Evidence-based practice in dentistry: benefit or hindrance.
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Dental Research↗
Biomedical subjects
M Edgerton
Publications and source records attributed to M Edgerton.
Calcium blocks fungicidal activity of human salivary histatin 5 through disruption of binding with Candida albicans.
Salivary histatin 5 (Hst 5) kills the fungal pathogen C. albicans via a mechanism that involves binding and subsequent efflux of cellular ATP. Our aims were to identify inorganic ions found in saliva that influence Hst 5 fungicidal activity. Increasing ionic strength with relevant salivary anions (Cl(-) and CO(3)(-)) did not reduce Hst 5 binding or uptake by yeast cells, but reduced the Hst-induced efflux of ATP. Extracellular MgCl(2) (25 mM) maximally inhibited 30-40% of Hst 5 killing with 40% reduction in ATP efflux, while pre-treatment of cells with only 2 mM CaCl(2) inhibited 80-90% of killing, and prevented ATP efflux. Loss of fungicidal activity by the addition of CaCl(2) or MgCl(2) was a result of inhibition of binding of Hst 5 to C. albicans cells. Calcium is a potent inhibitor of Hst 5 candidacidal activity at physiological concentrations and may be the primary salivary ion responsible for the masking effect of saliva.
Adenosine Triphosphate↗
Genetically engineered human salivary histatin genes are functional in Candida albicans: development of a new system for studying histatin candidacidal activity.
Histatins are a structurally related family of salivary proteins known as histidine-rich proteins that are produced and secreted by the human major salivary glands. In vitro, histatins are potent cytotoxic proteins with selectivity for pathogenic yeasts including Candida albicans. Studies that investigate the mechanism of action of histatin proteins upon this important human pathogen have used a candidacidal assay in which the histatin is applied extracellularly. In order to develop a model system to study the mechanism of histatin action independently from binding and translocation events, the authors constructed C. albicans strains that contain chromosomally encoded human salivary histatin genes under the control of a regulated promoter. Intracellular expression of either histatin 5 or histatin 3 induced cell killing and ATP release in parallel. Since histatin killing can be initiated solely from intracellular sites, extracellular binding and internalization are preceding transport events. Thus the mechanism of histatin-induced ATP release does not require extracellular binding, and intracellular targets alone can activate ATP release. By employing a codon-optimization strategy it was shown that expression of heterologous sequences in C. albicans can be a useful tool for functional studies.
Adenosine Triphosphate↗
A chimeric MIP-1alpha/RANTES protein demonstrates the use of different regions of the RANTES protein to bind and activate its receptors.
Human RANTES (CCL5) and MIP-1alpha (CCL3) bind and activate several CC chemokine receptors. RANTES is a high-affinity ligand for CCR1 and CCR5, and it binds CCR3 with moderate affinity and CCR4 with low affinity. MIP-1alpha has similar binding characteristics to RANTES except that it does not bind to CCR3. Here we have generated a chimera of human MIP-1alpha and RANTES, called MIP/RANTES, consisting of the eight amino terminal residues of MIP-1alpha preceding the CC motif, and the remainder of the sequence is RANTES. The chimera is able to induce chemotaxis of human monocytes. MIP/RANTES has >100-fold reduction in binding to CCR1 and does not bind to CCR3 but retains full, functional binding to CCR5. It has equivalent affinity for CCR5 to MIP-1alpha and RANTES, binding with an IC(50) of 1.12 nM, and is able to mobilize calcium and induce endocytosis of CCR5 in PBMC in a manner equi-potent to RANTES. It also retains the ability to inhibit R5 using HIV-1 strains. Therefore, we conclude that the amino terminus of RANTES is not involved in CCR5 binding, but it is essential for CCR1 and CCR3.
Amino Acid Sequence↗
Salivary histatin 5 and human neutrophil defensin 1 kill Candida albicans via shared pathways.
Salivary histatins are a family of basic histidine-rich proteins in which therapeutic potential as drugs against oral candidiasis is apparent, considering their potent in vitro antifungal activity and lack of toxicity to humans. Histatin 5 (Hst 5) kills the fungal pathogen Candida albicans via a mechanism that involves binding to specific sites on the yeast cell membrane and subsequent release of cellular ATP in the absence of cytolysis. We explored the killing pathway activated by Hst 5 and compared it to those activated by other antifungal agents. The candidacidal activity of human neutrophil defensin 1 (HNP-1) shared very similar features to Hst 5 cytotoxic action with respect to active concentrations and magnitude of induction of nonlytic ATP efflux, depletion of intracellular ATP pools, and inhibitor profile. Hst 5 and HNP-1 are basic proteins of about 3 kDa; however, they have unique primary sequences and solution structures that cannot explain how these two molecules act so similarly on C. albicans to induce cell death. Our finding that HNP-1 prevented Hst 5 binding to the candidal Hst 5 binding protein suggests that the basis for the overlapping actions of these two naturally occurring antimicrobial proteins may involve interactions with shared yeast components.
