Proteins of the Bcl-2 family are intracellular membrane-associated proteins that regulate programmed cell death (apoptosis) either positively or negatively by as yet unknown mechanisms. Bax, a pro-apoptotic member of the Bcl-2 family, was shown to form channels in lipid membranes. Bax triggered the release of liposome-encapsulated carboxyfluorescein at both neutral and acidic pH. At physiological pH, release could be blocked by Bcl-2. Bcl-2, in contrast, triggered carboxyfluorescein release at acidic pH only. In planar lipid bilayers, Bax formed pH- and voltage-dependent ion-conducting channels. Thus, the pro-apoptotic effects of Bax may be elicited through an intrinsic pore-forming activity that can be antagonized by Bcl-2.

Bcl-2 family members either promote or repress programmed cell death. Bax, a death-promoting member, is a pore-forming, mitochondria-associated protein whose mechanism of action is still unknown. During apoptosis, cytochrome C is released from the mitochondria into the cytosol where it binds to APAF-1, a mammalian homologue of Ced-4, and participates in the activation of caspases. The release of cytochrome C has been postulated to be a consequence of the opening of the mitochondrial permeability transition pore (PTP). We now report that Bax is sufficient to trigger the release of cytochrome C from isolated mitochondria. This pathway is distinct from the previously described calcium-inducible, cyclosporin A–sensitive PTP. Rather, the cytochrome C release induced by Bax is facilitated by Mg2+ and cannot be blocked by PTP inhibitors. These results strongly suggest the existence of two distinct mechanisms leading to cytochrome C release: one stimulated by calcium and inhibited by cyclosporin A, the other Bax dependent, Mg2+ sensitive but cyclosporin insensitive.

Immunoglobulin E (IgE) is central to the induction of allergic diseases through its binding to the high-affinity receptor (Fc epsilon R1) on mast cells and basophils. Crosslinking by allergens of the bound IgE leads to the release of various inflammatory mediators. IgE production by B cells requires a physical interaction with T cells, involving a number of surface adhesion molecules, as well as the soluble factors interleukin-4 (IL-4) and IL-13 (ref. 5) produced by T cells, basophils and mast cells. Here we report that, in the presence of IL-4, mast and basophilic cell lines can provide the cell contact signals that are required for IgE synthesis. The human cell lines HMC-1 (mast) and KU812 (basophilic) both express the ligand for CD40 (CD40L) which is shown to be responsible for the IgE production. Moreover, freshly isolated purified human lung mast cells and blood basophils are also shown to express CD40L and to induce IgE production. This evidence suggests that mast cells and basophils may therefore play a key role in allergy not only by producing inflammatory mediators, but also by directly regulating IgE production independently of T cells.

CD40 ligand (CD40L) is expressed on the surface of activated CD4+ T cells, basophils, and mast cells. Binding of C40L to its receptor, CD40, on the surface of B cells stimulates B cell proliferation, adhesion and differentiation. A preparation of soluble, recombinant CD40L (Tyr-45 to Leu-261), containing the full-length 29-kDa protein and two smaller fragments of 18 and 14 kDa, has been shown to induce differentiation of B cells derived either from normal donors or from patients with X-linked hyper-IgM syndrome (Durandy, A., Schiff, C., Bonnefoy, J.-Y., Forveille, M., Rousset, F., Mazzei, G., Milili, M., and Fischer, A. (1993) Eur. J. Immunol. 23, 2294-2299). We have now purified each of these fragments to homogeneity and show that only the 18-kDa fragment (identified as Glu-108 to Leu-261) is biologically active. When expressed in recombinant form, the 18-kDa protein exhibited full activity in B cell proliferation and differentiation assays, was able to rescue of B cells from apoptosis, and bound soluble CD40. Sucrose gradient sedimentation shows that the 18-kDa protein sediments as an apparent homotrimer, a result consistent with the proposed trimeric structure of CD40L. This demonstrates that a soluble CD40L can stimulate CD40 in a manner indistinguishable from the membrane-bound form of the protein.

