RESUMEN
We identified a discrete number of microRNAs differentially expressed in benign or malignant mesothelial tissues. We focused on mir-145 whose levels were significantly downregulated in malignant mesothelial tissues and malignant pleural mesothelioma (MPM) cell lines as compared to benign tissues (pleura, peritoneum or cysts). We show that promoter hyper-methylation caused very low levels in MPM cell lines and specimens. Treatment of MPM cell lines with mir-145 agonists negatively modulated some protumorigenic properties of MPM cells, such as clonogenicity, cell migration and resistance to pemetrexed treatment. The main effector mechanism of the clonogenic death induced by mir-145 was that of accelerated senescence. We found that mir-145 targeted OCT4 via specific binding to its 3'-UTR. Increased intracellular levels of mir-145 decreased the levels of OCT4 and its target gene ZEB1, thereby counteracting the increase of OCT4 induced by pemetrexed treatment which is known to favor the development of chemoresistant cells. In line with this, reintroduction of OCT4 into mimic-145 treated cells counteracted the effects on clonogenicity and replicative senescence. This further supports the relevance of the mir-145-OCT4 interaction for the survival of MPM cells. The potential use of mir-145 expression levels to classify benign vs malignant mesothelial tissues and the differences between pemetrexed-induced senescence and that induced by the re-expression of mir-145 are discussed.
Asunto(s)
Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/genética , Mesotelioma/genética , MicroARNs/genética , Neoplasias Pleurales/genética , Regiones no Traducidas 3'/genética , Animales , Antineoplásicos/farmacología , Secuencia de Bases , Línea Celular , Línea Celular Tumoral , Movimiento Celular/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Senescencia Celular/genética , Metilación de ADN , Regulación hacia Abajo , Técnicas de Silenciamiento del Gen , Glutamatos/farmacología , Guanina/análogos & derivados , Guanina/farmacología , Células HEK293 , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino , Mesotelioma/metabolismo , Mesotelioma/patología , Mesotelioma Maligno , Ratones SCID , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Pemetrexed , Neoplasias Pleurales/metabolismo , Neoplasias Pleurales/patología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Ácido NucleicoRESUMEN
Micro RNAs (miRs) are small non-coding RNAs aberrantly expressed in human tumors. Here, we aim to identify miRs whose deregulated expression leads to the activation of oncogenic pathways in human gastric cancers (GCs). Thirty nine out of 123 tumoral and matched uninvolved peritumoral gastric specimens from three independent European subsets of patients were analyzed for the expression of 851 human miRs using Agilent Platform. The remaining 84 samples were used to validate miRs differentially expressed between tumoral and matched peritumoral specimens by qPCR. miR-204 falls into a group of eight miRs differentially expressed between tumoral and peritumoral samples. Downregulation of miR-204 has prognostic value and correlates with increased staining of Bcl-2 protein in tumoral specimens. Ectopic expression of miR-204 inhibited colony forming ability, migration and tumor engraftment of GC cells. miR-204 targeted Bcl-2 messenger RNA and increased responsiveness of GC cells to 5-fluorouracil and oxaliplatin treatment. Ectopic expression of Bcl-2 protein counteracted miR-204 pro-apoptotic activity in response to 5-fluorouracil. Altogether, these findings suggest that modulation of aberrant expression of miR-204, which in turn releases oncogenic Bcl-2 protein activity might hold promise for preventive and therapeutic strategies of GC.
Asunto(s)
MicroARNs/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Neoplasias Gástricas/genética , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Fluorouracilo/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Ratones Desnudos , MicroARNs/metabolismo , Compuestos Organoplatinos/farmacología , Oxaliplatino , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/metabolismo , Regulación hacia ArribaRESUMEN
Here we show that pemetrexed-treated mesothelioma cells undergo accelerated senescence. This is characterized by the secretion of proinflammatory and mitogenic cytokines, reminiscent of an SASP (senescence-associated secretory phenotype). Conditioned media from senescent MPM (malignant pleural mesothelioma) cells trigger the emergence of EMT (epithelial-to-mesenchymal)-like, clonogenic and chemoresistant cell subpopulations, expressing high levels of ALDH (aldehyde dehydrogenase) activity (ALDH(bright) cells). We show by fluorescence-activated cell sorting of purified ALDH(bright) and ALDH(low) cells, that both cell-autonomous and cell-non-autonomous mechanisms converge to maintain the SASP-induced, EMT-like cell subpopulations. Chemoresistant ALDH(bright) cells exist within primary MPM specimens and enrichment for ALDH(bright) cells correlates with an earlier tumor onset into NOD/SCID mice. We show that RAS(v12) expression induces SASP-like changes in untransformed human mesothelial cells, and that p53 ablation increases the effect of RAS(v12) expression. We identify STAT3 activation as a crucial event downstream to SASP signaling. In fact, small hairpin RNA-mediated ablation of STAT3 deeply attenuates the induction of EMT genes and the increase of ALDH(bright) cells induced by SASP-cytokines. This strongly affects the chemoresistance of MPM cells in vitro and leads to anticancer effects in vivo.
