Determination of Polypeptide Antibiotic Residues in Food of Animal Origin by Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry.

A novel UHPLC-MS/MS method for the determination of polypeptide antibiotic residues in animal muscle, milk, and eggs was developed and validated. Bacitracin A, colistin A, colistin B, polymyxin B1, and polymyxin B2 were extracted from the samples with a mixture of acetonitrile/water/ammonia solution 25%, 80/10/10 (v/v/v), and put through further evaporation, reconstitution, and filtration steps. The chromatographic separation was performed on a C18 column in gradient elution mode. Mass spectral acquisitions were performed in selective multiple reaction monitoring mode by a triple quadrupole mass spectrometer. The method was validated according to the criteria of Commission Decision 2002/657/EC. The method quantifies polypeptides in a linear range from 10 to 1000 µg kg-1, where the lowest concentration on the calibration curve refers to the limit of quantification (LOQ). The recoveries ranged from 70 to 99%, the repeatability was below 13%, and within-laboratory reproducibility was lower than 15%. The decision limit (CCα) and detection capability (CCß) values were calculated, and ruggedness and stability studies were performed, to fulfill the criteria for confirmatory methods. Moreover, the developed method may also be used for screening purposes by its labor efficiency.

Analysis of Major Components of Bacitracin, Colistin and Virginiamycin in Feed Using Matrix Solid-phase Dispersion Extraction by Liquid Chromatography-electrospray Ionization Tandem Mass Spectrometry.

A quantitative LC-MS/MS method has been developed for simultaneous determination of bacitracin A, bacitracin B, colistin A, colistin B and virginiamycin M1 in feed. This rapid simple and effective extraction method was based on matrix solid-phase dispersion. Qualitative and quantitative analyses were performed by LC-ESI-MS/MS. CCß of polypeptide antibiotics upon the method ranged from 9.6 to 15.8 µg kg-1 and 19.4 to 27.5 µg kg-1, respectively. The limit of quantification of polypeptide antibiotics was 25 µg kg-1 in feed samples. The recoveries of polypeptide antibiotics spiked in feed samples at a concentration range of 25-100 µg kg-1 were found above 75.9-87.9% with relative standard deviations within days less than 15.7% and between days less than 20.6%. This rapid and reliable method can be used to efficiently separate, characterize and quantify the residues of polypeptide antibiotics in feed with advantages of simple pretreatment and environmental friendly.

Self-assembly of bacitracin-gold nanoparticles and their toxicity analysis.

As the widely use of gold nanoparticles (AuNPs) in drug delivery, the precise control on the size and morphology of the AuNPs is urgently required. In this scenario, traditional synthesis methods cannot meet current requirement because of their inherent defects. We have depicted here a novel method for fabricating monodispersed large size gold nanoparticles, based on the self-assembly of bacitracin. The AuNPs could be facilely, low-cost, and green synthesized with repeatability and controllability in this method. The Bac gold nanoparticles (Bac-AuNPs), composed by bacitracin core and gold shell, exhibited a spherical morphology in TEM and a face-centered cubic crystal structure in X-Ray diffraction and selected area electron diffraction. The mean diameter of the Bac-AuNPs was 89nm. The nanoparticles were mono-dispersed and the zeta potential of the nanoparticles was 4.1±0.64mV. Notably, in cell viability assay, the Bac-AuNPs showed less toxicity to HepG2 cells and HEK293 cells compared to small size AuNPs. Collectively, the size, rheological characteristic and the biocompatibility supported the use of the gold nanoparticles as intracellular delivery vehicles for drug delivery, especially for tumor therapy. And this study could provide a maneuverable, controllable and green strategy for the synthesis of AuNPs, which would be applied in disease diagnosis and therapy with biosafety.

Accelerated Stability Modeling for Peptides: a Case Study with Bacitracin.

