NudC associates with Lis1 and the dynein motor at the leading pole of neurons.

NUDC is a highly conserved protein important for nuclear migration and viability in Aspergillus nidulans. Mammalian NudC interacts with Lis1, a neuronal migration protein important during neocorticogenesis, suggesting a conserved mechanism of nuclear movement in A. nidulans and neuronal migration in the developing mammalian brain (S. M. Morris et al., 1998). To further investigate this possibility, we show for the first time that NudC, Lis1, and cytoplasmic dynein intermediate chain (CDIC) colocalize at the microtubule organizing center (MTOC) around the nucleus in a polarized manner facing the leading pole of cerebellar granule cells with a migratory morphology. In neurons with stationary morphology, NudC is distributed throughout the soma and colocalizes with CDIC and tubulin in neurites as well as at the MTOC. At the subcellular level, NudC, CDIC, and p150 dynactin colocalize to the interphase microtubule array and the MTOC in fibroblasts. The observed colocalization is confirmed biochemically by coimmunoprecipitation of NudC with CDIC and cytoplasmic dynein heavy chain (CDHC) from mouse brain extracts. Consistent with its expression in individual neurons, a high level of NudC is detected in regions of the embryonic neocortex undergoing extensive neurogenesis as well as neuronal migration. These data suggest a biochemical and functional interaction of NudC with Lis1 and the dynein motor complex during neuronal migration in vivo.

Early events in the formation of a tissue structure from dispersed bovine adrenocortical cells following transplantation into scid mice.

The early events that follow the transplantation of dispersed bovine adrenocortical cells into scid mice were investigated. We introduced adrenocortical cells into a small cylinder inserted beneath the kidney capsule, where they form a tissue structure that becomes vascularized and secretes steroids that replace those from the animal's own adrenal glands, which are removed during the transplantation surgery. We studied cell proliferation, cell survival, apoptosis, and the role of p21WAF1/CIP1/SDI1 over the first 6 days following transplantation. Additionally we examined the invasion of the tissue by host macrophages and endothelial cells. The data show that there is healthy survival of most of the transplanted cells, and that this is related to their position in the cell transplantation cylinder. In the layer of cells that was adjacent to the kidney parenchyma there was a higher rate of cell proliferation and a lower rate of apoptosis than in cells that were located in the upper part of the cylinder. In the lower layer cells were more likely to have nuclear p21, and macrophages and endothelial cells were observed only in this region. Cells that incorporated bromodeoxyuridine administered to animals 2 or 4 days following transplantation were not more likely than other cells to be undergoing a second division when the animals were killed at 6 days, suggesting that proliferation in the lower layer is not confined to a small subpopulation of cells. Among different animals, the extent to which the spaces between the transplanted cells became lined by host endothelial cells was correlated with higher levels of proliferation and nuclear p21, suggesting that vascularization is the critical step for the continued survival and proliferation of the transplant. The present experiments show that bovine adrenocortical cells transplanted into scid mice form a useful model for the study of tissue formation from dispersed cells and the interaction of the transplanted cells with the host.

Relationship of p21(WAF1/CIP1/SDI1) to cell proliferation in primary cultures of adrenocortical cells.

p21(WAF1/CIP1/SDI1) was originally described as a protein expressed at high levels in senescent human fibroblasts. We have studied the expression of p21 in adrenocortical cells, p21 is not expressed under most circumstances in the intact adrenal gland in vivo, except when the gland is damaged. When human and bovine adrenocortical cells are isolated and placed in both short-term and long-term culture, p21 levels are much higher. These levels did not show a large increase when the cells senesce after long-term proliferation. Thus, these observations raise the question of whether the elevated p21 in primary cultures of adrenocortical cells is caused by damage or whether p21 is elevated because the cells are dividing rather than quiescent, because it has been reported that p21 levels peak in G1 and G2 in dividing cells. In the present experiments on bovine and human adrenocortical cells in primary culture, labeling techniques that correlated nuclear p21 with measures of cell proliferation (bromodeoxyuridine incorporation and nuclear Ki-67 antigen) supported the hypothesis that p21 is associated with cell division and not with damage. This is consistent with recent data showing that, when adrenocortical cells are transplanted into immunodeficient mice, p21 is associated with healthy dividing cells in the transplant, p21 is not a unique marker for senescence, and more studies are required both to clarify its role in cell biology and to determine molecular features which characterize the senescent state of cells both in vitro and in vivo.

Biotin-labeled hairpin oligonucleotides: probes to detect double-strand breaks in DNA in apoptotic cells.

Hairpin oligonucleotides were synthesized with stems ending in a double-stranded structure, which can be ligated to double-strand breaks in DNA, and with loops that contain nucleotides modified by the attachment of biotin. These probes specifically and sensitively detect double-strand breaks in apoptotic cells. Localization of these probes is restricted to areas of chromatin characteristic of apoptosis, whereas much more diffuse labeling was obtained when all available 3' DNA ends were labeled by terminal transferase. In principle, hairpin oligonucleotide probes can be designed with any type of 3' or 5' overhang complementary to double-strand DNA termini being detected.

Cell transplantation: a tool to study adrenocortical cell biology, physiology, and senescence.

We have established a mouse model for the growth and function of bovine and human adrenocortical cells in immunodeficient animals. We used the technique of cell transplantation, in which dispersed cells are introduced into an appropriate host in vivo to form a functional tissue. The ability to regenerate vascularized tissue, of normal histology and ultrastructure, is an inherent property of transplanted adrenocortical cells. Steroids secreted by the transplants replace the essential functions of the animals' own adrenal glands. Successful methods of transplantation described here have in common that the adrenocortical cells are permitted to aggregate in a space or matrix that provides adequate extracellular fluid and appropriate nutrients and oxygen. The present experiments show the potential of cell transplantation as a tool for the investigation of adrenocortical cell biology, molecular biology and physiology. The complete potential of the system will become apparent as new uses of the technique are devised, particularly with respect to human adrenocortical cells and to genetically modified cells.