RESUMEN
Various extracts from the seeds of Nigella sativa have been used in traditional folk medicine to treat inflammation, liver disorders and arthritis. These seeds have been experimentally shown to possess antioxidant and hepatoprotective properties. Beside the hypoglycaemic and hypolipidemic effects, this study was carried out to evaluate, in vitro, toxicological effect of lipid extracts from the Nigella sativa seeds. The tested fractions were: (i) defatted methanolic extract, (ii) total lipid extract obtained by hexane extraction from methanolic extract and (iii) neutral and polar lipid fractions. The fractions were assessed, in vitro, for their inhibitory activity potential on the enzyme alpha-glucosidase as suppressing the enzyme activity is one among the therapeutic approaches to attenuate postprandial hyperglycemia. High inhibition of alpha-glucosidase by the two polar lipid fractions (F6 and F7) was reflected by their IC50 (0.51±0.04mg/ml and 0.55±0.09mg/ml, respectively), compared to acarbose (0.53±0.06mg/ml) and thymoquinone (0.65±0.05mg/ml). The hypoglycaemic effect of the polar lipid fraction of Nigella sativa could be explained by the inhibition of alpha-glucosidase, which is one of early steps of carbohydrate metabolism. Toxicological evaluation was investigated on precision-cut rat liver slices (PCLS). On PCLS, lipid extracts reduced ATP levels by 27 to 35%. Results indicate suggest that Nigella sativa extracts don't show a hepatoprotective effect against acetaminophen, but don't exhibit a major hepatotoxicity when tested alone.
Asunto(s)
Adenosina Trifosfato/metabolismo , Inhibidores de Glicósido Hidrolasas/farmacología , Hígado/efectos de los fármacos , Nigella sativa , Extractos Vegetales/farmacología , Animales , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Experimental/tratamiento farmacológico , Femenino , Hígado/metabolismo , Ratas , Ratas Wistar , Semillas , Estreptozocina , alfa-Glucosidasas/metabolismoRESUMEN
Polymorphonuclear neutrophils are key players in host defense against pathogens through the robust production of superoxide anion by the NADPH oxidase and the release of antibacterial proteins from granules. However, inappropriate release of these agents in the extracellular environment induces severe tissue injury, thereby contributing to the physiopathology of acute and chronic inflammatory disorders. Many studies have been carried out to identify molecules capable of inhibiting phagocyte functions, in particular superoxide anion production, for therapeutic purposes. In the present study, we show that thymoquinone (TQ), the major component of the volatile oil from Nigella sativa (black cumin) seeds strongly inhibits fMLF-induced superoxide production and granules exocytosis in neutrophils. The inhibition of superoxide anion was not due to a scavenger effect, as TQ did not inhibit superoxide anion produced by the xanthine/xanthine oxidase system. Interestingly, TQ impaired the phosphorylation on Ser-304 and Ser-328 of p47(PHOX), a cytosolic subunit of the NADPH oxidase. TQ also attenuated specific and azurophilic granule exocytosis in fMLF-stimulated neutrophils as evidenced by decreased cell surface expression of gp91(PHOX) and CD11b, and release of myeloperoxidase. Furthermore, both the PKC and MAPK pathways, which are involved in p47(PHOX) phosphorylation and granules exocytosis, respectively, were inhibited by TQ in fMLF-stimulated neutrophils. Finally, in a model of pleurisy induced by λ-carrageenan in rats, TQ reduced neutrophil accumulation in the pleural space, showing that it not only inhibits PMN functions in vitro, but also exhibits anti-inflammatory properties in vivo. Thus, TQ possesses promising anti-inflammatory therapeutic potential.
