Método de extracción y detección de antígenos de Anisakis en alimentos destinados al consumo humano o animal

Método de extracción y detección de antígenos de Anisakis en alimentos destinados al consumo humano o animal. La presente invención se refiere a un método de extracción y detección de alérgenos de parásitos de pescado en muestras alimentarias para el consumo humano o animal. La extracción se basa en aplicar soluciones con baja fuerza iónica, homogeneización, sonicación y diferentes pH a diversos tipos de pescado ya sean frescos o tratados. La detección se basa en métodos inmunoquímicos mediante el uso de anticuerpos policlonales que permiten detectar proteínas antigénicas del parásito así como anticuerpos policlonales que permiten detectar el alérgeno Ani s 4, que por sus características físico-químicas resiste el tratamiento térmico del alimento. El método es sensible, ya que se puede detectar Ani s 4 en cantidades inferiores a 1ppm con tasas de recuperación mayores a un 65%. El método descrito es específico ya que no muestra reactividad cruzada con componentes de las distintas matrices ensayadas.Peer reviewedConsejo Superior de Investigaciones Científicas (España), Fundación para la Investigación Biomédica del Hospital Carlos IIIB1 Patente sin examen previ

Método de extracción y detección de antígenos de Anisakis en alimentos destinados al consumo humano o animal

Método de extracción y detección de antígenos de Anisakis en alimentos destinados al consumo humano o animal. La presente invención se refiere a un método de extracción y detección de alérgenos de parásitos de pescado en muestras alimentarias para el consumo humano o animal. La extracción se basa en aplicar soluciones con baja fuerza iónica, homogeneización, sonicación y diferentes pH a diversos tipos de pescado ya sean frescos o tratados. La detección se basa en métodos inmunoquímicos mediante el uso de anticuerpos policlonales que permiten detectar proteínas antigénicas del parásito así como anticuerpos policlonales que permiten detectar el alérgeno Ani s 4, que por sus características físico-químicas resiste el tratamiento térmico del alimento. El método es sensible, ya que se puede detectar Ani s 4 en cantidades inferiores a 1ppm con tasas de recuperación mayores a un 65%. El método descrito es específico ya que no muestra reactividad cruzada con componentes de las distintas matrices ensayadas.Consejo Superior de Investigaciones Científicas (España), Fundación para la Investigación Biomédica del Hospital Carlos IIIA1 Solicitud de patente con informe sobre el estado de la técnic

Pectic galactan affects cell wall architecture during secondary cell wall deposition

Despite recent advances regarding the role of pectic β-(1,4)-galactan neutral side chains in primary cell wall remodelling during growth and cell elongation, little is known about the specific function of this polymer in other developmental processes. We have used transgenic Arabidopsis plants overproducing chickpea βI-Gal β-galactosidase under the 35S CaMV promoter (35S::βI-Gal) with reduced galactan levels in the basal non-elongating floral stem internodes to gain insight into the role of β-(1,4)-galactan in cell wall architecture during the cessation of elongation and the beginning of secondary growth. The loss of galactan mediated by βI-Gal in 35S::βI-Gal plants is accompanied by a reduction in the levels of KOH-extracted xyloglucan and an increase in the levels of xyloglucan released by a cellulose-specific endoglucanase. These variations in cellulose–xyloglucan interactions cause an altered xylan and mannan deposition in the cell wall that in turn results in a deficient lignin deposition. Considering these results, we can state that β-(1,4)-galactan plays a key structural role in the correct organization of the different domains of the cell wall during the cessation of growth and the early events of secondary cell wall development. These findings reinforce the notion that there is a mutual dependence between the different polysaccharides and lignin polymers to form an organized and functional cell wall

ABCG5/G8 gene is associated with hypercholesterolemias without mutation in candidate genes and non-cholesterol sterols

Context Approximately 20% to 40% of clinically defined familial hypercholesterolemia (FH) cases do not show a causative mutation in candidate genes (mutation-negative FH), and some of them may have a polygenic origin. Objective The aim of this work was to study the prevalence of ABCG5/G8 genetic variants in mutation-negative FH, as defects in these genes relate to intestinal hyperabsorption of cholesterol and thus ABCG5/G8 variants could explain in part the mechanism of hypercholesterolemia. Design, setting, and patients We sequenced the ABCG5/G8 genes in 214 mutation-negative FH and 97 controls. Surrogate markers of cholesterol absorption (5?-cholestanol, ?-sitosterol, campesterol, stigmasterol, and sitostanol) were quantified by high-performance liquid chromatography?tandem mass spectrometry in both studied groups. Results We found 8 mutation-negative FH patients (3.73%) with a pathogenic mutation in ABCG5/G8 genes. We observed significantly higher concentration of surrogate markers of cholesterol absorption in mutation-negative FH than in controls. In addition, we found significantly higher concentrations of cholesterol absorption markers in mutation-negative FH with ABCG5/G8 defects than in mutation-negative, ABCG5/G8-negative FH. A gene score reflecting the number of common single nucleotide variants associated with hypercholesterolemia was significantly higher in cases than in controls (P = .032). Subjects with a gene score above the mean had significantly higher 5?-cholestanol and stigmasterol than those with a lower gene score. Conclusions Mutation-negative FH subjects accumulate an excess of rare and common gene variations in ABCG5/G8 genes. This variation is associated with increased intestinal absorption of cholesterol, as determined by surrogate makers, suggesting that these loci contribute to hypercholesterolemia by enhancing intestinal cholesterol absorption.This study was supported by grants from the Spanish Ministry of Economy and Competitiveness PI15/01983, PI13/02507, PI12/01321, CIBERCV, CIBEROBN, and Cuenca Villoro Foundation. These projects are co-financed by Instituto de Salud Carlos III and the European Regional Development Fund (ERDF) of the European Union “A way to make Europe.