Adenosine Triphosphate↗
Released ATP is an extracellular cytotoxic mediator in salivary histatin 5-induced killing of Candida albicans.
Salivary histatins (Hsts) are antifungal peptides with promise as therapeutic agents against candidiasis. Hst 5 kills the fungal pathogen Candida albicans via a mechanism that involves release of cellular ATP in the absence of cytolysis. Here we demonstrate that released ATP has a further role in Hst 5 killing. Incubation of the cells with ATP analogues induced cell death, and addition of the ATP scavenger apyrase to remove extracellular ATP released during Hst 5 treatment resulted in a reduction in cell killing. Experiments using anaerobically grown C. albicans with decreased susceptibility to Hst 5 confirmed that depletion of cellular ATP as a result of ATP efflux was not sufficient to cause cell death. In contrast to Hst-susceptible aerobic cultures, anaerobically grown cells were not killed by exogenously applied ATP. These findings established that Hst binding, subsequent entry into the cells, and ATP release precede the signal for cytotoxicity, which is mediated by extracellular ATP. In a higher-eukaryote paradigm, released ATP acts as a cytotoxic mediator by binding to membrane nucleotide P2X receptors. Based on a pharmacological profile and detection of a C. albicans 60-kDa membrane protein immunoreactive with antibody to P2X(7) receptor, we propose that released ATP in response to Hst 5 activates candidal P2X(7)-like receptors to cause cell death.
Adenosine Triphosphate↗
Salivary histatin 5 and its similarities to the other antimicrobial proteins in human saliva.
Non-immune salivary proteins--including lactoperoxidase, lysozyme, lactoferrin, and histatins--are key components of the innate host defense system in the oral cavity. Many antimicrobial proteins contain multiple functional domains, with the result that one protein may have more than one mechanism of antimicrobial activity. These domains may be separated by proteolytic cleavage, creating smaller proteins with functional antimicrobial activity in saliva as described for lysozyme, lactoferrin, and histatins. These small cationic proteins then exert cytotoxic activity to oral bacteria and fungi. Salivary histatin 5 initiates killing of C. albicans through binding to yeast membrane proteins and non-lytic release of cellular ATP. Extracellular ATP may then activate fungal ATP receptors to induce ultimate cell death. This mechanism for fungal cytotoxicity may be shared by other antimicrobial cationic proteins. Microbicidal domains of salivary and host innate proteins should be considered as potential therapeutic agents in the oral cavity.
Adenosine Triphosphate↗
Salivary histatin 5 induces non-lytic release of ATP from Candida albicans leading to cell death.
Salivary histatins are potent in vitro antifungal proteins and have promise as therapeutic agents against oral candidiasis. We performed pharmacological studies directed at understanding the biochemical basis of Hst 5 candidacidal activity. Three inhibitors of mitochondrial metabolism: carbonyl cyanide p-chlorophenylhydrazone, dinitrophenol, and azide inhibited Hst 5 killing of Candida albicans, while not inhibiting cellular ATP production. In contrast, Hst 5 caused a drastic reduction of C. albicans intracellular ATP content, which was a result of an efflux of ATP. Carbonyl cyanide p-chlorophenylhydrazone, dinitrophenol, and azide inhibited Hst 5-induced ATP efflux, thus establishing a correlation between ATP release and cell killing. Furthermore, C. albicans cells were respiring and had polarized membranes at least 80 min after ATP release, thus implying a non-lytic exit of cellular ATP in response to Hst 5. Based on evidence that transmembrane ATP efflux can occur in the absence of cytolysis through a channel-like pathway and that released ATP can act as a cytotoxic mediator by binding to membrane purinergic receptors, we evaluated whether extracellular ATP released by Hst 5 may have further functional role in cell killing. Consistent with this hypothesis, purinergic agonists BzATP and adenosine 5'O-(thiotriphosphate) induced loss of C. albicans cell viability and purinergic antagonists prevented Hst 5 killing.
Adenosine Triphosphate↗
Myeloperoxidase deficiency associated with atypical oral candidiasis: A clinical report.
