Zaragoza, Spain
Zaragoza, Spain

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Nunez D.,Institute Carboquimica ICB | Nunez D.,Aragon Health Research Institute IIS Aragon | Domingo M.P.,Institute Carboquimica ICB | Domingo M.P.,Aragon Health Research Institute IIS Aragon | And 11 more authors.
Process Biochemistry | Year: 2013

The interaction of the adhesion molecule of the immunoglobulin family intercellular adhesion molecule 1 (ICAM-1) with its ligands such that the integrins LFA-1 and Mac-1 is crucial for the regulation of several physiological and pathophysiological processes like cell mediated-elimination of tumor or virus infected cells, cancer metastasis or inflammatory autoimmune processes. Thus, production of milligrams of protein is required to perform structural and functional studies as well as design novel approaches to find out new inhibitors of ICAM-1/LFA-1 interaction. Here we report on the production of a recombinant human ICAM-1 chimera comprising the first two extracellular domains of ICAM-1 linked to the Fc fraction of a human IgG1. To this aim we have used a cost-effective method based on the expression of a His-tagged protein in Escherichia coli followed by a single step of refolding and purification on immobilized metal affinity columns. This method is able to produce 3 mg/l of bacterial culture in just 72 h with purity greater than 95%. The identity and the native structure of refolded human ICAM-1 chimera were confirmed by biochemical and biophysical studies including SDS-electrophoresis, immunoblot, circular dichroism, isothermal titration calorimetry and fluorescence spectroscopy. Native folding and functional activity of the chimera were further confirmed by different cell biology studies, including B cell adhesion, T cell binding and inhibition of NK cell function. These studies indicate a high biological activity of the protein since it induces a 200-fold increase/mg of protein in B cell adhesion and the inhibitory dose 50 to block cell-mediated cytotoxicity is 10 pg/effector cell. These analyses show that our protocol is able to produce a recombinant human ICAM-1 chimera fully active and useful to analyze the biological processes in which ICAM-1/LFA-1 interaction is critically involved. © 2013 Published by Elsevier Ltd. All rights reserved.


Delgado-Camon A.,Institute Carboquimica ICB | Jarne C.,Institute Carboquimica ICB | Cebolla V.L.,Institute Carboquimica ICB | Larranaga O.,Donostia International Physics Center | And 8 more authors.
Tetrahedron | Year: 2015

Abstract Berberrubine has been synthesized by microwave assisted selective demethylation of berberine. The high selectivity observed in this reaction has been explained and justified by means of computational calculations using Density Functional Theory (DFT) and Natural Resonance Theory (NRT). The existence of two resonant structures of berberrubine is the driving force of regioselective demethylation. Berberrubine is a chemosensor of alkanes, and may have practical applications in petrochemical analysis as a 'mass' detector because fluorescent response of saturated hydrocarbons does not depend on hydrocarbon chain length. Berberrubine operates via dipole-induced dipole interactions. Likewise, it has two fluorescent forms in acidic and basic media, which correspond to a keto-enol tautomerism. The fluorescent signal for berberrubine and the amplification of berberrubine-alkane signals by heating can be rationalized from the predominance of enol form when berberrubine is adsorbed onto silica gel. © 2015 Elsevier Ltd.


Sanz-Vicente I.,University of Zaragoza | Lapieza M.P.,University of Zaragoza | Lapieza M.P.,Institute Carboquimica ICB | Cebolla V.L.,Institute Carboquimica ICB | Galban J.,University of Zaragoza
Microchemical Journal | Year: 2015