The Accelerated Stability Assessment Program (ASAP) was applied for the first time to a peptide, the antibiotic active pharmaceutical ingredient bacitracin. Bacitracin and its complex with zinc were exposed to temperature and relative humidity conditions from 50 to 80°C and from 0 to 63% for up to 21 days. High-performance liquid chromatography was used to analyze the stressed samples for both degradant formation and loss of the active (bacitracin A) and two inactive isoforms, with identities confirmed by mass spectrometry. These data were then analyzed using a humidity-corrected Arrhenius equation and isoconversion approach to create a shelf-life predicting model for typical storage conditions. Model fitting was found to be good with low residuals in both temperature and relative humidity axes for all parameters examined. The generated model's predictions for both the native and zinc complex of bacitracin for both formation of the major degradation product (F) and loss of the active isoform (A) were consistent with longer-term measured values at 30°C/53%RH and 40°C/75%RH, validating this approach for accelerating the determination of long-term stability of a peptide.

Immobilizing bacitracin on titanium for prophylaxis of infections and for improving osteoinductivity: An in vivo study.

Bacitracin immobilized on the titanium (Ti) surface significantly improves anti-bacterial activity and biocompatibility in vitro. In the current study, we investigated the biologic performance (bactericidal effect and bone-implant integration) of bacitracin-modified Ti in vivo. A rat osteomyelitis model with femoral medullary cavity placement of Ti rods was employed to analyze the prophylactic effect of bacitracin-modified Ti (Ti-BC). Thirty-six female Sprague Dawley (SD) rats were used to establish the Ti implant-associated infection. The Ti and Ti-BC rods were incubated with and without Staphylococcus aureus to mimic the contaminated Ti rod and were implanted into the medullary cavity of the left femur, and sterile Ti rods were used as the blank control. After 3 weeks, the bone pathology was evaluated using X-ray and micro-computed tomography (micro-CT) analysis. For the investigation of the Ti-BC implant osseointegration in vivo, fifteen SD rats were divided into three groups (N=5), namely Ti, Ti-dopamine immobilized (Ti-DOPA), and Ti-BC. Ti rods were implanted into the left femoral cavity and micro-CT and histological evaluation was conducted after 12 weeks. The in vivo study indicated that Ti-immobilized bacitracin owned the prophylaxis potential for the infection associated with the Ti implants and allowed for the osseointegration. Thus, the multiple biofunctionalized Ti implants could be realized via immobilization of bacitracin, making them promising candidates for preventing the Ti implant-associated infections while retaining the osseointegration effects.

Biofunctionalization of titanium with bacitracin immobilization shows potential for anti-bacteria, osteogenesis and reduction of macrophage inflammation.

Titanium has been widely used in the orthopedic and dental fields, however, the inert nature of Ti makes it unsuitable for application in promoting bone cell growth，osteogenic differentiation and antibacterial ability. The aims of the current study were to investigate the antimicrobial activity and biofunction of the polypeptide antibiotic bacitracin, and obtain a multi-biofunctional titanium implant by covalently-immobilizing titanium with the bacitracin. The results showed that the bacitracin possessed low minimum inhibitory concentration (MIC) to both Staphylococcus aureus and Methicillin-resistant Staphylococcus aureus (MRSA), with the non-cytotoxicity concentration up to 500µg/mL to human bone marrow mesenchymal stem cells (hBMSCs), furthermore, the bacitracin could improve the osteogenic differentiation of hBMSCs. The results of Scanning electron microscope (SEM) and X-ray photoelectron spectroscopy (XPS) indicated that bacitracin had been covalently immobilized on the surface of titanium. Immobilized bacitracin could improve the hydrophilic of immobilized titanium. The results of antimicrobial assay demonstrated that the covalently-immobilized bacitracin also had excellent antimicrobial property, and the bacitracin immobilized titanium could inhibit bacterial adhesion and colonization. The results of cell biology experiments proved that the bacitracin immobilized titanium could improve hBMSCs' adhesion, proliferation and osteogenic differentiation. We also found that the macrophages were difficult to spread or activate on the surface of bacitracin immobilized titanium, and the secretion of inflammatory factors had been inhibited. In conclusion, the novel bacitracin immobilized titanium has multi-biofunctions including outstanding antibacterial properties, excellent cell biology performance, and restraining inflammation, which has exciting application prospect.

Fluorescein-labeled Bacitracin and Daptomycin Conjugates: Synthesis, Fluorescence Imaging and Evaluation.

