RESUMEN
We describe a flexible system for gene expression profiling using arrays of tens of thousands of oligonucleotides synthesized in situ by an ink-jet printing method employing standard phosphoramidite chemistry. We have characterized the dependence of hybridization specificity and sensitivity on parameters including oligonucleotide length, hybridization stringency, sequence identity, sample abundance, and sample preparation method. We find that 60-mer oligonucleotides reliably detect transcript ratios at one copy per cell in complex biological samples, and that ink-jet arrays are compatible with several different sample amplification and labeling techniques. Furthermore, results using only a single carefully selected oligonucleotide per gene correlate closely with those obtained using complementary DNA (cDNA) arrays. Most of the genes for which measurements differ are members of gene families that can only be distinguished by oligonucleotides. Because different oligonucleotide sequences can be specified for each array, we anticipate that ink-jet oligonucleotide array technology will be useful in a wide variety of DNA microarray applications.
Asunto(s)
Expresión Génica , Hibridación in Situ/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Oligonucleótidos/química , Células Cultivadas , Cromatografía Líquida de Alta Presión , ADN Complementario/metabolismo , Humanos , Procesamiento de Imagen Asistido por Computador , Células Jurkat , Células K562 , Oligonucleótidos/síntesis química , Sistemas de Lectura Abierta , Reacción en Cadena de la Polimerasa , ARN Complementario/metabolismo , ARN Mensajero/metabolismo , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Saccharomyces cerevisiae , Sensibilidad y Especificidad , Factores de Tiempo , Transcripción Genética , Tretinoina/química , Células Tumorales CultivadasRESUMEN
The most important product of the sequencing of a genome is a complete, accurate catalogue of genes and their products, primarily messenger RNA transcripts and their cognate proteins. Such a catalogue cannot be constructed by computational annotation alone; it requires experimental validation on a genome scale. Using 'exon' and 'tiling' arrays fabricated by ink-jet oligonucleotide synthesis, we devised an experimental approach to validate and refine computational gene predictions and define full-length transcripts on the basis of co-regulated expression of their exons. These methods can provide more accurate gene numbers and allow the detection of mRNA splice variants and identification of the tissue- and disease-specific conditions under which genes are expressed. We apply our technique to chromosome 22q under 69 experimental condition pairs, and to the entire human genome under two experimental conditions. We discuss implications for more comprehensive, consistent and reliable genome annotation, more efficient, full-length complementary DNA cloning strategies and application to complex diseases.
Asunto(s)
Cromosomas Humanos Par 22 , Biología Computacional , Genoma Humano , Análisis de Secuencia por Matrices de Oligonucleótidos , Algoritmos , Empalme Alternativo , Línea Celular , ADN Complementario , Exones , Proyecto Genoma Humano , Humanos , Sondas de OligonucleótidosRESUMEN
Ascertaining the impact of uncharacterized perturbations on the cell is a fundamental problem in biology. Here, we describe how a single assay can be used to monitor hundreds of different cellular functions simultaneously. We constructed a reference database or "compendium" of expression profiles corresponding to 300 diverse mutations and chemical treatments in S. cerevisiae, and we show that the cellular pathways affected can be determined by pattern matching, even among very subtle profiles. The utility of this approach is validated by examining profiles caused by deletions of uncharacterized genes: we identify and experimentally confirm that eight uncharacterized open reading frames encode proteins required for sterol metabolism, cell wall function, mitochondrial respiration, or protein synthesis. We also show that the compendium can be used to characterize pharmacological perturbations by identifying a novel target of the commonly used drug dyclonine.
