Abstract:

Proteins present in the archaeal cell envelope play key roles in a variety of processes necessary for survival in extreme environments. The haloarchaeon Haloferax volcanii is a good model for membrane proteomic studies because its genome sequence is known, it can be genetically manipulated, and a number of studies at the “omics” level have been performed in this organism. This work reports an easy strategy to improve the resolution of acidic membrane proteins from H. volcanii by 2DE. The method is based on the solubilization, delipidation, and salt removal from membrane proteins. Due to the abundance of the S-layer glycoprotein (SLG) in membrane protein extracts, other proteins from the envelope are consequently underrepresented. Thus, a protocol to reduce the amount of the SLG by EDTA treatment was applied and 11 cm narrow range pH (3.9–5.1) IPG strips were used to fractionate the remaining proteins. Using this method, horizontal streaking was substantially decreased and at least 75 defined spots (20% of the predicted membrane proteome within this pI/Mw range) were reproducibly detected. Two of these spots were identified as thermosome subunit 1 and NADH dehydrogenase from H. volcanii, confirming that proteins from the membrane fraction were enriched. Removal of the SLG from membrane protein extracts can be applied to increase protein load for 2DE as well as for other proteomic methods.

Author affiliation: Paggi, Roberto A.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata; Argentina

Author affiliation: Gimenez, Maria Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata; Argentina

Author affiliation: de Castro, Rosana Esther. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata; Argentina

Author affiliation: Cesari, Andreina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata; Argentina

Abstract:

Proteins present in the archaeal cell envelope play key roles in a variety of processes necessary for survival in extreme environments. The haloarchaeon Haloferax volcanii is a good model for membrane proteomic studies because its genome sequence is known, it can be genetically manipulated, and a number of studies at the “omics” level have been performed in this organism. This work reports an easy strategy to improve the resolution of acidic membrane proteins from H. volcanii by 2DE. The method is based on the solubilization, delipidation, and salt removal from membrane proteins. Due to the abundance of the S-layer glycoprotein (SLG) in membrane protein extracts, other proteins from the envelope are consequently underrepresented. Thus, a protocol to reduce the amount of the SLG by EDTA treatment was applied and 11 cm narrow range pH (3.9–5.1) IPG strips were used to fractionate the remaining proteins. Using this method, horizontal streaking was substantially decreased and at least 75 defined spots (20% of the predicted membrane proteome within this pI/Mw range) were reproducibly detected. Two of these spots were identified as thermosome subunit 1 and NADH dehydrogenase from H. volcanii, confirming that proteins from the membrane fraction were enriched. Removal of the SLG from membrane protein extracts can be applied to increase protein load for 2DE as well as for other proteomic methods.

Author affiliation: Paggi, Roberto A.. Universidad Nacional de Mar del Plata. Facultad de Cs.exactas y Naturales. Instituto de Invest.biologicas; Argentina. Universidad Nacional de Mar del Plata; Argentina

Author affiliation: Gimenez, Maria Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata; Argentina

Author affiliation: de Castro, Rosana Esther. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata; Argentina

Author affiliation: Cesari, Andreina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Mar del Plata. Instituto de Investigaciones Biológicas; Argentina. Universidad Nacional de Mar del Plata; Argentina

Abstract:

This study investigated the structural and biophysical characteristics of GumB and GumC, two Xanthomonas campestris membrane proteins that are involved in xanthan biosynthesis. Xanthan is an exopolysaccharide that is thought to be a virulence factor that contributes to bacterial in planta growth. It also is one of the most important industrial biopolymers. The first steps of xanthan biosynthesis are well understood, but the polymerization and export mechanisms remain unclear. For this reason, the key proteins must be characterized to better understand these processes. Here we characterized, by biochemical and biophysical techniques, GumB, the outer membrane polysaccharide export protein, and GumC, the polysaccharide co-polymerase protein of the xanthan biosynthesis system. Our results suggested that recombinant GumB is a tetrameric protein in solution. On the other hand, we observed that both native and recombinant GumC present oligomeric conformation consistent with dimers and higher-order oligomers. The transmembrane segments of GumC are required for GumC expression and/or stability. These initial results provide a starting point for additional studies that will clarify the roles of GumB and GumC in the xanthan polymerization and export processes and further elucidate their functions and mechanisms of action.

