Characterization of microcarrier cultures of neurons and astrocytes from cerebral cortex and cerebellum

In the present investigation a method is described for culturing cerebellar granule cells (glutamatergic neurons), cerebral cortical neurons (GABAergic neurons) and cortical astrocytes on Cytodex 3 microcarriers. It was possible to obtain a high yield of attached neurons and astrocytes on the microcarriers and the cell specific characteristics such as the ability to release neurotransmitter (neurons) and a high activity of glutamine synthetase (astrocytes) were preserved. This system, allowing mixtures of neurons and astrocytes at any given ratio to be produced, may constitute an attractive model system by which the interaction between neurons and astrocytes with regard to exchange of neurotransmitter precursors as well as other compounds may be studied.     

Three-dimensional architecture of chromosome fibres in the crane fly: co-oriented autosomal bivalents and amphitelic sex univalents during prometaphase

A technique for the fixation of cells during live observation (Nicklas et al. 1979) was used to investigate chromosomes which were moving at the time of fixation. Chromosome fibres were reconstructed by tracking their microtubules in longitudinal serial sections. A considerable proportion of non-kinetochoric microtubules (free microtubules, fMTs) is skewed with respect to the fibre axis. These skew fMTs contribute to the degree of disorder. It was found that the difference in the relative proportion of skew fMTs between active fibres (oriented in the direction of movement) and passive fibres (oriented backwards) is significantly correlated with the chromosome velocity (correlation coefficient r=0.796, P=0.01). It can be concluded that the pulling force generated in the chromosome fibre is a function of skew fMTs.     

Purification of transferrin and albumin from mouse ascites fluid

Mouse ascites fluid, which is readily obtained when cell lines and hybridomas are maintained in host mice, is a convenient source of several plasma proteins. This paper describes procedures for the purification of albumin and transferrin from mouse ascites fluid. Mouse transferrin was prepared from a 50-75% ammonium sulfate fraction of mouse ascites fluid by CM- and DEAE-cellulose chromatography. Mouse albumin was obtained by the same purification route, but required an additional chromatography step on Cibacron Blue F3GA-agarose. Both proteins were shown to be homogeneous by polyacrylamide gel electrophoresis and immunoelectrophoresis. Characterization, which included a determination of amino acid composition, partial N-terminal sequence, molecular weight and extinction coefficient, correlated well with known values reported for human transferrin and albumin. The purified mouse proteins may be useful for biochemical studies, antibody preparation, and as growth factors for hybridomas or other mouse cell lines maintained in culture.     

Shoot regeneration of mesophyll protoplasts transformed by Agrobacterium tumefaciens,not achievable with untransformed protoplasts

Summary Alternative methods for shoot regeneration in protoplast derived cultures were developed in Nicotiana paniculata and Physalis minima. In both species protoplast derived callus is not regeneratable to shoots by conventional methods, e.g. hormone treatment. Leaf discs and stem segments of N. paniculata and P. minima were incubated with Agrobacterium tumefaciens shooter strains harbouring pGV 2215 or pGV 2298 or wildtype strain B6S3. After 36 h of co-incubation protoplasts were prepared. (Leaf disc and stem segment cloning). Co-cultivation experiments were also undertaken with protoplasts of both species. Transformed clones, characterized by their hormone independent growth and octopine production, could be isolated after about two months. Transformation frequencies of leaf disc and stem segment cloning and co-cultivation experiments varied from 5×10^–3 to 5×10^–5. After about one year of cultivation on hormone-free culture medium, shoots could be recovered from colonies of N. paniculata, transformed by the strain harbouring pGV 2298. In protoplast derived colonies of P. minima, shoot induction was obtained only after transformation by bacteria carrying pGV 2215. This demonstrates the importance of the particular shooter mutant, as well as the response of the host plant. Transformed shoots of P. minima produced octopine, whereas octopine production in transformed shoots and callus of N. paniculata was undetectable after one year of cultivation, though T-DNA was still present in the plant genome. Transformed shoots of N. paniculata and P. minima do not produce any roots. Shoots of N. paniculata have an especially tumerous phenotype. Shoots of both species were successfully grafted to normal donor plants of N. tabacum.Abbreviations B5-h Gamborg medium without hormones (Gamborg 1968) - V47 protoplast medium (Binding 1974) - D2a protoplast medium (Li et al. 1980) - MS-h Murashige and Skoog medium without hormones (Murashige and Skoog 1962) Dedicated to Professor Dr. G. Melchers in occasion of his 80th birthday     

Immunostaining of spindle components in tipulid spermatocytes using a serum against pericentriolar material

First meiotic division of tipulid (Pales ferruginea) spermatocytes was investigated by double immunostaining with anti-tubulin IgG and scleroderma 5051 serum against pericentriolar material (PCM). PCM-like material became visible in late diakinesis in centrosomal areas as well as in kinetochores. Anti-PCM fluorescence was most pronounced in metaphase and diminished again in anaphase. Displacement of one of the centrosomes from the nucleus at diakinesis in Pales spermatocytes leads to the formation of a bipolar, normally functioning spindle possessing one aster and centriole-free spindle pole (AFP). In about 80% of the AFPs observed there were no traces of anti-PCM staining detectable. This finding supports the assumption based on previous studies that polar PCM is not obligatory for spindle pole formation. The chromosomes seem to be able to induce the organization of a half-spindle. The strong anti-PCM fluorescence of the kinetochores observed here may be taken as further indication of tipulid chromosome autonomy regarding spindle formation.     

Fluorescence properties of catecholamines in isolated storage organelles of adrenal medulla

THe quantum yield, the life time and the degree of polarization of the fluorescence of intact chromaffin granules isolated from bovine adrenal medulla were compared to those of catecholamines solutions and catecholamine/ATP mixtures. Rising concentrations of catecholamines in aqueous solutions exhibited increasing quenching and decreasing life times indicating that the quenching was collision induced. Similar effects occurred in mixtures of catecholamines with ATP and Ca²⁺ showing that the nucleotide did not remarkedly hinder the mobility of the catechol group. In suspensions of whole granules stron quenching and shortening of life time was observed compared with solutions of disrupted granules. Fluorescence yield and life time were decreased by about the same factor suggesting that storage of the amines was not correlated with a major immobilization of the catechol group. The degree of polarization of intact granules was higher than that of solutions of catecholamines alone, but similar to catecholamine/ATP mixtures with concentrations corresponding to those found in the granules. This indicates an interaction of catecholamines with ATP in the granules. The results are in agreement with a storage model for catecholamines in the chromaffin granules of adrenal medulla in which catecholamines are bound to ATP, but in a non-rigid way.