Cerium Perhydroxide Deposits Research Articles

H2O2 localization in the green alga Micrasterias after salt and osmotic stress by TEM-coupled electron energy loss spectroscopy

Reactive oxygen species (ROS), including hydrogen peroxide (H2O2), are constantly generated as by-products of normal metabolic cellular pathways and can be overproduced in response to stress. In this study, we investigated ROS production and localization of H2O2 after salt (200 mM KCl) and osmotic (iso-osmotic sorbitol concentration) stress in the unicellular green alga Micrasterias. By means of the dye H2DCFDA and confocal laser scanning microscopy, most ROS production could be detected in KCl-treated cells when compared to sorbitol-exposed cells and controls. For ultrastructural detection of H2O2, CeCl3, which reacts with H2O2 and produces cerium perhydroxide deposits, has been used. Cerium was identified by transmission electron microscopy (TEM)-coupled electron energy loss spectroscopy (EELS) in organelles of KCl- and sorbitol-treated cells and in controls. Statistical measurements of the presence of the cerium M4,5 edge were performed in mitochondria, chloroplasts, cell walls, and cytoplasmic sites of five individual cells after each treatment. The most pronounced increase in H2O2 production was found in chloroplasts of KCl- and sorbitol-treated cells. This shows that the chloroplast reveals the strongest response in H2O2 production after stress induction in Micrasterias. Significant elevation of H2O2 production also occurred in mitochondria and cytoplasm, whereas H2O2 levels remained unchanged or even slightly decreased in cell walls of treated cells. Additionally, TEM micrographs and EELS analyses provided indirect evidence for an increased H2O2 production at the plasma membrane of KCl-treated cells, indicating an involvement of the plasma membrane NADPH oxidase in H2O2 generation.

Localization of hydrogen peroxide in pumpkin (Cucurbita ficifolia bouché) seedlings exposed to high-dose gamma ray

Hydrogen peroxide (H2O2) was detected cytochemically, via transmission electron microscopy (TEM), in pumpkin tissues exposed to high-dose gamma ray. Its reaction with cerium chloride produced electron-dense precipitates of cerium perhydroxides. Their patterns of deposition in the tissues of both control plants and those irradiated with gamma ray (PIG) were typically found in the plasma membranes and cell walls. However, gamma irradiation remarkably increased the intensities of cerium perhydroxide deposits (CPDs) in the plasma membranes and cell walls for all tissue types, but especially the leaves. The only exception was for vessels in the cotyledons. After gamma irradiation, the (H2O2) content in all tissues was higher than in the control samples, except for the cotyledons of PIG, where the (H2O2) content was lower than for all others. The increased appearance of CPDs may have been due to the enhancement of (H2O2) accumulation by gamma radiation. This accumulation also varied according to the cell or tissue type examined.

Reactive oxygen species in the elongation zone of maize leaves are necessary for leaf extension.

The production and role of reactive oxygen species (ROS) in the expanding zone of maize (Zea mays) leaf blades were investigated. ROS release along the leaf blade was evaluated by embedding intact seedlings in 2',7'-dichlorofluorescein-containing agar and examining the distribution of 2',7'-dichlorofluorescein fluorescence along leaf 4, which was exposed by removing the outer leaves before embedding the seedling. Fluorescence was high in the expanding region, becoming practically non-detectable beyond 65 mm from the ligule, indicating high ROS production in the expansion zone. Segments obtained from the elongation zone of leaf 4 were used to assess the role of ROS in leaf elongation. The distribution of cerium perhydroxide deposits in electron micrographs indicated hydrogen peroxide (H(2)O(2)) presence in the apoplast. 2',7'-Dichlorofluorescein fluorescence and apoplastic H(2)O(2) accumulation were inhibited with diphenyleneiodonium (DPI), which also inhibited O*(2)(-) generation, suggesting a flavin-containing enzyme activity such as NADPH oxidase was involved in ROS production. Segments from the elongation zone incubated in water grew 8% in 2 h. KI treatments, which scavenged H(2)O(2) but did not inhibit O*(2)(-) production, did not modify growth. DPI significantly inhibited segment elongation, and the addition of H(2)O(2) (50 or 500 microM) to the incubation medium partially reverted the inhibition caused by DPI. These results indicate that a certain concentration of H(2)O(2) is necessary for leaf elongation, but it could not be distinguished whether H(2)O(2), or other ROS, are the actual active agents.

