Animals ; Humans ; MicroRNAs ; genetics ; Neoplasms ; genetics ; pathology ; RNA Interference ; RNA, Neoplasm ; genetics ; RNA, Small Interfering ; genetics

In order to confirm the role that the 49th amino acid residue plays in enzymatic inactivity of Glutamine 49 phospholipase A2(Gln49-PLA2),site-directed mutagenesis of its 49th amino acid gene codon was conducted using PCR.Aspartic acid 49 phospholipase A2(Asp49-PLA2-Q49D-PLA2),the mutant of Gln49-PLA2 was expressed in E.coli with pET32a+ vector.The fusion protein,expressed as inclusion body,after being denatured,was on-column refolded and purified by immobilized metal affinity chromatography(IMAC),and then cleaved by Factor Xa.The mature Q49D-PLA2 mutant was obtained by Hitrap SP cation exchange and Superdex 75 gel filtration chromatography,with the recovery rate of 1.3%,and the specific activity of the mature Q49D-PLA2 mutant was 72 U/mg.It has been demonstrated that the 49th glutamine amino acid residue is the main reason in enzymatic inactivity of Gln49-PLA2 and the results are helpful for denatured protein refolding,especially in rich disulfide bonds conditions.

AlM: To monitor long - term changes of matrix metalloproteinase-2 ( MMP-2 ) in human tears fluid after laser in situ keratomileusis ( LASlK) .
METHODS: Thirty - two myopia cases ( 64 eyes ) underwent uneventful LASlK were enrolled in the study. Tear fluid were collected and MMP-2 expression was analyzed by Western - bolt assay preoperatively and postoperatively on 15d, at 1, 3mo, and 1a.
RESULTS:LASlK increased the concentration of MMP-2 in human tear fluid. At 15d postoperatively, the magnitude of MMP-2 was 1. 4 times that of preoperative, thereafter subsided, but didn't return to preoperative level by 3mo ( P < 0. 05 ). Up to 1a after surgery, the concentration of MMP-2 almost recovered (P>0. 05).
CONCLUSlON: MMP- 2 is significantly expressed in human tear fluid after LASlK, then subsided with time, but didn't return to preoperative level by 3mo and almost recovered up to 1a, indicating wound healing of LASlK would continue up at least 3mo after surgery and almost recovered 1a postoperatively.

OBJECTIVETo profile the protein expression in activated rat hepatic stellate cells (HSCs).

METHODSPrimary rat HSCs were isolated and cultured in vitro. After 10 days in vitro culture, the HSCs were activated. Total protein extracted from these activated HSCs were digested, and the obtained peptides were analyzed by using online 2D nanoLC-MS/MS. The identified proteins were classified according to their distributions and functions.

RESULTS1014 proteins were identified from 50 microg HSCs protein extract, the molecular weights of these proteins ranged from 7832 Da to 588,364 Da. Most of these proteins resided in nucleus, cytoskeleton, mitochondrion and endoplasmic reticulum. And these proteins were mainly involved in nucleic acid metabolism, organelle organization, signal transduction and energy generation. Among these proteins, alpha-smooth muscle actin, vimentin and desmin were specifically expressed in activated HSCs.

CONCLUSIONTo the best of our knowledge, this is the most comprehensive protein expression profile of activated rat HSCs.

Actins ; analysis ; metabolism ; Animals ; Cell Nucleus ; metabolism ; Cells, Cultured ; Chromatography, High Pressure Liquid ; methods ; Desmin ; analysis ; metabolism ; Hepatic Stellate Cells ; metabolism ; Male ; Proteome ; analysis ; metabolism ; Proteomics ; Rats ; Rats, Sprague-Dawley ; Tandem Mass Spectrometry ; Vimentin ; analysis ; metabolism

Aged ; Appendiceal Neoplasms ; diagnostic imaging ; Cystadenoma, Mucinous ; diagnostic imaging ; Female ; Humans ; Male ; Retrospective Studies ; Tomography, X-Ray Computed ; methods

OBJECTIVETo study mechanisms of reduction of the malignant activities of human naso-pharyngeal carcinoma cell CNE-2L2 induced by ectopic expression of BCSC-1 gene.

