Interaction of the salivary low-molecular-weight mucin (MG2) with Actinobacillus actinomycetemcomitans

Periodontitis is associated with the presence of certain Gram-negative bacteria in the oral cavity, among these Actinobacillus actinomycetemcomitans. In order to determine which types of salivary components interact with A. actinomycetemcomitans two strains (HG 1175 and FDC Y4) were incubated with whole saliva and individual glandular secretions, viz. parotid, submandibular, and sublingual saliva. Immunochemical analysis by immunoblotting of bacteria-bound salivary proteins showed that IgA, the low-molecular mucin MG2, parotid agglutinin, and a 300 kDa sublingual and submandibular glycoprotein, were bound to the bacterial strains tested. In addition, adherence of A. actinomycetemcomitans to salivary proteins in a solid-phase was studied. After electrophoresis and transfer of salivary proteins to nitrocellulose membranes A. actinomycetemcomitans adhered only to MG2. In this assay periodate treatment, mild acid hydrolysis or neuraminidase digestion of the saliva glycoproteins abolished binding of two clinical isolates (HG 1175 and NY 664), suggesting that sialic acid residues on MG2 are involved in the binding. In contrast, adherence of the smooth laboratory strain Y4 was not affected by removal of sialic acid residues or even periodate treatment of MG2.Abbreviations S-IgA Secretory IgA - MG1 high-molecular-weight mucin - MG2 low-molecular-weight mucin - EP-GP extra parotid-glycoprotein - PRPs proline-rich proteins - SNA Sambucus nigra agglutinin - MAA Maackia amurensis agglutinin - PNA peanut agglutinin - UEA Ulex europaeus agglutinin     

Crystalline anatase-rich titanium can reduce adherence of oral streptococci

Dental implant abutments that emerge through the mucosa are rapidly covered with a salivary protein pellicle to which bacteria bind, initiating biofilm formation. In this study, adherence of early colonizing streptococci, Streptococcus gordonii, Streptococcus oralis, Streptococcus mitis and Streptococcus sanguinis to two saliva-coated anodically oxidized surfaces was compared with that on commercially pure titanium (CpTi). Near edge X-ray absorption (NEXAFS) showed crystalline anatase was more pronounced on the anodically oxidized surfaces than on the CpTi. As revealed by fluorescence microscopy, a four-species mixture, as well as individual bacterial species, exhibited lower adherence after 2?h to the saliva-coated, anatase-rich surfaces than to CpTi. Since wettability did not differ between the saliva-coated surfaces, differences in the concentration and/or configuration of salivary proteins on the anatase-rich surfaces may explain the reduced bacterial binding effect. Anatase-rich surfaces could thus contribute to reduced overall biofilm formation on dental implant abutments through diminished adherence of early colonizers.     

Involvement of human mucous saliva and salivary mucins in the aggregation of the oral bacteria Streptococcus sanguis,Streptococcus oralis,and Streptococcus rattus

The contribution of human parotid (Par) and submandibular/sublingual (SM/SL) saliva and of the human whole salivary mucin fraction (HWSM) to saliva-induced bacterial aggregation was studied for S. sanguis C476, S. oralis I581, and S. rattus HG 59. The mucous SM/SL saliva showed a much higher aggregation potency towards the S. sanguis and S. oralis strain than did the serous Par saliva. The SM/SL saliva-induced aggregation was observed after 30 min, at 60 min followed by the Par saliva-induced aggregation, and showed a 4-fold higher aggregation titer of 128 for S. sanguis, and an 8-fold higher titer of 516 for S. oralis. In contrast, the Par saliva showed a slightly higher aggregation activity than the SM/SL saliva towards S. rattus as judged by a twofold higher titer of 64. Morphologically, however, the SM/SL saliva-induced aggregation of S. rattus was far more pronounced as was also found for S. sanguis. Finally, the HWSM-induced aggregation showed a 4 to 8-fold higher titer than the originating salivary source, measuring 2048 for S. oralis and 128 for S. rattus. Moreover, no difference was observed in aggregation activity between the HWSM from whole saliva of a blood group O donor and the HWSM from SM/SL saliva of a blood group A donor. All the data point to an important, though not exclusive role of the human salivary mucin fraction in the saliva-induced aggregation of these strains.     

