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We read with interest the paper by Mak et al. (1) on asthma in a Hong Kong Chinese population. They reported a protective effect of the catalase (CAT)–262 T allele in non­smoking asthma patients. This result is of importance, as it suggests for the first time a potential role of the CAT–262 polymorphism in the etiology of asthma. At variance with previous publications was the lack of association of erythrocyte catalase activity with the CAT–262 polymorphism within controls and cases. In a cohort of 196 coal miners, we observed lower erythrocyte catalase activities in miners heterozygous and in those homozygous for the CAT–262 T allele, heterozygotes having an intermediate activity (2). Although the difference was not statistically significant, a 28 percent decrease in catalase activity was observed in the non-smoker T carrier controls by Mak et al. (1), similar to the 31 and 33 percent of decrease reported by us (2), and by Ahn et al. (3) in 18 women from a population-based case-control study respectively. As Mak et al. (1) observed a decrease in catalase activity related to CAT–262 polymorphism in non-smokers only, we stratified on smoking status in our coal miner cohort. We found again a significant decrease in catalase activity with the CAT–262 T allele in current and in ex- or non-smokers (see figure), as we found previously in miners with current high and low exposure to coal dust (2), which demonstrates the consistency of the association. The lack of significance of the association in the study by Mak et al. (1) may be due to a low statistical power as 28 subjects were heterozygous and only one was homozygous for the CAT–262 T allele out of the 308 Chinese controls. Figure Catalase activity according to smoking status and to CAT–262 genotype It is interesting to note that Mak et al (1) observed a protective effect of the CAT–262 T allele in non-smoking asthma patients only. Regardless of the disease, an oxidant-rich environment has been found to increase risk only in subjects with the low-activity catalase CAT–262 T allele (4). Factors previously found to be protective, such as an antioxidant-rich dietary environment or a low level of circulating proinflammatory cytokines, seemed to be beneficial only in subjects with the high catalase activity CC genotype (5). Similar to the oxidant-rich environment in our occupational study, asthma may generate reactive oxygen species, and catalase activity significantly increases in asthma patients compared to controls (1). As catalase is involved in the first response to oxidative stimuli, the increase in its activity may be interpreted as a compensatory response to the changes in the oxidant/antioxidant imbalance occuring in asthmatics. In the study of Mak et al. (1), the level of the oxidant state related to asthma may partly mask the a priori deleterious effect of the CAT–262 T allele. Further, the CAT–262 T allele frequency in this Chinese population (1) was five times lower than that observed in Caucasians (mean 26 percent [20–56] from 7 studies), and was similar to the frequency observed in 40 African Americans (6 percent) by Zhou et al. (6). The differences in reported CAT–262 T allele frequency between studies indicates significant variation between populations, and could also partly explain the a priori deleterious effect of the CAT–262 T allele, as patterns of linkage disequilibrium across populations were different. In summary, these results highlight the complexity of the potential role of the CAT–262 polymorphism in the etiology of oxidant-related diseases.

International audience; Eels are submitted to hydrostatic pressure (HP) during their spawning migration (about 6000 Km). Before migration, they change from the yellow to the silver stage (silvering process). The effects of HP in relation to the silvering process have been studied on aerobic metabolism and more precisely on reactive oxygen species (ROS) metabolism. HP acclimatization of yellow eels improves oxidative phosphorylation together with supposed concomitant changes in electron leak and ROS production. Therefore hydroxyl radical (OH*) production, superoxyde dismutase and catalase activities, malondialdehyde content and in parallel oxygen consumption were measured in the red muscle of long-term pressure exposed and control group yellow and silver eels. At atmospheric pressure, yellow eels exhibited significantly higher oxygen consumption and OH* production than silver eels; and significantly lower malondialdehyde content. This could be due to the increase in membrane fluidity induced by the silvering process. Long-term HP exposure decreases yellow eel oxygen consumption which becomes similar to that of the silver stage. In parallel there is a decrease in OH* production and concomitantly antioxidant enzyme activities follow the same tendency. Thus the respiratory chain improvement in pressure acclimatized yellow eels is accompanied by a ROS production decrease which could mean an electron leak decrease.

