Simultaneous determination of selected ionophoric coccidiostats and amino acids in feed premixes using high-performance liquid chromatography-ultraviolet detection method.

The combination of ionophoric coccidiostats and amino acids (AAs) is important in poultry feeding to enhance immunity and improve the growth and feed efficiency of birds suffering from coccidiosis. A simple, rapid, and economical high-performance liquid chromatography-ultraviolet detection (HPLC-UV) method for the simultaneous determination of three ionophoric coccidiostats, namely salinomycin (SAL), maduramicin (MAD), and monensin (MON) in addition to three AAs; L-tryptophan (L-TRP), alpha-ketoleucin (KLEU), and L-valine (L-VAL) in feed premixes was developed and validated. Chromatographic separation was achieved in less than 12 min using a phenyl hexyl column with a mobile phase consisting of acetonitrile/methanol/water (25:20:55, v/v/v) adjusted to pH 3 using phosphoric acid. Isocratic elution was performed at a flow rate of 1 mL/min with UV detection at 210 nm. The method showed good linearity in the ranges 0.50-5.0 mg/mL for MON, 0.20-2.0 mg/mL for MAD and SAL, 10.0-100.0 µg/mL for L-TRP and KLEU, and 50.0-500.0 µg/mL for VAL. The developed method was successfully applied to determine the studied analytes in feed premixes with good recoveries and precision. The good validation criteria of the proposed method allow its utilization in quality control laboratories.

Inhibition of platelet-activating factor (PAF)-induced platelet aggregation by fatty acids from human saliva.

Experiments were undertaken to identify the nature of a previously identified inhibitor of PAF-induced platelet aggregation (PA) in human saliva. Human saliva fractionated by preparative thin layer chromatography (TLC) yielded a fraction that co-migrated with fatty acids (FAs) and inhibited PAF-induced aggregation of platelets. Synthetic FAs tested for their capacities to inhibit 0.1 nM PAF-induced PA showed that only the cis-unsaturated compounds were inhibitory with activities of some of the polyunsaturated FAs (PUFA) reaching almost 100% at 20 µM. Eicosapentanoic acid (EPA) and 8,11,14-eicosatrienoic acid also deaggregated the PAF-induced aggregates. With the exception of oleic acid (OLA), cis-monounsaturated FAs, and elaidic acid, the trans isomer of OLA, were poor inhibitors. In a direct comparison with other platelet agonists, ADP, thrombin, and ionophore A23187, the active saliva fraction and selected individual FAs inhibited, to greater or lesser extent, PA induced by each of the agonists. EPA, OLA, linoleic acid (LNA), and the active saliva fraction were potent inhibitors of ADP-induced PA, EPA completely inhibited thrombin-induced PA and the saliva fraction showed only weak - moderate inhibitory activity to both thrombin- and ionophore A23187-induced PA. Other reports of endogenous PAF inhibitors in mammalian tissues are compared to the present results. PAF can trigger and amplify inflammatory cascades suggesting a possible modulation role for cis-unsaturated FAs in some diseases.

The Chloride Anion Acts as a Second Messenger in Mammalian Cells - Modifying the Expression of Specific Genes.

BACKGROUND/AIMS: Cystic Fibrosis (CF) is caused by mutations in the CFTR gene, encoding a cAMP-activated chloride (Cl-) channel. We have previously demonstrated that the expression of several genes can be modulated by the CFTR activity; among them, SRC, MTND4, CISD1, and IL1B. However, the CFTR signalling mechanism involved in the expression of CFTR-dependent genes is unknown. The aim of this work was to determine if intracellular chloride (Cl-)i might function as a second messenger modulating the expression of specific genes. METHODS: Differential display (DD) was applied to IB3-1 cells (CF cells), cultured under conditions that produce different intracellular Cl- concentrations ([Cl-]i), to analyse their expression profile. RESULTS: Several differentially expressed gene products were observed by using DD, suggesting the presence of chloride-dependent gene expression. Two cDNA fragments, derived from differentially expressed mRNAs and showing opposed response to Cl-' were isolated, cloned, sequenced and its Cl- dependency validated by reverse transcription quantitative-PCR (RT-qPCR). We identified the gene RPS27, which encodes the multifunctional ribosomal protein RPS27, also known as metallopanstimulin-1 (MPS-1), and the gene GLRX5, encoding glutaredoxin-related protein 5, as chloride-dependent genes. RPS27 was negatively regulated with increased [Cl-]i, approximately from 25-75 mM Cl- (EC50 = 46 ± 7 mM), and positively regulated from 75-125 mM Cl- (EC50 = 110 ± 11 mM) (biphasic response). In contrast, GLRX5 was positively modulated by [Cl-]i, showing a typical sigmoidal dose-response curve from 0-50 mM Cl-, reaching a plateau after 50 mM Cl- (EC50 ∼ 34 mM). CONCLUSION: The results suggest the existence of chloride-dependent genes. The Cl- anion, therefore, might act as a second messenger for channels or receptors able to modulate the intracellular Cl- concentration, regulating in turn the expression of specific genes.

