RESUMEN

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease spread throughout the world. The most frequent causes of death in NAFLD patients are due both to liver and cardiovascular damage. Several pathways, including the dimethylarginine dimethylaminohydrolase (DDAH)/asymmetric dimethylarginine (ADMA) pathway, are involved in the pathogenesis of NAFLD. It has been reported that ADMA plasmatic levels are increased in patients with hepatic dysfunction such as NAFLD. Although many studies demonstrated that some foods are effective in the treatment of NAFLD, few studies have evaluated their effects with respect to the prevention of the disease. It has been reported that sweet orange juice (OJ) consumption may be associated with potential health benefits. However, some varieties of sweet orange are more effective than others. The aim of the present paper was to investigate the effect of blond and blood sweet orange juice in prevention of NAFLD by evaluating its ability to improve liver steatosis in mice with diet-induced obesity, reducing oxidative stress and affecting the DDAH/ADMA pathway. Results obtained in our experimental conditions evidenced that blood orange juice rather than blond orange juice was more effective. Blood orange juice or blond orange juice enriched in anthocyanins may represent a promising dietary option for the prevention of fatty liver disease.

RESUMEN

This paper describes an automated method for extracting the apparent positions of fiducial points from 2D or 3D images of a phantom. We consider a 3D-lattice phantom for two main reasons: first, ease of manufacture and isotropy of its structure with respect to coordinate projections; second, a connected structure allowing to uniquely assess the adjacency relationship between fiducial points even if geometric distortions arising from main magnet inhomogeneity and gradient fields non-linearity is severe as observed in open-bore systems. In order to validate our proposed method and compare different choices for the parameters of our phantom (i.e. number and distance between grids and thickness of its branches) we developed in-house a software for simulating 2D or 3D volume images of the phantom, using customizable MRI sequence parameters and Spherical Harmonic Coefficients for the fields. We deem worthy of note that using simulated images is the only way to evaluate the estimated position error, since it allows to compare the estimates to their theoretical counterparts. Furthermore, the use of simulated images allows to evaluate the robustness of the method with respect to image quality in terms of Signal-to-Noise Ratios and geometric distortion, and allows to evaluate different phantom geometries without having to manufacture them. The proposed method can be easily extended to phantoms having an arbitrary overall shape, as long as it is a fully connected structure. Specifically, it is easy to design a phantom with fiducial points laying outside of the homogeneity sphere, so that indirect measurement of the fields becomes possible, for example by using the recent method proposed by Acquaviva et al. To the best of our knowledge, the proposed method outperforms other state-of-the-art methods, with an average positioning offset of 0.052 mm (with a 0.99 quantile of 0.12 mm) when working on images featuring a differential Signal-to-Noise Ratio within Region-of-Interest (ROI) equal to 105 (20.2 dB) and a ROI-to-background SNR of 20 dB. Estimating the positions of 6859 fiducial points in a volume, our highest density case, was carried out in less than 30 min on a desktop personal computer.

Asunto(s)

Imagenología Tridimensional/normas , Imagen por Resonancia Magnética/normas , Reconocimiento de Normas Patrones Automatizadas/métodos , Fantasmas de Imagen , Aire , Simulación por Computador , Procesamiento Automatizado de Datos , Humanos , Modelos Teóricos , Relación Señal-Ruido , Programas Informáticos

RESUMEN

OBJECTIVE: Moringa oleifera Lam., a multipurpose tree, is used traditionally for its nutritional and medicinal properties. It has been used for the treatment of a variety of conditions, including inflammation, cancer and metabolic disorders. MATERIALS AND METHODS: We investigated the effect of Moringa oleifera Lam. on adipogenic differentiation of human adipose-derived mesenchymal stem cells and its impact on lipid metabolism and cellular antioxidant systems. RESULTS: We showed that Moringa oleifera Lam. treatment during adipogenic differentiation reduces inflammation, lipid accumulation and induces thermogenesis by activation of uncoupling protein 1 (UCP1), sirtuin 1 (SIRT1), peroxisome proliferator-activated receptor alpha (PPARα), and coactivator 1 alpha (PGC1α). In addition, Moringa oleifera Lam. induces heme oxygenase-1 (HO-1), a well established protective and antioxidant enzyme. Finally Moringa oleifera Lam. significantly decreases the expression of molecules involved in adipogenesis and upregulates the expression of mediators involved in thermogenesis and lipid metabolism. CONCLUSIONS: Our results suggest that Moringa oleifera Lam. may promote the brown remodeling of white adipose tissue inducing thermogenesis and improving metabolic homeostasis.