Asunto(s)
Senescencia Celular , Resistencia a Antineoplásicos , Mesotelioma/patología , Fenotipo , Aldehído Deshidrogenasa/metabolismo , Animales , Recuento de Células , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Transformación Celular Neoplásica/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Medios de Cultivo Condicionados/metabolismo , Citocinas/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Genes ras/genética , Glutamatos/farmacología , Guanina/análogos & derivados , Guanina/farmacología , Humanos , Masculino , Mesodermo/efectos de los fármacos , Mesodermo/patología , Mesotelioma/genética , Mesotelioma/metabolismo , Ratones , Mitógenos/metabolismo , Pemetrexed , ARN Interferente Pequeño/genética , Factor de Transcripción STAT3/deficiencia , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
The metabolism of polycyclic aromatic hydrocarbons (PAHs) was studied in vivo and in vitro in systems consisting of Rigidoporus lignosus and its laccase, in the presence of so-called "mediator" compounds. The static culture of the native fungal strain was able to metabolize anthracene and 2-methylanthracene, but not 9-nitroanthracene. The addition of redox mediators 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 1-hydroxybenzotriazole (HBT) or violuric acid (VA) led to a significant increase in the degradation of substrates. The oxidation of PAHs was not significant when purified laccase was used without the addition of mediators. The addition of these compounds increased the oxidation of all substrates by approximately 70-80% after 72 h of incubation. The degradation rate was highest for 2-methylanthracene in the presence of VA.
Asunto(s)
Lacasa/metabolismo , Hidrocarburos Policíclicos Aromáticos/metabolismo , Polyporales/metabolismo , Biodegradación Ambiental , Lacasa/aislamiento & purificación , Oxidación-Reducción , Polyporales/enzimología , Especificidad por Sustrato , Factores de TiempoRESUMEN
ADAMTS13, the von Willebrand factor-cleaving protease, is constitutively produced by endothelial cells. The aim was to quantify ADAMTS13 production in proliferating and nonproliferating human umbilical vein endothelial cells (HUVECs) and to determine if such production was regulated. HUVECs were plated at 50,000 cells/cm(2) or 10,000 cells/cm(2), densities yielding confluence or subconfluence, respectively. Subconfluent HUVEC supernatants and cell lysates contained at least 2-fold more ADAMTS13 antigen than those of confluent HUVECs (p < 0.05). Immunohistochemistry supported this increase and indicated no dramatic shifts in subcellular localization of HUVEC ADAMTS13 by HUVEC proliferation. The activity of the ADAMTS13 produced by subconfluent HUVECs was increased about 2-fold also. HUVECs rapidly increased ADAMTS13 mRNA in response to subconfluent conditions. This change in regulation, in turn, leads to increased ADAMTS13 protein production and activity by these proliferating cells.
Asunto(s)
Proteínas ADAM/genética , Proliferación Celular , Células Endoteliales/metabolismo , Regulación de la Expresión Génica , Proteína ADAMTS13 , Células Cultivadas , Células Endoteliales/citología , Endotelio Vascular/citología , Humanos , ARN Mensajero/análisisRESUMEN
A molecular modelling study was performed using the CATALYST software package on a dataset of 100 thiosemicarbazide and thiazole derivatives acting as MAO-B irreversible inhibitors in order to, (i) better elucidate the possible role of the ligand features which are significant for binding and (ii) generate chemical features based pharmacophore models which were subsequently used as 3D queries for database searching. Based on known MAO-B inhibitors, pharmacophore hypotheses were created in order to find similarities between the thiazoles and thiosemicarbazides and identify the key sub-structures most likely to be significant for high MAO-B inhibitory activity.
