mtCLIC is up-regulated and maintains a mitochondrial membrane potential in mtDNA-depleted L929 cells.

To explain why mitochondrial DNA (mtDNA)-depleted or rho0 cells still keep a mitochondrial membrane potential (Delta(psi)m) in the absence of respiration, several hypotheses have been proposed. The principal and well accepted one involves a reverse of action for ANT combined to F1-ATPase activity. However, the existence of other putative electrogenic channels has been speculated. Here, using mRNA differential display reverse transcriptase-polymerase chain reaction on L929 mtDNA-depleted cells, we identified mtCLIC as a differentially expressed gene in cells deprived from mitochondrial ATP production. Mitochondrial chloride intracellular channel (mtCLIC), a member of a recently discovered and expanding family of chloride intracellular channels, is up-regulated in mtDNA-depleted and rho0 cells. We showed that its expression is dependent on CREB and p53 and is sensitive to calcium and tumor necrosis factor alpha. Interestingly, up- or down-regulation of mtCLIC protein expression changes Delta(psi)m whereas the chloride channel inhibitor NPPB reduces the Delta(psi)m in mtDNA-depleted L929 cells, measured with the fluorescent probe rhodamine 123. Finally, we demonstrated that purified mitochondria from mtDNA-depleted cells incorporate, in a NPPB-sensitive manner, more 36chloride than parental mitochondria. These findings suggest that mtCLIC could be involved in mitochondrial membrane potential generation in mtDNA-depleted cells, a feature required to prevent apoptosis and to drive continuous protein import into mitochondria.

CREB activation induced by mitochondrial dysfunction is a new signaling pathway that impairs cell proliferation.

We characterized a new signaling pathway leading to the activation of cAMP-responsive element-binding protein (CREB) in several cell lines affected by mitochondrial dysfunction. In vitro kinase assays, inhibitors of several kinase pathways and overexpression of a dominant-negative mutant for calcium/calmodulin kinase IV (CaMKIV), which blocks the activation of CREB, showed that CaMKIV is activated by a mitochondrial activity impairment. A high calcium concentration leading to the disruption of the protein interaction with protein phosphatase 2A explains CaMKIV activation in these conditions. Transcrip tionally active phosphorylated CREB was also found in a rho0 143B human osteosarcoma cell line and in a MERRF cybrid cell line mutated for tRNA(Lys) (A8344G). We also showed that phosphorylated CREB is involved in the proliferation defect induced by a mitochondrial dysfunction. Indeed, cell proliferation inhibition can be prevented by CaMKIV inhibition and CREB dominant-negative mutants. Finally, our data suggest that phosphorylated CREB recruits p53 tumor suppressor protein, modifies its transcriptional activity and increases the expression of p21(Waf1/Cip1), a p53-regulated cyclin-dependent kinase inhibitor.

Quantitative assay for group M (subtype A-H) and group O HIV-1 RNA detection in plasma.

A quantitative HIV-1 test is described based on a competitive RT-PCR assay combined with a sandwich hybridization as a detection system. The internal RNA standard (IS) was designed specifically to be competitive during the amplification and during the hybridization step. Sample viral load determination was carried out with one RT-PCR in the presence of 10(3) IS copies. The HIV-1 copy number was calculated by reference to an external standard curve performed on known and increasing amounts of the reference HIV-1 (Ref HIV-1) RNA co-amplified with a constant amount of the IS RNA. The assay had a linear range from 10(1) to 10(6) HIV-1 copies. HIV-1 strains belonging to the different subtypes from group M, but also group O, were all detected. Absolute quantification of purified HIV-1 RNA copies gave identical results as the AMPLICOR HIV-1 Monitor assay. The quantification of patient's samples was evaluated according to different criteria such as dynamic range, sensitivity, efficacy of material recovery, reproducibility and convenience of sample handling. The microplate format of the assay combined with the colorimetric detection provides a convenient tool and fulfills the requirement for routine molecular diagnostic laboratories.

Development of a sensitive multi-well colorimetric assay for active NFkappaB.

The transcription factor nuclear factor kappaB (NFkappaB) is a key factor in the immune response triggered by a wide variety of molecules such as inflammatory cytokines, or some bacterial and viral products. This transcription factor represents a new target for the development of anti-inflammatory molecules, but this type of research is currently hampered by the lack of a convenient and rapid screening assay for NFkappaB activation. Indeed, NFkappaB DNA-binding capacity is traditionally estimated by radioactive gel shift assay. Here we propose a new DNA-binding assay based on the use of multi-well plates coated with a cold oligonucleotide containing the consensus binding site for NFkappaB. The presence of the DNA-bound transcription factor is then detected by anti-NFkappaB antibodies and revealed by colorimetry. This assay is easy to use, non-radioactive, highly reproducible, specific for NFkappaB, more sensitive than regular radioactive gel shift and very convenient for high throughput screening.

