The membrane bound LRR lipoprotein Slr, and the cell wall-anchored M1 protein from Streptococcus pyogenes both interact with type I collagen

Streptococcus pyogenes is an important human pathogen and surface structures allow it to adhere to, colonize and invade the human host. Proteins containing leucine rich repeats (LRR) have been identified in mammals, viruses, archaea and several bacterial species. The LRRs are often involved in protein-protein interaction, are typically 20-30 amino acids long and the defining feature of the LRR motif is an 11-residue sequence LxxLxLxxNxL (x being any amino acid). The streptococcal leucine rich (Slr) protein is a hypothetical lipoprotein that has been shown to be involved in virulence, but at present no ligands for Slr have been identified. We could establish that Slr is a membrane attached horseshoe shaped lipoprotein by homology modeling, signal peptidase II inhibition, electron microscopy (of bacteria and purified protein) and immunoblotting. Based on our previous knowledge of LRR proteins we hypothesized that Slr could mediate binding to collagen. We could show by surface plasmon resonance that recombinant Slr and purified M1 protein bind with high affinity to collagen I. Isogenic slr mutant strain (MB1) and emm1 mutant strain (MC25) had reduced binding to collagen type I as shown by slot blot and surface plasmon resonance. Electron microscopy using gold labeled Slr showed multiple binding sites to collagen I, both to the monomeric and the fibrillar structure, and most binding occurred in the overlap region of the collagen I fibril. In conclusion, we show that Slr is an abundant membrane bound lipoprotein that is co-expressed on the surface with M1, and that both these proteins are involved in recruiting collagen type I to the bacterial surface. This underlines the importance of S. pyogenes interaction with extracellular matrix molecules, especially since both Slr and M1 have been shown to be virulence factors.     

Structure-activity relationships of 1,3,5-triazine-2,4,6-triones as human gonadotropin-releasing hormone receptor antagonists

SAR studies of 1,3,5-triazine-2,4,6-triones as human gonadotropin-releasing hormone receptor antagonists resulted in potent compounds. The best compound from the series had a binding affinity of 2 nM.     

Identification of mouse brain proteins associated with isoform 3 of metallothionein

Using immunological approaches and mass spectrometry, five proteins associated with metallothionein-3 in mouse brains have been identified. Metallothionein-3 and associated proteins were isolated using immunoaffinity chromatography over immobilized anti-mouse brain MT3 antibody. Proteins in the recovered pool were separated by SDS-polyacrylamide gel electrophoresis, and distinct bands were excised and the proteins digested using trypsin. Peptides were extracted and analyzed using electrospray ionization mass spectrometry. Initial identification was done comparing the identified peptide mass:charge ratios to the MASCOT database. Confirmation of proteins was accomplished by sequencing of selected peptides using tandem mass spectrometry and comparison to the MASCOT database. The proteins were heat-shock protein 84 (mouse variant of heat-shock protein 90), heat-shock protein 70, dihydropyrimidinase-like protein 2, creatine kinase, and beta actin. Independently using antibodies against metallothionein-3, creatine kinase, and heat-shock protein 84 showed that all three proteins were coimmunoprecipitated from whole mouse brain homogenates with each of the three antibodies. Mixing purified samples of metallothionein and human brain creatine kinase also generated a complex that could be immunoprecipitated either by anti-metallothionein-3 or anticreatine kinase antibody. These data are consistent with metallothionein-3 being present in the mouse brain as part of a multiprotein complex providing new functional information for understanding the role of metallothionein-3 in neuronal physiology.     

Phylogenetic effects, the loss of complex characters, and the evolution of development in calyptraeid gastropods

