Tablysin-15 inhibits osteoclastogenesis and LPS-induced bone loss via attenuating the integrin αvß3 pathway.

Excessive osteoclast leads to the imbalance in bone reconstruction and results in osteolytic diseases, such as osteoporosis and rheumatic arthritis. Integrin αvß3 abundantly expresses on osteoclast and plays a critical role in the formation and function of osteoclast, therefore, blockage of αvß3 has become an attractive therapeutic option for osteolytic diseases. In this study, we find that Tablysin-15, a RGD motif containing disintegrin, concentration-dependently suppresses RANKL-induced osteoclastogenesis, F-actin ring formation and bone resorption without affecting the cell viabilities. Tablysin-15 binds to integrin αvß3 and inhibits the activation of FAK-associated signaling pathways. Tablysin-15 also suppresses the activation of NF-кB, MAPK, and Akt-NFATc1 signaling pathways, which are crucial transcription factors during osteoclast differentiation. Moreover, Tablysin-15 decreases the osteoclastogenesis marker gene expression, including MMP-9, TRAP, CTSK, and c-Src. Finally, Tablysin-15 significantly inhibits LPS-induced bone loss in a mouse model. Taken together, our results indicate that Tablysin-15 significantly suppresses osteoclastogenesis in vitro and in vivo, thus it might be a excellent candidate for treating osteolytic-related diseases.

Salivary acquired pellicle-inspired DpSpSEEKC peptide for the restoration of demineralized tooth enamel.

Salivary acquired pellicle (SAP) is a layer of proteins and glycoproteins of salivary origin that tightly coat the tooth surface. Statherin is an important part of the SAP. The initial six-peptide sequence DpSpSEEK (where pS denotes phosphorylated serine) of the N-terminus of statherin can be immobilized on a hydroxyapatite (HAP) surface and the negatively charged domains of the DpSpSEEK side chain can catch free Ca2+ in saliva due to the charge adsorption effect. In order to prepare more functional materials based on DpSpSEEK, we designed a cysteine-labeled peptide sequence DpSpSEEKC, which could conjugate other macromolecules by forming a sulfur-based linkage. In this work, we measured the adsorption of DpSpSEEKC to HAP by various methods. We also coated DpSpSEEKC on a demineralized tooth enamel surface to evaluate its biomineralization capacity. The DpSpSEEKC-coated samples were characterized after immersion in artificial saliva for 2 weeks. The results showed that DpSpSEEKC has a strong adsorption capacity to HAP and could induce remineralization on the demineralized tooth enamel surface due to its carboxyl and phosphate groups. Compared with the control samples, the mechanical properties of the DpSpSEEKC-coated samples were obviously improved. In conclusion, DpSpSEEKC can provide a potential method for restoring demineralized tooth enamel.

Black fly salivary gland extract inhibits proliferation and induces apoptosis in murine splenocytes.

Black flies are known to be vectors of pathogens including Onchocerca volvulus, which causes human onchocerciasis, and Vesicular Stomatitis Virus. Their salivary secretion has been shown to contain a complex cocktail of anti-haemostatic factors and immunomodulatory activities, which may contribute to efficient transmission of the pathogens. Black fly salivary gland extract (SGE) inhibits mitogen-stimulated mouse splenocyte proliferation, including proliferation of both CD4(+) and CD8(+) T cells. The factor responsible for the inhibition was determined to be a protein (or protein complex) of a size larger than 50 kDa. Moreover, exposure to SGE results in activation of caspase 3 and characteristic morphological changes in CD4(+) and CD8(+) T cells, suggesting that induction of apoptosis could, at least in part, be responsible for this inhibition.

Structure and mechanism in salivary proteins from blood-feeding arthropods.

