The Experts below are selected from a list of 1917 Experts worldwide ranked by ideXlab platform

Frank G. Oppenheim - One of the best experts on this subject based on the ideXlab platform.

  • Direct assessment of the antioxidant property of salivary Histatin.
    Journal of clinical biochemistry and nutrition, 2019
    Co-Authors: Tomoko Komatsu, Frank G. Oppenheim, Eva J. Helmerhorst, Kyo Kobayashi, Masaichi-chang-il Lee
    Abstract:

    Histatin, a salivary protein, affects oral homeostasis through preservation of tooth integrity and protection against caries and fungal infections. However, the effects of Histatin in the generation of oxidative stress induced by reactive oxygen species and in the oral cavity remain unclear. In this study, the effects of Histatin on direct reactive oxygen species scavenging activity were examined using electron spin resonance. We demonstrated, for the first time, that Histatin exhibits antioxidant activity against hydroxyl radicals generated by Fenton's reaction by metal chelation or binding. The direct antioxidant effects of Histatin, along with its antimicrobial activity, may be important in the oral protection of salivary proteins.

  • anti candidal activity of genetically engineered Histatin variants with multiple functional domains
    PLOS ONE, 2012
    Co-Authors: Frank G. Oppenheim, Eva J. Helmerhorst, Gwynneth D Offner, Urs Lendenmann
    Abstract:

    The human bodily defense system includes a wide variety of innate antimicrobial proteins. Histatins are small molecular weight proteins produced by the human salivary glands that exhibit antifungal and antibacterial activities. While evolutionarily old salivary proteins such as mucins and proline-rich proteins contain large regions of tandem repeats, relatively young proteins like Histatins do not contain such repeated domains. Anticipating that domain duplications have a functional advantage, we genetically engineered variants of Histatin 3 with one, two, three, or four copies of the functional domain by PCR and splice overlap. The resulting proteins, designated reHst3 1-mer, reHist3 2-mer, reHis3 3-mer and reHist3 4-mer, exhibited molecular weights of 4,062, 5,919, 7,777, and 9,634 Da, respectively. The biological activities of these constructs were evaluated in fungicidal assays toward Candida albicans blastoconidia and germinated cells. The antifungal activities per mole of protein increased concomitantly with the number of functional domains present. This increase, however, was higher than could be anticipated from the molar concentration of functional domains present in the constructs. The demonstrated increase in antifungal activity may provide an evolutionary explanation why such domain multiplication is a frequent event in human salivary proteins.

  • concentration and fate of Histatins and acidic proline rich proteins in the oral environment
    Archives of Oral Biology, 2009
    Co-Authors: Melanie Campese, Frank G. Oppenheim, Xiuli Sun, Jos A Bosch, Eva J. Helmerhorst
    Abstract:

    Saliva plays a critical role in the protection of oral hard and soft tissues and contains a multitude of constituents with well-characterized biological activities in vitro. Among these are Histatins and acidic proline-rich proteins (PRPs). Nevertheless, few functional studies have recognized the structural instability of these proteins in the proteolytic environment of whole saliva. The aim of this investigation was to determine Histatin and acidic PRP levels in parotid secretion (PS) and in whole saliva (WS) as well as to establish their susceptibility to proteolysis in these salivary fluids. Using cationic polyacrylamide gel electrophoresis and densitometric analysis the average total Histatin concentration (Histatin 1+3+5) in WS was determined to be 33.3+/-16.7 microg/ml (n=22) and the average total acidic PRP concentration (PRP1/PIF-s+PRP3/PIF-f) was 427.9+/-123.3 microg/ml (n=22). Histatin and acidic PRP concentrations in PS were 6 and 1.5 times higher than in WS (n=7), respectively. WS Histatin and acidic PRP levels each correlated significantly with WS total protein concentrations (P<0.01 and P<0.05, respectively), as well as with each other (P<0.01). Stability studies of Histatin 3 and PRP1/Pif-s in PS revealed t(1/2) times of 7.2+/-5.5 and 50.3+/-24.8h, respectively (n=7). Histatin 3 (40 microg/ml) and PRP1 (400 microg/ml), added to WS in concentrations equivalent to their concentrations in PS, disappeared at a much faster rate, with t(1/2) values of 1.7+/-1.6 min and 29.3+/-15.3 min, respectively (n=7). The data indicate that proteolysis in WS is an important factor in explaining the substantially lower concentrations of Histatins and acidic PRPs in WS as compared to in glandular secretions.

