RESUMO
Objective:To investigate whether KtrA was a binding protein of c-di-AMP, the second messenger in Leptospira, and to explore the function and regulatory mechanism of the c-di-AMP-KtrA/B system. Methods:KtrA gene was amplified by PCR and cloned into pET42a plasmid to construct the pET42a ktrA prokaryotic expression vector. Then the vector was transferred into E. coli BL21DE3 to construct an engineering bacterium E. coli BL21DE3 pET42a-ktrA for the expression of recombinant KtrA (rKtrA). The expressed rKtrA was purified by affinity chromatography. BIAcore technology was used to detect the binding ability of rKtrA to c-di-AMP. Bacterial two-hybrid analysis was used to analyze the interaction between KtrA and KtrB in the leptospiral Ktr system with or without exogenetic c-di-AMP. The above genes were then complemented into the potassium transport-deficient E. coli mutants to analyze the function of the c-di-AMP-KtrA/B pathway. Results:An prokaryotic engineering bacterium for the expression of ktrA gene of Leptospira was constructed successfully. The purified rKtrA could specifically bind to c-di-AMP. There was interaction between KtrA and KtrB, but the interaction could be dissociated by c-di-AMP. The KtrA/B system was involved in potassium ion uptake and it was negatively regulated by c-di-AMP. Conclusions:Leptospiral KtrA was a c-di-AMP-binding protein and the c-di-AMP-KtrA/B system was involved in potassium ion transport.
RESUMO
Objective To determine the expression and distribution of Disabled-2(Dab2) in normal human adrenal glands, and further to study the expression of Dab2 in tissues of different adrenocortical adenomas, and to elucidate whether Dab2 can be a specific molecular marker in the pathology of primary aldosteronism. Methods Real-time PCR and immunohistochemical staining were used to detect Dab2 expression in 10 aldosterone-producing adenoma (APA) samples, 8 cortisol-producing adenoma ( CPA) samples, 8 non-functioning adenoma ( NFA) samples and 6 normal adrenal samples. Results Immunohistochemical staining showed that Dab2 was significantly highly expressed in zona glomerulosa of normal human adrenal glands. Sporadical cluster of ZG cells with moderate Dab2 staining were demonstrated in APAs. In all CPA and NFA tumors, weak dab2 staining was detected. According to the results of real-time PCR, Dab2 mRNA expression was increased significantly in APAs compared with normal adrenal glands. There was no significant difference between normal adrenal glands, CPAs, and NFAs in regard to Dab2 mRNA expression. Compared to nontumor portions, APAs also showed higher Dab2 mRNA expression in the tumor( P<0. 05). Conclusion Dab2 was predominantly localized in zona glomerulosa in normal adrenal gland. Increased Dab2 mRNA expression was detected in APAs compared with normal adrenal glands. Whereas, Dab2 protein expression was just moderate increased in APAs. Weather Dab2 can be a specific molecular marker in the pathology of primary aldosteronism has to be further studied.