Physiology and pathology of neutral amino acid transport in renal and intestinal epithelial cells

About 95% of nutrient protein is absorbed in the mammalian intestine as amino acids and trl-and dipeptides. Once absorbed by the intestine amino acids are distributed by the circulation throughout the body. In the kidney an ultrafiltrate of the blood plasma is generated from which all amino acids are reabsorbed in the proximal tubule. System B{u2070} is the main amino acid transport system in these two tissues for broad neutral amino acids. Four transporters B{u2070}AT1 (SLC6A19), B{u2070}AT2 (SLC6A15), B{u2070}AT3 (SLC6A18) and ASTC2 (SCL1A5) have properties of this system when studied in isolation. B{u2070}ATl and B{u2070}AT2 co-transport one Na{u207A} -ion and one amino acid substrate. The low-affinity B{u2070}ATl transporter accepts most neutral amino acids, while the high-affinity transporter B{u2070}AT2 prefers branch-chained amino acids and proline. B{u2070}AT3 prefers alanine and glycine, but has also been reported to transport a variety of neutral amino acids. ASCT2 is a Na{u207A}-dependent antiporter, preferring neutral amino acids except those with an aromatic side-chain. However, in two studies it has been reported that amino acid transport of ASCT2 was inhibited also by phenylalanine. Analysis of ASCT2 function in vivo and in vitro, has led to the proposal that it represents the molecular correlate of system B{u2070} in kidney and intestine. Mutations in B{u2070}ATl, however, cause Hartnup disorder, a defect of neutral amino acid transport in kidney and intestine. Thus it is important to reconcile differences and to clarify the contribution of these amino acid transporters to system B{u2070}-like transport in kidney and intestine. Therefore, the overall aim for this thesis was to investigate the distribution and contribution of B{u2070}AT1 (SlC6A19), B{u2070}AT2 (SlC6A15), and ASTC2 (SCL1AS) to neutral amino acid transport in the kidney. Immunofluorescence studies revealed localisation of B{u2070}AT1 in the apical membrane of early (S1-S2) segments of the proximal tubule, while SOAT2 was localised to the apical membrane of the later (S2-S3) segments. This is consistent with physiological data reporting low-affinity transport in early segments and high-affinity transport in later segments of the proximal tubule. ASCT2 was localised in the apical membrane of S2 segments and in the basolateral membrane of the distal tubule. Transport studies with renal brush border membrane vesicles revealed a dominant transport activity that is consistent with the properties of B{u2070}AT1. This transport activity was lacking in B{u2070}AT1-deficient mice. B{u2070}AT2 activity was excluded by competition studies with selective substrates. ASCT2 activity was excluded due to the lack of additional amino acid transport in brush border membrane vesicles preloaded with A5CT2 amino acid substrates. Therefore, it is concluded that B{u2070}AT2 and ASCT2 do not contribute significantly to neutral amino acid transport in the kidney. B{u2070}AT1 is the main transport activity for neutral amino acid transport in the kidney

Impaired nutrient signaling and body weight control in a Na⁺ neutral amino acid cotransporter (Slc6a19)-deficient mouse

Amino acid uptake in the intestine and kidney is mediated by a variety of amino acid transporters. To understand the role of epithelial neutral amino acid uptake in whole body homeostasis, we analyzed mice lacking the apical broad-spectrum neutral (0) amino acid transporter BᴼAT1 (Slc6a19). A general neutral aminoaciduria was observed similar to human Hartnup disorder which is caused by mutations in SLC6A19. Na⁺ -dependent uptake of neutral amino acids into the intestine and renal brush-border membrane vesicles was abolished. No compensatory increase of peptide transport or other neutral amino acid transporters was detected. Mice lacking BᴼAT1 showed a reduced body weight. When adapted to a standard 20% protein diet, BᴼAT1-deficient mice lost body weight rapidly on diets containing 6 or 40% protein. Secretion of insulin in response to food ingestion after fasting was blunted. In the intestine, amino acid signaling to the mammalian target of rapamycin (mTOR) pathway was reduced, whereas the GCN2/ATF4 stress response pathway was activated, indicating amino acid deprivation in epithelial cells. The results demonstrate that epithelial amino acid uptake is essential for optimal growth and body weight regulation.This work was supported by National Health and Medical Research Council Grant 525415, Australian Research Council Grant DP0877897, University of Sydney Bridging Grant RIMS2009-02579), and by an anonymous foundatio

