The radioprotective effect of a wheat germ diet on rat myocardial tissue exposed to X-rays / Efecto radioprotector de una dieta de germen de trigo en tejido miocárdico de ratas expuestas a rayos X

Introduction: To determine whether a diet of wheat (Triticum aestivum) germ has a radioprotective effect on albino rat (Rattus rattus var. Albinus) myocardial tissue. Method: Rats between 200 and 250 g were divided into 4 groups of 6 each: Two groups were fed either a wheat diet or regular diet 16 days before and after a single exposure to 18 mSv of X-rays. The other two groups were fed the same diets but not exposed to X-rays. The animals were sacrificed, and heart tissue was submitted to histopathological study. Results: Rats fed a standard diet and exposed to X-rays presented marked hyperemia of blood vessels, necrosis, presence of connective tissue fibrocytes, loss of muscle architecture and radial arrangement. Exposed rats fed a wheat diet presented with only light necrosis and the presence of fibrocytes. Rats not exposed to X-rays had healthy myocardia. Conclusions: Wheat germ diet may have a radioprotective effect on rat myocardium

Introducción: Determinar si una dieta con germen de trigo (Triticum aestivum) tiene un efecto radio protector sobre el tejido miocárdico de ratas albinas (Rattus rattus var. Albinus) Método: Ratas con un peso entre 200 - 250 g fueron divididas en 4 grupos de 6 cada uno: Dos grupos fueron alimentadas con una dieta regular de germen de trigo durante 16 días antes y después de una única exposición a un grupo con 18 mSv de rayos X. Los otros dos grupos fueron alimentados con una dieta estándar, uno de ellos, fue expuesto a los rayos X. Los animales fueron sacrificados y se realizó un estudio histopatológico del tejido cardiaco. Resultados: Las ratas alimentadas con una dieta estándar y expuesta a los rayos X, presentaron marcada hiperemia de vasos sanguíneos, necrosis, presencia de fibrocitos en el tejido conectivo, pérdida de la arquitectura y disposición radial muscular. Las ratas expuestas y alimentadas con dieta de trigo presentaron solo una ligera necrosis y presencia de fibrocitos. Las ratas no expuestas a rayos X presentaron un miocardio saludable. Conclusiones: La dieta del germen de trigo puede tener un efecto radioprotector sobre el miocardio de rata

CdS/TiO2-fluorescein isothiocyanate nanoparticles as fluorescence resonance energy transfer probe for the determination of trace alkaline phosphatase based on affinity adsorption assay.

The CdS/TiO(2)-fluorescein isothiocyanate (FITC) luminescent nanoparticles (CdS/TiO(2)-FITC) with the particle size of 20 nm have been synthesized by sol-gel method. CdS/TiO(2)-FITC could emit the fluorescence of both FITC and CdS/TiO(2). The fluorescence resonance energy transfer (FRET) occurred between the donor CdS/TiO(2) and the acceptor FITC in the CdS/TiO(2)-FITC. Taking advantages of the excellent characteristics of FRET, a new CdS/TiO(2)-FITC FRET labeling reagent and a CdS/TiO(2)-FITC-wheat germ agglutinin (CdS/TiO(2)-FITC-WGA) fluorescent probe have been developed. The FRET occurring between the donor CdS/TiO(2) and the acceptor FITC in the labelled product CdS/TiO(2)-FITC-WGA-AP, formed in the affinity adsorption reaction between the WGA in this CdS/TiO(2)-FITC-WGA fluorescent probe and alkaline phosphatase (AP), sharply enhanced the fluorescence signal of FITC and quench the fluorescence signal of CdS/TiO(2). Moreover, the ΔF (the change of the fluorescence signal) of FITC and CdS/TiO(2) were proportional to the content of AP, respectively. Thus, a new method that CdS/TiO(2)-fluorescein isothiocyanate nanoparticles for the determination of trace AP based on FRET-affinity adsorption assay has been established. The limit of quantification (LOQ) of the method was 1.3×10(-17) g AP mL(-1) for CdS/TiO(2) and 1.1×10(-17) g AP mL(-1) for FITC, respectively. This sensitive, rapid, high selective and precise method has been applied to the determination of AP in human serum and the prediction of human disease with the results agreed well with enzyme-linked immunosorbent assay (ELISA) in Zhangzhou Municipal Hospital of Fujian Province. Simultaneously, the reaction mechanism for the determination of AP was also discussed.

WGA-grafted PLGA-nanospheres: preparation and association with Caco-2 single cells.

Biotech drugs are poorly absorbed, highly susceptible to loss of activity, and require formulations with protective effects. Their incorporation into nanospheres is a promising approach since colloidal formulations were shown to be absorbed. Due to rather low absorption rates the surface engineering with absorption enhancing agents is a current challenge. Thus, an optimized protocol relying on carbodiimide-mediated covalent binding of biorecognitive ligands to the surface of biodegradable and biocompatible poly(D,L-lactic-co-glycolic)acid (PLGA) nanospheres was established. Important parameters such as retainment of particle size distribution and biorecognitive characteristics of the ligand as well as storage stability were considered. As exemplified by nanoparticles grafted with wheat germ agglutinin (WGA) as a carbohydrate binding ligand and Caco-2 single cells, flow cytometry as well as confocal laser scanning microscopy revealed improved cytoassociation of the nanoscaled carriers as compared to the unmodified ones. As detected by flow cytometry, the binding of WGA-decorated nanospheres considerably increases the roughness of the cell surface with increasing ligand density. Additionally, the biorecognitive nanoparticles were not toxic in the Caco-2 model. The protocol presented for surface modification of nanospheres promises high versatility of application in search for biorecognitive ligands enhancing the cytoadhesion, cytoinvasion as well as transcellular transport of colloidal carriers.

