Charybdotoxin block of a Shaker K+ channel was studied in Xenopus oocyte macropatches. Toxin on rate increases linearly with toxin concentration in an ionic strength-dependent fashion and is competitively diminished by tetraethylammonium. On rate is insensitive to transmembrane voltage and to K+ on the opposite side of the membrane. Conversely, toxin off rate is insensitive to toxin concentration, ionic strength, and added tetraethylammonium but is enhanced by membrane depolarization or K+ (or Na+) in the trans solution. Charge neutralization of charybdotoxin Lys27, however, renders off rate voltage insensitive. Our results argue that block of voltage-gated K+ channels results from the binding of one toxin molecule, so that Lys27 enters the pore and interacts with K+ (or Na+) in the ion conduction pathway.

The gating of ClC-0, the voltage-dependent Cl- channel from Torpedo electric organ, is strongly influenced by Cl- ions in the external solution. Raising external Cl- over the range 1-600 mM favors the fast-gating open state and disfavors the slow-gating inactivated state. Analysis of purified single ClC-0 channels reconstituted into planar lipid bilayers was used to identify the role of Cl- ions in the channel's fast voltage-dependent gating process. External, but not internal, Cl- had a major effect on the channel's opening rate constant. The closing rate was more sensitive to internal Cl- than to external Cl-. Both opening and closing rates varied with voltage. A model was derived that postulates (a) that in the channel's closed state, Cl- is accessible to a site located at the outer end of the conduction pore, where it binds in a voltage-independent fashion, (b) that this closed conformation can open, whether liganded by Cl- or not, in a weakly voltage-dependent fashion, (c) that the Cl(-)-liganded closed channel undergoes a conformational change to a different closed state, such that concomitant with this change, Cl- ion moves inward, conferring voltage-dependence to this step, and (d) that this new Cl(-)-liganded closed state opens with a very high rate. According to this picture, Cl- movement within the pre-open channel is the major source of voltage dependence, and charge movement intrinsic to the channel protein contributes very little to voltage-dependent gating of ClC-0. Moreover, since the Cl- activation site is probably located in the ion conduction pathway, the fast gating of ClC-0 is necessarily coupled to ion conduction, a nonequilibrium process.

A gene for a slowly activating, voltage-dependent K(+) -selective channel was designed and synthesized on the basis of its known amino acid sequence. The synthetic gene was cloned into a transcription vector, and in vitro transcribed mRNA was injected into Xenopus oocytes for electrophysiological assay of the resulting ionic currents. The currents are voltage-dependent and highly selective for K+ over Na+. The selectivity among monovalent cations follows a familiar K(+)- channel sequence: K+ greater than Rb+ greater than NH4+ greater than Cs+ much greater than Na+, Li+. The currents are inhibited by Ba2+, Cs+, and tetraethylammonium (TEA), common pore blockers of K+ channels. Open-channel blockade by Cs+ (but not by Ba2+ or TEA) depends on applied voltage. The major inhibitory effect of Ba2+ is to alter channel gating by favoring the closed state; this effect is specific for Ba2+ and is relieved by external K+. The results argue that although the polypeptide expressed is very small for a eukaryotic ion channel, 130 amino acid residues in length, the ionic currents observed are indeed mediated by a genuine K(+) -channel protein. This synthetic gene is therefore well suited for a molecular analysis of the basic mechanisms of K(+) -channel function.

Background

Bortezomib is a proteasome inhibitor with minimal clinical activity as a monotherapy in solid tumours, but its combination with other targeted therapies is being actively investigated as a way to increase its anticarcinogenic properties. Here, we evaluate the therapeutic potential of co-treatment with bortezomib and indole-3-carbinol (I3C), a natural compound found in cruciferous vegetables, in human ovarian cancer.

Methods

We examined the effects of I3C, bortezomib and cisplatin in several human ovarian cancer cell lines. Synergy was determined using proliferation assays and isobologram analysis. Cell cycle and apoptotic effects were assessed by flow cytometry. The mechanism of I3C and bortezomib action was determined by RNA microarray studies, quantitative RT-PCR and western blotting. Antitumour activity of I3C and bortezomib was evaluated using an OVCAR5 xenograft mouse model.

