A gradient of epidermal growth factor receptor signaling determines the sensitivity of rbf1 mutant cells to E2F-dependent apoptosis

The inactivation of retinoblastoma (Rb) family members sensitizes cells to apoptosis. This cell death affects the development of mutant animals and also provides a critical constraint to the malignant potential of Rb mutant tumor cells. The extent of apoptosis caused by the inactivation of Rb is highly cell type and tissue specific, but the underlying reasons for this variation are poorly understood. Here, we characterize a specific time and place during Drosophila melanogaster development where rbf1 mutant cells are exquisitely sensitive to apoptosis. During the third larval instar, many rbf1 mutant cells undergo E2F-dependent cell death in the morphogenetic furrow. Surprisingly, this pattern of apoptosis is not caused by inappropriate cell cycle progression but instead involves the action of Argos, a secreted protein that negatively regulates Drosophila epidermal growth factor receptor (EGFR [DER]) activity. Apoptosis of rbf1 mutant cells is suppressed by the activation of DER, ras, or raf or by the inactivation of argos, sprouty, or gap1, and inhibition of DER strongly enhances apoptosis in rbf1 mutant discs. We show that RBF1 and a DER/ras/raf signaling pathway cooperate in vivo to suppress E2F-dependent apoptosis and that the loss of RBF1 alters a normal program of cell death that is controlled by Argos and DER. These results demonstrate that a gradient of DER/ras/raf signaling that occurs naturally during development provides the contextual signals that determine when and where the inactivation of rbf1 results in dE2F1-dependent apoptosis.     

Calibration of a flow cytometer against a microphotometer for morphologic cell identification

The calibration of a flow cytometer against a microphotometer, to allow the correlation of cell morphology with fluorescence intensity, is described. Using three human lymphoblastoid cell lines, the photomultiplier amplification of the microphotometer and the flow cytometer that gave optimum linearity between fluorescence intensity and DNA content for the two instruments was established. Thereafter, at these settings, there was satisfactory linear agreement between the fluorescence intensity profiles, as measured by the flow cytometer and the microphotometer, of stained cell populations. Day-to-day variation was also minimal, and it was demonstrated that the application of this procedure can provide an alternative to the employment of the sorting facility of a flow cytometer for the morphologic identification of cell subpopulations during flow cytometric analysis.     

A novel GC-rich dispersed repeat sequence in Drosophila melanogaster.

A novel family of dispersed repeat sequences from Drosophila melanogaster is described. Sequence analysis of two members of this family show them to contain greater than 75% GC bases. These are comprised of multiple repeats of GGX triplets interspersed occasionally with CGPy and TTPy. Southern blotting shows that these repeats are not transposable elements. Twenty four homologous recombinants have been localised by in situ hybridization to seven sites in the Drosophila genome. Polyadenylated RNAs homologous to this repeat family are expressed in a complex pattern which is developmentally regulated. We suggest that this family encodes a set of glycine-rich domains in Drosophila proteins.     

Short term metabolism of urea and purine cytokinins

Approximately 20 to 25% of the cytokinin benzyladenine (BA) taken up by soybean tissues in culture is converted to a stable, long-lived derivative which contains BA as part of its structure. This derivative may be metabolically related to 6-benzylamino-9-β-d-ribofuranosylpurine 5′-monophosphate (BAMP). In in vivo incubations of 2 hours or less, we recover only BA, benzyladenosine, and BAMP. Benzyladenosine never accounts for more than 10% of the total radioactivity while BAMP builds up to about 20% of the total within 2 to 4 hours. After this period it begins to disappear, and a new, unidentified substance arises at a rate which roughly parallels the loss of BAMP. After about 48 hours this substance, which has good cytokinin activity, accounts for some 20 to 25% of the total radioactivity and persists at this level for at least 60 days. In the meantime the remainder of the BA, as well as benzyladenosine and BAMP, disappear completely. In addition, evidence is presented which suggests that the urea cytokinins are not active as such but first are metabolically transformed into other substances.     

