Selachian tooth development: II. Immunolocalization of amelogenin polypeptides in epithelium during secretory amelogenesis in Squalus acanthias

We have determined the distribution of amelogenin polypeptides in an order of elasmobranchs using indirect immunofluorescence with rabbit polyclonal antibodies prepared to purified murine amelogenins. We find that amelogenins are definitely present within the inner enamel epithelium prior to the production of the extracellular matrix component termed "enameloid" (row II developing tooth organs). During subsequent stages of selachian tooth development (row III tooth organs), immunofluorescence staining data indicated localization of amelogenin antigens within epithelium as well as the enameloid extracellular matrix. The results from these immunohistochemical studies suggest that the 16-20 kdalton amelogenins, which are characteristic of murine inner enamel epithelial cells undergoing terminal biochemical differentiation into secretory ameloblasts, may also be regarded as molecular markers for amelogenesis in developing teeth in the spiny dogfish, Squalus acanthias.     

Fine structural and immunohistochemical detection of collar enamel in the teeth of <Emphasis Type="Italic">Polypterus senegalus</Emphasis>, an actinopterygian fish

This is the first detailed report about the collar enamel of the teeth of Polypterus senegalus. We have examined the fine structure of the collar enamel and enamel organ of Polypterus during amelogenesis by light and transmission electron microscopy. An immunohistochemical analysis with an antibody against bovine amelogenin, an antiserum against porcine amelogenin and region-specific antibodies or antiserum against the C-terminus, middle region and N-terminus of porcine amelogenin has also been performed to examine the collar enamel matrix present in these teeth. Their ameloblasts contain fully developed Golgi apparatus, rough endoplasmic reticulum and secretory granules. During collar enamel formation, an amorphous fine enamel matrix containing no collagen fibrils is found between the dentin and ameloblast layers. In non-demineralized sections, the collar enamel (500 nm to 1 μm thick) is distinguishable from dentin, because of its higher density and differences in the arrangement of its crystals. The fine structural features of collar enamel in Polypterus are similar to those of tooth enamel in Lepisosteus (gars), coelacanths, lungfish and amphibians. The enamel matrix shows intense immunoreactivity to the antibody and antiserum against mammalian amelogenins and to the middle-region- and C-terminal-specific anti-amelogenin antibodies. These findings suggest that the proteins in the enamel of Polypterus contain domains that closely resemble those of bovine and porcine amelogenins. The enamel matrix, which exhibits positive immunoreactivity to mammalian amelogenins, extends to the cap enameloid surface, implying that amelogenin-like proteins are secreted by ameloblasts as a thin matrix layer that covers the cap enameloid after enameloid maturation.     

Selective photooxidation of methionine, tyrosine and tryptophane using thionine and toluidine blue as sensitizers (author's transl)

Thionine and toluidine blue were used as sentizers on photooxidation processes of methionine, tryosine and tryptophane. They were more effective than methylene blue. Methionine was photooxidized to sulfoxide and tryptophane to kinurenine. A tyrosine-sensitizer addition compound was postulated. Dye concentration, pH, temperature and EDTA presence conditions were determined on each one of the modification reactions. Methionine at acid pH was selectively modified. On the basis of obtained results and published references, a direct interaction of singlet oxygen with methionine and tryptophase and the excited dye with tyrosine was respectively discussed.     

The ultrastructure of odontogenesis in larval and adult urodeles; differentiation of the dental epithelial cells

Summary Sections of undemineralized tooth germs ofAmbystoma andTriturus were examined. The ultrastructure of early germs, both larval and adult, and of dentinogenesis, resembled that of mammals. In adult bicuspid teeth, once the dentine of the cusps was mineralized, mineral crystals of a similar size to early mammalian enamel crystals, appeared between the dentine and the inner dental epithelium (i.d.e). Concomitantly, the i.d.e showed features of mammalian secreting ameloblasts. This new layer, regarded as true enamel, lacked collagen, possessed an ordered arrangement of crystals and reached a maximum thickness of 6 m.In larval monocuspid teeth, once dentine mineralization had reached the plasma membranes of the i.d.e at the tip of the cusp, the i.d.e developed a ruffled border. At this stage the dentine of the tip, regarded as enameloid, was very hard and difficult to section. The ruffled border, characteristic of other cells which transport materials, was regarded as indicating that the i.d.e was removing organic matter from the enameloid. The differences in development between larval and adult teeth support the concept that there is a change in cellular activity of the i.d.e which occurs during metamorphosis from the larval to the adult urodele.     

