RESUMO
The main objective of the present study was to find suitable DNA-targeting sequences (DTS) for the construction of plasmid vectors to be used to treat ischemic diseases. The well-known Simian virus 40 nuclear DTS (SV40-DTS) and hypoxia-responsive element (HRE) sequences were used to construct plasmid vectors to express the human vascular endothelial growth factor gene (hVEGF). The rate of plasmid nuclear transport and consequent gene expression under normoxia (20 percent O2) and hypoxia (less than 5 percent O2) were determined. Plasmids containing the SV40-DTS or HRE sequences were constructed and used to transfect the A293T cell line (a human embryonic kidney cell line) in vitro and mouse skeletal muscle cells in vivo. Plasmid transport to the nucleus was monitored by real-time PCR, and the expression level of the hVEGF gene was measured by ELISA. The in vitro nuclear transport efficiency of the SV40-DTS plasmid was about 50 percent lower under hypoxia, while the HRE plasmid was about 50 percent higher under hypoxia. Quantitation of reporter gene expression in vitro and in vivo, under hypoxia and normoxia, confirmed that the SV40-DTS plasmid functioned better under normoxia, while the HRE plasmid was superior under hypoxia. These results indicate that the efficiency of gene expression by plasmids containing DNA binding sequences is affected by the concentration of oxygen in the medium.
Assuntos
Animais , Humanos , Masculino , Camundongos , Sequência de Bases/genética , Hipóxia Celular/genética , Expressão Gênica/genética , /genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Marcação de Genes , Vetores Genéticos/genética , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase , Plasmídeos/genética , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
A construct (AT1R-NF) containing a "Flag" sequence added to the N-terminus of the rat AT1 receptor was stably expressed in Chinese hamster ovary cells and quantified in the cell membrane by confocal microscopy after reaction with a fluorescein-labeled anti-Flag monoclonal antibody. Angiotensin II bound to AT1R-NF and induced endocytosis with a half-time of 2 min. After 60-90 min, fluorescence accumulated around the cell nucleus, suggesting migration of the ligand-receptor complex to the nuclear membrane. Angiotensin antagonists also induced endocytosis, suggesting that a common step in the transduction signal mechanism occurring after ligand binding may be responsible for the ligand-receptor complex internalization
Assuntos
Animais , Cricetinae , Ratos , Angiotensina II/fisiologia , Células CHO , Endocitose , Receptores de Angiotensina/fisiologia , Angiotensina II/antagonistas & inibidores , Northern Blotting , Membrana Celular , Endocitose/fisiologia , Ligantes , Microscopia Confocal , Transdução de Sinais , TransfecçãoRESUMO
1. In the filamentous mold Aspergillus nidulans, net nitrite uptake is inducible by nitrate and nitrite, is probably different from the nitrate uptake system and is partially repressed by ammonium. 2. The concentration dependence of net nitrite uptake by the biA1 facA303 strain of A. nidulans shows a saturation kinetics with an apparent Km value of 0.64 mM. 3. Strains of A. nidulans carrying the nihA1 and nihA1 chlA14 mutations considerably reduced the affinity of the nitrite uptake system for the substrate when nitrite concentrations ranging from 2.5 mM to 10.0 mM were tested (apparent Km values of 5.9 mM and 23.6 mM, respectively). 4. These results suggest that the toxic effect of nitrite on A. nidulans is due to enhanced nitrite uptake by nihA strains when high concentrations are present in the medium
Assuntos
Aspergillus nidulans/metabolismo , Nitritos/farmacocinética , Amônia/farmacologia , Nitratos/farmacocinética , Nitritos/antagonistas & inibidores , Fatores de TempoRESUMO
The mycelial Pi-repressible acid phosphatase presented p-nitrophenylphosphatase activity with negative cooperativity and Michaelian behavior when synthesized by the wild-type and pho-2A mutant strains of Neurospora crassa, respectively. The major acid phosphatase present in cell extracts of the pho-2A mutant of N. crassa grown in low Pi medium is more thermolabile (t½= 4 min at 54 grade C, pH 5.4) than that of the wild strain (stable for at least 80 min at 54 grade C, pH 5.4). The pho-2A mutant of N. crassa secreted a more thermobabile acid phosphatase (t½=30 min at 50 grade C, pH 5.4) than the wild strain (t½ of at least 80 min at 50 grade C, pH 5.4). The pho-2A mutant of N. crassa synthesized a more thermolabile acid phosphatase (t½=37 min at 54 grade C, pH 5.4) than the wild strain in high Pi medium (t½=14 min at 54 grade C, pH 5.4). The pleiotropic nature of the pho-2 locus and its possible involement in the mechanism of phosphatase secretion by N. crassa are proposed
Assuntos
4-Nitrofenilfosfatase , Fosfatase Ácida , Fosfatase Alcalina , Enzimas/metabolismo , Neurospora crassa , Genes , MutaçãoRESUMO
1. Even though altered forms of acid phosphatase II were wynthesixed by the mutant strains nuc-1A and nuc-2A of N. crassa, their synthesis was independent of exogenous phosphate concentratiosn. 2. Synthesis of acid phosphatase I by nuc-2A was also insensitive to exogenous phosphate concentrations. When nuc-1A was grown on a low-phosphate medium, it also produced a heat-labile acid phosphatase in addition to a I-like acid phosphatase. I-like acid phosphatase was not detected in the mycelium of the preg*c mutant strain grown on low-phosphate medium. 3. These results are consitent with participation of the nuc-2, preg and nuc-1 genes in regulating the transport and/or secretion of acid phosphatase and probably other phosphatases by Neurospora crassa