Zinc ionophores isolated from Terminalia bellirica fruit rind extract protect against cardiomyocyte hypoxia/reoxygenation injury.

The study aimed to isolate and characterize zinc ionophores from Terminalia bellirica fruit using a liposome assay and test its utility in H9c2 rat cardiomyoblasts cells subjected to hypoxia/reoxygenation. Ethyl acetate extract that exhibited zinc ionophore activity was resolved to yield three polyphenols that were characterized as epicatechin-3-gallate (ECG), epigallocatechin-3-gallate (EGCG) and epigallocatechin (EGC) by nuclear magnetic resonance and electrospray ionization-mass spectra. The polyphenols enhanced the uptake of zinc into the liposomes and increased FluoZin-3 fluorescence. These polyphenols in the presence of 10 µM ZnCl2 enhanced the zinc import into H9c2 cells, whose intracellular zinc levels were otherwise lowered upon hypoxia/reoxygenation. EGCG proved to be more potent than ECG, which indeed was more effective than EGC in improving cellular zinc levels and in attenuating the apoptosis of H9c2 cells after hypoxia/reoxygenation injury. The polyphenols required zinc for anti-apoptotic effect. The cardioprotective effect is indeed due to enhanced zinc uptake mediated by these polyphenols.

A new sample treatment strategy based on simultaneous supramolecular solvent and dispersive solid-phase extraction for the determination of ionophore coccidiostats in all legislated foodstuffs.

A single-step sample treatment, for the simultaneous extraction and clean-up for the determination of ionophore coccidiostats in EU legislated foodstuffs, is here proposed. The treatment is based on the combination of: (i) a supramolecular solvent with restricted access properties (SUPRAS-RAM), spontaneously formed by the addition of hexanol, water and THF to the sample; and (ii) dispersive solid phase extraction (dSPE). The SUPRAS-RAM extract was directly compatible with LC-MS/MS and no further re-extraction, evaporation or cleanup procedures were necessary. SUPRAS-RAM efficiently extracted the ionophores (recoveries in milk, eggs, fat, liver, kidney, and chicken and beef muscle were in the range 71-112%) and removed proteins and carbohydrates, whereas dSPE removed fats and other lipophilic compounds. The method was validated following the European Commission Decision 2002/657/EC. Detection limits (0.004-0.07 µg kg-1) were far below the maximum residue limits (1-150 µg kg-1). Method analytical and operational characteristics were suitable for routine determination of ionophores.

Colorimetric ionophore-based coextraction titrimetry of potassium ions.

Potassium ion concentration can be successfully determined volumetrically by moving the titration from a homogeneous phase to a two phase solvent system. This is because potassium can be readily complexed in a selective and thermodynamically stable manner by ionophores such as valinomycin. Previous work demonstrated the successful titration of potassium by ion-exchange into an organic phase containing valinomycin, but the sample itself served as titrant, which is not sufficiently practical for routine applications. This problem is overcome here by a co-extraction based approach, with the sodium salt of the water soluble lipophilic anion tetraphenylborate as titrant. The extraction of potassium tetraphenylborate must be preferred over that of the hydrogen ion-tetraphenylborate pair, which is used to indicate the endpoint by the presence of a lipophilic indicator in the organic phase. This is controlled by the sample pH, which for the conditions chosen here is around 7 for optimal sharpness and accuracy of the endpoint. The approach is demonstrated in a colorimetric detection approach, by use of a tethered digital camera and subsequent automated analysis of the resulting image files. The potassium analysis in a variety of samples is successfully demonstrated, including blood serum.

Chemical Genomics, Structure Elucidation, and in Vivo Studies of the Marine-Derived Anticlostridial Ecteinamycin.