Asunto(s)
Antiinflamatorios/farmacología , Benzoquinonas/farmacología , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/efectos de los fármacos , Nigella sativa/química , Adulto , Animales , Antiinflamatorios/aislamiento & purificación , Antiinflamatorios/uso terapéutico , Benzoquinonas/aislamiento & purificación , Benzoquinonas/uso terapéutico , Western Blotting , Calcio/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Citometría de Flujo , Humanos , Masculino , NADP/metabolismo , Neutrófilos/inmunología , Neutrófilos/metabolismo , Pleuresia/tratamiento farmacológico , Pleuresia/inmunología , Pleuresia/metabolismo , Ratas Sprague-Dawley , Superóxidos/antagonistas & inhibidores , Superóxidos/metabolismoRESUMEN
In order to further understand and assess the validity of herbal medicine, we investigated the potential inhibitory effect of various extracts from Fraxinus angustifolia and Pistacia lentiscus, two plants used traditionally in Algeria against several inflammatory diseases such as rheumatism, arthritis, and gout, on purified bovine milk xanthine oxidase (XO) activity. The total phenolic contents of the leaves and bark of F. angustifolia and the leaves and seeds of P. lentiscus were estimated. P. lentiscus aqueous fractions from hexane and chloroform extractions and F. angustifolia aqueous fraction from ethyl acetate extraction inhibited XO activity by 72.74 +/- 2.63% (50% inhibitory concentration [IC(50)] = 27.52 microg/mL), 68.97 +/- 3.89% (IC(50) = 42.46 microg/mL) and 53.92 +/- 3.17% (IC(50) = 58.84 mmicroug/mL), respectively, at 100 microg/mL, compared to that of reference drug, allopurinol (98.18% [IC(50) = 6.34 microg/mL]). Moreover, at a concentration of 50 microg/mL, both P. lentiscus extracts showed inhibition rates higher than 50%. F. angustifolia leaf extracts showed only mild inhibition. Lineweaver-Burk analysis showed that the inhibitory activity exerted by F. angustifolia bark aqueous extract and P. lentiscus aqueous extracts is of mixed type, whereas the leaf extracts from F. angustifolia inhibited XO noncompetitively. Positive correlations were established between XO inhibition and total phenols (r = 0.89) and flavonoids (r = 0.93) for P. lentiscus and with total phenols (r = 0.72) and tannins (r = 0.54) for F. angustifolia. Our findings suggest that the therapeutic use of these plants may be due to the observed XO inhibition, thereby supporting their use in traditional folk medicine against inflammatory-related diseases, in particular, gout.
Asunto(s)
Regulación hacia Abajo , Inhibidores Enzimáticos/química , Fraxinus/química , Pistacia/química , Extractos Vegetales/química , Xantina Oxidasa/química , Argelia , Animales , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/enzimología , Artritis Reumatoide/inmunología , Bovinos , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Gota/tratamiento farmacológico , Gota/enzimología , Gota/inmunología , Humanos , Cinética , Leche/enzimología , Extractos Vegetales/farmacología , Extractos Vegetales/uso terapéutico , Xantina Oxidasa/inmunologíaRESUMEN
Diabetes mellitus is associated with vascular complications including an impairment of vascular function and alterations in the reactivity of blood vessels to vasoactive hormones. However, the signaling mechanisms leading to vascular dysfunction in diabetes are not fully understood. This microarray-based study was designed to identify differential gene expression between the normal and diabetic mesenteric vasculature and to investigate the effect of inhibiting epidermal growth factor receptor (EGFR) signaling on global gene expression in the mesenteric bed of streptozotocin (STZ)-induced diabetic rats. Transcriptome analysis was performed in triplicate using oligonucleotide microarrays housing 10,000 rat genes on the mesenteric bed of normal, diabetic, and diabetic rats treated with AG1478, a selective inhibitor of EGFR. Four weeks of diabetes led to a profound alteration in gene expression within the mesenteric bed with 1167 of the 3074 annotated genes being up-regulated and 141 genes down-regulated by at least 2-fold. The up-regulated gene ontologies included receptor tyrosine kinases, G-protein coupled receptors and ion channel activity. In particular, significant overexpressions of colipase, phospholipase A2, carboxypeptidases, and receptor tyrosine kinases such as EGFR, erbB2 and fibroblast growth factor receptor were observed in diabetes mesenteric vasculature. A 4-week intraperitoneal treatment of diabetic animals with AG1478 (1.2 mg/kg/alt diem) beginning on the same day as STZ injection prevented up-regulation of the majority (approximately 95%) of the genes associated with STZ diabetes including those apparently "unrelated" to the known EGFR pathway without correction of hyperglycemia. These results suggest that activation of EGFR signaling is a key initiating step that leads to induction of multiple signaling pathways in the development of diabetes-induced vascular dysfunction. Thus, therapeutic targeting of EGFR may represent a novel strategy for the prevention and/or treatment of vascular dysfunction in diabetes.