  • Kinetics of Histatin proteolysis in whole saliva and the effect on bioactive domains with metal-binding, antifungal, and wound-healing properties
    FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 2009
    Co-Authors: Xiuli Sun, Frank G. Oppenheim, Erdjan Salih, Eva J. Helmerhorst
    Abstract:

    The present study was undertaken to investigate the rate and mode of degradation of individual Histatin proteins in whole saliva to establish the impact on its functional domains. Pure synthetic Histatins 1, 3, and 5 were incubated with whole saliva supernatant as the enzyme source, and peptides in the resultant digests were separated by reverse-phase-HPLC and structurally characterized by electrospray ionization-tandem mass spectrometry. The overall Vmax/Km ratios, a measure of proteolytic efficiency, were on the order of Histatin-5 > Histatin-3 > Histatin-1. Mathematical models predict that Histatins 1, 3, and 5 levels in whole saliva stabilize at 5.1, 1.9, and 1.2 μM, representing 59, 27, and 11% of glandular Histatins 1, 3, and 5 levels, respectively. Monitoring of the appearance and disappearance of Histatin fragments yielded the identification of the first targeted enzymatic cleavage sites as K13 and K17 in Histatin 1, R22, Y24, and R25 in Histatin 3, and Y10, K11, R12, K13, H15, E16, K17, and H18 in Histatin 5. The data indicate that metal-binding, antifungal, and wound-healing domains are largely unaffected by the primary cleavage events in whole saliva, suggesting a sustained functional activity of these proteins in the proteolytic environment of the oral cavity.—Sun, X., Salih, E., Oppenheim, F. G., Helmerhorst, E. J. Kinetics of Histatin proteolysis in whole saliva and the effect on bioactive domains with metal-binding, antifungal, and wound-healing properties.

  • Concentration and fate of Histatins and acidic proline-rich proteins in the oral environment.
    Archives of oral biology, 2009
    Co-Authors: Melanie Campese, Frank G. Oppenheim, Xiuli Sun, Jos A Bosch, Eva J. Helmerhorst
    Abstract:

    Saliva plays a critical role in the protection of oral hard and soft tissues and contains a multitude of constituents with well-characterized biological activities in vitro. Among these are Histatins and acidic proline-rich proteins (PRPs). Nevertheless, few functional studies have recognized the structural instability of these proteins in the proteolytic environment of whole saliva. The aim of this investigation was to determine Histatin and acidic PRP levels in parotid secretion (PS) and in whole saliva (WS) as well as to establish their susceptibility to proteolysis in these salivary fluids. Using cationic polyacrylamide gel electrophoresis and densitometric analysis the average total Histatin concentration (Histatin 1+3+5) in WS was determined to be 33.3+/-16.7 microg/ml (n=22) and the average total acidic PRP concentration (PRP1/PIF-s+PRP3/PIF-f) was 427.9+/-123.3 microg/ml (n=22). Histatin and acidic PRP concentrations in PS were 6 and 1.5 times higher than in WS (n=7), respectively. WS Histatin and acidic PRP levels each correlated significantly with WS total protein concentrations (P

Enno C. I. Veerman - One of the best experts on this subject based on the ideXlab platform.

  • Salivary peptide Histatin 1 mediated cell adhesion: a possible role in mesenchymal-epithelial transition and in pathologies.
    Biological chemistry, 2018
    Co-Authors: Irene A. Van Dijk, Enno C. I. Veerman, Eric Reits, Jan G. M. Bolscher, Jan Stap
    Abstract:

    Histatins are histidine-rich peptides present in the saliva of humans and higher primates and have been implicated in the protection of the oral cavity. Histatin 1 is one of the most abundant Histatins and recent reports show that it has a stimulating effect on cellular adherence, thereby suggesting a role in maintaining the quality of the epithelial barrier and stimulating mesenchymal-to-epithelial transition. Here we summarize these findings and discuss them in the context of previous reports. The recent findings also provide new insights in the physiological functions of Histatin 1, which are discussed here. Furthermore, we put forward a possible role of Histatin 1 in various pathologies and its potential function in clinical applications.

  • human salivary peptide Histatin 1 stimulates epithelial and endothelial cell adhesion and barrier function
    The FASEB Journal, 2017
    Co-Authors: Irene A. Van Dijk, Enno C. I. Veerman, Jan G. M. Bolscher, Kamran Nazmi, Maria Laura Ferrando, Anneeva Van Der Wijk, Ron A Hoebe, Wouter J De Jonge, Przemek M Krawczyk, Jan Stap
    Abstract:

    Histatins are multifunctional histidine-rich peptides secreted by the salivary glands and exclusively present in the saliva of higher primates, where they play a fundamental role in the protection of the oral cavity. Our previously published results demonstrated that Histatin-1 (Hst1) promotes cell–substrate adhesion in various cell types and hinted that it could also be involved in cell–cell adhesion, a process of fundamental importance to epithelial and endothelial barriers. Here we explore the effects of Hst1 on cellular barrier function. We show that Hst1 improved endothelial barrier integrity, decreased its permeability for large molecules, and prevented translocation of bacteria across epithelial cell layers. These effects are mediated by the adherens junction protein E-cadherin (E-cad) and by the tight junction protein zonula occludens 1, as Hst1 increases the levels of zonula occludens 1 and of active E-cad. Hst1 may also promote epithelial differentiation as Hst1 induced transcription of the epit...

