C-Terminal Truncation of α 1,6-Fucosyltransferase from Rhizobium Sp. does not Annul the Transferase Activity of the Enzyme

6 pages.-- PMID: 11814863 [PubMed].Recently we have over-expressed the enzyme a 1,6-fucosyltransferase from Rhizobium sp. in Escherichia coli. In this heterologous system the enzyme was mainly expressed as inclusion bodies and the one that was expressed soluble showed a shortlasting activity in solution due to precipitation of the protein. A structural analysis of the sequence using the TMpred program predicted a highly hydrophobic region of 19 aa close to the C-terminal of the protein. In order to investigate the influence of this region on the formation of inclusion bodies and the precipitation from solution, we cloned a truncated version of the protein where a C-terminal fragment of 65 aa, including the predicted transmembrane-like region, was removed. The resulting protein was expressed in a soluble form without formation of inclusion bodies. The truncated protein catalyzed the transfer of a fucopyranosyl moiety from GDP-b-l-Fucose to chitobiose. Comparison of the acceptor specificity between the truncated a 1,6-fucosyltransferase and the wild-type enzyme, showed a similar behavior for both enzymes. Our results indicate that the active center is not located in the C-terminal extreme of the protein in contrast to the case of the mammalian glycosyltransferases. Also, these results indicate that the a-6-motif III is not directly involved in the catalytic activity of the enzyme.This work was supported by the Spanish DGES (Grant PB96-0828) and Comunidad de Madrid (Grant 07B/0027/1999).Peer reviewe

Development of a new method for D-xylose detection and quantification in urine, based on the use of recombinant xylose dehydrogenase from Caulobacter crescentus.

The gene xylB from Caulobacter crescentus has been cloned and expressed in Escherichia coli providing a high yield of xylose dehydrogenase (XylB) production and excellent purity (97%). Purified recombinant XylB showed an absolute dependence on the cofactor NAD+ and a strong preference for d-xylose against other assayed mono and disaccharides. Additionally, XylB showed strong stability when stored as freeze-dried powder at least 250 days both at 4 °C and room temperature. In addition, more than 80% of the initial activity of rehydrated freeze-dried enzyme remained after 150 days of incubation at 4 °C. Based on these characteristics, the capability of XylB in d-xylose detection and quantification was studied. The linearity of the method was maintained up to concentrations of d-xylose of 10 mg/dL and the calculated limits of detection (LoD) and quantification (LoQ) of xylose in buffer were 0.568 mg/dL and 1.89 mg/dL respectively. Thus, enzymatic detection was found to be an excellent method for quantification of d-xylose in both buffer and urine samples. This method can easily be incorporated in a new test for the diagnosis of hypolactasia through the measurement of intestinal lactase activity.This work was supported by a grant from Venter Pharma SL (Spain) and partially supported by the Spanish Ministerio de Economía y Competitividad (Grant MAT2015-65184-C2-2-R, MINECO/FEDER).Peer reviewe

Oligosacáridos utilizados para inhibir la mitosis de los astrocitos y de las células tumorales del sistema nervioso; y procedimiento de obtención de estos oligosacáridos

Traducción de Patente Europea E92923821 (fecha de solicitud, 13/11/1992).-- Prioridad: ES199111139102522.-- Titular: Consejo Superior de Investigaciones Científicas (CSIC).La invención se refiere a oligosacáridos y a preparaciones medicinales que contienen ingredientes orgánicos activos.Peer reviewe

Detection of metabolite changes in C6 glioma cells cultured with antimitotic oleyl glycoside by1H MAS NMR

The synthetic glycoside, oleyl N-acetyl-α-D-glucosaminide (1), was previously shown to exhibit antimitotic activity on rat (C6) and human (U-373) glioma lines. To obtain information about its mechanism of action, metabolite changes in C6 glioma cells were analyzed after treatment with 1 using high-resolution magic angle spinning 1H NMR. Compound 1 caused either a decrease or an increase in the intensity of the signal assigned to coenzyme A (CoA) metabolites depending on the concentration used. The data obtained from the 1H NMR spectra of cells cultured with 1, combined with those obtained after treatment with oleic acid (an inhibitor of acetyl-CoA carboxylase) and phenyl butyrate (a known antineoplastic agent), suggest that 1 may be altering the metabolism of fatty acids and induce apoptosis of C6 glioma cells. These results point to NMR spectroscopy as an efficient technique for monitoring the response of the cells to therapeutic agents.Peer Reviewe

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

Preparation and Characterization of Aminoglycoside-Loaded Chitosan/Tripolyphosphate/Alginate Microspheres against E. coli

Although aminoglycosides are one of the common classes of antibiotics that have been widely used for treating infections caused by pathogenic bacteria, the evolution of bacterial resistance mechanisms and their inherent toxicity have diminished their applicability. Biocompatible carrier systems can help sustain and control the delivery of antibacterial compounds while reducing the chances of antibacterial resistance or accumulation in unwanted tissues. In this study, novel chitosan gel beads were synthesized by a double ionic co-crosslinking mechanism. Tripolyphosphate and alginate, a polysaccharide obtained from marine brown algae, were employed as ionic cross-linkers to prepare the chitosan-based networks of gel beads. The in vitro release of streptomycin and kanamycin A was bimodal; an initial burst release was observed followed by a diffusion mediated sustained release, based on a Fickian diffusion mechanism. Finally, in terms of antibacterial properties, the particles resulted in growth inhibition of Gram-negative (E. coli) bacteria

