Structural basis for Myf and Psa fimbriae‐mediated tropism of pathogenic strains of Yersinia for host tissues

Three pathogenic species of the genus Yersinia assemble adhesive fimbriae via the FGL‐chaperone/usher pathway. Closely related Y. pestis and Y. pseudotuberculosis elaborate the pH6 antigen (Psa), which mediates bacterial attachment to alveolar cells of the lung. Y. enterocolitica, instead, assembles the homologous fimbriae Myf of unknown function. Here, we discovered that Myf, like Psa, specifically recognizes β1‐3– or β1‐4–linked galactose in glycosphingolipids, but completely lacks affinity for phosphatidylcholine, the main receptor for Psa in alveolar cells. The crystal structure of a subunit of Psa (PsaA) complexed with choline together with mutagenesis experiments revealed that PsaA has four phosphatidylcholine binding pockets that enable super‐high‐avidity binding of Psa‐fibres to cell membranes. The pockets are arranged as six tyrosine residues, which are all missing in the MyfA subunit of Myf. Conversely, the crystal structure of the MyfA‐galactose complex revealed that the galactose‐binding site is more extended in MyfA, enabling tighter binding to lactosyl moieties. Our results suggest that during evolution, Psa has acquired a tyrosine‐rich surface that enables it to bind to phosphatidylcholine and mediate adhesion of Y. pestis/pseudotuberculosis to alveolar cells, whereas Myf has specialized as a carbohydrate‐binding adhesin, facilitating the attachment of Y. enterocolitica to intestinal cells.     

Association of short DNA fragments: Steady state fluorescence polarization study

We have studied aggregation/association of monodisperse DNA fragments (ranging from 30–90 base pairs) by steady-state fluorescence polarization of intercalculated ethidium. The method of excitation at different wavelengths in the ethidium absorption spectrum provides information about anisotropic twisting and tumbling mobility of the fragments. We find that end-over-end tumbling rather than axial spinning and internal twisting motions are affected by aggregation/association. The critical concentration for observing the effects of intermolecular interactions is approximately 5 mg DNA/mL at room temperature, independent of fragment length. Association is favored by low temperature and high (> 10 mM) concentration of Mg²⁺. From temperature-and salt-dependence experiments we infer that the “aggregates” are similar to those observed in a recently discovered DNA sol–gel transition [M. G. Fried and V. A. Bloomfield (1984) Biopolymers 23 , 2141–2155]. We also discuss possible arrangements of the fragments within the aggregates and their possible relation to formation of DNA liquid crystals.     

Characterization of functionally important domains in human vitamin K-dependent protein S using monoclonal antibodies.

Vitamin K-dependent protein S is an anticoagulant plasma protein functioning as a cofactor to activated protein C in the degradation of coagulation factors Va and VIIIa. To determine which regions in protein S are important for its cofactor activity, we have raised and characterized a large panel of monoclonal antibodies against human protein S. Several of the antibodies were directed against Ca2(+)-dependent epitopes, and they were found to be located either in the domain containing gamma-carboxyglutamic acid (Gla), the thrombin-sensitive region, or in the first epidermal growth factor (EGF)-like domain. The first two types of epitopes were exposed at approximately 1 mM Ca2+, whereas the epitope(s) in the EGF-like domains required less than 1 microM Ca2+, suggesting the presence of one or more high affinity Ca2(+)-binding site(s). The antibodies, as well as their Fab' fragments, against all three types of Ca2(+)-dependent epitopes efficiently inhibited the activated protein C cofactor function of protein S, but through different mechanisms. The antibodies against the Gla domain exerted their effects through inhibition of protein S binding to negatively charged phospholipid. Fab'-fragments of antibodies against the thrombin-sensitive region and the first EGF-like domain were the most potent inhibitors of the activated protein C cofactor function but did not inhibit phospholipid binding of protein S. In conclusion, we have identified the domains in protein S that are important for the activated protein C cofactor activity. The Gla domain is instrumental in the binding of protein S to phospholipid, whereas the thrombin-sensitive region and the first EGF-like domain may be directly involved in protein-protein interactions on the phospholipid surface.     

