Intravascular plug formation induced by poly-APS is the principal mechanism of the toxin's lethality in rats/rat tissues

Toxic water soluble polymeric 3-alkylpyridinium salts isolated from the sponge Raniera sarai strongly inhibited AChE in vitro. In vivo, experimental animals died due to plugs formed in microcirculation. The mechanism of this plug formation is unknown. In vitro, the toxin did not affect the coagulation rate, but the rate of platelet aggregation was accelerated in a dose-dependent manner. The hemolytic activity of poly-APS was diminished by the addition of serum proteins in a dose-dependent manner. These results support the conclusion that non-specific binding to proteins is the underlying mechanism of the lethality of poly APS.     

DNA damage induced by indirect and direct acting mutagens in catalase-deficient transgenic tobacco. Cellular and acellular Comet assays

We have measured the level of DNA damage induced by treating roots (cellular Comet assay) and isolated root nuclei (acellular Comet assay) of catalase-deficient (CAT1AS) and wild-type (SR1) tobacco with the promutagen o-phenylenediamine (o-PDA) and the direct acting genotoxic agents hydrogen peroxide and ethyl methanesulphonate (EMS). The roots of CAT1AS have about 60% less catalase activity compared to the roots of SR1. The promutagen o-PDA applied on tobacco roots induced significantly higher levels of DNA damage in the CAT1AS transgenic line than in SR1, while after application of o-PDA on isolated root nuclei, no DNA damage could be detected. In the catalase-deficient line CAT1AS about six-fold lower concentrations of H(2)O(2) are sufficient to induce the same levels of DNA damage as in SR1. By contrast, after treatment of isolated root nuclei with H(2)O(2) no difference in the induced levels of DNA damage was observed between CAT1AS and SR1. The DNA damaging effect of EMS was not affected by the presence of catalase in the tobacco roots and the levels of DNA damage measured by the cellular and acellular assay were similar.Comparing the effects of genotoxic agents in both the cellular and acellular Comet assays may help to elucidate their mechanism of action. Differences in both systems may reveal the participation of scavengers and of repair and metabolic enzymes on the activity of the genotoxic agent and the role of the cell wall in preventing the agent from reacting with nuclear DNA.     

Analysis of the substrate specificity of α-L-arabinofuranosidases by DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis

Applied Microbiology and Biotechnology - Carbohydrate-active enzyme discovery is often not accompanied by experimental validation, demonstrating the need for techniques to analyze substrate...     

Structural and Physiological Analyses of the Alkanesulphonate-Binding Protein (SsuA) of the Citrus Pathogen Xanthomonas citri

Background

The uptake of sulphur-containing compounds plays a pivotal role in the physiology of bacteria that live in aerobic soils where organosulfur compounds such as sulphonates and sulphate esters represent more than 95% of the available sulphur. Until now, no information has been available on the uptake of sulphonates by bacterial plant pathogens, particularly those of the Xanthomonas genus, which encompasses several pathogenic species. In the present study, we characterised the alkanesulphonate uptake system (Ssu) of Xanthomonas axonopodis pv. citri 306 strain (X. citri), the etiological agent of citrus canker.

Methodology/Principal Findings

A single operon-like gene cluster (ssuEDACB) that encodes both the sulphur uptake system and enzymes involved in desulphurisation was detected in the genomes of X. citri and of the closely related species. We characterised X. citri SsuA protein, a periplasmic alkanesulphonate-binding protein that, together with SsuC and SsuB, defines the alkanesulphonate uptake system. The crystal structure of SsuA bound to MOPS, MES and HEPES, which is herein described for the first time, provides evidence for the importance of a conserved dipole in sulphate group coordination, identifies specific amino acids interacting with the sulphate group and shows the presence of a rather large binding pocket that explains the rather wide range of molecules recognised by the protein. Isolation of an isogenic ssuA-knockout derivative of the X. citri 306 strain showed that disruption of alkanesulphonate uptake affects both xanthan gum production and generation of canker lesions in sweet orange leaves.

Conclusions/Significance

The present study unravels unique structural and functional features of the X. citri SsuA protein and provides the first experimental evidence that an ABC uptake system affects the virulence of this phytopathogen.     

Variation in Apical Hook Length Reflects the Intensity of Sperm Competition in Murine Rodents

Background

Post-copulatory sexual selection has been shown to shape morphology of male gametes. Both directional and stabilizing selection on sperm phenotype have been documented in vertebrates in response to sexual promiscuity.

Methodology

Here we investigated the degree of variance in apical hook length and tail length in six taxa of murine rodents.

