OBJECTIVETo explore interaction proteins affect functions of connexin 30 (Cx30) by screening and identification interaction proteins of Cx30.

METHODSThe fusion expression vecto of CX30-C-terminal functional domain-pGEX-4T-2-GST was constructed, and then, fusion protein and GST were purified. They were incubated with the proteins of the foetus brain tissue disruption to pull down interaction proteins. The interaction proteins were separated by SDS-PAGE. Differential straps were cut to enzymolysis to prepare for mass chromatographic analysis, and then to index and screen interaction proteins in NCBInr database. The interaction proteins were identified by immunolocalization.

RESULTSThe four interaction proteins of Cx30 were screened in the foetus brain tissue, as follow, Keratin 16, Camk2b, Tubulin beta-3 and alpha-tubulin. Cx30 was proved to coexist with Keratin 16 and Tubulin beta-3.

CONCLUSIONSKeratin 16, Camk2b, Tubulin beta-3 and alpha-tubulin are the interaction proteins of Cx30. The interaction proteins affect the assembly, intracellular transport, and channel switch of Cx30.

Connexin 30 ; Connexins ; genetics ; metabolism ; Genetic Vectors ; Glutathione Transferase ; Humans ; Mutagenicity Tests ; Protein Interaction Mapping ; Recombinant Proteins ; genetics ; metabolism

OBJECTIVETo study the metastasis-associated molecules differentially expressed in highly and poorly metastatic sublines and the mechanism of metastasis in lung giant cell carcinoma.

METHODSHighly and poorly metastatic sublines (PLA801D and PLA801C)were used as metastasis model. Cell motility and invasion assay in vitro were first compared between the two sublines. Then, gelatin zymography analysis was used to determine the MMP-2 and MMP-9 activity. The protein expression level of secreted MMP-2, MMP-9, TIMP-1, TIMP-2 and intracellular expression level of p53, p16, PCNA, CD44(V6) isomeride, E-cadherin, CK18, nm23-H1 as well as the mRNA expression level of MMP-2, MMP-9, TIMP-1, TIMP-2, VEGF were compared through Western blot. Semi-quantitative RT-PCR analysis was used to determine the intracellular mRNA expression of MMP-2, MMP-9, TIMP-1, TIMP-2 and VEGF.

RESULTSThe in vitro cell invasion potential of highly metastatic subline PLA801D was significantly higher than that of poorly metastatic subline PLA801C by about 4 folds, while the cell motility potential was similar. The secreted MMP-2 activity was notably higher in PLA801D, which was initiated by the higher expression of MMP-2 at protein and mRNA level. In addition, the expression level of p53, PCNA, CK18 protein and VEGF mRNA were significantly higher, while the expression level of p16, E-cadherin and nm23-H1 protein were significantly lower in PLA801D. Some molecules such as MMP-9, TIMP-1, TIMP-2, CD44(V6) isomeride, which had been reported to be associated with tumor metastasis, were not observed to change significantly between the two sublines.

CONCLUSIONThere are significant differences in metastatic potential and phenotypes between highly and poorly metastatic sublines of lung giant cell carcinoma. Some differentially expressed molecules might be playing roles in promoting or inhibiting metastasis of lung giant cell carcinoma, which may be useful to elucidate the mechanism of metastasis.

Carcinoma, Giant Cell ; metabolism ; pathology ; Cell Line, Tumor ; Humans ; Interleukin-8 ; genetics ; Lung Neoplasms ; metabolism ; pathology ; Matrix Metalloproteinase 2 ; genetics ; metabolism ; Matrix Metalloproteinase 9 ; genetics ; metabolism ; Neoplasm Invasiveness ; Neoplasm Metastasis ; RNA, Messenger ; analysis ; Tissue Inhibitor of Metalloproteinase-1 ; analysis ; Vascular Endothelial Growth Factor A ; genetics

