Clostridium perfringens epsilon toxin rapidly decreases membrane barrier permeability of polarized MDCK cells

Epsilon toxin is produced by Clostridium perfringens types B and D which are responsible for fatal intestinal diseases in animals. The main biological activity of epsilon toxin is the production of oedema in various organs. We have previously found that epsilon toxin forms a large membrane complex in MDCK cells which is not internalized into cell, and induces cell volume enlargement and loss of cell viability (Petit, L., Gibert, M., Gillet, D., Laurent-Winter, C., Boquet, P., Popoff, M. R. (1997) J Bacteriol 179, 6480-6487). Here, we show that epsilon toxin is very potent to decrease the trans-epithelial electrical resistance of polarized MDCK cells grown on filters without altering the organization of the junctional complexes. The dose-dependent decrease in trans-epithelial electrical resistance, more marked when the toxin was applied to the apical side than to the basal side of MDCK cells, was associated with a moderate increase of the paracellular permeability to low-molecular-weight compounds but not to macromolecules. Epsilon toxin probably acts by forming large membrane pores which permit the flux of ions and other molecules such as the entry of propidium iodide and finally to the loss of cell viability.     

Comparison of protein patterns of Listeria monocytogenes grown in biofilm or in planktonic mode by proteomic analysis

The proteome of a Listeria monocytogenes strain isolated from a food plant was investigated to study the differential protein pattern expressed by biofilms and planktonic bacteria. The approach used in this study was a combination of two-dimensional electrophoresis, matrix-assisted laser desorption ionization-time of flight and database searches for the protein identification. Thirty-one proteins varied significantly between the two growth conditions. Twenty-two and nine proteins were up- and down-regulated respectively and nine proteins were successfully identified. The variations of the protein patterns indicated that the biofilm development is probably controlled by specific regulation of protein expression involved at various levels of cellular physiology.     

Proteome analysis of the plasma membrane of Mycobacterium tuberculosis

The plasma membrane of Mycobacterium tuberculosis is likely to contain proteins that could serve as novel drug targets, diagnostic probes or even components of a vaccine against tuberculosis. With this in mind, we have undertaken proteome analysis of the membrane of M. tuberculosis H37Rv. Isolated membrane vesicles were extracted with either a detergent (Triton X114) or an alkaline buffer (carbonate) following two of the protocols recommended for membrane protein enrichment. Proteins were resolved by 2D-GE using immobilized pH gradient (IPG) strips, and identified by peptide mass mapping utilizing the M. tuberculosis genome database. The two extraction procedures yielded patterns with minimal overlap. Only two proteins, both HSPs, showed a common presence. MALDI-MS analysis of 61 spots led to the identification of 32 proteins, 17 of which were new to the M. tuberculosis proteome database. We classified 19 of the identified proteins as 'membrane-associated'; 14 of these were further classified as 'membrane-bound', three of which were lipoproteins. The remaining proteins included four heat-shock proteins and several enzymes involved in energy or lipid metabolism. Extraction with Triton X114 was found to be more effective than carbonate for detecting 'putative' M. tuberculosis membrane proteins. The protocol was also found to be suitable for comparing BCG and M. tuberculosis membranes, identifying ESAT-6 as being expressed selectively in M. tuberculosis. While this study demonstrates for the first time some of the membrane proteins of M. tuberculosis, it also underscores the problems associated with proteomic analysis of a complex membrane such as that of a mycobacterium.     

On the interchain associations in aqueous solutions of a succinoglycan polysaccharide

Formation of interchain associations between succinoglycan chains have been studied by comparing weight average molecular weight, intrinsic viscosity of succinoglycan as a function of the conditions to prepare the solutions (polymer concentration, the heating temperature adopted compared with T_m). The different solutions obtained were characterized by their Newtonian viscosity, the storage and loss moduli and their sensitivity to the temperature. It was found that interchain associations, first stabilized mainly during the disorder-order transition convert to more stable associations by aging at temperatures below but not too far from T_m. These associations appear from succinoglycan solutions characterized by an overlap parameter higher than about 8 and modify only very slightly the conformational transition parameters obtained from microcalorimetry measurements.     