Asunto(s)

Diferenciación Celular/efectos de los fármacos , Metabolismo de los Lípidos/efectos de los fármacos , Moringa oleifera , Extractos Vegetales/farmacología , Células Madre , Antioxidantes/farmacología , Hemo-Oxigenasa 1 , Humanos

RESUMEN

AIM: To verify the involvement of free radicals in tumor progression and to investigate the effects of an ethanolic extract of Ruta Chalepensis L. and of rutin in blood of patients with colon cancer. MATERIALS AND METHODS: Leaves of Ruta Chalepensis L. were collected in the area around Catania (Italy). For the preparation of the ethanol extract of leaves, an exhaustive extraction of 100 g of the drug was carried out in Soxhlet with 800 ml of 95% ethanol. Fifty-six patients with colorectal cancer were randomly selected for this study; among these, 34 were affected by an early stage (T1 N0 M0 according to scale), while 22 were affected by an advanced stage (T4, N1-2, M0) of cancer. Data obtained from these patients were compared with those of a control group consisting of 20 healthy subjects. Plasma of each sample was used for determining non-proteic antioxidant capacity, thiol groups, lipid hydroperoxides and nitrite/nitrate levels, evaluated by spectrophotometric tests. In addition, percentage of haemolysis was evaluated incubating (for 2 hours at 37 degrees C) erythrocyte suspension with a free radical donor (50 mM 2,2'-azobis-amidino propane chloridrate), in the presence or absence of ethanolic extract of Ruta Chalepensis L. (250 microg/ml) or rutin (1 mM). RESULTS: Non-proteic antioxidant capacity was significantly lower in cancerous patients than in healthy subjects (p < 0.001). This decrease was stage-related. In fact, non-proteic antioxidant capacity resulted lower in advanced than in early colorectal cancer (p < 0.001). The same significant stage-related decrease was observed in plasma thiol groups (p < 0.001). Coherently with the decrease in non-proteic antioxidant capacity and thiol groups, higher levels of lipid hydroperoxides and nitrite/nitrate were observed in patients with colorectal cancer with respect to healthy subjects (p < 0.001) and the increase in these markers of oxidative stress was related to the cancer stadiation. Neoplastic patients also showed an increased percentage of oxidative hemolysis respect to controls and the haemolytic damage was correlated with the stage of colon cancer. Both the extract of Ruta Chalepensis L. and rutin were able to protect erythrocytes from oxidative stress induced by the free radical donor, but the extract of Ruta Chalepensis L. was more effective than rutin. This protective effect was significant only in erythrocytes from patients with early colorectal group, whereas no significant modification was induced by Ruta Chalepensis L. or rutin in red blood cells from advanced colorectal cancer patients exposed to the same experimental conditions. CONCLUSION: Oxidative stress correlates with colon cancer stadiation and both the extract of Ruta chalepensis and rutin are able to protect red blood cells from radical-induced damage. However, their effects are significant in early stages of cancer. So these natural antioxidants might be usefull to prevent carcinogenesis and/or tumor progression.

