Storage elicits a fast antioxidant enzyme activity in <Emphasis Type="Italic">Araucaria angustifolia</Emphasis> embryos

Storage of recalcitrant seeds leads to the initiation of subcellular damage or to the initiation of germination process, and both may result in viability loss. This study aimed to elucidate the biochemical basis of embryos survival of Araucaria angustifolia recalcitrant seeds during storage. After harvesting, seeds were stored at ambient conditions (without temperature and humidity control) and in a cold chamber (temperature of 10 ± 3 °C, and relative humidity of 45 ± 5 %). Moisture content, viability, H₂O₂ content, lipid peroxidation, protein content, and activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX), at 0, 15, 45 and 90 days of storage, were evaluated. Seed viability reduced about 40 % during the storage period accompanied by a reduction in soluble protein (about 64 % of reduction) in both storage conditions, and increased lipid peroxidation (about 115 % and 66 % for ambient and cold chamber conditions, respectively). H₂O₂ content used as a marker of oxidative stress was reduced during the period, possibly controlled by the action of CAT and APX, for which increased activities were observed. The results allowed the identification of seven SOD isoenzymes (one Mn-SOD, five Fe-SOD and one Cu/Zn-SOD), whose activities also increased in response to storage. Some biochemical damage resulting from storage was observed, but viability reduction was not due to failure of enzymatic protection mechanisms.     

Optimization of protein–protein docking for predicting Fc–protein interactions

The antibody crystallizable fragment (Fc) is recognized by effector proteins as part of the immune system. Pathogens produce proteins that bind Fc in order to subvert or evade the immune response. The structural characterization of the determinants of Fc–protein association is essential to improve our understanding of the immune system at the molecular level and to develop new therapeutic agents. Furthermore, Fc‐binding peptides and proteins are frequently used to purify therapeutic antibodies. Although several structures of Fc–protein complexes are available, numerous others have not yet been determined. Protein–protein docking could be used to investigate Fc–protein complexes; however, improved approaches are necessary to efficiently model such cases. In this study, a docking‐based structural bioinformatics approach is developed for predicting the structures of Fc–protein complexes. Based on the available set of X‐ray structures of Fc–protein complexes, three regions of the Fc, loosely corresponding to three turns within the structure, were defined as containing the essential features for protein recognition and used as restraints to filter the initial docking search. Rescoring the filtered poses with an optimal scoring strategy provided a success rate of approximately 80% of the test cases examined within the top ranked 20 poses, compared to approximately 20% by the initial unrestrained docking. The developed docking protocol provides a significant improvement over the initial unrestrained docking and will be valuable for predicting the structures of currently undetermined Fc–protein complexes, as well as in the design of peptides and proteins that target Fc.     

A synergistic trio of invasive mammals? Facilitative interactions among beavers,muskrats, and mink at the southern end of the Americas

With ecosystems increasingly having co-occurring invasive species, it is becoming more important to understand invasive species interactions. At the southern end of the Americas, American beavers (Castor canadensis), muskrats (Ondatra zibethicus), and American mink (Neovison vison), were independently introduced. We used generalized linear models to investigate how muskrat presence related to beaver-modified habitats on Navarino Island, Chile. We also investigated the trophic interactions of the mink with muskrats and beavers by studying mink diet. Additionally, we proposed a conceptual species interaction framework involving these invasive species on the new terrestrial community. Our results indicated a positive association between muskrat presence and beaver-modified habitats. Model average coefficients indicated that muskrats preferred beaver-modified freshwater ecosystems, compared to not dammed naturally flowing streams. In addition, mammals and fish represented the main prey items for mink. Although fish were mink’s dominant prey in marine coastal habitats, muskrats represented >50 % of the biomass of mink diet in inland environments. We propose that beavers affect river flow and native vegetation, changing forests into wetlands with abundant grasses and rush vegetation. Thus, beavers facilitate the existence of muskrats, which in turn sustain inland mink populations. The latter have major impacts on the native biota, especially on native birds and small rodents. The facilitative interactions among beavers, muskrats, and mink that we explored in this study, together with other non-native species, suggest that an invasive meltdown process may exist; however further research is needed to confirm this hypothesis. Finally, we propose a community-level management to conserve the biological integrity of native ecosystems.     

Cryopreserved CD90+ cells obtained from mobilized peripheral blood in sheep: a new source of mesenchymal stem cells for preclinical applications

Mobilized peripheral blood (MPB) bone marrow cells possess the potential to differentiate into a variety of mesenchymal tissue types and offer a source of easy access for obtaining stem cells for the development of experimental models with applications in tissue engineering. In the present work, we aimed to isolate by magnetic activated cell sorting CD90+ cells from MPB by means of the administration of Granulocyte-Colony Stimulating Factor and to evaluate cell proliferation capacity, after thawing of the in vitro culture of this population of mesenchymal stem cells (MSCs) in sheep. We obtained a median of 8.2 ± 0.6 million of CD90+ cells from the 20-mL MPB sample. After thawing, at day 15 under in vitro culture, the mean CD90+ cells determined by flow cytometry was 92.92 ± 1.29 % and cell duplication time determined by crystal violet staining was 47.59 h. This study describes for the first time the isolation, characterization, and post-in vitro culture thawing of CD90+ MSCs from mobilized peripheral blood in sheep. This population can be considered as a source of MSCs for experimental models in tissue engineering research.     

