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Biomedical subjects

C Lemaire

Publications and source records attributed to C Lemaire.

At least 37 records · Page 2Linked to original sources
PubMed1999

Specific dual effect of cycloheximide on B lymphocyte apoptosis: involvement of CPP32/caspase-3.

Cycloheximide (CHX) is known to stimulate or to prevent apoptosis, according to the cell type used. We found that CHX, in a dose-dependent way, exerted the two opposite effects in B lymphocytes. CHXhigh (2.5 microg/mL) inhibited protein synthesis (>90%) and greatly increased B cell apoptosis but failed to prevent apoptosis induction by dexamethasone (DEX) or dibutyryl-cAMP (dbcAMP), which is in opposition with CHX activity in thymocytes. On the contrary, CHXlow (0.05 microg/mL), modestly inhibited protein synthesis (<15%) and reduced spontaneous as well as drug-induced apoptosis and further augmented the protection conferred by interleukin-4 or lipopolysaccharide. To examine the role of caspases in CHX effects, we used the broad spectrum peptide caspase inhibitor Z-VAD-fmk: it totally abrogated spontaneous as well as drug- and CHXhigh-induced cell death. Apoptosis was associated with CPP32/caspase-3 activation, since cleavage of CPP32/caspase-3 and caspase-3 activity were increased by DEX, dbcAMP as well as by CHXhigh treatment. Meanwhile, caspase-3 activity was reduced by CHXlow treatment. Therefore, CHX exerts opposite effects on B lymphocyte apoptosis which are associated with a dual action on caspase-3 activation.

Animals↗
PubMed1999

Comparison of impaired subcortico-frontal metabolic networks in normal aging, subcortico-frontal dementia, and cortical frontal dementia.

Normal aging, progressive supranuclear palsy (PSP), and frontotemporal dementia (FTD) are characterized by different degrees of decline in frontal lobe functions. We used (18)FDG-PET and statistical parametric mapping (SPM96) to compare relative subcorticofrontal metabolic impairment at rest in 21 healthy elderly subjects (HES), 20 PSP patients, and 6 FTD patients. When HES were compared to 22 healthy young subjects, widespread decrease in metabolism was observed in bilateral medial prefrontal areas including anterior cingulate cortices, in dorsolateral prefrontal areas, in left lateral premotor area, in Broca's area, and in left insula. In PSP compared to the 43 healthy subjects (HS), we observed subcorticofrontal metabolic impairment including both motor and cognitive neural networks. Impairment of functional connections between midbrain tegmentum and cerebellar, temporal and pallidal regions was demonstrated in PSP as compared to HS. When comparing FTD to HS, glucose uptake was primarily reduced in dorsolateral and ventrolateral prefrontal cortices and in frontopolar and anterior cingulate regions. There was also bilateral anterior temporal, right inferior parietal, and bilateral striatal hypometabolism. Finally, FTD showed more severe striatofrontal metabolic impairment than PSP, while mesencephalothalamic involvement was only observed in PSP. Our data suggest that subcorticofrontal metabolic impairment is distributed in distinct subcorticocortical networks in normal aging, PSP, and FTD. Subcorticofrontal dementia in PSP is related to hypometabolism in discrete frontal areas, which are probably disconnected from certain subcortical structures. The concept of subcortical dementia is reinforced by our data, which show disrupted functional connections between mesencephalon and cerebellar cortex, inferior and medial temporal regions, and pallidum.

Adult↗
PubMed1999

Fluorodopa uptake and glucose metabolism in early stages of corticobasal degeneration.

Fluorodopa (FDOPA) and fluorodeoxyglucose (FDG) PET was performed in six patients in early stages of corticobasal degeneration (CBD) and compared to Parkinson's disease (PD) patients with a similar degree of bradykinesia and rigidity and to healthy controls. Statistical parametric mapping analysis comparing CBD to controls showed metabolic decrease in premotor, primary motor, supplementary motor, primary sensory, prefrontal, and parietal associative cortices, and in caudate and thalamus contralateral to the side of clinical signs. Except for the prefrontal regions a similar metabolic pattern was observed when CBD was compared to PD. Putamen FDOPA uptake was decreased in both CBD and PD. Caudate FDOPA uptake in CBD patients was decreased contralateral to clinical signs when compared to controls, but was higher than in PD. In early stages of CBD, FDOPA and FDG PET patterns differed from those observed in PD. In CBD the asymmetry in FDOPA uptake was less pronounced than that of clinical signs or metabolic impairment.