Asunto(s)
Diabetes Mellitus/metabolismo , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/fisiología , Regulación de la Expresión Génica , Arterias Mesentéricas/fisiología , Venas Mesentéricas/fisiología , Transducción de Señal/genética , Regulación hacia Arriba/genética , Animales , Diabetes Mellitus/genética , Diabetes Mellitus/fisiopatología , Receptores ErbB/genética , Masculino , Ratas , Ratas Wistar , Transducción de Señal/fisiología , Factores de Tiempo , Regulación hacia Arriba/fisiologíaRESUMEN
Renal dysfunction is a major cause of morbidity and mortality in patients with hypertension. In an attempt to understand the molecular mechanisms leading to renal dysfunction and in particular that of epidermal growth factor receptor (EGFR) and RasGTPase signaling, we analyzed global gene expression changes in the kidneys of deoxycorticosterone acetate (DOCA)-salt-induced hypertensive rats with and without treatment with AG1478, a selective inhibitor of EGFR tyrosine kinase, or FPTIII, a farnesyl transferase inhibitor known to inhibit RasGTPase. Microarray-based global gene expression analysis was performed in triplicate for each rat kidney taken from normotensive Wistar rats, DOCA-salt hypertensive (DH) rats, DH rats treated with AG1478, or DH rats treated with FPTIII. From the initial data set of 10,163 gene spots per group, upregulation of 2398 genes and downregulation of only 50 genes by more than 2-fold was observed in hypertensive rat kidneys compared to non-diseased controls. Interestingly, treatment of animals with AG1478 or FPTIII prevented upregulation of more than 97% of genes associated with hypertension in the rat kidney. Analysis of proteinuria, renal artery responsiveness and histopathology studies confirmed that DOCA-salt hypertensive rats had developed kidney damage over the study period and that this kidney dysfunction could be significantly prevented upon AG1478 or FPTIII treatment without normalising blood pressure. Taken together, our data imply that signaling cascades involving EGFR and/or RasGTPase pathways are key contributors to the induction of renal damage in hypertension and these and potentially other downstream effector molecules may serve as novel targets for therapeutic intervention.