Cellular localization and metabolic function of n-butylamine-induced amine oxidases in the fungus Aspergillus niger AKU 3302.

Using transmission electron microscopy, the amine oxidase activity in Aspergillus niger AKU 3302 was localized to the outer side of the cell wall but not inside the cell using the cerium perhydroxide deposition method. The presence of cerium in the deposit was confirmed by energy-dispersive microanalysis of X-rays. Interestingly, immunocytochemical localization using gold labeling with a specific antibody indicated the presence of amine oxidase protein inside the cell wall and not only on the outer surface. Besides labeling of the cell wall, a high level of labeling was also observed inside the cell in what seemed to be secretory vesicle structures. It is proposed that the highly active amine oxidase AO-I is located in the cell wall and serves primarily as a detoxifying agent, preventing amines from entering and damaging the cell. The amine oxidation exhibits an interesting spatial orientation involving a release of toxic hydrogen peroxide into the extracellular space. The inactive amine oxidase protein located inside the cell is most probably the amine oxidase AO-II, found in cell homogenates. It is also likely that the less active AO-II is an improperly folded precursor of AO-I, which acquired low-level activity after cell homogenization in the presence of Cu(II) and oxygen due to autooxidative formation of topaquinone.

Localization of hydrogen peroxide production in Zinnia elegans L. stems

Abstract Hydrogen peroxide is required for a variety of physiological processes associated with plant cell wall biosynthesis. Several recent studies have suggested that starch-KI could be used as an effective histochemical stain for studying the spatial and temporal regulation of H2O2 production during plant development. In this study, we found that detection of H2O2 in Zinnia elegans stems using starch-KI staining was highly dependent upon plant age, developmental status, and the manner in which the tissue was handled prior to staining. An alternative staining technique based on cerium perhydroxide deposition clearly showed that most of the H2O2 detected by starch-KI resulted from wounding at the cut surfaces of stem sections. However, with cerium staining, substantial H2O2 production was apparent well below the cut surface in collenchyma cell walls where previous work had demonstrated localization of peroxidase activity. Co-localization of H2O2 and peroxidase in collenchyma tissues may suggest an involvement in crosslinking of the pectins and hemicelluloses that predominate the walls of these cells. Cerium deposition also suggested that H2O2 production in xylem vessel walls may lag slightly behind secondary cell wall thickening.

Cytochemical energy-filtering transmission electron microscopy of mitochondrial free radical formation in paraquat cytotoxicity.

The generation of oxygen free radicals was investigated using cytochemistry and its energy-filtering transmission electron microscopy in reference to the toxic mediator for the herbicide paraquat. When isolated intact mitochondria from rat livers were incubated in a medium containing paraquat and NADH, a mitochondrial NADH-quinone oxidoreductase activity generated superoxide anions to cause the destruction of mitochondria which resulted in cell death. The superoxide anions were immediately converted into hydrogen peroxide, which then formed cerium perhydroxide deposits in the presence of cerium ions and precipitated on the outer surface of the mitochondrial outer membrane. This localization was also specifically identified by energy spectral imaging and image-electron energy loss spectral analyses. Precipitation reaction was scavenged by the addition of either cytochrome c or catalase and inhibited by dicoumarol (an inhibitor of NAD(P)H-quinone oxidoreductases). These cytochemical energy-filtering transmission electron microscopic results indicated that paraquat generated free radicals from the outer membrane of mitochondria.

Localization of Hydrogen Peroxide Production in Pisum sativum L. Using Epi-Polarization Microscopy to Follow Cerium Perhydroxide Deposition.

Cerium is becoming an increasingly popular reagent for histochemical localization of oxidases and phosphatases because it combines directly with reaction products to form fine precipitates of electron-dense materials that can be easily detected using transmission electron microscopy or laser confocal scanning microscopy. We used epi-polarization microscopy to detect cerium perhydroxide deposits formed when H2O2 was produced by diamine oxidase in pea (Pisum sativum L.) epicotyls exposed to exogenous putrescine. Diamine oxidase activity was abundant in cortical cell walls but showed little, if any, association with vascular tissues. Maps of cerium deposition generated using scanning electron microscopy/x-ray microanalysis verified these observations. This study demonstrates the use of epi-polarization microscopy to follow cerium deposition, and the ready accessibility of this microscopy technique should facilitate more widespread use of cerium for plant histochemistry and cytochemistry.

