Global oral health inequalities: the view from a research funder.

Despite impressive worldwide improvements in oral health, inequalities in oral health status among and within countries remain a daunting public health challenge. Oral health inequalities arise from a complex web of health determinants, including social, behavioral, economic, genetic, environmental, and health system factors. Eliminating these inequalities cannot be accomplished in isolation of oral health from overall health, or without recognizing that oral health is influenced at multiple individual, family, community, and health systems levels. For several reasons, this is an opportune time for global efforts targeted at reducing oral health inequalities. Global health is increasingly viewed not just as a humanitarian obligation, but also as a vehicle for health diplomacy and part of the broader mission to reduce poverty, build stronger economies, and strengthen global security. Despite the global economic recession, there are trends that portend well for support of global health efforts: increased globalization of research and development, growing investment from private philanthropy, an absolute growth of spending in research and innovation, and an enhanced interest in global health among young people. More systematic and far-reaching efforts will be required to address oral health inequalities through the engagement of oral health funders and sponsors of research, with partners from multiple public and private sectors. The oral health community must be "at the table" with other health disciplines and create opportunities for eliminating inequalities through collaborations that can harness both the intellectual and financial resources of multiple sectors and institutions.

Substrate-induced conformational changes and dynamics of UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferase-2.

O-Glycan biosynthesis is initiated by the transfer of N-acetylgalactosamine (GalNAc) from a nucleotide sugar donor (UDP-GalNAc) to Ser/Thr residues of an acceptor substrate. The detailed transfer mechanism, catalyzed by the UDP-GalNAc polypeptide:N-acetyl-alpha-galactosaminyltransferases (ppGalNAcTs), remains unclear despite structural information available for several isoforms in complex with substrates at various stages along the catalytic pathway. We used all-atom molecular dynamics simulations with explicit solvent and counterions to study the conformational dynamics of ppGalNAcT-2 in several enzymatic states along the catalytic pathway. ppGalNAcT-2 is simulated both in the presence and in the absence of substrates and reaction products to examine the role of conformational changes in ligand binding. In multiple 40-ns-long simulations of more than 600 ns total run time, we studied systems ranging from 45,000 to 95,000 atoms. Our simulations accurately identified dynamically active regions of the protein, as previously revealed by the X-ray structures, and permitted a detailed, atomistic description of the conformational changes of loops near the active site and the characterization of the ensemble of structures adopted by the transferase complex on the transition pathway between the ligand-bound and ligand-free states. In particular, the conformational transition of a functional loop adjacent to the active site from closed (active) to open (inactive) is correlated with the rotameric state of the conserved residue W331. Analysis of water dynamics in the active site revealed that internal water molecules have an important role in enhancing the enzyme flexibility. We also found evidence that charged side chains in the active site rearrange during site opening to facilitate ligand binding. Our results are consistent with the single-displacement transfer mechanism previously proposed for ppGalNAcTs based on X-ray structures and mutagenesis data and provide new evidence for possible functional roles of certain amino acids conserved across several isoforms.

Characterization of the gene encoding the salivary Gln/Glu-rich C-terminal variant A protein.

The rat submandibular gland-specific GRP-Ca gene (encoding C-terminal variant A of the glutamine/glutamic acid-rich protein) has been cloned from a male Wistar/Furth genomic library. The complete sequence, including 2.0 kb of 5' flanking and 0.5 kb of 3' flanking DNA has been determined. Electron microscopic heteroduplex analysis and sequence analysis established that transcripts coding for GRP-Ca and GRP-Cb are encoded by separate genes. The GRP-Ca gene is approx. 4.5 kb in size and is comprised of four exons and three introns. Comparison of this gene with several rodent and human salivary proline-rich protein-encoding genes (PRP) indicates that GRP-Ca shares this exon-intron structure with the rat SMR-2 gene, the hamster H29 gene, and the human PRP genes. In addition, a 28-bp element found in the proximal promoter region of GRP-Ca was found to be highly conserved among the superfamily of PRP genes.

Light and electron microscopic immunolocalization of rat submandibular gland mucin glycoprotein and glutamine/glutamic acid-rich proteins.

