Molecular basis of anticoagulant and anticomplement activity of the tick salivary protein Salp14 and its homologs.

During feeding, a tick's mouthpart penetrates the host's skin and damages tissues and small blood vessels, triggering the extrinsic coagulation and lectin complement pathways. To elude these defense mechanisms, ticks secrete multiple anticoagulant proteins and complement system inhibitors in their saliva. Here, we characterized the inhibitory activities of the homologous tick salivary proteins tick salivary lectin pathway inhibitor, Salp14, and Salp9Pac from Ixodesscapularis in the coagulation cascade and the lectin complement pathway. All three proteins inhibited binding of mannan-binding lectin to the polysaccharide mannan, preventing the activation of the lectin complement pathway. In contrast, only Salp14 showed an appreciable effect on coagulation by prolonging the lag time of thrombin generation. We found that the anticoagulant properties of Salp14 are governed by its basic tail region, which resembles the C terminus of tissue factor pathway inhibitor alpha and blocks the assembly and/or activity of the prothrombinase complex in the same way. Moreover, the Salp14 protein tail contributes to the inhibition of the lectin complement pathway via interaction with mannan binding lectin-associated serine proteases. Furthermore, we identified BaSO4-adsorbing protein 1 isolated from the tick Ornithodoros savignyi as a distant homolog of tick salivary lectin pathway inhibitor/Salp14 proteins and showed that it inhibits the lectin complement pathway but not coagulation. The structure of BaSO4-adsorbing protein 1, solved here using NMR spectroscopy, indicated that this protein adopts a noncanonical epidermal growth factor domain-like structural fold, the first such report for tick salivary proteins. These data support a mechanism by which tick saliva proteins simultaneously inhibit both the host coagulation cascade and the lectin complement pathway.

Non-destructive visualisation of protective proteins in the in situ pellicle.

Several salivary anti-microbial and buffering components are part of the acquired in vivo pellicle. The purpose of the present in situ study was to visualise these proteins within the in situ formed pellicle and to investigate their distribution with respect to pellicle formation time and intra-oral localisation. Bovine enamel slabs were fixed on individual splints. They were carried by 6 subjects buccally and palatally in the region of the upper first molar teeth over 30 and 120 min, respectively, for in situ pellicle formation. After intra-oral exposure, enamel specimens were processed for transmission electron microscopy. Secretory immunoglobulin A (sIgA), lactoferrin, lysozyme, carbonic anhydrase (CA) I and II were visualised successfully in the in situ pellicle layer by gold immuno-labelling. All components were found to be distributed randomly within all layers of the pellicle. Significantly higher amounts of the proteins were detected after 120 min of formation time. Furthermore, significantly more labelled lactoferrin and lysozyme were found on buccal surfaces compared with palatal sites. For CA I, CA II and sIgA, no significant influence of the localisation was detected. All investigated anti-bacterial and buffering proteins are distributed randomly in the in situ formed pellicle layer and thus could contribute to its protective properties as an early defence barrier.

Electron microscopic detection of salivary alpha-amylase in the pellicle formed in situ.

Immunological and biochemical analyses have shown that alpha-amylase is an essential component of the acquired pellicle. After adsorption, this enzyme might act as a receptor for bacterial adherence. However, data indicating that amylase is bound to the pellicle surface in vivo and thus available for adhering bacteria are rare. Therefore, the present study focused on alpha-amylase within the pellicle formed in situ, using gold-immunolabeling electron microscopic techniques. Pellicles were formed by intra-oral exposure of enamel specimens for 30 and 120 min in six subjects. The results obtained by transmission electron microscopy indicate that amylase was randomly distributed in the pellicle layer without any preferential localization within the pellicle. Thus, salivary alpha-amylase might be considered as an important structural component that is even involved in the early stages of pellicle formation. The findings of field emission in-lens scanning electron microscopy provided evidence that the enzyme is located on the pellicle surface. It could be concluded that alpha-amylase might act as a receptor for bacterial adherence to the pellicle in vivo.

Initial salivary pellicle formation on solid substrates studied by AFM.

