Bidirectional, iterative approach to the structural delineation of the functional "Chemoprint" in GPR40 for agonist recognition

GPR40, free fatty acid receptor 1 (FFAR1), is a member of the GPCR superfamily and a possible target for the treatment of type 2 diabetes. In this work, we conducted a bidirectional iterative investigation, including computational modeling and site-directed mutagenesis, aimed at delineating amino acid residues forming the functional "chemoprint" of GPR40 for agonist recognition. The computational and experimental studies revolved around the recognition of the potent synthetic agonist GW9508. Our experimentally supported model suggested that H137(4.56), R183(5.39), N244(6.55), and R258(7.35) are directly involved in interactions with the ligand. We have proposed a polarized NH-pi interaction between H137(4.56) and GW9508 as one of the contributing forces leading to the high potency of GW9508. The modeling approach presented in this work provides a general strategy for the exploration of receptor-ligand interactions in G-protein coupled receptors beginning prior to acquisition of experimental data.

Modeled structure of a G-protein-coupled receptor:The cholecystokinin-1 receptor

The Cholecystokinin-1 receptor (CCK1R) mediates actions of CCK in areas of the central nervous system and of the gut. It is a potential target to treat a number of diseases. As for all G-protein-coupled receptors, docking of ligands into modeled CCK1R binding site should greatly help to understand intrinsic mechanisms of activation. Here, we describe the procedure we used to progressively build a structural model for the CCK1R, to integrated, and on the basis of site-directed mutagenesis data on its binding site. Reliability of the CCK1R model was confirmed by interaction networks that involved conserved and functionally crucial motifs in G-protein-coupled receptors, such as Glu/Asp-Arg-Tyr and Asn-Pro-Xaa-Xaa-Tyr motifs. In addition, the 3-D structure of CCK1R-bound CCK resembled that determined by NMR in a lipid environment. The derived computational model was also used for revealing binding modes of several nonpeptide ligands and for rationalizing ligand structure-activity relationships known from experiments. Our findings indeed support that our "validated CCK1R model" could be used to study the intrinsic mechanism of CCK1R activation and design new ligands.

Binding site of AMPA-receptor allosteric modulator

The computer molecular docking of piperonyl acid piperidide (BDP) and some its analogs already known as ampakins was conducted for estimating their possible binding with AMPA-receptor glutamate domains in cyclothiazide binding area and for further design of new structures maximally complimentary to the receptor. On the base of the conducted docking it can be suggested that the binding site of BDP (amides of benzodioxane-6-carboxylic and piperonyl acids) analogs is located in AMPA-receptor cyclothiazide binding pocket. It is shown that formation of protein-ligand complexes of AMPA-receptor with benzodioxane-6-carboxylic and piperonyl acid derivatives, similarly to cyclothiazide, proceeds with interaction with Ser497, Leu751, which significance is confirmed by site-specific mutagenesis.

Mass spectrometric analysis of muscle samples to detect potential antibiotic growth promoter misuse in broiler chickens

Mass spectrometric methods were developed and validated for the analysis in chicken muscle of a range of antibiotic growth promoters: spiramycin, tylosin, virginiamycin and bacitracin, and separately for two marker metabolites of carbadox (quinoxaline-2-carboxylic acid and 1,4-bisdesoxycarbadox), and a marker metabolite of olaquindox (3-methyl-quinoxaline-2-carboxylic acid). The use of these compounds as antibiotic growth promoters has been banned by the European Commission. This study aimed to develop methods to detect their residues in muscle samples as a means of checking for the use of these drugs during the rearing of broiler chickens. When fed growth-promoting doses for 6 days, spiramycin (31.4 mu g kg(-1)), tylosin (1.0 mu g kg(-1)), QCA (6.5 mu g kg(-1)), DCBX (71.2 mu g kg(-1)) and MQCA (0.2 mu g kg(-1)) could be detected in the muscle 0 days after the withdrawal of fortified feed. Only spiramycin could consistently be detected beyond a withdrawal period of 1 day. All analytes showed stability commercial cooking process, therefore raw or cooked muscle could be used for monitoring purposes.

