Objective To construct the inducible vector carrying green fluorescence protein(GFP). Methods We constructed an inducible vector pMD-GFP, including GFP cDNA, which allowed controlled expression of protein upon addition of 100 ?mol/L Zinc as an external inducer. The whole length of GFP cDNA were transfected into human hepatocellular cancer cells HCC-9204 by the method of lipofectin transfection. The expression of GFP was observed under fluorescence microscopy. Results The inducible vector carrying green fluorescence protein was successfully constructed. The observation under fluorescence microscopy showed that green fluorescence was spread in entire HCC-9204 cells transfected with GFP gene. Conclusion This new kind of the inducible vector could serve as a new tool and method for observing the growth and metastasis of neoplasm.

ObjectiveTo investigate the effect of p27 KIP1 transfection on cell cycle and apoptosis of hepatocellular carcinoma cells(HCC). MethodsWe used an inducible expression system pMD neo, which allowed controlled expression of protein upon addition of zinc as an external inducer. p27 KIP1 cDNA was transfected into human HCC 9204 cell line. Expression of p27 KIP1 was analyzed and cell growth was observed. ResultsExpression of p27 KIP1 in protein and mRNA increased significantly in HCC 9204 cell line transfected with p27 KIP1 . The cell growth reduced by 35%, p27 KIP1 over expression caused cell growth arrest at G 1 by 35% ( P =0 000). Apoptotic cell index significantly increased ( P =0 000).Conclusionp27 KIP1 may cause cell cycle arrest in G 1 phase and subsequently lead to apoptosis.

To elucidate the effect of p27 KIP1 on cell cycle and proliferation of gastric carcinoma cells, we transfected the full e length cDNA of p27 KIP1 into human SGC7910 gastric cancer cells by the method of lipofectin transfection. Expression of p27 KIP1 at protein or mRNA level was analyzed by Western blotting, and RNA dot blotting, respectively. Effect of p27 KIP1 on cell growth was observed by trpan blue exclusion assay and anchorage independent growth in soft agar. Flow cytometry was applied to assess the effect of p27 KIP1 on cell cycle. The results showed that the expression of p27 KIP1 at protein or mRNA level increased evidently in SGC7901 cells transfected with p27 KIP1 . The cell growth was reduced by 42% 48h post induction with zinc as determined by cell viability assay. The rate of anchorage independent growth in soft agar decreased significantly. p27 KIP1 over expression caused cell arrest at G 1 by 36%(from 33 68% to 69 29%, P

Objective To determine the biological effect of p27 KIP1 on gastric carcinoma cells SGC7901. Methods The total length of p27 KIP1 cDNA was transfected into human gastric cancer cells SGC7901 by lipofectin transfection. Expression of p27 KIP1 in protein or mRNA level was examined by Western blotting and RNA dot blotting respectively. Effect of p27 KIP1 on cell growth was observed by trpan blue exclusion assay. Tumorigenicity test in nude mice was applied to assess the biological effect of p27 KIP1 in vitro. Results Expression of p27 KIP1 in protein or mRNA increased evidently in SGC7901 cells transfected with p27 KIP1 . The cell growth was reduced by 42% about 48h after the induction with Zn 2+ ,which was determined by cell viability assay. The tumorigenicity of nude mice was reduced evidently(P

Research on novel pullulanase has major significance on the domestic industrialization of pullulanase and the breakdown of foreign monopoly. A thermophilic bacteria LM 18-11 producing thermostable pullulanase was isolated from Lunma hot springs of Yunnan province. It was identified as Anoxybacillus sp. by 16S rDNA phylogenetic analysis. Full-length pullulanase gene was cloned from Anoxybacillus sp. LM18-11. The optimum temperature of the pullulanase was between 55 and 60 degrees C with a half-life as long as 48 h at 60 degrees C; and its optimum pH was between 5.6 and 6.4. V(max) and K(m) of the pullulanase was measured as 750 U/mg and 1.47 mg/mL, which is the highest specific activity reported so far. The pullulanase crystals structure showed a typical alpha-amylase family structure. The N-terminal has a special substrate binding domain. Activity and substrate binding were decreased when the domain was deleted, the V(max) and K(m) were 324 U/mg and 1.95 mg/mL, respectively. The pullulanase was highly heterologous expressed in Bacillus subtilis by P43 promoter. The extracellular enzyme activity was 42 U/mL, which increased more than 40 times compared to the initial strain. This pullulanase has good application prospects.
Anoxybacillus ; classification ; enzymology ; China ; Glycoside Hydrolases ; metabolism ; Hydrogen-Ion Concentration ; Phylogeny ; RNA, Ribosomal, 16S ; genetics ; Temperature

