Synthesis and Application of Dye-Ligand Affinity Adsorbents

Dye-ligand affinity chromatography is a widely used technique in protein purification. The utility of the reactive dyes as affinity ligands results from their unique chemistry, which confers wide specificity towards a large number of proteins. They are commercially available, are inexpensive, and can easily be immobilized. Important factors that contribute to the successful operation of a dye-ligand chromatography include adsorbent properties, such as matrix type and ligand concentration, the buffer conditions used in the adsorption and elution stages, and contacting parameters like flow rate and column geometry. In general, with dye-ligand affinity chromatography, the specificity is provided by the adsorption and elution conditions employed in a particular purification, and these must often be worked out by trial and error. The present chapter provides protocols for the synthesis of dye-ligand affinity adsorbents as well as protocols for screening, selection, and optimization of a dye-ligand purification step. The purification of the glutathione transferases from Phaseolus vulgaris crude extract on Cibacron Blue 3GA-Sepharose is given as an example

Isoenzyme- and Allozyme-Specific Inhibitors: 2,20-Dihydroxybenzophenones and Their Carbonyl N-Analogues that Discriminate between Human Glutathione Transferase A1-1 and P1-1 Allozymes

The selectivity of certain benzophenones and their carbonyl N-analogues was investigated towards the human GSTP1-1 allozymes A, B and C involved in MDR. The allozymes were purified from extracts derived from E. coli harbouring the plasmids pEXP5-CT/TOPO-TAhGSTP1* A, pOXO4-hGSTP1*B or pOXO4-hGSTP1*C. Compound screening with each allozyme activity indicated three compounds with appreciable inhibitory potencies, 12 and 13 with P1-1A 62% and 67%, 11 and 12 with P1-1C 51% and 70%, whereas that of 15 fell behind with P1-1B (41%). These findings were confirmed by IC50 values (74–125 lM). Enzyme inhibition kinetics, aided by molecular modelling and docking, revealed that there is competition with the substrate CDNB for the same binding site on the allozyme (Ki(13/ A) = 63.6 +- 3.0 lM, K (15/B) = 198.6 +- 14.3 lM, and Ki(11/ C) = 16.5 +- 2.7 lM). These data were brought into context by an in silico structural comparative analysis of the targeted proteins. Although the screened compounds showed moderate inhibitory potency against hGSTP1-1, remarkably, some of them demonstrated absolute isoenzyme and/or allozyme selectivity

Antioxidant capacity and immunomodulatory effects of a crysolaminarinenriched extract in Senegalese sole

The microalgae are an important source of bioactive molecules including &beta;-glucans that can be used as immunostimulants in aquaculture. In the present study, the antioxidant capacity, cytotoxicity and immunomodulatory activity of a chrysolaminarin-enriched extract obtained from the diatom Phaeodactylum tricornutum was evaluated. The extract showed a higher total antioxidant activity as determined by ORAC and FRAP assays and a lower DPPH scavenging activity than particulate yeast-&beta;-glucan. The cytotoxicity test indicated that extract concentrations higher than 0.01% w/v could impair cell viability of human dermal fibroblasts. To evaluate the immunomodulatory activity, juvenile soles were intraperitoneally injected with the chrysolaminarin-enriched extract suspended in coconut oil (1 mg/fish) followed by a reinjection at 7 days. A sham group injected with the carrier solution was maintained as a negative control. Cumulated mortality of fish injected with the chrysolaminarin-enriched extract was 29.4% after six days and no mortality was recorded after extract reinjection. Expression analyses of fifteen genes related to the innate immune system in kidney, spleen and intestine showed temporal and organ-specific responses. A rapid (2 days post-injection; dpi) and strong induction of the pro-inflammatory il1b and the antimicrobial peptide hamp1 in the three immunological organs, the hsp90aa in kidney and spleen, irf3 in intestine and c3 in spleen was observed indicating a potent inflammatory response. The recovery of steady-state levels for all activated genes at 5 dpi, and the down-regulation of c-lectin receptor as well as some interferon-related genes (ifn1, irf1, irf3, irf8, irf9 and mx) in kidney and cxc10 in spleen indicated that the soles were able to activate a homeostatic response against the &beta;-glucan insult. The reinjection of the chrysolaminarin-enriched extract did not activate a new inflammatory response but reduced the mRNA levels of hsp90aa and irf3 indicating that soles developed some resistance to &beta;-glucans. Overall, these results reveal this enriched extract as a novel and potent source of &beta;-glucans with antioxidant and immunomodulatory capacity suitable for immunostimulation in aquaculture. </p

High throughput mutagenesis for identification of residues regulating human prostacyclin (hIP) receptor

