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Generation of a Human Neuronal Stable Cell Model for Niemann-Pick C Disease by RNA Interference

Laura Rodríguez-Pascau, Maria Josep Coll, Josefina Casas, Lluïsa Vilageliu, and Daniel Grinberg

JIMD Rep., 4, 29–37, 2012

Niemann–Pick type C (NPC) disease is a fatal autosomal recessive neurodegenerative disorder caused, most commonly, by mutations in the NPC1 gene. At the cellular level, the disease is characterized by the storage of multiple lipids in the endosomal–lysosomal system, including free cholesterol, glycosphingolipids, sphingomyelin and the catabolic product of sphingolipids, sphingosine. Therapeutic options for NPC disease are relatively limited. One drawback for the development of novel therapies is the lack of suitable human neuronal cell models. In this work, a stable SH-SY5Y cell model for NPC disease was generated using short hairpin RNAs. An inhibition of the NPC1 expression of around 90% was obtained at the RNA level. The NPC1 knockdown was confirmed at the protein level. To characterize the stable cell line generated, cholesterol levels were analyzed in the NPC1-knockdown SH-SY5Y cells by filipin staining and gas chromatography–mass spectrometry. A characteristic NPC pattern and a twofold increase of the free cholesterol levels, related to intact SH-SY5Y cells, were found. Moreover, sphingolipids were analyzed by liquid chromatography–mass spectrometry and an increase in ganglioside GM2 levels was observed. The stable NPC1-knockdown SH-SY5Y cell line generated in the present study provides a human neuronal cell model for this lethal disease that could be a valuable tool for the study of future therapeutic approaches.

 

D-Fagomine lowers postprandial blood glucose and modulates bacterial adhesion

Livia Gómez, Eunice Molinar-Toribio, María Ángeles Calvo-Torras, Carles Adelantado, M. Emília Juan, Joana M. Planas, Xavier Cañas, Carles Lozano, Sergio Pumarola, Pere Clapés and Josep Lluís Torres

British Journal of Nutrition, 107, 1739–1746, 2012

D-Fagomine is an iminosugar originally isolated from seeds of buckwheat (Fagopyrum sculentum Moench), present in the human diet and now available as a pure crystalline product. We tested D-fagomine for activities connected to a reduction in the risk of developing insulin resistance, becoming overweight and suffering from an excess of potentially pathogenic bacteria. The activities were: intestinal sucrase inhibition in vitro (rat mucosa and everted intestine sleeves), modulation of postprandial blood glucose in rats, bacterial agglutination and bacterial adhesion to pig intestinal mucosa. When ingested together with sucrose or starch, D-fagomine lowered blood glucose in a dose-dependent manner without stimulating insulin secretion. D-Fagomine reduced the area under the curve (0–120 min) by 20% (P<0·01) and shifted the time to maximum blood glucose concentration (Tmax) by 15 min at doses of 1–2 mg/kg body weight when administered together with 1 g sucrose/kg body weight. Moreover, D-fagomine (0·14mM) agglutinated 60% of Enterobacteriaceae (Escherichia coli, Salmonella enterica serovar Typhimurium) populations (P<0·01), while it did not show this effect on Bifidobacterium spp. or Lactobacillus spp. At the same concentration, D-fagomine significantly (P<0·001) inhibited the adhesion of Enterobacteriaceae (95–99% cells in the supernatant) and promoted the adhesion of Lactobacillus acidophilus (56% cells in the supernatant) to intestinal mucosa. D-Fagomine did not show any effect on bacterial cell viability. Based on all this evidence, D-fagomine may be used as a dietary ingredient or functional food component to reduce the health risks associated with an excessive intake of fast-digestible carbohydrates, or an excess of potentially pathogenic bacteria.

 

C6-Ceramide and targeted inhibition of acid ceramidase induce synergistic decreases in breast cancer cell growth

Margaret Flowers, Gemma Fabriás, Antonio Delgado, Josefina Casas, Jose Luis Abad, Myles C. Cabot

