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Proteoglicanos/metabolismo , Animales , Células CHO , Células COS , Cricetinae , Expresión Génica , Células HeLa , Humanos , Inmunohistoquímica , Microscopía Inmunoelectrónica , Proteoglicanos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fracciones Subcelulares/metabolismo , TransfecciónRESUMEN
In this article, we report the misdirected targeting of expressed aggrecan domains. Aggrecan, the chondroitin sulfate (CS) proteoglycan of cartilage, normally progresses through the exocytic pathway. Proteins expressed from constructs containing the putative aggrecan signal sequence (i.e., the first 23 N-terminal amino acids), specified globular (G) domains G1 and/or G3, and a segment of the CS domain were detected in the endoplasmic reticulum (ER) and Golgi complex. Although proteins expressed from constructs containing the putative signal and G3, but lacking G1, were detected to a limited extent in the secretory pathway, they primarily accumulated in nuclei. Discrete nuclear inclusions were seen when G3 was expressed. Immunoelectron microscopic characterization of the inclusions suggested the association of nuclear G3 with other proteins. When signal-free G3 constructs and those with G3 immediately following the N-terminal signal were expressed, abundant dispersed accumulations filled the nucleoplasm. The data suggest first, that signal-free and signal-containing G3 proteins enter the nucleus from the cytosol, and second, that the entry of signal-containing G3 proteins into the ER lumen is inefficient. Hsp25, Hsp70, and ubiquitin were colocalized with nuclear G3, indicating the involvement of chaperones and the degradative machinery in the formation and/or attempted disposal of the abnormal nuclear inclusions. Overall, the results focus attention on (1) intracellular protein trafficking at the ER membrane and the nuclear envelope and (2) chaperone interactions and mechanisms leading to abnormal protein deposition in the nucleus.
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Núcleo Celular/metabolismo , Proteínas de la Matriz Extracelular , Procesamiento Proteico-Postraduccional , Señales de Clasificación de Proteína , Estructura Terciaria de Proteína , Proteoglicanos/metabolismo , Agrecanos , Animales , Western Blotting , Células CHO , Núcleo Celular/química , Núcleo Celular/ultraestructura , Cricetinae , Exocitosis , Genes Reporteros , Proteínas HSP70 de Choque Térmico/metabolismo , Inmunohistoquímica , Cuerpos de Inclusión/química , Laminas , Lectinas Tipo C , Microscopía Confocal , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/química , Octoxinol/química , Pruebas de Precipitina , Transporte de Proteínas , Proteoglicanos/química , Proteoglicanos/genética , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Ubiquitinas/metabolismoRESUMEN
The prp2 gene of fission yeast has previously been shown to encode the large subunit of the splicing factor spU2AF. SpU2AF(59) is an evolutionarily conserved protein that has an arginine/serine-rich region and three RNA recognition motifs (RRMs). We have sequenced three temperature-sensitive alleles of prp2 and determined that the mutations result in single amino acid changes within one of the RRMs or between RRMs. All mutant alleles of prp2 have pre-mRNA splicing defects at the non-permissive temperature. Although the mutant strains are growth-arrested at 37 degrees C, they do not elongate like typical fission yeast cell cycle mutants. The DNA of the prp2(-) strains stains more intensely than a wild-type strain, suggesting that the chromatin may be condensed. Ultrastructural studies show differences in the mutant nuclei including a prominent distinction between the chromatin- and non-chromatin-enriched regions compared to the more homogenous wild-type nucleus. Two-hybrid assays indicate that some of the wild-type protein interactions are altered in the mutant strains. These results suggest that normal functioning of spU2AF(59) may be essential not only for pre-mRNA splicing but also for the maintenance of proper nuclear structure and normal cell cycle progression.
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Mutación , Proteínas Nucleares , Precursores del ARN/metabolismo , Empalme del ARN , Ribonucleoproteínas/genética , Proteínas de Saccharomyces cerevisiae , Schizosaccharomyces/genética , Alelos , Ciclo Celular/fisiología , Núcleo Celular/ultraestructura , ARN Helicasas DEAD-box , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Hidroxiurea/farmacología , ARN de Hongos/genética , ARN de Hongos/metabolismo , Ribonucleoproteínas/metabolismo , Schizosaccharomyces/citología , Schizosaccharomyces/crecimiento & desarrollo , Schizosaccharomyces/metabolismo , Factor de Empalme U2AF , Temperatura , Técnicas del Sistema de Dos HíbridosRESUMEN
Osteopetrosis is a heterogeneous group of bone diseases characterized by an excess accumulation of bone and a variety of immune defects. Osteopetrosis (op) and incisors absent (ia) are two nonallelic mutations in the rat which demonstrated these skeletal defects as a result of reduced bone resorption. Osteopetrotic (op) rats have severe sclerosis as a result of reduced numbers of osteoclasts which are structurally abnormal. The sclerosis in ia rats is not as severe as in op mutants; they have elevated numbers of osteoclasts, but they are also morphologically abnormal, lacking a ruffled border. Both of these mutations have defects in the inflammation-primed activation of macrophages. They demonstrate independent defects in the cascade involved in the conversion of vitamin D binding protein (DBP) to a potent macrophage activating factor (DBP-MAF). Because this factor may also play a role in the pathogenesis of osteoclastic dysfunction, the effects of ex vivo-generated DBP-MAF were evaluated on the skeletal system of these two mutations. Newborn ia and op rats and normal littermate controls were injected with DBP-MAF or vehicle once every 4 days from birth until 2 weeks of age, at which time bone samples were collected to evaluate a number of skeletal parameters. DBP-MAF treated op rats had an increased number of osteoclasts and the majority of them exhibited normal structure. There was also reduced bone volume in the treated op animals and an associated increased cellularity of the marrow spaces. The skeletal sclerosis was also corrected in the ia rats; the bone marrow cavity size was significantly enlarged and the majority of the osteoclasts appeared normal with extensive ruffled borders.
