The historical development of salivary research in The Netherlands.
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Cystatins↗
Biomedical subjects
P A Roukema
Publications and source records attributed to P A Roukema.
Aggregation of oral bacteria by human salivary mucins in comparison to salivary and gastric mucins of animal origin.
Seventeen strains of oral bacteria of the genera Actinomyces (5), Bacteroides (3), and Streptococcus (9) were tested for aggregation by the human whole salivary mucin fraction (HWSM) in comparison to three types of animal mucin preparations from submandibular glands of cow (BSM) and sheep (OSM), and from the stomach of pig (PGM). Considerable variation was seen with respect to the rate and titer of aggregation induced by these mucins. The aggregating activity of HWSM varied widely among the different bacterial strains. The Bacteroides group showed hardly any induced aggregation, whereas the final aggregation titers varied for S. sanguis (3 strains) between 12 and 48, for S. oralis (3 strains) between 6 and 48, for the S. mutans group (3 strains) between 6 and 96, and for the five Actinomyces strains even between 6 and 192. For a particular strain, similar differences in titer were seen between the four mucins. For a human salivary mucin (MG-2) it has been described that sialic acid in the sequence NeuAc (alpha 2,3)Gal(beta 1,3)GalNac- was specifically involved in the interaction with S. sanguis strains, in contrast to S. rattus BHT. Our results, however, indicate that this sugar sequence is not a prerequisite for the aggregation of S. sanguis, as animal mucins, devoid of this structure, were equally well or even better capable of inducing aggregation. On the other hand, desialization of BSM and OSM largely abolished their aggregating capability towards S. rattus BHT. Moreover, it was found that BSM and OSM, which are comparable with respect to their major oligosaccharide structure, show considerable differences in aggregating activity towards the same bacterial strain.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
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.
Blood Group Antigens↗
Aggregation of 27 oral bacteria by human whole saliva. Influence of culture medium, calcium, and bacterial cell concentration, and interference by autoaggregation.
Twenty-seven oral strains of the genera Actinomyces (5), Bacteroides (3), and Streptococcus (19) were tested for aggregation by human whole saliva, as well as the effect of culture medium, Ca-ions, and bacteria concentration thereupon. Of the media tested, GF-broth gave rise to less interference by autoaggregation or higher aggregation titers than BHI and TSB, and was used throughout this study. In most cases, Ca-ions (1 mM) only enhanced the rate of induced aggregation, whereas raising the bacteria concentration increased the rate of both induced- and autoaggregation. The final titers, ranging from 1-64, were hardly affected by these parameters, except those of S. rattus HG 59 and S. mutans HG 199, which were respectively increased and decreased by Ca-ions. Saliva-induced aggregation was observed for 21 strains of A. viscosus, A. naeslundii, A. israelii, B. gingivalis, B. intermedius, S. cricetus, S. mutans, S. rattus, S. sanguis, and S. sobrinus, mostly within 15 min to 3 h. Seventeen of these strains also showed autoaggregation, usually well after the onset of induced aggregation. Any potential induced aggregation of B. gingivalis HG 91 was always masked by autoaggregation, as well as that of the S. mutans strains under a particular set of conditions. The aggregation rate and titer varied considerably in a mutually unrelated and strain-dependent way. These microtiterplate data were matched by the 5 spectrophotometric patterns observed for saliva-bacterial interaction, which moreover, gave the better differentiation between induced and autoaggregation. In conclusion, most strains tested can show rapid saliva-induced aggregation in a strain-dependent way, yet strongly affected by the experimental conditions and interference from autoaggregation.
Animals↗
Comparison of different assays for the aggregation of oral bacteria by human whole saliva.
