Identification of the prooxidant site of human ceruloplasmin: A model for oxidative damage by copper bound to protein surfaces

Free transition metal ions oxidize lipids and lipoproteins in vitro; however, recent evidence suggests that free metal ion-independent mechanisms are more likely in vivo. We have shown previously that human ceruloplasmin (Cp), a serum protein containing seven Cu atoms, induces low density lipoprotein oxidation in vitro and that the activity depends on the presence of a single, chelatable Cu atom. We here use biochemical and molecular approaches to determine the site responsible for Cp prooxidant activity. Experiments with the His-specific reagent diethylpyrocarbonate (DEPC) showed that one or more His residues was specifically required. Quantitative [14C]DEPC binding studies indicated the importance of a single His residue because only one was exposed upon removal of the prooxidant Cu. Plasmin digestion of [14C]DEPC-treated Cp (and N-terminal sequence analysis of the fragments) showed that the critical His was in a 17-kDa region containing four His residues in the second major sequence homology domain of Cp. A full length human Cp cDNA was modified by site-directed mutagenesis to give His-to-Ala substitutions at each of the four positions and was transfected into COS-7 cells, and low density lipoprotein oxidation was measured. The prooxidant site was localized to a region containing His426 because CpH426A almost completely lacked prooxidant activity whereas the other mutants expressed normal activity. These observations support the hypothesis that Cu bound at specific sites on protein surfaces can cause oxidative damage to macromolecules in their environment. Cp may serve as a model protein for understanding mechanisms of oxidant damage by copper-containing (or -binding) proteins such as Cu, Zn superoxide dismutase, and amyloid precursor protein.

Comparison of the defensive elicitation activity of cerato-populin and cerato-platanin: a structural model

Plants can defend themselves from potential pathogenic microorganisms relying on a complex interplay of signaling pathways: activation of the MAPK cascade, transcription of defense related genes, production of reactive oxygen species, nitric oxide and synthesis of other defensive compounds such as phytoalexins. These events are triggered by the recognition of pathogen’s effectors (effector-triggered immunity) or PAMPs (PAMP-triggered immunity). The Cerato Platanin Family (CPF) members are Cys-rich proteins secreted and localized on fungal cell walls, involved in several aspects of fungal development and pathogen-host interactions. Although more than hundred genes of the CPF have been identified and analyzed, the structural and functional characterization of the expressed proteins has been restricted only to few members of the family. Interestingly, those proteins have been shown to bind chitin with diverse affinity and after foliar treatment they elicit defensive mechanisms in host and non-host plants. This property turns cerato platanins into interesting candidates, worth to be studied to develop new fungal elicitors with applications in sustainable agriculture. This study focus on cerato-platanin (CP), core member of the family and on the orthologous cerato-populin (Pop1). The latter shows an identity of 62% and an overall homology of 73% with respect to CP. Both proteins are able to induce MAPKs phosphorylation, production of reactive oxygen species and nitric oxide, overexpression of defense’s related genes, programmed cell death and synthesis of phytoalexins. CP, however, when compared to Pop1, induces a faster response and, in some cases, a stronger activity on plane leaves. Aim of the present research is to verify if the dissimilarities observed in the defense elicitation activity of these proteins can be associated to their structural and dynamic features. Taking advantage of the available CP NMR structure, Pop1’s 3D one was obtained by homology modeling. Experimental residual dipolar couplings and 1H, 15N, 13C resonance assignments were used to validate the model. Previous works on CPF members, addressed the highly conserved random coil regions (loops b1-b2 and b2-b3) as sufficient and necessary to induce necrosis in plants’ leaves: that region was investigated in both Pop1 and CP. In the two proteins the loops differ, in their primary sequence, for few mutations and an insertion with a consequent diversification of the proteins’ electrostatic surface. A set of 2D and 3D NMR experiments was performed to characterize both the spatial arrangement and the dynamic features of the loops. NOE data revealed a more extended network of interactions between the loops in Pop1 than in CP. In addition, in Pop1 we identified a salt bridge Lys25/Asp52 and a strong hydrophobic interaction between Phe26/Trp53. These structural features were expected not only to affect the loops’ spatial arrangement, but also to reduce the degree of their conformational freedom. Relaxation data and the order parameter S2 indeed highlighted reduced flexibility, in particular for loop b1-b2 of Pop1. In vitro NMR experiments, where Pop1 and CP were titrated with oligosaccharides, supported the hypothesis that the loops structural and dynamic differences may be responsible for the different chitin-binding properties of the two proteins: CP selectively binds tetramers of chitin in a shallow groove on one side of the barrel defined by loops b1-b2, b2-b3 and b4-b5, Pop1, instead, interacts in a non-specific fashion with oligosaccharides. Because the region involved in chitin-binding is also responsible for the defense elicitation activity, possibly being recognized by plant's receptors, it is reasonable to expect that those structural and dynamic modifications may also justify the different extent of defense elicitation. To test that hypothesis, the initial steps of a protocol aimed to the identify a receptor for CP, in silico, are presented.

