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Biomedical subjects

E C Veerman

Publications and source records attributed to E C Veerman.

At least 19 recordsLinked to original sources
PubMed2006

Design of a peptibody consisting of the antimicrobial peptide dhvar5 and a llama variable heavy-chain antibody fragment.

Immunoconjugates have been widely studied as potential therapeutics for infectious diseases to direct unspecific antimicrobials to pathogens. In this study, the recombinant approach was used for expression of the immunoconjugate composed of the variable domain of a llama heavy-chain antibody (VHH) against Streptococcus mutans and dhvar5, a synthetic antimicrobial peptide. Before cloning, the impact of the elongation of the peptide termini on its biological activity was evaluated by chemical synthesis of the N- or C-termini extended dhvar5 peptides. As the elongation of the C-terminus had a greater influence on decline of the antimicrobial activity, the N-terminal fusion was designed. To promote in vivo release of the active peptide, a factor Xa cleavage site was inserted between VHH and dhvar5. Propagation of transformed Escherichia coli with the constructed plasmid was only possible in the absence of isopropyl beta-D-thiogalactoside (IPTG). Although these data demonstrate that the diminished antimicrobial activity of dhvar5 by the N-terminal fusion to VHH was not sufficient for the protection of the bacterial host cells against the peptide lethal effect, an insight into propeptides biological activities may be beneficial not only for new and more successful rearrangement of the VHH-dhvar5 immunoconjugate construct, but also design of the other recombinant molecules composed of peptides toxic to host cells.

Amino Acid Sequence↗
PubMed2004

[Influence of teas with fruit aroma and ice teas on pH and buffer capacity of saliva].

A number of teas with fruit aroma and ice teas has been tested on erosivity. The teas with a fruit taste have at their drinking temperature (45 degrees C) a pH between 6.2 to 7.4. Even teas with a citron or citrus flavour are neutral. The most acidic tea tested has a mango-peach-taste, but contains no buffer system. After mixing 1 ml of total saliva up to 10 ml tea or by rinsing the oral cavity with tea the pH of oral fluid was hardly lowered (minimum 6.3). In contrast, ice teas are rather acidic (pH < 4) and have a strong buffer capacity. Rinsing the oral cavity with 5 ml ice tea resulted in a decrease of the pH of the oral fluid to 4.1-4.9. However, within 2 minutes the pH in the oral fluid was already increased to 5.9-7.1 and after 5 minutes to 6.3-7.1. In other words, the teas with fruit aroma, based on organic taste components do not have an erosive potential. On the other hand, ice teas decrease the pH of the oral fluid drastically to a level that they become erosive. Therefore, from a dental point of view, teas with only a taste of fruit can be advised to replace drinking of soft drinks, but ice teas not.

Beverages↗
PubMed2001

Human salivary agglutinin binds to lung surfactant protein-D and is identical with scavenger receptor protein gp-340.

Salivary agglutinin is a 300-400 kDa salivary glycoprotein that binds to antigen B polypeptides of oral streptococci, thereby playing a role in their colonization and the development of caries. A mass spectrum was recorded of a trypsin digest of agglutinin. A dominant peak of 1460 Da was sequenced by quadrupole time-of-flight (Q-TOF) tandem MS. The sequence showed 100% identity with part of the scavenger receptor cysteine-rich ('SRCR') domain found in gp-340/DMBT1 (deleted in malignant brain tumours-1). The mass spectrum revealed 11 peaks with an identical mass as a computer-simulated trypsin digest of gp-340. gp-340 is a 340 kDa glycoprotein isolated from bronchoalveolar lavage fluid that binds specifically to lung surfactant protein-D. DMBT1 is a candidate tumour suppressor gene. A search in the human genome revealed only one copy of this gene. The molecular mass, as judged from SDS/PAGE and the amino acid composition of agglutinin, was found to be nearly identical with that of gp-340. It was shown by Western blotting that monoclonal antibodies against gp-340 reacted with salivary agglutinin, and monoclonals against agglutinin reacted with gp-340. It was demonstrated that gp-340 and agglutinin bound in a similar way to Streptococcus mutans and surfactant protein-D. Histochemically, the distribution of gp-340 in the submandibular salivary glands was identical with the agglutinin distribution, as shown in a previous paper [Takano, Bogert, Malamud, Lally and Hand (1991) Anat. Rec. 230, 307-318]. We conclude that agglutinin is identical with gp-340, and that this molecule interacts with S. mutans and surfactant protein-D.