The saliva of blood-feeding arthropods contains rich mixtures of ligand binding proteins targeted at inhibiting hemostasis and inflammation in the host. Since blood feeding has evolved many times, different taxonomic groups utilize completely different families of proteins to perform similar tasks. Structural studies performed on a number of these proteins have revealed biologically novel and sophisticated mechanisms used to perform their functions. Here, the results of these structural and mechanistic studies are reviewed.

Fusion of fungicidal peptide dhvar4 to enterococcal peptide pheromone increases its bactericidal activity against Enterococcus faecalis.

Bacterial peptide pheromone has a high affinity to its membrane receptor. Fusion of these peptides to pore-forming antimicrobial peptide might enhance its bactericidal activity against pheromone-sensing bacteria. We constructed two chimeric peptides by fusing the pore-forming fungicidal peptide dhvar4 to the C-terminus of enterococcal peptide pheromones cCF10 and cOB1 individually. Comparison on the bactericidal activities against pheromone-sensing bacteria Enterococcus faecalis demonstrates that the chimeric peptides cCF10-dhvar4 and cOB1-dhvar4 are more potent than the parent peptide dhvar4. The LD(50)s of both chimeric peptides (1.0 microm) are 10 times lower than that of dhvar4 (10.8 microm). Free peptide pheromone could inhibit E. faecalis killing mediated by both chimeric peptides. As same as that of the parent peptide, both chimeric peptides kill bacteria by disrupting its cell membrane. These results indicate that fused enterococcal peptide pheromone increases the bactericidal activity of fungicidal peptide against E. faecalis by improving its ability to reach the cell membrane.

Human salivary mucin MUC7 12-mer-L and 12-mer-D peptides: antifungal activity in saliva, enhancement of activity with protease inhibitor cocktail or EDTA, and cytotoxicity to human cells.

MUC7 12-mer-L exhibits potent in vitro antifungal activity in low-ionic-strength buffers. In this study, we investigated the anticandidal activity and stability of MUC7 12-mer-L and its all-D-amino-acid isomer, along with Hsn5 12-mer (P113) and magainin-II, in human clarified and unclarified saliva in the absence or presence of protease inhibitor cocktail (PIC, which includes EDTA) or EDTA alone. In the absence of PIC or EDTA in saliva, only MUC7 peptides showed significant candidacidal activity. At a 100 microM concentration in clarified saliva and unclarified saliva, MUC7 12-mer-D demonstrated 94 versus 64% killing, respectively; MUC7 12-mer-L showed 57 versus 32% killing; Hsn5 12-mer showed 16 versus 0% killing; and magainin-II showed no killing. Addition of PIC or EDTA to either saliva caused the enhancement of antifungal activities of all peptides, although to different degrees. Taken together, the results suggest that EDTA (a metal-dependent protease inhibitor and/or divalent cation chelator) enhanced the antifungal activity of all four peptides mainly by chelation of divalent cations present in saliva (known to inhibit peptide antifungal activity), and PIC enhanced the activity of the three L peptides above that achievable by EDTA alone through inhibition of all classes of proteases. Peptide stability in saliva monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed no degradation of MUC7 12-mer-D and 23, 60, and 75% degradation of MUC7 12-mer-L, Hsn5 12-mer, and magainin-II, respectively. Cytotoxicity assays determined that, at 100 microM peptide concentrations, MUC7 12-mer-D and 12-mer-L caused 3.5 and 4.3% hemolysis in phosphate-buffered saline and no toxicity to the HOK-16B cell line (derived from normal human oral keratinocytes). In summary, MUC7 12-mer peptides appear to be excellent candidates for investigation of antifungal activity in in vivo models of oral candidiasis.

Cobra venom contains a pool of cysteine-rich secretory proteins.