Nuclear stains with soluble metachrome metal mordant dye lakes. The effect of chemical endgroup blocking reactions and the artificial introduction of acid groups into tissues.

Following our study on the effect of deoxyribonucleic acid (DNA) extraction on nuclear staining with soluble metal mordant dye lakes covering 29 dye lakes we chose a series of lakes representing the three groups: (1) readily prevented by DNA removal, (2) weakened by DNA extraction but not prevented, (3) unaffected by DNA removal, for application of other endgroup blockade reactions. The lakes selected were alum and iron hematoxylins, iron alum and ferrous sulfate galleins, Fe2+ gallo blue E, iron alum celestin blue B, iron alum fluorone black and the phenocyanin TC-FeSO4 sequence. Azure A with and without an eosin B neutral stain, was used as a simple cationic (and anionic) dye control. Methylation was less effective than with simple cationic dyes, but did weaken celestin blue, gallo blue E and phenocyanin Fe2+ nuclear stains. These dyes also demonstrate other acid groups: acid mucins, cartilage matrix, mast cells, central nervous corpora amylacea and artificially introduced carboxyl, sulfuric and sulfonic acid groups. Alum hematoxylin stained cartilage weakly and demonstrated sulfation and sulfonation sites. The iron galleins, iron fluorone black and acid iron hematoxylin do not. A pH 4 iron alum hematoxylin gave no staining of these sites; an alum hematoxylin acidified with 1% 12 N HCl gave weaker results. Deamination prevented eosin and orange G counterstains but did not impair nuclear stains with any of the mordant dye lakes. The simple acetylations likewise did not alter mordant dye nuclear staining, the Skraup reagent gave its usual sulfation effect on other tissue elements, but did not alter nuclear stains by mordant dyes. The mordant dyes do not bind to periodic acid engendered aldehyde sites and p-toluidine/acetic acid and borohydride aldehyde blockades did not alter mordant dye lake nuclear staining. Nitration by tetranitromethane, which blocks azo coupling of tyrosine residues, did not alter nuclear staining by the mordant dye lakes. Benzil at pH 13, which prevents the beta-naphthoquinone-4-Na sulfonate (NQS) arginine reaction and the Fullmer reaction of basic nucleoprotein, did not affect iron gallein, iron or alum hematoxylin stains of nuclei or lingual keratohyalin.     

Shear Bond Strength and Fracture Analysis of Human vs. Bovine Teeth

Purpose

To evaluate if bovine enamel and dentin are appropriate substitutes for the respective human hard tooth tissues to test shear bond strength (SBS) and fracture analysis.

Materials and Methods

80 sound and caries-free human erupted third molars and 80 freshly extracted bovine permanent central incisors (10 specimens for each group) were used to investigate enamel and dentine adhesion of one 2-step self-etch (SE) and one 3-step etch and rinse (E&R) product. To test SBS the buccal or labial areas were ground plane to obtain appropriate enamel or dentine areas. SE and E&R were applied and SBS was measured prior to and after 500 thermocycles between +5 and +55°C. Fracture analysis was performed for all debonded areas.

Results

ANOVA revealed significant differences of enamel and dentin SBS prior to and after thermocycling for both of the adhesives. SBS- of E&R-bonded human enamel increased after thermocycling but SE-bonded did not. Bovine enamel SE-bonded showed higher SBS after TC but E&R-bonded had lower SBS. No differences were found for human dentin SE- or E&R-bonded prior to or after thermocycling but bovine dentin SE-bonded increased whereas bovine dentine E&R-bonded decreased. Considering the totalized and adhesive failures, fracture analysis did not show significances between the adhesives or the respective tooth tissues prior to or after thermocycling.