Effect of venotropic drugs on the respiratory activity of isolated mitochondria and in endothelial cells.

Several drugs used in the treatment of chronic peripheral ischaemic and venous diseases, i.e. aescine, Cyclo 3, Ginkor Fort, hydroxyethylrutosides, naftidrofuryl, naphthoquinone and procyanidolic oligomers, were tested on the mitochondrial respiratory activity. The results show that all these drugs protected human endothelial cells against the hypoxia-induced decrease in ATP content. In addition, they all induced a concentration-dependent increase in respiratory control ratio (RCR) of liver mitochondria pre-incubated with the drugs for 60 min. The drugs were divided into two groups according to their effects. The first group (A), comprising aescine, Ginkor Fort, naftidrofuryl and naphthoquinone, increased RCR by decreasing state 4 respiration rate. The second group of drugs (B), comprising hydroxyethylrutosides, procyanidolic oligomers and Cyclo 3, increased RCR by increasing state 3 respiration rate. The drugs of group A were able to prevent the inhibition of complexes I and III respectively by amytal and antimycin A while the first two drugs of group B increased adenine nucleotide translocase activity. Cyclo 3 inhibited the carbonylcyanide m-chlorophenyl hydrazone (mCCP)-induced uncoupling of mitochondrial respiration. None of these seven drugs could protect complexes IV and V, respectively, from inhibition by cyanide and oligomycin. When tested on endothelial cells the drugs of group A, in contrast to group B, prevented the decrease in ATP content induced by amytal or antimycin A. The present results suggest that the protective effects on mitochondrial respiration activity by these venotropic drugs may explain their protective effect on the cellular ATP content in ischaemic conditions and some of their beneficial therapeutic effect in chronic vascular diseases.

Growth and cytotoxic activity by Mycobacterium ulcerans in protein-free media.

The pathogenic slow-growing Mycobacterium ulcerans has, until now, been cultured in liquid media containing albumin. Here, we report the first description of use of Sauton medium and modified Reid medium, two protein-free media, in which M. ulcerans was able to grow and produce its toxin, a major virulence factor of this environmental organism which causes a skin disease commonly called Buruli ulcer. These results suggest that Sauton and modified Reid may be useful for certain fields of M. ulcerans research requiring protein-free growth conditions.

Effects of modulations of the energetic metabolism on the mortality of cultured cells.

Since cells are open systems which exchange material with their surroundings, they can be considered as open systems far from equilibrium and in this way, they follow the principles of thermodynamics of open systems. This approach stresses the fact that cells optimize their use of energy according to their functions. However, with time and/or under environmental challenges, cells can reorganize themselves at other lower levels of energy production and utilization (Toussaint et al. (1991) Mech. Ageing Dev. 61, 45-64). Considered as optimized systems, cells can adapt their behaviours according to the balance between, on one side, their energetic potential and the level of their defence systems, and on the other side, the intensity of the stress. Mainly three types of behaviour can be theoretically predicted. If the stresses are very low, the damages generated are instantaneously repaired and the cellular system remains at its steady state of energy production and utilization. If the stresses are of an intermediary intensity, it is predicted that the cell can leave its steady state of energy production and utilization and find a new one characterized by a lower level of entropy production and a higher level of errors. Third, if the stresses are of a very high intensity which can be cytotoxic, the level of the energetic potential of the cell is directly related to cell survival. We tested the latter prediction in the present work in two ways. First, the level of energy production was lowered by partially uncoupling the mitochondria. Then the effect of stresses under tert-butylhydroperoxide or ethanol was investigated in order to look for a synergistic effect on cell death with the mitochondria uncoupling. Secondly, the effect of a modification of the energetic sources during the stress was tested. Besides a protective effect found with specific defence systems, the presence of energetic metabolites such as D-glucose, pyruvate/malate, glutamate/malate, was tested and found to be protective. The effect of a stimulator of the energetic metabolism, naftidrofuryl oxalate, was also investigated and found protective. The experimental data provide good evidence that energetic factors can modulate the resistance of cells to various stresses.

Induction of cyclooxygenase by interleukin 1: comparative study between human synovial cells and chondrocytes.

