Accessible sialic acid content of oral epithelial cells from healthy and gingivitis subjects.

The purpose of the present study was to determine if there were differences in the quantity of accessible sialic acid on superficial epithelial cells collected from different areas of the mouth, and from healthy subjects with good oral hygiene, as compared to subjects with gingivitis. Superficial epithelial cells were collected by gently scraping the tongue dorsum, hard palate, free gingiva and buccal epithelium. The cells were washed and treated with clostridial neuraminidase to release accessible sialic acid; this was quantitated using a fluorometric assay. Buccal cells released an average of 62.6 ng sialic acid per 10,000 cells, which was nearly 3-fold more than cells from the hard palate (24.1 ng), free gingiva (21.9 ng), or tongue (15.4 ng). Buccal and free gingival cells collected from 5 healthy subjects had significantly higher levels of accessible sialic acid on their surface than cells collected from 5 subjects with gingivitis. These differences were significant at the p less than 0.001 and p less than 0.01 levels, respectively. The data obtained suggest that the oral hygiene status of an individual can influence the quantity of accessible sialic acid residues on oral epithelium; this would be expected to influence the attachment and colonization of bacteria which bind to sialic acid-containing receptors.

Differentiation of cyclophosphamide-treated hamster secondary palate: ultrastructural and biochemical observations.

A study was undertaken to analyze the ultrastructural aspects and the enzyme acid phosphatase cytochemistry and biochemistry of the pathogenesis of cyclophosphamide (CP)-induced cleft palate in hamster fetuses. The initial CP-induced alterations were the appearance of lysosomes in the mesenchymal cells of the vertically developing palatal primordia within 8 hr of drug administration. The mesenchymal lysosomal activity, which increased during the next 16 hr, was abnormal and interpreted as a sub-lethal response to CP treatment. Subsequently, the lysosomal activity in the mesenchyme diminished gradually and, 48 hr after CP treatment, was absent. At this time, lysosomes were seen in the epithelial cells of the vertical palate. Fifty-six hours after CP treatment, unlike controls where palatal shelves were already fused, lysosomal activity subsided in the epithelial cells. Changes, however, continued to be seen at the epithelial-mesenchymal interface. These changes were characterized by discontinuity in the basal lamina, and by epithelial-mesenchymal contacts. They persisted for 8 hr but were absent thereafter. Sixty-four hours after CP administration, the vertical shelves became horizontal and remained so until term. Following analysis of data, both from the literature and from the present study, it was suggested that CP first affected mesenchymal cell proliferation, and then its cytodifferentiation, during the critical phase of early vertical development; consequently the reorientation of the shelves to a horizontal plane was delayed, inducing cleft palate.

Cytokeratins in hamster cheek pouch epithelium during DMBA-induced carcinogenesis.

The pattern of keratin expression in hamster cheek pouch epithelium during 15-wk of DMBA-induced carcinogenesis was studied. The sequential changes in cytokeratins of premalignant and malignant tissues and comparative investigation of normal epithelial tissues were examined during a weekly sequential DMBA-induced chemical carcinogenesis. Keratin polypeptides of normal pouch epithelium appear in a molecular weight range of 43-67 kd and 5-6 proteins can be identified. The disappearance of high molecular weight keratin (61-67 kd) was observed from the 6-wk DMBA-treated premalignant group to the 15-wk DMBA-treated malignant group. An additional keratin polypeptide was noted initially on the 11th-wk-DMBA-treated group and remained to the 15th-wk-DMBA treated group.

A biochemical study of glycosaminoglycans in the palatal rugae of the monkey (Macaca fascicularis).

Glycosaminoglycans (GAG) were extracted from the connective tissue of the palatal rugae, separated by electrophoresis and compared with the results obtained for the remaining palatal mucosal and gingival connective tissues. The GAG content of the rugae (3.01 mg/g defatted dry weight) was higher than in the remaining palatal mucosa (2.33 mg/g defatted dry weight) or gingiva (1.68 mg/g defatted dry weight). Dermatan sulphate was the predominant GAG in both the palatal rugae (48% of total GAG) and the remaining palatal mucosa (50%) followed by hyaluronic acid (33 and 31% respectively). The results do not support previous histochemical observations in which the rugae appeared to be rich in hyaluronic acid.

Calmodulin quantification and immunolocalization in developing embryonic orofacial tissue.

