Is the effect of interleukin-1 on glutathione oxidation in cultured human fibroblasts involved in nuclear factor-kappaB activation?

Our understanding of the interleukin-1 (IL-1) signaling molecular mechanisms has recently made considerable progress, with the discovery of the IL-1 receptor-associated kinase and the downstream enzymatic cascade that leads to the activation of nuclear factor-kappaB (NF-kappaB). IL-1 signaling and especially NF-kappaB activation are thought to be redox-sensitive, even though the precise nature and the molecular targets of the oxidants/antioxidants involved remain largely unknown. Here, we investigated the possible role of cellular oxidized/reduced glutathione (GSSG/GSH) balance in IL-1 signaling. We describe a quantitative method based on capillary electrophoresis designed to assay both intracellular GSH and GSSG in adhering fibroblasts. This method allows the GSSG/GSH balance to be followed during IL-1 stimulation. Our data show that IL-1 induces rapid and transient oxidation of intracellular glutathione in human fibroblasts. Using various antioxidants, including pyrrolidine dithiocarbamate and curcumin, we were unable to show a direct relationship between this IL-1-induced glutathione oxidation and NF-kappaB activation. Of the five antioxidants tested, only curcumin was able to inhibit IkappaBalpha degradation upstream and, hence, NF-kappaB DNA-binding activity and NF-kappaB-dependent expression of IL-6 downstream.

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.

Protection by bilobalide of the ischaemia-induced alterations of the mitochondrial respiratory activity.

Ischaemia is a common feature of most vascular diseases. There is evidence from experimental and clinical studies that Ginkgo biloba extract protects tissues from ischaemia/reperfusion damages. Bilobalide seems to be responsible, at least in part, for this activity. However, the mechanism of the protection afforded by bilobalide is not yet known. In this work, the effects of bilobalide on mitochondrial respiration were investigated during liver and brain ischaemia, since mitochondria alteration is an early event in ischaemia-induced damage. Bilobalide could prevent the decrease in respiratory activity induced by ischaemia in liver and in brain, both when glutamate/malate or succinate was used as substrate. Ischaemia decreased state 3 respiration rate and bilobalide prevented this decrease. While bilobalide was not able to prevent the decrease in adenine translocase activity, it protected complex I activity. Bilobalide allows mitochondria to maintain their respiratory activity in ischaemic conditions by protecting complex I and probably complex III activities. Hence, the energetic pool of tissues is preserved during the ischaemic period as well as its viability. This mechanism provides, a possible explanation for the anti-ischaemic properties of bilobalide and of Ginkgo biloba extract in therapeutic interventions.

Identification of lysine 74 in the pyruvate binding site of alanine dehydrogenase from Bacillus subtilis. Chemical modification with 2,4,6-trinitrobenzenesulfonic acid, n-succinimidyl 3-(2-pyridyldithio)propionate, and 5'-(p-(fluorosulfonyl)benzoyl)adenosine.

L-Alanine dehydrogenase from Bacillus subtilis was inactivated with two different lysine-directed chemical reagents, i.e. 2,4, 6-trinitrobenzenesulfonic acid and N-succinimidyl 3-(2-pyridyldithio)propionate. In both cases, the inactivation followed pseudo first-order kinetics, with a 1:1 stoichiometric ratio between the reagent and the enzyme subunits. Partial protection of the active site from inactivation could be obtained by each of the substrates, NADH or pyruvate, but complete protection could only be achieved in the presence of the ternary complex E.NADH. pyruvate. The nucleotide analogue of NADH, 5'-(p-(fluorosulfonyl)benzoyl)adenosine was also used for affinity labeling of the enzyme active site. Differential peptide mapping, performed both in the presence and in the absence of the substrates, followed by reversed phase high performance liquid chromatography separation, diode-array analysis, mass spectrometry, and N-terminal sequencing of the resulting peptides, allowed the identification of lysine 74 in the active site of the enzyme. This residue, which is conserved among all L-alanine dehydrogenases, is most likely the residue previously postulated to be necessary for the binding of pyruvate in the active site. Surprisingly, this residue and the surrounding conserved residues are not found in amino acid dehydrogenases like glutamate, leucine, phenylalanine, or valine dehydrogenases, suggesting that A-stereospecific amino acid dehydrogenases such as L-alanine dehydrogenase could have evolved apart from the B-stereospecific amino acid dehydrogenases.

Automated solid-phase synthesis of cyclic peptides bearing a side-chain tail designed for subsequent chemical grafting.

Recent developments in allyl chemistry and palladium solubilization allow automated continuous-flow solid-phase synthesis of cyclic or branched peptides, with specific side-chain cleavage and on-line cyclization. In this paper, we adapted the method to the synthesis of cyclic peptides bearing an anchoring tail on a side chain of the cycle. Side products were obtained with the standard procedure and an additional washing step had to be introduced in the synthesis protocol to remove side products resulting from the palladium allyl cleavage step. The method is illustrated by the automated synthesis of cyclo[-DVal-Arg-Gly-Asp-Glu (-epsilon Ahx-Cys-NH2)-] which contains the Arg-Gly-Asp adhesion motif (RGD) recognized by cellular integrins. The tail of the peptide was designed with a thiol at the carboxylic end to allow subsequent grafting by covalent attachment. Such tailed cyclic peptides can be grafted on different supports for new applications in biomaterial design, cell adhesion assays, affinity chromatography, immunization, vaccine development, ELISA kits, and the building of libraries of conformationally constrained peptides.

