OBJECTIVE: Synthesis and characterization of disulfiram (DSF) derivatives,and preliminary study on their antitumor activities in vitro. METHODS: Two derivatives were synthesized in two steps,and their structures were confirmed using ¹H NMR,¹³C NMR and LC-MS. CCK-8 cytotoxicity assay was used to compare the inhibitory effects of DSF and its derivatives on proliferation of MCF-7 and A549 cells with or without FBS. Effects of NAC on anti-tumor activities of these compounds were detected in the same way. Whether DSF and its derivatives could induce apoptosis were analyzed using flow cytometry. Expressions of related apoptotic proteins in MCF-7 and A549 cells were detected using Western blot. RESULTS: Results from the spectral analyses show that the structures of the derivatives were consistent with the expectation. The CCK-8 results show that the inhibitory effects of the two derivatives on these two kinds of tumor cells were stronger than that of DSF,and there were significant differences on the value of IC₅₀. There was an obvious decline in the proliferation inhibitory effect of the derivatives in the absence of FBS. In addition,the inhibitory effects on the proliferation of MCF-7 and A549 cells was significantly weakened after the addition of NAC (P < 0.05). Flow cytometry analyses show that the apoptosis rates of MCF-7 and A549 cells which were treated with the two derivatives increased compared with DSF (P < 0.05 or P < 0.01). The results from Western blot analyses show that the two derivatives increased the expression of BAX and decreased the expression of PARP in the MCF-7 and A549 cells. CONCLUSION: The two derivatives showed anti-tumor activities. In addition,the inhibitory effects on the two kinds of tumor cells in the presence of FBS were better than that of DSF. The mechanisms may be related to the expression of apoptosis-related proteins and the increase of intracellular ROS production.

OBJECTIVE: To investigate the effect of demethylation of endothelin converting enzyme 1 (ECE-1) gene on left ventricular hypertrophy in rats undergoing abdominal aortic constriction (AAC) and its effect on the PI3K/PKB signaling pathway. METHODS: 30 male SD rats were randomly divided into three groups:control,model and sham groups,with 10 rats in each group. Pressure overload myocardial hypertrophy model was established by AAC in the model group,abdominal aorta was isolated in the sham group but not ligated,and conventional operation was not performed in the control group. Left ventricular end-diastolic diameter (LVED),left ventricular posterior wall thickness (LVPWT),ejection fractions (EF),and fractional shortening (FS) were measured using echocardiography. Heart weight indices (HWI) were calculated after weighing. The areas of myocardial cells (AMC) were measured using hematoxylin-eosin staining. Demethylation levels of ECE-1 genes were detected using methylation-specific PCR. Expressions of atrial natriuretic peptide (ANP) and ECE-1 were detected using Real-time quantitative PCR. Plasma endothelin 1 (ET-1) levels were detected using enzyme-linked immunosorbent assay. Protein expressions of p-PI3K,PI3K,p-PKB and PKB were detected using Western blot. RESULTS: Compared with the control group,there were no statistically significant differences in the indicators of the sham group. Compared with the sham group,LVPWT,HWI,AMC,the demethylation level of ECE-1 gene in ventricular myocardium,plasma ET-1 level,ECE-1 and ANP mRNA in model group were increased (P < 0.01). LVED,EF,FS and the protein expression of p-PI3K and p-PKB in myocardium were decreased (P < 0.01 or P < 0.05). CONCLUSION: ECE-1 gene demethylation is involved in the formation of pressure overload cardiac hypertrophy,which may be related to the inhibition of PI3K/PKB signaling pathway.

OBJECTIVE: To evaluate in vitro genotoxicity of three representative Polybrominated Diphenyl Ethers (PBDEs):BDE-3,BDE-47 and BDE-209. METHODS: In vitro alkaline comet assay,in vitro cytokinesis-block micronucleus (CBMN) test and TK gene mutation assay were conducted using TK6 cells. These assays detect DNA damage,chromosome aberrations and gene mutation. Five doses of each PBDEs were used:60,90,120,180 and 240 μmol/L for BDE-3;60,120,180,200 and 240 μmol/L for BDE-47;and 24,40,120,180 and 240 μmol/L for BDE-209;while DMSO was used as negative control. RESULTS: Comparing with the negative control groups,exposure to the three PBDEs did not cause DNA damage,i.e. an increase in comets tail length,tail DNA intensity and tail moment (P > 0.05);and also did not increase the frequency of micronucleus (P > 0.05). However,the same exposures increased TK gene mutation frequencies significantly (P < 0.05) and in a dose-dependent manner ([R_BDE-3²]=0.85,[R_BDE-47²]=0.85,[R_BDE-209²]=0.90). The mutagenicity of BDE-47 was strongest. CONCLUSION: BDE-3,BDE-47 and BDE-209 were mutagenic to TK6 cell.