BACKGROUND: Previously, glycopeptides antibiotics such as vancomycin, ramoplanin and an antifungal antibiotic nystatin have been studied for their diagnostic and therapeutic potential. OBJECTIVE: To further explore the diagnostic and chemotherapeutic potential of other antibiotics we have now employed daptomycin, a lipopetide antibiotic and bacitracin, a polypeptide antibiotic in uptake and vitality tests on human cell lines. METHOD: Fluorescent conjugates of bacitracin and daptomycin were synthesized using fluorescein isothiocynate (FITC) for confocal laser scanning microscopy (CLSM) and fluorescence activated cell sorting (FACS). The cellular uptake of the synthesized daptomycin and bacitracin conjugates was studied on seven human cell lines, two healthy and five malignant using CLSM and FACS. To examine the cell membrane damage caused by the conjugates FACS experiments were carried out using propidium iodide. RESULTS: The uptake pattern was different for both antibiotics for all the cell lines. The cytoplasmic uptake of daptomycin conjugate was lower than the bacitracin conjugate, resulting in decreased cell membrane damage. CONCLUSION: No preferential uptake into malignant or healthy cells was found for the two different antibiotic conjugates and the uptake patterns were also different between the two antibiotics. However, the lower cytotoxicity and different uptake mechanism makes daptomycin conjugate a prospective candidate for further study as a diagnostic agent for various intracellular infections.

The role of protein disulfide isomerase in the post-ligation phase of ß3 integrin-dependent cell adhesion.

INTRODUCTION: Protein disulfide isomerase (PDI) catalyzes disulfide bond exchange. It is crucial for integrin-mediated platelet adhesion and aggregation and disulfide bond exchange is necessary for αIIbß3 and αvß3 activation. However, the role of disulfide bond exchange and PDI in the post-ligation phase of αIIbß3 and αvß3 mediated cell adhesion has yet to be determined. METHODS: To investigate a possible such role, we expressed wild type (WT) human αIIb and either WT human ß3, or ß3 harboring single or double cysteine to serine substitutions disrupting Cys473-Cys503 or Cys523-Cys544 bonds, in baby hamster kidney (BHK) cells, leading to expression of both human αIIbß3 and a chimeric hamster/human αvß3. Adhesion to fibrinogen-coated wells was studied in the presence or absence of bacitracin, a PDI inhibitor, with and without an αvß3 blocker. RESULTS: Flow cytometry showed WT and mutant αIIbß3 expression in BHK cells and indicated that mutated αIIbß3 receptors were constitutively active while WT αIIbß3 was inactive. Both αIIbß3 and αvß3 integrins, WT and mutants, mediated adhesion to fibrinogen as shown by reduced but still substantial adhesion following treatment with the αvß3 blocker. Mutated αIIbß3 integrins disrupted in the Cys523-Cys544 bond still depended on PDI for adhesion as shown by the inhibitory effect of bacitracin in the presence of the αvß3 blocker. Mutated integrins disrupted in the Cys473-Cys503 bond showed a similar trend. CONCLUSIONS: PDI-mediated disulfide bond exchange plays a pivotal role in the post-ligation phase of αIIbß3-mediated adhesion to fibrinogen, while this step in αvß3-mediated adhesion is independent of disulfide exchange.

New silica nanostructure for the improved delivery of topical antibiotics used in the treatment of staphylococcal cutaneous infections.

In this paper, we report the synthesis, characterization (FT-IR, XRD, BET, HR-TEM) and bioevaluation of a novel γ-aminobutiric acid/silica (noted GABA-SiO2 or γ-SiO2) hybrid nanostructure, for the improved release of topical antibiotics, used in the treatment of Staphylococcus aureus infections. GABA-SiO2 showed IR bands which were assigned to Si-O-Si (stretch mode). The XRD pattern showed a broad peak in the range of 18-30° (2θ), indicating an amorphous structure. Based on the BET analysis, estimations about surface area (438.14 m²/g) and pore diameters (4.76 nm) were done. TEM observation reveals that the prepared structure presented homogeneity and an average size of particles not exceeding 10nm. The prepared nanostructure has significantly improved the anti-staphylococcal activity of bacitracin and kanamycin sulfate, as demonstrated by the drastic decrease of the minimal inhibitory concentration of the respective antibiotics loaded in the GABA-SiO2 nanostructure. These results, correlated with the high biocompatibility of this porous structure, are highlighting the possibility of using this carrier for the local delivery of the antimicrobial substances in lower active doses, thus reducing their cytotoxicity and side-effects.

