Expression of soluble, active fragments of the morphogenetic protein SpoIIE from Bacillus subtilis using a library-based construct screen

SpoIIE is a dual function protein that plays important roles during sporulation in Bacillus subtilis. It binds to the tubulin-like protein FtsZ causing the cell division septum to relocate from mid-cell to the cell pole, and it dephosphorylates SpoIIAA phosphate leading to establishment of differential gene expression in the two compartments following the asymmetric septation. Its 872 residue polypeptide contains a multiple-membrane spanning sequence at the N-terminus and a PP2C phosphatase domain at the C-terminus. The central segment that binds to FtsZ is unlike domains of known structure or function, moreover the domain boundaries are poorly defined and this has hampered the expression of soluble fragments of SpoIIE at the levels required for structural studies. Here we have screened over 9000 genetic constructs of spoIIE using a random incremental truncation library approach, ESPRIT, to identify a number of soluble C-terminal fragments of SpoIIE that were aligned with the protein sequence to map putative domains and domain boundaries. The expression and purification of three fragments were optimised, yielding multimilligram quantities of the PP2C phosphatase domain, the putative FtsZ-binding domain and a larger fragment encompassing both these domains. All three fragments are monomeric and the PP2C domain-containing fragments have phosphatase activity

Correlation between genotype and phenotype in patients with cystic fibrosis

The members of the Cystic Fibrosis Genotype-Phenotype Consortium are listed in the AppendixBackground. Cystic fibrosis is the most common lethal autosomal recessive disorder among whites. Seventy-two percent of patients with this disease are homozygotes or compound heterozygotes for eight mutations of the cystic fibrosis transmembrane conductance regulator gene on chromosome 7: ΔF 508, G542X, R553X, W1282X, N1303K, 621 + 1G → T, 1717-1G → A, and R117H. We studied the relation between genotype and phenotype in patients from 14 countries. Methods. Each of 399 patients who were compound heterozygotes for ΔF 508 and one other mutation was matched with the ΔF 508 homozygote of the same sex who was the closest in age from the same center. A paired analysis was performed of the following outcome variables: age at diagnosis, sweat chloride concentration, growth percentiles, pulmonary-function values, chest-film score, pseudomonas colonization, nasal polyps, pancreatic sufficiency, pancreatitis, diabetes mellitus, meconium ileus, distal intestinal obstruction syndrome, rectal prolapse, cirrhosis, and gallbladder disease. Results. The compound heterozygotes having the genotype R117H/ΔF 508 clearly differed from the age- and sex-matched ΔF 508 homozygotes: they more often had pancreatic sufficiency (87 percent vs. 4 percent, P<0.001), were older when the diagnosis was first made (mean [±SD] age, 10.2±10.5 vs. 2.5±4.3 years; P = 0.002), and had lower sweat chloride concentrations (80±18 vs. 108±14 mmol per liter, P<0.001). There were no statistically significant differences between ΔF 508 homozygotes and other compound heterozygotes with regard to any variable tested. Conclusions. Prenatal and prognostic counseling for patients with the R117H/ΔF 508 genotype should include the likelihood that they will have long-term pancreatic sufficiency. Patients with the other genotypes should expect the early onset of pancreatic insufficiency. For none of the genotypes studied can predictions be made about the occurrence of common complications or the severity or course of pulmonary disease.link_to_OA_fulltex

Replication of restless legs syndrome loci in three European populations

Restless legs syndrome (RLS) is associated with common variants in three intronic and intergenic regions in MEIS1, BTBD9, and MAP2K5/LBXCOR1 on chromosomes 2p, 6p and 15q. Methods: Our study investigated these variants in 649 RLS patients and 1230 controls from the Czech Republic (290 cases and 450 controls), Austria (269 cases and 611 controls) and Finland (90 cases and 169 controls). Ten single nucleotide polymorphisms (SNPs) within the three genomic regions were selected according to the results of previous genome-wide scans. Samples were genotyped using Sequenom platforms. Results: We replicated associations for all loci in the combined samples set (rs2300478 in MEIS1, p=1.2661025, odds ratio (OR)=1.47, rs3923809 in BTBD9, p=4.1161025, OR=1.58 and rs6494696 in MAP2K5/LBXCOR1, p=0.04764, OR=1.27). Analysing only familial cases against all controls, all three loci were significantly associated. Using sporadic cases only, we could confirm the association only with BTBD9. Conclusion: Our study shows that variants in these three loci confer consistent disease risks in patients of European descent. Among the known loci, BTBD9 seems to be the most consistent in its effect on RLS across populations and is also most independent of familial clustering

Rheology of lime paste - A comparison with cement paste

International audienceThe rheological properties of a suspension of lime in water (lime putty) are studied with the help of creep tests in a wide range of deformations including very small values. The results are compared with those obtained with a cement paste and several similarities between the two systems are observed. It is shown that the apparent yield stress of a lime suspension is the sum of two components: one due to standard reversible colloidal interactions and one due to the formation of a brittle structure associated with the formation of links due to dissolution-precipitation mechanisms. This second component increases with the time of rest as the square root of time, and the corresponding structure irreversibly breaks as soon as some significant deformation has been imposed. We show that similar structures are formed at concentrations between 25 and 34% (solid volume fraction) and evolve in a similar way when the time is scaled by a factor decreasing with the solid fraction