Derivados oxidados del tiol de la albúmina humana

La albúmina sérica humana (HSA), es una proteína monomérica compuesta por tres dominios, conocidos como dominios I, II y III. Presenta en su estructura 17 puentes disulfuro y una única cisteína libre (Cys34) que se localiza en el dominio I (DomI). La HSA es la proteína mayoritaria del plasma y su tiol se encuentra principalmente reducido (HSA-SH), constituyendo el 80% de los tioles reducidos en el compartimiento intravascular. Una fracción menor se encuentra formando disulfuros con tioles de bajo peso molecular (HSA-SSR) u oxidado a ácidos sulfínico (HSA-SO2H) y sulfónico (HSA-SO3H). En esta tesis se profundizó en el análisis de las isoformas de la HSA en plasma mediante cromatoenfoque y se trabajó en la expresión, purificación y caracterización del dominio I (DomI) de la HSA producido en Pichia pastoris. En cuanto al cromatoenfoque, se realizó una puesta a punto del análisis de muestras de plasma de donantes sanos mediante controles de precipitación de HSA en el amortiguador de trabajo y estableciendo la cantidad de HSA a inyectar para obtener una buena resolución. El cromatoenfoque es reproducible y puede utilizarse tal como se puso a punto, para estudiar el aumento de las fracciones oxidadas de la HSA de personas con sepsis. Por otra parte, se expresó el DomI en P. pastoris a partir de dos construcciones. Una conteniendo dos mutaciones sinónimas en su secuencia (DomIMS) y otra generada a partir de la secuencia de referencia de la HSA (DomISR). Ambos DomI se expresaron y secretaron correctamente hacia el medio de cultivo, y mostraron la presencia de un pigmento marrón. Respecto al DomIMS, la eliminación de la mayor parte del pigmento se logró con una cromatografía de intercambio catiónico a pH 5.0 y elución con NaCl 1 M. La reducción selectiva de dicha cisteína se logró por incubación con 2-mercaptoetanol, como se vio por reacción con 5,5-ditiobis(2-nitrobenzoato) (DTNB) y espectrometría de masa. Además se determinó la constante de reacción de la Cys34 de DomI con DTNB. El valor de 229 M-1 s-1 resultó 16 veces mayor que el de la HSA deslipidada, en iguales condiciones experimentales. En relación al DomISR, se transformaron células de la cepa GS115 His- Mut+, se seleccionaron primero transformantes, y luego transformantes multicopia. Se prepararon crioinóculos que expresaron el DomISR correctamente. En un ensayo preliminar se vio que la Cys34 del DomISR se reduce en iguales condiciones que la del DomIMS y reacciona con DTNB con una constante similar, sugiriendo que no existen diferencias estructurales entre ambo

Expression, purification and initial characterization of human serum albumin domain I and its cysteine 34

Human serum albumin presents in its primary structure only one free cysteine (Cys34) which constitutes the most abundant thiol of plasma. An antioxidant role can be attributed to this thiol, which is located in domain I of the protein. Herein we expressed domain I as a secretion protein using the yeast Pichia pastoris. In the initial step of ammonium sulfate precipitation, a brown pigment co-precipitated with domain I. Three chromatographic methods were evaluated, aiming to purify domain I from the pigment and other contaminants. Purification was achieved by cation exchange chromatography. The protein behaved as a noncovalent dimer. The primary sequence of domain I and the possibility of reducing Cys34 to the thiol state while avoiding the reduction of internal disulfides were confirmed by mass spectrometry. The reactivity of the thiol towards the disulfide 5,5´-dithiobis(2-nitrobenzoate) was studied and compared to that of full-length albumin. A ~24-fold increase in the rate constant was observed for domain I with respect to the entire protein. These results open the door to further characterization of the Cys34 thiol and its oxidized derivatives

Methodik

Die vegetationskundliche und strukturelle Zuordnung der Lebensraumtypen erfolgt nach der vorrangig von Braun-Blanquet entwickelten Vegetationsklassifizierung, einer hierarchischen Gliederung der Vegetationstypen (Syntaxonomie), die die Ebenen der Assoziation, des Verbandes, der Ordnung und der Klasse umfasst. Hierbei ist die Assoziation die grundlegende Einheit, in der die Pflanzengesellschaften zusammengefasst werden, die sich durch gleiche charakteristische Arten(gruppen)kombinationen auszeichnen. Der Verband vereinigt ähnliche Assoziationen. Das sind bereits umfassendere, jedoch standörtlich noch recht einheitliche Vegetationseinheiten. In Ordnungen werden ähnliche Verbände zusammengefasst. Die Klasse vereinigt ähnliche Ordnungen

Expression, purification and initial characterization of human serum albumin domain I and its cysteine 34.

