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Activation of denovo GSH synthesis pathway in mouse spleen ..
PURPOSE. Lens glutathione synthesis knockout (LEGSKO) mouse lenses lack de novo glutathione (GSH) synthesis but still maintain >1 mM GSH. We sought to determine the source of this residual GSH and the mechanism by which it accumulates in the lens. METHODS. Levels of GSH, glutathione disulfide (GSSG), and GSH-related compounds were measured in vitro and in vivo using isotope standards and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. RESULTS. Wild-type (WT) lenses could accumulate GSH from γ-glutamylcysteine and glycine or from intact GSH, but LEGSKO lenses could only accumulate GSH from intact GSH, indicating that LEGSKO lens GSH content is not due to synthesis by a salvage pathway. Uptake of GSH in cultured lenses occurred at the same rate for LEGSKO and WT lenses, could not be inhibited, and occurred primarily through cortical fiber cells. In contrast, uptake of GSH from aqueous humor could be competitively inhibited and showed an enhanced Km in LEGSKO lenses. Mouse vitreous had >1 mM GSH, whereas aqueous had
To examine the role of cellular GSH in the attenuation of oxidative stress by curcumin, the level of cellular GSH was altered by either N-acetyl-cysteine (NAC), a precursor of GSH by supplying cysteine , or by curcumin, or by BSO. HSC were treated for 24 hr with BSO (0.25mM), or curcumin (20μM), or NAC (5mM), with or without the pre-exposure to BSO (0.25mM) for 1 hr. The levels of total cellular GSH, cellular ROS as well as LPO were determined. As demonstrated in , compared to the untreated control, curcumin as well as NAC significantly increased the level of cellular GSH by 203% and 153%, respectively. The pre-treatment of cells with BSO, which inhibited GCL activity and depleted cellular GSH (), dramatically diminished the stimulatory effect of curcumin or NAC on cellular GSH content. Further analyses indicated that NAC, mimicking curcumin, scavenged cellular ROS () and reduced the level of LPO () in HSC, suggesting the role of GSH in the attenuation of oxidative stress. The pretreatment of cells with BSO dramatically eliminated the ability of curcumin and NAC in scavenging cellular ROS () and in reducing cellular LPO (). Taken together, these results demonstrated that the attenuation of oxidative stress by curcumin in activated HSC required de novo synthesis of GSH.
ligase catalytic subunit and thioredoxin in de novo GSH synthesis
Because the activity of GCL and the promoters of both GCLC and GCLM will influence GSH content, and since we have previously demonstrated that GSH synthesis plays an important protective role in mediating DEP-induced inflammation, it is important to investigate the potential of genetic variations in GSH synthesis genes to influence cardiovascular response to DE inhalation. It has been previously demonstrated that single nucleotide polymorphisms in the 5′ promoter regions of both GCLC and GCLM lead to compromised promoter activity and are associated with an increased risk of myocardial infarction, impaired vasomotor function and dilated cardiomyopathy in a Japanese population (; , ; ). Although the effects of these polymorphisms are relatively small (one copy of the −588T GCLM allele results in ~2-fold increased risk of MI), their frequency (e.g. ~20% of the population have at least one GCLM −588T allele) highlights their importance to public health in the context of air pollution and cardiovascular disease.
Total RNA was isolated by TRI-Reagent (Sigma), following the protocol provided by the manufacturer. Real-time PCR was carried out using SYBR green, as we previously described . mRNA fold changes of target genes relative to the endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control were calculated as suggested by Schmittgen et. al . mRNA levels were expressed as fold changes after normalization with GAPDH. Primers used for real-time PCR were in the following:
Lec10 Dickinson GSH Synt | Glutathione | Biosynthesis
The secretion of a peptide called glutathione by lung cells is impaired in cystic fibrosis, and there is good evidence to suggest that the lack of glutathione in lung fluid plays a key role in the chronic inflammation and infection that occurs.
Results:taVNS did not affect circulating glucose, free fatty acids, insulin, glucagon, or pancreatic polypeptide. Rates of endogenous glucose production (P = 0.79), hepatic HCL, ATP, and Pi were also not different (P = 0.91, P = 0.48 and P = 0.24) between taVNS or sham stimulation. Hepatic HCL, ATP, and Pi remained constant during prolonged fasting for 3 h. No changes in heart rate or shift in cardiac autonomic function from HRV towards sympathetic or parasympathetic predominance were detected.
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Glutathione (GSH) and the GSH synthesis gene Gclm …
To elucidate the role of the elevated level of cellular GSH stimulated by curcumin in the attenuation of oxidative stress, it was hypothesized that the antioxidant capability of curcumin might mainly result from de novo synthesis of GSH. To begin to test the hypothesis, pilot experiments were conducted to determine the optimal conditions to deplete cellular GSH in activated HSC using L-buthionine sulfoximine (BSO), a specific inhibitor of glutamate-cysteine ligase (GCL). The latter is the key rate-limiting enzyme in GSH synthesis . As shown in , the treatment of HSC with BSO at 0.25mM for 24 hr caused a dramatic decrease in the level of total GSH by 69%. Higher doses of BSO resulted in no further apparent decrease in the level of GSH. Additional experiments demonstrated that BSO caused a time-dependent decrease in the content of total cellular GSH in HSC treated with BSO at 0.25mM for various hours ().
De novo serine biosynthesis diverges from glycolysis
To answer the question how curcumin could induce the de novo synthesis of GSH and increase the level of cellular GSH in activated HSC, we hypothesized that curcumin might stimulate the activity of GCL, the rate-limiting enzyme in GSH synthesis, in HSC. To test the hypothesis, HSC were treated with curcumin at indicated concentrations for 24 hr. As demonstrated in , compared with the untreated control (the 1st column on the left), curcumin dramatically increased the activity of GCL in a dose-dependent manner in HSC. However, curcumin at the concentrations higher than 30μM showed no such a dose-dependent manner (data not shown). This result indicated that the induction of de novo synthesis of GSH and the increase in the level of cellular GSH by curcumin might mainly result from the stimulation of the activity of GCL.
Age-associated perturbations in glutathione synthesis …
To determine if DE exposure resulted in uptake of DEP by alveolar macrophages, we performed BAL on male WT mice following a 6-h exposure to either FA or DE as described above. Fifty microliter samples of washed resuspended cells were further diluted with 450 µL PBS, placed into Shandon Cytospin cartridges (ThermoScientific, Waltham, MA), and the cartridges were centrifuged at 600 rpm for 10 min. Deposited cells were stained using a Diff-Quik Fixative (Siemens Healthcare Diagnostics, Newark, DE). Bright field images of representative alveolar macrophages were taken at 40× magnification using a Nikon Optiphot microscope (Nikon USA, Melville, NY).
The glutathione biosynthetic pathway of Plasmodium …
We showed in vitro that flavonoids increase expression of gamma-glutamylcysteine synthetase and, by using a unique transgenic reporter mouse strain, we showed increased expression in vivo, with a concomitant increase in the intracellular glutathione concentrations in muscles.
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