Interlaboratory ring test of time-resolved fluoroimmunoassays for zeranol and alpha-zearalenol and comparison with zeranol test kits

Many zeranol immunoassay test kits cross-react with toxins formed by naturally occurring Fusarium spp. fungi, leading to false-positive screening results. This paper describes the evaluation and application of recently published, dry reagent time-resolved fluoroimmunoassays (TR-FIA) for zeranol and the toxin alpha-zearalenol. A ring test of bovine urine fortified with zeranol and/or alpha-zearalenol in four European Union National Reference Laboratories demonstrated that the TR-FIA tests were accurate and robust. The alpha-zearalenol TR-FIA satisfactorily quantified alpha-zearalenol in urine fortified at 10-30 ng ml(-1) . The specificity-enhanced zeranol TR-FIA accurately quantified zeranol in the range 2-5 ng ml(-1) and gave no false-positive results in blank urine, even in the presence of 30 ng ml(-1) alpha-zearalenol. Zeranol TR-FIA specificity was demonstrated further by analysing incurred zeranol-free urine samples containing natural Fusarium spp. toxins. The TR-FIA yielded no false-positive results in the presence of up to 22 ng ml(-1) toxins. The performance of four commercially available zeranol immunoassay test kits was more variable. Three kits produced many false-positive results. One kit produced only one potential false-positive using a protocol that was longer than that of the TR-FIA. These TR-FIAs will be valuable tools to develop inspection criteria to distinguish illegal zeranol abuse from contamination arising from in vivo metabolism of Fusarium spp. toxins.

Depletion of four nitrofuran antibiotics and their tissue-bound metabolites in porcine tissues and determination using LC-MS/MS and HPLC-UV

Depletion of the nitrofuran antibiotics furazolidone, furaltadone, nitrofurantoin and nitrofurazone and their tissue-bound metabolites AOZ, AMOZ, AHD and SEM from pig muscle, liver and kidney tissues is described. Groups of pigs were given feed medicated with one of the nitrofuran drugs at a therapeutic concentration (400 mg kg(-1)) for ten days. Animals were slaughtered at intervals and tissue samples collected for analysis for six weeks following withdrawal of medicated feed. These samples were analysed both for parent nitrofurans (using LC-MS/MS and HPLC-UV), and for tissue-bound metabolites (using LC-MS/MS). The parent drugs were detectable only sporadically and only in pigs subjected to no withdrawal period whatsoever. This confirms the instability of the four major nitrofuran antibiotics in edible tissues. In contrast, the metabolites accumulated to high concentrations in tissues (ppm levels) and had depletion half lives of between 5.5 and 15.5 days. The metabolites of all four drugs were still readily detectable in tissues six weeks after cessation of treatment. This emphasizes the benefits of monitoring for the stable metabolites of the nitrofurans.

Stability studies of the metabolites of nitrofuran antibiotics during storage and cooking

Nitrofuran antibiotics cannot be used in food production within the European Union because of their potential health risks to consumers. The recent discovery of their widespread use in global food industries and the finding of semicarbazide in baby food as a result of packaging contamination have focused attention on the toxicity and stability of these drugs and their metabolites. The stability of the nitrofuran marker residues 3-amino-2-oxazolidinone (AOZ), 3-amino-5-morpholinomethyl-2-oxazolidone (AMOZ), 1-aminohydantoin (AHD) and semicarbazide (SEM) were tested. Muscle and liver of nitrofuran treated pigs were cooked by frying, grilling, roasting and microwaving. Between 67 and 100% of the residues remained after cooking, demonstrating that these metabolites are largely resistant to conventional cooking techniques and will continue to pose a health risk. The concentration of metabolites in pig muscle and liver did not drop significantly during 8 months of storage at -20 degrees C. Metabolite stock and working standard solutions in methanol were also stable for 10 months at 4 degrees C. Only a 10 ng ml(-1) solution of SEM showed a small drop in concentration over this extended storage period.

Emergency slaughter of casualty cattle increases the prevalence of anthelmintic drug residues in muscle

The ProSafeBeef project studied the prevalence of residues of anthelmintic drugs used to control parasitic worms and fluke in beef cattle in Ireland. Injured (casualty) cattle may enter the human food chain under certain conditions, verified by an attending veterinarian and the livestock keeper. An analytical survey was conducted to determine if muscle from casualty cattle contained a higher prevalence of anthelmintic drug residues than healthy (full slaughter weight) cattle as a result of possible non-observance of complete drug withdrawal periods. A validated analytical method based on matrix solid-phase dispersive extraction (QuEChERS) and ultra-performance liquid chromatography-tandem mass spectrometry was used to quantify 37 anthelmintic drugs and metabolites in muscle (assay decision limits, CCa, 0.15-10.2 µg kg -1). Of 199 control samples of beef purchased in Irish shops, 7% contained detectable anthelmintic drug residues but all were compliant with European Union Maximum Residue Limits (MRL). Of 305 muscle samples from injured cattle submitted to abattoirs in Northern Ireland, 17% contained detectable residues and 2% were non-compliant (containing either residues at concentrations above the MRL or residues of a compound unlicensed for use in cattle). Closantel and ivermectin were the most common residues, but a wider range of drugs was detected in muscle of casualty cattle than in retail beef. These data suggest that specific targeting of casualty cattle for testing for anthelmintic residues may be warranted in a manner similar to the targeted testing for antimicrobial compounds often applied in European National Residues Surveillance Schemes. © 2012 Copyright Taylor and Francis Group, LLC.