  • The influence of chronic wound extracts on inflammatory cytokine and Histatin stability
    PloS one, 2016
    Co-Authors: Mireille A. Boink, Enno C. I. Veerman, Jan G. M. Bolscher, Kamran Nazmi, Sanne Roffel, Catherine Van Montfrans, Amit Gefen, Susan Gibbs
    Abstract:

    Chronic ulcers represent a major health burden in our society. Despite many available therapies, a large number of ulcers do not heal. Protein based therapies fail in part due to proteolytic activity in the chronic wound bed. The aim of this in vitro study was to determine whether typical inflammatory cytokines and human salivary Histatins remain stable when incubated with chronic wound extracts. Furthermore we determined whether a short exposure of Histatins or cytokines was sufficient to exert long term effects on fibroblast migration. Stability of human recombinant cytokines IL-6 and CXCL8, and Histatin variants (Hst1, Hst2, cyclic Hst1, minimal active domain of Hst1) in the presence of chronic wound extracts isolated from non-healing ulcers, was monitored by capillary zone electrophoresis. Migration-stimulating activity was assessed using a dermal fibroblast wound healing scratch assay. Histatins and cytokines stayed stable in saline for > 24 h at 37°C, making them ideal as an off-the-shelf product. However, incubation with chronic wound extracts resulted in serious breakdown of Hst1 and Hst2 (~50% in 8 h) and to lesser extent cyclic Hst1 and the minimal active domain of Hst1 (~20% in 8 h). The cytokines IL-6 and CXCL8 were more stable in chronic wound extracts (~40% degradation in 96 h). An initial 8-hour pulse of Histatins or cytokines during a 96-hour study period was sufficient to stimulate fibroblast migration equally well as a continuous 96-hour exposure, indicating that they may possibly be used as novel bioactive therapeutics, exerting their activity for up to four days after a single exposure.

  • Histatin 1 a histidine rich peptide in human saliva promotes cell substrate and cell cell adhesion
    The FASEB Journal, 2015
    Co-Authors: Irene A. Van Dijk, Enno C. I. Veerman, Jan G. M. Bolscher, Kamran Nazmi, Jan Stap
    Abstract:

    Histatins (Hsts) are histidine-rich peptides exclusively present in the saliva of higher primates. In this study, we explored the effects of Hsts on cell-substrate and cell-cell adhesion. Histatin (Hst)-1 caused a significant (>2-fold) increase (EC50 = 1 µM) in the ability of human adherent cells to attach and spread, even in conditions that impaired cell spreading. Other tested Hsts did not stimulate cell spreading, indicating a specific effect of Hst1. The effect of Hst1 on cell-cell adhesion was investigated by using transepithelial resistance (TER) measurements in the human cell line Caco-2, a widely used model for the epithelial layer. We found that 10 µM Hst1 caused a 20% increase in TER compared to the negative control, indicating a function for Hst1 in intercellular cell adhesion and epithelial integrity. A role for Hst1 in both cell-substrate and cell-cell adhesion is highly conceivable, because these 2 modes of adhesion are closely related via shared components and connected signaling pathways.

  • The role of salivary Histatin and the human cathelicidin LL-37 in wound healing and innate immunity.
    Biological chemistry, 2010
    Co-Authors: Menno J. Oudhoff, Jan G. M. Bolscher, Marjolein E. Blaauboer, Kamran Nazmi, Nina Scheres, Enno C. I. Veerman
    Abstract:

    Antimicrobial peptides are multifunctional in innate immunity and wound repair of multicellular organisms. We were the first to discover that Histatins, a family of salivary antimicrobial peptides, enhance epithelial cell migration, suggesting a role in oral wound healing. It is unknown whether Histatins display innate-immunity activities, similar to other antimicrobial peptides such as LL-37. Therefore, we compared the effect of Histatin-2 and LL-37 on several activities within the context of wound healing and innate immunity. We found that Histatin-2 enhances fibroblast migration, but only weakly induces proliferation. LL-37 enhances both fibroblast migration and proliferation, but only at a narrow concentration optimum (approximately 1 μm). At higher concentrations LL-37 causes cell death, whereas Histatin-2 is not cytotoxic. Both peptides do not alter fibroblast-to-myofibroblast differentiation. Histatin-2 does not alter interleukin-8 (IL-8) expression and lipopolysaccharide (LPS)-elevated cytokine and chemokine expression. In contrast, LL-37 induces IL-8 expression, but dampens the LPS-induced immune response. Neither Histatin-2 nor LL-37 affects human-neutrophil migration. Histatins are, unlike other antimicrobial peptides, not cytotoxic or proinflammatory. It seems that they are important for the initial stage of wound healing in which fast wound coverage is important for healing without infection, inflammation, or fibrosis development. Interestingly, these characteristics are more typical for the mouth than for skin. © 2010 by Walter de Gruyter Berlin New York.

