced apoptosis of midgut cells also activated expression of four ookinete-induced peroxidase genes, suggesting their involvement in general apoptotic responses. The two-step nitration reaction provides a mechanism to precisely localize and circumscribe the toxic products generated by defense reactions involving nitration. The present study furthers our understanding of the biochemistry of midgut defense reactions to parasite invasion and how these may influence the efficiency of malaria transmission by anopheline mosquitoes.

3,3'-Diaminobenzidine↗

Potential applications of the oxidoreductive enzymes in the decolorization and detoxification of textile and other synthetic dyes from polluted water: a review.

Recently, the enzymatic approach has attracted much interest in the decolorization/degradation of textile and other industrially important dyes from wastewater as an alternative strategy to conventional chemical, physical and biological treatments, which pose serious limitations. Enzymatic treatment is very useful due to the action of enzymes on pollutants even when they are present in very dilute solutions and recalcitrant to the action of various microbes participating in the degradation of dyes. The potential of the enzymes (peroxidases, manganese peroxidases, lignin peroxidases, laccases, microperoxidase-11, polyphenol oxidases, and azoreductases) has been exploited in the decolorization and degradation of dyes. Some of the recalcitrant dyes were not degraded/decolorized in the presence of such enzymes. The addition of certain redox mediators enhanced the range of substrates and efficiency of degradation of the recalcitrant compounds. Several redox mediators have been reported in the literature, but very few of them are frequently used (e.g., 1-hydroxybenzotriazole, veratryl alcohol, violuric acid, 2-methoxy-phenothiazone). Soluble enzymes cannot be exploited at the large scale due to limitations such as stability and reusability. Therefore, the use of immobilized enzymes has significant advantages over soluble enzymes. In the near future, technology based on the enzymatic treatment of dyes present in the industrial effluents/wastewater will play a vital role. Treatment of wastewater on a large scale will also be possible by using reactors containing immobilized enzymes.

Coloring Agents↗

Evaluation of white-rot fungi for detoxification and decolorization of effluents from the green olive debittering process.

Wastewater produced by the debittering process of green olives (GOW) is rich in polyphenolics and presents high chemical oxygen demand and alkalinity values. Eight white-rot fungi ( Abortiporus biennis, Dichomitus squalens, Inonotus hispidus, Irpex lacteus, Lentinus tigrinus, Panellus stipticus, Pleurotus ostreatus and Trametes hirsuta) were grown in GOW for 1 month and the reduction in total phenolics, the decolorization activity and the related enzyme activities were compared. Phenolics were efficiently reduced by P. ostreatus (52%) and A. biennis (55%), followed by P. stipticus (42%) and D. squalens (36%), but only P. ostreatus had high decolorization efficiency (49%). Laccase activity was the highest in all of the fungi, followed by manganese-independent peroxidase (MnIP). Substantial manganese peroxidase (MnP) activity was observed only in GOW treated with P. ostreatus and A. biennis, whereas lignin peroxidase (LiP) and veratryl alcohol oxidase (VAOx) activities were not detected. Early measurements of laccase activity were highly correlated ( r(2)=0.91) with the final reduction of total phenolics and could serve as an early indicator of the potential of white-rot fungi to efficiently reduce the amount of total phenolics in GOW. The presence of MnP was, however, required to achieve efficient decolorization. Phytotoxicity of GOW treated with a selected P. ostreatus strain did not decline despite large reductions of the phenolic content (76%). Similarly, in GOW treated with purified laccase from Polyporus pensitius, a reduction in total phenolics which exceeded 50% was achieved; however, it was not accompanied by a decline in phytotoxicity. These results are probably related to the formation of phenoxy radicals and quinonoids, which re-polymerize in the absence of VAOx but do not lead to polymer precipitation in the treated GOW.

Alcohol Oxidoreductases↗

A comparison of the heme electronic states in equilibrium and nonequilibrium protein conformations of high-spin ferrous hemoproteins. Low temperature magnetic circular dichroism studies.

