Cloning and initial characterization of a new subunit for mammalian serine-palmitoyltransferase

Serine-palmitoyltransferase (SPT) catalyzes the rate-limiting step of the de novo synthesis of sphingolipids. SPT is considered to be a heterodimer composed of two subunits, SPTLC1 and SPTLC2. Here we report the identification of a novel, third, SPT subunit (SPTLC3) that shows 68% homology to the SPTLC2 subunit. Quantitative real-time PCR revealed that SPTLC3 expression is highly variable between different human tissues and cell lines. The highest expression was observed in placenta tissue and human trophoblast cell lines. The overexpression of SPTLC3 in Hek293 cells, which otherwise have very little endogenous SPTLC3, led to a 2- to 3-fold increase in cellular SPT activity. Silencing of SPTLC3 expression in HepG2 cells or human trophoblast cells by transfecting SPTLC3-specific siRNA resulted in a significant reduction of cellular SPT activity. The expression of two SPT isoforms could be a cellular mechanism to adjust SPT activity to tissue-specific requirements of sphingolipid synthesis.     

Function and evolution of plasmid-borne genes for pyrimidine biosynthesis in Borrelia spp

The thyX gene for thymidylate synthase of the Lyme borreliosis (LB) agent Borrelia burgdorferi is located in a 54-kb linear plasmid. In the present study, we identified an orthologous thymidylate synthase gene in the relapsing fever (RF) agent Borrelia hermsii, located it in a 180-kb linear plasmid, and demonstrated its expression. The functions of the B. hermsii and B. burgdorferi thyX gene products were evaluated both in vivo, by complementation of a thymidylate synthase-deficient Escherichia coli mutant, and in vitro, by testing their activities after purification. The B. hermsii thyX gene complemented the thyA mutation in E. coli, and purified B. hermsii ThyX protein catalyzed the conversion of dTMP from dUMP. In contrast, the B. burgdorferi ThyX protein had only weakly detectable activity in vitro, and the B. burgdorferi thyX gene did not provide complementation in vivo. The lack of activity of B. burgdorferi's ThyX protein was associated with the substitution of a cysteine for a highly conserved arginine at position 91. The B. hermsii thyX locus was further distinguished by the downstream presence in the plasmid of orthologues of nrdI, nrdE, and nrdF, which encode the subunits of ribonucleoside diphosphate reductase and which are not present in the LB agents B. burgdorferi and Borrelia garinii. Phylogenetic analysis suggested that the nrdIEF cluster of B. hermsii was acquired by horizontal gene transfer. These findings indicate that Borrelia spp. causing RF have a greater capability for de novo pyrimidine synthesis than those causing LB, thus providing a basis for some of the biological differences between the two groups of pathogens.     

Revisiting the localisation of Zn cations sorbed on pathological apatite calcifications made through X-ray absorption spectroscopy

The role of oligo-elements such as Zn in the genesis of pathological calcifications is widely debated in the literature. An essential element of discussion is given by their localisation either at the surface or within the Ca apatite crystalline network. To determine the localisation, X-ray absorption experiments have been performed at SOLEIL. The Exafs results suggest that Zn atoms, present in the Zn²⁺ form, are bound to about 4 O atoms at a distance of 2.00 Å, while the interatomic distance R_CaO ranges between 2.35 Å and 2.71 Å. Taking into account the content of Zn (around 1000 ppm) and the difference in ionic radius between Zn²⁺ (0.074 nm) and Ca²⁺ (0.099 nm), a significant longer interatomic distance would be expected in the case of Zn replacing Ca within the apatite crystalline network. We thus conclude that Zn atoms are localised at the surface and not in the apatite nanocrystal structure. Such structural result has essential biological implications for at least two reasons. Some oligoelements have a marked effect on the transformation of chemical phases, and may modify the morphology of crystals. These are both major issues because, in the case of kidney stones, the medical treatment depends strongly on the precise chemical phase and on the morphology of the biological entities at both macroscopic and mesoscopic scales.     

Changes in the Treatment Responses to Artesunate-Mefloquine on the Northwestern Border of Thailand during 13 Years of Continuous Deployment

Background

Artemisinin combination treatments (ACT) are recommended as first line treatment for falciparum malaria throughout the malaria affected world. We reviewed the efficacy of a 3-day regimen of mefloquine and artesunate regimen (MAS₃), over a 13 year period of continuous deployment as first-line treatment in camps for displaced persons and in clinics for migrant population along the Thai-Myanmar border.

Methods and Findings

3,264 patients were enrolled in prospective treatment trials between 1995 and 2007 and treated with MAS₃. The proportion of patients with parasitaemia persisting on day-2 increased significantly from 4.5% before 2001 to 21.9% since 2002 (p<0.001). Delayed parasite clearance was associated with increased risk of developing gametocytaemia (AOR = 2.29; 95% CI, 2.00–2.69, p = 0.002). Gametocytaemia on admission and carriage also increased over the years (p = 0.001, test for trend, for both). MAS₃ efficacy has declined slightly but significantly (Hazards ratio 1.13; 95% CI, 1.07–1.19, p<0.001), although efficacy in 2007 remained well within acceptable limits: 96.5% (95% CI, 91.0–98.7). The in vitro susceptibility of P. falciparum to artesunate increased significantly until 2002, but thereafter declined to levels close to those of 13 years ago (geometric mean in 2007: 4.2 nM/l; 95% CI, 3.2–5.5). The proportion of infections caused by parasites with increased pfmdr1 copy number rose from 30% (12/40) in 1996 to 53% (24/45) in 2006 (p = 0.012, test for trend).

