Detection of ivermectin residues in bovine liver using an enzyme immunoassay

Ivermectin, a member of the avermectin group, is frequently used to control parasites in many food producing animal species. A method for the detection and quantification of ivermectin residues in bovine liver has been developed. Liver samples (4 g) were extracted with acetonitrile and applied to a competitive enzyme immunoassay using a polyclonal antiserum raised in rabbits against an ivermectin-transferrin conjugate, The limit of detection of the assay (mean +/- 3s) calculated from the analysis of 24 known negative samples was 1.6 ng g(-1), Intra- and inter-assay RSDs were determined as 8.8 and 14.6%, respectively, using a negative bovine liver sample fortified with 100 ng g(-1) of ivermectin. Four Friesian steers were treated with a pour-on application of ivermectin at a dose rate of 0.5 mg kg(-1) body mass then withdrawn and killed at 7, 14, 21 and 28 d, Livers mere removed and ivermectin residue concentrations determined using the proposed immunoassay procedure, Seven days post-treatment the ivermectin liver concentration was determined as 52.7 ng g(-1), decreasing to 4.1 ng(-1) at 28 d, All immunoassay results were confirmed using high-performance liquid chromatography (HPLC), The immunoassay and HPLC results for invermectin ranged from 1 to 58 ng g(-1) and were in close correlation (r = 0.99).

PATHOLOGY AND RESIDUES IN VEAL CALVES TREATED EXPERIMENTALLY WITH CLENBUTEROL

Six veal calves were medicated with clenbuterol at 20 mu g kg bodyweightl day(-1) for 42 days before they were slaughtered, to evaluate the lesions and residues in target organs. Compared with six unmedicated calves the most noticeable changes were tracheal dilatation, decreased uterine weight, slight mucous hypersecretion in the uterus and vagina and depletion of liver glycogen. The highest concentrations of clenbuterol (62 to 128 ng/g(-1)) were recorded in the choroid/retina, and the aqueous humour had the lowest concentration (0.5 to 2.4 ng ml(-1)). The residue concentrations were higher than the maximum residue level set for clenbuterol (0.5 ng g(-1))

N-acetylgalactosamine kinase: a naturally promiscuous small molecule kinase

N-acetylgalactosamine kinase is a member of the GHMP family of small molecule kinases which catalyses the ATP-dependent phosphorylation of N-acetylgalactosamine. It is highly similar in structure and sequence to galactokinase. Alteration of galactokinase at a key tyrosine residue (Tyr-379 in the human enzyme) has been shown to dramatically enhance the substrate range of this enzyme. Here, we investigated the substrate specificity of the wild type N-acetylgalactosamine kinase and demonstrated that it can also catalyse the phosphorylation of N-acetylglucosamine and N-acetylmannosamine. In human N-acetylgalactosamine kinase, the equivalent residue to Tyr-379 in galactokinase is Phe-444. Alteration of this residue did not result in dramatic changes to the specificity of the enzyme. The more relaxed substrate specificity of N-acetylgalactosamine kinase, compared to galactokinase, can be explained by the greater flexibility of a glycine rich loop in the active site of the enzyme. These results suggest that N-acetylgalactosamine kinase is a potential biocatalyst for the phosphorylation of N-acetyl sugars. However, it is unlikely that it will be possible to further broaden the substrate range by alteration of Phe-444.

Structure-activity relationships of FMRFamide-related peptides contracting Schistosoma mansoni muscle

This study reports the potent myoactivity of flatworm FMRFamide-related peptides (FaRPs) on isolated muscle fibers of the human blood fluke, Schistosoma mansoni. The turbellarian peptides YIRFamide (EC50 4 eta M), GYIRFamide (EC50 1 eta M). and RYIRFamide (EC50 7 eta M), all induced muscle contraction more potently than the cestode FaRP GNFFRFamide (EC50 500 eta M). Using a series of synthetic analogs of the flatworm peptides YIRFamide, GYIRFamide and RYIRFamide, the structure-activity relationships of the muscle FaRP receptor were examined. With a few exceptions, each residue in YIRFamide is important in the maintenance of its myoactivity. Alanine scans resulted in peptides that were inactive (Ala(1), Ala(2), Ala(3) and Ala(4) YIRFamide; Ala(4) and Ala(5) RYIRFamide) or had much reduced potencies (Ala(1), Ala(2) and Ala(3) RYIRFamide). Substitution of the N-terminal (Tyr(1)) residue of YIRFamide with the non-aromatic residues Thr or Arg produced analogs with greatly reduced potency. Replacement of the N-terminal Tyr with aromatic amino acids resulted in myoactive peptides (FIRFamide, EC50 100 eta M; WIRFamide, EC50 0.5 eta M). The activity of YIRFamide analogs which possessed a Leu(2), Phe(2) or Met(2) residue (EC50's 10, 1 and 3 eta M, respectively) instead of Ile(2) was not significantly altered, whereas, YVRFamide had a greatly reduced (EC50 200 eta M) activity. Replacement of the Phe(4) with a Tyr(4) (YIRYamide) also greatly lowered potency. Truncated analogs were either inactive (FRFamide, YRFamide, HRFamide, RFamide, Famide) or had very low potency (IRFamide and MRFamide), with the exception of nLRFamide (EC50 20 eta M). YIRF free acid was inactive. In summary, these data show the general structural requirements of this schistosome muscle FaRP receptor to be similar, but not identical, to those of previously characterized molluscan FaRP receptors. (C) 1997 Elsevier Science Inc.