Hypoxia induces PMN adherence to umbilical vein endothelium.

OBJECTIVE: In vitro incubation of cultured endothelial cells under hypoxia leads to the activation of these cells and results in an increase of their adhesiveness for neutrophils (PMN). Because of the possible relevance of these observations for pathological situations, we investigated whether adherence of PMN also occurs in an entire vein after its incubation in hypoxic conditions. METHODS: Human umbilical veins in complete cords were incubated for 2 h in normoxic or hypoxic conditions and the adherence of unstimulated human 51Cr-labelled-PMN was measured under flow conditions. Experiments with human umbilical vein endothelial cells (HUVEC) were performed in parallel for comparison. Morphological studies in scanning electron microscopy were carried out in both in vitro and ex vivo situations. RESULTS: Hypoxia induced an increase in the adherence of PMN either to HUVEC or to the umbilical vein endothelium up to 5- to 6-fold when compared to normoxic conditions (P < 0.001). In both cases, this hypoxia-induced adherence was inhibited by anti-ICAM-1 antibodies or when the PAF (platelet-activating factor) synthesis was blocked during hypoxia by oleic acid. Furthermore, the adherence of PMN was inhibited when PMN were pre-incubated with WEB 2086 (a selective PAF receptor antagonist). These results indicate a crucial role of PAF in this process. Morphological studies confirmed that the number of PMN adherent to hypoxic HUVEC or to the hypoxic umbilical vein endothelium was much greater than the number of PMN on normoxic endothelial cells. Both in vitro and ex vivo, PMN adherent to the hypoxic endothelial cells to the contrary of the ones adherent to normoxic endothelial cells demonstrated membrane foldings typical of an activated state. CONCLUSION: These results show that in a complete vein, hypoxia induced an increased adhesiveness of endothelial cells for PMN by a similar mechanism to the one observed for cultured endothelial cells. They suggest an active role of endothelial cells in the initiation of the inflammatory response often described in ischemic-reperfused organs.

Protection of hypoxia-induced ATP decrease in endothelial cells by ginkgo biloba extract and bilobalide.

Due to their localization at the interface between blood and tissue, endothelial cells are the first target of any change occurring within the blood, and alterations of their functions can seriously impair organs. During hypoxia, which mimics in vivo ischemia, a cascade of events occurs in the endothelial cells, starting with a decrease in ATP content and leading to their activation and release of inflammatory mediators. EGb 761 and one of its constituents, bilobalide, were shown to inhibit the hypoxia-induced decrease in ATP content in endothelial cells in vitro. Under these conditions, glycolysis was activated, as evidenced by increased glucose transport, as well as increased lactate production. Bilobalide was found to increase glucose transport under normoxic but not hypoxic conditions. In addition, EGb and bilobalide prevented the increase in total lactate production observed after 60 min of hypoxia. However, after 120 min of hypoxia, the total lactate production was similar under normoxic and hypoxic conditions, and both compounds increased this production. These results indicate that glycolysis slowed down between the 60th and 120th minute of hypoxia, while EGb and bilobalide delayed the onset of glycolysis activation. In another experimental model, both compounds were shown to increase the respiratory control ratio of mitochondria isolated from liver of rats treated orally. Since ischemia is known to uncouple mitochondria, the protection of ATP content and the delay in glycolysis activation observed during hypoxia in the presence of EGb 761 or bilobalide is best explained by a protection of mitochondrial respiratory activity, at least during the first 60 min of hypoxia incubation. Both products retain the ability to form ATP, thereby reducing the cell's need to induce glycolysis, probably by preserving ATP regeneration by mitochondria as long as oxygen is available.

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.

Importance of the structural zinc atom for the stability of yeast alcohol dehydrogenase.

Yeast alcohol dehydrogenase is a tetrameric enzyme containing zinc. Initially we confirmed the presence of two zinc atoms per subunit. Incubation of the enzyme with increasing concentrations of dithiothreitol, a method for partial chelation, allowed first the reduction of four disulphide bridges per enzyme, but eventually was sufficient to chelate the structural zinc atom without having any effect on the zinc located in the active site. The enzyme activity was not affected but the enzyme became very sensitive to heat denaturation. Chelation by EDTA was also performed. Given its location at an external position in the globular protein, protected in each subunit by one disulphide bridge, the results establish that the second zinc atom present on each enzymic subunit plays a prominent conformational role, probably by stabilizing the tertiary structure of yeast alcohol dehydrogenase. Recovery experiments were performed by incubation of the native enzyme, or the dithiothreitol-treated enzyme, with a small amount of Zn2+. A stabilization effect was found when the structural zinc was re-incorporated after its removal by dithiothreitol. In all cases a large increase in activity was also observed, which was much greater than that expected based on the amount of re-incorporated zinc atom, suggesting the re-activation of some inactive commercial enzyme which had lost some of its original catalytic zinc atoms.

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.

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.

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.