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Costimulatory blockade is one of the most promising therapeutic targets in autoimmune diseases as well as in transplant recipients, and inhibition of the cluster of differentiation (CD)40-CD154 interaction, which is required for T cell activation and development of an effective immune response, is particularly promising in islet transplant recipients. Here, we report the ability of several small-molecule organic dyes to concentration dependently inhibit this interaction with IC(50) values in the low-micromolar range. They were found to be considerably more active in inhibiting this interaction than the tumor necrosis factor (TNF)-R1-TNF-alpha or B cell-activating factor (BAFF)-R-BAFF interaction, which are members of the same family. They specifically inhibited CD154-induced cell responses in human B cells as well as in THP-1 myeloid cells, which can serve as surrogate dendritic cells, at concentrations well below their cytotoxic concentrations determined in the same cells. Flow cytometry experiments confirmed their ability to inhibit the CD154-induced, but not the Staphylococcus aureus Cowan I- or phorbol 12-myristate 13-acetate-induced increase in the surface expression of CD54, CD40, and major histocompatibility complex class II. Accordingly, these compounds can be useful not only for experimental investigations involving the inhibition of the CD40-CD154 costimulatory interaction but can also provide important structure-activity relationship information and can serve as the starting point of a targeted drug discovery program.

Three cx-galactosidase genes, agll, ag12 and ag13, were isolated from a cDNA expression library of Trichoderma reesei RutC-30 constructed in the yeast Saccharomyces cerevisiae by screening the library on plates containing the substrate 5-bromo-4-chloro-3-indolyl-a-D-gnlactopyranoside. The genes ugll, ag12 and agl3 encode 444, 746 and 624 amino acids, respectively, including the signal sequences. The deduced amino acid sequences of AGLI and AGLIII showed similarity with the a-galactosidases of plant, animal, yeast and filamentous fungal origin classified into family 27 of glycosyl hydrolases whereas the deduced amino acid sequence of AGLII showed similarity with the bacterial a-galactosidases of family 36. The enzymes produced by yeast were analysed for enzymatic activity against different substrates. AGLI, AGLII and AGLIII were able to hydrolyse the synthetic substrate p-nitrophenyl-a-D-galactopyranoside and the small galactose-containing oligosaccharides, melibiose and raffinose. They liberated galactose from polymeric galacto(g1uco)mannan with different efficiencies. The action of AGLI towards polymeric substrates was enhanced by the presence of the endo-l,4-/?-mannanase of T reesei. AGLII and AGLIII showed synergy in galacto(g1uco)mannan hydrolysis with the endo-l,4-/?-mannanase of 7: reesei and a B-mannosidase of Aspergillus niger. The calculated molecular mass and the hydrolytic properties of AGLI indicate that it corresponds to the a-galactosidase previously purified from 7: reesei.

The axel gene encoding acetyl xylan esterase was isolated from an expression library of the filamentous fungus Trichoderma reesei using antibodies raised against the purified enzyme. Apparently axel codes for the two forms, PI 7 and PI 6.8, of acetyl xylan esterase previously characterized. The axel encodes 302 amino acids including a signal sequence and a putative propeptide. The catalytic domain has no amino acid similarity with the reported acetyl xylan esterases but has a clear similarity, especially in the active site, with fungal cutinases which are serine esterases. Similarly to serine esterases, the axel product was inactivated with phenylmethylsulfonyl fluoride. At its C-terminus it carries a cellulose binding domain of fungal type, which is separated from the catalytic domain by a region rich in serine, glycine, threonine and proline. The binding domain can be separated from the catalytic domain by limited proteolysis without affecting the activity of the enzyme towards acetylated xylan, but abolishing its capability to bind cellulose.