Heterogeneous genetic invasions of three insecticide resistance mutations in Indo‐Pacific populations of Aedes aegypti (L.)

Nations throughout the Indo‐Pacific region use pyrethroid insecticides to control Aedes aegypti, the mosquito vector of dengue, often without knowledge of pyrethroid resistance status of the pest or origin of resistance. Two mutations (V1016G + F1534C) in the sodium channel gene (Vssc) of Ae. aegypti modify ion channel function and cause target‐site resistance to pyrethroid insecticides, with a third mutation (S989P) having a potential additive effect. Of 27 possible genotypes involving these mutations, some allelic combinations are never seen whereas others predominate. Here, five allelic combinations common in Ae. aegypti from the Indo‐Pacific region are described and their geographical distributions investigated using genome‐wide SNP markers. We tested the hypothesis that resistance allele combinations evolved de novo in populations versus the alternative that dispersal of Ae. aegypti between populations facilitated genetic invasions of allele combinations. We used latent factor mixed‐models to detect SNPs throughout the genome that showed structuring in line with resistance allele combinations and compared variation at SNPs within the Vssc gene with genome‐wide variation. Mixed‐models detected an array of SNPs linked to resistance allele combinations, all located within or in close proximity to the Vssc gene. Variation at SNPs within the Vssc gene was structured by resistance profile, whereas genome‐wide SNPs were structured by population. These results demonstrate that alleles near to resistance mutations have been transferred between populations via linked selection. This indicates that genetic invasions have contributed to the widespread occurrence of Vssc allele combinations in Ae. aegypti in the Indo‐Pacific region, pointing to undocumented mosquito invasions between countries.     

Interactions ofCandida albicans with bacteria and salivary molecules in oral biofilms

The yeastCandida albicans coaggregates with a variety of streptococcal species, an interaction that may promote oral colonization by yeast cells.C. albicans andCandida tropicalis are the yeasts most frequently isolated from the human oral cavity and our data demonstrate that both these species bind toStreptococcus gordonii NCTC 7869 while two otherCandida species (Candida krusei andCandida kefyr) do not. Adherence ofC. albicans was greatest when the yeast had been grown at 30° C to mid-exponential growth phase. For 21 strains ofC. albicans there was a positive correlation between the ability to adhere toS. gordonii and adherence to experimental salivary pellicle. Whole saliva either stimulated or slightly inhibited adherence ofC. albicans toS. gordonii depending on the streptococcal growth conditions. The results suggest that the major salivary adhesins and coaggregation adhesins ofC. albicans are co-expressed.     

Laminin-1 activates Cdc42 in the mechanism of laminin-1-mediated neurite outgrowth

Here, we investigated the role of the small Rho GTPases Rac, Cdc42, and Rho in the mechanism of laminin-1-mediated neurite outgrowth in PC12 cells. PC12 cells were transfected with plasmids expressing wild-type and dominant-negative mutants of Rac (RacN17), Cdc42 (Cdc42N17), or Rho (RhoN19). Over 90% of the dominant-negative Rho- and Rac-transfected cells extended neurites when plated on laminin-1; however, none of the PC12 cells transfected with the dominant-negative Cdc42 mutant extended neurites. In cells cotransfected with plasmids expressing c-Jun N-terminal kinase and wild-type Cdc42, laminin-1 treatment stimulated detectable levels of c-Jun phosphorylation. Further, cotransfection with c-Jun N-terminal kinase and the dominant-negative Cdc42 mutant blocked laminin-1-mediated c-Jun phosphorylation. Transfection with either wild-type Rac or the dominant-negative Rac did not effect c-Jun phosphorylation. These data demonstrate that Cdc42 is activated by laminin-1 and that Cdc42 activation is required in the mechanism of laminin-1-mediated neurite outgrowth.     