Most Salmonella enterica serovars produce two periplasmic [Cu,Zn] superoxide dismutases, SodCI, which is prophage encoded, and SodCII, encoded by a conserved chromosomal gene. Both enzymes were proposed to enhance Salmonella virulence by protecting bacteria against products of macrophage oxidative burst. However, we previously found SodCI, but not SodCII, to play a role during mouse infection by S. enterica serovar Typhimurium. Here we have extended these findings to another serovar of epidemiological relevance: sv Enteritidis. In both serovars, the dominant role of SodCI in virulence correlates with its higher levels in bacteria proliferating in mouse tissues, relative to SodCII. To analyze the basis of these differences, the coding sequences of sodCI and sodCII genes were exchanged with the reciprocal 5'-regions (in serovar Typhimurium). The accumulation patterns of the two proteins in vivo were reversed as a result, indicating that the regulatory determinants lie entirely within the regions upstream from the initiation codon. In the construct with the sodCI gene fused to the sodCII 5'-region, SodCI contribution to virulence was reduced but remained significant. Thus, both, high-level expression and some unidentified qualities of the enzyme participate in the phenotypic dominance of SodCI over SodCII in Salmonella pathogenicity.

International audience; In ectotherms as well as in endotherms, the mitochondrial respiratory chain is the major source of reactive oxygen species (ROS) including the highly reactive, hydroxyl radical (OH*). It is known that the different steps involved in ROS production and the antioxidant systems are comparable in ectotherms and endotherms. But regulatory mechanisms in ROS production are less known especially in fish submitted to environmental changes. Firstly, we performed a ROS muscle metabolism interspecies study, using trout and eels as ectotherms and rats as endotherms, measuring OH* production, superoxide dismutase and catalase activities and in parallel oxygen consumption (MO2). Secondly, we studied the effects of an environmental factor (hydrostatic pressure) on ROS metabolism in both fish species. The results show that, at atmospheric pressure, fish have a higher OH*/MO2 ratio than rats which exhibit a higher superoxide dismutase activity. In fish exposed to hydrostatic pressure, this ratio is reversed so that for the same MO2 value, fish produce less OH* under hydrostatic pressure than at atmospheric pressure.

International audience; Although ions play important roles in the cell and chloroplast metabolism, little is known about ion transport across the chloroplast envelope. Using a proteomic approach specifically targeted to the Arabidopsis chloroplast envelope, we have identified HMA1, which belongs to the metal-transporting P1B-type ATPases family. HMA1 is mainly expressed in green tissues, and we validated its chloroplast envelope localization. Yeast expression experiments demonstrated that HMA1 is involved in copper homeostasis and that deletion of its N-terminal His-domain partially affects the metal transport. Characterization of hma1 Arabidopsis mutants revealed a lower chloroplast copper content and a diminution of the total chloroplast superoxide dismutase activity. No effect was observed on the plastocyanin content in these lines. The hma1 insertional mutants grew like WT plants in standard condition but presented a photosensitivity phenotype under high light. Finally, direct biochemical ATPase assays performed on purified chloroplast envelope membranes showed that the ATPase activity of HMA1 is specifically stimulated by copper. Our results demonstrate that HMA1 offers an additional way to the previously characterized chloroplast envelope Cu-ATPase PAA1 to import copper in the chloroplast.

International audience; A total of 212 coagulase-negative Staphylococcus strains recovered prospectively during 119 surgeries for proven or suspected bone and joint infection (BJI) were identified by sodA sequencing. These strains were identified as 151 Staphylococcus epidermidis isolates, 15 S. warneri isolates, 14 S. capitis isolates, 9 S. hominis isolates, 6 S. lugdunensis isolates, 5 S. haemolyticus isolates, 4 S. caprae isolates, 4 S. pasteuri isolates, 3 S. simulans isolates, and 1 S. cohnii isolate. Only S. epidermidis, S. lugdunensis, S. capitis, and S. caprae were found to be infecting organisms and were involved, respectively, in 35 (81.4%), 3 (7.0%), 3 (7.0%), and 2 (4.6%) cases of BJI.