Aged↗
PubMed1999

IL-4 inhibits apoptosis and prevents mitochondrial damage without inducing the switch to necrosis observed with caspase inhibitors.

We previously demonstrated that the broad-spectrum caspase inhibitor, zVAD-fmk, totally deviated apoptosis to necrosis in B lymphocytes. We report here that, in contrast with zVAD-fmk, IL-4 protected B cells from spontaneous and from dexamethasone-induced apoptosis and actually maintained cell viability. This was assessed by morphological and biochemical criteria and accompanied by the maintenance of mitochondrial transmembrane potential (DeltaPsiCm) and elevated glutathione (GSH) levels. Under these conditions, zVAD-fmk also totally inhibited apoptosis in thymocytes, but it partly preserved cell viability with a parallel increase in the percentage of cells exhibiting high DeltaPsiCm and elevated GSH levels. Nevertheless, non-rescued cells were deviated to necrosis. Therefore, the pathway leading to either apoptosis or necrosis appears to involve common mitochondrial dysfunctions which could not be reversed by caspase inhibition, suggesting that the pharmacological inhibition of cell death should occur at an earlier stage.

Amino Acid Chloromethyl Ketones↗
PubMed1998

Age-associated modulation of apoptosis and activation in murine B lymphocytes.

We have investigated the influence of age on B-cell responsiveness. The present study showed that the B-cell mitogen, lipopolysaccharide (LPS), similarly stimulated the proliferation of purified B lymphocytes obtained from either young mice (3 months) or old mice (24 months). In contrast, expression of the differentiation marker, alkaline phosphatase (ALP), was about fourfold higher in young mice than in older mice upon stimulation with LPS or with dextran sulfate (DXS) and interleukin-5 (IL-5). The occurrence of apoptosis during aging was then studied: unexpectedly, spontaneous cell death was double in B lymphocytes from young mice compared to older animals. Stimulation with DXS with or without IL-5 rescued B lymphocytes from cell death in young mice but protection decreased with aging, and no longer occurred in 24-month-old mice B cells. Meanwhile, the protective activity conferred by IL-4 was maintained at similar levels throughout aging. However, B cells from old mice were more responsive to apoptosis induction with cycloheximide, dibutyryl cAMP and dexamethasone. Together, the present results indicate an age-associated alteration in apoptosis and activation of B lymphocytes which could contribute to the age-related decline of the immune response.

Aging↗
PubMed1998

Inhibition of caspase activity induces a switch from apoptosis to necrosis.

The role of caspases in B lymphocyte cell death was investigated by using two broad spectrum inhibitors of the caspase family, Z-Asp-cmk and Z-VAD-fmk. They totally prevented spontaneous and drug-induced apoptosis and inhibited the CPP32/caspase-3-like activity exhibited by apoptotic cells. However, the suppression of apoptosis was not associated with a long-term increase of cell survival, but conversely, with a switch from apoptotic death to the necrotic form. These results strongly suggest that apoptosis and necrosis share common initiation pathways, the final issue being determined by the presence of an active caspase.

Amino Acid Chloromethyl Ketones↗
PubMed1998

Molecular and biochemical analysis of Saccharomyces cerevisiae cox1 mutants.

We report on the molecular and biochemical analysis of a set of 13 respiratory deficient mutants of Saccharomyces cerevisiae which are specifically altered in COX1, the gene encoding the subunit Cox1p of cytochrome c oxidase. DNA sequence analysis shows that three are due to frameshift mutations, two to nonsense mutations, and eight to missense mutations. All, except the missense mutant S157L, have impaired electron transfer and respiratory activity. Analysis of the mitochondrial translation products shows that when Cox1p is absent, Cox2p and Cox3p are still synthesized. In the missense mutants, the steady state levels in the mitochondrial membranes of the three mitochondrially encoded subunits Cox1p, Cox2p and Cox3p and the nuclear-encoded subunit Cox4p are reduced. In the frameshift and nonsense mutants, Cox1p is absent and Cox2p, Cox3p and Cox4p are considerably decreased or undetectable. A comparison of the steady state levels of Cox1p through Cox4p in the COX1, COX2, COX3 and COX4 mutants shows the interdependance of the accumulation of these four subunits in the mitochondrial membranes.