Eva J. Helmerhorst - One of the best experts on this subject based on the ideXlab platform.

  • Direct assessment of the antioxidant property of salivary Histatin.
    Journal of clinical biochemistry and nutrition, 2019
    Co-Authors: Tomoko Komatsu, Frank G. Oppenheim, Eva J. Helmerhorst, Kyo Kobayashi, Masaichi-chang-il Lee
    Abstract:

    Histatin, a salivary protein, affects oral homeostasis through preservation of tooth integrity and protection against caries and fungal infections. However, the effects of Histatin in the generation of oxidative stress induced by reactive oxygen species and in the oral cavity remain unclear. In this study, the effects of Histatin on direct reactive oxygen species scavenging activity were examined using electron spin resonance. We demonstrated, for the first time, that Histatin exhibits antioxidant activity against hydroxyl radicals generated by Fenton's reaction by metal chelation or binding. The direct antioxidant effects of Histatin, along with its antimicrobial activity, may be important in the oral protection of salivary proteins.

  • anti candidal activity of genetically engineered Histatin variants with multiple functional domains
    PLOS ONE, 2012
    Co-Authors: Frank G. Oppenheim, Eva J. Helmerhorst, Gwynneth D Offner, Urs Lendenmann
    Abstract:

    The human bodily defense system includes a wide variety of innate antimicrobial proteins. Histatins are small molecular weight proteins produced by the human salivary glands that exhibit antifungal and antibacterial activities. While evolutionarily old salivary proteins such as mucins and proline-rich proteins contain large regions of tandem repeats, relatively young proteins like Histatins do not contain such repeated domains. Anticipating that domain duplications have a functional advantage, we genetically engineered variants of Histatin 3 with one, two, three, or four copies of the functional domain by PCR and splice overlap. The resulting proteins, designated reHst3 1-mer, reHist3 2-mer, reHis3 3-mer and reHist3 4-mer, exhibited molecular weights of 4,062, 5,919, 7,777, and 9,634 Da, respectively. The biological activities of these constructs were evaluated in fungicidal assays toward Candida albicans blastoconidia and germinated cells. The antifungal activities per mole of protein increased concomitantly with the number of functional domains present. This increase, however, was higher than could be anticipated from the molar concentration of functional domains present in the constructs. The demonstrated increase in antifungal activity may provide an evolutionary explanation why such domain multiplication is a frequent event in human salivary proteins.

  • concentration and fate of Histatins and acidic proline rich proteins in the oral environment
    Archives of Oral Biology, 2009
    Co-Authors: Melanie Campese, Frank G. Oppenheim, Xiuli Sun, Jos A Bosch, Eva J. Helmerhorst
    Abstract:

    Saliva plays a critical role in the protection of oral hard and soft tissues and contains a multitude of constituents with well-characterized biological activities in vitro. Among these are Histatins and acidic proline-rich proteins (PRPs). Nevertheless, few functional studies have recognized the structural instability of these proteins in the proteolytic environment of whole saliva. The aim of this investigation was to determine Histatin and acidic PRP levels in parotid secretion (PS) and in whole saliva (WS) as well as to establish their susceptibility to proteolysis in these salivary fluids. Using cationic polyacrylamide gel electrophoresis and densitometric analysis the average total Histatin concentration (Histatin 1+3+5) in WS was determined to be 33.3+/-16.7 microg/ml (n=22) and the average total acidic PRP concentration (PRP1/PIF-s+PRP3/PIF-f) was 427.9+/-123.3 microg/ml (n=22). Histatin and acidic PRP concentrations in PS were 6 and 1.5 times higher than in WS (n=7), respectively. WS Histatin and acidic PRP levels each correlated significantly with WS total protein concentrations (P<0.01 and P<0.05, respectively), as well as with each other (P<0.01). Stability studies of Histatin 3 and PRP1/Pif-s in PS revealed t(1/2) times of 7.2+/-5.5 and 50.3+/-24.8h, respectively (n=7). Histatin 3 (40 microg/ml) and PRP1 (400 microg/ml), added to WS in concentrations equivalent to their concentrations in PS, disappeared at a much faster rate, with t(1/2) values of 1.7+/-1.6 min and 29.3+/-15.3 min, respectively (n=7). The data indicate that proteolysis in WS is an important factor in explaining the substantially lower concentrations of Histatins and acidic PRPs in WS as compared to in glandular secretions.

