Celiac Disease: Immunochemical and Diagnosis Aspects

Fil: Aleanzi, Mabel Cristina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina.La enfermedad celíaca se caracteriza por daño a la mucosa de intestino delgado y mala absorción de nutrientes, en individuos genéticamente susceptibles, en respuesta a la ingestión de ciertas proteínas presentes en gluten de trigo y cereales relacionados (cebada, centeno y avena). La susceptibilidad genética está asociada a los alelos que codifican para el heterodímero HLAII DQ2/ DQ8. Los objetivos principales de esta tesis fueron desarrollar herramientas de diagnóstico inmunoquímico sensibles y específicas tanto para la detección de gliadinas en alimentos destinados a celíacos, como para el diagnóstico de la patología. Hasta 1997 los marcadores serológicos eran los anticuerpos IgA e IgG anti-gliadinas y los autoanticuerpos antiendomisio. A partir de esa fecha, la identificación de la enzima transglutaminasa de tejido como el blanco principal de los anticuerpos antiendomisio, condujo a un gran avance en el conocimiento de la patología a nivel molecular. Fue posible vincular la predisposición genética con la acción de esta enzima sobre las gliadinas y el desorden inmunológico. Sobre esa base desarrollamos ensayos de ELISA para detectar anticuerpos, utilizando alternativamente como antígenos de captura transglutaminasa o péptidos de gliadina, en sus secuencias nativas y selectivamente deamidadas. Evaluados sobre un panel de sueros de individuos sanos y celíacos, los tres antígenos resultaron específicos y sensibles. Consideramos que la especificidad de la respuesta de los anticuerpos de enfermos celíacos frente los péptidos de gliadina selectivamente deamidados, es el hallazgo más relevante de este trabajo, para su aplicación al diagnóstico serológico de la patología.Celiac disease is characterized by small-intestinal mucosal injury and nutrient malabsorption in genetically susceptible individuals in response to the dietary ingestion of wheat gluten and similar proteins in barley, rye and oat. Genetic susceptibility is associated with alleles encoding HLA II heterodimers DQ2 / DQ8. The main objectives of this thesis were to develop sensitive and specific immunochemical tools, both for gliadin detection in foods destined to celiac patients, and also for the diagnosis of the pathology. Until 1997 main serological markers were IgA and IgG anti-gliadin antibodies and anti-endomysial auto-antibodies. Since that date, identification of the enzyme tissue transglutaminase as the main antigen target of antiendomysial antibodies, achieved to a great advance in the knowledge of the pathology at the molecular level. It was possible to correlate the genetic predisposition with the action of the enzyme on the gliadins and the immunological disorder. On this base we developed ELISA assays to detect antibodies, using alternatively as capture antigens, tissue transglutaminase or gliadin peptides, in their native and selectively deamidated sequences. Evaluated over a panel of healthy and celiac individual the three antigens resulted specific and sensitive .We consider that the specificity of the response of celiac patients antibodies against the selectively deamidated peptides, is the most relevant finding of this work, in order to be applied to the laboratory diagnosis of the pathology.Universidad Nacional del Litora

Plastidic Phosphoglycerate Kinase from Phaeodactylum tricornutum: On the Critical Role of Cysteine Residues for the Enzyme Function

