A site-directed mutant DNA fragment was synthesized and transfected into clinical Neisseria gonorrhoeae (NG) stains to construct the transformants that contained the corresponding mutagenesis of regulation region of mtrR gene. According to the technique of gene splicing by overlap extension (SOEing), a DNA segment with specific mutagenesis was constructed by two-step polymerase chain reaction (PCR). The mutation fragments EF could be used for the next experiment in which the mutation NG strains were induced. By comparing the recombinant EF fragments to the corresponding DNA fragments of clinical NG strains, 2 of these were not compatible completely. The results of sequencing revealed that there was a 9 bp deletion between the 45 to 54 inverted repeat sequence localized within the mtrR promoter. It can be confirmed that the fragments EF are the specifically designed mutant fragments.
Bacterial Proteins/*genetics ; DNA Fragmentation ; DNA, Bacterial/genetics ; Mutagenesis, Site-Directed ; Neisseria gonorrhoeae/*genetics ; Neisseria gonorrhoeae/metabolism ; Recombination, Genetic ; Repressor Proteins/*genetics ; Sequence Deletion ; Transfection ; Transformation, Bacterial

AIM:To investigate the differentiation-inducing effects of baicalin on HL-60 leukemia cells.METHODS:The effect of baicalin on differential induction in AML cell line HL-60 was evaluated by cellular morphology,clone formation assay,CD11b and CD33 expression and NBT assay.RESULTS:Baicalin inhibited the proliferation of HL-60 cells.It enhanced the expression of CD11b on HL-60 cells,also increased the expression of CD33.HL-60 cells showed differentiation morphology after the drug treatment examined by Wright-Gimesa staining and NBT assay.CONCLUSION:Baicalin possessed differentiation-inducing effects on HL-60 cells.

Objective To study the influence of mesenchymal stem cells(MSCs) implantation on ventricular remodeling and heart function after myocardial infarcion. Methods Bone marrow was aspirated from Gui-zhou Xiang swines.After being isolated,cultured and co-cultured with 5-azacytidine,either autologous MSCs(experiment group) or a comparable volume of physiologic saline(control group) were injected into the infarcted myocardium.Three and six weeks later,echocardiographic measurement was performed to assess the myocardial structure and heart function,and single photon emission computed tomography(SPECT) was employed for myocardial imaging.Implanted stem cells were detected by the anti-Brdv antibody DAB with HE staining. Results Left ventricular ejection fraction(EF),fractional shortening and wall thickening were higher in experiment group than control group.The thickness of the ventricular wall and septum was found increased while the left ventricular chamber size was smaller in experiment group.It was indicated by SPECT that three and six weeks after implantation,there was obvious image defect in control group while in experiment group there were some imaging areas in the infarcted area.Brdv-labelled cells were observed in the central part of and around the infarcted area.Conclusion Implantation of MSCs into the infarcted myocardium is believed to attenuate the remodeling process,inhibit the extent of wall thinning and dilatation of the ventricular chamber.MSCs implantation may also improve the contractile ability of the myocardium and heart function.

Biliary Atresia ; diagnosis ; etiology ; therapy ; Bilirubin ; blood ; Biomarkers ; blood ; Calcium-Binding Proteins ; deficiency ; Cholangiopancreatography, Magnetic Resonance ; Cholestasis, Intrahepatic ; diagnosis ; etiology ; therapy ; Citrullinemia ; diagnosis ; etiology ; therapy ; DNA Mutational Analysis ; Humans ; Infant ; Jaundice ; diagnosis ; etiology ; therapy ; Liver Function Tests ; Male ; Mitochondrial Membrane Transport Proteins ; genetics ; Mutation ; Organic Anion Transporters ; deficiency

Objective To investigate the effect of Ulinastatin on the delivery of cytokines in patients with septic shock.Methods It was a prospective and controlled clinical study.Seventy-eight patients with septic shock were randomly divided into control group and treatment group and thirty-nine in every group.Patients in treatment group received Ulinastatin 200 000 units intravenous everyday for 3 days,while those in control group received equal volume of normal saline as placebo.At different time points (at 24 th,48 th,72 th hour after start of treatment),the levels of tumor necrosis factor-alpha (TNF-?),interleukin-1 (IL-1),interleukin-6 (IL-6 ),interleukin-8 (IL-8) and superoxide dismutase (SOD) in serum were assayed.Results In comparison with control group,the levels of TNF-?,IL-1,IL-6,IL- 8 of treatment group decreased markedly (P＜0.05,P＜0.01) at different time points,whereas the level of SOD was higher markedly (P＜0.05,P＜0.01) at various time points.Conclusion Ulinastatin has protective effect on patients with septic shock through decreasing the levels of TNF-?,IL-1,IL-6,IL-8 and increasing in the level of SOD.