ISOLATION AND PRIMARY STRUCTURE OF A NOVEL AVIAN PANCREATIC-POLYPEPTIDE FROM 5 SPECIES OF EURASIAN CROW

Chicken pancreatic polypeptide is the prototype of the neuropeptide Y (NPY)/PP superfamily of regulatory peptides. This polypeptide was appended the descriptive term avian, despite the presence of some 8600 extant species of bird. Additional primary structures from other avian species, including turkey, goose and ostrich, would suggest that the primary structure of this polypeptide has been highly-conserved during avian evolution. Avian pancreatic polypeptides structurally-characterised to date have distinctive primary structural features unique to this vertebrate group including an N-terminal glycyl residue and a histidyl residue at position 34. The crow family, Corvidae, is representative of the order Passeriformes, generally regarded as the most evolutionarily recent and diverse avian taxon. Pancreatic polypeptide has been isolated from pancreatic tissues from five representative Eurasian species (the magpie, Pica pica; the jay, Garrulus glandarius; the hooded crow, Corvus corone; the rook, Corvus frugilegus; the jackdaw, Corvus monedula) and subjected to structural analyses. Mass spectroscopy estimated the molecular mass of each peptide as 4166 +/- 2 Da. The entire primary structures of 36 amino acid residue peptides were established in single gas-phase sequencing runs. The primary structures of pancreatic polypeptides from all species investigated were identical: APAQPAYPGDDAPVEDLLR-FYNDLQQYLNVVTRPRY. The peptides were deemed to be amidated due to their full molar cross-reactivity with the amide-requiring PP antiserum employed. The molecular mass (4165.6 Da), calculated from the sequences, was in close agreement with mass spectroscopy estimates. The presence of an N-terminal alanyl residue and a prolyl residue at position 34 differentiates crow PP from counterparts in other avian species. These residues are analogous to those found in most mammalian analogues. These data suggest that the term avian, appended to the chicken peptide, is no longer tenable due to the presence of an Ala1, Pro34 peptide in five species from the largest avian order. These data might also suggest that, in keeping with the known structure/activity requirements of this peptide family, crow PP should interact identically to mammalian analogues on mammalian receptors.

THE CORRECTED PRIMARY STRUCTURE OF CHICKEN (AVIAN) PANCREATIC-POLYPEPTIDE

Chicken (avian) pancreatic polypeptide was the first member of the pancreatic polypeptide (PP)/neuropeptide Y (NPY) superfamily to be discovered and structurally-characterised. In this 36 amino acid residue, C-terminally amidated peptide, residues 22 and 23 were identified as Asp and Asn, respectively. However, sequencing of chicken PP using modem automated gas-phase sequencing technology has revealed that the original primary structure is incorrect in that residue 22 is Asn and that residue 23 is Asp. After digestion of chicken PP with endoproteinase Asp-N, fragments of chicken PP corresponding in molecular mass to residues 16-22 and 23-36, were unequivocally identified. The corrected primary structure of chicken PP is therefore: Gly-Pro-Ser-Gln-Pro-Thr-Tyr-Pro-Gly-Asp-Asp-Ala-Pro-Val-Glu-Asp-Leu-Ile-Arg-Phe-Tyr-Asn-Asp-Leu-Gln-Gln-Tyr-Leu-Asn-Val-Val-Thr-Arg-His-Arg-Tyr-NH2.