Agglutinins↗
PubMed2001

Effects of histatin 5 and derived peptides on Candida albicans.

Three anti-microbial peptides were compared with respect to their killing activity against Candida albicans and their ability to disturb its cellular and internal membranes. Histatin 5 is an anti-fungal peptide occurring naturally in human saliva, while dhvar4 and dhvar5 are variants of its active domain, with increased anti-microbial activity. dhvar4 has increased amphipathicity compared with histatin 5, whereas dhvar5 has amphipathicity comparable with that of histatin 5. All three peptides caused depolarization of the cytoplasmic and/or mitochondrial membrane, indicating membranolytic activity. For the variant peptides both depolarization and killing occurred at a faster rate. With FITC-labelled peptides, no association with the cytoplasmic membrane was observed, contradicting the formation of permanent transmembrane multimeric peptide pores. Instead, the peptides were internalized and act on internal membranes, as demonstrated with mitochondrion- and vacuole-specific markers. In comparison with histatin 5, the variant peptides showed a more destructive effect on mitochondria. Entry of the peptides and subsequent killing were dependent on the metabolic state of the cells. Blocking of the mitochondrial activity led to complete protection against histatin 5 activity, whereas that of dhvar4 was hardly affected and that of dhvar5 was affected only intermediately.

Amino Acid Sequence↗
PubMed2001

Salivary cystatin activity and cystatin C in natural and experimental gingivitis in smokers and non-smokers.

BACKGROUND: Recent studies show that subjects with natural gingivitis or periodontitis have elevated levels of salivary cystatins compared to periodontally healthy individuals. Increased glandular output of cystatins in inflammatory conditions suggests an active, most likely protective, rôle for these proteins in inflammatory processes. Furthermore, it has been shown that the development of gingival inflammation is suppressed in smokers during experimental gingivitis. AIMS: The purpose of the present study was to investigate whether (i) the levels of salivary cystatins in natural gingivitis are related to smoking status, and (ii) to study whether experimentally induced gingivitis is associated with changes in salivary cystatin levels, in both smokers and non-smokers. MATERIAL AND METHODS: Whole saliva samples were taken in relation to natural gingivitis, gingival health and 14-day experimental gingivitis in 25 non-dental students (14 non-smokers and 11 smokers). The salivary flowrate was determined. Samples were analyzed for levels of protein, cystatin and cystatin-C. RESULTS: Salivary flow and protein concentrations in cleared human whole saliva samples of non-smokers and smokers were not different from each other at any timepoint during the trial. With regard to cystatins, the results showed that in the state of natural gingivitis cystatin activity is lower in smokers as compared to non-smokers. In smokers, the resolution of natural gingivitis to the state of gingival health did not result in a change of cystatin activity and levels of cystatin C. At the end of the 14-day experimental gingivitis period, smokers showed a decrease in cystatin activity and cystatin C as well as lower outputs of cystatin activity and cystatin C. CONCLUSION: Smoking is associated with lower cystatin activity and output of cystatin C during gingival inflammation.

Adolescent↗
PubMed2001

Antimicrobial peptides: properties and applicability.

All organisms need protection against microorganisms, e. g. bacteria, viruses and fungi. For many years, attention has been focused on adaptive immunity as the main antimicrobial defense system. However, the adaptive immune system, with its network of humoral and cellular responses is only found in higher animals, while innate immunity is encountered in all living creatures. The turning point in the appreciation of the innate immunity was the discovery of antimicrobial peptides in the early eighties. In general these peptides act by disrupting the structural integrity of the microbial membranes. It has become clear that membrane-active peptides and proteins play a crucial role in both the innate and the adaptive immune system as antimicrobial agents. This review is focused on the functional and structural features of the naturally occurring antimicrobial peptides, and discusses their potential as therapeutics.