Functionalization Of T Lymphocytes With Citrate-Coated Superparamagnetic Iron Oxide Nanoparticles For Magnetically Controlled Immune Therapy

Purpose: Immune activation with T cell tumor infiltration is beneficial for the prognosis of patients suffering from solid cancer. Depending on their immune status, solid tumors can be immunologically classified into three groups: “hot” tumors are infiltrated with T lymphocytes, “cold” tumors are not infiltrated and “immune excluded” tumors are only infiltrated in the peripheral tumor tissue. Checkpoint inhibitors provide new therapeutic options for “hot” tumors by triggering the immune response of T cells. In order to enable this for cold tumors as well, T cells must be enriched in the tumor. Therefore, we use the principle of magnetic targeting to guide T cells loaded with citrate-coated superparamagnetic iron oxide nanoparticles (SPIONCitrate) to the tumor by an externally applied magnetic field. Methods: SPIONCitrate were produced by alkaline coprecipitation of iron(II) and iron(III) chloride and in situ coating with sodium citrate. The concentration-dependent cytocompatibility of the particles was determined by flow cytometry and blood stability assays. Atomic emission spectroscopy was used for the quantification of the particle uptake into T lymphocytes. The attractability of the loaded cells was observed by live-cell imaging in the presence of an externally applied magnetic field. Results: SPIONCitrate displayed good cytocompatibility to T cells and did not show any sign of aggregation in blood. Finally, SPIONCitrate-loaded T cells were strongly attracted by a small external magnet. Conclusion: T cells can be “magnetized” by incorporation of SPIONCitrate for magnetic targeting. The production of the particle-cell hybrid system is straightforward, as the loading process only requires basic laboratory devices and the loading efficiency is sufficient for cells being magnetically controllable. For these reasons, SPIONCitrate are potential suitable candidates for magnetic T cell targeting

Molecular cloning of the mouse IMINO system: an Na+- and CI--dependent proline transporter

Neurotransmitter transporters of the SLC6 family play an important role in the removal of neurotransmitters in brain tissue and in amino acid transport in epithelial cells. Here we demonstrate that the mouse homologue of slc6a20 has all properties of th

The orphan transporter v7-3 (slc6a15) is a Na(+)-dependent neutral amino acid transporter (B(0)AT2)

Transporters of the SLC6 (solute carrier 6) family play an important role in the removal of neurotransmitters in brain tissue and in amino acid transport in epithelial cells. In the present study, we demonstrate that mouse v7-3 (slc6a15) encodes a transporter for neutral amino acids. The transporter is functionally and sequence related to B(0)AT1 (slc6a19) and was hence named B(0)AT2. Leucine, isoleucine, valine, proline and methionine were recognized by the transporter, with values of K(0.5) (half-saturation constant) ranging from 40 to 200 μM. Alanine, glutamine and phenylalanine were low-affinity substrates of the transporter, with K(0.5) values in the millimolar range. Transport of neutral amino acids via B(0)AT2 was Na(+)-dependent, Cl(−)-independent and electrogenic. Superfusion of mouse B(0)AT2-expressing oocytes with amino acid substrates generated robust inward currents. Na(+)-activation kinetics of proline transport and uptake under voltage clamp suggested a 1:1 Na(+)/amino acid co-transport stoichiometry. Susbtrate and co-substrate influenced each other's K(0.5) values, suggesting that they share the same binding site. A mouse B(0)AT2-like transport activity was detected in synaptosomes and cultured neurons. A potential role of B(0)AT2 in transporting neurotransmitter precursors and neuromodulators is proposed