Asunto(s)
Receptores ErbB/metabolismo , Regulación de la Expresión Génica , Hipertensión/genética , Enfermedades Renales/genética , Riñón/metabolismo , Transducción de Señal , Animales , Desoxicorticosterona/análogos & derivados , Inhibidores Enzimáticos/farmacología , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Farnesiltransferasa/antagonistas & inhibidores , Hipertensión/inducido químicamente , Hipertensión/complicaciones , Hipertensión/fisiopatología , Riñón/efectos de los fármacos , Riñón/patología , Enfermedades Renales/etiología , Enfermedades Renales/fisiopatología , Proteínas de Unión al GTP Monoméricas/antagonistas & inhibidores , Proteínas de Unión al GTP Monoméricas/genética , Proteínas de Unión al GTP Monoméricas/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Organofosfonatos/farmacología , Quinazolinas , Ratas , Ratas Wistar , Arteria Renal/efectos de los fármacos , Arteria Renal/fisiología , Cloruro de Sodio , Tirfostinos/administración & dosificación , Tirfostinos/farmacología , Vasoconstricción/efectos de los fármacos , Vasodilatación/efectos de los fármacosRESUMEN
In order to characterize the roles of tyrosine kinases (TKs) and epidermal growth factor receptor (EGFR) in diabetes-induced vascular dysfunction, we investigated the ability of a chronic administration of genistein, a broad-spectrum inhibitor of TKs and AG1478, a specific inhibitor of EGFR TK activity to modulate the altered vasoreactivity of the perfused mesenteric bed to common vasoconstrictors and vasodilators in streptozotocin (STZ)-induced diabetes in rats. The vasoconstrictor responses induced by norepinephrine (NE), endothelin-1 (ET-1) and angiotensin II (Ang II), were significantly increased, whereas vasodilator responses to carbachol and histamine were significantly reduced in the perfused mesenteric bed of STZ-induced diabetic rats in comparison with healthy rats. Treatment of diabetic animals with genistein or AG1478 produced a significant normalization of the altered agonist-induced vasoconstrictor and vasodilator responses without affecting blood glucose levels. In contrast, neither inhibitor had any effect on the vascular responsiveness of control (nondiabetic) animals. Treatment of diabetic animals with diadzein, an inactive analogue of genistein, did not affect the vasoconstrictor and vasodilator responses in control or diabetic animals. Phosphorylated EGFR levels were markedly raised in the mesenteric bed from diabetic animals and were normalized upon treatment with AG1478 or genistein. These data suggest that activation of TK-mediated pathways, including EGFR TK signalling are involved in the development of diabetic vascular dysfunction.
Asunto(s)
Diabetes Mellitus Experimental/fisiopatología , Receptores ErbB/antagonistas & inhibidores , Genisteína/farmacología , Tirfostinos/farmacología , Vasoconstricción/efectos de los fármacos , Vasodilatación/efectos de los fármacos , Angiotensina II/farmacología , Animales , Carbacol/farmacología , Línea Celular , Diabetes Mellitus Experimental/inducido químicamente , Endotelina-1/farmacología , Receptores ErbB/metabolismo , Femenino , Histamina/farmacología , Humanos , Técnicas In Vitro , Arterias Mesentéricas/efectos de los fármacos , Arterias Mesentéricas/fisiología , Venas Mesentéricas/efectos de los fármacos , Venas Mesentéricas/fisiología , Norepinefrina/farmacología , Quinazolinas , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Estreptozocina , Vasoconstrictores/farmacología , Vasodilatadores/farmacologíaRESUMEN
Small interfering RNA (siRNA), antisense oligonucleotides (ODNs), ribozymes and DNAzymes have emerged as sequence-specific inhibitors of gene expression that may have therapeutic potential in the treatment of a wide range of diseases. Due to their rapid degradation in vivo, the efficacy of naked gene silencing nucleic acids is relatively short lived. The entrapment of these nucleic acids within biodegradable sustained-release delivery systems may improve their stability and reduce the doses required for efficacy. In this study, we have evaluated the potential in vitro and in vivo use of biodegradable poly (D,L-lactide-co-glycolide) copolymer (PLGA) microspheres as sustained delivery devices for ODNs, ribozyme, siRNA and DNA enzymes. In addition, we investigated the release of ODN conjugates bearing 5'-end lipophilic groups. The in vitro sustained release profiles of microsphere-entrapped nucleic acids were dependent on variables such as the type of nucleic acid used, the nature of the lipophilic group, and whether the nucleic acid used was single or double stranded. For in vivo studies, whole body autoradiography was used to monitor the bio-distribution of either free tritium-labelled ODN or that entrapped within PLGA microspheres following subcutaneous administration in Balb-c mice. The majority of the radioactivity associated with free ODN was eliminated within 24 h whereas polymer-released ODN persisted in organs and at the site of administration even after seven days post-administration. Polymer microsphere released ODN exhibited a similar tissue and cellular tropism to the free ODN. Micro-autoradiography analyses of the liver and kidneys showed similar bio-distribution for polymer-released and free ODNs with the majority of radioactivity being concentrated in the proximal convoluted tubules of the kidney and in the Kupffer cells of the liver. These findings suggest that biodegradable PLGA microspheres offer a method for improving the in vivo sustained delivery of gene silencing nucleic acids, and hence are worthy of further investigation as delivery systems for these macromolecules.
