Animals ; Gene Expression ; Mice ; Mice, Inbred Strains ; Spinal Cord ; metabolism ; Spinal Cord Injuries ; genetics ; metabolism ; p21-Activated Kinases ; genetics

OBJECTIVETo construct a recombinant lentiviral vector for manganese superoxide dismutase (MnSOD) gene expression, and observe its effect on the proliferation of esophageal cancer cells in vitro.

METHODSChemical methods were employed for synthesis of the MnSOD cDNA sequence sections, along with the attB sites. Target gene fragment was constructed on the pMD-18T vector, and the recombinant plasmid pDONR221 was obtained after BP recombination reaction. Sequencing was followed by LR recombination reaction between the plasmid and DEST to obtain the lentiviral vector, which worked with helper plasmid for co-transfection of human embryonic kidney epithelial cells (293T cells). Amplification was done to determine its titer, and both transfection and selection procedures were made to get two stable transfected esophageal cancer TE-1 cell lines with medium MnSOD expression (TE-1Mm cells) and high MnSOD expression (TE-1Mh cell), and empty vector cell (TE-1Mn cells). Reverse transcription polymerase chine reaction (RT-PCR), immunofluorescence, immunocytochemistry and Western blot were used to detect the target gene with respect to its expression in the TE-1 cells. Additionally, colorimetric 3-[4,5-dimethy thiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, agar colony formation assay, annexin V-FITC/PI staining and flow cytometry experiments were also conducted as to observe the influence of the medium and high MnSOD overexpressions on the proliferation of esophageal cancer cells.

RESULTSRT-PCR indicated that the transfected TE-1 cells showed positive MnSOD expression at different levels. Immunofluorescence, immunocytochemistry and Western blot suggested that TE-1Mm cells and TE-1Mh cells had MnSOD protein expression at different levels. MTT assay indicated that TE-1Mm cells had a significantly decreased survival rate compared with that of the two control cells (TE-1 cells and TE-1Mn cells), and TE-1 Mh cells had an significantly increased survival rate (P<0.05). The colony formation ability of TE-1Mm cells was (23.0 +/- 2.7)%, and that of TE-1Mh cells was (45.3 +/- 4.5)%, significantly different form the (34.7 +/- 4.2)% in TE-1 cells and (33.7 +/- 4.7)% in TE-1Mn cells (P<0.05). Annexin V-FITC/PI double staining experiment of the stably transfected cells cultured for 48 h showed that the early apoptosis rate in TE-1Mm cells was (10.6 +/- 1.0)%, significantly higher than (2.6 +/- 0.2)% in the TE-1 cells, (2.5 +/- 0.6)% in the empty vector cells and (1.0 +/- 0.1)% in the TE-1Mh cels (P<0.05). The fluorescence index (FI) of mitochondrial apoptosis of TE-1Mm cells was 0.948 +/- 0.019, significantly lower than that of TE-1 cell (1.000 +/- 0.022) and empty vector The fluorescence index of TE-1Mn cells (0.997 +/- 0.023) and TE-1 cells (1.000 +/- 0.022) were significant different from that of 0.948 +/- 0.019 in TE-1Mm cells and 1.076 +/- 0.022 in TE-1Mh cells, indicating a significant difference of mitochondrial apoptosis between the cell groups. FCM results indicated that the ROS fluorescence index of TE-1Mm cells was 0.859 +/- 0.040, that of TE-1Mh cells was 0.763 +/- 0.039, significantly lower than that of TE-1 cells (1.000 +/- 0. 042) and empty vector cells (1.002 +/- 0.047) (P<0.05).

CONCLUSIONSStably transfected cell lines with MnSOD expression have been successfully established. MnSOD overexpression shows bidirectional effect on the proliferation of esophageal cancer cells.

Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Esophageal Neoplasms ; metabolism ; pathology ; Gene Expression Regulation, Neoplastic ; Genetic Vectors ; HEK293 Cells ; Humans ; Lentivirus ; genetics ; Mitochondria ; pathology ; Plasmids ; RNA, Messenger ; metabolism ; Reactive Oxygen Species ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Superoxide Dismutase ; genetics ; metabolism ; Transfection

A method for the determination of Fe, Co, Mn and Ni in synthetic diamonds by inductively coupled plasma atomic emission spectrometry ( ICP-AES) was proposed. The synthetic diamond sample was decomposed completely, while the sample was burned in air at 1000 ℃ for 10 h, and then a mixed acid of H2 SO4 , aqua regia and HClO4 was used for the dissolving the residue of the sample. In this method, the limits of detection of Fe, Co, Mn and Ni were 0. 0147, 0. 0018, 0. 0006 and 0. 0027 mg/L, respectively. Under the optimum condition, Fe, Co, Mn and Ni in synthetic diamond sample were determined. The values of RSDs (n=7) were less than 0. 5%. The recoveries of added standard were 94. 0%-105. 0%.

A method for the simultaneous determination of oxygen and nitrogen in synthetic diamonds by inert gas high temperature extraction-impulse heating method was proposed. The sample weight, the selection of analysis power and the calibration curves of oxygen and nitrogen were discussed. Oxygen and nitrogen in analytical samples are determined. Values of RSDs (n=7) for oxygen and nitrogen were less than 4. 5% and 4. 0% respectively. The analytical results of oxygen and nitrogen obtained by the proposed method were in good agreement with those by inert gas fusion-impulse heating method.

The infant (a girl aged 6 months) was admitted to the hospital because of oliguria and acute renal dysfunction. The laboratory examination results showed serious metabolic acidosis and increased blood urea nitrogen and serum creatinine levels. The patient continued to be anuric after 10 days of treatment with continuous renal replacement therapy (CRRT). she died a day later. The family history showed that the patient's sister died of acute renal failure 6 months after birth. The genomic sequencing results showed AGXT mutation in the patient and confirmed the diagnosis of primary hyperoxaluria type 1 (PH1). Her parents were heterozygous carriers. PH1 should be considered when the children have abnormal renal function or recurrent renal calculi or have a family history of these symptoms. AGXT gene analysis is an important method for PH1 diagnosis.
Acute Kidney Injury ; etiology ; Female ; Humans ; Hyperoxaluria, Primary ; complications ; Infant ; Mutation ; Oliguria ; etiology ; Transaminases ; genetics

A boy aged 11 years was admitted due to intermittent weakness and difficulty in walking for 6 years, and hepatomegaly, glycopenia and unconsciousness for 4 years. The laboratory examinations showed severe metabolic acidosis, hypoglycemia, and abnormal liver function. CT scan showed marked liver enlargement with fat density shadow. The boy was given fluid infusion, correction of acidosis, intravenous injection of glucose, L-carnitine, compound vitamin B, and coenzyme Q10, but he was in a persistent coma and it was difficult to correct refractory metabolic acidosis and hypoglycemia. The boy died. Blood and urinary organic acid screening and gene detection confirmed that the boy had late-onset glutaric aciduria type II (GAIIc) caused by electron-transferring-flavoprotein dehydrogenase (ETFDH) gene defect. GAIIc is an inherited metabolic disease with a low incidence, resulting in a high misdiagnosis rate. GAIIc should be considered for children with recurrent weakness or reduced activity endurance, hypoglycemia, and marked liver enlargement with abnormal liver function. Urinary organic acid analysis and blood tandem mass spectrometry can help with the early diagnosis of GAIIc, and ETFDH gene analysis helps to make a confirmed diagnosis.
Child ; Hepatomegaly ; etiology ; Humans ; Hypoglycemia ; etiology ; Male ; Multiple Acyl Coenzyme A Dehydrogenase Deficiency ; diagnosis ; Muscle Weakness ; etiology

OBJECTIVETo investigate the expressions of phosphorylated protein kinase B (p-AKT), phosphorylated glycogen synthase kinase 3β (p-GSK3β) and β-catenin proteins and to evaluate their relationship with the clinical pathological characteristics in epithelial tumors of the ovary.

