Martin Best-Belpomme

Crucial role of the amino-terminal tyrosine residue 42 and the carboxyl-terminal PEST domain of I kappa B alpha in NF-kappa B activation by an oxidative stress.

Activation of transcription factor NF-κB involves the signal-dependent degradation of basally phosphorylated inhibitors such as IκBα. In response to proinflammatory cytokines or mitogens, the transduction machinery has recently been characterized, but the activation mechanism upon oxidative stress remains unknown. In the present work, we provide several lines of evidence that NF-κB activation in a T lymphocytic cell line (EL4) by hydrogen peroxide (H2O2) did not involve phosphorylation of the serine residues 32 and 36 in the amino-terminal part of IκBα. Indeed, mutation of Ser32 and Ser36 blocked IL-1β- or PMA-induced NF-κB activation, but had no effect on its activation by H2O2. Although IκBα was phosphorylated upon exposure to H2O2, tyrosine residue 42 and the C-terminal PEST (proline-glutamic acid-serine-threonine) domain played an important role. Indeed, mutation of tyrosine 42 or serine/threonine residues of the PEST domain abolished NF-κB activation by H2O2, while it had no effect on activation by IL-1β or PMA-ionomycin. This H2O2-inducible phosphorylation was not dependent on IκB kinase activation, but could involve casein kinase II, because an inhibitor of this enzyme (5,6-dichloro-1-β-d-ribofuranosyl-benzimidazole) blocks NF-κB activation. H2O2-induced IκBα phosphorylation was followed by its degradation by calpain proteases or through the proteasome. Taken together, our findings suggest that NF-κB activation by H2O2 involves a new mechanism that is totally distinct from those triggered by proinflammatory cytokines or mitogens.

Heat shock proteins are induced by cadmium in Drosophila cells

Drosophila cells were treated with increasing concentrations of CdCl2 (10 microM-1 mM). The toxicity of cadmium, as observed by cellular death and the ability of the cells to survive after removal of CdCl2, depended on concentration and duration of treatment. The overall synthesis of protein, measured by incorporation of [35S]methionine, decreased. It fell to 66% of the controls after 24 h of exposition to 50 microM CdCl2 and to 29% after 48 h. We showed that cadmium induced the synthesis of heat shock proteins (hsps), which started after 6 h and was maximal after 24 h of 50-100 microM CdCl2 treatment.

N-Acetylcysteine Prevents the Deleterious Effect of Tumor Necrosis Factor-α on Calcium Transients and Contraction in Adult Rat Cardiomyocytes

Background—The negative effect of tumor necrosis factor-&agr; (TNF-&agr;) on heart contraction, which is mediated by sphingosine, is a major component in heart failure. Because the cellular level of glutathione may limit sphingosine production via the inhibition of the Mg-dependent neutral sphingomyelinase (N-SMase), we hypothesized that cardiac glutathione status might determine the negative contractile response to TNF-&agr;. Methods and Results—We examined the effects of TNF-&agr; in isolated cardiomyocytes obtained from control rats or rats that were given the glutathione precursor N-acetylcysteine (NAC, 100 mg IP per animal). In cardiomyocytes obtained from control rats, 25 ng/mL TNF-&agr; increased reactive oxygen species generation and N-SMase activity (500% and 34% over basal, respectively) and decreased the amplitude of [Ca2+]i in response to electrical stimulation (22% below basal). NAC treatment increased cardiac glutathione content by 42%. In cardiomyocytes obtained from NAC-treated rats, 25 ng/mL TNF-&agr; had no effect on reactive oxygen species production or N-SMase activity but increased the amplitude of [Ca2+]i transients and contraction in response to electrical stimulation by 40% to 50% over basal after 20 minutes. This was associated with a hastened relaxation (20% reduction in t1/2 compared with basal) and an increased phosphorylation of both Ser16- and Thr17-phospholamban residues (260% and 115% of maximal isoproterenol effect, respectively). Conclusions—It is concluded that cardiac glutathione status, by controlling N-SMase activation, determines the severity of the adverse effects of TNF-&agr; on heart contraction. Glutathione supplementation may therefore provide therapeutic benefits for vulnerable hearts.

Importance of post-transcriptional regulation of chemokine genes by oxidative stress.

The transcription factor, nuclear factor kappa B (NF-kappa B), is activated by various stimuli including cytokines, radiation, viruses and oxidative stress. Here we show that, although induction with H(2)O(2) gives rise to NF-kappa B nuclear translocation in both lymphocyte (CEM) and monocyte (U937) cells, it leads only to the production of mRNA species encoding interleukin-8 (IL-8) and macrophage inflammatory protein 1 alpha in U937 cells. Under similar conditions these mRNA species are not observed in CEM cells. With the use of a transient transfection assay of U937 cells transfected with reporter constructs of the IL-8 promoter and subsequently treated with H(2)O(2), we show that (1) IL-8-promoter-driven transcription is stimulated in both U937 and CEM cells and (2) the NF-kappa B site is crucial for activation because its deletion abolishes activation by H(2)O(2). The production of IL-8 mRNA in U937 cells is inhibited by the NF-kappa B inhibitors clasto-lactacystin-beta-lactone and E-64D (l-3-trans-ethoxycarbonyloxirane-2-carbonyl-L-leucine-3-methyl amide) but requires protein synthesis de novo. Moreover, inhibition of the p38 mitogen-activated protein kinase also decreases the IL-8 mRNA up-regulation mediated by H(2)O(2). Taken together, these results show the importance of post-transcriptional events controlled by a p38-dependent pathway in the production of IL-8 mRNA in U937. The much lower activation of p38 in CEM cells in response to H(2)O(2) could explain the lack of stabilization of IL-8 mRNA in these cells.