Conclusion

Artesunate-mefloquine remains a highly efficacious antimalarial treatment in this area despite 13 years of widespread intense deployment, but there is evidence of a modest increase in resistance. Of particular concern is the slowing of parasitological response to artesunate and the associated increase in gametocyte carriage.     

Calcium Is a Major Determinant of Xylem Vulnerability to Cavitation

Xylem vulnerability to cavitation is a key parameter in the drought tolerance of trees, but little is known about the control mechanisms involved. Cavitation is thought to occur when an air bubble penetrates through a pit wall, and would hence be influenced by the wall''s porosity. We first tested the role of wall-bound calcium in vulnerability to cavitation in Fagus sylvatica. Stems perfused with solutions of oxalic acid, EGTA, or sodium phosphate (NaPO₄) were found to be more vulnerable to cavitation. The NaPO₄-induced increase in vulnerability to cavitation was linked to calcium removal from the wall. In contrast, xylem hydraulic conductance was unaffected by the chemical treatments, demonstrating that the mechanisms controlling vulnerability to cavitation and hydraulic resistance are uncoupled. The NaPO₄ solution was then perfused into stems from 13 tree species possessing highly contrasted vulnerability to cavitation. Calcium was found to be a major determinant of between-species differences in vulnerability to cavitation. This was evidenced in angiosperms as well as conifer species, thus supporting the hypothesis of a common mechanism in drought-induced cavitation.In plants, long-distance sap transport occurs under negative pressures in xylem conduits. Sap flows between adjoining conduits through pits that form pores in the walls, and that facilitate the flow of water while preventing the passage of air bubbles. Under water stress conditions, xylem tensions increase and the conduits become vulnerable to cavitation. Cavitation provokes an air embolism that leads to a loss of hydraulic conductance, thus exacerbating plant water deficit.Species resistance to cavitation has been intensively studied over the past two decades, and is now ranked among the traits with the highest functional and ecological significance. In woody species for instance, xylem vulnerability to cavitation correlates tightly with species-specific drought tolerance (Pockman and Sperry, 2000; Tyree et al., 2003; Maherali et al., 2004), with more xerophilous species proving less vulnerable to cavitation. Substantial variations have also been found between genotypes of a different species (Cochard et al., 2007; Dalla-Salda et al., 2009). This implies that this trait could potentially be used in breeding programs to identify more drought-tolerant species or genotypes. However, efforts in this direction are still strongly impeded by a lack of understanding of the molecular and genetic basis of cavitation resistance. Our work represents a first significant step toward resolving this challenging issue.Understanding the fine mechanism of cavitation formation is a pivotal step toward identifying the key structures and the key genes coding for these structures, yet we currently have only partial insights. According to a hypothesis first formulated by Zimmermann (1983), water stress-induced cavitation is thought to occur when a tiny air bubble penetrates through a pit membrane, and would consequently be strongly influenced by the porosity of the membrane (Tyree and Sperry, 1988; Cochard, 2006). There is also experimental evidence for a role of the mechanical properties of the pit membrane in this cavitation process (Choat et al., 2004; Sperry and Hacke, 2004). Clearly, the structural, physical, and chemical properties of pit membranes are central to the determinism of cavitation.Pit membranes are modified primary cell walls made of tightly interwoven cellulose microfibrils in a matrix of hydrated hemicelluloses and pectins. Pectins consist of a complex set of GalUA (GalA)-rich polysaccharides, and four pectic domains can be distinguished: homogalacturonan (HG), rhamnogalacturonan I, rhamnogalacturonan II, and xylogalacturonan (Willats et al., 2001). The high degree of structural complexity and heterogeneity across the pectin family is the result of both biosynthesis in the endomembrane system and the action of an array of wall-based pectin-modifying enzymes (Willats et al., 2001). HG units are synthesized in the Golgi apparatus and deposited in the cell wall in a form containing 70% to 80% methyl-esterified GalA residues (O''Neill et al., 1990; Mohnen, 1999). The removal of methyl ester groups by pectin methyl esterase (PME) within the cell wall matrix produces free carboxyl groups capable of being cross-linked by calcium cations in an “egg-box” structure (Grant et al., 1973; Pelloux et al., 2007). These calcium-dependent cross-linkages are dependent both on the degree and the distribution of methyl-esterified GalA units through the HG network (Willats et al., 2001). Calcium therefore plays a central role as it determines the supramolecular assembly of the pectic chains and the formation of a pectate gel.Pectins capable of Ca²⁺ cross-linking are particularly common in bordered pit membranes (Chaffey et al., 1997; Hafren et al., 2000). Moreover, pectin-bound calcium influences wall elasticity (Ezaki et al., 2005; Proseus and Boyer, 2006; Derbyshire et al., 2007), and could therefore influence the stretching properties of the pit membranes and, consequently, the mechanism of cavitation. We tested the hypothesis that calcium plays a major role in the determinism of cavitation. This hypothesis was formulated long ago (Sperry and Tyree, 1988) but has not yet been thoroughly tested. We designed a series of experiments to demonstrate the specific role of calcium in this mechanism, and analyzed a large number of woody species to establish the role of calcium cross-linkage in across- and within-species variation in cavitation resistance. The data strongly support our hypothesis.     

