N-formyl peptide receptors cluster in an active raft-associated state prior to phosphorylation

In response to ligand binding, G protein-coupled receptors undergo phosphorylation and activate cellular internalization machinery. An important component of this process is the concentration of receptors into clusters on the plasma membrane. Aside from organizing the receptor in anticipation of internalization, little is known of the function of ligand-mediated G protein-coupled receptor clustering, which has traditionally been thought of as being a phosphorylation-dependent event prior to receptor internalization. We now report that following receptor activation, the N-formyl peptide receptor (FPR) forms distinct membrane clusters prior to its association with arrestin. To determine whether this clustering is dependent upon receptor phosphorylation, we used a mutant form of the FPR, DeltaST-FPR, which lacks all phosphorylation sites in the carboxyl-terminal domain. We found that activation of the signaling-competent DeltaST-FPR resulted in rapid receptor clustering on the plasma membrane independent of Gi protein activation. This clustering required receptor activation since the D71A mutant receptor, which binds ligand but is incapable of transitioning to an active state, failed to induce receptor clustering. Furthermore we demonstrated that FPR-mediated clustering and signaling were cholesterol-dependent processes, suggesting that translocation of the active receptor to lipid rafts may be required for maximal signaling activity. Finally we showed that FPR stimulation in the absence of receptor phosphorylation resulted in translocation of FPR to GM1-rich clusters. Our results demonstrate for the first time that formation of a clustered activated receptor state precedes receptor phosphorylation, arrestin binding, and internalization.     

Catalytic superoxide scavenging by metal complexes of the calcium chelator EGTA and contrast agent EHPG

Metal ion chelators widely used in experimental protocols and clinical diagnosis are generally assumed to be inert. We previously reported that the ubiquitous chelator EDTA has high levels of superoxide suppressing activity. Here, we report that the common chelators calcium chelator EGTA and contrast agent EHPG have significant activities in suppressing superoxide levels depending on the nature of metal ion chelated. The most active species is Mn(II)-EGTA which exhibited an IC50 value of 0.19 microM for superoxide destruction. In addition, IC50 values for Mn(II)-EHPG and 2Cu(II)-EGTA were 0.69 and 0.60 microM, respectively. In conclusion, Mn(II) and Cu(II) complexes of the common chelators EGTA and EHPG exhibit considerable superoxide scavenging activities. Caution should be employed in their use in biological systems where superoxide has a key role and they may be useful for the development of catalytic anti-oxidants.     

Modulation of KCNQ4 channel activity by changes in cell volume

KCNQ4 channels expressed in HEK 293 cells are sensitive to cell volume changes, being activated by swelling and inhibited by shrinkage, respectively. The KCNQ4 channels contribute significantly to the regulatory volume decrease (RVD) process following cell swelling. Under isoosmotic conditions, the KCNQ4 channel activity is modulated by protein kinases A and C, G protein activation, and a reduction in the intracellular Ca2+ concentration, but these signalling pathways are not responsible for the increased channel activity during cell swelling.     

Regulation of intercellular adhesion strength in fibroblasts

The regulation of adherens junction formation in cells of mesenchymal lineage is of critical importance in tumorigenesis but is poorly characterized. As actin filaments are crucial components of adherens junction assembly, we studied the role of gelsolin, a calcium-dependent, actin severing protein, in the formation of N-cadherin-mediated intercellular adhesions. With a homotypic, donor-acceptor cell model and plates or beads coated with recombinant N-cadherin-Fc chimeric protein, we found that gelsolin spatially co-localizes to, and is transiently associated with, cadherin adhesion complexes. Fibroblasts from gelsolin-null mice exhibited marked reductions in kinetics and strengthening of N-cadherin-dependent junctions when compared with wild-type cells. Experiments with lanthanum chloride (250 microm) showed that adhesion strength was dependent on entry of calcium ions subsequent to N-cadherin ligation. Cadherin-associated gelsolin severing activity was required for localized actin assembly as determined by rhodamine actin monomer incorporation onto actin barbed ends at intercellular adhesion sites. Scanning electron microscopy showed that gelsolin was an important determinant of actin filament architecture of adherens junctions at nascent N-cadherin-mediated contacts. These data indicate that increased actin barbed end generation by the severing activity of gelsolin associated with N-cadherin regulates intercellular adhesion strength.     

Phospholipid complexation and association with apolipoprotein C-II: insights from mass spectrometry

The interactions between phospholipid molecules in suspensions have been studied by using mass spectrometry. Electrospray mass spectra of homogeneous preparations formed from three different phospholipid molecules demonstrate that under certain conditions interactions between 90 and 100 lipid molecules can be preserved. In the presence of apolipoprotein C-II, a phospholipid binding protein, a series of lipid molecules and the protein were observed in complexes. The specificity of binding was demonstrated by proteolysis; the resulting mass spectra reveal lipid-bound peptides that encompass the proposed lipid-binding domain. The mass spectra of heterogeneous suspensions and their complexes with apolipoprotein C-II demonstrate that the protein binds simultaneously to two different phospholipids. Moreover, when apolipoprotein C-II is added to lipid suspensions formed with local concentrations of the same lipid molecule, the protein is capable of remodeling the distribution to form one that is closer to a statistical arrangement. These observations demonstrate a capacity for apolipoprotein C-II to change the topology of the phospholipid surface. More generally, these results highlight the fact that mass spectrometry can be used to probe lipid interactions in both homogeneous and heterogeneous suspensions and demonstrate reorganization of the distribution of lipids upon surface binding of apolipoprotein C-II.     