Temporally and quantitatively coordinated synthesis of cyclic adenosine monophosphate appears to be critical for normal development of the mammalian secondary palate. Calmodulin has been implicated as being involved in mediating the activity of a number of fundamental calcium-regulated intracellular enzyme systems including phosphodiesterases, adenylate cyclase, and a variety of kinases, all of which may regulate or be regulated by intracellular cAMP. Calmodulin levels were thus quantified, and endogenous calmodulin was immunolocalized in developing palatal tissue in vivo and in embryonic palatal mesenchymal cells in vitro. Endogenous palatal calmodulin levels, determined by radioimmunoassay, showed little variation during the period of murine palatal ontogenesis and averaged 0.23 ng/micrograms protein. Murine palate mesenchymal cells in monolayer, either in logarithmic growth or at confluency, contained similar levels of calmodulin. In palate mesenchymal cells in primary culture, specific anti-calmodulin staining was confined to the cell cytoplasm and was concentrated in the perinuclear region. Since immunostaining for calmodulin appeared to be associated with discrete cytoplasmic filaments, distribution of actin and tubulin were investigated. Immunostaining for tubulin in these cells was also localized to the perinuclear region, while immunolocalization of actin showed staining patterns, reflective of stress fibers, which were quite different from those seen for calmodulin. Immunostaining was seen in vivo in all regions of the palatal epithelium with superficial peridermal cells staining most intensely. Specific immunostaining was also evident in palatal mesenchyme, where a pericellular distribution was seen. Staining patterns were similar throughout the period of palatal ontogeny. In addition, a sharply defined localization of calmodulin to cartilagenous extracellular matrix was noted. This study provides a useful initial approach toward understanding the role calmodulin may play in embryonic orofacial development.

Histological localization of myxoid tissue in normal human palatal mucosa and its glycosaminoglycans.

Eighteen specimens of palatal mucosa were taken from 17 human subjects. Paraffin-wax sections were stained by routine methods and with various techniques to demonstrate glycosaminoglycans (GAG). In some sections, GAG were removed by selective degradative procedures before staining. Beneath all rugae, there were myxoid areas varying in size and marginal definition. Collagen fibres were few; elastic and reticulin fibres were numerous in a minority of sections. Alcianophilia at pH 2.5, preventable by streptomyces hyaluronidase digestion, suggested the presence of hyaluronic acid beneath the rugae. Alcian-blue staining at pH 1.0 and with the critical electrolyte concentration method using 0.5 M MgCl2 did not distinguish the myxoid tissue from the surrounding connective tissue and could be prevented by digestion with testicular hyaluronidase or chondroitinase ABC. Chondroitin sulphate and, or dermatan sulphate thus may be present but were not localized to the myxoid tissue. This unusual zone of loose connective tissue may act as a physical buffer resisting the local effects of high loads by allowing reversible extrusion of the water.

Mechanical factors in the evolution of the mammalian secondary palate: a theoretical analysis.

The secondary palate of mammals is a bony shelf that closes the ventral aspect of the rostrum. The rostrum, therefore, approximates to a tapered semicylindrical tube that is theoretically a mechanically efficient structure for resisting the forces of biting, including the more prolonged bouts of mastication typical of mammals. Certain mammal-like reptiles illustrate stages in the development of the palate in which the shelves projecting medially from each premaxilla and maxilla do not meet in the midline. We evaluate several geometric properties of sections through the rostrum of the American opossum (Didelphis virginiana). For loading at the incisors and canines, these properties indicate the structural strength and stiffness in both bending and torsion of the rostrum and of single maxillae. We then repeat the analysis but progressively omit segments of the palatal shelf, a procedure which simulates, in reverse, the evolutionary development of the structure. The results demonstrate that the secondary palate contributes significantly to the torsional strength and stiffness of the rostrum of Didelphis and to the strength of each maxilla in lateromedial bending. The major evolutionary implications of the results are that the rapid increase in rostral strength with small increments of the palatal shelves may have been a significant factor in the development of the complete structure. The results indicate that there was a marked jump in torsional strength and stiffness when the shelves met in the midline, which is likely to have been important in the subsequent development of the diverse masticatory mechanisms of cynodonts and mammals. On the basis of this analysis the mammalian secondary palate may be interpreted as one of a number of methods, seen in the mammal-like reptiles, for strengthening the rostrum.

Lipids of epidermis and keratinized and non-keratinized oral epithelia.