Human neutrophils produce free radicals from the cell-zymosan interface during phagocytosis and from the whole plasma membrane when stimulated with calcium ionophore A23187

The production of free radicals, superoxide anions (O 2 −, and hydrogen peroxide (H 2O 2) was histochemically investigated in human neutrophils that were stimulated by either phagocytosis or the calcium ionophore A23187. To demonstrate O 2 −, peripheral neutrophils from healthy donors were incubated at 37 °C in a medium containing nitroblue tetrazolium and glucose in the presence of either opsonized zymosan A and/or A23187. To demonstrate H 2O 2, neutrophils pretreated with a stimulant for 10 min were washed and incubated in a cerium medium containing CeCl 3 and glucose in a Tris-maleate buffer. In cells engaged in phagocytosis, diformazan (for O 2 −) and cerium perhydroxide deposits (for H 2O 2) were restricted to the neutrophil-particle interface and on the inner surface of phagosomes. The remaining free surface of the plasma membrane was devoid of reaction products. In the case of neutrophils stimulated with A23187, the production of O 2 − and H 2O 2 was visualized over the whole surface of the plasma membrane. These histochemical reactions were inhibited by p-benzoquinone, superoxide dismutase, ferricytochrome c or catalase, and p-diazobenzenesulfonate (a membrane-impermeable protein denaturant). The results showed that human neutrophils produce free radicals exocellularly and that the site of production varies with different stimuli.

Neutrophils Adherent to a Nonphagocytosable Surface (Glomerular Basement Membrane) Produce Oxidants Only at the Site of Attachment

Adherence of neutrophils to glomerular basement membrane containing immunoglobulin G aggregates was accompanied by a marked increase in oxygen uptake (eightfold). Very little of the O2 consumed was recovered as superoxide, measured by cytochrome c reduction, or as H2O2, measured with horseradish peroxidase and scopoletin. When neutrophils were incubated with the basement membrane preparation in the presence of cerium chloride to detect H2O2, electron micrographs showed cerium perhydroxide deposits in the contact area between the cells and the basement membrane, but not on the remainder of the cell surface. The results imply that superoxide is produced only where the plasma membrane is in contact with the basement membrane matrix, and that it mostly breaks down to H2O2 or undergoes other reactions at this site. The longer lifetime of H2O2 compared with that of superoxide allows some of the H2O2 produced to be detected in the medium. The results also suggest that the area of contact between the neutrophil and surfaces such as basement membrane is inaccessible to proteins in the medium, eg, cytochrome c. Circulating scavengers such as superoxide dismutase or catalase, or proteolytic inhibitors, may therefore be unable to control events occurring at this site.

Neutrophils adherent to a nonphagocytosable surface (glomerular basement membrane) produce oxidants only at the site of attachment

Adherence of neutrophils to glomerular basement membrane containing immunoglobulin G aggregates was accompanied by a marked increase in oxygen uptake (eightfold). Very little of the O2 consumed was recovered as superoxide, measured by cytochrome c reduction, or as H2O2, measured with horseradish peroxidase and scopoletin. When neutrophils were incubated with the basement membrane preparation in the presence of cerium chloride to detect H2O2, electron micrographs showed cerium perhydroxide deposits in the contact area between the cells and the basement membrane, but not on the remainder of the cell surface. The results imply that superoxide is produced only where the plasma membrane is in contact with the basement membrane matrix, and that it mostly breaks down to H2O2 or undergoes other reactions at this site. The longer lifetime of H2O2 compared with that of superoxide allows some of the H2O2 produced to be detected in the medium. The results also suggest that the area of contact between the neutrophil and surfaces such as basement membrane is inaccessible to proteins in the medium, eg, cytochrome c. Circulating scavengers such as superoxide dismutase or catalase, or proteolytic inhibitors, may therefore be unable to control events occurring at this site.