Objective To investigated the effects of combined arsenic trioxide(ATO) and resveratrol(Res)on the viability of NB4 human leukemia cells. Methods NB4 human leukemia cell was used in this experiment.Cells were cultured in ATO (0,0.1875,0.3750,0.7500, 1.1250, 1.5000,2.2500,3.0000,5.0000 μmol/L) and Res (0, 1.5625,3.1250,6.2500, 12.5000, 18.7500,25.0000,37.5000,50.0000 μmol/L). Cell viabilities were measured by MTT in different treatment groups. Half inhibitory concentration(IC50) was calculated. The ratio of concentration of ATO and Res 1.5∶ 18,1.5∶ 25,1.5∶ 35 was added to cells, and the combination index(CI) was calculated. The level of ROS in control, ATO( 1.5000 μmol/L), Res(25.0000 μmol/L) and ATO(0.9000 μmol/L) + Res( 12.5000μmol/L) groups was measured by chemiluminescence assay. Results ①ATO( ≥0.7500 μmol/L) reduced the viability of NB4 cells in a concentration-dependent manner(P ＜ 0.05 ), and IC50 was (1.78 ± 0.11 )μmol/L. ②)Res (≥18.7500 μ mol/L) dose-dependently decreased the viability of NB4 cells (P ＜ 0.05 ), and IC50 was ( 18.71 ±0.18)μ mol/L. ③Combination of ATO and Res showed an antagonistic effect on NB4 cells viability. ④The ROS in Res group( 1670.55 ± 13.97) was significantly lower than that in control group(2345.88 ± 14.48,P ＜ 0.05). The ROS in ATO group (3092.42 ± 94.84) was significantly higher than that in control group(P ＜ 0.05). The ROS in ATO + Res group (1860.27 ± 15.99) was significantly lower than that in ATO group(P ＜ 0.05). Conclusions NB4 cell survival rate can be decreased by ATO and Res. The combination of arsenic trioxide and Res presents an antagonistic effect on NB4 cell viability, in part by reducing intracellular ROS formation.

Objective To study the expression of plasma oxidized low-density lipoprotein (oxLDL) in children with acute phase Kawasaki disease (KD), and investigate its value for early prediction of coronary artery lesions in KD. Methods Totally 80 children with KD were collected. Children were divided into four groups by the results of echocardiogram of coronary artery in different periods: CAL1 group (children with coronary artery lesions (CAL+) both in acute and sub-acute phase, 8 cases), CAL2 group (children with CAL+in acute phase but recovery normal (CAL-) in sub-acute phase, 10 cases), NCAL1 group (children with CAL-in acute phase but occur CAL+ in sub-acute phase, 10 cases) and NCAL2 group (children with CAL- both in acute and sub-acute phase, 52 cases). The serum samples (before the use of intravenous immunoglobulin) were collected in acute phase. Twenty healthy controls and twenty fever controls were enrolled into the study, and their serum samples were collected. OxLDL was measured by enzyme linked immunosorbent assay (ELISA). They were compared using ANOVA, pairwise comparison LSD-t test. And ROC curve analysis was used to determine the threshold. Results Compared with the control groups,plasma oxLDL levels were higher in children with KD, both CA+and CAL-[(15.0±3.3) mU/L, (12.3±3.5) mU/L vs (9.2±2.2) mU/L, (8.0±2.3) mU/L, F=20.435, P<0.05]. Plasma oxLDL levels were increased more significantly in children with CAL+ than children with CAL- in KD [(15.0 ±3.3) mU/L vs (12.3 ±3.5) mU/L, t=2.28, P=0.002]. There was significant difference in the concentration of oxLDL between the groups of Kawasaki disease (F=5.068, P=0.003). Plasma oxLDL levels were significantly higher in the NCAL1 group than those in the NCAL2 group [(14.5 ±3.8) mU/L vs (11.9±3.3) mU/L, t=2.29, P=0.02], but there were no statistically significant difference between the NCAL1 group and CAL1 or CAL2 group [(14.5±3.8) mU/L vs (15.9±3.9) mU/L, (14.5±3.8) mU/L vs (14.2±2.7) mU/L, t=0.73, 0.20;P=0.41, 0.84]. ROCs analysis indicated that oxLDL≥13.83 mU/L, could be the threshold for the prediction of coronary artery lesions with the sensitivity of 0.607 and a specificity of 0.75. Conclusion OxLDL plays an important role in coronary artery lesions in KD. The coronary endothelial dysfunction is earlier than coronary dilatation, and oxLDL is expected to become a reliable early predictor of coronary artery lesions in KD.

Although previous studies have shown functional efficacy of myelotomy for the treatment of spinal cord injury (SCI), the underlying mechanism remained unknown. This study aimed to determine the relationship between myelotomy-mediated neuroprotection and autophagy following SCI by evaluating the expression of microtubule-associated protein light chain 3 (LC3-II) and mammalian target of rapamycin complex 1 (mTORC1). Ninety-nine adult female rats were randomly assigned to either sham-operated group (SG), model group (MG), or 24 h-myelotomy group (MTG). SCI at T10 was induced with a New York University impactor, and myelotomy was performed 24 h after SCI. Functional recovery was evaluated via the open-field test. The protein expression of LC3-II was analyzed by Western blot, and the mRNA expression of LC3-II and mTORC1 were detected by real-time quantitative reverse transcriptase polymerase chain reaction. Rats in the MTG exhibited significantly better performance in the hind limbs compared to those in the MG on day seven and fourteen post-injury. Myelotomy suppressed the protein and mRNA expression of LC3-II on day three, seven and fourteen post-injury and increased the mRNA expression of mTORC1 in the MTG on day three and seven post-injury. The LC3-II protein expression was significantly and negatively correlated with BBB scores at day seven and fourteen post-injury. These results showed that myelotomy-induced neuroprotection in a rat model of SCI was likely mediated by inhibition of autophagy by activation of the mTORC1 signaling pathway

AIMTo investigate the effect of taurine on nitric oxide synthase (NOS) activity and nitric oxide products (NO2 /NO3 ) content in myocardium and plasma during shock resuscitation.

