PubMed Health⌕ Search

Biomedical subjects

J Remacle

Publications and source records attributed to J Remacle.

At least 109 records · Page 6Linked to original sources
PubMed1992

The importance of antioxidant enzymes in cellular aging and degeneration.

Aerobic cells contain various amounts of the three main antioxidant enzymes: superoxide dismutase (SOD), catalase and GSH peroxidase. These three enzymes are necessary for cell survival since inhibition of their activity leads to the arrest of cell mitosis and to cell death. Amongst them, GSH peroxidase was shown to be more efficient than catalase and much more than SOD. This result was obtained by comparing the cell protection against oxidative stress after their microinjection in the cytoplasm. With age, the level of these antioxidant enzymes does not change in several experimental models, so that it is not possible to explain the aging process by a lack of protection due to a decrease in the activity of these three enzymes. However, tissues and cells are more susceptible to free radical attacks with age. In order to understand the importance of free radicals in this process, we have to distinguish between their respective effects on cell mitosis, cell death and cell aging. The effects on mitosis and cell death are well described, and the results clearly show a threshold of response which is determined by the antioxidant content of the cell. There is now evidence that short free radical stresses can also speed up the aging of in vitro cultured human fibroblasts. However, such effects are not typical of free radicals but are also obtained with many other deleterious substances so that free radicals have to be considered as one amongst other factors responsible for influencing the evolution of a cell to an older stage or to cell death. The lowering of the general metabolism and of the free energy in old cells are probably the main factors responsible for the increased susceptibility of these cells to stresses such as oxidative stresses.

Aerobiosis↗
PubMed1992

Aging as a multi-step process characterized by a lowering of entropy production leading the cell to a sequence of defined stages. II. Testing some predictions on aging human fibroblasts in culture.

The concepts of irreversible thermodynamics have been used in order to develop a theory of aging considered as a multi-step process leading the cell through a sequence of defined stages characterized by a lower level of entropy production and finally to a critical level of errors involving cell death (Toussaint et al., 1991). One of the predictions of this model is that external stresses which can be considered as fluctuations would accelerate the evolution of the cell from one state to the other according to the intensity of the stress. Seven morphotypes have been observed in the serially cultivated human fibroblasts, cells passing progressively from one morphotype to the other. In this paper, we experimentally tested the effect of two different molecules, tert-butylhydroperoxide and ethanol, in order to determine their influence on the shift from one morphotype to the other. When applied for a single period of time on cultivated cells, both molecules effectively showed a modification in the pattern of the different morphotypes which was dependent on the stress intensity: a decreased proportion of the early morphotypes and an increased proportion of the late and post-mitotic morphotypes were observed within three days after the stresses. Similar results were obtained when successive stresses were performed at every subculture. The results also indicated that all stages are not equally stable with morphotypes III and IV being the most stable. The positive effect on the increased shift of these cells from one morphotype to the other by two different stresses firms one of the prediction of the thermodynamic model which states that cellular aging can be considered as a multi-step process which can be speeded up by various external modifications.

Cell Division↗
PubMed1992

The effect of intra- and intermolecular disulfide bonds after peptide grafting on the properties of yeast alcohol dehydrogenase.

Hydrophilic peptides including cysteine residues were grafted on activated yeast alcohol dehydrogenase. The grafted enzyme preparation was then submitted to oxidation at various concentrations in order to favour the formation of intramolecular or intermolecular disulfide bonds. Intermolecular bonds led to enzyme inactivation. But a rigidification of the enzyme was observed with intramolecular bonds. However, thiol groups also chelated the catalytic and structural zinc atoms, leading to the corresponding enzyme inactivation and thermolability. Formation of intramolecular disulfide bridges after peptide grafting strengthens enzyme conformation and can induce enzyme stabilization, but it has to take into account the possible interference with the naturally occurring cysteine bridges.

Alcohol Dehydrogenase↗
PubMed1991

Aging as a multi-step process characterized by a lowering of entropy production leading the cell to a sequence of defined stages.

