Twenty-nine antagonistic bacillus strains, isolated from some Chinese traditional medicine and fermented food , inhibit the growth of Fusarium oxysporum f. sp. Vasinfectum and Verticillium dahliae Kleb. And twelve of them are able to produce antagonistic proteins. Among the twelve strains, five (H110, H184, H216, B316 and B382) showed higher antibacterial activity. Furthermore, H110 and H184 were identified as Bacillus subtilis, and H216, B316 and B382 as Bacillus licheniformis based on physiological and biochemistry experiments. The antagonistic proteins of five strains were all thermostable, resistant to proteinase K and trypsin, while H184 and H216 partially sensitive to pepsin.

0.05).Conclusions The results by GH exercise test is identified with GH provocative test.Because the GH exercise test is safe and simple,it is appropriate to use the GH exercisetest to screen for growth hormone deficiency during childhood.

Objective To evaluate the image quality of 64-multi detector computed tomography (MDCT)and the clinical accuracy in detecting coronary artery lesions.Methods One hundred and five patients were studied by MDCT.The results were compared with invasive coronary angiography(ICA). Patients were excluded for atrial fibrillation,but not for high heart rate,coronary calcification,or obesity. MDCT was analyzed with regard to image quality and presence of coronary artery lesions.Results The data evaluation of the image quality was based on a total of 1365 segments(13 coronary segments for each patient),of which 1144 segments were considered to have diagnostic image quality,but 221 segments (16.2%)could not be sufficiently evaluated because of severe calcifications(153 segments)and motion artifacts(68 segments).The median calcium score[Agatston score equivalent(ASE)]was 154(range 0—1983).87 of the 105 patients had an ASE of less than 1,000[median 105(range 0—994)],and 18 patients had an ASE greater than 1000[median 1477(range 1115—1983)].For detecting lesions with 50% or greater narrowing(without any exclusion criteria),the overall sensitivity,specificity,positive predictive value,and negative predictive value were 85.7%,97.9%,93.0%,and 95.5%,respectively. When limiting the number of patients to those with a calcium score of less than 1000 ASE,the threshold- corrected sensitivity for lesions with 50% or greater narrowing was 96.0%;specificity,98.9%;positive predictive value,95.3%;and negative predictive value,99.0%.Conclusion Our results indicate high quantitative and qualitative diagnostic accuracy of 64-slice MSCT in comparison to QCA in a broad spectrum of patients.

To study the effect of pulchinenoside (PULC) on the Frizzled (FZD) expression of adjuvant arthritis ( AA) rats. AA rats were prepared through the toe injection with complete Freund's adjuvant to culture fibroblast-like synoviocytes (FLS). The effect of the oral administration with PULC on the FZD8 expression was detected by the real time qPCR. The effect of FZD8 knockout on the expressions of IL-1, IL-6, IL-8 were detected by MTT and ELISA. The role of miR-375 in the abnomal expression of FZD8 was detected by the real time qPCR. The results showed signfiicant decrease in the FZD8 expression among AA rats, FLS proliferation ater FZD8 knockout and IL-1, IL-6, IL-8 expressions and notable increase in miR-375 expression after the oral administration with PULC. The up-regulated miR-375 expression can inhibit the FZD8 expression. PULC may inhibit the FZD8 expression by up-regulating the miR-375 expression.
Animals ; Arthritis, Experimental ; drug therapy ; genetics ; metabolism ; Disease Models, Animal ; Drugs, Chinese Herbal ; administration & dosage ; Humans ; Male ; Rats ; Rats, Sprague-Dawley ; Receptors, Cell Surface ; genetics ; metabolism ; Saponins ; administration & dosage

OBJECTIVETo construct the acid-sensitive potassium hannel-3(TASK3) eukaryotic expression plasmid and to establish a stable SH-SY5Y cell line expressing enhanced green fluorescent protein (EGFP)-tagged TASK3.

METHODSTASK3 coding region was subcloned into pEGFP-N1 plasmid to construct a recombinant vector alled pEGFP-TASK3. The correct recombinant expressing plasmid was transfected with X-feet transfection reagent to SH-SY5Y cells. The cell line stably expressiing EGFP tagged-TASK3 gene was established by screening with antibiotic G418 and fluorescence microscope. The expression and localization of the EGFP tagged-TASK3 fusion protein was detected by Western blot and confocal microscope. Exposure of the SH-SY5Y cell line expressing stably TASK3-eGFP fusion proteins was exposed to different pH media (7.0, 6.7, 6.4, 6.1) for 24 h, the cell viability was assessed with cell counting Kit-8 (CCK-8).

