In silico and in vivo protective effect of Morus nigra leaves on oxidative damage induced by iron overload.

Morus nigra L. is a plant popularly known as 'amoreira preta', very used in folk medicine. Iron overload (hemochromatosis) is a clinical condition that causes damage to various tissues due to oxidative stress. Therapy to control iron overload is still unsatisfactory. The protective effect on oxidative stress induced by iron overload was verified. Phytochemical characterization was evaluated by UHPLC-MS/MS. The in silico toxicity predictions of the main phytochemicals were performed via computer simulation. To induce iron overload, the animals received iron dextran (50 mg/kg/day). The test groups received doses of 500 and 1000 mg/kg of M. nigra extract for six weeks. Body weight, organosomatic index, serum iron, hepatic markers, cytokines, interfering factors in iron metabolism, enzymatic and histopathological evaluations were analyzed. Vanillic acid, caffeic acid, 6-hydroxycoumarin, p-coumaric acid, ferulic acid, rutin, quercitrin, resveratrol, apigenin and kaempferol were identified in the extract. In addition, in silico toxic predictions showed that the main compounds presented a low probability of toxic risk. The extract of M. nigra showed to control the mediators of inflammation and to reduce iron overload in several tissues. Our findings illustrate a novel therapeutic action of M. nigra leaves on hemochromatosis caused by iron overload.

Evaluation of anti-biofouling potential of Viburnum opulus extracts / Evaluación del potencial anti bioincrustante de extractos de Viburnum opulus

In this study the in vitro investigation of the inhibitory effect of ethanol extract of Viburnum opulus L. bark sample on Streptococcus mutans planctonic cells and biofilm has been intended. A Scanning electron microscopy analysis has been performed in order to investigate the inhibitory effect of the extract on Streptococcus mutans biofilms. Furthermore, the Exopolysaccharide and dextran production of this bacteria have been identified in the presence of the extract. It has been found out that the bark extract with the concentration of 2,5 mg/mL is able to inhibit more than 50% of the cells in the different times development phases. According to this, the exopolymeric matrix on the biofilm surface disperses and the Exopolysaccharide and dextran production get lowered in the presence of bark extract compared to the control group. It is considered that this extract can be used as an alternative approach for the new chemotherapeutic strategies against tooth decay.

En este estudio se investigó el efecto inhibitorio in vitro del extracto de etanólico de una muestra de corteza de Viburnum opulus L. en biopelículas de células planctónicas de Streptococcus mutans. Se realizó un análisis de microscopía electrónica de barrido para investigar el efecto inhibitorio del extracto sobre las biopelículas de Streptococcus mutans. Además, se identificó la producción de exopolisacárido y dextrano de esta bacteria en presencia del extracto. Se descubrió que el extracto de corteza con una concentración de 2,5 mg/ml inhibió más del 50% de las células en las diferentes fases de desarrollo. Consecuentemente, la matriz exopolimérica en la superficie de la biopelícula se dispersa y la producción de exopolisacárido y dextrano se reduce en presencia de extracto de corteza en comparación con el grupo de control. Se sugiere que este extracto puede ser usado como un enfoque alternativo para las nuevas estrategias quimioterapéuticas contra la carie dental.

Silymarin prevents diabetes-induced hyperpermeability in human retinal endothelial cells / La silimarina previene la hiperpermeabilidad inducida por la diabetes en células endoteliales de retina humana

