Microinjection of antioxidant enzymes to protect cells from oxygen derived free radicals.
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Catalase↗
Biomedical subjects
J Remacle
Publications and source records attributed to J Remacle.
Microinjection of antibodies against superoxide dismutase and glutathione peroxidase.
Antibodies were prepared against glutathione peroxidase, superoxide dismutase, and catalase. Inhibition of the enzyme activity was obtained with anti-Gpx and anti-SOD antibodies but not with anti-CAT antibodies. The antibodies were then injected into human fibroblasts and bovine chondrocytes in culture either under normal conditions or under 1 atm of oxygen. The injected anti-Gpx and anti-SOD antibodies increased the mortality rate of the fibroblasts incubated under 1 atm of oxygen. However, when cells were incubated under normal atmosphere, anti-Gpx antibodies inhibited the division while anti-SOD antibodies increased this capacity. Anti-Gpx antibodies injected into chondrocytes decreased their viability. Injection of control antiserum had no effect. These data stress the primary importance of Gpx as antioxidant under all conditions and the relative efficiency of SOD according to the balance between the radical production and the activity of the other antioxidant systems.
Animals↗
Effect of procaine on cultivated human WI-38 fibroblasts.
Procaine is a local anesthetic, also used in experimental gerontology and has been tested in cultivated human WI-38 fibroblasts. This molecule was found to enhance growth rate and cell densities in actively dividing cultures. As the cells aged, however, this stimulatory effect diminished and finally vanished. In a long term experiment the enhancement of growth of procaine treated cultures was finally replaced by a toxic effect even at low concentration. The amount of the thermolabile enzyme found in phase III cells did not change when procaine was added to the culture medium. In this cellular aging model, procaine behaved like a metabolic stimulator of actively dividing cells but not as an "antiaging" molecule as it is sometimes assumed.
Cell Division↗
Affinity and stability modifications of immobilized alcohol dehydrogenase through multipoint copolymerization.
Yeast alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1), a potentially useful enzyme for cofactor regeneration processes, was covalently immobilized in a multipoint fashion by activation with acryloyl chloride and subsequent copolymerization in a polyacrylamide gel. Several properties such as the activity and stability were systematically studied for the free enzyme, the acryloate-enzyme and the immobilized enzyme. The activation energy was significantly lowered upon immobilization. The thermal stability of the immobilized enzyme was, however, greatly increased. But its maximum activity was observed at a lower temperature. These results suggest an important effect of the diffusional restrictions and of the mode of activation and immobilization on the activity and the stability of the enzyme.
Acrylates↗
A new technique for highly sensitive detection of superoxide dismutase activity by chemiluminescence.
A stable enzymatic free radical generation system has been developed which allows a precise production of 02-. and its detection by chemiluminescence between 2 pmol and 8 nmol. This test has been used for assaying superoxide dismutase (SOD) by inhibition of the chemiluminescence (CL) signal. No inhibition was observed with catalase, which excludes the participation of H2O2 in lucigenin CL. N,N-Diethyldithiocarbamate gives 100% inhibition of SOD activity either from a purified enzymatic preparation or from biological samples, which confirms the specificity of the CL assay. SOD assay can be performed either on a purified enzymatic preparation or on biological materials such as cultured cells.
Acridines↗
Alteration of the microtubule organization in aging WI-38 fibroblasts. A comparative study with embryonic hamster lung fibroblasts.
The microtubule organization in human WI-38 fibroblasts subcultivated in vitro has been investigated using nocodazole, a reversible inhibitor of the microtubules. Two phenotypes were observed. The typical fibroblast cells, called Type 1 cells, showed, after nocodazole treatment, a centripetal depolymerization wave of the microtubules and the giant Type 2 cells which have a more heterogeneous behaviour. Some of the cells clearly showed a centrifugal depolymerization of the microtubules, others a mixed behavior and less than 1% displayed the same behavior as the Type 1 cells. Confirming previous data obtained with Hamster fibroblasts (Raes et al., 1983, 1984), these results suggest a modification in the microtubule organization which could account for the aberrant division of some WI-38 cells in aged cultures. The relevance of this observation for the emergence of the morphologically different Type 2 cells and for cell division impairment in serially in vitro cultivated cells is discussed.
Aging↗
Comparative study of the enzymatic defense systems against oxygen-derived free radicals: the key role of glutathione peroxidase.
Human WI-38 diploid fibroblasts have been cultivated under high toxic O2 pressure, and their survival curves are reported. Superoxide dismutase, catalase, or glutathione peroxidase provided some protection when injected in the cells exposed to O2. This protective effect, recorded after 3 or 4 days of incubation, was the most pronounced when cells were injected just before oxygen exposure. Quantitative injection assays have been performed for the three enzymes. Surprisingly, glutathione peroxidase was found to be much more effective than both catalase and superoxide dismutase, the latter being particularly inefficient.