We studied the subcellular localization of two major secretory products of adult rat submandibular gland (RSMG), blood group A-reactive mucin glycoprotein and glutamine/glutamic acid-rich protein (GRP), by light and electron microscopic immunocytochemistry. The structure of the major neutral oligosaccharide of the mucin was shown to be: GalNAc alpha 1,3(Fuc alpha 1,2)Gal beta 1,3GalNAc. A mouse monoclonal antibody (1F9) with specificity for blood group A determinants was prepared against the mucin. The antibody recognized a single band of approximately 114 KD on Western blots of RSMG extract. A previously characterized monoclonal antibody (59) against GRP (Mirels et al.: J Biol Chem 262: 7289, 1987) reacted with a doublet of 45-50 KD on Western blots of extraparotid saliva. Immunofluorescence and immunoperoxidase staining of cryostat sections of RSMG with anti-mucin antibodies and anti-GRP antibodies revealed reactivity in acinar cells of the gland. No specific labeling was seen in duct cells of RSMG or in mucous acinar cells of the adjacent sublingual gland. Post-embedding immunogold labeling of thin sections of glutaraldehyde-fixed RSMG with anti-mucin showed strong labeling of the Golgi apparatus and secretory granules of acinar cells. Gold particles were seen mainly over electron-lucent areas of the granules. No labeling occurred over the endoplasmic reticulum. The labeling pattern with the anti-GRP antibodies was similar, except that both electron-dense and -lucent areas of the granules were labeled, and the endoplasmic reticulum was reactive. Double labeling with two different sizes of gold particles showed that both mucin and GRP co-localized in the same granules. Pre-absorption of the antibodies with their respective antigens eliminated immunolabeling of the acinar cells. These antibodies will be useful in studies of cell differentiation in RSMG and of synthesis, processing, and packaging of RSMG secretory products.

A comparison of serine and threonine O-glycosylation by UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase.

O-glycosylated proteins are ubiquitous in eukaryotes and are responsible for a variety of biological functions. O-glycosylation is initiated by the addition of N-acetylgalactosamine to serine or threonine residues, though it is not clear how specific residues are selected for modification. We have compared serine and threonine glycosylation using peptide substrates based on sequences from erythropoietin (EPO) and von Willebrand factor (HVF) that are glycosylated in vivo. UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase was derived from rat parotid, submandibular, and sublingual glands, liver and kidney as well as from human colostrum. The threonine-containing substrates were glycosylated to a much greater extent than those containing serine for all the enzyme sources. Changes in reaction pH, donor concentration, or divalent cation were unable to increase glycosylation of serine. When the incubation time was extended, serine in the EPO-based peptide was found to incorporate GalNAc at a low level, in contrast to the serine-containing HVF peptide, which did not glycosylate at all. By circular dichroism, the non-glycosylating peptide was the only one of the series that did not exhibit random coil structure. Our data suggest that although the structural and sequence requirements for O-glycosylation of serine and threonine residues are similar, serine sites are glycosylated less effectively than are threonine sites in vitro.

Artificial salivas: present and future.

Modern technology has allowed us to understand better the functions of saliva and now provides a rationale for developing: (1) diagnostic reagents for monitoring oral and systemic health status and (2) replacement therapies for individuals with salivary dysfunctions. Several areas of dental research are directed at augmenting or enhancing both the quality and quantity of saliva for individuals with dry mouth. An "intrinsic" approach is being explored which utilizes medications such as pilocarpine and bromhexine to stimulate the salivary glands to produce more saliva. An "extrinsic" approach proposes to use topically applied artificial saliva. Studies in our laboratory have been directed toward developing artificial salivas which incorporate many of the protective features of "native" saliva. An ideal artificial saliva should be "long-lasting", provide lubrication, inhibit colonization of microflora responsible for dental caries and gingivitis, and coat the oral soft tissues for protection against environmental insult and desiccation. Studies are currently under way to determine the structural requirements of salivary molecules responsible for these protective functions. Composite salivary molecules consisting of multiple biologically active or "functional domains" could then be designed and synthesized based upon primary sequence and conformational analyses, computer-assisted structural predictions, and in vitro testing. These supersalivary substances could then be used as saliva substitutes for targeting to selected oral surfaces to promote mineralization, hydration, and/or regulate microbial-mediated disease.