The visible and near infrared magnetic circular dichroism (MCD) spectra of equilibrium high-spin ferrous derivatives of myoglobin, hemoglobin, horseradish peroxidase and mitochondrial cytochrome c oxidase at 15 K are compared with those of the corresponding proteins in nonequilibrium conformations produced by low-temperature photodissociation of CO-complexes of these proteins as well as of O2-complexes of myoglobin and hemoglobin. Over all the spectral region (450-800 nm) the intensities of MCD bands of hemoproteins studied in equilibrium conformation are shown to be strongly temperature-dependent, including a negative band at ca. 630 nm and positive bands at ca. 690 nm and at ca. 760 nm. In contrast to the absorption spectra, the low-temperature MCD spectra of high-spin ferrous hemoproteins differ significantly, reflecting the peculiarities in the heme iron coordination sphere which are created by a protein conformation. The MCD spectra reveal clearly the structural changes in the heme environment which occur on ligand binding. On the basis of assignment of d leads to d and charge-transfer transitions in the near infrared region the correlation is suggested between the wavelength position of the MCD band at approx. 690 nm and the value of iron out-of-plane displacement as well as between the location of the band at approx. 760 nm and the Fe-N epsilon (proximal histidine) bond strength (length) in equilibrium and nonequilibrium conformations of the hemoproteins studied. The high sensitivity of low-temperature MCD spectra to geometry at heme iron is discussed.

Animals↗

Root growth and lignification of two wheat species differing in their sensitivity to NaCl, in response to salt stress.

Application of a 100-mM NaCl salt stress to wheat seedlings of a salt-tolerant (Triticum durum var. Ben Béchir) and a salt-sensitive (Triticum aestivum var. Tanit) species decreases the fresh and dry weights of roots especially in the salt-sensitive species, and slightly increases the ratio of dry to fresh weight, especially in the salt-resistant species. All peroxidase activities are increased by salt stress, the water-soluble peroxidase activity being increased much more in the salt-sensitive than in the salt-tolerant species, while the opposite result is observed with the cell-wall peroxidase activity. Some water-soluble peroxidases have been hypothesised to have auxin oxidase activity (which might explain the effect observed on the root biomass), while the cell-wall peroxidases would be involved in lignification. Histochemical observation confirms a more intense lignification in the root cells of the salt-tolerant species compared to the sensitive species, under the effect of NaCl.

Kinetics↗

Contribution of peroxidases in host-defense, diseases and cellular functions.

Peroxidases figure prominently in biology and contribute significantly to cell biology, host defense against infection, and pathogenesis of several inflammatory diseases. These varied and diverse aspects of peroxidase biochemistry and its clinical implications will be the subjects of in-depth analysis at the 4th International peroxidase meeting held in Kyoto. Specific topics range from the molecular basis of peroxidase structure and function to the clinical consequences of autoantibodies generated against myeloperoxidase (MPO), the peroxidase present in circulating neutrophils. Consideration of novel aspects of peroxidase biology, both unanticipated biochemical properties of MPO and the potential role of MPO in the pathogenesis of inflammatory diseases such as atherosclerosis, will also be included. In addition to peroxidases, the newly expanded family of NADPH oxidases will be discussed. We hope that this collection of scientists who share a common interest in peroxidase biology but each possess expertise in distinctly different aspects of the subject will provide a setting for spirited discussion and a lively exchange of views to yield advances in understanding and to create new applications of those insights to benefit clinical medicine, agriculture and industry.

Animals↗

Response to ischemia-reperfusion injury in hypertrophic heart. Role of free-radical metabolic pathways.

Clinical and experimental evidence demonstrates that hypertrophied cardiac tissue is more sensitive to ischemic injury than is normal myocardium. Recent studies indicate that cardiac ischemia-reperfusion injury involves the generation of toxic oxygen free radicals. We used the spontaneously hypertensive rat (SHR) model, with its otherwise genetically identical control (the Wistar-Kyoto [WKY] rat), to investigate the potential role of enzymes that generate and detoxify oxygen radicals in the sensitivity of hypertrophied heart to ischemia and reperfusion. Because hypertension develops progressively with age in SHRs, we assayed xanthine oxidase, superoxide dismutase, catalase, and glutathione peroxidase at three different time points and found significant fluctuations at different ages. At age 26 weeks, physiological measurements demonstrated hypertension and increased sensitivity to ischemia and reperfusion, measured as significantly decreased left ventricular recovery after injury. At this age, xanthine oxidase, which may generate oxygen radicals, was significantly increased in SHR compared with WKY rats (p = 0.003). Superoxide dismutase, which is a principal step in oxygen-radical detoxification, was significantly lower (p = 0.044). These data suggest that differences in the constitutive levels of oxygen-radical metabolic pathways are different in hypertrophied myocardium, and it is suggested that this finding may play a role in the response of these hearts to ischemia-reperfusion injury.

Animals↗

Oxidative stress: a role in the pathogenesis of Parkinson's disease.

