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New thiol protease inhibitors, estatins A and B, were isolated from the culture filtrate of Myceliophthora thermophila M4323. The basic, water-soluble inhibitors were characterized as having an agmatine, /m^-epoxysuccinic acid and L-phenylalanine or L-tyrosine moieties in the structure. The molecular formulas C18H25N5O5and C18H25N5O6for A and B were indicated by elemental analysis and fast atom bombardment MS. Estatins were specific inhibitors against thiol proteases such as papain, ficin and bromelain. They suppressed IgE antibody production in mice, but not IgG.Lowmolecular weight protease inhibitors are useful at least as medicines or reagents for research.During our screening for thiol protease-specific inhibitors, a fungal culture produced inhibitors against papain. The inhibitors were isolated and named estatins A and B (Fig.

Moderate concentrations of bredinin (1.2 x 10-; M) strongly inhibited growth of L5178Y cells, with the effect being reversed by guanylic acid (GMP). However, at higher concentrations of bredinin the inhibition was not reversed completely by GMP added in excess. Bredinin was cytocidal at concentrations above 2 x 10-1 M, but 5 x 10-1 M bredinin in the presence of excess GMP, bredinin was cytostatic.Bredinin inhibited nucleic acid synthesis of L5178Y cells, but bredinin itself was not incorporated into the nucleic acid. Inhibition of nucleic acid synthesis was clearly reversed by GMP. Similarly chromosomal aberrations in L5178Y cells caused by bredinin were reversed by GMP. In contrast, the effect of a high concentration of bredinin on cell multiplication was not reversed by GMP. The modal volume of L5178Y cells increased during incubation in the presence of bredinin and GMP for 24 hours, 5 x 10-1 M bredinin with GMP causing a 70% increase in cell volume. This increase in cell volume was mainly due to an increase in the protein content of the cells.The cytostatic effect of bredinin with GMP was reversed completely by adenosine-3',5'-cyclic monophosphate (cyclic AMP). Other cyclic nucleotides and nucleotides were ineffective. The reversing effect of cyclic AMP on cell survival depended upon the concentration of GMP, and was not seen in the absence of GMP. It was concluded that cyclic AMP influences the secondary cytostatic effect of bredinin, and not the primary cytotoxic effect reversed by GMP.As reported in previous papers' 2,,,, the new antibiotic bredinin (4-carbamoyl-l-/3-n-ribofuranosyl imidazolium-5-olate) is a derivative of AICA-riboside. Bredinin strongly inhibits the growth of tumor cells in tissue culture, this inhibition being reversed by GMP. The cytolytic activity of bredinin is due to inhibition of the conversion of IMP to GMP in the purine nucleotide biosynthetic pathway.GMP does not completely reverse the inhibitory activity of high concentrations of bredinin, an observation explained by bredinin acting at another site. This paper reports studies exploring the secondary effect of bredinin on L5178Y cells. Materials and MethodsBredinin was obtained as described previously".Other materials were purchased as follows: adenosine-3',5'-cyclic monophosphate from Sigma Chemical Company, St. Louis, U.S.A.; 3H-thymidine and 3H-uridine from New England Nuclear. 14C-Bredinin (2.12 mCi/mM) was synthesized in our laboratory by the method described previously4'.The mouse leukemia cell line, L5178Y, was grown at 37°C in FISCHER'S medium supplemented with 10% dialyzed bovine serum, penicillin and streptomycin. In this medium, the L5178Y cells grew logarithmically, with a generation time of about 11 hours until they reached a cell density of 101 cells/ml. FISCHER'S medium and colcemid for chromosome study were purchased from Grand Island Biology Co., Ltd., U.S.A. Bovine serum was prepared in our laboratory. Routine tests for Mycoplasma in the serum gave negative results.

The aglycone of the nucleoside antibiotic, bredinin, was as strongly cytotoxic to L5178Y cells as bredinin. The cytotoxic properties of the aglycone were very similar to those of bredinin and the minimum inhibitory concentrations of both were 10-5.M.

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