RESUMO
The brain of all higher organisms has a modular architecture. Processing of various tasks, such as learning, olfaction, or motor control is performed in specialized brain areas, characterized by morphological and molecular peculiarities. To identify those genes that are transcribed in only one region of the insect brain, we chose two different approaches, differential display PCR and DNA array hybridization, with two different insect species, the desert locust Schistocerca gregaria and the fruitfly Drosophila melanogaster. The optic lobes (centers of visual information processing), the midbrain (the region of the brain where almost all "higher" centers are localized), and the thoracic ganglia (regions required to control various peripheral organs) were compared in both types of experiments. Both, the differential display PCR screen of the different parts of the locust brain as well as the DNA array screen of the Drosophila brain revealed almost identical numbers of transcripts exclusively present in either of the three above-mentioned brain areas. Interestingly, the brain areas with the largest number of differential transcripts are the thoracic ganglia and not the midbrain.
Assuntos
Encéfalo/metabolismo , Drosophila melanogaster/metabolismo , Gafanhotos/metabolismo , Transcrição Gênica/fisiologia , Animais , Northern Blotting/métodos , Encéfalo/anatomia & histologia , Mapeamento Encefálico , Drosophila melanogaster/anatomia & histologia , Expressão Gênica , Gafanhotos/anatomia & histologia , Hibridização In Situ/métodos , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Lobo Óptico de Animais não Mamíferos/metabolismo , Reação em Cadeia da Polimerase/métodos , RNA/metabolismoRESUMO
Human herpesvirus 8 (HHV-8) encodes several putative oncogenes, which are homologues to cellular host genes known to function in cell cycle regulation, control of apoptosis, and cytokine signaling. Viral interleukin (vIL-6) is believed to play an important role in the pathogenesis of Kaposi's sarcoma as well as primary effusion lymphoma and multicentric Castleman's disease. Therefore, vIL-6 is a promising target for novel therapies directed against HHV-8-associated diseases. By phage display screening of human synthetic antibody libraries, we have selected a specific recombinant antibody, called monoclonal anti-vIL-6 (MAV), binding to vIL-6. The epitope recognized by MAV was localized on the top of the D helix of the vIL-6 protein, which is a part of receptor binding site III. Consequently, MAV specifically inhibits vIL-6-mediated growth of the primary effusion lymphoma-derived cell line BCBL-1 and blocks STAT3 phosphorylation in the human hepatoma cell line HepG2. Since it was previously found that vIL-6 can also induce signals from within the cell, presumably within the endoplasmic reticulum, we fused the recombinant antibody MAV with the endoplasmic retention sequence KDEL (MAV-KDEL). As a result, COS-7 cells expressing MAV-KDEL and synthesizing vIL-6 ceased to secrete the cytokine. Moreover, we observed that vIL-6 that was bound to MAV-KDEL and retained in the endoplasmic reticulum did not induce STAT3 phosphorylation in HepG2 cells. We conclude that the activity of the intracellularly retained vIL-6 protein is neutralized by MAV-KDEL. Our results might represent a novel therapeutic strategy to neutralize virally encoded growth factors or oncogenes.
Assuntos
Anticorpos Antivirais/metabolismo , Retículo Endoplasmático/metabolismo , Interleucina-6/imunologia , Biblioteca de Peptídeos , Proteínas Virais/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Antivirais/imunologia , Especificidade de Anticorpos , Células COS , Linhagem Celular , Chlorocebus aethiops , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Região Variável de Imunoglobulina/imunologia , Interleucina-6/genética , Interleucina-6/metabolismo , Testes de Neutralização , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Transdução de Sinais/imunologia , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Interleukin-6 (IL-6) activates cells by binding to the membrane-bound IL-6 receptor (IL-6R) and subsequent formation of a glycoprotein 130 homodimer. Cells that express glycoprotein 130, but not the IL-6R, can be activated by IL-6 and the soluble IL-6R which is generated by shedding from the cell surface or by alternative splicing. Here we show that cholesterol depletion of cells with methyl-beta-cyclodextrin increases IL-6R shedding independent of protein kinase C activation and thus differs from phorbol ester-induced shedding. Contrary to cholesterol depletion, cholesterol enrichment did not increase IL-6R shedding. Shedding of the IL-6R because of cholesterol depletion is highly dependent on the metalloproteinase ADAM17 (tumor necrosis factor-alpha-converting enzyme), and the related ADAM10, which is identified here for the first time as an enzyme involved in constitutive and induced shedding of the human IL-6R. When combined with protein kinase C inhibition by staurosporine or rottlerin, breakdown of plasma membrane sphingomyelin or enrichment of the plasma membrane with ceramide also increased IL-6R shedding. The effect of cholesterol depletion was confirmed in human THP-1 and Hep3B cells and in primary human peripheral blood monocytes, which naturally express the IL-6R. For decades, high cholesterol levels have been considered harmful. This study indicates that low cholesterol levels may play a role in shedding of the membrane-bound IL-6R and thereby in the immunopathogenesis of human diseases.