A polyether antibiotic, ecteinamycin (1), was isolated from a marine Actinomadura sp., cultivated from the ascidian Ecteinascidia turbinata. 13C enrichment, high resolution NMR spectroscopy, and molecular modeling enabled elucidation of the structure of 1, which was validated on the basis of comparisons with its recently reported crystal structure. Importantly, ecteinamycin demonstrated potent activity against the toxigenic strain of Clostridium difficile NAP1/B1/027 (MIC = 59 ng/µL), as well as other toxigenic and nontoxigenic C. difficile isolates both in vitro and in vivo. Additionally, chemical genomics studies using Escherichia coli barcoded deletion mutants led to the identification of sensitive mutants such as trkA and kdpD involved in potassium cation transport and homeostasis supporting a mechanistic proposal that ecteinamycin acts as an ionophore antibiotic. This is the first antibacterial agent whose mechanism of action has been studied using E. coli chemical genomics. On the basis of these data, we propose ecteinamycin as an ionophore antibiotic that causes C. difficile detoxification and cell death via potassium transport dysregulation.

Liquid to liquid extraction and liquid chromatography-tandem mass spectrometry determination of hainanmycin in feed.

Hainanmycin is a new veterinary polyether antibiotic and has few sensitive analytical method in present days. In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) relying on multiple reaction monitoring (MRM) detection was developed for analysis of hainanmycin in animal feed. Feed samples were extracted with ethyl acetate and purified by two steps of liquid-liquid extraction (LLE) to get rid of water solvable matrix and lipids one by one. The final simple was analyzed by LC-MS/MS. The LC mobile phase was composed of 0.1% aqueous formic acid and 0.1% formic acidified acetonitrile by gradient elution. Average recoveries ranged from 74.22% to 87.85%, as determined by spiking with 2.0 (LOQ) ∼2500µgkg-1 of hainanmycin. The inter-day and intra-day coefficient of variation was 9.21% to 11.77% and 7.67% to 13.49%, respectively. The limit of detection (LOD) and the limit of quantitation (LOQ) were 0.36µgkg-1 and 2.0µgkg-1, respectively.

Comparison of triple quadrupole mass spectrometry and Orbitrap high-resolution mass spectrometry in ultrahigh performance liquid chromatography for the determination of veterinary drugs in sewage: benefits and drawbacks.

This paper presents a comparison of triple quadrupole tandem mass spectrometry (MS/MS) and Orbitrap high-resolution mass spectrometry (HRMS) combined to ultrahigh performance liquid chromatography for the determination of glucocorticoids and polyether ionophores in sewage, in order to show the major benefits and drawbacks for each mass spectrometry analyser. Overall, HRMS measurements have enhanced performance in terms of confirmatory capabilities than MS/MS measurements. Moreover, similar limits of quantification, limits of detection, linear range and repeatability for glucocorticoids with both the MS/MS and HRMS methods were compared, but in the case of polyether ionophores, slightly better limits of detection and limits of quantification were obtained with the HRMS method because of the high sensitivity obtained when diagnostic ions are used for quantification instead of selected reaction monitoring transitions for these compounds. The two methods have been applied to the analysis of several influent and effluent sewage samples from sewage treatment plants located in the Tarragona region (Catalonia, Spain), showing an excellent correlation between the two methods.

Quantification of four ionophores in soil, sediment and manure using pressurised liquid extraction.

A multi-residue pressurised liquid extraction (PLE) methodology has been established for the determination of the four ionophores: lasalocid, monensin, salinomycin and narasin in solid environmental matrices. The PLE methodology is combined with solid phase extraction as clean-up using liquid chromatography coupled to tandem mass spectrometry applying electrospray ionisation for detection. The samples were freeze-dried prior to extraction. The absolute recoveries for soil and sediment ranged from 71 to 123% (relative standard deviation (RSDs) below 16%) and in the range 94-133% (RSDs 9-35%) for poultry manure. The final method allowed for the detection of four ionophores down to a few hundred ngkg(-1) in natural solid matrices with limit of quantifications (LOQs) being 0.96, 0.87, 0.98, and 0.64µgkg(-1) in soil for lasalocid, monensin, salinomycin, and narasin, respectively. Corresponding LOQs in sediment were 1.28, 1.34, 1.39, and 0.78µgkg(-1) for the respective ionophores, while in manure the LOQs were 0.98, 1.01, 1.45, and 1.01µgkg(-1).

Determination of polyether ionophores in urban sewage sludge by pressurised liquid extraction and liquid chromatography-tandem mass spectrometry: study of different clean-up strategies.

