RESUMEN
The acceleration of drug discovery due to combinatorial chemistry and high-throughput screening methods has increased the numbers of candidate pharmaceuticals entering the drug development phase, and the capability to accurately predict whether drug candidates will induce various members of the drug-metabolizing cytochrome P450 (CYP) enzyme superfamily is currently of great interest to the pharmaceutical industry. In the present study, we describe the rapid and reliable analysis of CYP induction in a readily obtained model system (cultured rat hepatocytes) using both real-time quantitative reverse transcription-polymerase chain reaction (real-time RT-PCR) and the RNA invasive cleavage assay. The levels of members in the three primary inducible rat CYP subfamilies (CYP1A1, CYP2B1/2, and CYP3A1) were analyzed in untreated and induced (beta-naphthoflavone, phenobarbital, and hydrocortisone) hepatocyte cultures under various media conditions to screen for optimal CYP induction profiles. The fold inductions measured by real-time RT-PCR and the RNA invasive cleavage assay were also compared with enzyme activity measurements in parallel cultures using liquid chromatography/double mass spectrometry-based assays, and the sensitivity and the specificity of the two RNA analysis methods were compared. Using these techniques, various culture conditions were examined for optimizing induction of the three CYP subfamily members. Both real-time RT-PCR and the RNA invasive cleavage assay prove to be effective methods for determining the effects of drugs on specific CYPs in primary rat hepatocytes.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/biosíntesis , Hepatocitos/enzimología , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Animales , Citocromo P-450 CYP3A , Sistema Enzimático del Citocromo P-450/genética , Dexametasona/farmacología , Inducción Enzimática , Masculino , Espectrometría de Masas , Pliegue de Proteína , Ratas , Ratas Sprague-DawleyRESUMEN
A rugged, stable liquid sheath interfacing procedure for on-line CE/MS is described. This procedure combines optimized component sizes, tapered capillary tips, and adjustment of the capillary with respect to the liquid sheath tube during electrospray operation (active capillary positioning) to establish a stable electrospray quickly and reliably. The interface is especially effective at low flow rates, and CE/MS with a liquid sheath flow rate of 250 nL/min has been achieved. Active capillary positioning also allows on-line capillary isoelectric focusing mass spectrometry with in-probe focusing. In this application, the capillary is retreated into the liquid sheath tube, which becomes a microreservoir. After focusing, the capillary is returned to its proper position for electrospray, and focused zones are mobilized into the mass spectrometer for on-line detection. Both active capillary positioning and in-probe focusing readily lend themselves to automation.
Asunto(s)
Electroforesis Capilar/métodos , Hemoglobinas/análisis , Espectrometría de Masas/métodos , Electroforesis Capilar/instrumentaciónRESUMEN
The microscale techniques of CZE, cIEF and SDS capillary electrophoresis have been evaluated for the analysis of a complex glycoprotein, recombinant tissue plasminogen activator (rtPA). A series of omega-amino acid buffers (pH approximately 5) was found suitable for the CZE separation of rtPA on coated capillaries. rtPA could be resolved into a series of major and minor peaks in an epsilon-aminocaproic acid buffer containing 0.01% (v/v) Tween 80. For cIEF, a two step method with pressure mobilization was utilized. Using a commercial instrument, either a polymer solution with a 50 microns I.D. capillary or narrow bore capillaries without a polymer solution (25 microns I.D.) were employed. rtPA was resolved into at least eight species within a pI range of 6.4-9.2 using Ampholine 3.5-10. Migration time precision for the major peaks ranged from 0.2% for CZE to < or = 2-3% R.S.D. for cIEF. Total recovery of rtPA from the capillary was also demonstrated for both methods. Analysis of rtPA, rtPA Type I, rtPA Type II and the desialylated forms resulted in the expected elution profiles. Finally, the potential of SDS capillary electrophoresis using a coated capillary for an rtPA Type I/Type II purity assay was shown.
Asunto(s)
Electroforesis/métodos , Activador de Tejido Plasminógeno/análisis , Activador de Tejido Plasminógeno/química , Acetatos/química , Acrilamidas/química , Adsorción , Secuencia de Aminoácidos , Mezclas Anfólitas/química , Animales , Electroforesis Capilar/métodos , Electroforesis en Gel de Poliacrilamida/métodos , Ensayo de Inmunoadsorción Enzimática , Cabras/inmunología , Sueros Inmunes/inmunología , Focalización Isoeléctrica/métodos , Punto Isoeléctrico , Datos de Secuencia Molecular , Neuraminidasa/metabolismo , Plasminógeno/metabolismo , Alcohol Polivinílico/química , Estructura Secundaria de Proteína , Proteínas Recombinantes/análisis , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Reproducibilidad de los Resultados , Dodecil Sulfato de Sodio/química , Tensoactivos/química , Activador de Tejido Plasminógeno/inmunología , Activador de Tejido Plasminógeno/metabolismoRESUMEN
We have measured the response of WC/C multilayers to x-ray fluxes on the order of 200 MW/cm2 using laser-generated plasmas and found that these multilayers will maintain near peak reflectivity for at least 1 ns but are eventually destroyed. A description of the experiments and data analysis methods is given. Transmission electron micrographs of WC/C multilayers before and after irradiation show melting to be the dominant damage mechanism. The results of the experiments will be compared with simulations.
RESUMEN
A theoretical model for the laser-induced thermal lens effect in weakly absorbing media is derived. The model predicts the intensity variation in the far field of the laser beam in the presence of the lensing medium and takes into account the aberrant nature of the thermal lens. Some experimental results which support the validity of this approach are presented.