RESUMEN
Based on the multivalent binding capability of streptavidin (SA) to biotin, a multifunctional quantum dot probe (QD-(AS-ODN+p160)) coupled with antisense oligonucleotide (AS-ODN) and peptide p160 is designed for real-time tracking of targeted delivery of AS-ODN and regulation of folate receptor-α (hFR-α) in MCF-7 breast cancer cells. Fluorescence spectra, capillary electrophoresis (CE) and dynamic light scattering (DLS) are used to characterize the conjugation of AS-ODN and p160 with quantum dots (QDs), DLS results confirm the well stability of the probe in aqueous media. Confocal imaging and quantitative flow cytometry show that QD-(AS-ODN+p160) is able to specifically target human breast cancer MCF-7 cells. Low temperature and ATP depletion treatments reveal the cellular uptake of QD-(AS-ODN+p160) is energy-dependent, and the effects of inhibition agents and co-localization imaging further confirm the endocytic pathway is mainly receptor-mediated. Transmission electron microscopy (TEM) shows the intracellular delivery and endosomal escape of QD probe along with incubation time extended. Two transfection concentrations of QD probe (10 nM and 50 nM) below half inhibitory concentration (IC50 ) value are chosen according to MTT assay. Real-time PCR shows at these two concentration cases the relative mRNA expression levels of hFR-α reduce to 72.5 ± 3.9% and 17.6 ± 1.0%, respectively. However, western blot and quantitative ELISA analysis show the expression level of hFR-α protein has a significant decrease only at 50 nM, indicating that gene silence is concentration-dependent. These results demonstrate that the QD-(AS-ODN+p160) probe not only achieves gene silence in a cell-specific manner but also achieves real-time tracking during AS-ODN intracellular delivery.
Asunto(s)
Receptor 1 de Folato/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias/metabolismo , Oligonucleótidos Antisentido/química , Péptidos/química , Puntos Cuánticos/química , Sistemas de Liberación de Medicamentos , Citometría de Flujo , Células HeLa , Células Hep G2 , Humanos , Concentración 50 Inhibidora , Células MCF-7 , Microscopía Confocal , Microscopía Electrónica de Transmisión , Nanotecnología , ARN Interferente Pequeño/metabolismo , Estreptavidina/químicaRESUMEN
Size-controllable micron or nano-disk carbon fiber electrode (CFE) is prepared and demonstrated to be excellent for extra-cellular transmitter release detection at tiny structures and vesicle fusion kinetics analysis with high spatio-temporal resolution. An improved electrochemical etching procedure was employed, for the first time, to fabricate cylindrical fiber with controlled micron or nano-diameter. Afterwards, a facile insulation with polypropylene sheath was employed to completely insulate the whole body of the thinned fiber, and an ultrasmall-disk sensing area was finally produced by cutting of the insulated fibers. Scanning electron microscopy (SEM) was employed to characterize the ultrasmall geometry size of the fabricated electrode and to show the tight adherence of the insulation sheath on the fiber. The cut ends of the electrodes were also shown to be smooth, clean and without obvious jagged layer. The fabricated micron or nano-disk carbon electrodes show ideal steady-state voltammetric behavior with satisfying reversibility. Subsequently, the performance of the ultrasmall-disk CFE for amperometric detection of cell secretion was characterized. Results showed that, compared to the conventional micro-disk CFE, the etched small disk CFE possesses higher sensitivity due to its obviously improved signal-to-noise level, which enables minute amounts of 3000 oxidizable molecules to be detectable. The nano-disk CFE was shown to be particularly ideal for analysis of fusion kinetics, due to its avoidance of diffusion broadening of the detected spikes, which is the inherent defect of the conventional micro-CFE technique.
Asunto(s)
Electroquímica/instrumentación , Microelectrodos , Neuronas/metabolismo , Neurotransmisores/metabolismo , Vesículas Sinápticas/metabolismo , Animales , Caenorhabditis elegans , Células Cultivadas , Diseño de Equipo , Análisis de Falla de Equipo , Cinética , Ratas , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Synaptotagmins (Syts) are calcium-binding proteins which are conserved from nematodes to humans. Fifteen Syts have been identified in mammalian species. Syt I is recognized as a Ca(2+) sensor for the synchronized release of synaptic vesicles in some types of neurons, but its role in the secretion of dense core vesicles (DCVs) remains unclear. The function of Syt IV is of particular interest because it is rapidly up-regulated by chronic depolarization and seizures. Using RNAi-mediated gene silencing, we have explored the role of Syt I and IV on secretion in a pituitary gonadotrope cell line. Downregulation of Syt IV clearly reduced Ca(2+)-triggered exocytosis of dense core vesicles (DCVs) in LbetaT2 cells. Syt I silencing, however, had no effect on vesicular release.