A method for the determination of five polyether ionophores in urban sewage sludge has been developed. The extraction of compounds was performed by pressurised liquid extraction using acetone, while florisil was used for in-cell clean-up to minimise the matrix effect in the sludge extracts. An amide polar-embedded reversed-phase column was used for the chromatographic separation with a rapid-resolution liquid chromatograph coupled to a tandem mass spectrometer. Moreover, several clean-up strategies such as in-cell and on-cell clean-up and solid-phase extraction clean-up, among others, were tested and their results are discussed in the present paper. Recoveries (10 and 250 µg/kg in dry weight (d.w.), n=6) were close to 90%, repeatability and reproducibility (%RSD, 10 and 250 µg/kg (d.w.), n=6) were less than 10% and 12%, respectively. Limits of detection (LODs) and limits of quantification (LOQs) ranged between 0.5 and 1 µg/kg (d.w.) and between 1 and 5 µg/kg (d.w.), respectively. The method was applied to samples collected in five sewage treatment plants in Catalonia. Monensin and narasin were determined in some sludge samples at concentrations from

Revisiting the enniatins: a review of their isolation, biosynthesis, structure determination and biological activities.

Enniatins are cyclohexadepsipeptides isolated largely from Fusarium species of fungi, although they have been isolated from other genera, such as Verticillium and Halosarpheia. They were first described over 60 years ago, and their range of biological activities, including antiinsectan, antifungal, antibiotic and cytotoxic, drives contemporary interest. To date, 29 enniatins have been isolated and characterized, either as a single compound or mixtures of inseparable homologs. Structurally, these depsipeptides are biosynthesized by a multifunctional enzyme, termed enniatin synthetase, and are composed of six residues that alternate between N-methyl amino acids and hydroxy acids. Their structure elucidation can be challenging, particularly for enniatins isolated as inseparable homologs; however, several strategies and tools have been utilized to solve these problems. Currently, there is one drug that has been developed from a mixture of enniatins, fusafungine, which is used as a topical treatment of upper respiratory tract infections by oral and/or nasal inhalation. Given the range of biological activities observed for this class of compounds, research on enniatins will likely continue. This review strives to digest the past studies, as well as, describe tools and techniques that can be utilized to overcome the challenges associated with the structure elucidation of mixtures of enniatin homologs.

Simultaneous determination of five polyether ionophores using liquid chromatography with one-step fluorescent derivatization.

We present a selective method for simultaneous determination of five polyether ionophores such as salinomycin (SAL), monensin (MON), narasin (NAR), semduramicin (SEM) and lasalocid (LAS) in aquatic samples using a liquid chromatography with one-step fluorescent derivatization of 2-(4-hydrazinocarbonyl-phenyl) 4,5-diphenylimidazole (HCPI) and 4-(4,5-diphenyl-1H-imidazol-2-yl) benzoyl chloride hydrochloride (DIB-Cl). Fluorescent one-step derivatization for SAL, MON, NAR and SEM using HCPI and for LAS using DIB-Cl was monitored by an LC/fluorescence detector (E(x), 340 nm; E(m), 465 nm). Chromatographic separation was performed on a TSK-GEL ODS-120T (4.6 × 150 mm, 3 µm) column using a mobile phase of 0.1% formic acid in acetonitrile and 0.5 mM ammonium formate in water (70/30, v/v). The limits of detections were 0.01 µg/mL (50 pg) for LAS, 0.05 µg/mL (250 pg) for SAL, NAR and SEM, and 0.1 µg/mL (500 pg) for MON, respectively. The recoveries for water samples were indicated to be the range of 79.6 ± 6.4 - 99.0 ± 4.4% with associated precision values (between-day for 3 days) for repeatability. Based on solid-phase extraction, the limit of quantitation values indicated 0.1 ng/mL for SAL, MON, NAR and SEM, and 0.01 ng/mL for LAS in water samples.

Isolation, structure elucidation and biological activities of trichofumins A, B, C and D, new 11 and 13mer peptaibols from Trichoderma sp. HKI 0276W.

Trichofumins A-D were isolated from cultures of Trichoderma sp. HKI 0276 as new 11 and 13mer peptaibols. Similar to 15mer peptaibols they promote morphogenesis of the fungus Phoma destructiva and cause hypothermia in mice as a characteristic of neuroleptic activity. Membrane measurements using a synthetic BLM model showed that A, B, C and D increased membrane permeability for cations in a similar manner as was shown for larger peptaibols but with comparably lower efficiency.

