Variation in Structure and Process of Care in Traumatic Brain Injury: Provider Profiles of European Neurotrauma Centers Participating in the CENTER-TBI Study.

INTRODUCTION: The strength of evidence underpinning care and treatment recommendations in traumatic brain injury (TBI) is low. Comparative effectiveness research (CER) has been proposed as a framework to provide evidence for optimal care for TBI patients. The first step in CER is to map the existing variation. The aim of current study is to quantify variation in general structural and process characteristics among centers participating in the Collaborative European NeuroTrauma Effectiveness Research in Traumatic Brain Injury (CENTER-TBI) study. METHODS: We designed a set of 11 provider profiling questionnaires with 321 questions about various aspects of TBI care, chosen based on literature and expert opinion. After pilot testing, questionnaires were disseminated to 71 centers from 20 countries participating in the CENTER-TBI study. Reliability of questionnaires was estimated by calculating a concordance rate among 5% duplicate questions. RESULTS: All 71 centers completed the questionnaires. Median concordance rate among duplicate questions was 0.85. The majority of centers were academic hospitals (n = 65, 92%), designated as a level I trauma center (n = 48, 68%) and situated in an urban location (n = 70, 99%). The availability of facilities for neuro-trauma care varied across centers; e.g. 40 (57%) had a dedicated neuro-intensive care unit (ICU), 36 (51%) had an in-hospital rehabilitation unit and the organization of the ICU was closed in 64% (n = 45) of the centers. In addition, we found wide variation in processes of care, such as the ICU admission policy and intracranial pressure monitoring policy among centers. CONCLUSION: Even among high-volume, specialized neurotrauma centers there is substantial variation in structures and processes of TBI care. This variation provides an opportunity to study effectiveness of specific aspects of TBI care and to identify best practices with CER approaches

Passive water permeability of some wild type and mutagenized amino acid cotransporters of the SLC6/NSS family expressed in Xenopus laevis oocytes

In this paper passive water movement across the cell membrane mediated by wild type and mutagenized cotransporters was investigated. We evaluated water movement and, in parallel, amino acid uptake induced by some members of the SLC6/NSS family belonging to different kingdoms, namely the rat GABA transporter GAT1, the insect amino acid transporters KAAT1 and CAATCH1 and the bacterial leucine transporter LeuT, whose structure was recently solved. We also tested whether mutated proteins in which the solute translocation mechanism is altered or even abolished were able to induce water movement across cell membrane. The proteins of interest were expressed in Xenopus laevis oocytes and osmotic water permeabilities were estimated from the rate of cell volume change induced by an osmotic gradient in the absence of cotransported solutes. Under osmotic stress all the studied wild type amino acid cotransporters increased the water permeability of the membrane. The GABA transport inhibitor SKF 89976A inhibited both GABA transport and water movement induced by the expression of GAT1. Interestingly, the capacity of mutant proteins to induce water movement was not predictable on the basis of their substrate transport ability. In particular the GAT1 mutant Q291N, void of any transport activity, induced a water permeability similar to that induced by the wt protein. The KAAT1 mutant T339C, which showed a higher transport activity, induced a water permeability not significantly different from the wild type transporter. Interestingly, the bacterial leucine cotransporter LeuT, whose binding site for leucine and Na(+) is void of water, induced water movement through the plasma membrane

Ionic selectivity of the coupled and uncoupled currents carried by the amino acid transporter KAAT1

The ability of the intestinal amino acid co-transporter KAAT-1 expressed in Xenopus oocytes to transport different cations in either amino acid coupled or uncoupled manner was studied using voltage-clamp conditions. KAAT1-expressing oocytes exhibit a transporter-related current in the absence of organic substrate (uncoupled current). In the presence of various alkali cations the amplitude of this current follows the sequence: ILi > INa > IK approximately equal to IRb approximately equal to ICs. Addition of 1 mM leucine causes large increases in K+ and Na+ currents, while the Li+ current undergoes a more complex change and Rb+ and Cs+ currents are only marginally affected. Pre-steady-state currents in the absence of organic substrate are apparent when Na+, K+, or Li+ are the bathing ions; analysis of these currents in terms of charge movement reveals that Na+, K+, and Li+ interact differently with the transporter. The uncoupled current in mixtures of Na+ and Li+ fails to exhibit anomalous mole-fraction behavior. Kinetic analysis of ion binding and uncoupled permeation argues against a multi-ion single-file mechanism in the KAAT1 cotransporter