Asunto(s)

Antioxidantes/uso terapéutico , Neoplasias del Colon/tratamiento farmacológico , Fitoterapia , Extractos Vegetales/uso terapéutico , Ruta , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias del Colon/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estrés Oxidativo

RESUMEN

AIM OF THE STUDY: Roots of Helleborus bocconei Ten. subsp. siculus (Schiffner) Merxm. & Podl. are widely used in veterinary folk medicine in Sicily (Italy) to diagnose and cure lower respiratory tract infections in cattle. This study intended to evaluate the in vitro antibacterial activity of the methanolic root extract of Helleborus bocconei Ten. subsp. siculus, and of the bufadienolide and ecdysteroid fractions extracted from its roots, against strains belonging to species commonly associated with respiratory tract infections. MATERIALS AND METHODS: The phytochemical screening of the previously prepared plant extracts was carried out by chemical, thin-layer chromatography and spectroscopic methods. The in vitro antibacterial activity of the extracts against seven different standard bacterial strains was evaluated by broth microdilution. RESULTS: According to the present study, Helleborus bocconei Ten. subsp. siculus roots contain bufadienolides and ecdysteroids, and the extracts containing these compounds, as well as the crude methanolic root extract, show antibacterial activity against microorganisms responsible for respiratory infections. In particular, the bufadienolide extract has the highest inhibitory activity against all the tested organisms, and, as the other extracts, shows the lowest MIC values (100mug/ml) against Moraxella catarrhalis and Streptococcus pneumoniae. CONCLUSIONS: Since the roots of Helleborus bocconei Ten. subsp. siculus contain substances with antibacterial activity, the traditional use of this plant may also derive from its antibacterial properties.

Asunto(s)

Antibacterianos/farmacología , Helleborus/química , Extractos Vegetales/farmacología , Raíces de Plantas/efectos de los fármacos , Cromatografía en Capa Delgada , Pruebas de Sensibilidad Microbiana

RESUMEN

Nitric oxide (NO), produced by nitric oxide synthase, is implicated in the pathophysiology of renal ischemia/reperfusion (I/R) injury. This study sought to elucidate the impact of pharmacological induction of heme oxygenase-1 (HO-1) on renal I/R injury. Rats were subjected to 45 minutes of renal ischemia followed by various times of reperfusion (30 minutes, 1 hour, or 3 hours). Plasma from sacrificed rats was obtained, and the kidneys processed for the expression of iNOS, cleaved caspase-3, p38MAPK and for immunohistochemical analysis. Furthermore, we determined renal and plasma levels of lipid hydroperoxides, total thiol groups, and plasmatic NO2-/NO3- formation. Our results showed a time-dependent increase in iNOS expression, which was also confirmed by increased plasma formation of NO2-/NO3-. Interestingly, this effect was reversed by pretreatment (12 hours) with SnCl2, a potent and specific inducer of renal HO-1 expression and activity, or by intraperitoneal injection of biliverdin (10 mg/kg). Furthermore, we observed a concomitant reduction in plasma and renal LOOH formation, a normalization of renal total thiol content, a reduction of caspase-3-mediated apoptosis, and a significant increase in p38MAPK phosphoration. Taken together, these results suggested that HO-1 and its byproduct biliverdin play major roles in the pathophysiological cascade leading to renal I/R injury.

Asunto(s)

Inhibidores de la Ciclooxigenasa/farmacología , Hemo-Oxigenasa 1/biosíntesis , Óxido Nítrico Sintasa de Tipo II/metabolismo , Estrés Oxidativo/fisiología , Circulación Renal , Daño por Reperfusión/fisiopatología , Animales , Modelos Animales de Enfermedad , Inducción Enzimática/efectos de los fármacos , Isoenzimas/biosíntesis , Nitratos/metabolismo , Óxido Nítrico Sintasa de Tipo II/genética , Nitritos/metabolismo , Ratas , Compuestos de Sulfhidrilo/metabolismo

