Anion Conducting States of Excitatory Amino Acid Transporters

Excitatory amino acid transporters (EAATs) are secondary active, electrogenic transporters which translocate L-glutamate (glu) against its concentration gradient using the co-transport of 3 Na+, 1 H+, and the counter-transport of 1 K+ ion. In addition, these carriers possess a thermodynamically uncoupled anion channel that fluxes Cl- but is promiscuous with several permeant anionic species. The roles of EAATs are to shape the spatio-temporal profile of released glu in both the synaptic cleft and extra-synaptic regions as well as maintaining a low ambient extracellular concentration of glu. This transport activity regulates activation of glu receptors and thus regulates excitatory neurotransmission. Using a combination of techniques, we were successful in identifying inward oriented transporter conformations which allow transitions to open channels states. This observation was enabled by our development of a novel method to isolate EAAT1 in the inward facing conformation. While constrained to these conformations, currents with the same macroscopic amplitudes as conducting states mediated by the outward facing, Na+ bound states were observed. The persistence of currents is indicative of a channel gating mechanism that is insensitive to transporter orientation and that the anion channel is open during the majority of the transport cycle. Additional conducting states allows for a larger contribution of the anion channel function of EAATs to shape cellular function then previously assumed. Next we investigated the gating mechanism of the anion channel. We assayed for the ability of Na+ to gate the anion channel in both glial (EAAT1 and EAAT2) and neuronal (EAAT3 and EAAT4) isoforms. We discovered that the glial isoforms are not gated by Na+ but are leak channels with an open probability and single channel conductance that is insensitive to Na+ concentrations. In contrast, neuronal EAAT isoforms EAAT3 and EAAT4 both display Na+ dependent channel activity. This is the first example of a significant functional difference between glial and neuronal transporter isoforms of the solute carrier 1 (SLC1) family. The research presented here allows for a greater understanding of low open probability channel states and the possible contributions of the EAAT anion channel to the functioning of the nervous system

Analysis Strategies for Bioactive, Polar Fatty Amides in Complex Samples

Bioactive lipids are known to exert physiological effects and interact with neuroreceptors. Little is known about the bioregulation of primary fatty acid amides, though N-acyl glycines are thought to be their anabolic precursors. Chapter 1 details the current metabolic, physiological, and receptor interactions of primary fatty acid amides and their related congeners and reviews current strategies for isolation and detection. Chapter 2 outlines mass spectrometry collision induced fragmentation assignments pertaining to method development of multiple reaction monitoring detection of these species. Chromatographic separation methods of fatty acyls, with a focus on primary fatty acid amides and N-acyl glycines, are outlined in chapter 3 and these results are used to develop a two dimensional liquid chromatography tandem mass spectrometry analysis method. Multiple reaction monitoring was utilized as the detection mode due to the enhanced limit of detection obtained from mass filtering background and matrix components. The implementation of our developed analytical separation and detection approach for quantitation of these bioactive lipids in vertebrate samples and future implications of primary fatty acid amide and N-acyl glycine analysis are reviewed in chapter 4. In addition, the appendix summarizes a method for selective, sensitive multiple reaction monitoring detection of palmitoylethanolamine, an N-acylethanolamine with analgesic and anti-inflammatory properties

Learning from the 21st century fight: insights from operations and training

Advanced militaries, optimised for conventional conflicts, are experiencing significant change as they attempt to adapt to the demands of a new defence and security paradigm. The effectiveness of that transformation will depend largely on their ability to fully absorb the lessons of history and of recent operations. The paper begins by juxtaposing the four key factors for ensuring that these lessons are identified and absorbed ('the environment', 'the culture', 'the leaders, and 'the process') within today's Australian Army. The paper then goes on to outline some of the insights (lessons) gained from operations and training activities, with an emphasis on force protection and urban operations. This is followed by an analysis of the way ahead for the Australian Army and why the Army must institutionalise a learning process and, perhaps more importantly, a true learning culture where all have an openness to new ideas and a thirst for constant improvement

A Formal Model of Partitioning for Integrated Modular Avionics

The aviation industry is gradually moving toward the use of integrated modular avionics (IMA) for civilian transport aircraft. An important concern for IMA is ensuring that applications are safely partitioned so they cannot interfere with one another. We have investigated the problem of ensuring safe partitioning and logical non-interference among separate applications running on a shared Avionics Computer Resource (ACR). This research was performed in the context of ongoing standardization efforts, in particular, the work of RTCA committee SC-182, and the recently completed ARINC 653 application executive (APEX) interface standard. We have developed a formal model of partitioning suitable for evaluating the design of an ACR. The model draws from the mathematical modeling techniques developed by the computer security community. This report presents a formulation of partitioning requirements expressed first using conventional mathematical notation, then formalized using the language of SRI'S Prototype Verification System (PVS). The approach is demonstrated on three candidate designs, each an abstraction of features found in real systems

