Proline residue-modified polycationic analogs of gramicidin S with high antibacterial activity against both Gram-positive and Gram-negative bacteria and low hemolytic activity.

Novel polycationic analogs of the cyclic decapeptide antibiotic, gramicidin S, possessing NH(2), D/L-Phe-NH or L-Lys-NH groups at the 4alpha- or 4beta-positions of the L-Pro residues, were synthesized. While L-Pro(4alpha/beta-NH(2))-containing analogs exhibited much weaker antibacterial activity, the D/L-Phe and L-Lys-substituted analogs exhibited higher antibacterial activity against Gram-negative bacteria than the parent gramicidin S. All of these additional amino group-containing analogs showed substantially reduced toxicity against human blood cells.     

Studies on the structure of HCN oligomers

The structure of the water-insoluble fraction of HCN oligomers (azulmic acid) was studied by IR, NMR, gel permeation chromatography, and chemical methods. The results show that nearly half of the nitrogen atoms contained in the oligomers are of the primary amino type and the other half are involved in -C = N- type bonding. It was found that the oligomers are easily and almost quantitatively acetylated, and the acetylated oligomers show characteristic acetyl amide IR absorption and NMR spectra. Owing to the greatly improved solubility due to acetylation, the molecular weight distribution was determined for the acetylated oligomers by gel permeation chromatography. Composite peaks were obtained ranging from 300 to 900 in molecular weight. Our results are essentially consistent with the structure proposed by Völker, but we point out there may be other possible structures also consistent with our experimental results.     

Molecular changes in UV-induced and γ-ray-induced mutations in human lymphoblastoid cells

We have characterized the structural changes in the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene of 14 UV-induced, 15 γ-ray-induced and 17 spontaneous mutants of human lymphoblastoid cells selected for 6-thioguanine (6TG) resistance. Southern blot analysis using the full-length HPRT cDNA as a probe revealed that 29% (5/17) of the spontaneous mutants contained detectable alterations in their restriction fragment patterns. Among the 15 mutants induced by γ rays, 7 (47%) had such alterations indicative of large deletions in the HPRT gene. In contrast, all 14 UV-induced mutants exhibited hybridization patterns indistinguishable from those of the wild-type cells. These results suggest that UV is likely to induce point mutations at the HPRT locus on the human chromosome and that the molecular mechanism of UV-induced mutation is quite different from that of ionizing radiation-induced mutation or spontaneous mutation in human cells.     

Involvement of oxidative DNA damage and apoptosis in antitumor actions of aminosugars.

We investigated the mechanisms of apoptosis and DNA damage induced by aminosugars in relation to their antitumor actions. The order of cytotoxic effects of aminosugars was D-mannosamine (ManN) > D-galactosamine (GalN) > D-glucosamine (GlcN). A comparison of the frequency of apoptotic cells showed the same order. DNA ladders were formed by only ManN and the formation of DNA ladders was inhibited by a caspase inhibitor. Pulsed-field gel electrophoresis showed that ManN caused cellular DNA cleavage at a lower concentration than those causing apoptosis. Cellular DNA cleavage was inhibited by catalase and enhanced by a catalase inhibitor. Flow cytometry showed that ManN enhanced the production of intracellular peroxides. These results suggest that ManN-induced apoptosis is preceded by H2O2-mediated DNA damage. The order of the extent of damage to 32P-labeled DNA fragments by aminosugars plus Cu(II) was ManN > GalN > GlcN. The DNA damage was inhibited by catalase and bathocuproine, suggesting that H2O2 reacts with Cu(I) to form the metal-peroxide complex capable of causing DNA damage. Two mechanisms of H2O2 generation from aminosugars were proposed: one is the major pathway to form a dioxo compound and NH4+; the other is the minor pathway to form a pyrazine derivative through the condensation of two molecules of an aminosugar. The order of reactivity to generate these products was ManN > GalN > GlcN. On the basis of these results, it is concluded that aminosugars, especially ManN, produce H2O2 to cause DNA damage, which mediates apoptosis resulting in tumor growth inhibition.     

Amplification of bleomycin-induced DNA cleavage by pyrrole triamide.

We investigated the amplification of bleomycin-induced DNA cleavage by synthetic pyrrole triamide (PyPyPy) using 32P-labeled DNA fragments obtained from human genes. Peplomycin, a kind of bleomycins, plus Fe(II) caused DNA cleavage at the 5'-GC-3' and 5'-GT-3' sequences (damaged bases are underlined). The addition of PyPyPy enhanced the cleavage at cytosine and thymine residues 3' to consecutive guanines, particularly at the 5'-GGGGC-3' and 5'-GGGGT-3' sequences. These results suggest that PyPyPy binds to DNA to induce its conformational change, resulting in alteration of the site specificity and amplification of DNA cleavage. The present study on amplifiers of antitumor drugs would show a novel approach to the establishment of more effective chemotherapy.