Conclusions

Tail sperm length and apical hook length were positively associated with relative testis mass, our proxy for levels of sperm competition, thus indicating directional post-copulatory selection on sperm phenotypes. Moreover, our study shows that increased levels of sperm competition lead to the reduction of variance in the hook length, indicating stabilizing selection. Hence, the higher risk of sperm competition affects increasing hook length together with decreasing variance in the hook length. Species-specific post-copulatory sexual selection likely optimizes sperm morphology.     

Below- and aboveground traits explain local abundance,and regional,continental and global occurrence frequencies of grassland plants

Plants vary widely in how common or rare they are, but whether commonness of species is associated with functional traits is still debated. This might partly be because commonness can be measured at different spatial scales, and because most studies focus solely on aboveground functional traits. We measured five root traits and seed mass on 241 central European grassland species, and extracted their specific leaf area, height, mycorrhizal status and bud-bank size from databases. Then we tested if trait values are associated with commonness at seven spatial scales, ranging from abundance in 16-m² grassland plots, via regional and European-wide occurrence frequencies, to worldwide naturalization success. At every spatial scale, commonness was associated with at least three traits. The traits explained the greatest proportions of variance for abundance in grassland plots (42%) and naturalization success (41%) and the least for occurrence frequencies in Europe and the Mediterranean (2%). Low root tissue density characterized common species at every scale, whereas other traits showed directional changes depending on the scale. We also found that many of the effects had significant non-linear effects, in most cases with the highest commonness-metric value at intermediate trait values. Across scales, belowground traits explained overall more variance in species commonness (19.4%) than aboveground traits (12.6%). The changes we found in the relationships between traits and commonness, when going from one spatial scale to another, could at least partly explain the maintenance of trait variation in nature. Most importantly, our study shows that within grasslands, belowground traits are at least as important as aboveground traits for species commonness. Therefore, belowground traits should be more frequently considered in studies on plant functional ecology.     

RNA binding is more critical to the suppression of silencing function of Cucumber mosaic virus 2b protein than nuclear localization

Previously, we found that silencing suppression by the 2b protein and six mutants correlated both with their ability to bind to double-stranded (ds) small RNAs (sRNAs) in vitro and with their nuclear/nucleolar localization. To further discern the contribution to suppression activity of sRNA binding and of nuclear localization, we have characterized the kinetics of in vitro binding to a ds sRNA, a single-stranded (ss) sRNA, and a micro RNA (miRNA) of the native 2b protein and eight mutant variants. We have also added a nuclear export signal (NES) to the 2b protein and assessed how it affected subcellular distribution and suppressor activity. We found that in solution native protein bound ds siRNA, miRNA, and ss sRNA with high affinity, at protein:RNA molar ratios ~2:1. Of the four mutants that retained suppressor activity, three showed sRNA binding profiles similar to those of the native protein, whereas the remaining one bound ss sRNA at a 2:1 molar ratio, but both ds sRNAs with 1.5-2 times slightly lower affinity. Three of the four mutants lacking suppressor activity failed to bind to any sRNA, whereas the remaining one bound them at far higher ratios. NES-tagged 2b protein became cytoplasmic, but suppression activity in patch assays remained unaffected. These results support binding to sRNAs at molar ratios at or near 2:1 as critical to the suppressor activity of the 2b protein. They also show that cytoplasmically localized 2b protein retained suppressor activity, and that a sustained nuclear localization was not required for this function.     

Subfamily-Specific Adaptations in the Structures of Two Penicillin-Binding Proteins from Mycobacterium tuberculosis

Beta-lactam antibiotics target penicillin-binding proteins including several enzyme classes essential for bacterial cell-wall homeostasis. To better understand the functional and inhibitor-binding specificities of penicillin-binding proteins from the pathogen, Mycobacterium tuberculosis, we carried out structural and phylogenetic analysis of two predicted D,D-carboxypeptidases, Rv2911 and Rv3330. Optimization of Rv2911 for crystallization using directed evolution and the GFP folding reporter method yielded a soluble quadruple mutant. Structures of optimized Rv2911 bound to phenylmethylsulfonyl fluoride and Rv3330 bound to meropenem show that, in contrast to the nonspecific inhibitor, meropenem forms an extended interaction with the enzyme along a conserved surface. Phylogenetic analysis shows that Rv2911 and Rv3330 belong to different clades that emerged in Actinobacteria and are not represented in model organisms such as Escherichia coli and Bacillus subtilis. Clade-specific adaptations allow these enzymes to fulfill distinct physiological roles despite strict conservation of core catalytic residues. The characteristic differences include potential protein-protein interaction surfaces and specificity-determining residues surrounding the catalytic site. Overall, these structural insights lay the groundwork to develop improved beta-lactam therapeutics for tuberculosis.