OBJECTIVE: To investigate the regulation of the prostaglandin (PG) synthesis by interleukin 1 (IL-1) in human synovial cells and chondrocytes. METHODS: Both cell types stimulated by human recombinant IL-1 synthesized PGE2, PGF2 alpha and 6-keto-PGF1 alpha. RESULTS: PGE2 was the major PG synthesized. When arachidonic acid was added exogenously at the end of the stimulation, an increase in the prostaglandin synthesis was observed after 6 and 24 h suggesting that cyclooxygenase is the limiting enzyme. Using actinomycin D and cycloheximide, PG synthesis was shown to be protein synthesis dependent. Inhibition of the constitutive cyclooxygenase by aspirin before the IL-1 stimulation confirmed that the increased prostaglandin synthesis was due to a de novo synthesis of cyclooxygenase. CONCLUSION: This enzyme induction by IL-1 was found to be similar in both cell types.

Effects of naftidrofuryl on hypoxia-induced activation and mortality of human endothelial cells.

The present study was designed to elucidate the possible beneficial effects of naftidrofuryl on ischemia-induced endothelium damage. For this purpose, an in vitro model was developed wherein human endothelial cells isolated from umbilical vein were submitted to hypoxia. Long-term hypoxia incubation (6 h) induced cell mortality, and naftidrofuryl strongly protected endothelial cells against this mortality in a dose-dependent manner and at concentrations as low as 10(-9) M. 66% protection was still observed after 16 h of hypoxia. Naftidrofuryl had to be present during the hypoxia incubation to exert its action; preincubation up to 24 h in the presence of naftidrofuryl could not protect endothelial cells incubated under hypoxia without naftidrofuryl. Short-term hypoxia, which does not induce mortality, strongly activates the endothelial cells with an increase in the cytosolic calcium concentration, in the phospholipase A2 activity, and in the synthesis of prostaglandin and of platelet-activating factor. It also enhances the adherence of polymorphonuclear neutrophils. Naftidrofuryl was able to markedly inhibit this whole cascade of events in a dose-dependent manner. We also demonstrated that naftidrofuryl could block the decrease in ATP concentration that results from the hypoxic conditions. These results indicate that by preserving the energetic level of the cells, naftidrofuryl prevents the activation of endothelial cells and the cell mortality induced by hypoxia. By maintaining an intact endothelium in vivo during ischemia, naftidrofuryl could prevent the further damage induced by leukocyte recruitment and activation.

Relationship between the critical level of oxidative stresses and the glutathione peroxidase activity.

The production and removal of the various oxygen-derived free radicals is a dynamic and complex process which normally results in a steady state of very low concentrations of these reactive molecules in the cell. The mathematical modelling of this process showed that any lowering of the glutathione peroxidase activity will increase the steady state level of the hydroperoxides and will decrease the level of organic peroxides necessary to destabilize the system. In this paper, we experimentally tested this relationship by the estimation of the level of peroxidative stresses which lead to cell degeneration in the presence of more or less active selenium-dependent glutathione peroxidase (GPX). The enzyme was inhibited by mercaptosuccinate (MS) and the cells were submitted to various extents of oxidative stress using tert-butylhydroperoxide (TBHP). Critical levels of this peroxidative molecule could be determined by the determination of the concentration leading to 50% cell death. A relationship between this critical level of TBHP and the GPX activity was established. The critical level strongly decreased with the inhibition of GPX and was found to be zero when 44% of the GPX activity is inhibited. Presented in this way, the results clearly show the pattern of the inverse relationship between the susceptibility of the cell to oxidative stress and the GPX activity.

Human umbilical vein endothelial cells submitted to hypoxia-reoxygenation in vitro: implication of free radicals, xanthine oxidase, and energy deficiency.

Ischemia-reperfusion is observed in various diseases such as myocardium infarct. Different theories have been proposed to explain the reperfusion injury, among them that the free radical generation plays a crucial role. To study the mechanisms of the reperfusion injury, a hypoxia (H)-reoxygenation (R) model upon human umbilical vein endothelial cells in culture was developed in order to mimic the in vivo situation. Different parameters were quantified and compared under H or H/R, and we found that oxygen readmission led to damage amplification after a short hypoxia period. To estimate the importance of various causes of toxicity, the effects of various protective molecules were compared. Different antioxidant molecules, iron-chelating agent, xanthine oxidase inhibitors, and energy-supplying molecules were very efficient protectors. Synergy could also be observed between the antioxidants and the energy-supplying molecules or the xanthine oxidase inhibitors. The toxic effect of O2.(-) could be lowered by the presence of SOD or glutathione peroxidase in the culture medium, whereas glutathione peroxidase was the most efficient enzyme when injected into the cells. The production of O2.(-) and of H2O2 by endothelial cells was directly estimated to be, respectively, of 0.17 and 0.035 mumol/min/mg prot during the R period. O2.(-) production was completely inhibited when allopurinol was added during H and R. In addition, a xanthine oxidase activity of 21.5 10(-6) U/mg prot could be observed by a direct assay in cells after H but not in control cells, thus confirming the previous conclusions of xanthine oxidase as a potent source of free radicals in these conditions. Thanks to the use of cultured human endothelial cells, a clear picture was obtained of the overall process leading to cell degenerescence during the reoxygenation process. We particularly could stress the importance of the low energetic state of these cells, which is a critical factor acting synergistically with the oxidant molecules to injure the cells. These results also open new possibilities for the development of new therapeutics for ischemia.

