Capillary zone electrophoresis investigation of interactions between granulocyte-colony stimulating factor and dextran sulfate/carrageenan oligosaccharide

The interactions between granulocyte-colony stimulating factor (G-CSF) and dextran sulfate/kappa-carrageenan oligosaccharide were studied by capillary zone electrophoresis. Dextran sulfate could strongly interact with G-CSF and the complex was detected. The binding constant and stoichiometry were determined to be 1.2x10(6) (mol/L)(-1) and 3:1, respectively. However, the interaction between K-carrageenan oligosaccharide and G-CSF was not found.

Synthesis, Kinetic Evaluation and Utilization of a Biotinylated Dipeptide Proline Diphenyl Phosphonate for the Disclosure of Dipeptidyl Peptidase IV-like Serine Proteases

In this study, we report on the synthesis, kinetic characterisation, and application of a novel biotinylated and active site-directed inactivator of dipeptidyl peptidase IV (DPP-IV). Thus, the dipeptide-derived proline diphenyl phosphonate NH(2)-Glu(biotinyl-PEG)-Pro(P)(OPh)(2) has been prepared by a combination of classical solution- and solid-phase methodologies and has been shown to be an irreversible inhibitor of porcine DPP-IV, exhibiting an over all second-order rate constant (k(i)/K(i)) for inhibition of 1.57 x 10(3) M(-1) min(-1). This value compares favourably with previously reported rates of inactivation of DPP-IV by dipeptides containing a P(1) proline diphenyl phosphonate grouping [B. Boduszek, J. Oleksyszyn, C.M. Kam, J. Selzler, R.E. Smith, J.C. Powers, Dipeptide phophonates as inhibitors of dipeptidyl peptidase IV, J. Med. Chem. 37 (1994) 3969-3976; B.F. Gilmore, J.F. Lynas, C.J. Scott, C. McGoohan, L. Martin, B. Walker, Dipeptide proline diphenyl phosphonates are potent, irreversible inhibitors of seprase (FAPalpha), Biochem, Biophys. Res. Commun. 346 (2006) 436-446.], thus demonstrating that the incorporation of the side-chain modified (N-biotinyl-3-(2-(2-(3-aminopropyloxy)-ethoxy)-ethoxy)-propyl) glutamic acid residue at the P(2) position is compatible with inhibitor efficacy. The utilisation of this probe for the detection of both purified dipeptidyl peptidase IV and the disclosure of a dipeptidyl peptidase IV-like activity from a clinical isolate of Porphyromonas gingivalis, using established electrophoretic and Western blotting techniques previously developed by our group, is also demonstrated.

In vitro release of dextran sulfate from silicone intravaginal rings

Abstract 02665

A surface plasmon resonance biosensor assay for the simultaneous determination of thiamphenicol, florefenicol, florefenicol amine and chloramphenicol residues in shrimps

A rapid and sensitive screening qualitative method using a surface plasmon resonance (SPR) biosensor was developed which can detect of all fenicol antibiotic residues in shrimps from a single sample extract. This method requires ethyl acetate extraction followed by a single wash with isooctane/chlorofonrm. Each sample extract is injected over the surfaces of two biosensor chip flow cells, one surface having the capability to detect florefenicol amine (FF amine), florefenicol (FF), and thiamphenicol (TAP) and the second surface for chloramphenicol (CAP) detection. The estimated detection capabilities (CC beta) were 0. 1, 0.2, 250, and 0.5 ppb for CAP, FF, FF amine, and TAP, respectively. This quick, simple test allowed the detection of CAP residues in shrimps at the minimum required performance limit (MRPL) of 0.1 mu g kg(-1) for this compound and of FF, FF amine, and TAP below their maximum residue limits (MRLs). (c) 2006 Elsevier B.V. All rights reserved.

Expedited Solid-Phase Synthesis of Fluorescently Labeled and Biotinylated Aminoalkane Diphenyl Phosphonate Affinity Probes for Chymotrypsin- and Elastase-Like Serine Proteases

In this study, we report on a novel, expedited solid-phase approach for the synthesis of biotinylated and fluorescently tagged irreversible affinity based probes for the chymotrypsin and elastase-like serine proteases. The novel solid-phase biotinylation or fluorescent labeling of the aminoalkane diphenyl phosphonate warhead using commercially available Biotin-PEG-NovaTag or EDANS NovaTag resin permits rapid, facile synthesis of these reagents. We demonstrate the kinetic evaluation and utilization of a number of these irreversible inactivators for chymotrypsin-like (chymotrypsin/human cathepsin G) and elastase-like serine proteases. Encouragingly, these compounds display comparable potency against their target proteases as their N-benzyloxycarbonyl (Cbz)-protected parent compounds, from which they were derived, and function as efficient active site-directed inactivators of their target proteases. We subsequently applied the biotinylated reagents for the sensitive detection of protease species via Western blot, showing that the inactivation of the protease was specifically mediated through the active site serine. Furthermore, we also demonstrate the successful detection of serine protease species with the fluorescently labeled derivatives “in-gel”, thus avoiding the need for downstream Western blotting. Finally, we also show the utility of biotinylated and pegylated affinity probes for the isolation/enrichment of serine protease species, via capture with immobilized streptavidin, and their subsequent identification via de novo sequencing. Given their selectivity of action against the serine proteases, we believe that these reagents can be exploited for the direct, rapid, and selective identification of these enzymes from biological milieu containing multiple protease subclasses.