Interactions ofCandida albicans with bacteria and salivary molecules in oral biofilms

The yeastCandida albicans coaggregates with a variety of streptococcal species, an interaction that may promote oral colonization by yeast cells.C. albicans andCandida tropicalis are the yeasts most frequently isolated from the human oral cavity and our data demonstrate that both these species bind toStreptococcus gordonii NCTC 7869 while two otherCandida species (Candida krusei andCandida kefyr) do not. Adherence ofC. albicans was greatest when the yeast had been grown at 30° C to mid-exponential growth phase. For 21 strains ofC. albicans there was a positive correlation between the ability to adhere toS. gordonii and adherence to experimental salivary pellicle. Whole saliva either stimulated or slightly inhibited adherence ofC. albicans toS. gordonii depending on the streptococcal growth conditions. The results suggest that the major salivary adhesins and coaggregation adhesins ofC. albicans are co-expressed.     

Comparison of N-linked Glycoproteins in Human Whole Saliva, Parotid, Submandibular, and Sublingual Glandular Secretions Identified using Hydrazide Chemistry and Mass Spectrometry

INTRODUCTION: Saliva is a body fluid that holds promise for use as a diagnostic fluid for detecting diseases. Salivary proteins are known to be heavily glycosylated and are known to play functional roles in the oral cavity. We identified N-linked glycoproteins in human whole saliva, as well as the N-glycoproteins in parotid, submandibular, and sublingual glandular fluids. MATERIALS AND METHODS: We employed hydrazide chemistry to affinity enrich for N-linked glycoproteins and glycopeptides. PNGase F releases the N-peptides/proteins from the agarose-hydrazide resin, and liquid chromatography-tandem mass spectrometry was used to identify the salivary N-glycoproteins. RESULTS: A total of 156 formerly N-glycosylated peptides representing 77 unique N-glycoproteins were identified in salivary fluids. The total number of N-glycoproteins identified in the individual fluids was: 62, 34, 44, and 53 in whole saliva, parotid fluid, submandibular fluid, and sublingual fluid, respectively. The majority of the N-glycoproteins were annotated as extracellular proteins (40%), and several of the N-glycoproteins were annotated as membrane proteins (14%). A number of glycoproteins were differentially found in submandibular and sublingual glandular secretions. CONCLUSIONS: Mapping the N-glycoproteome of parotid, submandibular, and sublingual saliva is important for a thorough understanding of biological processes occurring in the oral cavity and to realize the role of saliva in the overall health of human individuals. Moreover, identifying glycoproteins in saliva may also be valuable for future disease biomarker studies.     

Effect of parotid submandibular and sublingual saliva on wound healing in rats.

1. The effectiveness of wound licking with parotid, submandibular or sublingual saliva on wound healing was evaluated in selectively sialadenectomized rats. 2. The rate of healing of experimentally induced cutaneous wounds was evaluated macroscopically by photography at 0, 2, 4 and 6 days after surgery. 3. Sialadenectomy of all major glands significantly slowed down wound healing compared to sham-operated controls. 4. Parotid licking had no effect compared to controls; submandibular licking and sublingual licking appeared to be very effective. 5. The results suggest that saliva promotes wound healing in experimentally induced cutaneous wounds by communal licking; this is a result of the submandibular and sublingual saliva and not the parotid saliva.     

