Drawbacks of dialysis procedures for removal of EDTA

9 p.-3 fig.Ethylenediaminetetraacetic acid (EDTA) is a chelating agent commonly used in protein purification, both to eliminate contaminating divalent cations and to inhibit protease activity. For a number of subsequent applications EDTA needs to be exhaustively removed. Most purification methods rely in extensive dialysis and/or gel filtration in order to exchange or remove protein buffer components, including metal chelators. We report here that dialysis protocols,even as extensive as those typically employed for protein refolding, may not effectively remove EDTA, which is reduced only by approximately two-fold and it also persists after spin-column gel filtration, as determined by NMR and by colorimetric methods. Remarkably, the most efficient removal was achieved by ultrafiltration, after which EDTA became virtually undetectable. These results highlight a potentially widespread source of experimental variability affecting free divalent cation concentrations in protein applications.This work has been funded by the European Union's Horizon 2020 research and innovation programme under the Marie Sklowdowska-Curie grant agreement number 675132 (http://cordis.europa.eu/project/rcn/198275_en.html), and by grants from the Spanish Ministerio de Economía y Competitividad (MINECO/FEDER, http://www.mineco.gob.es/ portal/site/mineco/idi) SAF2015-68590R to DPS and CTQ2015-64597-C2-2-P to FJC.Peer reviewe

Molecular Recognition in C-Type Lectins: The Cases of DC- SIGN, Langerin, MGL, and L-Sectin

Carbohydrates play a pivotal role in intercellular communication processes. In particular, glycan antigens are key for sustaining homeostasis, helping leukocytes to distinguish damaged tissues and invading pathogens from healthy tissues. From a structural perspective, this cross-talk is fairly complex, and multiple membrane proteins guide these recognition processes, including lectins and Toll-like receptors. Since the beginning of this century, lectins have become potential targets for therapeutics for controlling and/or avoiding the progression of pathologies derived from an incorrect immune outcome, including infectious processes, cancer, or autoimmune diseases. Therefore, a detailed knowledge of these receptors is mandatory for the development of specific treatments. In this review, we summarize the current knowledge about four key C-type lectins whose importance has been steadily growing in recent years, focusing in particular on how glycan recognition takes place at the molecular level, but also looking at recent progresses in the quest for therapeutics.We thank the European Research Council (RECGLYCA NMR, advanced grant no. 788143), and the Agencia Estatal de Investigacion (Spain) for grants RTI2018-094751-B-C21 and B-C22, Ramon y Cajal contract to A.A. and the Severo Ochoa Excellence Accreditation (SEV-2016-0644)

Enzymatic method of producing 4-O- β-D-galactopyranosyl-D-xylose, 4-O- β-D-galactopyranosyl-D-xylose obtained using said method, compositions containing said and the use thereof in evaluating intestinal lactase

Filing Date: 2002-06-14.-- Priority Data: ES 200101419 (2001-06-18).-- International Publication Number: WO_2002103038 (20021227).An enzymatic process to obtain 4-O-β-D-galactopyranosyl-D-xylose useful in compositions or solutions in the in vivo evaluation of intestinal lactose activity in humans, that comprises the steps of preparing a reaction mixture of D-xylose, a β-D-galactopyranoside and a reaction medium that comprises water buffered to a pH between 5.0 and 9.0; adding 10 to 1,000 units of β-D-galactosidase per gram of β-D-galactopyranoside; subjecting the reaction mixture to a reaction or a temperature between a temperature higher than the freezing point of the reaction mixture and 45ºC, for 2 to 48 hours; for the reaction by deactivation of the β-D-galactosidase; and to isolate and crystallize the fractions that contain 4-O-β-D-galactopyranosyl-D-xylose from a crystallization mixture selected between mixtures of acetone/methanol in a ratio between 5/1 to 20/1 and mixtures of acetone/water in a ratio between 5/1 to 20/1

Vimentin filament organization and stress sensing depend on its single cysteine residue and zinc binding

17 pág., 10 figs.The vimentin filament network plays a key role in cell architecture and signalling, as well as in epithelial–mesenchymal transition. Vimentin C328 is targeted by various oxidative modifications, but its role in vimentin organization is not known. Here we show that C328 is essential for vimentin network reorganization in response to oxidants and electrophiles, and is required for optimal vimentin performance in network expansion, lysosomal distribution and aggresome formation. C328 may fulfil these roles through interaction with zinc. In vitro, micromolar zinc protects vimentin from iodoacetamide modification and elicits vimentin polymerization into optically detectable structures; in cells, zinc closely associates with vimentin and its depletion causes reversible filament disassembly. Finally, zinc transportdeficient human fibroblasts show increased vimentin solubility and susceptibility to disruption, which are restored by zinc supplementation. These results unveil a critical role of C328 in vimentin organization and open new perspectives for the regulation of intermediate filaments by zinc. DOI: 10.1038/ncomms8287 OPEN 1This work was supported by grants SAF2012–36519, MINECO, Spain and RD12/0013/0008, ISCIII to D.P.-S., and CTQ2012–32025, MINECO, y CAM MHIT S2010/BMD-2353 to F.J.C. C.L.O. and B.G. have been recipients of fellowships BES-2010–033718 and BES-2007–15806, respectively (FPI, MINECO). We acknowledge support from COST Action CM1001.Peer reviewe

