Immunohistochemical localization of the differentiation marker E11 in dental development of rats

E11 antigen, originally characterized in a rat osteosarcoma cell line, is known to be expressed during late stages of the osteogenic cell lineage both in vitro and in vivo. The aim of the present study was to monitor the occurrence and distribution patterns of the E11 antigen using monoclonal antibodies (mAb E11 and MEP-1) during different stages of tooth germ development of new-born rats by means of immunohistochemistry. Both antibodies strongly bound to plasma membranes of ameloblasts in presecretory and secretory stages. In addition, odontoblasts and cells of the periodontium were immunoreactive for E11 and MEP-1. During maturation, the immunoreactivity of ameloblast plasma membranes decreased significantly. Our data suggest that E11 and MEP-1 might be important as markers for cell differentiation and mineralization processes during tooth germ development.     

Betamethasone effects on fetal sheep cerebral blood flow are not dependent on maturation of cerebrovascular system and pituitary–adrenal axis

Synthetic glucocorticoids are administered to pregnant women in premature labour to accelerate fetal lung maturation at a time when fetal cerebrovascular and endocrine systems are maturing. Exposure to glucocorticoids at 0.8–0.9 of gestation increases peripheral and cerebrovascular resistance (CVR) in fetal sheep. We examined whether the increase of CVR and its adverse effect on cerebral blood flow (CBF) depend on the current level of maturation of the pituitary–adrenal axis and the cerebrovascular system. Using fluorescent microspheres, regional CBF was measured in 11 brain regions before and 24 h and 48 h after the start of 3.3 μg kg⁻¹ h⁻¹ betamethasone ( n = 8) or vehicle ( n = 7) infusions to fetal sheep at 0.73 of gestation. Hypercapnic challenges were performed before and 24 h after the onset of betamethasone exposure to examine betamethasone effects on cerebrovascular reactivity. Betamethasone exposure decreased CBF by approximately 40% in all brain regions after 24 h of infusion ( P < 0.05). The decline in CBF was mediated by a CVR increase of 111 ± 16% in the cerebral cortex and 129 ± 29% in subcortical regions ( P < 0.05). Hypercapnic cerebral vasodilatation and associated increase in CBF were blunted ( P < 0.05). Fetal CBF recovered after 48 h of betamethasone administration. There were no differences in glucocorticoid induced CBF and CVR changes compared with our previous findings at 0.87 of gestation. We conclude that the cerebrovascular effects of antenatal glucocorticoids are independent of cerebrovascular maturation and preparturient increase in activity of the fetal pituitary–adrenal axis.     

Suppression of local and systemic responses in streptococcal cell wall-induced acute inflammation of the air pouch by cyclosporine A. Comparison with the effects of two anti-inflammatory bis-benzimidazoles.

Injection of streptococcus group A cell wall-derived peptidoglycan polysaccharide into a subcutaneous air pouch causes local outpouring of neutrophils and macrophages and distant hemopoietic proliferation in spleen and bone marrow. Cyclosporine A (CyA) suppressed neutrophil accumulation and all cell lines of hemopoiesis. trans-1,2-Bis(5-amidino-2-benzimidazolyl)ethene (BBE) also interfered with neutrophil exudation, yet reduced only the erythroid component of the hemopoietic process. The ethane analogue of BBE, on the other hand, did not prevent neutrophil emigration, but held down splenic erythropoiesis and myelopoiesis. All three compounds stimulated streptococcus group A cell wall-derived peptidoglycan polysaccharide uptake by pouch macrophages. CyA being the least active, BBE and its ethane analogue also produced a shift of wear-and-tear pigment from large numbers of small splenic macro-phages into small numbers of large macrophages. The pouch model is very useful in the study of anti-inflammatory compounds and has furnished the first evidence of CyA interference with massive neutrophilic infiltration and with hemopoietic signals.     

Migration of human melanoma cells depends on extracellular pH and Na+/H+ exchange

Their glycolytic metabolism imposes an increased acid load upon tumour cells. The surplus protons are extruded by the Na⁺/H⁺ exchanger (NHE) which causes an extracellular acidification. It is not yet known by what mechanism extracellular pH (pH_e) and NHE activity affect tumour cell migration and thus metastasis. We studied the impact of pH_e and NHE activity on the motility of human melanoma (MV3) cells. Cells were seeded on/in collagen I matrices. Migration was monitored employing time lapse video microscopy and then quantified as the movement of the cell centre. Intracellular pH (pH_i) was measured fluorometrically. Cell–matrix interactions were tested in cell adhesion assays and by the displacement of microbeads inside a collagen matrix. Migration depended on the integrin α2β1. Cells reached their maximum motility at pH_e∼7.0. They hardly migrated at pH_e 6.6 or 7.5, when NHE was inhibited, or when NHE activity was stimulated by loading cells with propionic acid. These procedures also caused characteristic changes in cell morphology and pH_i. The changes in pH_i, however, did not account for the changes in morphology and migratory behaviour. Migration and morphology more likely correlate with the strength of cell–matrix interactions. Adhesion was the strongest at pH_e 6.6. It weakened at basic pH_e, upon NHE inhibition, or upon blockage of the integrin α2β1. We propose that pH_e and NHE activity affect migration of human melanoma cells by modulating cell–matrix interactions. Migration is hindered when the interaction is too strong (acidic pH_e) or too weak (alkaline pH_e or NHE inhibition).     

