The Structural Domains of Pseudomonas aeruginosa Phosphorylcholine Phosphatase Cooperate in Substrate Hydrolysis: 3D Structure and Enzymatic Mechanism

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen. It colonizes different tissues by the utilization of diverse mechanisms. One of these may involve the breakdown of the host cell membrane through the sequential action of hemolytic phospholipase C and phosphorylcholine phosphatase (PchP). The action of hemolytic phospholipase C on phosphatidylcholine produces phosphorylcholine, which is hydrolyzed to choline (Cho) and inorganic phosphate by PchP. The available biochemical data on this enzyme demonstrate the involvement of two Cho-binding sites in the catalytic cycle and in enzyme regulation. The crystal structure of P. aeruginosa PchP has been determined. It folds into three structural domains. The first domain harbors all the residues involved in catalysis and is well conserved among the haloacid dehalogenase superfamily of proteins. The second domain is characteristic of PchP and is involved in the recognition of the Cho moiety of the substrate. The third domain stabilizes the relative position of the other two. Fortuitously, the crystal structure of PchP captures molecules of Bistris (2‐[bis(2‐hydroxyethyl)amino]‐2‐(hydroxymethyl)propane‐1,3‐diol) at the active site and at an additional site. This represents two catalytically relevant complexes with just one or two inhibitory Bistris molecules and provides the basis of the PchP function and regulation. Site‐directed mutagenesis along with biochemical experiments corroborates the structural observations and demonstrates the interplay between different sites for Cho recognition and inhibition. The structural comparison of PchP with other phosphatases of the haloacid dehalogenase family provides a three‐dimensional picture of the conserved catalytic cycle and the structural basis for the recognition of the diverse substrate molecules.     

Large-Scale Variation in Combined Impacts of Canopy Loss and Disturbance on Community Structure and Ecosystem Functioning

Ecosystems are under pressure from multiple human disturbances whose impact may vary depending on environmental context. We experimentally evaluated variation in the separate and combined effects of the loss of a key functional group (canopy algae) and physical disturbance on rocky shore ecosystems at nine locations across Europe. Multivariate community structure was initially affected (during the first three to six months) at six locations but after 18 months, effects were apparent at only three. Loss of canopy caused increases in cover of non-canopy algae in the three locations in southern Europe and decreases in some northern locations. Measures of ecosystem functioning (community respiration, gross primary productivity, net primary productivity) were affected by loss of canopy at five of the six locations for which data were available. Short-term effects on community respiration were widespread, but effects were rare after 18 months. Functional changes corresponded with changes in community structure and/or species richness at most locations and times sampled, but no single aspect of biodiversity was an effective predictor of longer-term functional changes. Most ecosystems studied were able to compensate in functional terms for impacts caused by indiscriminate physical disturbance. The only consistent effect of disturbance was to increase cover of non-canopy species. Loss of canopy algae temporarily reduced community resistance to disturbance at only two locations and at two locations actually increased resistance. Resistance to disturbance-induced changes in gross primary productivity was reduced by loss of canopy algae at four locations. Location-specific variation in the effects of the same stressors argues for flexible frameworks for the management of marine environments. These results also highlight the need to analyse how species loss and other stressors combine and interact in different environmental contexts.     

Global Cardiovascular Research Output,Citations, and Collaborations: A Time-Trend,Bibliometric Analysis (1999–2008)

Introduction

Health research is one mechanism to improve population-level health and should generally match the health needs of populations. However, there have been limited data to assess the trends in national-level cardiovascular research output, even as cardiovascular disease [CVD] has become the leading cause of morbidity and mortality worldwide.

Materials and Methods

We performed a time trends analysis of cardiovascular research publications (1999–2008) downloaded from Web of Knowledge using a iteratively-tested cardiovascular bibliometric filter with >90% precision and recall. We evaluated cardiovascular research publications, five-year running actual citation indices [ACIs], and degree of international collaboration measured through the ratio of the fractional count of addresses from one country against all addresses for each publication.