METHODSThe expression of p-AKT, p-GSK3β, and β-catenin was detected with immunohistochemical staining (EnVision method) in 10 cases of benign epithelial neoplasia, 10 cases of borderline epithelial neoplasia and 70 cases of ovarian carcinoma. The relationship of the expression of p-AKT, p-GSK3β and β-catenin with the clinical pathological features was analyzed.

RESULTSThe positive expression rates of p-AKT, p-GSK3β and β-catenin in epithelial ovarian carcinoma were 67.1% (47/70), 60.0% (42/70) and 71.4% (50/70), respectively. Compared to the results of benign and borderline epithelial neoplasia, the expression of the three proteins in carcinoma of the ovary was significantly different (all P < 0.05).Positive correlation was found between p-AKT and p-GSK3β, p-GSK3β and β-catenin, and p-AKT and β-catenin in epithelial ovarian carcinoma (r = 0.546, 0.581, 0.500, respectively; all P < 0.05). Compared to the results of benign and borderline epithelial neoplasia, the expression of p-AKT protein in epithelial ovarian carcinoma was significantly different (all P < 0.05). The expression of p-AKT was correlated with the differentiation of epithelial ovarian carcinoma (P < 0.05), but no relationship was found between its expression and histological classification and FIGO staging (P > 0.05). The expression of p-GSK3β and β-catenin in epithelial ovarian carcinoma were both higher than that in benign and borderline epithelial neoplasia (P < 0.05), and correlated with tumor differentiation and FIGO staging (P < 0.05), but no relationship were found between their expression with histological classification (P > 0.05).

CONCLUSIONSPositive correlations are found between p-AKT, p-GSK3β and β-catenin in epithelial ovarian carcinoma. The activation of β-catenin is possibly correlated with inactivation of p-GSK3β that binds to p-AKT.

Adult ; Aged ; Carcinoma, Endometrioid ; metabolism ; pathology ; Cell Differentiation ; Cystadenocarcinoma, Mucinous ; metabolism ; pathology ; Cystadenocarcinoma, Serous ; metabolism ; pathology ; Cystadenoma, Mucinous ; metabolism ; pathology ; Cystadenoma, Serous ; metabolism ; pathology ; Female ; Glycogen Synthase Kinase 3 ; metabolism ; Glycogen Synthase Kinase 3 beta ; Humans ; Middle Aged ; Neoplasm Staging ; Ovarian Neoplasms ; metabolism ; pathology ; Phosphorylation ; Proto-Oncogene Proteins c-akt ; metabolism ; beta Catenin ; metabolism

AIM To fit the moisture adsorption models for Astragali Radix aqueous extract.METHODS For the aqueous extract prepared by decocting,the moisture adsorption kinetics curve and adsorption/desorption isotherms were drawn by gravimetry,and then the data on moisture adsorption were fitted.The fitted models were optimized according to evaluation indices of root mean square error (RMSE),residual sum of squares (RSS) and coefficient of determination (R2).RESULTS The double exponential model for moisture adsorption kinetics curve and Peleg model for adsorption/desorption isotherms were considered as the two best models with regard to their fitting effects (R2 values were the closest to 1,RMSE and RSS values were the closest to 0).Among them,various adsorption/desorption isotherms all belonged to type Ⅱ ("S" isotherm),while the higher equilibrium moisture content of desorption isotherm than that of adsorption isotherm contributed to obvious type H3 hysteresis phenomenon.CONCLUSION Double exponential model and Peleg model can well fit the moisture adsorption kinetics curve and adsorption/desorption isotherms of Astragali Radix aqueous extract,respectively.

OBJECTIVETo investigate the expression of E-cadherin in nasopharyngeal carcinoma (NPC) and its relationship with cervical lymph node metastasis.

METHODSThe expression of E-cadherin in 80 patients with NPC was detected by immunohistochemistry.