Region-specific appearance of myelin constituents in the developing rat spinal cord

Summary The appearance of myelin-specific glycolipids and of myelin basic protein was studied with regard to the detailed anatomy of the rat cervical spinal cord. The expression of these constituents in particular fibre tracts and regions occurs at specific times of development between postnatal days 1 and 14. This mosaic-like appearance started in the ventral funiculus (day 1) followed by fasciculus cuneatus and ventro-lateral funiculus (day 2), and fasciculus gracilis and dorso-lateral funiculus (days 3 to 4). Cortico-spinal tract (day 11), Lissauer tract (day 14) and the commissures started to acquire myelin very late. In the grey matter, myelin constituents appeared around days 11 to 14 in a patchy pattern. These results support a concept of highly local interactions regulating oligodendrocyte differentiation. In addition, a general rostro-caudal gradient of myelin development exists in the spinal cord, which is independent of the ascending or descending nature of the fibre tracts. Appearance of myelin constitutents in the caudal spinal cord was not prevented by a neonatal transection at mid-thoracic levels.     

Extracellular detection of K+ release during migration of transformed Madin-Darby canine kidney cells

 Madin Darby canine kidney cells transformed by alkaline stress (MDCK-F cells) constitutively migrate at a rate of about 1 μm·min^–1. Migration depends on the intermittent activity of a Ca²⁺-stimulated, 53-pS K⁺ channel (K_Ca channel) that is inhibitable by charybdotoxin. In the present study we examined whether this intermittent K_Ca channel activity results in a significant K⁺ loss across the plasma membrane. K⁺ efflux from MDCK-F cells should result in a transient increase of extracellular K⁺ ([K⁺]_e) in the close vicinity of a migrating cell. However, due to the rapid diffusion of K⁺ ions into the virtually infinite extracellular space, such a transient increase in [K⁺]_e was too small to be detected by conventional K⁺-selective electrodes. Therefore, we developed a ”shielded ion-sensitive microelectrode” (SIM) that limited diffusion to a small compartment, formed by a shielding pipette which surrounded the tip of the K⁺-sensitive microelectrode. The SIM improved the signal to noise ratio by a factor of at least three, thus transient increases of [K⁺]_e in the vicinity of MDCK-F cells became detectable. They occurred at a rate of 1.3 min^–1. The cell releases 40 fmol K⁺ during each burst of intermittent K_Ca channel activity, which corresponds to about 15% of the total cellular K⁺ content. Since transmembrane K⁺ loss must be accompanied by anion loss and therefore leads to a decrease of cell volume, these findings support the hypothesis that intermittent volume changes are a prerequisite for the migration of MDCK-F cells. Received: 15 April 1996 / Received after revision: 18 June 1996 / Accepted: 23 July 1996     

Interaction of group A streptococcal peptidoglycan polysaccharide with human polymorphonuclear leukocytes: implications for pathogenesis of chronic inflammation.

Injection of sterile aqueous preparations of the peptidoglycan polysaccharide of group A streptococci produces chronic inflammation in several animal models. Accordingly, the effect of peptidoglycan and group A-specific polysaccharide (PG-APS) polymers on human polymorphonuclear neutrophil oxidative metabolism was studied with the supposition that this interaction may contribute to the inflammation observed. PG-APS in concentrations of 1.0 to 100 micrograms/ml stimulated oxygen consumption and hexose monophosphate shunt activity in the presence of 10% normal serum in a dose-related manner. Stimulation did not occur in serum-free media and was reduced in media with heat-treated serum. The stimulation of hexose monophosphate shunt activity by PG-APS opsonized with normal serum (bound complement components) and the activated supernatant from which PG-APS had been removed by centrifugation (presumably containing the soluble complement component, C5a) demonstrated 79 and 75%, respectively, of the stimulation with PG-APS in the presence of 10% normal serum. PG-APS triggered release of O2- (3.4 +/- 2.0 nmol by 10(6) human polymorphonuclear neutrophils over 30 min), which was significantly enhanced (9.6 +/- 2.9 nmol O2-) by treatment of cells with cytochalasin B. These results show that PG-APS interacts with serum in such a fashion as to activate human polymorphonuclear neutrophil metabolism and increase secretion of O2-.