Asunto(s)
Inhibidores de la Monoaminooxidasa/química , Inhibidores de la Monoaminooxidasa/farmacología , Monoaminooxidasa/metabolismo , Animales , Bases de Datos Factuales , Ligandos , Hígado/citología , Hígado/metabolismo , Matemática , Mitocondrias/enzimología , Modelos Químicos , Modelos Moleculares , Estructura Molecular , Monoaminooxidasa/química , Inhibidores de la Monoaminooxidasa/metabolismo , Unión Proteica , Relación Estructura-Actividad Cuantitativa , Ratas , Semicarbacidas/química , Tiazoles/químicaRESUMEN
The interaction between monomeric insulin and monosaccharides has been investigated through circular dichroism, fluorescence spectroscopy and two dimensional nuclear magnetic resonance. CD spectra indicate that D-glucose interacts with monomeric insulin whereas D-galactose, D-mannose and 2-deoxy-D-glucose have a lower effect. Fluorescence emission was quenched at sugar concentrations of 5-10 mM. Titration with the different sugars produces a quenching of the tyrosine spectrum from which a binding free energy value for the insulin-sugar complexes has been evaluated. Transfer nuclear Overhauser enhancement NMR experiments indicate the existence of dipolar interactions at short interatomic distances between C-1 proton of D-glucose in the beta form and the monomeric insulin. Further, NMR total correlation spectra experiments revealed that the hormone is in the monomeric form and that upon addition of glucose no aggregation occurs. The interaction does not involve relevant changes in the secondary structure of insulin suggesting that the interaction occur at the side chain level. Molecular dynamics simulations and modeling studies, based on the dynamic fluctuations of potential binding moiety sidechains, argued from results of NMR spectroscopy, provide additional informations to locate the putative binding sites of D-glucose to insulin.
Asunto(s)
Glucosa/química , Insulina/química , Sitios de Unión , Dicroismo Circular , Glucosa/metabolismo , Humanos , Insulina/metabolismo , Espectroscopía de Resonancia Magnética/métodos , Modelos Moleculares , Unión Proteica , Protones , Programas Informáticos , Espectrometría de Fluorescencia/métodosRESUMEN
The isoenzyme I of cytosolic Cu,Zn-superoxide dismutase (SOD) from Nicotiana plumbaginifolia (tobacco) leaves has been purified to apparent homogeneity. The relative molecular mass of the native isoenzyme, determined by gel filtration chromatography, is about 33.2 kDa. SDS-polyacrylamide gel electrophoresis shows that the enzyme is composed of two equal subunits of 16.6 kDa The isolectric point, assayed by isoelectric focusing, in the pH range of 3.5-6.5, is 4.3. The enzyme stability was tested at different temperatures, pH, and concentration of inhibitors (KCN and H(2)O(2)). The catalytic constant (k(cat)) was 1.17 +/- 0.14 x 10(9) M(-1) s(-1) at pH 9.9 and 0.1 M ionic strength. The activation energy of the thermal denaturation process is 263 kJ mol(-1). The electrostatic surface potential of the modeled tobacco Cu,Zn-SOD I was calculated showing that the functional spatial network of charges on the protein surface has been maintained, independently of the amino acid substitution around the active sites.
Asunto(s)
Nicotiana/enzimología , Superóxido Dismutasa/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Bovinos , Citosol/enzimología , Electroquímica , Electroforesis en Gel de Poliacrilamida , Inhibidores Enzimáticos/farmacología , Estabilidad de Enzimas , Fluoruros/metabolismo , Calor , Concentración de Iones de Hidrógeno , Punto Isoeléctrico , Isoenzimas/antagonistas & inhibidores , Isoenzimas/química , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Datos de Secuencia Molecular , Peso Molecular , Hojas de la Planta/enzimología , Superóxido Dismutasa/antagonistas & inhibidores , Superóxido Dismutasa/aislamiento & purificación , Superóxido Dismutasa/metabolismo , Propiedades de SuperficieRESUMEN
Molecular dynamics simulation indicates that the dynamical behaviour of the insulin dimer is asymmetric. Atomic level knowledge of the interaction modes and protein conformation in the solvation state identifies dynamical structures, held by hydrogen bonds that stabilize, mainly in one monomer, the interaction between the chains. Dynamic cross-correlation analysis shows that the two insulin monomers behave asymmetrically and are almost independent. Solvation energy, calculated to evaluate the contribute of each interface residue to the dimer association pattern, well compares with the experimental association state found in protein mutants indicating that this parameter is an important factor to explain the association properties of mutated insulin dimers.