Use of mixed micelles for presentation of building blocks in a new combinatorial discovery methodology: proof-of-concept studies.

We describe a new method of combinatorial screening in which building blocks, instead of being linked together chemically, are placed on the surface of nanoparticles. Two- or three-dimensional structures form on the surface of these particles through the close approach of different building blocks, with sufficient flexibility to be able to adapt and interact with putative binding sites in biological systems. The particles assemble without the need for formation of chemical bonds, so libraries comprised of many structures can be prepared rapidly, with large quantities of material available for testing. Screening methods can include solid and solution-phase binding assays, or tissue culture models, for example looking for structures which can change the behaviour of cells in a disease-modifying manner.

Dereplicating nonribosomal peptides using an informatic search algorithm for natural products (iSNAP) discovery.

Nonribosomal peptides are highly sought after for their therapeutic applications. As with other natural products, dereplication of known compounds and focused discovery of new agents within this class are central concerns of modern natural product-based drug discovery. Development of a chemoinformatic library-based and informatic search strategy for natural products (iSNAP) has been constructed and applied to nonribosomal peptides and proved useful for true nontargeted dereplication across a spectrum of nonribosomal peptides and within natural product extracts.

Bacitracin-conjugated superparamagnetic iron oxide nanoparticles: synthesis, characterization and antibacterial activity.

Bacitracin-conjugated superparamagnetic iron oxide (Fe(3)O(4)) nanoparticles were prepared by click chemistry and their antibacterial activity was investigated. After functionalization with hydrophilic and biocompatible poly(acrylic acid), water-soluble Fe(3)O(4) nanoparticles were obtained. Propargylated Fe(3)O(4) nanoparticles were then synthesized by carbodiimide reaction of propargylamine with the carboxyl groups on the surface of the iron oxide nanoparticles. By further reaction with N(3)-bacitracin in a Cu(I)-catalyzed azide-alkyne cycloaddition, the magnetic Fe(3)O(4) nanoparticles were modified with the peptide bacitracin. The functionalized magnetic nanoparticles were characterized by powder X-ray diffraction, X-ray photoelectron spectroscopy, TEM, zeta-potential analysis, FTIR spectroscopy and vibrating-sample magnetometry. Cell cytotoxicity tests indicate that bacitracin-conjugated Fe(3)O(4) nanoparticles show very low cytotoxicity to human fibroblast cells, even at relatively high concentrations. In view of the antibacterial activity of bacitracin, the biofunctionalized Fe(3)O(4) nanoparticles exhibit an antibacterial effect against both Gram-positive and Gram-negative organisms, which is even higher than that of bacitracin itself. The enhanced antibacterial activity of the magnetic nanocomposites allows the dosage and the side effects of the antibiotic to be reduced. Due to the antibacterial effect and magnetism, the bacitracin-functionalized magnetic nanoparticles have potential application in magnetic-targeting biomedical applications.

Acid-denatured Green Fluorescent Protein (GFP) as model substrate to study the chaperone activity of protein disulfide isomerase.

Green fluorescent protein (GFP) has been widely used in several molecular and cellular biology applications, since it is remarkably stable in vitro and in vivo. Interestingly, native GFP is resistant to the most common chemical denaturants; however, a low fluorescence signal has been observed after acid-induced denaturation. Furthermore, this acid-denatured GFP has been used as substrate in studies of the folding activity of some bacterial chaperones and other chaperone-like molecules. Protein disulfide isomerase enzymes, a family of eukaryotic oxidoreductases that catalyze the oxidation and isomerization of disulfide bonds in nascent polypeptides, play a key role in protein folding and it could display chaperone activity. However, contrasting results have been reported using different proteins as model substrates. Here, we report the further application of GFP as a model substrate to study the chaperone activity of protein disulfide isomerase (PDI) enzymes. Since refolding of acid-denatured GFP can be easily and directly monitored, a simple micro-assay was used to study the effect of the molecular participants in protein refolding assisted by PDI. Additionally, the effect of a well-known inhibitor of PDI chaperone activity was also analyzed. Because of the diversity their functional activities, PDI enzymes are potentially interesting drug targets. Since PDI may be implicated in the protection of cells against ER stress, including cancer cells, inhibitors of PDI might be able to enhance the efficacy of cancer chemotherapy; furthermore, it has been demonstrated that blocking the reductive cleavage of disulfide bonds of proteins associated with the cell surface markedly reduces the infectivity of the human immunodeficiency virus. Although several high-throughput screening (HTS) assays to test PDI reductase activity have been described, we report here a novel and simple micro-assay to test the chaperone activity of PDI enzymes, which is amenable for HTS of PDI inhibitors.