G protein-coupled receptor systems and their lipid environment in health disorders during aging

Cells, tissues and organs undergo phenotypic changes and deteriorate as they age. Cell growth arrest and hyporesponsiveness to extrinsic stimuli are all hallmarks of senescent cells. Most such external stimuli received by a cell are processed by two different cell membrane systems: receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs). GPCRs form the largest gene family in the human genome and they are involved in most relevant physiological functions. Given the changes observed in the expression and activity of GPCRs during aging, it is possible that these receptors are directly involved in aging and certain age-related pathologies. On the other hand, both GPCRs and G proteins are associated with the plasma membrane and since lipid-protein interactions regulate their activity, they can both be considered to be sensitive to the lipid environment. Changes in membrane lipid composition and structure have been described in aged cells and furthermore, these membrane changes have been associated with alterations in GPCR mediated signaling in some of the main health disorders in elderly subjects. Although senescence could be considered a physiologic process, not all aging humans develop the same health disorders. Here, we review the involvement of GPCRs and their lipid environment in the development of the major human pathologies associated with aging such as cancer, neurodegenerative disorders and cardiovascular pathologies.     

c-Jun N-terminal protein kinase signalling pathway mediates lovastatin-induced rat brain neuroblast apoptosis

We have previously shown that lovastatin, an HMG-CoA reductase inhibitor, induces apoptosis in rat brain neuroblasts. c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) are implicated in regulation of neuronal apoptosis. In this work, we investigated the role of JNK and p38 MAPK in neuroblast apoptosis induced by lovastatin. We found that lovastatin induced the activation of JNK, but not p38 MAPK. It also induced c-Jun phosphorylation with a subsequent increase in activator protein-1 (AP-1) binding, AP-1-mediated gene expression and BimEL protein levels. The effects of lovastatin were prevented by mevalonate. Pre-treatment with iJNK-I (a selective JNK inhibitor) prevented the effect of lovastatin on both neuroblast apoptosis and the activation of the JNK cascade. Furthermore, we found that the activation of the JNK signalling pathway triggered by lovastatin is accompanied by caspase-3 activation which is also inhibited by iJNK-I pre-treatment. Finally, a specific inhibitor of p38 MAPK, SB203580, had no effect on lovastatin-induced neuroblast apoptosis. Taken together, our data suggest that the activation of the JNK/c-Jun/BimEL signalling pathway plays a crucial role in lovastatin-induced neuroblast apoptosis. Our findings may also contribute to elucidate the intracellular mechanisms involved in the central nervous system side effects associated with statin therapy.     

Translational machinery and protein folding: evidence of conformational variants of the estrogen receptor alpha

As an approach to understand how translation may affect protein folding, we analyzed structural and functional properties of the human estrogen receptor alpha synthesized by different eukaryotic translation systems. A minimum of three conformations of the receptor were detected using limited proteolysis and a sterol ligand-binding assay. The receptor in vitro translated in rabbit reticulocyte lysate was rapidly degraded by protease, produced major bands of about 34 kDa and showed a high affinity for estradiol. In a wheat germ translation system, the receptor was more slowly digested. Two soluble co-existing conformations were evident by different degradation patterns and estradiol binding. Our data show that differences in the translation machinery may result in alternative conformations of the receptor with distinct sterol binding properties. These studies suggest that components of the cellular translation machinery itself might influence the protein folding pathways and the relative abundance of different receptor conformers.     

The l-isoform but not d-isoforms of a JNK inhibitory peptide protects pancreatic beta-cells

The activation of c-jun N-terminal kinase (JNK) in pancreatic islets is associated with impaired function and viability, and JNK inhibitory peptides (JNKIs) are cytoprotective. In particular, l-isoforms of JNKIs were shown to improve islets viability, while the d-retroinverso isoform of JNKI (RI-JNKI), with a higher therapeutic potential due to longer half-life, has not been studied. We compared the cytoprotective properties of L-JNKI and RI-JNKI. Treatment of murine islets with L-JNKI resulted in preservation of islet equivalents and greater percentage of viable beta-cells in culture. In contrast, RI-JNKI was not protective. We found that L-JNKI but not RI-JNKI prevents endogenous c-jun phosphorylation in insulinoma cells. Moreover, RI-JNKI induced islet cells necrosis and activates the p-38 kinase. In conclusion, L-JNKI directly affects beta-cells and ameliorates islet viability and function, while RI-JNKI has toxic effects, limiting its biological application to islet cell biology.