Effect of anoxia and reoxygenation on antioxidant enzyme activities in immortalized brain endothelial cells

Summary The effects of anoxia and reoxygenation on major antioxidant enzyme activities were investigatedin vitro in immortalized rat brain endothelial cells (RBE₄ cells). A sublethal anoxic period of 12 h was assessed for RBE₄ cells using the neutral red uptake test. Anoxia markedly influenced the specific activity of catalase and superoxide dismutase, with no major effect on glutathione peroxidase or glutathione reductase. After 24 h postanoxia, the superoxide dismutase activity modulated by the presence or absence of oxygen returned to control value. Damage and recovery of RBE₄ immortalized rat brain endothelial cells in culture after exposure to free radicals and other oxygen-derived species provides a usefulin vitro model to study anoxia-reoxygenation trauma at the cellular level.     

Oxygen-Dependent Inhibition of Neutrophil Respiratory Burst by Nitric Oxide

Effect of nitric oxide (NO) on the respiratory burst of neutrophils was examined under different oxygen tensions. Phorbol myristate acetate (PMA) stimulated oxygen consumption and superoxide (O₂^-) generation in neutrophils by a mechanism which was inhibited reversibly by NO. The inhibitory effect of NO increased significantly with a decrease in oxygen tension in the medium. The inhibitory effect of NO was suppressed in medium containing oxyhemoglobin (HbO₂), a NO scavenging agent. In contrast, 3-morpholinosydnonimine (SIN-1), a compound that rapidly generates peroxynitrite (ONOO^-) from the released NO and O₂^-, slightly stimulated the PMA-induced respiratory burst. These results suggested that NO, but not ONOO, might reversibly inhibit superoxide generation by neutrophils especially at physiologically low oxygen tensions thereby decreasing oxygen toxicity particularly in and around hypoxic tissues.     

In vitro anti- and pro-oxidative effects of natural polyphenols

The anti- and pro-oxidative effects of phenolic compounds and antioxidants were studied in two different in vitro model systems utilizing ethyl linoleate and 2′-deoxyguanosine (2′-dG) as oxidative substrates, and a Fenton reaction (H₂O₂, Fe²⁺) to initiate oxidation. Oxidation of the biomolecules in both model systems exhibited dose dependency. In the 2′-dG assay, oxidation was closely related to H₂O₂ generation, which occurred during autoxidation of the phenolics. Hydroxylating activity was greatly enhanced by Mn²⁺ and Cu²⁺, but not by Zn²⁺ or Co²⁺. Ethyl linoleate peroxidation was inhibited by low concentrations of catechol, quercitin, and instant coffee. However, peroxidation was promoted by high concentrations of the same compounds, probably by recycling of chelated inactive Fe³⁺ to the active Fe²⁺ state.     

Occurrence of hemoglobin in the nitrogen-fixing root nodules of Alnus glutinosa

A true hemoglobin (Hb) was shown to be present in the root nodules of Alnus glutinosa L. After purification by gel filtration and ion exchange, the Hb formed a stable complex with oxygen. This oxygen complex could then be converted to carboxyhemoglobin by treatment with CO. Optical absorption spectra typical of Hb were observed. The molecular weight was estimated to be 15 100 by gel filtration, and 18 300 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Hb was largely insoluble when the initial homogenization was done in the absence of a detergent. Under these conditions much of the Hb appears to be associated with clusters of Frankia , the nitrogen-fixing actinomycete that infects plant cells within the nodules. The exact localization of the Hb in vivo is uncertain. The relatively low average concentration of Hb in Alnus nodules suggests that it is either confined to a relatively small fraction of total nodule volume, or has a function other than facilitation of O₂ transport.     

Liming induced stimulation of the amino acid metabolism in mycorrhizal roots of Norway spruce (Picea abies [L.] Karst.)

Localization and activity of three enzymes involved in the amino acid metabolism of ectomycorrhizas were investigated within an interdisciplinary experiment performed in a mature Norway spruce stand in Southern Germany (Höglwald). The enzymes NAD-glutamate dehydrogenase and aspartate aminotransferase were present in root cells, whereas aminopeptidase was found in mycorrhizas of Norway spruce such as Piceirhiza nigra and those with the fungi Cenococcum geophilum, Elaphomyces sp., Russula ochroleuca and Tylospora sp. Mycorrhizas growing in the humus layer contained about double the amount of protein found in those taken from the upper mineral soil (0–5 cm).Acid irrigation of the soil had no effect on the activity of any of the investigated enzymes, soluble protein or total N-contents irrespective of whether roots were taken from the organic layer or from the upper mineral soil. Liming, however, stimulated the activity of the three enzymes in mycorrhizas of the organic layer (O_f+O_h) whereas it had no effect on the activity of the investigated enzymes of mycorrhizas in the upper mineral soil. This effect is attributed to increased contents of soluble organic nitrogen compounds in the soil of the limed plots as compared to the unlimed plots.     

