RESUMEN
Lysosome-associated membrane protein (LAMP)-1, one of the major protein components of the lysosomal membrane, is upregulated in the human glioblastoma cell lines, U-373 MG and LN-Z308, which undergo cisplatin-induced apoptosis. These human brain tumor cell lines demonstrated apoptosis in response to cisplatin/nifedipine treatment. Both cell lines demonstrated an apoptotic response by more than one criterion. Apoptosis was demonstrated by DNA fragmentation techniques such as DNA laddering, ApopTag in situ labeling, and an ELISA-based method of detecting liberated oligosomes. These cells also had characteristic morphologic changes and upregulation of bax consistent with apoptosis. LAMP-1 expression at the protein and mRNA level was examined and found to increase with cisplatin/nifedipine treatment. LAMP-1 expression was examined using indirect immunofluorescent staining, Northern blot analysis and Western blot analysis. The finding of an augmentation of LAMP-1 in these cells induced to die is enigmatic. These findings raise the possibility of LAMP-1 involvement in the apoptotic process.
Asunto(s)
Antígenos CD/metabolismo , Apoptosis , Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Glicoproteínas de Membrana/metabolismo , Regulación hacia Arriba , Apoptosis/efectos de los fármacos , Secuencia de Bases , Neoplasias Encefálicas/patología , Cisplatino/farmacología , Cartilla de ADN , Electroforesis en Gel de Agar , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente Indirecta , Glioblastoma/patología , Humanos , Proteínas de Membrana de los Lisosomas , Nifedipino/farmacología , Células Tumorales CultivadasRESUMEN
Many patients with AIDS have a myelopathy characterized by vacuolization of spinal cord white matter. The biochemical and molecular changes underlying this myelin disturbance have not yet been characterized. Myelin basic protein (MBP) is potentially important because it is a key structural protein of myelin with roles in compaction and stabilization. In the present study, we describe the steady-state protein concentration of MBP in 46 patients with AIDS and 12 control subjects at autopsy. Patients with myelopathy exhibited no change in the abundance of the predominant 18.5- and 17.2-kd isoforms, but a 14-kd MBP-immunoreactive degradation fragment was increased significantly. MBP degradation correlated significantly with the severity of histopathologic changes, including neutral lipid deposition, the density of vacuolated fibers, and the number of ferritin-stained activated microglia. Alkaline gel electrophoresis of isolated MBP showed preferential loss of the least cationic isomer (C-8). The concentration of MBP RNA in slot blots was normal in cords exhibiting myelopathy, and the ratio of mRNA corresponding to the 18.5- and 17.2-kd MBP isoforms, measured using reverse transcriptase-PCR, was not altered. This study suggests that mononuclear phagocyte-mediated degradation of MBP may play a role in AIDS myelopathy, and the preferential loss of the C-8 component of MBP may have mechanistic implications.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/metabolismo , Síndrome de Inmunodeficiencia Adquirida/patología , Proteína Básica de Mielina/metabolismo , Síndrome de Inmunodeficiencia Adquirida/genética , Adulto , Western Blotting , Enfermedades Desmielinizantes/genética , Enfermedades Desmielinizantes/metabolismo , Enfermedades Desmielinizantes/patología , Humanos , Hidrólisis , Inmunohistoquímica , Isomerismo , Masculino , Persona de Mediana Edad , Proteína Básica de Mielina/genética , ARN Mensajero/análisis , Médula Espinal/metabolismo , Médula Espinal/patologíaRESUMEN
We utilized an AFP-HPRT transgene, i.e. the HPRT coding sequences under the regulation of AFP enhancer and promoter sequences, to localize the AFP extinguisher locus in intertypic somatic cell hybrids (hepatoma X fibroblast). This hybrid gene construct, which directly links AFP regulation to a reversibly selective gene, enabled the selection of stably transfected cells which express AFP, as well as cells showing extinction of AFP. Mouse hepatoma cells stably transfected with and expressing the transgene were fused to human fibroblasts, and the resulting somatic cell hybrids were characterized using Southern, Northern and karyotypic analyses. That several hybrids exhibited the proper extinction of AFP, AFP-HPRT and albumin suggests coregulation of these genes by an extinguisher. Segregant lines derived from these hybrids were selected for the loss of extinguisher activity and for reexpression of the transgene. Karyotypic analysis of hybrid and segregant lines, exhibiting proper AFP, albumin and AFP-HPRT phenotypes, revealed that the presence of human chromosome 7 was most closely associated with the AFP-extinguished state. The hybrids generated in these studies now make it possible to isolate the sequences responsible for AFP and albumin extinction.
Asunto(s)
Albúminas/genética , Cromosomas Humanos Par 7/genética , Regulación de la Expresión Génica , Células Híbridas/fisiología , alfa-Fetoproteínas/genética , Animales , Carcinoma Hepatocelular , Cromosomas Humanos , ADN/análisis , Elementos de Facilitación Genéticos/genética , Fibroblastos , Humanos , Hipoxantina Fosforribosiltransferasa/genética , Cariotipificación , Ratones , Regiones Promotoras Genéticas/genética , ARN Mensajero/análisis , Transfección , Transgenes/genéticaRESUMEN
We describe a sequential staining technique for the karyotypic analysis of interspecific mouse x human somatic cell hybrids. Fluorescence in situ hybridization of samples, previously stained using standard trypsin/Giemsa protocols, was instrumental in the identification of human chromosomes present in hybrid lines. This procedure not only provided a simple distinction between human and mouse chromosomes, but it also allowed the visualization and monitoring of human sequences present in interspecific translocations and subchromosomal fragments.
Asunto(s)
Bandeo Cromosómico/métodos , Células Híbridas/ultraestructura , Hibridación in Situ/métodos , Cariotipificación/métodos , Animales , Colorantes Azulados , Fijadores , Humanos , RatonesRESUMEN
The histidyl-tRNA synthetase gene (hisS) from Streptococcus equisimilis was cloned and sequenced. The gene for this aminoacyl-tRNA synthetase has an open reading frame of 1278 nucleotides. The deduced amino acid sequence encodes a protein of 426 amino acids with MW = 47,932. The protein is predicted to be soluble with a pl = 5.27. The protein sequence has extensive overall identity/similarity with the Escherichia coli and the yeast histidyl-tRNA synthetases (approximately 58% and approximately 20%, respectively). A putative promoter for gene transcription lies within two hundred nucleotides of the polypeptide start codon. The enzyme was overexpressed, to a level of about 18% of total cellular protein, as a fusion protein (containing an additional 15 amino acids) in E. coli using the pT7 expression system containing the T7 RNA polymerase/promoter (Tabor and Richardson, Proc. Natl. Acad. Sci. U.S.A. 82:1074-1078, 1985). The predicted MW for the hisS gene product is in good agreement with the size of the fusion protein determined by SDS-PAGE (M(r) = 53,700). Amino acid sequencing of the intact fusion protein and proteolytic fragments confirmed the deduced sequence of the synthetase at many positions throughout the protein. The expressed protein catalyzed the specific aminoacylation of tRNA(His) in vitro.