For comparison, human whole saliva-induced aggregation was studied by phase-contrast microscopy, spectrophotometry combined with macroscopic observations, and in microtiterplate assay under identical experimental conditions for Actinomyces viscosus HG 85 (T14-V) and HG 380 (T14-AV), Bacteroides gingivalis HG 66 (W 83), Streptococcus rattus HG 59 (BHT), and Streptococcus sanguis I HG 169. The entire process of formation, extension, and sedimentation of aggregates could merely be observed by the combination of these assays. The very first stages of aggregation could only be detected and quantitated by phase-contrast microscopy. Within 2 1/2 min, 50% of the A. viscosus, S. rattus, and S. sanguis cells were aggregated, denoted as T50. In microtiterplates, however, aggregates were observed in general only after sedimentation at 30-45 min of incubation, expressed as TA. For interpretation of the spectrophotometric curves, additional microscopic and macroscopic data were a prerequisite. The small decline in absorbance during the first 30-45 min (phase 1) corresponded to the formation and extension of nonsedimenting aggregates, whereas the subsequent pronounced fall in absorbance (phase 2) was caused by the massive sedimentation of aggregates. The moment of inflexion between both phases, TI, marked the onset of sedimentation of aggregates and corresponded very well with TA, at which time already 92-98% of the cells were aggregated as quantitated by microscopy. In conclusion, only by microscopy the formation and extension of aggregates could be observed within a few minutes and quantitated in terms of aggregation rate. From 30-45 min, merely the sedimentation of aggregates was visualized in microtiterplates, whereas the time course of the overall process was recorded indirectly by spectrophotometry.
Actinomyces↗
Primary structure of O- and N-glycosylic carbohydrate chains derived from murine submandibular mucin (MSM).
The carbohydrate moiety of mouse submandibular mucin (MSM) contains mainly D-mannose and 2-acetamido-2-deoxy-D-glucose together with sialic acid, D-galactose, and 2-acetamido-2-deoxy-D-galactose. O-Glycosylically bound saccharides, obtained by treatment of MSM with alkaline borohydride, were shown by methylation analysis to have the structure: alpha-NeuAc-(2----3)-beta-Gal-(1----3)-GalNAc-ol. N-Glycosylically bound saccharides obtained from MSM by hydrazinolysis, and analysed by 500-MHz 1H-n.m.r. spectroscopy, were shown to have the following comprehensive structures. (Formula: see text).
Animals↗
Biochemical and immunochemical characteristics of the major protein of the murine parotid-granule membrane.
The membrane fraction (ParB) of the secretory granules of mouse parotid gland was isolated and characterized. The major phospholipids were phosphatidylcholine and sphingomyelin. The membranes contained one major protein, PMC, constituting at least 30 per cent of the total protein. PMC was purified: it is a small acidic protein with molecular weight of 12,000, containing one residue of phosphate per molecule. Using anti-PMC serum, PMC was detected only in the mouse parotid and saliva. Immunochemical characterization of organelle fractions indicated that PMC was mainly present in the secretory granule fraction; it was in part tightly bound to granule membranes. PMC was also present in both the 100,000 g parotid-tissue supernatant and the water-extract of the ParB granules. This dual localization was corroborated by immunofluorescent studies with anti-PMC serum which demonstrated that PMC was distributed uniformly over the acinar cells. The major protein component of these membranes is absent from other exocrine organs, e.g. pancreas, submandibular and sublingual glands.
Animals↗
Influence of human whole saliva on bacterial aggregation.
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Bacterial Adhesion↗
Biochemical characteristics and protein composition of murine saliva evoked in vivo by receptor-selective agonists.
We have demonstrated that the proteins of mouse saliva secreted in vivo are dependent on the nature of the stimulus, both qualitatively and quantitatively. The electrophoretic pattern of salivary proteins obtained by stimulation with phenylephrine is different from that evoked by carbamylcholine or isoproterenol. The electrophoretic pattern of alpha-adrenergic saliva largely resembles that of the proteins secreted in vitro by male submandibular glands, indicating that these proteins are predominantly derived from the granular convoluted tubular cells of submandibular glands. Secretion of proteins from acinar cells of parotid and submandibular glands was particularly stimulated by beta-adrenergic agonists and, to a lesser extent, by cholinergic agonists. The data from the in vivo induced salivas are in agreement with the morphological data on the regulation of the secretory process from parotid and submandibular glands. On the other hand, the secretory process in vitro does not occur specifically, whereas that in vivo does.