Asunto(s)
Bases de Datos Factuales , Perfilación de la Expresión Génica , Saccharomyces cerevisiae/fisiología , Pared Celular/fisiología , Ergosterol/biosíntesis , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Genes Reporteros , Prueba de Complementación Genética , Variación Genética , Humanos , Mitocondrias/metabolismo , Modelos Genéticos , Mutagénesis , Sistemas de Lectura Abierta , Fenotipo , Propiofenonas/farmacología , Receptores sigma/genética , Ribosomas , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Esteroide Isomerasas/genética , Transcripción GenéticaRESUMEN
Genome-wide transcript profiling was used to monitor signal transduction during yeast pheromone response. Genetic manipulations allowed analysis of changes in gene expression underlying pheromone signaling, cell cycle control, and polarized morphogenesis. A two-dimensional hierarchical clustered matrix, covering 383 of the most highly regulated genes, was constructed from 46 diverse experimental conditions. Diagnostic subsets of coexpressed genes reflected signaling activity, cross talk, and overlap of multiple mitogen-activated protein kinase (MAPK) pathways. Analysis of the profiles specified by two different MAPKs-Fus3p and Kss1p-revealed functional overlap of the filamentous growth and mating responses. Global transcript analysis reflects biological responses associated with the activation and perturbation of signal transduction pathways.
Asunto(s)
Proteínas de Ciclo Celular , Perfilación de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Sistema de Señalización de MAP Quinasas , Proteínas Represoras , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Proteínas Inhibidoras de las Quinasas Dependientes de la Ciclina , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/fisiología , Fase G1 , Genoma Fúngico , Lipoproteínas/farmacología , Lipoproteínas/fisiología , Factor de Apareamiento , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Familia de Multigenes , Análisis de Secuencia por Matrices de Oligonucleótidos , Péptidos/farmacología , Péptidos/fisiología , Feromonas , Proteína Quinasa C/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/fisiología , Factores de Transcripción/metabolismo , Activación TranscripcionalRESUMEN
We describe here a method for drug target validation and identification of secondary drug target effects based on genome-wide gene expression patterns. The method is demonstrated by several experiments, including treatment of yeast mutant strains defective in calcineurin, immunophilins or other genes with the immunosuppressants cyclosporin A or FK506. Presence or absence of the characteristic drug 'signature' pattern of altered gene expression in drug-treated cells with a mutation in the gene encoding a putative target established whether that target was required to generate the drug signature. Drug dependent effects were seen in 'targetless' cells, showing that FK506 affects additional pathways independent of calcineurin and the immunophilins. The described method permits the direct confirmation of drug targets and recognition of drug-dependent changes in gene expression that are modulated through pathways distinct from the drug's intended target. Such a method may prove useful in improving the efficiency of drug development programs.
Asunto(s)
Calcineurina/genética , Ciclosporina/farmacología , Regulación Fúngica de la Expresión Génica , Inmunofilinas/genética , Inmunosupresores/farmacología , Saccharomyces cerevisiae/genética , Tacrolimus/farmacología , Diseño de Fármacos , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Genotipo , Modelos Biológicos , Mutación , Reacción en Cadena de la Polimerasa , Reproducibilidad de los Resultados , Saccharomyces cerevisiae/efectos de los fármacos , Transducción de SeñalRESUMEN
Data-adaptive algorithms are presented for separating overlapping signatures of heterozygotic allele pairs in electrophoresis data. Application is demonstrated for human microsatellite CA-repeat polymorphisms in LiCor 4000 and ABI 373 data. The algorithms allow overlapping alleles to be called correctly in almost every case where a trained observer could do so, and provide a fast automated objective alternative to human reading of the gels. The algorithm also supplies an indication of confidence level which can be used to flag marginal cases for verification by eye, or as input to later stages of statistical analysis.
