AIM: To construct human eosinophil-derived neurotoxin (hEDN) and HBV core protein (HBVc) eukaryotic fusion expression vector with a linker (Gly4 Ser)3 between them to optimize the molecule folding, which will be used ...AIM: To construct human eosinophil-derived neurotoxin (hEDN) and HBV core protein (HBVc) eukaryotic fusion expression vector with a linker (Gly4 Ser)3 between them to optimize the molecule folding, which will be used to inhibit HBV replication in vitro.METHODS: Previously constructed pcDNA3. 1(-)/TR was used as a template. Linker sequence was synthesized and annealed to form dslinker, and cloned into pcDNA3.1(-)/TR to produce plasmid pcDNA3.1(-)/HBc-linker. Then the hEDN fragment was PCR amplified and inserted into pcDNA3.1(-)/HBc-linker to form pcDNA3.1(-)/TNL in which the effector molecule and the target molecule were separated by a linker sequence. pcDNA3.1(-)/TNL expression was identified by indirect immunofluorescence staining. Radioimmunoassay was used to analyse anti-HBV activity of pcDNA3.1(-)/TNL.Meanwhile, metabolism of cells was evaluated by NTT colorimetry.RESULTS: hEDN and HBVc eukaryotic fusion expression vector with a linker (Gly4Ser)3 between them was successfully constructed. pcDNA3.1(-)/TNL was expressed in HepG2.2.15 cells efficiently. A significant decrease of HBsAg concentration from pcDNA3.1(-)/TNL transfectant was observed compared to pcDNA3. 1(-)/TR (P=0.036, P＜0.05).MTT assay suggested that there were no significant differences between groups (P=0.08, P＞0.05).CONCLUSION: Linker introduction enhances the inhibitory effect of HBV targeted ribonuclease significantly.展开更多
AIM: To investigate the effect of glutamine (Gln)-containing parenteral nutrition on phagocytic activity and to elucidate the possible roles of Gln in the secretion of anabolic hormones and nitrogen balance in rats un...AIM: To investigate the effect of glutamine (Gln)-containing parenteral nutrition on phagocytic activity and to elucidate the possible roles of Gln in the secretion of anabolic hormones and nitrogen balance in rats undergoing a gastrectomy. METHODS: Rats with an internal jugular catheter were divided into 2 experimental groups and received total parenteral nutrition (TPN). The TPN solutions were isonitrogenous and identical in nutrient compositions except for differences in amino acid content. One group received conventional TPN (control), and in the other group, 25% of the total amino acid nitrogen was replaced with Gin. After receiving TPN for 3 d, one-third of the rats in each experimental group were sacrificed as the baseline group. The remaining rats underwent a partial gastrectomy and were killed 1 and 3 d, respectively, after surgery. Plasma, peritoneal lavage fluid (PLF), and urine samples were collected for further analysis. RESULTS: The Gln group had fewer nitrogen losses 1 and 2 d after surgery (d1, 16.6±242.5 vs-233.4±205.9 mg/d, d2, 31.8±238.8 vs-253.4±184.6 mg/d, P<0.05). There were no differences in plasma growth hormone (GH) and insulin-like growth factor-1 levels between the 2 groups before or after surgery. The phagocytic activity of peritoneal macrophages was higher in the Gln group than in the control group 1 d after surgery (A 1185±931 vs323±201, P<0.05). There were no differences in the phagocytic activities of blood polymorphonuclear neutrophils between the 2 groups at the baseline or on the postoperative days. No significant differences in interleukin-1β or interleukin-6 concentrations in PLF were observed between the 2 groups. However, tumor necrosis factor-α level in PLF was significantly lower in the Gln group than in the control group on postoperative d 3. CONCLUSION: TPN supplemented with Gln can improve the nitrogen balance, and enhance macrophage phagocytic activity at the site of injury. However, Gin supplementation has no effect on phagocytic cell activity in the systemic circulation, GH and insulin-like growth factor-1 might not be responsible for attenuating nitrogen losses in rats with a partial gastrectomy.展开更多