Preparation, characterization and application of artificial Caco-2 cell surfaces in the silver nanoparticle enhanced fluorescence technique.

To approach in vivo conditions during real time monitoring of biorecognitive interactions, biomimetic surfaces were prepared by fusion of purified plasma membrane fractions from Caco-2 cells with self-assembled monolayers attached to silver colloid coated standard microplates. Proper orientation and integrity of the membrane coating was confirmed by binding of fluorescein-labelled wheat germ agglutinin (F-WGA) which interacts with certain carbohydrates of the glycocalyx. Additionally, the competition with the complementary carbohydrate decreased the F-WGA binding to 15%. The assay setup offers information about the real time binding kinetics and the affinity of the interaction with the cell membrane excluding interfering events such as internalization and metabolism. As exemplified by F-WGA-binding, the mean velocity of the interaction is 627.07 mFU/s and the working range is 40-240 nM with a detection limit of 1.6 pmol F-WGA. The storage stability of the ready-to-use plates exceeded at least 1 month. The real time monitoring of lectin-prodrug binding to the biomimetic membranes revealed that high conjugation numbers reduces the affinity. The assays are simple and fast one-step reactions without any washing steps as this new technique discriminates between membrane bound and bulk fluorescence. Thus, biomimetic membranes on silver colloid layers represent a versatile tool for high throughput screening at early stages of drug discovery and development.

Lectin-mediated bioadhesion: preparation, stability and caco-2 binding of wheat germ agglutinin-functionalized Poly(D,L-lactic-co-glycolic acid)-microspheres.

To take advantage of the cytoadhesive characteristics of Wheat germ agglutinin (WGA) for improved particulate drug delivery, the interaction between WGA-grafted poly(D,L-lactic-co-glycolic acid)-microspheres and Caco-2 monolayers was investigated using bovine serum albumin (BSA) or glycine coated microspheres as a control. Covalent immobilization of WGA by the carbodiimide/N-hydroxysuccinimide-method on 4 microm microspheres yielded a surface density of 9.67+/-1.21x10(6) molecules/particle, whereas 0.22+/-0.04x10(6) WGA-molecules were bound by physical adsorption. After storage for 21 days in HEPES-buffer and treatment of the particles with 5 M urea, 86% of covalently linked lectin was still attached to the particles. At 4 degrees C the Caco-2 binding rate of both, WGA- and BSA-modified particles increased with addition of increasing numbers of particles until saturation was reached at 38150+/-1740 (WGA) or 12066+/-1195 (BSA) microspheres bound/mm(2) Caco-2 monolayer. Inhibition of Caco-2 binding of WGA-functionalized microspheres by chitotriose indicated for specificity of the interaction. As observed by confocal laser scanning microscopy, the fluorescein-loading of the particles was accumulated intracellularly after incubation of Caco-2 monolayers with WGA-modified microspheres contrary to glycine-grafted microspheres. Additionally, in case of WGA-functionalized microspheres the amount of cell associated fluorescein was 200-fold higher than that of the free solution. In conclusion, WGA-modified microspheres are expected to enhance intestinal transport of incorporated drugs due to cytoadhesion provided by the lectin coating.

Synthesis of bioadhesive lectin-HPMA copolymer-cyclosporin conjugates.

An amino group containing cyclosporin A (CsA) derivative has been synthesized and conjugated to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer via an aromatic azo bond, which can be specifically cleaved by azoreductase activity in colon to release the drug for the treatment of colon diseases. Lectins, peanut (Arachis hypogea) agglutinin (PNA) and wheat germ agglutinin (WGA), have been conjugated to HPMA copolymer-CsA derivative conjugates (PCsA), respectively, to give bioadhesive conjugates. The PNA and WGA are the targeting proteins that can bind to diseased colon tissue and healthy tissue, respectively. There were on average four P(CsA) copolymer chains attached on one WGA molecule with a drug content of 16.0 wt % and five P(CsA) copolymer chains attached on one PNA molecule with a drug content of 11.5 wt %. The incubation of a P(CsA) copolymer with the rat cecal contents resulted in the cleavage of the azo bond and release of the cyclosporin derivative. The biological evaluation of the conjugates is under way.

Use of colloidal gold complexes of wheat germ agglutinin as a label for neural cells.

We have made stable complexes between wheat germ agglutinin and either 5 or 10 nm particles of colloidal gold. These complexes were phagocytosed by neuronal and glial cells in embryonic rat hippocampal cultures and the incorporated gold gave intense, low-background staining in the light microscope either directly, for the most heavily labelled cells, or after intensification by physical development of silver. Cells were labelled in a punctate fashion over perikarya and processes. In the electron microscope, particles of gold were observed in lysosomal vesicles, frequently in an aggregated form. Gold complex incorporated into cells in culture was retained by those cells over periods up to 20 days. Embryonic hippocampal cells were labelled in suspension culture by incorporation of wheat germ agglutinin-gold complexes and transplanted into the brains of syngeneic adult host rats. Grafted neurons and glia were observed in the electron microscope to retain high levels of gold label over periods up to 30 days. Receipt of synaptic connections by transplanted neurones was observed. Complexes of wheat germ agglutinin with 10 nm gold particles were injected unilaterally into field CA3 of the hippocampus of adult rats. Specific retrograde transport of gold was observed in the light and electron microscopes to pyramidal and hilar neurones of the contralateral hippocampus and to neurones of the medial septal nucleus. Colloidal gold-wheat germ agglutinin complexes appear to be useful cellular markers that can be visualized at both light and electron microscope levels.