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Antifungal Agents↗
Long-term outcome analysis of two treatment methods for cleft palate: combined levator retropositioning and pharyngeal flap versus double-opposing Z-plasty.
OBJECTIVE: Two surgical techniques for repair of a cleft palate include levator retropositioning in combination with a pharyngeal flap and the Furlow double-opposing Z-plasty. This study compared morbidity and speech results from the use of these two methods in an effort to determine which was the superior technique. DESIGN: Patient records from 1986 to 1996 were retrospectively reviewed, and 10 patients with a cleft palate who underwent repair with a levator retropositioning and pharyngeal flap were compared to 14 patients who underwent a double-opposing Z-plasty repair. Postoperative complications including fistula formation, obstructive sleep apnea, and residual velopharyngeal insufficiency were recorded. Speech was assessed perceptually and through the use of nasometry. RESULTS: Both surgical techniques resulted in good speech in the majority of patients. Only two patients in the study, both in the Z-plasty group, had severe postoperative hypernasality. Two patients in the levator retropositioning and pharyngeal flap group developed severe postoperative obstructive sleep apnea, requiring additional surgery. CONCLUSION: The levator retropositioning and pharyngeal flap technique was successful in achieving good speech results, but it also caused more serious postoperative complications when compared to the double-opposing Z-plasty technique.
Cleft Palate↗
Candidacidal activity of salivary histatins. Identification of a histatin 5-binding protein on Candida albicans.
Candida albicans is the predominant species of yeast isolated from patients with oral candidiasis, which is frequently a symptom of human immunodeficiency virus infection and is a criterion for staging and progression of AIDS. Salivary histatins (Hsts) are potent in vitro antifungal agents and have great promise as therapeutic agents in humans with oral candidiasis. The molecular mechanisms by which Hsts kill yeast cells are not known. We report here, that unlike other antimicrobial proteins, Hsts do not display lytic activities to lipid membranes, measured by release and dequenching of the fluorescent dye calcein. Analysis of the magnitude and time course of Hst-induced calcein release from C. albicans cells further showed that loss of cell integrity was a secondary effect following cell death, rather than the result of primary disruption of the yeast cell membrane. 125I-Hst 5 binding studies indicated that C. albicans expressed a class of saturable binding sites (KD = 1 microM), numbering 8.6 x 10(5) sites/cell. Both Hst 3 and Hst 4 competed for these binding sites with similar affinities, which is consistent with the micromolar concentration of Hsts required for candidacidal activity. Specific 125I-Hst 5 binding was not detected to C. albicans spheroplasts, which were 14-fold less susceptible to Hst 5 killing, compared with intact cells in candidacidal assays. In overlay experiments, 125I-Hst 5 bound to a 67-kDa protein detected in C. albicans whole cell lysates and crude membrane fractions, but not in the yeast cell wall fraction. Consistent with the overlay data, cross-linking of 125I-Hst 5 to C. albicans resulted in the appearance of a specific 73-kDa 125I-Hst 5-containing complex that was not detected in the cell wall. 125I-Hst 5-binding protein of similar size was also observed in susceptible S. cerevisiae strain TI#20. This is the first description of Hst 5 binding sites on C. albicans which mediate cell killing and identification of a 67-kDa yeast Hst 5-binding protein. The binding characteristics of Hst 5 are in agreement with the observed potency of its biological effect and provide crucial information to the use of Hst 5 as a therapeutic agent. The presence of a specific C. albicans Hst 5-binding protein provides further insight into the potential mechanism of yeast killing and suggests a basis for differential activity between yeast killing and the nontoxic nature of Hsts to humans.
Acquired Immunodeficiency Syndrome↗
Delineation of an active fragment and poly(L-proline) II conformation for candidacidal activity of bactenecin 5.