Asunto(s)
ADN Catalítico/administración & dosificación , Silenciador del Gen , Ácido Láctico/química , Oligonucleótidos Antisentido/administración & dosificación , Ácido Poliglicólico/química , Polímeros/química , ARN Catalítico/administración & dosificación , ARN Interferente Pequeño/administración & dosificación , Animales , Autorradiografía , ADN Catalítico/química , Sistemas de Liberación de Medicamentos , Fluorouracilo/administración & dosificación , Fluorouracilo/farmacocinética , Técnicas In Vitro , Riñón/metabolismo , Hígado/metabolismo , Ratones , Ratones Endogámicos BALB C , Microesferas , Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/farmacocinética , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , ARN Catalítico/química , ARN Interferente Pequeño/química , Bazo/metabolismo , Factores de TiempoRESUMEN
Exposure of cells to paraquat leads to production of superoxide anion (O2*-). This reacts with hydrogen peroxide to give the hydroxyl radical (*OH), leading to lipid peroxidation and cell death. In this study, we investigated the effects of cellular prion protein (PrPC) overexpression on paraquat-induced toxicity by using an established model system, rabbit kidney epithelial A74 cells, which express a doxycycline-inducible murine PrPC gene. PrPC overexpression was found to significantly reduce paraquat-induced cell toxicity, DNA damage, and malondialdehyde acid levels. Superoxide dismutase (total SOD and CuZn-SOD) and glutathione peroxidase activities were higher in doxycycline-stimulated cells. Our findings clearly show that PrPC overexpression plays a protective role against paraquat toxicity, probably by virtue of its superoxide dismutase-like activity.
Asunto(s)
Daño del ADN , Paraquat/toxicidad , Proteínas PrPC/fisiología , Animales , Células Cultivadas/química , Células Cultivadas/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Doxiciclina/farmacología , Células Epiteliales/química , Células Epiteliales/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Glutatión Peroxidasa/análisis , Riñón , Malondialdehído/análisis , Ratones , Estrés Oxidativo , Proteínas PrPC/genética , Conejos , Proteínas Recombinantes de Fusión/fisiología , Superóxido Dismutasa/análisis , TransfecciónRESUMEN
Xanthine oxidoreductase (XOR) was purified for the first time from sheep's milk. The ultraviolet-visible absorption spectrum was essentially identical to those of the corresponding bovine, human, and goats' milk enzymes and showed an A280/A450 ratio of 5.35 +/- 0.24, indicating a high degree of purity. Like milk XOR from other species, sheep's milk enzyme showed a single band on SDS-PAGE corresponding to a subunit with approximate Mr 150,000. Xanthine oxidase activity of purified sheep's milk XOR (0.69 +/- 0.04 micromole urate min(-1) mg(-1)) was low relative to that of the bovine milk enzyme (1.83 +/- 0.02 micromole urate min(-1) mg(-1)), but higher than those of human or goats' milk XOR. As in the latter 2 cases, the low activity of sheep's milk XOR can be attributed to its relatively low molybdenum content (0.18 atoms per subunit), compared with that of the bovine milk enzyme (0.56 atoms Mo per subunit). Consistent with this, NADH oxidase activity of sheep's milk XOR was similar to that of enzymes purified from bovine, human, or goats' milk. The presence of desulpho-enzyme in sheep's milk XOR was demonstrated by resulfuration experiments, whereby xanthine oxidase activity was increased by approximately 75%.