The WaaL O-antigen lipopolysaccharide ligase has features in common with metal ion-independent inverting glycosyltransferases(*)

WaaL is a membrane enzyme that catalyzes a key step in lipopolysaccharide (LPS) synthesis: the glycosidic bonding of a sugar at the proximal end of the undecaprenyl-diphosphate (Und-PP) O-antigen with a terminal sugar of the lipid A-core oligosaccharide (OS). Utilizing an in vitro assay, we demonstrate here that ligation with purified Escherichia coli WaaL occurs without adenosine-5'-triphosphate (ATP) and magnesium ions. Furthermore, E. coli and Pseudomonas aeruginosa WaaL proteins cannot catalyze ATP hydrolysis in vitro. We also show that a lysine substitution of the arginine (Arg)-215 residue renders an active protein, whereas WaaL mutants with alanine replacements in the periplasmic-exposed residues Arg-215, Arg-288 and histidine (His)-338 and also the membrane-embedded aspartic acid-389 are nonfunctional. An in silico approach, combining predicted topological information with the analysis of sequence conservation, confirms the importance of a positive charge at the small periplasmic loop of WaaL, since an Arg corresponding to Arg-215 was found at a similar position in all the WaaL homologs. Also, a universally conserved H[NSQ]X(9)GXX[GTY] motif spanning the C-terminal end of the predicted large periplasmic loop and the membrane boundary of the transmembrane helix was identified. The His residue in this motif corresponds to His-338. A survey of LPS structures in which the linkage between O-antigen and lipid A-core OS was elucidated reveals that it is always in the beta-configuration, whereas the sugars bound to Und-PP are in the alpha-configuration. Together, our biochemical and in silico data argue that WaaL proteins use a common reaction mechanism and share features of metal ion-independent inverting glycosyltransferases.

Different sugar residues of the lipopolysaccharide outer core are required for early interactions of Salmonella enterica serovars Typhi and Typhimurium with epithelial cells

The role of lipopolysaccharide (LPS) in entry of Salmonella Typhimurium into epithelial cells remains unclear. In this study, we tested the ability of a series of mutants with deletions in genes for the synthesis and assembly of the O antigen and the outer core of LPS to adhere to and invade HeLa, BHK, and IB3 epithelial cells lines. Mutants devoid of O antigen, or that synthesized only one O antigen unit, or with altered O antigen chain lengths were as able as the wild type to enter epithelial cells, indicating that this polysaccharide is not required for invasion of epithelial cells in vitro. In contrast, the LPS core plays a role in the interaction of S. Typhimurium with epithelial cells. The minimal core structure required for adherence and invasion comprised the inner core and residues Glc I Gal I of the outer core. A mutant of S. Typhimurium that produced a truncated LPS core lacking the terminal galactose residue had a significant lower level of adherence to and ingestion by the three epithelial cell lines than did strains with this characteristic. Complementation of the LPS production defect recovered invasion to parental levels. Heat-killed bacteria with a core composed of Glc 1 Gal I. but not bacteria with a core composed of Glc 1, inhibited uptake of the wild type by HeLa cells. A comparison of the chemical structure of the S. Typhi core with the published chemical structure of that of S. Typhimurium indicated that the Glc I Gal 1 Glc 11 backbone is conserved in both serovars. However, S. Typhi requires a terminal glucose for maximal invasion. Therefore, our data indicate that critical saccharide residues of the outer core play different roles in the early interactions of serovars Typhi and Typhimurium with epithelial cells. (C) 2010 Elsevier Ltd. All rights reserved.

Characterization of the highly conserved VFMGD motif in a bacterial polyisoprenyl-phosphate N-acetylaminosugar-1-phosphate transferase

Polyisoprenyl-phosphate N-acetylaminosugar-1-phosphate transferases (PNPTs) constitute a family of eukaryotic and prokaryotic membrane proteins that catalyze the transfer of a sugar-1-phosphate to a phosphoisoprenyl lipid carrier. All PNPT members share a highly conserved 213-Valine-Phenylalanine-Methionine-Glycine-Aspartic acid-217 (VFMGD) motif. Previous studies using the MraY protein suggested that the aspartic acid residue in this motif, D267, is a nucleophile for a proposed double-displacement mechanism involving the cleavage of the phosphoanhydride bond of the nucleoside. Here, we demonstrate that the corresponding residue in the E. coli WecA, D217, is not directly involved in catalysis, as its replacement by asparagine results in a more active enzyme. Kinetic data indicate that the D217N replacement leads to more than twofold increase in V(max) without significant change in the K(m) for the nucleoside sugar substrate. Furthermore, no differences in the binding of the reaction intermediate analog tunicamycin were found in D217N as well as in other replacement mutants at the same position. We also found that alanine substitutions in various residues of the VFMGD motif affect to various degrees the enzymatic activity of WecA in vivo and in vitro. Together, our data suggest that the highly conserved VFMGD motif defines a common region in PNPT proteins that contributes to the active site and is likely involved in the release of the reaction product.

