ClC proteins are chloride channels and transporters that are found in a wide variety of prokaryotic and eukaryotic cell-types. The mammalian chloride channel ClC-1 is an important modulator of the electrical excitability of skeletal muscle. The Torpedo electric-organ chloride channel, ClC-0 is structurally and functionally similar to ClC- 1. These proteins are referred to as the muscle-type ClC channels. The present work identifies several functional differences between the muscle type channels, and explores the structural basis of these and other previously reported differences. First the temperature dependence of ClC-1 channels was quantified. These calculations revealed distinct contrasts to previously published measurements of ClC-0 temperature sensitivity, indicating differences between the channels in the structural rearrangements associated with channel gating. Next the effect of extracellular ion substitution on ClC-0 function was examined. These measurements suggested that occupancy of an anion binding-site on the extracellular side of the selectivity-filter stabilises the open state of the channel, and that the diameter of the channel pore increases during channel opening. Three-dimensional models of the muscle-type channels were constructed based on the atomic coordinates of prokaryotic homologues. Differences in selectivity between ClC-0 and ClC-1 could be rationalised, in part, by differences in the chemistry of the narrow constriction of the channel pore. The major structural divergence between the muscle-type channels occurs in the expansive intracellular carboxy terminus. Replacing this region of ClC-1 with the corresponding region from ClC-0 resulted in distinct changes in common gating of the channel. These experiments rigorously characterise the dependence of ClC-1 function on temperature and the effect of foreign anionic-substrates on ClC-0 function. The results identify important residues involved in ionic selectivity of the channels, and validate the use of high-resolution prokaryotic channel structures as a predictive tool for studying the muscle-type channels. They also demonstrate that the carboxy-terminal of the channels is an important determinant of common gating. / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008

Whole document restricted, see Access Instructions file below for details of how to access the print copy. / Huntington’s disease [HD] is a debilitating adult onset inherited neurodegenerative disorder with primary degeneration in the striatum and widespread secondary degeneration throughout the brain. There are currently no clinical treatments to prevent onset, delay progression or replace lost neurons. Striatal cell transplantation strategies under clinical evaluation appear viable and effective for the treatment of HD. However, the future of regenerative medicine lies in developing renewable, expandable multipotent neural cell sources for transplantation. This Thesis has investigated a range of novel developments for enhancing the therapeutic potential of neural progenitor cell transplantation in a quinolinic acid [QA] lesion rat model of HD using two cell sources, adult neural progenitor cells and human embryonic stem cell [hESC] derived neural progenitor cells. Chapter Three identified a novel method for in vitro lithium priming of adult neural progenitor cells which enhances their neurogenic potential at the expense of glial formation. Chapter Four demonstrated that lithium priming of adult neural progenitor cells altered their phenotypic fate in vivo after transplantation, enhancing regional specific differentiation and efferent projection formation. The therapeutic potential of this strategy was demonstrated by accelerated acquisition of motor function benefits in the QA model. Chapter Five then demonstrated the ability for post transplantation environmental enrichment to modify therapeutic functional outcomes in the QA lesion model, and through lithium priming and enrichment demonstrated that adult neural progenitors are amenable to combinatorial interventions which can alter their phenotypic fate and enhance anatomical integration. Chapter Six investigated the in vivo effects of in vitro noggin priming of hESC derived neural progenitor cells and identified enhanced safety and neuronal differentiation in the QA lesioned striatum after noggin priming. Furthermore Chapter Seven provided evidence for functional reconstruction and therapeutic functional benefits from transplantation of noggin primed hESC derived neural progenitor cells and also highlighted the need for systematic evaluations of hESC derived transplants to optimise their safety in vivo. These results are beneficial in demonstrating the realistic therapeutic potential held by these two cell sources. They demonstrate how transient interventions can enhance therapeutic outcomes of neural progenitor cell transplantation for HD and have developed the understanding of neural progenitor cell transplantation as a therapeutic tool, bringing transplantation from different cell sources closer to eventual translation for HD sufferers.

