Streptococcus sanguis 12 adhesins mediating attachment to saliva-coated hydroxyapatite beads (S-HA) were isolated and characterized. Cell surface fibrils were released from this organism by a method of freeze-thawing followed by brief homogenization. Fibrils in the homogenate were precipitated by ultracentrifugation or ammonium sulphate precipitation. This precipitate was shown to contain fibrils by electron microscopy. Sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) analysis of fibrils showed a single band which stained with Coomassie blue and periodate-Schiff. The molecule had a Mr in excess of 300,000. This protein has been given the name long-fibril protein (LFP). Antibody raised against the LFP reacted with long fibrils of S. sanguis 12. LFP was degraded by subtilisin, pronase, papain, and trypsin, but not by chymotrypsin and muramidases. Fibrils were hydrolyzed by subtilisin into discrete lower Mr protein bands which reacted with both anti-fibril and anti-LFP serum. F(ab')₂ prepared from anti-fibril IgG inhibited adhesion of S. sanguis 12 to pH modified S-HA, indicating that fibrils were acting as an adhesin mediating attachment via the neuraminidase-sensitive receptor on S-HA. Five recombinant clones expressing surface antigens of S. sanguis 12 were isolated by ligating a partial digest of S. sanguis 12 chromosomal DNA with the plasmid vector pUC 18, and transforming into Escherichia coli JM83. Recombinant clones were screened by a colony immunoassay with antisera raised against either S. sanguis 12 whole cells or with anti-fibril serum. Positive clones were then analyzed by SDS-PAGE, Western blotting and restriction endonuclease digestion of recombinant plasmids. One recombinant plasmid, pSA2 expressed two proteins of Mrs of 20,000 and 36,000. The 36,000-Mr protein has been designated as SsaB (S. sanguis adhesin B). Both proteins were purified to homogeneity by gel filtration and ion exchange chromatography. Anti-SsaB serum was used in an immunogold bead labelling experiment to demonstrate that this protein was present on the surfaces of S. sanguis 12 and in the non-saliva-aggregating variant 12na, but not on the non-adhering non-aggregating hydrophilic variant 12L. Western blot analysis with anti-SsaB and anti-20 kd sera showed that both SsaB and the 20 kd proteins were present in cell extracts of S. sanguis 12 and its variants. SsaB inhibited adhesion of S. sanguis 12na to S-HA, indicating that it was the adhesin which mediates the binding to the pH-sensitive receptor. SsaB was found to be present on all S. sanguis strains tested, but not on other oral streptococci. Chemical cross-linking studies of SsaB on S. sanguis 12 cell surface suggested that this protein may be present in a higher Mr complex. This study provides direct evidence that binding of S. sanguis 12 to S-HA involves at least two adhesin-receptor interactions. The adhesin mediating binding to the neuraminidase-sensitive receptor on S-HA involves the long fibrils and the adhesin binding to the acid labile receptor is a 36,000 Mr protein. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Focal adhesion kinase (FAK) controls the assembly of focal adhesion sites and transduces signals from several membrane receptors. Controlled activation and localization of FAK functionally links cell adhesion, migration and survival. FAK is overexpressed in many cancer types, promoting tumor invasiveness and metastasis. The molecular mechanisms allowing FAK to fulfil numerous different functions and act as versatile ‘nanomachines’ are poorly understood. We have previously revealed that ligand-induced dimerization along with intramolecular interactions control FAK activation and localization where the C-terminal focal adhesion targeting (FAT) domain is strictly involved. In this study, we combine NMR with X-ray crystallography, as well as biophysical and computational methods to understand the molecular mechanisms that link the large-scale dynamics and intramolecular and intermolecular interactions of FAT into FAK’s capacity to integrate various stimuli into a site-specific function. Our results reveal FAT-mediated dynamical interplays between binding of known and newly discovered FAT ligands, and multimerization and autoactivation of FAK. Additionally, we investigate the impact of neuronal alternative splicing on FAT dynamics and interactions. Collectively, our results elucidate FAT’s role in allosterically controlling various FAK functions, and might inspire allosteric protein-protein interaction inhibitors against FAK-dependent cancer cell proliferation.

The diploma thesis in the first part deals with a theoretical description of coating technologies, textile materials used in coatings, types of coated polymers and properties of coatings - especially adhesion. The experimental part of the work is devoted to the preparation of textile coatings from polyester fabric and coating based on addition silicone. Using the developed method of sample preparation for T-peel testing of the adhesive strength, the improvement of the adhesion of the coating by chemical adhesion with the support of adhesive agents was studied. The second method studied the change in compactness and adhesion of the coating to the fabric after shaking as a function of breathability. The influence of side reactions of reagents on silicone cohesion was studied by preparing dogbones for testing tensile-deformation properties. The research also included the characterization of silicone samples using ATR-FTIR, monitoring the weight gain and thickness of the fabric after coating, the feel and color stability of the applied fabric and observing the coating under an optical microscope.

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