Catalase↗
[The effect of benzydamine on the generation and interaction of reactive oxygen species and cyclo- and lipoxygenase].
Pharmacological mechanisms of benzydamine (Tantum) are studied which are of relevance for the antiinflammatory properties of this non-steroidal antiinflammatory drug (NSAID). Benzydamine most effectively inhibits the generation of reactive oxygen species by murine neutrophils (IC50 1.7 X 10(-5) mol/l). Piroxicam, indomethacin and acetylsalicylic acid are ineffective. Benzydamine, however, does not interfere with xanthine oxidase-dependent superoxide anion radical formation or epinephrine oxidation. The other tested NSAID are as well inactive. The findings confirm the missing cyclooxygenase inhibition of benzydamine (IC50 greater than 10(-3) mol/l), contrary to the other NSAID which are strong (indomethacin IC50 6 X 10(-8) mol/l; piroxicam IC50 2 X 10(-7) mol/l) or moderate cyclooxygenase inhibitors (acetylsalicylic acid IC50 10(-5) mol/l). LTB4 generation via the lipoxygenase is only inhibited by indomethacin (EC50 3.6 X 10(-5) mol/l). Benzydamine appears unique among other NSAID by its relatively strong interference with the generation of reactive oxygen radicals and the lack of cyclooxygenase inhibition.
Animals↗
Simple-kinetic descriptions of alcohol dehydrogenase after immobilization on tresyl-chloride-activated agarose.
Yeast alcohol dehydrogenase was successfully immobilized on tresyl-chloride-activated agarose; the optimized conditions allowed an enzyme activity recovery of over 90%. Comparison of free and immobilized enzyme properties showed an unchanged intrinsic activation energy of the reaction and a shift of optimum activity to a higher pH medium after immobilization. Comparison of the kinetic parameters for both substrates of the reaction showed that the Michaelis-Menten model could not take into consideration all the constraints induced by the immobilization on the enzyme properties but that the Theorell-Chance model was more appropriate. These results are discussed taking into consideration the factors affecting the immobilized enzyme. Finally, we discuss the possibilities of cofactor regeneration with this immobilized alcohol dehydrogenase.
Alanine Dehydrogenase↗
A new experimental model to study oxygen toxicity.
An experimental model was developed in order to study the protective effect of antioxidant molecules. Human diploid WI-38 fibroblasts were cultivated under 2 atm of 95% O2. Antioxidants like alpha-tocopherol or superoxide dismutase (SOD) were added respectively in the culture medium or directly inside the cell through a microinjection technique. With both antioxidant molecules a protection was observed. In the control experiment, cells died within 6 or 8 days depending on the confluency and the malonaldehyde content increased sharply. This model represents a new tool in order to test other antioxidant systems towards an oxidative stress.
Antioxidants↗
Subcellular localization and modification with ageing of glutathione, glutathione peroxidase and glutathione reductase activities in human fibroblasts.
Differential centrifugation and isopycnic equilibration in density gradients were used to localize glutathione (GSH), glutathione peroxidase and glutathione reductase in the subcellular organelles of WI-38 fibroblasts. GSH was present in all the subcellular fractions, whereas the glutathione peroxidase and reductase activities were restrained to the cytoplasm and the mitochondrial fractions. After equilibration in density gradients, the results showed the presence of GSH, glutathione peroxidase and glutathione reductase in both the cytoplasm and mitochondria. GSH was also located in plasma membranes and probably in peroxisomes, endoplasmic reticulum and lysosomal membranes. Evolution of GSH in ageing fibroblasts showed a sudden increase of its concentration just before cell death. The glutathione peroxidase activity already decreases in the early passages, while the decrease of the glutathione reductase activity was constant and reached a drastic low level at the end of the culture. In conclusion, GSH is probably involved in the cell degeneration associated with ageing but because of its multiple functions and its ubiquitous localization, it is difficult to assert to which extent this metabolite is implicated in the ageing process.
Cell Line↗
Alteration of enzymes in ageing human fibroblasts in culture. III. Modification of superoxide dismutase as an environmental and reversible process.
The alteration of superoxide dismutase (SOD) defined as the change occurring in its thermostability and observed in ageing cells, concerned the cytoplasmic but not the mitochondrial enzymes. Altered SOD disappeared if it was incubated in a supernatant from the young cells whereas supernatants from the old cells induced the alteration. The alteration induced on purified SOD was found to be associated with the appearance of thermolabile tetramers. Cytoplasmic SOD tetramers were also observed in the supernatants of the old cells. The addition of NADPH into the incubation medium could reverse this alteration; also when cultivated in the presence of vincamine the alteration, normally present in the old cells, disappears. The alteration of SOD is therefore associated with the formation of tetramers; it is a reversible process influenced by the cytoplasmic composition of the old cells.