RESUMEN

The interaction of a series of amphiphilic 2-alkyl aminoacids (lipoamino acids, LAAs) with different cell cultures and biomembrane models was investigated. LAAs can be useful promoieties to modify the physico-chemical properties of many drugs, and in particular their lipophilicity. Tests were performed in vitro on mammalian cells (murine astrocytes) and human red blood cells (haemolysis), and in vivo on rabbit eye as alternative models to assess the tolerability or the potential damaging effects of these compounds on different biological systems. The mode of interaction of LAAs with pure phospholipid multilamellar liposomes, taken as a biomembrane model, was also analysed by differential scanning calorimetry experiments. Different tolerability/toxicity patterns were obtained in the various models; in particular, the most lipophilic terms of the series, methyl 2-aminohexadecanoate (LAA16), displayed haemolytic activity and toxicity for mouse astrocyte cultures. A specific assay confirmed that LAA16 acted at level of cell membranes, while neither any damaging effects on nucleus or apoptotic induction were observed. The shorter-chain LAAs and the tetradecyl homologue (LAA14) showed the best compatibility with the various cell models.

Asunto(s)

Aminoácidos/farmacología , Membrana Celular/efectos de los fármacos , Lípidos/farmacología , Liposomas , Tensoactivos/farmacología , Aminoácidos/química , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Rastreo Diferencial de Calorimetría , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Dimiristoilfosfatidilcolina/química , Relación Dosis-Respuesta a Droga , Membrana Eritrocítica/efectos de los fármacos , Eritrocitos/efectos de los fármacos , Ojo/efectos de los fármacos , Ojo/patología , Hemólisis/efectos de los fármacos , Humanos , Lípidos/química , Masculino , Fluidez de la Membrana/efectos de los fármacos , Ratones , Ácido Mirístico , Ácido Palmítico , Conejos , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Solubilidad , Tensoactivos/química

RESUMEN

The reperfusion of ischemic tissue often delays its physiological and functional recovery; this paradoxical effect is ascribed to increased release of free radicals including O(2)(-) and NO. For these reasons, scavenging reactive oxygen species or inhibition the NO synthesis has been shown to result in an enhanced neuronal survival after cerebral ischemia. Many authors believe that therapy for stroke patients would be a cocktail of drugs with various mechanisms of action. Combination therapy is a difficult and complicated avenue for drug development because of the possibility of drug-drug interactions. An alternative approach would be to combine multiple activities within the same compound. In consideration of the free-radical scavenging and inhibitory effect on NOS of various natural and synthetic compounds, the aim of this study was to analyze the antioxidant properties of some imidazole derivatives previously synthesized in our laboratory. Results obtained in the present study provide evidence that tested compounds exhibit interesting antioxidant properties, expressed either by their capacity to scavenge free radicals or their ability to reduce lipid peroxidation. In particular, compounds A and B represent chemical structures which can be easily modified to improve the observed antioxidant properties and to provide new therapeutic strategies focused on multiple downstream events.

Asunto(s)

Antioxidantes/farmacología , Inhibidores Enzimáticos/farmacología , Imidazoles/farmacología , Óxido Nítrico Sintasa de Tipo I/antagonistas & inhibidores , Peróxidos Lipídicos/metabolismo , Solubilidad

RESUMEN

Anthocyanins, colored flavonoids, are water-soluble pigments present in the plant kingdom; in fact they are secondary plant metabolites responsible for the blue, purple, and red color of many plant tissues. Present in beans, fruits, vegetables and red wines, considerable amounts of anthocyanins are ingested as constituents of the human diet (180-215 mg daily). There is now increasing interest in the in vivo protective function of natural antioxidants contained in dietary plants against oxidative damage caused by free radical species. Recently, the antioxidant activity of phenolic phytochemicals, has been investigated. Since the antioxidant mechanism of anthocyanin pigments is still controversial, in the present study we evaluated the effects of cyanidin and cyanidin 3-O-beta-D-glucoside on DNA cleavage, on their free radical scavenging capacity and on xanthine oxidase activity. Cyanidin and cyanidin 3-O-beta-D-glucoside showed a protective effect on DNA cleavage, a dose-dependent free radical scavenging activity and significant inhibition of XO activity. These effects suggest that anthocyanins exhibit interesting antioxidant properties, and could therefore represent a promising class of compounds useful in the treatment of pathologies where free radical production plays a key role.