Solution NMR of polypeptides hyperpolarized by dynamic nuclear polarization.

Hyperpolarization of nuclear spins through techniques such as dynamic nuclear polarization (DNP) can greatly increase the signal-to-noise ratio in NMR measurements, thus eliminating the need for signal averaging. This enables the study of many dynamic processes which would otherwise not be amenable to study by NMR spectroscopy. A report of solid- to liquid-state DNP of a short peptide, bacitracin A, as well as of a full-length protein, L23, is presented here. The polypeptides are hyperpolarized at low temperature and dissolved for NMR signal acquisition in the liquid state in mixtures of organic solvent and water. Signal enhancements of 300-2000 are obtained in partially deuterated polypeptide when hyperpolarized on (13)C and of 30-180 when hyperpolarized on (1)H. A simulated spectrum is used to identify different resonances in the hyperpolarized (13)C spectra, and the relation between observed signal enhancement for various groups in the protein and relaxation parameters measured from the hyperpolarized samples is discussed. Thus far, solid- to liquid-state DNP has been used in conjunction with small molecules. The results presented here, however, demonstrate the feasibility of hyperpolarizing larger proteins, with potential applications toward the study of protein folding or macromolecular interactions.

Bacitracin inhibits the reductive activity of protein disulfide isomerase by disulfide bond formation with free cysteines in the substrate-binding domain.

The peptide antibiotic bacitracin is widely used as an inhibitor of protein disulfide isomerase (PDI) to demonstrate the role of the protein-folding catalyst in a variety of molecular pathways. Commercial bacitracin is a mixture of at least 22 structurally related peptides. The inhibitory activity of individual bacitracin analogs on PDI is unknown. For the present study, we purified the major bacitracin analogs, A, B, H, and F, and tested their ability to inhibit the reductive activity of PDI by use of an insulin aggregation assay. All analogs inhibited PDI, but the activity (IC(50) ) ranged from 20 µm for bacitracin F to 1050 µm for bacitracin B. The mechanism of PDI inhibition by bacitracin is unknown. Here, we show, by MALDI-TOF/TOF MS, a direct interaction of bacitracin with PDI, involving disulfide bond formation between an open thiol form of the bacitracin thiazoline ring and cysteines in the substrate-binding domain of PDI.

Tissue factor coagulant function is enhanced by protein-disulfide isomerase independent of oxidoreductase activity.

Protein-disulfide isomerase (PDI) switches tissue factor (TF) from coagulation to signaling by targeting the allosteric Cys186-Cys209 disulfide. Here, we further characterize the interaction of purified PDI with TF. We find that PDI enhances factor VIIa-dependent substrate factor X activation 5-10-fold in the presence of wild-type, oxidized soluble TF but not TF mutants that contain an unpaired Cys186 or Cys209. PDI-accelerated factor Xa generation was blocked by bacitracin but not influenced by inhibition of vicinal thiols, reduction of PDI, changes in redox gradients, or covalent thiol modification of reduced PDI by N-ethylmaleimide or methyl-methanethiosulfonate, which abolished PDI oxidoreductase but not chaperone activity. PDI had no effect on fully active TF on either negatively charged phospholipids or in activating detergent, indicating that PDI selectively acts upon cryptic TF to facilitate ternary complex formation and macromolecular substrate turnover. PDI activation was reduced upon mutation of TF residues in proximity to the macromolecular substrate binding site, consistent with a primary interaction of PDI with TF. PDI enhanced TF coagulant activity on microvesicles shed from cells, suggesting that PDI plays a role as an activating chaperone for circulating cryptic TF.

