RESUMEN
The use of an appropriate reference gene to ensure accurate normalisation is crucial for the correct quantification of gene expression using qPCR assays and RNA arrays. The main criterion for a gene to qualify as a reference gene is a stable expression across various cell types and experimental settings. Several reference genes are commonly in use but more and more evidence reveals variations in their expression due to the presence of on-going neuropathological disease processes, raising doubts concerning their use. We conducted an analysis of genome-wide changes of gene expression in the human central nervous system (CNS) covering several neurological disorders and regions, including the spinal cord, and were able to identify a number of novel stable reference genes. We tested the stability of expression of eight novel (ATP5E, AARS, GAPVD1, CSNK2B, XPNPEP1, OSBP, NAT5 and DCTN2) and four more commonly used (BECN1, GAPDH, QARS and TUBB) reference genes in a smaller cohort using RT-qPCR. The most stable genes out of the 12 reference genes were tested as normaliser to validate increased levels of a target gene in CNS disease. We found that in human post-mortem tissue the novel reference genes, XPNPEP1 and AARS, were efficient in replicating microarray target gene expression levels and that XPNPEP1 was more efficient as a normaliser than BECN1, which has been shown to change in expression as a consequence of neuronal cell loss. We provide herein one more suitable novel reference gene, XPNPEP1, with no current neuroinflammatory or neurodegenerative associations that can be used for gene quantitative gene expression studies with human CNS post-mortem tissue and also suggest a list of potential other candidates. These data also emphasise the importance of organ/tissue-specific stably expressed genes as reference genes for RNA studies.
Asunto(s)
Sistema Nervioso Central , Expresión Génica/genética , ARN/genética , Autopsia , Sistema Nervioso Central/metabolismo , Europa (Continente) , Perfilación de la Expresión Génica , Humanos , Enfermedades Neurodegenerativas/genética , Estándares de Referencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
The receptor tyrosine kinase product of the anaplastic lymphoma kinase (ALK) gene has been implicated in oncogenesis as a product of several chromosomal translocations, although its endogeneous role in the hematopoietic and neural systems has remained poorly understood. We describe that the generation of animals homozygous for a deletion of the ALK tyrosine kinase domain leads to alterations in adult brain function. Evaluation of adult ALK homozygotes (HOs) revealed an age-dependent increase in basal hippocampal progenitor proliferation and alterations in behavioral tests consistent with a role for this receptor in the adult brain. ALK HO animals displayed an increased struggle time in the tail suspension test and the Porsolt swim test and enhanced performance in a novel object-recognition test. Neurochemical analysis demonstrates an increase in basal dopaminergic signalling selectively within the frontal cortex. Altogether, these results suggest that ALK functions in the adult brain to regulate the function of the frontal cortex and hippocampus and identifies ALK as a new target for psychiatric indications, such as schizophrenia and depression, with an underlying deregulated monoaminergic signalling.
Asunto(s)
Conducta Animal/fisiología , Química Encefálica/fisiología , Proteínas Tirosina Quinasas/genética , Quinasa de Linfoma Anaplásico , Animales , Ansiedad/genética , Ansiedad/psicología , Química Encefálica/genética , Bromodesoxiuridina , Proliferación Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Depresión/genética , Depresión/psicología , Dopamina/metabolismo , Femenino , Citometría de Flujo , Suspensión Trasera , Inmunohistoquímica , Masculino , Ratones , Ratones Noqueados , Actividad Motora , Proteínas Tirosina Quinasas Receptoras , Reconocimiento en Psicología/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Serotonina/metabolismo , Natación/psicología , Timidina/análogos & derivados , Timidina/farmacologíaRESUMEN
The use of neural precursor cells (NPCs) represents a promising repair strategy for many neurological disorders. However, the molecular events and biological features that control NPC proliferation and their differentiation into neurons, astrocytes, and oligodendrocytes are unclear. In the present study, we used a comparative proteomics approach to identify proteins that were differentially regulated in NPCs after short-term differentiation. We also used a subcellular fractionation technique for enrichment of nuclei and other dense organelles to identify proteins that were not readily detected in whole cell extracts. In total, 115 distinct proteins underwent expression changes during NPC differentiation. Forty one of these were only identified following subcellular fractionation. These included transcription factors, RNA-processing factors, cell cycle proteins, and proteins that translocate between the nucleus and cytoplasm. Biological network analysis showed that the differentiation of NPCs was associated with significant changes in cell cycle and protein synthesis machinery. Further characterization of these proteins could provide greater insight into the mechanisms involved in regulation of neurogenesis in the adult central nervous system (CNS) and potentially identify points of therapeutic intervention.