Pore-forming properties and antibacterial activity of proteins extracted from epidermal mucus of fish.

Among several biological functions, the epidermal mucus of fish may play an important role in host defense, particularly in the prevention of colonization by parasites, bacteria and fungi. In previous work, two hydrophobic proteins of 27 and 31 kDa were isolated from carp mucus. This study identified a strong antibacterial activity (0.16-0.18 microM) well correlated with pore-forming properties. Here this work was extended to other fish species, four fresh water fish and one sea water fish. After a first step of purification, water-soluble and hydrophobic material were separated, and both fractions were analyzed by SDS-PAGE and capillary electrophoresis. Only the hydrophobic component induced pore-forming activity, when reconstituted in planar lipid bilayers. This pore-forming activity was well correlated to a strong antibacterial activity against several bacteria strains. These results suggest that fish secrete antibacterial proteins able to permeabilize the membrane of the target cell and thus act as a defense barrier.

Physical stability of ethyl diatrizoate nanocrystalline suspension in steam sterilization.

PURPOSE: To study the effects of formulation variables on the physical stability of a submicron crystal (nanocrystal) suspension under steam sterilization conditions. METHODS: Suspensions of ethyl diatrizoate nanocrystals were prepared by wet milling in the presence of the surfactant poloxamine 908. Particle size distribution and zeta potential were measured by photon correlation spectroscopy. RESULTS: On heating, the mean particle size of the nanocrystal suspension remained essentially unchanged up to 110 degrees C, the cloud point of the stabilizing surfactant, but increased significantly above that temperature. The increase in particle size was a result of particle aggregation rather than crystal growth. Adding a cloud point booster to the suspension significantly minimized the particle aggregation at high temperatures. The purity of poloxamine 908 and the tonicity agent and buffer salt used also affected the heat stability of the suspension, the latter agents apparently through altering the surfactant cloud point. CONCLUSIONS: The aggregation of the ethyl diatrizoate nanocrystalline suspension under steam sterilization conditions was a result of phase separation of the stabilizing surfactant at its cloud point. When formulated with a cloud point booster to prevent the phase-separation, the suspension maintained its physical stability under steam sterilization without any significant change in particle size distribution.

[Gradient extraction of ammonium specific ionophore antibiotics from mycelium]. / Gradientnaia ékstraktsiia ammoniispetsifichnykh ionofornykh antibiotikov iz mitseliia produtsenta.

Streptomyces brunneofungs 118 and S.griseolus 224 were isolated from natural objects and shown to synthesize ammonium specific products belonging to macrotetrolide compounds. Gradient extraction was applied to the mycelium and it was demonstrated that the compounds were rather labile both in the native cells and on synthetic carriers and could be hydrolyzed by aqueous solutions of acetone and ethanol to various linear oligomers of narctinic acids. Acetone mainly stabilized the monomer and dimer fragments whereas in the ethanol extracts a complete set of the oligomers (from the monomer to the tetramer) was detectable. Graident extract of suspension of the microbial intact cells is useful in the study of some properties and the primary identification of biologically active hydrophobic products even at the early stages of their isolation.

Liquid chromatographic determination of monensin in premix and animal feeds: collaborative study.

An interlaboratory study of a liquid chromatographic (LC) method for determining monensin in premix (60-80 g/lb or 132-176 mg/g) and animal feeds (5-200 g/ton or 0.0055-0.22 mg/g) was conducted in laboratoriesin the United States, Canada, France, and Germany. The LC system used a reversed-phase column, postcolumn derivatization with vanillin, and UV detection. The method separates monensin from other ionophores such as narasin and salinomycin. Each laboratory analyzed a total of 20 samples of premix, liquid feed supplements, poultry, and cattle feeds. Concentrations of monensin in all samples ranged from 0 to 176 mg/g (80 g/lb). Reproducibility relative standard deviation (RSDR) for premix ranged from 2.8 to 3.4%. For feed samples containing monensin, repeatability standard deviation (sr) ranged from 0.9 to 7.0. Reproducibility standard deviation (sR) ranged from 1.2 to 11. Repeatability relative standard deviation (RSDr) ranged from 6.1 to 21% and RSDR values-ranged from 8.6 to 25%. Sample preparation for the LC method is less labor intensive than that for the microbiological assays. The LC assay is more efficient than the microbiological assays. This LC method for determination of monensin in premix and animal feeds has been adopted first action by AOAC INTERNATIONAL.

