FLP/FRT-mediated conditional mutagenesis in pre-erythrocytic stages of Plasmodium berghei

We describe here a highly efficient procedure for conditional mutagenesis in Plasmodium. The procedure uses the site-specific recombination FLP-FRT system of yeast and targets the pre-erythrocytic stages of the rodent Plasmodium parasite P. berghei, including the sporozoite stage and the subsequent liver stage. The technique consists of replacing the gene under study by an FRTed copy (i.e., flanked by FRT sites) in the erythrocytic stages of a parasite clone that expresses the flip (FLP) recombinase stage-specifically--called the 'deleter' clone. We present the available deleter clones, which express FLP at different times of the parasite life cycle, as well as the schemes and tools for constructing new deleter parasites. We also outline and discuss the various strategies for exchanging a wild-type gene with an FRTed copy and for generating conditional gene knockout or knockdown parasite clones. Finally, we detail the protocol for obtaining sporozoites that lack a protein of interest and for monitoring sporozoite-specific DNA excision and depletion of the target protein. The protocol should allow the functional analysis of any essential protein in the sporozoite, liver stage or hepatic merozoite stages of rodent Plasmodium parasites.     

Derivation of scenario-specific water quality guidelines for acid mine drainage in South Africa,using a risk-based approach

Acid mine drainage (AMD) continues to threaten water quality in many mining regions globally. Data paucity renders it challenging to inform appropriate water quality management strategies for a succinct scientific understanding of the effects of AMD on freshwater ecosystems. The current study investigated the effects of AMD collected from a defunct coalmine in Mpumalanga, South Africa, on freshwater ecosystems using a risk-based approach on five indigenous species, Adenophlebia auriculata, Burnupia stenochorias, Caridina nilotica, Pseudokirchneriella subcapitata and Oreochromis mossambicus in 2016. Species responded differently to AMD after 96 hours and 240 hours of exposure in static experimental test designs. Burnupia stenochorias was more sensitive to AMD after 96 and 240 hours of exposure, whereas O. mossambicus was tolerant during short-term exposure, but became more sensitive after 240 hours of exposure than the other species tested. The availability of metals in AMD was directly associated with dilution rate. Scenario-specific water quality guidelines for AMD have been derived as 0.122% for short-term and 0.014% for long-term exposure. These may form important indicative dilutions for other AMDs that do not match the scenarios of this study. The toxicity of AMD to a wide range of aquatic species, including field validations, requires further investigation.     

Intracellular ethanol accumulation in Saccharomyces cerevisiae during fermentation.

An intracellular accumulation of ethanol in Saccharomyces cerevisiae was observed during the early stages of fermentation (3 h). However, after 12 h of fermentation, the intracellular and extracellular ethanol concentrations were similar. Increasing the osmotic pressure of the medium caused an increase in the ratio of intracellular to extracellular ethanol concentrations at 3 h of fermentation. As in the previous case, the intracellular and extracellular ethanol concentrations were similar after 12 h of fermentation. Increasing the osmotic pressure also caused a decrease in yeast cell growth and fermentation activities. However, nutrient supplementation of the medium increased the extent of growth and fermentation, resulting in complete glucose utilization, even though intracellular ethanol concentrations were unaltered. These results suggest that nutrient limitation is a major factor responsible for the decreased growth and fermentation activities observed in yeast cells at higher osmotic pressures.     

Selectivity in enzyme catalysed reaction: Preferential hydrolysis of saturated methyl ester over the corresponding unsaturated ester by pig liver esterase

Summary Saturated methyl ester is preferentially hydrolysed in presence of corresponding ,-unsaturated ester by Pig Liver Esterasse. This selectivity is known for aliphatic, aromatic and heterocyclic esters.Chemo, regio and stereoselective transformations are an important goal towards synthetic efficiency (Bartman and Trost, 1984). Among the various methods that are employed to achieve such reactions, enzymatic transformations are in the forefront of current research activities (Jones, 1986) because of several advantages over the chemical methods. These include extremely high degree of selectivity, easier isolation of products, specially with immobilized enzymes and their reusability. In the past decade this class of biocatalysts has provided a plathora of valuable selective transformations (Boland et.al, 1991). Pig Liver Esterase (PLE) is one such enzyme which has been thoroughly studied and used for asymmetric (Zhu and Tedford, 1990) and regioselective (Adachi et.al, 1986) hydrolysis of esters. Several chiral building blocks for the synthesis of complex natural products (Imori et.al., 1983) have been synthesized through PLE. In this communication, we report the preferential hydrolysis of a saturated ester over its unsaturated counterpart. The preference is total and works well for aliphatic, aromatic as well as heterocyclic systems.     