Asunto(s)

Antocianinas/farmacología , Antioxidantes/farmacología , ADN/efectos de los fármacos , Glucósidos/farmacología , Antocianinas/metabolismo , Compuestos de Bifenilo/farmacología , ADN/química , Radicales Libres , Hidrazinas/farmacología , Modelos Químicos , NAD/metabolismo , Picratos , Plásmidos/efectos de los fármacos , Xantina Oxidasa/metabolismo

RESUMEN

Flavonoids have recently aroused considerable interest because of their broad pharmacological activity. In fact, flavonoids have been reported to have antiviral, antiallergic, antiplatelet, anti-inflammatory and antitumoral activities. The pharmacological properties of bioflavonoids have been ascribed both to the concomitant inhibition of enzymes involved in the production of free radicals and to their free-radical scavenging and iron chelating capacity. However the antioxidant capacity of bioflavonoids due to free-radical scavenging and/or to iron chelating is still controversial. In this study, we have investigated the free-radical scavenging capacity of bioflavonoids (rutin, catechin, and naringin). In addition, the effects of these polyphenols on xanthine oxidase activity, spontaneous lipid peroxidation, and DNA cleavage were investigated. The bioflavonoids under examination showed a dose-dependent free-radical scavenging effect, a significant inhibition of xanthine oxidase activity, and an antilipoperoxidative capacity. In addition, they showed a protective effect on DNA cleavage.

Asunto(s)

Antioxidantes/farmacología , Daño del ADN/efectos de los fármacos , Flavanonas , Flavonoides/farmacología , Depuradores de Radicales Libres/farmacología , Animales , Antioxidantes/metabolismo , Catequina/metabolismo , Catequina/farmacología , Daño del ADN/fisiología , Activación Enzimática/efectos de los fármacos , Flavonoides/metabolismo , Depuradores de Radicales Libres/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Peroxidación de Lípido/fisiología , Ratas , Ratas Wistar , Rutina/metabolismo , Rutina/farmacología , Xantina Oxidasa/metabolismo

RESUMEN

L-Propionylcarnitine, a propionyl ester of L-carnitine, increases the intracellular pool of L-carnitine. It exhibits a high affinity for the enzyme carnitine acetyltransferase (CAT) and, thus, is readily converted into propionyl-coenzyme A and free carnitine. It has been reported that L-propionylcarnitine possesses a protective action against heart ischemia-reperfusion injury; however, the antioxidant mechanism is not yet clear. L-Propionylcarnitine might reduce the hydroxyl radical production in the Fenton system, by chelating the iron required for the generation of hydroxyl radicals. To obtain a better insight into the antiradical mechanism of L-propionylcarnitine, the present research analyzed the superoxide scavenging capacity of L-propionylcarnitine and its effect on linoleic acid peroxidation. In addition, the effect of L-propionylcarnitine against DNA cleavage was estimated using pBR322 plasmid. We found that L-propionylcarnitine showed a dose-dependent free-radical scavenging activity. In fact, it was able to scavenge superoxide anion, to inhibit the lipoperoxidation of linoleic acid, and to protect pBR322 DNA from cleavage induced by H2O2 UV-photolysis.

Asunto(s)

Antiinflamatorios no Esteroideos/farmacología , Antioxidantes/farmacología , Carnitina/análogos & derivados , Carnitina/farmacología , Daño del ADN/efectos de los fármacos , Superóxidos/metabolismo , Antiinflamatorios no Esteroideos/metabolismo , Antioxidantes/metabolismo , Carnitina/metabolismo , Relación Dosis-Respuesta a Droga , Peróxido de Hidrógeno/farmacología , Ácido Linoleico/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Oxidantes/farmacología , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismo , Rayos Ultravioleta

RESUMEN

The genus Cistus includes many typical species of Mediterranean flora; Cistus species are used as antidiarrhetics, as general remedies for treatment of various skin diseases in folk medicine and as anti-inflammatory agents. These species contain flavonoids that are considered to be chain-breaking antioxidants. In this work, we have investigated the effects of crude aqueous extracts from Cistus incanus and Cistus monspeliensis on DNA cleavage and their free-radical scavenging capacity. In addition, their effect on lipid peroxidation in rat liver microsomes was evaluated. These extracts showed a protective effect on DNA cleavage and a dose-dependent free-radical scavenging capacity; Cistus monspeliensis was more active than Cistus incanus; these results were confirmed by a significant inhibition of lipid peroxidation in rat liver microsomes. The experimental evidence, therefore, suggests that because of their antioxidant activity these extracts may offer excellent photoprotection for skin and may be useful in the treatment of human diseases where oxidative stress plays a key role.

