The cloning, high level expression in Escherichia coli of human beta-defensin 3 and its antimicrobial activity analysis / 生物工程学报

In recent years, Bacterial resistance is more and more serious for the irrational use of antibiotics produces resistant strains and other reasons. Human are trying to solve the problem from different ways, including the study of antimicrobial peptides. Defensin is one of the most important of antimicrobial peptides. A novel antimicrobial peptide, human beta-defensin 3, was isolated and demonstrated a salt-insensitive broad spectrum of potent antimicrobial activity against many potentially pathogenic microbes. The total RNA was extracted from human tonsil and the hbetaD-3 specific cDNA sequence was amplified with RT-PCR. After sequenced, the target DNA fragment was cloned into pQE-80L vector together with the DNA fragment encoding carrier protein DHFR. The recombinant vectors were transformed into E. coli M15 and the expression was induced based on the optimal values of the IPTG concentration incubation temperature and induction time determined in the previous section. The expressed proteins were analyzed by SDS-PAGE and Western-blotting. The mass of the recombinant protein was about 40% of total bacteria protein. Isolate and purify the target protein. The recombinant hbetaD-3 fusion proteins possess the antimicrobial activity to staphylococcus aureus, multiresistant staphylococcus aureus (only vancomycin-sensitive) and Candida albicans in the assay of drug susceptibility. Advanced study can be continued based on our experiments.

The isolation and purification of 19S regulator compound and the change of its protein level in rat skeletal muscle after severe scalding / 中华烧伤杂志

<p><b>OBJECTIVE</b>To explore the role of 19S regulator compound protein in the degradation of skeletal muscle protein in scalded rats.</p><p><b>METHODS</b>Wistar rats were scalded and they were randomly divided into normal and 1, 2, 3, 5, and 7 postburn day (PBD) groups with 8 rats in each group. The 19S regulator compound of skeletal muscle in scalded rats was isolated and purified with chromatography. Rabbit-anti-rat antibody IgG of 19S regulator compound was prepared conventionally. The antibody was injected to rats in injection group (I) in which 19S antibody in dose of 3 mg/kg BW was injected for two times via tail vein with 6-hour interval. The rats in I group were decapitated on 1, 2 and 3 post-injection days, respectively. The scalded rats in control group (C) were treated in the same way, except that the antibody was replaced by normal saline. The change in content of 19S regulator compound was determined by western-blot. Meanwhile, the releasing rate of tyrosine from the skeletal muscle of scalded rats was also detected by fluorescent photography.</p><p><b>RESULTS</b>19S regulator compound with high purity was obtained. The content of 19S regulator compound in rat skeletal muscle was increased significantly after 2 PBD. But the protein degradation rate was also obviously increased on 2 PBD. The antibody of 19S compound might inhibit the enhancement of protein degradation.</p><p><b>CONCLUSION</b>Burn injury might up-regulate the protein level of skeletal muscle 19S regulator compound, which therefore activated the protein degradation by 26S protease compound. This might be an important factor leading to postburn negative nitrogen balance.</p>

Mechanism of TNFα and IL-1 enhancing protein catabolism in skeletal muscles of mice in early stage after severe burn / 第三军医大学学报

Objective　To observe the effects and mechanism of TNFα and IL-1 on protein catabolism of skeletal muscles of mice in the early stage of severe burn. Methods　A BALB/c mice model was established with full thickness scalded of 18%～20% TBSA including the back and one hind limb. The catabolic rate of protein of soleus muscle was reflected with the net tyrosine releasing rate. The changes of the tyrosine releasing rates of the injured and uninjured limbs, the levels of TNFα and IL-1 of the plasma and soleus muscle, and the activities of lysosomal proteolytic enzymes were observed in 72 h after scald injury of the mice. Results　After injury, the levels of TNFα and IL-1 of plasma, injured and uninjured limbs were all increased, and reached the summit at the 48th h, but those of the injured limb were much higher than those of the uninjured one, then gradually the levels were decreased in 72 h. The protein catabolic rate of uninjured limb was higher than the normal at h 48, but it came to normal level at h 72. The rate of the injured limb was higher than that of normal and uninjured limbs significantly 72 h after the scalding. hrTNFα and hrIL-1 enhanced the protein catabolic rate and the activities of lysosomal protrolytic enzymes in the soleus muscle of normal mice in vivo and in vitro respectively. Conclusion　In the early stage of scald injury, TNFα and IL-1 could enhance the lysosomal protease activity, increase the protein catabolic rate in skeletal muscle and promote the negative nitrogen balance directly. These effects may not depend on the actions of emergent hormones.

Mechanism of TNFα and IL-1 enhancing protein catabolism in skeletal muscles of mice in early stage after severe burn / 第三军医大学学报

Objective　To observe the effects and mechanism of TNFα and IL-1 on protein catabolism of skeletal muscles of mice in the early stage of severe burn. Methods　A BALB/c mice model was established with full thickness scalded of 18%～20% TBSA including the back and one hind limb. The catabolic rate of protein of soleus muscle was reflected with the net tyrosine releasing rate. The changes of the tyrosine releasing rates of the injured and uninjured limbs, the levels of TNFα and IL-1 of the plasma and soleus muscle, and the activities of lysosomal proteolytic enzymes were observed in 72 h after scald injury of the mice. Results　After injury, the levels of TNFα and IL-1 of plasma, injured and uninjured limbs were all increased, and reached the summit at the 48th h, but those of the injured limb were much higher than those of the uninjured one, then gradually the levels were decreased in 72 h. The protein catabolic rate of uninjured limb was higher than the normal at h 48, but it came to normal level at h 72. The rate of the injured limb was higher than that of normal and uninjured limbs significantly 72 h after the scalding. hrTNFα and hrIL-1 enhanced the protein catabolic rate and the activities of lysosomal protrolytic enzymes in the soleus muscle of normal mice in vivo and in vitro respectively. Conclusion　In the early stage of scald injury, TNFα and IL-1 could enhance the lysosomal protease activity, increase the protein catabolic rate in skeletal muscle and promote the negative nitrogen balance directly. These effects may not depend on the actions of emergent hormones.