Author affiliation: Bianco, María Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina

Author affiliation: Jacobs, Melisa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina

Author affiliation: Salinas, Silvina Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina

Author affiliation: Salvay, Andrés Gerardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Física de Líquidos y Sistemas Biológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Física de Líquidos y Sistemas Biológicos; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina

Author affiliation: Ielmini, M. V.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina

Author affiliation: Ielpi, Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina

Abstract:

Los polisacáridos juegan un papel esencial en el metabolismo celular y en el funcionamiento de los organismos. Son sintetizados empleando enzimas glicosiltransferasas (GTs), cuyas propiedades determinan el tamaño y la estructura del producto final. Muchas GTs están localizadas en membranas celulares, dificultando su purificación y caracterización. En este trabajo de Tesis se estudian bioquímica y biofísicamente dos GTs que participan en la síntesis del polisacárido xantano producido por Xanthomonas campestris. La primer GT es GumI, de la cual sólo había escasos antecedentes genéticos. En esta Tesis se presenta la primera caracterización bioquímica y funcional de GumI, demostrando su actividad glicosiltransferasa previamente propuesta. Se realizaron ensayos de complementación funcional, demostrando su actividad manosiltransferasa in vivo. Además, se demostró que GumI está unida a la membrana, y que la interacción está mediada por interacciones hidrofóbicas y electrostáticas. GumI fue purificada y su actividad fue estudiada in vitro, obteniéndose los parámetros óptimos de reacción. GumI dio origen a una nueva familia, GT-94, en la clasificación Carbohydrate Active EnZymes. Finalmente, mediante ensayos de cristalización se obtuvieron agujas bidimensionales que al presente no fueron aptas para resolver la estructura de GumI por cristalografía de rayos-X. La segunda GT es GumK, cuya estructura cristalina se resolvió en el laboratorio y, como GumI, es una GT monotópica de membrana. Se profundizó sobre las bases moleculares de la unión a sus sustratos y a la membrana. Se realizaron diferentes mutaciones sobre la zona propuesta de unión a la membrana, demostrando la complejidad de dicha interacción. Además, se realizaron estudios por simulación computacional con el fin de obtener la estructura del complejo GumK/UDP-GlcA. El modelo obtenido explicaría la especificidad por este sustrato. Estudios realizados en este trabajo mediante diversas técnicas -tales como proteólisis limitada, fluorescencia, dicroísmo circular, calorimetría isotérmica de titulación, resonancia magnética nuclear y dispersión de rayos-X a bajo ángulo- sugieren fuertemente la presencia de cambios conformacionales disparados por la unión de UDP-GlcA.

Polysaccharides, which play a crucial role in cellular metabolism and in the performance of organisms, are synthesized by glycosyltransferase (GT) enzymes, whose properties determine the size and structure of the final product. Many GTs are localized in cellular membranes, difficulting their purification and characterization. In this Thesis, two GTs (GumI and GumK) which participate in the synthesis of the polysaccharide xanthan produced by Xanthomonas campestris were studied biochemically and biophysically. The genetic background data on GumI is limited. This Thesis reports the first functional and biochemical characterization of GumI, demonstrating its previously proposed glycosyltransferase activity. Functional complementation demonstrated its in vivo mannosyltransferase activity. Results also show that GumI is a membrane-associated protein, and that the interaction is mediated by hydrophobic and electrostatic forces. GumI was purified and its activity was studied in vitro, obtaining optimal reaction parameters. Finally, crystallization experiments allowed obtaining bidimensional needles, which were not suitable to resolve GumI structure by X-ray crystallography. GumK, whose structure was resolved in the laboratory, is a monotopic GT, like GumI. This Thesis deepened on the molecular basis of the binding to its substrate and the membrane. Mutagenesis of residues of the region proposed for membrane binding was performed, resulting in no-soluble protein and thus demonstrating the complexness of membrane interaction. Moreover, computational simulation studies were carried out with the aim to obtain a model of the GumK/UDP-GlcA structure. The model obtained may explain the GumK specificity for this substrate. Studies performed by various techniques, such as fluorescence, circular dichroism, isothermal titration calorimetry, nuclear magnetic resonance, and small-angle X-ray scattering, strongly suggest the presence of conformational changes in GumK triggered by the UDP-GlcA binding.