Asunto(s)

Antioxidantes/farmacología , Daño del ADN/efectos de los fármacos , Extractos Vegetales/farmacología , Plantas Medicinales , Animales , Antioxidantes/metabolismo , Depuradores de Radicales Libres/metabolismo , Depuradores de Radicales Libres/farmacología , Peróxido de Hidrógeno/farmacología , Peroxidación de Lípido/efectos de los fármacos , Peroxidación de Lípido/fisiología , Hígado/metabolismo , Microsomas/efectos de los fármacos , Microsomas/metabolismo , Oxidantes/farmacología , Extractos Vegetales/metabolismo , Ratas , Piel/metabolismo , Rayos Ultravioleta

RESUMEN

The thyroid transcription factor 1 homeodomain (TTF-1 HD) shows a peculiar DNA-binding specificity which is partially dictated by several amino acids of the recognition helix. TTF-1 preferentially recognizes sequences containing the 5'-CAAG-3' core motif while most other homeodomains, such as Antennapedia (Antp), recognizes sites containing the 5'-TAAT-3' core motif. Since phenomena of 'induced fit' may occur during protein/DNA interaction, a primary role for high affinity binding and target discrimination has to be searched in the effect played by subtle structural determinants in these proteins. By using spectroscopic analysis in aqueous solution, we compared the structural stability of TTF-1 and Antp homeodomains. Although the three-dimensional structural architecture of homeodomains is conserved, some differences are detectable in terms of their structural stability. At 24 degrees C the TTF-1 HD is less structured than the Antp HD with 24 and 34% of the residues in the alpha-helical conformation, respectively. This poor folded structure reflects into different thermal and isothermal stability between the two homeodomains. TTF-1 HD exhibits a Tm of 39 degrees C and is stabilized by a delta GDH2O of +1487 cal/mol, calculated by Urea unfolding, while Antp HD exhibits a Tm of 48 degrees C and is stabilized by a delta GDH2O of +2742 cal/mol. By using mutants of both TTF-1 and Antp HDs we demonstrate that one of the major determinants in controlling the structural stability of the recognition helix is the residue at position 54. Since previous studies have shown that also residue at position 56 is involved in stabilization of the recognition helix, we conclude that the structure of this critical element is controlled by an interplay between residues at position 54 and 56 of the homeodomain.

Asunto(s)

Proteínas de Homeodominio/química , Proteínas Nucleares/química , Factores de Transcripción/química , Secuencia de Aminoácidos , Animales , Proteína con Homeodominio Antennapedia , Secuencia de Bases , Dicroismo Circular , Cartilla de ADN/genética , Estabilidad de Medicamentos , Proteínas de Homeodominio/genética , Calor , Técnicas In Vitro , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Desnaturalización Proteica , Estructura Secundaria de Proteína , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Glándula Tiroides/metabolismo , Factor Nuclear Tiroideo 1 , Factores de Transcripción/genética

RESUMEN

The ability of asialo-thyroglobulin to bind the thyroid RHL-1 subunit of the asialoglycoprotein receptor has been investigated. Ligand blot assays show that the recombinant carbohydrate recognition domain of the thyroid RHL-1 subunit specifically interacts with rat desialated thyroglobulin. Moreover, RT-PCR and Western blot assays show that TSH deprivation decreases RHL-1 expression in PC C13 thyroid differentiated cells whereas insulin deprivation does not have any effect. The simultaneous absence of both TSH and insulin dramatically decreases the level of RHL-1 expression.

