Structural stabilization of botulinum neurotoxins by tyrosine phosphorylation

AbstractTyrosine phosphorylation of botulinum neurotoxins augments their proteolytic activity and thermal stability, suggesting a substantial modification of the global protein conformation. We used Fourier-transform infrared (FTIR) spectroscopy to study changes of secondary structure and thermostability of tyrosine phosphorylated botulinum neurotoxins A (BoNT A) and E (BoNT E). Changes in the conformationally-sensitive amide I band upon phosphorylation indicated an increase of the α-helical content with a concomitant decrease of less ordered structures such as turns and random coils, and without changes in β-sheet content. These changes in secondary structure were accompanied by an increase in the residual amide II absorbance band remaining upon H-D exchange, consistent with a tighter packing of the phosphorylated proteins. FTIR and differential scanning calorimetry (DSC) analyses of the denaturation process show that phosphorylated neurotoxins denature at temperatures higher than those required by non-phosphorylated species. These findings indicate that tyrosine phosphorylation induced a transition to higher order and that the more compact structure presumably imparts to the phosphorylated neurotoxins the higher catalytic activity and thermostability

HGUE-C-1 is an atypical and novel colon carcinoma cell line

Background: Colorectal carcinoma is a common cause of cancer. Adjuvant treatments include: 5-fluorouracil administered together with folinic acid, or more recently, oral fluoropyrimidines such as capecitabine, in combination with oxaliplatin or irinotecan. Metastatic colorectal cancer patients can benefit from other additional treatments such as cetuximab or bevacizumab. Methods: Using cell culture techniques, we isolated clonal populations from primary cultures of ascitic effusion derived from a colon cancer patient and after several passages an established cell line, HGUE-C-1, was obtained. Genetic analysis of HGUE-C-1 cells was performed by PCR of selected exons and sequencing. Cell proliferation studies were performed by MTT assays and cell cycle analyses were performed by flow cytometry. Retinoblastoma activity was measured by luciferase assays and proteins levels and activity were analysed by Western blot or immunohistochemistry. Results: We have established a new cell line from ascitic efussion of a colon cancer patient who did not respond to 5-fluorouracil or irinotecan. HGUE-C-1 cells did not show microsatellite instability and did not harbour mutations in KRAS, BRAF, PI3KCA or TP53. However, these cells showed loss of heterozygosity affecting Adenomatous Polyposis Coli and nuclear staining of β-catenin protein. The HGUE-C-1 cell line was sensitive to erlotinib, gefitinib, NVP-BEZ235, rapamycin and trichostatin, among other drugs, but partially resistant to heat shock protein inhibitors and highly resistant to AZD-6244 and oxaliplatin, even though the patient from which this cell line was derived had not been exposed to these drugs. Molecular characterization of this cell line revealed low expression levels and activity of Retinoblastoma protein and elevated basal levels of Erk1/2 activity and p70S6K expression and activity, which may be related to chemoresistance mechanisms. Conclusions: HGUE-C-1 represents a novel and peculiar colon carcinoma model to study chemoresistance to chemotherapeutic agents and to novel anti-neoplasic drugs that interrupt signalling pathways such as the APC/βcatenin, Ras/Raf/Mek/Erk, PI3K/mTOR/p70S6K pathways as well as histone regulation mechanisms.This article has been funded by grants from the Instituto de Salud Carlos III FIS PI080901and FIS PI01202025 to Miguel Saceda

Is metabolomics reachable? Different purification strategies of human colon cancer cells provide different CE-MS metabolite profiles

13 páginas, 3 figuras, 1 tabla.--El pdf del artículo es la versión de autor.In this work, four different metabolite purification approaches are investigated prior to metabolomics of human HT29 colon cancer cells. Namely, methanol deproteinization, ultrafiltration and two SPE methods using C18 and polymer-based cartridges were studied. The extracts were characterized via a metabolomic approach based on the application of CE TOF MS (CE-MS). CE-MS analysis time was less than 20 min per sample and allowed the simultaneous and reproducible analysis of more than 80 metabolites in a single run with a minimum consumption of sample and reagents. Metabolome analysis revealed in some cases important differences among the studied metabolite purification procedures. No significant differences were observed in the metabolite profile using C18 and polymer-based cartridges, or between ultrafiltration and methanol deproteinization. However, important differences were observed in the metabolomic profiles obtained from SPE and methanol deproteinization samples. These results demonstrate the crucial role of the metabolite purification strategy in metabolomics since it can bias (and in some cases mislead) the conclusions achieved by the metabolomic studyThis work was supported by AGL2008-05108-C03-01 and 200870I185 (Ministerio de Ciencia e Innovación, Spain), and CSD2007-00063 FUN-C-FOOD (Programa CONSOLIDER, Ministerio de Educacion y Ciencia, Spain). C.I. thanks the Ministerio de Ciencia e Innovación for her FPI pre-doctoral fellowship.Peer reviewe