Introduction: Vascular endothelial growth factor (VEGF) plays an essential role in development of diabetic macular edema (DME). While there is evidence suggesting that silymarin, a flavonoid extracted from Silybum marianum, could be useful for prevention and treatment of diabetic nephropathy, no studies have been conducted in diabetic retinopathy (DR). The aim of this study was to assess the effect of silymarin on disruption of inner blood retinal barrier (BRB), the primary cause of DME. Materials and methods: Human retinal endothelial cells (HRECs) were cultured under standard (5.5mM D-glucose) and diabetogenic conditions (25mM D-glucose and 25mM D-glucose + recombinant vascular endothelial growth factor [rVEGF, 25mg/mL]). To assess cell viability, three concentrations of silymarin were tested (2, 4 and 10μg/mL). The effect of silymarin on HREC disruption was determined using a dextran (70kD) permeability asssay. Results: No differences were found in the viability of HRECs treated with 2 or 4μg/mL of silymarin as compared to untreated cells, but viability significantly decreased after using 10 μg/mL. The concentration of 4 μg/mL was therefore selected. Silymarin (4μg/mL) caused a significant decrease in VEGF-induced permeability in both media with 5.5nM (422±58 vs. 600±72 ng/mL/cm2; p<0.03) and 25nM of D-glucose (354 ± 28 vs. 567 ± 102 ng/mL/cm2; p<0.04). Discussion: Our results show that silymarin is effective for preventing hyperpermeability induced by diabetic conditions in HRECs. Further studies are needed to assess whether silymarin could be useful to treat DME (AU)

Introducción: El Vascular endothelial growth factor (VEGF) juega un papel esencial en el desarrollo del edema macular diabético (EMD). Existe evidencia que indica que el uso de la silimarina, extracto flavonoide del Silybum marianum, podría ser útil en la prevención y el tratamiento de la nefropatía diabética pero no se dispone de datos en retinopatía diabética (RD). El objetivo del estudio es evaluar el efecto de la silimarina sobre la disrupción de la barrera hematorretininana, que es la causa primaria del EMD. Material y métodos: Células endoteliales de retina humana (HRECs) se cultivaron en condiciones estándar (5.5mM de D-glucosa) y en condiciones suprafisiológicas de glucosa (25mM de D-glucosa y 25mM de D-glucosa + VEGF 25mg/dl). Para evaluar la viabilidad de las células se probaron 3 concentraciones de silimarina (2, 4 y 10μg/ml). El efecto de la silimarina sobre la disrupción de las HRECs se determinó mediante análisis de permeabilidad a dextrano (70kD). Resultados: No se observaron diferencias en la viabilidad de las HRECs tratadas con 2 o 4μg/ml de silimarina en comparación con las células no tratadas, pero se observó una reducción de la viabilidad con la concentración de 10μg/ml. Por consiguiente, se seleccionó la concentración de 4μg/ml de silimarina. La silimarina (4μg/ml) produjo un descenso significativo de la permeabilidad inducida por VEGF tanto en medio con 5.5mM de D-glucosa (422 ±58 vs. 600 ±72 ng/ml/cm2; p<0.03) como en medio con 25mM de D-glucosa (354±28 vs. 567±102 ng/ml/cm2; p<0.04). Discusión: Nuestros resultados demuestran que la silimarina es efectiva para prevenir la hiperpermeabilidad inducida por condiciones suprafisiológicas de glucosa en HRECs. Son necesarios más estudios para evaluar si la silimarina podría ser útil para el tratamiento del EMD (AU)

Resolving the detailed structure of cortical and thalamic neurons in the adult rat brain with refined biotinylated dextran amine labeling.

Biotinylated dextran amine (BDA) has been used frequently for both anterograde and retrograde pathway tracing in the central nervous system. Typically, BDA labels axons and cell somas in sufficient detail to identify their topographical location accurately. However, BDA labeling often has proved to be inadequate to resolve the fine structural details of axon arbors or the dendrites of neurons at a distance from the site of BDA injection. To overcome this limitation, we varied several experimental parameters associated with the BDA labeling of neurons in the adult rat brain in order to improve the sensitivity of the method. Specifically, we compared the effect on labeling sensitivity of: (a) using 3,000 or 10,000 MW BDA; (b) injecting different volumes of BDA; (c) co-injecting BDA with NMDA; and (d) employing various post-injection survival times. Following the extracellular injection of BDA into the visual cortex, labeled cells and axons were observed in both cortical and thalamic areas of all animals studied. However, the detailed morphology of axon arbors and distal dendrites was evident only under optimal conditions for BDA labeling that take into account the: molecular weight of the BDA used, concentration and volume of BDA injected, post-injection survival time, and toning of the resolved BDA with gold and silver. In these instances, anterogradely labeled axons and retrogradely labeled dendrites were resolved in fine detail, approximating that which can be achieved with intracellularly injected compounds such as biocytin or fluorescent dyes.