International audience; Mutations in Cu/Zn superoxide dismutase (encoded by SOD1), one of the causes of familial amyotrophic lateral sclerosis (ALS), lead to progressive death of motoneurons through a gain-of-function mechanism. RNA interference (RNAi) mediated by viral vectors allows for long-term reduction in gene expression and represents an attractive therapeutic approach for genetic diseases characterized by acquired toxic properties. We report that in SOD1(G93A) transgenic mice, a model for familial ALS, intraspinal injection of a lentiviral vector that produces RNAi-mediated silencing of SOD1 substantially retards both the onset and the progression rate of the disease.

International audience; Friedreich ataxia (FRDA) results from a generalized deficiency of mitochondrial and cytosolic iron-sulfur protein activity initially ascribed to mitochondrial iron overload. Recent in vitro data suggest that frataxin is necessary for iron incorporation in Fe-S cluster (ISC) and heme biosynthesis. In addition, several reports suggest that continuous oxidative damage resulting from hampered superoxide dismutases (SODs) signaling participates in the mitochondrial deficiency and ultimately the neuronal and cardiac cell death. This has led to the use of antioxidants such as idebenone for FRDA therapy. To further discern the role of oxidative stress in FRDA pathophysiology, we have tested the potential effect of increased antioxidant defense using an MnSOD mimetic (MnTBAP) and Cu,ZnSOD overexpression on the murine FRDA cardiomyopathy. Surprisingly, no positive effect was observed, suggesting that increased superoxide production could not explain by itself the FRDA cardiac pathophysiology. Moreover, we demonstrate that complete frataxin-deficiency neither induces oxidative stress in neuronal tissues nor alters the MnSOD expression and induction in the early step of the pathology (neuronal and cardiac) as previously suggested. We show that cytosolic ISC aconitase activity of iron regulatory protein-1 progressively decreases, whereas its apo-RNA binding form increases despite the absence of oxidative stress, suggesting that in a mammalian system the mitochondrial ISC assembly machinery is essential for cytosolic ISC biogenesis. In conclusion, our data demonstrate that in FRDA, mitochondrial iron accumulation does not induce oxidative stress and we propose that, contrary to the general assumption, FRDA is a neurodegenerative disease not associated with oxidative damage.

International audience; In a prospective, double-blind, randomised placebo-controlled study, we tested the hypothesis that a new formulation consisting of wheat gliadin chemically combined with a vegetal (thus orally effective) preparation of superoxide dismutase (SOD) allows to prevent hyperbaric oxygen (HBO)-induced oxidative cell stress. Twenty healthy volunteers were exposed to 100% oxygen breathing at 2.5 ATA for a total of 60 min. DNA strand breaks (tail moments) were determined using the alkaline version of the comet assay. Whole blood concentrations of reduced (GSH) and oxidised (GSSG) glutathione and F2-isoprostanes, SOD, glutathione peroxidase (GPx) and catalase (Cat) activities and red cell malondialdehyde (MDA) content were determined. After HBO exposure the tail moment (p = 0.03) and isoprostane levels (p = 0.049) were significantly lower in the group that received the vegetal formulation. Neither SOD and Cat nor GSH and GSSG were significantly affected by this preparation or HBO exposure. By contrast, blood GPx activity, which tended to be lower in the SOD-group already before the HBO exposure (p = 0.076), was significantly lower afterwards (p = 0.045). We conclude that an orally effective SOD-wheat gliadin mixture is able to protect against DNA damage, which coincided with reduced blood isoprostane levels, and may therefore be used as an antioxidant.