Las Nuevas Tecnologías en la Universalización. Retos y perspectivas

En este artículo se hace una reflexión acerca del impacto que provoca el uso de las Nuevas Tecnologías de la Informática y las Comunicaciones en la formación del futuro profesional, de forma clara y sencilla se explica en qué consiste la NTIC, así como su influencia en las nuevas tendencias de la educación a distancia en los tiempos actuales, lo que propicia, en gran medida, la elevación y preparación de los nuevos profesionales de la educación en el manejo de estas novedosas técnicas para poder hacer un uso eficiente de ellas y aprovechar al máximo todas sus potencialidades

New Technologies in the universalization. Challenges and Prospects

This article reflects on the impact caused by the use of New Technologies of Information and Communication in the formation of future professional, clear and simple way is explained what the NTIC and its influence on new trends in distance education in modern times, which favors large extent, elevation and preparation of new education professionals in the management of these new techniques to make efficient use of them and make the most their full potential

Different distribution of daunomycin in plasma membranes from drug-sensitive and drug-resistant P388 leukemia cells

When the anthracycline daunomycin (DNM) is incorporated into isolated plasma membranes from P388 murine leukemia cells, the drug partitions between ‘deep’ and ‘surface’ membrane domains. Such domains have been characterized on the basis of: (1) fluorescence resonance energy transfer between 1,6-diphenylhexa-1,3,5-triene or 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene as energy donors, which are well known in their positioning within the membrane, and daunomycin as the energy acceptor, and (2) quenching of the fluorescence of the membrane-associated drug by the water-soluble quencher iodide. The distribution of DNM between the two plasma membrane domains is different depending on the cellular phenotype. Thus, in membranes from drug-sensitive cells, DNM is preferentially confined to ‘surface’ domains, while in membranes from drug-resistant cells, the drug distributes more homogeneously between ‘surface’ and ‘deep’ domains. Experiments using artificial lipid vesicles suggest that differences in the relative levels of certain lipids in the plasma membranes from drug-sensitive and drug-resistant cells, namely phosphatidylserine and cholesterol, are partly responsible for the observed differences in the distribution of DNM. Since drug-membrane interactions are important in anthracycline cytotoxicity, it is possible that our observations on a different membrane distribution of daunomycin, may be related to the different sensitivity to the drug exhibited by these cells.This work has been partly supported by Grants PB87-07qO (to J.M.G.-R.) and PB87-0791 (to J.A.F.) from the DGICT of Spain.Peer reviewe

Trimers of N-alkylglycines are potent modulators of the multidrug resistance phenotype

9 pages, 7 figures, 1 table.-- PMID: 15644429 [PubMed].-- Printed version published Jan 11, 2005.-- Supporting information available at: http://jpet.aspetjournals.org/cgi/content/full/jpet.104.078014/DC1The multidrug resistance (MDR) phenotype is considered a major cause of the failure of cancer chemotherapy. The acquisition of MDR is usually mediated by the overexpression of drug efflux pumps such as glycoprotein P (P-gp) or multidrug resistance-related protein 1 (MRP1). Thus, the identification, validation, and development of compounds that mitigate the MDR phenotype by modulating the activity of these transport proteins is an important yet elusive target. Here, we have addressed this issue and screened an N-trialkylglycine-based combinatorial library composed of 5120 compounds to search for modulators of the MDR phenotype. The screening identified 20 trimers of N-alkylglycine that increased the intracellular accumulation of daunomycin (DNM) in drug-resistant L1210R tumor cells that overexpressed the P-gp. These compounds seem to act as P-gp antagonists, as evidenced by the augmentation of DNM accumulation in the L1210P-gp cell line, a drug-sensitive L1210 cell stably expressing the murine P-gp protein. Similarly, several of the active N-trialkylglycines also produced an increment in DNM uptake in human HL60R cells, which primarily express the MRP1 protein. Trialkylglycines notably sensitized L1210R and HL60R tumor cells to DNM with a potency that rivaled that of verapamil. These findings provide new molecular scaffolds for the development of effective chemosensitizers against the MDR phenotype that, in due turn, could be used as adjuvant drugs in cancer chemotherapy.This work was supported by grants from La Fundación La Caixa (to A.F.- M.); Fundació La Marató de TV3 (to A.M.); the Spanish Interministerial Commission of Science and Technology (CICYT) and the European Commission (to A.F.-M. and E.P.-P.).Peer reviewe

Formation of 5-(hydroxymethyl)furfural during subcritical water extraction of natural matrices. Is it bioactivity-relevant?

