Identification of a Plasmodium falciparum Phospholipid Transfer Protein

Infection of erythrocytes by the human malaria parasite Plasmodium falciparum results in dramatic modifications to the host cell, including changes to its antigenic and transport properties and the de novo formation of membranous compartments within the erythrocyte cytosol. These parasite-induced structures are implicated in the transport of nutrients, metabolic products, and parasite proteins, as well as in parasite virulence. However, very few of the parasite effector proteins that underlie remodeling of the host erythrocyte are functionally characterized. Using bioinformatic examination and modeling, we have found that the exported P. falciparum protein PFA0210c belongs to the START domain family, members of which mediate transfer of phospholipids, ceramide, or fatty acids between membranes. In vitro phospholipid transfer assays using recombinant PFA0210 confirmed that it can transfer phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin between phospholipid vesicles. Furthermore, assays using HL60 cells containing radiolabeled phospholipids indicated that orthologs of PFA0210c can also transfer phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine. Biochemical and immunochemical analysis showed that PFA0210c associates with membranes in infected erythrocytes at mature stages of intracellular parasite growth. Localization studies in live parasites revealed that the protein is present in the parasitophorous vacuole during growth and is later recruited to organelles in the parasite. Together these data suggest that PFA0210c plays a role in the formation of the membranous structures and nutrient phospholipid transfer in the malaria-parasitized erythrocyte.     

The DroughtBox: A new tool for phenotyping residual branch conductance and its temperature dependence during drought

Xylem hydraulic failure is a major driver of tree death during drought. However, to better understand mortality risk in trees, especially during hot-drought events, more information is required on both rates of residual water-loss from small branches (g_res) after stomatal closure, as well as the phase transition temperature (T_p), beyond which g_res significantly increases. Here, we describe and test a novel low-cost tool, the DroughtBox, for phenotyping g_res and T_p across species. The system consists of a programmable climatically controlled chamber in which branches dehydrate and changes in the mass recorded. Test measurements show that the DroughtBox maintains stable temperature and relative humidity across a range of set points, a prerequisite for getting accurate g_res and T_p values. Among a study group of four conifer and one angiosperm species, we observed a range of g_res (0.44–1.64 mmol H₂O m⁻² s⁻¹) and T_p (39.4–43.8°C) values. Furthermore, the measured time to hydraulic failure varied between two conifers species and was shortened in both species following a heatwave event. The DroughtBox is a reliable and customizable tool for phenotyping g_res and T_p, as well as for testing models of time to hydraulic failure that will improve our ability to assess climate change impacts on plants.     

Chloral hydrate decreases gap junction communication in rat liver epithelial cells

Gap junction communication (GJC) is involved in controlling cell proliferation and differentiation. Alterations in GJC are associated with carcinogenesis, but the mechanisms involved are unknown. Chloral hydrate (CH), a by-product of chlorine disinfection of water, is carcinogenic in mice, and we demonstrated that CH reduced GJC in a rat liver epithelial cell line (Clone 9). To examine the mechanism(s) by which CH inhibits GJC, Clone 9 cells treated with CH were examined using Western blot, real-time polymerase chain reaction, immunocytochemical, and dye-communication techniques. Treatment with CH (0.1–5 mM for 24 h) resulted in a dose-dependent inhibition of GJC as measured by Lucifer yellow dye transfer. Western blot analysis demonstrated expression of connexin (Cx) 43 and 26 in control cells and reduced expression of Cx 43 but not Cx 26 protein from 0.1 to 1 mM CH. CH treatment from 2.5 to 5 mM caused an apparent increase in expression of both connexins that was concomitant with a reduction in mRNA expression for both connexins. Similarly, with immunocytochemistry, a dose-dependent decrease in Cx 43 staining at sites of cell–cell contact was apparent in CH (0.5–5 mM)-treated cultures, whereas no Cx 26 staining was observed. Thus, Clone 9 cells contain two types of connexins but only one type of plasma membrane channel. Understanding of the regulation of connexin may shed light on mechanisms responsible for inhibition of GJC by chemical carcinogens.     