  • Kinetics of Histatin proteolysis in whole saliva and the effect on bioactive domains with metal-binding, antifungal, and wound-healing properties
    FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 2009
    Co-Authors: Xiuli Sun, Frank G. Oppenheim, Erdjan Salih, Eva J. Helmerhorst
    Abstract:

    The present study was undertaken to investigate the rate and mode of degradation of individual Histatin proteins in whole saliva to establish the impact on its functional domains. Pure synthetic Histatins 1, 3, and 5 were incubated with whole saliva supernatant as the enzyme source, and peptides in the resultant digests were separated by reverse-phase-HPLC and structurally characterized by electrospray ionization-tandem mass spectrometry. The overall Vmax/Km ratios, a measure of proteolytic efficiency, were on the order of Histatin-5 > Histatin-3 > Histatin-1. Mathematical models predict that Histatins 1, 3, and 5 levels in whole saliva stabilize at 5.1, 1.9, and 1.2 μM, representing 59, 27, and 11% of glandular Histatins 1, 3, and 5 levels, respectively. Monitoring of the appearance and disappearance of Histatin fragments yielded the identification of the first targeted enzymatic cleavage sites as K13 and K17 in Histatin 1, R22, Y24, and R25 in Histatin 3, and Y10, K11, R12, K13, H15, E16, K17, and H18 in Histatin 5. The data indicate that metal-binding, antifungal, and wound-healing domains are largely unaffected by the primary cleavage events in whole saliva, suggesting a sustained functional activity of these proteins in the proteolytic environment of the oral cavity.—Sun, X., Salih, E., Oppenheim, F. G., Helmerhorst, E. J. Kinetics of Histatin proteolysis in whole saliva and the effect on bioactive domains with metal-binding, antifungal, and wound-healing properties.

  • Concentration and fate of Histatins and acidic proline-rich proteins in the oral environment.
    Archives of oral biology, 2009
    Co-Authors: Melanie Campese, Frank G. Oppenheim, Xiuli Sun, Jos A Bosch, Eva J. Helmerhorst
    Abstract:

    Saliva plays a critical role in the protection of oral hard and soft tissues and contains a multitude of constituents with well-characterized biological activities in vitro. Among these are Histatins and acidic proline-rich proteins (PRPs). Nevertheless, few functional studies have recognized the structural instability of these proteins in the proteolytic environment of whole saliva. The aim of this investigation was to determine Histatin and acidic PRP levels in parotid secretion (PS) and in whole saliva (WS) as well as to establish their susceptibility to proteolysis in these salivary fluids. Using cationic polyacrylamide gel electrophoresis and densitometric analysis the average total Histatin concentration (Histatin 1+3+5) in WS was determined to be 33.3+/-16.7 microg/ml (n=22) and the average total acidic PRP concentration (PRP1/PIF-s+PRP3/PIF-f) was 427.9+/-123.3 microg/ml (n=22). Histatin and acidic PRP concentrations in PS were 6 and 1.5 times higher than in WS (n=7), respectively. WS Histatin and acidic PRP levels each correlated significantly with WS total protein concentrations (P

Robert F. Troxler - One of the best experts on this subject based on the ideXlab platform.

  • Human salivary gland-specific daily variations in Histatin concentrations determined by a novel quantitation technique.
    Archives of oral biology, 2004
    Co-Authors: Heloisa Gusman, Robert F. Troxler, Eva J. Helmerhorst, Cataldo W. Leone, Martha E. Nunn, Bianca Flora, Frank G. Oppenheim
    Abstract:

    Histatins constitute a distinct family of human salivary antimicrobial peptides, of which Histatins 1, 3 and 5 are the most abundant. To evaluate salivary gland-specific differences in Histatin secretion, we used the recently developed Histatin-zinc precipitation method to quantify Histatins and to assess daily variations in secretions. Stimulated pure secretions from parotid glands (HPS) and submandibular/sublingual glands (SMSL) were collected from 10 different subjects at four different times of the day (9:35 a.m.; 12:40 p.m.; 2:50 p.m. and 5:00 p.m.). Zinc precipitation and subsequent reversed phase HPLC analysis were performed to determine concentrations of Histatins 1, 3 and 5 with reference to purified Histatin standards. Both HPS and SMSL secretions displayed daily variations in Histatin concentrations. HPS values showed a maximum at mid-day and SMSL samples showed a maximum in the morning. Mean daily Histatin concentrations were almost three fold higher in SMSL than in HPS. Mean Histatin 1, 3 and 5 concentrations in HPS from 10 subjects ranged from 0.7 to 2.8, 0.6 to 4.3 and 1.0 to 4.3mg%, respectively. The corresponding means in SMSL were 2.8-12.2, 1.5-7.5 and 2.6-9.0mg%, respectively. Remarkably, although Histatins constitute only 3-10% of total protein in these secretions, an almost perfect correlation between total protein and total Histatin concentrations was observed for both glands. Despite a broad range in Histatin concentrations between individuals, this study demonstrated a hitherto unidentified daily variation in Histatin concentrations in HPS and SMSL secretions and a differential expression pattern which might have functional implications.

