120 research outputs found
A new sphincter-splitting operation for surgical access to lying tumours of the rectum
No full textFifty-eight patients underwent anterior anorectotomy for removal of intrarectal tumor. The mean age of patients was 69 years: 33 were over 70.26 tumors were benign and 32 malignant. There were no postoperative deaths during hospital stay or the 30 days following discharge. Six abscesses and 2 self-limiting urethral fistulas were diagnosed in 2 male patients. The wound healed at day 14 on the average. Sphincter function was judged to be poor or average in 2 cases each and normal in the remaining 54. One patient had local recurrence at 6 years, amenable to reoperation. Five patients died of their cancer between 10 months and 2 years. Anterior anorectotomy permits exposition up to 14 cm from the anal verge, is not mutilating, can be performed under spinal anesthesia, and is recommended for removal of benign rectal tumors as well as small carcinomas in patients physically or mentally unfit for major abdominal surgery
Time-resolved measurements of sugar-binding-induced conformational changes in the melibiose permease from Escherichia coli
No full textThe melibiose permease (MelB) of E.coli functions as a secondary-active symporter by using the electrochemical H+, Na+, or Li+ gradient to accumulate, e.g., melibiose [review in Pourcher et al. 1990a]. The global and primary objective of this thesis was to apply pre-steady state methods for the investigation of reaction rates of individual steps in the cycle of MelB. Especially the melibiose binding induced transition was investigated by the solid-supported membrane (SSM) technique [Seifert et al. 1993] in combination with a rapid solution exchange system [Pintchovius and Fendler 1999] and with the Stopped-flow technique [Roughton 1934]. To approach this goal, either wild-type or mutated MelB were purified and reconstituted into liposomes as described [Pourcher et al. 1995]. Although the orientation of the proteins is a critical factor for the activity of MelB, it was, so far, unknown. To determine the orientation of the proteins in the liposomes, single Cys mutants R139C and R141C [Abdel-Dayem et al. 2003] were selectively labeled with 3-(N-maleimidylpropionyl)biocytin (MPB) and analyzed by SDS-PAGE and Western Blot. The assay indicated that most of the proteins are inside-out (ISO) oriented permitting to relate the pre-steady state electrical and fluorescence signals to the reverse transport activity of MelB. The melibiose induced electrical signal was investigated in wild-type MelB with the SSM technique. The transporter was activated by a substrate concentration jump, and transient currents were measured. When the transporter was preincubated with Na+ at saturating concentrations, a charge translocation in the protein upon melibiose binding could still be observed. This result demonstrates that binding of the uncharged substrate melibiose triggers a charge displacement in the protein. Further analysis showed that the charge displacement is neither related to extra Na+ binding to the transporter, nor to the displacement of already bound Na+ within MelB. Electrogenic melibiose binding is explained by a conformational change with concomitant displacement of charged amino acid side chains and/or a reorientation of helix dipoles. A kinetic model is suggested, in which Na+ and melibiose binding are distinct electrogenic processes associated with approximately the same charge displacement. Melibiose binding is fast in the presence of Na+ (k > 50 s-1). Furthermore, two previously identified transport deficient mutants of loop 4-5, R141C and E142C [Abdel-Dayem et al. 2002, Séry 2002], were purified and extensively studied with the SSM. Whereas the electrical signals from control cysteine-less mutant showed a bi-exponential time course of decay, those from