Correlative evidence links stress, accumulation of oxidative cellular damage and ageing in lower organisms and in mammals. We investigated their mechanistic connections in p66Shc knockout mice, which are characterized by increased resistance to oxidative stress and extended life span. We report that p66Shc acts as a downstream target of the tumour suppressor p53 and is indispensable for the ability of stress-activated p53 to induce elevation of intracellular oxidants, cytochrome c release and apoptosis. Other functions of p53 are not in¯uenced by p66Shc expression. In basal conditions, p66Shc7/7 and p537/7 cells have reduced amounts of intracellular oxidants and oxidation-damaged DNA. We propose that steady-state levels of intracellular oxidants and oxidative damage are genetically determined and regulated by a stress-induced signal transduction pathway involving p53 and p66Shc.

Pathogenic strains of Helicobacter pylon cause progressive vacuolatlon and death of epithelial cells. To identify the nature of vacuoles, the distribution of markers of various membrane traffic compartments was studied. Vacuoles derive from the endocytic pathway since they include the fluid-phase marker Lucifer yellow. Early endosome markers such as rab5, trerrin, and trnsferrin receptor, as well as the lysosomal hydrolase cathepsin D, are excluded from these structures. In contrast, the vacuolar membrane is specifically stained by ity-purified antibodies against rab7, a small GTPase, localized to late endosomal compartments. The labeling of rab7 on vacuolar membranes increases as vacuolation progresses, without a concomitant increase of cellular rab7. Cell vacuolation is inhibited by the microtubule-depolymerizing agents nocodazole and colchicine. Taken together, these findins indicate that the vacuoles specifically originate from late endosomal compartments.Strong evidence indicates that toxigenic strains of Helicobacter pylori play a major role in the development of type B gastritis, peptic ulcers, and gastric adenocarcinoma (1-3). Biopsies of H. pylori-colonized stomach epithelium show cellular swelling, expansion of endosomal compartments, and extensive vacuolation (4). A vacuolar degeneration of epithelial cells, followed by cell death, is induced in vitro by H. pylori bacterial extracts (5-7). The luminal pH of these vacuoles is acidic, as deduced from the uptake of neutral red, a membrane-permeant amine that becomes protonated in the vacuolar lumen, and from the potentiating effect of ammonia (8). Two virulence factors, produced by pathogenic strains of H. pylori are thought to be mainly responsible for this cell degeneration and death: a urease and a cytotoxin (3,4,7,8). The urease hydrolyzes urea and the ammonia produced permeates membranes, as neutral red does, and accumulates as ammonium ions inside intracellular acidic compartments, thus causing their osmotic swelling (9-11). However important, this process alone cannot account for the manyfold increase in vacuole volume, because, without membrane addition, it would lead to vacuole rupture.Recently a cytotoxin has been isolated from culture supernatants of a virulent strain of H. pylori (12) and shown to cause vacuolation in vitro (12) and in vivo (13). The genes encoding this protein (vacA; H. pylori vacuolating toxin A), and a bacterial antigen (cagA) characteristic of H. pylori pathogenic strains, have been recently cloned and sequenced, and they share no similarity with any other known protein (13,14). Thus, the molecular mechanism of action of vacA and the sequence of events leading to vacuolar cell degeneration and death remain largely obscure.The inhibition of vacuole formation by bafilomycins, specific inhibitors of the vacuolar-type ATPase proton pump (V-ATPase) (15), indicates that vacuoles originate from intracellular compartments endowed with a V-ATPase, such as endosomes, lysosomes, or the trans-Golgi network (TGN) (16)(17)(18)....

Mutant alphabeta TCRs were generated by replacing domains of the alpha and beta chain constant regions with homologous domains from TCR delta and gamma chains, respectively. Chimeric TCRs in which the alpha chain contains TCR delta chain sequences within the connecting peptide domain are unresponsive to alloantigens and superantigens, and have defective interactions with the CD3/zeta complex. Although these antigen-unresponsive TCRs undergo zeta chain phosphorylation upon stimulation with superantigen, they do not generate a full signal capable of producing IL-2. Mutant TCRs acquire signaling activity with a combination of superantigen and calcium ionophore, indicating a defect in calcium-mediated signaling. Finally, a conserved motif, FETDxNLN, present in the alpha chain connecting peptide domain, is disrupted in all signaling-defective TCRs. This conserved alpha chain connecting peptide motif might mediate the transfer of signals from the alphabeta heterodimer to the CD3/zeta complex.

The CD4 coreceptor interacts with non-polymorphic regions of major histocompatibility complex class II molecules on antigen-presenting cells and contributes to T cell activation. We have investigated the effect of CD4 triggering on T cell activating signals in a lymphoma model using monoclonal antibodies (mAb) which recognize different CD4 epitopes. We demonstrate that CD4 triggering delivers signals capable of activating the NF-AT transcription factor which is required for interleukin-2 gene expression. Whereas different anti-CD4 mAb or HIV-1 gp120 could all trigger activation of the protein tyrosine kinases p56lck and p59fyn and phosphorylation of the Shc adaptor protein, which mediates signals to Ras, they differed significantly in their ability to activate NF-AT. Lack of full activation of NF-AT could be correlated to a dramatically reduced capacity to induce calcium flux and could be complemented with a calcium ionophore. The results identify functionally distinct epitopes on the CD4 coreceptor involved in activation of the Ras/protein kinase C and calcium pathways.

Interaction of the CD4 co-receptor with major histocompatibility complex (MHC) class II molecules during antigen presentation results in enhancement of antigen receptor signaling. The synergism between the two receptors is believed to result from the juxtaposition of the CD4-associated tyrosine kinase p56lck with the cytoplasmic domains of CD3 complex components. Here, we report that cross-linking of CD4 on the surface of Jurkat cells using monoclonal antibodies results in activation of the CD3-associated kinase p59fyn. Co-cross-linking of CD4 and CD3 results in synergistic activation of p59fyn. The p59fyn kinase is also hyperactive in a Jurkat cell line stably transfected with a constitutively active p56lck mutant, indicating that p56lck mediates CD4 activation of p59fyn. In support of this hypothesis, expression of a dominant inhibitory mutant of p59fyn blocks CD4 signals involved in gene activation. In addition, the p59fyn dominant inhibitor mutant blocks gene-activating signals induced by expression of a constitutively active mutant of p56lck. Overexpression of the regulatory kinase p50csk, which attenuates TcR signaling by inactivation of p59fyn, inhibits signaling from the constitutively active form of p56lck. Taken together, these data suggest that CD4/p56lck enhancement of TcR signaling is, at least in part, mediated by activation of p59fyn, and may be regulated by p50csk.