[Research on salivary proteins: from properties to functions]. / Onderzoek naar speekseleiwitten: van eigenschappen naar functies.

Saliva does more than merely contribute to the digestion of food. It is essential to the health of the oral cavity and with that, indirectly, of the entire body. In the 1970s and 1980s, the most important proteins and peptides in saliva were identified and characterised. For example, mucins MUC5B and MUC7, proline-rich proteins, cystatins and histatins are now known to the level of the amino acid sequence and molecular structure. The associated physical properties indicate how these proteins carry out their protective function. Sometimes, however, this information can mislead science because the relationship between property and function is not necessarily unambiguous. In addition, unexpected properties are sometimes discovered compelling scientists to re-evaluate critically the transition from physical property to physiological function. In certain cases, this has led to perceiving the (possible) function of these proteins in a completely different light.

Bacillus globigii cell size is influenced by variants of the quorum sensing peptide extracellular death factor.

Toxin-antitoxin modules are necessary for the mode of action of several antibiotics. One of the most studied toxin-antitoxin modules is the quorum sensing-dependent MazEF system in Escherichia coli. The quorum sensing factor in this system is called the extracellular death factor (EDF), a linear pentapeptide with the sequence NNWNN. In spite of the extensive research on the mazEF system and the involvement of the quorum sensing factor EDF, the effect of EDF itself on bacteria has not yet been studied. In this research, we determined the effect of EDF and variants on cell growth in the Gram-negative bacterium E. coli and the Gram-positive Bacillus globigii. By aligning the zwf gene (from where EDF originates) of different bacterial species, we found 27 new theoretical variants of the peptide. By evaluating growth curves and light microscopy we found that three EDF variants reduced bacterial cell size in B. globigii, but not in E. coli. The D-peptides did not affect cell size, indicating that the effect is stereospecific. Peptides wherein tryptophan was substituted by alanine also did not affect cell size, which indicates that the effect seen is mediated by an intracellular target.

[Salivary stones: aetiology, composition and treatment]. / Speekselstenen: etiologie, samenstelling en behandeling.

Salivary stones or sialoliths, are calcified concrements which are most frequently located in the submandibular glands and their ducts. Their size and weight show considerable variation. The aetiology is unknown. It has been suggested that salivary stones could be related to an altered saliva composition, the anatomy of the ducts of the salivary gland and/or the fusion of microsialoliths. Salivary stones consist mainly of anorganic material such as hydroxyapatite, whitlockite and calciumphosphate, but they also contain organic components such as proteins and lipids. Treatment can consist of salivary gland massage combined with an acid diet, ultrasonic pulverisation, and surgical or sialendoscopical removal.

[Dextrorotary protease substrates could be suitable for oral microbiological diagnostics]. / Rechtsdraaiende proteasesubstraten lijken bruikbaar voor orale microbiologische diagnostiek.

The currently available methods for the identification of micro-organisms in a clinical sample are time consuming, complex or lack sensitivity and specificity. Consequently, no tests are available for rapid microbiological diagnostics in dental clinics so far. Based on the fact that bacteria posess unique enzymes for processing dextrorotary amino acids, a rapid method has been developed to detect metabolically active bacteria. Using dextrorotary amino acid containing substrates, Porphyromonas gingivalis could be detected in clinical samples with a specificity of 96-100% within 10 minutes. This new method opens the door for the development of a rapid oral diagnostic microbiological test.

Comparing culture, real-time PCR and fluorescence resonance energy transfer technology for detection of Porphyromonas gingivalis in patients with or without peri-implant infections.

