THE NMR CONFORMATIONAL STUDIES OF BACITRACIN A

Bacitracin consists of a mixture of several closely related polypeptide antibiotics produced from Bacillus licheniformis.The major component, Bacitracin A, is a cyclic dodecapeptide（Fig, 1）, which has strong bacteriocidal effect on the G+ bacteria. The bacteriocidal activity requires the presence of divalent cations such as Zn;, As we know, the biological function of ploypeptides and proteins are closely related to their three-dimensional structure. Bacitracin A is very important for the demonstration of the coordination site of metal and the relationship between

Analysis of bacitracin and its related substances by liquid chromatography tandem mass spectrometry

A suitable liquid chromatography quadrupole time-of-flight mass spectrometric(LC–Q-TOF–MS) method was developed for separation and characterization of related substances in bacitracin test drug. The separation was performed on Li Chrospher RP-18 column using methanol as mobile phase A and 0.2% ammonium acetate buffer solution as mobile phase B in gradient elution. A total of 12 related substances were detected through high resolution mass spectrometric determination in a positive electrospray ionization mode. They were identified as co-existing active components and degradation products of bacitracin through the analysis and elucidation of both the protonated parents and the product ions of all the related substances and their fragmentation pathways were also proposed.

Effect of Enzymes in Buccal Mucous Membrane on Buccal Absorption of Insulin

To evaluate the effect of proteolytic enzymes on the absorption of insulin in the buccal mucosa, the trichloroacetic acid (TCA) method was used to estimate the degradation of insulin under different conditions in the buccal mucosal homogenates. In vivo experiments estimating the enhancement of hypoglycaemic effect by enzyme inhibitors were also conducted. The results showed that proteolytic enzymes in the buccal mucosa were less active than in the intestine. Bacitracin, aprotinin and sodium deoxycholate could inhibit the degradation of insulin in the buccal mucosal homogenates. The degradation of insulin in buccal mucosal homogenates of normal hamsters was smaller than that of diabetic hamsters. In vivo experiments of hypoglycaemia supported the in vitro results. When given buccally, bacitracin, aprotinin and sodium deoxycholate could increase the relative pharmacological bioavailability of insulin. When co-administered with aprotinin(0.1%), bacitracin(0.5%) and sodium deoxycholate(5%), the relative pharmacological bioavailabilities of insulin were 4.84%, 6.60% and 14.95% respectively. The in vitro and in vivo results suggest that proteolytic enzymes are present in the buccal mucosa, which limit absorption of insulin. Co-administration with some enzyme inhibitors can improve the bioavailability of insulin via buccal delivery and sodium deoxycholte is more efficient than some enzyme inhibitors used for improving buccal absorption.

Livestock Antibiotic Effects on Nitrification, Denitrification, and Microbial Community Composition in Soils

The broiler (Gallus gallus domesticus) industry in the United States and several other countries routinely includes subtherapeutic levels of antibiotics such as roxarsone, virginiamycin, and bacitracin in the feed to improve bird growth yields. Large fractions of the antibiotics fed to the birds are excreted in manure (litter), which is often applied to soils to improve fertility. Some concerns with this practice are antibiotic-induced alterations in microbially-mediated nutrient cycling, which could influence plant productivity and environmental quality. To investigate this possibility, a series of lab experiments were conducted to determine the effects of increasing levels of the three livestock antibiotics on nitrification, denitrification, and microbial community composition (fatty acid methyl ester profiles) of soils collected along a catena. Roxarsone and virginiamycin significantly influenced microbial community composition and inhibited nitrification in the soils, but only at levels that were several-fold higher than expected in poultry litter-applied soils. Bacitracin did not affect microbial growth, microbial community composition, or nitrification at any concentration tested (up to 500 mg·kg-1). None of the antibiotics influenced denitrification at environmentally-relevant concentrations. Amounts of antibiotics in soil solution were greatly reduced by sorption, which followed Freundlich models in the concentration range of 1 -?500 mg·L-1. Results from this study indicated that addition of roxarsone, virginiamycin, or bacitracin to these soils at environmentally-relevant concentrations would not likely impact microbial community composition, nitrification or denitrification due to intrinsic resistance/insensitivity of microorganisms to these antibiotics and reductions in the bioavailable amounts due to sorption by soil surfaces.

Effect of Zeolite on Small Intestine Microbiota of Broiler Chickens:A Case Study

After ban of antibiotics growth promoters (AGP) in Europe in 2006, use of non-medicated gut microbiota regulators as feed additives has dramatically increased. This study aimed at describing the effects of a copper-exchanged zeolite on broiler growth performance, small intestine morphology and microbiota composition. Illumina Sequencing of the V3-V4 region of the 16S rRNA gene was employed to study the small intestine microbiota. This microbiota with copper-exchanged zeolite treated-chickens was significantly less diverse with an almost exclusive presence of Lactobacillus johnsonii and Lactobacillus reuteri. These Lactobacilli are correlated with increased diameter, length and weight of the three segments of the small intestine and decreased viscosity of the intestinal content, suggesting probiotic action. The tested copper-exchanged zeolite would act as a prebiotic, selecting a “favorable” flora for the healthy broilers development.