METHODSTwenty-four rabbits were divided randomly into 3 groups (n=8): control group, shock group, taurine group. The model of hemorrhagic shock resuscitation was used. The activities of nitric oxide synthase (NOS), lactate dehydrogenase (LDH) and the contents of nitric oxide products (NO2- /NO3-) in plasma were observed before shock and shock 1.5 hours, after resuscitation 1 hour, 2 hours and 3 hours. The activities of NOS and the contents of NO2-/NO3- in myocardium homogenate were measured after resuscitation 3 hours. Meanwhile, pathologic samples treated routinely.

RESULTS(1) During resuscitation, the activities of NOS, LDH and the contents of NO2- /NO3- in plasma of shock group were significantly higher than that of before shock and shock 1.5 hours (P < 0.01). (2) After resuscitation 3 hours, the activity of NOS and the contents of NO2- / NO3 in myocardium of shock group were significantly higher than that of control group (P < 0.01). The cardiac myocyte appeared edema, fatty degeneration. (3) All the changes of above mentioned could be attenuated by intravenous injection taurine (40 mg/kg) (P < 0.01).

CONCLUSIONThese results suggest that the NOS activation and NO release may mediated myocardium injury induced by shock resuscitation, taurine can ameliorate the myocardium injury, which may be related to decreasing the generation of NO.

Animals ; Myocardium ; metabolism ; pathology ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase ; metabolism ; Plasma ; metabolism ; Rabbits ; Resuscitation ; Shock, Hemorrhagic ; blood ; metabolism ; Taurine ; pharmacology

AIMTo approach the relationship between lung injury induced by shock/reperfusion and nitric oxide as well as the beneficial effect of taurine.

METHODSTwenty four rabbits were divided randomly into 3 groups (n = 8): control group, shock group, taurine group. The model of lung injury induced by shock/reperfusion was used. The activities of nitric oxide synthase (NOS), superoxide dismutase (SOD), the contents of malondialdehyde (MDA), nitric oxide products (NO2-/NO3-) in plasma and lung homogenate, lung wet/dry weight, lung water content, lung permeability index, and protein content in the pulmonary alveolar lavage fluid were measured. Meanwhile, pathologic samples treated routinely.

RESULTS(1) At 3 hours after reperfusion, the activities of SOD in plasma and lung homogenate decreased markedly, but the other indexes above mentioned were increased significantly compared with the control group (P < 0.01). (2) A close correlation was shown between MDA content and NO2-/NO3- content in plasma and lung. Furthermore, the content of NO2-/NQ3- in lung homogenate showed strong positive correlation with the lung injury parameters. (3) Taurine (40 mg x kg(-1) i.v.) could attenuate all the changes above mentioned at the same time points of reperfusion.

CONCLUSIONNO may play an important role in lung injury induced by shock/reperfusion. Taurine can ameliorate the lung injury, mechanism of which may be related to decreasing the generation of NO and anti-lipoperoxidation.

Animals ; Lung ; drug effects ; metabolism ; Lung Injury ; etiology ; prevention & control ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase ; metabolism ; Rabbits ; Reperfusion Injury ; complications ; drug therapy ; Shock, Hemorrhagic ; complications ; drug therapy ; Superoxide Dismutase ; metabolism ; Taurine ; pharmacology ; therapeutic use

OBJECTIVETo study the function of IL-18 in promoting metastasis of lung cancer.

METHODSThe differential expression of IL-18 protein or mRNA level between highly and poorly metastatic sublines of human lung giant cell carcinoma metastatic model was detected by Western blot, semi-quantitative RT-PCR and northern blot analysis. The poorly metastatic PLA801C subline or highly metastatic PLA801D subline was transfected with constructed IL-18 sense or IL-18 antisense expressed plasmid by lipofectamine stable transfection technique. The metastasis-related effect mediated by IL-18, the metastatic phenotype differences, cell motility and cell invasion potential in vitro determined by MICS system and the expression level of metastasis-associated biomarkers detected by Western blot analysis, were compared between IL-18 stably transfectants and mock control, i.e. between PLA801C/IL-18(S) and PLA801C/pcDNA3.1, or between PLA801D/IL-18(As) and PLA801D/pcDNA3.