Organic layers of salivary biopolymers adsorbed on soft and hard oral tissues, referred to also as salivary pellicle, play a critical role with respect to all surface phenomena taking place in the oral cavity. The initial stages of pellicle formation are of great interest since they determine the ensuing processes of salivary biopolymer adsorption and subsequent adherence of bacteria. In spite of the important physiological role of the pellicle in protecting the enamel surface against short-term acidic attacks, the composition and ultrastructure of the pellicle layer are not yet understood and resolved in detail. The present study utilized atomic force microscopy (AFM), for the first time, to elucidate the morphogenesis and ultrastructural pattern of initial salivary pellicle formation taking place in situ on solid substrates of mica, silicon wafer and graphite. Using tapping mode AFM, salivary pellicles were found in all intraorally exposed specimens and revealed a globular surface morphology of the adsorbed protein layer. The average diameter and height of the adsorbed salivary proteins were determined to be 15 +/- 3 nm and 2.0 +/- 0.5 nm, respectively. It was also found that the surface energy of the substrates affects the rate of pellicle formation, while the overall size of the adsorbed salivary proteins appears to be identical on all studied substrates.

Bacterial adhesion on commercially pure titanium and zirconium oxide disks: an in vivo human study.

BACKGROUND: Little is known about the mechanisms of bacterial interaction with implant materials in the oral cavity. A correlation between plaque accumulation and progressive bone loss around implants has been reported. Bacterial adhesion shows a direct positive correlation with surface roughness. Other surface characteristics also seem to be extremely important with regard to plaque formation. Different adhesion affinities of bacteria have been reported for different materials. The aim of this study was to characterize the percentage of surface covered by bacteria on commercially pure titanium and zirconium oxide disks. METHODS: Ten patients participated in this study. A removable acrylic device was adapted to the molar-premolar region, and commercially pure titanium (control) and zirconium oxide (test) disks were glued to the buccal aspect of each device. The surface roughness of titanium and test specimens was similar. After 24 hours, all disks were removed and processed for scanning electron microscopy, for the evaluation of the portion of surface covered by bacteria. RESULTS: In control specimens, the area covered by bacteria was 19.3% +/- 2.9; in test specimens, the area was 12.1% +/- 1.96. The disk surface covered by bacteria on test specimens was significantly lower than that of control specimens (P = 0.0001). CONCLUSION: Our results demonstrate that zirconium oxide may be a suitable material for manufacturing implant abutments with a low colonization potential.

Bacterial adhesion on titanium nitride-coated and uncoated implants: an in vivo human study.

Titanium nitride (TiN) has been used in many fields as a surgical instrument coating that makes the surgical materials more resistant to wear and corrosion. The aim of the present study was an in vivo evaluation of the bacterial adhesion to TiN-coated (test) and uncoated (control) titanium implants. Six patients aged between 21 and 25 years and in excellent systemic health participated in the study. All of the participants gave their informed consent. The participants were selected on the basis of good periodontal health and no signs of mouth breathing. In each of the 6 participants, a removable acrylic device was adapted to the molar-premolar region of each quadrant of the jaws. One 4 x 13 mm titanium implant was glued to the buccal aspect of each device. The plasma spray covered 11.5 mm of the body of the implant, whereas the neck was machined titanium. Test implants were glued to the right devices and control implants were glued to the left devices. After 24 hours, the implants were removed from each device and processed for scanning electron microscopy for evaluation of the machined portion of the implant covered by bacteria. A total of 24 implants were used in this study, 12 test and 12 control. Surface characterization of the machined portion of the neck of the implant was performed on an additional 10 implants (5 test and 5 control). On test implants the implant surface covered by bacteria was significantly lower compared with that of control implants (P = .0001). The surface roughness was similar in both groups. TiN surfaces showed a significant reduction of the presence of bacteria, and this fact could probably be important in the decrease of the inflammation of the peri-implant soft tissues.

Isolation and physical characterization of the MUC7 (MG2) mucin from saliva: evidence for self-association.

