Src family kinases Fyn and Lyn are constitutively activated and mediate plasmacytoid dendritic cell responses.

Plasmacytoid dendritic cells (pDC) are type I interferon-producing cells with critical functions in a number of human illnesses; however, their molecular regulation is incompletely understood. Here we show the role of Src family kinases (SFK) in mouse and human pDCs. pDCs express Fyn and Lyn and their activating residues are phosphorylated both before and after Toll-like receptor (TLR) stimulation. Fyn or Lyn genetic ablation as well as treatment with SFK inhibitors ablate pDC (but not conventional DC) responses both in vitro and in vivo. Inhibition of SFK activity not only alters TLR-ligand localization and inhibits downstream signalling events, but, independent of ex-vivo TLR stimulation, also affects constitutive phosphorylation of BCAP, an adaptor protein bridging PI3K and TLR pathways. Our data identify Fyn and Lyn as important factors that promote pDC responses, describe the mechanisms involved and highlight a tonic SFK-mediated signalling that precedes pathogen encounter, raising the possibility that small molecules targeting SFKs could modulate pDC responses in human diseases.

Arsenic-induced responses in Pityrogramma calomelanos (L.) Link: Arsenic speciation, mineral nutrition and antioxidant defenses.

Arsenic (As) hyperaccumulation trait has been described in a limited number of fern species. The physiological basis of hyperaccumulation remains unclear, especially in non-Pteris species such as Pityrogramma calomelanos. Aiming at a better understanding of As-induced responses, P. calomelanos plants were exposed to 1 mM As for 21 days and compared with control plants. Chemical analyses revealed that As accumulation was ten times higher in pinnae then in roots and stipes. In pinnae, As was present mainly as arsenite, whereas arsenate was the dominant form in stipes and roots. Arsenic promoted an increase in antioxidant enzyme activities in both fern parts and several alterations in mineral nutrition, especially with regard to P and K. A higher content of non-protein thiols was observed in pinnae of plants exposed to As, whereas As induced the increase in lipid peroxidation in roots. The results showed that Pityrogramma calomelanos shares with Pteris vittata several aspects of As metabolism. High root-shoot As translocation showed to be essential to avoid toxic effects in roots, since the root is more sensitive to the metalloid. The higher capacity of P. calomelanos to sequester arsenite in the pinna and its efficient antioxidant system maintain the reactive oxygen species at a low level, thus enhancing the continuous accumulation of As. Molecular investigations are needed to elucidate the evolution of As-tolerance mechanisms in Pteridaceae species, especially with regard to membrane transporters and ROS signaling.

Differences in phosphorus translocation contributes to differential arsenic tolerance between plants of Borreria verticillata (Rubiaceae) from mine and non-mine sites.

We have identified new arsenic-tolerant plant species Borreria verticillata (Rubiaceae) that has mine and non-mine populations at a highly contaminated site (CS) and an uncontaminated site (UCS), respectively, in Brazil. Plants of B. verticillata from both sites were cultivated at different As and P concentrations. At low P concentration, CS plants showed reduced As uptake, higher P translocation to shoots, higher constitutive levels of phenolic compounds in roots, and higher tolerance to this metalloid. At the lowest P and highest As concentration, CS plants showed higher biomass. In addition, CS plants showed higher P uptake in the absence of As, suggesting that more efficient P translocation could contribute more to tolerance than decreased As uptake. In contrast, at low P concentration, UCS plants showed higher As content in shoot and root, increase in phenol levels in roots, reduction in dry biomass, and decrease of the effective efficiency of photochemical reactions and the electron transport rate. Under higher P concentrations, the decrease in As uptake was similar in both populations. The differences between the two populations with respect to As and P uptake suggest that altered kinetic properties or expression of P transporters contribute to higher As tolerance in B. verticillata from CS. As a ruderal and As-tolerant plant, B. verticillata could be successfully used for the revegetation of contaminated soils.

Properties of cryptic epitopes and their corresponding antibodies as indicated by the study of human and ovine growth hormones.