Differences in saliva collection location and disparities in baseline and diurnal rhythms of alpha-amylase: a preliminary note of caution

Identified in the early 1980s as a surrogate marker of the sympathetic nervous system component of the stress response, there has been renewed interest in measuring salivary alpha-amylase (sAA) to test biosocial models of stress vulnerability. This brief report presents studies that document that oral fluids from the parotid and submandibular gland areas had higher sAA values than did whole saliva specimens, and sAA values in whole saliva were higher than levels measured in oral fluids from the sublingual gland area. sAA in oral fluids from the parotid and submandibular gland areas showed the highest and more pronounced diurnal variation than levels in whole saliva, and sAA in sublingual saliva showed the lowest and shallowest diurnal variation. When this source of inherent variability in sAA activity levels is not controlled for by collecting oral fluids consistently from specific gland areas, the detection of individual differences, associations between sAA and “behavioral” variables, and intra-individual change in sAA levels may be compromised. Awareness, and management, of this ubiquitous source of measurement error in sAA are essential to ensure the success of future research on the correlates and concomitants of sAA levels, stress-related reactivity and recovery, and diurnal variation.     

A functional and chemical study of radiation effects on rat parotid and submandibular/sublingual glands.

The aim of this study was to monitor composition and rate of secretion of rat parotid and submandibular/sublingual saliva following local single doses of X-rays ranging from 5 to 20 Gy. Pilocarpine-stimulated samples of parotid and submandibular/sublingual saliva were simultaneously collected with miniaturized Lashley cups before and 1-30 days after irradiation. The lag phase (period between injection of pilocarpine and start of the secretion) and flow rate were recorded and the concentrations of sodium, potassium, calcium, phosphate, and amylase were measured. With increasing dose and time, the salivary flow rate as well as sodium concentration decreased, while potassium concentrations increased throughout the follow-up period. The lag phase and the concentration of amylase reached their maximum at 3 and 10 days after irradiation, respectively. The changes in lag phase and flow rate were most obvious after doses of 15 or 20 Gy and showed a great similarity for parotid and submandibular/sublingual saliva. No dose-response relationship was observed for the changes in concentrations of calcium and phosphate. It is concluded that for radiation doses of 10 Gy and above, irreversible changes (lag phase, flow rate, potassium, sodium) were observed. A saturation of the irradiation effects (lag phase, flow rate) seems to exist at doses larger than 15 Gy. No significant differences were observed between the radiation-induced functional changes in parotid and submandibular/sublingual salivary gland tissue.     

Postprandial transformations of enzymatic and hormonal properties of saliva and blood

In 14 volunteers, saliva from both parotid, submandibular and sublingual glands were collected by capsules under stimulation of sialosis with citric acid or alimentary trial breakfast. It was taken immediately and on the 1st and 3rd hours of postprandial response. In saliva and the blood serum, alpha-amylases, trypsin, common protein, thyrotropin, thyroxine, triiodthyronin, luteinizing hormone, follicle-stimulating hormone, prolactin, progesterone, oestradiol and hydrocortisone were assessed by means of immuno-assay technique. All but oestradiol hormones had a lower concentration in the saliva than in the blood serum. The concentration and deficits of hormones and trypsin in saliva of submandibular and sublingual glands is higher, than in saliva of parotid glands, the latter having a higher alpha-amylolytic activity. The share of p-amylase in comparison with s-amylase in saliva of parotid glands is lesser than in saliva of submandibular and sublingual glands. In alimentary stimulation of sialosis, the saliva with higher amylolytic and tryptic activity, higher concentration of thyrotropin and thyroxine was found than under a non-alimentary stimulation. After the 1st and the 3rd hours following a trial breakfast, in response to a non-alimentary stimulation of sialosis the saliva was found to preserve properties of a postprandial saliva.     