International audience; Hyperhomocysteinemia is often associated with an increase in blood pressure. However our previous study has shown that methionine supplementation induced an increase in blood pressure in Wistar-Kyoto (WKY) rats and a decrease in blood pressure in spontaneously hypertensive rats (SHR) with significant differences in plasma homocysteine (Hcy) metabolites levels. Previously liver antioxidant status has been shown to be decreased in SHR compared to WKY rats. It has been suggested that oxidative stress may predispose to a decrease in NO bioavailability and induce the flux of Hcy through the liver transsulfuration pathway. Thus the aim of this study was 1) to investigate the effect of methionine supplementation on NO-derived metabolites in plasma and urine 2) to investigate whether abnormalities in Hcy metabolism may be responsible for the discrepancies observed between WKY rats and SHR concerning blood pressure and 3) to investigate whether a methionine-enriched diet, differently modified plasma and liver antioxidant status in WKY rats an SHR. We conclude that the increase in blood pressure in WKY rats is related to high plasma cysteine levels and is not due to a decrease in NO bioavailability and that the decrease in blood pressure in SHR is associated with high plasma GSH levels after methionine supplementation. So GSH synthesis appears to be stimulated by liver oxidative stress and GSH is redistributed into blood in SHR. So the great GSH synthesis can be rationalized as an autocorrective response that leads to a decreased blood pressure in SHR.

Because oxidative stress is involved in arterial hypertension, impairment of hepatic antioxidant defences could develop in the course of this disease. Metallothionein (MT), an antioxidant protein, is present in high rates in the liver. The aim of this study was to investigate the effect of a mineralocorticoid-salt treatment on blood pressure, hepatic antioxidant enzyme activities, and cardiac MT levels in transgenic MT null mice compared with control mice to further clarify the role of MT during the experimental development of arterial hypertension. Control and transgenic MT –/– mice were submitted to an 8-week mineralocorticoid-salt treatment. Hepatic glutathione peroxidase, glutathione reductase, superoxide dismutase, and catalase activities and cardiac MT and mineral levels were measured. Mineralocorticoid-salt treatment induced an increase in blood pressure in both transgenic MT –/– and control mice that was associated with an impairment of liver antioxidant status. MT deficiency was associated with modifications of hepatic antioxidant enzyme activities and with a decrease in cardiac iron levels. Adaptive processes of antioxidant systems may explain the absence of an effect of metallothionein deficiency on the development of mineralocorticoid-salt hypertension. The interactions that occur between the in vivo antioxidant systems probably produce a complex regulation of the oxidative balance and consequently prevent antioxidant deficiency.Key words: hepatic antioxidant enzymes, metallothionein, transgenic mice, DOCA-salt hypertension.

International audience; Chemical preconditioning using the mitochondrial toxin, 3-nitropropionic acid (3-NP) has been reported to induce neuroprotection against subsequent global ischemia. To investigate the underlying mechanisms, Mongolian gerbils were pretreated with either vehicle or 3-NP at the dose of 3 or 10 mg/kg, intraperitoneal, 3 days prior to a 5-min bilateral carotid artery occlusion followed by either 48 h or 7 days of blood recirculation. Neuronal damage was assessed by a cresyl violet/fuchsin acid staining. Induction of heat shock protein 72 (HSP72) and manganese superoxide dismutase (MnSOD) expression was evaluated by Western blotting. Astroglial and microglial activation was detected by immunohistochemistry (glial fibrillary acid protein) and by histochemistry (isolectin B4 staining), respectively. Present data show that the hippocampal neuronal damage induced by ischemia were of similar extent between the vehicle- and 3-NP-treated gerbils, whatever the dose tested, indicating that 3-NP did not induce tolerance to transient forebrain ischemia under our experimental conditions. The lack of difference in the post-ischemic level of HSP72 and MnSOD protein expression and in the intensity of astroglial and microglial activation represents further indirect indications of the absence of 3-NP preconditioning effect. In conclusion, although chemical preconditioning with 3-NP is a well-established phenomenon at least in vitro and in models of focal ischemia, the relevance of 3-NP as a preconditioning molecule towards global brain ischemia remains an open question.