Antibodies (Ab) directed to hidden antigenic determinants (cryptotopes) are undesirable because they are not neutralizing. Additionally, we have previously demonstrated a close association between the extent of Ab to cryptic determinants and the expression of autoantibodies (autoAb) under some experimental conditions. Thus, the first objective of this work was to establish the physicochemical characteristics of Ab to cryptotopes and the second one was to examine the structural features of cryptic epitopes themselves. Using human and ovine growth hormones (hGH and oGH) as antigenic models and competition ELISA under different conditions of temperature, pH or ionic strength, we did not find any difference between the binding properties of anti-cryptic epitope antibodies (Ab) and anti-native epitope Ab. Then, using synthetic peptides and tryptic digests and direct and competition ELISAs we studied the structures of cryptic hGH and oGH epitopes. Isolated peptides either in solution or adsorbed on microplates failed to react. Partially digested hGH was recognized only when insolubilized on microplates, and anti-oGH Ab only reacted with a large fragment of the hormone either in solution or insolubilized. These results indicate that, at least in the case of hGH and oGH, cryptic epitopes are not simple linear sequences, as commonly referred without any evidence, but new exposed conformational structures different from those found in the native antigen.

Effects of various adjuvants and a viral infection on the antibody specificity toward native or cryptic epitopes of a protein antigen.

An immunization protocol that induces antibodies (Abs) directed to cryptic epitopes of a protein antigen (Ag) reduces the efficacy of vaccines that ideally should induce Abs against native epitopes. We have shown earlier that viral infections concomitant with immunization against a protein tend to shift the Ab specificity toward cryptic epitopes and tend to induce the production of autoantibodies (autoAbs). Here, we show the effects of three adjuvants on the Ab specificity in the absence or presence of a viral infection (lactate dehydrogenase-elevating virus or LDV), with human growth hormone (hGH) being, as before, the protein Ag. Pathogen-free CBA/Ht and BALB/c mice were immunized with hGH in the presence of complete Freund's adjuvant (CFA), monophosphoryl lipid A (MPL) or alum, with the animals being either infected with LDV or not infected with LDV. Conventional and competition enzyme-linked immunosorbent assays (ELISAs) indicated that in noninfected mice, CFA induced higher titres of anti-hGH Ab than did MPL or alum, with the Ab being almost totally directed to cryptic hGH epitopes. Strikingly, CFA plus LDV infection in CBA/Ht mice shifted the specificity of the anti-hGH Ab toward native epitopes, whereas the virus decreased the Ab titre when MPL or alum was used. Our Western blot results showed that 70% of mice immunized with hGH in the presence of any adjuvant produced autoAbs against a variety of tissue Ags. The amount of autoAb and the concentration of Ab to hGH cryptic epitopes did correlate, suggesting a relationship between both kinds of Ab. Significant differences were observed in the various effects of adjuvants and the viral infection between the two mouse strains used in this work.

Differential responses of superoxide dismutase in freezing resistant Solanum curtilobum and freezing sensitive Solanum tuberosum subjected to oxidative and water stress.

In photosynthetic tissues superoxide dismutase (SOD) plays an important role by scavenging the superoxide radical whose production is an usual reaction in chloroplast thylakoids. To test the differential response of SOD, two Andean potato species differing in frost resistance, Solanum curtilobum (frost resistant) and Solanum tuberosum (frost sensitive), were subjected to methyl viologen-mediated oxidative stress and polyethylene glycol (PEG)-induced water stress. A significant increment (approximately two-fold) in total SOD and FeSOD activity, which occupied about 50% of the total activity, was found when leaves of S. curtilobum were exposed to water stress. In contrast, the SOD activity in leaves of S. tuberosum remained unchanged. The exposure of leaves of S. curtilobum to oxidative stress increased total SOD and FeSOD activity by 350%. High correlation between SOD activity and the F(v)/F(m )ratio under both PEG induced water stress and MV-mediated oxidative stress was observed. This suggests that SOD can protect PSII from superoxide generated by oxidative and water stress. The higher SOD activity could be an important mechanism to explain why some natives Andean potato like S. curtilobum are more resistant to abiotic stresses than S. tuberosum.