RESULTSAll the results of identification by PCR, digestion with restriction endonuclease and sequencing indicated that the recombinant eukaryotic expression plasmid pEGFP-TASK3 was constructed correctly. The stable SH-SY5Y cell line expressing EGFP tagged-TASK3 fusion protein was successfully established. Exposure of the wild type SH-SY5Y cells and the stable SH-SY5Y-GFP tag-TASK3 cell line to different pH media (7.0, 6.7, 6.4, 6.1) for 24 h, the cell viability of two group cells significantly reduced with pH declining, and the difference was statistically significant (P < 0.05). Compared with wild type SH-SY5Y cells, the cell viability of stable SH-SYSY-GFP tag-TASK3 cell line increased significantly with the same pH media, and the difference was statistically significant (P < 0.05).

CONCLUSIONThe eukaryotic expression vector pEGFP-TASK3 is successfully constructed and the cell line stably expressing TASK3-eGFP fusion is established which is important for their fundamental research and potential applications.

Blotting, Western ; Cell Line ; Gene Expression ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; Humans ; Plasmids ; Polymerase Chain Reaction ; Potassium Channels, Tandem Pore Domain ; genetics ; Transfection

The role of pulchinenoside (PULC) in the regulation of MeCP2 expression was investigated in RA model rats. Adjuvant arthritis rats were used as RA model rats, and fibroblast-like synoviocytes (FLS) from the RA model rats were cultured. The effect of 100 mg x kg(-1) PULC gavage treatment on the MeCP2 expression and the effect of MeCP2 siRNA on the expression of SFRP2 and β-catenin were detected by real time qPCR and Western blotting. The role of PULC in the FLS proliferation was detected by MTT. The results showed that the MeCP2 expression was down-regulated, the SFRP2 expression was up-regulated and the FLS proliferation was inhibited in FLS after therapy. MeCP2 siRNA significantly inhibited the MeCP2 expression, up-regulated the SFRP2 expression and inhibited the β-catenin expression in FLS from RA model rats. PULC may increase the SFRP2 expression, inhibit the Wnt signaling and inhibit the FLS proliferation in FLS from the RA model rats by inhibiting the MeCP2 expression.
Animals ; Arthritis, Rheumatoid ; drug therapy ; genetics ; metabolism ; Disease Models, Animal ; Drugs, Chinese Herbal ; administration & dosage ; Fibroblasts ; drug effects ; metabolism ; Gene Expression Regulation ; drug effects ; Humans ; Male ; Methyl-CpG-Binding Protein 2 ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Synovial Membrane ; cytology ; drug effects ; metabolism ; Wnt Signaling Pathway ; drug effects ; beta Catenin ; genetics ; metabolism

OBJECTIVE: To develop a specific, sensitive, reproducible SPE-HPLC method for the determination of 37 drugs in whole blood. METHODS: With the doxapram as internal standard, Oasis column was used to extract drugs from whole blood. Two kinds of mobile phases were used in this study. Separations were achieved by a LiChrospher 100 RP-C18 (250 mm x 4.0 mm x 5 microm) column kept at 50 degrees C, the DAD detector was set at 230 nm and 250 nm. RESULTS: The limit of detection were 1-30 ng/mL. The method showed excellent linearity and the linear correlation coefficient was > or =0.997 98. The relative standard deviation for between-day and within-day assay were <10%. CONCLUSION The method is effective, simple, reliable and has been used in real cases.
Chromatography, High Pressure Liquid/methods* ; Doxapram/isolation & purification* ; Doxepin/isolation & purification* ; Estazolam/isolation & purification* ; Forensic Medicine ; Humans ; Morphine/isolation & purification* ; Papaverine/isolation & purification* ; Pharmaceutical Preparations/isolation & purification* ; Prazosin/isolation & purification* ; Procaine/isolation & purification* ; Reproducibility of Results ; Sensitivity and Specificity ; Solid Phase Extraction/methods* ; Solvents/chemistry*

AIMTo investigate the role of MAPK in the migration of human coronary artery smooth muscle cells(HCASMC ) into three-dimensional fibrin gels.

METHODSHCASMC were primarily cultured. HCASMC migration was measured with a phase-contrast microscope in the presence or absence of PD98059, SB203580, and SP600125, the inhibitors of ERK, p38, and JNK, respectively. Phosphorylation of ERK, p38 and JNK were analyzed by Western blotting in the presence or absence of PD98059, SB203580 or SP600125.

RESULTSHCASMC that migrated into the three-dimensional fibrin gel exhibited a characteristic elongated spindle-shaped appearance and formed vessel-like structure. The number of migrated HCASMC increased with incubation time and concentration of fibrinogen in the range between 0.8 g/L and 6.4 g/L. Western blot showed that fibrin induced phosphorylation of ERK, p38 and JNK time dependently and PD98059, SB203580 and SP600125 could inhibit their activation, respectively. Migration of HCASMC into the fibrin gels was inhibited by SP600125 20 micromol/L and SB203580 10 micromol/L, respectively. Furthermore, inhibition of SP600125 20 micromol/L had a more profound effect. PD98059 50 ,mol/L, however, failed to influence migration of HCASMC. Hence, migration of HCASMC into the fibrin gels is JNK- and p38-dependent, but not ERK-dependent.