Asunto(s)

Asialoglicoproteínas/metabolismo , Receptores de Superficie Celular/metabolismo , Tiroglobulina/análogos & derivados , Glándula Tiroides/metabolismo , Tirotropina/metabolismo , Animales , Receptor de Asialoglicoproteína , Línea Celular , Conejos , Ratas , Receptores de Superficie Celular/genética , Tiroglobulina/metabolismo

RESUMEN

Oxidative damage occurring in the lenses of patients with senile cataract may be due to partially reduced forms of oxygen. We assayed the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione reductase (GSH-Red), and glucose-6-phosphate dehydrogenase (G6PD) in rat lenses at different ages (1, 4, and 24 months), and also evaluated lens glutathione (GSH) levels and the effects of chronic administration of vitamin E and sodium ascorbate. We observed a significant age-related decrease in GSH-Px, GSH-Red and G6PD activities, but no age-related change in SOD activity. Chronic treatment with both vitamin E and sodium ascorbate failed to restore enzymatic activities to the levels of younger rats. An age-related reduction in GSH content was also observed; however, chronic administration of vitamin E, but not of sodium ascorbate, restored GSH levels to those of younger rats.

Asunto(s)

Envejecimiento/metabolismo , Antioxidantes/farmacología , Ácido Ascórbico/farmacología , Cristalino/enzimología , Oxidorreductasas/metabolismo , Vitamina E/farmacología , Animales , Antioxidantes/administración & dosificación , Ácido Ascórbico/administración & dosificación , Glucosafosfato Deshidrogenasa/metabolismo , Glutatión Peroxidasa/metabolismo , Glutatión Reductasa/metabolismo , Cristalino/efectos de los fármacos , Masculino , Ratas , Ratas Wistar , Superóxido Dismutasa/metabolismo , Factores de Tiempo , Vitamina E/administración & dosificación

RESUMEN

The topology of the thyroid transcription factor 1 homeodomain (TTF-1HD)-DNA complex was investigated by a strategy which combines limited proteolysis and selective chemical modification experiments with mass spectrometry methodologies. When limited proteolysis digestions were carried out with the protein in the absence or presence of its target oligonucleotide, differential peptide maps were obtained from which the amino acid residues involved in the interaction could be inferred. Similarly, selective acetylation of lysine residues in both the isolated and the complexed homeodomain allowed us to identify the amino acids protected by the interaction with DNA. Surface topology analysis of isolated TTF-1HD performed at neutral pH was in good agreement with the three-dimensional structure of the molecule as determined by NMR studies under acidic conditions. Minor differences were detected in the C-terminal region of the protein which, contrary to NMR data, showed no accessibility to proteases. Analysis of the complex provided an experimental validation of the model proposed on the basis of the homology with the homeodomain structures described so far. An increased accessibility of the C-terminal region was observed following the interaction, suggesting its displacement from the protein core by the oligonucleotide molecule. Comparative experiments with DNA fragments differing in sequence and binding capabilities highlighted structural differences among the complexes, mainly located in the N-terminal region of the homeodomain, thus accounting for their different dissociation constants.

Asunto(s)

ADN/química , Proteínas de Homeodominio/química , Proteínas Nucleares/química , Glándula Tiroides/química , Factores de Transcripción/química , Acetilación , Animales , Simulación por Computador , ADN/metabolismo , Endopeptidasas/metabolismo , Proteínas de Homeodominio/metabolismo , Hidrólisis , Lisina/metabolismo , Sustancias Macromoleculares , Espectrometría de Masas , Modelos Moleculares , Proteínas Nucleares/metabolismo , Oligonucleótidos/metabolismo , Unión Proteica , Ratas , Factor Nuclear Tiroideo 1 , Factores de Transcripción/metabolismo