Animals↗
Study on membrane fragments released from the sublingual glands of the mouse during secretion in vitro and in vivo.
During in vitro secretion membrane fragments are released by the sublingual glands (SL) of the mouse. After stimulation of saliva with 1 microM carbamylcholine, these membranes have been isolated by centrifugation at 100,000 X g for 1 h. The release of 0.56% of the total tissue content of DNA during a 3 h period denotes that the amount of damaged tissue is very low. After 3 h, based on the determination of sialic acid, 25.4% of the secretory product, the sublingual mucin, has been released. The amount of membrane-bound alkaline phosphatase, released during a period of 3 h is 0.63%. As at least three quarters of this amount is due to broken cells, the amount of membrane-bound alkaline phosphatase released during the secretory process (0.16%) is very low compared to the amount of secretory product (25.4%). The low amount of alkaline phosphatase is in accordance with EM observations, which show that secretory granule membranes lack alkaline phosphatase activity. However, at those locations, where luminal membranes fuse with the granule membranes, alkaline phosphatase has been detected. So, the low alkaline phosphatase activity may be due to the presence of some luminal membranes in the secretory product. The involvement of alkaline phosphatase in the secretory process is also indicated by the complete inhibition of the secretory process by tetramisole (5 mM). The SL membrane preparation, isolated from the incubation fluid, had a relatively simple electrophoretic pattern and some antigenic determinants in common with the granular membranes from the Par and SM glands. The phospholipid composition of the released SL membranes differed strongly from the Par and SM granule membranes, especially in their relatively low amount of sphingomyelin (6.8%) and their high amount of phosphatidylethanolamine (26.4%). The lysophosphatidylcholine was only 3.5%. Among others, the phospholipid composition of the SL membranes may be responsible for the specific properties and behaviour of the sublingual granular membranes during the secretory process.
Alkaline Phosphatase↗
Immunochemical study and acinar localization of AM1, a murine submandibular glycoprotein with esterolytic activity.
Glycoprotein AM1, a glycoprotein from the submandibular glands of the mouse was isolated from the 100 000 X g tissue extract by polyacrylamide gel electrophoresis. An antiserum to purified glycoprotein AM1 was prepared, and its specificity was tested by immunodiffusion and immunoelectrophoresis. Glycoprotein AM1 could be detected in large quantity only in the submandibular glands of the mouse and in very small amounts in the parotid and sublingual glands and in serum. No glycoprotein AM1 was found in the murine brain, heart, lung, liver, spleen, kidney, pancreas, spinal cord and testis. In addition, glycoprotein AM1 was not detectable in the submandibular glands of the rat and rabbit, and in whole human saliva. No cross-reactivity was found with murine submandibular proteinase A and porcine pancreatic kallikrein. The cellular localization of glycoprotein AM1 was determined by the indirect immunofluorescence technique. In the submandibular glands bright fluorescence was only present in the acinar cells, throughout the whole gland. In the sublingual glands faint fluorescence was detectable as a diffuse network around the acini and possibly in the serous acinar demilune cells. On a subcellular level, glycoprotein AM1 could be demonstrated in the extract of the SMC secretory granular fraction, which originates largely from the acinar cells. On the other hand, glycoprotein AM1 was hardly detectable in the SMB secretory granular fraction, which originates predominantly from the granular convoluted tubular cells. Concomitantly, glycoprotein AM1 was secreted in vivo and could be detected in whole saliva, particularly after stimulation with isoproterenol and carbamylcholine, and also with phenylephrine, but to a much lesser extent.
Animals↗
AM1, a glycoprotein from the submandibular glands of the mouse, has esterolytic and amidolytic activities.