<p>Chloride ion channels are macromolecular pores providing for passage of chloride ions (and certain other inorganic and organic anions) through the cell membrane, down their electrochemical gradients. Chloride channels are differentially expressed in various cells, to best suit specific cellular activities. They are present in practically all living cells, and regardless of cell specialization they play an important role in vital housekeeping functions of cell-volume and pH regulation and in membrane potential stabilization. Regulation of cell volume underlies the structural integrity and constancy of the intracellular milieu. A variety of metabolic pathways have been shown to be sensitive to cell volume, and alterations of cell volume and osmoregulation processes can influence various intracellular signaling and organizing factors.</p><p>Volume-regulated anion channels (VRACs) are believed to play a pivotal role in cell-volume regulating processes. In this report I present data from macroscopic patch-clamp studies of VRACs performed in a fibroblast cell line and from single channel studies of chloride channels (tentatively related to VRACs) in mouse brown adipocytes in primary culture.</p><p>One of the characteristic features of the VRACs is their dependence on the presence of cytoplasmic ATP. In whole-cell experiments, removal of ATP from the pipette solution almost completely prevented activation of VRACs, whereas substitution of ATP with the nonhydrolyzable analog ATPγS did not alter the activation of VRACs. The inhibitors of protein tyrosine kinases (PTK) tyrphostin A25 and B46 depressed VRAC currents in both cases (ATP and ATPγS), but a PTK ineffective analog (tyrphostin A1) did not affect VRAC currents. We infer that in the cell preparation we used, ATP has a dual role in VRAC regulation: it is required for channel-protein phosphorylation and it can influence channel activity through non-hydrolytic binding in a ligand-receptor manner. It can additionally be suggested that tyrosine-specific protein kinases can be involved in the regulation of VRACs, independently of the effects of ATP. We also studied cell cycle-related changes in activation of VRACs by osmotic swelling of cells chemically arrested at different phases of the cell cycle. We found no significant changes during most of the cell cycle, except short periods before and after mitosis and in the quiescent G0 state.</p><p>The single Cl^-channels of brown adipocytes resemble in their electrophysiological phenotype outwardly rectifying Cl^- channels (ORCCs). We investigated the sensitivity of these channels to intracellular Ca²⁺. It appeared that the commonly used Ca²⁺-chelators EGTA and BAPTA could influence the ORCCs currents by themselves, independently of their calcium chelating effects. In some channels, these chelators induced classical flickery-type block of activity, whereas in others there was quasi-blockage, i.e. a peculiar combination of flickery blockage and overall channel activation. The chloride channel blocking agents DIDS and SITS mimicked the true/quasi blockage of EGTA and BAPTA. These phenomena add to the structure-function characteristics of the ORCC molecule. Moderate inhibitory effect of Ca²⁺ within a physiological range of intracellular concentrations (sub-µM) was also detected; however, the biological relevance of this observation, as well as of these Cl^- channels in general, remains to be clarified.</p>

This study evaluated the potential of the aerobic Mycobacterium strain JS6O to grow on a variety of organic acid substrates, and the possible effects an organic acid would have on the degradation rate of vinyl chloride (VC). A series of batch growth tests were designed to determine the time it took to consume the substrate and the overall increase in biomass. Strain JS6O was found capable of growth on acetate, propionate, and butyrate, but could not grow on formate or lactate. Acetate was chosen for further study because strain JS6O consumed acetate the most rapidly of all the organic acids tested, and acetate is a common product of fermentation reactions in the subsurface. Strain JS6O was confirmed to grow on both ethylene and vinyl chloride as the sole carbon and energy source. Comparatively, strain JS6O's rate of growth on VC is much slower than that of ethylene. With acetate as an augmenting growth substrate, ethylene and VC utilization rates increased by 30% and 48%, respectively. Since acetate and VC are often found together in contaminated chlorinated ethene plumes, this makes a strong case for natural attenuation of VC by strain JS6O. A series of kinetic tests were implemented to determine the K[subscript s] and k[subscript max] of strain JS6O for ethylene, VC, and c-DCE. The K[subscript s] and k[subscript max] for ethylene determined through NLSR methods was similar to the values published in Coleman et al. (2002), supporting the maintenance of a pure culture throughout the experimental work. When strain JS6O was exposed to the isomers of DCE (trans-1,2-dichloroethylene (t-DCE), cis-1,2-dichloroethylene (c-DCE), and 1,1-dichloroethylene (1,1-DCE)) the cells were unable to grow on these compounds. However, when growing on acetate, strain JS6O cometabolized c-DCE and t-DCE, but not 1,1-DCE, with c-DCE transformed more rapidly than t-DCE. Transformation of c-DCE was also observed with growth on VC and ethylene. The presence of c-DCE was shown to partially inhibit VC degradation, but had no effect on ethylene degradation. The cometabolism results with acetate further indicate that strain JS6O is a good candidate for natural attenuation of multiple chlorinated ethenes in the subsurface. / Graduation date: 2006

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 77-87).