Variability of the ocean heat content during the last millennium - an assessment with the ECHO-g Model

Studies addressing climate variability during the last millennium generally focus on variables with a direct influence on climate variability, like the fast thermal response to varying radiative forcing, or the large-scale changes in atmospheric dynamics (e. g. North Atlantic Oscillation). The ocean responds to these variations by slowly integrating in depth the upper heat flux changes, thus producing a delayed influence on ocean heat content (OHC) that can later impact low frequency SST (sea surface temperature) variability through reemergence processes. In this study, both the externally and internally driven variations of the OHC during the last millennium are investigated using a set of fully coupled simulations with the ECHO-G (coupled climate model ECHAMA4 and ocean model HOPE-G) atmosphere-ocean general circulation model (AOGCM). When compared to observations for the last 55 yr, the model tends to overestimate the global trends and underestimate the decadal OHC variability. Extending the analysis back to the last one thousand years, the main impact of the radiative forcing is an OHC increase at high latitudes, explained to some extent by a reduction in cloud cover and the subsequent increase of short-wave radiation at the surface. This OHC response is dominated by the effect of volcanism in the preindustrial era, and by the fast increase of GHGs during the last 150 yr. Likewise, salient impacts from internal climate variability are observed at regional scales. For instance, upper temperature in the equatorial Pacific is controlled by ENSO (El Nino Southern Oscillation) variability from interannual to multidecadal timescales. Also, both the Pacific Decadal Oscillation (PDO) and the Atlantic Multidecadal Oscillation (AMO) modulate intermittently the interdecadal OHC variability in the North Pacific and Mid Atlantic, respectively. The NAO, through its influence on North Atlantic surface heat fluxes and convection, also plays an important role on the OHC at multiple timescales, leading first to a cooling in the Labrador and Irminger seas, and later on to a North Atlantic warming, associated with a delayed impact on the AMO.

Tropical climate and vegetation simulations during the Heinrich event 1 using an Earth System Model of Intermediate Complexity (EMIC) - the University of Victoria Earth System-Climate Model (UVic ESCM)

Abrupt climate changes from 18 to 15 thousand years before present (kyr BP) associated with Heinrich Event 1 (HE1) had a strong impact on vegetation patterns not only at high latitudes of the Northern Hemisphere, but also in the tropical regions around the Atlantic Ocean. To gain a better understanding of the linkage between high and low latitudes, we used the University of Victoria (UVic) Earth System-Climate Model (ESCM) with dynamical vegetation and land surface components to simulate four scenarios of climate-vegetation interaction: the pre-industrial era, the Last Glacial Maximum (LGM), and a Heinrich-like event with two different climate backgrounds (interglacial and glacial). We calculated mega-biomes from the plant-functional types (PFTs) generated by the model to allow for a direct comparison between model results and palynological vegetation reconstructions. Our calculated mega-biomes for the pre-industrial period and the LGM corresponded well with biome reconstructions of the modern and LGM time slices, respectively, except that our pre-industrial simulation predicted the dominance of grassland in southern Europe and our LGM simulation resulted in more forest cover in tropical and sub-tropical South America. The HE1-like simulation with a glacial climate background produced sea-surface temperature patterns and enhanced inter-hemispheric thermal gradients in accordance with the "bipolar seesaw" hypothesis. We found that the cooling of the Northern Hemisphere caused a southward shift of those PFTs that are indicative of an increased desertification and a retreat of broadleaf forests in West Africa and northern South America. The mega-biomes from our HE1 simulation agreed well with paleovegetation data from tropical Africa and northern South America. Thus, according to our model-data comparison, the reconstructed vegetation changes for the tropical regions around the Atlantic Ocean were physically consistent with the remote effects of a Heinrich event under a glacial climate background.