Synthesis and Characterization of Citrate-Stabilized Gold-Coated Superparamagnetic Iron Oxide Nanoparticles for Biomedical Applications

Surface-functionalized gold-coated superparamagnetic iron oxide nanoparticles (Au-SPIONs) may be a useful tool in various biomedical applications. To obtain Au-SPIONs, gold salt was precipitated onto citrate-stabilized SPIONs (Cit-SPIONs) using a simple, aqueous one-pot technique inspired by the Turkevich method of gold nanoparticle synthesis. By the further stabilization of the Au-SPION surface with additional citrate (Cit-Au-SPIONs), controllable and reproducible Z-averages enhanced long-term dispersion stability and moderate dispersion pH values were achieved. The citrate concentration of the reaction solution and the gold/iron ratio was found to have a major influence on the particle characteristics. While the gold-coating reduced the saturation magnetization to 40.7% in comparison to pure Cit-SPIONs, the superparamagnetic behavior of Cit-Au-SPIONs was maintained. The formation of nanosized gold on the SPION surface was confirmed by X-ray diffraction measurements. Cit-Au-SPION concentrations of up to 100 µg Fe/mL for 48 h had no cytotoxic effect on Jurkat cells. At a particle concentration of 100 µg Fe/mL, Jurkat cells were found to take up Cit-Au-SPIONs after 24 h of incubation. A significantly higher attachment of thiol-containing L-cysteine to the particle surface was observed for Cit-Au-SPIONs (53%) in comparison to pure Cit-SPIONs (7%)

Intellectuels de l’islam contemporain

Qui sont les nouvelles générations intellectuelles dans le monde musulman sunnite aujourd’hui ? Quels débats proposent-elles dans la sphère publique ? Ce recueil couvre un ensemble large et diversifié : le monde arabe, l’islam de l’Asie du Sud, aussi bien que les diasporas musulmanes en Occident. Il détaille la configuration et l’évolution des champs intellectuels en les reliant au contexte politique et social mais aussi à l’histoire proche en faisant dialoguer sociologie et histoire. Dans certains cas, de nouvelles générations intellectuelles se définissent depuis la fin des années 1990 comme « réformistes », mais dans un sens différent du réformisme libéral qui s’était développé dans l’entre-deux-guerres. Ces nouvelles générations viennent en effet chronologiquement après l’émergence et la routinisation des islamismes entre la fin des années 1970 et le tout début du XXIe siècle. Ces intellectuels ne sont pas forcément et uniquement animés par le religieux, et construisent l’islam sur un mode fort différent des islamistes, en s’opposant explicitement aux interprétations que ceux-ci ont développées depuis le début du XXe siècle. Ces acteurs et ces débats, qui participent d’un paradigme post-identitaire, sont donc à la fois un produit de l’islamisme et une réponse à celui-ci. Ces intellectuels se prononcent contre la lecture littérale des textes et pour une interprétation contextuelle et historicisée des textes religieux. Ils préfèrent voir l’islam comme partie des valeurs universelles plutôt que comme terme d’une alternative qui oppose l’identité islamique à l’Occident. Mais certains sont aussi pris dans des contradictions internes liées aux contextes politiques nationaux qui leur interdisent parfois d’exister publiquement. Ces contradictions sont issues par ailleurs de la définition même du libéralisme religieux, qui rend leurs productions difficiles à ancrer dans la réalité. Par ailleurs, ils n’agissent pas de manière autonome, et sont souvent en situation de dialogue implicite ou explicite dans la sphère publique avec les intellectuels qui appartiennent au monde des oulémas ou à ceux qui s’autoproclament islamistes. Ce recueil fait ainsi le point sur les évolutions récentes des champs intellectuels musulmans au-delà de l’islamisme politique