A conserved phenylalanine of motif IV in superfamily 2 helicases is required for cooperative, ATP-dependent binding of RNA substrates in DEAD-box proteins

We have identified a highly conserved phenylalanine in motif IV of the DEAD-box helicases that is important for their enzymatic activities. In vivo analyses of essential proteins in yeast showed that mutants of this residue had severe growth phenotypes. Most of the mutants also were temperature sensitive, which suggested that the mutations altered the conformational stability. Intragenic suppressors of the F405L mutation in yeast Ded1 mapped close to regions of the protein involved in ATP or RNA binding in DEAD-box crystal structures, which implicated a defect at this level. In vitro experiments showed that these mutations affected ATP binding and hydrolysis as well as strand displacement activity. However, the most pronounced effect was the loss of the ATP-dependent cooperative binding of the RNA substrates. Sequence analyses and an examination of the Protein Data Bank showed that the motif IV phenylalanine is conserved among superfamily 2 helicases. The phenylalanine appears to be an anchor that maintains the rigidity of the RecA-like domain. For DEAD-box proteins, the phenylalanine also aligns a highly conserved arginine of motif VI through van der Waals and cation-pi interactions, thereby helping to maintain the network of interactions that exist between the different motifs involved in ATP and RNA binding.     

Bacillus cereus produces several nonproteinaceous insecticidal exotoxins

Bacillus cereus is mainly known as a human food-borne opportunistic pathogen. Here, we used biological assays and HPLC to investigate the ability of B. cereus to produce insecticidal exotoxins during the stationary growth phase. None of the 575 B. cereus strains screened produced detectable levels of β-exotoxin I, a small, heat-stable insecticidal nucleotide analogue. However, six out of a subset of 270 B. cereus strains produced several small, nonproteinaceous insecticidal exotoxins different from β-exotoxin I. Thus, B. cereus can secrete a large array of proteinaceous and nonproteinaceous toxins acting on insects and mammals.     

CTLA-4 regulates expansion and differentiation of Th1 cells following induction of peripheral T cell tolerance

Intravenous treatment with Ag (peptide)-coupled, ethylene carbodiimide-fixed syngeneic splenocytes (Ag-SP) is a powerful method to induce anergy in vitro and peripheral T cell tolerance in vivo. In this study, we examined the effects of Ag-SP administration on T cell activity ex vivo and in vivo using OVA-specific DO11.10 TCR transgenic T cells. Although treatment with OVA323-339-SP resulted in a strong inhibition of peptide-specific T cell recall responses in vitro, examination of the immediate effects of Ag-SP treatment on T cells in vivo demonstrated that tolerogen injection resulted in rapid T cell activation and proliferation. Although there was an increase in the number of OVA-specific DO11.10 T cells detected in the lymphoid organs, these previously tolerized T cells were strongly inhibited in mounting proliferative or inflammatory responses upon rechallenge in vivo with peptide in CFA. This unresponsiveness was reversible by treatment with anti-CTLA-4 mAb. These results are consistent with the hypothesis that Ag-SP injection induces a state of T cell anergy that is maintained by CTLA-4 engagement.     

Sulfato/thiosulfato reducing bacteria characterization by FT-IR spectroscopy: a new approach to biocorrosion control

Sulfato and Thiosulfato Reducing Bacteria (SRB, TRB) can induce corrosion process on steel immersed in seawater. This phenomenon, called biocorrosion, costs approximatively 5 billion euros in France each year. We provide the first evidence that Fourier Transformed InfraRed (FTIR) spectroscopy is a competitive technique to evaluate the sulfurogen flora involved in biocorrosion in comparison with time consuming classical identification methods or PCR analyses. A great discrimination was obtained between SRB, TRB and some contamination bacteria known to be present in seawater and seem to be able to reduce sulfate under particular conditions. Moreover, this preliminary study demonstrates that FTIR spectroscopic and genotypic results present a good correlation (these results are confirmed by other data obtained before or later, data not shown here). The advantages gained by FTIR spectroscopy are to give information on strain phenotype and bacterial metabolism which are of great importance in corrosion processes.     

