Biologic activities of recombinant human-beta-defensin-4 toward cultured human cancer cells.

AIM: The aim of the study was in vitro analysis of biological activity of recombinant human beta-defensin-4 (rec-hBD-4). METHODS: hBD-4 cDNA was cloned into pGEX-2T vector, and recombinant plasmid was transformed into E. coli BL21(DE3) cells. To purify soluble fusion GST-hBD-4 protein, affinity chromatography was applied. Rec-hBD-4 was cleaved from the fusion protein with thrombin, and purified by reverse phase chromatography on Sep-Pack C18. Effects of rec-hBD-4 on proliferation, viability, cell cycle distribution, substrate-independent growth, and mobility of cultured human cancer cells of A431, A549, and TPC-1 lines were analyzed by direct cell counting technique, MTT assay, flow cytofluorometry, colony forming assay in semi-soft medium, and wound healing assay. RESULTS: Rec-hBD-4 was expressed in bacterial cells as GST-hBD-4 fusion protein, and purified by routine 3-step procedure (affine chromatography on glutathione-agarose, cleavage of fusion protein by thrombin, and reverse phase chromatography). Analysis of in vitro activity of rec-hBD-4 toward three human cancer cell lines has demonstrated that the defensin is capable to affect cell behaviour in concentration-dependent manner. In 1-100 nM concentrations rec-hBD-4 significantly stimulates cancer cell proliferation and viability, and promotes cell cycle progression through G2/M checkpoint, greatly enhances colony-forming activity and mobility of the cells. Treatment of the cells with 500 nM of rec-hBD-4 resulted in opposite effects: significant suppression of cell proliferation and viability, blockage of cell cycle in G1/S checkpoint, significant inhibition of cell migration and colony forming activity. CONCLUSION: Recombinant human beta-defensin-4 is biologically active peptide capable to cause oppositely directed effects toward biologic features of cancer cells in vitro dependent on its concentration.

[Interlocus associations and their variability in cattle]. / Mezhlokusnye assotsiatsii i ikh izmenchivost' u krupnogo rogatogo skota.

The results of analysis of interloci associations between two pairs of syntenic loci (transferrin and ceruloplasmin, receptor for vitamin D and kappa-casein) and two non-syntenic ones (amylase-1 and post-transferrin 2) in two cattle groups of Red Steppe breed (infected and uninfected by bovine leukosis virus) and in two groups of Black-and-White Holsteins (from relatively "pure" zone and from the 10 km zone of Chernobyl NPP) were presented. It is found that "linkage disequilibrium" between loci is observed independent of their synteny. The data obtained allowed the authors to suppose, that the interloci associations are rather controlled by different factors of artificial and natural selection than by the genetic linkages between genes.