Results of Heinrich event 1 simulation from the University of Victoria Earth System-Climate Model (UVic ESCM) and the Community Climate System Model (CCSM3)

We investigated changes in tropical climate and vegetation cover associated with abrupt climate change during Heinrich Event 1 (HE1, ca. 17.5 ka BP) using two different global climate models: the University of Victoria Earth System-Climate Model (UVic ESCM) and the Community Climate System Model version 3 (CCSM3). Tropical South American and African pollen records suggest that the cooling of the North Atlantic Ocean during HE1 influenced the tropics through a southward shift of the rain belt. In this study, we simulated the HE1 by applying a freshwater perturbation to the North Atlantic Ocean. The resulting slowdown of the Atlantic Meridional Overturning Circulation was followed by a temperature seesaw between the Northern and Southern Hemispheres, as well as a southward shift of the tropical rain belt. The shift and the response pattern of the tropical vegetation around the Atlantic Ocean were more pronounced in the CCSM3 than in the UVic ESCM simulation. For tropical South America, opposite changes in tree and grass cover were modeled around 10° S in the CCSM3 but not in the UVic ESCM. In tropical Africa, the grass cover increased and the tree cover decreased around 15° N in the UVic ESCM and around 10° N in the CCSM3. In the CCSM3 model, the tree and grass cover in tropical Southeast Asia responded to the abrupt climate change during the HE1, which could not be found in the UVic ESCM. The biome distributions derived from both models corroborate findings from pollen records in southwestern and equatorial western Africa as well as northeastern Brazil.

Solar sensitivity experiments for the pre-industrial time period using the comprehensive global climate model CCSM3 (Community Climate System Model 3)

The Southern Hemisphere Westerly Winds (SWW) constitute an important zonal circulation that influences large-scale precipitation patterns and ocean circulation. Variations in their intensity and latitudinal position have been suggested to exert a strong influence on the CO2 budget in the Southern Ocean, thus making them a potential factor affecting the global climate. The possible influence of solar forcing on SWW variability during the Holocene is addressed. Solar sensitivity experiments with a comprehensive global climate model (CCSM3) are carried out to study the response of SWW to solar variability. In addition, It is shown that a high-resolution iron record from the Chilean continental slope (41° S), which is interpreted to reflect changes in the position of the SWW, is significantly correlated with reconstructed solar activity during the past 3000 years. Taken together, the proxy and model results suggest that centennial-scale periods of lower (higher) solar activity caused equatorward (southward) shifts of the annual mean SWW.

Mixed model formulations for multi-environment trials

When studying genotype X environment interaction in multi-environment trials, plant breeders and geneticists often consider one of the effects, environments or genotypes, to be fixed and the other to be random. However, there are two main formulations for variance component estimation for the mixed model situation, referred to as the unconstrained-parameters (UP) and constrained-parameters (CP) formulations. These formulations give different estimates of genetic correlation and heritability as well as different tests of significance for the random effects factor. The definition of main effects and interactions and the consequences of such definitions should be clearly understood, and the selected formulation should be consistent for both fixed and random effects. A discussion of the practical outcomes of using the two formulations in the analysis of balanced data from multi-environment trials is presented. It is recommended that the CP formulation be used because of the meaning of its parameters and the corresponding variance components. When managed (fixed) environments are considered, users will have more confidence in prediction for them but will not be overconfident in prediction in the target (random) environments. Genetic gain (predicted response to selection in the target environments from the managed environments) is independent of formulation.