RESULTSIL-18 was only present in highly metastatic PLA801D subline at either protein or mRNA level, which implied that IL-18 might play a role in promoting metastasis of lung cancer. After IL-18 sense expressed plasmid was transfected into poorly metastatic PLA801C subline, IL-18 fused protein with myc tag detected by Western blot analysis using either IL-18 or myc tag monoclonal antibody. In addition, cell motility ability in vitro was significantly increased about 3 times and E-cadherin protein was significantly down-regulated at about 50% in PLA801C/IL-18(S) transfectants compared with mock control. While IL-18 expressed plasmid was transfected into highly metastatic PLA801D subline, IL-18 protein and mRNA were simultaneously decreased by 30%. In addition, cell invasion ability in vitro was significantly decreased at about 75% and E-cadherin protein was significantly up-regulated in PLA801D/IL-18(As) transfectants compared with mock control.

CONCLUSIONIL-18 might play a role in enhancing tumor metastasis of lung cancer by down-regulating E-cadherin protein expression.

Cadherins ; metabolism ; Carcinoma, Giant Cell ; metabolism ; secondary ; Cell Line, Tumor ; Cell Movement ; DNA, Antisense ; genetics ; Gene Expression Regulation, Neoplastic ; Humans ; Interleukin-18 ; biosynthesis ; genetics ; Lung Neoplasms ; metabolism ; pathology ; Neoplasm Invasiveness ; Neoplasm Metastasis ; genetics ; Plasmids ; RNA, Messenger ; biosynthesis ; genetics ; Transfection

OBJECTIVE To study the differentiation of PC12 cells induced by total salvianolic acid (Tsa) and the mechanism. METHODS MTT assay was used to detect the effect of Tsa 0.01, 0.1 and 1.0 μg·L-1on proliferation of PC12 cells and on the cells damaged by oxygen and glucose deprivation (OGD).The number of projections of PC12 cells was statistically analyzed.Western blotting was applied to detect the levels of microtubule-associated protein2 (MAP-2), extracellular signal-regulated kinase1/2 (ERK1/2), phosphorylated ERK1/2(p-ERK1/2), mitogen-activated protein kinase kinase1/2(MEK1/2) and p-MEK1/2 proteins.MEK inhibitor U0126 was examined for its effect on expressions of p-ERK1/2 and ERK1/2 protein in PC12 cells induced by Tsa 1.0 μg·L-1.RESULTS Compared with normal control group, Tsa 1.0 μg·L-1could promote PC12 cell proliferation, and the survival rate was increased by 90%, but the survival rate of PC12 cells was not affected by Tsa 0.01 or 0.1 μg·L-1. Compared with OGD injured group,PC12 cells injured by OGD could be repaired by Tsa 0.1 or 1.0 μg·L-1,and the survival rate was increased to (47.7±1.8)% and (63.2±13.0)%, respectively (P<0.05, P<0.01). Compared with normal control group,Tsa 0.01,0.1 and 1.0 μg·L-1could promote the growth of PC12 cell projections (P<0.01). Western blotting results showed that Tsa could promote the expressions of MAP-2, p-ERK1/2 and p-MEK1/2 proteins, and this effect could be blocked by U0126 inhibitor (P<0.01). CONCLUSION Tsa can induce the proliferation and differentiation of PC12 cells, the mechanism of which is possibly the activation of p-MEK and p-ERK1/2.

Objective To investigate the characteristics and distribution of human eehinococcosis in Hobukesar Mongolian Autonomous County (HMAC) in Xinjiang. Methods Using cluster sampling methods, the 2 counties (Tiebukenwusa and Narenhebuke) in HMAC were chosen as focusing areas for investigation. A survey of human echinococcosis including questionnaire, serological test and abdominal ultrasonic scan was carried out. Results The prevalence of human echinococcosis was 9.0% (64/712) by ultrasound and surgical history, including 8.7% (62/712) for cystic eehinococcosis(CE), 0.3%(2/712) for alveolar echinococcosis(AE) and 15.6%(111/712) for total of serological positives in HMAC. CE prevalence rate of different occupations, age, family slaughtering livestock and drinking water source had significant differences(P<0.05). Herdsmen as the highest risk group showed a CE prevalence of the 13.4% (27/201) in comparison with other occupations. The ages between 20 to<40 year-old were at the highest risk stage with 12.8% incidence. But CE prevalence rate of different gender, ethnic and education groups had not significant differences(P>0.05). Conclusions HMAC could be considered as a high endemic human CE region in Xinjiang. The current study reported the main risk factors may include occupations, age difference and drinking water source.