Characterization of the differentiated antioxidant profile of human saliva

Saliva is armed with various defense mechanisms, such as the immunological and enzymatic defense systems. In addition, saliva has the ability to protect the mucosa against mechanical insults and to promote its healing via the activity of epidermal growth factor. However, another defense mechanism, the antioxidant system, exists in saliva and seems to be of paramount importance. The most interesting finding of the present study was the demonstration of the existence of much higher concentrations of the various salivary molecular and enzymatic antioxidant parameters in the parotid saliva compared with the submandibular/sublingual saliva. For example, peroxidase, superoxide dismutase, uric acid, and total antioxidant status were higher in resting parotid saliva compared with resting submandibular/sublingual saliva by 2405, 235, 245, and 147%, respectively. Another important finding was the distinction between the salivary antioxidant system and the immunological and enzymatic protective systems, as represented by the salivary concentrations of secretory IgA and lysozyme, respectively. These findings suggest that the profound antioxidant capacity of saliva secreted from parotid glands is related either to the different physiological demands related to eating (parotid predominance), to oral integrity maintenance (submandibular/sublingual predominance), or to the high content of deleterious redox-active transitional metal ions present in parotid saliva. This also may signify that our oral cavity environment is only partially protected against oxidative stress during most of the day and night.     

Purification of saliva agglutinin of<Emphasis Type="Italic"> Streptococcus intermedius</Emphasis> and its association with bacterial aggregation and adherence

Streptococcus intermedius strain 1208-1 cells were aggregated in the presence of saliva. The saliva agglutinin was purified by centrifugation, filtration, and gel filtration. SDS-PAGE analyses indicated that the purified agglutinin consisted of two high-molecular-mass proteins. Aggregation was dependent on calcium over pH 5.5, with 1 mM being the most effective concentration. Boiling inactivated purified agglutinin. S. intermedius strain 3 and Streptococcus mutans strain 1 were aggregated in the purified agglutinin. After adsorption with strain 1208-1 cells, the saliva sample did not exhibit any aggregation activity, and the agglutinin bands were no longer visible by SDS-PAGE. Adherence analyses demonstrated that the purified agglutinin immobilized on the surfaces of polystyrene wells, actinomyces cells, and apatite beads accounted for the binding of streptococcus cells. Agglutinin also effectively inhibited adherence to apatite beads coated with native saliva.     

Effect of Porphyromonas gingivalis ATCC 33277 Vesicle on Adherence of Streptococcus mutans OMZ 70 to the Experimental Pellicle

The vesicles of Porphyromonas gingivalis ATCC 33277 strongly aggregated Streptococcus cricetus, S. rattus, and S. mutans, but poorly aggregated S. sobrinus. The adherence of S. mutans OMZ 70 to hydroxyapatite (HA) coated with whole saliva was increased in parallel with the quantity of the vesicles. The significant increase of adherence of S. mutans OMZ 70 by the vesicles was also observed on the HA coated with parotid saliva, submandibular saliva, serum, and type I collagen. These findings suggest that the vesicles may act as a bridge between mutans streptococcus and the tooth surface.     

Differences in saliva collection location and disparities in baseline and diurnal rhythms of alpha-amylase: A preliminary note of caution

Identified in the early 1980s as a surrogate marker of the sympathetic nervous system component of the stress response, there has been renewed interest in measuring salivary alpha-amylase (sAA) to test biosocial models of stress vulnerability. This brief report presents studies that document that oral fluids from the parotid and submandibular gland areas had higher sAA values than did whole saliva specimens, and sAA values in whole saliva were higher than levels measured in oral fluids from the sublingual gland area. sAA in oral fluids from the parotid and submandibular gland areas showed the highest and more pronounced diurnal variation than levels in whole saliva, and sAA in sublingual saliva showed the lowest and shallowest diurnal variation. When this source of inherent variability in sAA activity levels is not controlled for by collecting oral fluids consistently from specific gland areas, the detection of individual differences, associations between sAA and “behavioral” variables, and intra-individual change in sAA levels may be compromised. Awareness, and management, of this ubiquitous source of measurement error in sAA are essential to ensure the success of future research on the correlates and concomitants of sAA levels, stress-related reactivity and recovery, and diurnal variation.     

