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Serum ultrafiltrates (SUF) from human patients with different types of cancer contain a blocking factor (BF) that inhibits the cytolytic activity ofhuman tumor necrosis factor a (TNF-a) in vitro. BF is a protein with a molecular mass of28 kDa on reducing sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE). The active material was purified to homogeneity by a combination of affinity chromatography, PAGE, and high-pressure liquid chromatography. Amino add sequence analysis revealed that BF is derived from the membrane TNF receptor. Purified BF blocks the lytic activity ofrecombinant human and mouse TNF-a and recombinant human lymphotoxin on murine L929 cells in vitro. However, BF inhibits the lytic activity of TNF-a more effectively than it does that of lymphotoxin. The BF also inhibits the necrotizing activity ofrecombinant human TNF-a when cou jected into established cutaneous Meth A tumors in BALB/c mice. The BF may have an important role in (i) the regulation and control of TNF-a and lymphotoxin activity in cancer patients, (ii) interaction between the tumor and the host antitumor mechanisms, and (ii) use of systemically administered TNF-a in clinical trials with human cancer patients.

The postsynaptic molecule gephyrin is involved in clustering neurotransmitter receptors. To test for protein variants that correspond to alternatively spliced gephyrin mRNAs, antibodies were made against 1) an amino-terminal domain of gephyrin (GN(N)) and 2) its invariant carboxy-terminus (GN(C)). Both antibodies recognized an antigen with the expected molecular weight of 93-95 kDa in rat and human brain tissue, as well as five additional proteins between 90 and 108 kDa. Most of these variants were found distributed throughout the brain, and their developmental profiles paralleled those of synaptic markers. Interestingly, the pattern of antigens immunostained across brain regions by anti-GN(N) was markedly distinct from that labeled by anti-GN(C), a difference consistent with carboxy-terminal modification. In control experiments in which hippocampal membranes were treated to activate endogenous proteases, there was no evidence that certain gephyrin variants originate from proteolysis. A subset of the antigens was, however, rapidly degraded during the treatment. A corresponding production of stable, carboxy-terminal gephyrin fragments of 48-50 kDa occurred within 1 min of proteolytic activation and was blocked by the selective calpain inhibitor CX295. These findings suggest that multiple gephyrin proteins are active in the brain and that some of their roles may require functional modulation by limited proteolysis.

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