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The effects of serum deprivation on several general cellular biochemical processes ("pleiotypic response") related to the growth of normal fibroblasts can be mimicked by treatment of these cells with prostaglandin El in the presence of serum. N8,02'-Dibutyryl adenosine 3': 5'-cyclic monophosphate and theophylline inhibit the membrane transport processes without much effect on other pleiotypic reactions such as overall protein and RNA synthesis and protein degradation. The amount of intracellular cyclic AMP increases during serum starvation and returns to the initial concentration in unstarved cells when growth is initiated again upon addition of serum. Fibroblasts transformed by simian virus 40 have a lower cyclic AMP content than their untransformed parents. Serum deprivation neither increases cyclic AMP content nor significantly affects the pleiotypic reactions in transformed cells. Cycloheximide causes a decrease in cyclic AMP content of normal fibroblasts coincidentally with the ability of this inhibitor to stimulate uridine transport and slow protein degradation in cells deprived of serum.In normal cells, several biochemical parameters, including membrane transport, overall rates of protein and RNA synthesis, and protein degradation, fluctuate coordinately with changes in cell-growth rate. These mechanistically unrelated processes and their coordinated response to environmental changes comprise what we have defined as the "pleiotypic program" and "pleiotypic control," respectively. In malignant cells, the pleiotypic program and the rate of growth are relatively insensitive to changes in cultural conditions; we thus picture transformed cells as having a defect in the pleiotypic control mechanism (1).In cultured fibroblasts, insulin as well as serum activates the pleiotypic program (1, 2). Moreover, the same set of reactions is regulated in many other cell types by specific hormones or growth factors. Thus, certain general features of hormonal regulation may also be exerted through the pleiotypic mechanism (1).We have postulated that a "mediator", probably formed at the cell membrane, coordinates the responses of the different reactions under pleiotypic control. Because of the resemblance of this regulatory program to "stringent control" in bacteria, we inquired whether guanosine 5'-diphosphate, 2'-or 3'-diphosphate (ppGpp) (a possible mediator of stringent control) (3, 4) could be involved in control in animal cells. However, this nucleotide was not detected in cultured 3T3 cells under conditions that should have elicited its appearance (5).In the meantime, numerous reports have implicated cAMP as a regulator of growth, morphology, and contact inhibition in cultured cells (6-11). We report here that altering the inAbbreviations: Bu2cAMP, N6,02'-dibutyryl adenosine 3':5'-cyclic monophosphate; PGE,, prostaglandin El; SV40, simian virus 40. tracellular cAMP content by various means can affect all the reactions under pleiotypic control in a way, suggesting that cAMP is very likely the pleiotypic mediator...

A biphasic increase of putrescine concentration occurs in rat hepatorna tissue culture cells induced to proliferate. DL-a-Methyl ornithine, a competitive inhibitor of ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.7) of hepatoma tissue culture cells, blocks the usual increases of putrescine and spermidine concentrations in these cells, and causes a rapid fall in the levels of putrescine which is followed by a striking decrease of spermidine. In parallel with the depletion of these amines, incorporation of [3H]thymidine into DNA and cell proliferation are inhibited. Addition of putrescine, spermidine, or spermine results in an immediate resumption of cell proliferation. Cell proliferation is also restored by L-ornithine presumably due to in situ competitive inhibition of ornithine decarboxylase. These findings in hepatoma tissue culture cells support the concept that polyamines play an essential function in the cell division processes.

Formation of the 4-kDa peptides, which are essential constituents of the extracellular plaques in Alzheimer's disease, involves the sequential cleavage of the amyloid precursor protein (APP) by p-and y-secretases. The carboxy-terminal 99-amino-acid peptide which is liberated from APP by p-secretase was used as a potential native substrate of the y-secretase(s). With the addition of an initiator Met and a FLAG sequence at the C-terminus (PAPP100-FLAG), it was expressed in Escherichia coli under the control of the T7 promotor. The preferred site(s) of cleavage in the N-terminal 40-amino-acid P-amyloid peptide and PAPPI 00-FLAG by potential y-secretase(s) were rapidly identified using matrix-assisted Iaser-desorptionlionization time-of-flight mass spectroscopy in addition to peptide mapping followed by protein sequence analysis. Since y-secretases seem to be active at acidic pH, three cathepsins (D, E and B) were selected for testing. Studies using different detergents indicated that the cleavage preference of cathepsin D for the PAPP100-FLAG is highly dependent on the surfactant used to solubilize this substrate. All three cathepsins were found to be capable of catabolizing both p-amyloid peptides and the PAPPIOO-FLAG. As cathepsin D was found to cleave the PAPP100-FLAG in the vicinity of the C-terminus of the /l-aniyloid peptides and cathepsin B has a high carboxypeptidase activity at low pH, the possibility cannot be excluded that cathepsins D and B are involved in the amyloidogenic processing of APP.