Influence of increased concentration of Ca2+, La3+, PVP and urea on primary adhesivity of embryonic brain cells. Ultrastructural pattern of adhering cells

With the help of a previously described experimental arrangement the influence of increased external concentration of Ca2+, La3+, PVP and urea was tested on the initial stages of brain cell adhesivity and its kinetics. Urea, an inhibitor of hydrogen bonds, significantly inhibited the adhesivity of the treated cells. PVP significantly increased cell adhesivity. The adhesivity was enhanced and speeded up by increased concentrations of Ca2+ and La3+. It is evident that the membrane surface potential, zeta potential and formation of H+ bonds and bridges are highly important for cellular adhesivity. EM control of freshly dissociated cells disclosed that a part of the cells had been damaged. According to the ultrastructural organisation, the surface membrane is damaged to a small or greater extent. Intercellular contacts were formed in vitro either between non damaged surfaces of membranes, or between fragments of membranes, or contacts were mediated by membrane debris. Because cellular debris disappeared during rotation from single adhesive complexes, it is probable, that disrupted membranes are used for restoration of membranes, or serve as a metabolic substrates, or are catabolized.     

Evidence for a mechanical coupling of glycoprotein microfibrils with collagen fibrils in Wharton's jelly

Wharton's jelly of human umbilical cord is known to contain hyaluronic acid and sulphated glycosaminoglycans (probably as proteoglycans) immobilized in an insoluble collagen fibril network. A secondary, independent, insoluble network based on glycoprotein microfibrils of 13 nm diameter and interpenetrated with the collagen network has now been found in amounts corresponding to 9% of the weight of collagen. Elastin, however, is absent. Tissue slices placed in physiological buffer swell to two-fold their in vivo volume. This is due to the influence of the polysaccharides since treatment with either testicular hyaluronidase, Streptomyces hyaluronidase or chondroitinase ABC, causes their quantitative removal and abolishes the swelling tendency of tissue. Tissue so treated remains close to its in vivo volume indicating that for this state the fibrillar network, overall, is in its relaxed unstressed configuration. Subsequent treatment with a protease causes the degradation of the glycoprotein microfibril network and a two-fold increase in tissue volume while treatment with bacterial collagenase, resulting in the solubilization of 46% of the collagen, causes only a slight deswelling. These results suggest that the unstressed configuration of the network system at the in vivo volume of tissue is due to the collagen network being held in compression by the microfibril network. With intact tissue protease digestion with trypsin, in addition, causes a preferential release of sulphated glycosaminoglycans. Hyaluronic acid, however, remains largely immobilized.     

The effect of short-term temperature changes on the adhesivity of nerve cells. Participation of DNA molecules on non-specific cellular adhesion

Brain cells from 16 to 18-day-old mice embryos were dissociated by mild trypsinization and sieving. Immediately after dissociation the cells were preincubated in a PBS solution at -6 to +54 degrees C for 3 and 20 min. After this preincubation cells were rotated for 60 min at 37 degrees C in the PBS solution. Cellular adhesivity was estimated during this time period and EM pictures of organized in vitro aggregates after 24-28 h were taken. In a separate series of experiments, freshly dissociated were treated with DNAase before the rotation procedure. Preincubation in a cold or a warm medium did not alter the inhibition of cellular adhesivity significantly. Distinct inhibition of cellular adhesion was observed in cells preincubated above 53 degrees C. Adhesion was also inhibited below -5 degrees C, however, this effect was mainly dependent on the rate of freezing and thawing. Digestion of dissociated cells with DNAase (20 micrograms/ml) decreased cell adhesion. At 37 degrees C the adhesivity decreased by about 20%. Aggregates of cells preincubated at 0 degrees C for 20 min did not exhibit marked EM changes after 24-28 h in vitro. The present results have shown the rather high resistance of molecules responsible for cellular adhesion and its reversibility to temperature changes. Furthermore, non-specific cellular adhesion was shown on physically active DNA molecules.     

The effect of dibutyryl cyclic AMP on cultivated glial cells from corpus callosum of 30-day-old rats.

1) Fragments of corpus callosum of 30-day-old rats were cultivated in Rose chambers. Mature astrocytes and predominantly differentiated oligodendroglial cells are present in the corpus callosum of rats at this age. 2) In vitro, astrocytes dedifferentiated into epithelial--like flattened cells, which highly adhered to the surface. Cells exhibited signs of phagocytosis, and their ultrastructure differed from differentiated cells, especially by the absence of microtubules and microfilaments. 3) After dibutyryl cyclic AMP (db-c-AMP) application, epithelial--like cells became rounded and multipolar. Cells were less liable to adhere to the surface. Bundles of microfilaments and microtubules appeared in cell perikarya and their processes. 3--4 hour after application, the morphological changes were well developed and disappeared after 24--48 hours. 4) It is being suggested that the increased intracellular concentration of db-c-AMP induced the formation of microfilamentous structures. The formation of the cyto-skeleton has a direct influence on the shape of cells and appearance of processes and cell adhesivity. 5) These results have demonstrated the high structural plasticity of mature astrocytes, their dedifferentiation in vitro and convertion into mature cells after db-c-AMP application.