Subcellular distribution of selenium during uptake and its influence on mitochondrial oxidations in germinatingVigna radiata L

The metabolic significance of Se in plants is not well documented, though the presence of many selenoenzymes in bacteria and the essentiality of Se in higher animals is established. Since germination is an active process in plant growth and metabolism, the effect of Se was investigated in germinatingVigna radiata L, a nonaccumulating Sedeficient legume. Growth and protein were enhanced in seedlings supplemented with selenium (Se) as sodium selenite in the medium up to 1 μg/mL. The pattern of uptake of⁷⁵Se in the differentiating tissues and the subcellular distribution were investigated. The percentage of incorporation of⁷⁵Se was greater in the mitochondria at the lowest level (0.5 μg/mL) of Se supplementation compared to higher levels of Se exposure. Proteins precipitated from the postmitochondrial supernatant fractions, when separated by means of polyacrylamide gel electrophoresis (PAGE), indicated a major selenoprotein in the seedlings germinated at 2.0 μg/mL Se. In seedlings grown with supplemented Se, enhanced respiratory control ratio and succinate dehydrogenase activity were observed in the mitochondria of tissues, indicative of a role for Se in mitochondrial membrane functions.     

Lack of metabolic temperature compensation in the intertidal gastropods,Littorina saxatilis (Olivi) and L. obtusata (L.)

Two intertidal snails, Littorina saxatilis (Olivi, 1972) (upper eulittoral fringe/maritime zone) and Littorina obtusata (Linnaeus, 1758) (lower eulittoral) were collected from a boulder shore on Nobska Point, Cape Cod, Massachusetts, in July and acclimated for 15–20 days at 4 ° or 21 °C. Oxygen consumption rate (Vo₂) was determined for 11–15 subsamples of individuals at 4 °, 11 ° and 21 °C with silver/platinum oxygen electrodes. Multiple factor analysis of variance (MFANOVA) of lo₁₀ transformed values of whole animal Vo₂ with log₁₀ dry tissue weight (DTW) as a covariant revealed that increased test temperature induced a significant increase in Vo₂ in both species (P<0.00001). In contrast, MFANOVA revealed that temperature acclimation did not affect Vo₂ in either L. saxatilis (P= 0.35) or L. obtusata (P= 0.095). Thus, neither species displayed a capacity for the typical metabolic temperature compensation marked by an increase in Vo₂ at any one test temperature in individuals acclimated to a lower temperature that is characteristic of most ectothermic animals. Lack of capacity for metabolic temperature acclimation has also been reported in other littorinid snail species, and may be characteristic of the group as a whole. Lack of capacity for respiratory temperature acclimation in these two species and other littorinids may reflect the extensive semi-diurnal temperature variation that they are exposed to in their eulittoral and eulittoral fringe/maritime zone habitats. In these habitats, any metabolic benefits derived from longer-term temperature compensation of metabolic rates are negated by extreme daily temperature fluctuations. Instead, littorinid species appear to have evolved mechanisms for immediate metabolic regulation which, in L. saxatilis and L. obtusata and other littorinids, appear to centre on a unique ability for near instantaneous suppression of metabolic rate and entrance into short-term metabolic diapause at temperatures above 20–35 °C, making typical seasonal respiratory compensation mechanisms characteristic of most ectotherms of little adaptive value to littorinid species.     

Gas phase composition effects on suspension cultures of Taxus cuspidata

The effect of different concentrations and combinations of oxygen, carbon dioxide, and ethylene on cell growth and taxol production in suspension cultures of Taxus cuspidata was investigated using several factorial design experiments. Low head space oxygen concentration (10% v/v) promoted early production oftaxol. High carbon dioxide concentration (10% v/v) inhibited taxol production. The most effective gas mixture composition in terms of taxol production was 10% (v/v) oxygen, 0.5% (v/v) carbon dioxide, and 5 ppm ethylene. Cultures grown underambient concentration of oxygen had a delayed uptake of glucose and fructose compared to cultures grown under 10% (v/v) oxygen. Average calcium uptake rates into the cultured cells decreased and average phosphate uptake rates increased as ethylene was increased from 0 to 10 ppm. These results may indicate that gas composition alters partitioning of nutrients, which in turn affects secondary metabolite production. (c) 1995 John Wiley & Sons, Inc.     

Extracellular matrix components and proteolytic enzymes in uterine cervical carcinoma

The important components of mucopolysaccharides and collagen have been analyzed in tissues of control and carcinoma of uterine cervix. Among these components hyaluronic acid and chondroitin sulphate levels were found to be increased, whereas decreased level of collagen was observed in uterine cervical carcinoma. Serum cathepsin B, D and acid and alkaline phosphatases have also been analyzed in controls and carcinoma patients before and after treatments. The activities of these enzymes have been found to increase prominently in advanced stages. Among these enzymes cathepsin B and alkaline phosphatase have exhibited remarkable increase in activity in uterine cervical carcinoma. Different modes of treatment exerted reversion of the elevated activities of these enzymes. However, combined therapy type II (radiation combined with cisplatin and cyclophosphomide) seems to be more effective in reverting the activities of these enzymes to normal levels.     

A comparison of screening methods for antioxidant activity in seaweeds

The inhibition of lipid peroxidation and radical scavenging effects were studied to evaluate the antioxidant activity for extracts of 17 species of seaweed. The antioxidant effect was evaluated by determination of lipoxygenase activity and by α, α-diphenyl-β-picrylhydrazyl (DPPH) decolorization. Lipoxygenase activity was depressed in the presence of aqueous and ethanol extracts of 4 algal species; Sargassum species had the highest antioxidant activity of all the species examined. The ethanol extracts of one Sargassum species showed competitive inhibition with the substrate. The same species also showed radical scavenging activity in the DPPH decolorization test. Comparison of these results shows no relationship between enzyme inhibition and radical scavenging activity. This revised version was published online in August 2006 with corrections to the Cover Date.