Cocoa flavonoids up-regulate antioxidant enzyme activity via the ERK1/2 pathway to protect against oxidative stress-induced apoptosis in HepG2 cells

Oxidative stress is widely recognized as an important mediator of apoptosis in liver cells and plays a pivotal role in the pathogenesis of several diseases. Cocoa flavonoids have shown a powerful antioxidant activity providing protection against oxidation and helping prevent oxidative stress-related diseases. However, the molecular mechanisms responsible for this protection are not fully understood. Thus, in this study we investigated the protective effect of a cocoa polyphenolic extract (CPE) against tert-butyl hydroperoxide (t-BOOH)-induced apoptosis and the molecular mechanisms involved in this process. Incubation of HepG2 cells with t-BOOH induced apoptosis as evidenced by caspase-3 activation. This effect was accompanied by increased reactive oxygen species formation and by transient activation of the extracellular regulated kinases (ERKs) as well as sustained activation of the c-Jun N-terminal kinases (JNKs). On the contrary, pretreatment of HepG2 cells with CPE prevented apoptosis through the reduction of reactive oxygen species generation and the modulation of the apoptotic pathways activated by t-BOOH. CPE treatment also activated survival signaling proteins, such as protein kinase B (AKT) and ERKs, and increased the activities of two antioxidant enzymes, glutathione peroxidase (GPx) and glutathione reductase (GR). ERK's implication on GPx and GR induction and the protective effect of CPE against t-BOOH-induced oxidative stress and apoptosis were confirmed through experiments with selective inhibitors. These findings suggest that CPE is an effective inductor of GPx and GR activities via ERK activation and that this up-regulation seems to be required to attenuate t-BOOH-induced injury.     

Resource manipulation reveals flexible allocation rules to growth and reproduction in a Mediterranean evergreen oak

Aims In plants, resource allocation to growth and reproduction may depart from trade-off expectations if (i) investment in growth and reproduction relies on different resource pools, (ii) allocation to reproduction is dependent upon reaching some growth threshold or (iii) reproduction is developmentally linked to growth, both functions relying on the same resource pool. We examined the effects of enhanced resource level on patterns of resource allocation to growth and reproduction in holm oak (Quercus ilex sbsp. ballota), a Mediterranean evergreen tree.Methods In the experimental year (2003), we manipulated the amount of soil nutrients in autumn (to increase nutrient uptake during shoot elongation in the following spring) and soil water in summer (to increase water uptake during acorn growth). Indicators of growth and male and female reproduction were estimated in the pre-experimental (2002), experimental (2003) and post-experimental (2004) years.Important findings Fertilized trees produced significantly longer shoots, but the number of female flowers per shoot was not affected by treatments. The production of male catkins was also enhanced by fertilization. Irrigation did not affect the production of female flowers or abortion rates. Growth and female reproduction showed no consistent relationship in untreated trees, but resource addition elicited a growth-female reproduction trade-off in the experimental year. The sign of this significant relationship changed in the post-experimental year, indicating the existence of lagged effects of resource manipulation on acorn production. Overall, patterns of allocation to growth and reproduction varied as a function of sex, resource availability and year, a result consistent with extreme allocational plasticity in holm oak.     

The sulfate-binding site structure of the human eosinophil cationic protein as revealed by a new crystal form

