RESUMEN
ATP and other purine nucleotides are important biomarkers for ischemia and may have considerable potential as targets for management of ischemic heart disease and stroke. The main objective of the study is to develop a rapid HPLC assay, which has adequate sensitivity and specificity for measuring concentrations of ATP, ADP, AMP, GTP, GDP and GMP in erythrocytes (RBC). The assays used ion-pair chromatography coupled with ultraviolet detection at 254 nm to separate and detect the purine nucleotides. Using 50-100 microL of RBC lysate as blank biologic matrix, the assay was linear from 100 to 2000 microg/mL for ATP and ADP, and 20-400 microg/mL for AMP, GTP, and GDP with coefficients of determination (r(2)) >0.99. GDP and GMP were not measurable in the study because of low concentrations and interference from endogenous materials, respectively. The intra-assay and inter-assay variations over a period of 1 year were less than 10% and 20%, respectively for most of the nucleotides. The assay was successfully applied to two pilot biomarker studies to measure RBC concentrations of the purine nucleotides in rats under restraining and exercise conditions. Preliminary results showed that the RBC concentrations of ATP and GTP were higher in the spontaneously hypertensive rats (SHR) compared to the Sprague-Dawley (SD) rats, and that exercise increased RBC concentrations of ATP in rats treated with the calcium channel blocker diltiazem.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Eritrocitos/metabolismo , Nucleótidos de Purina/sangre , Espectrofotometría Ultravioleta/métodos , Adenosina Difosfato/sangre , Adenosina Monofosfato/sangre , Adenosina Trifosfato/sangre , Animales , Bioensayo , Biomarcadores/sangre , Bloqueadores de los Canales de Calcio/farmacología , Diltiazem/farmacología , Guanosina Trifosfato/sangre , Masculino , Condicionamiento Físico Animal , Proyectos Piloto , Ratas , Ratas Endogámicas SHR , Ratas Sprague-Dawley , Estándares de Referencia , Sensibilidad y Especificidad , Extracción en Fase Sólida , Especificidad de la EspecieRESUMEN
In order to identify a suitable rodent model for preclinical study of calcium antagonists, pharmacokinetics and metabolism of diltiazem (DTZ) were compared in normotensive SD and hypertensive SHR rat models following multiple doses (5 mg/kg twice daily for 5 doses). Plasma concentrations of DTZ and its major metabolites appeared to be higher in the SHR than the SD rats, although the differences did not reach statistical significance (p > 0.05). The preliminary results suggest metabolism profile of DTZ in the SHR may be closer to humans than the SD rats and may be more preferred in pre-clinical drug development studies for DTZ.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacocinética , Diltiazem/farmacocinética , Modelos Animales , Animales , Bloqueadores de los Canales de Calcio/administración & dosificación , Diltiazem/administración & dosificación , Evaluación Preclínica de Medicamentos/métodos , Inyecciones Subcutáneas , Masculino , Ratas , Ratas Endogámicas SHR , Ratas Sprague-Dawley , Especificidad de la EspecieRESUMEN
Ketoconazole is a widely prescribed antifungal drug, which has also been investigated as an anticancer therapy in both clinical and pre-clinical settings. However, severe hepatic injuries were reported to be associated with the use of ketoconazole, even in patients routinely monitored for their liver functions. Several questions concerning ketoconazole-induced hepatic injury remain unanswered, including (1) does ketoconazole alter cytochrome P450 expression at the transcriptional level?, (2) what types of gene products responsible for cytotoxicity are induced by ketoconazole?, and (3) what role do the major metabolites of ketoconazole play in this pathophysiologic process? A mouse model was employed to investigate hepatic gene expression following hepatotoxic doses of ketoconazole. Hepatic gene expression was analyzed using a toxicogenomic microarray platform, which is comprised of cDNA probes generated from livers exposed to various hepatoxicants. These hepatoxicants fall into five well-studied toxicological categories: peroxisome proliferators, aryl hydrocarbon receptor agonists, noncoplanar polychlorinated biphenyls, inflammatory agents, and hypoxia-inducing agents. Nine genes encoding enzymes involved in Phase I metabolism and one Phase II enzyme (glutathione S-transferase) were found to be upregulated. Serum amyloid A (SAA1/2) and hepcidin were the only genes that were downregulated among the 2364 genes assessed. In vitro cytotoxicity and transcription analyses revealed that SAA and hepcidin are associated with the general toxicity of ketoconazole, and might be usefully explored as generalized surrogate markers of xenobiotic-induced hepatic injury. Finally, it was shown that the primary metabolite of ketoconazole (de-N-acetyl ketoconazole) is largely responsible for the hepatoxicity and the downregulation of SAA and hepcidin.