Rhizoferrin: a complexone type siderophore of the Mucorales and entomophthorales (Zygomycetes).

The present investigation presents evidence that rhizoferrin, a novel polycarboxylate or complexone-type siderophore, originally isolated from Rhizopus microsporus, represents the common siderophore within the Zygomycetes. Thus, rhizoferrin could be detected by HPLC analysis in various families of the Mucorales, e.g., Rhizopus microsporus var. rhizopodiformis, Mucor mucedo and Phycomyces nitens (Mucoraceae), Chaetostylum fresenii and Cokeromyces recurvatus (Thamnidiaceae), Cunninghamella elegans and Mycotypha africana (Choanephoraceae) and Mortierella vinacea (Mortierellaceae) and in Basidiobolus microsporus (Entomophthorales). The function of rhizoferrin as a siderophore in the fungus R. microsporus var. rhizopodiformis was demonstrated by time- and concentration-dependent uptake of [55Fe]-labelled rhizoferrin, yielding saturation kinetics with values of Km = 8 microM and V(max) = 1.2 nmol min-1 (mg dry wt)-1.

Isolation of a novel siderophore from Pseudomonas cepacia.

A novel iron-binding compound was identified in ethyl acetate extracts of the supernates from Pseudomonas cepacia cultures. This compound, named azurechelin, was produced by 88% of P. cepacia strains isolated from the respiratory tract. Production of azurechelin was regulated by the iron concentration in the culture medium. Azurechelin enhanced the growth of P. cepacia in a medium containing transferrin 200 mg/L. Azurechelin released iron from transferrin in an equilibrium dialysis assay, suggesting that it could complete with transferrin for iron. Azurechelin could also stimulate iron uptake by P. cepacia. This siderophore appeared to have a novel structure with neither the typical characteristics of catechol nor of hydroxamate compounds.

CP-60,993, a new dianemycin-like ionophore produced by Streptomyces hygroscopicus ATCC 39305: fermentation, isolation and characterization.

CP-60,993, 19-epi-dianemycin, is a novel polycyclic ether antibiotic produced by Streptomyces hygroscopicus ATCC 39305. Fermentation recovery, purification and crystallization were achieved using standard procedures. CP-60,993 was characterized as a monocarboxylic acid. Elemental analysis suggested a molecular formula of C47H78O14 for the free acid and C47H77O14 Na for the sodium salt. Crystalline from CP-60,993 sodium salt shows the following properties: m.p. 193-205 degrees C, E1%(1 cm) = 157 at 232 nm, [alpha]25 degrees C(D) + 11.0 (c 1, methanol). The structure, determined by MS, PMR and CMR, differs from dianemycin only in the stereochemistry at position 19. This was confirmed by X-ray crystallography carried out on the rubidium salt of CP-60,993. It exhibited activity in vitro against Gram-positive and anaerobic bacteria, efficacy against Eimeria coccidia in vivo in poultry, and stimulation in vitro of rumen propionic acid production.

Identification of extracellular siderophores of pathogenic strains of Aspergillus fumigatus.

Clinical and environmental isolates of Aspergillus fumigatus synthesized extracellular siderophores when grown in defined medium. Six hydroxamate siderophores were purified from culture filtrates and identified by thin layer chromatography. The most prominent siderophore was identified as N,N',N"-triacetylfusarinine C and the second most prominent siderophore was identified as ferricrocin. In addition, a hydrolytic product of N,N',N"-triacetylfusarinine C was identified. Three other siderophores were present in smaller amounts and were not identified. Since the same siderophores were produced by isolates from diseases of varying severity and from environmental material, it is unlikely that the extracellular siderophores function as virulence factors during infection. However, they may function as growth factors by mediating iron uptake by the fungus in the micro-environment of the inflammatory focus.