Human serum albumin presents in its primary structure only one free cysteine (Cys34) which constitutes the most abundant thiol of plasma. An antioxidant role can be attributed to this thiol, which is located in domain I of the protein. Herein we expressed domain I as a secretion protein using the yeast Pichia pastoris. In the initial step of ammonium sulfate precipitation, a brown pigment co-precipitated with domain I. Three chromatographic methods were evaluated, aiming to purify domain I from the pigment and other contaminants. Purification was achieved by cation exchange chromatography. The protein behaved as a non-covalent dimer. The primary sequence of domain I and the possibility of reducing Cys34 to the thiol state while avoiding the reduction of internal disulfides were confirmed by mass spectrometry. The reactivity of the thiol towards the disulfide 5,5´-dithiobis(2-nitrobenzoate) was studied and compared to that of full-length albumin. A ~24-fold increase in the rate constant was observed for domain I with respect to the entire protein. These results open the door to further characterization of the Cys34 thiol and its oxidized derivatives

Applicazioni del modello APEX per la simulazione dei sistemi colturali della collina centro italiana

I modelli matematici di simulazione opportunamente calibrati e validati, possono rappresentare strumenti utili per la pianificazione e la gestione dei sistemi colturali rispetto alla più dispendiosa sperimentazione di campo (Yu et al., 2006) che comunque è imprescindibile per una corretta interpretazione delle simulazioni (Bouma et al., 2008). Il modello Agricultural Policy/environmental EXtender (APEX) è stato sviluppato per applicazioni a scala di microbacino (Williams and Izaurralde, 2006). APEX estende le capacità di EPIC (Environmental Policy Integrated Climate model) (Williams et al., 1989) su più ampia scala offrendo la possibilità di suddividere il microbacino in subaree omogenee dal punto di vista pedoclimatico e di utilizzo del suolo così da simularne il deflusso superficiale e sottosuperficiale, il trasporto di nutrienti, del sedimento e dei pesticidi dalle subaree all’outlet di bacino. Principalmente APEX è stato impiegato per la gestione dei reflui zootecnici al fine di preservare la qualità delle risorse idriche in Texas (Flowers et al., 1996) e per modellizzare l’impatto della gestione del suolo sul deflusso superficiale, l’erosione del suolo, la produttività delle colture e la dinamica del carbonio nel suolo (Wang et al., 2008). Il presente lavoro rientra in un progetto di ricerca finanziato dal Ministero delle Politiche Agricole Alimentari e Forestali - MiPAAF dal titolo: "Scenari di adattamento dell'agricoltura italiana ai cambiamenti climatici (AGROSCENARI)". Il progetto mira ad individuare, valutandone la sostenibilità, le modalità di adattamento ai cambiamenti climatici dei principali sistemi produttivi agricoli italiani per valutare l’effetto di differenti scenari climatici sulla produttività e l’impatto ambientale dei sistemi colturali. In particolare la ricerca descritta si pone l’obiettivo di valutare la capacità di APEX di simulare i sistemi colturali cerealicolo-industriali centro italiani al fine di identificare strategie gestionali che consentano di ridurne l’impatto ambientale anche in relazione ad ipotetici scenari di cambiamento climatico

APEX model applications for the mediterranean cropping systems simulation

The Agricultural Policy/Environmental eXtender model (APEX) extends the EPIC (Environmental Policy Integrated Climate model) capabilities for applications at the microcatchment scale. The research described aims to calibrate APEX to simulate industrial-cereal cropping systems in order to identify management strategies capable of reducing the environmental impact in relation to hypothetical scenarios of climate change and to develop mitigation strategies that allow for direct agricultural activities towards sustainable production. Good correlations between observed and simulated values were obtained for crop productivity as shown by the low Root Mean Square Error (RMSE) obtained. APEX provided valuable results on the runoff quantification, in particular for the higher levels. In the analyzed context APEX can be a useful tool for environmental planning and implementation of sustainable cropping systems

Target-related coupling in bimanual reaching movements

Weigelt M, Rieger M, Mechsner F, Prinz W. Target-related coupling in bimanual reaching movements. Psychological Research. 2007;71(4):438-447