Lipid compositions have been determined by quantitative thin-layer chromatography for epidermis, floor of mouth, gingiva, palate and buccal epithelium of the pig (Sus scrofa). All of the epithelia contained phospholipids, glycosylceramides, cholesteryl sulfate, cholesteryl esters, free sterols, fatty acids and triglycerides. Epidermis, palate and gingiva had similar lipid compositions, which were characterized by the presence of O-acylglycosylceramides, O-acylceramides and relatively high proportions of ceramides. Floor of mouth and buccal epithelium, which are not keratinized, contained no O-acylglucosylceramides or O-acylceramides and very little ceramide. The higher water permeability of the non-keratinized oral regions may reflect the absence of acylglucosylceramides and the low proportions of ceramides.

Embryogenesis of stratified squamous epithelium. II. Developmental stage-specific immunoreactivity of keratins.

Throughout embryogenesis of the rat palate, from the early fetal to adult stages, a consistent subset of keratin proteins is synthesized in the epithelial lining cells. Although the relative abundance of particular keratins has been shown to vary with ongoing palatogenesis, the expression of finite keratins appears to be genetically predetermined. In order to preliminarily ascertain whether conformational changes accompanied intermediate filament 'maturation' from monomeric to polymeric keratin formation, we screened cytokeratins with polyclonal and monoclonal antibodies generated against adult-type keratins. Until epithelial stratification occurred on the 16th day of gestation, the keratin proteins were weakly immunoreactive. On the other hand, subsequent to epithelial thickening, adult-type immunoreactivity was initiated and progressed concomitantly with ongoing palatal development. These findings suggest that the cytokeratin intermediate filaments may progress through conformational 'maturation' during polymerization, and play a role in the eventual acquisition of the adult-type epithelial structure and function.

Difference picture algorithms for the analysis of extracellular components of histological images.

A computer-assisted method for objectively identifying and displaying the distribution of molecules that can only be positively identified by a combination of staining characteristics and susceptibility to specific enzymatic digestion or chemical degradation is presented. The visual image of an enzymatically digested tissue section is subtracted from that of an adjacent buffer-incubated control section and the distribution of the extracellular molecules removed from the tissue section displayed. Photomicrographs are taken using white light and narrow bandwidth filters of wavelengths at or near the maximum absorbance for the dye products used to visualize the extracellular matrix and cells. Each negative is standardized using reference gray levels. The cell and matrix images of both digested and undigested sections are then registered. The locations of cells in both control and digested sections are identified and set to an undefined gray level value in the matrix images. The cell-removed images of the control and digested sections are then registered and the difference in gray levels between the two images calculated and displayed. The validity of results obtained is primarily dependent on the soundness of the histological visualization and digestion techniques used, but is independent of investigator interpretation.

Electrophoretic study of keratin polypeptides in chemically-induced oral carcinomas in the hamster.

Comparison was made of the electrophoretic keratin polypeptide patterns of normal hard palate epithelia from three hamsters and of eight palatal squamous cell carcinomas induced by 7,12-dimethylbenz(a)anthracene (DMBA) treatment. Keratin polypeptides from normal epithelia had a molecular weight range of about 48,000 to 70,000. In the tumour extracts, the large polypeptides (above 61,000) found in the normal epithelia were absent, but the majority of other small polypeptides below 61,000 were expressed. Three as yet undefined polypeptides, in the range of 40,000 to 70,000, were detected in tumour extracts, but not in extracts of normal palatal mucosa. The keratin polypeptide electrophoretic alterations in carcinomas of hamster palatal mucosa are similar to those reported for extra-oral carcinomas in other animal species.

Differentiation of the avian secondary palate.

The avian secondary palate exhibits the unique feature of a midline cleft. Cryostat sections indicated that although extensive contact between homologous shelves was present, chick palatal medial edge epithelium (MEE) failed to fuse. The failure of fusion and subsequent clefting of the avian palate were correlated with continued proliferation of the avian MEE, a failure of selective MEE cell death, and an absence of elevated levels of intracellular cAMP. Moreover, immunohistochemical staining for cAMP and microspectrophotometric quantitation of staining intensity indicated that staining of chick MEE was significantly (p less than .01) less than murine MEE at comparable gestational ages. These data indicate that differentiation of the avian secondary palate is fundamentally different than reported for the mammalian palate in that many developmental events known to be associated with normal mammalian palate formation (cessation of MEE proliferation, MEE cell death, elevated levels of MEE cAMP) fail to occur in the chick. The developing avian secondary palate, with its midline cleft, thus provides an interesting and useful model system with which to compare mammalian palate formation where the palate is normally fused in the midline.