Chemoenzymatic preparation of Chondroitin sulfates with a defined sulfation pattern

Comunicación Oral impartida en el XVII European Congress on BiotechnologyN

The Effect of Antitumor Glycosides on Glioma Cells and Tissues as Studied by Proton HR-MAS NMR Spectroscopy

Abstract The effect of the treatment with glycolipid derivatives on the metabolic profile of intact glioma cells and tumor tissues, investigated using proton high resolution magic angle spinning ( 1 H HR-MAS) nuclear magnetic resonance (NMR) spectroscopy, is reported here. Two compounds were used, a glycoside and its thioglycoside analogue, both showing anti-proliferative activity on glioma C6 cell cultures; however, only the thioglycoside exhibited antitumor activity in vivo. At the drug concentrations showing anti-proliferative activity in cell culture (20 and 40 µM), significant increases in choline containing metabolites were observed in the 1 H NMR spectra of the same intact cells. In vivo experiments in nude mice bearing tumors derived from implanted C6 glioma cells, showed that reduction of tumor volume was associated with significant changes in the metabolic profile of the same intact tumor tissues; and were similar to those observed in cell culture. Specifically, the activity of the compounds is mainly associated with an increase in choline and phosphocholine, in both the cell cultures and tumoral tissues. Taurine, a metabolite that has been considered a biomarker of apoptosis, correlated with the reduction of tumor volume. Thus, the results indicate that the mode of action of the glycoside involves, at least in part, alteration of phospholipid metabolism, resulting in cell death

Novel sulfoglycolipid IG20 causes neuroprotection by activating the phase II antioxidant response in rat hippocampal slices

Los datos de investigación asociados a este artículo están disponibles en http://dx.doi.org/10.1016/j.neuropharm.2016.12.016Compound IG20 is a newly synthesised sulphated glycolipid that promotes neuritic outgrowth and myelinisation, at the time it causes the inhibition of glial proliferation and facilitates exocytosis in chromaffin cells. Here we have shown that IG20 at 0.3–10 μM afforded neuroprotection in rat hippocampal slices stressed with veratridine, glutamate or with oxygen plus glucose deprivation followed by reoxygenation (OGD/reox). Excess production of reactive oxygen species (ROS) elicited by glutamate or ODG/reox was prevented by IG20 that also restored the depressed tissue levels of GSH and ATP in hippocampal slices subjected to OGD/reox. Furthermore, the augmented iNOS expression produced upon OGD/reox exposure was also counteracted by IG20. Additionally, the IG20 elicited neuroprotection was prevented by the presence of inhibitors of the signalling pathways Jak2/STAT3, MEK/ERK1/2, and PI3K/Akt, consistent with the ability of the compound to increase the phosphorylation of Jak2, ERK1/2, and Akt. Thus, the activation of phase II response and the Nrf2/ARE pathway could explain the antioxidant and anti-inflammatory effects and the ensuing neuroprotective actions of IG20This study was supported by a grant from Ministerio de Economía y Competitividad, Spain (MINECO SAF2013-44108-P to AGG and LG; MAT2015-65184-C2-2-R to AFM, CABICYC UAM-Bioiberica and European Commission-ERC, People (Marie Curie Actions) FP7 under REA grant agreement n PCIG11-GA-2012-322156; Spanish Ministry of Health (Instituto de Salud Carlos III) (grant PI14/00372) and Miguel Servet (CP11/00165); Bayer A.G., “From Targets to Novel Drugs” program (grant 2015-03-1282) and Fundacion FIPSE (grant 12-00001344-15) to RL. RL thanks IS Carlos III for research contract under Miguel Servet Program. P.M. thanks MECD for FPU fellowship (AP2010-1219

Transplantation of Predegenerated Peripheral Nerves after Complete Spinal Cord Transection in Rats: Effect of Neural Precursor Cells and Pharmacological Treatment with the Sulfoglycolipid Tol-51

Following spinal cord injury (SCI), the regenerative capacity of the central nervous system (CNS) is severely limited by the failure of axonal regeneration. The regeneration of CNS axons has been shown to occur by grafting predegenerated peripheral nerves (PPNs) and to be promoted by the transplantation of neural precursor cells (NPCs). The introduction of a combinatorial treatment of PPNs and NPCs after SCI has to address the additional problem of glial scar formation, which prevents regenerating axons from leaving the implant and making functional connections. Previously, we discovered that the synthetic sulfoglycolipid Tol-51 inhibits astrogliosis. The objective was to evaluate axonal regeneration and locomotor function improvement after SCI in rats treated with a combination of PPN, NPC, and Tol-51. One month after SCI, the scar tissue was removed and replaced with segments of PPN or PPN+Tol-51; PPN+NPC+Tol-51. The transplantation of a PPN segment favors regenerative axonal growth; in combination with Tol-51 and NPC, 30% of the labeled descending corticospinal axons were able to grow through the PPN and penetrate the caudal spinal cord. The animals treated with PPN showed significantly better motor function. Our data demonstrate that PPN implants plus NPC and Tol-51 allow successful axonal regeneration in the CNS.Grant PID2021-128446NB funded by MICIU/AE/10.13039/501100011033 and ERDF7EU to JM. VBA received a postdoctoral fellowship, CVU 33557, from the Consejo Nacional de Ciencia, Humanidades y Técnología CONACHYT México. AAC received a scholarschip “Yo Investigo” from the Consejería de Educación Castilla-La Mancha, Spain.Peer reviewe