OBJECTIVE: To investigate effects of Jiangzhitong (JZT) on glucolipid metabolism and oxidative stress level in insulin resistance (IR) cells,and to study the protective effect of JZT on IR. METHODS: The rat hepatic cell line,BRL-3A,was sub-divided to the control,Palmitic Acid and JZT groups. Cell survival rates were determined using MTT. Levels of lucose consumption and insulin-stimulated glucose consumption were detected using GOD-POD method. ROS levels were detected using the DCFH-DA method. Triglycerides,total cholesterol,high density lipoprotein cholesterol,low-density lipoprotein cholesterol levels,catalase activity,T-AOC,MDA content and GSH content were detected using different kits. RESULTS: Exposure to 25 mg/mL JZT significantly decreased the survival rates of cell. JZT exposure increased levels of basic glucose consumption,high density lipoprotein cholesterol,while decreased triglycerides,total cholesterol,low-density lipoprotein cholesterol in IR cells. JZT also significantly decreased levels of ROS and malondialdehyde,while increased catalase activity and total antioxidant capacity in IR cells. CONCLUSION: JZT demonstrated protective effects on IR cells which could be due to reduced oxidative stress and improved glucolipid metabolism.

OBJECTIVE: To explore the expression of miR-143,miR-145 and Survivin mRNAs,and their clinical significance in esophageal cancers among Kazaks in Xinjiang. METHODS: Expression levels of miR-143,miR-145 and Survivin mRNAs in 47 cases of Kazak esophageal cancer were detected by qPCR. RESULTS: The expression of miR-143 and miR-145 in cancer tissues was lower than that in the distal non-cancerous tissues,and the difference was significant (P < 0.01). On the other hand,survivin expression in cancer tissues was higher than that in the distal non-cancerous tissues (P=0.000). Expression of miR-143 was correlated with degree of differentiation,lymph node metastasis and clinical stages (P=0.018,0.004,0.022,respectively). Expression of miR-145 was correlated with degree of differentiation and lymph node metastasis (P=0.007,0.039,respectively). Expression of Survivin mRNA was correlated with lymph node metastasis and clinical stages (P=0.042,0.034,respectively). Expression of miR-143 was positively correlated with miR-145 (r=0.662,P=0.000),and negatively correlated with Survivin mRNA (r=-0.313,P=0.002). CONCLUSION: miR-143,miR-145,and Survivin mRNA are involved in the development and progression of Kazak esophageal cancer.

OBJECTIVE: To use gene chip technology and bioinformatics to investigate PM_2.5-exposure on expression of genes related to carcinogenesis and mutagenesis in human bronchial epithelial (HBE) cells. METHODS: HBE cells were treated with 50 μg/mL water-soluble PM_2.5 for 24 h. Untreated cells were used as a blank control group,and three parallel samples were established. Extracted RNA samples were subjected to fluorescent-labeling,purification and hybridization to the chip. Collected data were pre-processed and statistically calculated using the Rosetta Resolver^® System (Rosetta Biosoftware) to analyze differentially expressed genes. The data were analyzed using the Cluster Profiler software for principal component and cluster analysis,and pathway and GO (Gene Ontolog) analysis. Differences in gene expression of HBE cells after PM_2.5 exposure were preliminarily investigated. Protein interaction relationships of differentially expressed genes were analyzed using the String protein interaction database,then the core protein with the largest number of nodes was selected. RESULTS: According to the preset screening conditions,245 differentially expressed genes were screened,including 27 up-regulated genes and 218 down-regulated genes. After further analyses,the top 10 up-regulated and down-regulated core genes such as PHACTR2,SFRP1,WFDC1 were screened out. The top 10 core differential gene by GO functional annotation enrichment analysis show that the differentially expressed genes were mainly enriched in molecular functions such as transmembrane receptor activity,receptor activity,cell signaling,and cell molecular conduction. Their mechanisms involved biological transmembrane transport,translocation of cells to lipopolysaccharide inorganic ions,and transport of cellular nutrients. The KEGG enrichment analyses show that the differentially expressed genes were mainly enriched in seven core pathways involved in tumor cell migration,bile metabolism related,neural activity,and genotoxicity. Protein interaction networking mapped out 5 core proteins including SST,BDNF,NCAM1,SSTR1 and Bcl2L11. CONCLUSION: Exposure of HBE cells to PM_2.5 showed differential expression of genes which are involved with carcinogenesis and mutagenesis. The information is useful for better understanding the carcinogenic effect of PM_2.5.

