Showing papers by "BIA Separations (Slovenia) published in 2014"

Glycidyl methacrylate and ethylhexyl acrylate based polyHIPE monoliths: Morphological, mechanical and chromatographic properties

Abstract: Using water-in-oil emulsions with a high volume share of aqueous (droplet) phase as precursors (High Internal Phase Emulsions; HIPEs), highly porous polymers (polyHIPEs) were prepared from glycidyl methacrylate (GMA) and ethylhexyl acrylate (EHA), their morphology investigated and mechanical and chromatographic characteristics evaluated. All polyHIPE monoliths had open cellular porous morphology with primary pores (cavities) between 4.8 μm and 26.2 μm and secondary level of interconnecting pores. Introduction of EHA into the oil phase and consequently into the polymer matrix of polyHIPEs had a significant effect on the mechanical properties; both tensile strength and elasticity were increased. On the other hand, chromatographic properties, such as protein binding capacity and back pressure, did not dramatically change.

Identification of proteins interacting with ammodytoxins in Vipera ammodytes ammodytes venom by immuno-affinity chromatography.

Abstract: In order to perform their function, proteins frequently interact with other proteins. Various methods are used to reveal protein interacting partners, and affinity chromatography is one of them. Snake venom is composed mostly of proteins, and various protein complexes in the venom have been found to exhibit higher toxicity levels than respective components separately. Complexes can modulate envenomation activity of a venom and/or potentiate its effect. Our previous data indicate that the most toxic components of the Vipera ammodytes ammodytes (Vaa) venom isolated so far—ammodytoxins (Atxs)—are contributing to the venom’s toxicity only moderately; therefore, we aimed to explore whether they have some interacting partner(s) potentiating toxicity. For screening of possible interactions, immuno-affinity chromatography combined with identification by mass spectrometry was used. Various chemistries (epoxy, carbonyldiimidazole, ethylenediamine) as well as protein G functionality were used to immobilize antibodies on monolith support, a Convective Interaction Media disk. Monoliths have been demonstrated to better suit the separation of large biomolecules. Using such approach, several proteins were indicated as potential Atx-binding proteins. Among these, the interaction of Atxs with a Kunitz-type inhibitor was confirmed by far-Western dot-blot and surface plasmon resonance measurement. It can be concluded that affinity chromatography on monolithic columns combined with mass spectrometry identification is a successful approach for screening of protein interactions and it resulted with detection of the interaction of Atx with Kunitz-type inhibitor in Vaa venom for the first time.

Modulation of Activity of Known Cytotoxic Ruthenium(III) Compound (KP418) with Hampered Transmembrane Transport in Electrochemotherapy In Vitro and In Vivo

Abstract: To increase electrochemotherapy (ECT) applicability, the effectiveness of new drugs is being tested in combination with electroporation. Among them two ruthenium(III) compounds, (imH)[trans-RuCl4(im)(DMSO-S)] (NAMI-A) and Na[trans-RuCl4(ind)2] (KP1339), proved to possess increased antitumor effectiveness when combined with electroporation. The objective of our experimental work was to determine influence of electroporation on the cytotoxic and antitumor effect of a ruthenium(III) compound with hampered transmembrane transport, (imH)[trans-RuCl4(im)2] (KP418) in vitro and in vivo and to determine changes in metastatic potential of cells after ECT with KP418 in vitro. In addition, platinum compound cisplatin (CDDP) and ruthenium(III) compound NAMI-A were included in the experiments as reference compounds. Our results show that electroporation leads to increased cellular accumulation and cytotoxicity of KP418 in murine melanoma cell lines with low and high metastatic potential, B16-F1 and B16-F10, but not in murine fibrosarcoma cell line SA-1 in vitro which is probably due to variable effectiveness of ECT in different cell lines and tumors. Electroporation does not potentiate the cytotoxicity of KP418 as prominently as the cytotoxicity of CDDP. We also showed that the metastatic potential of cells which survived ECT with KP418 or NAMI-A does not change in vitro: resistance to detachment, invasiveness, and re-adhesion of cells after ECT is not affected. Experiments in murine tumor models B16-F1 and SA-1 showed that ECT with KP418 does not have any antitumor effect while ECT with CDDP induces significant dose-dependent tumor growth delay in the two tumor models used in vivo.