Aging↗
Alteration of enzymes in ageing human fibroblasts in culture. IV. Effect of glutathione on the alteration of glucose-6-phosphate dehydrogenase.
Alteration and inactivation of glucose-6-phosphate dehydrogenase (G6PD) can be induced in human fibroblasts by incubation of a cell supernatant at 4 degrees C and pH 7.4. When added in such conditions, glutathione (GSH) had a stabilizing effect on the enzyme. On the other hand, substances which are known to deplete the cells of their GSH content, dramatically increase the inactivation rate. When analysed by gel filtration after 24 h of incubation at 4 degrees C, the inactive G6PD appears as a dimeric protein when GSH is present, while as a monomer in the control experiment. Reactivation of the monomers was stimulated with GSH. The heat inactivation of the dimeric fraction first started with a sharp activity increase of 20%. This increase vanished when the enzyme was first reactivated before the thermolability experiment. We propose that what is called altered G6PD is the expression of a quick reactivation of an inactive, labile dimer. Finally, a schematic view of the G6PD alteration is proposed.
Cell Line↗
Comparative study of conjugation between horseradish peroxidase and D-cytochrome b5. Incorporation into subcellular membranes.
Horseradish peroxidase was conjugated to D-cytochrome b5 by three different two-step methods. The yield of conjugates based on the peroxidase enzymatic activity recovered after gel filtration was very low in the glutaraldehyde method, but higher in the N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) and periodate methods. The molecular size of the conjugates was analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Monomeric conjugates were mostly formed via the glutaraldehyde and SPDP methods in the presence of appropriate molar ratios of proteins. Most of the conjugates formed via the periodate method were polymers. The conjugate preparations of the three methods could be incorporated into microsomal membranes. Conjugate polymers, however, appeared less able to be incorporated then monomers. There was a nonpreferential incorporation of free or conjugated D-cytochrome b5 contained in the conjugate preparation of the glutaraldehyde method. In conclusion, this study gives preference to the glutaraldehyde method for the preparation of conjugates that will subsequently be used as an in vivo marker of the D-cytochrome b5 incorporation into membranes.
Animals↗
Removal of cadmium by microorganisms in a two-stage chemostat.
A two-stage chemostat was used to study removal of cadmium by microorganisms in continuous culture. The medium was contaminated with 0.8 mg of Cd per liter. At 20 degrees C, most of the microbial biomass formed aggregates which settled in the second stage of the chemostat. Effluent was free of bacteria. Up to 80% of the metal contained in the inlet flux was removed by the biomass, with 20% remaining in solution. At 10 degrees C and with a shorter retention time, flocculation was poorer and metal removal by settling biomass did not exceed 35%.
Journal Article↗
Polyploid cells in ageing hamster fibroblasts in vitro: possible implication of the centrosome.
Fibroblasts from hamster embryos cultivated in vitro present the typical ageing process of other fibroblastic lines, but they also suddenly give rise to giant non dividing cells which could be considered to represent terminally differentiated cells [36]. We investigated the latter mechanism, first by showing that microtubules in these cells depolymerized from the centrosome and not from the cell periphery as in other cells; secondly we analysed the structure of the centrosome on serial sections and found a diminished pericentriolar material; finally time lapse sequence studies of cell division confirmed that this process sometimes aborts giving rise to these giant polyploid cells. As a consequence, what first appeared as a differentiation process is in fact the result of an environmental deterioration which probably reaches a critical level thus creating a catastrophic consequence for the cell.
Animals↗
Disappearance of paternal histocompatibility antigens from hybrid mouse blastocyst at the time of implantation.
Products of the major histocompatibility complex (H-2) are important in allograph rejection. In view of the close relationship between mother and foetus, we can consider the latter as an allograph which is however not rejected by an immunological reaction. We studied the presence of H-2 antigens on embryo membranes at the time of implantation, by immunochemical labeling using gold particles coupled with protein A. Results showed that the expression of H-2 antigens is different before and after implantation. It seems that after implantation, H-2 antigens disappear from trophoblastic membranes. This could explain the absence of immunological reaction of the mother against the foetus.
Animals↗
Ageing of hamster embryo fibroblasts as the result of both differentiation and stochastic mechanisms.
Fibroblasts from hamster embryos were serially cultivated in vitro and their evolution followed from a morphological, physiological and biochemical point of view. After an exponential growth for about 20 passages, cells entered the ageing phase which ended up after 29-34 passages. From our observations, it seems that the arrest of growth results from two different phenomena: first, the typically fibroblastic cells may undergo a stochastic ageing process; second, some of these cells evolve into a terminal differentiation process, characterized by a different non-fibroblastic phenotype.
Aging↗