Unprecedented regio and stereocontrol in Povarov reaction of benzylidene-(3-nitrophenyl) amine

Typically, Povarov reactions of imines derived from aromatic amines and aromatic aldehydes show poor exo/endo-stereoselectivity and to date no data is available on the regioselectivity of the cyclisation when 3-substituted imines are employed. We have demonstrated that reaction using acyclic enamides as the alkene component with 3-nitro substituted imines is completely regioselective and gave only the 5-nitro substituted tetrahydroquinoline. As a bonus the reaction also became completely exo-selective with the stereochemistry of the E-alkene preserved in the tetrahydroquinoline product.

Reduction of severe bovine serum associated matrix effects on carboxymethylated dextran coated biosensor surfaces

Surface plasmon resonance (SPR) based biosensor technology has been widely used in life science research for many applications. While the advantages of speed, ruggedness, versatility, sensitivity and reproducibility are often quoted, many researchers have experienced severe problem of non-specific binding (NSB) to chip surfaces when performing analysis of biological samples Such as bovine serum. Using the direct measurement of the bovine protein leptin, present in bovine serum samples as a model, a unique buffering system has been developed and optimised which was able to significantly reduce the non-specific interactions of bovine serum components with the carboxymethyl dextran chip (CM5) surface on a Biacore SPR The developed NSB buffering system comprised of HBS-EP buffer, containing 0.5 M NaCl, 0.005% CM-dextran pH 9.0. An average NSB reduction (n = 20) of 85.9% and 87.3% was found on an unmodified CM5 surface and a CM5 with bovine leptin immobilised on the chip surface, respectively. A reduction in NSB of up to 94% was observed on both surfaces. The concentration of the constitutive components and pH of the buffer were crucial in achieving this outcome. (C) 2008 Elsevier B.V. All rights reserved.

The trace amine associated receptor (TAAR6) gene is not associated with schizophrenia in the Irish Case-Control Study of Schizophrenia (ICCSS) sample

To replicate previous association between TAAR6 and schizophrenia, including our own finding in the Irish Study of High Density Schizophrenia Families (ISHDSF) sample, we genotyped 12 single nucleotide polymorphisms (SNPs) in the Irish Case-Control Study of Schizophrenia (ICCSS) sample. Only rs9389020 provided nominal evidence for association (p

FASCIOLA-HEPATICA - ULTRASTRUCTURAL-CHANGES TO THE TEGUMENT OF JUVENILE FLUKES FOLLOWING INCUBATION IN-VITRO WITH THE DEACETYLATED (AMINE) METABOLITE OF DIAMPHENETHIDE

Ultrastructural changes to the tegument of 5-week-old, 3-week-old and freshly-excysted Fasciola hepatica following in vitro incubation with the deacetylated (amine) metabolite of diamphenethide (DAMD, 10 mu gml(-1)) were examined by transmission electron microscopy, A similar sequence of tegumental changes occurred in all three age groups of fluke, although, with increasing fluke age, the time before onset increased and the damage became more extensive. The 5-week-old flukes showed an initial stress response after 3 h, typified by blebbing of the apical plasma membrane, formation of microvilli and an accumulation and accelerated release of secretory bodies at the tegumental apex, as well as swelling of the basal infolds, The swelling increased in extent with progressively longer periods of incubation in DAMD, leading to extreme edema and sloughing of the tegument after 9 h. The 3-week-old flukes showed a stress response and swelling of the basal infolds after only 1.5 h, although sloughing of the tegument did not occur until after 9 h. In the freshly-excysted metacercaria, a stress response and some sloughing of the tegument were evident after only 0.5 h. At all stages of development, the ventral tegument was more severely affected than the dorsal, Changes also occurred to the tegumental cells which were indicative of a disruption in the synthesis and release of tegumental secretory bodies: the amount of GER became reduced, the cisternae became swollen and their ribosomal covering decreased, the Golgi complexes disappeared from the cells and the numbers of secretory bodies in the cells also decreased, The heterochromatin content of the nuclei increased and eventually the tegumental cells began to break down, Again, the changes became apparent more rapidly at the earlier stages of development. The ultrastructural changes to the tegument are linked to a possible mode of action for diamphenethide as an inhibitor of protein synthesis. In turn, the results may help to explain the drug's high efficacy against juvenile stages of F. hepatica.

THE IONIC RESPONSE OF THE LIVER FLUKE, FASCIOLA-HEPATICA TO OUABAIN, MONENSIN AND THE DEACETYLATED (AMINE) METABOLITE OF DIAMPHENETHIDE

1. The ionic response of the liver fluke, Fasciola hepatica to perturbation of Na,K-pump activity has been determined by atomic absorption spectrophotometry.