Bactenecin 5 and its fragments [BN22 (1-22), BN16 (7-22), and BC24 (20-43)] were synthesized by solid-phase methods. Their antifungal activities on Candida albicans have been studied and compared with those of the native bactenecin 5. The conformational preferences of these peptides in aqueous and nonaqueous solutions and in lipid vesicles were examined by circular dichroism. The highly active N-terminal fragment (BN16) was examined in aqueous solution using 500 MHz two-dimensional NMR. Bactenecin 5 and its fragments are potent candidacidal agents against C. albicans. The N-terminal fragments (BN22 and BN16) of bactenecin 5 are relatively more active than the C-terminal fragment BC24, especially at lower concentrations. The N-terminal region (7-22) which retains the activity of the whole molecule appears to be the functional domain for candidacidal activity. The CD spectra of bactenecin 5 and its fragments are reminiscent of the CD spectrum of poly(L-proline) type II structure in aqueous and nonaqueous solutions and also in lipid vesicles. The temperature dependence of NH chemical shifts and 1H/2H exchange effect on amide resonances suggest the absence of intramolecularly hydrogen-bonded NH groups. The coupling constant (JNH-CalphaH) values, conformational restriction offered by the Pro residues (phi = -60 degrees +/- 15 degrees), the set of medium- and short-range nuclear Overhauser effects observed for the active N-terminal fragment (BN16), and the restrained structure calculation using DIANA suggest that poly(L-proline) type II conformers of the peptide molecules could be significantly populated in aqueous solution. The ability of bactenecin peptides to induce disruption of lipid vesicles correlates well with their activity. Our results suggest that poly(L-proline) type II structure may, indeed, be the biologically active conformation for candidacidal activity of bactenecin peptides.
Amino Acid Sequence↗
Co-expression of the neurokinin NK2 receptor and G-protein components in the fission yeast Schizosaccharomyces pombe.
The fission yeast Schizosaccharomyces pombe has proven useful for studying molecular interactions between a range of signal transduction components. We now report the first co-expression of a mammalian seven-transmembrane receptor and G-protein components in S. pombe. We selected the human neurokinin NK2 receptor together with its G-protein-signalling partner Gq for this study. Yeast membrane fractions showed high levels of NK2 receptor-binding activity (1159 +/- 534 (n = 3) fmol/mg protein) although initial experiments with intact cells revealed an absence of receptors at the cell surface. Using a construct comprising the NK2 coding sequence fused with the signal sequence from an endogenous phosphatase (phoI), we detected approximately 400 NK2 receptors/cell in unbroken yeast. Successful co-expression of the NK2 receptor with the G-protein subunits G alpha q, beta 1 or beta 2 and gamma 3 failed to modulate agonist binding, suggesting the absence of functional interaction between these components. As an alternative test of G alpha q function, we next expressed its downstream effector target phospholipase C-beta 1 (PLC beta 1) in S. pombe. Although PLC beta 1 undergoes powerful in vitro activation by G alpha q derived from baculovirus-infected Sf9 cells and mammalian cells, G alpha q expressed in S. pombe is totally ineffective. Similar results were also achieved with the G-protein subunit G alpha 16. Together, these data suggest that seven-transmembrane receptors can be expressed in S. pombe at high levels and directed to the cell surface although their interaction with co-expressed G-proteins in undetectable. Production of inactive G alpha-chains in S. pombe may account for these observations.
Amino Acid Sequence↗
Functional domain and poly-L-proline II conformation for candidacidal activity of bactenecin 5.
The functional domain for candidacidal activity of bactenecin 5 has been determined by synthesizing bactenecin 5 and its fragments [1-22 (BN22), 7-22 (BN16) and 20-43 (BC24)]. The N-terminal sequence BN16 retained the candidacidal potency of the parent molecule and this region appears to be the candidacidal domain. The circular dichroism spectra of these peptides indicate the presence of largely poly-L-proline II conformations in aqueous solutions and in lipid vesicles. The coupling constant (JNH-C alpha H) values, and a set of medium- and short-range nuclear Overhauser effects observed for the N-terminal peptide (BN16) in the two-dimensional nuclear magnetic resonance suggest that poly-L-proline II helix could be the biologically active conformation.
Amino Acid Sequence↗
Surface-modified poly(methyl methacrylate) enhances adsorption and retains anticandidal activities of salivary histatin 5.
Denture-induced stomatitis is a common intraoral disease which is associated with high levels of Candida albicans adhesion to a denture surface. The aim of this study was to produce a surface-modified denture resin, which is usually manufactured from poly(methyl methacrylate) (PMMA), carrying an immobilized anticandidal protein. PMMA was modified by surface polymerization of methyl methacrylic acid to enhance adsorption of a potent candidacidal salivary protein, histatin 5. The modified PMMA showed higher surface adsorption and desorption of histatin 5 than the unmodified material. Because histatin 5 destabilizes C. albicans cell membranes and allows efflux of intracellular molecules, candidacidal activity was monitored by dye release from fungal cells. Adsorbed histatin 5 did not release dye from the yeast cells; however, dye was detected as histatin was desorbed from the surface. In an adhesion assay, modified PMMA decreased human submandibular-sublingual saliva (HSMSL) mediated adherence of yeast cells to the polymer. Precoating histatin 5 onto unmodified PMMA also abolished HSMSL-mediated adhesion. These experiments show that dental acrylic may be surface modified and loaded with histatin 5 as a means of controlled release of histatin 5 to an affected area. This surface modification may additionally reduce adhesion of C. albicans cells to the saliva-coated material.
