A Target Repurposing Approach Identifies N-myristoyltransferase as a New Candidate Drug Target in Filarial Nematodes

Myristoylation is a lipid modification involving the addition of a 14-carbon unsaturated fatty acid, myristic acid, to the N-terminal glycine of a subset of proteins, a modification that promotes their binding to cell membranes for varied biological functions. The process is catalyzed by myristoyl-CoA:protein N-myristoyltransferase (NMT), an enzyme which has been validated as a drug target in human cancers, and for infectious diseases caused by fungi, viruses and protozoan parasites. We purified Caenorhabditis elegans and Brugia malayi NMTs as active recombinant proteins and carried out kinetic analyses with their essential fatty acid donor, myristoyl-CoA and peptide substrates. Biochemical and structural analyses both revealed that the nematode enzymes are canonical NMTs, sharing a high degree of conservation with protozoan NMT enzymes. Inhibitory compounds that target NMT in protozoan species inhibited the nematode NMTs with IC₅₀ values of 2.5–10 nM, and were active against B. malayi microfilariae and adult worms at 12.5 µM and 50 µM respectively, and C. elegans (25 µM) in culture. RNA interference and gene deletion in C. elegans further showed that NMT is essential for nematode viability. The effects observed are likely due to disruption of the function of several downstream target proteins. Potential substrates of NMT in B. malayi are predicted using bioinformatic analysis. Our genetic and chemical studies highlight the importance of myristoylation in the synthesis of functional proteins in nematodes and have shown for the first time that NMT is required for viability in parasitic nematodes. These results suggest that targeting NMT could be a valid approach for the development of chemotherapeutic agents against nematode diseases including filariasis.     

Effects of chronic heart failure on skeletal muscle capillary hemodynamics at rest and during contractions.

Chronic heart failure (CHF) reduces muscle blood flow at rest and during exercise and impairs muscle function. Using intravital microscopy techniques, we tested the hypothesis that the speed and amplitude of the capillary red blood cell (RBC) velocity (VRBC) and flux (FRBC) response to contractions would be reduced in CHF compared with control (C) spinotrapezius muscle. The proportion of capillaries supporting continuous RBC flow was less (P < 0.05) in CHF (0.66 +/- 0.04) compared with C (0.84 +/- 0.01) muscle at rest and was not significantly altered with contractions. At rest, VRBC (C, 270 +/- 62; CHF, 179 +/- 14 microm/s) and FRBC (C, 22.4 +/- 5.5 vs. CHF, 15.2 +/- 1.2 RBCs/s) were reduced (both P < 0.05) in CHF vs. C muscle. Contractions significantly (both P < 0.05) elevated VRBC (C, 428 +/- 47 vs. CHF, 222 +/- 15 microm/s) and FRBC (C, 44.3 +/- 5.5 vs. CHF, 24.0 +/- 1.2 RBCs/s) in C and CHF muscle; however, both remained significantly lower in CHF than C. The time to 50% of the final response was slowed (both P < 0.05) in CHF compared with C for both VRBC (C, 8 +/- 4; CHF, 56 +/- 11 s) and FRBC (C, 11 +/- 3; CHF, 65 +/- 11 s). Capillary hematocrit increased with contractions in C and CHF muscle but was not different (P > 0.05) between CHF and C. Thus CHF impairs diffusive and conductive O2 delivery across the rest-to-contractions transition in rat skeletal muscle, which may help explain the slowed O2 uptake on-kinetics manifested in CHF patients at exercise onset.     

Confocal analysis of the molecular heterogeneity in the pericellular microenvironment produced by adult canine chondrocytes cultured in agarose gel

Adult articular chondrocytes are each surrounded by a heterogeneous microenvironment and together form the chondron. Since little is known of chondron development, agarose gel culture, confocal immunohistochemistry and image analysis have been used to characterize the molecular anatomy and temporal development of the chondrocyte pericellular microenvironment in vitro. Two structurally distinct domains were identified during the 12-week culture period. The first comprised a narrow glycocalyx, 1–3 ·m in width, which consolidated over time and was rich in collagen types II, VI, IX and XI, fibronectin, decorin and the aggrecan epitopes, 5D4 and HABR. The second region emerged after 4–6 weeks in culture and progressively developed a broad territorial region up to 12 ·m wide around the chondrocyte and pericellular glycocalyx. Co-localization studies confirmed the dominance of aggrecan epitopes 2B6, EFG-4, 5D4 and HABR in the territorial domain, whereas surface density mapping with NIH image revealed two patterns of staining, one punctate and stippled, the other more uniform in distribution. The pericellular differentiation identified appeared analogous to the chondrons of adult articular cartilage, and provides an appropriate in vitro model for further studies of cell surface receptor function in the orchestration of pericellular matrix assembly This revised version was published online in November 2006 with corrections to the Cover Date.     

The translocation and post release settlement of red squirrels Sciurus vulgaris to a previously uninhabited woodland

Red squirrels have undergone a 30% contraction of their range in the last 10 years in Ireland, a decline attributed to the introduced grey squirrel. Large regions in the west of Ireland are free of both species of squirrel, due to the isolation of forests there and their relatively recent planting. The potential of these forests as translocation sites for red squirrels was investigated to ascertain the possibility of increasing the range of red squirrels in Ireland. Nineteen red squirrels were moved into Derryclare wood, Co. Galway using a soft release into enclosures, and their subsequent survival and settlement in the wood was monitored, using trapping and radio-tracking. The successful release of 94.7% of squirrels from the enclosure, and 68.4% survival to the start of the following year’s breeding season were in excess of the target survival rates of 75 and 50%, respectively. Five of the females were found to be lactating in May 2006, and seven offspring captured in August 2006. A squirrel was observed in a follow-up visit in October 2007. Radio-tracked red squirrels tended to remain in the general vicinity of release, incorporating supplementary feeders as part of their home ranges. Squirrels also fed on the natural food available in the wood, with feeding signs readily observed in the wider woodland. The techniques used proved very successful in survival, retention and future reproduction of the translocated population. Timing of release may be an important consideration for future translocations with survival better in summer-released animals.     

