Resveratrol increases vascular oxidative stress resistance

Epidemiological studies suggest that Mediterranean diets rich in resveratrol are associated with reduced risk of coronary artery disease. However, the mechanisms by which resveratrol exerts its vasculoprotective effects are not completely understood. Because oxidative stress and endothelial cell injury play a critical role in vascular aging and atherogenesis, we evaluated whether resveratrol inhibits oxidative stress-induced endothelial apoptosis. We found that oxidized LDL and TNF-alpha elicited significant increases in caspase-3/7 activity in endothelial cells and cultured rat aortas, which were prevented by resveratrol pretreatment (10(-6)-10(-4) mol/l). The protective effect of resveratrol was attenuated by inhibition of glutathione peroxidase and heme oxygenase-1, suggesting a role for antioxidant systems in the antiapoptotic action of resveratrol. Indeed, resveratrol treatment protected cultured aortic segments and/or endothelial cells against increases in intracellular H(2)O(2) levels and H(2)O(2)-mediated apoptotic cell death induced by oxidative stressors (exogenous H(2)O(2), paraquat, and UV light). Resveratrol treatment also attenuated UV-induced DNA damage (comet assay). Resveratrol treatment upregulated the expression of glutathione peroxidase, catalase, and heme oxygenase-1 in cultured arteries, whereas it had no significant effect on the expression of SOD isoforms. Resveratrol also effectively scavenged H(2)O(2) in vitro. Thus resveratrol seems to increase vascular oxidative stress resistance by scavenging H(2)O(2) and preventing oxidative stress-induced endothelial cell death. We propose that the antioxidant and antiapoptotic effects of resveratrol, together with its previously described anti-inflammatory actions, are responsible, at least in part, for its cardioprotective effects.     

Adaptive radiation with regard to nutrient sequestration strategies in the carnivorous plants of the genus Nepenthes

Carnivorous pitcher plants of the genus Nepenthes have evolved a great diversity of pitcher morphologies. Selective pressures for maximizing nutrient uptake have driven speciation and diversification of the genus in a process known as adaptive radiation. This leads to the evolution of pitchers adapted to specific and often bizarre source of nutrients, which are not strictly animal-derived. One example is Nepenthes ampullaria with unusual growth pattern and pitcher morphology what enables the plant to capture a leaf litter from the canopy above. We showed that the plant benefits from nitrogen uptake by increased rate of photosynthesis and growth what may provide competitive advantage over others co-habiting plants. A possible impact of such specialization toward hybridization, an important mechanism in speciation, is discussed.     

Macromolecular docking restrained by a small angle X-ray scattering profile

While many structures of single protein components are becoming available, structural characterization of their complexes remains challenging. Methods for modeling assembly structures from individual components frequently suffer from large errors, due to protein flexibility and inaccurate scoring functions. However, when additional information is available, it may be possible to reduce the errors and compute near-native complex structures. One such type of information is a small angle X-ray scattering (SAXS) profile that can be collected in a high-throughput fashion from a small amount of sample in solution. Here, we present an efficient method for protein–protein docking with a SAXS profile (FoXSDock): generation of complex models by rigid global docking with PatchDock, filtering of the models based on the SAXS profile, clustering of the models, and refining the interface by flexible docking with FireDock. FoXSDock is benchmarked on 124 protein complexes with simulated SAXS profiles, as well as on 6 complexes with experimentally determined SAXS profiles. When induced fit is less than 1.5 Å interface C_α RMSD and the fraction residues of missing from the component structures is less than 3%, FoXSDock can find a model close to the native structure within the top 10 predictions in 77% of the cases; in comparison, docking alone succeeds in only 34% of the cases. Thus, the integrative approach significantly improves on molecular docking alone. The improvement arises from an increased resolution of rigid docking sampling and more accurate scoring.     

Assembly and Molecular Architecture of the Phosphoinositide 3-Kinase p85α Homodimer

Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that are activated by growth factor and G-protein-coupled receptors and propagate intracellular signals for growth, survival, proliferation, and metabolism. p85α, a modular protein consisting of five domains, binds and inhibits the enzymatic activity of class IA PI3K catalytic subunits. Here, we describe the structural states of the p85α dimer, based on data from in vivo and in vitro solution characterization. Our in vitro assembly and structural analyses have been enabled by the creation of cysteine-free p85α that is functionally equivalent to native p85α. Analytical ultracentrifugation studies showed that p85α undergoes rapidly reversible monomer-dimer assembly that is highly exothermic in nature. In addition to the documented SH3-PR1 dimerization interaction, we identified a second intermolecular interaction mediated by cSH2 domains at the C-terminal end of the polypeptide. We have demonstrated in vivo concentration-dependent dimerization of p85α using fluorescence fluctuation spectroscopy. Finally, we have defined solution conditions under which the protein is predominantly monomeric or dimeric, providing the basis for small angle x-ray scattering and chemical cross-linking structural analysis of the discrete dimer. These experimental data have been used for the integrative structure determination of the p85α dimer. Our study provides new insight into the structure and assembly of the p85α homodimer and suggests that this protein is a highly dynamic molecule whose conformational flexibility allows it to transiently associate with multiple binding proteins.     

