Background:NOD-like receptor family CARD domain containing 3(NLRC3)plays an important role in both innate and adaptive immunity.This study was to explore the function and related mechanisms of NLRC3 in a hypoxia/reoxy...Background:NOD-like receptor family CARD domain containing 3(NLRC3)plays an important role in both innate and adaptive immunity.This study was to explore the function and related mechanisms of NLRC3 in a hypoxia/reoxygenation(H/R)-induced inflammatory response in RAW264.7 cells.Methods:Liver ischemia-reperfusion(I/R)model in mice and H/R model in RAW264.7 cells were constructed.Western blotting was used to determine the protein expression level of NLRC3 in liver tissue and NLRC3,TRAF6,p–p65,p65,IκB–α,and the K63-linked ubiquitination level of TRAF6 in cells.The immunofluorescence assay was performed to evaluate the nuclear level of the NF–κB(p65).ELISA was conducted to measure the content of IL–1βin serum and cell supernatant.The interaction between NLRC3 and TRAF6 in cells was analyzed by the Co-IP assay.Results:The NLRC3 protein level in liver tissue was decreased with the prolongation of reperfusion time(P<0.05).The expression of NLRC3 and IκB–αprotein in RAW264.7 was decreased gradually,while the expression of p–p65 and TRAF6 proteins and K63-linked ubiquitination of TRAF6 were increased gradually with the prolongation of reoxgenation time(P<0.05).The Co-IP assay revealed that NLRC3 and TRAF6 can bind to each other directly.However,NLRC3 had no effect on the expression of TRAF6 protein.The ubiquitination test results showed that the K63-linked ubiquitination level of TRAF6 in H/R+Lv–NLRC3 group was significantly lower than that in the H/R+negative control(NC)group(P<0.05).Moreover,the activation of NF–κB in H/R+Lv–NLRC3 group was inhibited compared with that in the H/R+NC group,and the level of the inflammatory factor IL–1βin the cell culture supernatant was also decreased accordingly(P<0.05).Conclusions:NLRC3 might alleviate H/R-induced inflammation in RAW264.7 cells by inhibiting K63-linked ubiquitination of TRAF6.展开更多
To investigate the chemical structure of cell wall mannan obtained from pathogenic yeast, Candida tropicalis NBRC 1400 (former antigenic standard strain, IFO 1400). As a result of two-dimensional NMR analysis, it was ...To investigate the chemical structure of cell wall mannan obtained from pathogenic yeast, Candida tropicalis NBRC 1400 (former antigenic standard strain, IFO 1400). As a result of two-dimensional NMR analysis, it was shown that the mannan of this strain is composed of α-1,6-, α-1,3-, α-1,2- and β-1,2-linked mannose residues. In this research, the mannan was subjected to three degradation procedures, acid-treatment, α-mannosidase, and acetolysis under two conditions in order to determine the chemical structure of the antigenic oligomannosyl side chains in this molecule. The 1H-nuclear magnetic resonance spectra of resultant oligosaccharides, pentaose and hexaose, demonstrated the existence of the oligomannosyl side chains corresponding to Manα1-3Manα1-2Manα1-2Manα1-2Man and Manα1-3Manα1-2Manα1-2Manα1-2Manα1-2Man, respectively, which have previously also been found in Candida albicans serotype A strain mannans. These findings indicate that C. tropicalis and C. albicans serotype A have no significant difference in the chemical structure of these cell wall mannans. Therefore, it can be interpreted that it is extremely difficult to distinguish both species by targeting the antigenic group in these mannans.展开更多
基金This study was supported by grants from the National Natural Science Foundation of China(81873592)the graduate tutor team construction project of Chongqing Municipal Education Commission Foundation(dstd201801).
文摘Background:NOD-like receptor family CARD domain containing 3(NLRC3)plays an important role in both innate and adaptive immunity.This study was to explore the function and related mechanisms of NLRC3 in a hypoxia/reoxygenation(H/R)-induced inflammatory response in RAW264.7 cells.Methods:Liver ischemia-reperfusion(I/R)model in mice and H/R model in RAW264.7 cells were constructed.Western blotting was used to determine the protein expression level of NLRC3 in liver tissue and NLRC3,TRAF6,p–p65,p65,IκB–α,and the K63-linked ubiquitination level of TRAF6 in cells.The immunofluorescence assay was performed to evaluate the nuclear level of the NF–κB(p65).ELISA was conducted to measure the content of IL–1βin serum and cell supernatant.The interaction between NLRC3 and TRAF6 in cells was analyzed by the Co-IP assay.Results:The NLRC3 protein level in liver tissue was decreased with the prolongation of reperfusion time(P<0.05).The expression of NLRC3 and IκB–αprotein in RAW264.7 was decreased gradually,while the expression of p–p65 and TRAF6 proteins and K63-linked ubiquitination of TRAF6 were increased gradually with the prolongation of reoxgenation time(P<0.05).The Co-IP assay revealed that NLRC3 and TRAF6 can bind to each other directly.However,NLRC3 had no effect on the expression of TRAF6 protein.The ubiquitination test results showed that the K63-linked ubiquitination level of TRAF6 in H/R+Lv–NLRC3 group was significantly lower than that in the H/R+negative control(NC)group(P<0.05).Moreover,the activation of NF–κB in H/R+Lv–NLRC3 group was inhibited compared with that in the H/R+NC group,and the level of the inflammatory factor IL–1βin the cell culture supernatant was also decreased accordingly(P<0.05).Conclusions:NLRC3 might alleviate H/R-induced inflammation in RAW264.7 cells by inhibiting K63-linked ubiquitination of TRAF6.
文摘To investigate the chemical structure of cell wall mannan obtained from pathogenic yeast, Candida tropicalis NBRC 1400 (former antigenic standard strain, IFO 1400). As a result of two-dimensional NMR analysis, it was shown that the mannan of this strain is composed of α-1,6-, α-1,3-, α-1,2- and β-1,2-linked mannose residues. In this research, the mannan was subjected to three degradation procedures, acid-treatment, α-mannosidase, and acetolysis under two conditions in order to determine the chemical structure of the antigenic oligomannosyl side chains in this molecule. The 1H-nuclear magnetic resonance spectra of resultant oligosaccharides, pentaose and hexaose, demonstrated the existence of the oligomannosyl side chains corresponding to Manα1-3Manα1-2Manα1-2Manα1-2Man and Manα1-3Manα1-2Manα1-2Manα1-2Manα1-2Man, respectively, which have previously also been found in Candida albicans serotype A strain mannans. These findings indicate that C. tropicalis and C. albicans serotype A have no significant difference in the chemical structure of these cell wall mannans. Therefore, it can be interpreted that it is extremely difficult to distinguish both species by targeting the antigenic group in these mannans.