CONCLUSIONFibrin gel induces HCASMC migration into itself by activation of JNK and p38, but not ERK, which may play an important role in pathogenesis of atherothrombosis and restenosis.

Anthracenes ; pharmacology ; Cell Movement ; Cells, Cultured ; Coronary Vessels ; cytology ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Fibrin ; metabolism ; Flavonoids ; pharmacology ; Humans ; Imidazoles ; pharmacology ; JNK Mitogen-Activated Protein Kinases ; metabolism ; MAP Kinase Signaling System ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; Pyridines ; pharmacology ; p38 Mitogen-Activated Protein Kinases ; metabolism

OBJECTIVETo investigate the potential regulatory role played by the hormone resistin in lipid metabolism and expression of nuclear factor (NF)-kB and tumor necrosis factor (TNF)-a during hepatic steatosis.

METHODSA non-alcoholic fatty liver disease (NAFLD) cell model was established by treating the normal human hepatic cell line, L02, with palmitic acid. Four research groups of L02 cells were generated: C group (control, no palmitic acid treatment), P group (NAFLD model, treated with 20 microg/ml palmitic acid), CR group (C group treated with 50 microg/L recombinant human resistin), and PR group (P group treated with 50 microg/L recombinant human resistin). All treatments were carried out for 72 hours. Oil red O staining was used to detect the intracellular changes in lipid drops. Biochemical assays were used to measure triglycerides (TGs), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma-glutamyl transpeptidase (GGT) levels in culture medium. The mRNA and protein expression levels of insulin receptor substrate (IRS)-2, NF-kB, and TNF-a were determined by reverse transcription-polymerase chain reaction and Western blot analysis, respectively.

RESULTSThe TG, ALT, AST, and GGT levels were higher in the P, CR, and PR groups than in the C group. The NF-kB mRNA level was also higher in the P, CR, and PR groups (Student's t = 17.64, 22.03, 26.06 respectively) than in the C group, as was the TNFa mRNA level ( t = 5.67, 5.38, 11.64), but the IRS-2 mRNA level was lower ( t = 8.19, 9.23, 20.93) (all, P less than 0.05). In addition, no significant difference in these mRNA levels were found between the P group and the CR group (NF-kB: t = 1.75, TNFa: t = 0.58, IRS-2: t = 2.14; all, P more than 0.05). The detected protein levels of NF-kB, TNFa, and IRS-2 were consistent with the mRNA levels.

CONCLUSIONResistin can promote steatosis in LO2 cells through the NF-kB signaling pathway, thereby contributing to the NAFLD pathogenic process.

Cell Line ; Fatty Liver ; metabolism ; Humans ; Liver ; metabolism ; NF-kappa B ; metabolism ; Non-alcoholic Fatty Liver Disease ; Resistin ; metabolism ; Signal Transduction ; Tumor Necrosis Factor-alpha ; metabolism

AIMTo construct short hairpin RNA (shRNA) eukaryotic expression vectors targeting TGF-beta1 for further research on the effects of TGF-beta1 on vasculogenesis and angiogenesis.

METHODSThree pairs of siRNA target sequences coding from the mRNA of TGF-beta1 gene were designed and three pairs of nucleotides were synthesized. After annealing, the double-strand DNA products were ligated into the pEN_mH1c entry vector, and in turn into the shRNA eukaryotic expression vector pDS_hpEy labled by GFP through the LR recombination reaction. After sequencing successfully, the three resulting TGF-beta1 shRNA expression vectors were transfected into the mouse fibroblast cell line (NIH/3T3), and then cell clones stably expressing TGF-beta1 shRNA were screened. Reverse Transcript-Polymerase Chain Reaction (RT-PCR) and Western blot were used to detect the mRNA and protein expression.

RESULTSRT-PCR and Western blot showed that one of the TGF-beta1 shRNA expression vectors pDS_Tc downregulated TGF-pl mRNA and protein expression markedly in NIH/3T3 cells.

CONCLUSIONShRNA eukaryotic expression vectors targeting TGF-beta1 are successfully constructed which can be used for further investigation on the mechanism through which TGF-beta1 regulates vasculogenesis and angiogenesis.

Animals ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; metabolism ; Mice ; NIH 3T3 Cells ; Neovascularization, Physiologic ; RNA Interference ; RNA, Messenger ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; Transfection ; Transforming Growth Factor beta1 ; genetics ; metabolism