The previously isolated female submandibular glycoprotein AM1 ( Nieuw Amerongen , A.V., Vreugdenhil , A.P. and Roukema , P.A. (1977) Biochim. Biophys. Acta 495, 324-335) has been shown to have hydrolytic activity using N-alpha-benzoyl-L-arginine ethylester (BAEE) and ChromozymR PK as a substrate. AM1 can be secreted in vivo by isoproterenol, and to a lesser extent by carbamylcholine and phenylephrine. Based on BAEE as a substrate, AM1 has an optimum pH of 7.8. In female submandibular glands, about one-third of total esterolytic activity resided in glycoprotein AM1, but in male submandibular glands, less than 3%. The Km value of glycoprotein AM1 is 50 microM and its Vmax is 117 mumol/min per mg glycoprotein AM1. The enzymatic activity is not inhibited by Ca2+, Mg2+ and Na+, slightly by Cu2+ and strongly by Hg2+ and phenylmethylsulfonyl fluoride. Glycoprotein AM1 is capable of hydrolyzing ChromozymR PK with a turnover value 20-fold lower than that for BAEE. With ChromozymR PK as a test substrate, glycoprotein AM1 was purified by a factor of 11. With this substrate, glycoprotein AM1 has an optimum pH between 6.6 and 7.6. Also with chromozymR PK as a substrate, the submandibular glands of female mouse showed a much higher activity of glycoprotein AM1 than the submandibular glands of the male mouse. About 75% of all enzymatic activity of female submandibular glands resided in glycoprotein AM1 and in male submandibular glands 22%. The Km value is 57 microM and its Vmax 6.7 mumol/min per mg glycoprotein AM1. From the biochemical characteristics and the localization of glycoprotein AM1, it has been concluded that glycoprotein AM1 is not identical to any of the other described murine submandibular esteroproteinases , such as kallikrein, gamma-subunit of the nerve growth factor, proteinase A and proteinase F.
Amidohydrolases↗
Influence of autonomic agonists on the in vitro incorporation of [3H]leucine and N-acetyl[14C]mannosamine into submandibular action of the mouse.
The influence of isoproterenol and pilocarpine on the in vitro incorporation of [3H]leucine and N-acetyl[14C]mannosamine into the proteins of the submandibular glands of the mouse has been investigated during a 10 h period. The total uptake of both labelled precursors into the glands was hardly affected by isoproterenol and pilocarpine during the first 2 h of incubation, thereafter both agonists decreased the uptake slightly. The incorporation of [3H]leucine into secreted proteins was largely similar for the control, isoproterenol and pilocarpine during an incubation of 10 h. [14C]ManNAc incorporation showed a lag period of about 2 h and could be observed in the secreted proteins after 2 h. Particularly after 6 h a strong increase was observed for the control and isoproterenol, whereas pilocarpine showed a much lower increase. The secreted protein components were separated by electrophoresis to study the incorporation of the labelled precursors in separate secretory proteins such as submandibular mucin. Apparently, both agonists increased the incorporation of [14C]ManNAc relative to [3H]leucine into submandibular mucin of the mouse. During a period of 10 h the [14C]ManNAc incorporation into the mucin was enhanced 2-3-fold by isoproterenol and 3-4-fold by pilocarpine. A non-radioactive experiment in vitro showed that the molar ratio of the sugar residues did not change. However, the total amount of sugars relative to the amino acids increased by 50%, pointing to an increase in the degree of glycosylation. This suggests that both adrenergic and cholinergic agonists regulate the total number of carbohydrate chains attached to one and the same polypeptide core of the submandibular mucin of the mouse.
Amino Acids↗
Murine submandibular mucin (MSM): a mucin carrying N-and O-glycosylically bound carbohydrate-chains.
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Animals↗
Ultracentrifugation of salivary mucins.
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Amino Acids↗
Localization of amylase and mucins in the major salivary glands of the mouse.