The principles of the thermodynamics of irreversible processes which occur in biological cells considered as open systems, have been reviewed and applied in order to describe a possible evolution of cells during aging. The main feature of such an approach is that cells operating in a steady state, optimize their free energy production by lowering their entropy production which is kept to a minimum. Instabilities can however occur which can lead the cell from one steady state to another characterized by a lower production of entropy. Concomitantly, the level of errors or entropy of the system will increase. The process will continue and the cell will go from one state to another until a critical level is attained where the cell can not cope any more with keeping its organization and will die. The effect of stresses and mitosis have been also considered in this model. Such an approach stresses that cells can only subsist as a whole in certain states which are the result of the genetic constitution but also of the optimalization of cellular functions given their requirement in energy and the fluctuations from the environmental changes. In this respect, it reconciles both programmed and stochastic theories of aging.

Aging↗
PubMed1991

Alteration of enzymes in ageing human fibroblasts in culture. V. Mechanisms of glutathione peroxidase modification.

Ageing of WI-38 fibroblasts in culture was used as a model in order to investigate the evolution and the alteration of the key antioxidant enzyme glutathione peroxidase. The activity of glutathione peroxidase is influenced by the presence of selenium in the culture medium and we have also shown that the specific activity of this enzyme does not decrease during ageing, but rather slightly increases. No alteration could be detected by immunotitration. Also the kinetic parameter Km for tert-butyl hydroperoxide has not changed. However, the heat resistance of the enzyme dramatically decreases with ageing. Dilutions of the enzyme preparations had the same influence on the thermosensitivity of the enzyme. This dilution effect is most probably linked to the dissociation of the enzyme subunits into dimers and monomers. Moreover, the kinetic of thermoinactivation curves are best explained by consecutive reactions of inactivation with an intermediary enzyme form. These observations strongly support the hypothesis that ageing is associated with an increased dissociation constant of the tetrameric glutathione peroxidase leading to an easier dissociation of the enzyme in old cells.

Cell Line↗
PubMed1991

Cytotoxicity of linoleic acid peroxide, malondialdehyde and 4-hydroxynonenal towards human fibroblasts.

Lipid peroxidation occurs during oxidative stress and leads to the formation of various active compounds. However, controversy remains about its importance in the events leading to cell death. One approach to estimate their role in cell death would be to test the toxicity of oxidative products generated during the stress. In this work, three of these products were incubated with human fibroblasts and their toxicities were compared. The three compounds tested are: linoleic acid peroxide (LOOH), malondialdehyde (MDA) and 4-hydroxynonenal (HNE). Three cellular parameters were assayed: viability, DNA synthesis estimated by thymidine incorporation and protein synthesis measured by leucine incorporation. Protection against cellular damages was also tested adding alpha-tocopherol in the culture medium. The results showed that the peroxide was more toxic than HNE and much more than MDA. The possibility of initiation and propagation of the free radical chain reaction could explain this highest toxicity. The fibroblasts seem to be protected by alpha-tocopherol against LOOH. These effects emphasize the crucial role of this lipophilic antioxidant to protect cells against peroxidation damages.

Aldehydes↗
PubMed1991

Susceptibility of glutathione peroxidase to proteolysis after oxidative alteration by peroxides and hydroxyl radicals.

Glutathione peroxidase is a key enzyme in the antioxidant system of the cells. This enzyme has been shown to be irreversibly inactivated by H2O2, tert-butyl hydroperoxide (tert-BHP) and hydroxyl radicals when incubated without GSH. We observed that in our experimental conditions glutathione peroxidase was not degraded by trypsin or chymotrypsin while degraded by pronase, papaïn, pepsin, and lysosomal proteases. Hydroxyl radicals and superoxide anions but not H2O2 or tert-BHP could also fragment the enzyme on their own. A former incubation with H2O2, tert-BHP, or hydroxyl radicals also increased the proteolytic susceptibility of glutathione peroxidase. Like superoxide dismutase (SOD) and other oxidatively denatured proteins, glutathione peroxidase inactivated by peroxides or free radicals seems to be degraded preferentially by proteases. As hydroxyl radicals can fragment the enzyme by themselves, the increased proteolytic susceptibility afterwards is easily understood while the increased susceptibility induced by H2O2 and tert-BHP seems to be more specific.

