Chunlong Liu

The Central Hinge Link Truncation of the Antimicrobial Peptide Fowlicidin-3 Enhances Its Cell Selectivity without Antibacterial Activity Loss

ABSTRACT Antimicrobial peptides (AMPs) have been paid considerable attention because of their broad-spectrum antimicrobial activity and a reduced possibility of the development of bacterial drug resistance. Fowlicidin-3 (Fow-3) is an identified type of chicken cathelicidin AMP that has exhibited considerable antimicrobial activity and cytotoxicity. To reduce cell toxicity and improve cell selectivity, several truncated peptides of fowlicidin-3, Fow-3(1-15), Fow-3(1-19), Fow-3(1-15-20-27), and Fow-3(20-27), were synthesized. Our results indicated that neither the N- nor C-terminal segment alone [Fow-3(1-15), Fow-3(1-19), Fow-3(20-27)] was sufficient to confer antibacterial activity. However, Fow-3(1-19) with the inclusion of the central hinge link (-AGIN-) retained substantial cell toxicity, which other analogs lost. Fow-3(1-15-20-27) displayed potent antimicrobial activity for a wide range of Gram-negative and Gram-positive bacteria and no obvious hemolytic activity or cytotoxicity. The central link region was shown to be critically important in the function of cell toxicity but was not relevant to antibacterial activity. Fow-3(1-15-20-27) maintained antibacterial activity in the presence of physiological concentrations of salts. The results from fluorescence spectroscopy, scanning electron microcopy, and transmission electron microcopy showed that Fow-3(1-15-20-27) as well as fowlicidin-3 killed bacterial cells by increasing membrane permeability and damaging the membrane envelope integrity. Fow-3(1-15-20-27) could be a promising antimicrobial agent for clinical application.

High-yield recombinant expression of the chicken antimicrobial peptide fowlicidin-2 in Escherichia coli

The antimicrobial peptide fowlicidin‐2 identified in chicken is a member of the cathelicidins family. The mature fowlicidin‐2 possesses high antibacterial efficacy and lipopolysaccharide (LPS) neutralizing activity, and also represents an excellent candidate as an antimicrobial agent. In the present study, the recombinant fowlicidin‐2 was successfully produced by Escherichia coli (E. coli) recombinant expression system. The gene encoding fowlicidin‐2 with the codon preference of E. coli was designed through codon optimization and synthesized in vitro. The gene was then ligated into the plasmid pET‐32a(+), which features fusion protein thioredoxin at the N‐terminal. The recombinant plasmid was transformed into E. coli BL21(DE3) and cultured in Luria‐Bertani (LB) medium. After isopropyl‐β‐D‐thiogalactopyranoside (IPTG) induction, the fowlicidin‐2 fusion protein was successfully expressed as inclusion bodies. The inclusion bodies were dissolved and successfully released the peptide in 70% formic acid solution containing cyanogen bromide (CNBr) in a single step. After purification by reverse‐phase high‐performance liquid chromatography (RP‐HPLC), ∼6.0 mg of fowlicidin‐2 with purity more than 97% was obtained from 1 litre of bacteria culture. The recombinant peptide exhibited high antibacterial activity against the Gram‐positive and Gram‐negative bacteria, and even drug‐resistant strains. This system could be used to rapidly and efficiently produce milligram quantities of a battery of recombinant antimicrobial peptides as well as for large‐scale production.

Effect of Levels of Yucca Schidigera Extract on Ruminal Fermentation Parameters, Digestibility of Nutrients and Growth Performance in Sheep

An in vivo fermentation experiment (Expt.1) and a digestibility and growth trial(Expt.2) were conducted to determine the effect of levels of Yucca Schidigera Extract (YSE) on ruminal fermentation parameters ,digestibility of nutrients and growth performance in Sheep. Three same levels of YSE supplementation were studied in both experiments. In the Expt.1,four rumen fistulated male sheep with initial body weight of 33±1.8 ㎏were randomly assigned according to a 4×4 Latin square design. The dietary treatments were YSE based offering at 0,100, 200 and 300 mg/kg diet. It was found that ruminal pH was not significantly different among treatments. Relative to control, ruminal propionate concentration was increased by YSE addition in a dose-dependent manner by up to 29.79%（P<0.05）,the acetic concentration was decreased by up to2 1.23%（P<0.05）.Ruminal ammonia concentration was larger (P<0.05) in sheep receiving no YSE (increased by 18.86 mg/dl) than in those receiving 200mg/kg (2.85 mg/dl increase in NH3) or 300mg/kg (2.72 mg/dl increase). Protozoa populations in the rumen were lower (P<0.05) with YSE of 200-300 mg/kg than without. In the digestibility and growth trial(Expt.2),increasing levels of YSE resulted in liner increase in daily body weight gain rate (P<0.05) and feed conversion efficiency(P<0.05). Additionally ,apparent digestibilities(%) of DM,OM,CP,and NDF were significantly different in all treatment .In conclusion, the 200 and 300 mg/kg YSE groups have a particular suppressing effect on ruminal ammonia concentration, ammonia N concentrations and protozoa populations were decreased, The effects on ruminal propionate and acetic concentration were probably a result of a selective inhibitory effect of YSE on rumen microbial species. The effect of YSE on ruminal ammonia concentration likely resulted from a decreased population of protozoa, presumably, from ammonia binding by YSE. YSE can significantly improved the apparent digestibility of nutrient and sheep growth performance.