  • Physical parameters of hydroxyapatite adsorption and effect on candidacidal activity of Histatins.
    Archives of oral biology, 2003
    Co-Authors: A. Yin, James Grogan, Robert F. Troxler, H.c. Margolis, Y. Yao, Frank G. Oppenheim
    Abstract:

    Histatins 1, 3 and 5 are the major members of a histidine-rich protein family present in human salivary secretions. These proteins are distinct from many salivary proteins in their high positive charge density at neutral pH, and their antibacterial and antifungal properties. In this study, the hydroxyapatite adsorption characteristics of Histatin 1, containing a single phosphoserine residue, recombinantly expressed Histatin 1, native Histatin 3, synthetic Histatin 5 and an internal 12-residue sequence of Histatin 5 were investigated. A Langmuir-type model was used to analyse the adsorption. A comparison of the affinities and binding sites of phosphorylated and recombinant Histatin 1 provided an estimate of the positive influence of the single phosphoseryl group on mineral adsorption. Furthermore, an apparent correlation was shown to exist between peptide chain length and the number of binding sites. The influence of Histatin 5 adsorption on its anticandidal activity was also investigated by performing Candida albicans killing assays with Histatin 5 and Histatin 5/hydroxyapatite suspensions. A decrease in killing activity was observed with the increase of hydroxyapatite present. The results suggest that the anticandidal properties of Histatin 5 could be impaired by the conformations resulting from mineral adsorption, or that putative cellular receptors necessary for candidacidal activity are inaccessible when Histatin 5 is adsorbed on hydroxyapatite.

  • Inhibitory effect of synthetic Histatin 5 on leukotoxin from Actinobacillus actinomycetemcomitans.
    Oral microbiology and immunology, 2002
    Co-Authors: Y. Murakami, Edward T. Lally, Robert F. Troxler, Eva J. Helmerhorst, G. Ori, Frank G. Oppenheim
    Abstract:

    Actinobacillus actinomycetemcomitans is a gram-negative bacterium strongly implicated in the pathogenesis of juvenile periodontitis. This periodontal pathogen synthesizes a leukotoxin that destroys human polymorphonuclear leukocytes (PMNs), and this toxin is thought to be responsible for the virulence of A. actinomycetemcomitans. It was therefore of interest to assess whether major virulence factors of periodontal pathogens were neutralized by salivary components. This study focuses on the effect of Histatins, components of the nonimmune oral defense system, on leukotoxin activity. Leukotoxin was extracted with polymyxin B from freshly grown anaerobic cultures of A. actinomycetemcomitans strain Y4. PMNs isolated from blood of healthy human volunteers were incubated in a cytotoxicity assay containing PMNs (10(7) cells/ml) and leukotoxin preparation (0-500 microg/ml) in Hanks' balanced salt solution at 37 degrees C for 0-120 min with or without synthetic Histatin 5 (0-500 microM). Cytotoxicity was measured by release of lactate dehydrogenase (LDH) at different time intervals. Histatin 5 neutralized the toxic effect of the leukotoxin preparation in a concentration-dependent manner, with an IC(50) value of 150 microM. When PMNs were preincubated with Histatin 5 (300 microM), washed and subsequently exposed to leukotoxin, no protective effect was observed. This observation suggests a mechanism of inhibition whereby Histatin 5 either directly neutralizes the leukotoxin or interferes with the leukotoxin-PMN interaction. The inhibitory effect of Histatin 5 on leukotoxic activity may suggest a new biological function of Histatins in the oral cavity as a naturally occurring secondary antibiotic.

  • The human salivary peptide Histatin 5 exerts its antifungal activity through the formation of reactive oxygen species
    Proceedings of the National Academy of Sciences of the United States of America, 2001
    Co-Authors: Eva J. Helmerhorst, Robert F. Troxler, Frank G. Oppenheim
    Abstract:

    Previous studies have shown that the human salivary antifungal peptide Histatin 5 is taken up by Candida albicans cells and associates intracellularly with mitochondria. The purpose of the present study was to investigate the biological consequence of this specific subcellular targeting. Histatin 5 inhibited respiration of isolated C. albicans mitochondria as well as the respiration of intact blastoconidia in a dose and time-dependent manner. A nearly perfect correlation was observed between Histatin-induced inhibition of respiration and cell killing with either logarithmic- or stationary-phase cells, but stationary-phase cells were less sensitive. Because nonrespiring yeast cells are insensitive to Histatin 5, the potential mechanistic relationship between Histatin 5 interference with the respiratory apparatus and cell killing was explored by using an oxygen radical sensitive probe (dihydroethidium). Fluorimetric measurements showed that Histatin 5 induced the formation of reactive oxygen species (ROS) in C. albicans cells as well as in isolated mitochondria and that ROS levels were highly correlated with cell death. In the presence of an oxygen scavenger (l-cysteine), cell killing and ROS formation were prevented. In addition, the membrane-permeant superoxide dismutase mimetic 2,2,6,6-tetramethylpiperidine-N-oxyl, abolished Histatin-induced ROS formation in isolated mitochondria. In contrast to Histatin 5, the conventional inhibitors of the respiratory chain, sodium cyanide or sodium azide, neither induced ROS nor killed yeast cells. These data provide strong evidence for a comprehensive mechanistic model of Histatin-5-provoked yeast cell death in which oxygen radical formation is the ultimate and essential step.

  • Killing of Candida albicans by Histatin 5: cellular uptake and energy requirement.
    Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology, 2001
    Co-Authors: Csilla Gyurko, Robert F. Troxler, Eva J. Helmerhorst, Urs Lendenmann, Frank G. Oppenheim
    Abstract:

    Histatins, a group of histidine-rich proteins in human saliva, exhibit antimicrobial activity and are therefore considered to be important in the prevention of infections in the oral cavity. Although killing of C. albicans by Histatins has been extensively studied, little is known about the processes responsible for this antifungal activity. Recent studies show the requirement of metabolic activity and ATP production for Histatin 5 killing activity. Therefore, the goal of this study was to investigate the kinetics of Histatin 5 interaction at different temperatures with C. albicanswild type cells and with respiratory deficient mutants of C. albicans. Synthetic Histatin 5 was labeled with fluorescein-5-isothiocyanate (FITC) and its association with C. albicans cells was followed by epi-fluorescence microscopy and fluorescence confocal microscopy. At 37 °C, Histatin 5 accumulates intracellularly, and both killing activity and uptake of unlabeled and FITC-labeled Histatin 5 are time- and concentration-dependent. At 4 °C, no killing is observed and FITC-Histatin 5 is only associated with the cytoplasmic membrane. Internalization and killing activity only occurs after cells are transferred to 37 °C. In addition, cellular accumulation of Histatin 5 is concomitant with a moderate alteration of membrane integrity leading to the release of UV-absorbing cell components into the medium. The uptake of Histatin 5, the release of UV-absorbing materials and killing of C. albicans are markedly decreased by the respiratory inhibitor sodium azide. Concomitantly, respiratory deficient mutants of C. albicans are also less susceptible to Histatin 5. These results indicated that Histatin 5 killing activity could be directly correlated to Histatin 5 internalization. Both of these processes are prevented by modulators of cellular metabolic activity.

Brian O'connell - One of the best experts on this subject based on the ideXlab platform.

  • Saliva affects the antifungal activity of exogenously added Histatin 3 towards Candida albicans
    FEMS microbiology letters, 2005
    Co-Authors: Hisako Yamagishi, Thomas J. Walsh, Tin Sein, Deirdre H. Fitzgerald, Brian O'connell
    Abstract:

    Antifungal activity of Histatin 3 against two Candida albicans clinical isolates was determined in assays containing rabbit submandibular gland saliva. Histatin 3 inhibited the cell growth and germination of both isolates dose-dependently (10–100 μg ml−1) with maximum inhibition occurring after 60 min incubation. Adding fresh Histatin 3 after 60 min caused further reduction in the viable cell count. Higher Histatin 3 concentrations (50–100 μg ml−1) and prolonged exposure to peptide were required to inhibit germination. Histatin 3 was rapidly degraded in rabbit submandibular gland saliva and this may explain why fresh addition of Histatin 3 increases candidacidal activity.

  • Binding, internalisation and degradation of Histatin 3 in Histatin-resistant derivatives of Candida albicans.
    FEMS microbiology letters, 2003
    Co-Authors: Deirdre H. Fitzgerald, David C. Coleman, Brian O'connell
    Abstract:

    The antifungal mechanism of salivary Histatin has been studied in Candida albicans and involves binding to a specific receptor, translocation across the membrane and targeting intracellularly. Cell death correlates with non-lytic release of ATP that may function as a cytotoxic mediator extracellularly. By sequential exposure to increasing concentrations of Histatin 3, we generated Histatin-resistant derivatives of C. albicans strain CA132A that show five-fold less killing at physiological concentrations of Histatin 3. Protection against Histatin killing in Histatin-resistant derivatives is not due to alterations in binding, internalisation or degradation of Histatin or efflux of ATP. These results indicate that protective mechanisms activated by exposure to Histatin 3 may involve unidentified pathways downstream of binding and internalisation events.