OBJECTIVE: To investigate the effect of ethephon and its mechanisms on precocious puberty of prepubertal SD female rats. METHODS: Postnatal day-15 SD female rats were randomly divided into 4 groups according to their body weights. Rats in the control group were given distilled water and the low,medium and high doses groups were given the ethephon at 134,269 and 538 mg/kg,respectively. Water and ethephon were given orally,once a day for 5 days. The first batch of animals was used to observe the time for vaginal opening. After the second batch of animals were killed,blood,hypothalamus and pituitary tissues were taken,and serum FSH and LH levels were detected by enzyme-linked immunosorbent assay (ELASA). Real-time quantitative PCR (qPCR) was performed to examine the mRNA expression levels of the hypothalamic-pituitary-gonadal axis (HPGA)-related genes KISS-1,GPR54,GnRH,and GnRHR. RESULTS: Compared with the control group,vaginal opening time of the ethephon middle dose group was advanced (P < 0.05) and that for the high dose group was significantly prolonged (P < 0.05). In addition,qPCR analyses show that expression of HPGA axis-related genes in the ethephon middle dose group was increased,and the high-dose group was decreased,and the difference was statistically significant (P < 0.05). There was no significant difference in serum LH and FSH levels (P > 0.05). CONCLUSION: Ethephon exposure was effective in promoting precocious puberty in prepubertal SD rats at the medium dose,and its mechanism may be related to the expression of genes related to the HPGA axis.

OBJECTIVE: To clone VP1 genes from enterovirus 71 (EV71) and coxsackievirus A16 (CA16) and to express the genes in eukaryotic cells. METHODS: VP1 coding sequences were obtained by PCR,and then inserted into pcFlag vector to construct FLAG-fused eukaryotic expression plasmids. The recombinant plasmids were used to transfect 293T cells and RD cells,and Western blotting was performed to detect the recombinant proteins. RESULTS: The plasmids containing VP1 were confirmed by sequencing. Recombinant proteins were detected in transfected cells using Western blotting. CONCLUSION: Exogenous expression of plasmids containing VP1 coding sequences of EV71 and CA16 were constructed successfully,and the recombinant proteins was detected in transfected cells.

OBJECTIVE: To investigate effects of long-term exposure to di-(2-ethylhexyl) phthalate (DEHP) on thyroid hormone levels in adult male rats. METHODS: Thirty-six healthy adult male Wistar rats were randomly divided into four groups according to body weight:control group and the 150,300 and 600 mg/kg DEHP exposure group. Rats were given normal food and water,but the rats in the exposed group were given DEHP by continuous gavage for 3 months. Before the rats were sacrificed,body masses of the rats were measured and the viscera ratio of each tissue was calculated. Urine iodine content,serum thyroid hormone content and serum TTR,TRH and TSAb levels were detected. One-way analysis of variance (ANOVA) was used to analyze the differences among the groups. SNK test was used for further multiple comparisons. RESULTS: After 3 months of exposure to DEHP,there was no significant change in body mass. Compared with the control group,there was no significant difference in the wet weight of thyroid and the ratio of viscera in each dose group. The ratios of liver viscera in the 150,300 and 600 mg/kg groups increased and showed a dose-effect relationship (P < 0.05). Urine iodine level of rats in the 600 mg/kg group was significantly decreased (P < 0.05). Serum FT4 level of rats in the 600 mg/kg group was decreased (P < 0.05). CONCLUSION: Exposure of rats to DEHP for 3 months had no significant effect on the body masses of rats. However,the exposure affected the function of thyroid and liver organs via inhibiting the synthesis,secretion and transport of TSH,and disrupting the thyroid hormone levels.