Animals↗
PubMed1991

Association of antioxidant systems in the protection of human fibroblasts against oxygen derived free radicals.

The protection of human diploid fibroblasts against high oxygen tension was investigated using various combinations of the three major antioxidant enzymes: superoxide dismutase, catalase and glutathione peroxidase. alpha-Tocopherol, a well-known hydrophobic antioxidant, was also tested in combination with the different enzymes. Microinjection of solutions containing different combinations of the three enzymes was compared with the injection of each single enzyme. We observed that the protections given by catalase or superoxide dismutase on the one hand, and by glutathione peroxidase on the other hand, were additive. Surprisingly, the combinations of catalase and superoxide dismutase were less effective than catalase alone and was even toxic at low SOD concentrations. Addition of alpha-tocopherol following the injection of any of the three enzymes was highly beneficial, but the strongest synergistic effect was obtained with glutathione peroxidase. These results stress the importance of membrane protection by alpha-tocopherol and indirectly by glutathione peroxidase. They also showed that any injection leading to the decrease in the O2.- or H2O2 concentration combined with one of these two protectors is very beneficial for the cells probably by decreasing the OH concentration. This is also proven by the very good protective effect obtained with desferrioxamine.

Antioxidants↗
PubMed1990

Respiratory activity of isolated rat liver mitochondria following in vitro exposure to oxygen species: a threshold study.

Respiratory activity of isolated rat liver mitochondria was assayed following in vitro exposure to oxygen radicals. Our results show that mitochondrial respiration is more sensitive to O2.(-) than to H2O2. However, ferrous ions drastically enhance the toxicity of the enzymatic system generating H2O2 because of the production of the hydroxyl radicals. A protection against those oxygen species could be given by SOD in the xanthine/xanthine oxidase system and by catalase with the glucose/glucose oxidase system. The most damaging system was the combination of Fe2+ with H2O2. In this case, OH. is formed in a Fenton-like reaction. The fact that the OH. is the most damaging molecule accounts for the finding that catalase and desferrioxamine were efficient protectors in this system. Threshold levels of O2.(-) and H2O2 able to inhibit the mitochondrial respiration have been estimated. It is concluded that under normal respiration such thresholds are not reached in vivo and that the impairment of the mitochondrial respiratory activity does not seem to originate only from the natural free radical production in those organelles. However, if the production of free radicals is such to exceed the defense capability, like under oxidative stress, then the critical threshold can be surpassed and the respiration impaired leading to irreversible damages.

Aging↗
PubMed1990

Glutathione peroxidase, superoxide dismutase, and catalase inactivation by peroxides and oxygen derived free radicals.

Glutathione peroxidase (GPX), superoxide dismutase (SOD) and catalase are the most important enzymes of the cell antioxidant defense system. However, these molecules are themselves susceptible to oxidation. The aim of this work was to estimate to what extent this system could be inactivated by its own substrates. We tested the effect of hydrogen peroxide, cumene hydroperoxide, t-butyl hydroperoxide and hydroxyl and superoxide radicals on GPX, SOD and catalase. For GPX, a 50% inactivation was observed at 10(-1) M (30 min, 37 degrees C) for hydrogen peroxide, 3 x 10(-4) M (15 min, 37 degrees C) for cumene hydroperoxide and 5 x 10(-5) M (11 min, 37 degrees C) for t-butyl hydroperoxide. Unlike the hydroxyl radicals, superoxide anions did not inactivate this enzyme. Catalase was inactivated by hydroxyl radicals and by superoxide anions but organic peroxides had no effect. SOD was inactivated by 50% by hydrogen peroxide at 4 x 10(-4) M (20 min, 37 degrees C), but organic peroxides and hydroxyl radicals were ineffective on this enzyme. Since the three enzymes of the antioxidant system are susceptible to at least one of the oxidative reactive molecules, in the case of high oxidative stresses such an inhibition could take place, leading to an irreversible autocatalytical process in which the production rate of the oxidants will continuously increase, leading to cell death.

