RESUMO
Small interfering RNAs (siRNAs) delivery remains a bottleneck for RNA interference (RNAi) - based therapies in the clinic. In the present study, a fusion protein with two cell-penetrating peptides (CPP), Hph1-Hph1, and a double-stranded RNA binding domain (dsRBD), was constructed for the siRNA delivery: dsRBD was designed to bind siRNA, and CPP would subsequently transport the dsRBD/siRNA complex into cells. We assessed the efficiency of the fusion protein, Hph1-Hph1-dsRBD, as a siRNA carrier. Calcium-condensed effects were assessed on GAPDH and green fluorescent protein (GFP) genes by western blot, real time polymerase chain reaction (RT-PCR), and flow cytometry analysis in vitro. Evaluations were also made in an in vivo heart transplantation model. The results demonstrated that the fusion protein, Hph1-Hph1-dsRBD, is highly efficient at delivering siRNA in vitro, and exhibits efficiency on GAPDH and GFP genes similar to or greater than lipofectamine. Interestingly, the calcium-condensed effects dramatically enhanced cellular uptake of the protein-siRNA complex. In vivo, Hph1-Hph1-dsRBD transferred and distributed ^ targeted siRNA throughout the whole mouse heart graft. Together, these results indicate that Hph1-Hph1-dsRBD has potential as an siRNA carrier for applications in the clinic or in biomedical research.
Assuntos
Peptídeos Penetradores de Células/metabolismo , Técnicas de Transferência de Genes , Terapia Genética/métodos , Transplante de Coração , Fragmentos de Peptídeos/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Transporte Biológico , Cálcio/metabolismo , Peptídeos Penetradores de Células/química , Regulação da Expressão Gênica , Genes Reporter , Gliceraldeído-3-Fosfato Desidrogenases/biossíntese , Gliceraldeído-3-Fosfato Desidrogenases/genética , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Células HeLa , Humanos , Masculino , Camundongos Endogâmicos C57BL , Fragmentos de Peptídeos/química , Domínios e Motivos de Interação entre Proteínas , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Reperfusion injury is characterized by significant oxidative stress. F(2)-isoprostanes (F(2)-IsoP's) and isofurans (IsoF's), the latter preferentially produced during increased oxygen tension, are recognized markers of in vivo oxidative stress. We aimed to determine whether increasing oxygen tension during reperfusion modified levels of plasma total IsoF's and F(2)-IsoP's. Forty-five patients undergoing upper-limb surgery were randomized to receive inspired oxygen concentrations of 30, 50, or 80% during the last 15 min of surgery. Venous blood samples were taken before the change in inspired oxygen, after 10 min (before reperfusion), and after 15 min (5 min after reperfusion). IsoF's and F(2)-IsoP's were measured by gas chromatography-mass spectrometry. Venous oxygen tension and hemoglobin concentrations were also measured. Plasma IsoF and F(2)-IsoP levels in the 50 and 80% O(2) groups were not significantly different from those of the 30% O(2) group. In secondary analyses, using data combining all groups, levels of IsoF's, but not F(2)-IsoP's, associated with higher venous oxygen tension (P=0.038). Hemoglobin negatively modified the influence of oxygen tension on levels of IsoF's (P=0.014). This study has shown, for the first time, that plasma IsoF levels associate with higher oxygen tension in a human model of reperfusion, and this effect is significantly attenuated by hemoglobin.
Assuntos
Biomarcadores/sangue , Furanos/sangue , Hemoglobinas/metabolismo , Isoprostanos/sangue , Oxigênio/metabolismo , Traumatismo por Reperfusão/metabolismo , Extremidade Superior/cirurgia , Adulto , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Hemoglobinas/farmacologia , Humanos , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Consumo de Oxigênio/fisiologia , Traumatismo por Reperfusão/sangue , Extremidade Superior/patologiaRESUMO
BACKGROUND/AIMS: Heteromeric KCNEx/KCNQ1 (=KvLQT1, Kv7.1) K(+) channels are important for repolarization of cardiac myocytes, endolymph secretion in the inner ear, gastric acid secretion, and transport across epithelia. They are modulated by pH in a complex way: homomeric KCNQ1 is inhibited by external acidification (low pH(e)); KCNE2/KCNQ1 is activated; and for KCNE1/KCNQ1, variable effects have been reported. METHODS: The role of KCNE subunits for the effect of pH(e) on KCNQ1 was analyzed in transfected COS cells and cardiac myocytes by the patch-clamp technique. RESULTS: In outside-out patches of transfected cells, hKCNE2/hKCNQ1 current was increased by acidification down to pH 4.5. Chimeras with the acid-insensitive hKCNE3 revealed that the extracellular N-terminus and at least part of the transmembrane domain of hKCNE2 are needed for activation by low pH(e). hKCNE1/hKCNQ1 heteromeric channels exhibited marked changes of biophysical properties at low pH(e): The slowly activating hKCNE1/hKCNQ1 channels were converted into constitutively open, non-deactivating channels. Experiments on guinea pig and mouse cardiac myocytes pointed to an important role of KCNQ1 during acidosis implicating a significant contribution to cardiac repolarization under acidic conditions. CONCLUSION: External pH can modify current amplitude and biophysical properties of KCNQ1. KCNE subunits work as molecular switches by modulating the pH sensitivity of human KCNQ1.