Anthelmintic drug residues in beef: UPLC-MS/MS method validation, European retail beef survey, and associated exposure and risk assessments

Anthelmintic drugs are widely used to control parasitic infections in cattle. The ProSafeBeef project addressed the need for data on the exposure of European consumers of beef to potentially harmful drug residues. A novel analytical method based on matrix solid-phase dispersive extraction and ultra-performance liquid chromatography-tandem mass spectrometry was validated for 37 anthelmintic drugs and metabolites in muscle (assay decision limits, CCa, = 0.15-10.2 µg kg -1). Seven European countries (France, Spain, Slovenia, Ireland, Italy, Belgium and Portugal) participated in a survey of retail beef purchased in local shops. Of 1061 beef samples analysed, 26 (2.45%) contained detectable residues of anthelmintic drugs (0.2-171 µg kg -1), none above its European Union maximum residue limit (MRL) or action level. Residues detected included closantel, levamisole, doramectin, eprinomectin, moxidectin, ivermectin, albendazole and rafoxanide. In a risk assessment applied to mean residue concentrations across all samples, observed residues accounted for less than 0.1% of the MRL for each compound. An exposure assessment based on the consumption of meat at the 99th percentile of consumption of adults in 14 European countries demonstrated that beef accounted for less than 0.02% of the acceptable daily intake for each compound in each country. This study is the first of its kind to apply such a risk-based approach to an extensive multi-residue survey of veterinary drug residues in food. It has demonstrated that the risk of exposure of the European consumer to anthelmintic drug residues in beef is negligible, indicating that regulation and monitoring is having the desired effect of limiting residues to non-hazardous concentrations. © 2012 Copyright Taylor and Francis Group, LLC.

Stability during cooking of anthelmintic veterinary drug residues in beef

Anthelmintic drugs are widely used for treatment of parasitic worms in livestock, but little is known about the stability of their residues in food under conventional cooking conditions. As part of the European Commissionfunded research project ProSafeBeef, cattle were medicated with commercially available anthelmintic preparations, comprising 11 active ingredients (corresponding to 21 marker residues). Incurred meat and liver were cooked by roasting (40 min at 190°C) or shallow frying (muscle 8-12 min, liver 14-19 min) in a domestic kitchen. Raw and cooked tissues and expressed juices were analysed using a novel multi-residue dispersive solid-phase extraction method (QuEChERS) coupled with ultra-performance liquid chromatography-tandem mass spectrometry. After correction for sample weight changes during cooking, no major losses were observed for residues of oxyclozanide, clorsulon, closantel, ivermectin, albendazole, mebendazole or fenbendazole. However, significant losses were observed for nitroxynil (78% in fried muscle, 96% in roast muscle), levamisole (11% in fried muscle, 42% in fried liver), rafoxanide (17% in fried muscle, 18% in roast muscle) and triclabendazole (23% in fried liver, 47% in roast muscle). Migration of residues from muscle into expressed cooking juices varied between drugs, constituting 0% to 17% (levamisole) of total residues remaining after cooking. With the exception of nitroxynil, residues of anthelmintic drugs were generally resistant to degradation during roasting and shallow frying. Conventional cooking cannot, therefore, be considered a safeguard against ingestion of residues of anthelmintic veterinary drugs in beef. © 2011 Taylor & Francis.

Kinetics of semicarbazide and nitrofurazone in chicken eggs and egg powders

The accumulation, depletion and partitioning of semicarbazide (SEM) and its parent compound nitrofurazone (NFZ) in eggs were studied using hens fed NFZ at therapeutic and sub-therapeutic levels. Dietary NFZ correlated strongly with NFZ and total SEM in eggs, while 28% of observed SEM was present in the form of parent NFZ. Depletion half-life in eggs was 2.4 days for SEM and 1.1 days for NFZ. NFZ accumulated preferentially in yolk (57-63%) as opposed to albumen, while 71-80% of SEM was found in yolk. In whole egg, 29% of SEM was present as tissue-bound residues compared with 80% in breast muscle. Whilst NFZ and SEM were partly degraded by pasteurization and spray drying, sufficient NFZ remained to suggest it might be detectable in egg powders when SEM is observed at low µg kg -1 concentrations. NFZ was detectable in whole eggs during ingestion of only 0.1% of the therapeutic NFZ dose, making detection of intact NFZ in eggs a feasible means to prove conclusively the administration of this banned compound.