New Tools for Investigating the Comparative Biology of Caenorhabditis briggsae and C. elegans

Comparative studies of Caenorhabditis briggsae and C. elegans have provided insights into gene function and developmental control in both organisms. C. elegans is a well developed model organism with a variety of molecular and genetic tools to study gene functions. In contrast, there are only very limited tools available for its closest relative, C. briggsae. To take advantage of the full potential of this comparative approach, we have developed several genetic and molecular tools to facilitate functional analysis in C. briggsae. First, we designed and implemented an SNP-based oligonucleotide microarray for rapid mapping of genetic mutants in C. briggsae. Second, we generated a mutagenized frozen library to permit the isolation of targeted deletions and used the library to recover a deletion mutant of cbr-unc-119 for use as a transgenic marker. Third, we used the cbr-unc-119 mutant in ballistic transformation and generated fluorescently labeled strains that allow automated lineaging and cellular resolution expression analysis. Finally, we demonstrated the potential of automated lineaging by profiling expression of egl-5, hlh-1, and pha-4 at cellular resolution and by detailed phenotyping of the perturbations on the Wnt signaling pathway. These additions to the experimental toolkit for C. briggsae should greatly increase its utility in comparative studies with C. elegans. With the emerging sequence of nematode species more closely related to C. briggsae, these tools may open novel avenues of experimentation in C. briggsae itself.     

An integrative in silico methodology for the identification of modulators of macrophage migration inhibitory factor (MIF) tautomerase activity

Macrophage migration inhibitory factor (MIF) is a major proinflammatory cytokine that has been increasingly implicated in the pathogenesis of several inflammatory, autoimmune, infectious and oncogenic diseases. Accumulating evidence suggests that the tautomerase activity of MIF plays a role in modulating some of its intra- and extra-cellular activities. Therefore, the identification and development of small-molecule inhibitors targeting the catalytic activity of MIF has emerged as an attractive and viable therapeutic strategy to attenuate its function in health and disease. Herein we report a novel virtual screening protocol for the discovery of new inhibitors of MIF’s tautomerase activity. Our protocol takes into account the flexibility and dynamics of the catalytic site by coupling molecular dynamics (MD) simulations aimed at modeling the protein’s flexibility in solution to (i) docking with FlexX, or (ii) docking with FlexX and pharmacophoric filtering with Unity. In addition, we applied in parallel a standalone docking using the new version of Surflex software. The three approaches were used to screen the ChemBridge chemical library and the inhibitory activity of the top-ranked 333 compound obtained from each approach (1000 compound in total) was assessed in vitro using the tautomerase assay. This biochemical validation process resulted in the identification of 12 novel MIF inhibitors corresponding to a 1.2% hit rate. Six of these hits came from Surflex docking; two from FlexX docking with MD simulations and four hits were identified with MDS and pharmacophore filtering with minimal overlap between the hits from each approach. Six hits were identified with IC₅₀ values lower than 10 μM (three hits with IC₅₀ lower than 1 μM); four were shown to be suicide inhibitors and act via covalent modification of the N-terminal catalytic residues Pro1. One additional inhibitor, N-phenyl-N-1,3,4-thiadiazol-2-yl-thiourea, (IC₅₀ = 300 nM) was obtained from FlexX docking combined to pharmacophoric filtering on one of the eight MD structures. These results demonstrate the power of integrative in silico approaches in the discovery of new modulator of MIF’s tautomerase activity. The chemical diversity and mode of action of these compounds suggest that they could be used as molecular probes to elucidate the functions and biology of MIF and as lead candidates in drug developments of anti-MIF drugs.     

The potential of earthworms to restore ecosystem services after opencast mining – A review

Opencast coal mining has several environmental impacts, which require land rehabilitation when mining operations are finished. For that reason, restoration after such extractive industries’ work is common and has been well studied. However, many ecological restoration schemes do not examine to what extent complete and functioning ecosystems have been restored above and below ground. While the aim should be to restore functioning ecosystems, most restoration plans focus only on vegetation and above ground macro-fauna.Among the potential species that are likely to be important early in mine land restoration, earthworms are particularly good candidates. They provide several ecosystem services that are likely to accelerate soil restoration, improve primary production and facilitate the restoration of a functional ecosystem in mining areas. These services include the following: increase in topsoil fertility, food for a wide range of predators and recycling of waste organic materials on rehabilitated areas.Here, we outline some of the challenges specifically facing opencast mining restoration and describe how the ecosystem services provided by earthworms may address some of these challenges.