3 research outputs found
Positive correlation between the generation of reactive oxygen species and activation/reactivation of transgene expression after hydrodynamic injections into mice.
Purpose : Hydrodynamic injection has been shown to reactivate silenced transgene expression in mouse liver. In this study, the roles of inflammatory cytokines and reactive oxygen species (ROS) in the reactivation were examined. Methods : Production of inflammatory cytokines and ROS by hydrodynamic injection of saline was examined in mice that had received a hydrodynamic injection of a plasmid expressing Gaussia luciferase. The level of reporter gene expression was used as an indicator of the reactivation. The involvement of cytokines and ROS was examined by depleting Kupffer cells or by pre-administration of antioxidants, respectively. Results : A hydrodynamic injection of saline induced a significant production of interleukin (IL)-6. Depleting Kupffer cells using clodronate liposomes markedly reduced the IL-6 production but had no significant effect on the transgene expression. On the other hand, an injection of catalase or N-acetylcysteine significantly inhibited the hydrodynamic injection-induced reactivation of silenced transgene expression. The silenced expression was also reactivated by carbon tetrachloride, an inducer of oxidative stress in the liver, in a dose-dependent manner, and this reactivation was significantly inhibited by catalase. Conclusions : These findings show a positive correlation between the generation of ROS and the reactivation of silenced transgene expression after hydrodynamic injections
Near-Infrared Fluorescence Probes for Enzymes Based on Binding Affinity Modulation of Squarylium Dye Scaffold
We present a novel design strategy for near-infrared
(NIR) fluorescence
probes utilizing dye–protein interaction as a trigger for fluorescence
enhancement. The design principle involves modification of a polymethine
dye with cleavable functional groups that reduce the dye’s
protein-binding affinity. When these functional groups are removed
by specific interaction with the target enzymes, the dye’s
protein affinity is restored, protein binding occurs, and the dye’s
fluorescence is strongly enhanced. To validate this strategy, we first
designed and synthesized an alkaline phosphatase (ALP) sensor by introducing
phosphate into the squarylium dye scaffold; this sensor was able to
detect ALP-labeled secondary antibodies in Western blotting analysis.
Second, we synthesized a probe for β-galactosidase (widely used
as a reporter of gene expression) by means of β-galactosyl substitution
of the squarylium scaffold; this sensor was able to visualize β-galactosidase
activity both in vitro and in vivo. Our strategy should be applicable
to obtain NIR fluorescence probes for a wide range of target enzymes