Automated solid-phase extraction method for the determination of piperaquine in capillary blood applied onto sampling paper by liquid chromatography

A bioanalytical method for the determination of piperaquine in 100 microL blood applied onto sampling paper, by solid-phase extraction and liquid chromatography, has been developed and validated. Blood spots were cut into small pieces prior to addition of 0.3M perchloric acid, acetonitrile and phosphate buffer containing an internal standard. The liquid phase was loaded onto a mixed phase cation-exchange (MPC) solid-phase extraction column. Piperaquine and the internal standard were analysed by liquid chromatography and separated on a Chromolith Performance (100 mm x 4.6 mm) column with acetonitrile:phosphate buffer pH 2.5, I = 0.1 (8:92, v/v) at the flow of 3.5 mL/min. The UV detection was performed at 345 nm. The intra-assay precision was 12.0% at 0.150 microM, 7.3% at 1.25 microM and 7.3% at 2.25 microM. The inter-assay precision was 1.8% at 0.150 microM, 5.2% at 1.25 microM and 2.8% at 2.25 microM. The lower limit of quantification (LLOQ) was determined to 0.050 microM where the precision was 14.7%.     

Testing the monophyly of the New Zealand and Australian endemic family Conoesucidae Ross based on combined molecular and morphological data (Insecta: Trichoptera: Sericostomatoidea)

Conoesucidae (Trichoptera, Insecta) are restricted to SE Australia, Tasmania and New Zealand. The family includes 42 described species in 12 genera, and each genus is endemic to either New Zealand or Australia. Although monophyly has been previously assumed, no morphological characters have been proposed to represent synapomorphies for the group. We collected molecular data from two mitochondrial genes (16S and cytochrome oxidase I), one nuclear gene (elongation factor 1-α) (2237–2277 bp in total), and 12 morphological characters to produce the first phylogeny of the family. We combined the molecular and morphological characters and performed both a maximum parsimony analysis and a Bayesian analysis to test the monophyly of the family, and to hypothesize the phylogeny among its genera. The parsimony analysis revealed a single most parsimonious tree with Conoesucidae being a monophyletic taxon and sistergroup to the Calocidae. The Bayesian inference produced a distribution of trees, the consensus of which is supported with posterior probabilities of 100% for 15 out of 22 possible ingroup clades including the most basal branch of the family, indicating strong support for a monophyletic Conoesucidae. The most parsimonious tree and the tree from the Bayesian analysis were identical except that the ingroup genus Pycnocentria changed position by jumping to a neighbouring clade. Based on the assumption that the ancestral conoesucid species was present on both New Zealand and Australia, a biogeographical analysis using the dispersal-vicariance criteria demonstrated that one or two (depending on which of the two phylogenetic reconstructions were applied) sympatric speciation events took place on New Zealand prior to a single, late dispersal from New Zealand to Australia.     

Hydrophobic properties of gram-negative rods colonizing upper respiratory tract of healthy people

The cell surface hydrophobicity is one of the non specific factors of adhesion influencing the ability of microorganisms to colonize nasopharynx. The aim of this paper was to evaluate via salt aggregation test (SAT) the cell surface hydrophobicity of 150 strains of gram-negative rods isolated from the throat or/and nasal specimens of healthy people. It has been found that among the nonfermenting rods hydrophobic strains were predominant. In contrast, the isolates of Enterobacteriaceae family were characterized by the distinctive features of the cell surface within particular genera or even species. The obtained results show that, despite differences in cell surface hydrophobicity, numerous species of gram-negative rods have the ability to colonize the mucous membrane of upper respiratory tract. This suggests that the cell surface hydrophobicity is rather a feature of species or genus, but it is not related to the ecological niche of microorganisms in human body.     

Effect of Cd2+ on growth of the cadmium-resistant and -sensitive Staphylococcus aureus

The effect of Cd2+ on aerobic and anaerobic growth was studied in the Cd2+-resistant Staphylococcus aureus 17810R which harbours the cadA and cadB markers on a penicillinase plasmid pII17810. Also the effect of Cd2+ on growth of the plasmidless strain 17810S, sensitive to Cd2+ was investigated. The results indicate that under all growth conditions the Cd2+-resistant S. aureus 17810R is protected against Cd2+ toxicity up to 100 microM Cd2+ by the 2H+/Cd2+ antiporter, the product of the cadA gene. Energetics of growth of both strains under various conditions is also discussed.