Fujiang Chu

Expression of the antimicrobial peptide cecropin fused with human lysozyme in Escherichia coli.

Lysozyme is an abundant, cationic antimicrobial protein that plays an important role in host defense. It targets the β (1–4) glycosidic bond between N-acetylglucosamine and N-acetylmuramic residues that make up peptidoglycan, making lysozyme highly active against Gram-positive bacteria. However, lysozyme alone is inactive against Gram-negative bacteria because it cannot reach the peptidoglycan layer. Cecropins are cationic molecules with a wide range of antimicrobial activities. The main target for these peptides is the cytoplasmic membrane. We resume that cecopin may disrupt the outer membrane, giving the enzyme access to the peptidoglycan in cell wall. So in the present study, novel hybrid protein combining Musca domestica cecropin (Mdc) with human lysozyme (Hly) was designed. The DNA sequence encoding recombination fusion protein Mdc–hly was cloned into the pET-32a vector for protein expression in Escherichia coli strain BL21 (DE3). The protein was expressed as a His-tagged fusion protein, and the Mdc–hly was released from the fusion by enterokinase cleavage and separated from the carrier thioredoxin. Antimicrobial activity assays showed that the recombinant fusion protein Mdc–hly has improved in vitro antimicrobial activity and action spectrum compared to Mdc and hly. Mdc–hly may have important potential application as a future safely administered human drug and food additive.

Cecropin Suppresses Human Hepatocellular Carcinoma BEL- 7402 Cell Growth and Survival in vivo without Side-Toxicity

Conventional chemotherapy against hepatocellular carcinoma typically causes various side effects. Our previous study showed that cecropin of Musca domestica can induce apoptosis in human hepatocellular carcinoma BEL-7402 cells in vitro. However, whether cecropin inhibits BEL-7402 cell in vivo and the question of possible side effects remained undentified. The present study confirmed tumor-inhibitory effects of cecropin in vivo, and furthermore strongly suggested that cecropin cytotoxicity in BEL-7402 cells in vivo may be mainly derived from its pro-apoptotic action. Specifically, we found that cecropin exerted no obvious side effects in tumor-bearing mice as it had no significant hematoxicity as well as visceral toxicity. Therefore, cecropin may be a potential candidate for further investigation as an antitumor agent against hepatocellular carcinoma.

Expression pattern of antibacterial genes in the Musca domestica.

This work studied the transcriptional patterns of three antibacterial genes, attacin, defensin and cecropin, during the development of Musca domestica. Quantitative analysis by real-time PCR was performed on mRNA levels in different development stages and challenged 3rd-instar larva at different time points after challenge of Musca domestica. The results revealed a predominance of the transcripts of all three genes during the 3rd-instar larvae and the adults. In the meanwhile, it revealed the greatest increase in mRNA. The transcript levels increased to 801 times, 1009 times and 2500 times respectively for cecropin, attacin and defensin in 3rd-instar larvae after challenging susceptible bacterium. The results suggested that the transcriptional patterns of Musca domestica antibacterial genes were different during the different growth stages as well as the microbial challenge encountered in 3rd-instar larvae.

Scientific evaluation of the subchronic toxicity of Musca domestica larvae extracts in Sprague Dawley rats.

Musca domestica larvae extracts (MDLE) is a potential drug used to treat lipopolysaccharide-induced atherosclerosis pro-inflammatory responses. The purpose of the study was to evaluate the safety of MDLE via a 13-week repeated dose subchronic toxicity test in rats. Both male and female Sprague Dawley rats were divided into four groups, eight animals each from the control and high-dose group (33.0 g/kg) were allocated into recovery groups. The four groups of rats were administrated with MDLE (0, 13.2, 22.0, 33.0 g/kg) in the diet for 13weeks respectively. During the experimental period, the rats were observed for symptoms and signs of gross toxicity daily, food consumption and body weight were measured weekly. Urinalysis, thrombotest, blood biochemical and hematological analyses were performed regularly; Expression of peroxide dismutase gene in liver was quantified and a histopathological examination was also performed. There were no MDLE-induced abnormalities in any of the groups during or after the 13 weeks except the relative weight of liver of high-dose group and middle-dose group was significantly higher than that of control group in male rats (P<0.05). The results indicate a no observed adverse effect level for MDLE is 13.2 and 33.0 g/kg bw/day in male and female rats, respectively.

Effect of Musca domestic maggot polypeptide extract on HUVEC dysfunction induced by early-activated macrophages

Abstract Context: Musca domestica Linn. maggot is a traditional Chinese medicine. In our previous studies, Musca domestica maggot protein-enriched fraction and polypeptide extract (molecular weight <30 kD) were found to reverse endothelial cell dysfunction in atherosclerotic lesions. Objective: This study determines whether and how M. domestica maggot polypeptide extract affects the dysfunction of human umbilical vein endothelial cells (HUVEC) induced by macrophages (Mϕ). Materials and methods: HUVEC and early-activated THP-1 Mϕ (incubated with LPS of 1 μg/ml for 2 h) were co-cultured in a Transwell system. The effects of Musca domestica maggot polypeptide extract (with increasing concentrations, i.e., 1.0, 2.5, 5.0, 10.0, 20.0, and 40.0 µg/ml) on the proliferation and migration HUVEC and their secretion of vascular endothelial growth factor (VEGF) were determined by flow cytometry, modified Boyden chamber assay, and enzyme-linked immunosorbent assay (ELISA) after incubation for 24 h. Results: Musca domestica maggot polypeptide extract decreased the proliferation of HUVEC in a concentration-dependent manner, with a 50% effective concentration (EC50) of 22.16 ± 1.48 µg/ml, and effectively inhibited HUVEC migration (EC50 = 35.15 ± 2.03 µg/ml) and VEGF secretion (EC50 = 28.64 ± 1.29 µg/ml). Discussion and conclusion: Musca domestica maggot polypeptide extract can inhibit the dysfunction of HUVEC induced by early-activated THP-1 Mϕ.