Activation in the family of Candida rugosa isolipases by polyethylene glycol

Disponible en: http://www.xtal.iqfr.csic.es/publications/jmolcat2005.pdfWe have investigated activation of two isoenzymes (lip1 and lip3) from Candida rugosa in polyethylene glycol (PEG) media. Aqueous solutions of PEG 8000 and 20,000 activate lip3 but not lip1 from C. rugosa. Maximum activation (260%) of lip3 requires 6 h of pre-incubation with PEG 8000 (4%, w/v). PEG seems to shift the equilibrium between the open and the closed forms of lip3 towards the active conformation. Inhibition experiments demonstrate that ligands have easier access to the lip3 active site than to the lip1 active site, both in the presence and the absence of PEG. The presence of PEG in the crystallization medium is responsible for reported differences in the crystal structures of lip1 and lip3. A comparative analysis of crystallographic models of lip1 and lip3 suggests a role for PEG in activation of lip3 and further stabilization of the activated/open form via dimerization in aqueous media.This work has been financed by the Spanish CICYT (PB92/0495 and BIO 96/0837). A predoctoral fellowship for M.F.P. was provided by the Autonomous Community of Madrid (Spain).Peer reviewe

Structural and Phylogenetic Analysis of Rhodobacter capsulatus NifF: Uncovering General Features of Nitrogen-fixation (nif)-Flavodoxins

Analysis of the crystal structure of NifF from Rhodobacter capsulatus and its homologues reported so far reflects the existence of unique structural features in nif flavodoxins: a leucine at the re face of the isoalloxazine, an eight-residue insertion at the C-terminus of the 50’s loop and a remarkable difference in the electrostatic potential surface with respect to non-nif flavodoxins. A phylogenetic study on 64 sequences from 52 bacterial species revealed four clusters, including different functional prototypes, correlating the previously defined as “short-chain” with the firmicutes flavodoxins and the “long-chain” with gram-negative species. The comparison of Rhodobacter NifF structure with other bacterial flavodoxin prototypes discloses the concurrence of specific features of these functional electron donors to nitrogenase.Fil: Perez Dorado, Inmaculada. Consejo Superior de Investigaciones Cientificas; EspañaFil: Bortolotti, Ana. 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; ArgentinaFil: 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; ArgentinaFil: Hermoso, Juan A.. Consejo Superior de Investigaciones Cientificas; Españ

La hidroxiapatita asociada al sulfato cálcico como material de substitución ósea. Aspectos biológicos.

La cirugía ortopédica y máxilo-facial necesitan realizar en muchas ocasiones resecciones masivas de tejido óseo. Esto ha hecho que se haya propuesto la utilización de materiales inertes como substitutos óseos, gracias a la habilidad que tienen de permitir la regeneración del hueso, tanto en el campo de la medicina como en el de la odontología. Nuestro trabajo tiene por objetivo estudiar un biomaterial compuesto de hidroxiapatita y sulfato cálcico como material de substitución ósea en forma de cilindros. Se han intervenido 75 animales de experimentación (conejo Albino de Nueva Zelanda), divididos en tres grupos, cada uno de ellos compuesto por 25 animales. En el grupo I o control, los animales fueron intervenidos quirúrgicamente realizándoseles una cavidad a nivel metafisodiafisario en el fémur sin realizar implantes. A los animales del grupo II y III se les realizó la misma intervención, pero se les implantó un cilindro de hidroxiapatita (HA) y sulfato cálcico a dos concentraciones diferentes, 50% - 50% y 70% - 30% respectivamente. El estudio ha demostrado una regeneración ósea de la cavidad rellenada a expensas de la rápida reabsorción del yeso, y de una lenta reabsorción de la hidroxiapatita.Peer Reviewe

Estudio epizootiológico de las dermatofitosis en perros y gatos

Se ha investigado la etiología de 141 casos de dermatitis en las especies canina (121 casos) y felina (17 casos). Mediante procedimientos dermatológicos de rutina, se comprobó la etiología micótica de veintisiete de ellos, encontrando una incidencia superior en gatos (58,82%) que en perros (13,71%). Las formas clínicas implicadas fueron: anular (60%), fávica (24%), kerion (12%) y onicomicosis (4%), no presentando una zona corporal específica de localización.A study was made of the aetiology of 141 cases of dermatitis in dogs (121 cases) and cats (17 cases). Rutine dermatological procedures revealed a mycotic aetiology in 27 animals, though it was more prevalent in cats (58,82%) than in dogs (13, 71%). Tbe clinicalforms involved uiere. annular (60%), favic (24%), kerion (12%) and onicomycosis (4%). No specific body site in the body was determined

A complement to the modern crystallographer's toolbox: Caged gadolinium complexes with versatile binding modes