R141C or E142C consisted of only a single fast exponential component, and the slow decaying component associated with substrate translocation was missing. The electrical signals evoked by a melibiose concentration jump in the presence of Na+ were much smaller than the corresponding signals in C-less MelB. Furthermore, R141C lost the stimulating effect of melibiose on Na+ binding. Steady-state Trp fluorescence spectroscopy revealed impaired conformational changes after melibiose binding in the mutants and fluorescence resonance energy transfer (FRET) measurements indicated that the mutants still show cooperative modification of their sugar binding sites by Na+. These data suggest that loop 4-5 contributes to the coordinated interactions between the ion- and sugar binding site and participates in conformational changes after melibiose binding that are essential for the subsequent obligatory coupled translocation of substrates. By using the Stopped-flow technique, three different approaches were followed. First, the intrinsic Trp fluorescence of MelB, known to increase upon melibiose binding [Mus-Veteau et al. 1995], revealed a signal with a T 1 of ~15 ms in C-less. This time constant is of the same order of magnitude as that determined with the SSM method suggesting that Trp fluorescence and electrical signal are related processes. Conformation for this assumption came from the fact that the activation energies Ea for both processes are similar (around 45 KJ/mol). Second, by using the fluorescent sugar analog Dns2-S-Gal, which monitors events close to the sugar binding site [Maehrel et al. 1998], a signal with a T 1 of ~18 ms was recorded upon Na+ addition. Finally, the fluorescent dye MIANS was used to selectively label the single Cys mutant E365C of loop 10-11. Stopped-flow measurements revealed a melibiose-induced fluorescent signal with a T 1 of 45 ms. Since electrical measurements with the MIANS-labeled E365C excluded the possibility that the label is responsible for the slower kinetics, the conformational change detected by the MIANS fluorescence was assigned to a slow transition in the cycle of MelB after melibiose binding. Ea was determined to be 96 KJ/mol corroborating, thus, the hypothesis of a different process. In conclusion, it was possible to correlate the electrical and fluorescence signals to partial reactions of the transport cycle and to determine their rate constants. According to this new model, the melibiose-induced signal detected with the Trp and electrical measurements corresponds to a step preceding the carriers’ reorientation (3 3*, k ~ 65s-1), and the melibiose-induced signal detected with the MIANS fluorescence to the reorientation itself (3* 4, k ~ 20s-1).Um die Aufnahme und Abgabe von organischen Substraten und Ionen in die Zelle zu ermöglichen, besitzt die Plasmamembran eine Reihe von verschiedenen Transportsystemen. Die Melibiose Permease (MelB) in der zytoplasmatischen Membran von Escherichia Coli ist ein solches Transportsystem und gehört zur Familie der Glykosid-Pentosid-Hexuronid-Transporter. MelB besteht aus 473 Aminosäuren und hat ein Molekulargewicht von 53 kDa. Das Protein ist sehr hydrophob (70% apolar) und hat zwölf Transmembrandomänen mit alpha-helikaler Konformation [Botfield et al. 1992, Gwizdek et al. 1997, Hacksel et al. 2002, Pourcher et al. 1990a]. Der Transport verschiedenster Substrate durch biologische Membranen ist an bereits existierende Gradienten anderer Substrate oder Ionen gekoppelt (sekundärer Transport). So nutzt zum Beispiel MelB den durch andere Systeme geschaffenen elektrochemischen Natriumgradienten, um akkumulierend ein Substrat (zum Beispiel alpha-Galaktoside wie Melibiose oder beta-Galaktoside wie Methyl-1-thio-beta-D-galaktopyranosid) in die Zelle zu befördern. MelB kann als kotransportierende Ionen neben Natrium auch Lithium und Protonen verwenden [Bassilana et al. 1985, Lopilato et al. 1978, Pourcher et al. 1995, Tsuchiya et al. 1978, Tsuchiya et al. 1980, Tsuchiya et al. 1983, Tsuchiya & Wilson 1978, Wilson & Wilson 1987]. Die Kosubstrate binden an den Transporter mit einer Stoichiometrie von 1:1. Des weiteren erhöht die Bindung des Kations die Affinität des Transporters für den Zucker, wobei Na+ und Li+ bessere Aktivatoren als H+ sind. MelB katalysiert die gekoppelte Translokation von Na+ (H+ oder Li+) und Zuckern ebenfalls mit einer Stoichiometrie von 1:1. Die Substratbindung an der Außenseite und die Substratdissoziation ins Zytoplasma erfolgen geordnet, d. h. Na+ bindet zuerst gefolgt von Zucker, wohingegen der Zucker zuerst dissoziiert gefolgt von Na+ [Bassilana et al. 1987, Damiano-Forano et al. 1986, 1988, Pourcher et al. 1990a]. Dabei erhöht das Membranepotential den aktiven Transport, indem es die Rate der Na+-Dissoziation in das Zytoplasma erhöht. Wenn Melibiose in der Zelle ist, wird es von der alpha-Galaktosidase in Glukose und Galaktose gespalten, die beide von der Zelle verstoffwechselt werden und der Energieproduktion dienen. MelB ist ein besonders interessantes Protein für die Untersuchung des Transport-mechanismus, da es verschiedene Zucker und Kationen als Substrate nutzt. Außerdem läßt das Protein sich in großen Mengen herstellen, aufreinigen und in Liposomen rekonstituieren [Pourcher et al. 1995]. Die Melibiose Permease besitzt strukturelle und funktionelle Homologie zu anderen Na+-/Substrat-Kotransportern, die bei biologisch und medizinisch relevanten Prozessen wichtige Funktionen einnehmen. Trotz ihrer Bedeutung sind die Kenntnisse zur Struktur und Funktionsweise dieser Transportproteine sehr begrenzt und deren Aufklärung bleibt weiterhin eine große Herausforderung. MelB dient dabei als Modell, die zugrundeliegenden molekularen Mechanismen sekundär aktiver Na+-Kotransporter besser zu verstehen. Damit können generelle Prinzipien zur Funktionsweise formuliert werden, die auch auf eukaryotische Transporter angewandt werden können, die bei pathophysiologischen Prozessen eine Rolle spielen. Bisher wurden hauptsächlich stationäre Messungen durchgeführt, die einen Einblick in den Gesamtreaktionsmechanismus von MelB gegeben haben [Pourcher et al. 1990a]. Vor kurzem wurden elektrogene Vorgänge, die durch die Aktivität von MelB verursacht worden sind, zum ersten Mal untersucht [Ganea et. al. 2001]. Hierzu wurden Proteoliposomen, die den gereinigten MelB-Transporter enthielten, auf eine festkörperunterstützte Membran (SSM) adsorbiert. Diese Technik ist besonders interessant, da mit Hilfe eines schnellen Lösungs-wechsels Konzentrationssprünge von nahezu jedem beliebigen Substrat an der SSM durchgeführt werden können. Bei dieser ersten Charakterisierung wurde festgestellt, daß die Na+-Bindung an MelB eine Ladungsverschiebung innerhalb des Proteins auslöst, die sich in einer schnell abfallenden Komponente eines transienten Stromsignals widerspiegelt. Mehrere Zwischenschritte innerhalb des Reaktionszyklus wurden bisher nicht oder nur ungenügend charakterisiert. Deswegen bestand die grundlegende Zielsetzung dieser Arbeit darin, vorstationäre, oder pre-steady state Methoden, zur Untersuchung einzusetzen. Diese erlauben es, Reaktionsgeschwindigkeitskonstanten von einzelnen Schritten im Reaktions-zyklus von MelB aufzulösen. Um dieses Ziel zu erreichen, wurden der Wildtyp und verschiedene mutierte Transporter solubilisiert, aufgereinigt und anschließend in Liposomen rekonstitutiert, wie es bereits für MelB beschrieben worden ist [Pourcher et al. 1995, Rigaud et al. 1995]. Die Methode der Aufreinigung und Rekonstitution hat den Vorteil, daß man ohne Wechselwirkung mit anderen zellulären Proteinen die Eigenschaften von MelB untersuchen kann. Die Proteoliposomen wurden in dieser Arbeit hauptsächlich mit der SSM und der Stopped-Flow-Technik untersucht. ..