In this paper we present a rapid method for determining phosphorylcholine (ChoP). ChoP was first hydrolysed by the enzyme alkaline phosphatase (AP); the choline formed was then submitted to a reaction with Choline Oxidase (ChOx). Both reactions were carried out simultaneously, in the same test without previous steps of incubation. The analytical signals used were the intrinsic fluorescence of ChOx due to FAD and that corresponding to a fluorescein derivative bonded to ChOx (ChOx-FS); both can be related with the concentration of ChoP. Once the conditions were optimized, the response range for ChoP was 5.2×10-7-1.0×10-5M using the peak area as the analytical parameter with a precision of about 4% (RSD) at both ChOx and ChOx-FS wavelengths.In the experimental conditions found, it was also possible to determine free choline (Ch) with figures of merit similar to those obtained for ChoP. These results have made possible the sequential determination of Ch and ChoP in milk powder, using only one aliquot of the mixture, with good results. The recoveries obtained for both analytes were close to 100 %. The method is rapid because an incubation step is not necessary. Moreover, the enzymatic reaction is autoindicating and thus the additional detection step required by other published enzymatic methods is avoided. © 2015 Elsevier B.V.


Jarne C.,Institute Carboquimica ICB | Cebolla V.L.,Institute Carboquimica ICB | Membrado L.,Institute Carboquimica ICB | Le Mapihan K.,Total S.A. | Giusti P.,Total S.A.
Energy and Fuels | Year: 2011

Automated multiple development (AMD) is a gradient elution technique for high-performance thin-layer chromatography (HPTLC), in which successive runs are performed with decreasing solvent strength and increasing developing distance. A method for obtaining separations or distributions of heavy petroleum products according to their number of aromatic rings has been developed using AMD together with caffeine-impregnated silica gel HPTLC layers and selective detection by fluorescence scanning densitometry. A gradient of dichloromethane-heptane, starting from a composition of 70:30 (v/v) to 65:35 (v/v), in 34 elution steps with a migration of 2 mm per step, over a total migration distance of 68 mm, has been used. Calibration of elution has been carried out using polycyclic aromatic compounds (PACs) with a molecular weight (MW) from 200 to 1000 atomic mass units (amu), which have been separated under the described conditions. Other parameters on AMD separation have also been discussed, such as the effect of mobile and stationary phases. The application of this technique to different heavy petroleum products is discussed. © 2011 American Chemical Society.


Adanez-Rubio I.,Institute Carboquimica ICB | Gayan P.,Institute Carboquimica ICB | Abad A.,Institute Carboquimica ICB | De Diego L.F.,Institute Carboquimica ICB | And 2 more authors.
Energy and Fuels | Year: 2012

The chemical looping with oxygen uncoupling (CLOU) process is a chemical looping combustion (CLC) technology that allows for the combustion of solid fuels with inherent CO 2 separation. As in CLC technology, in the CLOU process, the oxygen necessary for fuel combustion is supplied by a solid oxygen carrier, which is moving between two reactors: the fuel and air reactors. The CLOU technology uses the property of some metal oxides (CuO, Mn 2O 3, and Co 3O 4), which can generate gaseous oxygen at high temperatures. The oxygen generated by the oxygen carrier reacts directly with the solid fuel, which is mixed with the oxygen carrier in the fuel reactor. The reduced oxygen carrier is transported to the air reactor, where it is oxidized by air. In this work, a material prepared by spray drying containing 60 wt % CuO and 40 wt % MgAl 2O 4 as supporting material was evaluated as an oxygen carrier for the CLOU process using different installations. First, the oxygen release rate and the fluidization behavior, with regard to the agglomeration and attrition rate, were analyzed in a thermogravimetric analyzer (TGA) and in a batch fluidized bed, respectively. Then, the effects of the main operating conditions, such as the temperature, solids flow rate, and gas velocity in the fuel reactor, on the oxygen-carrier capability to release gaseous oxygen were analyzed in a continuous CLOU unit using N 2 and CO 2 as fluidization media. In addition, the effect of the oxygen concentration in the air reactor on the capability of the oxygen carrier to be regenerated was evaluated. The results obtained showed that this oxygen carrier has suitable characteristics for the CLOU process. Nevertheless, after 40 h of continuous operation at high temperatures, the particle integrity decreased significatively, indicating the need to improve the lifetime of this kind of material for use in an industrial CLOU process. © 2012 American Chemical Society.

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