RESUMEN
Influence of morphine self-administration on gene expression in the rat amygdala was studied using rat genome DNA arrays U34A from Affymetrix. Animals were trained to self-administer morphine, each having two 'yoked' control animals, receiving passive injections of either morphine or saline. After 40 sessions of self-administration, amygdalae were removed, total RNA was isolated and used to prepare probes for Genechip arrays. The treatment was found to significantly change abundance of 29 transcripts. Analysis by means of reverse transcription real-time PCR showed significant changes in abundance of five transcripts: gamma protein kinase C (PKC), upstream binding factor 2 (UBF2), lysozyme, noggin and heat shock protein 70 (hsp70). After 30 days of forced abstinence from morphine self-administration, abundance of hsp70 and lysozyme returned to basal levels. Changes in abundance of UBF2 persisted, and abundance of three additional genes, namely nuclear factor I/A, gamma1 subunit of GABAA receptor and the neuronal calcium sensor 1, changed. Additionally, acute as well as chronic intraperitoneal morphine administration changed the abundance of PKC gamma, gamma1 subunit of GABAA and hsp70 genes.
Asunto(s)
Amígdala del Cerebelo/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Morfina/administración & dosificación , Narcóticos/administración & dosificación , Amígdala del Cerebelo/metabolismo , Animales , Proteínas Portadoras , Esquema de Medicación , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Inyecciones Intraperitoneales/métodos , Masculino , Muramidasa/genética , Muramidasa/metabolismo , Neurofibromina 1/genética , Neurofibromina 1/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Proteínas/genética , Proteínas/metabolismo , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Autoadministración/métodos , Factores de TiempoRESUMEN
The juvenile-hormone-binding protein (JHBP) from Galleria mellonella hemolymph, which is a member of the high-affinity/low-molecular-mass group of JHBP proteins, was found to be glycosylated. Glycosylation was confirmed by the following evidence. Carbohydrate gas-liquid chromatography analysis of the purified JHBP preparations showed the presence of a low amount of sugars (Man and GlcNAc were the major components). The JHBP electrophoretic band blotted onto nitrocellulose was stained with GlycoTrack (a reagent kit used for the detection of protein glycosylation) and showed strong binding of concanavalin A (ConA). JHBP was fractionated on a ConA-Sepharose 4B column into ConA-bound (strongly stained with ConA) and ConA-unbound (hardly stained with ConA) portions. Both fractions showed juvenile-hormone-binding activity and were glycosylated, as revealed by staining both of them with GlycoTrack. Electrospray-ionization mass spectrometry of JHBP suggested the presence of a small amount of presumably nonglycosylated protein (24988 Da) and five glycoforms, two of which (containing Man2GlcNAc, or Man2Fuc1GlcNAc2 chain) were not bound or were weakly bound to ConA, and three (with Man3GlcNAc2, Man5Fuc1GlcNAc2, or Man5GlcNAc2, chain) were present in the fraction strongly bound to ConA. In conclusion, the monosugar composition, GlycoTrack staining, ConA-binding properties and molecular mass analyses of JHBP supplied convincing evidence for its glycosylation and some information on the character of the oligosaccharide chains.