State-dependent block of CNG channels by dequalinium

Cyclic nucleotide-gated (CNG) ion channels are nonselective cation channels with a high permeability for Ca(2+). Not surprisingly, they are blocked by a number of Ca(2+) channel blockers including tetracaine, pimozide, and diltiazem. We studied the effects of dequalinium, an extracellular blocker of the small conductance Ca(2+)-activated K(+) channel. We previously noted that dequalinium is a high-affinity blocker of CNGA1 channels from the intracellular side, with little or no state dependence at 0 mV. Here we examined block by dequalinium at a broad range of voltages in both CNGA1 and CNGA2 channels. We found that dequalinium block was mildly state dependent for both channels, with the affinity for closed channels 3-5 times higher than that for open channels. Mutations in the S4-S5 linker did not alter the affinity of open channels for dequalinium, but increased the affinity of closed channels by 10-20-fold. The state-specific effect of these mutations raises the question of whether/how the S4-S5 linker alters the binding of a blocker within the ion permeation pathway.     

Expression of ATP6V1C1 during oral carcinogenesis

We investigated the gene and protein expressions of V-type ATPase protein subunit C1 (ATP6V1C1) in cases of oral squamous cell carcinoma (OSCC) and contralateral normal mucosa in smokers, nonsmokers and former smokers. Subjects were separated into five groups of 15: group 1, smokers with OSCC; group 2, normal contralateral mucosa of OSCC patients; group 3, chronic smokers; group 4, former smokers who had stopped smoking 1 year earlier; group 5, individuals who had never smoked. Exfoliative cytology specimens from oral mucosa of smokers, former smokers and nonsmokers showed normal gene and protein expression. We found significantly greater gene expression in the OSCC group than in the nonsmoker groups. No difference in gene expression was observed between normal contralateral mucosa and nonsmoker groups, smoker and nonsmoker groups or former smoker and nonsmoker groups. We observed intense immunostaining for ATP6V1C1 protein in all cases of OSCC and weak or no staining in smoker, former smoker and nonsmoker groups. Significantly greater expression of ATP6V1C1 protein was observed in the OSCC group compared to the other groups, which supports the role of ATP6V1C1 in effecting changes associated with oral cancer. Analysis of the mucosae of chronic smokers, former smokers and the normal contralateral mucosa of patients with OSCC showed unaltered ATP6V1C1 gene and protein expression. Early stages of carcinogenesis, represented by altered epithelium of chronic smokers, had neither gene nor protein alterations as seen in OSCC. Therefore, we infer that the changes in ATP6V1C1 occur during later stages of carcinogenesis. Our preliminary study provides a basis for future studies of using ATP6V1C1 levels for detecting early stage OSCC.     

Properties of the inner pore region of TRPV1 channels revealed by block with quaternary ammoniums

The transient receptor potential vanilloid 1 (TRPV1) nonselective cationic channel is a polymodal receptor that activates in response to a wide variety of stimuli. To date, little structural information about this channel is available. Here, we used quaternary ammonium ions (QAs) of different sizes in an effort to gain some insight into the nature and dimensions of the pore of TRPV1. We found that all four QAs used, tetraethylammonium (TEA), tetrapropylammonium (TPrA), tetrabutylammonium, and tetrapentylammonium, block the TRPV1 channel from the intracellular face of the channel in a voltage-dependent manner, and that block by these molecules occurs with different kinetics, with the bigger molecules becoming slower blockers. We also found that TPrA and the larger QAs can only block the channel in the open state, and that they interfere with the channel's activation gate upon closing, which is observed as a slowing of tail current kinetics. TEA does not interfere with the activation gate, indicating that this molecule can reside in its blocking site even when the channel is closed. The dependence of the rate constants on the size of the blocker suggests a size of around 10 Å for the inner pore of TRPV1 channels.     

Normalization references for Europe and North America for application with USEtox? characterization factors

Purpose

In life cycle impact assessment, normalization can be a very effective tool for the life cycle assessment practitioner to interpret results and put them into perspective. The paper presents normalization references for the recently developed USEtox? model, which aims at calculating globally applicable characterization factors. Normalization references for Europe and North America are determined, and guidance for expansions to other geographical regions is provided.

Materials and methods

The base years of the European and North American inventories are 2004 and 2002/2008, respectively. Emission data were extracted from two literature sources referring to each of the considered regions. The inventory for North America was adapted to avoid extrapolation of data from other regions and thus bring consistency with the emission inventory for Europe. In spite of different inventory assumptions, a similar coverage of substances was obtained for both regions with relatively high representation of metals and a number of organic compounds, mainly consisting of non-methane volatile organic compounds and pesticides. The two inventory sets were eventually characterized with the characterization factors (CFs) calculated with the version 1.0 of the USEtox? model and substance database; both interim and recommended CFs were used.

Results and discussion

Normalization references are provided for Europe and North America for the three USEtox? toxic impact categories; ratios between the normalization references for the two regions in all cases lie below a factor of 3. Causes for the observed discrepancies are found to be different inventory assumptions as well as variations in the type and intensity of actual emissions between the two regions. Additional causes are inventories that only cover a limited number of substances, and the characterization model, which can only provide interim factors for certain substances like metal compounds. Based on these causes and on a review of recent studies on normalization references, a list of substances to be prioritized when collecting emission data was built, demonstrating the importance of metals.

