RESUMO
PURPOSE: We developed a prokaryotic expression system to express the major capsid protein of Polyomavirus, VP1. Furthermore, we investigated the transport of single stranded (ss) and double stranded (ds) DNA, mediated through VP1 as drug delivery system into mouse fibroblasts. METHODS: To study DNA delivery we used two kinds of DNA, a ssDNA fragment (19mer) and dsDNA (plasmid pEGFPN1, 4.7 kb or a FITC-labelled dsDNA fragment, 1.8 kb). RESULTS: The uptake of VP1 capsoids loaded with FITC-labelled oligodeoxynucleotides (FODNs) was observed. VP1 pentamers loaded with condensates of dendrimer/dsDNA fragments (FITC-labelled) resulted in significantly higher fluorescence signal in the cytoplasm of NIH 3T3 cells in comparison to control experiments without VP1. Additionally, VP1 capsoids loaded with plasmid pEGFPN1 without dendrimers resulted in an approximately 10 fold higher transfection rate in comparison to blank DNA controls. CONCLUSIONS: Our results demonstrated the potential of VP1 capsoids as DNA delivery system. EGFP expression was significantly enhanced when plasmid DNA was delivered via VP1 capsoids, compared to control experiments with naked DNA.
Assuntos
Proteínas de Ligação a DNA/genética , DNA/metabolismo , Oligonucleotídeos Antissenso/genética , Proteínas de Plantas/genética , Plasmídeos/genética , Fatores de Transcrição/genética , Transfecção/métodos , Células 3T3 , Animais , DNA/genética , Proteínas de Ligação a DNA/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Camundongos , Oligonucleotídeos Antissenso/metabolismo , Proteínas de Plantas/metabolismo , Plasmídeos/metabolismo , Polyomavirus/genética , Polyomavirus/metabolismo , Transativadores , Fatores de Transcrição/metabolismoRESUMO
Oligodendrocytes (OL) isolated from adult pig brains regenerate their processes and form a network of fibers after 14-18 days in vitro (DIV). Stimulation of protein kinase C (Pk-C) by tumour promoters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) produces at day 7 in vitro a similar network after only 20 hr. H-7, an inhibitor of Pk-C, as well as amiloride, which inhibits the subsequent Na+/H+ exchange, reversibly suppress this effect. Activation of the protein kinase A or calmodulin pathway do not result in an increased OL process production. Furthermore, TPA induced proliferation in a subpopulation of OL. We conclude that the stimulation of Pk-C is of utmost importance for OL regeneration.
Assuntos
Encéfalo/fisiologia , Isoquinolinas/farmacologia , Regeneração Nervosa/efeitos dos fármacos , Oligodendroglia/citologia , Piperazinas/farmacologia , Proteína Quinase C/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina , Amilorida/farmacologia , Animais , Encéfalo/citologia , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Proteína Glial Fibrilar Ácida/análise , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/ultraestrutura , Proteína Quinase C/antagonistas & inibidores , SuínosRESUMO
Oligodendrocytes were isolated from adult pig brain and cultivated for 18-24 days. [(14)C]acetate, [(3)H]galactose or [(35)S]sulfate were added to the medium for an additional 24 h. Lipids were extracted and separated by high-performance thin-layer chromatography. The labeled lipids were studied by fluorography and scintillation counting. [(14)C]acetate was incorporated in decreasing order into neutral lipids, phosphatidylcholine, ethanolamine phosphatides, galactocerebrosides, phosphatidylinositol, phosphatidylserine, sulfatides and sphingomyelin. From the [(14)C]acetate incorporated into ethanolamine and choline phosphatides, 71.6 and 14.8%, respectively, were found in plasmalogens. Among neutral lipids, [(14)C]acetate labeled not only cholesterol but also large amounts of triglycerides. No cholesterol esters were synthesized. [(3)H]galactose primarily labeled galactocerebrosides, sulfatides, and monogalactosyl diglyceride. [(35)S]sulfate incorporation was restricted to sulfatides. Together with our previous results concerning proteins, these data show that: (1) oligodendrocytes remain highly differentiated in long-term cultures; (2) they are able to synthesize the major components of myelin; (3) they synthesize surprisingly high amounts of triglycerides and of monogalactosyl diglyceride, a marker for myelination.
