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PubMed2006

[The mechanism of ABA-induced apoplastic H2O2 accumulation in maize leaves].

In maize (Zea mays L.) cells, the sources of apoplast hydrogen peroxide (H(2)O(2)) induced by abscisic acid (ABA) has been proposed. Histochemical and cytochemical localization of ABA-induced H(2)O(2) production in leaves of maize was examined, using 3, 3-diaminobenzidine (DAB) and cerium chloride (CeCl(3)) staining respectively. Pretreatment with two different inhibitors for each enzymes, with diphenylene iodonium (DPI) and imidazole against NADPH oxidase, NaCN and NaN(3) against peroxidases (POD), and quinacrine and guazatine against polyamine oxidases (PAO), reduced H(2)O(2) accumulation induced by ABA. Among them, NADPH oxidase inhibitors arrested the production of apoplastic H(2)O(2) most strongly (Figs.1,2). Further studies showed that exogenous ABA treatment could enhance the activities of plasma membrane (PM) NADPH oxidase, cell wall POD and apoplast PAO significantly. Moreover, the activity of PM NADPH oxidase kept increasing before 1.5 h, while the other two enzymes began to decrease at 0.5 h within 2 h by ABA treatment (Fig.3). The results suggest that exogenous ABA treatment can lead to significant increases in H(2)O(2) accumulation in apoplast through up-regulating the activities of the PM NADPH oxidases, cell wall POD and apoplast PAO, of which the first one plays a central role in ABA-induced apoplastic H(2)O(2) accumulation.

Abscisic Acid↗
PubMed2000

Chemiluminescent choline biosensor using histidine-modified peroxidase immobilised on metal-chelate substituted beads and choline oxidase immobilised on anion-exchanger beads co-entrapped in a photocrosslinkable polymer.

A novel sensing layer design is presented based on the non-covalent immobilisation of enzymes on derivatized Sepharose beads subsequently entrapped in PVA-SbQ photopolymer. Two different modified Sepharose beads were used, IDA- and DEAE-Sepharose, for the immobilisation, respectively, of horseradish peroxidase (HRP) modified with histidine, and choline oxidase (Chx). The HRP-IDA-Sepharose-based sensing layer was used in a flow injection analysis chemiluminescent system as the basis of an H2O2 biosensor. It was shown that the pre-immobilisation on IDA-Sepharose beads enhanced the sensing layer stability and enabled the immobilisation of a larger amount of enzyme. A 1.8 mg charge of HRP-IDA-Sepharose beads in the sensing layer produced the most sensitive H2O2 biosensor. Such an analytical system exhibited very good performances, with a cycle time of 2 min and a detection limit of 15 pmol (detection ranging over four decades at least), and an unusual long operational stability of 200 measurements (CV, 3.5%). The HRP-IDA-Sepharose beads were then combined with Chx-DEAE-Sepharose. With this modified Sepharose-based biosensor the limit of detection for choline (S/N, 3) was equal to 0.5 pmol and the working range was 0.35 pmol-10 nmol. Moreover, the cycle time was only 2.5 min with the new sensing layer, and a long operational stability of 150 successive assays was found, with a variation coefficient of 2.6%.

Alcohol Oxidoreductases↗
PubMed1983

The enteric nervous system in tissue culture. III. Studies on neuronal survival and the retention of biochemical and morphological differentiation.

The maintenance of differentiated properties and survival rates of enteric neurons, grown in explant cultures for periods of up to 3 weeks, was studied. Using catecholamine fluorescence, immunohistochemistry and autoradiography, it was found that adrenergic neurons, VIP-containing neurons and putative GABAergic neurons, which constitute small subpopulations of guinea pig myenteric neurons in vivo, were all represented in plexus explants after maintenance in culture for 2-3 weeks. The pattern of expression of the transmitter-related enzymes, acetylcholinesterase and monoamine oxidase, paralleled that found in in situ studies. Investigation of neuronal structure by intracellular injection of horseradish peroxidase revealed that the cultured neurons continue to express the wide diversity in gross morphology which characterizes these cells in vivo. Employing autoradiography following uptake of [3H]GABA to label putative GABAergic neurons, their survival rate from days 1 to 15 of culturing was determined. No neuronal death was detected between days 1 and 5, while the number of neurons decreased between days 5 and 15. These observations suggest that enteric neurons maintained in explant cultures survive well and maintain to a high degree their histochemical and morphological properties.