Comparison of DNA extraction kits and modification of DNA elution procedure for the quantitation of subdominant bacteria from piggery effluents with real-time PCR
No full textInternational audienceFour commercial DNA extraction kits and a minor modification in the DNA elution procedure were evaluated for the quantitation of bacteria in pig manure samples. The PowerSoil®, PowerFecal®, NucleoSpin® Soil kits and QIAamp® DNA Stool Mini kit were tested on raw manure samples and on lagoon effluents for their ability to quantify total bacteria and a subdominant bacteria specific of pig manure contamination: Lactobacillus amylovorus. The NucleoSpin® Soil kit (NS kit), and to a lesser extent the PowerFecal® kit were the most efficient methods. Regardless of the kit utilized, the modified elution procedure increased DNA yield in the lagoon effluent by a factor of 1.4 to 1.8. When tested on 10 piggery effluent samples, compared to the QIAamp kit, the NS kit combined with the modified elution step, increased by a factor up to 1.7 log10 the values of the concentration of L. amylovorus. Regardless of the type of manure, the best DNA quality and the highest concentrations of bacteria were obtained using the NS kit combined with the modification of the elution procedure. The method recommended here significantly improved quantitation of subdominant bacteria in manure. Four commercial DNA extraction kits and a minor modification in the DNA elution procedure were evaluated for the quantitation of bacteria in pig manure samples. Modification of the elution procedure significantly improved quantitation of subdominant bacteria in manure. © 2014 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd
Que Reste-T-Il Des H�ritiers Et De La Reproduction (1964-1971) Aujourd'hui? Questions, M�thodes, Concepts Et R�ception D'une Sociologie De L'�ducation
No full textJ\'ai essay� de restituer ici quelques informations historiographiques, � partir de mes notes d\'enseignement, de documents de recherche des ann�es 60 et, le plus souvent, de simples souvenirs de discussions avec d\'autres chercheurs ; souvenirs aussi de lectures d\'ouvrages et de compte-rendus de cette �poque ou de confrontations avec des auditoires (universitaires ou non) dans des d�bats publics. Je me suis donc plac� dans le cadre des questions pos�e par J.-L. Robert dans son introduction � la Journ�e d\'�tudes sur \' les concepts �labor�s dans la p�riode 1945-1975 ,\' ainsi que des d�marche qui se voulaient scientifiques avec leurs m�thodes, leurs questions, leurs objets, leurs sources et conclusions \' ; mais aussi dans le cadre du suivi, sur deux d�cennies, de \' la diffusion et de l\'appropriation des concepts \' qui concernaient au premier chef la sociologie de l\'�ducation et de la culture . Je r�ponds ainsi � plusieurs questions de fait que posait J.-M. Chapoulie dans son introduction ou sur lesquelles il a sollicit� ma m�moire. Mais je n\'entre pas dans le rappel de ce qu\'ont �t� mes rapports personnels avec Bourdieu durant la douzaine d\'ann�es de collaboration �troite o� s\'est �labor�e une sociologie de l\'�ducation qui nous �tait en gros commune et qui l\'est rest�e, en d�pit de notre divergence �pist�mologique apr�s 1972, puisque je viens d\'en donner un aper�u dans un article r�cent[No keywords]
Composition pour le traitement aérobie de résidus ligno-cellulosiques, procédé associé et leur utilisation pour un prétraitement biologique desdtis résidus
No full text[Departement_IRSTEA]Ecotechnologies [TR1_IRSTEA]TEDComposition pour le traitement de résidus agricoles ligno-cellulosiques (11), de type paille, caractérisée en ce qu'elle comprend un mélange liquide d'une solution aqueuse renfermant les éléments minéraux NPK, apte à former un milieu nutritif pour des micro-organismes endogènes desdits résidus agricoles à traiter, et d'une source de lignine issue d'un procédé de délignification alcaline de biomasses ligno-cellulosiques. Le traitement en milieu aérobie desdits résidus avec la composition conduit à une augmentation de l'accessibilité de la cellulose aux cellulases. Utilisation pour le prétraitement biologique desdits résidusligno-cellulosiques
Experimental design for the optimization of propidium monoazide treatment to quantify viable and non-viable bacteria in piggery effluents
No full text[Departement_IRSTEA]Ecotechnologies [TR1_IRSTEA]TED [Axe_IRSTEA]TED-SAFIRInternational audienceBackground: Distinguishing between viable and dead bacteria in animal and urban effluents is a major challenge. Among existing methods, propidium monoazide (PMA)-qPCR is a promising way to quantify viable cells. However, its efficiency depends on the composition of the effluent, particularly on total suspended solids (TSS)) and on methodological parameters. The aim of this study was evaluate the influence of three methodological factors (concentration of PMA, incubation time and photoactivation time) on the efficiency of PMA-qPCR to quantify viable and dead cells of Listeria monocytogenes used as a microorganism model, in two piggery effluents (manure and lagoon effluent containing 20 and 0.4 TSS g.kg-1, respectively). An experimental design strategy (Doehlert design and desirability function) was used to identify the experimental conditions to achieve optimal PMA-qPCR results. Results: The quantification of viable cells of L. monocytogenes was mainly influenced by the concentration of PMA in the manure and by the duration of photoactivation in the lagoon effluent. Optimal values differed with the matrix: 55 ?M PMA, 5 min incubation and 56 min photoactivation for manure and 20 ?M PMA, 20 min incubation and 30 min photoactivation for lagoon effluent. Applied to five manure and four lagoon samples, these conditions resulted in satisfactory quantification of viable and dead cells. Conclusion: PMA-qPCR can be used on undiluted turbid effluent with high levels of TSS, provided preliminary tests are performed to identify the optimal conditions. © 2015 Desneux et al