Asunto(s)
Leche/enzimología , Complejos Multienzimáticos/metabolismo , NADH NADPH Oxidorreductasas/metabolismo , Ovinos/metabolismo , Xantina Deshidrogenasa/química , Xantina Oxidasa/química , Absorción , Animales , Bovinos , Electroforesis en Gel de Poliacrilamida/veterinaria , Femenino , Cabras , Humanos , Cinética , Leche Humana/enzimología , Molibdeno/análisis , Complejos Multienzimáticos/aislamiento & purificación , NADH NADPH Oxidorreductasas/aislamiento & purificación , Especificidad de la Especie , Azufre/metabolismo , Xantina Deshidrogenasa/aislamiento & purificación , Xantina Deshidrogenasa/metabolismo , Xantina Oxidasa/aislamiento & purificación , Xantina Oxidasa/metabolismoRESUMEN
PURPOSE: To evaluate low generation, G2 and G3, poly(propylenimine) dendrimers for the potential cellular delivery of antisense oligonucleotides (ODNs) targeting the epidermal growth factor receptor (EGFR) in A431 epidermoid carcinoma cells. METHODS: Cell cytotoxicity of the dendrimers was evaluated using trypan blue exclusion assays. Cellular uptake studies of fluorescently labeled ODNs were performed using fluorescence-activated cell sorting analysis. Intracellular fate of dendrimer-delivered ODNs was assessed in both fixed and live cells using fluorescent microscopy. Antisense ODN activity was assessed in terms of cancer cell growth, inhibition of target EGFR protein, and reduction in mRNA levels. RESULTS: G2 dendrimer (DAB-8) was less toxic than G3 (DAB-16) dendrimer in A431 cells, with IC50 of >175 and approximately 30 microg/ml, respectively. Uptake of fluorescently labeled ODN:dendrimer complexes was increased by up to 100-fold compared to a marker of fluid-phase endocytosis and up to 9-fold over free ODN at the optimal dendrimer:ODN (w/w) ratio of 5:1. Uptake of dendrimer:ODN complexes was significantly reduced at 4 degrees C (p < 0.05). Live cell fluorescent microscopy resulted in an intracellular distribution of dendrimer:ODN complexes that was suggestive of endocytic uptake; in contrast, cell fixation resulted in an artefactual nuclear localization. Treatment of A431 cells with anti-EGFR antisense ODN:dendrimer complexes inhibited cell growth, protein, and mRNA expression to levels comparable to Oligofectamine-mediated delivery. CONCLUSIONS: G2 and G3 poly(propylenimine) dendrimers markedly improved the delivery and activity of ODNs and thus may represent general reagents for the delivery of ODNs to cells in culture.
Asunto(s)
Dendrímeros , Oligonucleótidos Antisentido , Receptores ErbB , Citometría de Flujo , Humanos , OligonucleótidosRESUMEN
Xanthine oxidoreductase (XOR) was purified from goats' milk. The u.v.-visible absorption spectrum was essentially identical to those of the corresponding bovine and human milk enzymes and showed an A280/A450 ratio of 5.20+/-0.12, indicating a high degree of purity. Like bovine and human milk XORs, enzyme purified from goats' milk showed a single band on SDS-PAGE corresponding to a subunit with approximate Mr 150,000. On Western blotting, mouse monoclonal anti-human XOR antibody cross-reacted with purified caprine and bovine XORs. The specific xanthine oxidase activity of goats' milk XOR, however, was very much lower than that of bovine XOR, although NADH oxidase activities of XOR from the two sources were similar. In these respects, the caprine milk XOR mirrors the human milk enzyme, in which case the kinetic effects have previously been attributed to relatively low molybdenum content. The molybdenum content of goats' milk XOR also was shown to be relatively low, with 0.09 atoms Mo per subunit, compared with 055 atoms Mo per subunit for the bovine enzyme. A parallel purification of human milk XOR showed 0.03 atoms Mo per subunit. The possible physiological significance of the low molybdenum content of the caprine milk enzyme and of its correspondingly low enzymic activity is discussed.