Chloroplastidic phosphoglycerate kinase (PGKase) plays a key role in photosynthetic organisms, catalyzing a key step in the Calvin cycle. We performed the molecular cloning of the gene encoding chloroplastidic PGKase-1 in the diatom Phaeodactylum tricornutum. The recombinant enzyme was expressed in Escherichia coli, purified and characterized. Afterward, it showed similar kinetic properties than the enzyme studied from other organisms, although the diatom enzyme displayed distinctive responses to sulfhydryl reagents. The activity of the enzyme was found to be dependent on the redox status in the environment, determined by different compounds, including some of physiological function. Treatment with oxidant agents, such as diamide, hydrogen peroxide, glutathione and sodium nitroprusside resulted in enzyme inhibition. Recovery of activity was possible by subsequent incubation with reducing reagents such as dithiothreitol and thioredoxins (from E. coli and P. tricornutum). We determined two midpoint potentials of different regulatory redox centers, both values indicating that PGKase-1 might be sensitive to changes in the intracellular redox environment. The role of all the six Cys residues found in the diatom enzyme was analyzed by molecular modeling and site-directed mutagenesis. Results suggest key regulatory properties for P. tricornutum PGKase-1, which could be relevant for the functioning of photosynthetic carbon metabolism in diatoms.Fil: Bosco, Maria Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Aleanzi, Mabel Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentin

UDPglucose pyrophosphorylase from Xanthomonas spp. Characterization of the enzyme kinetics, structure and inactivation related to oligomeric dissociation

The genes encoding for UDPglucose pyrophosphorylase in two Xanthomonas spp. were cloned and overexpressed in Escherichia coli. After purification to electrophoretic homogeneity, the recombinant proteins were characterized, and both exhibited similar structural and kinetic properties. They were identified as dimeric proteins of molecular mass 60 kDa, exhibiting relatively high specific activity (∼80 Units/mg) for UDPglucose synthesis. Both enzymes utilized UTP or TTP as substrate with similar affinity. The purified Xanthomonas enzyme was inactivated after dilution into the assay medium. Studies of crosslinking with the bifunctional lysyl reagent bisuberate suggest that inactivation occurs by enzyme dissociation to monomers. UTP effectively protects the enzyme against inactivation, from which a dissociation constant of 15 μM was calculated for the interaction substrate-enzyme. The UTP binding to the enzyme would induce conformational changes in the protein, favoring the subunits interaction to form an active dimer. This view was reinforced by protein modeling of the Xanthomonas enzyme on the basis of the prokaryotic UDPglucose pyrophosphorylase crystallographic structure. The in silico approach pointed out two main critical regions in the enzyme involved in subunit-subunit interaction: the region surrounding the catalytic-substrate binding site and the C-term. © 2008 Elsevier Masson SAS. All rights reserved.Fil: Bosco, Maria Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Machtey, Matías. Universidad Nacional del Litoral; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Aleanzi, Mabel Cristina. Universidad Nacional del Litoral; Argentin

Biocombustibles y biorrefinerías: una asociación entre la biotecnología y la ingeniería química - II. Glicerol: Valorización por vía química, biológica y por enzimas

En los albores del siglo XX, la economía mundial estaba basada principalmente en recursos renovables. Los alimentos y demás bienes se obtenían mayoritariamente de la biomasa. Eranrelativamente buenos tiempos para la Argentina, ya que, en este escenario, su fortaleza se sustentaba en la producción de carnes y granos. Al avanzar el siglo, la economía mundial cambió completamente de eje: el carbón y el petróleo se convirtieron en los recursos primordiales, y la obtención de bienes pasó a estar centrada en estos recursos no renovables(o, estrictamente, de renovación muy lenta).Fil: Beccaria, Alejandro José. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; ArgentinaFil: Guerrero, Sergio Adrian. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; ArgentinaFil: Aleanzi, Mabel Cristina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; ArgentinaFil: Comelli, Raul Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Investigaciones en Catálisis y Petroquímica "Ing. José Miguel Parera". Universidad Nacional del Litoral. Instituto de Investigaciones en Catálisis y Petroquímica "Ing. José Miguel Parera"; ArgentinaFil: Iglesias, Alberto Alvaro. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentin

A colorimetric method for the assay of ADP-glucose pyrophosphorylase

The purpose of the current work was to develop a relatively simple method to assay ADPGlcPPase activity having high sensitivity, accuracy, and reliability. We sought a procedure allowing the assay in both of the reaction directions, based on a technique suitable for the screening of numerous samples. The method quantifies inorganic orthophosphate released from the specific hydrolysis of the enzyme activity products. For ADPGlc synthesis, the method measures Pi after hydrolysis of PPi by inorganic pyrophosphatase. Pyrophosphorolysis is assayed by determining Pi derived from CF1-ATPase-mediated hydrolysis of ATP. Pi dosage is performed by the technique based in the formation of a phosphomolybdate?Malachite Green complex [14,15]. We optimized the procedure to a microscale grade, reaching convenient sensitivity and the possibility of automation, by using a microplate (multiwell) absorbance readerFil: Fusari, Corina. Universidad Nacional del Litoral; ArgentinaFil: Demonte, Ana María Magdalena. Universidad Nacional del Litoral; ArgentinaFil: Figueroa, Carlos Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Aleanzi, Mabel Cristina. Universidad Nacional del Litoral; ArgentinaFil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentin

Insights into Glycogen Metabolism in Chemolithoautotrophic Bacteria from Distinctive Kinetic and Regulatory Properties of ADP-Glucose Pyrophosphorylase from Nitrosomonas europaea

Nitrosomonas europaea is a chemolithoautotroph that obtains energy by oxidizing ammonia in the presence of oxygen and fixes CO2 via the Benson-Calvin cycle. Despite its environmental and evolutionary importance, very little is known about the regulation and metabolism of glycogen, a source of carbon and energy storage. Here, we cloned and heterologously expressed the genes coding for two major putative enzymes of the glycogen synthetic pathway in N. europaea, ADP-glucose pyrophosphorylase and glycogen synthase. In other bacteria, ADP-glucose pyrophosphorylase catalyzes the regulatory step of the synthetic pathway and glycogen synthase elongates the polymer. In starch synthesis in plants, homologous enzymes play similar roles. We purified to homogeneity the recombinant ADP-glucose pyrophosphorylase from N. europaea and characterized its kinetic, regulatory, and oligomeric properties. The enzyme was allosterically activated by pyruvate, oxaloacetate, and phosphoenolpyruvate and inhibited by AMP. It had a broad thermal and pH stability and used different divalent metal ions as cofactors. Depending on the cofactor, the enzyme was able to accept different nucleotides and sugar phosphates as alternative substrates. However, characterization of the recombinant glycogen synthase showed that only ADP-Glc elongates the polysaccharide, indicating that ATP and glucose-1-phosphate are the physiological substrates of the ADP-glucose pyrophosphorylase. The distinctive properties with respect to selectivity for substrates and activators of the ADP-glucose pyrophosphorylase were in good agreement with the metabolic routes operating in N. europaea, indicating an evolutionary adaptation. These unique properties place the enzyme in a category of its own within the family, highlighting the unique regulation in these organisms.Fil: Machtey, Matías. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Kuhn, Misty L.. Northwestern University Feinberg School Of Medicine; . Loyola University Of Chicago;Fil: Flasch, Diane A.. University Of Michigan, Ann Arbor; . Loyola University Of Chicago;Fil: Aleanzi, Mabel Cristina. Universidad Nacional del Litoral; ArgentinaFil: Ballicora, Miguel A.. Loyola University Of Chicago;Fil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentin

Biocombustibles y Biorrefinerías: Una Asociación Entre la Biotecnología y la Ingeniería Química. Sobre microalgas y otras hierbas