Asunto(s)
Algoritmos , Alelos , Polimorfismo Genético , Secuencias Repetitivas de Ácidos Nucleicos , Animales , Composición de Base , Electroforesis , Humanos , Matemática , Ratones , Reacción en Cadena de la PolimerasaRESUMEN
Osmotically stimulated vasopressin and oxytocin release were measured in pinealectomized and sham operated male rats infused with hypertonic sodium chloride. Neuronal activation in the hypothalamic regions associated with oxytocin and vasopressin release was investigated by quantitative assessment of Fos protein production. The osmotically stimulated release of both vasopressin and oxytocin was significantly lower in pinealectomized animals as compared to sham operated controls. The slope of regression lines between plasma osmolality and hormone concentrations in the sham animals showed a 1.0 +/- 0.1 pmol per mosm/kg rise in vasopressin and 2.0 +/- 0.4 pmol per mosm/kg rise in oxytocin whilst in the pinealectomized animals these values were significantly lower at 0.4 +/- 0.1 pmol vasopressin per mosm/kg and 0.8 +/- 0.2pmol oxytocin per mosm/kg. The osmotic thresholds for hormone release were unaffected by pinealectomy. Fos production was also significantly lower in the supraoptic nucleus and organ vasculosum of the lamina terminalis in the pinealectomized rat at 62 +/- 20 and 59 +/- 9 Fos immunoreactive cells/section as compared to corresponding values of 202 +/- 31 and 123 +/- 20 Fos immunoreactive cells/section in the shams. These observations suggest that reduced hormone release in the pinealectomized animal is due to lowered responsiveness of central osmoregulatory mechanisms and that melatonin may therefore influence the activation of the magnocellular system.
Asunto(s)
Hipotálamo/fisiología , Proteínas Oncogénicas v-fos/biosíntesis , Oxitocina/metabolismo , Glándula Pineal/fisiología , Vasopresinas/metabolismo , Animales , Presión Sanguínea/fisiología , Volumen Sanguíneo/fisiología , Hipotálamo/metabolismo , Masculino , Neuronas/metabolismo , Concentración Osmolar , Oxitocina/sangre , Glándula Pineal/cirugía , Ratas , Ratas Sprague-Dawley , Vasopresinas/sangreRESUMEN
Pinealectomy has been shown to alter daily rhythms of neurohypophysial hormone release, with plasma hormone concentrations being elevated in the morning, as compared to intact rats. To determine whether pineal removal also altered the response to known stimuli of hormone release, vasopressin concentrations were measured in control, sham-operated, and pinealectomized animals during extracellular fluid hypertonicity produced by an intraperitoneal (i.p.) injection of hypertonic saline or hypovolaemia produced by an i.p. injections of polyethylene glycol. In the combined sham-operated and unoperated groups, injection of hypertonic saline produced a marked increase in plasma vasopressin concentrations from 2.18 +/- 0.28 to 7.2 +/- 1.24 pmol/liter, but the response was attenuated in pinealectomized animals, concentrations increasing to only 3.4 +/- 1.2 pmol/liter. Similarly, following infusion of hypertonic saline, the increase in plasma vasopressin per unit increase in plasma sodium was lower in pinealectomized animals than the pineal intact controls. The response to hypovolaemia was also attenuated, plasma hormone concentrations following reduction in blood volume of approximately 10% increasing to only 3.6 +/- 0.6 pmol/liter as compared to 7.3 +/- 2.2 pmol/liter in the control groups. There were no significant differences in pituitary vasopressin content in any of the groups studied. Thus, the pineal may influence the vasopressin response to physiological stimuli.