Animals↗
PubMed2004

Rosiglitazone, an agonist of peroxisome proliferator-activated receptor gamma, protects against gastric ischemia-reperfusion damage in rats: role of oxygen free radicals generation.

Peroxisome proliferator-activated receptor gamma (PPAR-gamma) is a nuclear hormone receptor super family that has recently been implicated in atherosclerosis, inflammation, cancer, infertility, and demyelination. Oxidative stress, neutrophil infiltration, proinflammatory cytokines, and the exhibition of luminal acid play a role in the pathogenesis of gastric injury induced by ischemia-reperfusion. Rosiglitazone, a specific PPAR-gamma ligand, has been shown to have antiinflammatory activity, but its effects on experimental ischemia-reperfusion gastric injury remain unknown. We have investigated the effects of the rosiglitazone on gastric injury caused by ischemia following reperfusion in rats. Tumour necrosis factor-alpha (TNF-alpha) levels and changes in enzymatic activities of myeloperoxidase, as a marker of neutrophils infiltration, xanthine oxidase, superoxide dismutase, and glutathione peroxidase, were determined. Histological analysis of the lesions was also carried out. Pretreatment with 1 or 4 mg/kg of rosiglitazone ameliorated the gastric damage induced by clamping the celiac artery for 30 min followed by 60 min of reperfusion. It significantly (P<0.05) reduced the index of neutrophil infiltration and the levels of the cytokine. Rosiglitazone did not revert the reduced glutathione peroxidase activity but enhanced significantly (P<0.01) the decreased xanthine oxidase and superoxide dismutase activities in gastric mucosa of ischemic rats. In conclusion, rosiglitazone reduces the damage in ischemia-reperfusion gastric injury and alleviates the inflammatory response and the oxidative events.

Animals↗
PubMed1987

Interference in an electrochemical detection system for peroxidase-linked reactions based on a fluoride ion-selective electrode.

Use of the fluoride ion-selective electrode as a detector in a linked glucose oxidase (EC 1.1.3.4)/peroxidase (EC 1.11.1.7) method for glucose was investigated for possible interference from sugars, metabolites, drugs, and anticoagulants. The CV for the method was 1.5% (SD 0.37 mmol/L) at a glucose concentration of 25.0 mmol/L. Interference was studied with glucose at this concentration, interference being defined as any result differing by more than +/- 3 SD. When present in concentrations of clinical relevance, interference from urea, uric acid, or acetaminophen would preclude the use of this detection system for routine analysis.

Electrochemistry↗
PubMed1993

Intrinsic connections of layer III of striate cortex in squirrel monkey and bush baby: correlations with patterns of cytochrome oxidase.

This study used biocytin and horseradish peroxidase (HRP) to examine the intrinsic connections of the cytochrome oxidase (CO) rich blob and CO poor nonblob zones within layer III of striate cortex in two primate species, nocturnal prosimian bush babies (Galago crassicaudatus) and diurnal simian squirrel monkeys (Saimiri sciureus). Our main objective was to determine whether separate classes of lateral geniculate nucleus (LGN) cells projected to separate superficial layer zones or layers in either species. There were three significant findings. First, we confirm that layer III consists of three sublayers, IIIA, IIIB, and IIIC in both species. Layer IIIA receives input from layers IIIB, IIIC, and V, with little or no input from LGN recipient layers IV and VI. Layer IIIB receives its input from nearly every cortical layer. Layer IIIC, receives input principally from layers IV alpha [which receives its input from magnocellular (M) LGN cells] and from layers V and VI. Taken together with other findings on the extrinsic connections of these layers, our data suggest that IIIA and IIIC provide output to separate hierarchies of visual areas and IIIB acts as a set of interneurons. Second, we find that, as in macaque monkeys, cells in both IV beta and IV alpha of bush babies and squirrel monkeys project to layer IIIB, converging within the blobs. These results suggest that information from all LGN cell classes [parvocellular (P), M, and the Koniocellular (K) or their equivalents] may be integrated within the blobs. Thus, blobs in all of these primates may perform a function that transcends visual niche differences. Third, our data show a species specific difference in the connections of the IIIB nonblobs; nonblobs receive indirect input via IV alpha from the LGN M pathway in bush babies but receive indirect input via IV beta from the LGN parvocellular (P) pathway in squirrel monkeys. These findings indicate that the role of nonblob zones within striate cortex differs from that of blob zones and takes into account visual niche differences.