METHODSDNA was stained with propidium iodide and assayed upon a flow cytometer. Chromosomes were stained with Hoechest 33258. Adhesion of CNE-2L2 cells was detected by cell aggregation test. Protein expression on CNE-2L2 cells was examined by Western blot.

RESULTSCell cycle analysis showed that the percentage of CNE-2L2 cells was 55.1%, 43.4%, and 39.4% in G0/G1 phase, 25.2%, 28.7%, and 30.9% in S phase, and 19.7%, 27.9%, and 29.7% in G2/M phase for the cell with ectopic expression of BCSC-1 gene, wild type cell (W cells), and the cell transduced with the mock (M cell). Many mitotic cells were found in W cells and M cells. In contrast, almost no mitotic cell was observed in the cells with ectopic expression of BCSC-1 gene. Ectopic BCSC-1 expression resulted in cell aggregation, enhanced expression of E-cadherin, cx-catenin, and p53.

CONCLUSIONSEctopic BCSC-1 expression causes enhancement of adhesion of CNE-2L2 cells associated with enhanced expression of E-cadherin and alpha-catenin, arrest of cell in G1 phase, which may be associated with enhanced expression of p53. These alteration may play a role in the reduction of malignant activities of the cells with ectopic expression of BCSC-1 gene.

Cell Adhesion ; Cell Cycle ; physiology ; Cell Line, Tumor ; Humans ; Nasopharyngeal Neoplasms ; Neoplasm Proteins ; biosynthesis ; genetics

Objective To investigate the effects of ZNF331 overexpression on proliferation and apoptosis of human colon cancer cell HCT116, and the relevant apoptotic mechanism.Methods The lentivirus vector of overexpressed ZNF331,Flag-pLV-Neo-ZNF331,was constructed and packaged.HCT116/p53 +/+(wild type p53)and HCT116/p53 -/-(deficient p53)cells were infected.Clones with ZNF331 overexpression were identified by Western blotting.Cell proliferation assay,colony formation assay and flow cytometry analysis were used to examine the effects of ZNF 331 on cell proliferation and apoptosis.Immunoprecipitation,luciferase reporter gene assay and real-time PCR were performed to detect interactions between ZNF331 and p53, p53 transcriptional activity and the expression of p 53 apoptotic target genes, respectively.Results The lentivirus vector of overexpressed ZNF 331 was successfully generated.Stable clones of ZNF331 overexpression were established.ZNF331 showed no significant effect on cell proliferation of HCT 116/p53 +/+, but inhibited cell proliferation of HCT116/p53 -/-(P<0.01).ZNF331 could interact with p53,dose-dependently inhibit the transcriptional activity of p53 and downregulate the mRNA levels of pro-apoptotic p53 target genes, Puma and p53AIP1 (P<0.05).ZNF331 could suppress p53-induced apoptosis(P <0.01).Conclusion The influence of ZNF331 overexpression on colon cancer cell proliferation is dependent on p 53 status.ZNF331 overexpression can suppress colon cancer cell apoptosis by interacting with p 53 and inhibiting the transcriptional activity of p 53.

Adult ; Female ; Humans ; Image Enhancement ; Magnetic Resonance Imaging ; Male ; Middle Aged ; Tuberculoma ; diagnosis ; Tuberculosis, Hepatic ; diagnosis

OBJECTIVETo evaluate the effect of a 10-day course of triptolide (TP) on rat cytochrome (CY) P3A4 activity, and on the pharmacokinetics of cyclophosphamide (CPA).

METHODSIn the pharmacokinetics experiment, rats were orally given 0.9% NaCl solution (n=5) and TP [1.2 (mg/kg·d)] for 10 days and a single dose of CPA was administered intravenously (100 mg/kg) to rats on day 11. Blood samples were collected up to 4 h at predetermined time intervals, the plasma concentration of CPA was determined by high performance liquid chromatography (HPLC) and pharmacokinetic parameters were determined. In the in vitro CYP3A4 activity inhibition research, rat blank liver microsomes were divided into 3 groups: a control group, a TS (5 μL, 200 μmol/L) with TP (5 μL, 12.5 μmol/L) group, a TS with ketoconazole (5 μL, 1 μmol/L) group. Concentration of 6β-hydroxylated testosterone (6β-OHT) in liver microsomes was measured by HPLC and the activity of CYP 3A4 was calculated through the following formula: Einhibitor/Econtrol × 100%=Cinhibitor/Ccontrol × 100%.

