Human hepatocarcinoma HepG2 cells are known to be insensitive to tumor necrosis factor (TNF) cytotoxicity. In this report, preliminary washing of HepG2 cells with serum-free medium to remove endogenous and exogenous α-fetoprotein (AFP) from the cultivation medium transfers cells from the TNF-resistant to the TNF-sensitive state without addition of any transcriptional inhibitors. HepG2 cells sensitized to by washing again became TNF-resistant after their treatment with exogenous AFP. Protective AFP activity against TNF-induced cytotoxicity directly depends on the AFP/TNF concentration ratio, demonstrating biphasic AFP activity. Our data show that 0.2 mg/ml of AFP acts synergistically to enhance cytotoxicity of suboptimal TNF doses. In contrast, the same AFP dose significantly attenuates the cytotoxicity of high TNF doses. It is concluded that AFP can function as a protective factor against TNF cytotoxicity in human hepatoma cells. These observations suggest that AFP secretion by certain tumor cells allows a highly flexible regulation of TNF cytotoxicity, dependent on the amount of endogenous AFP.

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The almost complete sequence of a region of the 2 3 4 ribosomal RNA from Escherichia coli, 175 nucleotides in length, located between the 550th and the 1000th nucleotide at the 3' end of 23-S RNA and containing the binding site of protein L 1, is given. Possible models of secondary structure are proposed.It has been shown in the preceding papers [1,2] that under suitable conditions ribonucleoproteins are released upon ribonuclease digestion of a reconstituted complex between ribosomal protein L1 and the 2 3 3 RNA from Escherichia coli. These ribonucleoproteins contain an RNA region encompassing 150 -200 nucleotides according to the enzyme : RNA ratio used, located between the 550th and the 1000th nucleotide at the 3' end of 23-S RNA. This RNA region was (together with proteins L1 and L9) also found in ribonucleoproteins released by ribonuclease digestion of the 50-S subunit 131.The knowledge of the primary structure of this region is necessary to understand how it interacts with proteins L1 and L9. In the preceding paper [2] we described the sequence analysis of its T1 RNase digestion products. In this paper we describe the determination of the relative order of these T1 oligonucleotides within the region. The almost complete primary structure of a section encompassing 175 nucleotides and containing the L1 binding site is presented. This sequence enabled us to establish the relative positions of the different RNA subfragments contained in the ribonucleoproteins [2] and thus to localise which parts of the region are more strongly protected in the presence of protein L1.

F1 antigen (Caf1) of Yersinia pestis is assembled via the Caf1M chaperone/Caf1A usher pathway. We investigated the ability of this assembly system to facilitate secretion of full-length heterologous proteins fused to the Caf1 subunit in Escherichia coli. Despite correct processing of a chimeric protein composed of a modified Caf1 signal peptide, mature human interleukin-1␤ (hIL-1␤), and mature Caf1, the processed product (hIL- The chaperone/usher protein-assisted assembly pathway is the major pathway of fimbria assembly in the family of gramnegative bacteria, Enterobacteriaceae (29). In contrast to the complex general secretory (type II) (14) and contact-mediated (type III) (18) pathways, the chaperone/usher export machinery involves only two specific proteins, a periplasmic chaperone and usher protein, for export across the outer membrane. The periplasmic chaperone ensures correct folding of structural subunits and transports the folded subunit to the outer membrane usher protein, which mediates surface localization, apparently by forming a large gated channel (29).1␤Secretion systems utilizing the chaperone/usher pathway can be divided into two families based on structural features of the chaperones and cell surface structures (9, 36). A prototype of the first family is the pap gene cluster encoding the PapD chaperone and PapC usher, which mediate assembly of the composite rigid Pap pili of Escherichia coli (29). PapD contains two domains, each with a ␤-barrel and an immunoglobulin (Ig)-like fold (8). The caf gene cluster that produces and assembles the capsular F1 (Caf1) antigen of Yersinia pestis is the best-characterized representative of the second family (2,6,7,12,22,37). The genes encode a 26.5-kDa periplasmic chaperone (Caf1M) (7) and a 90.4-kDa outer membrane protein (Caf1A) (12), which together can mediate the surface assembly of Caf1 antigen (6) in recombinant E. coli cells (2, 13). Caf1M-like periplasmic chaperones are characterized by an extended variable sequence between the proposed F1 and G1 ␤-strands, a disulfide bond connecting these two strands, and an accessory N-terminal sequence (2, 36, 37). Together, these three features may form an extension to the binding domain, which is important for chaperone function (2,22,37). In contrast to pap-like gene clusters, all members of caf-like gene clusters are involved in the assembly of structures with a simple composition and a less rigid structure (fibrillae or capsule-like morphology) (9, 36).The crystal structures of the PapD-PapK chaperone-adapter subunit complex (28) and the type 1 pilin FimC-FimH chaperone-adhesin complex (3) have revealed that these pilin structural subunits also have immunoglobulin-like folds, except that the seventh ␤-strand is missing, leaving part of the hydrophobic core of the subunit exposed. Binding of the chaperone G1 ␤-strand to the C-terminal ␤-strand of the pilin within this hydrophobic groove completes the pilin immunoglobulin fold (3,28). This donor strand complementation interaction between periplasmic chaperone ...