Asunto(s)
Simulación por Computador , Insulina/química , Modelos Moleculares , Dimerización , Estructura Secundaria de ProteínaRESUMEN
The adhesion of lipid vesicles (liposomes) having controlled chemical and physical structure to polymer supported human serum albumin (HSA) thin layers was investigated by a spectrofluorimetric technique. The vesicle lipid bilayer was labeled with a small amount of an apolar fluorescent probe (diphenylexathriene) and the vesicle suspension was set in contact with the protein film. After washing and drying, the adhering vesicles containing sample was dissolved in chloroform and the homogeneous solution was analyzed by standard spectrofluorimetric techniques. Different parameters of the lipid bilayer, suspending solution, and protein film were varied and their influence on the liposome binding was investigated. Concerning the lipid bilayer, we studied the effect of liposome surface charge by using different mixtures of neutral (dipalmitoyl-phosphatidylcholine) and charged (dipalmitoyl-phosphatidic acid) phospholipids and the fluid or gel nature of the lipid bilayer (switched on and off by temperature variation). Variations of the local environment involve Ca(2+) and H(+) changes in the millimolar range as well as different hydrodynamical flows (in the range 0.1-10 cm/s). Preliminary measurements using different protein layers were also performed. Results show: (a) negligible adhesion without the protein layer, (b) the presence of a maximum for the liposome adhesion vs ion concentration (depending on the liposome composition and kind of the adsorbed ions), (c) a much stronger adhesion for vesicles in the fluid phase (overcoming the entropy-driven desorption increase with temperature), and (d) a dramatic lowering of the adhesion capability under hydrodynamic flow. Points a-c have been interpreted on the basis of a simple mechanoelectrical model. Copyright 2000 Academic Press.
RESUMEN
We have investigated the mechanism of inhibition and site of action of the novel human metabotropic glutamate receptor 5 (hmGluR5) antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP), which is structurally unrelated to classical metabotropic glutamate receptor (mGluR) ligands. Schild analysis indicated that MPEP acts in a non-competitive manner. MPEP also inhibited to a large extent constitutive receptor activity in cells transiently overexpressing rat mGluR5, suggesting that MPEP acts as an inverse agonist. To investigate the molecular determinants that govern selective ligand binding, a mutagenesis study was performed using chimeras and single amino acid substitutions of hmGluR1 and hmGluR5. The mutants were tested for binding of the novel mGluR5 radioligand [(3)H]2-methyl-6-(3-methoxyphenyl)ethynyl pyridine (M-MPEP), a close analog of MPEP. Replacement of Ala-810 in transmembrane (TM) VII or Pro-655 and Ser-658 in TMIII with the homologous residues of hmGluR1 abolished radioligand binding. In contrast, the reciprocal hmGluR1 mutant bearing these three residues of hmGluR5 showed high affinity for [(3)H]M-MPEP. Radioligand binding to these mutants was also inhibited by 7-hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester (CPCCOEt), a structurally unrelated non-competitive mGluR1 antagonist previously shown to interact with residues Thr-815 and Ala-818 in TMVII of hmGluR1. These results indicate that MPEP and CPCCOEt bind to overlapping binding pockets in the TM region of group I mGluRs but interact with different non-conserved residues.
Asunto(s)
Cromonas/metabolismo , Antagonistas de Aminoácidos Excitadores/metabolismo , Piridinas/metabolismo , Receptores de Glutamato Metabotrópico/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células CHO , Células COS , Cricetinae , Modelos Moleculares , Datos de Secuencia Molecular , Ratas , Receptores de Glutamato Metabotrópico/química , Receptores de Glutamato Metabotrópico/metabolismo , Relación Estructura-ActividadRESUMEN
Laccase from Rigidoporus lignosus, a white-rot basidiomycete, has been isolated from culture filtrates. The enzyme was purified to homogeneity and some of its structural and kinetic parameters have been determined. The effects of pH, temperature, and organic solvents on the activity and stability of the enzyme, under different conditions, were also assayed. The results we have obtained, including the rather broad substrate specificity of enzyme, combined with their relatively easy production and purification, suggest that laccase may be efficiently employed in a variety of biotechnology applications.