BACKGROUND AND OBJECTIVE: The aim of the study was to compare the detection of Porphyromonas gingivalis using a fluorescence resonance energy transfer (FRET) technology with commonly used diagnostic methods in salivary and subgingival plaque samples from subjects with dental implants. P. gingivalis was considered as a marker for a pathogenic microbiota. MATERIAL AND METHODS: Ninety-seven adult subjects were recruited, including periodontally healthy controls with no dental implants, implant controls with no peri-implant disease and patients with peri-implant disease. Saliva and subgingival/submucosal plaque samples were collected from all subjects and were analyzed using culture, real-time PCR and FRET technology employing P. gingivalis-specific substrates. RESULTS: It was found that the P. gingivalis-specific substrates were highly suitable for detecting the presence of P. gingivalis in saliva and in subgingival plaque samples, showing comparable specificity to culture and real-time PCR. CONCLUSION: We applied the FRET technology to detect P. gingivalis in implant patients with or without an implant condition and in controls without implants. The technique seems suitable for detection of P. gingivalis in both plaque and saliva samples. However, with all three techniques, P. gingivalis was not very specific for peri-implantitis cases. Future work includes fine-tuning the FRET technology and also includes the development of a chair-side application.

[Risks associated with erosive beverages in secondary school canteens in the Netherlands]. / Risico van erosie door dranken in schoolkantines.

The consumption of acidic beverages may cause dental erosion. The consumption of such beverages during school hours may contribute to the development of erosion. In order to study the extent to which children of secondary schools are exposed to potentially erosive beverages, 43 locations in 37 schools in the Netherlands were visited. In each school, all available types of beverages were registered and classified as non-erosive or potentially erosive. In addition, the number of students, type of school, opening times of the canteens and the number of vending machines were recorded. It was revealed that a wide variety of beverages is available in school canteens, of which a large number are potentially erosive (71.8 +/- 12.9%). The number of different products available, the number of different beverages available and the number of potentially erosive drinks all correlated with the number of students per school, but not with the type of education. The number of drinks available and the number of potentially erosive drinks correlated with the opening hours of the canteens and the number of vending machines.

[Unhealthy weight loss. Erosion by apple cider vinegar]. / Ongezond afslanken. Erosie door appelazijn.

Erosive tooth wear was diagnosed in the dentition of a 15-year-old girl with a Moroccan background. After an anamnesis, extensive analysis of possible risk factors and a study of the pattern of erosion, it was concluded that the erosive tooth wear was induced by daily consumption of a glass of apple cider vinegar Further investigation revealed that in North-African culture, women have used apple cider vinegar to achieve weight loss for generations. Bodybuilders are also known to make use of this method of weight reduction.

[Saliva and wound healing]. / Speeksel en wondgenezing.

The oral mucosa is frequently exposed to mechanical forces, which may result in tissue damage. Saliva contributes to the repair of the oral mucosa in several ways. In the first place, it creates a humid environment to improve the function of inflammatory cells. During the last few years, it has been shown that saliva also contains a large number of proteins with a role in wound healing. Saliva contains growth factors, especially Epidermal Growth FACTOR, which promotes the proliferation of epithelial cells. Trefoil factor 3 and histatin promote the process of wound closure. The importance of Secretory Leucocyte Protease Inhibitor is demonstrated by the fact that in the absence of this salivary protein, oral wound healing is considerably delayed. Understanding these salivary proteins opens the way for the development of new wound healing medications.

The erosive potential of jawbreakers, a type of hard candy.

OBJECTIVES: To explore the consumption pattern of a specific type of acidic solid candy, the so-called jawbreakers, by primary school children and determine the erosive potential of this type of candy in vivo. METHODS: A questionnaire about jawbreaker consumption was distributed among 10-12 year-old-children (n = 302). Subsequently, 19 healthy volunteers tested four different jawbreakers in vivo. Whole saliva was collected 5 min before, 3 min during and 11 min after consumption. Salivary flow rate and pH were determined. RESULTS: Two-thirds of the children reported a history of jawbreaker consumption, 18% during the last week. More than half of the children estimated their average time for consumption of one jawbreaker to be more than 15 min. In vivo, the jawbreakers induced 8.6-13.9-fold increase in salivary flow rate. Sucking on sour, jumbo and strawberry jawbreakers induced a drop in salivary pH to values below pH 5.5. During consumption of fireball jawbreakers, the intra-oral pH hardly changed. CONCLUSIONS: Jawbreakers are frequently used by children, who keep this candy in their mouth for a long time. Jawbreakers differ considerable in erosive potential, with sour and jumbo jawbreakers > strawberry jawbreaker >> fireball jawbreaker. This information is of use for dental hygienists counselling juvenile patients with dental erosion.

