Inhibition of human telomerase by PNA-cationic peptide conjugates.

The inhibition of human telomerase has been explored using peptide conjugated derivatives of a PNA pentamer directed at the RNA template of telomerase. It is demonstrated that the presence of cationic peptides at the N-terminus of the PNA results in enhanced inhibition of telomerase activity. Furthermore, inhibition depended on the specificity of PNA recognition.

The mechanism and substrate specificity of the NADPH:flavin oxidoreductase from Escherichia coli.

The NAD(P)H:flavin oxidoreductase from Escherichia coli, Fre, is a monomer of 26.2 kDa that catalyzes the reduction of free flavins by NADPh or NADH. Overexpression in E. coli now allows the preparation of large amounts of pure protein. Structural requirements for recognition of flavins as substrates and not as cofactors were studied by steady-state kinetics with a variety of flavin analogs. The entire isoalloxazine ring was found to be the essential part of the flavin molecule for interaction with the polypeptide chain. Methyl groups at C-7 and C-8 of the isoalloxazine ring and the N-3 of riboflavin also play an important role in that interaction, whereas the ribityl chain of the riboflavin is not required for binding to the protein. On the other hand, the presence of the 2'-OH of the ribityl chain stimulates the NADPH-dependent reaction significantly. Moreover, a study of competitive inhibitors for both substrates demonstrated that Fre follows a sequential ordered mechanism in which NADPH binds first followed by riboflavin. Lumichrome, a very good inhibitor of Fre, may be used to inhibit flavin reductase in E. coli growing cells. As a consequence, it can enhance the antiproliferative effect of hydroxyurea, a cell-specific ribonucleotide reductase inactivator.

Recognition and receptor-mediated uptake of phosphorylated high mannose-type oligosaccharides by cultured human fibroblasts.

The intracellular transport of newly synthesized lysosomal hydrolases to lysosomes requires the presence of one or more phosphorylated high mannose-type oligosaccharides per enzyme. A receptor that mediates mannose-6-PO4-specific uptake of lysosomal enzymes is expressed on the surface of fibroblasts and presumably accounts for the intracellular transport of newly synthesized enzymes to the lysosome. In this study, we examined the internalization of lysosomal enzyme-derived phosphorylated oligosaccharides by cultured human fibroblasts. Oligosaccharides of known specific activity bearing a single phosphate in monoester linkage were internalized with Kuptake of 3.2 X 10(-7) M, whereas oligosaccharides bearing two phosphates in monoester linkage were internalized with a Kuptake of 3.9 X 10(-8) M. Thus, phosphorylated high mannose-type oligosaccharides appear to be the minimal structure required for recognition and uptake by the fibroblast receptor. The finding that the Kuptake for monophosphorylated oligosaccharides is 100-fold less than the reported Ki for mannose-6-phosphate indicates that the fibroblast phosphomannosyl receptor contains a binding site that recognizes features of the oligosaccharide in addition to mannose-6-phosphate.