Kasstasin: A novel potent vasoconstrictor peptide from the skin secretion of the African red-legged running frog, Kassina maculata

Amphibian skin secretions are established sources of bioactive peptides. Here we describe the isolation, structural and pharmacological characterisation of a novel vasoconstrictor peptide from the skin secretion of the African hyperoliid frog, Kassina maculata, which exhibits no structural similarity to any known class of amphibian skin peptide. The peptide consists of 21 amino acid residues, FIKELLPHLSGIIDSVANAIK, and is C-terminally amidated. The provisional structure was obtained by MS/MS fragmentation using an Orbitrap mass spectrometer and L/I ambiguities were resolved following molecular cloning of biosynthetic precursor-encoding cDNA. A synthetic replicate of the peptide was found to possess weak antimicrobial and haemolytic activities but was exceptionally effective in constricting the smooth muscle of rat tail artery (EC50 of 25pM). In reflection of its exceptional potency in constricting rat arterial smooth muscle, the peptide was named kasstasin, a derivation of Kassina and “stasis” (stoppage of flow). These data illustrate the continuing potential of amphibian skin secretions to provide novel natural peptide templates for biological evaluation.

Apo J/clusterin expression and secretion: Evidence for 15-deoxy-Δ12,14-PGJ2-dependent mechanism

Cyclooxygenase-2 (Cox-2) and Apo J/clusterin are involved in inflammatory resolution and have each been reported to inhibit NF-?B signalling. Using a well-validated rat pheochromocytoma (PC12) cell culture model of Cox-2 over-expression the current study investigated inter-dependence between Cox-2 and clusterin with respect to induction of expression and impact on NF-?B signalling. Both gene expression and immunoblot analysis confirmed that intracellular and secreted levels of clusterin were elevated in Cox-2 over-expressing cells (PCXII). Clusterin expression was increased in control (PCMT) cells in a time- and dose-dependent manner by 15-deoxy-? 12,14-prostaglandin J 2 (15d-PGJ 2), but not PGE 2, and inhibited in PCXII cells by pharmacological Cox inhibition. In PCXII cells, inhibition of two transcription factors known to be activated by 15d-PGJ 2, heat shock factor 1 (HSF-1) and peroxisome proliferator activated receptor (PPAR)?, by transcription factor oligonucleotide decoy and antagonist (GW9662) treatment, respectively, reduced clusterin expression. While PCXII cells exhibited reduced TNF-a-induced cell surface ICAM-1 expression, IkB phosphorylation and degradation were similar to control cells. With respect to the impact of Cox-2-dependent clusterin upregulation on NF-?B signalling, basal levels of I?B were similar in control and PCXII cells, and no evidence for a physical association between clusterin and phospho-I?B was obtained. Moreover, while PCXII cells exhibited reduced NF-?B transcriptional activity, this was not restored by clusterin knock-down. These results indicate that Cox-2 induces clusterin in a 15d-PGJ 2-dependent manner, and via activation of HSF-1 and PPAR?. However, the results do not support a model whereby Cox-2/15d-PGJ 2-dependent inhibition of NF-?B signalling involves clusterin.

Functional peptidomics of amphibian skin secretion: A novel Kunitz-type chymotrypsin inhibitor from the African hyperoliid frog, Kassina senegalensis

Amphibian skin secretions are, for the most part, complex peptidomes. While many peptide components have been biologically- and structurally-characterised into discrete "families", some of which are analogues of endogenous vertebrate regulatory peptides, a substantial number are of unique structure and unknown function. Among the components of these secretory peptidomes is an array of protease inhibitors. Inhibitors of trypsin are of widespread occurrence in different taxa and are representative of many established structural classes, including Kunitz, Kazal and Bowman-Birk. However, few protease inhibitors with activity against other specific proteases have been described from this source. Here we report for the first time, the isolation and structural characterisation of an inhibitor of chymotrypsin of Kunitz-type from the skin secretion of the African hyperoliid frog, Kassina senegalensis. To this end, we employed a functional peptidomic approach. This scheme involves fractionation of the peptidome, functional end-point screening, structural characterisation of resultant actives followed by molecular cloning of biosynthetic precursor-encoding cDNA(s). The novel mature and active polypeptide identified consisted of 62 amino acid residues (average molecular mass 6776.24 Da), of which 6 were positionally-conserved cysteines. The P(1) position within the active site was occupied by a phenylalanyl residue. Bioinformatic analysis of the sequence using BLAST, revealed a structural similarity to Kunitz-type chymotrypsin inhibitors from other organisms, ranging from silkworms to snakes.

