Antimicrobial/cytolytic peptides from the venom of the North African scorpion, Androctonus amoreuxi: Biochemical and functional characterization of natural peptides and a single site-substituted analog

The venoms of scorpions are complex cocktails of polypeptide toxins that fall into two structural categories: those that contain cysteinyl residues with associated disulfide bridges and those that do not. As the majority of lethal toxins acting upon ion channels fall into the first category, most research has been focused there. Here we report the identification and structural characterization of two novel 18-mer antimicrobial peptides from the venom of the North African scorpion, Androctonus amoreuxi. Named AamAP1 and AamAP2, both peptides are C-terminally amidated and differ in primary structure at just two sites: Leu?Pro at position 2 and Phe?Ile at position 17. Synthetic replicates of both peptides exhibited a broad-spectrum of antimicrobial activity against a Gram-positive bacterium (Staphylococcus aureus), a Gram-negative bacterium (Escherichia coli) and a yeast (Candida albicans), at concentrations ranging between 20µM and 150µM. In this concentration range, both peptides produced significant degrees of hemolysis. A synthetic replicate of AamAP1 containing a single substitution (His?Lys) at position 8, generated a peptide (AamAP-S1) with enhanced antimicrobial potency (3-5µM) against the three test organisms and within this concentration range, hemolytic effects were negligible. In addition, this His?Lys variant exhibited potent growth inhibitory activity (ID(50) 25-40µm) against several human cancer cell lines and endothelial cells that was absent in both natural peptides. Natural bioactive peptide libraries, such as those that occur in scorpion venoms, thus constitute a unique source of novel lead compounds with drug development potential whose biological properties can be readily manipulated by simple synthetic chemical means.

Challenges of UK/Irish Contractors Regarding Material Management and Logistics in Confined Site Construction

The aim of this paper is to identify the various managerial issues encountered by UK/Irish contractors in the management of materials in confined urban construction sites. Through extensive literature review, detailed interviews, case studies, cognitive mapping, causal loop diagrams, questionnaire survey and documenting severity indices, a comprehensive insight into the materials management concerns within a confined construction site environment is envisaged and portrayed. The leading issues highlighted are: that contractors’ material spatial requirements exceed available space, it is difficult to coordinate the storage of materials in line with the programme, location of the site entrance makes delivery of materials particularly difficult, it is difficult to store materials on-site due to the lack of space, and difficult to coordinate the storage requirements of the various sub-contractors. With the continued development of confined urban centres and the increasing high cost of materials, any marginal savings made on-site would translate into significant monetary savings at project completion. Such savings would give developers a distinct competitive advantage in this challenging economic climate. As on-site management professionals successfully identify, acknowledge and counteract the numerous issues illustrated, the successful management of materials on a confined urban construction site becomes attainable.

Confined site construction: An empirical analysis of factors impacting health and safety management

Purpose – The purpose of this paper is to identify, clarify and tabulate the various managerial issues encountered, to aid in the management of the complex health and safety concerns which occur within a confined construction site environment.

Design/methodology/approach – This is achieved through conducting extensive qualitative and qualitative research in the form of case studies, interviews and questionnaire survey.

Findings – The leading managerial issues in the management of health and safety on a confined construction site are found to be: “Difficulty to move materials around site safely”; “Lack of adequate room for the effective handling of materials”; “Difficulty in ensuring site is tidy and all plant and materials are stored safely”; “Close proximity of individuals to operation of large plant and machinery”; and joint fifth “Difficulty in ensuring proper arrangement and collection of waste materials on-site” along with “Difficulty in controlling hazardous materials and equipment on site”.

Practical implications – The resulting implication for practice of these results can be summarised by identifying that with sustained development of urban centres on a global scale, coupled with the increasing complexity of architectural designs, the majority of on-site project management professionals are faced with the onerous task of completing often intricate designs within a limited spatial environment, under strict health and safety parameters.

Originality/value – The subsequent value of the findings are such that just as on-site management professionals successfully identify the various managerial issues highlighted, the successful management of health and safety on a confined construction site is attainable.

Critical factors affecting management of site operatives and personal in confined construction site

The aim of this paper is to identify and classify the numerous managerial issues encountered in the management of personnel in confined site construction. For the purpose of this research, a confined construction site is defined as a site where permanent works fit the site footprint, extending to levels above and/or below ground level, leaving spatial restrictions for other operations (e.g. plant and material movements, materials storage and temporary accommodation etc.) and require effective resource co-ordination beyond normal on-site management input. A literature review and analysis, case studies incorporating interviews and focus groups along with a questionnaire survey were used in order to gain a comprehensive insight into the issues in the management of personnel in a confined construction site environment. The following are the top five leading issues highlighted in the management of personnel in confined site construction; (1) Accidents due to an untidy site, (2) One contractor holding up another because of the lack of space, (3) A risk to personnel because of vehicular traffic on-site, (4) Difficult to facilitate several contractors at one work location, and (5) Numerous personnel working within the one space. In today’s modern environment, spatial restrictions are quickly becoming the norm in the industry. Therefore, the management of personnel on-site becomes progressively more difficult with the decrease in available space on-site. Where such environments exist, acknowledging the numerous issues highlighted above, aids site management in the supervision and co-ordination of personnel on-site, thus reducing accidents, increasing productivity and increase profit margins, in spatially restricted environments. As on-site management professionals successfully identify, acknowledge and counteract the numerous issues illustrated, the successful management of personnel on a confined construction site is achievable. By identifying the numerous issues, on-site management can proactively mitigate such issues through adopting counteractive measures and through successful identification of the traits identified.