Breast Cancer Res Treat, 133, 447–458, 2012

The sphingolipid ceramide is known to play a central role in chemo- and radiation-induced cell death. Acid ceramidase (AC) hydrolyzes ceramide, and thus reduces intracellular levels of this proapoptotic lipid. The role of AC as a putative anticancer target is supported by reports of upregulation in prostate cancer and in some breast tumors. In this study, we determined whether the introduction of an AC inhibitor would enhance the apoptosisinducing effects of C6-ceramide (C6-cer) in breast cancer cells. Cultured breast cancer cells were treated with DM102 [(2R,3Z)-N-(1-hydroxyoctadec-3-en-2-yl)pivalamide, C6-cer, or the combination. Cell viability and cytotoxic synergy were assessed. Activation of apoptotic pathways, generation of reactive oxygen species, and mitochondrial transmembrane potential were determined. DM102 was a more effective AC inhibitor than N-oleoylethanolamine (NOE) and (1R,2R)-2-N-(tetradecanoylamino)-1-(40-nitrophenyl)-1,3-propandiol (B-13) in MDA-MB-231, MCF-7, and BT-474 cells. As single agents, C6-cer (IC50 5–10 lM) and DM102 (IC50 20 lM) were only moderately cytotoxic in MDA-MB-231, MCF-7, and SK-BR-3 cells. Co-administration, however, produced synergistic decreases in viability (combination index\0.5) in all cell lines. Apoptosis was confirmed in MDA-MB-231 cells by detection of caspase 3 cleavage and a[3-fold increase in caspase 3/7 activation, PARP cleavage, and a[70% increase in Annexin-V positive cells. C6-cer/DM102 increased ROS levels 4-fold in MDAMB-231 cells, shifted the ratio of Bax:Bcl-2 to[9-fold that of control cells, and resulted in mitochondrial membrane depolarization. DM102 also increased the synthesis of 3Hpalmitate-labeled long-chain ceramides by 2-fold when C6-cer was present. These data support the effectiveness of targeting AC in combination with exogenous short-chain ceramide as an anticancer strategy, and warrant continued investigation into the utility of the C6-cer/DM102 drug duo in human breast cancer.
 

 

Optimisation of the operational conditions of trichloroethylene degradation using Trametes versicolor under quinone redox cycling conditions using central composite design methodology

Marcel Vilaplana, Ana Belén García, Gloria Caminal, Francisco Guillén, Montserrat Sarrà

Biodegradation, 23 (2), 333–341, 2012

Extracellular radicals produced by Trametes versicolor under quinone redox cycling conditions can degrade a large variety of pollutant compounds, including trichloroethylene (TCE). This study investigated the effect of the agitation speed and the gas–liquid phase volume ratio on TCE degradation using central composite design (CCD) methodology for a future scale-up to a reactor system. The agitation speed ranged from 90 to 200 rpm, and the volume ratio ranged from 0.5 to 4.4. The results demonstrated the important and positive effect of the agitation speed and an interaction between the two factors on TCE degradation. Although the volume ratio did not have a significant effect if the agitation speed value was between 160 and 200 rpm, at lower speed values, the specific pollutant degradation was clearly more extensive at low volume ratios than at high volume ratios. The fitted response surface was validated by performing an experiment using the parameter combination in the model that maximised TCE degradation. The results of the experiments carried out using different biomass concentrations demonstrated that the biomass concentration had a positive effect on pollutant degradation if the amount of biomass present was lower than 1.6 g dry weight l-1. The results show that the maximum TCE degradation was obtained at the highest speed (200 rpm), gas–liquid phase volume ratio (4.4), and a biomass concentration of 1.6 g dry weight l-1.

 

Three-Dimensional Interdigitated Electrode Array as a Tool for Surface Reactions Registration

Andrey Bratov, Natalia Abramova, M. Pilar Marco, Francisco Sanchez-Baeza

Electroanalysis 24 (1), 69 – 75, 2012

Possible applications of a new transducer based on a three dimensional interdigitated electrode array (3D-IDEA) with electrode digits separated by an insulating barrier are discussed. Due to the presence of insulating barriers that separate the adjacent digits of the electrodes the main portion of the probing electrical current goes close to the surface of the barrier. Chemical modification of the barrier surface with the probe molecules permits to realise direct detection of subsequent target analytes in solution. The functional mechanism of the device is based on registration of changes in conductivity at the surface of the barrier provoked by electrical charge redistribution caused by surface chemical reactions. Three-dimensional sensor shows considerable improvement in sensitivity compared with a standard planar IDEA design sensors. The potential of the developed device as a sensor transducer to detect various chemical and bio chemical reactions is demonstrated. Examples include enzyme immobilisation, polyelectrolytes layer-by-layer deposition, covalent chemical modification with molecules containing reactive thiol groups.

 


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