Abstract Despite considerable theoretical and empirical work on the population genetic effects of mode of development in benthic marine invertebrates, it is unclear what factors generate and maintain interspecific variation in mode of development and few studies have examined such variation in a phylogenetic context. Here I combine data on mode of development with a molecular phylogeny of 72 calyptraeid species to test the following hypotheses about the evolution of mode of development: (1) Is the loss of feeding larvae irreversible? (2) Is there a phylogenetic effect on the evolution of mode of development? (3) Do embryos of direct‐developing species lose the structures necessary for larval feeding and swimming and, if so, is the degree of embryonic modification correlated with the genetic distance between species? The results of these analyses suggest that mode of development evolves rapidly and with little phylogenetic inertia. There are three cases of the possible regain of feeding larvae, in all cases from direct development with nurse eggs. It appears that species with planktotrophic, lecithotrophic, or direct development with nurse eggs all have equal evolutionary potential and retain the possibility of subsequent evolution of a different mode of development. However, species with direct development from large yolky eggs appear to be subject to phylogenetic constraints and may not be able to subsequently evolve a different mode of development. Finally, species that have more recently evolved direct development have less highly modified embryos than older direct‐developing species. Since species with nurse eggs generally have fewer embryonic modifications than those from large yolky eggs, this embryological difference may be the underlying cause of the difference in evolutionary potential.     

Using latent effects to determine the ecological importance of dissolved organic matter to marine invertebrates

The uptake and utilization of dissolved organic matter (DOM)by marine invertebrates is a field that has received significantattention over the past 100 years. Although it is well establishedthat DOM is taken up by marine invertebrates, the extent towhich it contributes to an animal's survival, growth, and reproduction(that is, the ecological benefits) remains largely unknown.Previous work seeking to demonstrate the putative ecologicalbenefits of DOM uptake have examined them within a single lifestage of an animal. Moreover, most of the benefits are demonstratedthrough indirect approaches by examining (1) mass balance, or(2) making comparisons of oxyenthalpic conversions of transportrates to metabolic rate as judged by oxygen consumption. Wesuggest that directly examining delayed metamorphosis or thelatent effects associated with nutritional stress of larvaeis a better model for investigating the ecological importanceof DOM to marine invertebrates. We also provide direct evidencethat availability of DOM enhances survival and growth of thebryozoan Bugula neritina. That DOM offsets latent effects inB. neritina suggests that the underlying mechanisms are at leastin part energetic.     

Interaction between non-anionic phospholipids and cytochrome c induced by reactive oxygen species

The oxidative interaction of cytochrome c (Cyt c) with liposomes of Palmitoyl Linoleyl Phosphatidyl Choline (PLPC) initiated by radio-induced free radicals was investigated. Results showed that the peroxidation of PLPC is decreased in the presence of Cyt c, meaning that this latter is the preferential target of hydroxyl radicals. In addition, when Cyt c was incubated with peroxidized PLPC, it was found to be able to decompose hydroperoxides of PLPC into hydroxides. The peroxidase activity of Cyt c proceeded via the opening of the tertiary structure of Cyt c, as suggested by the loss of the sixth coordination bond of the heme-iron. Even if it is known to preferentially interact with cardiolipin, this work shows that Cyt c is also able to interact with hydroperoxide species of non-anionic phospholipids.     

Plant glutathione peroxidases are functional peroxiredoxins distributed in several subcellular compartments and regulated during biotic and abiotic stresses

We provide here an exhaustive overview of the glutathione (GSH) peroxidase (Gpx) family of poplar (Populus trichocarpa). Although these proteins were initially defined as GSH dependent, in fact they use only reduced thioredoxin (Trx) for their regeneration and do not react with GSH or glutaredoxin, constituting a fifth class of peroxiredoxins. The two chloroplastic Gpxs display a marked selectivity toward their electron donors, being exclusively specific for Trxs of the y type for their reduction. In contrast, poplar Gpxs are much less specific with regard to their electron-accepting substrates, reducing hydrogen peroxide and more complex hydroperoxides equally well. Site-directed mutagenesis indicates that the catalytic mechanism and the Trx-mediated recycling process involve only two (cysteine [Cys]-107 and Cys-155) of the three conserved Cys, which form a disulfide bridge with an oxidation-redox midpoint potential of -295 mV. The reduction/formation of this disulfide is detected both by a shift on sodium dodecyl sulfate-polyacrylamide gel electrophoresis or by measuring the intrinsic tryptophan fluorescence of the protein. The six genes identified coding for Gpxs are expressed in various poplar organs, and two of them are localized in the chloroplast, with one colocalizing in mitochondria, suggesting a broad distribution of Gpxs in plant cells. The abundance of some Gpxs is modified in plants subjected to environmental constraints, generally increasing during fungal infection, water deficit, and metal stress, and decreasing during photooxidative stress, showing that Gpx proteins are involved in the response to both biotic and abiotic stress conditions.