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Células Madre Adultas/citología , Ventrículos Laterales/citología , Células Madre Multipotentes/citología , Neuronas/citología , Proteómica , Células Madre Adultas/metabolismo , Animales , Western Blotting , Técnicas de Cultivo de Célula , Ciclo Celular , Diferenciación Celular , Electroforesis en Gel Bidimensional/métodos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ventrículos Laterales/metabolismo , Redes y Vías Metabólicas , Ratones , Ratones Endogámicos C57BL , Células Madre Multipotentes/metabolismo , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Mapeo Peptídico , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The decrease in plasticity that occurs in the central nervous system during postnatal development is accompanied by the appearance of perineuronal nets (PNNs) around the cell body and dendrites of many classes of neuron. These structures are composed of extracellular matrix molecules, such as chondroitin sulfate proteoglycans (CSPGs), hyaluronan (HA), tenascin-R, and link proteins. To elucidate the role played by neurons and glial cells in constructing PNNs, we studied the expression of PNN components in the adult rat cerebellum by immunohistochemistry and in situ hybridization. In the deep cerebellar nuclei, only large excitatory neurons were surrounded by nets, which contained the CSPGs aggrecan, neurocan, brevican, versican, and phosphacan, along with tenascin-R and HA. Whereas both net-bearing neurons and glial cells were the sources of CSPGs and tenascin-R, only the neurons expressed the mRNA for HA synthases (HASs), cartilage link protein, and link protein Bral2. In the cerebellar cortex, Golgi neurons possessed PNNs and also synthesized HASs, cartilage link protein, and Bral2 mRNAs. To see whether HA might link PNNs to the neuronal cell surface by binding to a receptor, we investigated the expression of the HA receptors CD44, RHAMM, and LYVE-1. No immunolabelling for HA receptors on the membrane of net-bearing neurons was found. We therefore propose that HASs, which can retain HA on the cell surface, may act as a link between PNNs and neurons. Thus, HAS and link proteins might be key molecules for PNN formation and stability.
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Cerebelo/metabolismo , Matriz Extracelular/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuroglía/metabolismo , Neuronas/metabolismo , Animales , Cerebelo/citología , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Femenino , Glucuronosiltransferasa/metabolismo , Receptores de Hialuranos/metabolismo , Hialuronano Sintasas , Ácido Hialurónico/metabolismo , Hibridación in Situ , Plasticidad Neuronal/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
Some patients with Major Depression and other neurological afflictions display hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis. HPA hyperactivity may be due to impaired feedback inhibition and manifested as increased levels of circulating cortisol. Subcutaneous implants of corticosterone pellets were used to mimic this situation in mice to gain insight into any effects on brain function by comparative proteomic analysis using two-dimensional Differential In-Gel Electrophoresis. A total of 150 different protein spots were altered by corticosterone treatment in the hypothalamus, hippocampus and cerebral cortex. Of these, 117 spots were identified by matrix-assisted laser desorption/ionization-time of flight mass fingerprinting equating to 51 different proteins. Association of these corticosterone-modulated proteins with biological functions using the Ingenuity Pathways Analysis tool showed that cell morphology was significantly altered in the hippocampus and cerebral cortex, whereas the hypothalamus showed significant changes in cell death. Ingenuity Pathways Analysis of the canonical signaling pathways showed that glycolysis and gluconeogenesis were altered in the hypothalamus and the hippocampus and all three brain regions showed changes in phenylalanine, glutamate and nitrogen metabolism. Further elucidation of these pathways could lead to identification of biomarkers for the development of pharmacological therapies targeted at neuropsychiatric disorders.