Indirect N2O emissions from shallow groundwater in an agricultural catchment (Seine Basin, France)

Production and accumulation of nitrous oxide (N₂O), a major greenhouse gas, in shallow groundwater might contribute to indirect N₂O emissions to the atmosphere (e.g., when groundwater flows into a stream or a river). The Intergovernmental Panel on Climate Change (IPCC) has attributed an emission factor (EF_5g) for N₂O, associated with nitrate leaching in groundwater and drainage ditches—0.0025 (corresponding to 0.25% of N leached which is emitted as N₂O)—although this is the subject of considerable uncertainty. We investigated and quantified the transport and fate of nitrate (NO₃ ^?) and dissolved nitrous oxide from crop fields to groundwater and surface water over a 2-year period (monitoring from April 2008 to April 2010) in a transect from a plateau to the river with three piezometers. In groundwater, nitrate concentrations ranged from 1.0 to 22.7 mg NO₃ ^?–N l^?1 (from 2.8 to 37.5 mg NO₃ ^?–N l^?1 in the river) and dissolved N₂O from 0.2 to 101.0 μg N₂O–N l^?1 (and from 0.2 to 2.9 μg N₂O–N l^?1 in the river). From these measurements, we estimated an emission factor of EF_5g = 0.0026 (similar to the value currently used by the IPCC) and an annual indirect N₂O flux from groundwater of 0.035 kg N₂O–N ha^?1 year^?1, i.e., 1.8% of the previously measured direct N₂O flux from agricultural soils.     

Microheterogeneity of human galactose-1-Phosphate uridylyl transferase. Isoelectrofocusing results

Using thin-layer acrylamide gel isoelectrofocusing, several bands of galactose-1-Phosphate uridylyl transferase were found in various human tissues. Liver transferase, as well as that of some other tissues, was resolved into several bands with pHi between 5.30 and 5.80; red cell enzyme was resolved into five bands with pHi between 5.0 and 5.45. The comparison of erythrocytes with their precursors, reticulocytes and erythroblasts, showed a striking difference: the pHi of the erythroblast enzyme was between 5.55 and 5.90 and that of reticulocytes between 5.30 and 5.50. It is possible that molecular aging is the cause of the anodisation of the erythrocyte transferase and the microheterogeneity of the enzyme observed in other tissues.     

Melon resistance to the aphid Aphis gossypii: behavioural analysis and chemical correlations with nitrogenous compounds

In the melon, the Vat (monogenic, dominant) resistance gene governs both an antixenotic reaction to the melon aphid Aphis gossypii Glover (Homoptera, Aphididae) and a resistance to non-persistent virus transmission, restricted to this vector species. We investigated the behavioural features and tissue localisation of the antixenosis resistance by the electrical penetration graph technique (EPG, DC system). We also compared the chemical composition in amino compounds and proteins of the phloem sap collected from two isogenic lines of melon (Cucumis melo L.), carrying the Vat gene or not. All behavioural and chemical data indicated that this resistance is constitutive. EPG analysis clearly showed that access to phloem, although delayed by alterations in pathway activities, was not impaired in terms of frequency of access or initiation of feeding. The most striking feature was, however, a very reduced duration of ingestion from phloem of resistant plants, making this compartment one of the tissues where the effects of the Vat gene are unambiguously expressed. This was confirmed by clear differential activity of phloem extracts in artificial no-choice bioassays. Chemical analyses have shown that phloem saps from the two isogenic lines were extremely similar in profiles of ninhydrin positive compounds, and contained a low total amount of free amino acids (less than 10 mM). Out of more than 40 distinguishable peaks in the chromatograms (protein and non-protein amino acids, as well as small peptides), only five differentiated the two genotypes. Two of them were increased in the resistant genotype: glutamic acid and a major unknown peak, probably a non-protein amino acid (different from pyrazolyl-alanine, a Cucumis-specific amino acid). The three others were depressed in resistant plants, and included the sulphur amino acid cystine and a peptide peak partly composed of the cysteine-containing peptide glutathione (reduced form). Sap collection also showed that phloem exudation rates, as well as total protein and glutathione levels, were depressed in phloem sap from resistant plants. Such data are all indicative of a modified phloem-sealing physiology, linked to sulfhydryl oxidation processes, in plants carrying the Vat gene. The originality of the mechanism of Vat resistance to aphids is discussed.