International audience; 1. Heat stress (HS) is known to protect the myocardium against ischaemic damage. It has been reported that reactive oxygen species (ROS) are abundantly produced during this stress. Since mechanisms triggering the HS-induced cardioprotection remain unknown, we investigated the role of ROS in the genesis of this protective phenomenon. 2. Rats were divided into four groups (n=8 in each group), subjected to either hyperthermia (42 degrees C internal temperature for 15 min) or sham anaesthesia and treated or not with N-2-mercaptopropionyl glycine (MPG), a synthetic antioxidant, 10 min before HS. Twenty-four hours later, their hearts were isolated, retrogradely perfused, and subjected to a 30-min occlusion of the left coronary artery followed by 120 min of reperfusion. Myocardial Hsp 27 and 70 expression was assessed by Western blot analysis (n=4). Cardiac activities of antioxidant enzymes (superoxide dismutase and glutathione peroxidase) were also examined (n=4). 3. Infarct-to-risk zone ratio was significantly reduced in HS (17+/-1.3%) compared to Sham (34.3+/-1.7%) hearts. This effect was abolished by MPG pretreatment (40.6+/-1.9% in HS+MPG vs. 39.8+/-2.5% in Sham+MPG hearts). This cardioprotection was associated with an enhanced Hsp 27 and 70 expression, which was not modified by MPG pretreatment. Antioxidant enzyme activities was not modified by heat stress or MPG pretreatment. 4. Free radical production following hyperthermia appears to play a role in the heat stress induced cardioprotection, independently of Hsp levels. Antioxidant enzyme activities do not seem to be implicated in this cardioprotective mechanism.

International audience; Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterised by selective degeneration of motoneurones. Familial ALS is an age-dependent autosomal dominant disorder in which mutations in the homodimeric enzyme Cu/Zn superoxide dismutase 1 (SOD1) is linked to the disease. An animal model for this disease is a transgenic mouse expressing the mutated human SOD1(G93A) gene. Recent electrophysiological data emphasised that the striking selective vulnerability of motoneurones might be due to their differential calcium buffering capacities. Therefore we have investigated, using immunohistochemistry, the expression of different calcium binding proteins in brainstem and spinal cord from normal and SOD1 mutated mice. Among the 13 calcium-binding proteins screened, only one, S100A6, a homodimeric calcium-binding protein able to bind four Zn(2+), appeared to be highly expressed in the SOD1 mutated mice. In brainstem, reactive astrocytes, but not motoneurones, from several regions, including nerve 12 root, were highly S100A6-positive. Hypoglossal nucleus was negative for S100A6. In dorsal root, reactive astrocytes from both white matter and anterior horn were highly reactive. If overexpression of S100A6 is specific for ALS, it will be a valuable diagnostic marker for this disease.

International audience; The effects of 8-week diethylmaleate (DEM) and clofibric acid (CFA) supplemented diet on blood pressure, body and liver weights, liver antioxidant status and nitric oxide synthase (NOS) activity were investigated in 8-week DOCA-salt treated and untreated Sprague-Dawley male rats. It appeared that DEM and particularly CFA treatments were associated with a significant decrease in blood pressure in DOCA-salt treated rats, and an accentuation of the decreases in body weights in both diet supplemented groups. This was not associated with increases in NO production in the liver. In contrast, hepatic lipid peroxidation was significantly decreased in both DOCA-salt treated and untreated groups on DEM and particularly on CFA supplemented diet. The protective effects of CFA and DEM against hepatic cellular damage could be involved in the decreases in blood pressure in DOCA-salt treated rats, where CFA was more efficient than DEM. In CFA supplemented groups, there was a strong increase in hepatic superoxide dismutase (SOD), glutathione-peroxidase (GSH-Px), and catalase (CAT) activities and in DEM supplemented groups, increases in SOD and CAT activities and in GSH levels were observed. Our data suggest that normalization of blood pressure in DOCA-salt treated rats by CFA was due to an enhancement of the half-life of NO while DEM increased its availability.