Author affiliation: Salinas, Silvina R.. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

Abstract:

The purpose of this work was to obtain information about conformational changes of the plasma membrane Ca2+-pump (PMCA) in the membrane region upon interaction with Ca2+, calmodulin (CaM) and acidic phospholipids. To this end, we have quantified labeling of PMCA with the photoactivatable phosphatidylcholine analog [125I]TID-PC/16, measuring the shift of conformation E2 to the auto-inhibited conformation E1I and to the activated E1A state, titrating the effect of Ca2+ under different conditions. Using a similar approach, we also determined the CaM-PMCA dissociation constant. The results indicate that the PMCA possesses a high affinity site for Ca2+ regardless of the presence or absence of activators. Modulation of pump activity is exerted through the C-terminal domain, which induces an apparent auto-inhibited conformation for Ca2+ transport but does not modify the affinity for Ca2+ at the transmembrane domain. The C-terminal domain is affected by CaM and CaM-like treatments driving the auto-inhibited conformation E1I to the activated E1A conformation and thus modulating the transport of Ca2+. This is reflected in the different apparent constants for Ca2+ in the absence of CaM (calculated by Ca2+-ATPase activity) that sharply contrast with the lack of variation of the affinity for the Ca2+ site at equilibrium. This is the first time that equilibrium constants for the dissociation of Ca2+ and CaM ligands from PMCA complexes are measured through the change of transmembrane conformations of the pump. The data further suggest that the transmembrane domain of the PMCA undergoes major rearrangements resulting in altered lipid accessibility upon Ca2+ binding and activation.

Author affiliation: Mangialavori, Irene Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina

Author affiliation: Ferreira Gomes, Mariela Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina

Author affiliation: Pignataro, María Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina

Author affiliation: Strehler, Emanuel E.. Mayo Clinic College of Medicine; Estados Unidos

Author affiliation: Rossi, Juan Pablo Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina

Abstract:

Candida fukuyamaensis RCL-3 yeast strain isolated from a copper filter plant is able to lower copper concentration in culture medium. In the present study, effect of copper in proteins expression and mechanisms involved in copper resistance were explored using comparative proteomics. Mono-dimensional gel electrophoresis revealed differential band expressions between cells grown with or without copper. 2-DE analysis of C. fukuyamaensis RCL-3 revealed that copper exposure produced at least an over-expression of 40 proteins. Sixteen proteins were identified and grouped in four categories according to their functions: glycolysis and ATP production, synthesis of proteins, oxidative stress response, and processing and transport of proteins. Integral membrane proteins and membrane-associated proteins were analyzed, showing nine protein bands overexpressed in Cu-supplemented medium. Four proteins were identified, namely nucleoporin pom152, elongation factor 2, copper chaperone Sod1 Ccs1, and eiosome component Lsp1. The proteomic analysis performed allowed the identification of different metabolic pathways and certain proteins involved in metal input and storage related to cell ability to bioremediate copper. These proteins and mechanisms could be used for future applications of C. fukuyamaensis RCL-3 in biotechnological processes such as remediation of heavy metals.

