RESUMO
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.
Assuntos
Tonsila do Cerebelo/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Morfina/administração & dosagem , Entorpecentes/administração & dosagem , Tonsila do Cerebelo/metabolismo , Animais , Proteínas de Transporte , Esquema de Medicação , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Injeções Intraperitoneais/métodos , Masculino , Muramidase/genética , Muramidase/metabolismo , Neurofibromina 1/genética , Neurofibromina 1/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Proteínas/genética , Proteínas/metabolismo , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Autoadministração/métodos , Fatores de TempoRESUMO
It has been previously suggested that juvenile hormone binding protein(s) (JHBP) belongs to a new class of proteins. In the search for other protein(s) that may contain structural motifs similar to those found in JHBP, hemolymph from Galleria mellonella (Lepidoptera) was chromatographed over a Sephadex G-200 column and resulting fractions were subjected to SDS-PAGE, transferred onto nitrocellulose membrane and scanned with a monoclonal antibody, mAb 104, against hemolymph JHBP. Two proteins yielded a positive reaction with mAb 104, one corresponding to JHBP and the second corresponding to a transferrin, as judged from N-terminal amino acid sequencing staining. Transferrin was purified to about 80% homogeneity using a two-step procedure including Sephadex G-200 gel filtration and HPLC MonoQ column chromatography. Panning of a random peptide display library and analysis with immobilized synthetic peptides were applied for finding a common epitope present in JHBP and the transferrin molecule. The postulated epitope motif recognized by mAb 104 in the JHBP sequence is RDTKAVN, and is localized at position 82-88.
Assuntos
Proteínas de Transporte/química , Proteínas de Insetos , Lepidópteros/química , Transferrina/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Antígenos/química , Antígenos/genética , Antígenos/imunologia , Proteínas de Transporte/genética , Proteínas de Transporte/imunologia , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Compostos Cromogênicos/química , Eletroforese em Gel de Poliacrilamida , Mapeamento de Epitopos , Epitopos/análise , Hemolinfa/química , Immunoblotting , Lepidópteros/genética , Lepidópteros/imunologia , Dados de Sequência Molecular , Filogenia , Análise de Sequência de Proteína , Transferrina/genética , Transferrina/imunologia , Triazinas/químicaRESUMO
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.
Assuntos
Proteínas de Transporte/química , Hemolinfa/química , Proteínas de Insetos/química , Mariposas/química , Animais , Cromatografia de Afinidade , Concanavalina A/química , Glicoproteínas/química , Glicosídeo Hidrolases/metabolismo , Glicosilação , Espectrometria de MassasRESUMO
Previously described methods of purification of hemolymph juvenile hormone-binding protein (hJHBP) from Lepidoptera were tedious and required multiple steps. These methods resulted in low protein yield (Kramer et al., 1976; Goodman et al., 1978; Peterson et al., 1982; Park et al., 1993; Ozyhar & Kochman, 1987). In this report a simple method of purification of hJHBP from Galleria mellonella (L.) larvae is described. Monoclonal antibodies against hJHBP were obtained and crosslinked to CNBr-activated Sepharose 4B. The hemolymph of G. mellonella was centrifuged and then chromatographed on Sephadex G-200 gel filtration column. Juvenile-hormone-binding activity containing material from Sephadex G-200 column was subjected to purification on an immunoaffinity column. Bound protein was eluted from anti-hJHBP Sepharose 4B gel by lowering pH to 3.0 with 200 mM citric acid 200 mM Na2HPO4 buffer. This method resulted in 320-fold purification of G. mellonella hJHBP with 56% yield.