Amino Acid Sequence↗
PubMed1998

Induction of apoptosis by dexamethasone in the B cell lineage.

The susceptibility to induction of apoptosis by the synthetic glucocorticoid, dexamethasone (Dex), was analysed at different stages of B cell maturation. Cells of the 70Z/3 pre-B cell line, expressing cytoplasmic mu chains, and LPS-stimulated 70Z/3 cells, expressing surface IgM, were used as a model of differentiation of pre-B cells into immature B cells. Cell proliferation and cell cycle progression were similarly inhibited by Dex (100 nM) in both naive 70Z/3 pre-B cells and in LPS-stimulated 70Z/3 cells. In contrast, Dex failed to affect apoptosis of naive 70Z/3 cells while it increased that of LPS-stimulated 70Z/3 cells. Splenic mature B lymphocytes were highly susceptible to Dex-induced apoptosis since subphysiological doses (5 nM) increased the frequency of apoptotic cells to more than 80%. On the other hand, the treatment of B lymphocytes with LPS, which led to proliferation and differentiation into immunoblasts, decreased the susceptibility to Dex-induced apoptosis. These effects were mediated by the glucocorticoid receptor since they were abrogated by the RU 486 antagonist. The response of B cells to glucocorticoids is thus dependent on their stage of differentiation.

Animals↗
PubMed1998

[Bronchial lobar obstruction from a sucralfate tablet: a rare cause of acute respiratory insufficiency].

We report two cases of patients with chronic respiratory disease who experienced an asphyxia after aspirating a sucralfate tablet that occluded a lobar bronchus. In adults, a foreign body is a rare cause of acute respiratory failure from tracheobronchial occlusion. The sucralfate tablet has the physical property of expanding rapidly when wet (contact with mucosa). After aspiration, the tablet expands to a larger size and can occlude a lobar bronchus, causing acute respiratory failure. In patients at risk of aspiration, we recommend the use of sucralfate in liquid or suspension form.

Aged↗
PubMed1998

High-yield radiosynthesis and preliminary in vivo evaluation of p-[18F]MPPF, a fluoro analog of WAY-100635.

No-carrier-added 4-[18F]fluoro-N-[2-[1-(2-methoxyphenyl)-1 piperazinyl]ethyl-N-2-pyridinyl-benzamide (p-[18F]MPPF) was synthesized by nucleophilic substitution of the corresponding nitro compound in the presence of Kryptofix 222 and K2CO3 by microwave heating (3 min, 500 W) using a remotely controlled radiosynthesis. Baseline separation of p-[18F]MPPF from the nitro derivative was performed on a semipreparative HPLC C18 column. After Sep-Pak formulation, the radiopharmaceutical was obtained with a radiochemical yield of 25% (EOS) in about 70 min. Specific radioactivity averaged between 1-5 Ci/micromol EOS. Labelling of the ortho and meta derivatives was also attempted. Brain uptake of p-[18F]MPPF was studied with PET on fluothane-anesthetized cats. Following intravenous injection of p-[18F]MPPF, high accumulation of radioactivity was observed in the hippocampus and cerebral cortex. Low levels of radioactivity were observed in cerebellum. At 30 min, the mean hippocampus/cerebellum and cortex/cerebellum ratios were 5 and 3.8, respectively. The accumulation of the tracer was blocked by prior administration of reference WAY-100635, demonstrating the specificity of the ligand.

Animals↗
PubMed1998

Mutations in the membrane anchor of yeast cytochrome c1 compensate for the absence of Oxa1p and generate carbonate-extractable forms of cytochrome c1.

Oxa1p is a mitochondrial inner membrane protein that is mainly required for the insertion/assembly of complex IV and ATP synthase and is functionally conserved in yeasts, humans, and plants. We have isolated several independent suppressors that compensate for the absence of Oxa1p. Molecular cloning and sequencing reveal that the suppressor mutations (CYT1-1 to -6) correspond to amino acid substitutions that are all located in the membrane anchor of cytochrome c1 and decrease the hydrophobicity of this anchor. Cytochrome c1 is a catalytic subunit of complex III, but the CYT1-1 mutation does not seem to affect the electron transfer activity. The double-mutant cyt1-1,164, which has a drastically reduced electron transfer activity, still retains the suppressor activity. Altogether, these results suggest that the suppressor function of cytochrome c1 is independent of its electron transfer activity. In addition to the membrane-bound cytochrome c1, carbonate-extractable forms accumulate in all the suppressor strains. We propose that these carbonate-extractable forms of cytochrome c1 are responsible for the suppressor function by preventing the degradation of the respiratory complex subunits that occur in the absence of Oxa1p.