RESULTSLower expression of E-cadherin was associated with advanced N-stage of the tumor (P = 0.018). There was no significant correlation between the expression of E-cadherin and lymph node size (P = 0.435). The expression of E-cadherin was higher in patients with cervical lymph node metastasis limited to a single area than that distributing in some scattered areas (P = 0.000). There was a trend that the expression of E-cadherin in the cases with the tumor and lymph nodes in the same side was higher (56.5%) than that in the patients with bilateral lymph node metastases (32.6%), however, the difference was not significant (P = 0.059). The expression rates of E-cadherin in patients with lymph node metastasis in levels II, III and Va were higher than that in levels I, IV, Vb and VI, but with a non-significant difference (P = 0.059).

CONCLUSIONThe expression of E-cadherin has influence on the lymph node metastasis in nasopharyngeal carcinoma. E-cadherin expression is negatively correlated with the numbers of the lymph node metastases and the metastasis distance, i.e. a lower expression of E-cadherin leads to an advanced N-stage. The lymph node metastasis of nasopharyngeal cancer from above to below is more considerably influenced by E-cadherin expression than the metastasis towards contralateral lymph nodes.

Adolescent ; Adult ; Aged ; Cadherins ; metabolism ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Female ; Humans ; Lymph Nodes ; pathology ; Lymphatic Metastasis ; pathology ; Male ; Middle Aged ; Nasopharyngeal Neoplasms ; metabolism ; pathology ; Neoplasm Staging ; Young Adult

OBJECTIVETumor necrosis factor-α (TNF-α) is known to induce changes in endothelial cell morphology and permeability. The aim of this study is to determine the underlying signaling mechanisms involved in these responses.

METHODSCultured human umbilical vein endothelial cells (HUVECs) were exposed to TNF-α, and HUVEC cytoskeletal changes were evaluated by observing fluorescence of F-actin following ligation with labeled antibodies. Endothelial permeability was detected by measuring the flux of horseradish peroxidase (HRP)-albumin across the EC monolayers. To explore the signaling pathways behind TNF-α-induced changes in HUVEC morphology and permeability, HUVECs were treated with either the Rho GTPase inhibitor Y27632 or the mitogen-activated protein kinases (MAPK) inhibitors PD98059 and SB203580 before TNF-α administration. To further elucidate possible involvement of the RhoA and ERK pathways in TNF-α-induced HUVEC changes, retrovirus-carried recombinant dominant-negative forms and constitutive-activative forms of RhoA, namely T19NRhoA and Q63LRhoA, were pre-infected into HUVECs prior to TNF-α exposure.

RESULTSTNF-α induced F-actin cytoskeleton rearrangement and increased HUVEC permeability in a dose and time-dependent manner. The maximal increase in the HRP-BSA flux (40 ng/ml) was seen in cells exposed to TNF-α at 100 ng/ml after 2 h. Preconditioning of HUVEC monolayer with Y27632 or PD98059 significantly reduced TNF-α induced permeability increase (HRP concentration from 40 ng/ml decreased to 12.5 ng/ml, P < 0.05) and F-actin cytoskeleton rearrangement, HUVEC pre-infection with activated forms of Q63LRhoA increased HUVEC permeability and upregulated pERK compared to GFP infection, while HUVEC pre-infection with inhibited forms of T19NRhoA attenuated the effects of TNF-α on HUVEC permeability.

CONCLUSIONThese results indicate that TNF-α-induced EC barrier dysfunction and morphological changes of the F-actin via activating RhoA-ERK/MAPK signal pathway.

Cell Membrane Permeability ; Cells, Cultured ; Cytoskeleton ; metabolism ; Endothelial Cells ; cytology ; drug effects ; metabolism ; Endothelium, Vascular ; cytology ; Human Umbilical Vein Endothelial Cells ; drug effects ; Humans ; Mitogen-Activated Protein Kinases ; metabolism ; Signal Transduction ; Tumor Necrosis Factor-alpha ; pharmacology ; rhoA GTP-Binding Protein ; metabolism