Beyond a Fluorescent Probe: Inhibition of Cell Division Protein FtsZ by mant-GTP Elucidated by NMR and Biochemical Approaches

© 2015 American Chemical Society. FtsZ is the organizer of cell division in most bacteria and a target in the quest for new antibiotics. FtsZ is a tubulin-like GTPase, in which the active site is completed at the interface with the next subunit in an assembled FtsZ filament. Fluorescent mant-GTP has been extensively used for competitive binding studies of nucleotide analogs and synthetic GTP-replacing inhibitors possessing antibacterial activity. However, its mode of binding and whether the mant tag interferes with FtsZ assembly function were unknown. Mant-GTP exists in equilibrium as a mixture of C2′- and C3′-substituted isomers. We have unraveled the molecular recognition process of mant-GTP by FtsZ monomers. Both isomers bind in the anti glycosidic bond conformation: 2′-mant-GTP in two ribose puckering conformations and 3′-mant-GTP in the preferred C2′ endo conformation. In each case, the mant tag strongly interacts with FtsZ at an extension of the GTP binding site, which is also supported by molecular dynamics simulations. Importantly, mant-GTP binding induces archaeal FtsZ polymerization into inactive curved filaments that cannot hydrolyze the nucleotide, rather than straight GTP-hydrolyzing assemblies, and also inhibits normal assembly of FtsZ from the Gram-negative bacterium Escherichia coli but is hydrolyzed by FtsZ from Gram-positive Bacillus subtilis. Thus, the specific interactions provided by the fluorescent mant tag indicate a new way to search for synthetic FtsZ inhibitors that selectively suppress the cell division of bacterial pathogens.BFU2011-23416 and BFU2014-51823-R (J.M.A), CTQ2012-32065 (J.J.B.), CM 2010/BMD-2353 (J.J.B. and J.M.A.), FCT SFRH/BPD/65462/2009 and UID/Multi/04378/2013 (F.M.) and a FPI fellowship (L.B.R.A).Peer Reviewe

Glycosylated Cell Penetrating Peptides, GCPPs

This is the peer reviewed version of the following article: Gallego, I. , Rioboo, A. , Reina, J. ., Díaz, B. , Canales, Á., Cañada, F. ., Guerra-Varela, J. , Sánchez, L. and Montenegro, J. (2019), Glycosylated Cell Penetrating Peptides, GCPPs. ChemBioChem. doi:10.1002/cbic.201800720, which has been published in final form at https://doi.org/10.1002/cbic.201800720. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived VersionsThe cell membrane regulates the exchange of molecules and information with the external environment. However, this control barrier hinders the delivery of exogenous bioactive molecules that can be applied to correct cellular malfunctions. Therefore, the traffic of macromolecules across the cell membrane represents a great challenge for the development of the next generation of therapies and diagnostic methods. Cell penetrating peptides are short peptide sequences capable of delivering a broad range of biomacromolecules across the cellular membrane. However, penetrating peptides still suffer from limitations mainly related with their lack of specificity and potential toxicity. Glycosylation has emerged as a potential promising strategy for the biological improvement of synthetic materials. In this work we have developed a new convergent strategy for the synthesis of penetrating peptides functionalized with glycan residues by an oxime bond connection. We have systematically characterized the uptake efficiency and the intracellular distribution of these glycopeptides by flow cytometry, confocal microscopy and in zebrafish animal models. The incorporation of these glycan residues into the peptide structure influenced the internalization efficiency and the cellular toxicity of the resulting glycopeptide hybrids in the different cell lines tested. The results reported here highlight the potential of the glycosylation of penetrating peptides to modulate their activityWe acknowledge funding from the Spanish Agencia Estatal de Investigación (AEI) [CTQ2014-59646-R, SAF2017-89890-R, CTQ2016-76263-P, CTQ2015-64597-C2-2P], the Xuntade Galicia (ED431G/09, ED431C 2017/25 and 2016-AD031) and the ERDF. I. G. received a predoctoral fellowship from the Xunta de Galicia (ED481A-2018/116). A. R. received a predoctoral fellowship from the Fundación Segundo Gil Dávila. J.G.-V. and L.S. acknowledge the financial support received from the Xunta de Galicia (Galicia, Spain) under the Grupos de Referencia Competitiva Programme: Project GRC2014/010. J. M. received a Ramón y Cajal (RYC-2013-13784), an ERC Starting Investigator Grant (DYNAP-677786) and a Young Investigator Grant from the HFSP (RGY0066/2017)S

