Construction ofClostridium butyricum hybrid plasmids and transfer toBacillus subtilis

Summary Small plasmids were isolated from type strains ofClostridium butyricum. Strain NCIB 7423 carries one plasmid (pCBU1) of 6.4 kb, whereas strain NCTC 7423 carries two unrelated plasmids of 6.3 kb (pCBU2) and 8.4 kb (pCBU3). Cleavage sites for 18 restriction endonucleases have been mapped on these plasmids and detailed physical maps are presented. For the purpose of developing vector plasmids for gene cloning in saccharolytic clostridia these crypticC. butyricum plasmids were joined to a selectable marker that will likely be expressed in clostridia. This was achieved by cloning the clostridial plasmids into theE. coli vector pBR322 carrying the chloramphenicol acetyltransferase (CAT) gene from theStaphylococcus aureus plasmid pC194. The recombinant plasmids were tested for their ability to confer chloramphenicol resistance toBacillus subtilis. Hybrid plasmids (pHL105, pHL1051) derived from pCBU2 were identified, which are capable of replication and expression of theS. aureus drug resistance marker in bothE. coli andB. subtilis. No structural instability was detected upon retransformation of pHL105 fromB. subtilis intoE. coli. The recombinant plasmids might thus be useful as shuttle vectors for the gene transfer betweenE. coli and a wide range of bacilli and clostridia.     

Growth of an anaerobic sulfate-reducing bacterium sustained by oxygen respiratory energy conservation after O2-driven experimental evolution

Desulfovibrio species are representatives of microorganisms at the boundary between anaerobic and aerobic lifestyles, since they contain the enzymatic systems required for both sulfate and oxygen reduction. However, the latter has been shown to be solely a protective mechanism. By implementing the oxygen-driven experimental evolution of Desulfovibrio vulgaris Hildenborough, we have obtained strains that have evolved to grow with energy derived from oxidative phosphorylation linked to oxygen reduction. We show that a few mutations are sufficient for the emergence of this phenotype and reveal two routes of evolution primarily involving either inactivation or overexpression of the gene encoding heterodisulfide reductase. We propose that the oxygen respiration for energy conservation that sustains the growth of the O₂-evolved strains is associated with a rearrangement of metabolite fluxes, especially NAD⁺/NADH, leading to an optimized O₂ reduction. These evolved strains are the first sulfate-reducing bacteria that exhibit a demonstrated oxygen respiratory process that enables growth.     

Effects of water-deficit on lipids of safflower aerial parts

Three-week-old plants of safflower (Carthamus tinctorius L.) were subjected to a water-deficit stress. The lipid composition of the shoot parts of both control (well-watered) and water stressed plants was analyzed. Experimental data revealed that moderate stress induced an increase in total lipid content within all lipidic classes. However, severe water-deficit induced a sharp decrease in the total lipid content and specially in polar lipids, particularly in phosphatidylethanolamine, phosphatidylcholine, monogalactosyl-diacylglycerol and digalactosyl-diacylglycerol. Also, the content of neutral lipids was increased. Concerning the fatty acid composition, water-deficit induced a decrease in their degree of unsaturation expressed by a reduction in the proportions of linolenic (18:3) and linoleic (18:2) acids and most of lipidic classes.     

mau-2 acts cell-autonomously to guide axonal migrations in Caenorhabditis elegans

The gene mau-2 has been found to be required for the guidance of cellular and axonal migrations along both the anteroposterior and the dorsoventral body axes during the development of the nematode C. elegans. We show that mau-2 encodes a novel, previously uncharacterized protein that is highly conserved among animals. Maternal mau-2 gene expression is sufficient for normal development until the fourth larval stage, and a MAU-2::GFP fusion protein localizes to the cytoplasm of neurones. mau-2 is ubiquitously expressed in embryos by late gastrulation and becomes predominantly expressed in the nervous system as morphogenesis progresses. Expression of mau-2 within individual neurones rescues the guidance defects of mau-2 mutants, indicating that mau-2 functions cell-autonomously. Altering the activity of both the dorsal repellent slt-1 and mau-2 leads to the abnormal dorsal projection of the AVM axon, a phenotype that is novel and specific to the interaction of these two genes, indicating that mau-2 participates in the guidance of AVM by a slt-1-independent mechanism. Taken together, mau-2 defines a novel guidance factor that might be involved in the intracellular processing of guidance cues encountered by migrating cells and axons during development.     

Human TH17 lymphocytes promote blood-brain barrier disruption and central nervous system inflammation

T(H)17 lymphocytes appear to be essential in the pathogenesis of numerous inflammatory diseases. We demonstrate here the expression of IL-17 and IL-22 receptors on blood-brain barrier endothelial cells (BBB-ECs) in multiple sclerosis lesions, and show that IL-17 and IL-22 disrupt BBB tight junctions in vitro and in vivo. Furthermore, T(H)17 lymphocytes transmigrate efficiently across BBB-ECs, highly express granzyme B, kill human neurons and promote central nervous system inflammation through CD4+ lymphocyte recruitment.     