Conclusion

Although SBS was different on human and bovine teeth, no differences were found for fracture analysis. This indicates that solely conducted SBS on bovine substrate are not sufficient to judge the perfomance of adhesives, thus bovine teeth are questionnable as a substrate for shear bond testing.     

The structure and cytochemistry of the neurosecretory cells of Fasciola gigantica Cobbold and Fasciola hepatica L.

Histochemical studies of the nervous system of Fasciola gigantica and Fasciola hepatica were undertaken. Neurosecretory cells were detected by Gomori's aldehydefuchsin, Bargmann's chrome hematoxylin-phloxin, Mallory's triple stain, periodic acid-Schiff, Heidenhain's Azan and alcian blue after potassium permanganate oxidation. Two types of neurosecretory cells were recognized and designated as "A" and "B". Type "A" cells occurred in small numbers in the brain and subesophageal mass and type "B" cells ubiquitous in distribution. The reactions of these cells to the standard stains for neurosecretory substance generally, were less intense than the neurosecretory cells of other animals such as crustaceans and insects. The structure, organisation, distribution and cytochemistry of neurosecretory cells in Fasciola gigantica and Fasciola hepatica is discussed.     

Selective Demonstration of Histidine

Mounted paraffin sections of formalin-fixed tissue are treated for 24 hr at room temperature in an iodine solution (0.3% iodine, 0.6% potassium iodide) at pH 10 to block the aromatic nuclei of tyrosine and tryptophane. A coupled tetrazonium reaction using naphthanil diazo blue B (tetrazotized o-dianisidine) as a 0.1% solution at pH 9.2 for 15 min at 4°C, as the first coupling agent, and H acid (8-amino-1-naphthol-3, 6-dissulfonic acid), as a 2% solution at pH 9.2 for 15 min at 4°C, as the second coupling agent, stains sites of histidine a red-brown to red-purple color.     

Ameloblastic secretion and calcification of the enamel layer in shark teeth

Tooth primordia at early stages of mineralization in the sharks Negaprion brevirostris and Triaenodon obesus were examined electron microscopically for evidence of ameloblastic secretion and its relation to calcification of the enamel (enameloid) layer. Ameloblasts are polarized with most of the mitochondria and all of the Golgi dictyosomes localized in the infranuclear end of the cell toward the squamous outer cells of the enamel organ. Endoplasmic reticular membranes and ribosomes are also abundant in this region. Ameloblastic vesicles bud from the Golgi membranes and evidently move through perinuclear and supranuclear zones to accumulate at the apical end of the cell. The vesicles secrete their contents through the apical cell membrane in merocrine fashion and appear to contribute precursor material both for the basal lamina and the enameline matrix. The enamel layer consists of four zones: a juxta-laminar zone containing newly polymerized mineralizing fibrils (tubules); a pre-enamel zone of assembly of matrix constituents; palisadal zones of mineralizing fibrils (tubules); and interpalisadal zones containing granular amorphous matrix, fine unit fibrils, and giant cross-banded fibers with a periodicity of 17.9 nm. It seems probable that amorphous, non-mineralizing fibrillar and mineralizing fibrillar constituents of the matrix are all products of ameloblastic secretion. Odontoblastic processes are tightly embedded in the matrix of the palisadal zones and do not appear to be secretory at the stages investigated. The shark tooth enamel layer is considered homologous with that of other vertebrates with respect to origin of its mineralizing fibrils from the innerental epithelium. The term enameloid is appropriate to connote the histological distinction that the enamel layer contains odontoblastic processes but should not signify that shark tooth enamel is a modified type of dentine. How amelogenins and/or enamelins secreted by amelo- blasts in the shark and other vertebrates are related to nucleation and growth of enamel crystallites is still not known.     

Selective Demonstration of Histidine

Mounted paraffin sections of formalin-fixed tissue are treated for 24 hr at room temperature in an iodine solution (0.3% iodine, 0.6% potassium iodide) at pH 10 to block the aromatic nuclei of tyrosine and tryptophane. A coupled tetrazonium reaction using naphthanil diazo blue B (tetrazotized o-dianisidine) as a 0.1% solution at pH 9.2 for 15 min at 4°C, as the first coupling agent, and H acid (8-amino-1-naphthol-3, 6-dissulfonic acid), as a 2% solution at pH 9.2 for 15 min at 4°C, as the second coupling agent, stains sites of histidine a red-brown to red-purple color.     

