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A novel proteinase inhibitor, Lentinus proteinase inhibitor, has been purified from the fruiting bodies of the edible mushroom, Lentinus edodes, by buffer extraction and affinity chromatography on immobilized anhydrotrypsin. The protein simultaneously inhibits bovine beta-trypsin and alpha-chymotrypsin at independent sites, with apparent dissociation constants of 3.5 x 10(-10) M and 4 x 10(-8) M, respectively. The purified protein is eluted as two well-separated peaks on reversed-phase HPLC, one of which is inhibitory-active and the other inactive, and they are interconvertible under folding/unfolding conditions. Among the mammalian and microbial serine proteinases examined, including human enzymes of blood coagulation and fibrinolysis, activated factor XI was inhibited by the Lentinus proteinase inhibitor. Chemical modification studies suggest involvement of one or more arginine residues in the inhibition of trypsin. The complete primary structure composed of 142 amino acids with an acetylated N-terminus was determined by protein analysis. The theoretical molecular mass (15999.2) from the sequence is close to the experimental value of 15999.61 +/- 0.61 determined by mass spectrometry. Although there are no apparently homologous proteinase inhibitors in the protein database, there is a rather striking similarity to the propeptide segment of a microbial serine proteinase, as well as to the N-terminal region of the mature enzyme.

BACKGROUND We have recently reported that simultaneous determination of the fucosylation and glucosaminylation of alpha-fetoprotein (AFP) was useful in the differential diagnosis of various liver diseases. Here, we measured these indices in germ cell tumor and compared them with those in various diseases to reveal the characteristic feature of the carbohydrate moiety of AFP in this tumor. METHODS Fucosylation and glucosaminylation indices of AFP were determined for 743 serum samples from 10 patients with germ cell tumor, 437 with hepatocellular carcinoma (HCC), 241 with benign liver diseases (24 patients with acute viral hepatitis, 99 with chronic hepatitis, and 118 with hepatic cirrhosis), and 55 with carcinoma metastatic to the liver from digestive organs (41 from stomach, 10 from gallbladder, 2 from pancreas, and 2 from colon) by crossed immunoaffinoelectrophesis in the presence of concanavalin A and Lens culinaris agglutinin. RESULTS The fucosylation index of AFP in germ cell tumors (99% +/- 2%, mean +/- SD) was higher than those in HCC (39% +/- 31%), carcinoma metastatic to the liver (76% +/- 27%), and benign liver diseases (4% +/- 7%); the differences were statistically significant (P < 0.001). The glucosaminylation index of AFP in germ cell tumor (45% +/- 20%) also was significantly higher than those in HCC (4% +/- 4%) and in benign liver diseases (1% +/- 4%) (P < 0.001). The difference in mean percentage of the glucosaminylation index, however, was not significant between germ cell tumors (45% +/- 20%) and carcinoma metastatic to the liver (39% +/- 23%). CONCLUSIONS The current study indicates that the most characteristic feature of glycosylation of AFP in germ cell tumors is fucosylation rather than glucosaminylation.

We studied the effects of 5 kinds of catechins on the adhesion of mouse lung carcinoma 3LL cells to the monolayer of bovine lung endothelial cells. (-)-Epicatechin gallate and (-)-epigallocatechin gallate were active in inhibiting the 3LL cell adhesion, while (+)-catechin and (-)-epicatechin were inactive. (-)-Epigallocatechin showed a considerable cytotoxicity. These data suggest that the specific chemical structure is required to exert the inhibitory activity of catechins and the search for the cellular binding protein(s) bound to these inhibitory catechins would provide a clue to clarify the mechanism of interactions between tumor cells and endothelial cells.