The Escherichia coli AlkA protein is a base excision repair glycosylase that removes a variety of alkylated bases from DNA. The 2.5 A crystal structure of AlkA complexed to DNA shows a large distortion in the bound DNA. The enzyme flips a 1-azaribose abasic nucleotide out of DNA and induces a 66 degrees bend in the DNA with a marked widening of the minor groove. The position of the 1-azaribose in the enzyme active site suggests an S(N)1-type mechanism for the glycosylase reaction, in which the essential catalytic Asp238 provides direct assistance for base removal. Catalytic selectivity might result from the enhanced stacking of positively charged, alkylated bases against the aromatic side chain of Trp272 in conjunction with the relative ease of cleaving the weakened glycosylic bond of these modified nucleotides. The structure of the AlkA-DNA complex offers the first glimpse of a helix-hairpin-helix (HhH) glycosylase complexed to DNA. Modeling studies suggest that other HhH glycosylases can bind to DNA in a similar manner.

Cancer cells invade by secreting degradative enzymes, which are sequestered in lysosomal vesicles. In this study, the impact of an acidic extracellular environment on lysosome size, number, and distance from the nucleus in human mammary epithelial cells (HMECs) and breast cancer cells of different degrees of malignancy was characterized because the physiological microenvironment of tumors is frequently characterized by extracellular acidity. An acidic extracellular pH (pH(e)) resulted in a distinct shift of lysosomes from the perinuclear region to the cell periphery irrespective of the HMECs' degree of malignancy. With decreasing pH, larger lysosomal vesicles were observed more frequently in highly metastatic breast cancer cells, whereas smaller lysosomes were observed in poorly metastatic breast cancer cells and HMECs. The number of lysosomes decreased with acidic pH values. The displacement of lysosomes to the cell periphery driven by extracellular acidosis may facilitate exocytosis of these lysosomes and increase secretion of degradative enzymes. Filopodia formations, which were observed more frequently in highly metastatic breast cancer cells maintained at acidic pH(e), may also contribute to invasion.

Sialyl Lewis x and derivatives have been synthesized using £-1,4-galactosyltransferase and recombinant a-2,3sialyltransferase and a-l,3-fucosyltransferase. The enzymatic glycosylations have been achieved on preparative scales with in situ regeneration of UDP-galactose, CMP-7V-acetylneuraminic acid, and GDP-fucose. Additionally, galactosyltransferase and fucosyltransferases have been studied with respect to their substrate specificity and inhibition. The enzymatic procedures have also been used in the synthesis of 2'-deoxy-LacNAc, 2'-amino-2'-deoxy-LacNAc, 2-azido-Lac, Lewis x, the Lewis x analog with GlcNAc replaced with 5-thioglucose, [Gal-l-13C]-LacNAc, [Gal-1 -13C]-sialyl Lewis x, and the corresponding terminal glycal. The synthesized 13C-labeled sialyl Lewis x and intermediates (including Lewis x and sialyl LacNAc) were used for conformational study using NMR techniques combined with calculations based on GESA and MM2 programs. GESA calculation of sialyl Lewis x gave four minimum-energy conformers, and the two (A and B) consistent with NMR results were further refined with MM2 calculation. The one (A') with lower energy was picked as the preferred conformer which had all intemuclear distances and glycosidic torsional angles consistent with the NMR analysis. The glycosidic torsional angle \p of Gal-GlcNAc, for example, was determined to be 18°on the basis of the coupling between Gal-l-13C and GlcNAc, while the predicted value was 15°. The tetrasaccharide appears to form a well-defined hydrophilic surface along NeuAc-Gal-Fuc, and a hydrophobic face underneath NeuAc-Gal-GlcNAc. Comparing the conformation of sialyl Lewis x to sialyl Lewis a indicates that the recognition domain of sialyl Lewis x mainly comes from the sialic acid-galactose-fucose residues.