A set of seven caged gadolinium complexes were used as vectors for introducing the chelated Gd3+ ion into protein crystals in order to provide strong anomalous scattering for de novo phasing. The complexes contained multidentate ligand molecules with different functional groups to provide a panel of possible interactions with the protein. An exhaustive crystallographic analysis showed them to be nondisruptive to the diffraction quality of the prepared derivative crystals, and as many as 50% of the derivatives allowed the determination of accurate phases, leading to high-quality experimental electron-density maps. At least two successful derivatives were identified for all tested proteins. Structure refinement showed that the complexes bind to the protein surface or solvent-accessible cavities, involving hydrogen bonds, electrostatic and CH-π interactions, explaining their versatile binding modes. Their high phasing power, complementary binding modes and ease of use make them highly suitable as a heavy-atom screen for high-throughput de novo structure determination, in combination with the SAD method. They can also provide a reliable tool for the development of new methods such as serial femtosecond crystallography. © 2014 International Union of Crystallography.Peer Reviewe

Structural insights into the synthesis of FMN in prokaryotic organisms

Riboflavin kinases (RFKs) catalyse the phosphorylation of riboflavin to produce FMN. In most bacteria this activity is catalysed by the C-terminal module of a bifunctional enzyme, FAD synthetase (FADS), which also catalyses the transformation of FMN into FAD through its N-terminal FMN adenylyl transferase (FMNAT) module. The RFK module of FADS is a homologue of eukaryotic monofunctional RFKs, while the FMNAT module lacks homologyto eukaryotic enzymes involved in FAD production. Previously, the crystal structure of Corynebacterium ammoniagenes FADS (CaFADS) was determined in its apo form. This structure predicted a dimer-of-trimers organization with the catalytic sites of two modules of neighbouring protomers approaching each other, leading to a hypothesis about the possibility of FMN channelling in the oligomeric protein. Here, two crystal structures of the individually expressed RFK module of CaFADS in complex with the products of the reaction, FMN and ADP, are presented. Structures are complemented with computational simulations, binding studies and kinetic characterization. Binding of ligands triggers dramatic structural changes in the RFK module, which affect large portions of the protein. Substrate inhibition and molecular-dynamics simulations allowed the conformational changes that take place along the RFK catalytic cycle to be established. The influence of these conformational changes in the FMNAT module is also discussed in the context of the full-length CaFADS protomer and the quaternary organization.This work has been supported by MINECO, Spain (BIO2013-42978-P to MM and BFU2014-59389-P to JAH), the Aragonian Government-FEDER (B18), Autonomous Community of Madrid (S2010/BMD-2457), Departamento Administrativo de Ciencia, Tecnología e Innovación (COLCIENCIAS) and Universidad Industrial de Santander (project 1818 to IL).Peer Reviewe

Disulfide Engineered Lipase to Enhance the Catalytic Activity: A Structure-Based Approach on BTL2

Enhancement, control, and tuning of hydrolytic activity and specificity of lipases are major goals for the industry. Thermoalkaliphilic lipases from the I.5 family, with their native advantages such as high thermostability and tolerance to alkaline pHs, are a target for biotechnological applications. Although several strategies have been applied to increase lipases activity, the enhancement through protein engineering without compromising other capabilities is still elusive. Lipases from the I.5 family suffer a unique and delicate double lid restructuration to transition from a closed and inactive state to their open and enzymatically active conformation. In order to increase the activity of the wild type Geobacillus thermocatenulatus lipase 2 (BTL2) we rationally designed, based on its tridimensional structure, a mutant (ccBTL2) capable of forming a disulfide bond to lock the open state. ccBTL2 was generated replacing A191 and F206 to cysteine residues while both wild type C64 and C295 were mutated to serine. A covalently immobilized ccBTL2 showed a 3.5-fold increment in esterase activity with 0.1% Triton X-100 (2336 IU mg-1) and up to 6.0-fold higher with 0.01% CTAB (778 IU mg-1), both in the presence of oxidizing sulfhydryl agents, when compared to BTL2. The remarkable and industrially desired features of BTL2 such as optimal alkaliphilic pH and high thermal stability were not affected. The designed disulfide bond also conferred reversibility to the enhancement, as the increment on activity observed for ccBTL2 was controlled by redox pretreatments. MD simulations suggested that the most stable conformation for ccBTL2 (with the disulfide bond formed) was, as we predicted, similar to the open and active conformation of this lipase.Financial and logistic support from Colombian Universidad del Valle and COLCIENCIAS (CI 71083-Grant 745-2016-Project 110671250425), Spanish CICYT project BIO-2005-6018576, BFU2017-90030-P, and BFU2011-25326, B. Di G. In addition, thanks to the Spanish MINECO for a FPU fellowship.S

Mejora del Tratamiento de las Personas con Enfermedad Mental en las Universidades Públicas

