Oxidative stress in the root nodules of Phaseolus vulgaris is induced under conditions of phosphorus deficiency

One of the most adverse effects of phosphorus (P) deficiency on N₂-fixing legumes is the generation of harmful active oxygen species which cause oxidative stress. And although oxidative stress has been widely studied in roots and shoots of various plant species, it has not yet sufficiently been documented in bean nodules so far. In this study, two recombinant inbred lines RIL115 (P-deficiency tolerant) and RIL147 (P-deficiency sensitive) of common bean and Concesa (local variety) were inoculated separately with the reference strain R. tropici CIAT899, RhM₁₁ (R. gallicum) or RhM₁₄ (R. tropici); two local strains of the Marrakesh region of Morocco. Nodulated plants were grown under semi-hydroponic conditions with sufficient or deficient P supply and analyzed for their oxidative responses at the flowering stage. The results indicated that P-deficiency decreased the growth of shoots (48 %) and nodules (32 %), particularly with RhM₁₄ exhibiting the highest decrease (52 %) of nodulation. This constraint increased electrolyte leakage of nodules (40 %) as compared to leaves (20 %), especially for plants inoculated with RhM₁₄ and CIAT899. Moreover, high H₂O₂ and malondialdehyde contents were noticed in P-deficient nodules of RhM₁₄ and RhM₁₁. These variations were associated with peroxidase activity stimulation in P-deficient nodules induced by CIAT899 and RhM₁₄. In symbiosis with RIL115, these last strains exhibited the highest nodule phenol content. Overall, phenol content was mainly enhanced in P-deficient nodules (35 %) as compared to the leaves (16 %). It was concluded that the genotypes inoculated with CIAT899 and RhM₁₁ are relatively P-deficiency tolerant combinations as compared to those inoculated with RhM₁₄. Increase of oxidative stress in nodules rather than in leaves points to the need for further investigations of mechanisms that improve the root-nodule efficiency under adverse conditions.     

Crystal structure of the HIV-2 neutralizing Fab fragment 7C8 with high specificity to the V3 region of gp125

7C8 is a mouse monoclonal antibody specific for the third hypervariable region (V3) of the human immunodeficiency virus type 2 (HIV-2)-associated protein gp125. The three-dimensional crystal structure of the Fab fragment of 7C8, determined to 2.7 Å resolution, reveals a deep and narrow antigen-binding cleft with architecture appropriate for an elongated epitope. The highly hydrophobic cleft is bordered on one side by the negatively charged second complementarity determining region (CDR2) and the unusually long positively charged CDR3 of the heavy chain and, on the other side, by the CDR1 of the light chain. Analysis of 7C8 in complex with molecular models of monomeric and trimeric gp125 highlights the importance of a conserved stretch of residues FHSQ that is localized centrally on the V3 region of gp125. Furthermore, modeling also indicates that the Fab fragment neutralizes the virus by sterically impairing subsequent engagement of the gp125 trimer with the co-receptor on the target cell.     

Pre-analytical Factors Influence Accuracy of Urine Spot Iodine Assessment in Epidemiological Surveys

Urinary iodine concentration (UIC) is commonly used to assess iodine status of subjects in epidemiological surveys. As pre-analytical factors are an important source of measurement error and studies about this phase are scarce, our objective was to assess the influence of urine sampling conditions on UIC, i.e., whether the child ate breakfast or not, urine void rank of the day, and time span between last meal and urine collection. A nationwide, two-stage, stratified, cross-sectional study including 1560 children (6–12 years) was performed in 2012. UIC was determined by the Sandell-Kolthoff method. Pre-analytical factors were assessed from children’s mothers by using a questionnaire. Association between iodine status and pre-analytical factors were adjusted for one another and socio-economic characteristics by multivariate linear and multinomial regression models (RPR: relative prevalence ratios). Skipping breakfast prior to morning urine sampling decreased UIC by 40 to 50 μg/L and the proportion of UIC?<?100 μg/L was higher among children having those skipped breakfast (RPR?=?3.2[1.0–10.4]). In unadjusted analyses, UIC was less among children sampled more than 5 h from their last meal. UIC decreased with rank of urine void (e.g., first vs. second, P?<?0.001); also, the proportion of UIC?<?100 μg/L was greater among 4th rank samples (vs. second RPR?=?2.1[1.1–4.0]). Subjects’ breakfast status and urine void rank should be accounted for when assessing iodine status. Providing recommendations to standardize pre-analytical factors is a key step toward improving accuracy and comparability of survey results for assessing iodine status from spot urine samples. These recommendations have to be evaluated by future research.     