Chloride ion channels are macromolecular pores providing for passage of chloride ions (and certain other inorganic and organic anions) through the cell membrane, down their electrochemical gradients. Chloride channels are differentially expressed in various cells, to best suit specific cellular activities. They are present in practically all living cells, and regardless of cell specialization they play an important role in vital housekeeping functions of cell-volume and pH regulation and in membrane potential stabilization. Regulation of cell volume underlies the structural integrity and constancy of the intracellular milieu. A variety of metabolic pathways have been shown to be sensitive to cell volume, and alterations of cell volume and osmoregulation processes can influence various intracellular signaling and organizing factors. Volume-regulated anion channels (VRACs) are believed to play a pivotal role in cell-volume regulating processes. In this report I present data from macroscopic patch-clamp studies of VRACs performed in a fibroblast cell line and from single channel studies of chloride channels (tentatively related to VRACs) in mouse brown adipocytes in primary culture. One of the characteristic features of the VRACs is their dependence on the presence of cytoplasmic ATP. In whole-cell experiments, removal of ATP from the pipette solution almost completely prevented activation of VRACs, whereas substitution of ATP with the nonhydrolyzable analog ATPγS did not alter the activation of VRACs. The inhibitors of protein tyrosine kinases (PTK) tyrphostin A25 and B46 depressed VRAC currents in both cases (ATP and ATPγS), but a PTK ineffective analog (tyrphostin A1) did not affect VRAC currents. We infer that in the cell preparation we used, ATP has a dual role in VRAC regulation: it is required for channel-protein phosphorylation and it can influence channel activity through non-hydrolytic binding in a ligand-receptor manner. It can additionally be suggested that tyrosine-specific protein kinases can be involved in the regulation of VRACs, independently of the effects of ATP. We also studied cell cycle-related changes in activation of VRACs by osmotic swelling of cells chemically arrested at different phases of the cell cycle. We found no significant changes during most of the cell cycle, except short periods before and after mitosis and in the quiescent G0 state. The single Cl- channels of brown adipocytes resemble in their electrophysiological phenotype outwardly rectifying Cl- channels (ORCCs). We investigated the sensitivity of these channels to intracellular Ca2+. It appeared that the commonly used Ca2+-chelators EGTA and BAPTA could influence the ORCCs currents by themselves, independently of their calcium chelating effects. In some channels, these chelators induced classical flickery-type block of activity, whereas in others there was quasi-blockage, i.e. a peculiar combination of flickery blockage and overall channel activation. The chloride channel blocking agents DIDS and SITS mimicked the true/quasi blockage of EGTA and BAPTA. These phenomena add to the structure-function characteristics of the ORCC molecule. Moderate inhibitory effect of Ca2+ within a physiological range of intracellular concentrations (sub-µM) was also detected; however, the biological relevance of this observation, as well as of these Cl- channels in general, remains to be clarified.

No description available.

Cardiac ischemic preconditioning (IPC) is defined as brief periods of ischemia and reperfusion that protect the heart against longer ischemia and reperfusion. IPC triggers Cl- efflux and protein kinase C epsilon (PKCe) translocation to the particulate fraction. Chloride channel 2 (ClC-2) is volume regulated and is a potential end effector of IPC. The goal of my study was to investigate the involvement of PKCε and ClC-2 protein module in IPC of isolated adult rabbit ventricular myocytes. Co-immunoprecipitation (co-IP) assays on HEK 293 cells, transfected with ClC-2-Flag, confirmed that ClC-2 interacts with PKCe. Subcellular fractionation showed that PKCe/ClC-2 protein module is localized to the sarcolemma of cardiomyocytes. Lastly, ischemia/reperfusion injury was simulated in cardiomyocytes with 45min simulated ischemia (SI)/60min simulated reperfusion (SR) and IPC was induced by pre-treatment with 10min SI/20min SR. Co-IP after each time interval showed that IPC transiently enhanced PKCe/ClC-2 interaction. PKC inhibitor, GF109203X, abrogated the enhanced interaction.