Pathways of chemical degradation of polypeptide antibiotic bacitracin.

We described the main pathways of bacitracin (Bc) decomposition, chromatographically set the position of its major degradation products and evaluated microbiological activity of isolated components of Bc and its degradation products. All processes of Bc decomposition under stress and accelerated test conditions were monitored with HPLC, performed mainly on a new type reversed-phase (RP-18e) monolithic silica column (Chromolith) enabling fast separation times and some of them also on conventional HPLC columns. Diode array detection, preparative HPLC and FAB mass spectrometry were used for identification of individual Bc components. We found that the major decomposition mechanism in water solutions of Bc is oxidation, and in alkaline solutions, deamidation. In oxidation process the components B1, B2 and B3 and A are oxidized into their corresponding oxidative products H1, H2, H3 and F respectively by the same mechanism. A detailed study of oxidative degradation products revealed that HPLC separation with an acid mobile phase caused splitting of peaks of components H2, H3 and F into two peaks but the peak of component H1 did not split due to its special structural properties. For the component A we confirmed gradual formation of desamido product through an intermediate. We found oxidative degradation products of Bc to be relatively stable, and desamido degradation products to be rather unstable. The estimation of kinetics of Bc decomposition was presented with a semi-quantitative model. Microbiological activity of individual isolated active components of Bc was established and the negligible antimicrobial activity of the degradation products was confirmed.

The metallopeptide antibiotic bacitracin inhibits interleukin-12 alphabeta and beta2 secretion.

The metalloantibiotic bacitracin is a known inhibitor of protein disulfide isomerase (PDI). The disulfide-linked interleukin-12 (IL-12) alphabeta-heterodimer and beta2-homodimer forms are crucial mediators of cell-mediated immune responses and inflammatory reactions. Bacitracin was found to potently block secretion of both the alphabeta- and beta2-dimer forms of IL-12, while it did not affect secretion of the beta-monomer. This inhibition coincided with a reduction in the intracellular amount of PDI found in complex with the beta-chain during intracellular transit. Bacitracin did not affect mRNA levels of the alphabeta- and beta-chain. Similar to bacitracin, N-acetylcysteine blocked alphabeta- and beta2-secretion as well as PDI-beta-chain complex formation. Thus, blocking PDI or shifting the endoplasmic reticulum towards a more reduced status disrupts the oxidative folding pathway or assembly of IL-12 dimer forms. The assembly stage of cytokines in the endoplasmic reticulum may represent a novel target for pharmacological intervention.

Hyphenation of liquid chromatography to ion trap mass spectrometry to identify minor components in polypeptide antibiotics.

The application of liquid chromatography-ion trap mass spectrometry for the characterization of linear and cyclic polypeptide antibiotics was investigated. The aim was on-line identification of impurities in those antibiotic complexes without recourse to time-consuming isolation and purification procedures. Hyphenated techniques, such as liquid chromatography coupled to mass spectrometry, are ideally suited for this purpose. Characterization was performed with an ion trap mass spectrometer offering MS(n) capability; this enables more structural information to be obtained. Liquid chromatography in combination with ion trap mass spectrometry was successfully applied for the characterization of impurities in gramicidin, polymyxin B, polymyxin E, and bacitracin and the study of the degradation products of polymyxins B and E.

Structure and function of "metalloantibiotics".

Although most antibiotics do not need metal ions for their biological activities, there are a number of antibiotics that require metal ions to function properly, such as bleomycin (BLM), streptonigrin (SN), and bacitracin. The coordinated metal ions in these antibiotics play an important role in maintaining proper structure and/or function of these antibiotics. Removal of the metal ions from these antibiotics can cause changes in structure and/or function of these antibiotics. Similar to the case of "metalloproteins," these antibiotics are dubbed "metalloantibiotics" which are the title subjects of this review. Metalloantibiotics can interact with several different kinds of biomolecules, including DNA, RNA, proteins, receptors, and lipids, rendering their unique and specific bioactivities. In addition to the microbial-originated metalloantibiotics, many metalloantibiotic derivatives and metal complexes of synthetic ligands also show antibacterial, antiviral, and anti-neoplastic activities which are also briefly discussed to provide a broad sense of the term "metalloantibiotics."