Validation of membrane protein topology models by oxidative labeling and mass spectrometry

Computer-assisted topology predictions are widely used to build low-resolution structural models of integral membrane proteins (IMPs). Experimental validation of these models by traditional methods is labor intensive and requires modifications that might alter the IMP native conformation. This work employs oxidative labeling coupled with mass spectrometry (MS) as a validation tool for computer-generated topology models. ·OH exposure introduces oxidative modifications in solvent-accessible regions, whereas buried segments (e.g., transmembrane helices) are non-oxidizable. The Escherichia coli protein WaaL (O-antigen ligase) is predicted to have 12 transmembrane helices and a large extramembrane domain (Pérez et al., Mol. Microbiol. 2008, 70, 1424). Tryptic digestion and LC-MS/MS were used to map the oxidative labeling behavior of WaaL. Met and Cys exhibit high intrinsic reactivities with ·OH, making them sensitive probes for solvent accessibility assays. Overall, the oxidation pattern of these residues is consistent with the originally proposed WaaL topology. One residue (M151), however, undergoes partial oxidation despite being predicted to reside within a transmembrane helix. Using an improved computer algorithm, a slightly modified topology model was generated that places M151 closer to the membrane interface. On the basis of the labeling data, it is concluded that the refined model more accurately reflects the actual topology of WaaL. We propose that the combination of oxidative labeling and MS represents a useful strategy for assessing the accuracy of IMP topology predictions, supplementing data obtained in traditional biochemical assays. In the future, it might be possible to incorporate oxidative labeling data directly as constraints in topology prediction algorithms.

Functional analysis of the large periplasmic loop of the Escherichia coli K-12 WaaL O-antigen ligase

WaaL is a membrane enzyme implicated in ligating undecaprenyl-diphosphate (Und-PP)-linked O antigen to lipid A-core oligosaccharide. We determined the periplasmic location of a large (EL5) and small (EL4) adjacent loops in the Escherichia coli K-12 WaaL. Structural models of the EL5 from the K-12, R1 and R4 E. coli ligases were generated by molecular dynamics. Despite the poor amino acid sequence conservation among these proteins, the models afforded similar folds consisting of two pairs of almost perpendicular alpha-helices. One alpha-helix in each pair contributes a histidine and an arginine facing each other, which are highly conserved in WaaL homologues. Mutations in either residue rendered WaaL non-functional, since mutant proteins were unable to restore O antigen surface expression. Replacements of residues located away from the putative catalytic centre and non-conserved residues within the centre itself did not affect ligation. Furthermore, replacing a highly conserved arginine in EL4 with various amino acids inactivates WaaL function, but functionality reappears when the positive charge is restored by a replacement with lysine. These results lead us to propose that the conserved amino acids in the two adjacent periplasmic loops could interact with Und-PP, which is the common component in all WaaL substrates.

Functional characterization of the initiation enzyme of S-layer glycoprotein glycan biosynthesis in Geobacillus stearothermophilus NRS 2004/3a

The glycan chain of the S-layer glycoprotein of Geobacillus stearothermophilus NRS 2004/3a is composed of repeating units [-->2)-alpha-l-Rhap-(1-->3)-beta-l-Rhap-(1-->2)-alpha-l-Rhap-(1-->], with a 2-O-methyl modification of the terminal trisaccharide at the nonreducing end of the glycan chain, a core saccharide composed of two or three alpha-l-rhamnose residues, and a beta-d-galactose residue as a linker to the S-layer protein. In this study, we report the biochemical characterization of WsaP of the S-layer glycosylation gene cluster as a UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase that primes the S-layer glycoprotein glycan biosynthesis of Geobacillus stearothermophilus NRS 2004/3a. Our results demonstrate that the enzyme transfers in vitro a galactose-1-phosphate from UDP-galactose to endogenous phosphoryl-polyprenol and that the C-terminal half of WsaP carries the galactosyltransferase function, as already observed for the UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase WbaP from Salmonella enterica. To confirm the function of the enzyme, we show that WsaP is capable of reconstituting polysaccharide biosynthesis in WbaP-deficient strains of Escherichia coli and Salmonella enterica serovar Typhimurium.