Role of the Cytosolic Heat Shock Protein 70 Ssa5 in the Ciliate Protozoan Tetrahymena thermophila

Heat shock protein 70 (Hsp70) is a member of a family of conserved chaperone proteins whose function is well investigated in many model organisms. Here we focus on an Hsp70 called Ssa5 in the ciliate protozoan Tetrahymena thermophila, and reveal that its translation is heat inducible as for general Hsps. Moreover, the protein is abundantly expressed in the cytoplasm during sexual reproduction (conjugation) as well as in response to heat‐stress. Knocking out of SSA5 (ΔSSA5) does not affect the survival of the cell under heat‐stress, likely due to other Hsp70 paralogs compensating for the defect. During conjugation, ΔSSA5 leads to a fertilization defect in which the two pronuclei are in close proximity but never fuse. The unfertilized pronuclei differentiate, resulting in a heterokaryon with developed haploid germline and somatic nuclei. In addition, degeneration of the parental somatic nucleus is not affected. These results suggest a specific involvement of Ssa5 in pronuclear fusion and fertilization.     

Thermal acclimation modulates the impacts of temperature and enrichment on trophic interaction strengths and population dynamics

Global change affects individual phenotypes and biotic interactions, which can have cascading effects up to the ecosystem level. However, the role of environmentally induced phenotypic plasticity in species interactions is poorly understood, leaving a substantial gap in our knowledge of the impacts of global change on ecosystems. Using a cladoceran–dragonfly system, we experimentally investigated the effects of thermal acclimation, acute temperature change and enrichment on predator functional response and metabolic rate. Using our experimental data, we next parameterized a population dynamics model to determine the consequences of these effects on trophic interaction strength and food‐chain stability. We found that (1) predation and metabolic rates of the dragonfly larvae increase with acute warming, (2) warm‐acclimated larvae have a higher maximum predation rate than cold‐acclimated ones, and (3) long‐term interaction strength increases with enrichment but decreases with both acclimation and acute temperatures. Overall, our experimental results show that thermal acclimation can buffer negative impacts of environmental change on predators and increase food‐web stability and persistence. We conclude that the effect of acclimation and, more generally, phenotypic plasticity on trophic interactions should not be overlooked if we aim to understand the effects of climate change and enrichment on species interaction strength and food‐web stability.     

Edge effects across boundaries between natural and restored jarrah (Eucalyptus marginata) forests in south‐western Australia

Edge effects are a widespread and ubiquitous ecological phenomenon, yet they remain poorly studied across edges between restored and natural forests. To address this lack of knowledge, we studied vertebrate communities across edges between 3‐year old restored mine‐pits and adjacent unmined forest in the jarrah (Eucalyptus marginata) forest of south‐western Australia. We found that mammal communities showed no edge response but reptile communities did. Overall reptile abundance and Morethia obscura abundance were higher in unmined forest along edges, Egernia napoleonis abundance was lower in unmined forest along edges, while Pogona minor abundance was lower in restored mine‐pits along edges. Predictive models were unable to predict species edge responses, due to the lack of knowledge of the ecology of jarrah forest reptiles, but proved useful in identifying potential ecological mechanisms behind observed edge responses and suggested that potential mechanisms were likely different for each species. Our study is the first to show edge responses in both habitats forming the edge between restored and natural forests, emphasizing the importance of studying both habitats forming the edge. Our results also suggest that, despite being poorly studied, edge responses are common across edges between restored and natural forest and result from a variety of ecological mechanisms. An increased understanding of the ecological mechanisms driving edge responses across edges between restored and natural forests will improve our ability to integrate restored areas into cross‐landscape management and, ultimately, improve our ability to manage landscapes for biodiversity conservation.     

Environmental variation and population responses to global change

Species' responses to environmental changes such as global warming are affected not only by trends in mean conditions, but also by natural and human‐induced environmental fluctuations. Methods are needed to predict how such environmental variation affects ecological and evolutionary processes, in order to design effective strategies to conserve biodiversity under global change. Here, we review recent theoretical and empirical studies to assess: (1) how populations respond to changes in environmental variance, and (2) how environmental variance affects population responses to changes in mean conditions. Contrary to frequent claims, empirical studies show that increases in environmental variance can increase as well as decrease long‐term population growth rates. Moreover, environmental variance can alter and even reverse the effects of changes in the mean environment, such that even if environmental variance remains constant, omitting it from population models compromises their ability to predict species' responses to changes in mean conditions. Drawing on theory relating these effects of environmental variance to the curvatures of population growth responses to the environment, we outline how species' traits such as phylogenetic history and body mass could be used to predict their responses to global change under future environmental variability.     

Survey of the year 2009: applications of isothermal titration calorimetry

Isothermal titration calorimetry (ITC) is now an established and invaluable method for determining the thermodynamic constants, association constant and stoichiometry of molecular interactions in aqueous solutions. The technique has become widely used by biochemists to study protein interaction with other proteins, small molecules, metal ions, lipids, nucleic acids and carbohydrates; and nucleic acid interaction with small molecules. The drug discovery industry has utilized this approach to measure protein (or nucleic acid) interaction with drug candidates. ITC has been used to screen candidates, guide the design of potential drugs and validate the modelling used in structure-based drug design. Emerging disciplines including nanotechnology and drug delivery could benefit greatly from ITC in enhancing their understanding and control of nano-particle assembly, and drug binding and controlled release.     