Schiff bases as cadmium(II) selective ionophores in polymeric membrane electrodes.

The construction and performance characteristics of polymeric membrane electrodes based on two neutral ionophores, N,N'-[bis(pyridin-2-yl)formylidene]butane-1,4-diamine (S1) and N-(2-pyridinylmethylene)-1,2-benzenediamine (S2) for quantification of cadmium ions, are described. The influences of membrane compositions on the potentiometric response of the electrodes have been found to substantially improve the performance characteristics. The best performance was obtained with the electrode having a membrane composition (w/w) of (S1) (2.15%):PVC (32.2%):o-NPOE (64.5%):KTpClPB (1.07%). The proposed electrode exhibits Nernstian response in the concentration range of 7.9x10(-8) to 1.0x10(-1) M Cd2+ with limit of detection 5.0x10(-8) M, performs satisfactorily over wide pH range (2.0-8.0) with a fast response time (10 s). The sensor has been found to work satisfactorily in partially non-aqueous media up to 30% (v/v) content of methanol, ethanol and acetonitrile and could be used for a period of 2 months. The analytical usefulness of the proposed electrode has been evaluated by its application in the determination of cadmium in real samples. The practical utility of the membrane electrode has also been observed in the presence of surfactants.

A comparison of data analysis methods for determining gas phase stabilities by CID: alkali metal complexes of polyether ionophore antibiotics.

The gas phase stabilities of Group I metal complexes of the polyether ionophore antibiotics lasalocid and monensin were investigated by collision induced dissociation mass spectrometry. Electrospray ionization was used with a triple quadrupole mass spectrometer for the determination of threshold dissociation energies upon application of increasing collision energies. Various data analysis techniques for the determination of dissociation energies are discussed to assess the most suitable method for determining the stabilities of the ionophore-metal complexes studied here. In all cases only the relative stabilities of different complexes may be obtained by the method presented in this study, which does not assess absolute gas phase dissociation energies. Correction factors have been applied, however, to account for the energy conversion during collisions of different metal complexes and the varying degrees of freedom of different sized ligands, allowing for the comparison of the stabilities of different ionophores with like-metals. The measured threshold dissociation energies were compared with respect to the ionic radius of the metal cation, revealing a maximum stability for the K+ complexes of both lasalocid and monensin. A striking decrease in the stabilities of the Rb+ and Cs+ complexes was observed and is believed to be related to a decreasing degree of coordination that the ionophores can accomplish with the larger metals.

A high-throughput screen for identifying transmembrane pore-forming peptides.

We have developed a visual microwell plate assay for rapid, high-throughput screening for membrane-disrupting molecules such as de novo designed pore formers, antibiotic peptides, bacterial toxins, and lipases. The detectability is based on the strong fluorescence emission of the lanthanide metal terbium(III) (Tb(3+)) when it interacts with the aromatic chelator dipicolinic acid (DPA). While Tb(3+) is not strongly fluorescent alone, the binary complex emits bright green fluorescence when irradiated with uv light. For the microwell plate assay, we prepared unilamellar phospholipid vesicles that had either Tb(3+) or DPA entrapped and the opposite molecule in the external solution. Disruption of the membranes allows the Tb(3+)/DPA complex to form, giving rise to a visibly fluorescent solution. In plates with 20-microl wells, the lower limit of visual detectability of the Tb(3+)/DPA complex in solution was about 2.5 microM. The lower limit of detectability using vesicles with entrapped Tb(3+) or DPA was about 50 microM phospholipid. We show that the membrane-disrupting effect of as little as 0.25 microM or 5 pmol of the pore-forming, antibiotic peptide alamethicin can be detected visually with this system. This sensitive, high-throughput assay is readily automatable and makes possible the visual screening of combinatorial peptide libraries for members that permeabilize lipid bilayer membranes.

A novel sensitive bioassay for detection of Bacillus cereus emetic toxin and related depsipeptide ionophores.