Author affiliation: Irazusta, Verónica Patricia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Planta Piloto de Procesos Industriales Microbiológicos (i); Argentina

Author affiliation: Michel, Lucas. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Salta. Instituto de Investigación para la Industria Química (i); Argentina

Author affiliation: Castellanos de Figueroa, Lucia Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Planta Piloto de Procesos Industriales Microbiológicos (i); Argentina. Universidad Nacional de Tucumán; Argentina

Abstract:

La Esferocitosis Hereditaria (ESH) es una anemia hemolítica de observación frecuente, caracterizada por alteraciones cuali y/o cuantitativas –genéticamente determinadas– de las proteínas de la membrana eritrocitaria. Consecuentemente, se debilita la unión entre la membrana eritrocitaria y el citoesqueleto subyacente, produciéndose la pérdida progresiva de la membrana con formación de hematíes de forma esférica, osmóticamente frágiles, que son selectivamente atrapados y destruidos en el bazo. Muchas alteraciones en diferentes genes pueden generar la misma enfermedad. Clínicamente se caracteriza por anemia, ictericia y esplenomegalia. La ESH es la anemia hemolítica hereditaria más frecuente en Argentina. A pesar de ello, es muy escasa la información sobre su comportamiento en nuestro país. Los objetivos de esta tesis son: a) Investigar avances en la metodología de diagnóstico a partir del análisis e implementación de nuevas pruebas que han sido desarrolladas durante la última década; b) Establecer aspectos demográficos de la ESH en la población atendida por nuestro grupo de trabajo; c) Dilucidar algunos mecanismos involucrados en la enfermedad. En este trabajo se utilizaron las pruebas tradicionales (fragilidad osmótica eritrocitaria y prueba de autohemólisis) y se desarrollaron por primera vez en nuestro país las nuevas pruebas diagnósticas (citometría de flujo con 5’eosina maleimida, criohemólisis hipertónica y fragilidad osmótica por citometría de flujo) estableciendo sus puntos de corte, sensibilidad, especificidad y valores predictivos. El análisis de los resultados de los ensayos electroforéticos de las membranas eritrocitarias permitió establecer que las deficiencias de espectrina y ankirina son las prevalentes en nuestra población. En los casos en que fue posible, el estudio se extendió al grupo familiar primario pudiendo detectar portadores sanos y establecer el patrón de herencia. Para poder realizar el diagnóstico en forma precoz, se desarrollaron las nuevas pruebas utilizando sangre capilar, estableciendo la presencia de la patología en neonatos de hasta 2 días de vida. El seguimiento clínico permitió observar la alta frecuencia de crisis hemolíticas asociadas a episodios febriles por lo que se decidió evaluar la posibilidad de que el agravamiento de la anemia fuera debido, en parte, a un efecto directo de la temperatura sobre los esferocitos. Se pudo observar que la hipertermia induce eriptosis mediada por la entrada de calcio a la célula, lo que podría contribuir al empeoramiento de la anemia o al desarrollo de la misma en individuos asintomáticos. La heterogeneidad clínica no pudo relacionarse al tipo de alteración proteica ni a los resultados de las pruebas diagnósticas. En la búsqueda de una alteración en común que pudiera correlacionar con la severidad de la anemia encontramos una expresión disminuida de acuaporina 1 (AQP-1), canal responsable del flujo de agua en el eritrocito, independientemente de la proteína de membrana afectada. El trabajo de tesis presentado implica un avance metodológico por haber desarrollado las nuevas técnicas diagnósticas en nuestro país y haber establecido la equivalencia para realizar los estudios con sangre capilar, conduciendo a un diagnóstico certero y precoz en los neonatos. Los antecedentes y el seguimiento clínico permitieron describir el comportamiento de la enfermedad en los pacientes pertenecientes a nuestra población. Por otra parte, los estudios in vitro, que mostraron sensibilidad de los eritrocitos de los pacientes a la temperatura y asociación de la disminución de AQP-1 en eritrocitos con la severidad de la enfermedad, abren un nuevo camino en la investigación de la esferocitosis hereditaria con una potencial aplicación terapéutica a futuro.