Residues of nitrofuran antibiotic parent compounds and metabolites in eyes of broiler chickens

Nitrofuran antibiotic residues in food continue to be of international concern. The finding of sources of semicarbazide (SEM), other than through the misuse of nitrofurazone, present a challenge to the use of SEM as a definitive marker residue for this drug. Detection of intact (parent) nitrofurazone would avoid confusion over the source of SEM residues. Broiler chickens were fed sub-therapeutic nitrofuran-containing diets and their tissues were analysed for parent compounds and metabolites by liquid chromatography coupled with tandem mass spectrometry detection (LC-MS/MS). Depletion half-lives in muscle were longer for tissue-bound metabolite residues, 3.4 days - 3-amino-2-oxazolidinone (AOZ), 3-amino-5-morpholinomethyl-2-oxazolidone (AMOZ) - to 4.5 days (SEM), than total metabolite residues, 2.0 days (AOZ) to 3.2 days (SEM). Metabolite concentrations were higher in eyes than in muscle. Metabolite half-lives in eyes ranged from 8.5 days (1-aminohydantoin (AHD)) to 20.3 days (SEM). Nitrofuran parent compounds were also detected in eyes. Furaltadone was detected in single eyes after 21 days' withdrawal of a 6 mg kg -1 furaltadone diet. When 50 eyes from broilers containing metabolites in muscle close to the 1 µg kg -1 minimum required performance level (MRPL) were pooled into single samples, 1.2 ng of furazolidone and 31.1 ng of furaltadone were detected, but nitrofurazone was not detected due to the long depletion half-life of SEM in muscle. Further studies are required to improve LC-MS/MS nitrofurazone sensitivity and refine the sample size necessary to use nitrofurazone detection in pooled eyes as a complement to SEM detection in muscle.

Structural and functional relationships in the virulence-associated cathepsin L proteases of the parasitic liver fluke, Fasciola hepatica

The helminth parasite Fasciola hepatica secretes cysteine proteases to facilitate tissue invasion, migration, and development within the mammalian host. The major proteases cathepsin L1 (FheCL1) and cathepsin L2 (FheCL2) were recombinantly produced and biochemically characterized. By using site-directed mutagenesis, we show that residues at position 67 and 205, which lie within the S2 pocket of the active site, are critical in determining the substrate and inhibitor specificity. FheCL1 exhibits a broader specificity and a higher substrate turnover rate compared with FheCL2. However, FheCL2 can efficiently cleave substrates with a Pro in the P2 position and degrade collagen within the triple helices at physiological pH, an activity that among cysteine proteases has only been reported for human cathepsin K. The 1.4-A three-dimensional structure of the FheCL1 was determined by x-ray crystallography, and the three-dimensional structure of FheCL2 was constructed via homology-based modeling. Analysis and comparison of these structures and our biochemical data with those of human cathepsins L and K provided an interpretation of the substrate-recognition mechanisms of these major parasite proteases. Furthermore, our studies suggest that a configuration involving residue 67 and the "gatekeeper" residues 157 and 158 situated at the entrance of the active site pocket create a topology that endows FheCL2 with its unusual collagenolytic activity. The emergence of a specialized collagenolytic function in Fasciola likely contributes to the success of this tissue-invasive parasite.

A novel toxicity fingerprinting method for pollutant identification with lux-marked biosensors

A novel technique is described for the identification and quantification of environmental pollutants based on toxicity fingerprinting with a metabolic lux-marked bacterial biosensor. This method involved characterizing the toxicity-based responses of the biosensor to seven calibration pollutants as acute temporal-dose response fingerprints. An algorithm is described to allow comparisons of responses of an unknown pollutant to be made against the calibration data. This is based on predicting pollutant concentration at each of six different time points over the course of a 5-min assay. If the prediction is consistent between the unknown pollutant and a calibration pollutant at the 95% test level, this is considered to be a positive identification. All seven calibration pollutants could be successfully distinguished from each other with this technique. Environmental samples, individually spiked with single concentrations of pollutants, were compared in this way against the calibration pollutants. An 83% identification success was achieved, with no false positives at the 95% test level. This is a simple and rapid technique that potentially can be applied to monitoring of industrial wastewater or as a screening tool for regulators.