The substrate specificity of a recombinant cysteine protease from Leishmania mexicana: application of a combinatorial peptide library approach

The substrate specificity of CPB2.8DeltaCTE, a recombinant cysteine protease from Leishmania mexicana, was mapped by screening a fluorescence-quenched combinatorial peptide library. Results from library screening indicated a preference for Arg or Lys in the S(3) subsite and for hydrophobic residues, both aliphatic and aromatic, in S(2). The S(1) subsite exhibited a specificity for the basic residues Arg and Lys. Generally, the specificity of the primed subsites was less strict compared with the non-primed side which showed preference for Arg, Lys and Ala in S'(1), Arg, Pro and Gly in S'(2) and Lys, Arg and Ser in S'(4). By contrast, a strict preference for the basic residues Arg and Lys was found for S'(3). Overall, there was a trend for basic residues in alternating subsites and smaller residues in the primed sites compared with the non-primed sites. In addition, there were strict requirements for the amino acids in subsites S(3)--S(1). Fluorescence-quenched peptides from the library with the highest on-resin cleavage were resynthesised and their kinetics of hydrolysis by CPB2.8DeltaCTE assessed in solution phase assays. Several good substrates containing the quintessential dipeptide particular to cathepsin-L-like enzymes, -F-R/K-, in P(2) and P(1) were identified (e.g. Y(NO(2))-EKFR down arrow RGK-K(Abz)G, Abz=2-aminobenzoyl; k(cat)K(m)(-1)=4298 mM(-1)s(-1)). However, novel substrates containing the dipeptide -L/I-Q- in P(2) and P(1) were also well hydrolysed (e.g. Y(NO(2))-YLQ down arrow GIQK-K(Abz)G; k(cat)K(m)(-1)=2583 mM(-1)s(-1)). The effect of utilising different fluorescent donor--quencher pairs on the value of k(cat)K(m)(-1) was examined. Generally, the use of the Abz/Q-EDDnp donor--quencher pair (EDDnp=N-(2,4-dinitrophenyl)ethylenediamine) instead of K(Abz)/Y(NO(2)) resulted in higher k(cat)K(m)(-1) values for analogous substrates.     

Short- and long-term structural properties of pultruded beam assemblies fabricated using adhesive bonding

Two beam assemblies fabricated using simple pultruded sections and adhesive bonding have been tested to determine their structural properties. The test configuration was three-point bending to simulate the most severe loading in a proposed application. Short-term stiffnesses are compared with those predicted using known section properties and linear elastic beam theory. Accelerated creep test data are used to determine long-term behaviour using Findley's linear viscoelastic theory. For the purpose of structural design, Findley's model is used to estimate the increase in maximum deflection due to a constant design loading of 1 week, 1 year and 10 years.     

Evidence that trypanothione reductase is an essential enzyme in Leishmania by targeted replacement of the tryA gene locus

Trypanothione reductase (TR), a flavoprotein oxidoreductase central to the unique thiol-redox system that operates in trypanosomatid protozoa, has been proposed as a potential target for the chemotherapy of trypanosomatid infections. In this study, targeted gene replacement was used to obtain evidence that TR is an essential cellular component and that its physiological function is crucial for parasite survival. Precise replacement of the Leishmania donovani tryA gene encoding TR was only possible upon simultaneous expression of the tryA coding region from an episome; in its absence, attempted removal of the last tryA allele invariably led to the generation of an extra copy of tryA, seemingly as a result of selective chromosomal polysomy. Partial replacement mutants were drastically affected in their ability to survive inside cytokine-activated macrophages in a murine model of Leishmania infection. As no compensatory mechanism for the partial loss of TR activity was observed in these mutants and as it was not possible to obtain viable Leishmania devoid of TR catalytic activity, specific inhibitors of this enzyme are likely to be useful anti-leishmanial agents for chemotherapeutic use.     

Doping gradients in layers of gallium phosphide grown by liquid epitaxy

Layers of epitaxial gallium phosphide doped with either tellurium, sulphur or zinc over the range 10¹⁶ to 10¹⁸ cm⁻³ have been grown from gallium solution using a vertical dipping system. These layers of thickness 60 to 80 μm have been produced on the (100) and (111)B faces of gallium phosphide single crystal substrates at high growth rates. Doping gradients have been investigated by a Schottky barrier technique over an angle lapped region of the grown slice, measuring the capacitance voltage characteristics of the individual barriers. Significant changes in doping level have been observed throughout the thickness of the layers and these are related to the variation of distribution coefficient, the losses of impurity from the system and the presence of competing background impurity systems.