Asunto(s)
Cabras , Leche/enzimología , Molibdeno/análisis , Xantina Deshidrogenasa/química , Xantina Oxidasa/química , Animales , Bovinos , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Cinética , Leche Humana/enzimología , Relación Estructura-Actividad , Xantina Deshidrogenasa/metabolismo , Xantina Oxidasa/metabolismoRESUMEN
Of the non-viral vectors, cationic lipid (CL) formulations are the most widely studied for the delivery of genes, antisense oligonucleotides and gene silencing nucleic acids such as small interfering RNAs. However, little is known about the impact of these delivery systems on global gene expression in target cells. In an attempt to study the geno-compatibility of CL formulations in target cells, we have used microarrays to examine the effect of Lipofectin and Oligofectamine on the gene expression profiles of human A431 epithelial cells. Using the manufacturer's recommended CL concentrations routinely used for gene delivery, cDNA microarray expression profiling revealed marked changes in the expression of several genes for both Lipofectin- and Oligofectamine-treated cells. Data from the 200 spot arrays housing 160 different genes indicated that Lipofectin or Oligofectamine treatment of A431 cells resulted in more than 2-fold altered expression of 10 and 27 genes, respectively. The downstream functional consequences of CL-induced gene expression alterations led to an increased tendency of cells to enter early apoptosis as assessed by annexin V-FITC flow cytometry analyses. This effect was greater for Oligofectamine than Lipofectin. Observed gene expression changes were not sufficient to induce any significant DNA damage as assessed by single cell gel electrophoresis (COMET) assay. These data highlight the fact that inadvertent gene expression changes can be induced by the delivery formulation alone and that these may, ultimately, have important safety implications for the use of these non-viral vectors in gene-based therapies. Also, the induced non-target gene changes should be taken into consideration in gene therapy or gene silencing experiments using CL formulations where they may potentially mask or interfere with the desired genotype and/or phenotype end-points.
Asunto(s)
Células Epiteliales/metabolismo , Expresión Génica/genética , Terapia Genética/efectos adversos , Vectores Genéticos/toxicidad , Lípidos/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosfatidiletanolaminas/genética , Anexina A5/genética , Apoptosis/genética , Células Cultivadas , Ensayo Cometa , ADN Complementario/biosíntesis , ADN Complementario/genética , Citometría de Flujo , Fluoresceína-5-Isotiocianato , Colorantes Fluorescentes , Perfilación de la Expresión Génica , Humanos , Hibridación in Situ , ARN/biosíntesis , ARN/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The molecular mechanism of neurodegeneration in transmissible spongiform encephalopathies (TSEs) remains unclear. Using radioactive copper ((64)Cu) at physiological concentration, we showed that prion infected cells display a marked reduction in copper binding. The level of full-length prion protein known to bind the metal ion was not modified in infected cells, but a fraction of this protein was not releasable from the membrane by phosphatidylinositol-specific phospholipase C. Our results suggest that prion infection modulates copper content at a cellular level and that modification of copper homeostasis plays a determinant role in the neuropathology of TSE.