Effects of environmental temperature on saliva flow rate and secretion of protein, amylase and mucin 5B.

OBJECTIVE: The aim of this study is to evaluate the short term effects of environmental temperature on saliva flow rate and composition. METHODS: In a cross-over study design 20 subjects (18-25 years old, 14 women, 6 men) were exposed in randomized order at different days to three temperatures (4 °C, 21 °C and 37 °C). Five minutes after a subject was exposed to the test temperature, collection of resting saliva was started for 5 min at the same temperature. Saliva flow rate, pH, viscosity, protein concentration, mucin 5B concentration and amylase activity were measured. RESULTS: Exposure to 4 °C resulted in an increase of the saliva flow rate (p < 0.01), protein output and amylase output (p < 0.001) compared to exposure to 21 °C or 37 °C. Although the figures for mucin 5B output at 4 °C were higher than at higher temperatures, this was not significant. There were no significant differences in the salivary mucin 5B concentration and viscosity between saliva samples collected at the indicated temperatures. CONCLUSIONS: Lowering of the temperature induces an increase in saliva flow rate, as well as protein and amylase output.

The erosive potential of lollipops.

AIM: To determine the erosive potential of several commercially available lollipops and the protective effect of saliva. METHODS: The erosive potential of lollipops was determined in vitro by measuring the pH and neutralisable acidity. Subsequently, 10 healthy volunteers tested different types of lollipops. Whole saliva was collected 5 minutes before, 15 minutes during and 10 minutes after consumption. Salivary flow rate and pH were determined. RESULTS: Fruit flavoured and cola flavoured lollipops have a very low pH (2.3 - 2.4). Yogurt-containing and salmiak (salty liquorice) flavoured lollipops have much higher pH values (3.8-4.7). The neutralisable acidity of 1g of lollipop showed a large variation from < 200 microl to > 1700 microl 0.1 M NaOH. In vivo, lollipops induced 2.5 to 4.7-fold increase in salivary flow rate with a concomitant drop in salivary pH. For fruit flavoured and cola flavoured lollipops the salivary pH dropped below pH 5.5. For strawberry yogurt and salmiak lollipops, the salivary pH remained above this critical value. The volunteers did not report significant differences in preferences for the lollipops. CONCLUSIONS: Lollipops differ considerable in erosive potential, with fruit flavoured and cola flavoured lollipops having the highest risk for inducing dental erosion. This information is of use for clinicians counselling juvenile patients with dental erosion.

[Heroin and oral health]. / Heroïnegebruik en mondgezondheid.

Heroin is a half synthetic opiate with. It is used by a relatively small number of the general population, but relatively frequently by homeless people and prisoners. Since heroin has an inhibitory effect on the nervous system and on breathing, an overdose of heroin can have fatal consequences. Sudden abstinence of heroin results in general discomfort, restlessness, muscle cramps, perspiration, nausea, shivers and goose bumps. Oral effects of heroin include increased risks of caries, periodontitis and bruxism. Saliva has the potential of detecting heroin abuse.

[Influence of sports on saliva]. / Invloed van sport op speeksel.

Physical exertion, such as practicing sports, may have an influence on the secretion and composition of saliva. As a result of intensive physical exertion, the saliva immunoglobulin concentration decreases and the concentration of protein--the output of protein (mg/min), however, remains constant--and the viscosity of the saliva increase, while the watery secretion decreases. Moreover, saliva testosterone levels decrease and salivary cortisol levels increase. The saliva Na+, K+, Ca2+ and PO4(3-) concentrations increase slightly or remain unchanged. After a single physical exertion, the alterations are reversible and the values return to baseline within a few hours. In the case of long lasting intensive physical exertions, for instance lasting several months, particularly the decrease in the concentration of the most prevalent immunoglobulins in saliva (s-IgA) seems to have a more permanent character. This has a detrimental effect on the immunological defence, which may reduce oral as well as upper respiratory tract health. Complaints about this are not uncommon among serious athletes who are active at a high competitive level. Among these athletes, the combination of changes in the composition and secretion of saliva, together with intense mouth-breathing and an adjusted diet and drinking habits carry a higher risk of developing caries and erosion. However, in case of decent oral health selfcare, the detrimental effects on oral health can be prevented.