New interfacial microtubule inhibitors of marine origin, PM050489/PM060184, with potent antitumor activity and a distinct mechanism

We have investigated the target and mechanism of action of a new family of cytotoxic small molecules of marine origin. PM050489 and its dechlorinated analogue PM060184 inhibit the growth of relevant cancer cell lines at subnanomolar concentrations. We found that they are highly potent microtubule inhibitors that impair mitosis with a distinct molecular mechanism. They bind with nanomolar affinity to unassembled αβ-tubulin dimers, and PM050489 binding is inhibited by known Vinca domain ligands. NMR TR-NOESY data indicated that a hydroxyl-containing analogue, PM060327, binds in an extended conformation, and STD results define its binding epitopes. Distinctly from vinblastine, these ligands only weakly induce tubulin self-association, in a manner more reminiscent of isohomohalichondrin B than of eribulin. PM050489, possibly acting like a hinge at the association interface between tubulin heterodimers, reshapes Mg2+-induced 42 S tubulin double rings into smaller 19 S single rings made of 7 ± 1 αβ-tubulin dimers. PM060184-resistant mutants of Aspergillus nidulans map to β-tubulin Asn100, suggesting a new binding site different from that of vinblastine at the associating β-tubulin end. Inhibition of assembly dynamics by a few ligand molecules at the microtubule plus end would explain the antitumor activity of these compounds, of which PM060184 is undergoing clinical trials.We wish to thank J. M. Fernandez Sousa (PharmaMar) for useful discussions and support, E. Hamel (NCI) for providing eribulin, C. Scazzocchio and G. Diallinas for useful advice on mutant screening, H. N. Arst for advice on mutant screening and mapping and for kindly providing strains MAD3688 and MAD4655, T. J. Fitzgerald (A&M University) for MTC and C. Alfonso (CIB) for AUC analysis. We also thank Rhône Poulenc Rorer Aventis for supplying docetaxel and Matadero Municipal Vicente de Lucas de Segovia for providing the calf brains for tubulin purification. B.P. had a contract from Comunidad de Madrid, and A.C. had a Ramon y Cajal contract, J.R.-S. had a fellowship from “Programa de Cooperación Científica entre el Ministerio de Ciencia, Tecnologías y Medio Ambiente de la República de Cuba (CITMA) y el CSIC”. This work was supported by grants BIO2010-16351 (J.F.D.), BQU2009-08536 (J.J.-B.), CAM S2010/BMD-2457 (J.F.D.), CAM S2010/BMD-2353 (J.J.-B., J.M.A.), IPT-2011-0752-900000 and BIO2012-30965 (M.A.P.), BFU2011-23416 (J.M.A.) and PharmaMar-CSIC contracts

Antigenic activity of Anisakis larvae is conserved after food processing and pepsin treatments

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Allergenic properties and cuticle microstructure of Anisakis simplex L3 after freezing and pepsin digestion

This article examines the viability of and the alterations to the larval cuticle and the pattern of the antigens released when live or frozen Anisakis simplex larvae were treated with acid and pepsin. The results showed that freezing did not greatly alter the larva body. If ruptures were observed, the antigen release to the incubation media was not enhanced, and most of the antigenic content was retained inside the bodies of the larvae. The immunoblotting assay demonstrated that most of the antigens released, including the allergen Ani s 4, were resistant to pepsin. Freezing killed the larvae, but their survival was not compromised by acid treatment or pepsin digestion when kept chilled. All these findings support recommendations about freezing fish for consumption raw or undercooked to prevent human infection by A. simplex larvae, However, our data show that the antigenicity of the larvae is preserved after freezing and may explain why some sensitized patients develop symptoms after ingestion of infested frozen fish.This work has been financed by the Spanish projects AGL2005-05699-C02-01/02 ALI, PIE 2004 7 0E 160, and PIE 2004 7 0E 340 CSIC.Peer Reviewe

Anisakis simplex antigens in fresh and frozen-thawed muscle of anchovies in vinegar

Marinated fish treatment using low pH to enlarge the storage life of fish as in anchovies in vinegar, does not kill Anisakis simplex larvae infesting fish muscle. To kill the larvae it is compulsory in many countries to freeze fish intended to be marinated raw, which prevents the consumer to be infested with the live larvae. However, it is not known if A. simplex antigens are released to the media after freezing and vinegar processing, which may cause allergic reaction to A. simplex sensitized consumers. Anchovy fillets were artificially infested with A. simplex L3, treated with a vinegar solution and chilled stored for 10 days. Infested frozen-thawed fillets were treated and stored in the same conditions. Viability of the larvae, SEM, immunoblotting and immunohistochemistry were performed on the treated fillets before and after pepsin treatment. Viability of the larvae was detected only in the chilled fillets; however, A. simplex antigens were detected in the chilled and in the frozen-thawed fillets even after pepsin treatment. This suggests that the consumption of anchovies in vinegar may be a potential hazard when ingested by sensitized consumers, even if freezing kills the larvae.This work has been supported by the Spanish projects AGL2005-05699-C02-01/02 ALI and PIE 2004 7 0E 160 and 340 CSIC.Peer reviewe