基金National Natural Scientific Foundation of China,No.30100157Innovation Project of FMMU,No.CX99005
文摘AIM: To construct human eosinophil-derived neurotoxin (hEDN) and HBV core protein (HBVc) eukaryotic fusion expression vector with a linker (Gly4 Ser)3 between them to optimize the molecule folding, which will be used to inhibit HBV replication in vitro.METHODS: Previously constructed pcDNA3. 1(-)/TR was used as a template. Linker sequence was synthesized and annealed to form dslinker, and cloned into pcDNA3.1(-)/TR to produce plasmid pcDNA3.1(-)/HBc-linker. Then the hEDN fragment was PCR amplified and inserted into pcDNA3.1(-)/HBc-linker to form pcDNA3.1(-)/TNL in which the effector molecule and the target molecule were separated by a linker sequence. pcDNA3.1(-)/TNL expression was identified by indirect immunofluorescence staining. Radioimmunoassay was used to analyse anti-HBV activity of pcDNA3.1(-)/TNL.Meanwhile, metabolism of cells was evaluated by NTT colorimetry.RESULTS: hEDN and HBVc eukaryotic fusion expression vector with a linker (Gly4Ser)3 between them was successfully constructed. pcDNA3.1(-)/TNL was expressed in HepG2.2.15 cells efficiently. A significant decrease of HBsAg concentration from pcDNA3.1(-)/TNL transfectant was observed compared to pcDNA3. 1(-)/TR (P=0.036, P＜0.05).MTT assay suggested that there were no significant differences between groups (P=0.08, P＞0.05).CONCLUSION: Linker introduction enhances the inhibitory effect of HBV targeted ribonuclease significantly.
基金Supported by Research Grant from National Science Council,Taipei, Taiwan, China No. 91-2815-C-038-007-B
文摘AIM: To investigate the effect of glutamine (Gln)-containing parenteral nutrition on phagocytic activity and to elucidate the possible roles of Gln in the secretion of anabolic hormones and nitrogen balance in rats undergoing a gastrectomy. METHODS: Rats with an internal jugular catheter were divided into 2 experimental groups and received total parenteral nutrition (TPN). The TPN solutions were isonitrogenous and identical in nutrient compositions except for differences in amino acid content. One group received conventional TPN (control), and in the other group, 25% of the total amino acid nitrogen was replaced with Gin. After receiving TPN for 3 d, one-third of the rats in each experimental group were sacrificed as the baseline group. The remaining rats underwent a partial gastrectomy and were killed 1 and 3 d, respectively, after surgery. Plasma, peritoneal lavage fluid (PLF), and urine samples were collected for further analysis. RESULTS: The Gln group had fewer nitrogen losses 1 and 2 d after surgery (d1, 16.6±242.5 vs-233.4±205.9 mg/d, d2, 31.8±238.8 vs-253.4±184.6 mg/d, P<0.05). There were no differences in plasma growth hormone (GH) and insulin-like growth factor-1 levels between the 2 groups before or after surgery. The phagocytic activity of peritoneal macrophages was higher in the Gln group than in the control group 1 d after surgery (A 1185±931 vs323±201, P<0.05). There were no differences in the phagocytic activities of blood polymorphonuclear neutrophils between the 2 groups at the baseline or on the postoperative days. No significant differences in interleukin-1β or interleukin-6 concentrations in PLF were observed between the 2 groups. However, tumor necrosis factor-α level in PLF was significantly lower in the Gln group than in the control group on postoperative d 3. CONCLUSION: TPN supplemented with Gln can improve the nitrogen balance, and enhance macrophage phagocytic activity at the site of injury. However, Gin supplementation has no effect on phagocytic cell activity in the systemic circulation, GH and insulin-like growth factor-1 might not be responsible for attenuating nitrogen losses in rats with a partial gastrectomy.