BACKGROUND:As the potent, specific immunosuppressants emerge, the survival rate after intestinal transplantation is improved to some extent. However, the adverse effects of immunosuppressants and expensive treatment costs are not tolerable for many patients. Therefore, it is clinical y meaningful to choose traditional Chinese medicine which presents immunosuppressive effects. Artesunate has immune suppression effect, reduces acute rejection fol owing smal intestine transplantation, and improves the success rate of smal intestine transplantation.
OBJECTIVE:To observe the effect and action mechanism of artesunate in acute rejection after smal intestine transplantation in rats.
METHODS:Al ogeneic smal intestine transplantation models were established in the closed group of
Sprague-Dawley rats and Wistar rats, and then were randomly divided into three groups, syngenic transplantation group (SD→SD), al ogeneic transplantation group (Wistar→SD), and artesunate treatment group (Wistar→SD+artesunate 60 mg/kg per day, intraperitoneal injection).
RESULTS AND CONCLUSION:Rats in syngenic transplantation group survived for more than 10 days and they were al kil ed on day 10. The average survival of rats in al ogeneic transplantation group and artesunate treatment group was respectively (6.73±0.58) days and (8.50±0.74) days, with significant differences between the two groups (P<0.01). Histopathological examination showed that, there was no apparent rejection in syngenic transplantation group specimens, but mild, moderate and severe rejections in al ogeneic transplantation group on days 3, 5, 7. In treatment group, some specimens had mild rejection, but appeared relatively late to a low degree. Enzyme linked immunosorbent assay results revealed that, serum interleukin-2 and interferon-gamma expression levels in al ogeneic transplantation group were significantly higher than other two groups after surgery (P<0.01), serum interleukin-2 gene expression level in treatment group was also higher than syngenic transplantation group, but there was no significant difference (P>0.05), serum interferon-gamma expression level in treatment group was higher than syngenic transplantation group (P<0.05). Artesunate can inhibit acute rejection after rat smal intestine transplantation, and its mechanism may be related to inhibition effect on the secretion and expression of interleukin-2, interferon-gamma and other cytokines.

Objective:To investigate the effects of CCR3 on Muc5ac in the airway of asthmatic mice.Methods: Fifty clean BALB/c mice were randomly divided into control group,asthmatic group,dexamethasone treatment group,SB328437 treatment group, vehicle-control group.Total cells and differential inflammatory cells were counted in BALF, the levels of IL-4 and TNF-αwere determined by ELISA,lung tissue HE staining the expressions of Mucin5ac(Muc5ac) and CCR3 in lung tissue were detected by immu-neohistochemical staining and RT-PCR.Results:The total cells,eosinophil,monocytes and lymphocyte cells in BALF,the levels of IL-4,TNF-αin BALF ,the goblet cell of airway wall,the expression of Muc5ac and CCR3 positive staining IOD in the airway and the expression of Muc5ac and CCR3 mRNA lung tissue obviously decreased in SB328437 treatment group and DEX treatment group which compared with asthmatic mice model group ( P<0.05 ) .Conclusion: SB328437 can inhibit the expression of CCR3 in pulmonary tissue,furtherly inhibit the expression and secretion of Muc5ac and control the airway inflammation.

Objective To investigate the apoptotic effect of microRNA-1 (miR-1) on hypoxemic cardiomyocytes. Methods The cultured H9C2 cells were divided into 5 groups:normal control group, negative control group, H2O2 group, miR-1 group and H2O2+miR-1 group. After verified the success of transfection by real time PCR, MTT and flow cytometry methods were used to test the cell vitality and apoptotic rate, while the mRNA and protein expression level of Bcl-2 were de-tected by real time PCR and Western blot methods. Results Compared with normal control group, there were no significant differences in all indexes in negative control group. The application of H2O2 and miR-1 respectively or together significantly increased the miR-1 level and apoptotic rate, and reduced the cell vitality and Bcl-2 expression level. Conclusion mi-croRNA-1 can induce cardiomyocyte apoptosis by downregulating anti-apoptosis factor Bcl-2.

0.05). ConclusionsAdriamycin can induce apoptosis of cancer cells, and this is an important mechanism for its anticancer effect. This effect may be related to the down regulation of Bel-2 (expression).

ObjectiveTo investigate the effect of alanyl-glutamine supplemented total parenteral nutrition (TPN) on immune function of patients with gastric cancer.MethodsFifty gastric cancer patients with radical gastrectomy were randomly divided into the control group (n=25) and experiment group (n=25). Patients in the control group received conventional TPN support, and patients in the experiment group received TPN support and alanyl-glutamine (0.34 g/kg). All patients were observed for 7 days. The levels of IgG, IgA, IgM, CD3, CD4, CD8 and CD4/CD8 were measured before operation and on first and eighth day after operation.ResultsAll the levels of immune function were decreased on first day after operation in two groups. But the levels of IgG, IgA, IgM, CD3, CD4 and CD4/CD8 were significantly different between two groups on eighth day after operation (P<0.05). No serious infectious complication occurred in both groups.ConclusionTPN supplemented with alanyl-glutamine can improve the immune function of patients with gastric cancer radical gastrectomy.