Antisera against phospholipid/Ca2+-dependent protein kinase (protein kinase C) were raised in rabbits. Immunospecificity of the polyclonal antibodies, as determined by immunoblot and ELISA, was shown by their reactivity to the enzyme but not to other protein kinases or any of many other proteins tested. Immunocytochemical localization of the kinase in rat brains revealed that although the enzyme was distributed broadly in different brain regions, it was highly restricted to the periphery of the nucleus of neurons in cerebral cortex and to axons and cells strongly resembling oligodendroglia in white-matter regions. Initial electron microscopy of cerebral cortex revealed that the enzyme was highly concentrated in the presynaptic terminals, and only rarely were labeled postsynaptic specialization elements seen. It is suggested that the discrete localization of the enzyme, which is distinct from that of the calmodulin/Ca2+-dependent system, may be related to certain biological and functional aspects of Phospholipid/Ca2+-dependent protein kinase (PL/Ca-PK, or protein kinase C) is a major class of protein-phosphorylating enzyme first found in brain (1) and subsequently shown to occur widely in tissues and phyla of the animal kingdom (2). The biological significance of this Ca2+ effector system is further suggested by the presence of its numerous but specific substrate proteins in tissues (for reviews, see refs. 3 and 4), such as myelin basic protein in brain (5, 6), and its key role in transduction of receptor-mediated signals (7,8), such as in the platelet activation induced by thrombin, platelet-activating factor, phorbol ester, diacylglycerol, and the Ca2+ ionophore A23187 (9). Some significant progress has been made during the last five years on the molecular, pharmacological, and regulatory aspects of this novel protein-phosphorylation system (3, 4). Immunological characterization of the enzyme, however, has not been possible. The major reasons for the lack of progress are the difficulties in the purification of the enzyme in large quantity for immunization, due to its instability, and the low antigenicity of the enzyme, probably because of its common occurrence in tissues. In this laboratory, monoclonal antibodies against the enzyme have been developed recently (10); unfortunately, they are of the IgM class of immunoglobulins and were found to be unsuitable for the intended immunological studies of the enzyme system. We now report the development of polyclonal antibodies against pig brain PL/ Ca-PK exhibiting a high immunospecificity toward the antigen and describe their use in immunocytochemical localization of the enzyme in rat brain. METHODSPurification of PL/Ca-PK. The enzyme from the crude extracts of pig brain (1 kg) was purified initially by DEAEcellulose (11) and Affi-Gel Blue chromatography (12), as previously described. The final step of the purification was affinity chromatography on polyacrylamide on which cholesterol and phosphatidylserine were immobilized, a procedure recently described by ...

CD40 ligand (CD40L), a surface molecule which can be expressed by T cells, mast cells and basophils, has been shown to be involved in the control of B cell proliferation, immunoglobulin class switching as well as in the activation of monocytes and T cells. We demonstrate that CD40L can also be expressed constitutively by eosinophils from an hypereosinophilic patient or, upon activation, by the eosinophilic cell line EOL-3 and normal blood eosinophils. Eosinophils were able to induce, in conjunction with IL-4, CD40L-dependent B cell proliferation in vitro. These results suggest that CD40L could play a role in the inflammatory processes during which eosinophil infiltration and activation are observed.

Crude extracts of a multiply peptidase-deficient strain of Salmonella typhimurium contain an aminopeptidase that specifically removes N-terminal methionine from peptides. This activity shows pronounced specificity for the peptide's second amino acid. Methionine is removed from peptides with alanine, threonine, or glycine in this position but not when the second amino acid is leucine or methionine. The activity is stimulated by Co2l and is inhibited by EDTA.Mutations that lead to overproduction (up to He concluded that N-terminal modification is a stepwise process, with deformylation preceding the removal of methionine by an aminopeptidase. Attempts to isolate this methionine-specific aminopeptidase have not been successful, however. Vogt (6) purified and characterized an activity capable of rapid cleavage of Met-Ala-Ser. He concluded that the specificity of this enzyme is not consistent with its involvement in N-terminal methionine removal. In addition, mutants of E. coli or Salmonella lacking this enzyme grow normally (7,8). Earlier reports of a ribosomal peptidase activity (9) almost certainly are based on the artifactual association of the enzyme described by Vogt with the ribosome fraction (6). Another attempt to identify a methionine-specific aminopeptidase led to the isolation of an enzyme that appears to have specificity for methionine but hydrolyzes no substrates larger than dipeptides (10).Clearly, a major problem in identifying a methioninespecific aminopeptidase in cell extracts is the presence of several broad-specificity enzymes capable of hydrolyzing N-terminal methionine peptides. We have shown that at least four such enzymes are present in crude extracts of Salmonella typhimurium and E. coli (7,8,11). We have also isolated mutants that lack all of these activities and observed that these mutants, although greatly restricted in their capacity to use peptides as amino acid sources, still use certain Nterminal methionine peptides (8,11). This paper makes use of these mutant strains to identify a methionine-specific aminopeptidase and to isolate mutants that substantially overproduce this activity. The substrate specificity ofthis enzyme is entirely consistent with the proposal that it is involved in the removal of N-terminal methionine from newly synthesized proteins.

Hete we report the cloning of the cDNA for human CD40-Ligand (CD4O-L) from a CD4positive T cell clone. The deduced amino acid sequence predicts a type II membrane protein of 261 amino acids. Northern blot and FACS analysis of PBMNC revealed that the human CD40-L can be detected on T cells and is absent from B cells and monocytes. The human CD40-L is expressed on both CD4-and CDS-positive T cells, (CD45RO') and (CD45RA+) subsets. We observed that IL-4, an inducer of IgE production, upregulated CD40-L mRNA level while IFNy, an inhibitor of IgE synthesis, reduced the expression of CD40-L mRNA. These data suggest a the correlation between human 0-L expression and IgE production.