Synthesis and in vitro evaluation of a biotinylated dextran-derived probe for molecular imaging.

Herein we report the design, synthesis, and in vitro evaluation of a gadolinium-containing biotinylated dextran-derived molecular imaging probe as a prospective neuroanatomical tracer by means of magnetic resonance imaging (MRI). The probe was effectively taken up by cultured differentiated murine neuroblastoma cells and significantly enhanced the contrast in T(1)- and T(2)-weighted MR images of labeled cells under physiological conditions. A significant longitudinal relaxation rate enhancement in the presence of avidin was observed allowing the verification of the results in the end of noninvasive longitudinal MRI connectivity studies by post-mortem histology. The in vitro results indicate that the probe has the potential to be used in vivo to identify the organization of global neuronal networks in the brain with MRI.

Surface-directed structure formation of ß-lactoglobulin inside droplets.

The morphology of ß-lactoglobulin structures inside droplets was studied during aggregation and gelation using confocal laser scanning microscopy (CLSM) equipped with a temperature stage and transmission electron microscopy (TEM). The results showed that there is a strong driving force for the protein to move to the interface between oil and water in the droplet, and the ß-lactoglobulin formed a dense shell around the droplet built up from the inside of the droplets. Less protein was found inside the droplets. The longer the ß-lactoglobulin was allowed to aggregate prior to gel formation, the larger the part of the protein went to the interface, resulting in a thicker shell and very little material being left inside the droplets. The droplets were easily deformed because no network stabilizes them. When 0.5% emulsifier, polyglycerol polyresinoleat (PGPR), was added to the oil phase, the ß-lactoglobulin was situated both inside the droplets and at the interface between the droplets and the oil phase; when 2% PGPR was added, the ß-lactoglobulin structure was concentrated to the inside of the droplets. The possibility to use the different morphological structures of ß-lactoglobulin in droplets to control the diffusion rate through a ß-lactoglobulin network was evaluated by fluorescence recovery after photobleaching (FRAP). The results show differences in the diffusion rate due to heterogeneities in the structure: the diffusion of a large water-soluble molecule, FITC-dextran, in a dense particulate gel was 1/4 of the diffusion rate in a more open particulate ß-lactoglobulin gel in which the diffusion rate was similar to that in pure water.

Biotinylated dextran amine as a marker for fetal hypothalamic homografts and their efferents.

We have explored the use of biotinylated dextran amine (BDA) as a marker for labeling fetal brain grafts and their connections with the host. As a model system we used transplantation of the hamster suprachiasmatic nucleus, the site of an endogenous biological clock governing circadian rhythms. Similar transplants into arrhythmic hosts have been shown to restore behavioral function with a period specific to the donor. For locomotor rhythms, efferent connections are not necessary. For other responses, including endocrine rhythms, efferent connections may be necessary. In order to visualize homografts and their efferents, injections of BDA, an anterograde tracer, were made into the anterior hypothalamic (AH) region containing the SCN or into the dorsal cortex (CTX) of fetal hamster brains. The fetal AH or CTX was microdissected out and stereotaxically implanted into the third ventricle of intact, adult hamsters. After 2, 4, 8, or 12 weeks, hosts were sacrificed and their brains were processed for detection of BDA by either histochemistry or immunofluorescence. BDA intensely labeled graft neurons, their dendrites, and axons with minimal or no spread to the adjacent host brain. Labeled graft axons could be followed for long distances (>1 mm) into the host brain and graft-derived varicosities formed close contacts with host neurons. BDA-labeled graft neurons, located at the perimeter of the graft, also extended dendrite-like processes into the host parenchyma. We conclude that BDA is a useful marker for fetal homografts and their efferents for survival times of less than 2 months.

Multiple neuroanatomical tracing in primates.