Influence of Various Acidic Beverages on Tooth Erosion. Evaluation by a New Method

Material & Methods

We have analyzed the loss of enamel and dentine after exposure to different non-alcoholic drinks with a simple new method using bovine teeth. 100 enamel and 100 dentine specimens from freshly extracted bovine incisors were randomly attributed to 10 groups (n=10 for enamel and dentine each). Prior to the start of the experiment all specimens were weighed using a precision balance. The mean initial masses (SD) were 35.8 mg (7.2) for enamel and 24.7 mg (7.0) for dentine. No statistically significant differences were found between groups for initial masses (p>0.05, ANOVA with Bonferroni post hoc test). Thereafter, all specimens of one group were simultaneously placed in 200 ml of the following fluids: Coca-Cola, Coca-Cola light, Sprite, apple juice, Red Bull, orange juice, Bonaqua Fruits (Mango-Acai), tap water, chlorinated swimming pool water, and lemon juice. Fluids were continuously ventilated at 37° C for 7 days. Thereafter the specimens were weighed again and the mean mass loss was calculated.

Results

The values were (enamel/dentine): Coca-Cola 7.5 mg/6.6 mg; Coca-Cola light 5.2 mg/3.5 mg, Sprite 26.1 mg/17.7 mg, apple juice 27.1 mg/15.2 mg, Red Bull 16.6 mg/17.0 mg, orange juice 24.3 mg/20.2 mg, Bonaqua Fruits (Mango-Acai) 17.8 mg/16.2 mg, tap water -0.2 mg/-0.3 mg, swimming pool water -0.3 mg/-0.2 mg, and lemon juice 32.0 mg/28.3 mg. From all drinks, Cola and Cola light showed the least erosivity (p<0.001, ANOVA with Bonferroni post hoc test) whereas lemon juice showed statistically significant higher erosivity than all other drinks except Sprite and apple juice (p<0.01, ANOVA with Bonferroni post hoc test).

Conclusions

In conclusion, erosivity of common non-alcoholic drinks varies widely. For example, Sprite, apple juice, and orange juice are about five times more erosive than Coca-Cola light. The findings from the present study should be taken into account in choosing a diet that provides satisfactory nutrition while minimizing tooth erosion.     

Histochemistry of the duodenal glands of the cat and horse.

The duodenal glands of cat and horse were studied using PAS, Alcian blue, dialysed iron, aldehyde fuchsin-Alcian blue and high iron diamine stains. It was found that the duodenal glands of the horse reacted positively to Alcian blue, dialysed iron stains and also took the Alcian blue stain in the combined aldehyde fuchsin-Alcian blue and high iron diamine-Alcian blue stains. Those of the cat gave negative results. These results suggest the presence of acidic groups in the mucosubstances secreted by the horse's duodenal glands. A suggestion is put forward on the strength of the high iron diamine-Alcian blue combined stains that the acidity is due to the presence of carboxyl groups. It is suggested that the acidity may be significant in either cellulose metabolism or the digestion of the bacterial microflora from the stomach of herbivores.     

Iron Hematoxylin Chelates: II. Histochemistry of Myelin Sheath Stains

Aldehyde blockage, methylation, acetylation, amine blockage, and 4 min, 30 min and 24 hr deamination of paraffin sections of cat spinal cord were followed by staining with Lendrum's phloxine-tartrazine, Luxol fast blue MBS, PAS, phosphotungstic acid-hematoxylin, and Weil stains. Only the effects on staining of myelin are reported. These histochemical procedures separated the 5 stains into 3 groups: (1) phloxine-tartrazine and Luxol fast blue, (2) PAS, and (3) Weil and phosphotungstic acid-hematoxylin. The 3 patterns indicate that these stains may attach to 3 different reactive molecular sites in the sections. For the Weil and phosphotungstic acid-hematoxylin, such reactive sites are probably a secondary or tertiary amine or both.     