OBJECTIVE: To investigate differential expressions of microRNAs in Xinjiang Kazak's esophageal cancers and their involvement in cancers. METHODS: Expression of microRNAs were evaluated using microRNA microarray and qPCR in 47 cases of Kazak esophageal carcinoma tissues and distal cancer-free tissues. RESULTS: The results from the microRNA microarray and qPCR analyses were consistent with each other. Compared with the distal cancer-free tissues,expressions of miR-21,miR-132 and miR-130b were up-regulated in the esophageal carcinoma tissues while expressions of miR-141,miR-143,miR-195 and miR-133b were down-regulated,and the difference was significant (P < 0.05). miR-21,miR-132,miR-141 and miR-143 were related to the clinical stagings;miR-21,miR-132,miR-143 and miR-133b were related to lymph node metastasis;miR-21,miR-141,miR-143 and miR-133b were related to the differentiation of the cancers,and the differences were significant (P < 0.05). CONCLUSION: The microRNA microarray analyses were useful in screening out differentially expressed genes which were related to the occurrence of esophageal cancers while the microRNA analyses were useful in the occurrence of esophageal cancers.

OBJECTIVE: The insect baculovirus expression system was used to express the early rapid response gene 5 protein,and to provide clues for subsequent protein crystallization and exploration of tertiary protein structure. METHODS: The recombinant transfer vector pFastBac1-IER5 was constructed via amplification of the IER5 gene fragment with HeLa cell cDNA as template. After confirmation by restriction enzyme digestion and DNA sequencing,the recombinant transfer vector was transformed into E.coli DH10BacTM competent cells to obtain the recombinant shuttle vector rBacmid-IER5. The recombinant shuttle vector was transfected into Sf9 cells and the recombinant baculovirus was collected when the cells showed obvious lesions. The expression products were analyzed and identified using indirect immunofluorescence,SDS-PAGE and Western blot. RESULTS: The expected two bands were observed after the recombinant transfer vector pFastBac1-IER5 was digested by restriction enzymes;a band with the excepted size of about 3300 bp was obtained from the recombinant shuttle vector rBacmid-IER5 by PCR amplification;the results from indirect immunofluorescence indicate that IER5 protein was expressed correctly in the Sf9 cells. The SDS-PAGE analyses reveal that the molecular weight of the expressed product was about 48k. The Western blot analyses show that the expressed product specifically reacted with the IER5 antibody. CONCLUSION: The recombinant IER5 protein from human was expressed successfully using the insect baculovirus expression system,and revealed the physical and chemical properties of the recombinant IER5 protein,like the molecular weight and dissolubility.

OBJECTIVE: Relationships between bacterial concentrations and optical density[D(λ),λ is the wavelength value] in the Salmonella typhimurium reverse mutation assay (Ames test) was investigated and a method for the determination of bacterial concentrations using optical density was developed. METHODS: TA102,a frequently-used auxotrophic Salmonella typhimurium in the Ames test,was selected as the test strain. The amplification conditions of the bacterial solution were strictly controlled. The wavelength of 540 and 600 nm were compared to choose the optimal wavelength for the determination of optical density,and the time of 0,2,4,6,8,10,12 and 14 hours were compared to choose the optimal times for amplification. Under the optimal wavelength and amplification time,the optical density of the bacterial solution was measured,and the bacterial concentration was obtained by plate colony-counting method. Then the regression equation between the bacterial concentration and the optical density was obtained. RESULTS: The optimal wavelength of strain TA102 was 540 nm,and the optimal culture condition was 100 r/min at 37℃ for 10 hours. The linear regression equation between the bacterial concentration and D(540) value was y=4×10⁹x (in the equation,x was D(540) value;y was bacterial concentration with the unit of/mL). CONCLUSION: Through the linear regression equation between the bacterial concentration and D(540) value,real-time control of the bacterial solution to reach the required concentration was achieved by measuring D(540) value,so as to ensure the quality of bacterial solution.