Conformational sampling of the botulinum neurotoxin serotype A light chain: implications for inhibitor binding

Botulinum neurotoxins (BoNTs) are the most potent of the known biological toxins, and consequently are listed as category A biowarfare agents. Currently, the only treatments against BoNTs include preventative antitoxins and long-term supportive care. Consequently, there is an urgent need for therapeutics to counter these enzymes--post exposure. In a previous study, we identified a number of small, nonpeptidic lead inhibitors of BoNT serotype A light chain (BoNT/A LC) metalloprotease activity, and we identified a common pharmacophore for these molecules. In this study, we have focused on how the dynamic movement of amino acid residues in and surrounding the substrate binding cleft of the BoNT/A LC might affect inhibitor binding modes. The X-ray crystal structures of two BoNT/A LCs (PDB refcodes=3BTA and 1E1H) were examined. Results from these analyses indicate that the core structural features of the examined BoNT/A LCs, including alpha-helices and beta-sheets, remained relatively unchanged during 1 ns dynamics trajectories. However, conformational flexibility was observed in surface loops bordering the substrate binding clefts in both examined structures. Our analyses indicate that these loops may possess the ability to decrease the solvent accessibility of the substrate binding cleft, while at the same time creating new residue contacts for the inhibitors. Loop movements and conformational/positional analyses of residues within the substrate binding cleft are discussed with respect to BoNT/A LC inhibitor binding and our common pharmacophore for inhibition. The results from these studies may aid in the future identification/development of more potent small molecule inhibitors that take advantage of new binding contacts in the BoNT/A LC.     

P2X7 receptor cell surface expression and cytolytic pore formation are regulated by a distal C-terminal region

The importance of the cytosolic C-terminal region of the P2X7 receptor (P2X7R) is unquestioned, yet little is known about the functional domains of this region and how they may contribute to the numerous properties ascribed to this receptor. A structure-function analysis of truncated and single-residue-mutated P2X7 receptors was performed in HEK-293 cells and Xenopus oocytes. Cells expressing receptors truncated at residue 581 (of 595) have negligible ethidium ion uptake, whereas those expressing the P2X7R truncated at position 582 give wild type ethidium ion uptake suggesting that pore formation requires over 95% of the C-terminal tail. Channel function was evident even in receptors that were truncated at position 380 indicating that only a small portion of the cytosolic region is required for channel activity. Surprisingly, truncations in the region between residues 551 and 581 resulted in non-functional receptors with no detectable cell surface expression in HEK-293 cells. A more detailed analysis revealed that mutations of single residues within this region could also abolish receptor function and cell surface expression, suggesting that this region may participate in regulating the surface expression of the pore-forming P2X7R.     

A conserved amphipathic helix in WASP/Scar proteins is essential for activation of Arp2/3 complex

Members of the Wiskott-Aldrich syndrome protein (WASP) family link Rho GTPase signaling pathways to the cytoskeleton through a multiprotein assembly called Arp2/3 complex. The C-terminal VCA regions (verprolin-homology, central hydrophobic, and acidic regions) of WASP and its relatives stimulate Arp2/3 complex to nucleate actin filament branches. Here we show by differential line broadening in NMR spectra that the C (central) and A (acidic) segments of VCA domains from WASP, N-WASP and Scar bind Arp2/3 complex. The C regions of these proteins have a conserved sequence motif consisting of hydrophobic residues and an arginine residue. Point mutations in this conserved sequence motif suggest that it forms an amphipathic helix that is required in biochemical assays for activation of Arp2/3 complex. Key residues in this motif are buried through contacts with the GTPase binding domain in the autoinhibited structure of WASP and N-WASP, indicating that sequestration of these residues is an important aspect of autoinhibition.