RESUMO
A transgenic cell line stably expressing the human organic anion transporting polypeptide (OATP1B1) was established. Human Embryonic Kidney 293 (HEK293) cell line stably expressing OATP1B1*1a sequence was amplified through PCR with the extracted total RNA as templates from human liver, then subcloned into the plasmid pMD19-T and verified by sequencing. OATP1B1*1b/OATP1B1*15 mutant sequences were obtained by site-directed mutation PCR with pMD19-T/ OATP1B1*1a as templates. The plasmids pcDNA3.1(+)/OATP1B1*1a, *1b and *15 were constructed and transfected into HEK293 cell line using Lipofectamine 2000 transfection reagent. Several stable transfected clones were obtained after selection with G418. Using rosuvastatin as a probe substrate of OATP1B1, the intracellular rosuvastatin accumulation in HEK293 and HEK-OATP1B1*1a, *1b and *15 monoclone cells were validated by a ultra-performance liquid chromatography-tandem mass spectrometry. OATP1B1 mRNA and protein expression were detected by RT-PCR and Western blot, respectively. The results from RT-PCR, rosuvastatin uptake and Western blot assay indicated that human OATP1B1 was highly expressed in transfected cells compared with controls. The HEK-293 cell lines stably expressing human OATP1B1-wild and variant (HEK-OATP1B1, *1b and *15) are potential models to study drug transport in vitro.
Assuntos
Células HEK293/metabolismo , Transportadores de Ânions Orgânicos/biossíntese , Transportadores de Ânions Orgânicos/genética , Transfecção/métodos , Membrana Celular/química , Membrana Celular/metabolismo , Variação Genética , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/metabolismo , Lipídeos , Fígado/metabolismo , Transportador 1 de Ânion Orgânico Específico do Fígado , Mutagênese Sítio-Dirigida , Plasmídeos/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Rosuvastatina Cálcica/metabolismoRESUMO
Genetic vaccines have progressed significantly since the first demonstration of the technology in 1992. When Sanford and Johnston first developed the idea, two applications were envisaged. One was as a new, simple, possibly more effective, method for delivering vaccines. The other was as a new tool to explore the immune system and to discover new vaccines. As there has been relatively little emphasis on the latter, we provide three examples of the potential uses of genetic immunization for discovery/manipulation. One of these technologies may have important implications for the safety of the vaccines. Finally, we propose that the clinical application of genetic vaccines may be limited by inadequate delivery systems and propose the characteristics of an ideal system.
Assuntos
Vacinas de DNA , Animais , Biolística , Expressão Gênica , Biblioteca Gênica , Vetores Genéticos , Humanos , Imunização/métodos , Imunização/tendências , Vacinas de DNA/administração & dosagemRESUMO
The endogenous opioid beta-endorphin, a derivative of proopiomelanocortin, stimulates the growth of cloned human small cell lung carcinoma. The present hypothesis states that mutations of the retinoblastoma gene (a tumor suppressor gene) associated to the malignant transformation of bronchial cells would trigger a cascade of biomolecular events leading to 'de novo' proopiomelanocortin expression in small cell lung carcinoma.
Assuntos
Carcinoma de Células Pequenas/genética , Regulação Neoplásica da Expressão Gênica , Genes do Retinoblastoma , Genes fos , Hormônios Ectópicos/biossíntese , Neoplasias Pulmonares/genética , Modelos Biológicos , Proteínas de Neoplasias/biossíntese , Pró-Opiomelanocortina/biossíntese , Síndrome de ACTH Ectópico/etiologia , Carcinoma de Células Pequenas/metabolismo , Síndrome de Cushing/etiologia , Síndrome de Cushing/genética , Feminino , Hormônios Ectópicos/genética , Humanos , Neoplasias Pulmonares/metabolismo , Proteínas de Neoplasias/genética , Pró-Opiomelanocortina/genética , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-fos/fisiologia , Proteína do Retinoblastoma/deficiência , Proteína do Retinoblastoma/fisiologia , beta-Endorfina/metabolismoRESUMO
1. The effect of beta-endorphin (beta-EP) and morphine sulfate (MS), in presence and absence of naloxone (NX), on chicken chorioallantoic membrane was studied as a function of blood vessel proliferation. 2. A 50% reduction in blood vessel proliferation occurred by 10 micrograms of beta-EP or by 5 micrograms of MS per egg compared to controls. 3. An individual dose, i.e. 5 micrograms of beta-EP, did not significantly inhibit blood vessel counts after initial 24 hr period of the drug application when given alone compared to inhibition occurring with combined use of NX. 4. NX (1 microgram) did not significantly reverse the angiostatic effects of MS (10 micrograms) or of beta-EP (5 micrograms). 5. The observed modulation of angiogenesis by opioids suggests involvement of beta-EP and MS in the proliferation of vascular endothelial cells. 6. This may be due to an effect of beta-EP and MS on cell-mediated immunity factors such as interferons, interleukins and prostaglandin E2.