Catalase↗
PubMed1990

Comparative study of oxygen toxicity in human fibroblasts and endothelial cells.

The resistance of human pulmonary fibroblasts (WI-38) and human umbilical vein endothelial cells to oxygen toxicity (1 atm O2) was compared. Endothelial cells were more sensitive than fibroblasts. They contained also less antioxidant enzymes except for SOD: respectively 132%, 96%, 70%, 59%, and 21% of the SOD, GSH peroxidase, GSH reductase, catalase, and G6PD content of fibroblasts. However, they contained 1.81-fold more GSH than fibroblasts. Their lower content of antioxidant enzymes can explain their higher sensitivity to oxygen. The efficiency of natural antioxidant molecules and enzymes in the protection of cells incubated 3 days under 1 atm O2 was studied. alpha-tocopherol added in the culture medium led to a significant protection, contrary to the result for ascorbic acid. Microinjection of catalase, SOD, and GSH peroxidase directly into the cells was also tested: the protection was concentration dependent for both types of cells but SOD did not protect the endothelial cells. Lower activities of the other enzymes were needed to achieve protection of the endothelial cells, compared to fibroblasts. Since endothelial cells were also shown to display lower antioxidant enzyme activities, it can be hypothesized that their content is optimized for survival in physiological conditions.

Ascorbic Acid↗
PubMed1990

Importance of a threshold for error accumulation in cell degenerative processes. I. Modulation of the threshold in a model of free radical-induced cell degeneration.

Antioxidant enzymes (catalase, superoxide dismutase and glutathione peroxidase) have been injected into human fibroblasts exposed to 2 atm O2 in order to test if the threshold of oxidative damage versus antioxidant defenses could be modulated and if the damage remains reversible beyond the threshold. Cell damage was estimated by thymidine incorporation and cell survival curves. The proportion of dividing cells, measured by thymidine incorporation, rapidly decreased after O2 incubation: no cells could divide after 15 h of hyperoxia. However, cells incubated for a short time and injected with a high concentration of any of the three enzymes divided like non-oxygen-incubated cells: the enzymes could protect the cells against their loss of division potential. However, when cells were incubated for a longer period and/or when the injected enzyme concentration was lower, cells were either less or not protected and could no longer divide. These results suggest the presence of a threshold for the oxidative damage which cannot be totally repaired and which impairs the cell division; this threshold can, however, be modulated by supplementation of antioxidant enzymes, glutathione peroxidase being the most efficient.

Catalase↗
PubMed1990

Involvement of mitochondria in cell degeneration.

Human WI-38 fibroblasts were microinjected with isolated mitochondria, and survival of the injected cells was followed. More than 95% of the cells were alive and able to divide when they were injected with fresh mitochondrial preparations having a high respiratory control ratio (RCR). The presence of lysosomes was found to be toxic to the cells, and hence mitochondria had to be isolated without being contaminated by lysosomes. The microinjection of isolated mitochondria from old rats induced a 20% degeneration of the injected cells. The proportion of dead cells was also found to be dependent on the metabolic control of the injected mitochondria. Moreover, an easily metabolized energy substrate such as D(-)-beta-hydroxybutyrate sodium salt was able to inhibit, in a dose-dependent manner, cell degeneration induced by microinjection of uncoupled mitochondria. These results suggest that modifications of mitochondria leading to their uncoupling are harmful to the cells, and this can explain some of the degenerative processes observed in natural or externally induced cell death.

3-Hydroxybutyric Acid↗
PubMed1989

Inhibition of platelet-activating factor biosynthesis via the acetyltransferase by arachidonic and oleic acids in ionophore A23187-stimulated bovine neutrophils.

