Carbamylation-induced inactivation of glyceraldehydes 3-phosphate dehydrogenase and thioltransferase in bovine lens / 国际眼科杂志(Guoji Yanke Zazhi)

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.

The Effect of Cyanate on Type I Collagen gene Expression by Dermal Fibroblasts in Culture / 대한피부과학회지

BACKGROUND: Fibrosis is a common response to various insults or injuries and can be the outcome of any perturbation in the cellular function of any tissue. Peritoneum is always exposed to the waste products during peritoneal dialysis. In aqueous solution, there is partial and spontaneous decomposition of urea to ammonia, carbonate and cyanate. Cyanate reacts irreversibly with the N-terminal groups of amino acid, peptides and many proteins by a process known as carbamylation and may contribute to peritoneal injury with fibrosis. But only little is known about the molecular and cellular mechanism underlying the effect of cyanate on the expression of type I collagen in cultured skin fibroblasts. OBJECTIVE: The purpose of this study was to examine the effect of cyanate on type I collagen gene expression in cultured skin fibroblasts. METHODS: In this study, the effects of cyanate were examined by Northern blot hybridization, chloramphenicol acetyltransferase(CAT) assay, and immunohistochemical stain in cultured human fibroblasts. RESULTS: In Northern blot hybridization, steady-state levels of alpha1(I) procollagen mRNA were increased 2-fold at 100 micromol of cyanate compared to untreated control. Cyanate caused an alteration in the alpha1(I) procollagen mRNA expression in a dose-related fashion. In CAT assay, the relative CAT activity was 1.0 in the untreated control, 0.9 at a concentration of 0.1 micromol, 2.3 at 10 micromol, and 2.6 at a 100 micromol. Cyanate caused a marked increase on type I collagen gene promotor activity. In imunohistochemical stain with anti-type I collagen antibody, type I collagen was markedly increased in cultured fibroblasts by cyanate. CONCLUSION: These results indicate that cyanate may be a powerful up-regulator of type I collagen production through the transcriptional activation of gene expression in cultured skin fibroblasts.

Effect of Cyanate on the Carbamylation and Biological Activity of Superoxide Dismutase / 대한신장학회잡지

The patients with end stage renal disease show several complications such as artherosclerosis, anemia and increased susceptibility to infection by damage due to oxygen free radicals. Superoxide dismutase(SOD) is directly linked to the fate of the highly reactive oxygen metabolites. If there is an alteration in the activity of SOD, this alteration may contribute to the complications by reactive oxygen species in patients with end stage renal disease. In this experiment, SOD activity and the effect of cyanate on the activity of SOD was studied to understand the mechanism of several complications mediated by oxygen free radicals in patients with end stage renal disease. SOD activity in the plasma and erythrocytes from patients with end stage renal disease was significantly lower than those from healthy controls. It is known that underproduction of SOD leads to excess production of superoxide and reduced iron favoring hydroxyl radical formation. The results in this experiment suggest that there is an overproduction of superoxide anion in patients with end stage renal disease. The overproduction of superoxide anion may contribute the patients with end stage renal disease susceptible to oxidant damages. To evaluate if cyanate could carbamylate SOD, SOD was incubated with cyanate. The level of carbamylated SOD increased as the time of exposure to cyanate increased from 0 hour to 72 hours. Furthermore, the degree of carbamylation of SOD increased as cyanate concentration in the incubation media rose from 20mM to 1M. There appears to be a maximum degree of carbamylation at a concentration of 1,000mM cyanate. To test the hypothesis that in vitro carbamylation of SOD alters its biological activity, SOD activity was measured after incubation with cyanate. The activity of carbamylated SOD decreased as the time of exposure to cyanate increased from 0 hour to 72 hours. Furthermore, the activity of carbamylated SOD decreased as cyanate concentration in the incubation media rose from 20mM to 1M and when albumin was added to the reaction mixture, the loss of SOD activity was prevented. These results are consistent with the hypothesis that SOD is also carbamylated and lost biological activity in end stage renal disease patients by cyanate, and that the degree of carbamylation depends on both the concentration of cyanate and the length of exposure. Also, these suggest that albumin may prevent carbamylation of SOD at least in vitro condition.

Effect of Cyanate on Lens Protein Carbamylation and Catalase Activuty

Proteins are known to be carbamylated as a result of reactions with ureaderived cyanate. We investigated the effect of carbamylation by cyanate on the catalase activity which is known that its decreased activity contributes to cataract formation, and on the lens protein. Catalase and lens protein were carbamylated by incubation with cyanate at 37degrees C. The extent of carbamylation was monitored by following the loss of free amino group using trinitrobenzenesulphonic acid. The level of carbamylated protein was 76% in patients with cataract. Carbamylated proteins in normal lens from rabbits and carbamylated catalase were increased as the time of exposure to cyanate activity. Our results suggest that cyanate is an inhibitor of catalase, and carbamylation of catalase as a result of reaction with ureaderived cyanate may contribute to cataract formation.