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Resorción Ósea/tratamiento farmacológico , Factores Activadores de Macrófagos/farmacología , Osteopetrosis/tratamiento farmacológico , Proteína de Unión a Vitamina D/farmacología , Animales , Médula Ósea/efectos de los fármacos , Médula Ósea/patología , Células de la Médula Ósea , Modelos Animales de Enfermedad , Factores Activadores de Macrófagos/administración & dosificación , Factores Activadores de Macrófagos/uso terapéutico , Microscopía Electrónica , Mutación/efectos de los fármacos , Mutación/genética , Osteoclastos/citología , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Osteoclastos/patología , Osteopetrosis/genética , Oxidación-Reducción , Ratas , Tibia/efectos de los fármacos , Tibia/patología , Tibia/ultraestructura , Proteína de Unión a Vitamina D/administración & dosificación , Proteína de Unión a Vitamina D/uso terapéuticoRESUMEN
The subcellular site of xylosylation, the first carbohydrate modification of the core protein that initiates glycosaminoglycan chain synthesis, was characterized in situ. Methods were developed to combine electron microscopic (EM) autoradiography and the radiolabeling of semi-intact chondrocytes. In the accompanying paper, Kearns et al. (Kearns, A. E., Vertel, B. M., and Schwartz, N. B. (1993) J. Biol. Chem. 268, 11097-11104) presented biochemical and subcellular fractionation studies that utilized semi-intact chondrocytes and radiolabeled UDP sugars to overcome obstacles to the direct analysis of xylosylation. The results suggested that xylosylation begins in the endoplasmic reticulum (ER) and continues in the Golgi. The site of xylosylation was not specified further due to the limitations of subcellular fractionation techniques. The studies described in this report were undertaken to localize these modifications directly in situ. Semi-intact cell preparations were optimized for ultrastructural preservation by modifications of permeabilization methods utilizing nitrocellulose filter overlays. Biochemical analysis demonstrated the exclusive incorporation of UDP-xylose into the cartilage chondroitin sulfate proteoglycan (aggrecan) core protein and 3'-phosphoadenosine 5'-phosphosulfate (PAPS) into the highly modified proteoglycan monomer. Immunolocalization studies showed the equivalence of cytoplasmic subcompartments in normal and semi-intact chondrocytes at the levels of light and electron microscopy. Once the biochemical and morphological equivalence of intact and semi-intact cells was established, EM autoradiographic studies were pursued using UDP-[3H]xylose and [35S]PAPS. Based on both qualitative and quantitative data, silver grains resulting from incorporated sulfate were concentrated in the perinuclear Golgi, while those resulting from incorporated xylose were found at the cis or forming face of the Golgi and in vesicular regions of the peripheral cytoplasm associated with the late ER. These data support the view that xylose addition begins in a late ER compartment and continues in intermediate compartments, perhaps including the cis-Golgi.
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Cartílago/metabolismo , Proteoglicanos Tipo Condroitín Sulfato/biosíntesis , Retículo Endoplásmico/metabolismo , Proteínas de la Matriz Extracelular , Aparato de Golgi/metabolismo , Proteoglicanos/biosíntesis , Uridina Difosfato Xilosa/metabolismo , Xilosa/metabolismo , Agrecanos , Animales , Autorradiografía , Radioisótopos de Carbono , Cartílago/ultraestructura , Núcleo Celular/ultraestructura , Células Cultivadas , Embrión de Pollo , Retículo Endoplásmico/ultraestructura , Glicosaminoglicanos/biosíntesis , Glicosilación , Aparato de Golgi/ultraestructura , Lectinas Tipo C , Microscopía Electrónica , Fosfoadenosina Fosfosulfato/metabolismo , Proteoglicanos/aislamiento & purificación , Radioisótopos de Azufre , TritioRESUMEN
Stable cell lines that synthesize heparin have been established from the Furth murine mastocytoma. The parental line (MST) divides in suspension every 14-18 h in growth medium supplemented with fetal bovine serum or defined growth factors. Adherent subclones were selected by adhesion to plastic culture vessels. Both adherent and nonadherent cells contain about 0.4 micrograms of glycosaminoglycan hexuronic acid per 10(6) cells, composed of 80% heparin and 20% chondroitin sulfate E. Deaminative cleavage of MST heparin by HNO2 at pH 1.5 released disaccharides that were similar in composition to those obtained from commercial heparin, except that disaccharides containing 3,6-O-desulfated GlcN units were not found. Greater than 90% of the glycosaminoglycans were stored in cytoplasmic granules, and challenge of the cells with dinitrophenylated bovine serum albumin and anti-dinitrophenyl IgE released a portion of the stored material. Growth studies of subclones showed that MST cells tolerate a 10-fold variation in glycosaminoglycan content. Incubation of cells with sodium chlorate reduced glycosaminoglycan sulfation by > 95% without affecting cell growth. Thus, granule glycosaminoglycans appear to be nonessential for growth of MST cells.