Platelet-activating factor (PAF) is a phospholipid mediator of inflammation and allergy that is synthesized by several inflammatory cells including neutrophils. Addition of exogenous arachidonic acid to ionophore A23187-stimulated bovine neutrophils led to the inhibition of PAF biosynthesis assayed by incorporation of [3H]acetate into PAF and by bioassay; under the same conditions, leukotriene B4 (LTB4) formation was not decreased. The activities of the PAF metabolism enzymes indicated that the PAF synthesis inhibition by arachidonic acid is mediated via the acetyltransferase inhibition which is the last enzyme of the PAF formation. Another unsaturated fatty acid, oleic acid, exhibited the same inhibitory effect on [3H]acetate-PAF formation; however, the saturated stearic acid did not lead to any inhibition. These findings suggest that liberation of unsaturated fatty acids from membrane phospholipids, as a consequence of phospholipase A2 activation, would modulate PAF formation via inhibition of the acetyltransferase. In addition, the utilization of arachidonic acid oleic acids in activated neutrophils furnishes an easy means of blocking PAF synthesis in order to understand the role of this mediator in cellular processes.

Acetates↗
PubMed1989

Effect of the microenvironment on the kinetic properties of immobilized enzymes.

A new immobilization method was developed in order to perform a systematic study of the influence of the microenvironment on the properties of immobilized enzymes. The enzyme, alcohol dehydrogenase, was first activated, then polypeptide arms of known composition were quantitatively grafted and finally the enzyme was covalently immobilized by co-polymerization of the activated ends of the peptide arms with acrylamide monomers. In this way, the polypeptide linker arms fully determine the properties of the microcavity of the gel in which the enzyme is immobilized by multipoint covalent linkages. The activation energy of the reaction was determined for different microenvironments, in solution as well as after immobilization. Kinetic parameters were also calculated and a new kinetic model was developed, allowing a correction for the diffusional restrictions. The results show that the diffusional restrictions on one hand, and the nature of the microenvironment on the other hand, interact in a dynamic way with the enzyme to determine its properties. Another key point to understanding the changes in the properties of the immobilized enzyme is to consider these proteins as dynamic structures, interacting physically and chemically with their microenvironment.

Alcohol Dehydrogenase↗
PubMed1988

Quantitative study of natural antioxidant systems for cellular nitrofurantoin toxicity.

The toxicity of nitrofurantoin was studied on human WI-38 fibroblasts: this chemical was lethal when added at concentrations higher than 5.10(-5) M in the culture medium. The protection afforded by antioxidants was then tested: alpha-tocopherol gave at 10(-4) M a light protection in contrast to ascorbic acid which even became toxic at high concentrations. We also tested catalase, superoxide dismutase and glutathione peroxidase introduced intracellularly by the microinjection technique. On a molecular basis, glutathione peroxidase was 23-times more efficient than catalase and 3000-times more than superoxide dismutase. The results also showed that a similar range of enzyme concentrations was found for the protection against high oxygen pressure. This suggests that, in the case of both oxygen and nitrofurantoin toxicity, the peroxide derivatives are the most toxic intermediates of the free radical attacks.

Antioxidants↗
PubMed1988

Use of the inhibition of enzymatic antioxidant systems in order to evaluate their physiological importance.

Chemical inhibitors of the different antioxidant enzymes were systematically testet either on purified enzymes of after incubation with human fibroblasts in culture. Inhibition values were obtained for catalase with aminotriazole, for superoxide dismutase with diethyldithiocarbamate, for glutathione peroxidase with mercaptosuccinate, for glutathione reductase with bischloroethylnitrosourea and for glutathione synthesis with buthionine sulfoximine. Viability of cells incubated with these inhibitors was then tested under normal conditions and under high oxygen pressure; the data were correlated with the above-mentioned inhibitory values. Cell viability was particularly affected when the glutathione-related enzymes, especially glutathione peroxidase, were inhibited.

Amitrole↗