Cloning and sequence analysis of an EnvCD homologue in Pseudomonas aeruginosa: regulation by iron and possible involvement in the secretion of the siderophore pyoverdine

Pseudomonas aeruginosa strain K437 is defective in the production of a 90 kDa ferripyoverdine receptor and is unable to grow in an iron-deficient medium in the presence of the non-metabolizable iron chelator 2,2′-dípyrídyl (0.25mM). An attempt to clone the ferripyoverdine receptor gene was made by complementing this growth defect. A number of clones restoring growth of K437 on dipyridyl-containing medium were obtained and several of these restored moderate expression of the 90 kDa receptor. A 5.5 kb Xhol-HindIII fragment derived from one of these clones was similarly capable of complementing the dipyridyl growth defect although it failed to restore expression of the 90 kDa ferripyoverdine receptor. Nucleotide sequencing of the 5.5 kb fragment revealed two large open reading frames (ORFs), designated ORFA and ORFB, which appeared to form an operon and were capable of encoding products of 41 kDa and 112 kDa, respectively. Using a phage T7-based expression system, products of 42 kDa and c. 108 kDa were produced from the cloned DNA, confirming that the ORFs were, indeed, expressed. The cloned ORFAB operon was inducible under conditions of iron limitation in both P. aeruginosa and Escherichia coli. In addition, mutants expressing ORFAB constitutively were constitutive for pyoverdine and ferripyoverdine receptor production suggesting that components of the pyoverdine-mediated iron-transport system are co-regulated with ORFAB. The predicted products of ORFA and ORFB showed significant homology to the Escherichia coli EnvC and EnvD polypeptides which are reportedly involved in septum formation. In addition, the ORFB product showed moderate homology to the CzcA polypeptide identified as a component of a membrane-associated plasmid-encoded cation efflux system in Alcaligenes eutrophus. Using in vitro mutagenesis and gene replacement, ORFA- and ORFB-deficient mutants of K372, the parent strain of K437, were constructed. These mutants were unable to grow on iron-deficient minimal medium containing 0.25 mM dipyridyl although they expressed the ferripyoverdine receptor and were proficient in pyoverdine-mediated iron uptake. Despite the homology of the ORFA and ORFB products to EnvC and EnvD, respectively, the ORFA-ORFB-deficient mutants were not defective in septum formation. Introduction of an ORFB mutation into the pyoverdine-producing strain ML5087 resulted in poor growth of the mutant in an iron-limited medium. The same mutation in a pyoverdine-deficient derivative of ML5087 did not adversely affect growth. These data are discussed in the context of a possible role in pyoverdine secretion for the ORFAB products.     

The cell cycle of the budding yeast Sterigmatomyces halophilus: culture fractionation by zonal centrifugation and the accumulation of DNA, RNA and protein

Sterigmatomyces halophilus is an unusual budding yeast in which daughter cells are formed, remote from the mother cell, on fine projections called sterigmata. Some fundamental properties of the cell cycle have been explored by separating cells from an exponentially growing culture into size, and thus age, classes by density-gradient centrifugation. Rate separations on high capacity, high resolution, equivolumetric gradients of sucrose, or, alternatively, isopycnic separations on gradients of Urografin revealed consistent and reproducible patterns of accumulation of DNA, RNA and protein through the cell cycle. Total DNA accumulation was stepwise, synthesis occurring late in the cycle, whilst protein accumulated continuously with no evidence for the discontinuities reported in some other lower eukaryotes. Total RNA accumulation, measured either colorimetrically or by long-term incorporation of radioactively-labelled uracil was transiently elevated early in the cycle and then accumulated continuously. A mathematical analysis of the volume distributions of the cells in fractions from the gradients showed that there is a hyperbolic relationship between cell age and size but that, to a first approximation, measurements of cell size (and density) are direct measures of age. The results are discussed with reference to (1) the unusually high buoyant density of this yeast, (2) the resolution of zonal cell separation methods and (3) macromolecular accumulation in the cell cycles of other eukaryotic micro-organisms.     

The plant microbiome

Plant genomes contribute to the structure and function of the plant microbiome, a key determinant of plant health and productivity. High-throughput technologies are revealing interactions between these complex communities and their hosts in unprecedented detail.     

Kinetics of substrate oxidation by whole cells and cell membranes of Helicobacter pylori

Abstract Oxygen uptake by Helicobacter pylori cells and membranes was determined. Cells from stirred broth cultures or agar plates, suspended in buffer, possessed a variable and apparently endogenous respiration which could be sustained for several hours. In contrast, oxygen consumption by cells from statically incubated broth cultures, in the absence of added substrate, was transient or undetectable. These latter cells, however, oxidised ethanol, fumarate, glucose, d-lactate, pyruvate and succinate, though glucose-oxidising ability declined rapidly. The K _m s for d-lactate, pyruvate and succinate metabolism were low (≤20 μM) and oxygen uptake was approximately 1.5, 2 and 2 mol per mol substrate respectively, indicating metabolism beyond acetate plus CO₂ and implying the presence of tricarboxylic acid cycle activity. Cell membranes oxidised fumarate, d-lactate, NADH, NADPH and succinate. NADPH oxidation was six times more rapid than that of NADH. Rates of oxygen uptake by cells suspended in buffer with metabolisable substrate were < 20% of those for cells suspended in a brain heart infusion medium. Uninoculated medium consumed significant quantities of oxygen.