Asunto(s)
Exocitosis , Vesículas Secretoras/metabolismo , Sinaptotagminas/fisiología , Animales , Calcio/farmacología , Línea Celular , Regulación hacia Abajo , Exocitosis/efectos de los fármacos , Exocitosis/genética , Ratones , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología , Vesículas Secretoras/efectos de los fármacos , Sinaptotagmina I/genética , Sinaptotagmina I/metabolismo , Sinaptotagmina I/fisiología , Sinaptotagminas/genética , Sinaptotagminas/metabolismoRESUMEN
Synaptotagmins (Syts) constitute a large family of at least 16 members and individual Syt isoforms exhibit distinct Ca(2+)-binding properties and subcellular localization. It remains to be demonstrated whether multiple Syt isoforms can function independently or cooperatively on certain type of vesicle. In the current study, we have developed NPY-pHluorin to specifically assess exocytosis of large dense core vesicles (LDCVs) and studied the requirement of Syt I and Syt IX for LDCV exocytosis in PC12 cells. We found that down-regulation of both Syt I and Syt IX resulted in a significant loss of Ca(2+)-dependent LDCV exocytosis. Moreover, our results suggest Syt I and Syt IX play redundant role in controlling the choice of fusion modes. Down-regulation of both Syt I and Syt IX renders more fusion in the kiss-and-run mode. We conclude that Syt I and Syt IX function redundantly in Ca(2+)-sensing and fusion pore dilation on LDCVs in PC12 cells.
Asunto(s)
Vesículas Sinápticas/metabolismo , Sinaptotagmina I/fisiología , Sinaptotagminas/fisiología , Animales , Catecolaminas/metabolismo , Exocitosis , Fusión de Membrana , Células PC12 , Interferencia de ARN , Ratas , Sinaptotagmina I/antagonistas & inhibidores , Sinaptotagmina I/genética , Sinaptotagminas/antagonistas & inhibidores , Sinaptotagminas/genéticaRESUMEN
Carbon fiber nanoelectrodes (tip diameter = ca. 100 nm) have been first used to monitor real-time dopamine release from single living vesicles of single rat pheochromocytoma (PC12) cells. The experiments show that active and inactive release sites exist on the surface of cells, and the spatial distributions have been differentiated even in the same active release zone. It is first demonstrated that multiple vesicles can sequentially release dopamine at the same site of the cell surface, which possibly plays the main role in the dopamine release from PC12 cells.
Asunto(s)
Dopamina/metabolismo , Nanotecnología , Potenciales de Acción/fisiología , Animales , Carbono/química , Fibra de Carbono , Dopamina/química , Electroquímica , Electrodos , Exocitosis/fisiología , Microelectrodos , Neurotransmisores/química , Células PC12 , RatasRESUMEN
Cyclic voltammetry (CV) and ultraviolet (UV) spectroscopy were used, for the first time, to study the interaction between aluminium(III) and calf thymus DNA under neutral pH conditions. Thus obtained data confirmed the existence of a relatively strong interaction between Al(III) and DNA. The binding site for aluminium(III) on DNA chains is not the bases, but the phosphate groups on the DNA backbones, the same as that for [Co(phen)3](3+/2+) that binds non-specifically and electrostatically to the deoxyribose phosphate backbone of DNA. When coexisting, Al(III) binds more favorably to DNA than [Co(phen)3](3+/2+), which implies the relatively strong binding of Al(III) to the phosphate backbone of DNA under neutral pH conditions. The nature of the binding of Al(III) to DNA is also discussed.
Asunto(s)
Aluminio/química , Aluminio/metabolismo , ADN/química , ADN/metabolismo , Animales , Sitios de Unión , Bovinos , Conductometría , Concentración de Iones de Hidrógeno , Sondas de Ácido Nucleico/química , Sondas de Ácido Nucleico/metabolismo , Espectrofotometría Ultravioleta , TimoRESUMEN
The immobilization of thiol-derivatized DNA on a Au (111) single crystal surface by self-assembly has been investigated by electrochemical scanning tunneling microscopy (EC-STM). Continuous potential-dependent orientation changes of double-stranded oligodeoxynucleotides (ODN) have been observed in a certain potential range from 200 to 600 mV (versus SCE). It is suggested that the DNA duplexes stand straight on the gold surface at potentials negative of the potential of zero charge (pzc) and then lay down on the surface when the potential shifts positively. These results are in agreement with the expectation based on the Coulombic interaction consideration between negatively charged DNA helices and gold surface. As the applied potential shifts positively, the surface charge changes from negative to positive, that is, the Coulombic force between negatively charged DNA helices and gold surfaces changes from repulsion to attraction. However, for the single-stranded oligodeoxynucleotides, no distinct changes in the surface structure were observed with the applied potential.