Docking and free energy simulations to predict conformational domains involved in hCG-LH receptor interactions using recombinant antibodies

Single chain fragment variables (ScFvs) have been extensively employed in studying the protein-protein interactions. ScFvs derived from phage display libraries have an additional advantage of being generated against a native antigen, circumventing loss of information on conformational epitopes. In the present study, an attempt has been made to elucidate human chorionic gonadotropin (hCG)-luteinizing hormone (LH) receptor interactions by using a neutral and two inhibitory ScFvs against hCG. The objective was to dock a computationally derived model of these ScFvs onto the crystal structure of hCG and understand the differential roles of the mapped epitopes in hCG-LH receptor interactions. An anti-hCG ScFv, whose epitope was mapped previously using biochemical tools, served as the positive control for assessing the quality of docking analysis. To evaluate the role of specific side chains at the hCG-ScFv interface, binding free energy as well as residue interaction energies of complexes in solution were calculated using molecular mechanics Poisson-Boltzmann/surface area method after performing the molecular dynamic simulations on the selected hCG-ScFv models and validated using biochemical and SPR analysis. The robustness of these calculations was demonstrated by comparing the theoretically determined binding energies with the experimentally obtained kinetic parameters for hCG-ScFv complexes. Superimposition of hCG-ScFv model onto a model of hCG complexed with the 51-266 residues of LH receptor revealed importance of the residues previously thought to be unimportant for hormone binding and response. This analysis provides an alternate tool for understanding the structure-function analysis of ligand-receptor interactions.     

Cell death via mitochondrial apoptotic pathway due to activation of Bax by lysosomal photodamage

Lysosomal photosensitizers have been used in photodynamic therapy. The combination of such photosensitizers and light causes lysosomal photodamage, inducing cell death. Lysosomal disruption can lead to apoptosis but its signaling pathways remain to be elucidated. In this study, N-aspartyl chlorin e6 (NPe6), an effective photosensitizer that preferentially accumulates in lysosomes, was used to study the mechanism of apoptosis caused by lysosomal photodamage. Apoptosis in living human lung adenocarcinoma cells (ASTC-a-1) after NPe6-photodynamic treatment (NPe6-PDT) was studied using real-time single-cell analysis. Our results demonstrated that NPe6-PDT induced rapid generation of reactive oxygen species (ROS). The photodynamically produced ROS caused a rapid destruction of lysosomes, leading to release of cathepsins, and the ROS scavengers vitamin C and NAC prevent the effects. Then the following spatiotemporal sequence of cellular events was observed during cell apoptosis: Bcl-2-associated X protein (Bax) activation, cytochrome c release, and caspase-9/-3 activation. Importantly, the activation of Bax proved to be a crucial event in this apoptotic machinery, because suppressing the endogenous Bax using siRNA could significantly inhibit cytochrome c release and caspase-9/-3 activation and protect the cell from death. In conclusion, this study demonstrates that PDT with lysosomal photosensitizer induces Bax activation and subsequently initiates the mitochondrial apoptotic pathway.     

Mass mortality in Pacific oysters is associated with a specific gene expression signature

Mass mortality events occur in natural and cultured communities of bivalve molluscs. The Pacific oyster, Crassostrea gigas, is a dominant species in many intertidal locations as well as an important aquacultured bivalve species, and for the last 50 years, adult oysters have suffered frequent and extreme mass mortality events during summer months. To investigate the molecular changes that precede these mortality events, we employed a novel nonlethal sampling approach to collect haemolymph samples from individual oysters during the period that preceded a mortality event. Microarray-based gene expression screening of the collected haemolymph was used to identify a mortality gene expression signature that distinguished oysters that survived the mortality event from those individuals that died during the event. The signature was cross-validated by comparing two separate episodes of mortality. Here, we report that near-mortality oysters can be distinguished from longer-lived oysters by the elevated expression of genes associated with cell death, lysosomal proteolysis, and cellular assembly and organization. These results show the potential utility of nonlethal sampling approaches for investigating the environmental causes of mortality in natural populations in the field, and for predicting when such events could occur and which individuals will be affected.     

Calorimetric study of a series of designed repeat proteins: modular structure and modular folding

Repeat proteins comprise tandem arrays of a small structural motif. Their structure is defined and stabilized by interactions between residues that are close in the primary sequence. Several studies have investigated whether their structural modularity translates into modular thermodynamic properties. Tetratricopeptide repeat proteins (TPRs) are a class in which the repeated unit is a 34 amino acid helix-turn-helix motif. In this work, we use differential scanning calorimetry (DSC) to study the equilibrium stability of a series of TPR proteins with different numbers of an identical consensus repeat, from 2 to 20, CTPRa2 to CTPRa20. The DSC data provides direct evidence that the folding/unfolding transition of CTPR proteins does not fit a two-state folding model. Our results confirm and expand earlier studies on TPR proteins, which showed that apparent two-state unfolding curves are better fit by linear statistical mechanics models: 1D Ising models in which each repeat is treated as an independent folding unit.