Animals↗
PubMed1985

Endogenous defenses against the cytotoxicity of hydrogen peroxide in cultured rat hepatocytes.

The catalase activity of cultured rat hepatocytes was inhibited by 90% pretreatment with 20 mM aminotriazole without effect on the activities of glutathione peroxidase or glutathione reductase, or on the viability of the cells over the subsequent 24 h. Glutathione reductase was inhibited by 85% by pretreatment with 300 microM 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) without effect on glutathione peroxidase, catalase, or on viability. Both pretreatments sensitized the hepatocytes to the cytotoxicity of H2O2 generated either by glucose oxidase (0.05-0.5 units/ml) or by the autoxidation of the one-electron-reduced state of menadione (50-250 microM). Aminotriazole pretreatment had no effect on the GSH content of the hepatocytes. BCNU reduced GSH levels by 50%. Depletion of GSH levels to less than 20% of control by treatment with diethyl maleate, however, did not sensitize the cells to either glucose oxidase or menadione, indicating that the effect of BCNU is related to inhibition of the GSH-GSSG redox cycle rather than to the depletion of GSH. With glucose oxidase, most of the cell killing in hepatocytes pretreated with either aminotriazole or BCNU occurred between 1 and 3 h. The antioxidant diphenylphenylenediamine (DPPD) had no effect on viability at 3 h. Catalase added to the culture medium 1 h after the addition of glucose oxidase prevented the cell killing measured at 3 h. The sulfhydryl reagents dithiothreitol (200 microM), N-acetyl-L-cysteine (4 mM), and alpha-mercaptopropionyl-L-glycine (2.5 mM) prevented the cell killing with exogenous H2O2 in hepatocytes sensitized by the inhibition of catalase or glutathione reductase. With menadione, there was no killing of nonpretreated hepatocytes at 1 h, and DPPD did not prevent the cell death after 3 h. Aminotriazole pretreatment enhanced the cell killing at 3 h but not at 1 h, and DPPD was not protective. Catalase added to the medium at 1 h inhibited the cell death measured at 3 h. In contrast, menadione killed hepatocytes pretreated with BCNU within 1 h. DPPD prevented cell death at 1 h, and there was evidence of lipid peroxidation in the accumulation of malondialdehyde in the culture medium. Catalase added with menadione did not prevent the cell killing at 1 h but did prevent it at 3 h. These data indicate that catalase and the GSH-GSSG cycle are active in the defense of hepatocytes against the toxicity of H2O2.(ABSTRACT TRUNCATED AT 400 WORDS)

Amitrole↗
PubMed1967

Indoleacetic acid oxidase activity of apoperoxidase.