Software Certification for Temporal Properties With Affordable Tool Qualification

It has been recognized that a framework based on proof-carrying code (also called semantic-based software certification in its community) could be used as a candidate software certification process for the avionics industry. To meet this goal, tools in the "trust base" of a proof-carrying code system must be qualified by regulatory authorities. A family of semantic-based software certification approaches is described, each different in expressive power, level of automation and trust base. Of particular interest is the so-called abstraction-carrying code, which can certify temporal properties. When a pure abstraction-carrying code method is used in the context of industrial software certification, the fact that the trust base includes a model checker would incur a high qualification cost. This position paper proposes a hybrid of abstraction-based and proof-based certification methods so that the model checker used by a client can be significantly simplified, thereby leading to lower cost in tool qualification

The Army lessons process: supporting individual and organisational learning in the Australian Army

This paper provides a brief outline of the Army lessons process that is applied when developing and applying lessons across all Army activities. The Army lessons process supports the conduct of operations and informs Army Raise-Train-Sustain functions, through the incorporation of lessons into doctrine, training and capability development. The paper focuses upon four distinct foci. Firstly, we examine the whole of Army responsibility in relation to the transfer of knowledge into lessons. Secondly, we consider the vital roles and responsibilities of knowledge authorities in this transfer. Thirdly, we examine the ways in which observations and insights transform into findings and lessons. Finally, we propose the establishment of the Army Lessons Network as a formal means of knowledge interaction

Using Formal Methods to Assist in the Requirements Analysis of the Space Shuttle GPS Change Request

We describe a recent NASA-sponsored pilot project intended to gauge the effectiveness of using formal methods in Space Shuttle software requirements analysis. Several Change Requests (CR's) were selected as promising targets to demonstrate the utility of formal methods in this application domain. A CR to add new navigation capabilities to the Shuttle, based on Global Positioning System (GPS) technology, is the focus of this report. Carried out in parallel with the Shuttle program's conventional requirements analysis process was a limited form of analysis based on formalized requirements. Portions of the GPS CR were modeled using the language of SRI's Prototype Verification System (PVS). During the formal methods-based analysis, numerous requirements issues were discovered and submitted as official issues through the normal requirements inspection process. Shuttle analysts felt that many of these issues were uncovered earlier than would have occurred with conventional methods. We present a summary of these encouraging results and conclusions we have drawn from the pilot project

Cysteine transport through excitatory amino acid transporter 3 (EAAT3)

Excitatory amino acid transporters (EAATs) limit glutamatergic signaling and maintain extracellular glutamate concentrations below neurotoxic levels. Of the five known EAAT isoforms (EAATs 1-5), only the neuronal isoform, EAAT3 (EAAC1), can efficiently transport the uncharged amino acid L-cysteine. EAAT3-mediated cysteine transport has been proposed to be a primary mechanism used by neurons to obtain cysteine for the synthesis of glutathione, a key molecule in preventing oxidative stress and neuronal toxicity. The molecular mechanisms underlying the selective transport of cysteine by EAAT3 have not been elucidated. Here we propose that the transport of cysteine through EAAT3 requires formation of the thiolate form of cysteine in the binding site. Using Xenopus oocytes and HEK293 cells expressing EAAT2 and EAAT3, we assessed the transport kinetics of different substrates and measured transporter-associated currents electrophysiologically. Our results show that L-selenocysteine, a cysteine analog that forms a negatively-charged selenolate ion at physiological pH, is efficiently transported by EAATs 1-3 and has a much higher apparent affinity for transport when compared to cysteine. Using a membrane tethered GFP variant to monitor intracellular pH changes associated with transport activity, we observed that transport of either L-glutamate or L-selenocysteine by EAAT3 decreased intracellular pH, whereas transport of cysteine resulted in cytoplasmic alkalinization. No change in pH was observed when cysteine was applied to cells expressing EAAT2, which displays negligible transport of cysteine. Under conditions that favor release of intracellular substrates through EAAT3 we observed release of labeled intracellular glutamate but did not detect cysteine release. Our results support a model whereby cysteine transport through EAAT3 is facilitated through cysteine de-protonation and that once inside, the thiolate is rapidly re-protonated. Moreover, these findings suggest that cysteine transport is predominantly unidirectional and that reverse transport does not contribute to depletion of intracellular cysteine pools

Critical Acceleration Levels for Free Standing Bridge Abutments

An analytic procedure for predicting threshold accelerations for movement of gravity wall bridge abutments due to earthquake loading is described. The method draws on previous work related to the sliding mode of failure, and a newly developed theory on seismic reduction of bearing capacity. The main contribution of this paper is to present laboratory observations verifying mode of failure and critical acceleration levels predicted by this procedure for model retaining wall bridge abutments subjected to seismic excitation on a shaking table. Three different test series were performed with different interface conditions between the wall, and the bridge deck, soil foundation, and backfill resulting in a variety of modes of wall deformation