Adsorption↗
Biocompatibility: its future in prosthodontic research.
The future of prosthodontic research will involve replacing lost tissues by using scientific methods that evaluate biomaterials and treatment designs based on desired biologic outcomes. The present concept of a biocompatible material is one that elicits an appropriate host response in a specific application. To design optimal biomaterials, three interactive components should be considered: the chemical nature of the surface, the mediating pellicle layer, and microbial and host response. Surface chemistry determines which molecules are selectively absorbed onto a surface from oral fluids. The pellicle-coated surface should be designed to elicit a more desirable host response. Pellicle composition can be altered by chemically changing the surface, precoating surfaces with biological molecules, or using synthetic materials designed to mimic natural tissues. Several surface-sensitive techniques are available to assess these modifications, including vibrational spectroscopy, electron microscopy for chemical analysis, and bioanalytical methods. To develop more biocompatible materials, a further understanding of pellicle formation as a function of surface composition, microbial adhesion to biomaterials, and cellular reaction to implant biomaterials is necessary. This knowledge will facilitate development of new biologically based rationales for treatment modalities in restorative dentistry.
Biocompatible Materials↗
Human submandibular-sublingual saliva promotes adhesion of Candida albicans to polymethylmethacrylate.
The purpose of this study was to identify components of saliva that interact with Candida albicans in solution and that may modulate adhesion to dental acrylic (polymethylmethacrylate [PMMA]) surfaces. Saliva-derived pellicles extracted from C. albicans blastoconidia and hyphal-form cells mixed with fresh human submandibular-sublingual saliva (HSMSL) contained predominantly high- and low-molecular-weight mucins (MG1 and MG2, respectively). In contrast, few components from fresh human parotid saliva were adsorbed to yeast cells. Coating PMMA beads with HSMSL significantly enhanced (10-fold) adhesion of both growth forms of C. albicans compared with human parotid saliva (2-fold), suggesting a role for mucins in adhesion. HSMSL-enhanced adhesion was completely abolished by preadsorbing HSMSL with either blastoconidia or hyphal-form cells prior to coating PMMA. However, coating PMMA with purified salivary mucins or the addition of mucin to preadsorbed saliva did not enhance or restore adhesion to levels found with fresh HSMSL. Adhesion assays employing guanidine-treated fresh HSMSL showed a complete lack of Candida binding, suggesting that subjecting HSMSL to dissociating conditions may alter a property of salivary mucins crucial for C. albicans adhesion. Protease and glycosidase treatment of yeast cells significantly reduced adhesion to HSMSL-coated PMMA. In addition, preincubation of C. albicans with mannose and galactose inhibited adhesion to HSMSL-coated PMMA. These results suggest that mucins may play a role in C. albicans adhesion to saliva-coated PMMA and that a glycoprotein on the yeast surface may be involved in these events.
Adsorption↗
Characterization of acquired denture pellicle from healthy and stomatitis patients.
Little information is available about the acquired pellicle layer that is formed on denture surfaces or its role in regulating microbial colonization of the prosthetic surface. Because denture-induced stomatitis is associated with increased numbers of Candida albicans and other microorganisms on the denture surface, the acquired denture pellicle (ADP) may play a role in modulating this colonization. This study examined and compared ADP from healthy patients and patients with stomatitis by chemical and immunochemical methods. The ADP was found to be composed of a selectively adsorbed layer containing salivary amylase, high molecular weight mucin (MG1), lysozyme, albumin, and sIgA. Salivary cystatins, proline-rich proteins, and low molecular weight mucin (MG2) were not detected. ADP amino acid composition was distinct from any of the ductal salivas, but had many similarities with enamel pellicle. Immunoblots of ADP from patients with stomatitis identified additional serum components, degradation products, and C. albicans cell components that were not detected in ADP from healthy patients. Quantification of these molecules in ADP could lead to a diagnostic test for oral mucosal disease underlying a denture base. Identification of specific molecules in denture pellicle that promote adhesion of C. albicans may elucidate a mechanism of fungal cell colonization on the denture surface. Future studies that chemically modify the denture acrylic resin surface to immobilize antimicrobial proteins may be a means of decreasing pathogenic plaque development.
Adhesiveness↗