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Antiinflamatorios/administración & dosificación , Encéfalo/efectos de los fármacos , Corticosterona/administración & dosificación , Vías Nerviosas/efectos de los fármacos , Proteómica/métodos , Animales , Western Blotting/métodos , Encéfalo/metabolismo , Encéfalo/patología , Encéfalo/fisiopatología , Mapeo Encefálico , Muerte Celular/efectos de los fármacos , Esquema de Medicación , Electroforesis en Gel Bidimensional/métodos , Expresión Génica/efectos de los fármacos , Masculino , Ratones , Modelos Biológicos , Vías Nerviosas/metabolismo , Vías Nerviosas/fisiopatologíaRESUMEN
1 Mammalian transient receptor potential (TRP) channels include the nonselective cation channel TRPV1, which is activated by a range of stimuli including low pH, vanilloids and heat. Previously, selective mutagenesis experiments identified an intracellular residue (S512Y) critical to discriminating between pH and vanilloid (capsaicin) gating of the rat TRPV1 receptor. 2 In this study, switching the equivalent residue in the human TRPV1 (which has some significant differences with the rat TRPV1) also rendered this channel relatively insensitive to activation by capsaicin and proved critical in determining the receptor's sensitivity to the putative endovanilloid N-arachidonoyl-dopamine (NADA), suggesting a similar mode of activation for these two agonists. 3 Potency of pH gating was reduced; however, voltage-dependent outward rectification properties of the pH-dependent current and gating by heat and pH sensitisation of the S512Y heat response remained unaffected. 4 Surprisingly, residual capsaicin gating was detected and could be sensitised by pH even in the presence of a competitive antagonist. Taken together, these findings indicate that effective functional interaction of capsaicin with the S512Y channel still occurred, although the vanilloid-dependent gating per se was severely compromised. 5 This observation provides additional evidence for capsaicin interacting at multiple sites, distinct from the S512 residue located close to the intracellular face of the pore.
Asunto(s)
Mutación , Canales Catiónicos TRPV/fisiología , Animales , Secuencia de Bases , Células CHO , Capsaicina/farmacología , Cricetinae , Cartilla de ADN , Humanos , Concentración de Iones de Hidrógeno , Activación del Canal Iónico/efectos de los fármacos , Canales Catiónicos TRPV/efectos de los fármacos , Canales Catiónicos TRPV/genéticaRESUMEN
Coassociation of the vanilloid transient receptor potential (Trp) ion channels, TrpV1 and TrpV2, was investigated by immunoprecipitation and immunofluorescence in transfected mammalian cell lines, rat dorsal root ganglia and spinal cord. TrpV1/TrpV2 heteromeric complexes were coimmunoprecipitated from human embryonic kidney cells and F-11 dorsal root ganglion hybridoma cells following their transient coexpression. Immunofluorescent labelling of transfected F-11 cells revealed colocalization of TrpV1 and TrpV2 at the cell surface. Immunoprecipitation from rat dorsal root ganglion lysates identified a minor population of receptor complexes composed of TrpV1/TrpV2 heteromers, consistent with a small proportion of cells double-labelled with TrpV1 and TrpV2 antibodies in rat dorsal root ganglion sections. TrpV1/TrpV2 receptor complexes may represent a functionally distinct ion channel complex that may increase the diversity observed within the Trp ion channel family.
Asunto(s)
Ganglios Espinales/metabolismo , Regulación de la Expresión Génica/fisiología , Canales Catiónicos TRPV/metabolismo , Animales , Western Blotting/métodos , Línea Celular/metabolismo , Células Cultivadas , Técnica del Anticuerpo Fluorescente/métodos , Ganglios Espinales/citología , Humanos , Inmunoprecipitación/métodos , Masculino , Ratas , Fracciones Subcelulares/metabolismo , Transfección/métodosRESUMEN
We describe a method for creating antibodies with a fluorescent reporter integrated into the antigen-binding site. A reporter molecule was chemically linked to a hypervariable loop of an antibody repertoire displayed on phage, and this repertoire was selected for antigen binding. In one selected antibody, the fluorescence of the probe responded quantitatively to antigen binding. The method may have application for the engineering of homogeneous immunoassays.