Trabajo presentado al 10th International Symposium on Supercritical Fluids celebrado del 13 al 16 de mayo de 2012 en San Francisco (US).Subcritical water extraction (SWE) has demonstrated a great potential for the extraction of bioactive compounds from natural matrices. The application of high extraction temperatures and pressures, while keeping the water in the liquid state, allows the attainment of high extraction yields in fast extraction processes. Although the applications of SWE to obtain bioactives from natural matrices are increasing, the studies dealing with the processes that might take place during the extraction are still scarce. Recently, we demonstrated how under SWE conditions not only the solubility of the analytes might be improved but also neoformed antioxidants can be obtained. Indeed, it was observed how the occurrence of reactions like Maillard and caramelization reactions during SWE resulted on the formation of neoantioxidants. This fact can be advantageous although the possible toxicity of the compounds formed during these non-enzymatic reactions should be carefully considered. Formerly, we observed that during the extraction of olive tree leaves at high extraction temperatures (200ºC), more active extracts in terms of antioxidant and in-vitro antiproliferative activities were obtained. Chemical characterization of those extracts revealed not only the presence of phenolic bioactives but also 5-(hydroxymethyl)furfural (HMF), a compound related to Maillard reaction. For this reason, in this work, a more exhaustive study has been devised to observe the influence of the temperature in the formation of HMF in SWE extracts of olive leaves. The final aim was to determine the relevance of the HMF present on different SWE extracts in terms of bioactivity. Thus, extracts obtained at different extraction temperatures were produced (50, 75, 100, 125, 150, 175 and 200 ºC) and chemically characterized by LC-MS. HMF was quantified in the extracts by HPLC-DAD. Moreover, the antioxidant activity, the amount of total phenols (Folin method) as well as the anticancer activity of both, extracts and HMF standard, were determined.This work was financed thanks to AGL2008-05108-C03-01 and AGL2011-29857-C03-01 (Ministerio de Educacion y Ciencia), CSD2007-00063 FUN-CFOOD (Programa CONSOLIDER-INGENIO 2010) and ALIBIRD, S2009/AGR-1469 (Comunidad de Madrid) projects. M.H. would like to thank MICINN for a “Ramón y Cajal” research contract. M.C.P. thanks MICINN for her “Juan de la Cierva” contract.Peer Reviewe

Formation and relevance of 5-hydroxymethylfurfural in bioactive subcritical water extracts from olive leaves

Although subcritical water extraction (SWE) has already shown its great potential for the attainment of natural bioactive extracts, concerns still remain on possible unexpected reactions that can arise during the extraction process, usually taking place at high pressure and temperature. It is already well-known that different components might be formed during the SWE extraction protocol due e.g. to Maillard reaction, which can improve the bioactivity of the obtained extracts. On the other hand, the formation of other compounds derived from these reactions, such as 5-hydroxymethylfurfural (HMF), has raised some concerns, mainly related to its safety. In this work, the formation of HMF during subcritical water extraction, at different conditions, from olive leaves has been monitored by using liquid chromatography with mass spectrometry (LC-MS) and diode array detection (LC-DAD). The possible influence of this compound in the overall antioxidant and antiproliferative activities against colon cancer cells has been also studied. Results showed an increase of HMF formation when increasing the extraction temperature, being the maximum concentration achieved at 200 °C (3.17 μg. HMF/mg extract); nevertheless, the HMF contained in the olive leave extracts did not influence the antioxidant capacity or the antiproliferative activity of the natural extracts, thus demonstrating the safety of the SWE process. © 2012 Elsevier Ltd.This work has been financed by AGL2008-05108-C03-01 and AGL2011-29857-C03-01 (Ministerio de Educacion y Ciencia), CSD2007-00063 FUN-CFOOD (Programa CONSOLIDER-INGENIO 2010) and by ALIBIRD, S2009/AGR-1469 (Comunidad de Madrid) projects. M.H. would like to thank MICINN for a “Ramón y Cajal” research contract. M.C.P. thanks MICINN for her “Juan de la Cierva” contract.Peer Reviewe