Deletion of EFL1 results in heterogeneity of the 60 S GTPase-associated rRNA conformation

Previous work suggested that the release of the nucleolar Tif6 from nascent 60 S subunits occurs in the cytoplasm and requires the cytoplasmic EF-2-like GTPase, Efl1. To check whether this release involves an rRNA structural rearrangement mediated by Efl1, we analyzed the rRNA conformation of the GTPase center of 80 S ribosomes in three contexts: wild-type, Deltaefl1 and a dominant suppressor R1 of Deltaefl1. This analysis was restricted to domain II and VI of 25 S rRNA. The rRNA analysis of R1 ribosomes allows us to distinguish the effects due to depletion of Efl1 from the resulting nucleolar deficit of Tif6. Efl1 inhibits the EF-2 GTPase activity, suggesting that the two proteins share a similar ribosome-binding site. The 80 S ribosomes from either type failed to show any difference of conformation in the two rRNA domains analyzed. However, the same analysis performed on the pool of free 60 S subunits reveals several rRNA conformational differences between wild-type and Deltaefl1 subunits, whereas that from the suppressor strain is similar to wild-type. This suggests that the nucleolar deficit of Tif6 during assembly of the 60 S preribosomes is responsible for the changes in rRNA conformation observed in Deltaefl1 60 S subunits. We also purified 60 S preribosomes from the three genetic contexts by TAP-tagging Tif6. The protein content of 60 S preribosomes associated with Tif6p in a Deltaefl1 strain are obtained at a lower yield but have, surprisingly, a protein composition that is a priori similar to that of wild-type and the suppressor strain.     

Découverte de charophytes et ostracodes de l’Yprésien inférieur dans les Monts des Ksour (Algérie) : biostratigraphie et paléoécologie

A stratigraphic, palaeontological and sedimentological study was carried out in the red beds cropping out on the left side of Oued Tafarahit, south-east of the Ksour Mountains (Algeria). The studied succession consists of a non-marine fining-upward detrital formation, including microconglomerate, sandstone and clay beds, which were previously attributed to the Cenozoic sensu lato, since no palaeontological evidence was available. Sedimentology suggests deposition in a fluvial environment. The clayey levels yielded the assemblage formed by the charophytes Peckichara atlasensis, Maedleriella cristellata, Nitellopsis (Tectochara) thaleri, Grovesichara sp., and Lamprothamnium papulosum and the ostracods Neocyprideis meguerchiensis, Herpetocypris? sp. and Cyprinotus? sp. This assemblage allows constraining the age of the detrital series from Oued Tafarahit to the Ypresian. Ostracods are typical of freshwater to euryhaline environments. The freshwater taxa (Herpetocypris? sp. and Cyprinotus? sp.) would indicate phases of desalinisation during periods of flood. However, the brackish water species (Neocyprideis meguerchiensis) is characteristic of saline phases related to low water table periods. Moreover the charophyte and ostracod assemblage confirms a close palaeobiogeographic relationship between North Africa and Southern Europe during the Lower Eocene.     

Isolation, Functional Characterization and Proteomic Identification of CC2-PLA2 from Cerastes cerastes Venom: A Basic Platelet-Aggregation-Inhibiting Factor

Three-step chromatography and proteomic analysis have been used to purify and characterize a new basic phospholipase A₂ named CC2-PLA₂ from the venom of Cerastes cerastes. This phospholipase A₂ has been isolated to an extent of about 50-folds and its molecular weight was estimated at 13,534 Da. For CC2-PLA₂ identification and LC-MALDI-MS/MS analysis, the protein was reduced, alkylated and double hydrolyzed by lysine-C endopeptidase and trypsin. Tryptic fragments of LC–MS/MS analyzed CC2-PLA₂ showed sequence similarities with other snake venom PLA₂. This presents only 51 % (61/120 amino acid residues) sequence homology with the first PLA₂ (gi |129506|) previously purified from the same venom. The isolated CC2-PLA₂ displayed anti-aggregative effect on platelets and induced an inflammatory response characterized by leukocytosis in the peripheral blood. This inflammatory response is accompanied by a release of inflammatory mediators such as IL-6, eosinophil peroxidase and complement system. Obtained results indicate that CC2-PLA₂ induced a release of high level of pro-inflammatory (IL-6) cytokine and no effect on the level of anti-inflammatory cytokine (IL-10) in blood sera. Furthermore, eosinophil peroxidase activity and hemolytic complement effect increased in peripheral blood. Mononuclear and neutrophil cells were found predominant in the induced leucocytosis following CC2-PLA₂ administration into animals.     

Tempol,a Superoxide Dismutase Mimetic Agent,Ameliorates Cisplatin-Induced Nephrotoxicity through Alleviation of Mitochondrial Dysfunction in Mice

BackgroundMitochondrial dysfunction is a crucial mechanism by which cisplatin, a potent chemotherapeutic agent, causes nephrotoxicity where mitochondrial electron transport complexes are shifted mostly toward imbalanced reactive oxygen species versus energy production. In the present study, the protective role of tempol, a membrane-permeable superoxide dismutase mimetic agent, was evaluated on mitochondrial dysfunction and the subsequent damage induced by cisplatin nephrotoxicity in mice.ConclusionThis study highlights the potential role of tempol in inhibiting cisplatin-induced nephrotoxicity without affecting its antitumor activity via amelioration of oxidative stress and mitochondrial dysfunction.