Asunto(s)
Anticuerpos/química , Técnicas Biosensibles/métodos , Secuencia de Aminoácidos , Anticuerpos/genética , Reacciones Antígeno-Anticuerpo , Sitios de Unión de Anticuerpos , Reactivos de Enlaces Cruzados , Colorantes Fluorescentes/química , Humanos , Inmunoensayo/métodos , Técnicas In Vitro , Modelos Moleculares , Óptica y Fotónica , Biblioteca de Péptidos , Ingeniería de Proteínas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMEN
Protein abundance changes during disease or experimental perturbation are increasingly analyzed by label-free LC/MS approaches. Here we demonstrate the use of LC/MALDI MS for label-free detection of protein expression differences using Escherichia coli cultures grown on arabinose, fructose or glucose as a carbon source. The advantages of MALDI, such as detection of only singly charged ions, and MALDI plate archiving to facilitate retrospective MS/MS data collection are illustrated. MALDI spectra from RP chromatography of tryptic digests of the E. coli lysates were aligned and quantitated using the Rosetta Elucidator system. Approximately 5000 peptide signals were detected in all LC/MALDI runs spanning over 3 orders of magnitude of signal intensity. The average coefficients of variation for all signals across the entire intensity range in all technical replicates were found to be <25%. Pearson correlation coefficients from 0.93 to 0.98 for pairwise comparisons illustrate high replicate reproducibility. Expression differences determined by Analysis of Variance highlighted over 500 isotope clusters ( p < 0.01), which represented candidates for targeted peptide identification using MS/MS. Biologically interpretable protein identifications that could be derived underpin the general utility of this label-free LC/MALDI strategy.
Asunto(s)
Proteínas/análisis , Proteoma/análisis , Proteómica/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Arabinosa/farmacología , Cromatografía Líquida de Alta Presión/métodos , Biología Computacional , Procesamiento Automatizado de Datos , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/análisis , Proteínas de Escherichia coli/metabolismo , Fructosa/farmacología , Glucosa/farmacología , Análisis de Componente Principal , Proteínas/metabolismo , Proteoma/metabolismo , Reproducibilidad de los Resultados , Programas Informáticos , Tripsina/químicaRESUMEN
Capsaicin (vanilloid) sensitivity has long served as the functional signature of a subset of nociceptive sensory neurons. Mutagenesis studies have revealed seemingly distinct regions involved in mediating ligand binding and channel activation at the capsaicin binding site. Residue 547 (transmembrane region 4) mediates significant species differences in resiniferatoxin (RTX) sensitivity, and the Ser(512) residue is critical in discriminating between pH and capsaicin gating. In the present study, the pharmacological profiles of a variety of ligands were studied to investigate cross-talk between these two regions. Exchange of residue 547 between species mediated a difference in capsaicin and RTX-dependent gating. Likewise, the potency of iodoresiniferatoxin (I-RTX) and a novel transient receptor potential vanilloid 1 antagonist were also altered. Experiments using the S512Y mutant channel have confirmed the importance of residue 512 for functional interaction of capsaicin and our novel antagonist. In this study, we were surprised to find that the mutation S512Y converted the activity of the antagonist I-RTX into an intrinsic agonist, albeit with a lower potency than its parent compound, RTX. Recent studies have proposed a novel model for the receptor, based on the X-ray crystal structure of the voltage-dependent potassium channel, in which both the 512 and 547 amino acid residues are in close proximity. Our data support the model whereby intracellular ligand interaction occurs within an S3-S4 "sensor" domain, enabling binding of ligands to be transduced to functional gating of the channel. The binding pocket also seems to be exquisitely sensitive to residue-specific interaction with ligands, because subtle changes in either ligand or channel structure can have profound effects on channel activity.