International audience; The erythroid differentiation of K562 cells could be achieved by exposure to several pharmacologic agents, including hemin, butyric acid (BA), and anthracycline antitumor drugs such as aclarubicin (ACLA) and doxorubicin (DOX). When used at subtoxic concentrations, these drugs induce the overexpression of erythroid genes, leading to hemoglobinization of cells. Because anthracyclines are known to generate oxidative damage, we intended to demonstrate the involvement of an oxidative stress in the chemically induced differentiation process. The addition of antioxidants to anthracycline- and BA-induced cells decreased their growth and dramatically reduced the percentage of differentiated cells at day 3. Northern blot analysis showed that antioxidants also decrease the expression of erythroid genes and related transcription factors in induced cells. Moreover, analyses of oxidative stress markers showed that treatment with BA, ACLA, and DOX lead to a decrease in reduced glutathione and antioxidant enzymes (glutathione peroxidase [GPx], glutathione reductase [GRase], CuZn superoxide dismutase [SOD], and catalase [CAT]). In addition, DOX increased thiobarbituric acid reactants (TBARs), and MnSOD activity was decreased by BA and DOX. Finally, the production of reactive oxygen species (ROS) by differentiating agents was demonstrated using the dihydroethidium probe in a microspectrofluorometric assay. Altogether, these results strongly suggest the involvement of an oxidative stress generated by BA or anthracyclines as the first step in the irreversible differentiation process. Additionally, these results underline the differences between BA, ACLA, and DOX molecular mechanisms.

When differentiated into mature macrophages by the combination of all-trans retinoic acid and 1,25-dihydroxyvitamin D3, the human promonocytic cell lines U937 and THP-1 expressed inducible nitric oxide synthase (iNOS) transcripts. During their differentiation, the cells acquired the capacity to produce not only superoxide anion (O2.-) but also nitric oxide (.NO) in response to IgG (or IgE)-opsonized zymosan. The inhibitors of the iNOS pathway, aminoguanidine and NG-monomethyl-L-arginine (L-NMMA), suppressed the production of .NO and enhanced the steady-state concentration of O2.- determined. Conversely, superoxide dismutase (SOD) scavenged the O2.- released and increased the .NO-derived nitrite concentration detected. These data suggested a possible interaction between O2.- and .NO. In differentiated U937 (or THP-1) cells, IgG or IgE-opsonized zymosan induced a strong time-dependent luminol-dependent chemiluminescence (LDCL), which was abrogated by SOD and partially inhibited by aminoguanidine or L-NMMA. Since the iNOS inhibitors did not directly scavenge O2.-, LDCL determination in the presence or absence of SOD and/or iNOS inhibitors demonstrated a concomitant production of O2.- and .NO. These radicals induced the formation of a .NO-derived product(s), probably peroxynitrite (ONOO-), which was required to elicit maximal LDCL. Finally, LDCL measurement provided a convenient tool to characterize iNOS triggering and demonstrated an interaction between NADPH oxidase and iNOS products in human macrophagic cells phagocytizing opsonized-zymosan. These findings show that in activated macrophages, iNOS activity can be involved in LDCL and support the debated hypothesis of iNOS participation to the microbicidal activity of human macrophages.

International audience; Dysregulation of free radical metabolism has been supposed to be involved in schizophrenia etiopathogeny. Recently, Wang et al. showed a red blood cell super oxide dismutase increase in positive schizophrenia (Crow's type I), but neither in negative schizophrenia (Crow's type II) nor in controls. The study included 28 in-patients suffering from acute positive psychosis who were compared with 15 controls. We confirmed the results of Wang. We found a significantly red blood cell Super oxide dismutase increase in positive psychosis, in comparison to negative psychosis and controls (p = 0.0001). This SOD increase was in relationship with the degree of clinical psychomotor excitement. After 21 days of neuroleptic treatment, SOD activity decreased and reached standard values. These results support the hypothesis of striking relationships between catecholaminergic hyper-metabolism and SOD increase, in positive psychosis. These could account for psychotic positive symptoms improvement with neuroleptic treatment, which blocks dopamine pathways.

