OBJECTIVE To investigate the apoptosis-inducing effect of esculetin （Esc） on acute myeloid leukemia （AML） HL-60 cells by regulating the protein kinase B （AKT）/S-phase kinase-associated protein 2 （SKP2）/MutT homolog 1 （MTH1） pathway. METHODS AML HL-60 cells were randomly divided into control group （routine culture）， Esc low-concentration group （L-Esc group， 25 μmol/L Esc）， Esc medium-concentration group （M-Esc group， 50 μmol/L Esc）， Esc high-concentration group （H-Esc group， 100 μmol/L Esc）， and high-concentration of Esc+ SC79 （AKT agonist） group （100 μmol/L Esc+5 μmol/L SC79）. Cell proliferation in each group was detected by MTT assay and colony formation assay. The level of reactive oxygen species （ROS） in cells was measured by using the CM-H2DCFDA fluorescent probe. Cell apoptosis was analyzed by flow cytometry. Western blot assay was performed to detect the expression levels of apoptosis-related proteins [B-cell lymphoma 2 （Bcl-2）， Bcl-2-associated X protein （Bax）， cleaved caspase-3]， AKT/SKP2/MTH1 pathway-related proteins （p-AKT， AKT， SKP2， MTH1）， along with the upstream and downstream proteins of AKT phosphatidylinositol 3-kinase （PI3K）， cyclin-dependent kinase inhibitor 1 （P21） and cyclin-dependent kinase inhibitor 1B （P27）. RESULTS Compared with control group， the cell viability， colony number， and the phosphorylation levels of AKT and PI3K proteins as well as protein expressions of SKP2， MTH1 and Bcl-2 were significantly decreased （P＜0.05）， while ROS level， apoptosis rate， and the expression levels of Bax， cleaved caspase-3， P21 and P27 proteins were significantly increased （P＜0.05）. Moreover， the effects of Esc exhibited concentration-dependence （P＜0.05）. Compared with H-Esc group， above indexes of high-concentration of Esc+ SC79 group were reversed significantly （P＜0.05）. CONCLUSIONS Esc may promote massive ROS production and induce activation of apoptosis in HL-60 cells by inhibiting the AKT/SKP2/MTH1 pathway， thus inhibiting the proliferation of HL-60 cells.

Mycoplasma capricolum subsp. capripneumoniae (Mccp) is the cause of contagious caprine pleuropneumonia (CCPP) in goats. Inactivated vaccines and capsular polysaccharide (CPS) indirect hemagglutination reagents are available for prevention and serological detection, but high culture costs and complex antigen quantification have been plagued by production staff. In order to solve these problems in production practice, a sugar fermentation medium with an initial pH value of 7.8, which could improve the production of two antigens simultaneously, was screened out by changing the initial pH value based on previous Mccp metabolomics analysis. Since phenol red can be identified by UV absorption spectrum and cetyltrimethylammonium bromide (CTAB) can bind to anionic capsular polysaccharide, a UV spectrum measurement method for analyzing the culture stage reached by Mccp and a CTAB precipitation test for relative quantification of capsular polysaccharide antigen content in the fermentation broth were established. The UV spectrum observation method can guide the production of Mccp according to the growth curve of Mccp, which greatly reduces the monitoring time of the traditional CCU method and improves the accuracy of the original eye-observation method. The established CTAB precipitation test can complete the monitoring of CPS content within 5 hours, which greatly reduces the time required compared with the traditional differential technique, and its accuracy was verified by the phenol-sulfuric acid method. The optimized culture medium and the two correlation comparison methods established in this study can effectively reduce the production cost of Mccp and improve the production efficiency. The two assays have been used in the research at our laboratory, which provides experimental data for further improvement of the production process of CCPP inactivated vaccine and capsular polysaccharide as well as rapid quantification.
Humans ; Animals ; Goats ; Cetrimonium ; Mycoplasma ; Polysaccharides

Objective: To investigate the expression of LIM domain binding 2 (LDB2) in lung cancer tissues and its correlation with sphingosine-1 phosphate receptor 1 (S1PR1). Methods: Lung cancer tissues and the corresponding adjacent tissues from 52 patients in Nantong Tumor Hospital during April 2010 and May 2011 were collected as the experimental group and the control group, respectively. The expression levels of LDB2 and S1PR1 were detected by the real-time PCR (qRT-PCR). The expression results of LDB2 gene were further verified by the Oncomine database, and its correlations with clinicopathological parameters were analyzed. The ROC curve was drawn to evaluate the diagnosis value of LDB2 expression in lung cancer. The correlation of LDB2 expression with the prognosis of lung cancer was analyzed by the “Kaplan-Meier Plotter” database. In addition, the relationship between LDB2 and S1PR1 was also analyzed. Results: The expression levels of LDB2 in lung cancer tissues (0.158 [0.062,0.383]) were significantly lower than that in the adjacent tissues (0.403 [0.261,0.711], U=700.0, P＜ 0.01). A total of 9 eligible studies were retrieved from the Oncomine database, and their expressions of LDB2 were also low (P＜0.01). The expressions of LDB2 in lung cancer tissues were not related to gender, age, smoking history, pathological type, tumor size, TNM staging and lymphatic metastasis (P＞0.05). The results of ROC curve showed that when the area under the ROC curve (AUC ROC ) was 0.741 (95% CI:0.643-0.839) and the cut-off value was 0.247, the sensitivity and specificity of LDB2 in the diagnosis of lung cancer were 80.8% and 61.5%, respectively. The Kaplan-Meier survival analysis showed that the 5-year overall survival time of the patients with low expression of LDB2 was shorter than that of the patients with high expression of LDB2(P＜0.01). In addition, the expression levels of S1PR1 in lung cancer tissues (0.710[0.337,1.523]) were significantly lower than that in the adjacent tissues (1.582[0.913,3.533],U=780.0, P＜0.01), and the expression levels of S1PR1 in lung cancer tissues were positively correlated with that of LDB2(r=0.827,P＜0.01). Conclusion The expressions of LDB2 and S1PR1 in lung cancer tissues are down-regulated, and have a positive correlation, and they may play an important role in the occurrence and development of lung cancer.