Asunto(s)
Canales Catiónicos TRPV/química , Canales Catiónicos TRPV/metabolismo , Aminoácidos/metabolismo , Animales , Sitios de Unión , Cricetinae , Cricetulus , Diterpenos/química , Relación Dosis-Respuesta a Droga , Electrofisiología , Humanos , Concentración 50 Inhibidora , Ligandos , Proteínas Mutantes/metabolismo , Ratas , Canales Catiónicos TRPV/agonistas , Canales Catiónicos TRPV/antagonistas & inhibidoresRESUMEN
Adult mouse subventricular zone (SVZ) neural progenitor cells (NPCs) retain the capacity to generate multiple lineages in vitro and in vivo. Thus far, the mechanisms involved in the regulation of these cells have not been well elucidated. We have carried out RNA profiling of adult SVZ cell cultures undergoing differentiation, to identify pathways that regulate progenitor cell proliferation and to define a set of transcripts that can be used as molecular tools in the drug discovery process. We carried out a stepwise stratification of the results to identify transcripts specifically enriched in NPCs and validated some of these using comparative literature analysis, quantitative polymerase chain reaction and immunological techniques. The results show a set of transcription factors, secreted molecules and plasma membrane markers that are differentially regulated during differentiation. Pathway analysis highlights alterations in insulin growth factor, Wnt and transforming growth factor beta signalling cascades. Further characterization of these components could provide greater insight into the mechanisms involved in the regulation of neurogenesis in the adult brain.
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Diferenciación Celular/fisiología , Sustancias de Crecimiento/metabolismo , Neuronas/metabolismo , Transducción de Señal/fisiología , Células Madre/metabolismo , Telencéfalo/embriología , Animales , Biomarcadores/metabolismo , Linaje de la Célula/genética , Células Cultivadas , Perfilación de la Expresión Génica , Sustancias de Crecimiento/genética , Inmunohistoquímica , Ventrículos Laterales/citología , Ventrículos Laterales/embriología , Ventrículos Laterales/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Factores de Crecimiento Nervioso/genética , Factores de Crecimiento Nervioso/metabolismo , Neuronas/citología , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Proteómica , Somatomedinas/genética , Somatomedinas/metabolismo , Células Madre/citología , Telencéfalo/citología , Telencéfalo/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismoRESUMEN
Selective antagonism of N-methyl-d-aspartate (NMDA) 2B subunit containing receptors has been suggested to have potential therapeutic application for multiple CNS disorders. The amino terminal NR2B residues 1 to 282 were found to be both necessary and sufficient for the binding and function of highly NR2B subunit specific antagonists like ifenprodil and CP-101,606. Using a genetic approach in mice, we successfully replaced the murine NR2B gene function by "knocking-in" (KI) a chimeric human NR2A/B cDNA containing the minimal domain abolishing ifenprodil binding into the endogenous NR2B locus. Patch-clamp recording from hippocampal cultures of the NR2B KI mice demonstrated that their NMDA receptors have reduced sensitivity to both ifenprodil and CP-101,606, as predicted, but also have a lower affinity for glycine. The NR2B KI mice exhibited normal locomotor activity making this ifenprodil-insensitive mouse model a valuable tool to test the specificity of NR2B selective antagonists in vivo.
Asunto(s)
Antagonistas de Aminoácidos Excitadores/metabolismo , Piperidinas/metabolismo , Subunidades de Proteína/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Células Cultivadas , Maleato de Dizocilpina/metabolismo , Agonistas de Aminoácidos Excitadores/metabolismo , Femenino , Marcación de Gen , Hipocampo/citología , Humanos , Masculino , Ratones , Ratones Transgénicos , Actividad Motora/fisiología , N-Metilaspartato/metabolismo , Neuronas/citología , Neuronas/metabolismo , Técnicas de Placa-Clamp , Subunidades de Proteína/genética , Receptores de N-Metil-D-Aspartato/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Xenopus laevisRESUMEN
Presenilins appear to form the active center of gamma-secretase but require the presence of the integral membrane proteins nicastrin, anterior pharynx defective 1, and presenilin enhancer 2 for catalytic function. We have simultaneously overexpressed all of these polypeptides, and we demonstrate functional assembly of the enzyme complex, a substantial increase in enzyme activity, and binding of all components to a transition state analogue gamma-secretase inhibitor. Co-localization of all components can be observed in the Golgi compartment, and further trafficking of the individual constituents seems to be dependent on functional assembly. Apart from its catalytic function, gamma-secretase appears to play a role in the trafficking of the beta-amyloid precursor protein, which was changed upon reconstitution of the enzyme but unaffected by pharmacological inhibition. Because the relative molecular mass and stoichiometry of the active enzyme complex remain elusive, we performed size exclusion chromatography of solubilized gamma-secretase, which yielded evidence of a tetrameric form of the complex, yet almost completely abolished enzyme activity. Gamma-secretase activity was reconstituted upon addition of an independent size exclusion chromatography fraction of lower molecular mass and nonproteinaceous nature, which could be replaced by a brain lipid extract. The same treatment was able to restore enzyme activity after immunoaffinity purification of the gamma-secretase complex, demonstrating that lipids play a key role in preserving the catalytic activity of this protease. Furthermore, these data show that it is important to discriminate between intact, inactive gamma-secretase complexes and the active form of the enzyme, indicating the care that must be taken in the study of gamma-secretase.