Deciphering protein complexes and protein interaction networks by tandem affinity purification and mass spectrometry: analytical perspective

We employed a combination of tandem affinity purification and mass spectrometry for deciphering protein complexes and the protein interaction network in budding yeast. 53 genes were epitope-tagged, and their interaction partners were isolated by two-step immunoaffinity chromatography from whole cell lysates. 38 baits pulled down a total of 220 interaction partners, which are members of 19 functionally distinct protein complexes. We identified four proteins shared between complexes of different functionality thus charting segments of a protein interaction network. Concordance with the results of genome-wide two-hybrid screening was poor (14% of identified interactors overlapped) suggesting that the two approaches may provide complementary views on physical interactions within the proteome.     

Structural Model of the Bilitranslocase Transmembrane Domain Supported by NMR and FRET Data

We present a 3D model of the four transmembrane (TM) helical regions of bilitranslocase (BTL), a structurally uncharacterized protein that transports organic anions across the cell membrane. The model was computed by considering helix-helix interactions as primary constraints, using Monte Carlo simulations. The interactions between the TM2 and TM3 segments have been confirmed by Förster resonance energy transfer (FRET) spectroscopy and nuclear magnetic resonance (NMR) spectroscopy, increasing our confidence in the model. Several insights into the BTL transport mechanism were obtained by analyzing the model. For example, the observed cis-trans Leu-Pro peptide bond isomerization in the TM3 fragment may indicate a key conformational change during anion transport by BTL. Our structural model of BTL may facilitate further studies, including drug discovery.     

Influence of spring and autumn phenological transitions on forest ecosystem productivity

We use eddy covariance measurements of net ecosystem productivity (NEP) from 21 FLUXNET sites (153 site-years of data) to investigate relationships between phenology and productivity (in terms of both NEP and gross ecosystem photosynthesis, GEP) in temperate and boreal forests. Results are used to evaluate the plausibility of four different conceptual models. Phenological indicators were derived from the eddy covariance time series, and from remote sensing and models. We examine spatial patterns (across sites) and temporal patterns (across years); an important conclusion is that it is likely that neither of these accurately represents how productivity will respond to future phenological shifts resulting from ongoing climate change. In spring and autumn, increased GEP resulting from an ‘extra’ day tends to be offset by concurrent, but smaller, increases in ecosystem respiration, and thus the effect on NEP is still positive. Spring productivity anomalies appear to have carry-over effects that translate to productivity anomalies in the following autumn, but it is not clear that these result directly from phenological anomalies. Finally, the productivity of evergreen needleleaf forests is less sensitive to phenology than is productivity of deciduous broadleaf forests. This has implications for how climate change may drive shifts in competition within mixed-species stands.     

Changes in redox states of respiratory pigments recorded from the eyes of live blowflies exposed to light stimuli and hypoxia

Time courses of mitochondrial responses to illumination-induced physiological loads and to hypoxia, were recorded optically from eyes of blowflies Calliphora vicina chalky. We isolated changes in redox states of haems a₃, a, c, and b. Two types of responses to light stimulation were observed. Haems b and a₃ responded with transient oxidation and haems a and c with reduction. The same two groups emerged in response to anoxic exposure. The onset of reduction of haems a and c had virtually no latency, while haems a₃ and b exhibited a transient oxidation followed by reduction only after 10–20 s. The dependence of the steady-state reduction level on P_textO2 P_{{{text{O}}_{2} }} produced the same groups. Haems a and c were significantly reduced at P_textO2 P_{{{text{O}}_{2} }} levels around 10 kPa while with haems b and a₃ load-induced oxidation was only replaced by reduction below 2 kPa. We propose haems respond to physiological loads in accordance with their steady-state reduction, which in turn depends largely on barriers for electron transport imposed by the mitochondrial membrane potential. We also propose it may be possible to assess the values of tissue P_textO2 P_{{{text{O}}_{2} }} and O₂ consumption by monitoring haems that are highly oxidized at rest such as haem a.