The conventional activity of electrophoretically purified horseradish peroxidase toward guaiacol, pyrogallol, 2,6-dimethoxyphenol, and benzidine is abolished by removal of the heme prosthetic group with a mixture of cold acetone and hydrogen chloride. The apoenzyme, though devoid of peroxidase activity, retains its activity as an indoleacetic acid oxidase when it is supplied with 10(-5) mole of manganous ion and 2,4-dachlorophenol per liter. This oxidase activity is cyanide-sensitive; azide also inhibits under specific conditions of both pH and cofactor concentration. Partial restoration of the peroxidase activity by recombination of apoprotein with heme produces no effect on the oxidase activity, except that cofactors are no longer absolutely required. Therefore, it appears that the activity of peroxidase as an indoleacetic acid oxidase need not directly involve the heme prosthetic group, or that manganous ions and dichlorophenol can substitute for the heme group in the reaction between indoleacetic acid and oxidase.

Azides↗
PubMed2006

Effects of salicylic acid and cold treatments on protein levels and on the activities of antioxidant enzymes in the apoplast of winter wheat leaves.

The effects of salicylic acid (SA) and cold on apoplastic protein levels and activities of apoplastic catalase (CAT), peroxidase (POX) and polyphenol oxidase (PPO) were investigated in winter wheat (Triticum aestivum cv. Dogu-88) leaves. The plants were grown with and without 10 microM SA treatment at both control (20/18 degrees C for 30 and 45-day) and cold (10/5 degrees C for 30-day and 5/3 degrees C for 45-day) acclimatisations. Molecular masses of the apoplastic polypeptides were shown ranging in size from 20 to 66 kDa on SDS-PAGE. Accumulation and pattern of the polypeptides were changed by both SA and cold. It is observed that CAT, POX and PPO activities at 45-day control leaves were higher than at 30-day. When the activities with SA and cold treatments are compared to their controls, CAT activities were decreased while POX and PPO activities were increased by both the treatments. When the activities with cold+SA treatment are compared to their cold treatments, CAT and POX activities were decreased while PPO activity was increased by SA. It is concluded that exogenous SA can be involved in cold tolerance by regulating apoplastic proteins and antioxidant enzyme activities.

Antioxidants↗
PubMed1982

An electrochemical assay system for peroxidase and peroxidase-couplers reactions based on a fluoride ion-selective electrode.

A sensitive and accurate potentiometric reaction-rate method for the assay of peroxidase (EC 1.11.1.7) and peroxidase-coupled reactions is described. The technique is based on the peroxidase-catalyzed rupture of the covalent C--F bond in certain organo-fluoro compounds in the presence of H2O2 and the measurement of liberated F- by a fluoride ion-selective electrode. In a study screening 26 organo-fluoro compounds, 12 were susceptible to C--F bond rupture. However, analytically useful reaction kinetics were observed only with 4-fluoroaniline, 5-fluor-2-methylaniline, 4-fluorophenol, 2,3,5,6-tetrafluorophenol, pentafluorophenol, and 3-fluoro-DL-tyrosine. With this assay system, peroxidase activity as low as 25 mU/L could be precisely measured within 2 min. The reaction was coupled to glucose oxidase and cholesterol oxidase for the determination of glucose and cholesterol. Glucose oxidase activity in solution (to 2 U/L) was measured to demonstrate the feasibility of the assay for oxidase enzymes. Only a few microliters of sample is required and most determinations can be completed within minutes. The assay system offers broad applications and is especially attractive for use in enzyme immunoassays.

Blood Glucose↗
PubMed1976

Effect of vitamin E deficiency on the level of superoxide dismutase, glutathione peroxidase, catalase and lipid peroxide in rat liver.

The activities of superoxide dismutase, glutathione peroxidase, catalase and xanthine oxidase were simultaneously studied in vitamin-E deficient and -supplemented rat liver and also measured the lipid peroxide content in liver. The lipid peroxide content of vitamin E-deficient rat liver, estimated by thiobarbituric acid, increased as compared with that of vitamin E-supplemented rat liver. No marked changes of activities of superoxide dismutase, glutathione peroxidase and catalase were observed, but the activity of xanthine oxidase which is strong superoxide generator increased in vitamin E-deficient rat liver. These results suggest that vitamin E prevents the accumulation of lipid peroxide, but not controls the level of peroxide scavenging system such as superoxide dismutase, glutathione peroxidase and catalase.

Animals↗