Asunto(s)
Volumen Sanguíneo , Glándula Pineal/fisiología , Glándula Pineal/cirugía , Polietilenglicoles/administración & dosificación , Solución Salina Hipertónica/administración & dosificación , Vasopresinas/sangre , Animales , Masculino , Concentración Osmolar , Ratas , Ratas Sprague-Dawley , Choque/sangre , Choque/inducido químicamenteRESUMEN
Plasma concentrations of neurohypophysial hormones show clear rhythms over 24 hr which can be suppressed by exposure to constant light, an observation consistent with pineal involvement. A study has therefore been performed on the changes in the hormone levels in the hypothalamus, posterior pituitary, and plasma over 24 hr in control, pinealectomised, and sham pinealectomised animals to determine if the pineal could play a role. Water intake, urine excretion, packed cell volume, plasma osmolality, and electrolytes were also monitored. Pinealectomy had little effect on fluid balance, but after 8 weeks for oxytocin and 2 weeks for vasopressin the morning values (0700-0800) for the circulating concentrations of the hormones were significantly higher in the pinealectomized group compared with the combined sham operated and unoperated groups (pineal intact). By contrast, the pituitary vasopressin was significantly lower in the pinealectomised group. The increase in plasma oxytocin and vasopressin seen over the hours of daylight and accompanying fall in plasma osmolality seen in the pineal intact group were absent in the pinealectomised group. Similarly, the evening fall in pituitary hormone concentrations and increase in hypothalamic hormone content were absent in the pinealectomised animals. After 10 days of exposure to constant light, the fall in plasma osmolality in the pineal-intact animals over the day was no longer significant; instead a significant increase in plasma osmolality and sodium was seen in the pinealectomised group. Exposure to constant light, while altering the patterns of neurohypophysial activity in the pineal intact group, had little effect on the pinealectomised animals.
Asunto(s)
Ritmo Circadiano/fisiología , Oxitocina/metabolismo , Glándula Pineal/fisiología , Neurohipófisis/metabolismo , Vasopresinas/metabolismo , Animales , Ingestión de Líquidos , Hipotálamo/metabolismo , Luz , Masculino , Melatonina/sangre , Concentración Osmolar , Oxitocina/sangre , Glándula Pineal/cirugía , Radioinmunoensayo , Ratas , Ratas Sprague-Dawley , Vasopresinas/sangre , Equilibrio HidroelectrolíticoRESUMEN
The vasoconstrictor assay, when properly performed, is a highly reliable method to determine bioequivalence of generic formulations. Recent research has resolved some of the remaining questions concerning the practical application of the assay. Significant vehicle-related differences have been observed between the potency of different, supposedly equivalent formulations now on the market. Large differences in concentrations of the active agent in similar vehicles usually have not resulted in corresponding differences in vasoconstrictor assay results. Finally, the time course of drug effects may differ among highly potent and less potent corticosteroids. In general, the higher the potency of the topical corticosteroid, the earlier the maximal effect is observed. This finding suggests that short application of highly potent agents might minimize systemic absorption without sacrificing efficacy.
Asunto(s)
Antiinflamatorios/farmacología , Antiinflamatorios/farmacocinética , Piel/efectos de los fármacos , Vasoconstrictores/farmacología , Administración Tópica , Glucocorticoides , Humanos , Vehículos Farmacéuticos , Equivalencia Terapéutica , Factores de TiempoRESUMEN
The vasoconstrictor assay in human beings was used to assess bioavailability during different time periods of exposure when 0.05% clobetasol propionate cream (Temovate) was applied and left on for periods of 0.5, 1.0, 1.5, and 16.0 hours and subsequently washed. Maximal responses were achieved by 1.5 hours of exposure, but there was no significant difference in intensity of vasoconstriction between 1.0, 1.5, and 16.0 hours of exposure before washing the sites. Exposures to 0.05% clobetasol propionate cream for 0.5 hour were not significantly different from 16-hour exposures to 0.05% fluocinonide cream, but exposures to 0.05% clobetasol propionate cream for 1.0, 1.5, and 16.0 hours all resulted in significant increases in vasoconstriction responses compared with fluocinonide cream applied and left on for 16 hours. Topical exposures to a superpotent topical steroid for a short time give vasoconstrictor responses equivalent to long time exposures.