Stalk cell formation in monolayers of Dictyostelium discoideum V12-M2

Stalk cell formation in low-cell-density monolayers of Dictyostelium discoideum, strain V12-M2, occurs following the sequential addition of cyclic AMP and the differentiation-inducing factor (DIF). Both cyclic AMP and DIF are essential for the appearance of the prestalk-specific isozyme alkaline phosphatase-II, which suggests that both factors are necessary for prestalk cell formation. The available evidence suggests that the cyclic AMP requirement for stalk cell formation is mediated through the cell surface cyclic AMP receptor. However, stalk cell formation is inhibited by caffeine and this inhibition is reversed by the cell-permeable analogue 8-Br-cyclic AMP, which suggests in addition a possible involvement for elevated intracellular cyclic AMP concentrations in stalk cell formation. During in vivo development cells first become independent of cyclic AMP at the tipped aggregate stage, but the acquisition of cyclic AMP independence is advanced by several hours when cells are incubated in the presence of cyclic AMP for 2 hours. Cells do not become independent of DIF until the culmination stage of development, which suggests the possibility that DIF is required for the conversion of prestalk cells to stalk cells. There is an absolute requirement for DIF for stalk cell formation in low-density monolayers of prestalk cells but only part of population exhibits a requirement for cyclic AMP, which suggests that the prestalk cell population consists of two distinct cell types. Stalk cell formation from prespore cells is totally dependent on both cyclic AMP and DIF.(ABSTRACT TRUNCATED AT 250 WORDS)     

SUMO fusions and SUMO-specific protease for efficient expression and purification of proteins

SUMO (small ubiquitin-related modifier) modulates protein structure and function by covalently binding to the lysine side chains of the target proteins. Yeast cells contain two SUMO proteases, Ulp1 and Ulp2, that cleave sumoylated proteins in the cell. Ulp1 (SUMO protease 1) processes the SUMO precursor to its mature form and also de-conjugates SUMO from side chain lysines of target proteins. Here we demonstrate that attachment of SUMO to the N-terminus of under-expressed proteins dramatically enhances their expression in E. coli. SUMO protease 1 was able to cleave a variety of SUMO fusions robustly and with impeccable specificity. Purified recombinant SUMO-GFPs were efficiently cleaved when any amino acid, except proline, was in the+1 position of the cleavage site. The enzyme was active over a broad range of buffer and temperature conditions. Purification of certain recombinant proteins is accomplished by production of Ub-fusions from which Ub can be subsequently removed by de-ubiquitinating enzymes (DUBs). However, DUBs are unstable enzymes that are difficult to produce and inexpensive DUBs are not available commercially. Our findings demonstrate that SUMO protease 1/SUMO-fusion system may be preferable to DUB/Ub-fusion. Enhanced expression and solubility of proteins fused to SUMO combined with broad specificity and highly efficient cleavage properties of the SUMO protease 1 indicates that SUMO-fusion technology will become a useful tool in purification of proteins and peptides.     

Crassulacean acid metabolism in the ZZ plant, Zamioculcas zamiifolia (Araceae)

Zamioculcas zamiifolia (Araceae), a terrestrial East African aroid, with two defining attributes of crassulacean acid metabolism (CAM) (net CO(2) uptake in the dark and diel fluctuations of titratable acidity) is the only CAM plant described within the Araceae, a mainly tropical taxon that contains the second largest number of epiphytes of any vascular plant family. Within the Alismatales, the order to which the Araceae belong, Z. zamiifolia is the only documented nonaquatic CAM species. Zamioculcas zamiifolia has weak CAM that is upregulated in response to water stress. In well-watered plants, day-night fluctuations in titratable acidity were 2.5 μmol H(+)·(g fresh mass)(-1), and net CO(2) uptake in the dark contributed less than 1% to daily carbon gain. Following 10 d of water stress, net CO(2) uptake in the light fell 94% and net CO(2) uptake in the dark increased 7.5-fold, such that its contribution increased to 19% of daily carbon gain. Following rewatering, dark CO(2) uptake returned to within 5% of prestressed levels. We postulate that CAM assists survival of Z. zamiifolia by reducing water loss and maintaining carbon gain during seasonal droughts characteristic of its natural habitat.