The human eosinophil cationic protein (ECP), also known as RNase 3, is an eosinophil secretion protein that is involved in innate immunity and displays antipathogen and proinflammatory activities. ECP has a high binding affinity for heterosaccharides, such as bacterial lipopolysaccharides and heparan sulfate found in the glycocalix of eukaryotic cells. We have crystallized ECP in complex with sulfate anions in a new monoclinic crystal form. In this form, the active site groove is exposed, providing an alternative model for ligand binding studies. By exploring the protein-sulfate complex, we have defined the sulfate binding site architecture. Three main sites (S1-S3) are located in the protein active site; S1 and S2 overlap with the phosphate binding sites involved in RNase nucleotide recognition. A new site (S3) that is unique to ECP is one of the key anchoring points for sulfated ligands. Arg 1 and Arg 7 in S3, together with Arg 34 and Arg 36 in S1, form the main basic clusters that assist in the recognition of ligand anionic groups. The location of additional sulfate bound molecules, some of which contribute to the crystal packing, may mimic the binding to extended anionic polymers. In conclusion, the structural data define a binding pattern for the recognition of sulfated molecules that can modulate the role of ECP in innate immunity. The results reveal the structural basis for the high affinity of ECP for glycosaminoglycans and can assist in structure-based drug design of inhibitors of the protein cytotoxicity to host tissues during inflammation.     

NLRP3 inflammasome suppression improves longevity and prevents cardiac aging in male mice

While NLRP3‐inflammasome has been implicated in cardiovascular diseases, its role in physiological cardiac aging is largely unknown. During aging, many alterations occur in the organism, which are associated with progressive impairment of metabolic pathways related to insulin resistance, autophagy dysfunction, and inflammation. Here, we investigated the molecular mechanisms through which NLRP3 inhibition may attenuate cardiac aging. Ablation of NLRP3‐inflammasome protected mice from age‐related increased insulin sensitivity, reduced IGF‐1 and leptin/adiponectin ratio levels, and reduced cardiac damage with protection of the prolongation of the age‐dependent PR interval, which is associated with atrial fibrillation by cardiovascular aging and reduced telomere shortening. Furthermore, old NLRP3 KO mice showed an inhibition of the PI3K/AKT/mTOR pathway and autophagy improvement, compared with old wild mice and preserved Nampt‐mediated NAD⁺ levels with increased SIRT1 protein expression. These findings suggest that suppression of NLRP3 prevented many age‐associated changes in the heart, preserved cardiac function of aged mice and increased lifespan.     

Intraspecific variation in heritable secondary metabolites and defensive strategies in a relict tree

Aims Key herbivory interaction traits such as plant defensive compounds may differ among populations of a single species due to the spatial variation in herbivore feeding guilds and the strength of the interaction. Moreover, the genealogy of population lineages could represent an additional source of variation interacting with the predominant eco-clinal trends. We tested for the existence of genetically based intraspecific variation in chemical defence profiles across the range of the relict tree Prunus lusitanica L. Additionally, we investigated geographical variation in defence inducibility and tested for the existence of a trade-off between qualitative and quantitative defences.Methods We conducted a greenhouse experiment where 210 plants were grown under a common environment, comprising 10 different populations throughout the distribution range of the species and spanning three separate regions: Iberia, Morocco and Macaronesia. To test for the inducibility of defences, we artificially defoliated plants. Three treatments were established within each population: undamaged, defoliated and sampled after 2 h, and defoliated and sampled after 72 h. The concentration of cyanogenic glycosides (prunasin) and phenolics was determined in leaf samples for all treatments.Important findings Basal levels of cyanogenics and phenolics significantly differed among populations and regions across the range of P. lusitanica, with this variation having a heritable basis. Cyanogenics (prunasin) were significantly higher in ancient Macaronesian populations, while phenolic concentrations were larger in Iberia. The higher cyanogenic levels found in Macaronesia could be a consequence of the known stronger herbivory pressure in the islands than in Iberia or the likely longer coevolutionary history with herbivores in this region. These findings indicate that the geographical variation of key ecological traits such plant chemical defences can be imprinted by phylogeographical signals, particularly in relict species. Regarding defence inducibility, prunasin increased after simulated herbivory whereas phenolics mostly decreased after defoliation. Variation in defence inducibility across populations and regions was evident, although no consistent patterns related to the variation in herbivore feeding guilds were observed, particularly among regions with and without ungulate browsing pressure. Finally, a trade-off among induced levels of qualitative (prunasin) and quantitative (phenolics) defences was detected in one of the defoliated treatments, likely as a result of a stronger resource limitation in damaged plants.