Asunto(s)
Antifúngicos/toxicidad , Expresión Génica/efectos de los fármacos , Cetoconazol/toxicidad , Hígado/efectos de los fármacos , Aldehído Deshidrogenasa/genética , Familia de Aldehído Deshidrogenasa 1 , Animales , Péptidos Catiónicos Antimicrobianos/genética , Proliferación Celular/efectos de los fármacos , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Perfilación de la Expresión Génica , Glutatión Transferasa/genética , Hepcidinas , Isoenzimas/genética , Hígado/metabolismo , Masculino , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/metabolismo , Retinal-Deshidrogenasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína Amiloide A Sérica/genéticaRESUMEN
A number of quantitative, real-time PCR methods have been developed for determining transgene copy numbers in plants. Here, we demonstrate that the Roche LightCycler system can be used to determine the zygosity of transgenic lines without the use of standard curves or efficiency correction calculations. We have developed a duplex PCR assay which permits the determination of zygosity, relative to a calibrator sample, in transgenic rice lines containing the gene for a viral glycoprotein. Our data demonstrate that unambiguous 2-fold discrimination of copy number can be attained by calculating relative copy number using the threshold crossing point (Ct) calculated by the LightCycler software combined with delta delta Ct calculations, provided that the appropriate calibrator sample is included in each run. The method presented here is rapid, sensitive, robust and easy to optimise.
Asunto(s)
Oryza/genética , Plantas Modificadas Genéticamente , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Genotipo , Estándares de Referencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/normasRESUMEN
Pharmacogenetic variation is an important factor in the therapeutic outcome of many drug treatments. The cytochrome P450 isoform CYP1A2 is involved in the metabolism of a number of antipsychotic drugs. Variable expression of this enzyme may result in idiosyncratic drug responses, including adverse reactions. A number of DNA sequence polymorphisms have been identified in the CYP1A2 gene. Of these, two alleles, CYP1A2*1C and CYP1A2*1F, have been linked to changes in gene expression among smokers. In addition, these polymorphisms have been linked to susceptibility to tardive dyskinesia in some patient populations receiving antipsychotic drug therapy. Here, we present a rapid and robust method for simultaneously genotyping the CYP1A2*1C and *1F alleles using fluorescent hybridization probes and a widely available real-time polymerase chain reaction platform. Such an assay would offer the opportunity to routinely establish the CYP1A2 genotype of a patient prior to commencing drug therapy.
Asunto(s)
Alelos , Citocromo P-450 CYP1A1/genética , ADN/genética , Desnaturalización de Ácido Nucleico , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo de Nucleótido Simple , Antipsicóticos/farmacocinética , Sistemas de Computación , ADN/análisis , Resistencia a Medicamentos/genética , Genotipo , Calor , Humanos , Reproducibilidad de los Resultados , Esquizofrenia/tratamiento farmacológico , Esquizofrenia/genética , Sensibilidad y Especificidad , Fumar/genéticaRESUMEN
BACKGROUND: The abundance of allergenic Penicillium species has been associated with an increased incidence of childhood asthma and pulmonary bleeding. Penicillium brevicompactum has been identified as the most prevalent indoor species of this genus. However, detailed studies on the allergens of the ubiquitous Penicillium species are still lacking. For the characterization of allergens of prevalent Penicillium species, molecular cloning of the allergen genes of P. brevicompactum was performed in the present study. METHODS: A phage cDNA library of P. brevicompactum was constructed in Uni-ZAP XR vector using mRNA isolated from the organism. The cDNA library of P. brevicompactum was screened using pooled atopic sera. RESULTS: Screening of P. brevicompactum cDNA library resulted in one positive clone encoding an estimated molecular weight of 11 kDa polypeptide, rich in acidic residues (>20%), with a pI of 3.87. This clone was designated as Pen b 26 and found to be reactive only against the atopic sera obtained from individuals sensitive to P. brevicompactum. The amino acid sequence analysis of Pen b 26 revealed that it had strong homology to the 60S acidic ribosomal protein P1 family from different eukaryotic sources, predominantly fungal aero-allergens. Other features of Pen b 26 including having high alpha-helical content (>50%), alanine-rich residues (>20%), and a well-conserved C-terminal epitope region fits well into the common properties of 60S acidic ribosomal proteins. CONCLUSIONS: The results obtained suggest that the allergenic clone, Pen b 26 is a 60S acidic ribosomal protein P1 of P. brevicompactum and shows strong similarity to other P1 family proteins.
Asunto(s)
Antígenos Fúngicos/genética , Clonación Molecular , ADN Complementario/aislamiento & purificación , Inmunoglobulina E/inmunología , Penicillium/genética , Penicillium/inmunología , Secuencia de Aminoácidos , Animales , Antígenos Fúngicos/inmunología , Secuencia de Bases , Northern Blotting , Biblioteca de Genes , Humanos , Hibridación in Situ , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
This white paper by eighty members of the Complex Trait Consortium presents a community's view on the approaches and statistical analyses that are needed for the identification of genetic loci that determine quantitative traits. Quantitative trait loci (QTLs) can be identified in several ways, but is there a definitive test of whether a candidate locus actually corresponds to a specific QTL?