Microstereological and histochemical studies of the salivary glands of the giant rat (Cricetomys gambianus, waterhouse)

The histology and histochemistry of the parotid, submandibular and sublingual glands were studied. The submandibular gland contained only serous acini as in the guinea pig, but unlike in many other mammals. The parotid gland contained only serous acini while the sublingual gland was mixed, mucous acini being the predominant secretory tissue interspersed by a few serous acini. Serous demilunes also commonly formed caps on the mucous acini. The ducts of the gland contributed over 30% of the volume of the submandibular gland, while those of the parotid and sublingual glands formed about 12 and 10% of the gland, respectively. The secretions of the parotid gland, as judged by histochemical methods, contained neutral mucins and some sialomucins. Neutral mucins, sulphomucins and sialomucins were detected in both the submandibular gland and sublingual gland.     

An in vitro study of initial adsorption from human parotid and submandibular/sublingual resting saliva at solid/liquid interfaces

The influence of saliva concentration, saliva total protein content and the wetting characteristics of exposed solids on in vitro film formation was studied by the technique of in situ ellipsometry. The rates and plateau values of adsorption (45 min) at solid/liquid interfaces (hydrophilic silica and hydrophobic methylated silica surfaces) were determinated for human parotid (HPS) and submandibular/sublingual (HSMSLS) resting saliva solutions (0.1 and 1.0%, (v/v), saliva in phosphate buffered saline). Adsorption rates were related to a model assuming mass transport through an unstirred layer adjacent to the surface. The results showed that the adsorption was rapid, concentration dependent and higher on hydrophobic than on hydrophilic surfaces. Analysis of the influence of protein concentration on the adsorbed amounts demonstrated an interaction between protein concentration and the two surfaces for HPS and HSMSLS, respectively. This may indicate differences in binding mode. Inter‐individual differences were found not to be significant at the 1% level of probability. Comparison of the observed adsorption and calculated diffusion rates suggest that on hydrophilic surfaces initial adsorption of proteins diffusing at rates corresponding to those of statherin and aPRPs takes place, whereas on hydrophobic surfaces lower molecular mass compounds appear to be involved.     

Expression and distribution of osteopontin and matrix metalloproteinase (MMP)-3 and -7 in mouse salivary glands

We investigated the expression and distribution of osteopontin in mouse salivary glands. Western blot analysis showed intense positive bands at the predicted molecular mass (about 60 kDa) in mouse parotid and sublingual glands. However, a cross-reacted band around 30 kDa was strongly detected in submandibular glands. Indirect immunofluorescent analysis showed that osteopontin was localized at the luminal (apical) membranes of the acinar cells in parotid and sublingual glands. However, it was not detected in acinar cells of submandibular glands. No expression was found in ductal cells of any glands. We also examined the expression of matrix metalloproteinase (MMP)-3 and -7. In parotid gland, MMP-3 was observed at 57 kDa, indicating a latent form, but MMP-7 was not detected. In contrast, MMP-7 definitely was observed at 28 kDa area in submandibular gland, whereas MMP-3 was not detected. These results suggest that osteopontin localizes at luminal sites of acinar cells and may be associated with saliva secretion in mouse salivary gland. It is also suggested that osteopontin may be cleaved by MMP-7 in mouse submandibular gland.     

Trafficking and postsecretory events responsible for the formation of secreted human salivary peptides: a proteomics approach