Results and Discussion

Global cardiovascular publication volume increased from 40 661 publications in 1999 to 55 284 publications in 2008, which represents a 36% increase. The proportion of cardiovascular publications from high-income, Organization for Economic Cooperation and Development [OECD] countries declined from 93% to 84% of the total share over the study period. High-income, OECD countries generally had higher fractional counts, which suggest less international collaboration, than lower income countries from 1999–2008. There was an inverse relationship between cardiovascular publications and age-standardized CVD morbidity and mortality rates, but a direct, curvilinear relationship between cardiovascular publications and Human Development Index from 1999–2008.

Conclusions

Cardiovascular health research output has increased substantially in the past decade, with a greater share of citations being published from low- and middle-income countries. However, low- and middle-income countries with the higher burdens of cardiovascular disease continue to have lower research output than high-income countries, and thus require targeted research investments to improve cardiovascular health.     

Host-Specific Enzyme-Substrate Interactions in SPM-1 Metallo-β-Lactamase Are Modulated by Second Sphere Residues

Pseudomonas aeruginosa is one of the most virulent and resistant non-fermenting Gram-negative pathogens in the clinic. Unfortunately, P. aeruginosa has acquired genes encoding metallo-β-lactamases (MβLs), enzymes able to hydrolyze most β-lactam antibiotics. SPM-1 is an MβL produced only by P. aeruginosa, while other MβLs are found in different bacteria. Despite similar active sites, the resistance profile of MβLs towards β-lactams changes from one enzyme to the other. SPM-1 is unique among pathogen-associated MβLs in that it contains “atypical” second sphere residues (S84, G121). Codon randomization on these positions and further selection of resistance-conferring mutants was performed. MICs, periplasmic enzymatic activity, Zn(II) requirements, and protein stability was assessed. Our results indicated that identity of second sphere residues modulates the substrate preferences and the resistance profile of SPM-1 expressed in P. aeruginosa. The second sphere residues found in wild type SPM-1 give rise to a substrate selectivity that is observed only in the periplasmic environment. These residues also allow SPM-1 to confer resistance in P. aeruginosa under Zn(II)-limiting conditions, such as those expected under infection. By optimizing the catalytic efficiency towards β-lactam antibiotics, the enzyme stability and the Zn(II) binding features, molecular evolution meets the specific needs of a pathogenic bacterial host by means of substitutions outside the active site.     

Solution and crystal structure of BA42, a protein from the Antarctic bacterium Bizionia argentinensis comprised of a stand‐alone TPM domain

The structure of the BA42 protein belonging to the Antarctic flavobacterium Bizionia argentinensis was determined by nuclear magnetic resonance and X‐ray crystallography. This is the first structure of a member of the PF04536 family comprised of a stand‐alone TPM domain. The structure reveals a new topological variant of the four β‐strands constituting the central β‐sheet of the αβα architecture and a double metal binding site stabilizing a pair of crossing loops, not observed in previous structures of proteins belonging to this family. BA42 shows differences in structure and dynamics in the presence or absence of bound metals. The affinity for divalent metal ions is close to that observed in proteins that modulate their activity as a function of metal concentration, anticipating a possible role for BA42. Proteins 2014; 82:3062–3078. © 2014 Wiley Periodicals, Inc.     

Exogenous disturbances and endogenous self-organized processes are not mutually exclusive drivers of spatial patterns in macroalgal assemblages