Hereditary spherocytosis (HS) is a common hemolytic anemia, characterized by genetically determined quantitative and/or qualitative abnormalities of the red cell membrane proteins. As a consequence, the connection of skeleton and bilayer gets weakened, loss of membrane surface occurs and changes in red cell lead to the formation of spherical, osmotically fragile erythrocytes, which are selectively trapped and destroyed in the spleen. Diverse mutations in different genes can generate the same disease. HS is characterized by anemia, jaundice, and splenomegaly. Although HS is the most common hereditary hemolytic anemia in Argentina, information concerning its behavior in our country is very scarce. Aims of this thesis are: a) To evaluate advances on diagnostic methodology since the development of new diagnostic tests described throughout the last decade; b) To establish demographics characteristics of HS in the population attended by our working group; c) To elucidate some mechanisms involved in the disease. In this study, standard screening tests –osmotic fragility and autohemolysis– and novel diagnostic tests –eosin-5’maleimide flow cytometry, hypertonic cryohemolysis, and flow cytometric osmotic fragility– were carried out. As these new diagnostic techniques were developed for the first time in our country, their cut-off points, sensitivity and specificity were established. Electrophoresis of membrane proteins established that spectrin and ankyrin are the most frequently found deficiencies in our population. As far as possible, direct relatives were also studied, allowing to detect silent carriers and to establish the inheritance pattern. Novel tests were also developed using capillary blood, to accomplish an earlier diagnosis. So, HS could be diagnosed in neonates aged only 2 days. Patients’ follow-up showed a high frequency of hemolytic crisis associated to febrile diseases. Therefore, we decided to evaluate whether the temperature increment on spherocytes may be one factor responsible for the worsening of hemolysis. Results showed that hyperthermia induced eriptosis mediated by calcium influx to the erythrocyte, which could play a role either for anemia worsening or for anemia development in previously asymptomatic individuals. No correlation between severity of the disease and either deficient protein or results of diagnostic tests could be demonstrated. Searching for an unique factor associated with severity of anemia, a decreased expression of aquaporin-1 (AQP-1), the channel responsible of water influx in the erythrocyte, was found irrespectively of the type of protein deficiency. This thesis implies a methodological advance due to the development for the first time in Argentina of the novel diagnostic tests, as well as the demonstration that they can also be performed with capillary blood, allowing an early and accurate diagnosis in neonates and small infants. Antecedents and clinical follow-up enabled to establish the behavior of the disease in the population of our country. Furthermore, results of in vitro studies showing a harmful effect of hyperthermia on erythrocytes of patients, and association of AQP-1 decrease with severity of the disease, open a new way on HS research with potential future applications.

Author affiliation: Crisp, Renée Leonor. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

Abstract:

Las membranas celulares son estructuras dinámicas complejas de composición bioquímica muy diversa. Las interacciones específicas lípidolípido y lípido-proteína pueden generar dominios estables o transientes de composición y función biológica diferenciales. En este trabajo, abordamos el estudio de la regulación de la actividad de una proteína integral de membrana, la bomba de calcio de membrana plasmática (PMCA) por anfifilos que integran el sistema en el cual se la reconstituye. En micelas de lípidos y detergente, pudimos verificar que la actividad depende de la proporción relativa de estas especies y postulamos un modelo que explica esta dependencia en base al intercambio de anfifilos a nivel del dominio transmembrana. El análisis realizado con diversos métodos espectroscópicos mostró que los cambios estructurales asociados a la activación por lípidos serían sutiles pero suficientes para transducir una señal de activación al dominio citoplasmático de PMCA. Utilizando espectroscopía de correlación de fluorescencia y la sonda fluorescente Laurdan observamos que la actividad depende además de la fluidez del entorno y del tamaño de las micelas. Para extender este estudio al entorno natural de las proteínas -la membrana celular- evaluamos diversas sondas fluorescentes y pudimos determinar que la sonda C-Laurdan permite estudiar por microscopía confocal la organización de membranas biológicas.

Cellular membranes are dynamic complex structures with a highly diverse biochemical composition. Specific lipid-lipid and lipid-protein interactions can generate transient or stable domains of particular composition and biological function. In this work, we study the regulation of the activity of an integral membrane protein, the plasma membrane calcium pump (PMCA), by phospholipids that make up the reconstitution system. Working in a mixed micelles lipid/detergent system, we verify that the activity depends on the relative proportion of these species and we posited a model that explains this relationship by the exchange between amphiphiles at the protein transmembrane domain. The analysis performed by spectroscopic methods showed that the structural changes associated with the activation by lipids are subtle but enough to transduce an activation signal to the cytoplasmic domain of PMCA. Using fluorescence correlation spectroscopy and the fluorescent probe Laurdan, we observed that the activity is also related to micelle size and the fluidity of the protein lipidic microenvironment. To extend this study to the natural environment of the protein –the cellular membrane- we assayed the performance of several fluorescent probes and we could determine that the probe CLaurdan allows studying the organization of biological membranes by confocal microscopy.