The basic keratin 10-binding domain of the virulence-associated pneumococcal serine-rich protein PsrP adopts a novel MSCRAMM fold

Streptococcus pneumoniae is a major human pathogen, and a leading cause of disease and death worldwide. Pneumococcal invasive disease is triggered by initial asymptomatic colonization of the human upper respiratory tract. The pneumococcal serine-rich repeat protein (PsrP) is a lung-specific virulence factor whose functional binding region (BR) binds to keratin-10 (KRT10) and promotes pneumococcal biofilm formation through self-oligomerization. We present the crystal structure of the KRT10-binding domain of PsrP (BR_187–385) determined to 2.0 Å resolution. BR_187–385 adopts a novel variant of the DEv-IgG fold, typical for microbial surface components recognizing adhesive matrix molecules adhesins, despite very low sequence identity. An extended β-sheet on one side of the compressed, two-sided barrel presents a basic groove that possibly binds to the acidic helical rod domain of KRT10. Our study also demonstrates the importance of the other side of the barrel, formed by extensive well-ordered loops and stabilized by short β-strands, for interaction with KRT10.     

Genotypic variation of nodules’ enzymatic activities in symbiotic nitrogen fixation among common bean (Phaseolus vulgaris L.) genotypes grown under salinity constraint

The effect of salt stress, under glasshouse conditions, was studied on plant biomass, nodulation, and activities of acid phosphatases (APase, EC 3.1.3.2) and trehalose 6-phosphate phosphatase (TPP, EC 3.1.3.12) in the symbiosis common bean (Phaseolus vulgaris L.)-rhizobia nodules. Four common bean recombinant inbred lines (147, 115, 104 and 83) were separately inoculated, with CIAT 899 or RhM11 strains and grown in hydroaeroponic culture. Two NaCl levels (0 and 25 mM NaCl plant^?1 week^?1 corresponding, respectively, to the control and the salt treatment) were applied and the culture was assessed during 42 days after their transplantation. The results showed that the nodulation of these lines was not affected by salinity except for the line 83 inoculated with CIAT 899, whose nodule dry weight decreased by 48.24 % compared with the corresponding controls. For the other symbiotic combinations, shoot and root biomasses were not significantly affected by salt constraint. Salinity stress generally reduced acid phosphatise and trehalose phosphate phosphatase activities in nodules that were less affected in plants inoculated with RhM11. Based on our data, it appears that nodule phosphatase activity may be involved in salinity tolerance in common beans and the levels of salt tolerance depend principally on specific combination of the rhizobial strain and the host cultivar.     

Purification and partial characterization of azoreductase from Enterobacter agglomerans

Azoreductase, an enzyme catalyzing the reductive cleavage of the azo bond of methyl red (MR) and related dyes, was purified to electrophoretic homogeneity from Enterobacter agglomerans. This bacterial strain, isolated from dye-contaminated sludge, has a higher ability to grow, under aerobic conditions, on culture medium containing 100mg/L of MR. The enzyme was purified approximately 90-fold with 20% yield by ammonium sulfate precipitation, followed by three steps of column chromatography (gel-filtration, anion-exchange, and dye-affinity). The purified enzyme is a monomer with a molecular weight of 28,000 Da. The maximal azoreductase activity was observed at pH 7.0 and at 35 degrees C. This activity was NADH dependent. The K(m) values for both NADH and MR were 58.9 and 29.4 microM, respectively. The maximal velocity (V(max)) was 9.2 micromol of NADH min(-1)mg(-1). The purified enzyme is inhibited by several metal ions including Fe(2+) and Cd(2+).     

Stretch-activated channels in pulmonary arterial smooth muscle cells from normoxic and chronically hypoxic rats

Stretch-activated channels (SACs) act as membrane mechanotransducers since they convert physical forces into biological signals and hence into a cell response. Pulmonary arterial smooth muscle cells (PASMCs) are continuously exposed to mechanical stimulations e.g., compression and stretch, that are enhanced under conditions of pulmonary arterial hypertension (PAH). Using the patch-clamp technique (cell-attached configuration) in PASMCs, we showed that applying graded negative pressures (from 0 to -60 mmHg) to the back end of the patch pipette increases occurrence and activity of SACs. The current-voltage relationship (from -80 to +40 mV) was almost linear with a reversal potential of 1 mV and a slope conductance of 34 pS. SACs were inhibited in the presence of GsMTx-4, a specific SACs blocker. Using microspectrofluorimetry (indo-1), we found that hypotonic-induced cell swelling increases intracellular Ca(2+) concentration ([Ca(2+)](i)). This [Ca(2+)](i) increase was markedly inhibited in the absence of external Ca(2+) or in the presence of the following blockers of SACs: gadolinium, streptomycin, and GsMTx-4. Interestingly, in chronically hypoxic rats, an animal model of PAH, SACs were more active and hypotonic-induced calcium response in PASMCs was significantly higher (nearly a two-fold increase). Moreover, unlike in normoxic rats, intrapulmonary artery rings from hypoxic rats mounted in a Mulvany myograph, exhibited a myogenic tone sensitive to SAC blockers. In conclusion, this work demonstrates that SACs in rat PASMCs can be activated by membrane stretch as well as hypotonic stimulation and are responsible for [Ca(2+)](i) increase. The link between SACs activation-induced calcium response and myogenic tone in chronically hypoxic rats suggests that SACs are an important element for the increased pulmonary vascular tone in PAH and that they may represent a molecular target for PAH treatment.