Endoscopic internal drainage for the management of leak, fistula, and collection after sleeve gastrectomy: our experience in 617 consecutive patients
No full textBackground: Endoscopy plays a pivotal role in the management of adverse events (AE) following bariatric surgery. Leaks, fistulae, and post-operative collection after sleeve gastrectomy (SG) may occur in up to 10% of cases. Objectives: To evaluate the efficacy and safety of endoscopic internal drainage (EID) for the management of leak, fistula, and collection following SG. Setting: Retrospective, observational, single center study on patients referred from several bariatric surgery departments to an endoscopic referral center. Methods: EID was used as first-line treatment for the management of leaks, fistulae, and collections. Leaks and fistulae were treated with double pigtail stent (DPS) deployment in order to guarantee internal drainage and second intention cavity obliteration. Collections were treated with endoscropic ultrasound (EUS)–guided deployment of DPS or lumen apposing metal stents. Results: A total of 617 patients (83.3% female; mean age, 43.1 yr) were enrolled in the study for leak (n = 300, 48.6%), fistula (n = 285, 46.2%), and collection (n = 32, 5.2%). Median follow-up was 19.5 months. Overall clinical success was 84.7% whereas 15.3% of cases required revisional surgery after EID failure. Clinical success according to type of AE was 89.5%, 78.5%, and 90% for leak, fistula, and collection, respectively. A total of 10 of 547 (1.8%) presented a recurrence during follow-up. A total of 28 (4.5%) AE related to the endoscopic treatment occurred. At univariate logistic regression predictors of failure were: fistula (OR 2.012), combined endoscopic approach (OR 2.319), need for emergency surgery (OR 1.755), and previous endoscopic treatment (OR 4.818). Conclusion: Early EID for the management of leak, fistula, and post-operative collection after SG seems a safe and effective first-line approach with good long-term results
Near-Linear Time Projection onto the ℓ1,∞ Ball; Application to Sparse Autoencoders
No full textLooking for sparsity is nowadays crucial to speed up the training of large-scale neural networks. Projections onto the ℓ1,2 and ℓ1,∞ are among the most efficient techniques to sparsify and reduce the overall cost of neural networks. In this paper, we introduce a new projection algorithm for the ℓ1,∞ norm ball. The worst-case time complexity of this algorithm is O(nm+Jlog(nm)) for a matrix in Rn×m. J is a term that tends to 0 when the sparsity is high, and to nm when the sparsity is low. Its implementation is easy and it is guaranteed to converge to the exact solution in a finite time. Moreover, we propose to incorporate the ℓ1,∞ ball projection while training an autoencoder to enforce feature selection and sparsity of the weights. Sparsification appears in the encoder to primarily do feature selection due to our application in biology, where only a very small part (<2%) of the data is relevant. We show that both in the biological case and in the general case of sparsity that our method is the fastest.The authors thank Thierry Pourcher (TIRO Laboratory) for providing the Lung dataset
Structure and function of amino acid and peptide transport proteins
No full textAll living cells are enclosed by biological membranes that separate the interior cytoplasm from the outer environment. Composed of a phospholipid bilayer with embedded proteins biological membranes act as insulators and filters. The transfer of selected substances and information across the membrane is controlled and mediated through membrane proteins. As a result, membrane proteins are central to almost all cellular processes. They play key roles in signalling between cells, in transport across cell membranes and in energy transduction processes. To accomplish these versatile cell functions, membrane proteins are available in an abundant diversity. Accordingly, it’s not astounding that about 30% of all genes encoded in the human genome are membrane proteins. However, only 158 (Status: May 2008) unique membrane protein structures, mainly from bacterial membrane proteins, have been deposited thus far in the Protein Data Bank (PDB), compared to the more than 10’000 soluble protein structures. These numbers highlight the enormous structural work that remains to be done in the field of membrane proteins. From a biomedical perspective, membrane proteins constitute about 50% of possible targets for novel drugs. The threedimensional (3D) structure of a protein is an essential starting point for the investigation of its molecular mechanisms of action, the basis for drug design. Therefore structural information of membrane proteins is of fundamental importance for human health. High-resolution structure determination of membrane proteins is currently one of the greatest challenges in cell biology. Membrane proteins possess a hydrophobic belt that is required for their incorporation into lipid membranes. For the extraction and purification of membrane proteins detergents are used to keep the proteins in a solubilized state. The lack of structural information on membrane