Functional Role of a Highly Conserved Sequence Motif in the Insect Amino Acid Transporter KAAT1

KAAT1 is an insect amino acid transporter, member of the NSS/SLC6 family of solute carriers, with peculiar functional properties, being activated by Na+ and by K+ (1). KAAT1 sequence shows a stretch of three consecutive glycines (Gly85 - Gly87) that is highly conserved between members of the family (2, 3) and that, according to topology prediction algorithms and to the crystal structure of the NSS/SLC6 member LeuT (4) is located in the extracellular loop 1 (EL1), near the access of the permeation pathway. In the present study we have investigated the functional role of this region by KAAT1 site directed mutagenesis and expression in X. laevis oocytes. The substitution of each glycine with alanine determined a reduction of transport activity of 40 - 50 % compared with wild-type (wt) (S.E. of 5%, n = 56). The shift of the glycine stretch toward the N or the C terminus of the protein, obtained by the synthesis of the double mutants N84G/G87A and G85A/A88G, reduced the activity respectively to 13% \ub1 1 (n = 25) and 8% \ub1 2 (n = 27) of wt, whereas the single mutants N84G and A88G showed a residual transport activity that was respectively 36 % \ub1 6 (n =31) and 5 % \ub1 1 (n = 38) of wt. Our results indicate that EL1 residues influence KAAT1 activity and that in particular, the conserved three-glycine stretch constitutes a \u201cfunctional motif\u201d in which not the single glycine residue, but the position of the entire motif plays an essential role in transport function. 1.Castagna et al., (1998), PNAS, 95: 5395-5400 2.Zhou and Kanner, (2005) J.Biol.Chem.280: 20316-20324. 3.Mao et al., (2008) Mol. Memb. Biol. 25: 115-127. 4.Yamashita et al., (2005) Nature. 437: 215-22

Extracellular ionic concentrations in Bombyx mori L. central nervous system

Aim of this study was to determine the ionic concentration gradients at the cellular membrane level in the central nervous system of an insect where the haemolymphatic sodium concentration relative to that of potassium is too low to allow action potential generation. The intracellular ionic concentrations determined by electrophysiological experiments performed at different Na and K concentrations in the perfusing solution, the Na and K concentrations in the nerve cord tissue water, and the extracellular spaces observed by electron microscopy, support the conclusion that the cells in the ventral nerve cord of Bombyx mori function in a medium with a relatively low potassium content and a high sodium concentration. This fluid is in a microenvironment protected by a barrier, the perineurium and associated glial elements, where must be located a regulation mechanism of cation composition. In order to aid the microelectrode penetration the preparations were exposed to Pronase. The Pronase action was investigated by electron microscopy and determinations of ionic concentrations

Inhibition of the lepidopteran amino acid co-transporter KAAT1 by phenylglyoxal : role of arginine 76

Phenylglyoxal (PGO), an arginine-modifying reagent, is an irreversible inhibitor of KAAT1-mediated leucine transport, expressed in Xenopus oocytes. The PGO effect was dose-dependent and 5 mM PGO determined a Vmax reduction to 24% of the control, consistent with the covalent binding to transporter arginine residues not located in the leucine binding site. The use of labelled [14C]PGO confirmed that the inhibitor binds KAAT1. The protein membrane domain contains seven arginine residues one of which, arginine 76, is conserved in the family of GABA transporters. Using site-directed mutagenesis we showed that only arginine 76 is crucial for KAAT1 activity and is involved in PGO binding

Pronase action on the sheath surrounding ventral nerve cord ganglia in Bombyx mori L.