The mutant prevention concentration (MPC) is defined as the lowest antimicrobial concentration required to inhibit the growth of the least susceptible bacterial cell based on an inoculum of ≥109 colony forming units (CFUs). The current protocol for MPC testing is technically demanding and time-consuming which limits its implementation into clinical microbiology laboratories. In an attempt to simplify the current MPC protocol we developed a modified MPC method, the microbroth dilution method, which requires two fewer days to complete than the current or traditional method. MPC values were consistent for all organisms and strains tested using both the traditional MPC method and the modified microbroth dilution MPC method.<p> Tigecycline is the first of a new class of compound glycylcyclines- with potent in vitro activity against Gram-positive organisms including penicillin-resistant and multi-drug resistant <i>Streptococcus pneumoniae</i> (SP) and methicillin-resistant <i>Staphylococcus aureus</i> (MRSA). We measured minimum inhibitory concentration (MIC) and MPC values for tigecycline against 47 clinical isolates of SP and found that the MPC90 values were >500 fold higher than the MIC90 values. To determine if MPC testing of tigecycline against SP is impacted by blood in the medium, we developed a new medium able to sustain the growth of SP without the need for blood; solidified Todd-Hewitt broth (sTHB). The MPC90 values of tigecycline against SP on sTHB were only 2 fold higher than the MIC90 values. When blood was added to the sTHB, the MPC90 values again became much greater than the MIC90 values (> 256 fold higher). MPC results for <i>Staphylococcus spp.</i> against tigecycline were not impacted by blood in the medium.<p> Benzalkonium chloride (BAK) is a cationic surface-acting agent that acts on bacterial cells by disrupting the intermolecular interaction of the lipid bilayer. To determine if the <i>fluoroquinolones gatifloxacin</i> (Gfx) and moxifloxacin (Mfx) are more active (lower MIC values) in the presence of BAK, we conducted MIC, MPC, and time-kill assays. MIC testing showed that in the presence of 3.125 to 50 µg/ml of BAK, the MIC of Gfx and Mfx decreased by 8- to 5000-fold against clinical isolates of methicillin-susceptible <i>Staphylococcus aureus</i> (MSSA), MRSA, Coagulase-negative <i>Staphylococci</i>(CNS), SP, <i>Escherichia coli</i> (EC), and <i>Pseudomonas aeruginosa</i> (PA). MPC testing showed that the presence of 7 to 10 µg/ml of BAK, the MPC of Gfx and Mfx decreased by 32- to 1000-fold against clinical isolates of MRSA. Conventional time-kill studies (using a bacterial load of 105 CFUs) showed that the killing activity of Gfx against clinical MRSA isolates was enhanced in the presence of BAK with a log10-reduction (percent kill) of 1.6 (76.08%) for Gfx alone at 180 minutes compared to a log10-redecution (percent kill) of 5.4 (100%) for Gfx plus BAK at 180 minutes.<p> Alexidine (Alx) is a bisbiguanide that has been used as an effective disinfectant in the dental industry and is potentially being developed for use as an antimicrobial agent for ocular infections. We conducted susceptibility testing of Alx using MIC testing, MPC testing, and time-kill assays against Gram-positive and Gram-negative pathogens. MIC testing showed that Alx is more active against Gram-positive pathogens than Gram-negative pathogens and showed better activity than the fluoroquinolones Gfx, Mfx, and levofloxacin (Lfx) against MRSA. The MPC values measured for MRSA and MSSA against Alx were non-reproducible using the traditional MPC method. Using the microbroth dilution MPC method, MPC90 values were found to be 32 fold higher than the MIC90 values. If the experimentally determined MPC values are true MPC values, initial MPC testing indicates that Alx may have a high likelihood for selecting for resistance, however, if the MPC values are not accurate it may be necessary to modify the MPC protocol in order to complete MPC testing of Alx against MRSA and MSSA. Conventional time-kill studies (using a bacterial load of 105 CFUs) measured bactericidal activity (> 3 log10-reduction) against MRSA, MSSA, SP, and PA.

Cardiac ischemic preconditioning (IPC) is defined as brief periods of ischemia and reperfusion that protect the heart against longer ischemia and reperfusion. IPC triggers Cl- efflux and protein kinase C epsilon (PKCe) translocation to the particulate fraction. Chloride channel 2 (ClC-2) is volume regulated and is a potential end effector of IPC. The goal of my study was to investigate the involvement of PKCε and ClC-2 protein module in IPC of isolated adult rabbit ventricular myocytes. Co-immunoprecipitation (co-IP) assays on HEK 293 cells, transfected with ClC-2-Flag, confirmed that ClC-2 interacts with PKCe. Subcellular fractionation showed that PKCe/ClC-2 protein module is localized to the sarcolemma of cardiomyocytes. Lastly, ischemia/reperfusion injury was simulated in cardiomyocytes with 45min simulated ischemia (SI)/60min simulated reperfusion (SR) and IPC was induced by pre-treatment with 10min SI/20min SR. Co-IP after each time interval showed that IPC transiently enhanced PKCe/ClC-2 interaction. PKC inhibitor, GF109203X, abrogated the enhanced interaction.