proteins is mainly related to their low expression levels, the instability in the detergent solution and their resistance to crystallization. The latter is a considerable limitation because X-ray crystallography, which at the present time is the most powerful technique for determining protein structures, requires highly ordered 3D protein crystals. Besides structure determination by X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy, the method of electron crystallography on 2D crystals has become increasingly important in membrane protein research. The striking advantage of 2D crystals is that the membrane protein is analyzed in its native environment, the lipid bilayer. However, as with 3D crystals, the production of highly ordered 2D crystals is a major barrier. Electron crystallography was first applied in 1975 by Henderson and Unwin to study the structure of bacteriorhodopsin in purple membranes (1, 2). Since then, substantial progress has been made in further development of electron crystallography, especially in sample preparation, cryo-transmission electron microscopy (cryoTEM) imaging and data processing. Thus, improved cryo-TEM in combination with electron diffraction of 2D crystals is used to establish the 3D protein structure (3). The recent structure determination of the mammalian aquaporin-0 (AQP-0) at the remarkable resolution of 1.9 Å demonstrated impressively the potential of the cryo-TEM approach. So far seven atomic models (<4 Å) of membrane proteins have been determined by high-resolution electron crystallography (plant light harvesting complex 2 (4), AQP-1 (5, 6), nicotinic acetylcholine receptor (7, 8), AQP-0 (9, 10), AQP-4 (11), glutathione transferase 1 (2)). Thereby one has to notice that less than two dozen groups are pursuing electron crystallography, compared to the hundreds of groups in X-ray crystallography. Even more mportant, 2D crystallization combined with TEM is not an all-or-nothing approach, meaning that also from poorly ordered crystals low resolution structures are obtainable. In conclusion, electron crystallography presents a highly favourable and successful method to explore the structures of membrane proteins (13). The atomic force microscope (AFM) is a powerful tool to investigate the surface topography of membrane proteins embedded in lipid bilayers under near-physiological conditions, i.e., in buffer solution, at room temperature and under normal pressure. The high lateral (≥5 Å) and vertical resolution (~1 Å), and the high signal-to-noise ratio of the topographs acquired by AFM make this instrument unique to study surface structure and dynamics of single functional membrane proteins. Besides high-resolution surface imaging, structural and mechanical properties of single membrane proteins can be studied by single molecule force spectroscopy (SMFS), an AFMrelated technique. In a typical SMFS experiment the cantilever tip approaches the membrane protein, pushes on it and then retracts. During this approach-retraction cycle the force acting on the molecule is measured and plotted as a function of the tip-surface distance: the so-called ‘force curve’ is thus obtained. Such force curves reveal details about inter- and intra-molecular interactions, unfolding barriers and energy landscapes in membrane proteins. Because these measurements take place in solution at physiological conditions, the binding of ligands and the subsequent alteration within the protein may be detected and visualized. This offers the unique possibility to directly monitor structural changes related to biological processes.
eugindat/). In Eugindat biological and medical scientists with various backgrounds were united in one consortium to concentrate the research on amino acid and peptide transporters. The transport of amino acids into cells is a crucial process for all living species from bacteria to humans. Defects on proteins involved in this transport lead to strong disturbances in the amino acid metabolism of the organism. Humans strongly rely on amino acids in their diet, since nine essential amino acids cannot be synthesized from other precursors. Consequently, it’s all the more important that systems for the uptake, distribution and reabsorption of amino acids work properly. Thereby, the proximal tubule plays a central role by reabsorbing over 95% of the filtered amino acid load. In the case where elevated levels (>5%) are detected in urine, the term aminoaciduria is applied. Primary Inherited Aminoacidurias (PIA) is a group of rare diseases arisen from genetic defects in amino acid transporters expressed in the plasma membrane of renal epithelial cells. PIA members are classified by the target amino acid or acids involved. The group includes Cystinuria, Lysinuric Protein Intolerance (LPI), Dicarboxylic Aminoaciduria (DA), Hartnup Disorder (HDis), Iminoglycinuria (IG) and unlabeled aminoacidurias. Cystinuria and LPI are the best studied PIAs. It was demonstrated that members of the heteromeric amino acid transporter family (HAT family) are the molecular base of cystinuria and LPI. HATs are composed of a heavy subunit (HSHAT) and the corresponding light subunit (LSHAT) that are liked together by a disulfide bridge. HSHATs are N-glycosylated type II membrane glycoproteins, whereas LSHATs are nonglycosylated polytopic membrane proteins with twelve putative transmembrane segments (TMS) (14-16). Two genes rBAT (HSHAT) and b0,+AT (LSHAT) could be identified responsible for cystinuria (17, 18) while mutations in the system 4F2hc (HSHAT) and y+LAT1 (LSHAT) lead to LPI (19, 20).