The human prostacyclin receptor (hIP receptor) is a seven-transmembrane G protein-coupled receptor (GPCR) that plays a critical role in vascular smooth muscle relaxation and platelet aggregation. hIP receptor dysfunction has been implicated in numerous cardiovascular abnormalities, including myocardial infarction, hypertension, thrombosis and atherosclerosis. Genomic sequencing has discovered several genetic variations in the PTGIR gene coding for hIP receptor, however, its structure-function relationship has not been sufficiently explored. Here we set out to investigate the applicability of high throughput random mutagenesis to study the structure-function relationship of hIP receptor. While chemical mutagenesis was not suitable to generate a mutagenesis library with sufficient coverage, our data demonstrate error-prone PCR (epPCR) mediated mutagenesis as a valuable method for the unbiased screening of residues regulating hIP receptor function and expression. Here we describe the generation and functional characterization of an epPCR derived mutagenesis library compromising >4000 mutants of the hIP receptor. We introduce next generation sequencing as a useful tool to validate the quality of mutagenesis libraries by providing information about the coverage, mutation rate and mutational bias. We identified 18 mutants of the hIP receptor that were expressed at the cell surface, but demonstrated impaired receptor function. A total of 38 non-synonymous mutations were identified within the coding region of the hIP receptor, mapping to 36 distinct residues, including several mutations previously reported to affect the signaling of the hIP receptor. Thus, our data demonstrates epPCR mediated random mutagenesis as a valuable and practical method to study the structurefunction relationship of GPCRs. © 2014 Bill et al

The Cysteine Protease α-Clostripain is Not Essential for the Pathogenesis of Clostridium perfringens-Mediated Myonecrosis

Clostridium perfringens is the causative agent of clostridial myonecrosis or gas gangrene and produces many different extracellular toxins and enzymes, including the cysteine protease α-clostripain. Mutation of the α-clostripain structural gene, ccp, alters the turnover of secreted extracellular proteins in C. perfringens, but the role of α-clostripain in disease pathogenesis is not known. We insertionally inactivated the ccp gene C. perfringens strain 13 using TargeTron technology, constructing a strain that was no longer proteolytic on skim milk agar. Quantitative protease assays confirmed the absence of extracellular protease activity, which was restored by complementation with the wild-type ccp gene. The role of α-clostripain in virulence was assessed by analysing the isogenic wild-type, mutant and complemented strains in a mouse myonecrosis model. The results showed that although α-clostripain was the major extracellular protease, mutation of the ccp gene did not alter either the progression or the development of disease. These results do not rule out the possibility that this extracellular enzyme may still have a role in the early stages of the disease process

Pharmaceutical And Biomedical Applications Of Affinity Chromatography: Recent Trends And Developments

Affinity chromatography is a separation technique that has become increasingly important in work with biological samples and pharmaceutical agents. This method is based on the use of a biologically-related agent as a stationary phase to selectively retain analytes or to study biological interactions. This review discusses the basic principles behind affinity chromatography and examines recent developments that have occurred in the use of this method for biomedical and pharmaceutical analysis. Techniques based on traditional affinity supports are discussed, but an emphasis is placed on methods in which affinity columns are used as part of HPLC systems or in combination with other analytical methods. General formats for affinity chromatography that are considered include step elution schemes, weak affinity chromatography, affinity extraction and affinity depletion. Specific separation techniques that are examined include lectin affinity chromatography, boronate affinity chromatography, immunoaffinity chromatography, and immobilized metal ion affinity chromatography. Approaches for the study of biological interactions by affinity chromatography are also presented, such as the measurement of equilibrium constants, rate constants, or competition and displacement effects. In addition, related developments in the use of immobilized enzyme reactors, molecularly imprinted polymers, dye ligands and aptamers are briefly considered

Pyruvate kinases have an intrinsic and conserved decarboxylase activity

The phosphotransfer mechanism of pyruvate kinases (PYKs) has been studied in detail, but the mechanism of the intrinsic decarboxylase reaction catalysed by PYKs is still unknown. 1H NMR was used in this work to follow oxaloacetate (OAA) decarboxylation by trypanosomatid and human PYKs confirming that the decarboxylase activity is conserved across distantly related species. Crystal structures of Trypanosoma brucei PYK (TbPYK) complexed with the product of the decarboxylase reaction (pyruvate), and a series of substrate analogues (D-malate, α-ketoglutarate and oxalate) show that the OAA analogues bind to the kinase active site with similar binding modes, confirming that both decarboxylase and kinase activities share a common site for substrate binding and catalysis. Decarboxylation of OAA as monitored by NMR for TbPYK is relatively slow with turn-over values of 0.86 s-1 and 1.47 s-1 in the absence and presence of fructose 2,6-bisphosphate (F26BP), respectively. Human M1PYK has a measured turn-over value of 0.50 s-1. The X-ray structures explain why the decarboxylation activity is specific for OAA and is not general for α-keto acid analogues. Conservation of the decarboxylase reaction across divergent species is a consequence of piggybacking on the conserved kinase mechanism which requires a stabilised enol intermediate

A Sound Propagation Model for Interagents Communication

In this paper, we will present a method for generating conversations between human-like agents and this will be done by proposing specific parameters for inter-agents messages with an approximation of virtual sound propagation. We will then be able to simulate appropriate human-like behaviours automatically. For instance, we will demonstrate how to create proper reactions for agents that are not able to understand, but only to hear the sentences. Finally, we will develop the notion of conversation privacy and inter-agents cooperation, as all our agents share the same virtual space