Amino Acid Sequence↗
PubMed2001

Differential effects of active versus passive coping on secretory immunity.

This study examined the acute immunological effects of two laboratory stressors, expected to evoke distinct patterns of cardiac autonomic activity; namely an "active coping" time-paced memory test, and a "passive coping" stressful video showing surgical operations. We measured salivary S-IgA, IgA-subclasses (IgA1, IgA2), and secretory component (SC). SC is responsible for the transport of S-IgA across the epithelium, and thus a rate-determining step in S-IgA secretion. Thirty-two male undergraduates were subjected to both stressors and a control video (a didactic television program). The memory test induced a typical "fight-flight" response, characterized by increases in heart rate and blood pressure in association with a decrease in cardiac preejection period (PEP) and vagal tone. The surgical video produced a "conservation-withdrawal"-like response, characterized by an enhanced vagal tone, a decrease in heart rate, and a moderate sympathetic coactivation (as indicated by a shortened PEP and an increased systolic pressure). The memory test induced an increase in the concentration and, to a lesser extent, in the output of S-IgA, IgA], and SC. The output of IgA2 was not significantly affected. For the surgical video, a different pattern emerged: During stressor exposure S-IgA remained unaffected, against the background of a small increase in SC output. However, 10 min after the surgical video S-IgA levels had decreased. This decrease in S-IgA was paralleled by a decrease in IgA1, but not IgA2. We conclude that acute stress can have both enhancing and suppressive effects on secretory immunity, the IgA1 subclass in particular. The mechanisms that underlie these divergent responses may include stressor-specific patterns of autonomic activation.

Adaptation, Psychological↗
PubMed2000

Characterization of histatin 5 with respect to amphipathicity, hydrophobicity, and effects on cell and mitochondrial membrane integrity excludes a candidacidal mechanism of pore formation.

Histatin 5 is a 24-residue peptide from human saliva with antifungal properties. We recently demonstrated that histatin 5 translocates across the yeast membrane and targets to the mitochondria, suggesting an unusual antifungal mechanism (Helmerhorst, E. J., Breeuwer, P., van't Hof, W., Walgreen-Weterings, E., Oomen, L. C. J. M., Veerman, E. C. I., Nieuw Amerongen, A. V., and Abee, T. (1999) J. Biol. Chem. 274, 7286-7291). The present study used specifically designed synthetic analogs of histatin 5 to elucidate the role of peptide amphipathicity, hydrophobicity, and the propensity to adopt alpha-helical structures in relation to membrane permeabilization and fungicidal activity. Studies included circular dichroism measurements, evaluation of the effects on the cytoplasmic transmembrane potential and on the respiration of isolated mitochondria, and analysis of the peptide hydrophobicity/amphipathicity relationship (Eisenberg, D. (1984) Annu. Rev. Biochem. 53, 595-623). The 14-residue synthetic peptides used were dh-5, comprising the functional domain of histatin 5, and dhvar1 and dhvar4, both designed to maximize amphipathic characteristics. The results obtained show that the amphipathic analogs exhibited a high fungicidal activity, a high propensity to form an alpha-helix, dissipated the cytoplasmic transmembrane potential, and uncoupled the respiration of isolated mitochondria, similar to the pore-forming peptide PGLa (Peptide with N-terminal Glycine and C-terminal Leucine-amide). In contrast, histatin 5 and dh-5 showed fewer or none of these features. The difference in these functional characteristics between histatin 5 and dh-5 on the one hand and dhvar1, dhvar4, and PGLa on the other hand correlated well with their predicted affinity for membranes based on hydrophobicity/amphipathicity analysis. These data indicate that the salivary protein histatin 5 exerts its antifungal function through a mechanism other than pore formation.

Amino Acid Sequence↗
PubMed2000

A role for Lewis a antigens on salivary agglutinin in binding to Streptococcus mutans.