Asociados al manejo del fuego por el hombre o sus ancestros inmediatos, los biocombustibles pueden definirse como aquellos materiales combustibles que son de origenbiológico. Así, se incluye la leña entre los biocombustibles de naturaleza sólida; los alcoholes obtenidos de que son de origen biológico. Si bien es claro el origen fósil de loshidrocarburos, éstos son excluidos del grupo de los biocombustibles por su remota formación. Un grupo importante de biocombustibles es producido por microorganismos.Estos procesos de producción transcurren en plantas industriales denominadas biorrefinerías, por analogía con la industria (petro) química, y se emplean diferentesmaterias primas de origen biológico. El conocido alcohol etílico es principalmente obtenidopor síntesis microbiana, empleando subproductos de la industria azucarera o almidones de maíz.Fil: Márquez, V.. Universidad Nacional del Litoral; ArgentinaFil: Beccaria, Alejandro José. Universidad Nacional del Litoral; ArgentinaFil: Aleanzi, Mabel Cristina. Universidad Nacional del Litoral; ArgentinaFil: Guerrero, Sergio Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Querini, Carlos Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Investigaciones en Catálisis y Petroquímica "Ing. José Miguel Parera". Universidad Nacional del Litoral. Instituto de Investigaciones en Catálisis y Petroquímica "Ing. José Miguel Parera"; ArgentinaFil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentin

Bi-national and interdisciplinary course in enzyme engineering

Higher education institutions and scientific funding agencies are emphasizing international projects that involve the integration and synergy between research groups, particularly if different disciplines are involved. Students with an education that reflects these trends will have more tools to succeed in the future, but it is challenging to provide this type of learning experience. Here we present the organization of a bi-national course with the goals to teach students protein structure/function relationships, which give them actual research experience in both computational and experimental laboratories, and engage them in an international networking experience. Two collaborative learning courses were organized at Loyola University Chicago (USA) and Universidad Nacional del Litoral (Argentina) for graduate and advanced undergraduate students. Multiple instructors at different stages in their careers gave lectures during the course and were able to interact with students on a one-on-one basis. Nearly every student from both institutions thoroughly enjoyed this approach, and they learned more about protein structure and gained important tools for their own research. We believe that this type of course design is applicable and transferable to other institutions and areas of science. We found that the combination of international networking and incorporation of actual research projects ignited the enthusiasm of students and instructors. Due to the success of these courses, we planned to incorporate them as regular series in our curriculum.Fil: Kuhn, Misty L.. Loyola University Chicago; Estados UnidosFil: Figueroa, Carlos Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Aleanzi, Mabel Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Olsen, Kenneth W.. Loyola University Chicago; Estados UnidosFil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Ballicora, Miguel A.. Loyola University Chicago; Estados Unido

Understanding the allosteric trigger for the fructose-1,6-bisphosphate regulation of the ADP-glucose pyrophosphorylase from Escherichia coli

ADP-glucose pyrophosphorylase is the enzyme responsible for the regulation of glycogen synthesis in bacteria. The enzyme N-terminal domain has a Rossmann-like fold with three neighbor loops facing the substrate ATP. In the Escherichia coli enzyme, one of those loops also faces the regulatory site containing Lys 39, a residue involved in binding of the allosteric activator fructose-1,6-bisphosphate and its analog pyridoxal-phosphate. The other two loops contain Trp 113 and Gln 74, respectively, which are highly conserved among all the ADP-glucose pyrophosphorylases. Molecular modeling of the E. coli enzyme showed that binding of ATP correlates with conformational changes of the latter two loops, going from an open to a closed (substrate-bound) form. Alanine mutants of Trp 113 or Gln 74 did not change apparent affinities for the substrates, but they became insensitive to activation by fructose-1,6-bisphosphate. By capillary electrophoresis we found that the mutant enzymes still bind fructose-1,6- bisphosphate, with similar affinity as the wild type enzyme. Since the mutations did not alter binding of the activator, they must have disrupted the communication between the regulatory and the substrate sites. This agrees with a regulatory mechanism where the interaction with the allosteric activator triggers conformational changes at the level of loops containing residues Trp 113 and Gln 74.Fil: Figueroa, Carlos Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina. Loyola University Chicago; Estados UnidosFil: Esper, María Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Bertolo, Ana. Cornell University; Estados UnidosFil: Demonte, Ana María Magdalena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Aleanzi, Mabel Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Ballicora, Miguel A.. Loyola University Chicago; Estados Unido