Of the toxins produced by Bacillus cereus, the emetic toxin is likely the most dangerous but, due to the lack of a suitable assay, the least well known. In this paper, a new, sensitive, inexpensive, and rapid bioassay for detection of the emetic toxin of B. cereus is described. The assay is based on the loss of motility of boar spermatozoa upon 24 h of exposure to extracts of emetic B. cereus strains or contaminated food. The paralyzed spermatozoa exhibited swollen mitochondria, but no depletion of cellular ATP or damage to plasma membrane integrity was observed. Analysis of the purified toxin by electrospray tandem mass spectrometry showed that it was a dodecadepsipeptide with a mass fragmentation pattern similar to that described for cereulide. The 50% effective concentration of the purified toxin to boar spermatozoa was 0.5 ng of purified toxin ml of extended boar semen-1. This amount corresponds to 10(4) to 10(5) CFU of B. cereus cells. No toxicity was detected for 27 other B. cereus strains up to 10(8) CFU ml-1. The detection limit for food was 3 g of rice containing 10(6) to 10(7) CFU of emetic B. cereus per gram. Effects similar to those provoked by emetic B. cereus toxin were also induced in boar spermatozoa by valinomycin and gramicidin at 2 and 3 ng ml of extended boar semen-1, respectively. The symptoms provoked by the toxin in spermatozoa indicated that B. cereus emetic toxin was acting as a membrane channel-forming ionophore, damaging mitochondria and blocking the oxidative phosphorylation required for the motility of boar spermatozoa.

A modified HPLC method for monensin analysis in liposomes and nanocapsules and its comparison with spectrophotometric and radioactive methods.

Monensin is a carboxylic ionophore which can potentiate the immunotoxin activity against human tumors in vitro and in vivo. Currently monensin is being encapsulated in liposomes and nanocapsules in our laboratory. The reported methods for monensin analysis by spectrophotometric and HPLC lack the required sensitivity. We have developed a sensitive HPLC method for analysis on monensin. Separation was achieved on a Beckman C18 reverse phase column with methanol-acetonitrile-methylene chloride-water-acetic acid (45:20:25:9.5: 0.5) as the mobile phase. The eluent was reacted with vanillin reagent in the post column reactor at 70 degrees C. The reagent reacted with monensin and formed a pink color, which was detected at 520 nm. The retention time of monensin was found to be 6 min. By using this method it was possible to quantify monensin down to 100 ng ml-1, with a signal to noise ration of > 17:1. Linearity was observed within the range of 10 to 100 ng (r2 > 0.99). Inter-day standard deviations for monensin samples of 20, 50 and 80 ng were 0.675, 0.543 and 0.736 respectively. Alternative methods of analysis include using radioactive [3H]monensin in liposomes which can be quantified by scintillation counter. The results from the HPLC, spectrophotometric and radioactive method were compared and were found to be within acceptable limits. The HPLC method is being utilized in our laboratory for quantitative analysis of monensin in liposomes and nanocapsules.

Different systems for iron supply of Salmonella typhimurium and Escherichia coli wild strains as tool for typing.

Hitherto the siderophore-pattern analysis included bioassays to detect enterobactin,aerobactin and other siderophores of Enterobacteriaceae. In addition, 2,3-dihydroxybenzoic acid, the precursor of enterobactin, and new hydroxamate siderophores could be examined by means of two new Salmonella mutants as indicator strains. In this manner the siderophore pattern analysis extends its discriminating ability. Among 167 Salmonella typhimurium strains tested, we detected 5 siderophore patterns. 6 siderophore patterns could be detected among a total of 204 E. coli strains. Using siderophore pattern analysis for clinical-epidemiological and ecological purposes appropriate technical methods can be recommended.

Oligomycin-dependent ionophoric protein subunit of mitochondrial adenosinetriphosphatase.

A proteolipid isolated from yeast mitochondrial adenosinetriphosphatase (subunit 9) (ATP phosphohydrolase; EC 3.6.1.3) by chloroform/methanol extraction has been shown to discharge photo-induced potentials across a planar phospholipid membrane containing bacteriorhodopsin. Oligomycin, a specific inhibitor of oxidative phosphorylation which binds to this protein, allows the potential gradient to be reestablished. When proteolipid was isolated from an oligomycin-resistant strain, ionophoric activity was still obtained but the effect was not reversed by oligomycin. These studies suggest that the hydrophobic subunit-9 polypeptide is the ionophoric component linking ATP synthesis (hydrolysis) with proton translocation.