Epithelial ultrastructure and the distribution of an estradiol sensitive antigen in the vagina of adult mice

Summary The distribution of an antigenic material specific for the cervicovaginal epithelium (CVA) was studied in the vaginal epithelium of the adult mouse with immunofluorescence and immunoferritin techniques. The antigen localization has been correlated to the fine structure of the vaginal epithelium in various states of functional activity. The antigen distribution in adult ovariectomized mice and in ovariectomized mice treated with estradiol was compared with that in normal cycling mice. CVA was found to be associated with the exterior of the cell membrane of vaginal epithelium cells, being part of the glycocalyx.Two cell types, mucous or keratinized, are derived from the germinative cell layer of the vaginal epithelium, depending on the hormonal environment. Mucous cells with morphological features that characterize cells about to cornify have been demonstrated. Fluorescence as well as ferritin particles, indicating the presence of antigen-antibody complexes, were seen within the mucous droplets of the mucous cells. The CVA production is apparently connected with vaginal mucus formation. The CVA distribution in the adult vaginal epithelium is discussed in relation to the distribution demonstrated earlier in the cervicovaginal epithelium of neonatal mice.This investigation was supported by grants from the Norwegian Cancer Society (Landsforeningen mot Kreft) and the Norwegian Research Council.     

Effect of traumatic occlusion on CGRP and SP immunoreactive nerve fibre morphology in rat molar pulp and periodontium

Summary Traumatic occlusion provides a trauma that affects the whole tooth and its supporting tissues. To study the effect of this trauma on CGRP and SP immunoreactive nerve morphology in pulp and periodontium, traumatic occlusion was induced in 2-months-old rats. The occlusal surface of the first maxillary molar in 30 rats were unilaterally raised 1 mm with a composite material. At different observation periods up to 30 days, the rats were transcardiacally perfused, the jaws demineralized, sectioned and processed for immunohistochemistry with the avidin-biotin-peroxidase method. Changes in nerve morphology, distribution and density in first and second molars and their supporting tissues were analyzed and compared in experimental (n=30) and control rats (n=14). Already after 5 days with traumatic occlusion, 22% of the experimental teeth had increased density of CGRP and SP immunoreactive nerves locally in gingiva, the periodontal ligament and the pulp, while in 15%, axonal proliferation and changed nerve morphology were found in the whole pulp (severe reaction). During a 20-day period, the pulpal nerve reactions progressed and included the whole pulp in 46% of the experimental teeth. The periodontal nerve responses were still localized only to the cervical and apical regions, and they remained local in these areas throughout the experimental periods. After 20 days the number of teeth with severe nerve changes seemed to decrease. The study shows that an unilateral change in occlusion of the first molar initiate nerve responses in the total molar dentition. In this experimental model the pulpal axons containing CGRP and SP reacted more serious to occlusal trauma than the nerves in the periodontium. The results indicate that the nerve changes in some cases might be transient.     

Identification of a novel putative signaling center,the tertiary enamel knot in the postnatal mouse molar tooth

The final shape of the molar tooth crown is thought to be regulated by the transient epithelial signaling centers in the cusp tips, the secondary enamel knots (SEKs), which are believed to disappear after initiation of the cusp growth. We investigated the developmental fate of the signaling center using the recently characterized Slit1 enamel knot marker as a lineage tracer during morphogenesis of the first molar and crown calcification in the mouse. In situ hybridization analysis showed that after Fgf4 downregulation in the SEK, Slit1 expression persisted in the deep compartment of the knot. After the histological disappearance of the SEK, Slit1 expression was evident in a novel epithelial cell cluster, which we call the tertiary enamel knot (TEK) next to the enamel-free area (EFA)-epithelium at the cusp tips. In embryonic tooth, Slit1 was also observed in the stratum intermedium (SI) and stellate reticulum cells between the parallel SEKs correlating to the area where the inner enamel epithelium cells do not proliferate. After birth, the expression of Slit1 persisted in the SI cells of the transverse connecting lophs of the parallel cusps above the EFA-cells. These results demonstrate the presence of a novel putative signaling center, the TEK, in the calcifying tooth. Moreover, our results suggest that Slit1 signaling may be involved in the regulation of molar tooth shape by regulating epithelial cell proliferation and formation of EFA of the crown.     

Radioimmunotherapy with alpha-particle emitters: microdosimetry of cells with a heterogeneous antigen expression and with various diameters of cells and nuclei

Intercellular variations in the level of antigen expression and in cellular and nuclear radii were taken into account in a model used to estimate cell survival for an in vitro experiment with antibodies containing alpha-particle emitters that target the cell surface. Using measured variations in these characteristics for cells of two human cancer cell lines, the model gave results for cell survival and the fundamental parameter of radiation sensitivity, z(0), that differ substantially from those obtained using only mean values. The cell survival may be underestimated by a factor of 100 if only mean values of these cellular parameters are used, and calculated values of z(0) may be overestimated by a factor of 2. Most of this effect stems from the variation in antigen expression. The magnitudes of the differences were found to be a function of the fractions of mean specific energy delivered by surrounding activity and by activity bound to the cells.     