The present report deals with a multiple tract-tracing procedure in non-human primates enabling the simultaneous visualization of retrogradely transported Fluoro-Gold (FG) and cholera toxin B subunit (CTB) in combination with anterogradely transported biotinylated dextran amine (BDA). Two issues have played key roles on the achievement of this reliable procedure: first, the recent development of a commercial antiserum against FG that allows us to convert the original fluorescent signal of this dye in a permanent precipitate via standard peroxidase-anti-peroxidase methods; second, the introduction of the novel peroxidase substrate Vector(R) VIP (V-VIP), resulting in a purple precipitate. The combination of these neuroanatomical tracers in one and the same histological section opens a possibility for the permanent visualization of the convergence of inputs from a particular brain area onto identified, two different subsets of projection cells of another area. Furthermore, this combination of three tracers emerges as a powerful technical tool for obtaining broad amounts of complementary data regarding the monkey brain connectivity, thus significantly reducing the number of animals needed to complete a particular study.

Intestinal transmission of macromolecules in newborn dairy calves of different ages at first feeding.

Four groups of eight newborn calves were used to study the intestinal transmission of colostral immunoglobulin from the intestinal lumen to the blood circulation. The first feed was given one, eight, 16 or 24 hours after birth. Thereafter, three feeds were given with eight hour intervals. All feeds were from the same pool of colostrum and the amount fed each time corresponded to 3 per cent of the calves birthweight. To estimate the transmission of macromolecules in each feed four different macromolecules were used as markers. For the first feed, the marker was bovine IgG, in the second FITC-dextran, in the third ovalbumin and in the fourth human serum albumin. Blood samples were taken eight hours after each feed and at one week old. There were no differences in transmission for the first feed although the calves varied in age between one and 24 hours, but in the second, third and fourth feeds the calves that received a first feed at one hour old, transmitted significantly more of the marker molecules than did the other three groups. The substantial transmission of macromolecules at the first feed in all four groups indicates that a base level of transmission capacity is maintained during the first 24 hours or longer and that, under certain conditions, acceptable passive immunisation is possible in calves given their first colostrum as late as 24 hours after birth.

Properties of oil/water emulsions stabilized by casein-acid polysaccharide mixtures.

Properties of oil/water emulsions stabilized with the soluble casein-acid polysaccharide mixtures were investigated. Compared to initial protein solutions, higher emulsifying properties of the mixtures are demonstrated. A study is made on the influence of the properties of the mixtures for obtaining thermostable emulsions of hard consistency which could be applied in production of a great variety of foodstuffs. The role of the formation of protein-acid polysaccharide complexes is discussed.

A bound form of silicon in glycosaminoglycans and polyuronides.

Silicon was found to be a constituent of certain glycosaminoglycans and polyuronides, where it occurs firmly bound to the polysaccharide matrix. 330-554 ppm of bound Si were detected in purified hyaluronic acid from umbilical cord, chondroitin 4-sulfate, dermatan sulfate, and heparan sulfate. These amounts correspond to 1 atom of Si per 50,000-85,000 molecular weight or 130-280 repeating units. 57-191 ppm occur in chondroitin 6-sulfate, heparin, and keratan sulfate-2 from cartilage, while hyaluronic acids from vitreous humor and keratan sulfate-1 from cornea were Si-free. Large amounts of bound Si are also present in pectin (2580 ppm) and alginic acid (451 ppm). The bound Si is not dialyzable, does not react with ammonium molybdate, is not liberated by autoclaving or 8 M urea, and is stable against weak alkali and acid. Strong alkali and acid hydrolyze the Si-polysaccharide bond. Free, direct-reacting, dialyzable silicate is obtained. Enzymatic hydrolysis of hyaluronic acid or pectin does not liberate silicic acid, but leads to products of low molecular weight still containing Si in bound form. It is concluded that Si is present as a silanolate, i.e., an ether (or esterlike) derivative of silicic acid, and that R(1)-O-Si-O-R(2) or R(1)-O-Si-O-Si-O-R(2) bridges play a role in the structural organization of glycosaminoglycans and polyuronides. Thus, Si may function as a biological crosslinking agent and contribute to architecture and resilience of connective tissue.