The morphological categorization of developing synaptic junctions

Summary The molecular layer of rat occipital cortex was studied at a number of ages throughout postnatal development in an attempt to formulate a quantitative morphogenetic scheme of synaptic development. As previous work had pointed to the potential usefulness of E-PTA stained material in synaptogenic studies, this technique was employed. Synaptic junctions were assigned to five categories, A-E, on the basis of variations in the organization of their presynaptic densities. Of these categories, Type A represented a mature junctional form with well-defined and discrete dense projections. Type E, by contrast, represented the immature end of the spectrum, its presynaptic apposition generally lacking recognizable densities. Types B-D represented intermediate forms.As development proceeded, the percentage of A type junctions increased from 0.3% at 7 days to 25% in the adult. Type E junctions showed the opposite trend falling from 42% at 7 days to 5% in the adult. On the basis of these results, and those relating to types B-D, possible morphogenetic schemes involving the five synaptic categories are put forward. These schemes incorporate and extend those previously suggested by other workers. By placing observations on a quantitative footing, the schemes proposed here lend themselves to use in experimental investigations.This work was supported by the Nuffield Foundation. We wish to thank Mr. W. Kendrick for technical assistance     

Some observations on the ultrastructure of developing rat cerebral capillaries

Summary Developing blood vessels in rat cerebral cortex were studied at a number of stages between 3 and 28 days postnatal, in an attempt to obtain data on the mechanisms by which the lumen is established within cords of mesodermal cells. A combination of techniques was utilized in an attempt to elucidate these mechanisms. These were: (a) aldehyde fixation and block staining with phosphotungstic acid; (b) aldehyde perfusion followed by perfusion of a lead solution and post-fixation in osmium tetroxide; (c) conventional preparation of tissue with aldehyde and osmium fixation.Support for interendothelial lumen formation was readily forthcoming, including vessels with junctions between two or more endothelial cells cut transversely. There was some support for intraendothelial lumen formation, in the form of seamless endothelial cells. Other features noted included the presence of free ribosomes and vacuoles in the endothelial cells, endothelial flaps, sprouts and tendrils, intraluminal debris, endothelial degeneration and a junction with a nonendothelial cell.Large numbers of endothelial vacuoles were noted, many of them occurring at the abluminal edge of the cells. These vacuoles may be involved in the formation of intraendothelial lumina and also in the enlargement of both types of lumina. This study provides evidence that besides the well-established inter-endothelial lumen formation, intraendothelial mechanisms may also be operative in rat cerebral cortex. The techniques employed in this study offer the potential for clarifying these and related issues.We would like to acknowledge the financial assistance of the Nuffield Foundation     

Properties of chicken skeletal muscle pyruvate kinase and a proposal for its evolutionary relationship to the other avian and mammalian isozymes.

Pyruvate kinase (EC 2.7.1.40) was isolated and purified from chicken and turkey breast muscle with a purification procedure very similar to that used for the bovine skeletal muscle isozyme (Cardenas, J., Dyson, R., and strandholm, J. (1973), J. Biol. Chem. 248,6931). A study of the chemical and physical properties of the chicken enzyme revealed that it is a tetramer of four apparently identical subunits, closely resembling in this and most other respects the mamalian type 7 isozyme. The properties of these two enzymes are similar enough to permit subunits of chicken type M pyruvate kinase to combine with subunits of mammalian type L (one of the three mammalian isozymes) to form interspecies tetrameric hybrid isozymes in relative quantities that do not differ makedly from those formed when both the M and L isozymes are of mammalian origin. The similarity between the mammalian and avian type M pyruvates kinases suggests a close evolutionary relationship. Further comparisons among the three mammalian and two avian isozymes of pyruvate kinase are consistent with a common evolutionary origin, perhaps from an ancestral form of the type K isozyme, which is the only pyruvate kinase identified in mammalian and avian embryos.     

Expression of the ASV src gene in hybrids between normal and virally transformed cells: Specific suppression occurs in some hybrids but not others

Somatic cell hybrids have been made between clones of rat cells transformed by avian sarcoma virus and rat or mouse cells that are untransformed. Intraspecies hybrids were either predominantly morphologically normal or predominantly transformed, some clones that formed transformed intraspecies hybrids yielding normal interspecies hybrids. Untransformed hybrids usually showed no detectable alteration in the structure or location of the integrated provirus, but viral RNA and pp60^src kinase activities were much reduced. No decrease in viral gene expression was seen in transformed hybrids. Thus hybrid suppression of viral transformation, mediated in trans by the untransformed parent, is a specific event that depends on both untransformed and transformed parental parameters.