Neuraminidase activity: a biochemical marker to distinguish Streptococcus mitis from Streptococcus sanguis.

Selected reference and freshly isolated strains of Streptococcus mitis (mitior) and Streptococcus sanguis were assayed for cell-associated neuraminidase activity by their ability to hydrolyze [3H-] sialyllactitol. A cell-associated neuraminidase was detected with S. mitis and S. sanguis serotype II (reclassified as S. mitis) but not with S. sanguis serotypes I and III. Neuraminidase activity of S. mitis correlated with this organism's inability to hydrolyze arginine, aesculin, and few, if any, sugars. The findings indicate that the presence of cell-associated neuraminidase activity is useful for the taxonomic classification of S. mitis.

Characterization of low-molecular-weight peptides in human parotid saliva.

The low-molecular-weight components of human saliva remain poorly characterized. Therefore, low-molecular-weight peptides (Mr < 3000) have been purified from human parotid saliva and characterized with respect to their amino acid sequence. From the sequences obtained, it is likely that these peptides are derived from proteolysis of the hydroxyapatite-interactive human salivary proteins, histatins, proline-rich proteins, and statherins. Since human parotid saliva is an amicrobial fluid, much of the low-molecular-weight peptide fraction of this secretion appears to be derived from the proteolytic processing of the larger proteins. Because of their small size, these peptides are likely to be in exchange with dental plaque fluid and may therefore help modulate events such as demineralization/remineralization, microbial attachment, and dental plaque metabolism at the tooth-saliva interface.

Characterization of cysteine-containing phosphoproteins from human submandibular-sublingual saliva.

Members of a family of acidic proteins taken from human submandibular-sublingual saliva were designated cysteine-containing phosphoproteins, since they could be distinguished from other salivary phosphoproteins by the presence of half-cystine. These molecules consisted of a single peptide chain of approximately 14,000 daltons. Their isoelectric points ranged from 4.3 to 5.9. Two groups (C2 and C3) were O-phosphorylated. Their charge heterogeneity was apparently due to variations in content of phosphate and acidic and basic amino acids.

Lipid composition of submandibular saliva from normal and cystic fibrosis individuals.

The submandibular saliva of patients with cystic fibrosis was found to contain about 66% more lipids/100 ml of saliva than that of normal individuals and exhibited elevated levels of neutral lipids, phospholipids, and glycolipids. No significant differences were noted in the proportions of individual neutral lipid and phospholipid components present in both types of samples. The glycolipids of normal saliva consisted entirely of glyceroglucolipids, whereas those of cystic fibrosis saliva, in addition to glyceroglucolipids, also contained small amounts of glycosphingolipids. These quantitative and qualitative differences may affect the physicochemical properties of the secretion.

Juvenile periodontitis as a model for neutrophil function: reduced binding of the complement chemotactic fragment, C5a.

The binding of the chemotactic complement fragment, C5a, to peripheral blood neutrophils of Localized Juvenile Periodontitis (LJP) patients and normal controls was quantitated using iodinated human C5a and a rapid centrifugation assay. There was a significant reduction in the number of binding sites per cell on neutrophils from the patient group, whereas the binding affinity remained the same as control values.

The effect of chronic atropine treatment on salivary composition and caries in rats.

Many instances of salivary dysfunction in humans can be traced to the use of medications that have hyposalivary side-effects. In this study, atropine, a cholinergic antagonist, was administered chronically to rats by use of osmotic mini-pumps. Steady-state blood levels, similar to levels obtained in human multiple oral dosing, were thus maintained. Atropine delivered in this manner for 24 days was found to decrease protein concentration of parotid saliva (p less than 0.05) elicited by pilocarpine, and to increase smooth-surface caries scores (p less than 0.05) in rats fed a cariogenic diet. Parotid saliva collected via ductal cannulation from rats subjected to chronic atropine administration (and stimulated to secrete by pilocarpine) exhibited increased levels of two basic proline-rich proteins (Peak A and SP-3), as evaluated by SDS-PAGE, compared with those observed in saliva from controls. Cannulation of sublingual glands in animals receiving high doses of atropine produced no measurable secretion upon pilocarpine stimulation. Carbachol stimulation of dispersed cell aggregates of sublingual glands from sham-operated and high-dose atropine groups indicated that the glands responded similarly once the antagonist was washed from the system, implying that the lack of secretion in vivo was caused by antagonism of the cholinergic receptor by atropine. Our observations suggest that this model system can be exploited for determination of the effects of chronic administration of hyposalivary drugs on salivary composition and caries rates.