A novel regeneration of iron citrate solution by biooxidation of iron-oxidizing bacteria

AbstractLiquid phase oxidation process using chelated iron solution is among the most promising techniques for the hydrogen sulfide removal due to its double advantage of waste minimization and resource recovery. Regeneration of chelated iron is a core reaction in this process. Regeneration of chelated iron in acidic solution is very difficult. In this paper, a novel regeneration of iron citrate in acidic solution by biooxidation of iron-oxidizing bacteria was reported firstly. By using such a process, the influence of iron-oxidizing bacteria on the regeneration rate was investigated. The results demonstrated the regeneration rate with the new technology was increased significantly. The process may contribute to the biooxidation of iron-oxidizing bacteria. Application of this novel process increased the regeneration rate under the optimum conditions, suggesting the iron citrate regeneration process may be a feasible and economical method in application.

Effects of Cold Stress on Porcine Fibroblasts HSP90 mRNA Expression

The stress response is a complex program of gene expression and biochemical adaptive. These cell stress response are of great interest both to basic biology and to medicine. Study of cold stress on cells and related gene expression contribute to understand the effects of cold stress on the biological mechanisms of the body. HSP90 is an important molecular chaperone ’ it has to maintain the protein conformation, promoting the wrong protein degradation. Stress can induce a significant increase in HSP90 expression ’ its expression are induced by cold exposure do not increase during the period of thermal stress itself but during the cell stress response that ensues after rewarming. We use real-time PCR detection expression of HSP90 mRNA of procine fibroblasts. We showed that preincubation of cells at 4 and 15° C induced the expression of HSP90 mRNA upon recovery at 38.5° C , and the degree of this induction was directly related to the time that the cells spend at 4 and 15° C (data not shown).We observation that 25 and 32° C (data not shown)were the temperature of the recovery incubation period, below which litter or no induction of stress response was observed, is consistent with a role of active cell metabolism in those induction.

Genetics Research and Advance on Development and Utilization of Wild Boars

Abstract Wild boar is one of the most important beast resources. It plays an important role in the maintenance of biological diversity. The genetic resources of wild boar can not only protect the genetic resources, but also improve the formation of new breeds in pigs. This paper summarized the advance on the main biological characteristics of wild boars, evolutionary origin between wild boars and domesticated pigs, and development and utilization of wild boars aimed to provide further insight into wild boars genetic research and its resource protection.

Effects of Daidzein or Genistein on Proliferation and Antioxidation of Mammary Epithelial Cell of Dairy Cow In Vitro

Effects of Daidzein or Genistein on the proliferation and antioxidation of mammary epithelial cells of dairy cow were investigated in vitro. 10 groups were assigned including blank control, estradiol (E2) group(10 ng/mL), different concentrations of Daidzein (1, 10, 100, 1 000 ng/mL) and Genistein (1, 10, 100, 1 000 ng/mL) groups. The MMT method was used to determine the proliferation effect of Genistein or Daidzein, and the results showed that Genistein at the concentration of 10 and 100 ng/mL, and Daidzein at the concentration of 100 and 1000 ng/mL significantly improved dairy cow mammary epithelial cell proliferation (P<0.05) , while significantly weaker than E2 group(P<0.05). In the antioxidation experiment, the T-SOD and GSH-PX activity, MDA and NO content of the mammary epithelial cells at the logarithmic growth phase treated with Daidzein or Genistein for 24 h were measured and the results showed that 100, 1000 ng/mL Daidzein, and 100, 1000 ng/ mL Genistein significantly increased the T-SOD activities and decreased MDA content (P<0.05).1 000 ng/ mL Daidzein and 100, 1 000 ng/mL Genistein significantly increased the GSH-PX activites (P<0.05). The results showed that proper levels of daidzein and genistein can improv the proliferation and antioxidation function of mammary epithelial cell of dairy cows.