Degradation of carbon disulphide (CS2) in soils and groundwater from a CS2 -contaminated site

This study is the first investigation of biodegradation of carbon disulphide (CS₂) in soil that provides estimates of degradation rates and identifies intermediate degradation products and carbon isotope signatures of degradation. Microcosm studies were undertaken under anaerobic conditions using soil and groundwater recovered from CS₂-contaminated sites. Proposed degradation mechanisms were validated using equilibrium speciation modelling of concentrations and carbon isotope ratios. A first-order degradation rate constant of 1.25 × 10-2 h-1 was obtained for biological degradation with soil. Carbonyl sulphide (COS) and hydrogen sulphide (H₂S) were found to be intermediates of degradation, but did not accumulate in vials. A 13C/12C enrichment factor of -7.5 ± 0.8 ‰ was obtained for degradation within microcosms with both soil and groundwater whereas a 13C/12C enrichment factor of -23.0 ± 2.1 ‰ was obtained for degradation with site groundwater alone. It can be concluded that biological degradation of both CS2-contaminated soil and groundwater is likely to occur in the field suggesting that natural attenuation may be an appropriate remedial tool at some sites. The presence of biodegradation by-products including COS and H₂S indicates that biodegradation of CS₂ is occurring and stable carbon isotopes are a promising tool to quantify CS₂ degradation.

CHARACTERIZATION OF THE CYSTEINE PROTEINASES OF THE COMMON LIVER FLUKE FASCIOLA-HEPATICA USING NOVEL, ACTIVE-SITE-DIRECTED AFFINITY LABELS

The excreted/secreted proteinases of adult and juvenile Fasciola hepatica maintained in vitro were found to hydrolyse the fluorogenic substrates Cbz-Phe-Arg- and Cbz-Arg-Arg-NHMec. This activity was demonstrated to have a classical cysteine proteinase inhibitor profile, with turn-over of both substrates being blocked by pre-incubation with E64 and peptidyl diazomethanes. The Cbz-Arg-Arg-NHMec hydrolysing activity of the mature fluke exhibited an alkaline stability not characteristic of its mammalian lysosomal counterparts. Further, the biotinylated affinity reagents biotin-Phe-Ala CHN2 and biotin-Phe-Cys(SBzyl)-CHN2 were used to label and characterize these cysteine proteinases in terms of apparent molecular weight and subsite specificity. Adult fluke media were found to contain four species of molecular weights 66, 58, 50 and 25-26 kDa; juvenile media contained three species of molecular weights 66, 54 and 25-26 kDa. The major 25-26 kDa cysteine proteinase common to both stages was shown to have a subsite specificity similar to that of mammalian cathepsin B.

Binding site of AMPA-receptor allosteric modulator

The computer molecular docking of piperonyl acid piperidide (BDP) and some its analogs already known as ampakins was conducted for estimating their possible binding with AMPA-receptor glutamate domains in cyclothiazide binding area and for further design of new structures maximally complimentary to the receptor. On the base of the conducted docking it can be suggested that the binding site of BDP (amides of benzodioxane-6-carboxylic and piperonyl acids) analogs is located in AMPA-receptor cyclothiazide binding pocket. It is shown that formation of protein-ligand complexes of AMPA-receptor with benzodioxane-6-carboxylic and piperonyl acid derivatives, similarly to cyclothiazide, proceeds with interaction with Ser497, Leu751, which significance is confirmed by site-specific mutagenesis.

CoMFA and homology-based models of the glycine binding site of N-methyl-D-aspartate receptor

Homology modeling was used to build 3D models of the N-methyl-D-aspartate (NMDA) receptor glycine binding site on the basis of an X-ray structure of the water-soluble AMPA-sensitive receptor. The docking of agonists and antagonists to these models was used to reveal binding modes of ligands and to explain known structure-activity relationships. Two types of quantitative models, 3D-QSAR/CoMFA and a regression model based on docking energies, were built for antagonists (derivatives of 4-hydroxy-2-quinolone, quinoxaline-2,3-dione, and related compounds). The CoMFA steric and electrostatic maps were superimposed on the homology-based model, and a close correspondence was marked. The derived computational models have permitted the evaluation of the structural features crucial for high glycine binding site affinity and are important for the design of new ligands.

Structural basis for understanding structure-activity relationships for the glutamate binding site of the NMDA receptor

We present new homology-based models of the glutamate binding site (in closed and open forms) of the NMDA receptor NR2B subunit derived from X-ray structures of the water soluble AMPA sensitive glutamate receptor. The models were used for revealing binding modes of agonists and competitive antagonists, as well as for rationalizing known experimental facts concerning structure-activity relationships: (i) the switching between the agonist and the antagonist modes of action upon lengthening the chain between the distal acidic group and the amino acid moiety, (ii) the preference for the methyl group attached to the a-amino group of ligands, (iii) the preference for the D-configuration of agonists and antagonists, and (iv) the existence of "superacidic" agonists.