TNF-a inducing protein (Tipa) is secreted from Helicobacter pylori (H. pylori): it is a potent inducer of TNF-a and chemokine genes, mediated through NF-jB activation, and it also induces tumorpromoting activity in Bhas 42 cells. To investigate the carcinogenic mechanisms of H. pylori with Tipa, we first examined how Tipa acts on gastric epithelial cells. We found that fluorescent-Tipa specifically bound to, and then entered, the cells in a dose-and temperature-dependent manner, whereas deletion mutant of Tipa (del-Tipa), an inactive form, neither bound to nor entered the cells, suggesting the presence of a specific binding molecule. Mutagenesis analysis of Tipa revealed that a dimer formation of Tipa with a disulfide bond is required for both specific binding and induction of TNF-a gene expression. A confocal laser scanning microscope revealed some Tipa in the nuclei, but del-Tipa was not present, which indicated that an active form of Tipa can penetrate the nucleus and may be involved in the induction of TNF-a gene expression. Examination of Tipa production and secretion in 28 clinical isolates revealed that H. pylori obtained from gastric cancer patients secreted Tipa in significantly higher amounts than did H. pylori from patients with chronic gastritis, suggesting that Tipa is an essential factor in H. pylori inflammation and cancer microenvironment in the human stomach. Tipa is thus a new carcinogenic factor of H. pylori that can enter the nucleus through a specific binding molecule, and its mechanism of action is completely different from that of CagA.

We present evidence supporting novel collaborations between the serine protease inhibitor (serpin) and the trefoil factor during the budding stage of the tunicate Polyandrocarpa misakiensis . Using a maltose-binding protein/P-serpin fusion protein, two polypeptides of 40 kDa and 45 kDa were pulled down from Polyandrocarpa homogenates. Based on their partial amino acid sequence data, a single cDNA (928 bp) was cloned. It encodes a polypeptide that has five tandem repeats of a trefoil consensus motif. Thus, we termed the cDNA P-trefoil . Both P-trefoil and P-serpin were expressed exclusively by coelomic cells during budding. P-Trefoil was expressed mainly by coelomic cells throughout the asexual life cycle of Polyandrocarpa , while P-Serpin was localized particularly in coelomic cells and in the extracellular matrix in developing buds. The native PTrefoil protein showed aminopeptidase activity. It induced cell growth in cultured Polyandrocarpa cells at a concentration of 8 µ g/mL. P-Serpin reinforced this activity of P-Trefoil. Further, a mixture of P-Trefoil and P-Serpin exhibited the in vitro induction of a gut-specific alkaline phosphatase. These results show for the first time that a serpin can interact with a trefoil factor to play a role in the cellular growth and differentiation of the gastric epithelium.