Age-related changes in mucins from human whole saliva.

The predominant mucins in human whole saliva, MG1 and MG2, serve to protect and to lubricate the oral cavity. In this study, both unstimulated and stimulated whole salivas were collected from two groups of subjects: young (18-35 years of age) and aged (65-83 years of age). The subjects were in apparent good health. Saliva samples from each subject were analyzed by SDS-PAGE. The gels were stained with Stains-all, and both MG1 and MG2 were quantitated by video-image densitometry. The protocol gave reproducible values for each mucin. The stimulated and unstimulated salivas from aged subjects showed significant reductions in concentrations of both MG1 and MG2, as quantitated in mucin dye-binding units. Possible associations of these reductions with the aging process are discussed.

The association of basic proline-rich peptides from human parotid gland secretions with caries experience.

To address whether there are associations between the peptide composition of human parotid saliva and dental decay (caries) experience, we have characterized the peptides from parotid ductal saliva collected from nine adults who have remained free from dental caries (mean age = 59.2; Decayed Missing Filled Surfaces index [DMFS] = 0) and nine individuals who have experienced caries (mean age = 51.2; mean DMFS = 38.4). Ethanol-soluble peptides were size-fractionated on columns of Bio-Gel P-2; the salivary peptides derived from caries-susceptible subjects appeared larger than those found in the saliva of caries-free subjects. Peptides were then resolved into 19 species by cation exchange HPLC. Sequence analysis identified 18 peptides that appear to be proteolytic cleavage products of the basic proline-rich proteins IB-4, IB-5, IB-7, IB-8b, and P-B. The peptides that were more abundant in saliva obtained from the caries-free group differed from those isolated from the caries-susceptible group. The median peptide concentration of one possible precursor protein, IB-7, was found to be higher in saliva collected from caries-free individuals than in that from caries-susceptible individuals. Although differences were found in the phenotypes of proline-rich proteins expressed by these groups of caries-free and caries-susceptible subjects, no statistically significant associations were observed among proline-rich phenotypes and the level of any peptide. Collectively, our results indicate that proteolytic processing of parotid salivary proteins differs among individuals who have remained caries-free and those who have experienced dental decay.

Association of free arginine and lysine concentrations in human parotid saliva with caries experience.

We determined the free-amino acid content of stimulated parotid (ductal) saliva from two groups of adult subjects whose caries experiences were markedly different. The levels of free arginine and free lysine in the parotid saliva of caries-free adults were significantly higher than those found in the parotid saliva of individuals with a history of dental decay. There was no correlation, however, between the levels of these amino acids and the DMFS score within the caries-susceptible groups. Microbial catabolism of dibasic amino acids contributes to the neutralization of plaque acids and may partially account for the higher resting plaque pH observed in caries-free subjects. Alternatively, the elevations observed in free levels of arginine and lysine may reflect a systemic alteration in amino acid metabolism which is common to the caries-free group of subjects.

Adherence of Streptococcus sanguis to salivary mucin bound to glass.

This study demonstrated that human submandibular-sublingual saliva (HSMSL) provided a better substrate than did whole saliva or parotid saliva for the binding of Streptococcus sanguis in a glass adherence assay. Additional evidence indicated that the lower molecular weight salivary mucin in HSMSL was involved in these interactions. Mucin's sialic acid residues were found to play a major role in mediating the binding of certain strains of Streptococcus sanguis.

Structural aspects of salivary glycoproteins.