The structural studies of amino acid and peptide transporters by TEM and AFM reported in this thesis became possible thank to an European initiative called Eugindat (European genomics initiative on disorders of plasma membrane To acquire a thorough knowledge of the amino acid transporters, http://www.ub.es/ structure of relevant transporters for PIA and renal reabsorption of amino acids, 2D and 3D crystallization of membrane and soluble proteins for structure determination was addressed within Eugindat. Recently, Eugindat members reported the X-ray structure of 4F2hc protein at 2.1 Å (monoclinic) and 2.8 Å (orthorhombic) resolutions (21). In contrast, there are no structures from eukaryotic or human amino acid transporters available. So far, only two structures of bacterial transport proteins with homology to their eukaryotic counterparts were successfully solved: the atomic structures of a bacterial leucine transporter (22) and of a bacterial glutamate transporter (23). Despite the awarded effort, these two transporters do not correspond to the class of LSHAT proteins. However, these examples indicate that important information to understand structure-function relationships of amino acid transporters is gained from prokaryotic homologues. In pursuit of our aim to reveal first structural information of PIA related amino acid transporters, we searched and studied prokaryotic homologues of LSHAT.
Within Eugindat, structural and functional studies were extended to cover other important transporters involved in human health, i.e. peptide transporters. Members of this second class of transport proteins were extensively studied in the past and belong to the peptide transporter (PTR) family (26, 27). Peptide transporters are integral membrane proteins that mediate the cellular uptake of di- and tripeptides. Similar to the amino acid transport, peptide transport is of fundamental importance in all species. In human peptide transport at the brush border membranes of small intestine, kidney and lung is handled by two members from the PTR family designated as PEPT1 and PEPT2. PEPT1 is considered as the major route by which protein digestion products enter the body. Previous studies demonstrated that peptide transporters have broad substrate specificity transporting essentially all 400 possible dipetides, 8000 possible tripeptides and a large spectrum of peptidomimetics into the cell (i.e. pharmacologically active compounds) (2831). Peptide transporters are therefore potent drug delivery systems. Substrate translocation The LSHAT membrane proteins b0,+AT and is coupled to the proton movement down y+LAT1 are members of the L-type amino acid an electrochemical proton gradient with the transporter family (LAT) that is a subfamily of membrane potential as the main driving force. the amino acid/polyamine/organocation (APC) transporter superfamily. The APC superfamily In contrast to the wealth of functional counts nearby 250 members in prokaryotes information, no structural information on peptide and eukaryotes that function as solute cation transporters is available. For our structural and symporters and solute cation antiporters (17). functional studies within Eugindat, we selected Most APC members are predicted to possess from several possible candidates the bacterial twelve α-helical transmembrane segments PTR family members YbgH, YdgR (TppB, (TMS) with cytosolic located N- and C-termini DtpA) and YhiP (DtpB) from Escherichia coli. (24, 25). According to the high sequence identity The structural work on these bacterial peptide and homology to eukaryotic and human APC transporters presented in this thesis represents transporters, we selected after an exhaustive search the first published structural information of these for structural (TEM/AFM) and functional studies important class of transport proteins. the two prokaryotic amino acid transporters AdiC and SteT. As shown in the presented thesis, the L-arginine/agmatine antiporter AdiC and the threonine/serine exchange transporter SteT represent excellent proteins to elucidate the molecular architecture of transporters from the APC superfamily
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