Microbial synthesis of bacitracin:Recent progress,challenges,and prospects

Microorganisms are important sources of various natural products that have been commercialized for human medicine and animal healthcare.Bacitracin is an important antibacterial natural product predominantly pro-duced by Bacillus licheniformis and Bacillus subtilis,and it is characterized by a broad antimicrobial spectrum,strong activity and low resistance,thus bacitracin is extensively applied in animal feed and veterinary medicine industries.In recent years,various strategies have been proposed to improve bacitracin production.Herein,we systematically describe the regulation of bacitracin biosynthesis in genus Bacillus and its associated mechanism,to provide a theoretical basis for bacitracin overproduction.The metabolic engineering strategies applied for bacitracin production are explored,including improving substrate utilization,using an enlarged precursor amino acid pool,increasing ATP supply and NADPH generation,and engineering transcription regulators.We also present several approaches of fermentation process optimization to facilitate the industrial large-scale production of bacitracin.Finally,the challenges and prospects associated with microbial bacitracin synthesis are discussed to facilitate the establishment of high-yield and low-cost biological factories.

Enhancement of precursor amino acid supplies for improving bacitracin production by activation of branched chain amino acid transporter BrnQ and deletion of its regulator gene lrp in Bacillus licheniformis

Bacitracin,a new type of cyclic peptide antibiotic,is widely used as the feed additive in feed industry.Branched chain amino acids(BCAAs)are the key precursors for bacitracin synthesis.In this research,soybean meal was served as the raw material to supply precursor amino acids for bacitracin synthesis,and enhanced production of bacitracin was attempted by engineering BCAA transporter BrnQ and its regulator Lrp in the bacitracin industrial production strain Bacillus licheniformis DW2.Firstly,our results confirmed that Lrp negatively affected bacitracin synthesis in DW2,and deletion of lrp improved intracellular BCAA accumulations,as well as the expression level of BCAA transporter BrnQ,which further led to a 14.71%increase of bacitracin yield,compared with that of DW2.On the contrary,overexpression of Lrp decreased bacitracin yield by 12.28%.Secondly,it was suggested that BrnQ acted as a BCAA importer in DW2,and overexpression of BrnQ enhanced the intracellular BCAA accumulations and 10.43%of bacitracin yield.While,the bacitracin yield decreased by 18.27%in the brnQ deletion strain DW2△brnQ.Finally,BrnQ was further overexpressed in lrp deletion strain DW2△lrp,and bacitracin yield produced by the final strain DW2△lrp::BrnQ was 965.34 U/mL,increased by 22.42%compared with that of DW2(788.48 U/mL).Collectively,this research confirmed that Lrp affected bacitracin synthesis via regulating the expression of BCAA transporter BrnQ and BCAA distributions,and provided a promising strain for industrial production of bacitracin.

One-Step Procedure for Direct Purification of Pediocin-Like Bacteriocins and Cationic Antimicrobial Peptides from Complex Culture Medium on an Analytical, Semipreparative, and Preparative Scale. A New Type of Liquid Chromatography—Colloidal High-Performance Liquid Chromatography (CHPLC)

A simple time- and labor-saving one-step procedure suitable for analytical-, semipreparative-, and preparative-scale preparation of bacteriocins and other cationic antibacterial peptides (for example as lysozyme) has been developed. Suspensions of bacterial cells, other colloidal- and fast-particle materials, and dissolved anionic molecules passed through the column;however, cationic bacteriocins were separated by chromatography. The purity of the target substance at more than 97% was achieved by the judgment of spectral, chromatographic, and antimicrobial activity. A many-fold concentration increase was achieved for bacteriocins, lysozyme, and bacitracin (a commercial antibiotic) in comparison with the starting solutions (see the text below). In the experiments, homemade cation-exchange columns have been used. The columns have been prepared by the grafting of glass fibers with a diameter of 0.2 μm. In this paper, applications of a new type of liquid chromatography—colloidal high-performance liquid chromatography (CHPLC)—were demonstrated.

EFFECT OF ENDOGENOUS LEU-ENKEPHALIN ON ACUPUNCTURE IMMUNE RESPONSE

Acupuncture can modulate immune responses and thus produce good therapeutical results when used as an subsidiary measure to treat some infectious diseases, tumors and unbalanced immune functions. ZHAO et al. held that endogenous opiates participate in the modulation of immune responses by acupuncture. It has been reported abroad

通过抑制蛋白质二硫键异构酶P4HB阻断NK细胞活化的研究

目的探究蛋白质二硫键异构酶脯氨酰-4-羟化酶β亚基(P4HB)在自然杀伤细胞(NK)活化中的功能。方法NK细胞在无血清和细胞因子的培养基中培养12 h后,白细胞介素(IL)-2、IL-12和IL-18联合刺激4 h,利用蛋白印迹检测P4HB的表达水平;将不同终浓度的P4HB特异性抑制剂bacitracin作用于原代NK细胞以及NK细胞系(N16),ELISA检测NK细胞的干扰素-γ(IFN-γ)分泌水平,流式细胞术(FACS)检测P4HB抑制剂对NK细胞杀伤K562细胞的影响;不同终浓度P4HB抑制剂预处理NK细胞后,与CD20抗体孵育,利用双荧光素酶报告系统检测其对Daudi-Luc细胞的杀伤作用。结果NK细胞活化时P4HB表达水平升高,抑制P4HB可降低NK细胞IFN-γ分泌水平并可减弱其对K562细胞的杀伤以及抗体依赖的细胞毒(ADCC)作用。结论蛋白质二硫键异构酶P4HB在NK细胞活化过程中发挥着重要作用。