Asunto(s)
Betametasona/análogos & derivados , Clobetasol/análogos & derivados , Piel/irrigación sanguínea , Vasoconstricción/efectos de los fármacos , Administración Cutánea , Adolescente , Adulto , Clobetasol/administración & dosificación , Clobetasol/farmacocinética , Femenino , Antebrazo , Humanos , Masculino , Persona de Mediana Edad , Distribución Aleatoria , Factores de TiempoRESUMEN
Many topical corticosteroid formulations are available as different concentrations of the steroid in a similar vehicle. We tested the existing assumption that higher concentrations give greater biologic activity. The vasoconstriction assay was used because of its known correlation with clinical activity. Statistical analyses of the different concentrations are as follows: Kenalog creams: 0.025% is equal to 0.1% is equal to 0.5%; Aristocort creams: 0.025% is equal to 0.1% is equal to 0.5%; Aristocort ointments: 0.1% is equal to 0.5%; Aristocort creams: 0.5% is equal to 0.025% but is less than 0.1%; Hytone cream: 1.0% is equal to 2.5%; Synalar creams: 0.01% is less than 0.025% which is less than 0.2%; Topicort creams: 0.25% is equal 0.05%; and Vallisone creams: 0.1% is greater than 0.01%. The assumption that increased concentration of the same steroid in the same vehicle type will give increased biologic activity is usually, but not always, incorrect for brand-name formulations now available.
Asunto(s)
Antiinflamatorios/administración & dosificación , Administración Tópica , Antiinflamatorios/farmacocinética , Disponibilidad Biológica , Relación Dosis-Respuesta a Droga , Composición de Medicamentos , Glucocorticoides , Humanos , Vehículos Farmacéuticos , Vasoconstricción/efectos de los fármacosRESUMEN
The topical antifungal agent naftifine has shown considerable potency against a broad spectrum of dermatophytes. In this study, an in vitro penetration test in human cadaver skin and an in vivo tape-stripping test were used to evaluate the penetration and antifungal activity of naftifine gel 1 percent and naftifine cream 1 percent compared with other antifungal agents. In both models, Trichophyton rubrum and T. mentagrophytes were the fungal species. Results show that naftifine gel 1 percent and naftifine cream 1 percent, in vitro and in vivo, penetrate the stratum corneum in concentrations that inhibit the growth of both fungal species. Following penetration in vitro, naftifine gel and cream were significantly more active against T. rubrum than econazole nitrate cream 1 percent. Following penetration in vivo, naftifine gel and cream were as active as econazole nitrate cream 1 percent and clotrimazole cream 1 percent against T. rubrum and T. mentagrophytes.
Asunto(s)
Alilamina/farmacocinética , Aminas/farmacocinética , Piel/metabolismo , Administración Tópica , Adolescente , Adulto , Alilamina/administración & dosificación , Antifúngicos/administración & dosificación , Antifúngicos/farmacología , Arthrodermataceae/crecimiento & desarrollo , Células Cultivadas , Emolientes , Geles , Humanos , Imidazoles/farmacocinética , Masculino , Persona de Mediana EdadRESUMEN
Studies were conducted to assess the bioequivalence of a new antimycotic formulation, ciclopirox olamine lotion 1%, to an established compound, ciclopirox olamine cream 1%. Results of in vitro studies, using skin samples from human cadavers and domestic pigs, demonstrated that the two formulations equally penetrate all layers of the stratum corneum and inhibit the growth of Trichophyton mentagrophytes and Candida albicans. In vivo studies in guinea pigs and in human volunteers demonstrated the comparable therapeutic efficacy of the lotion and the cream in experimental trichophytosis. In addition, a multicenter, double-blind clinical trial was undertaken to compare ciclopirox olamine lotion 1% with the vehicle alone in the treatment of patients with tinea pedis. Patients with plantar, interdigital, or vesicular tinea pedis were enrolled in the studies. Patients were treated for 28 days. Clinical and mycological responses were determined during treatment and two weeks posttreatment. Ciclopirox olamine lotion 1% was found to be significantly more effective than its vehicle in the treatment of patients with common tinea pedis. Minor localized side effects (pruritus, burning sensation) were reported in 2% of 89 patients treated with ciclopirox olamine lotion 1%. The results demonstrate the bioequivalence of ciclopirox olamine lotion 1% and ciclopirox olamine cream 1% and confirm the clinical effectiveness and safety of the lotion in the treatment of tinea pedis, a generally recalcitrant fungal infection. It is concluded that ciclopirox olamine lotion 1% can be used as an alternative to ciclopirox olamine cream 1% for treatment of tinea pedis, tinea versicolor, tinea cruris, tinea corporis, and cutaneous candidiasis when the convenience and/or cosmetic elegance of a lotion is desired.
