Recent studies have characterized the ABC half-transporter associated with mitoxantrone resistance in human cancer cell lines. Encoded by the ABCG2 gene, overexpression confers resistance to camptothecins, as well as to mitoxantrone. We developed four polyclonal antibodies against peptides corresponding to four different epitopes on the mitoxantrone resistance-associated protein, ABCG2. Three epitopes localized on the cytoplasmic region of ABCG2 gave rise to high-affinity antibodies, which were demonstrated to be specific for ABCG2. Western blot analysis of cells with high levels of ABCG2 showed a single major band of the expected 72-kDa molecular size of ABCG2 under denaturing conditions. Immunoblot analysis performed under non-reducing conditions and after treatment with cross-linking reagents demonstrated a molecular weight shift from 72 kDa to several bands of 180 kDa and higher molecular weight, suggesting detection of dimerization products of ABCG2. Evidence of N-linked glycosylation was also obtained using tunicamycin and N-glycosidase F. Finally, both by light, fluorescence and electron microscopic immunohistochemical staining, we demonstrate cytoplasmic and predominantly plasma membrane localization of ABCG2 in cell lines with high levels of expression. Plasma membrane staining was observed on the surface of the chorionic villi in placenta. These results support the hypothesis that ABCG2 is an ABC half-transporter that forms dimers in the plasma membrane, functioning as an ATP-dependent outward pump for substrate transport.

Anoplin is a decapeptide amide, GLLKRIKTLL-NH2 derived from the venom sac of the solitary spider wasp, Anoplius samariensis. It is active against Gram-positive and Gram-negative bacteria and is not hemolytic towards human erythrocytes. The present paper reports a structure-activity study of anoplin based on 37 analogues including an Ala-scan, C- and N-truncations, and single and multiple residue substitutions with various amino acids. The analogues were tested for antibacterial activity against both S. aureus ATCC 25923 and E. coli ATCC 25922, and several potent antibacterial analogues were identified. The cytotoxicity of the analogues against human erythrocytes was assessed in a hemolytic activity assay. The antibacterial activity and selectivity of the analogues against S. aureus and E. coli varied considerably, depending on the hydrophobicity and position of the various substituted amino acids. In certain cases the selectivity for Gram-positive and Gram-negative bacteria was either reversed or altogether eliminated. In addition, it was generally found that antibacterial activity coincided with hemolytic activity.

2-Acetamido-2-deoxy-β-d-glucopyranose (β-d-GlcpNAc), in O-glycosidic linkage to the side chain hydroxyls of serine (Ser) and threonine (Thr) residues, is often found in nuclear and cytoplasmic proteins. The “active ester” approach for solid phase glycopeptide synthesis calls for the direct glycosylation of N α-(9-fluorenylmethyloxycarbonyl)amino acid pentafluorophenyl esters (N α-Fmoc-AA-OPfp's). The synthesis of the required Ser(β-d-GlcpNAc) and Thr(β-d-GlcpNAc) building blocks poses special problems arising from the 2-amino substituent in the corresponding glycosyl donors. Activation of donors with a 2-N-acyl group provides relatively unreactive oxazoline intermediates, whereas the otherwise promising phthaloyl (Phth) group requires prolonged base treatment at elevated temperatures for its removal, and incomplete deprotection is often encountered. The dithiasuccinoyl (Dts) group provides bivalent protection in the same way as the Phth group, but has the advantage of being removed rapidly under mild conditions by thiolysis or other reductive procedures. The novel donor 3,4,6-tri-O-acetyl-2-deoxy-2-(dithiasuccinoylamino)-d-glucopyranosyl bromide (8) was prepared from d-glucosamine in four steps and overall 58% yield; this compound served for the fast and efficient glycosylation of N α-Fmoc-Ser-OPfp and N α-Fmoc-Thr-OPfp. The resultant glycosylated building blocks N α-Fmoc-Ser(Ac3-β-d-GlcpNDts)-OPfp (9) and N α-Fmoc-Thr(Ac3-β-d-GlcpNDts)-OPfp (10) were applied in the solid phase synthesis of several glycopeptides. Following incorporation of the glycosylated residue, the Dts function was removed quantitatively by thiolysis, the resultant free 2-amino group was acetylated, and stepwise chain elongation by Fmoc chemistry continued. Alternatively, it was possible to achieve selective Dts removal from 9 and 10 (without affecting the Pfp ester) by reduction with zinc under acidic conditions; in situ acetylation of the exposed amine provided the alternative building blocks N α-Fmoc-Ser(Ac3-β-d-GlcpNAc)-OPfp (11) and N α-Fmoc-Thr(Ac3-β-d-GlcpNAc)-OPfp (12).

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Phosphorylation is the only known in vivo substitution of starch, yet no structural evidence has been provided to explain its implications of the amylosidic backbone and its stimulating effects on starch degradation in plants. In this study, we provide evidence for a major influence on the glucosidic bond in starch specifically induced by the 3-O-phosphate. Two phosphorylated maltose model compounds were synthesized and subjected to combined molecular dynamics (MD) studies and 950 MHz NMR studies. The two phosphorylated disaccharides represent the two possible phosphorylation sites observed in natural starches, namely maltose phosphorylated at the 3'- and 6'-position (maltose-3'-O-phosphate and maltose-6'-O-phosphate). When compared with maltose, both of the maltose-phosphates exhibit a restricted conformational space of the alpha(1-->4) glycosidic linkage. When maltose is phosphorylated in the 3'-position, MD and NMR show that the glucosidic space is seriously restricted to one narrow potential energy well which is strongly offset from the global potential energy well of maltose and almost 50 degrees degrees from the Phi angle of the alpha-maltose crystal structure. The driving force is primarily steric, but the configuration of the structural waters is also significantly altered. Both the favored conformation of the maltose-3'-phosphate and the maltose-6'-phosphate align well into the 6-fold double helical structure of amylopectin when the effects on the glucosidic bond are not taken into account. However, the restrained geometry of the glucosidic linkage of maltose-3'-phosphate cannot be accommodated in the helical structure, suggesting a major local disturbing effect, if present in the starch granule semi-crystalline lattice.