RESUMEN

The thyroid transcription factor 1 (TTF-1) is a tissue-specific transcription factor involved in the development of thyroid and lung. TTF-1 contains two transcriptional activation domains (N and C domain). The primary amino acid sequence of the N domain does not show any typical characteristic of known transcriptional activation domains. In aqueous solution the N domain exists in a random-coil conformation. The increase of the milieu hydrophobicity, by the addition of trifluoroethanol, induces a considerable gain of alpha-helical structure. Acidic transcriptional activation domains are largely unstructured in solution, but, under hydrophobic conditions, folding into alpha-helices or beta-strands can be induced. Therefore our data indicate that the inducibility of alpha-helix by hydrophobic conditions is a property not restricted to acidic domains. Co-transfections experiments indicate that the acidic domain of herpes simplex virus protein VP16 (VP16) and the TTF-1 N domain are interchangeable and that a chimaeric protein, which combines VP16 linked to the DNA-binding domain of TTF-1, undergoes the same regulatory constraints that operate for the wild-type TTF-1. In addition, we demonstrate that the TTF-1 N domain possesses two typical properties of acidic activation domains: TBP (TATA-binding protein) binding and ability to activate transcription in yeast. Accordingly, the TTF-1 N domain is able to squelch the activity of the p65 acidic domain. Altogether, these structural and functional data suggest that a non-acidic transcriptional activation domain (TTF-1 N domain) activates transcription by using molecular mechanisms similar to those used by acidic domains. TTF-1 N domain and acidic domains define a family of proteins whose common property is to activate transcription through the use of mechanisms largely conserved during evolutionary development.

Asunto(s)

Proteínas Nucleares/química , Factores de Transcripción/química , Activación Transcripcional , Secuencia de Aminoácidos , Animales , Dicroismo Circular , Proteínas de Unión al ADN/metabolismo , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Hidrólisis , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Unión Proteica , Conformación Proteica , Ratas , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Espectrofotometría Ultravioleta , Proteína de Unión a TATA-Box , Factor Nuclear Tiroideo 1 , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

RESUMEN

Excessive activation of glutamate receptors via the N-methyl-D-aspartate (NMDA) subtype appears to play a role in the sequence of cellular events which lead to irreversible ischemic damage to neurons. Furthermore, NMDA receptor activation induces a stimulation of ornithine decarboxylase (ODC), the rate-limiting enzyme for polyamine (PA) biosynthesis. In order to better understand the role of PA we have measured ODC activity and the effect of methionine sulfoximine (MSO), a molecule able to stimulate ODC, on a model of transient cerebral ischemia. There was a significant increase in ODC activity in the rat cerebral cortex during post-ischemic reperfusion. The treatment with MSO induced a significant decrease in cerebral glutamine synthetase activity accompanied by a marked increase in ODC activity. In MSO-pretreated rats there was a significant decrease in the survival rate when compared to untreated ischemic rats.

Asunto(s)

Corteza Cerebral/efectos de los fármacos , Metionina Sulfoximina/farmacología , Ornitina Descarboxilasa/metabolismo , Daño por Reperfusión/tratamiento farmacológico , Animales , Corteza Cerebral/irrigación sanguínea , Masculino , Ratas , Ratas Wistar , Daño por Reperfusión/enzimología

RESUMEN

Considerable evidence indicates that reactive oxygen species play an etiological role in both cardiotoxicity and the skin necrosis induced by adriamycin (ADM). An increase in glutathione peroxidase activity on addition of selenium to cultured MCR-5 lung fibroblasts was observed; this increase was accompanied by enhanced cellular resistance to ADM toxicity. Moreover, the presence of exogenous antioxidant systems, such as superoxide dismutase, catalase, vitamin E, dimethylsulfoxide, and desferroxamine, an iron chelating agent, resulted in significant protection from ADM-mediated damage.

Asunto(s)

Antibióticos Antineoplásicos/toxicidad , Antioxidantes/farmacología , Doxorrubicina/toxicidad , Glutatión Peroxidasa/biosíntesis , Células Cultivadas , Deferoxamina/farmacología , Fibroblastos/efectos de los fármacos , L-Lactato Deshidrogenasa/metabolismo , Pulmón/efectos de los fármacos , Selenio/farmacología