RESULTSCompared with the control group, the area under the plasma concentration-time curve (AUC0-∞) of CPA was significantly increased by 229.05% pretreated with TP (P<0.01). Peak plasma concentrations (Cmax) of CPA was significantly increased and plasma half-life was correspondingly extended. The CYP3A4 activity was significantly inhibited by ketoconazole 93.5%±0.2% and TP 84.6%±0.3% compared with the control group (P<0.01 and P<0.05, respectively).

CONCLUSIONOur results strongly suggested that long-term oral intake of TP can distinctly inhibit the CYP3A4 activity and this inhibition evidently decrease the formation of toxic metabolites of CPA.

Animals ; Cyclophosphamide ; pharmacokinetics ; Cytochrome P-450 CYP3A ; metabolism ; Cytochrome P-450 CYP3A Inhibitors ; pharmacology ; Diterpenes ; pharmacology ; Epoxy Compounds ; pharmacology ; Herb-Drug Interactions ; Hydroxytestosterones ; metabolism ; Immunosuppressive Agents ; pharmacokinetics ; Injections, Intravenous ; Ketoconazole ; pharmacology ; Male ; Microsomes, Liver ; drug effects ; metabolism ; Phenanthrenes ; pharmacology ; Rats, Sprague-Dawley

OBJECTIVETo establish an animal model of sulfur mustard (SM)-induced acute lung injury in rats through different routes and compare the morphological changes in lung tissue and cells.

METHODSOne hundred and thirty-six male rats were selected and randomly divided into 5 groups, namely peritoneal cavity SM group (n=32), trachea SM group (n=32), peritoneal cavity propylene glycol group (n=32), trachea propylene glycol group (n=32), and normal control group (n=8). The rats in peritoneal cavity SM group were injected intraperitoneally with diluted SM (0.1 ml, 8 mg/kg), and the rats in trachea SM group were injected intratracheally with diluted SM (0.1 ml, 2 mg/kg). Once the rats were sacrificed at 6, 24, 48, and 72 h after SM treatment, morphological changes in lung tissue and cells were observed by light and electron microscopy.

RESULTSIn the peritoneal cavity SM group, the epithelial cells of bronchioles maintained intact with increased exudate and bleeding in alveolar cavity and large areas of pulmonary consolidation under the light microscope. In the tracheal SM group, focal ulcer formed in the epithelial cells of bronchioles with increased exudate and bleeding in alveolar cavity, partial pulmonary consolidation, and compensatory emphysema in peripheral alveolar space under the light microscope. The alveolar interval areas were widened obviously in both groups in a time-dependent manner. Under the electron microscope, we observed local loss of cellular membrane in type I alveolar epithelium, broken or lost microvilli in cells of typeⅡalveolar epithelium and fuzzy mitochondrial crista as well as the appearance of ribosome detached from rough endoplasmic reticulum in both two groups. Compared with those in the trachea SM group and the control group, the ratio of the alveolar septum average area to the visual field area in the peritoneal cavity SM group at 6, 24, 48, and 72 h was significantly higher (P<0.05).

CONCLUSIONThe lung tissue injury through the intraperitoneal route is more severe than that through the tracheal route, while focal ulceration of bronchioles epithelial cells appears in the case of tracheal route. The degree of injury increases over time in both groups, and the cellular damage is approximately the same in both groups.

Acute Lung Injury ; chemically induced ; pathology ; Animals ; Disease Models, Animal ; Lung ; pathology ; Male ; Mustard Gas ; toxicity ; Peritoneum ; Pulmonary Alveoli ; pathology ; ultrastructure ; Rats ; Trachea