Asunto(s)
Basidiomycota/química , Proteínas Fúngicas/química , Oxidorreductasas/química , Secuencia de Aminoácidos , Basidiomycota/enzimología , Basidiomycota/crecimiento & desarrollo , Cobre/química , Medios de Cultivo , Electroforesis en Gel de Poliacrilamida , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/aislamiento & purificación , Focalización Isoeléctrica , Cinética , Lacasa , Datos de Secuencia Molecular , Oxidorreductasas/biosíntesis , Oxidorreductasas/aislamiento & purificación , Análisis de Secuencia de Proteína , Homología de Secuencia de AminoácidoRESUMEN
Structure-activity relationships were performed on a new series of thiazole derivatives which selectively inactivate monoamine oxidase-B (MAO-B), purified from mitochondrial beef liver. All of the synthesized and tested compounds showed non-competitive inhibition, suggesting the formation of a stable adduct between the tertiary amine function, linked to the thiazolyl derivatives and the active site of the enzyme. The mechanism of MAO-B inhibition is discussed in terms of the Ionization Potential of the amine nitrogen atom and the conformational flexibility of the inhibitors.
Asunto(s)
Mitocondrias Hepáticas/enzimología , Inhibidores de la Monoaminooxidasa/farmacología , Monoaminooxidasa/efectos de los fármacos , Tiazoles/farmacología , Animales , Dominio Catalítico/efectos de los fármacos , Bovinos , Modelos Teóricos , Conformación Molecular , Relación Estructura-ActividadRESUMEN
Growth conditions influenced DNA methylation in cultured cerebellar granule cells, as indicated by immunocytochemical analysis with monoclonal antibodies raised against 5-methylcytidine. In cultures grown under suboptimal conditions, i.e. in medium containing 10 instead of 25 mM K+, a substantial reduction in both the number of immunopositive cells and the intensity of immunostaining occurred at 4 days in vitro (DIV), a time which preceded the appearance of the morphological features of apoptosis. These results suggest that a reduction in DNA methylation is one of the biochemical events associated with the 'condemned phase' of apoptosis, in which granule cells grown under suboptimal conditions become committed to death.
Asunto(s)
Apoptosis/fisiología , Cerebelo/crecimiento & desarrollo , Cerebelo/metabolismo , Metilación de ADN , Animales , Células Cultivadas , Cromatina/metabolismo , Medios de Cultivo , Citidina/análogos & derivados , Citidina/metabolismo , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Factor I del Crecimiento Similar a la Insulina/metabolismo , Potasio/metabolismo , RatasRESUMEN
Structure-activity relationship studies were carried out on a new series of hydrazino-thiosemicarbazide derivatives, which inhibit monoamino oxidase (MAO). Fifty-five compounds were synthesized and tested "in vitro" for their inhibitory effects on rat liver mitochondrial MAO. The most efficient MAO inhibitors were the benzylidene derivatives (sequence: see text] where R is the piperonyl radical and ethyl or isopropyl substituents are in R1 position. Correlation of MAO activity with hydrophobic, electronic and steric properties of tested compounds, evaluated by means of Quantum Mechanical calculations and calorimetric analysis (DSC) suggest that electronic and steric parameters give a better fit than hydrophobicity with the biological activity.
Asunto(s)
Hidrazinas , Inhibidores de la Monoaminooxidasa/farmacología , Semicarbacidas/farmacología , Relación Estructura-Actividad , 1,2-Dipalmitoilfosfatidilcolina/metabolismo , Animales , Sitios de Unión , Rastreo Diferencial de Calorimetría , Fenómenos Químicos , Química , Transporte de Electrón , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Hidrazinas/síntesis química , Hidrazinas/farmacología , Liposomas/metabolismo , Hígado/enzimología , Estructura Molecular , Monoaminooxidasa/metabolismo , Inhibidores de la Monoaminooxidasa/química , Ratas , Semicarbacidas/síntesis químicaRESUMEN
The purpose of this research is to study the relationship between chemical structure and inhibitory activity of some hydrazine-thiazole derivatives on rat liver mitochondria monoamine oxidase (MAO). Forty-five compounds belonging to three series of hydrazine-thiazole derivatives, with either alkylic or arylic substituents in the thiazole ring, were tested. The highest inhibitory activity was observed with piperonyl derivatives 25 and 40, which contain a 4-methyl group in the thiazole nucleus. The structure-activity relationship of MAO inhibitors was established in relation to hydrophobic, electronic and steric hindrance parameters. A mechanism of enzyme inhibition was proposed based on the calculation of HOMO energies.