Asunto(s)
Cobre/metabolismo , Neuronas/patología , Enfermedades por Prión/metabolismo , Células Cultivadas , Radioisótopos de Cobre , Humanos , Cinética , Enfermedades Neurodegenerativas/etiología , Enfermedades Neurodegenerativas/metabolismo , Fosfatidilinositol Diacilglicerol-Liasa , Fosfoinositido Fosfolipasa C , Enfermedades por Prión/etiología , Priones/metabolismo , Fosfolipasas de Tipo C/metabolismoRESUMEN
Gene silencing nucleic acids such as ribozymes, DNA enzymes (DNAzymes), antisense oligonucleotides (ODNs), and small interfering (si)RNA rely on hybridization to accessible sites within target mRNA for activity. However, the accurate prediction of hybridization accessible sites within mRNAs for design of effective gene silencing reagents has been problematic. Here we have evaluated the use of scanning arrays for the effective design of ribozymes, DNAzymes and siRNA sequences targeting the epidermal growth factor receptor (EGFR) mRNA. All three gene silencing nucleic acids designed to be complementary to the same array-defined hybridization accessible-site within EGFR mRNA were effective in inhibiting the growth of EGFR over-expressing A431 cancer cells in a dose dependent manner when delivered using the cationic lipid (Lipofectin) delivery system. Effects on cell growth were correlated in all cases with concomitant dose-dependent reduction in EGFR protein expression. The control sequences did not markedly alter cell growth or EGFR expression. The ribozyme and DNAzyme exhibited similar potency in inhibiting cell growth with IC50 values of around 750 nM. In contrast, siRNA was significantly more potent with an IC50 of about 100 nM when delivered with Lipofectin. The potency of siRNA was further enhanced when Oligofectamine was used to further improve both the cellular uptake and subcellular distribution of fluorescently labelled siRNA. Our studies show that active siRNAs can be designed using hybridization accessibility profiles on scanning arrays and that siRNAs targeting the same array-designed hybridization accessible site in EGFR mRNA and delivered using the same delivery system are more potent than ribozymes and DNAzymes in inhibiting EGFR expression in A431 cells.
Asunto(s)
ADN Catalítico/efectos de los fármacos , ADN/biosíntesis , Receptores ErbB/antagonistas & inhibidores , Silenciador del Gen/efectos de los fármacos , Ácidos Nucleicos/farmacología , ARN Catalítico/efectos de los fármacos , ARN Interferente Pequeño/metabolismo , Western Blotting , Línea Celular Tumoral , Sistemas de Liberación de Medicamentos , Citometría de Flujo , Humanos , Procesamiento de Imagen Asistido por Computador , Marcaje Isotópico , Liposomas , Hibridación de Ácido Nucleico , Ácidos Nucleicos/síntesis química , Análisis de Secuencia por Matrices de Oligonucleótidos , Oligonucleótidos/síntesis química , TransfecciónRESUMEN
Guillain-Barré syndrome following Campylobacter jejuni infection is frequently associated with anti-ganglioside autoantibodies mediated by molecular mimicry with ganglioside-like oligosaccharides on bacterial lipopolysaccharide (LPS). The regulation of antibody responses to these T-cell-independent antigens is poorly understood, and only a minority of Campylobacter-infected individuals develop anti-ganglioside antibodies. This study investigates the response to gangliosides and LPS in strains of mice by using a range of immunization strategies. In normal mice following intraperitoneal immunization, antibody responses to gangliosides and LPS are low level but can be enhanced by the antigen format or coadministration of protein to recruit T-cell help. Class switching from the predominant immunoglobulin M (IgM) response to IgG3 occurs at low levels, suggesting B1-cell involvement. Systemic immunization results in poor responses. In GalNAc transferase knockout mice that lack all complex gangliosides and instead express high levels of GM3 and GD3, generation of anti-ganglioside antibodies upon immunization with either complex gangliosides or ganglioside-mimicking LPS is greatly enhanced and exhibits class switching to T-cell-dependent IgG isotypes and immunological memory, indicating that tolerance to self gangliosides is a major regulatory factor. Responses to GD3 are suppressed in knockout mice compared with wild-type mice, in which responses to GD3 are induced specifically by GD3 and as a result of polyclonal B-cell activation by LPS. The anti-ganglioside response generated in response to LPS is also dependent on the epitope density of the ganglioside mimicked and can be further manipulated by providing secondary signals via lipid A and CD40 ligation.