Visualization of initial bacterial colonization on dentine and enamel in situ

Bacterial colonization of dentine is of high relevance in cariology, endodontology and periodontology. The aim of the present in situ study was to establish recent methods for visualization and quantification of initial bacterial adherence to dentine in comparison to enamel. For this purpose, bovine enamel and dentine slabs were fixed on buccal sites of individual upper jaw splints worn by 6 subjects for 30 min, 120 min and 360 min, respectively. Adherent bacteria on the slabs were visualized and quantified with DAPI-staining (4′,6-diamidino-2-phenylindole) and fluorescence in situ hybridization (FISH) of streptococci and eubacteria using the CLSM (confocal laser scanning microscopy) as well as an epifluorescence microscope. In addition, the number of colony forming units was quantified after desorption. Representative samples were processed for SEM (scanning electron microscopy) and TEM (transmission electron microscopy). All methods clearly indicated that a significantly higher number of bacteria adhered to dentine than to enamel. Furthermore, the amount of bacteria on the dentine increased with increasing oral exposure time, but remained rather constant on the enamel. The CLSM allowed visualization of bacteria in the dentinal tubules. Bacteria were found preferentially at the openings of the dentine tubules, but were distributed randomly on the enamel.In conclusion, the adopted methods are suitable for visualization and quantification of bacterial adhesion to dentine. Even the initial bacterial colonization of dentine is much more pronounced than bacterial adherence to the enamel.     

Luxol Fast Blue as a Stain for Mallory's Alcoholic Hyaline

Luxol fast blue MBS (du Pont), which has frequently been used as a stain for phospholipids, stains Mallory's “alcoholic” hyaline a deep purplish blue. The stain is stable and provides histological appearances far superior to other methods. It is used on paraffin sections of tissue fixed in formalin or formalin-sublimate as a 0.1% solution in 90% alcohol at 60°C for 8 hr. Differentiation is made with 0.05% Li₂CO₃ and a red counterstain applied.     

Staining Reactions of some Anionic Disazo Dyes and Histochemical Properties of the Red Impurity in Trypan Blue

Some staining properties of 10 anionic disazo dyes are clarified by comparison with previous chromatographic analysis. Trypan blue contains both blue and red components and the purified blue fraction displays no color shifts in tissue sections. Evans blue, Niagara blue 2B, Niagara sky blue, Niagara sky blue 4B and Niagara sky blue 6B generally resemble trypan blue. Congo red is a metachromatic dye and the only known example among anionic dyes of established purity whose color shows shifts in tissue sections and also in solutions with certain basic compounds. Other red dyes (Congo corinth, trypan red and vital red) are not metachromatic. The red dye impurity of trypan blue selectively stains nuclei which are pycnotic, degenerating or undergoing no further division. This reaction is apparently related to basic protein content. Other reactions of the red fraction of trypan blue (mammalian erythrocytes, blood plasma) are not fully explained on this basis.     

Mechanism of dye response and interference in the Bradford protein assay

Bradford Coomassie brilliant blue G-250 protein-binding dye exists in three forms: cationic, neutral, and anionic. Although the anion is not freely present at the dye reagent pH, it is this form that complexes with protein. Dye binding requires a macromolecular form with certain reactive functional groups. Interactions are chiefly with arginine rather than primary amino groups; the other basic (His, Lys) and aromatic residues (Try, Tyr, and Phe) give slight responses. The binding behavior is attributed to Van der Waals forces and hydrophobic interactions. Assay interference by bases, detergents, and other compounds are explained in terms of their effects upon the equilibria between the three dye forms.     