Author affiliation: Dodes Traian, Martín Miguel. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

Abstract:

Arsenic exists in natural systems in a variety of chemical forms, including inorganic arsenite (As [III]) and arsenate (As [V]). The majority of living organisms have evolved various mechanisms to avoid occurrence of arsenic inside the cell due to its toxicity. Common core genes include a transcriptional repressor ArsR, an arsenate reductase ArsC, and arsenite efflux pumps ArsB and Acr3. To understand arsenic resistance we have performed arsenic tolerance studies, genomic and bioinformatic analysis of two Exiguobacterium strains, S17 and N139, from the high-altitude Andean Lakes. In these environments high concentrations of arsenic were described in the water due to a natural geochemical phenomenon, therefore, these strains represent an attractive model system for the study of environmental stress and can be readily cultivated. Our experiments show that S17 has a greater tolerance to arsenite (10mM) than N139, but similar growth in arsenate (150mM). We sequenced the genome of the two Exiguobacterium and identified an acr3 gene in S17 as the only difference between both species regarding known arsenic resistance genes. To further understand the Acr3 we modeled the 3D structure and identified the location of relevant residues of this protein. Our model is in agreement with previous experiments and allowed us to identify a region where a relevant cysteine lies. This Acr3 membrane efflux pump, present only in S17, may explain its increased tolerance to As(III) and is the first Acr3-family protein described in Exiguobacterium genus.

Author affiliation: Ordoñez, Omar Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; Argentina

Author affiliation: Lanzarotti, Esteban Omar. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmaceuticas. Departamento de Química Biologica; Argentina

Author affiliation: Kurth, Daniel German. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; Argentina

Author affiliation: Cortez, Nestor Ricardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina

Author affiliation: Farias, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; Argentina

Author affiliation: Turjanski, Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmaceuticas. Departamento de Química Biologica; Argentina

Abstract:

Nicotinic receptors (AChRs) play key roles in synaptic transmission. We explored activation of neuronal alpha7 and mammalian muscle AChRs by morantel and oxantel. Our results revealed a novel action of morantel as a high-efficacy and more potent agonist than ACh of alpha7 receptors. The EC50 for activation by morantel of both alpha7 and alpha7-5HT3A receptors is 7-fold lower than that determined for ACh. The minimum morantel concentration required to activate alpha7-5HT3A channels is 6-fold lower than that of ACh, and activation episodes are more prolonged than in the presence of ACh. By contrast, oxantel is a weak agonist of alpha7 and alpha7-5HT3A, and both drugs are very low-efficacy agonists of muscle AChRs. The replacement of Gln57 in alpha7 by glycine, which is found in the equivalent position of the muscle AChR, decreases the efficacy for activation and turns morantel into a partial agonist. The reverse mutation in the muscle AChR (epsilonG57Q) increases 7-fold the efficacy of morantel. The mutations do not affect activation by ACh or oxantel, indicating that this position is selective for morantel. In silico studies show that the tetrahydropyrimidinyl group, common to both drugs, is close to W149 of the principal face of the binding site, whereas the other cyclic group is proximal to Q57 of the complementary face in morantel but not in oxantel. Thus, position 57 at the complementary face is a key determinant of the high selectivity of morantel for alpha7. These results provide new information for further progress in drug design.

Author affiliation: Bartos, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina

Author affiliation: Price, Kerry L.. University of Cambridge. Chemical Laboratory; Reino Unido

Author affiliation: Lummis, Sarah C.R.. University of Cambridge. Chemical Laboratory; Reino Unido

Author affiliation: Bouzat, Cecilia Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina