MDR-involved human glutathione transferases (hGSTs) are targets for inhibition by 2,2'-dihydroxybenzophenones and N-carbonyl analogues

Over expression of human GSTA1-1 in tumour cells is part of MDR mechanisms. Substituted 2-hydroxybenzophenones are ubiquitous in naturally occurring and synthetic compounds, exhibiting important biological activities. 2,2’-Dihydroxybenzophenones and N-carbonyl analogues, structurally, are ringopened forms of xanthone analogues which we reported recently as hGSTA1-1 inhibitors. The present study combined GST inhibition screening, in silico molecular docking and enzyme inhibition kinetics, revealing four analogues with strong inhibitory potency (IC50 = 0.18-1.8 μM) and modest cytotoxic activity for Caco2 cell line (LC50 = 35 to > 400 μM), thus being useful as lead structures for the design of new inhibitors against hGSTs

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

Mobile agents can benefit from standards efforts on interagent communication

On the road to the future success of mobile agents, we believe that interagent communication is an issue that has not been adequately addressed by the mobile agents community. Supplementing mobile agents with the ability to interact with other mobile or static agents, or agentified information sources is a necessity in the vastly heterogeneous arena in which mobile agents are called to compete. Thus, an agent communication language should be interpreted as a tool with the capacity to integrate disparate sources of information. In the first segment, we argue that mobile agents can benefit from current standards efforts on agent communication since the focus of such work is to address heterogeneity by defining a "common language" for communicating agents. In the second part, we discuss ongoing research on agent-to-agent communication and present current standards efforts relevant to agent communication