Investigating the effects of physiological bile acids on GLP-1 secretion and glucose tolerance in normal and GLP-1R(-/-) mice

Physiological secretion of bile acids has previously been linked to the regulation of blood glucose. GLP-1 is an intestinal peptide hormone with important glucose-lowering actions, such as stimulation of insulin secretion and inhibition of glucagon secretion. In this investigation, we assessed the ability of several bile acid compounds to secrete GLP-1 in vitro in STC-1 cells. Bile acids stimulated GLP-1 secretion from 3.3- to 6.2-fold but some were associated with cytolytic effects. Glycocholic and taurocholic acids were selected for in vivo studies in normal and GLP-1R(-/-) mice. Oral glucose tolerance tests revealed that glycocholic acid did not affect glucose excursions. However, taurocholic acid reduced glucose excursions by 40% in normal mice and by 27% in GLP-1R(-/-) mice, and plasma GLP-1 concentrations were significantly elevated 30 min post-gavage. Additional studies used incretin receptor antagonists to probe involvement of GLP-1 and GIP in taurocholic acid-induced glucose lowering. The findings suggest that bile acids partially aid glucose regulation by physiologically enhancing nutrient-induced GLP-1 secretion. However, GLP-1 secretion appears to be only part of the glucose-lowering mechanism and our studies indicate that the other major incretin GIP is not involved.

Screening and Quantitative Confirmatory Method for the Analysis of Glucocorticoids in Bovine Milk Using Liquid Chromatography-Tandem Mass Spectrometry

An LC/MS/MS method was developed and validated for the simultaneous identification, confirmation, and quantification of 12 glucocorticoids in bovine milk. The method was validated in accordance with the criteria defined in Commission Decision 2002/657/EC. The developed method can detect and confirm the presence of dexamethasone, betamethasone, prednisolone, flumethasone, 6 alpha-methylprednisolone, fluorometholone, triamcinolone acetonide, prednisone, cortisone, hydrocortisone, clobetasol propionate, and clobetasol butyrate in bovine milk. Milk samples are extracted with acetonitrile; sodium chloride is subsequently added to aid partition of the milk and acetonitrile mixture. The acetonitrile extract is then subjected to liquid-liquid purification by the addition of hexane. The purified extract is evaporated to dryness and reconstituted in a water acetonitrile mixture, and determination is carried out by LC/MS/MS. The method permits analysis of up to 30 samples in 1 day.

Benzimidazole carbamate residues in milk: Detection by Surface Plasmon Resonance-biosensor, using a modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) method for extraction

A surface plasmon resonance (SPR) biosensor screening assay was developed and validated to detect 11 benzimidazole carbamate (BZT) veterinary drug residues in milk. The polyclonal antibody used was raised in sheep against a methyl 5(6)-[(carboxypentyl)-thio]-2-benzimidazole carbamate protein conjugate. A sample preparation procedure was developed using a modified QuEChERS method. BZT residues were extracted from milk using liquid extraction/partition with a dispersive solid phase extraction clean-up step. The assay was validated in accordance with the performance criteria described in 2002/657/EC. The limit of detection of the assay was calculated from the analysis of 20 known negative milk samples to be 2.7 mu g kg(-1). The detection capability (CC beta) of the assay was determined to be 5 mu g kg(-1) for 11 benzimidazole residues and the mean recovery of analytes was in the range 81-116%. A comparison was made between the SPR-biosensor and UPLC-MS/MS analyses of milk samples (n = 26) taken from cows treated different benzimidazole products, demonstrating the SPR-biosensor assay to be fit for purpose. (C) 2009 Elsevier B.V. All rights reserved.

Development of a rapid, multi-class method for the confirmatory analysis of anti-inflammatory drugs in bovine milk using liquid chromatography tandem mass spectrometry

A rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the simultaneous identification, confirmation and quantitation of seven licensed anti-inflammatory drugs (AIDS) in bovine milk. The method was validated in accordance with the criteria defined in Commission Decision 2002/657/EC. Two classes of AIDS were investigated, corticosteroids and non-steroidal anti-inflammatory drugs (NSAIDs). The developed method is capable of detecting and confirming dexamethasone (DXM), betamethasone (BTM), prednisolone (FRED), tolfenamic acid (TV), 5-hydroxy flunixin (5-OH-FLU). meloxicam (MLX) and 4-methyl amino antipyrine (4-MAA) at their associated maximum residue limits (MRLs). These compounds represent all the corticosteroids and NSAIDs licensed for use in bovine animals producing milk for human consumption. These compounds have never been analysed before in the same method and also 4-methyl amino antipyrine has never been analysed with the other licensed NSAIDs. The method can be considered rapid as permits the analysis of up to 30 samples in one day. Milk samples are extracted with acetonitrile; sodium chloride is added to aid partition of the milk and acetonitrile mixture. The acetonitrile extract is then subjected to liquid-liquid purification by the addition of hexane. The purified extract is finally evaporated to dryness and reconstituted in a water/acetonitrile mixture and determination is carried out by LC-MS/MS. Decision limit (CC alpha) values and detection capability (CC beta) values have been established for each compound. (C) 2009 Elsevier B.V. All rights reserved.

Biosensor immunoassay of ivermectin in bovine milk

A rapid and sensitive biosensor immunoassay was developed for determination of ivermectin residues in bovine milk. A detection limit of 16.2 ng/mL was achieved. A Biacore optical biosensor based on surface plasmon resonance was used, and a range of extraction techniques was investigated. In the final assay procedure, ivermectin was extracted with acetonitrile followed by C-8 solid-phase extraction cleanup. It was proven experimentally that 2 methods of milk storage, freezing or addition of mercury-containing compounds as preservatives, could be used without considerable change in detected concentrations (samples were fortified with ivermectin after storage). The average values for milk samples spiked at 100 and 50 ng/mL concentrations were 102.6 and 51.5 ng/mL, respectively. Extraction and analysis of 20 milk samples were performed within a single working day.

Detection of streptomycin residues in whole milk using an optical immunobiosensor

The development of an assay for the detection of streptomycin residues in pasteurized whole milk using an optical biosensor (Biacore) is reported. Streptomycin-adipic hydrazide coupled to bovine thyroglobulin was used to produce a sheep polyclonal antibody. The antibody displayed excellent cross-reactivity with dihydrostreptomycin (106%). There was no significant cross-reaction with other aminoglycosides or common antibiotics. Streptomycin was also immobilized onto a CM5 sensor chip to provide a stable, reusable surface. The developed assay permitted the direct analysis of whole milk samples (similar to3.5% fat) without prior centrifugation and defatting. Results were available in 5 min. The limit of detection of the assay was determined as 4.1 ng/mL, well below the European maximum residue limit (MRL) of 200 ng/mL. Repeatability (or coefficient of variation) between runs was determined as 3.5% (100 ng/mL; 0.5 x MRL), 5.7% (200 ng/mL; MRL), and 7.6% (400 ng/mL; 2 x MRL).

Detection of unwanted residues of ivermectin in bovine milk by dissociation-enhanced lanthanide fluoroimmunoassay

Avermectins are frequently used to control parasitic infestations in many animal species. Previous studies have shown the long-term persistence of unwanted residues of these drugs in animal tissues and fluids. An immunoassay screening test for the detection acid quantification of ivermectin residues in bovine milk has been developed. After an extensive extraction procedure, milk samples were applied to a competitive dissociation-enhanced lanthanide fluoroimmunoassay using a monoclonal antibody against an ivermectin-transferrin conjugate, The monoclonal antibody, raised in Balb C mice, showed cross-reactivity with eprinomectin (92%), abamectin (82%) and doramectin (16%). The limit of detection of the assay (mean + 3 SD), calculated from the analysis of 17 known negative samples, was calculated as 4.6 ng/mL. Intra- and inter-assay RSDs were determined as 11.6% and 15.8%, respectively, using a negative bovine milk sample fortified with 25 ng/mL ivermectin. Six Friesian milking cows were treated with ivermectin, three with a pour-on formulation of the drug and three with an injectable solution at the manufacturer's recommended dose rate. An initial mean peak in ivermectin residue concentration was detected at day 4 (mean level = 47.5 ng/mL) and day 5 post-treatment (mean level = 26.4 ng/mL) with the injectable form and pour-on treatment, respectively. A second peak in residue concentration was observed using the DELFIA(R) procedure 28 days post-treatment in both treatment groups (23.1 ng/mL injectable and 51.9 ng/mL pour-on). These second peaks were not confirmed by HPLC and must at this Lime be considered to be false-positive results. By day 35 after treatment the mean ivermectin residue concentration of both groups fell below the limit of detection of the assay. Copyright (C) 2000 John Wiley & Sons, Ltd.