International audience; Our aim was to investigate the role of oxidative stress in elastase-induced pulmonary emphysema. C57BL/6 mice were subjected to pancreatic porcine elastase (PPE) instillation (0.05 or 0.5 U per mouse, i.t.) to induce pulmonary emphysema. Lungs were collected on days 7, 14, and 21 after PPE instillation. The control group was sham injected. Also, mice treated with 1% aminoguanidine (AMG) and inducible NO synthase (iNOS) knockout mice received 0.5 U PPE (i.t.), and lungs were analyzed 21 days after. We performed bronchoalveolar lavage, biochemical analyses of oxidative stress, and lung stereology and morphometry assays. Emphysema was observed histologically at 21 days after 0.5 U PPE treatment; tissues from these mice exhibited increased alveolar linear intercept and air-space volume density in comparison with the control group. TNF-α was elevated at 7 and 14 days after 0.5 U PPE treatment, concomitant with a reduction in the IL-10 levels at the same time points. Myeloperoxidase was elevated in all groups treated with 0.5 U PPE. Oxidative stress was observed during early stages of emphysema, with increased nitrite levels and malondialdehyde and superoxide dismutase activity at 7 days after 0.5 U PPE treatment. Glutathione peroxidase activity was increased in all groups treated with 0.5 U PPE. The emphysema was attenuated when iNOS was inhibited using 1% AMG and in iNOS knockout mice. Furthermore, proteolytic stimulation by PPE enhanced the expression of nitrotyrosine and iNOS, whereas the PPE+AMG group showed low expression of iNOS and nitrotyrosine. PPE stimulus also induced endothelial (e) NOS expression, whereas AMG reduced eNOS. Our results suggest that the oxidative and nitrosative stress pathways are triggered by nitric oxide production via iNOS expression in pulmonary emphysema.

International audience; Though the role of Hedgehog (Hh) signalling in patterning and differentiation during development is well established, the underlying signal transduction mechanisms remain obscure. This is the first report on the overexpression of the human Hh signalling receptor Smoothened (hSmo) in Saccharomyces cerevisiae and Pichia pastoris. We show that hSmo is expressed in both types of yeast in its native conformational state. The first purification presented here will allow the characterisation of hSmo expressed in yeast, and the scale-up of hSmo production enabling structural studies to develop new therapeutic approaches against tumors and neurodegenerative diseases induced by Hh signalling dysfunction.

International audience; Hypoxic preconditioning has been shown to induce neuroprotection against a subsequent damaging insult. In order to study the underlying molecular and cellular mechanisms of hypoxic preconditioning, we investigated, in gerbil hippocampus, the effects in vivo of transient exposure to hypoxia (4% O(2) for 6 min followed by either 48 h or 7 days of reoxygenation) (i) on the induction of 72 kDa heat shock protein (HSP72), heme oxygenase-1 (HO-1) and manganese superoxide dismutase (Mn SOD) as assessed by Western immunoblotting and (ii) on the astroglial and microglial activation as detected by both immunohistochemistry and Western immunoblotting for GFAP, and histochemistry for isolectin B4, respectively. Our data show that, although hypoxia and subsequent reoxygenation led to neither neuronal damage nor HSP72 induction in gerbil hippocampus, it induced a progressive and sustained expression of HO-1 and Mn SOD. As expected from the absence of neuronal death, hypoxia was not associated with microglial activation but led to a significant astrocytic activation. These findings demonstrate that transient hypoxia enhances the antioxidative enzymatic defenses of the brain, which are susceptible to increased tolerance against a subsequent damaging insult.

The reasons for the cellular specificity and slow progression of motoneuron diseases such as ALS are still poorly understood. We previously described a motoneuron-specific cell death pathway downstream of the Fas death receptor, in which synthesis of nitric oxide (NO) is an obligate step. Motoneurons from ALS model mice expressing mutant SOD1 showed increased susceptibility to exogenous NO as compared with controls. Here, we report a signaling mechanism whereby NO leads to death of mutant, but not control, motoneurons. Unexpectedly, exogenous NO triggers expression of Fas ligand (FasL) in cultured motoneurons. In mutant SOD1 G93A and SOD1 G85R , but not in control motoneurons, this up-regulation results in activation of Fas, leading through Daxx to phosphorylation of p38 and further NO synthesis. This Fas/NO feedback amplification loop is required for motoneuron death in vitro . In vivo , mutant SOD1 G93A and SOD1 G85R mice show increased numbers of positive motoneurons and Daxx nuclear bodies weeks before disease onset. Moreover, FasL up-regulation is reduced in the presence of transgenic dominant-negative Daxx. We propose that chronic low-level activation of the Fas/NO feedback loop may underlie the motoneuron loss that characterizes familial ALS and may help to explain its slowly progressive nature.