Adenosine Triphosphatases↗
PubMed1998

Towards a recombinant vaccine against diphtheria toxin.

Two recombinant fragments of diphtheria toxin (DT) were fused to an engineered tandem repeat of the immunoglobulin (Ig) binding domain of protein A, called ZZ. These fragments are (i) the receptor binding domain (DTR), which comprises amino acids 382 to 535 of DT, and (ii) a linear peptide (DT(168-220)) which comprises residues 168 to 220 of the loop between fragment A and fragment B of DT. The fusion proteins were produced in Escherichia coli and purified by affinity chromatography. In vitro experiments showed that the DTR domain is responsible for the capacity of ZZ-DTR to bind to Vero cells and is capable of inhibiting the cytotoxicity of DT for these cells. These findings suggest that DTR binds to the cell surface receptors of DT and hence adopts a conformation that is similar to that of the receptor binding domain of DT. We compared the capacities of ZZ-DTR, ZZ-DT(168-220), and a chemically detoxified form of DT currently used for vaccination to elicit antibodies in rabbits. The toxoid was more immunogenic than ZZ-DT(168-220), which in turn was more immunogenic than ZZ-DTR. However, ZZ-DT(168-220) antiserum was poorly efficient at neutralizing DT cytotoxicity on Vero cells, whereas ZZ-DTR antiserum was only 15-fold less potent than anti-DT antisera.

Animals↗
PubMed1998

Inhibition of thrombin-catalyzed factor V activation by bothrojaracin.

We have previously identified and characterized a potent and specific thrombin inhibitor, isolated from Bothrops jararaca, named bothrojaracin. Bothrojaracin interacts with the two positively charged recognition sites of thrombin referred to as exosite 1 and exosite 2, whereas it does not interact with the thrombin active site. Consequently, bothrojaracin inhibits thrombin-induced fibrinogen to fibrin conversion and platelet activation, without inhibition of thrombin-catalyzed cleavage of small synthetic substrates. In the present study, we show that bothrojaracin exerts an anticoagulant effect in plasma, illustrated by the prolongation of the aPTT. Using purified proteins, we observed that the anticoagulant effect of bothrojaracin was not only due to the inhibition of fibrinogen to fibrin conversion, but in addition to the inhibition of factor V activation by thrombin. Bothrojaracin decreased the rate of thrombin-catalyzed proteolysis of factor V and concurrently the generation of factor Va cofactor activity measured in a prothrombinase assay. We compared the effect of bothrojaracin with that of ligands binding specifically exosite 1 (hirudin C-terminal peptide SH54-65) or exosite 2 (heparin, prothrombin fragment 2). SH54-65 delayed thrombin catalyzed factor V activation whereas heparin or prothrombin fragment 2 did not. The thrombin derivatives beta- and gamma-thrombin, which are defective in their exosite 1, but present with a normally exposed exosite 2, had a reduced capacity to activate factor V, which was not further impaired by the exosite 2 ligands, bothrojaracin, heparin or prothrombin fragment 2. Altogether, our results provide further insight into the anticoagulant effect of bothrojaracin showing that it is a potent inhibitor of the feedback activation of factor V by thrombin, and thus of the up-regulation of its own production by thrombin. Inhibition of thrombin-catalyzed factor V activation by bothrojaracin is mainly mediated through the interaction of the inhibitor with thrombin exosite 1, whereas contribution of the interaction with exosite 2 does not appear to play a direct role in factor V recognition by thrombin.

Anticoagulants↗
PubMed1997

High-level production and isotope labeling of snake neurotoxins, disulfide-rich proteins.