The protective functions of saliva are attributed, in part, to its serous and mucous glycoproteins. We have studied, as representative molecules, the proline-rich glycoprotein (PRG) from human parotid saliva and the high (MG1) and low (MG2) molecular weight mucins from submandibular-sublingual saliva. PRG (38.9 kDa) contains 40% carbohydrate consisting of 6 triantennary N-linked units and a single peptide chain of 231 amino acids, 75% of which = PRO + GLY + GLN. PRG's secondary structure is comprised of 70% random coil (naked regions) and 30% beta-turns (glycosylated domains). MG1 (greater than 10(3) kDa) contains 15% protein (several disulfide linked subunits), 78% carbohydrate (290 units of 4-16 residues), 7% sulfate, and small amounts of covalently linked fatty acids. MG2 (200-250 kDa) contains 30% protein (single peptide chain), 68% carbohydrate (170 units of 2-7 residues), and 2% sulfate. The major carbohydrate units of MG2 are: NeuAc alpha 2,3Gal beta 1,3GalNAc,Gal beta 1,3GalNAc, and Fuc alpha 1,2Gal beta 1,3GalNAc. MG1 contains hydrophobic domains, as evidenced by its ability to bind fluorescent hydrophobic probes; MG2 does not. Collectively, the biochemical and biophysical comparisons between MG1 and MG2 indicate that these two mucins are structurally different. Several functional properties of MG1, MG2, and PRG have been examined, including their presence in two-hour in vivo enamel pellicle, binding to synthetic hydroxyapatite, lubricating properties, and interactions with oral streptococci. The data presented suggest that these glycoproteins may have multiple functions which are predicated, in part on their carbohydrate units. The potential significance of the structure-function relationships of these glycoproteins to the oral ecology is discussed.

Purification of a low-molecular-weight, mucin-type glycoprotein from human submandibular-sublingual saliva.

A low-molecular-weight, monomeric, mucin-type glycoprotein (MG2) has been isolated from human submandibular-sublingual saliva. Initial purification involved sequential gel-filtration on Sephadex G-200 and Sepharose CL-2B, the latter in the presence of 6M urea. Fractions containing MG2 were next separated from contaminating secretory IgA by immunoaffinity chromatography or recycling through Sephadex G-200. Mucin fractions were 14C-labeled by reductive methylation, and then the final purification-step entailed recycling radiolabeled materials through Sephadex G-200. Radiolabeling aided in the assessment of purity, as judged by SDS-PAGE and ion-exchange chromatography. The carbohydrate portion accounted for 69.6% of the recovered weight and was composed of N-acetyl-glucosamine, N-acetylgalactosamine, galactose, fucose, and N-acetylneuraminic acid. Sulfate was also present. The protein comprised 30.4% of the recovered weight with threonine, serine, proline, and glycine accounting for 75.2% of the total amino acids. The oligosaccharides were alkali-labile, indicating an O-glycosyl linkage to the peptide. The mucin was weakly acidic and had an estimated mol. wt. of 200 000-250 000.

Isolation and characterization of tracheobronchial mucin from a laryngectomee.

A tracheobronchial mucin was isolated from the tracheobronchial secretion of a laryngectomee. It was purified by gel filtration on Sepharose CL-6B in Tris-urea buffer and rechromatography of excluded materials through the same gel matrix. It was homogeneous in 0.7% agarose-2% polyacrylamide electrophoresis under nonreducing conditions. Comparable analysis with 2-mercaptoethanol revealed at least 3 subunits. Based upon recoverable weight, the mucin was composed of 75% carbohydrate, 21% protein, and 3% sulfate. Oligosaccharides obtained by alkaline beta-elimination indicated O-glycosyl linkage to the peptide component. Marked heterogeneity of the carbohydrate side-chains was reflected in the preparation of 20 distinct oligosaccharides ranging in size from 4 to 17 residues.

A new assessment in vitro of human salivary lubrication using a compliant substrate.

The lubrication effect of salivary secretions was assessed in terms of separating a rigid object from a compliant substrate. There was little difference among the various secretions of a single donor. The viscosity of salivas increased as a function of time. Neither the friction testing nor viscometry provided an adequate model of the tissue-coating function ascribed to saliva.