Streptococcus mutans is a major etiological agent in dental caries. Salivary agglutinin is one of the main salivary components binding to S. mutans. To learn more about the interaction of salivary agglutinin with S. mutans, parotid, submandibular, sublingual and palatal saliva samples were incubated with S. mutans suspension. Both depleted saliva samples and bacterial extracts were analyzed by SDS-PAGE and immunoblotting. Salivary agglutinin was present in all types of glandular saliva and in all cases bound to S. mutans, also to PC337C, a P1 mutant of S. mutans. Agglutinin was separated by SDS-PAGE under reducing and non-reducing conditions and then transferred to nitrocellulose. Non-reduced agglutinin bound S. mutans, but reduced agglutinin did not. Adhesion of S. mutans to agglutinin-coated microplates was inhibited by amine-containing components, 1 M NaCl or KCl and EDTA. Adhesion decreased with decreasing pH with no adhesion below pH 5.0. These data suggest that calcium-dependent electrostatic interactions play a role in binding. By immunoblotting was demonstrated that blood group antigens and Lewis antigens were present on agglutinin. Synthetic blood group antigens and Lewis antigens covalently coupled to polyacrylamide were tested for binding to S. mutans. Only Le(a)(Gal beta 1,3(Fuc alpha 1,4)GlcNAc) bound to S. mutans, whereas the blood group antigens Le(b), Le(x), Le(y), H1, H2, A, B and sialylated Le(a) did not. Lea without galactose (Fuc alpha 1,4GlcNAc) still bound to S. mutans, but Le(a) without fucose (Gal beta 1,3GlcNAc) did not. Binding of agglutinin to S. mutans was not inhibited by Le(a). In conclusion, S. mutans can bind to Le(a) carbohydrate epitopes in which the fucose is an essential residue. Le(a) carbohydrate epitopes are present on salivary agglutinin but play no major role in binding.

Agglutinins↗
PubMed2000

Synergistic effects of low doses of histatin 5 and its analogues on amphotericin B anti-mycotic activity.

The increase in the use of antifungal agents for prophylaxis and therapy has led to the development of antifungal drug resistance. Drug combinations may prevent or delay resistance development. The aim of the present study was to investigate whether naturally and designed cationic antifungal peptides act synergistically with commonly used antimycotics. No enhanced activity was found upon addition of dhvar4, a designed analogue of the human salivary peptide histatin 5, or PGLa to fluconazole or 5-flucytosine, respectively. In contrast, strong synergism of amphotericin B with the peptides was found against several Aspergillus, Candida, and Cryptococcus strains, and against an amphotericin B-resistant C. albicans laboratory mutant in the standardised broth microdilution assays according to the NCCLS standard method M27-T. Amphotericin B showed synergism with dhvar5, another designed analogue of histatin 5, and with magainin 2 against all seven tested strains. Combinations of amphotericin B with histatin 5, dhvar4, and PGLa showed synergism against four of the seven strains. The growth inhibitory activity of amphotericin B was enhanced by sub-MIC concentrations of peptide, but its haemolytic activity remained unaffected, suggesting that its cytotoxicity to host cells was not increased and that peptides may be suitable candidates for combination therapy.

Amphotericin B↗
PubMed2000

Antimicrobial activity of synthetic salivary peptides against voice prosthetic microorganisms.

OBJECTIVES: To investigate whether synthetic salivary antimicrobial peptides have an inhibitory effect on the growth of bacteria and yeasts isolated from used silicone rubber voice prostheses. METHODS: The antimicrobial activities of six synthetic salivary peptides (histatin 5, dhvarl, dhvar4, dhvar5, lactoferrin b 1730 [LFb 17-30], and cystatin S1-15) at concentrations of 2 and 4 mg/mL were determined against different oropharyngeal yeast (four) and bacterial (eight) strains and against a "total microflora" isolated from explanted voice prostheses using agar diffusion tests. The spectrum of susceptible microorganisms was determined qualitatively. RESULTS: Histatin 5 and cystatin S1-15 did not show any antimicrobial activity against the microorganisms involved in this study. Dhvar1 was active against some of the oropharyngeal microorganisms tested, including the yeast strains, but not against Rothia dentocariosa, Staphylococcus aureus, Escherichia coli, and the total microflora Dhvar4 was active against all microorganisms tested, including the total microflora. Dhvar5 lacked activity against E coli and the total microflora LFb 1730 did not inhibit the growth of any of the yeast strains involved and showed only minor activity against some of the bacterial strains. LFb 1730 slightly inhibited the growth of the total microflora from an explanted prosthesis. CONCLUSIONS: The synthetic salivary peptide dhvar4 has a broad antimicrobial activity against all microorganisms that are commonly isolated from explanted voice prostheses, including yeasts. Therewith, it may represent a useful drug, as an alternative for antibiotics and antimycotics employed in various ways to prolong the lifetime of voice prostheses in laryngectomees.