THE PARTIAL REACTIVATION OF FORMOLIZED TOBACCO MOSAIC VIRUS PROTEIN

A marked reactivation of tobacco mosaic virus protein that has been partially or completely inactivated by formaldehyde was obtained by dialysis at pH 3. The activity of partially inactivated virus proteins was generally increased about 10-fold by the reactivation process. It was also found possible to reactivate completely inactive preparations to an appreciable extent. It was shown that the inactivation and the subsequent reactivation cannot be explained by the toxicity of the formaldehyde or of the formolized protein or by aggregation. Inactivation was accompanied by a decrease in amino groups as indicated by Van Slyke gasometric determinations and by colorimetric estimations using ninhydrin. Inactivation also causes a decrease in the number of groups that react with Folin''s reagent at pH 7.7. The latter are probably the indole nuclei of tryptophane, for it was demonstrated that tryptophane, glycyltryptophane, and indole propionic acid react with formaldehyde in a similar manner, while tyrosine and glycyltyrosine do not. Evidence that reactivation is accompanied by an increase in amino nitrogen and in groups that react with Folin''s reagent was obtained by colorimetric estimation. The demonstration that the addition of formaldehyde to the virus protein results in a simultaneous decrease of activity, of amino groups, and of groups that react with Folin''s phenol reagent, and that under conditions favorable for the removal of formaldehyde the virus activity is regained and the number of such groups increases, indicates that certain of these groups play at least a partial role in the structure necessary for virus activity. These changes can best be interpreted on the basis of known chemical reactions and are considered as evidence that virus activity is a specific property of the protein.     

Nuclear stains with soluble metachrome metal mordant dye lakes

Summary Following our study on the effect of deoxyribonucleic acid (DNA) extraction on nuclear staining with soluble metal mordant dye lakes covering 29 dye lakes we chose a series of lakes representing the three groups: (1) readily prevented by DNA removal, (2) weakened by DNA extraction but not prevented, (3) unaffected by DNA removal, for application of other endgroup blockade reactions. The lakes selected were alum and iron hematoxylins, iron alum and ferrous sulfate galleins, Fe²⁺ gallo blue E, iron alum celestin blue B, iron alum fluorone black and the phenocyanin TC-FeSO₄ sequence. Azure A with and without an eosin B neutral stain, was used as a simple cationic (and anionic) dye control.Methylation was less effective than with simple cationic dyes, but did weaken celestin blue, gallo blue E and phenocyanin Fe²⁺ nuclear stains. These dyes also demonstrate other acid groups: acid mucins, cartilage matrix, mast cells, central nervous corpora amylacea and artificially introduced carboxyl, sulfuric and sulfonic acid groups. Alum hematoxylin stained cartilage weakly and demonstrated sulfation and sulfonation sites. The iron galleins, iron fluorone black and acid iron hematoxylin do not. A pH 4 iron alum hematoxylin gave no staining of these sites; an alum hematoxylin acidified with 1% 12 N HCl gave weaker results.Deamination prevented eosin and orange G counterstains but did not impair nuclear stains with any of the mordant dye lakes. The simple acetylations likewise did not alter mordant dye nuclear staining, the Skraup reagent gave its usual sulfation effect on other tissue elements, but did not alter nuclear stains by mordant dyes.The mordant dyes do not bind to periodic acid engendered aldehyde sites and p-toluidine/acetic acid and borohydride aldehyde blockades did not alter mordant dye lake nuclear staining. Nitration by tetranitromethane, which blocks azo coupling of tyrosine residues, did not alter nuclear staining by the mordant dye lakes¹. Benzil at pH 13, which prevents the -naphthoquinone-4-Na sulfonate (NQS) arginine reaction and the Fullmer reaction of basic nucleoprotein, did not affect iron gallein, iron or alum hematoxylin stains of nuclei or lingual keratohyalin.Assisted by Contract Nol-CB-43912 National Cancer Institute     

New aspects of the histology of the mandibular condyle in the rat

The function of the multipotential mesenchymal cells in the mandibular condyle was studied histochemically and histologically in 27 Long Evans/Turku rats. Sagittal sections from the temporomandibular joint were stained with haematoxylin and eosin, toluidine blue, or van Gieson's stain. A weakly orthochromatically stained fibrous layer was followed in the upper region by a weakly metachromatically stained mesenchymal cell layer. Deep within this was a strongly metachromatically stained layer of immature chondroblasts. The metachromasia of the matrix of these layers disappeared abruptly in an anterior direction and gradually in a posterior direction. The changes in the staining reactions are explained by the fact that mesenchymal cells can differentiate into chondrogenic or osteogenic cells depending on the environmental conditions. A new hypothesis is presented according to which regulation of the direction of condylar growth is achieved by choosing the cells for chondrogenesis more posteriorly or anteriorly from among the mesenchymal cells covering the whole condylar cartilage.