The CD95 signaling pathway comprises proteins that contain one or two death effector domains (DED), such as FADD/Mort1 or caspase-8. Here we describe a novel 37 kDa protein, DEDD, that contains an N-terminal DED. DEDD is highly conserved between human and mouse (98.7% identity) and is ubiquitously expressed. Overexpression of DEDD in 293T cells induced weak apoptosis, mainly through its DED by which it interacts with FADD and caspase-8. Endogenous DEDD was found in the cytoplasm and translocated into the nucleus upon stimulation of CD95. Immunocytological studies revealed that overexpressed DEDD directly translocated into the nucleus, where it co-localizes in the nucleolus with UBF, a basal factor required for RNA polymerase I transcription. Consistent with its nuclear localization, DEDD contains two nuclear localization signals and the C-terminal part shares sequence homology with histones. Recombinant DEDD binds to both DNA and reconstituted mononucleosomes and inhibits transcription in a reconstituted in vitro system. The results suggest that DEDD is a final target of a chain of events by which the CD95-induced apoptotic signal is transferred into the nucleolus to shut off cellular biosynthetic activities.

Certain B and T cell lines respond to activation signals, e.g. through the antigen receptor, by undergoing apoptotic cell death. In T cells it has been recently shown that TCR-mediated apoptosis involves APO-1/Fas(CD95) receptor-ligand interaction. To investigate whether the TCR-CD3 complex can trigger alternative apoptosis pathways we generated subclones of the T cell line Jurkat which were completely resistant towards APO-1-mediated apoptosis. These JurkatR cells differed phenotypically from sensitive parental JurkatS cells only by the lack of APO-1 protein expression. Although JurkatR cells responded normally to anti-CD3 stimulation by expression of APO-1 ligand they failed to undergo anti-CD3-induced apoptosis. Thus, in Jurkat cells APO-1-mediated apoptosis was the main, and might be the only, mechanism for anti-CD3-induced cell death. However, BL-60 B cells, highly sensitive to anti-IgM-induced apoptosis, did not use the APO-1 receptor-ligand system because they failed to express APO-1 ligand mRNA. Taken together, our results suggest that malignant T and B cell lines may use APO-1 receptor-ligand-dependent and -independent antigen receptor-induced apoptosis pathways respectively. Similarly, differential pathways may be used by T and B cell subsets.

Fresh specimens of human and rat neocortex were used to determine direct and indirect inhibitory potencies of choline esterase inhibitors (ChEIs) on ChE and the release of acetylcholine (ACh), respectively. Km values of ChE in homogenates of rat and human neocortex did not differ significantly, whereas the specific activity of ChE was > times higher in the rat. Butyryl ChE exhibited a higher Km and a lower specific activity than ACh esterase in human neocortex. Inhibition of ChE in rat and human tissue was similar [IC50 (nM; human): donepezil: 14, physostigmine: 22, tacrine: 95, galanthamine: 575, rivastigmine: 9120]. In neocortex slices preincubated with [3H]choline, the electrically evoked release of [3H]ACh was inhibited up to 60% by ChEIs (IC50 (nM, rat): donepezil: 30, physostigmine: 39, tacrine: 302, galanthamine: 646, rivastigmine: >10000). Similar IC50-values were also estimated for ACh release in human neocortex, although the maximal inhibitory effects were much smaller ( approximately 20%). We conclude that in comparison to rats: 1) neocortical ChE concentrations are lower and 2) that ChEIs have weaker indirect (muscarine receptor-mediated) presynaptic inhibitory effects in the human brain. We further suggest that a combination of ChEIs with brain-selective muscarine autoreceptor antagonists might help to improve their clinical efficacy.

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