The endothelia of microvessels isolated from mouse brain by mechanical means are rich in gamma-glutamyl transpeptidase; however, the enzyme often disappears when the cells migrate or proliferate from the microvessel isolates. In an endothelial cell line derived from similar isolates and negative for gamma-glutamyl transpeptidase, the enzyme could be induced in the endothelial cells when they were cocultured with glial cells. Thus there may be a requirement for continuous induction of gamma-glutamyl transpeptidase in brain microvessels by adjacent glial cells.
Transformation of Tetrahymena pyriformis to a rapid-swimming (presumably dispersal) form can be induced by washing cells and suspending them in distilled H2O, Dryl's solution or 10 mM Tris. Transformation is possible with high efficiency in mass cultures of axenically grown cells within approximately 5 h at 30 C. The radically different phenotype produced during transformation is characterized by a more elongate body form, increased numbers of somatic basal bodies and cilia, a long caudal cilium and oral membranelles positioned beneath the cell surface. DNA quantities characteristic of G1, S, and G2 cells are found in these transformed ciliates, suggesting that achievement of a particular stage in the DNA-division cycle is not a prerequisite for transformation. Preliminary observations on cells belonging to syngens 2-12 indicate that they also have a capacity to form a caudal cilium, but that the amicronucleate strain GL-C does not. Possible relevance of the transformed phenotype for taxonomy of Tetrahymena is discussed.
Microvessels isolated from mouse forebrain were used as the source material for the derivation of cerebral vascular endothelium and smooth-muscle cells in culture. The microvessels were isolated by a mechanical dispersion and filtration technique, and were maintained in vitro as organoid cultures. A microvessel classification system was developed and proved to be useful as a tool in monitoring culture progress and in predicting the type(s) of microvessel(s) that would give rise to migrating and/or proliferating cells. The isolated cerebral microvessels were heterogeneous in diameter, size of individual vascular isolate, and proliferative potential. The isolated microvessels ranged in diameter from 4 micron to 25 micron and in size from a single microvascular segment to a large multibranched plexus with mural cells. The initial viability, determined by erythrosin B exclusion, was approximately 50% on a per cell basis. All microvessel classes had proliferative potential although the rate and extent of proliferation were both microvessel class- and density-dependent. The smaller microvessels gave rise to endothelial cells, whereas the large microvessels gave rise to endothelial and smooth-muscle cells. The viability and progress of a microvessel toward derived cell proliferation seemed to be directly proportional to the number of mural cells present.
The mean DNA content of G2 macronuclei varies during the life cycle of the ciliate Tetrahymena thermophila. Early in the life cycle the mean is about 130 C; later it is about 94 C. In hybrids between strains A and B the decrease from 130 C to 94 C usually began after 60 fissions after conjugation. In B X B clones the decrease was complete by 50 fissions. The data suggest that there may be a genetic difference between strains A and B with respect to the onset of the decrease in DNA content. The downward regulation of the mean DNA content appears to be related to the mechanism which removes the variance in macronuclear DNA content which is added to macronuclei by unequal macronuclear division. Unequal macronuclear division regularly occurs at all stages of the life cycle, with larger macronuclei tending to divide more unequally. In the absence of regulation, unequal macronuclear division would constantly add variance to G1 macronuclei and their range would continue to increase. Analysis of the variances of G1 and G2 macronuclei suggests that at all stages of the life cycle the added variance is removed by acting upon nuclei which become too small or too large. According to this model, macronuclei with smaller amounts of DNA are regulated upward by an extra macronuclear S phase, while larger amounts are regulated downward by chromatin extrusion and the skipping of macronuclear S. The mean DNA content appears to change during the life cycle because the thresholds at which macronuclei become too small or too large are readjusted. It is postulated that these thresholds are a function of gene dosage.
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