Immunofluorescent localization of fibronectin in human oral mucosa.

Fibronectin (FN) with an immunohistochemical fluorescence technique using anti-human FN and anti-rat FN sera was localized in the basement membrane of lip, buccal mucosa, palate, tongue, around ectopic sebaceous glands and around acini and ducts of labial salivary glands though in different amounts. In the mucosal lamina propria, FN was present in a net-like pattern. The highest concentrations of FN were in the palatal and tongue connective tissues. Much FN was present in the walls of small blood vessels and in perineural sheaths of peripheral nerves. No fluorescence was seen in the acini of labial salivary glands. In their striated ducts however, some cells were intensely fluorescent; the significance of this is unknown.

Genetic aspects of the effects of methylmercury in mice: the incidence of cleft palate and concentrations of adenosine 3':5' cyclic monophosphate in tongue and palatal shelf.

Concentrations of adenosine 3':5' cyclic monophosphate (cAMP) were measured in the tongues and palates of 14.5-day-old fetuses from control and methylmercury-treated mothers of four inbred lines of mice which represent the four possible combinations of two H-2 alleles and two residual genetic backgrounds. The incidence of cleft palate in fetuses from control and methylmercury-treated mothers was also examined. The H-2 alleles significantly affected the degree of reduction of cAMP concentration in palates seen in fetuses from mothers treated with methylmercury. Neither the H-2 allele nor the residual genetic background played a role in the effect of methylmercury on cAMP concentrations in fetal tongues. The magnitude of increase in the incidence of cleft palate with methylmercury treatment was approximately the same for all lines. Thus, methylmercury-induced cleft palate may not be mediated by the reduction of cAMP. Finally, fetuses with cleft lip had increased palatal cAMP levels, whether or not they were from control or methylmercury treated mothers.

Trauma affects cyclic AMP and DNA levels at injured and non-injured distal oral mucosal sites.

Experiments in rats were conducted to test the hypothesis that gingival trauma affects cyclic AMP and DNA levels at the gingival wound, and non-injured distal (gingival, palatal) sites. Cyclic AMP and DNA levels rose and fell in a cyclic fashion during the time (0.5-24 h) periods analyzed. Significant increases in cAMP levels occurred at 8 and 20 h and at 8 and 16 h, respectively, at the wound and non-injured palatal site, peripheral to the wound. Similar increases (not significant) in cAMP levels were also noted at the non-injured gingival contralateral site at the same time intervals. DNA distributions were found to be significantly greater 10 and 16 h after injury at the gingival wound, and distal non-injured gingival and palatal sites.

Immunohistochemical localization of cyclic AMP in the developing rodent secondary palate.

During development of the mammalian secondary palate, medial-edge epithelia (MEE) from apposing palatal shelves adhere and undergo autolysis allowing palatal mesenchymal regions to unite. In a prior study (Greene & Pratt, 1979), we reported a transient increase in levels of cyclic AMP (cAMP) in the mouse and rat palate during epithelial adhesion and cell death. The objective of this study was to examine the distribution of cyclic AMP in the developing rodent secondary palate using immunohistochemistry to localize cyclic AMP. Staining for cAMP was observed in the epithelium just prior to and during epithelial fusion (day 16 in the rat; day 14 in the mouse). Cyclic AMP was distributed throughout the epithelial cytoplasm whereas no staining was seen in nuclei. Epithelial staining for cAMP was faint or absent 24 and 48 h prior to epithelial contact. Mesenchymal staining for cAMP was minimal and associated with the plasma membrane at all stages studied. These results demonstrate that elevated levels of cAMP in the rat and mouse palate during epithelial adhesion and cell death are mainly due to increases of the nucleotide in palatal epithelium. This observation suggests that a transient increase in epithelial cAMP may play a role in palatal epithelial differentiation.

Isolation and characterization of epithelial and connective tissue cells from rat palate.

Epithelial and connective tissue cells were isolated from rat palate by sequential enzymatic digestion. Differences between the two populations were noted with respect to proline uptake and incorporation, % collagen synthesized, effects of parathyroid hormone and metabolism of D-valine. From these studies it can be concluded that the cell populations are viable and distinct with respect to the biochemical parameters examined.