Complex spatial patterns are common in coastal marine systems, but mechanisms underlying their formation are disputed. Most empirical work has focused on exogenous spatially structured disturbances as the leading cause of pattern formation in species assemblages. However, theoretical and observational studies suggest that complex spatial patterns, such as power laws in gap-size distribution, may result from endogenous self-organized processes involving local-scale interactions. The lack of studies simultaneously assessing the influence of spatially variable disturbances and local-scale interactions has fuelled the idea that exogenous and endogenous processes are mutually exclusive explanations of spatial patterns in marine ecosystems. To assess the relative contribution of endogenous and exogenous processes in the emergence of spatial patterns, an intertidal assemblage of algae was exposed for two years to various combinations of intensity and spatial patterns of disturbance. Localized disturbances impinging at the margins of previously disturbed clearings and homogenous disturbances without any spatial pattern generated heterogeneous distributions of disturbed gaps and macroalgal patches, characterized by a power-law scaling. Spatially varying disturbances produced a spatial gradient in the distribution of algal patches and, to a lesser extent, also a power-law scaling in both patch- and gap-size distributions. These results suggest that exogenous and endogenous processes are not mutually exclusive forces that can lead to the formation of similar spatial patterns in species assemblages.     

Protein-membrane interaction and fatty acid transfer from intestinal fatty acid-binding protein to membranes. Support for a multistep process

Fatty acid transfer from intestinal fatty acid-binding protein (IFABP) to phospholipid membranes occurs during protein-membrane collisions. Electrostatic interactions involving the alpha-helical "portal" region of the protein have been shown to be of great importance. In the present study, the role of specific lysine residues in the alpha-helical region of IFABP was directly examined. A series of point mutants in rat IFABP was engineered in which the lysine positive charges in this domain were eliminated or reversed. Using a fluorescence resonance energy transfer assay, we analyzed the rates and mechanism of fatty acid transfer from wild type and mutant proteins to acceptor membranes. Most of the alpha-helical domain mutants showed slower absolute fatty acid transfer rates to zwitterionic membranes, with substitution of one of the lysines of the alpha2 helix, Lys27, resulting in a particularly dramatic decrease in the fatty acid transfer rate. Sensitivity to negatively charged phospholipid membranes was also reduced, with charge reversal mutants in the alpha2 helix the most affected. The results support the hypothesis that the portal region undergoes a conformational change during protein-membrane interaction, which leads to release of the bound fatty acid to the membrane and that the alpha2 segment is of particular importance in the establishment of charge-charge interactions between IFABP and membranes. Cross-linking experiments with a phospholipid-photoactivable reagent underscored the importance of charge-charge interactions, showing that the physical interaction between wild-type intestinal fatty acid-binding protein and phospholipid membranes is enhanced by electrostatic interactions. Protein-membrane interactions were also found to be enhanced by the presence of ligand, suggesting different collisional complex structures for holo- and apo-IFABP.     

Thermal Behavior, Microstructure, Polymorphism, and Crystallization Properties of Zero Trans Fats from Soybean Oil and Fully Hydrogenated Soybean Oil

Blends of soybean oil (SO) and fully hydrogenated soybean oil (FHSBO), with 10, 20, 30, 40, and 50% (w/w) FHSBO content were interesterified under the following conditions: 20 min reaction time, 0.4% sodium methoxide catalyst, and 500 rpm stirring speed, at 100 °C. The original and interesterified blends were examined for triacylglycerol composition, thermal behavior, microstructure, crystallization kinetics, and polymorphism. Interesterification produced substantial rearrangement of the triacylglycerol species in all the blends, reduction of trisaturated triacylglycerol content and increase in monounsaturated–disaturated and diunsaturated–monosaturated triacylglycerols. Evaluation of thermal behavior parameters showed linear relations with FHSBO content in the original blends. Blend melting and crystallization thermograms were significantly modified by the randomization. Interesterification caused significant reductions in maximum crystal diameter in all blends, in addition to modifying crystal morphology. Characterization of crystallization kinetics revealed that crystal formation induction period (τ _SFC) and maximum solid fat content (SFC_máx) were altered according to FHSBO content in the original blends and as a result of the random rearrangement. Changes in Avrami constant (k) and exponent (n) indicated, respectively, that—as compared with the original blends—interesterification decreased crystallization velocities and modified crystallization processes, altering crystalline morphology and nucleation mechanism. X-ray diffraction analyses revealed that interesterification altered crystalline polymorphism. The interesterified blends showed a predominance of the β′ polymorph, which is of more interest for food applications.