We have characterized the effects of BgII and BgIII, two sea anemone peptides with almost identical sequences (they only differ by a single amino acid), on neuronal sodium currents using the whole-cell patch-clamp technique. Neurons of dorsal root ganglia of Wistar rats (P5-9) in primary culture (Leibovitz′s L15 medium; 37°C, 95% air/5% CO₂) were used for this study (n = 154). These cells express two sodium current subtypes: tetrodotoxin-sensitive (TTX-S; K_i = 0.3 nM) and tetrodotoxin-resistant (TTX-R;K_i = 100 μM). Neither BgII nor BgIII had significant effects on TTX-R sodium current. Both BgII and BgIII produced a concentration-dependent slowing of the TTX-S sodium current inactivation (IC₅₀ = 4.1 ± 1.2 and 11.9 ± 1.4 μM, respectively), with no significant effects on activation time course or current peak amplitude. For comparison, the concentration-dependent action of Anemonia sulcata toxin II (ATX-II), a well characterized anemone toxin, on the TTX-S current was also studied. ATX-II also produced a slowing of the TTX-S sodium current inactivation, with an IC₅₀ value of 9.6 ± 1.2 μM indicating that BgII was 2.3 times more potent than ATX-II and 2.9 times more potent than BgIII in decreasing the inactivation time constant (τ_h) of the sodium current in dorsal root ganglion neurons. The action of BgIII was voltage-dependent, with significant effects at voltages below −10 mV. Our results suggest that BgII and BgIII affect voltage-gated sodium channels in a similar fashion to other sea anemone toxins and α-scorpion toxins.

Cytotoxicity of titanium dioxide (TiO₂) thin films on Chinese hamster ovary (CHO-K1) cells was evaluated after 24, 48 and 72 h of culture. The TiO₂ thin films were deposited using direct current magnetron sputtering. These films were post-deposition annealed at different temperatures (300, 500 and 800 °C) toward the anatase to rutile phase transformation. The root-mean-square (RMS) surface roughness of TiO₂ films went from 2.8 to 8.08 nm when the annealing temperature was increased from 300 to 800 °C. Field emission scanning electron microscopy (FESEM) results showed that the TiO₂ films’ thickness values fell within the nanometer range (290–310 nm). Based on the results of the tetrazolium dye and trypan blue assays, we found that TiO₂ thin films showed no cytotoxicity after the aforementioned culture times at which cell viability was greater than 98%. Independently of the annealing temperature of the TiO₂ thin films, the number of CHO-K1 cells on the control substrate and on all TiO₂ thin films was greater after 48 or 72 h than it was after 24 h; the highest cell survival rate was observed in TiO₂ films annealed at 800 °C. These results indicate that TiO₂ thin films do not affect mitochondrial function and proliferation of CHO-K1 cells, and back up the use of TiO₂ thin films in biomedical science.

We previously reported a multigene family of monodomain Kunitz proteins from Echinococcus granulosus (EgKU-1-EgKU-8), and provided evidence that some EgKUs are secreted by larval worms to the host interface. In addition, functional studies and homology modeling suggested that, similar to monodomain Kunitz families present in animal venoms, the E. granulosus family could include peptidase inhibitors as well as channel blockers. Using enzyme kinetics and whole-cell patch-clamp, we now demonstrate that the EgKUs are indeed functionally diverse. In fact, most of them behaved as high affinity inhibitors of either chymotrypsin (EgKU-2-EgKU-3) or trypsin (EgKU-5-EgKU-8). In contrast, the close paralogs EgKU-1 and EgKU-4 blocked voltage-dependent potassium channels (K_v); and also pH-dependent sodium channels (ASICs), while showing null (EgKU-1) or marginal (EgKU-4) peptidase inhibitory activity. We also confirmed the presence of EgKUs in secretions from other parasite stages, notably from adult worms and metacestodes. Interestingly, data from genome projects reveal that at least eight additional monodomain Kunitz proteins are encoded in the genome; that particular EgKUs are up-regulated in various stages; and that analogous Kunitz families exist in other medically important cestodes, but not in trematodes. Members of this expanded family of secreted cestode proteins thus have the potential to block, through high affinity interactions, the function of host counterparts (either peptidases or cation channels) and contribute to the establishment and persistence of infection. From a more general perspective, our results confirm that multigene families of Kunitz inhibitors from parasite secretions and animal venoms display a similar functional diversity and thus, that host-parasite co-evolution may also drive the emergence of a new function associated with the Kunitz scaffold. Parasite secretions are key players at host-parasite interfaces: parasite establishment and persistence rely, to a great extent, on interactions between these molecules and their host counterparts. We present the functional characterization of a multigene family of secreted Kunitz proteins from the cestode Echinococcus granulosus. Kunitz proteins are a class of metazoan high affinity serine peptidase inhibitors. In addition, families of Kunitz proteins are frequent components of animal venoms; besides inhibiting peptidases, some of these “Kunitz toxins” block cation channels and thus, provide a remarkable example of protein evolution where natural selection has driven the emergence of a new function associated with the same molecular scaffold. Using enzyme kinetics and electrophysiological assays, we demonstrated that the E. granulosus Kunitz family includes peptidase inhibitors as well as channel blockers. This diversity highlights an interesting similarity between animal toxins and parasite secretions that had not been previously described. Furthermore, the presence of analogous families of Kunitz proteins appears to be a distinctive trait of cestode genomes. We thus propose that these molecules contribute to a successful infection acting at the parasite-host interface. In addition, because they bear a strong specificity towards their targets, they are uniquely suited for the development of pharmaceuticals.