Results

I3C sensitised ovarian cancer cell lines to bortezomib treatment through potent synergistic mechanisms. Combination treatment with bortezomib and I3C led to profound cell cycle arrest and apoptosis as well as disruptions to multiple pathways, including those regulating endoplasmic reticulum stress, cytoskeleton, chemoresistance and carcinogen metabolism. Moreover, I3C and bortezomib co-treatment sensitised ovarian cancer cells to the standard chemotherapeutic agents, cisplatin and carboplatin. Importantly, in vivo studies demonstrated that co-treatment with I3C and bortezomib significantly inhibited tumour growth and reduced tumour weight compared with either drug alone.

Conclusion

Together, these data provide a novel rationale for the clinical application of I3C and bortezomib in the treatment of ovarian cancer.

Recent studies have correlated anticoagulant rodenticide exposure to the development of mange in wild felids. However, a causative association between anticoagulant exposure and the onset of mange or immune dysfunction that may increase susceptibility of wild felids to manage has not been established. To investigate the potential connection between anticoagulant rodenticide exposure and immune dysfunction, specific-pathogen-free domestic cats were exposed to brodifacoum over a six-week period and vaccinated with irrelevant antigens at different points during the course of the experiment to assess recall and direct immune responses. Measures of immune response included delayed-type hypersensitivity tests and cell proliferation assays. IgE and antigen-specific antibodies were quantified via ELISA assays. No cats developed coagulopathies despite having detectable levels of brodifacoum in the blood. Brodifacoum cats had significant decreases in the production of certain cytokines including IL-6 and IL-4. The results of this study suggest that cats may be less susceptible to anticoagulant rodenticide induced coagulopathy and that the effects of environmentally realistic brodifacoum exposure on humoral and cell-mediated immunity against foreign antigen exposures in domestic cats are minimal.

Regional climate change impact (CCI) studies have widely involved downscaling and bias correcting (BC) global climate model (GCM)-projected climate for driving land surface models. However, BC may cause uncertainties in projecting hydrologic and biogeochemical responses to future climate due to the impaired spatiotemporal covariance of climate variables and a breakdown of physical conservation principles. Here we quantify the impact of BC on simulated climate-driven changes in water variables (evapotranspiration (ET), runoff, snow water equivalent (SWE), and water demand for irrigation), crop yield, biogenic volatile organic compounds (BVOC), nitric oxide (NO) emissions, and dissolved inorganic nitrogen (DIN) export over the Pacific Northwest (PNW) region. We also quantify the impacts on net primary production (NPP) over a small watershed in the region (HJ-Andrews). Simulation results from the coupled ECHAM5-MPI-OM model with A1B emission scenario were first dynamically downscaled to 12 km resolution with the WRF model. Then a quantile-mapping-based statistical downscaling model was used to downscale them into 1/16° resolution daily climate data over historical and future periods. Two climate data series were generated, with bias correction (BC) and without bias correction (NBC). Impact models were then applied to estimate hydrologic and biogeochemical responses to both BC and NBC meteorological data sets. These impact models include a macroscale hydrologic model (VIC), a coupled cropping system model (VIC-CropSyst), an ecohydrological model (RHESSys), a biogenic emissions model (MEGAN), and a nutrient export model (Global-NEWS). Results demonstrate that the BC and NBC climate data provide consistent estimates of the climate-driven changes in water fluxes (ET, runoff, and water demand), VOCs (isoprene and monoterpenes) and NO emissions, mean crop yield, and river DIN export over the PNW domain. However, significant differences rise from projected SWE, crop yield from dry lands, and HJ-Andrews's ET between BC and NBC data. Even though BC post-processing has no significant impacts on most of the studied variables when taking PNW as a whole, their effects have large spatial variations and some local areas are substantially influenced. In addition, there are months during which BC and NBC post-processing produces significant differences in projected changes, such as summer runoff. Factor-controlled simulations indicate that BC post-processing of precipitation and temperature both substantially contribute to these differences at regional scales. We conclude that there are trade-offs between using BC climate data for offline CCI studies versus directly modeled climate data. These trade-offs should be considered when designing integrated modeling frameworks for specific applications; for example, BC may be more important when considering impacts on reservoir operations in mountainous watersheds than when investigating impacts on biogenic emissions and air quality, for which VOCs are a primary indicator. © 2014 Author(s).

© 2015 Chaisson et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credite