Bacteria↗
PubMed1999

Cationic amphipathic peptides, derived from bovine and human lactoferrins, with antimicrobial activity against oral pathogens.

Peptides derived from the N-terminal domain that comprises an amphipathic alpha-helix in human lactoferrin (LFh 18-31 and LFh 20-38) and bovine lactoferrin (LFb 17-30 and LFb 19-37) were chemically synthesised. Since many positively charged amphipathic alpha-helices contain antimicrobial activity, the peptides were tested for their antimicrobial activity against various oral pathogens. Both peptides from bovine lactoferrin had more potent antimicrobial activities than the human equivalents. Peptide LFb 17-30, containing the largest number of positively charged amino acids, showed the highest antimicrobial activity to both Gram-positive and Gram-negative bacteria. Since native lactoferrin molecules had no killing activity, release of these peptides from the native protein should be investigated to explore the use in oral care products.

Amino Acid Sequence↗
PubMed1999

Evaluation of the use of xanthan as vehicle for cationic antifungal peptides.

Oral candidiasis frequently occurs in individuals with dry mouth syndrome (xerostomia), in immunocompromised patients and in denture wearers. The aim of this study was to develop a formulation which will prolong the retention time of antimicrobial agents at the site of application. The activity against Candida albicans of a synthetic cationic peptide dhvar 1, based on the human fungicidal salivary peptide histatin 5, was tested either in a mixture with the bioadhesive polymer xanthan, or after covalent coupling to this polymer. The presence of xanthan resulted in an increase of the LC50 value of the peptide from 2.6 (S.D.=0.6) to 5.8 (S.D.=4.0). Covalent coupling caused an additional increase of the LC50 value to 18.4 (S. D.=6.7). Coupling caused a reduction of the viscosity and elasticity of the xanthan solution related to the applied concentration of the coupling agent. Incubation of the peptide with clarified human whole saliva resulted in proteolytic degradation of the peptide. In the presence of xanthan the degradation occurred more slowly. It was concluded that xanthan is an appropriate vehicle for antimicrobial peptides in a retention increasing formulation.

Antifungal Agents↗
PubMed1999

A critical comparison of the hemolytic and fungicidal activities of cationic antimicrobial peptides.

The hemolytic and fungicidal activity of a number of cationic antimicrobial peptides was investigated. Histatins and magainins were inactive against human erythrocytes and Candida albicans cells in phosphate buffered saline, but displayed strong activity against both cell types when tested in 1 mM potassium phosphate buffer supplemented with 287 mM glucose. The HC50/IC50 ratio, indicative of the therapeutic index, was about 30 for all peptides tested. PGLa was most hemolytic (HC50 = 0.6 microM) and had the lowest therapeutic index (HC50/IC50 = 0.5). Susceptibility to hemolysis was shown to increase with storage duration of the erythrocytes and also significant differences were found between blood collected from different individuals. In this report, a sensitive assay is proposed for the testing of the hemolytic activities of cationic peptides. This assay detects subtle differences between peptides and allows the comparison between the hemolytic and fungicidal potency of cationic peptides.

Amino Acid Sequence↗
PubMed1999

The cellular target of histatin 5 on Candida albicans is the energized mitochondrion.