Cross-linking phosphatidylinositol-specific phospholipase C traps two activating phosphatidylcholine molecules on the enzyme

Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC), a bacterial model for the catalytic domain of mammalian PI-PLC enzymes, was cross-linked by 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride to probe for the aggregation and/or conformational changes of PI-PLC when bound to activating phosphatidylcholine (PC) interfaces. Dimers and higher order multimers (up to 31% of the total protein when cross-linked at pH 7) were observed when the enzyme was cross-linked in the presence of PC vesicles. Aggregates were also detected with PI-PLC bound to diheptanoyl-PC (diC(7)PC) micelles, although the fraction of cross-linked multimers (19% at pH 7) was lower than when the enzyme was cross-linked in the presence of vesicles. PI-PLC cross-linked in the presence of a diC(7)PC interface exhibited an enhanced specific activity for PI cleavage. The extent of this cross-linking-enhanced activation was reduced in PI-PLC mutants lacking either tryptophan in the rim (W47A and W242A) of this (betaalpha)(8)-barrel protein. The higher activity of the native protein cross-linked in the presence of diC(7)PC correlated with an increased affinity of the protein for two diC(7)PC molecules as detected by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. In contrast to wild type protein, W47A and W242A had only a single diC(7)PC tightly associated when cross-linked in the presence of that activator molecule. These results indicate that (i) each rim tryptophan residue is involved in binding a PC molecule at interfaces, (ii) the affinity of the enzyme for an activating PC molecule is enhanced when the protein is bound to a surface, and (iii) this conformation of the enzyme with at least two PC bound that is stabilized by chemical cross-linking interacts more effectively with activating interfaces, leading to higher observed specific activities for the phosphotransferase reaction.     

In vitro culture: a simple and efficient way for salt-tolerant grapevine genotype selection

In order to quickly and efficiently evaluate the grapevine's salt tolerance, salinity tests were conducted on some grapevine varieties and rootstocks under in vitro conditions. Plant materials used in the salinity test were propagated using the axillary buds culture method. Single-node shoots were subjected to seven different NaCl concentrations (0, 20, 50, 100, 150, and 200 mM NaCl) in MS medium for 45 days. The different growth parameters analysed were: survival capacity, length of shoot, bud formation, and rooting capacity. Our results showed that salinity reduced in vitro growth and development of grapevine. Proliferation, growth, rooting and viability of explants decreased due to the increase in NaCl concentration. First symptoms of stress (leaves necrosis) appeared after 10 days of treatment with 80 mM NaCl, which may lead to total desiccation. It was determined that severity of salt treatment injury varied depending on the genotype and NaCl concentration. A positive correlation was found between the vigour of plants in saline medium and their faculty to tolerate salt. The most tolerant grapevine genotypes to salt treatment were Sejnene and Asli, followed by the moderately sensitive Saouadi and Sakasly genotypes, and last Razegui, 1103P, 41B, and SO(4), which were particularity sensitive. Thus, grapevine's salt tolerance seems to be linked to the genetic background. To cite this article: L. Hamrouni et al., C. R. Biologies 331 (2008).     

Expression of Heat Shock Protein 90 (hsp90) in Neural and Nonneural Tissues of the Control and Hyperthermic Rabbit

Tissue-specific differences were apparent in the constitutive level of hsp90 in various body tissues of the unstressed rabbit. Western blotting with monoclonal antibody 29A revealed very low levels in muscle and highest levels in neural regions (cerebellum, cerebral hemispheres, and retina) and in testes and thymus. Intermediate levels were apparent in other tissues such as liver, kidney, heart, and small intestine. Following hyperthermia, induction of hsp90 was not detected with 1-D Western blotting in tissues which demonstrated high constitutive levels; however, elevations were noted in tissues which showed lower constitutive amounts of the protein, such as kidney, heart, and muscle. Immunocytochemical studies revealed that hsp90 is preferentially localized to neuronal cell populations in the rabbit brain and that this pattern does not alter following hyperthermic conditions which result in glial induction of hsp70. In kidney, where constitutive levels of hsp90 are lower than in brain, an induction of hsp90 was noted in renal tubules following hyperthermia.     

Evaluation of site availability exploitation towards performance optimization in data grids

Data in distributed systems are often replicated into different storage elements in order to facilitate their access. This allows optimizing execution time and bandwidth consumption, ensures load balancing and increases data availability and quality of service. Several replication strategies have then been proposed in the literature. In this work, a new evaluation metric for replication strategies is introduced and experimentally evaluated. This metric, called SAvE, serves to tackle a key feature, although neglected in the literature, which is the ability of a replication strategy to exploit the most available sites in the system. The design of such a metric requires an accurate determination of the availability degree of each site. A new measurement of site availability, denoted SA, is then designed to be integrated into SAvE while overcoming the drawbacks experienced by existing measurements. Extensive experiments are performed using the OptorSim simulator to show the accuracy and the effectiveness of our contributions.