To elucidate the localization of post-translational modifications of different classes of human salivary proteins and peptides (acidic and basic proline-rich proteins (PRPs), Histatins, Statherin, P-B peptide, and "S type" Cystatins) a comparative reversed phase HPLC-ESI-MS analysis on intact proteins of enriched granule preparations from parotid and submandibular glands as well as parotid, submandibular/sublingual (Sm/Sl), and whole saliva was performed. The main results of this study indicate the following. (i) Phosphorylation of all salivary peptides, sulfation of Histatin 1, proteolytic cleavages of acidic and precursor basic PRPs occur before granule storage. (ii) In agreement with previous studies, basic PRPs are secreted by the parotid gland only, whereas all isoforms of acidic PRPs (aPRPs) are secreted by both parotid and Sm/Sl glands. (iii) Phosphorylation levels of aPRPs, Histatin 1, and Statherin are higher in the parotid gland, whereas the extent of cleavage of aPRP is higher in Sm/Sl glands. (iv) O-Sulfation of tyrosines of Histatin 1 is a post-translational modification specific for the submandibular gland. (v) The concentration of Histatin 3, Histatin 5, and Histatin 6, but not Histatin 1, is higher in parotid saliva. (vi) Histatin 3 is submitted to the first proteolytic cleavage (generating Histatins 6 and 5) during granule maturation, and it occurs to the same relative extent in both glands. (vii) The proteolytic cleavages of Histatin 5 and 6, generating a cascade of Histatin 3 fragments, take place after granule secretion and are more extensive in parotid secretion. (viii) Basic PRPs are cleaved in the oral cavity by unknown peptidases, generating various small proline-rich peptides. (ix) C-terminal removal from Statherin is more extensive in parotid saliva. (x) P-B peptide is secreted by both glands, and its relative quantity is higher in submandibular/sublingual secretion. (xi) In agreement with previous studies, S type Cystatins are mainly the product of Sm/Sl glands.     

Identification and characterization of tyrosylprotein sulfotransferase from human saliva

Tyrosylprotein sulfotransferase (TPST), the enzyme responsible for the sulfation of tyrosine residues, has been identified and characterized in submandibular salivary glands previously (William et al. Arch Biochem Biophys 338: 90-96). Tyrosylprotein sulfotransferase catalyses the sulfation of a variety of secretory and membrane proteins and is believed to be present only in the cell. In the present study, this enzyme was identified for the first time in human saliva. Analysis of human saliva and parotid saliva for the presence of tyrosylprotein sulfotransferase revealed tyrosine sulfating activity displayed by both whole saliva and parotid saliva at pH optimum of 6.8. In contrast to tyrosylprotein sulfotransferase isolated from submandibular salivary glands, salivary enzyme does not require the presence of Triton X-100, NaF and 5'AMP for maximal activity. Similar to the submandibular TPST, the enzyme from saliva also required MnCl2 for its activity. Maximum TPST activity was observed at 20 mM MnCl2. The enzyme from saliva was immunoprecipitated and purified by immunoaffinity column using anti-TPST antibody. Affinity purified salivary TPST showed a single band of 50-54 kDa. This study is the first report characterizing a tyrosylprotein sulfotransferase in a secretory fluid.     

Identification of protein components in human acquired enamel pellicle and whole saliva using novel proteomics approaches

Precursor proteins of the acquired enamel pellicle derive from glandular and non-glandular secretions, which are components of whole saliva. The purpose of this investigation was to gain further insights into the characteristics of proteins in whole saliva and in vivo formed pellicle components. To maximize separation and resolution using only micro-amounts of protein, a two-dimensional gel electrophoresis system was employed. Protein samples from parotid secretion, submandibular/sublingual secretion, whole saliva, and pellicle were subjected to isoelectric focusing followed by SDS-PAGE. Selected protein spots were excised, subjected to "in-gel" trypsin digestion, and examined by mass spectrometry (MS). The data generated, including peptide maps and tandem MS spectra, were analyzed using protein data base searches. Components identified in whole saliva include cystatins (SA-III, SA, and SN), statherin, albumin, amylase, and calgranulin A. Components identified in pellicle included histatins, lysozyme, statherin, cytokeratins, and calgranulin B. The results showed that whole saliva and pellicle have more complex protein patterns than those of glandular secretions. There are some similarities and also distinct differences between the patterns of proteins present in whole saliva and pellicle. MS approaches allowed identification of not only well characterized salivary proteins but also novel proteins not previously identified in pellicle.