PHA-stimulated human peripheral blood lymphocytes (PBL) contain an extractable cytoplasmic protein (ADR) that is capable of inducing DNA synthesis in isolated quiescent nuclei. This factor is not released from the cells, has no effect on intact cells, and is not detectable in the cytoplasm of normal resting PBL. These data suggest that ADR may serve as an intracellular signal ("second signal") for PHA-induced lymphocyte proliferation. The induction of ADR is not an early event associated with the expression of IL-2 receptors; rather the induction of ADR appears to be a later event resulting from the binding of IL-2 to IL-2 responsive cells. It was possible to utilize this system to study the impaired proliferative response to PHA seen in lymphocytes from elderly subjects. These poorly responsive cells expressed normal to above normal levels of ADR activity. However, their nuclei showed a marked impairment of response to exogenous ADR, with a linear relationship between the proliferative capacity of the intact cell and nuclear ADR responsiveness. These findings localize at least one defect in lymphocytes from old subjects to the nucleus. These observations will be extended to a study of responsiveness to B cell mitogens. Following this, cell proliferation will be examined directly, by inserting exogenous ADR into intact lymphocytes from old and young subjects utilizing erythrocyte ghost and electrofusion techniques. Next, the possible role of an intracellular inhibitor of ADR activity will be explored. In the course of these various studies, aged lymphocytes will be compared to aged fibroblasts, in order to determine whether similar or different mechanisms are operative in different cell types. A comparison will also be made between in vivo and in vitro aging. 2D-PAGE analysis will be performed on surface-labelled nuclei from old and young lymphocytes at various stages of activation, as well as nuclei that have been exposed to ADR in vitro. Finally, available cell lines from patients with diseases simulating premature aging (such as progeria) will be examined, in order to determine if they have intracellular defects similar to those seen in normal aging. It is hoped that these studies will provide insights into the intracellular defects associated with both "physiologic" and pathologic conditions of aging.