Objective To investigate whether elevated pre-procedural C-reactive protein (CRP) and Interleukin-6 (IL-6) concentrations may be relevant to early outcome in patients undergoing PCI.Method 100 consecutive patients undergoing pereutaneuous coronary intervention (PCI) were included in our study.Peripheral blood samples for CRP and IL-6 testing were withdrawn before PCI.Acute vascular complications resulted from PCI were determined by subsequently coronary angiography.The early coronary events during hospitalization were clinically followed.Results Thirty patients developed acute vessel occlusion,and another one developed subacute coronary thrombosis at 2 days after PCI.Increased levels of CRP correlated well with the occurrence of vascular complications as regards the significant difference existing amongⅠvsⅢandⅠvsⅣquartile groups,P

This research established an HPLC method for determination of six C-Glycoside flavones of warer-soluble total flavonoids from Isodon lophanthoides var. gerardianus (Benth.) H. Hara, and studied the antitumor activity of the warer-soluble total flavonoids. The HPLC system consisted of Kromasil 100-5 C18 (4.6 mm x 250 mm, 5 microm) column and a solution system of methanol, acetonitrile and 0.5% formic acid gradient elution at a flow rate of 0. 8 mL x min(-1) and the wavelength of detector was at 334 nm. The column temperature was 25 degrees C. The antitumor activity of water-soluble flavonoids was assayed using HepG2 cell as the tested cell. The linear ranges of vicenin II, vicenin III, isoschaftoside, schaftoside, vitexin, 6, 8-di-C-a-L-arabinosylapigenin were 0.25-2.53, 0.12-1.20, 0.37-3.69, 0.16-1.63, 0.19-1.92, 0.14-1.42 microg, respectively. The average recoveries (n = 6) were 99.6% (RSD 0.87%), 100.2% (RSD 2.0%), 99.6% (RSD 1.8%), 97.9% (RSD 1.5%), 98.8% (RSD 1.2%), 98.6% (RSD 1.2%), respectively. After exposure in 24, 48, 72 h, the total flavonoids showed inhibitory effect on the proliferation of HepG2 cells with IC50 as the evaluation index, the IC50 values of 1.89, 1.71, 1.51 g x L(-1), respectively. The method is quick, simple and accurate with good re- producibility, and can be used for determination of vicenin II, vicenin III, isoschaftoside, schaftoside, vitexin, 6, 8-di-C-a-L-arabino- sylapigenin in the warer-soluble total flavonoids from L lophanthoides var. gerardianus. The warer-soluble total flavonoids from L lophanthoides have inhibitory effect on the proliferation of HepG2 cells.
Antineoplastic Agents, Phytogenic ; analysis ; pharmacology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; analysis ; pharmacology ; Flavones ; analysis ; pharmacology ; Humans ; Isodon ; chemistry ; Monosaccharides ; analysis ; pharmacology

OBJECTIVETo observe the regulatory effects of psoralen, oleanolic acid, and stilbene glucoside, three active components of psoralea fruit, glossy privet fruit and tuber fleeceflower root respectively, on Aβ25-35induced self-renewal and neuron-like differentiation of neural stem cells (NSCs).

METHODSEmbryonic NSCs werein vitro isolated and cultured from Kunming mice of 14-day pregnancy, and randomly divided into the control group, the Aβ25-35 group, the Aβ25-35 +psoralen group, the Aβ25-35 +oleanolic acid group, and the Aβ25-35 + stilbene glucoside group. The intervention concentration of Aβ25-35 was 25 µmol/L, and the intervention concentration of three active components of Chinese medicine was 10(-7)mol/L. The effect of three active components of Chinese medicine on the proliferation of NSCs was observed by counting method. The protein expression of Tubulin was observed by Western blot and immunofluorescence. The ratio of Tubulin+/DAPI was caculated. Results Compared with the control group, the sperical morphology of NSCs was destroyed in the Aβ25-35 group, the counting of NSCs, the expression of Tubulin protein, and the ratio of Tubulin /DAPI all decreased (P <0.01, P <0.05). Compared with the Aβ25-35 group, the counting of NSCs, the expression of Tubulin protein, and the ratio of Tubulin + /DAPI all increased in the three Chinese medicine treated groups (P <0. 01, P <0. 05).

CONCLUSIONS25 µmol/L Aβ25-35 could inhibit self-renewal and neuron-like differentiating of NSCs. But psoralen, oleanolic acid, and stilbene glucoside could promote self-renewal of NSCs and neuron-like differentiation.

Amyloid beta-Peptides ; physiology ; Animals ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Drugs, Chinese Herbal ; pharmacology ; Embryo, Mammalian ; Female ; Mice ; Neural Stem Cells ; Neurogenesis ; drug effects ; Neurons ; cytology ; Peptide Fragments ; physiology ; Pregnancy

BACKGROUNDPeriostin originally designated osteoblast-specific factor 2 (OSF-2) is frequently found to be highly expressed in various types of human cancer cell lines in vitro and human cancer tissues in vivo. We proposed that periostin was a key factor during the process of proliferation and invasion in cancer cells. We investigated the effect of periostin on the function of human osteosarcoma cell line (U2OS), such as proliferation, apoptosis, invasion and the associated signal pathway.

