ABSTRACT
INTRODUCTION: The diagnosis and assessment of apical periodontitis by traditional periapical radiographs can be challenging and might yield false-negative results. The aim of this study was to determine whether interleukin-1beta (IL-1beta) and dentin sialoprotein (DSP) in gingival crevicular fluid (GCF) can be used as biological markers for apical periodontitis. METHODS: Forty healthy patients with teeth diagnosed with apical periodontitis of pulpal origin were included in the study. GCF samples were obtained from the diseased tooth and from a healthy contralateral control tooth. Total protein concentration in each sample was determined by using the Bio-Rad protein assay. Enzyme-linked immunosorbent assay was used to analyze the concentration of IL-1beta and DSP in the samples. RESULTS: Protein content of the GCF was statistically significantly higher in the disease group compared with the control group. The levels of IL-1beta and DSP were not statistically different between disease and control groups. CONCLUSIONS: Although this study was unable to demonstrate a significantly higher level of IL-1beta or DSP in the GCF of teeth with apical periodontitis, the observed presence of a significantly higher level of total protein in the GCF of diseased teeth suggests the possible role of total protein level as a marker for periapical disease.
Subject(s)
Extracellular Matrix Proteins/analysis , Gingival Crevicular Fluid/chemistry , Interleukin-1beta/analysis , Periapical Periodontitis/diagnosis , Phosphoproteins/analysis , Sialoglycoproteins/analysis , Biomarkers/analysis , Female , Humans , Male , Periapical Periodontitis/metabolism , Proteins/analysisABSTRACT
We have previously shown that MUC7 (human salivary low-molecular-mass mucin) 20-mer: LAHQKPFIRKSYKCLHKRCR (residues 32-51 of the parent MUC7, with a net positive charge of 7) possesses a broad-spectrum antimicrobial activity [Bobek and Situ (2003) Antimicrob. Agents Chemother. 47, 645-652]. The aims of the present study were to determine the minimum peptide chain length and its location within the 20-mer region that retains potent antifungal activity against Candida albicans and Cryptococcus neoformans and to examine the effect of net charge of the peptide as well as the role of cysteine residues on the fungicidal activity. First, several C-terminal truncated MUC7 20-mer fragments (16-mer, 12-mer, 11-mer, 10-mer and 8-mer) and one N-terminal fragment (8-mer-N) were synthesized and tested. The results showed that MUC7 12-mer, located at the C-terminal region of MUC7 20-mer, having a net charge of +6 and exhibiting an amphipathic helical conformation, not only retained but exceeded the antifungal activity of that of 20-mer. Secondly, several variants of the 12-mer peptide containing a lower or no net positive charge, or no cysteine residues were synthesized and tested. A clear correlation between the net positive charge of the 12-mer, its potency and initial interaction of peptide with fungal cells was found by killing assays, fluorescence microscopy and fungal cell-membrane potential measurements. Furthermore, cysteine residues, which play a critical role in bacterial binding, were found to be not important for the fungicidal activity of these peptides. These results identified MUC7 12-mer as a potential candidate for development into a novel antifungal therapeutic agent.
Subject(s)
Antifungal Agents/chemistry , Antifungal Agents/pharmacology , Mucins/chemistry , Mucins/pharmacology , Salivary Proteins and Peptides/chemistry , Salivary Proteins and Peptides/pharmacology , Amino Acid Sequence , Amino Acids, Basic/chemistry , Candida albicans/drug effects , Candida albicans/physiology , Circular Dichroism , Cryptococcus neoformans/drug effects , Cryptococcus neoformans/physiology , Cysteine/chemistry , Cysteine/physiology , Dose-Response Relationship, Drug , Humans , Membrane Potentials/drug effects , Molecular Sequence Data , Peptides/chemistry , Peptides/pharmacology , Protein Structure, Secondary , Static ElectricityABSTRACT
This study was aimed at examining the spectrum of antimicrobial activity of MUC7 20-mer (N-LAHQKPFIRKSYKCLHKRCR-C; residues 32 to 51 of MUC7, the low-molecular-weight human salivary mucin, comprised of 357 residues) and comparing its antifungal properties to those of salivary histatin 5 (Hsn-5). We also examined the secondary structure of the 20-mer and the possible mechanism of its antifungal action. Our results showed that MUC7 20-mer displays potent killing activity against a variety of fungi and both gram-positive and gram-negative bacteria at micromolar concentrations. Time-dependent killing of Candida albicans and Cryptococcus neoformans by MUC7 20-mer and Hsn-5 indicated differences in killing rates between MUC7 20-mer and Hsn-5. The secondary structure prediction showed that MUC7 20-mer adopts an amphiphilic helix with distinguishable hydrophilic and hydrophobic faces (a characteristic that is associated with antimicrobial activity). In comparison to that of Hsn-5, the fungicidal activity of MUC7 20-mer against C. albicans seems to be independent of fungal cellular metabolic activity, as evidenced by its killing potency at a low temperature (4 degrees C) and in the presence of inhibitors of oxidative phosphorylation in the mitochondrial system. Fluorescence microscopy showed the ability of MUC7 20-mer to cross the fungal cell membrane and to accumulate inside the cells. The internalization of MUC7 20-mer was inhibited by divalent cations. Confocal microscopy of cells doubly labeled with MUC7 20-mer and a mitochondrion-specific dye indicated that mitochondria are not the target of MUC7 20-mer for either C. albicans or C. neoformans.