Antibodies against murine submandibular and sublingual mucins have been raised in rabbits. Both antisera appeared to be specific. Using these antibodies, the mucins were localized in the acinar cells of the submandibular and sublingual glands respectively. The dyed amylopectin method was used to estimate the activity of amylase in the salivary glands. The enzyme was localized either by a starch-substrate film method or with antibodies against purified parotid amylase. The activity of amylase in parotid homogenates is about 1000-fold higher than that in homogenates of either submandibular or sublingual glands, in which the activity was comparable. Amylase was localized in the acinar cells of the parotid gland with both localization techniques. In the sublingual gland, amylase was found predominantly in the stroma around the acini, and there was some evidence that amylase was present in the demilune cells as well. In the submandibular gland, contradictory results were obtained with both techniques. With the starch-substrate film method, amylase activity was found in the granular convoluted tubular cells, whereas immuno-reactive amylase could only be demonstrated in the acinar cells of this gland. It is concluded that in the submandibular gland amylase and mucin are present in the same cell type.
Amylases↗
Influence of isoproterenol on the incorporation in vitro of [3H]-leucine and n-acetyl-[14C]-mannosamine into proteins and glycoproteins of the parotid glands of the mouse.
The in-vitro incorporation of [3H]-Leu in murine parotid glands started rapidly and continued during the whole incubation period of 10 h and was not stimulated significantly by isoproterenol, except during the first half hour. Secretion of [3H]-Leu incorporated was already observed after 30 min and continued up to 10 h. Isoproterenol stimulated the secretion of 3H-labelled protein during the whole period, but maximally after 4 h. Incorporation of [14C]-ManNAc proceeded at a much slower rate than that of [3H]-Leu. It was at least 1 h before incorporated [14C]-ManNAc appeared in the incubation medium. The secretion of [14C]-ManNAc incorporated was increased by isoproterenol between 2 and 6 h. A major part of [3H]-Leu was incorporated in amylase. The electrophoretically slowest moving isoenzyme of amylase incorporated substantial amounts of [14C]-ManNAc, in agreement with the presence of sialic acid in the isoenzyme. The onset of the [14C]-ManNAc uptake in amylase was slow compared to [3H]-Leu uptake. The [14C]-label could be detected in the secreted amylase after at least 4 h of incubation. It is suggested that isoproterenol decreased the incorporation of [14C]-ManNAc relative to that of [3H]-Leu into the secreted amylase.
Amylases↗
Secretory granules of murine salivary glands. A morphological, biochemical and morphological, biochemical and immunochemical study of submandibular and parotid granules.
Two secretory granular fractions from murine submandibular glands (SM) and one fraction from murine parotid glands (Par) were isolated by centrifugation on two discontinuous sucrose gradients. From the parotid glands the granular fraction was layered on 1.9 M sucrose (ParB), and in addition a second fraction was layered on 2.1 M sucrose (SMc). The contamination of the granular fractions by other cellular organelles was determined. The membranes of both the Parb and SMb granular fraction contained a major protein fraction with a molecular weight of 12-14,000 designated PMC and MMC, respectively. Their total amino acid composition was similar, but not their carbohydrate composition and immunochemical properties. The major protein within the Parb granules was amylase, and in addition AM2-glycoprotein was also present. Both secretory components had exclusively an acinar localization in the parotid glands, indicating the acinar origin of the Parb granules. In the SMb granular fraction, consisting of large secretory granules, amylase activity was detected by enzymatic method, and in addition the nerve growth factor and epidermal growth factor were demonstrated by immunochemical methods. These activities could only be localized in the SM granular tubular (GCT) cells, pointing to a GCT cellular origin of the SMb granules. On the other hand, immunoreactive amylase, AM2-glycoprotein, and submandibular mucin (MSM) were present in the SMc granular fraction. In the SM tissue sections these components were localized in the acinar cells. So, it is likely that the SMc granules are seromucous acinar granules.
Amylases↗