A large family of cysteine-rich secretory proteins (CRISPs) includes proteins of different origin, the function of the majority of CRISPs being unknown. For CRISPs isolated from snake venom, two types of activities were found: two proteins blocked cyclic nucleotide-gated ion channels, several others blocked potassium-stimulated smooth muscle contraction. Thus, snake CRISPs represent potentially valuable tools for studies of ion channels, which makes promising a search for new CRISPs. Here we report on the isolation of several novel CRISPs from the venoms of Asian cobra Naja kaouthia and African cobra Naja haje using a combination of different types of liquid chromatography. Four CRISP variants were identified in N. kaouthia venom and three proteins, one of them acidic, were found in N. haje venom. Acidic CRISP was found in a reptilian venom for the first time. Our data suggest that each cobra venom contains a pool of different CRISPs.

Safety and clinical effects of topical histatin gels in humans with experimental gingivitis.

BACKGROUND: Our research group has recently reported that exogenously applied histatins can inhibit plaque accumulation and gingival inflammation in a preclinical canine model (Paquette et al. 1997). OBJECTIVES: The aims of the present double-blinded, randomized, controlled clinical trial were to evaluate the safety and toxicity of three histatin (P-113) concentrations in gel formulations, and to assess potential clinical benefit on the development of gingivitis (partial mouth design). MATERIAL AND METHODS: One hundred and six healthy subjects were recruited and brought to optimal gingival health (GI < 0.5) prior to treatment initiation. At baseline, eligible subjects were randomized for one of the following treatments: (1) placebo; (2) 0.0625% P-113; (3) 0.125% P-113; and (4) 0.375% P-113. Patients self-applied gels twice daily for 29 days to the maxillary right quadrant with the use of customized stents. In addition, patients deferred all oral hygiene procedures within this quadrant for the duration of the treatment period. Safety was assessed in terms of physical and oral examinations, clinical laboratory testing and recording of adverse events. Clinical indices were measured weekly and included gingival index (GI), plaque index (PI) and %BOP. RESULTS: All study formulations were well tolerated by patients, and no differences in adverse event occurrences were noted among treatment groups, including taste alteration or staining. For the intent-to-treat population, significantly smaller %BOP changes were noted in subjects treated with 0.0625, 0.125 and 0.375% P-113 gels (17.4, 18.18 and 17.9%, respectively) versus placebo (28.0%) (p < 0.05) at day 29. When groups were compared in terms of per cent responders (change in %BOP < 15 or < 20%), P-113 treatment groups exhibited a higher frequency of response, especially for the 0.0625 and 0.125% P-113 formulations (p < 0.05). Although no statistically significant intergroup differences were noted for changes in GI or PI among all subjects (intent-to-treat population), significantly smaller changes in PI at day 22 were observed among compliant individuals (defined as subjects using > 60% of the target gel mass) administering P-113 gels as compared with compliant placebo subjects (p < 0.05). CONCLUSIONS: These data indicate safety and tolerance of P-113 gels for topical oral use in human subjects. These data also suggest that P-113 gels administered twice daily may reduce experimental gingivitis as measured with bleeding on probing in humans.

Toxins produced by arthropod parasites: salivary gland proteins of human body lice and venom proteins of chelonine wasps.

A review is presented of our ongoing research projects on the protein components of the saliva of human body lice and of the non-paralyzing venom of wasps in the subfamily Cheloninae. Sodium dodecyl sulfate-polyacryamide gel electrophoretic analysis of lice salivary gland proteins showed a predominance of high and intermediate mol. wt proteins. Immunoblotting with a low titer polyclonal antiserum to lice salivary proteins indicated that some, but not all, of the predominant high mol. wt salivary gland proteins are injected into the host during feeding. The venom of a Chelonus sp. wasp contains a chitinase, and a 33,000 mol. wt protein with a primary structure composed mostly of a series of 12 tandem repeats of a 14-residue sequence. The N-terminus of this protein and its homologs in a related species of Ascogaster share a conserved adjacent pair of acidic residues. Epitope mapping/immunoprecipitation experiments now in progress will provide information on which linear motifs are on the surface of the protein, and will thereby provide information on the tertiary structure of the protein.