The aim of this work was to produce and to label snake neurotoxins, disulfide-rich proteins. A mutant of a snake toxin, erabutoxin a, was used as a model. Its N-terminal part was fused to ZZ, a synthetic IgG-binding domain of protein A (B. Nilsson et al., 1987, Protein Eng. 1, 107-113), thus preventing degradation in the bacterial cytoplasm and providing a simple affinity-purification method on IgG Sepharose. A soluble fusion protein was obtained with a yield of 60 mg/L, corresponding to 20 mg/L toxin. The toxin moiety was folded on the column while the hybrid was still bound. The oxidoreducing conditions for the refolding were optimized and were found to be oxidative but with a need for reducing molecules. The concentration of the hybrid bound to the column could be increased up to 3.3 mg/ml without significantly altering the folding process. CNBr cleavage of the fusion protein followed by a purification step yielded about 2 mg of biologically active toxin mutant per gram of dry cell weight. This procedure was applied to produce 55 mg of a toxin uniformly labeled with 15N.

Binding, Competitive↗
PubMed1997

Expression of a Schistosoma mansoni 28-kilodalton glutathione S-transferase in the livers of transgenic mice and its effect on parasite infection.

Schistosomiasis is a debilitating tropical disease for which an effective vaccine is needed. A 28-kDa glutathione S-transferase from Schistosoma mansoni (Sm28GST) has been shown to induce protective immunity. Sm28GST possesses significant sequence identity to mammalian GST isoforms. In order to study self-reactivity in mice immunized with Sm28GST and the concomitant phenomena of immune tolerance and epitope suppression, as well as their consequences for the protective immunity induced by this vaccination, we developed transgenic (Tg) mice that express Sm28GST under the control of a part of the mouse transferrin gene promoter. A study of (P28)Tg mice showed that the expression of Sm28GST was strictly localized in pericentrolobular hepatocytes. No histological change, inflammatory infiltrates, or modification of seric L-aspartate: 2-oxoglutarate aminotransferase concentration was observed over an 18-month period, despite a cross-reactivity between Sm28GST and a mouse molecule of 30 kDa. The immunoglobulin G anti-Sm28GST response of (P28)Tg mice immunized with recombinant Sm28GST was lower (P < 0.001) than that observed in non-(P28)Tg littermates and inversely proportional of Sm28GST liver expression. The response of non-(P28)Tg mouse spleen cells to Sm28GST stimulation was greater (P < 0.01) than that observed with (P28)Tg mouse spleen cells. (P28)Tg mice infected with 40 S. mansoni furcocercariae harbored more worms (P < 0.05) than did non-(P28)Tg control mice. The increase in the level of infection in (P28)Tg mice was reflected in concomitant increases in the numbers of adult worms and schistosome eggs found in livers and intestines after whole-body perfusion at 56 days postinfection, but no relative increase in the fertility of individual female worms was observed. The results obtained argue for the involvement of Sm28GST in reducing levels of infection and support the view that this enzyme has a central role in the maintenance of parasite viability, at least during its migration through host tissues.

Animals↗
PubMed1997

[Endovascular closure of a foramen ovale after a right pneumonectomy].

We report the case of a patient, 62-year-old, with a non small cell lung cancer treated by right pneumonectomy followed by chemo and radiotherapy. After surgery appeared a refractory hypoxemia increasing with supine position. Cardiac catheterism showed a right left shunt by reopening of the "foramen ovale". We have performed foramen's occlusion by endovascular method with prothetic material with good result until the death, 14 months later, by neoplasic evolution.

Angioplasty↗
PubMed1997

UV irradiation of a B-cell hybridoma increases expression of alkaline phosphatase: involvement in apoptosis.

Expression of alkaline phosphatase (APase) by 7TD1 B-cell hybridoma was amplified by ultraviolet irradiation; cell growth was inhibited and cell death by apoptosis was increased. Irradiation induced high levels of APase activity in cycling as well as in apoptotic cells. In contrast, APase activity faded with time in nonirradiated cells and was no longer expressed in spontaneous apoptotic cells appearing after several days in culture. This was demonstrated by cell morphology, DNA fragmentation, and flow cytometry after simultaneous staining of DNA with Hoechst 33342 and APase with naphthol AS-TR phosphate--fast red RC fluorescent reagent. Levamisole, a specific inhibitor of APase activity, almost totally abrogated apoptosis induced by ultraviolet irradiation at doses that failed to affect 7TD1 cell survival. These data suggest that APase could play a role in the signalling cascade that mediates apoptosis in irradiated cells.

Alkaline Phosphatase↗