The interaction of La3+ complexes of DOTA/DTPA-glycoconjugates with the RCA120 lectin : a saturation transfer difference (STD) NMR spectroscopic study

The study of ligand-receptor interactions using high resolution NMR techniques, namely the Saturation Transfer Difference (STD), is presented for the recognition process between La(III) complexes of DOTA mono(amide) and DTPA bis(amide) glycoconjugates and the galactose specific lectin Ricinus Communis agglutinin (RCA120). This new class of Gd(III)-based potential targeted MRI contrast agents (CAs), bearing one or two terminal sugar (galactosyl or lactosyl) moieties, has been designed for in vivo binding to ASGPR (the asialoglycoprotein receptor), which is specifically expressed at the surface of liver hepatocytes, with the aim of leading to a new possible diagnosis of liver pathologies. The in vitro affinity constants of the divalent La(III)- glycoconjugate complexes to RCA120, used as a simple, water soluble receptor model, were higher than those of the monovalent analogues. The combination of the experimental data obtained from the STD NMR experiments with molecular modelling protocols (Autodock 4.1) allowed us to predict the binding mode of mono and divalent forms of these CAs to the galactose 1 binding sites of RCA120. The atomic details of the molecular interactions allowed corroborating and supporting the interaction of both the sugar moieties and the linkers with the surface of the protein and thus, their contribution to the observed interaction stabilities.Fundação para a Ciência e a Tecnologia (FCT

Enzymatic fine-tuning for 2-(6-hydroxynaphthyl) β-d-xylopyranoside synthesis catalyzed by the recombinant β-xylosidase BxTW1 from Talaromyces amestolkiae

This article is distributed under the terms of the Creative Commons Attribution 4.0 International License.-- et al.[Background]: Glycosides are compounds displaying crucial biological roles and plenty of applications. Traditionally, these molecules have been chemically obtained, but its efficient production is limited by the lack of regio- and stereo-selectivity of the chemical synthesis. As an interesting alternative, glycosidases are able to catalyze the formation of glycosides in a process considered green and highly selective. In this study, we report the expression and characterization of a fungal ß-xylosidase in Pichia pastoris. The transglycosylation potential of the enzyme was evaluated and its applicability in the synthesis of a selective anti-proliferative compound demonstrated. [Results]: The ß-xylosidase BxTW1 from the ascomycete fungus Talaromyces amestolkiae was cloned and expressed in Pichia pastoris GS115. The yeast secreted 8 U/mL of ß-xylosidase that was purified by a single step of cation-exchange chromatography. rBxTW1 in its active form is an N-glycosylated dimer of about 200 kDa. The enzyme was biochemically characterized displaying a K m and k cat against p-nitrophenyl-ß-d-xylopyranoside of 0.20 mM and 69.3 s¿1 respectively, and its maximal activity was achieved at pH 3 and 60 °C. The glycan component of rBxTW1 was also analyzed in order to interpret the observed loss of stability and maximum velocity when compared with the native enzyme. A rapid screening of aglycone specificity was performed, revealing a remarkable high number of potential transxylosylation acceptors for rBxTW1. Based on this analysis, the enzyme was successfully tested in the synthesis of 2-(6-hydroxynaphthyl) ß-d-xylopyranoside, a well-known selective anti-proliferative compound, enzymatically obtained for the first time. The application of response surface methodology, following a Box-Behnken design, enhanced this production by eightfold, fitting the reaction conditions into a multiparametric model. The naphthyl derivative was purified and its identity confirmed by NMR. [Conclusions]: A ß-xylosidase from T. amestolkiae was produced in P. pastoris and purified. The final yields were much higher than those attained for the native protein, although some loss of stability and maximum velocity was observed. rBxTW1 displayed remarkable acceptor versatility in transxylosylation, catalyzing the synthesis of a selective antiproliferative compound, 2-(6-hydroxynaphthyl) ß-d-xylopyranoside. These results evidence the interest of rBxTW1 for transxylosylation of relevant products with biotechnological interest.This work was carried out with funding from projects BIO2015-68387-R, RTC-2014-1777-3 and CTQ2015-64597-C2 from MINECO and S2013/MAE2972 from Comunidad de Madrid, as well as from the Natural Sciences and Engineering Research Council of Canada. M. Nieto-Domínguez thanks the MINECO for an FPU fellowship.We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).Peer Reviewe