Perturbation of B cell activation in SLAM-associated protein-deficient mice is associated with changes in gammaherpesvirus latency reservoirs

Signaling lymphocyte activation molecule (SLAM)-associated protein (SAP)) interactions with SLAM family proteins play important roles in immune function. SAP-deficient mice have defective B cell function, including impairment of germinal center formation, production of class-switched Ig, and development of memory B cells. B cells are the major reservoir of latency for both EBV and the homologous murine gammaherpesvirus, gammaherpesvirus 68. There is a strong association between the B cell life cycle and viral latency in that the virus preferentially establishes latency in activated germinal center B cells, which provides access to memory B cells, a major reservoir of long-term latency. In the current studies, we have analyzed the establishment and maintenance of gammaHV68 latency in wild-type and SAP-deficient mice. The results show that, despite SAP-associated defects in germinal center and memory B cell formation, latency was established and maintained in memory B cells at comparable frequencies to wild-type mice, although the paucity of memory B cells translated into a 10-fold reduction in latent load. Furthermore, there were defects in normal latency reservoirs within the germinal center cells and IgD(+)"naive" B cells in SAP-deficient mice, showing a profound effect of the SAP mutation on latency reservoirs.     

The Plasmodium berghei serine protease PbSUB1 plays an important role in male gamete egress

The Plasmodium subtilisin‐like serine protease SUB1 is expressed in hepatic and both asexual and sexual blood parasite stages. SUB1 is required for egress of invasive forms of the parasite from both erythrocytes and hepatocytes, but its subcellular localisation, function, and potential substrates in the sexual stages are unknown. Here, we have characterised the expression profile and subcellular localisation of SUB1 in Plasmodium berghei sexual stages. We show that the protease is selectively expressed in mature male gametocytes and localises to secretory organelles known to be involved in gamete egress, called male osmiophilic bodies. We have investigated PbSUB1 function in the sexual stages by generating P. berghei transgenic lines deficient in PbSUB1 expression or enzyme activity in gametocytes. Our results demonstrate that PbSUB1 plays a role in male gamete egress. We also show for the first time that the PbSUB1 substrate PbSERA3 is expressed in gametocytes and processed by PbSUB1 upon gametocyte activation. Taken together, our results strongly suggest that PbSUB1 is not only a promising drug target for asexual stages but could also be an attractive malaria transmission‐blocking target.     

The timing of molecular and morphological changes underlying reproductive transitions in wild tomatoes (Solanum sect. Lycopersicon)

Molecular mechanisms underlying the transition from genetic self‐incompatibility to self‐compatibility are well documented, but the evolution of other reproductive trait changes that accompany shifts in reproductive strategy (mating system) remains comparatively under‐investigated. A notable exception is the transition from exserted styles to styles with recessed positions relative to the anthers in wild tomatoes (Solanum Section Lycopersicon). This phenotypic change has been previously attributed to a specific mutation in the promoter of a gene that influences style length (style2.1); however, whether this specific regulatory mutation arose concurrently with the transition from long to short styles, and whether it is causally responsible for this phenotypic transition, has been poorly investigated across this group. To address this gap, we assessed 74 accessions (populations) from 13 species for quantitative genetic variation in floral and reproductive traits as well as the presence/absence of deletions at two different locations (StyleD1 and StyleD2) within the regulatory region upstream of style2.1. We confirmed that the putatively causal deletion variant (a 450‐bp deletion at StyleD1) arose within self‐compatible lineages. However, the variation and history of both StyleD1 and StyleD2 was more complex than previously inferred. In particular, although StyleD1 was statistically associated with differences in style length and stigma exsertion across all species, we found no evidence for this association within two species polymorphic for the StyleD1 mutation. We conclude that the previous association detected between phenotypic and molecular differences is most likely due to a phylogenetic association rather than a causal mechanistic relationship. Phenotypic variation in style length must therefore be due to other unexamined linked variants in the style2.1 regulatory region.