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@#AIM: To examine the morphological and biochemical alterations in the eyes of Thioltransferase knockout(TTase KO)mouse model as a function of age, and to explore the important function in redox homeostasis in the lens and in the age-related cataractogenesis.<p>METHODS: TTase KO model was established in this laboratory. TTase KO and WT mice were examined and the lens opacity was classified by using a slit lamp. Each lens was homogenized in lysis buffer and processed for measurement of glutathione(GSH)level. Examination of Protein-GSH mixed disulfides(PSSG)formation in the lens by Western blot analysis. Immunoprecipitation was used to identify the proteins formed PSSG. Dethiolation of lens proteins was carried out using purified recombinant human lens TTase(RHLT). <p>RESULTS: The slit lamp examination showed an age-dependent nuclear cataract development in both eyes of the WT and TTase KO mice. The onset of cataract was 4mo in the KO mice and 9mo in the WT mice. The GSH loss showed in both groups during aging and was prominent in the TTase KO mice after 9mo old. PSSG in the lenses of both groups showed progressive elevation, whereas the lenses of the KO group had a higher level of PSSG after 9mo. These GSH-conjugated proteins were confirmed as actin and glyceraldehyes 3-phosphate dehydrogenase(GAPDH)by immunoprecipitation and they could be eliminated when the homogenates were treated with RHLT. <p>CONCLUSION: The results showed that deletion of TTase gene in the mouse could lead to an early age-dependent cataract formation and the PSSG formation in these lenses appeared to link directly to lens opacity. The PSSG could be dethiolated by TTase. This data strengthens that TTase plays an essential role in maintaining lens clarity.
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Objective To discuss the protective effects of thioltransferase (TTase) on oxidative damaged human lens epithelial cells (HLEC) induced by ultraviolet radiation.Methods HLEC were cultured in vitro and then randomly divided into 4 groups:Normal group:normal cultured HLEC;UV group:normal cultured HLEC + UV radiation (with 302 nm UV radiation irradiation intensity 55.56 μW · cm-2 for 15 minutes,totaling irradiation volume 500 J · m-2);TTase siRNA group:HLEC transfected with TTase siRNA;TTase siRNA + UV group:HLEC transfected with TTase siRNA + UV radiation(with 302 nm UV radiation irradiation intensity 55.56 μW · cm-2 for 15 minutes,totaling irradiation volume 500 J · m-2).TTase mRNA expression was measured by qRT-PCR,the cell proliferation was detected by LDH Assay Kit,and the TTase activity was measured.TTase expression was detected by Western blotting.The levels of TGSH,GSH and GSSG of HLEC were measured,and then GSSG/T-GSH ratio was calculated.Results Cell proliferation ability in UV group,TTase siRNA group and TTase siRNA + UV group were decreased by 21.0％,17.0％ and 29.0％ compared with normal group (all P ＜ 0.05).TTase activity in UV group was 2.1 times of the normal group,TTlase siRNA group was 67.0％ of the normal group,Tlase siRNA + UV group was 1.3 times of TTase siRNA group (all P ＜ 0.05).TTase expression in UV group was 3.9 times of the normal group,TTase siRNA group was 35.0％ of the normal group,TTase siRNA + UV group was 3.0 times of siRNA group (all P ＜ 0.05).GSH content in UV group,TTase siRNA group and TTase siRNA + UV group were 68.4％,79.0％,61.7％ of the normal group (all P ＜ 0.05).GSSG content in UV group,TTase siRNA group and TTase siRNA + UV group were 2.3 times,1.4 times,3.7 times of the normal group (all P ＜ 0.05).GSSG/T-GSH in UV group,TTase siRNA group and TTase siRNA + UV group were 3.1 times,1.7 times,5.2 times of the normal group (all P ＜ 0.05).Conclusion TTase plays an important protective role in oxidative damaged HLEC induced by ultraviolet radiation.
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AIM:To observe the effects of advanced glycation end-products (AGEs) on thioltransferase (TTase) expression and activity in human lens epithelial cells. METHODS: Human lens epithelial cells B3 ( HLE B3 ) were treated with 1. 5mg/mL AGEs - BSA as the experimental groups cultured by fetal bovine serum of volume fraction 10% dulbecco modified eagle medium ( DMEM) and bovine serum albumin ( BSA) was added at the same concentrations as the negative control. The level of reactive oxygen species ( ROS ) was evaluated. Cells were collected at 0, 1, 2, 3, 4d and total RNA or protein was extracted. TTase mRNA levels were detected by qRT-RCR. TTase expression was detected by Western blot and its activity was measured. RESULTS: Compared with the control group, AGEs-BSA up-regulated the expression of ROS (P0. 05). Similarly, TTase activity peaked at 3d (nearly 2. 01-fold increase, P gradually, starting from the 3d TTase expression was reflected that there was statistically significant difference compared with control group (P CONCLUSION:AGEs-BSA significantly increases the production of ROS in human lens epithelial cells, and it then induces the oxidative stress which may promote the expression of TTase and enhances the activity of TTase.
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AIM: To investigate whether potassium cyanate can inactivate glyceraldehydes 3-phosphate dehydrogenase (GAPDH) and thioltransferase (TTase) in bovine lens.METHODS: Fresh intact bovine lenses were incubated with 100mmol/L potassium cyanate (KCNO) for 7 and 12 days respectively. Then all lens were incubated in 50mmol/L DMEM solution. The proteins in the watersoluble fractions from the normal control and the cyanate-modified lens were extracted. The activity of GAPDH and TTase in the water-soluble fraction after incubation at 37℃ was measured by spectrophotometer.RESULTS: GAPDH activity was significantly lower in the cyanate-modified lens proteins than that of the normal control (P<0.01), and considerably diminished in protein incubated with 100mmol/L potassium cyanate for 12 days. There were statistically significant differences in the activity of TTase between the normal control lenses and the carbamylated lenses incubated for 7 days (P<0.05) and 12 days (P<0.01). However. there was no statistical difference between the samples incubated with 100mmol/L KCNO for 7 and 12 days (P=0.19296).CONCLUSION: This study provides evidence to show carbamylation is able to inactivate GAPDH and TTase in bovine lenses. This may have implications for the susceptibility of lenticular GAPDH and TTase to carbamylation, and also for the research on pathogenesis of cataract.