Asunto(s)
Antifúngicos/uso terapéutico , Piridonas/uso terapéutico , Tiña del Pie/tratamiento farmacológico , Administración Tópica , Animales , Antifúngicos/administración & dosificación , Antifúngicos/farmacología , Ciclopirox , Ensayos Clínicos como Asunto , Método Doble Ciego , Cobayas , Humanos , Masculino , Pruebas de Sensibilidad Microbiana , Piridonas/administración & dosificación , Piridonas/farmacología , Distribución Aleatoria , Porcinos , Equivalencia Terapéutica , Trichophyton/efectos de los fármacosRESUMEN
Some practical applications of basic information in percutaneous absorption have been reviewed. Drug release from vehicles is discussed in relation to glucocorticosteroids. Penetration enhancers are reviewed with emphasis on the need for further investigations and applications of enhancers for clinical use. The role of the stratum corneum as a barrier to and a reservoir for drugs is discussed. Special problems in penetration as presented by regional anatomic variations, nails, and follicles are mentioned. Overall, we review some practical problems existing in the penetration of drugs through human skin.
Asunto(s)
Farmacocinética , Absorción Cutánea , Animales , Química Farmacéutica , HumanosRESUMEN
A gel formulation of erythromycin 2 percent was compared with its vehicle in a double-blind multicenter study involving patients with mild to moderate acne vulgaris. In an analysis of 187 patients treated twice daily for 8 weeks, erythromycin 2 percent gel proved to be significantly more effective than vehicle in reducing the numbers of both inflammatory and noninflammatory lesions. After 8 weeks, 60 percent of erythromycin-treated patients had good or excellent responses compared with 36 percent of those using vehicle (p = 0.001); the lesions in two patients using erythromycin were completely cleared. The majority of patients had a favorable impression of the cosmetic characteristics of the gel formulation.
Asunto(s)
Acné Vulgar/tratamiento farmacológico , Eritromicina/uso terapéutico , Administración Tópica , Adolescente , Adulto , Comportamiento del Consumidor , Eritromicina/administración & dosificación , Eritromicina/efectos adversos , Dermatosis Facial/tratamiento farmacológico , Femenino , Geles , Humanos , Masculino , Persona de Mediana EdadRESUMEN
The temporal pattern of ultrasonic vocalizations by mice in an undisturbed 'home' environment can now be assessed using a system based on amplitude discrimination. Within a chosen frequency band, vocalizations of sufficient intensity are detected by an amplitude discriminator. The output from a pulse generator is sent to a microcomputer which records the time of the incoming event. The system has been validated for monitoring ultrasonic vocalizations in the mouse.
Asunto(s)
Recolección de Datos/instrumentación , Microcomputadores , Vocalización Animal , Animales , Ratones , Factores de TiempoRESUMEN
Trade name glucocorticoid formulations triamcinolone acetonide, fluocinolone acetonide, and betamethasone valerate were compared with their generic equivalents because of increasing substitution of generic formulations for trade name formulations. The vasoconstrictor assay was the method used for these comparisons. Large differences were found between generic and trade name formulations containing the same steroid in the same concentration in both cream and ointment vehicles. If generic substitutions are to be used for trade name formulations, the physician must be aware that significant differences in therapeutic effectiveness may be expected.