Glutathione
(GSH) plays an important role in maintaining redox
homeostasis inside cells. Currently, there are no methods available
to quantitatively assess the GSH concentration in live cells. Live
cell fluorescence imaging revolutionized the field of cell biology
and has become an indispensable tool in current biological studies.
In order to minimize the disturbance to the biological system in live
cell imaging, the probe concentration needs to be significantly lower
than the analyte concentration. Because of this, any irreversible
reaction-based GSH probe can only provide qualitative results within
a short reaction time and will exhibit maximum response regardless
of the GSH concentration if the reaction is completed. A reversible
reaction-based probe with an appropriate equilibrium constant allows
measurement of an analyte at much higher concentrations and, thus,
is a prerequisite for GSH quantification inside cells. In this contribution,
we report the first fluorescent probe—ThiolQuant Green (TQ
Green)—for quantitative imaging of GSH in live cells. Due to
the reversible nature of the reaction between the probe and GSH, we
are able to quantify mM concentrations of GSH with TQ Green concentrations
as low as 20 nM. Furthermore, the GSH concentrations measured using
TQ Green in 3T3-L1, HeLa, HepG2, PANC-1, and PANC-28 cells are reproducible
and well correlated with the values obtained from cell lysates. TQ
Green imaging can also resolve the changes in GSH concentration in
PANC-1 cells upon diethylmaleate (DEM) treatment. In addition, TQ
Green can be conveniently applied in fluorescence activated cell sorting
(FACS) to measure GSH level changes. Through this study, we not only
demonstrate the importance of reaction reversibility in designing
quantitative reaction-based fluorescent probes but also provide a
practical tool to facilitate redox biology studies.
Graves' disease is a common autoimmune disorder characterized by thyroid stimulating hormone receptor autoantibodies (TRAb) and hyperthyroidism. To investigate the genetic architecture of Graves' disease, we conducted a genome-wide association study in 1,536 individuals with Graves' disease (cases) and 1,516 controls. We further evaluated a group of associated SNPs in a second set of 3,994 cases and 3,510 controls. We confirmed four previously reported loci (in the major histocompatibility complex, TSHR, CTLA4 and FCRL3) and identified two new susceptibility loci (the RNASET2-FGFR1OP-CCR6 region at 6q27 (P(combined) = 6.85 × 10(-10) for rs9355610) and an intergenic region at 4p14 (P(combined) = 1.08 × 10(-13) for rs6832151)). These newly associated SNPs were correlated with the expression levels of RNASET2 at 6q27, of CHRNA9 and of a previously uncharacterized gene at 4p14, respectively. Moreover, we identified strong associations of TSHR and major histocompatibility complex class II variants with persistently TRAb-positive Graves' disease.
This Letter presents a systematic study of effects of external electric fields (EEFs) on the key species and steps in the catalytic cycle of cytochrome P450 cam . The QM/MM/EEF results demonstrate that an EEF can exert significant effects on the entire catalytic cycle of P450. The EEF will control the initial gating of the cycle, its rate-determining step, its O 2 uptake, the geometry of its resting state, and the spin-state ordering and electronic structure of its various active species. Furthermore, the EEF has a potential of controlling the bond activation reactions of the active species as well. These effects are pronounced when the EEF is aligned perpendicular to the porphyrin plane.
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