Saliva contains two major families of mucins (MG1 and MG2); the polypeptide of the smaller of these glycoproteins (MG2) has been assigned as the product of the MUC7 gene. In this study we have devised a rapid two-step procedure that recovers this glycoprotein essentially free of other components and in sufficient quantity to enable physical and self-interaction studies. Raw saliva was solubilized in 4 M guanidinium chloride and thereafter subjected to Sepharose CL-4B chromatography. The MG2-rich fraction was recovered free from the larger MG1 glycoproteins and also smaller proteins/glycoproteins (molecular mass less than 100 kDa). MG2 glycoproteins were finally purified by anion-exchange chromatography on Mono Q. The purity of the preparation was assessed by SDS/PAGE after radiolabelling of the molecules with [14C]acetic anhydride. Peptide mapping, N-terminal sequencing and amino acid analysis verified the polypeptide of the mucins as the MUC7 gene product. The isolated molecules were examined by electron microscopy and appeared as short flexible worm-like structures 30-120 nm in length. The distribution was heterogeneous, containing a major component with number-average and weight-average lengths of 52 and 55 nm respectively and a minor component with number-average and weight-average lengths of 94 and 98 nm respectively. We propose that the two differently sized populations represent monomeric and dimeric species of the mucins. Gel chromatography performed in 0.2 M NaCl indicated the presence of monomers, dimers and tetramers; an average molecular mass for the preparation was 192 kDa. However, in 4 M guanidinium chloride the molecular mass was 158 kDa and a similar molecular mass (155 kDa) was determined for the mucin preparation after reduction. These results suggest that the mucins might self-associate via a protein-mediated interaction. On the basis of the results a model is proposed for the self-association of the MUC7 mucin, which might be important for its biological function.

Surface ultrastructure of intact and in situ chlorhexidine-treated human buccal cells. A method for scanning electron microscopy.

Air-dried and ethanol-fixed buccal epithelial cell smears from five subjects were observed by scanning electron microscopy. The mucous pellicle was precipitated as a smooth haze covering the cells, and outlines of bacteria were found embedded within it. Rinsing the preparations under running water gradually diminished the mucous pellicle but not the cell-adherent bacteria. A more complete dissolution of the pellicle was accomplished by washing the buccal epithelial cells before smearing. After a chlorhexidine mouthrinse the buccal cells appeared distorted, with only a few adherent bacteria. Three days after the rinsing, the denatured appearance still persisted on many cells, however, simultaneously with the emergence of undenatured epithelial cells with adherent bacteria. The method introduced in this study is useful to investigate the bacteria-mucus-epithelial cell interactions. A possible mode of antibacterial activity of chlorhexidine in vivo may be that it destroys bacterial adhesins. The substantivity of chlorhexidine in the oral cavity may be linked to the turnover rate of the oral epithelial cells.

[Patterns of highly glycosylated proteins from human saliva]. / Tekstury vysokoglikozilirovannykh proteinov rotovoi biologicheskoi zhidkosti cheloveka.

Mucine-like glycoproteins have been studied by structural indication. Textures of these compounds have been described. Possibility for using mucines as matrix structures in microcrystallographic method of analysis of complex multicomponent systems has been discussed.

Disassembly and reassembly in vitro of complexes of secretory proteins from Chironomus tentans salivary glands.

The secretory proteins of Chironomus tentans larvae form insoluble fibers that are spun into threads used to construct underwater feeding and pupation tubes. We began in vitro studies of the mechanism of assembly into fibers, the structure of the assembled proteins, and the contribution of individual proteins to the assembled structure. From measurements of turbidity and electron micrographs, we observed that the secretory proteins were isolated as complexes. These complexes are most likely at initial stages of assembly; further assembly into insoluble fibers must occur in vivo. Denaturation and reduction disrupted the complexes, and removal of the denaturing and reducing agents resulted in reassembly of the complexes. The circular dichroic spectrum of the complexes indicated that the assembled proteins had the tertiary structure alpha + beta. The largest secretory proteins were purified and shown to have both similar morphology, using electron microscopy, and a similar dichroic spectrum to that of the native complexes. We concluded that the large secretory proteins form the fibrous backbone of the complexes that we observe.