Tumour necrosis factor (TNF), jointly referring to TNF alpha and TNF beta, is a central mediator of immune and inflammatory responses; its activities are mediated by two distinct receptors, TNFR1 (p55) and TNFR2 (p75) (reviewed in refs 1-3). The cytoplasmic domains of the TNFRs are unrelated, suggesting that they link to different intracellular signalling pathways. Although most TNF responses have been assigned to one or the other of the TNF receptors (mostly TNFR1), there is no generally accepted model for the physiological role of the two receptor types. To investigate the role of TNFR1 in beneficial and detrimental activities of TNF, we generated TNFR1-deficient mice by gene targeting. We report here that mice homozygous for a disrupted Tnfr1 allele (Tnfr1(0)) are resistant to the lethal effect of low doses of lipopolysaccharide after sensitization with D-galactosamine, but remain sensitive to high doses of lipopolysaccharide. The increased susceptibility of Tnfr1(0)/Tnfr1(0) mutant mice to infection with the facultative intracellular bacterium Listeria monocytogenes indicates an essential role of TNF in nonspecific immunity.

The fluctuations of the membrane potential during mitochondrial Ca2+ transport have been monitored with an electrode sensitive to tetraphenylphosphonium. The following conclusions have been reached. 1. The membrane becomes depolarized during the influx of Ca2+. When the bulk of the Ca2+ pulse has been taken up, it repolarizes, but not completely. 2. If all of the accumulated Ca2+ is released from mitochondria and cycling is inhibited, the membrane repolarizes completely. 3. The accumulation of Ca2+ alone does not induce mitochondrial damage. In the presence of inorganic phosphate, the uptake of Ca2+ may lead to complete and irreversible depolarization, depending on the amount of Ca2+ and phosphate accumulated. The irreversible damage observed in the presence of phosphate is prevented by Mg2+.

IntroductionIn previous studies we showed that cultured human keratinocytes expressed the 55-kD TNF receptor (TNFR)

NAD' glycohydrolase activity is found at high levels in submitochondrial particles. It leads to the reaction products ADP-ribose, nicotinamide, and small amounts of 5'-AMP. Furthermore, submitochondrial particles catalyze the exchange reaction: [adenosine-14C]ADP-ribose + NAD+ -[adenosine-'4C]_ NAD+ + ADP-ribose. When submitochondrial particles are incubated with NAD+, mono(ADP-ribosyl)ation of protein molecules migrating with an apparent molecular weight of 30,000 in sodium dodecyl sulfate/polyacrylamide gel electrophoresis is demonstrable. Inhibitor studies suggest attachment of ADP-ribose to arginine residues. ADP-ribose bound to submitochondrial particles is rapidly turning over. The release of ADP-ribose from the protein is probably enzyme catalyzed. The rapid turnover, the specificity of the modification, and the inhibition of ADP-ribosylation by ATP and nicotinamide suggest a regulatory role of mono(ADP-ribosyl)ation of a protein in the inner mitochondrial membrane.ADP-ribosylation is a posttranslational protein modification receiving growing interest. Several reviews on both the biological and methodological aspects have been published (1-3). ADPribose is attached to acceptor proteins as a monomer, oligomer, or polymer. Mono(ADP-ribosyl)ation has so far been found to be catalyzed mainly by prokaryotic enzymes-e.g., diphtheria toxin (4), Pseudomnas exotoxin A (5), choleragen (6), and Escherichia coli heat-labile enterotoxin (7). Catalysis of mono-ADPribosylation by enzymes from rat liver (8) and erythrocytes from turkey and man (9, 10) has also been reported. Poly(ADP-ribosyl)ation, on the other hand, is found mainly in the nuclei of eukaryotic cells. The accumulating evidence indicates a close relationship between poly(ADP-ribosyl)ation and chromatin activities (11,12).During our studies on the mechanism of hydroperoxide-induced release of calcium from rat liver mitochondria we observed the hydrolysis of intramitochondrial pyridine nucleotides at the ,BN-glycosidic bond between nicotinamide and ADPribose in intact mitochondria (13,14). The hydrolysis of NAD+ is accompanied by a covalent protein modification at the inner side of the inner mitochondrial membrane by a NAD+-derived compound. Pyridine nucleotide hydrolysis, protein modification, and release of calcium from rat liver mitochondria are inhibited by ATP (15). On the basis of these findings we put forward the hypothesis that this covalent modification is part of a mechanism controlling calcium release.Kun et a! (16,17) have described ADP-ribosylation of a soluble protein derived from sonicated mitochondria. In recent papers Hilz and co-workers (18,19) analyzed the distribution of mono-and poly(ADP-ribose) in liver cells. We describe here a covalent modification by mono(ADP-ribose) of a protein in the inner mitochondrial membrane. The modification is found almost exclusively in protein molecules with a molecular weight of 30,000 in NaDodSO4/polyacrylamide gel electrophoresis. The modifying group has a high turnover. The fast removal of ADP...

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