RESUMO
In this study, we evaluated the NTPDases and ecto-5'-nucleotidase (CD73) expression profiles and the pattern of adenine nucleotide hydrolysis in rats submitted to the Walker 256 tumor model, 6, 10 and 15 days after the subcutaneous inoculation. Using RT-PCR analysis, we identified mRNA for all of the members of the ecto-nucleoside triphosphate diphosphohydrolase family investigated and a 5'-nucleotidase. By quantitative real-time PCR, Entpd1 (Cd39) and Entpd2 (Cd39L1) and CD73 were identified as the dominant genes expressed by the Walker 256 tumor, at all times studied. Extracellular adenine nucleotide hydrolysis by the Walker 256 tumor was estimated by HPLC analysis. Rapid hydrolysis of extracellular ATP by the tumor cells was observed, leading to the formation of adenosine and inosine in cells obtained from solid tumors at 6 and 10 days after inoculation. Cells obtained from solid tumors at 15 days of growth presented high levels of AMP and presented adenosine as a final product after 90 min of incubation. Results demonstrate that the presence of NTPDases and 5'-nucleotidase enzymes in Walker 256 tumor cells may be important for regulation of the extracellular adenine nucleotides/adenine nucleoside ratio, therefore leading to tumor growth.
Assuntos
5'-Nucleotidase/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Antígenos CD/metabolismo , Apirase/metabolismo , Carcinoma 256 de Walker/enzimologia , Animais , Linhagem Celular Tumoral , Masculino , Ratos , Ratos WistarRESUMO
Inflammatory and degenerative pathophysiological processes within the CNS are important causes of human disease. Astrocytes appear to modulate these reactions and are a major source of inflammatory mediators, e.g. extracellular adenine nucleotides, in nervous tissues. Actions following extracellular nucleotides binding to type 2 purinergic receptors are regulated by ectonucleotidases, including members of the CD39/ecto-nucleoside triphosphate diphosphohydrolase family. The ectonucleotidases of astrocytes expressed by rat brain rapidly convert extracellular ATP to ADP, ultimately to AMP. RT-PCR, immunocytochemistry as well as Western blotting analysis demonstrated expression of multiple ecto-nucleoside triphosphate diphosphohydrolase family members at both the mRNA and protein level. By quantitative real-time PCR, we identified Entpd2 (CD39L1) as the dominant Entpd gene expressed by rat hippocampal, cortical and cerebellar astrocytes. These data in combination with the elevated ecto-ATPase activity observed in these brain regions, suggest that NTPDase2, an ecto-enzyme that preferentially hydrolyzes ATP, is the major ecto-nucleoside triphosphate diphosphohydrolase expressed by rat astrocytes. NTPDase2 may modulate inflammatory reactions within the CNS and could represent a useful therapeutic target in human disease.
Assuntos
Adenosina Trifosfatases/genética , Astrócitos/enzimologia , Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Encéfalo/enzimologia , Cinética , Ratos , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Especificidade por Substrato , TransfecçãoRESUMO
Adenoviruses have been used for gene therapy or immunization due to their ability to efficiently infect a broad range of cells and tissues. These applications as well as specificity could be improved further by redirecting binding of the virus to specific cell types. In this regard, modification of viral genes encoding coat proteins is an option to achieve improvement in retargeting. In this report, we describe a substitution in the adenovirus type 2 fiber globular region by the 44 amino acid C4 domain of human immunodeficiency virus type 1 gp120. In vitro translation analysis and immunoprecipitation assays show that the incorporation of the C4 domain into the fiber protein does not ablate its trimerization property and demonstrates the availability of the C4 epitope for interaction with monoclonal anti-C4 antibody. The recombinant adenovirus containing this modified fiber was also characterized by immunoprecipitation with the same antibody, showing the viability of such kind of modification.
Assuntos
Adenoviridae/metabolismo , Capsídeo/química , Adenoviridae/química , Linhagem Celular , Epitopos , Genoma Viral , Proteína gp120 do Envelope de HIV/metabolismo , Ligantes , Modelos Biológicos , Reação em Cadeia da Polimerase , Testes de Precipitina , Ligação Proteica , Biossíntese de Proteínas , Estrutura Terciária de Proteína , Recombinação Genética , Transcrição GênicaRESUMO
The 18-kDa protein from Mycobacterium leprae is a major target for the immune response in leprosy. We have developed a system to express this antigen in yeast as a fusion protein with the C-terminal region of the yeast membrane protein GAS1, which would render the recombinant protein anchored to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. Cells lacking the GAS1 gene and transformed with the hybrid 18-kDa-GAS1 construct express a polypeptide that reacts with an 18-kDa-specific monoclonal antibody. In addition, these cells react with an alpha-CRD antibody after GPI-PLC treatment. The non-transformed cells are negative. These data indicate that our system may be suitable for the expression of foreign proteins in yeast in a GPI-anchored form.
Assuntos
Proteínas de Bactérias/genética , Glicosilfosfatidilinositóis/genética , Mycobacterium leprae/imunologia , Proteínas de Saccharomyces cerevisiae , Proteínas de Bactérias/imunologia , Proteínas Fúngicas/genética , Genes Fúngicos , Vetores Genéticos , Glicosilfosfatidilinositóis/imunologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Mycobacterium leprae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/imunologiaRESUMO
The 18-kDa protein from Mycobacterium leprae is a major target for the immune response in leprosy. We have developed a system to express this antigen in yeast as a fusion protein with the C-terminal region of the yeast membrane protein GAS1, which would render the recombinant protein anchored to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. Cells lacking the GAS1 gene and transformed with the hybrid 18-kDa-GAS1 construct express a polypeptide that reacts with an 18-kDa-specific monoclonal antibody. In addition, these cells react with an alpha-CRD antibody after GPI-PLC treatment. The non-transformed cells are negative. These data indicate that our system may be suitable for the expression of foreign proteins in yeast in a GPI-anchored form