Sono stati studiati differenti aspetti dell\u2019azione proteolitica dell\u2019enzima pronasi sui gangli della catena ventrale di Bombyx mori L. La penetrazione del microelettrodo nelle cellule nervose \ue8 facilitata dal trattamento del tessuto con l\u2019enzima. Osservazioni morfologiche ultrastrutturali indicano che l\u2019azione dell\u2019enzima si estrinseca principalmente a livello della guaina fibrosa del ganglio, provocando una parziale distruzione della stessa. Inoltre, il trattamento con pronasi non altera in modo significativo le concentrazioni ioniche presenti nella catena gangliare.1. Pronase action on ventral nerve cord ganglia in Bombyx mori L. larvae has been studied from different points of view. 2. Microelectrode penetration in the nervous cell is facilitated by exposure of the tissue to the enzyme. 3. Ultrastructural observations show that pronase action affects mainly the fibrous sheath of the tissue, leading to a partial desheathing of the ganglion. 4. Pronase treatment does not affect significantly the ionic concentrations in the nerve cord

Functional role of a highly conserved sequence motif in the insect amino acid transporter KAAT1

KAAT1 is an insect amino acid transporter, member of the NSS/SLC6 family of solute carriers, with peculiar functional properties, being activated by Na+ and by K+ (Castagna et al., PNAS, 1998). KAAT1 sequence shows a stretch of three consecutive glycines (Gly85 - Gly87) that is highly conserved between members of the family and that, according to topology prediction algorithms and to the crystal structure of the NSS/SLC6 member LeuT (Yamashita et al., Nature, 2005), is located in the extracellular loop 1 (EL1), near the access of permeation pathway. In the present study we have investigated the functional role of this region by KAAT1 site directed mutagenesis and expression in X. laevis oocytes. The substitution of each glycine with alanine determined a reduction of transport activity of 40 - 50 % compared with wild-type (wt), both in the presence of a Na+ or a K+ gradient. The shift of the glycine stretch toward the N or the C terminus of the protein, obtained by the synthesis of the double mutants N84G/G87A and G85A/A88G, reduced the activity respectively to 13 and 8 % of wt, whereas the single mutants N84G and A88G showed a residual transport activity that was respectively 40 % and 5 % of wt. Our results indicate that EL1 residues influence KAAT1 activity and that in particular, the conserved three-glycine stretch constitutes a \u201cfunctional motif\u201d in which not the single glycine residue, but the position of the entire motif plays an essential role in transport function

A PDZ target sequence controls the surface expression and recycling of the EAAC1/EAAT3 glutamate transporter

The neuronal glutamate transporter EAAC1/EAAT3(Excitatory Amino Acid Carrier-1) mediates the uptake of the excitatory neurotransmitter from the synaptic cleft. It is also expressed in epithelial cells where it provides the principal route of glutamate and aspartate absorption. The transporter activity and localization are modulated by auxiliary proteins that still have to be identified. In the C-terminus of the EAAC1/EAAT3 transporter, we observed a consensus sequence (-S-Q-F) for interaction with class I PDZ domains and we investigated the role of this motif in the transporter localization and activity. Mutant transporters were generated and overexpressed in the CV1, COS and MDCK (Madin Darby canine kidney) cell lines, and their localization and activity were tested by means of immunofluorescence, biotinylation, and uptake experiments. We found that removal of the PDZ-interacting sequence(T-S-Q-F) or substitution of the serine residue at -2 position with alanine or glutamate affected the cell surface stability of the transporter. Indeed, the steady state cell surface expression of mutant transporters was lower compared to wild type protein, but it was greatly increased by inhibition of the clathrindependent endocytosis (hyperosmotic stress). Double immunofluorescence experiments revealed that mutant transporters accumulated in an endocytic compartment which did not colocalize with transferrin, a marker of the recycling compartment. Instead, we found a partial colocalization with LAMP- 2, a marker of the lysosomal compartment, after inhibition of lysosomal degradation by means of leupeptine treatment. We suggest that the PDZ target sequence of EAAC1/EAAT3 transporter, and likely its interaction with PDZ proteins, may control the surface stability and/or recycling of the transporter. In the absence of this interaction, the transporter reaches the plasma membrane but instead of being retained in- or recycled to- the cell surface, it is internalized and degraded in a lysosomal compartment. We are now in the process to identify PDZ proteins interacting with the EAAC1/EAAT3 transporter in epithelial and neuronal cells