Asunto(s)
Hidrazinas/farmacología , Mitocondrias Hepáticas/enzimología , Inhibidores de la Monoaminooxidasa/farmacología , Tiazoles/farmacología , Animales , Femenino , Masculino , Ratas , Relación Estructura-ActividadRESUMEN
Cyclic adenosine diphosphate-ribose (cADPR) and ADPR were separated by high-performance liquid chromatography (HPLC) on a CarboPac PA-1 column at strong basic pH and quantitated by a pulsed amperometric detector. Although this HPLC method was quite sensitive and highly reproducible, it did not allow the separation of cADPR from guanosine monophosphate (GMP) which, when present, could be removed by ion-affinity chromatography, using gel-immobilized Fe3+ columns. Crude synaptic membranes from rat hippocampi were incubated with nicotinamide adenine dinucleotide (NAD) and acidic extracts were subject to HPLC analysis after neutralization. Incubation led to a time-dependent formation of ADPR, which was amplified when membranes were incubated in the presence of guanosine trisphosphate (GTP), guanosine-5'-0-(3-thiotrisphosphate) (GTP-gamma-S) or AlF3. cADPR did not accumulate in detectable amounts and only a minimal proportion (< 5%) of radioactivity originating from [3H]NAD co-eluted with authentic cADPR in extracts from hippocampal membranes. The simultaneous detection of cADPR and ADPR we have described may help the search for inhibitors of cADPR metabolism, which will allow to measure the cADPR that accumulates under basal conditions or in response to extracellular signals.
Asunto(s)
Adenosina Difosfato Ribosa/análogos & derivados , Adenosina Difosfato Ribosa/análisis , Hipocampo/metabolismo , NAD/metabolismo , Animales , Cromatografía Líquida de Alta Presión , ADP-Ribosa Cíclica , Masculino , Membranas/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
The three-dimensional structure of tomato P31 and T10 Cu,Zn superoxide dismutases (SODs) were computer modelled using the structure of the bovine enzyme as a template. The structure-essential residues retain in the models the position occupied in the other Cu,Zn SODs of known 3D structure and the overall packing of the beta-barrel is maintained. Formation of 'aromatic pairs' occurs between newly inserted aromatic residues. The number of total charges changes in the two variants and some charged residues located in the proximity of the active site in most Cu,Zn SODs disappear in tomato enzymes. Calculation of the electrostatic potential field, carried out by numerically solving the Poisson-Boltzmann equation, indicates that in both variants a negative potential field surrounds all the protein surface except the active site areas, characterized by positive potential values, as already observed in the bovine enzyme. This result confirms that coordinated mutations of charged residues have occurred in the evolution of this enzyme giving rise to a peculiar electrostatic potential distribution common to all members of this protein family.
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Hojas de la Planta/enzimología , Solanum lycopersicum/enzimología , Superóxido Dismutasa/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Bovinos , Electroquímica , Modelos Moleculares , Datos de Secuencia Molecular , Pliegue de Proteína , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Superóxido Dismutasa/genéticaRESUMEN
The 2-oxoglutarate carrier protein (OGCP) catalyzes the transport of the 2-oxoglutarate into the mitochondrial matrix by an electroneutral exchange for malate or some other dicarboxylic acids. Using primers based on the bovine heart cDNA sequence, overlapping cDNA clones encoding the rat OGCP were isolated from total rat heart poly(A+) cDNA. The entire rat cDNA is 1149 bp in length with 5' and 3' untranslated regions of 41 and 163 bp, respectively. The open reading frame encodes a protein consisting of 314 amino acids. The amino acid sequence of the rat 2-oxoglutarate carrier is 97% identical to that of the 2-oxoglutarate from cow and human. By Northern blot analysis, hybridizing transcripts were found in rat heart, liver and brain.
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Proteínas Portadoras/genética , Proteínas de Transporte de Membrana , Mitocondrias/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas Portadoras/metabolismo , Clonación Molecular , ADN Complementario , Humanos , Membranas Intracelulares/metabolismo , Ácidos Cetoglutáricos/metabolismo , Datos de Secuencia Molecular , RatasRESUMEN
Inositol hexakisphosphate (InsP6) stimulates 45Ca2+ influx in purified mitochondria from rat liver. The action of InsP6 is concentration-dependent, with an apparent EC50 value of 50 microM. Stimulation of 45Ca2+ influx may follow the interaction between InsP6 and specific membrane receptors. Accordingly, [3H]InsP6 binds to specific and saturable recognition sites in purified mitochondria. These results support the view that InsP6 acts as a signal molecule to regulate the intracellular homeostasis of Ca2+.