Macroevolutionary consequences of developmental mode in temnopleurid echinoids from the Tertiary of southern Australia

Abstract Taxonomic revision and cladistic analysis of a morphological dataset for Australian Tertiary temnopleurids resolve the phylogeny of the group and allow the testing of a series of hypotheses about the evolution of larval development and consequences of changes in development. Australian Tertiary temnopleurids encompass all three major developmental types found in marine invertebrates (planktotrophy, lecithotrophy, and brooding). Planktotrophy is plesiomorphic for this clade, and nonplanktotrophic larval development evolved independently at least three times during the Tertiary. The change to a nonplanktotrophic mode of larval development is unidirectional with no evidence of reversal. In addition, there is no evidence of an ordered transformation series from planktotrophy through planktonic lecithotrophy to brooding. In common with previous studies of other invertebrate groups, analysis of the raw data suggests that nonplanktotrophic taxa within this clade have significantly shorter species longevities, more restricted geographic ranges and higher speciation rates than taxa with planktotrophic development. However, analysis using phylogenetically independent contrasts is unable to confirm that the stratigraphic and geographic patterns are unbiased by the phylogenetic relationships of the included taxa.     

Influence of the Ca/Mg ratio on Cu resistance in three Silene armeria ecotypes adapted to calcareous soil or to different, Ni- or Cu-enriched, serpentine sites

This hydroponic study addresses the influence of low (0.3) and high (4.0) Ca/Mg molar ratios on Cu resistance of Silene armeria ecotypes from different habitats: a calcareous soil (ecotype Cadriano), a Ni-rich serpentine site (ecotype Prinzera), and an acid Cu-mine spoil soil containing serpentinite (ecotype Vigonzano). Under control conditions, without excess Cu, only Cadriano was negatively affected by the low Ca/Mg ratio. Under both low and high Ca/Mg ratios Cu resistance followed the order Vigonzano > Prinzera > Cadriano. More efficient Cu exclusion accounted for enhanced Cu resistance in Prinzera. The low Ca/Mg ratio increased Cu uptake in Prinzera but did not worsen toxicity effects; i.e. the plants had higher internal Cu effect concentrations. In Vigonzano Cu resistance was enhanced by the low Ca/Mg ratio. This was due only in part to better Cu exclusion. Magnesium-induced tolerance to higher Cu tissue concentrations appears to be in ecotypes from serpentine and acid mine spoils, but not in plants from calcareous soil, the exposure to low Ca/Mg ratio favours internal detoxification of Cu by means of more efficient chelation and compartmentation.     

Curvularia haloperoxidase: antimicrobial activity and potential application as a surface disinfectant

A presumed antimicrobial enzyme system, the Curvularia haloperoxidase system, was examined with the aim of evaluating its potential as a sanitizing agent. In the presence of hydrogen peroxide, Curvularia haloperoxidase facilitates the oxidation of halides, such as chloride, bromide, and iodide, to antimicrobial compounds. The Curvularia haloperoxidase system caused several-log-unit reductions in counts of bacteria (Pseudomonas spp., Escherichia coli, Serratia marcescens, Aeromonas salmonicida, Shewanella putrefaciens, Staphylococcus epidermidis, and Listeria monocytogenes), yeasts (Candida sp. and Rhodotorula sp.), and filamentous fungi (Aspergillus niger, Aspergillus tubigensis, Aspergillus versicolor, Fusarium oxysporum, Penicillium chrysogenum, and Penicillium paxilli) cultured in suspension. Also, bacteria adhering to the surfaces of contact lenses were killed. The numbers of S. marcescens and S. epidermidis cells adhering to contact lenses were reduced from 4.0 and 4.9 log CFU to 1.2 and 2.7 log CFU, respectively, after treatment with the Curvularia haloperoxidase system. The killing effect of the Curvularia haloperoxidase system was rapid, and 10(6) CFU of E. coli cells/ml were eliminated within 10 min of treatment. Furthermore, the antimicrobial effect was short lived, causing no antibacterial effect against E. coli 10 min after the system was mixed. Bovine serum albumin (1%) and alginate (1%) inhibited the antimicrobial activity of the Curvularia haloperoxidase system, whereas glucose and Tween 20 did not affect its activity. In conclusion, the Curvularia haloperoxidase system is an effective sanitizing system and has the potential for a vast range of applications, for instance, for disinfection of contact lenses or medical devices.