Conclusions

In the perspective of further refining the presented normalization references and of calculating new references for other regions, guidance is provided including a list of priority substances that should be considered when building emission inventories for normalization references.     

Aerated Shewanella oneidensis in continuously fed bioelectrochemical systems for power and hydrogen production

We studied the effects of aeration of Shewanella oneidensis on potentiostatic current production, hydrogen production in a microbial electrolysis cell, and electric power generation in a microbial fuel cell (MFC). The potentiostatic performance of aerated S. oneidensis was considerably enhanced to a maximum current density of 0.45 A/m² or 80.3 A/m³ (mean: 0.34 A/m², 57.2 A/m³) compared to anaerobically grown cultures. Biocatalyzed hydrogen production rates with aerated S. oneidensis were studied within the applied potential range of 0.3–0.9 V and were highest at 0.9 V with 0.3 m³ H₂/m³ day, which has been reported for mixed cultures, but is ～10 times higher than reported for an anaerobic culture of S. oneidensis. Aerated MFC experiments produced a maximum power density of 3.56 W/m³ at a 200‐Ω external resistor. The main reasons for enhanced electrochemical performance are higher levels of active biomass and more efficient substrate utilization under aerobic conditions. Coulombic efficiencies, however, were greatly reduced due to losses of reducing equivalents to aerobic respiration in the anode chamber. The next challenge will be to optimize the aeration rate of the bacterial culture to balance between maximization of bacterial activation and minimization of aerobic respiration in the culture. Biotechnol. Bioeng. 2010;105: 880–888. © 2009 Wiley Periodicals, Inc.     

Cell-specific caspase expression by different neuronal phenotypes in transient retinal ischemia

Emerging evidence supports an important role for caspases in neuronal death following ischemia-reperfusion injury. This study assessed whether cell specific caspases participate in neuronal degeneration and whether caspase inhibition provides neuroprotection following transient retinal ischemia. We utilized a model of transient global retinal ischemia. The spatial and temporal pattern of the active forms of caspase 1, 2 and 3 expression was determined in retinal neurons following ischemic injury. Double-labeling with cell-specific markers identified which cells were expressing different caspases. In separate experiments, animals received various caspase inhibitors before the induction of ischemia. Sixty minutes of ischemia resulted in a delayed, selective neuronal death of the inner retinal layers at 7 days. Expression of caspase 1 was not detected at any time point. Maximal expression of caspase 2 was found at 24 h primarily in the inner nuclear and ganglion cell layers of the retina and localized to ganglion and amacrine neurons. Caspase 3 also peaked at 24 h in both the inner nuclear and outer nuclear layers and was predominantly expressed in photoreceptor cells and to a lesser extent in amacrine neurons. The pan caspase inhibitor, Boc-aspartyl fmk, or an antisense oligonucleotide inhibitor of caspase 2 led to significant histopathologic and functional improvement (electroretinogram) at 7 days. No protection was found with the caspase 1 selective inhibitor, Y-vad fmk. These observations suggest that ischemia-reperfusion injury activates different caspases depending on the neuronal phenotype in the retina and caspase inhibition leads to both histologic preservation and functional improvement. Caspases 2 and 3 may act in parallel in amacrine neurons following ischemia-reperfusion. These results in the retina may shed light on differential caspase specificity in global cerebral ischemia.     

Structural insights into oligomerization and mitochondrial remodelling of dynamin 1‐like protein

Dynamin 1‐like protein (DNM1L) mediates fission of mitochondria and peroxisomes, and dysfunction of DNM1L has been implicated in several neurological disorders. To study the molecular basis of mitochondrial remodelling, we determined the crystal structure of DNM1L that is comprised of a G domain, a bundle signalling element and a stalk. DNM1L assembled via a central stalk interface, and mutations in this interface disrupted dimerization and interfered with membrane binding and mitochondrial targeting. Two sequence stretches at the tip of the stalk were shown to be required for ordered assembly of DNM1L on membranes and its function in mitochondrial fission. In the crystals, DNM1L dimers further assembled via a second, previously undescribed, stalk interface to form a linear filament. Mutations in this interface interfered with liposome tubulation and mitochondrial remodelling. Based on these results and electron microscopy reconstructions, we propose an oligomerization mode for DNM1L which differs from that of dynamin and might be adapted to the remodelling of mitochondria.     

Studies on the parameters controlling the stability of the TET peptidase superstructure from Pyrococcus horikoshii revealed a crucial role of pH and catalytic metals in the oligomerization process

The TET proteases from Pyrococcus horikoshii are metallopeptidases that form large dodecameric particles with high thermal stability. The influence of various physico-chemical parameters on PhTET3 quaternary structure was investigated. Analytical ultracentrifugation and biochemical analyses showed that the PhTET3 quaternary structure and enzymatic activity are maintained in high salt and that the complex is stable under extreme acidic conditions. Under basic pH conditions the complex disassembled into a low molecular weight species that was identified as folded dimer. Metal analyses showed that the purified enzyme only contains two equivalent of zinc per monomer, corresponding to the metal ions responsible for catalytic activity. When these metals were removed by EDTA treatment, the complex dissociated into the same dimeric species as those observed at high pH. Dodecameric TET particles were obtained from the metal free dimers when 2mM of divalent ions were added to the protein samples. Most of the dimers remained assembled at high temperature. Thus, we have shown that dimers are the building units in the TET oligomerization pathway and that the active site metals are essential in this process.