On the transposition of copia-like nomadic elements in cultured Drosophila cells

We studied the stability of the genomic distribution of six retrotransposon families in long-term and short-term cultures of Drosophila cells. In a subclone derived from Kc cells, no significant rearrangements were detected over an 8 year period. On the contrary, extensive reshuffling and amplification of transposon families were observed in recently established cell lines. These results show that in cultured Drosophila cells transposition appears to be restricted to the transition from the embryo to continuous cell lines.

Expression of adenylyl cyclase mRNAs in the denervated and in the developing mouse skeletal muscle

Changes in the activity and in the expression of adenylyl cyclase (AC) were examined in mouse skeletal muscle after denervation and during development. Four isoforms of AC (AC2, AC6, AC7, and AC9) were detected by Northern blot analysis in gastrocnemius muscle, AC9 being the most abundant. After denervation, the levels of AC2 and AC9 mRNA decreased, whereas those of AC6 and AC7 increased. AC activity in response to several neurotransmitters was increased after denervation. During development, AC activity was high in fetus and neonate and declined in the adult; the sensitivity of AC activity to various neurotransmitters was the highest on the third postnatal day. The levels of AC6 and AC7 mRNAs were high on the third postnatal day and then decreased in adult, paralleling the decline in AC activity. All the characteristics of AC expression and activity in fetus and neonate resembled those observed in denervated adult muscle. These results indicate that changes in AC activity and AC mRNAs play an important role in the various physiopathological states of skeletal muscle, especially during muscle atrophy.

Inhibition compétitive: Relations linéaires nouvellcs et générales

Summary Competitive inhibition obtained by usual means is based upon approximations entailing confusion between free and total ligand concentrations. The lack of reliability of these approximations may be a source of error. General equations and linear plots are thus proposed avoiding approximations and applicable to various fields such as enzymology, endocrinology, immunology and generally speaking any ligandinacromolecule interactions.

On the Mechanism of Action of H2O2 in the Cellular Stress

We propose a hypothesis according to which the reactive and reduced species of oxygen could be the intracellular inducers of the stress (or heat-shock) response. This hypothesis is based on the following observations on Drosophila cells: a) the return to normoxia after 24 h anaerobiosis is sufficient to induce the synthesis of the heat shock proteins without elevation of temperature together with a rapid increase of O2 consumption; b) hydrogen peroxide introduced in the culture medium induces the early transcriptional activation of the heat shock genes (maximal after 5 minutes); c) hydrogen peroxide added to cellular extracts in vitro (thus acting as an intracellular metabolite) activates instantaneously the binding capacity of a heat shock factor to a DNA heat shock regulatory element. Thus, hydrogen peroxide, and possibly other reactive reduced species of oxygen, could trigger the onset of the stress (or heat shock) response.

Early Activation of Heat Shock Genes in H2O2-Treated Drosophila Cells

Drosophila cells of a diploid clone derived from line Kc were treated with 1 mM H2O2 for 1 to 20 minutes. Dot blot and Northern blot analysis of RNAs extracted from control and treated cells showed that the transcriptional activation of the 6 heat-shock genes tested was early, and maximal within 5 minutes of H2O2 treatment. Analysis of the kinetics of induction of the heat-shock proteins (hsps) after an exposure to H2O2 of 2 or 5 minutes, followed by removal, suggests that this brief treatment was sufficient to trigger the synthesis of all the hsps, which was maximal 1.5 to 3h after this short H2O2 treatment.

Isolation and chromosomal localization of ecdysterone-responsive genes in a Drosophila cell line

Cultured Kc 0% cells of Drosophila melanogaster are responsive to ecdysterone treatment. A library of lambda phages carrying segments of Drosophila genomic DNA was differential screened using poly(A)+RNAs from control and ecdysterone-treated cells. Nine independent recombinant phages that hybridized more intensely with poly(A)+ RNA from treated cells and six that hybridized most strongly with poly(A)+RNA from untreated cells were selected. Genomic localization of these “inducible” and “repressible” sequences was determined by hybridization in situ. These results suggest that expression of several unique genes is increased by the hormone. The six “repressible” sequences each contained DNA that hybridized to multiple chromosomal sites and appeared to be mobile elements, suggesting that the steroid hormone might be acting on the transposable elements. These probes will be useful for the study of positive and negative steroid regulation within the same cell.