Histatin 5 is a human basic salivary peptide with strong fungicidal properties in vitro. To elucidate the mechanism of action, the effect of histatin 5 on the viability of Candida albicans cells was studied in relation to its membrane perturbing properties. It was found that both the killing activity and the membrane perturbing activity, studied by the influx of a DNA-specific marker propidium iodide, were inhibited by high salt conditions and by metabolic inhibitors, like sodium azide. In addition, exposure to histatin 5 resulted in a loss of the mitochondrial transmembrane potential in situ, measured by the release of the potential-dependent distributional probe rhodamine 123. Localization studies using tetramethylrhodamine isothiocyanate-labeled histatin 5 or fluorescein isothiocyanate-labeled histatin 5 showed a granular intracellular distribution of the peptide, which co-localized with mitotracker orange, a permeant mitochondria-specific probe. Like the biological effects, uptake of labeled histatin 5 was inhibited by mitochondrial inhibitors and high salt conditions. Our data indicate that histatin 5 is internalized, and targets to the energized mitochondrion.

Amino Acid Sequence↗
PubMed1999

Salivary lactoferrin and low-Mr mucin MG2 in Actinobacillus actinomycetemcomitans-associated periodontitis.

Concentrations and output of lactoferrin and of low-Mr mucin MG2 were determined in saliva of subjects suffering from Actinobacillus actinomycetemcomitans-associated periodontal disease and healthy subjects. Periodontal patients were clinically examined and a microbiological sample was taken from the deepest bleeding pockets in each quadrant. The number of viable A. actinomycetemcomitans was determined in the sampled sites of each patient. The MG2 output in the diseased subjects (13.6 microg protein/min) was decreased at least by a factor three compared to periodontal healthy subjects (44.3 microg protein/min). On the other hand, output of lactoferrin was not significantly different in healthy (9.5 microg/min) and diseased subjects (7.6 microg/min). Western analyses demonstrated a higher iron-saturation of lactoferrin in diseased subjects in comparison with the healthy subjects. Lactoferrin degrading enzymes, probably derived from microbial sources, could be detected in saliva of the periodontally diseased subjects, but not in saliva of healthy subjects. The combination of iron-saturation and degradation of lactoferrin suggests that anti-microbial properties of lactoferrin are diminished in periodontitis patients. Moreover, the low concentration of mucin MG2 suggests a decline in mucin defence and consequently a higher susceptibility for oral infection. A negative correlation (r= -0.4, p < 0.05) between the number of subgingival A. actinomycetemcomitans and lactoferrin in saliva suggested that low concentrations of lactoferrin favour the growth of the bacterium. These data indicate that a decline in the salivary defence system might increase the risk for oral infection by A. actinomycetemcomitans.

Actinobacillus Infections↗
PubMed1999

Chitinase in whole and glandular human salivas and in whole saliva of patients with periodontal inflammation.

In recent studies the existence of a chitinase in various mammals, like man, was described. The aim of the present study was to find out whether salivas of periodontally healthy and inflamed humans also contain chitinase activity. Chitinase activity, assayed with the substrate 4-methylumbelliferyl-beta-D-N,N',N"-triacetylchitotrioside, was shown to be present in human whole saliva, with an activity level and apparent molecular mass (35 kDa) that were comparable with those of the human serum enzyme. Both lysozyme and beta-N-acetylhexosaminidase could be separated from chitinase by means of Bio-Gel P-100 gel filtration chromatography. The enzyme was also present in glandular saliva of parotid, palatine, submandibular and sublingual glands. The chitinase activity was not of oral epithelial, bacterial or plaque bacterial origin and was not correlated with the activity of salivary amylase. A comparative study of whole salivas of periodontally healthy controls and gingivitis and periodontitis subjects showed that only in the case of periodontitis there was a significant increase of the specific chitinase activity. The latter enzyme showed a gel filtration pattern that was comparable with that of the enzyme from controls. The measured albumin levels in saliva and the absence of correlation between the chitinase activity levels in plasma and saliva from periodontitis patients indicated that the (increased) chitinase activities did not originate from blood leakage to the oral cavity.

Adolescent↗