Acute effects of hemodialysis on salivary flow rate and composition.

AIMS: To evaluate acute effects of hemodialysis (HD) on the salivary flow rate, pH and biochemical composition before, during and after completion of a dialysis session. MATERIAL AND METHODS: Unstimulated whole saliva (UWS) and chewing-stimulated whole saliva (CH-SWS) were collected in 94 HD patients. Salivary flow rate, pH, concentrations of total protein, albumin, cystatin C, secretory immunoglobulin A (S-IgA) and of sodium, potassium and urea were measured. RESULTS: HD had an acute stimulating effect on the salivary flow rate (UWSbefore = 0.30+/-0.22 ml/min, UWSduring = 0.39+/-0.25 ml/min, p < 0.005). The mean pH of UWS showed a small but significant increase during HD mainly due to an increased watery secretion from the salivary glands. The salivary biochemical constituents changed markedly, but no significant difference in output was found. The electrolyte concentration did not change significantly during dialysis. The level of urea in CH-SWS declined to 40% (Ureabefore = 25.+/-6.4 mmol/l, Ureaduring = 15.3+/-4.5 mmol/1). CONCLUSIONS: This study shows that HD has significant acute effects on both salivary secretion rate and protein concentrations in saliva. We conclude that the observed changes in salivary concentrations and proteins are mainly due to an increased watery secretion from the salivary glands.

Histatin 1 Enhances Cell Adhesion to Titanium in an Implant Integration Model.

Cellular adhesion is essential for successful integration of dental implants. Rapid soft tissue integration is important to create a seal around the implant and prevent infections, which commonly cause implant failure and can result in bone loss. In addition, soft tissue management is important to obtain good dental aesthetics. We previously demonstrated that the salivary peptide histatin 1 (Hst1) causes a more than 2-fold increase in the ability of human adherent cells to attach and spread on a glass surface. Cells treated with Hst1 attached more rapidly and firmly to the substrate and to each other. In the current study, we examine the potential application of Hst1 for promotion of dental implant integration. Our results show that Hst1 enhances the attachment and spreading of soft tissue cell types (oral epithelial cells and fibroblasts) to titanium (Ti) and hydroxyapatite (HAP), biomaterials that have found wide applications as implant material in dentistry and orthopedics. For improved visualization of cell adhesion to Ti, we developed a novel technique that uses sputtering to deposit a thin, transparent layer of Ti onto glass slides. This approach allows detailed, high-resolution analysis of cell adherence to Ti in real time. Furthermore, our results suggest that Hst1 has no negative effects on cell survival. Given its natural occurrence in the oral cavity, Hst1 could be an attractive agent for clinical application. Importantly, even though Hst1 is specific for saliva of humans and higher primates, it stimulated the attachment and spreading of canine cells, paving the way for preclinical studies in canine models.

[Saliva: more than just a factor in oral health]. / Speeksel: een mondoverstijgende gezondheidsfactor.

Several salivary proteins are involved in the antimicrobial defence within the oral cavity, and are able to eliminate or inhibit a great number of microorganisms that enter the body via the oral cavity. Thus changes in the composition of saliva may not only affect people's oral health, but also have consequences for their general health. Based on salivary proteins, antimicrobial peptides have been synthesized. Potentially, these could be used as a new class of antibiotics. The antimicrobial peptides are hardly cytotoxic to human cells and do not evoke any or only very little resistence in microorganisms.

The oxidation-reduction kinetics of the reaction of cytochrome c1 with non-physiological redox agents.