The interaction of fluorinated glycomimetics with DC-SIGN: multiple binding modes disentangled by the combination of NMR methods and MD simulations

Fluorinated glycomimetics are frequently employed to study and eventually modulate protein–glycan interactions. However, complex glycans and their glycomimetics may display multiple binding epitopes that enormously complicate the access to a complete picture of the protein–ligand complexes. We herein present a new methodology based on the synergic combination of experimental 19F-based saturation transfer difference (STD) NMR data with computational protocols, applied to analyze the interaction between DC-SIGN, a key lectin involved in inflammation and infection events with the trifluorinated glycomimetic of the trimannoside core, ubiquitous in human glycoproteins. A novel 2D-STD-TOCSYreF NMR experiment was employed to obtain the experimental STD NMR intensities, while the Complete Relaxation Matrix Analysis (CORCEMA-ST) was used to predict that expected for an ensemble of geometries extracted from extensive MD simulations. Then, an in-house built computer program was devised to find the ensemble of structures that provide the best fit between the theoretical and the observed STD data. Remarkably, the experimental STD profiles obtained for the ligand/DC-SIGN complex could not be satisfactorily explained by a single binding mode, but rather with a combination of different modes coexisting in solution. Therefore, the method provides a precise view of those ligand–receptor complexes present in solution.We thank Agencia Estatal de Investigación (Spain) for grants RTI2018-094751-B-C21 and B-C22, CTQ2015-68756-R, and for FPI and FPU fellowships to J.D.M. and P.V., respectively, and for the Severo Ochoa Excellence Accreditation (SEV-2016-0644). J.J.-B. also thanks to the European Research Council (RECGLYCANMR, Advanced Grant no. 788143). S.O. thanks the SFI Award 13/IA/1959Peer reviewe

La alimentación preescolar, educación para la salud de los 2 a los 6 años

The infant feeding and nutrition is one of the most important areas regarding the Health Education (HE). This health-promoting action must start at the early stage of the development and learning, such as the pre-school period. A valuable tool to initiate successfully the HE could be infant nutrition, which can and must be also approached in the educational area.Our objective of the investigation was to gather, from the main Spanish manuals of nutrition, the basic and nutritional characteristics of the stage from 2 to 6 years, in order to be used by educational personnel instead of health personnel in the school area. By the analysis of bibliographical documented review, related to the nutritional characteristics at this stage of growth.The results indicate that the Recommendations Dietary Allowance (RDA) from 2 years are very different with regard to the breast-fed stage, the contribution moves towards the carbohydrates in 50 %, in decline of fats <30 % and proteins. In addition, the preparation and incorporation of new ingredients to the daily foods is already regular, obtaining, in this way, a balanced and healthy diet. For that, we will follow a guideline of 3 daily foods, with 2 intermediate of minor quantity, and the breakfast will have a well-deserved importance.To conclude, we must indicate that the educator has to inculcate healthy habits of feeding in pre-school stage by means of the Health Education. The mentioned infant diet will be the base for a healthy adulthood, since during the infant stage, future pathologies may emerge such as diabetes, cancer of colon and hypertension.La alimentación y nutrición infantil constituye una de las áreas de mayor importancia dentro de la Educación para la Salud (EpS). Esta acción promotora de la salud debe iniciarse en la fase temprana del desarrollo y aprendizaje, como es la etapa preescolar. Una valiosa herramienta para iniciar la EpS es la alimentación infantil, la cual puede y debe ser abordada en el ámbito educativo. Por ello, nuestro objetivo de investigación fue recoger, de los principales manuales de nutrición españoles, las características, básicas, nutricionales de la etapa de 2 a 6 años, para ser utilizado por personal docente no sanitario en el ámbito escolar. Mediante análisis de revisión bibliográfica documentada, referente a las características nutricionales de esta etapa de crecimiento. Los resultados indican que las Recomendaciones Diarias Alimenticias (RDA) a partir de los 2 años son muy diferentes respecto a la etapa de lactante, el aporte se balancea hacía los carbohidratos  en un 50%, en decremento de grasas <30% y proteínas. Además, la preparación e incorporación de nuevos ingredientes a los platos diarios es ya una constate, obteniendo nuevas y atractivas características organolépticas, para conseguir una dieta equilibrada y saludable. Para ello, se seguirá una pauta de 3 comidas, con 2 intermedias de menor cantidad, y donde el desayuno ya tendrá una merecida  importancia. Para concluir, además debemos indicar que el educador a través de la EpS debe trasmitir e inculcar, desde esta temprana edad, hábitos de vida saludable a través de la alimentación infantil. Ya que esta dieta infantil será la base de una etapa adulta saludable, ya que es en la etapa infantil donde se fraguan futuras  patologías como diabetes, cáncer de colon e hipertensión