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.

[A comparative study of the protective effect of different phlebotonic agents on endothelial cells in hypoxia]. / Etude comparative de l'effet protecteur des différents phlébotoniques sur les cellules endothéliales en hypoxie.

Numerous reported findings indicate that the etiology of venous diseases is multifactorial. One of the chief factors is certainly stasis of blood in the veins of the lower limbs during long periods spent standing up. This stasis causes tissue hypoxia which first affects the venous wall. Because of their location at the interface between blood and vein wall and because of their fragility, endothelial cells are the first to suffer from the lack of oxygen. With the aim of understanding these events, the authors have developed a model of endothelial cells in culture subjected to hypoxia in vitro which mimics the conditions encountered clinically. This model has enabled us to test various drugs commonly used in venous disease. These included GbE, the active ingredient of GINKOR FORT, diosmin and procyanidol oligomers. It was thus shown that only GbE was not toxic to endothelial cells. GbE was also found to be capable of effectively protecting cells exposed to hypoxia. Protection under the influence of diosmin was obtained only at concentrations very close to toxic doses. In contrast, procyanidol oligomers offered no protection. The protective effect of GbE is believed to be due to the action of terpenes on the energy metabolism of the cell.

[A comparative study of the protective effect of various phlebotonic agents on hypoxic endothelial cells]. / Etude comparative de l'effet protecteur des différents phlébotoniques sur les cellules endothéliales en hypoxie.

Many findings indicate that the etiology of venous disease is multifactorial. One of the chief factors is certainly the stasis of blood in the lower limbs during long periods of standing. This stasis causes tissue hypoxia which first affects the venous wall. Because of their site at the blood/vein wall interface as well as their fragility, endothelial cells are the first to suffer from lack of oxygen. In order to understand these events, the authors have developed a model of endothelial cells in culture subjected to hypoxia in vitro which imitates conditions encountered clinically. This model was used to test different drugs commonly used in venous pathology. These included GbE, the active ingredient of GINKOR FORT, diosmine and procyanidolic oligomers. It was thus shown that only GbE was not toxic to endothelial cells. Furthermore, GbE was capable of effectively protecting cells subjected to hypoxia. Protection with diosmine was obtained only at concentrations very close to toxic doses. In contrast, procyanidolic oligomers afforded no protection. The protective effect of GbE is believed to be due to the action of terpenes on cellular energy metabolism.

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.

[Behavior of human endothelial cells in hyperoxia and hypoxia: effect of Ginkor Fort]. / Comportement des cellules endothéliales humaines sous hyperoxie et hypoxie: effet du Ginkor Fort.

Recent discoveries have shown that venous diseases have a multifactorial etiology. One of the factors which is definitely involved in this pathologic process is the change in the concentration of oxygen. An increase in the concentration of oxygen, hyperoxia, or reoxygenation following hypoxia, damages the tissues by stepping up the production of free radicals. In addition, a reduction in oxygen concentration, or hypoxia, is also damaging, probably through a reduction in ATP synthesis. From a therapeutic standpoint, the veins, and more particularly the endothelium, must be protected against the impact on the tissue of these changes in oxygen concentration. In this study, the effects of Ginkor Fort were tested on cultured endothelial cells subjected to varying oxygen pressures. The results show that Ginkor Fort can provide good protection of endothelial cells against hyperoxia and hypoxia-reoxygenation. These beneficial effects are probably due to the presence of flavonoids in the Ginko biloba extract; these flavonoids have an anti-oxidant effect. In addition, this substance also protects the cells against hypoxia, possibly by increasing the availability of oxygen for ATP synthesis. This dual protective effect, which is produced by two different mechanisms, may account for the wide spectrum of Ginkor Fort in its use in venous diseases.

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.

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.

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.