The kinetics of the oxidation-reduction reactions of cytochrome c1 with ascorbate, ferricyanide, triphenanthrolinecobalt(III) and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) have been examined using the stopped-flow technique. The reduction of ferricytochrome c1 by ascorbic acid is investigated as a function of pH. It is shown that at neutral and alkaline pH the reduction of the protein is mainly performed by the doubly deprotonated form of ascorbate. From the ionic-strength-dependence studies of the reactions of cytochrome c1 with ascorbate, ferricyanide and triphenanthrolinecobalt(III), it is demonstrated that the reactions rate is governed by electrostatic interactions. The second-order rate constants for the reaction of cytochrome c1 with ascorbate, ferricyanide, TMPD and triphenanthrolinecobalt(III) are 1.4 . 10(4), 3.2 . 10(3), 3.8 . 10(4) and 1.3 . 10(8) M-1 . s-1 (pH 7.9, I = 0, 10 degrees C), respectively. Application of the Debye-Hückel theory to the data of the ionic-strength-dependence studies of these redox reactions of cytochrome c1 yielded for ferrocytochrome c1 and ferricytochrome c1 a net charge of --5 and --4, respectively. The latter value is close to that of --3 for the oxidized enzyme, calculated from the amino acid sequence of the protein. This implies that not a local charge on the surface of the protein, but the overall net charge of cytochrome c1 governs the reaction rate with small redox molecules.

The pre-steady state reaction of ferrocytochrome c with the cytochrome c-cytochrome aa3 complex.

1. Using stopped-flow technique we have investigated the electron transfer form cytochrome c to cytochrome aa3 and to the (porphyrin) cytochrome c-cytochrome aa3 complex. 2. In a low ionic strength medium, the pre-steady state reaction occurs in a biphasic way with rate constants of at least 2.10(8) M-1.s-1 and about 10(7) M-1.S-1 (I=8.8 mM, pH 7.0, 10 degrees C), respectively. 3. A comparison of the rate constants, determined in the presence of an excess of cytochrome c with those found in the presence of an excess of cytochrome aa3 reveals the existence of two slower reacting sites on the functional unit (2 hemes and 2 coppers) of cytochrome aa3. On basis of these results we discuss various models. If no site-site interactions are assumed (non-cooperative model) cytochrome aa3 has 2 high and 2 low affinity sites available for the reaction with ferrocytochrome c. If negative cooperativity occurs, cytochrome aa3 has 2 high affinity sites which change into 2 low affinity sites upon binding of one cytochrome c molecule. The latter model is favoured.

The ionic strength dependence of the rate of a reaction between a small ion and a large ion with a dipole moment.

The ionic strength dependence of the rate constant of a reaction between a small ion and a large ion with a dipole moment (e.g. a protein) is described. This description takes into account only the electrostatic interactions between the two ions. This approach agrees with the Marcus theory treatment of the electrostatic interactions and also with the Debye-Hückel theory which is based on changes in the activity coefficients of the reactants. The contribution of the dipole moment of the protein to the ionic strength dependence of the rate constant has been calculated. A method is described whereby one can calculated the charge of the protein without knowing the precise ionic strength dependence of the rate constant. Two applications are mentioned to illustrate the usefulness of the method.

The reaction of cytochrome aa3 with (porphyrin) cytochrome c as studied by pulse radiolysis.

(1) Using the pulse-radiolysis and stopped-flow techniques, the reactions of iron-free (porphyrin) cytochrome c and native cytochrome c with cytochrome aa3 were investigated. The porphyrin cytochrome c anion radical (generated by reduction of porphyrin cytochrome c by the hydrated electron) can transfer its electron to cytochrome aa3. The bimolecular rate constant for this reaction is 2 x 10(7) M-1 . s-1 (5 mM potassium phosphate, 0.5% Tween 20, pH 7.0, 20 degrees C). (2) The ionic strength dependence of the cytochrome c-cytochrome aa3 interaction was measured in the ionic strength range between 40 and 120 mM. At ionic strengths below 30 mM, a cytochrome c-cytochrome aa3 complex is formed in which cytochrome c is no longer reducible by the hydrated electron. A method is described by which the contributions of electrostatic forces to the reaction rate can be determined. (3) Using the stopped-flow technique, the effect of the dielectric constant (epsilon) of the reaction medium on the reaction of cytochrome C with cytochrome aa3 was investigated. With increasing epsilon the second-order rate constant decreased.