Ku antigen interacts with abasic sites

One of the most abundant lesions in DNA is the abasic (AP) sites arising spontaneously or as an intermediate in base excision repair. Certain proteins participating in the processing of these lesions form a Schiff base with the deoxyribose of the AP site. This intermediate can be stabilized by NaBH(4) treatment. By this method, DNA duplexes with AP sites were used to trap proteins in cell extracts. In HeLa cell extract, along with a prevalent trap product with an apparent molecular mass of 95 kDa, less intensive low-molecular-weight products were observed. The major one was identified as the p80-subunit of Ku antigen (Ku). Ku antigen, a DNA binding component of DNA-dependent protein kinase (DNA-PK), participates in double-stranded break repair and is responsible for the resistance of cells to ionizing radiation. The specificity of Ku interaction with AP sites was proven by more efficient competition of DNA duplexes with an analogue of abasic site than non-AP DNA. Ku80 was cross-linked to AP DNAs with different efficiencies depending on the size and position of strand interruptions opposite to AP sites. Ku antigen as a part of DNA-PK was shown to inhibit AP site cleavage by apurinic/apyrimidinic endonuclease 1.     

Identification, cloning and functional characterization of a novel dermonecrotic toxin (phospholipase D) from brown spider (Loxosceles intermedia) venom

Brown spider bites are associated with lesions including dermonecrosis, gravitational spreading and a massive inflammatory response, along with systemic problems that may include hematological disturbances and renal failure. The mechanisms by which the venom exerts its noxious effects are currently under investigation. It is known that the venom contains a major toxin (dermonecrotic toxin, biochemically a phospholipase D) that can experimentally induce dermonecrosis, inflammatory response, animal mortality and platelet aggregation. Herein, we describe cloning, heterologous expression, purification and functionality of a novel isoform of the 33 kDa dermonecrotic toxin. Circular dichroism analysis evidenced correct folding for the toxin. The recombinant toxin was recognized by whole venom serum antibodies and by a specific antibody to a previously described dermonecrotic toxin. The identified toxin was found to display phospholipase activity and dermonecrotic properties. Additionally, the toxin caused a massive inflammatory response in rabbit skin dermis, evoked platelet aggregation, increased vascular permeability, caused edema and death in mice. These characteristics in combination with functional studies for other dermonecrotic toxins illustrate that a family of dermonecrotic toxins exists, and includes a novel member with high activity that may be useful for future structural and functional studies.     

Predicting species distributions from herbarium collections: does climate bias in collection sampling influence model outcomes?

Aim Species distribution models and geographical information system (GIS) technologies are becoming increasingly important tools in conservation planning and decision‐making. Often the rich data bases of museums and herbaria serve as the primary data for predicting species distributions. Yet key assumptions about the primary data often are untested, and violation of such assumptions may have consequences for model predictions. For example, users of primary data assume that sampling has been random with respect to geography and environmental gradients. Here we evaluate the assumption that plant voucher specimens adequately sample the climatic gradient and test whether violation of this assumption influences model predictions. Location Bolivia and Ecuador. Methods Using 323,711 georeferenced herbarium collections and nine climatic variables, we predicted the distribution of 76 plant species using maximum entropy models (MAXENT) with training points that sampled the climate environments randomly and training points that reflected the climate bias in the herbarium collections. To estimate the distribution of species, MAXENT finds the distribution of maximum entropy (i.e. closest to uniform) subject to the constraint that the expected value for each environmental variable under the estimated distribution matches its empirical average. The experimental design included species that differed in geographical range and elevation; all species were modelled with 20 and 100 training points. We examined the influence of the number of training points and climate bias in training points, elevation and range size on model performance using analysis of variance models. Results We found that significant parts of the climatic gradient were poorly represented in herbarium collections for both countries. For the most part, existing climatic bias in collections did not greatly affect distribution predictions when compared with an unbiased data set. Although the effects of climate bias on prediction accuracy were found to be greater where geographical ranges were characterized by high spatial variation in the degree of climate bias (i.e. ranges where the bias of the various climates sampled by collections deviated considerably from the mean bias), the greatest influence on model performance was the number of presence points used to train the model. Main conclusions These results demonstrate that predictions of species distributions can be quite good despite existing climatic biases in primary data found in natural history collections, if a sufficiently large number of training points is available. Because of consistent overprediction of models, these results also confirm the importance of validating models with independent data or expert opinion. Failure to include independent model validation, especially in cases where training points are limited, may potentially lead to grave errors in conservation decision‐making and planning.     

Sodium-dependent inactivation of sodium/calcium exchange in transfected Chinese hamster ovary cells