  • Susceptibility of Candida dubliniensis to Salivary Histatin 3
    Antimicrobial agents and chemotherapy, 2003
    Co-Authors: Deirdre H. Fitzgerald, David C. Coleman, Brian O'connell
    Abstract:

    Candida dubliniensis is a recently described Candida species associated with oral candidiasis in human immunodeficiency virus (HIV)-infected patients and patients with AIDS. The majority of C. dubliniensis clinical isolates tested to date are susceptible to the commonly used antifungal drugs, including fluconazole, ketoconazole, itraconazole, and amphotericin B. However, the appearance of fluconazole-resistant C. dubliniensis strains in this patient group is increasing. Histatins are a family of basic histidine-rich proteins present in human saliva which have therapeutic potential in the treatment of oral candidiasis. The mechanism of action of Histatin is distinct from that of commonly used azole and polyene drugs. Characterization of the antifungal activity of Histatin has mainly been carried out using C. albicans but it is also effective in killing C. glabrata and C. krusei. Here we report that C. dubliniensis is also susceptible to killing by Histatin 3. The concentration of Histatin 3 giving 50% killing (the IC(50) value) ranged from 0.043 to 0.196 mg/ml among different strains of C. dubliniensis. The least-susceptible C. dubliniensis strain, P9224, was found to internalize Histatin at a lower rate than the C. albicans reference strain CA132A. The dissociation constant (K(d)) for the least-susceptible strain (C. dubliniensis 9224) was ninefold higher than that for the C. albicans reference strain. These results suggest that Histatin 3 may have potential as an effective antifungal agent, particularly in the treatment of oral candidiasis in HIV-infected patients and patients with AIDS in which resistance to the commonly used antifungal drug fluconazole has emerged.

  • Histatin 3-Mediated Killing of Candida albicans: Effect of Extracellular Salt Concentration on Binding and Internalization
    Antimicrobial agents and chemotherapy, 1999
    Co-Authors: Indu S. Ambudkar, Hisako Yamagishi, William D. Swaim, Thomas J. Walsh, Brian O'connell
    Abstract:

    Human saliva contains histidine-rich proteins, Histatins, which have antifungal activity in vitro. The mechanism by which Histatins are able to kill Candida albicans may have clinical significance but is currently unknown. Using radiolabeled Histatin 3, we show that the protein binds to C. albicans spheroplasts in a manner that is dependent on time and concentration. Binding to the spheroplasts was saturable and could be competed with unlabeled Histatin 3. A single Histatin 3 binding site with a Kd = 5.1 μM was detected. Histatin 3 binding resulted in potassium and magnesium efflux, predominantly within the first 30 min of incubation. Studies with fluorescent Histatin 3 demonstrate that the protein is internalized by C. albicans and that translocation of Histatin inside the cell is closely associated with cell death. Histatin binding, internalization, and cell death are accelerated in low-ionic-strength conditions. Indeed, a low extracellular salt concentration was essential for cell death to occur, even when Histatin 3 was already bound to the cell. The interaction of Histatin 3 with C. albicans, and subsequent cell death, is inhibited at low temperature. These results demonstrate that the candidacidal activity of Histatin 3 is not due exclusively to binding at the cell surface but also involves subsequent interactions with the cell.

  • Transfer of a gene encoding the anticandidal protein Histatin 3 to salivary glands.
    Human gene therapy, 1996
    Co-Authors: Brian O'connell, Frank G. Oppenheim, Thomas J. Walsh, Tin Sein, Andrea Mastrangeli, Ronald G. Crystal, Bruce J. Baum
    Abstract:

    Mucosal candidiasis, the most common opportunistic fungal infection in human immunodeficiency virus (HIV)-infected patients, is an early sign of clinically overt acquired immunodeficiency syndrome (AIDS) and an important cause of morbidity, particularly in HIV-infected children. The appearance of azole-resistant strains of Candida albicans had made clinical management of candidiasis increasingly difficult. We propose a novel approach to the management of candidal infections that involves the use of naturally occurring antifungal proteins, such as the Histatins. Histatins are a family of small proteins that are secreted in human saliva. We have constructed recombinant adenovirus vectors that contain the Histatin 3 cDNA. These vectors are capable of directing the expression of Histatin 3 in the saliva of rats at up to 1,045 micrograms/ml, well above the levels found in normal human saliva. The adenovirus-directed Histatin demonstrated a 90% candidacidal effect in the timed-kill assay against both fluconazole-susceptible and fluconazole-resistant strains of C. albicans and inhibited germination by 45% in the same strains. These studies suggest that a gene transfer approach to overexpress naturally occurring antifungal proteins may be useful in the management of mucosal candidiasis.