METHODSA human PGCsi/U6 promoter-driven DNA template was adopted to induce short hairpin RNA (shRNA)-triggered RNA interference (RNAi) to block periostin gene expression in the cell line U2OS. U2OS cells were divided into three groups: cells transfected with phosphate buffered saline as control group (the U2OS group), cells transfected with pGCsi as negative control group (the NC group) and cells transfected with periostin/pGCsi as experimental group (the pGCsi-periostin group). Then, transfection efficiency of cell was observed under fluorescent microscope. The expressions of periostin and the related genes in cells were detected by reverse transcription polymerase chain reaction and Western Blotting. Cell viability was determined using the methyl-thiazolyl tetrazolium bromide (MTT) quantitative colorimetric assay. The invasion and migration capability of cells were tested by transwell plates with or without extracellular matrix gel. Furthermore, the changes of cell cycle and apoptosis were analyzed by flow cytometry.

RESULTSThe transfection efficiency of periostin/pGCsi to U2OS cells was about 70% - 80%. When compared with the NC group, the levels of mRNA and protein of periostin in the pGCsi-periostin group decreased by 82% (F = 564.71, P < 0.001) and 58% (F = 341.51, P < 0.001), respectively. Meantime, the earlier apoptosis value increased by 417% (F = 28.69, P < 0.001). The percentage of S phase pGCsi-periostin cells decreased by 21% (F = 47.00, P < 0.001), however, that of G0 - G1 phase cells increased by 12% (F = 14.50, P < 0.001). The capability of migration and invasion reduced by 41% (F = 17.79, P < 0.001) and 72% (F = 197.08, P < 0.001), respectively. The cell proliferation in the pGCsi-periostin group decreased by 59% and 72% at 48 and 120 hours after transfection, respectively. The mRNA expressions of transforming growth factor-β and vascular endothelial growth factor decreased by 17% (F = 73.99, P < 0.001) and 47% (F = 30.25, P < 0.001), respectively. A tendency of lower focal adhesion kinase (FAK) was shown in pGCsi-periostin cells but without any statistically significant difference. Otherwise the expression of p-FAK in those cells had markedly decreased by 21% (F = 16.81, P < 0.001).

CONCLUSIONSRNAi against periostin can effectively down-regulate periostin gene expression. Periostin increases the hyperplasia and invasion of cancer cells. Periostin might be involved in and served as a tumor promoter gene in the pathogenesis of osteosarcoma.

Apoptosis ; Bone Neoplasms ; etiology ; pathology ; Cell Adhesion Molecules ; antagonists & inhibitors ; genetics ; physiology ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Focal Adhesion Protein-Tyrosine Kinases ; metabolism ; Humans ; Integrin alphaVbeta3 ; physiology ; Neoplasm Invasiveness ; Osteosarcoma ; etiology ; pathology ; Phosphorylation ; RNA Interference ; Transfection

OBJECTIVETo investigate the etiologic value of diarrheagenic E. coil harboring genomic O island 28(OI-28) containing five putative virulence genes (Z0608, Z0609, Z0615, Z0634 and Z0635), which were related to RTX (Repeat in toxin) toxin family isolated from children with diarrheal disease in Taiyuan.

METHODSIn the study, 257 fecal samples from children with diarrheal disease collected in Shanxi Children's Hospital. Diarrheagenic E. coli and enteropathogenic bacteria were isolated and identified by conventional bacterial culture and typing specific diarrheagenic E. coli (EPEC, EIEC, ETEC and EHEC) diagnostic serum, while diarrheagenic E. coli harboring genomic 01-28 containing five putative virulence genes (Z0608, Z0609, Z0615, Z0634 and Z0635) were detected by PCR and DNA southern blot hybridization.

RESULTS206 strains (80.16%) of enteropathogenic bacteria were detected from 257 children with diarrhea disease, containing 149 strains (57.98%) of diarrheagenic E. coli and 57 strains(22.18%) of other entero-pathogenic bacteria. Among 3 strains (2.01%) of EPEC, 2 strains (1.34%) of ETEC, 2 strains (1.34%) EHEC were detected by typing specific serum, while all of the 142 strains (95.30%) isolated were suspected to be diarrheagenic E. coli. 21 strains (14.09%) of diarrheagenic E. coil harboring genomic O1-28 containing five putative virulence genes (Z0608, Z0609, Z0615, Z0634 and Z0635) were detected by polymerase chain reaction and DNA southen blot hybridization, 8 strains (5.37%) of diarrheagenic E. coli containing only one genomic OI-28 virulence gene, 2 strains (1.34%) of diarrheagenic E. coli containing two genomic OI-28 virulence gene. 21 children with diarrhea diseases caused OI-28-harboring E. coli containing five important putative virulence genes were among 0 to 3 years old (80.95%). These children correlating with OI-28-harboring E. coli did not present special clinical symptoms or signs.

CONCLUSIONThe diarrheagenic E. coil harboring genomic OI-28 was one of the important etiology for children with diarrheal disease in summer season.

Child ; China ; Diarrhea ; microbiology ; Escherichia coli ; genetics ; pathogenicity ; Escherichia coli Infections ; complications ; Genes, Bacterial ; Humans ; Virulence