The degeneration of nigro-striatal dopaminergic neurons is considered to be a predominant pathogenetic factor of Parkinson's disease (PD). However, the etiology of this degeneration is not known. Hypotheses assume accumulation of endogenous and/or exogenous toxins as trigger of the disease. An increase in the concentration of free radicals has been suggested to be toxic to cells, especially when combined with certain metals like free iron or copper. The role of melanin in the degenerative process is not clear, but autoxidative reactions such as the oxidation of dopamine (DA) to melanin generating radicals and toxic metabolites seem to enhance the vulnerability of neurons in the substantia nigra (SN). Disappearance of melanin in the SN, increase of total iron and ferric iron, extreme decrease of glutathione (GSH) levels, reduced activity of enzymes involved in the detoxification of hydrogen peroxide, hydroxyl and superoxide radicals (peroxidases, catalase, glutathione peroxidase), an increase of monoamine oxidase B (MAO B) activity and the substantial increase of malondialdehyde, a marker of lipid peroxidation, in the SN seem to indicate a role of an oxidative stress syndrome in the SN causing or aggravating PD.

Aged↗

Characterization of hydrogenosomes and their role in glucose metabolism of Neocallimastix sp. L2.

In the anaerobic fungus Neocallimastix sp. L2 fermentation of glucose proceeds via the Embden-Meyerhof-Parnas pathway. Enzyme activities leading to the formation of succinate, lactate, ethanol, and formate are associated with the cytoplasmic fraction. The enzymes 'malic enzyme,' NAD(P)H:ferredoxin oxidoreductase, pyruvate:ferredoxin oxidoreductase, hydrogenase, acetate:succinate CoA transferase and succinate thiokinase leading to the formation of H2,CO2, acetate, and ATP are localized in microbodies. Thus, these organelles are identified as hydrogenosomes. In addition, the microbodies contain the O2-scavenging enzymes NADH- and NADPH oxidase, while NAD(P)H peroxidase, catalase, or superoxide dismutase could not be detected. In cell-free extracts from zoospores of Neocallimastix sp. L2 the specific activities of hydrogenosomal enzymes as well as the quantities of these proteins are 2- to 6-fold higher than in mycelium extracts. These findings suggest that hydrogenosomes perform an important role--especially in zoospores--as H2-evolving, ATP-generating and O2-scavenging organelles.

Adenylate Kinase↗

Non-isotopic RNA probes. Comparison between different labels and detection systems.

Several studies have shown the use of non-radioactive labelled DNA probes for in situ hybridisation, mainly to identify cellular DNA. In this study mRNA in situ hybridisation was performed on rat pituitary with biotinylated complementary (c) RNA probes for rat prolactin and growth hormone (GH), and compared with radioactive 35S-radiolabelled probes. Biotinylated cRNA probes were labelled with either biotin-11-UTP or with allylamine-UTP, the latter method being able to produce a higher yield of labelled RNA. Different detection systems were tested, and hybridisation signal was seen in cells of anterior pituitary with both types of biotinylated probes. The signals were detected using either avidin-biotin-complex with peroxidase (ABC), peroxidase-anti-peroxidase (PAP) or gold-silver methods. ABC peroxidase detected using glucose oxidase-diaminobenzidine (DAB)-nickel solution appeared to be the best method for detecting labelled RNA probes, with very strong signal and low background. The biotinylated probes were comparable in sensitivity to the radiolabelled probes in detecting prolactin and GH mRNAs in the anterior lobe of the rat pituitary. These results indicate an alternative methods of labelling and detection of biotinylated probes which could have a potential role in research and diagnostic techniques.

Allylamine↗

Ruthenium(II) tris(bipyridyl) ion as a luminescent probe for oxygen uptake.

The present work describes an alternative technique for following the rate of oxygen uptake by chemical and enzymatic systems. This method is based on spectrofluorometric monitoring of the well-known quenching effect of molecular oxygen on the emission of the photoexcited [Ru(bpy)3]2+ ion, added to the reaction mixtures. The rate of oxygen consumption determined using the present method agrees with that obtained by conventional polarographic techniques in all of the following systems: ascorbate/Cu2+, glucose/glucose oxidase (EC 1.1.3.4), and propanal/horseradish peroxidase (EC 1.11.1.7); in the last case, agreement was observed both in the presence and absence of serum albumin and of chloroplasts. Spectrofluorometric data for amphotericin autoxidation in dimethyl sulfoxide are in accord with the rate of decay of the ESR signal of a spin trap added to the reaction mixture. The advantages and limitations of the present spectrofluorometric technique relative to conventional polarographic monitoring of dissolved oxygen are discussed.

2,2'-Dipyridyl↗