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Endopeptidasas/biosíntesis , Metabolismo de los Lípidos , Glicoproteínas de Membrana/metabolismo , Péptido Hidrolasas/metabolismo , Secretasas de la Proteína Precursora del Amiloide , Precursor de Proteína beta-Amiloide/metabolismo , Ácido Aspártico Endopeptidasas , Sitios de Unión , Western Blotting , Encéfalo/metabolismo , Catálisis , Línea Celular , Membrana Celular/metabolismo , Sistema Libre de Células/metabolismo , Cromatografía , Medios de Cultivo/metabolismo , Medios de Cultivo Condicionados/farmacología , ADN Complementario/metabolismo , Dimerización , Endopeptidasas/metabolismo , Aparato de Golgi/metabolismo , Humanos , Inmunohistoquímica , Péptidos/química , Estructura Terciaria de Proteína , Factores de TiempoRESUMEN
Interleukin (IL-) 2 and IL-15 share the IL-2 receptor betagamma c subunits (IL-2Rbetagamma c) but have specific, unique alpha receptor subunits. We studied species specificity of human (hu), simian (si), and mouse (mu) IL-15 and found that hu and si IL-15 behaved similarly in all systems investigated. Hu and mu IL-15 bound hu or mu IL-15Ralpha with equal high affinity in the presence or absence of IL-2Rbetagamma c and exhibited similar proliferative activities on cells containing all three subunits. However, quantitative differences were noted in the specific activity of hu and mu IL-15 in both in vitro and in vivo systems utilizing IL-2Rbetagamma c in the absence of IL-15Ralpha. These data show that hu IL-15 may be used in mouse model systems, however care must be taken when comparing the efficacy and toxicity of cytokines across species.
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Interleucina-15/metabolismo , Receptores de Interleucina-2/metabolismo , Animales , Unión Competitiva , División Celular/efectos de los fármacos , Células Cultivadas , Glicosilación , Haplorrinos , Humanos , Interleucina-15/farmacología , Ratones , Ratones Endogámicos C57BL , Unión Proteica , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Receptores de Interleucina-15 , Receptores de Interleucina-2/química , Solubilidad , Especificidad de la Especie , Bazo/citología , Bazo/efectos de los fármacos , Bazo/metabolismoRESUMEN
The aim of this study was to validate a low-density DNA microarray "Rat HepatoChip", which contains 59 genes from a range of potential toxic markers and drug metabolism-related genes. Liver mRNA was isolated from rats dosed with six different chemicals, dexamethasone, troleandomycin, miconazole, clotrimazole, and methylclofanapate, which are all known to induce different cytochrome P450 genes, and isoniazid, which does not cause histopathological changes. Replicate microarrays were used to measure the variability in the chips and in the process. The average variability in signal between different chips observed in triplicate experiments was 33% ranging from 21 to 39% depending on genes. We also demonstrated a strong correlation between the liver histopathology and the gene expression profiles indicating that the gene expression profile reflects histopathological changes. These results suggest that the Rat HepatoChip microarray may provide a fast and effective tool for assessing the toxicity profile of developmental drug candidates during the drug discovery process.