Estradiol-17 β and cAMP: in vitro studies on the cervicovaginal epithelium of neonatal mice

Summary In organ culture of the cervicovaginal epithelium from neonatal mice, the epithelium synthesizes a material with specific antigenic properties (CVA). CVA was studied with immunofluorescence and the amount estimated semiquantitatively. In line with earlier studies, adenosine 3,5-cyclic monophosphate (dibutyryl derivative) (dcAMP), increased the amount of CVA, but adenosine 2,3-cyclic monophosphate did not. Addition of histamine to the culture medium moderately increased the amount of CVA, whereas the antihistamine diphenhydramine (H₁-antagonist) slightly reduced the strong increase induced by dcAMP and estradiol in combination. No effect was seen under similar conditions using the H₂-antagonist metiamide. Taken together with earlier results it is considered possible that the action of histamine and diphenhydramine is related to effects on the cell membranes. Phentolamine had no effect. The dcAMP effect was inhibited by actinomycin D and cycloheximide.This investigation was supported by grants from the Norwegian Research Council for Science and the Humanities     

Adenylate cyclase cytochemistry: a methodological evaluation

Synopsis A cytochemical method for the demonstration of adenylate cyclase activity has been evaluated. Enzyme activity in the epithelium of the Müllerian part of the vaginal anlage in neonatal, oestradiol-treated mice has been studied under different experimental conditions. The effects of fixation, incubation conditions and post-fixation have been studied. Variations in the amount of impurities and acid content of the glutaraldehyde do not seem to influence the enzyme activity. High Pb²⁺ ion concentration seems to promote unspecific staining. Under standard conditions [2 mM Pb (NO₃)₂, pH 7.2, and incubation temperature 30°C], neither non-enzymatic nor nonsubstrate-dependent lead trapping in the tissue could be observed. The possible contribution of other enzymes utilizing ATP and AMP-P(NH)P as the formation precipitate, has been evaluated. Both ATP and AMP-P(NH)P have been used as substrates in this study. Provided the appropriate control experiments are performed, this cytochemical method is reliable for demonstration of adenylate cyclase activity.     

High-Level Production of Human Parathyroid Hormone (hPTH) by Induced Expression inSaccharomyces cerevisiae

Saccharomyces cerevisiaewas used as host for high-level production of intact human parathyroid hormone (hPTH). The yield increased about 30-fold by changing from the constitutive MFα promoter to the inducibleCUP1promoter in the expression cassettes, use of another host strain, and optimization of growth conditions where especially the pH value was crucial. The secreted products consisted mainly of intact hormone, hPTH(1-84). In addition, two C-terminally truncated forms that lacked the four or five last amino acid residues, hPTH(1-80) and hPTH(1-79), were identified. These hPTH forms migrated aberrantly by SDS–PAGE as 14-kDa proteins, while the real masses measured by mass spectrometry on HPLC-purified products were about 9 kDa. Availability of such easily purified truncated forms will be valuable for studies of how the C-terminal residues affect the structure and function of the hormone. Combination of mutations and disruptions of the host genes encoding proteinase A, B, carboxypeptidase Y, and Kex1p or Mkc7p did not influence the C-terminal deletions. The secretion of hPTH could be enhanced by overexpression of the yeast syntaxin geneSSO2, but the total level of the hormone was not improved due to impaired growth.     

Effect of traumatic occlusion on CGRP and SP immunoreactive nerve fibre morphology in rat molar pulp and periodontium.

Traumatic occlusion provides a trauma that affects the whole tooth and its supporting tissues. To study the effect of this trauma on CGRP and SP immunoreactive nerve morphology in pulp and periodontium, traumatic occlusion was induced in 2-months-old rats. The occlusal surface of the first maxillary molar in 30 rats were unilaterally raised 1 mm with a composite material. At different observation periods up to 30 days, the rats were transcardiacally perfused, the jaws demineralized, sectioned and processed for immunohistochemistry with the avidin-biotin-peroxidase method. Changes in nerve morphology, distribution and density in first and second molars and their supporting tissues were analyzed and compared in experimental (n = 30) and control rats (n = 14). Already after 5 days with traumatic occlusion, 22% of the experimental teeth had increased density of CGRP and SP immunoreactive nerves locally in gingiva, the periodontal ligament and the pulp, while in 15%, axonal proliferation and changed nerve morphology were found in the whole pulp (severe reaction). During a 20-day period, the pulpal nerve reactions progressed and included the whole pulp in 46% of the experimental teeth. The periodontal nerve responses were still localized only to the cervical and apical regions, and they remained local in these areas throughout the experimental periods. After 20 days the number of teeth with severe nerve changes seemed to decrease. The study shows that an unilateral change in occlusion of the first molar initiate nerve responses in the total molar dentition. In this experimental model the pulpal axons containing CGRP and SP reacted more serious to occlusal trauma than the nerves in the periodontium. The results indicate that the nerve changes in some cases might be transient.