High concentrations of cytosolic Na⁺ ions induce the time-dependent formation of an inactive state of the Na⁺/Ca²⁺ exchanger (NCX), a process known as Na⁺-dependent inactivation. NCX activity was measured as Ca²⁺ uptake in fura 2-loaded Chinese hamster ovary (CHO) cells expressing the wild-type (WT) NCX or mutants that are hypersensitive (F223E) or resistant (K229Q) to Na⁺-dependent inactivation. As expected, 1) Na⁺-dependent inactivation was promoted by high cytosolic Na⁺ concentration, 2) the F223E mutant was more susceptible than the WT exchanger to inactivation, whereas the K229Q mutant was resistant, and 3) inactivation was enhanced by cytosolic acidification. However, in contrast to expectations from excised patch studies, 1) the WT exchanger was resistant to Na⁺-dependent inactivation unless cytosolic pH was reduced, 2) reducing cellular phosphatidylinositol-4,5-bisphosphate levels did not induce Na⁺-dependent inactivation in the WT exchanger, 3) Na⁺-dependent inactivation did not increase the half-maximal cytosolic Ca²⁺ concentration for allosteric Ca²⁺ activation, 4) Na⁺-dependent inactivation was not reversed by high cytosolic Ca²⁺ concentrations, and 5) Na⁺-dependent inactivation was partially, but transiently, reversed by an increase in extracellular Ca²⁺ concentration. Thus Na⁺-dependent inactivation of NCX expressed in CHO cells differs in several respects from the inactivation process measured in excised patches. The refractoriness of the WT exchanger to Na⁺-dependent inactivation suggests that this type of inactivation is unlikely to be a strong regulator of exchange activity under physiological conditions but would probably act to inhibit NCX-mediated Ca²⁺ influx during ischemia. ischemia; cytosolic calcium concentration; cytosolic sodium concentration; cellular phosphatidylinositol-4,5-bisphosphate     

Galectin-loaded cells as a platform for the profiling of lectin specificity by fluorescent neoglycoconjugates: a case study on galectins-1 and -3 and the impact of assay setting

The involvement of galectins as pleiotropic regulators of cell adhesion and growth in disease progression explains the interest to define their ligand-binding properties. Toward this end, it is desirable to approach in vivo conditions to attain medical relevance. In order to simulate physiological conditions with cell surface glycans as recognition sites and galectins as mediators of intercellular contacts we developed an assay using galectin-loaded Raji cells. The extent of surface binding of fluorescent neoglycoconjugates depended on the lectin presence and the type of lectin, the nature of the probes' carbohydrate headgroup and the density of unsubstituted beta-galactosides on the cell surface. Using the most frequently studied galectins-1 and -3, application of this assay led to rather equal binding levels for linear and branched oligomers of N-acetyllactosamine. A clear preference of galectin-3 for alpha1-3-linked galactosylated lactosamine was noted. In parallel, a panel of 24 neoglycoconjugates was tested as inhibitors of galectin binding from solution to N-glycans of surface-immobilized asialofetuin. These two assays differ in presentation of the galectin and ligand, facilitating identification of assay-dependent properties. Under the condition of the cell assay, selectivity among oligosaccharides for the lectins was higher, and extraordinary affinity of galectin-1 to 3'-O-sulfated probes in a solid-phase assay was lost in the cell assay. Having introduced and validated a cell assay, the comprehensive profiling of ligand binding to cell-surface-presented galectins is made possible.     

How does forest landscape structure explain tree species richness in a Mediterranean context?

Although the strong relationship between vegetation and climatic factors is widely accepted, other landscape composition and configuration characteristics could be significantly related with vegetation diversity patterns at different scales. Variation partitioning was conducted in order to analyse to what degree forest landscape structure, compared to other spatial and environmental factors, explained forest tree species richness in 278 UTM 10 × 10 km cells in the Mediterranean region of Catalonia (NE Spain). Tree species richness variation was decomposed through linear regression into three groups of explanatory variables: forest landscape (composition and configuration), environmental (topography and climate) and spatial variables. Additionally, the forest landscape characteristics which significantly contributed to explain richness variation were identified through a multiple regression model. About 60% of tree species richness variation was explained by the whole set of variables, while their joint effects explained nearly 28%. Forest landscape variables were those with a greater pure explanatory power for tree species richness (about 15% of total variation), much larger than the pure effect of environmental or spatial variables (about 2% each). Forest canopy cover, forest area and land cover diversity were the most significant composition variables in the regression model. Landscape configuration metrics had a minor effect on forest tree species richness, with the exception of some shape complexity indices, as indicators of land use intensity and edge effects. Our results highlight the importance of considering the forest landscape structure in order to understand the distribution of vegetation diversity in strongly human-modified regions like the Mediterranean.     

Arabidopsis XXT5 gene encodes a putative alpha-1,6-xylosyltransferase that is involved in xyloglucan biosynthesis

The function of a putative xyloglucan xylosyltransferase from Arabidopsis thaliana (At1g74380; XXT5) was studied. The XXT5 gene is expressed in all plant tissues, with higher levels of expression in roots, stems and cauline leaves. A T-DNA insertion in the XXT5 gene generates a readily visible root hair phenotype (root hairs are shorter and form bubble-like extrusions at the tip), and also causes the alteration of the main root cellular morphology. Biochemical characterization of cell wall polysaccharides isolated from xxt5 mutant seedlings demonstrated decreased xyloglucan quantity and reduced glucan backbone substitution with xylosyl residues. Immunohistochemical analyses of xxt5 plants revealed a selective decrease in some xyloglucan epitopes, whereas the distribution patterns of epitopes characteristic for other cell wall polysaccharides remained undisturbed. Transformation of xxt5 plants with a 35S::HA-XXT5 construct resulted in complementation of the morphological, biochemical and immunological phenotypes, restoring xyloglucan content and composition to wild-type levels. These data provide evidence that XXT5 is a xyloglucan alpha-1,6-xylosyltransferase, and functions in the biosynthesis of xyloglucan.