El presente estudio, publicado por el Real Patronato sobre Discapacidad en colaboración con la Fundación ONCE, indaga en la situación de las personas con enfermedad mental en las universidades públicas españolas. La obra parte de una conceptualización de la enfermedad mental y la discapacidad para enmarcar el trabajo y la metodología empleada. Esta metodología combina tanto el uso de fuentes documentales y estadísticas como la realización de varias encuestas a cuatro tipos de destinatarios distintos: personas con enfermedad mental que han cursado estudios universitarios, personas con enfermedad mental que no lo han hecho, técnicos de asociaciones y técnicos de universidades. Del análisis se desprende una serie de conclusiones que los autores clasifican en dos dimensiones: una sustantiva y otra operativa, a partir de las cuales exponen las recomendaciones y propuestas más adecuadas para garantizar y mejorar el acceso de las personas con enfermedad mental a los estudios universitarios

Combined femoral and tibial component total knee arthroplasty device rotation measurement is reliable and predicts clinical outcome

Background The optimal total knee arthroplasty (TKA) rotational alignment and how best to obtain and measure it are debatable. The aim was to analyse the reliability of the Berger femoral, three different tibial and four different com- bined two-dimensional computer tomography (2D-CT) TKA component rotation measurements, and to ascertain which rotational values best predict a successful clinical outcome. Methods The 2D-CT scans were obtained post-operatively on 60 patients who had TKA. We determined one femoral [Berger’s femoral angle (BFA)], three tibial [Berger’s tibial angle (BTA), anatomical tibial angle (ATA) and bimalleolar posterior tibial component angle (BM_PTCA)] and four combined [transepicondylar posterior tibial component angle (TE_PTCA), bicondylar posterior tibial component angle (BC_PTCA, transepicondylar bimalleolar angle (TE_BM) and bicondylar bimalleolar angle (BC_BM)] TKA rotation angles. We made all measures in 23 patients twice by three observers and determined inter- and intra-observer agreement using the Bland–Altman plot method. We analysed measures of 55 patients using the area under the ROC curve (AUC) analysis to ascertain the discriminative capacity of BFA, ATA, TE_PTCA and BC_PTCA for predicting a successful clinical outcome according to the Knee Society Score (KSS) threshold. Results ATA showed the smaller inter- and intra-observer average of differences (-0.1° and 1.6°, respectively) of the studied methods followed by BFA (-0.9° and 1.4°), TE_PTCA (-2.1° and 2.7°) and BC_PTCA (-0.5° and 1.8°). BFA (-4° to 2.1° and -6.1° to 8.8°) and BC_PTCA (-4.4° to 3.4° and -7.9° to 4.4°) showed the narrower inter- and intra- observer limits of agreement. A TKA device rotation (BC_PTCA) < 0.8° of external rotation (ER) predicted a KSS and KSS knee successful outcome, and < 3.8° ER for KSS functional (AUC = 0.889; 0.907 and 0.764, respectively). BFA and ATA < 0.9° ER and < 3.9° internal rotation (IR) predicted a successful KSS knee outcome (AUC = 0.796 and 0.889, respectively). Conclusion The ATA tibial component rotation measurement was the most reliable of those studied. BFA, TE_PTCA and BC_PTCA were reliable measures for TKA femoral and combined rotation. The presence of a minimal rotation between the TKA components (BC_PTCA) and a small femoral ER or tibial IR predicted a successful KSS outcome.Peer ReviewedPostprint (published version

Different femoral rotation with navigated flexion-gap balanced or measured resection in total knee arthroplasty does not lead to different clinical outcomes

PURPOSE: Femoral rotation in total knee arthroplasty (TKA) is hypothesized to vary in the same knee depending on the method used to establish it. METHODS: Thirty-eight patients who underwent TKA surgery using a measured resection technique (RT) were compared with 40 patients who underwent a flexion-gap balancing technique with computer-assisted (for navigation) surgery (FB-CAS) to assess clinical and radiographic alignment differences at two years postoperatively. In 36 of the 40 patients in the FB-CAS group, both methods were used. Intraoperatively, the transepicondylar femoral rotation (TEFR) in reference to the transepicondylar axis was established as the rotation that balanced the flexion gap. Once the TEFR was obtained, an analogous rotation as measured by a posterior reference femoral rotation (PRFR) cutting guide was determined. RESULTS: Femoral component rotation determined by the TEFR and PRFR methods differed in each of the knees. The median TEFR was 0.08°±0.6° (range -¿1.5°, 1.5°), and the median PRFR was 0.06°±2.8° (range -¿6°, 5°). The mean difference in the rotational alignment between the TEFR and PRFR techniques was 0.01° ± 3.1°. The 95% limits of agreement between the mean differences in measurements were between 6.2° external rotation and -¿6.1° internal rotation. At 2 years postoperatively, we found no differences in the radiographic or clinical American Knee Society score between the two groups. CONCLUSION: Rotation of the femoral component in TKA can vary in the same knee depending on the surgical method used to establish it. This variation in femoral rotation is sufficiently small enough to have no apparent effect on the 2-year clinical outcome score.Postprint (author's final draft