Phosphorylation of the MAPKKK regulator Ste50p in Saccharomyces cerevisiae: a casein kinase I phosphorylation site is required for proper mating function

The Ste50 protein of Saccharomyces cerevisiae is a regulator of the Ste11p protein kinase. Ste11p is a member of the MAP3K (or MEKK) family, which is conserved from yeast to mammals. Ste50p is involved in all the signaling pathways that require Ste11p function, yet little is known about the regulation of Ste50p itself. Here, we show that Ste50p is phosphorylated on multiple serine/threonine residues in vivo. Threonine 42 (T42) is phosphorylated both in vivo and in vitro, and the protein kinase responsible has been identified as casein kinase I. Replacement of T42 with alanine (T42A) compromises Ste50p function. This mutation abolishes the ability of overexpressed Ste50p to suppress either the mating defect of a ste20 ste50 deletion mutant or the mating defect of a strain with a Ste11p deleted from its sterile-alpha motif domain. Replacement of T42 with a phosphorylation-mimetic aspartic acid residue (T42D) permits wild-type function in all assays of Ste50p function. These results suggest that phosphorylation of T42 of Ste50p is required for proper signaling in the mating response. However, this phosphorylation does not seem to have a detectable role in modulating the high-osmolarity glycerol synthesis pathway.     

Translokin is an intracellular mediator of FGF-2 trafficking

Basic fibroblast growth factor (bFGF or FGF-2) exerts its pleiotropic activities both as an exogenous and an intracellular factor. FGF-1 and FGF-2 are prototypes for this dual signalling, but the mechanisms of their intracellular actions remain unknown. Here we show that Translokin, a cytoplasmic protein of relative molecular mass 55,000 (M(r) 55K), interacts specifically with the 18K form of FGF-2. Translokin is ubiquitously expressed and colocalizes with the microtubular network. As Translokin does not interact with FGF-1, we used a strategy based on FGF-1-FGF-2 chimaeras to map the interacting regions in FGF-2 and to generate Nb1a2, a non-interacting variant of FGF-2. Although most of the FGF-2 properties are preserved in Nb1a2, this variant is defective in intracellular translocation and in stimulating proliferation. The fusion of a nuclear localization signal to Nb1a2 restores its mitogenic activity and its nuclear association. Inhibiting Translokin expression by RNA interference reduces the translocation of FGF-2 without affecting the intracellular trafficking of FGF-1. Our data show that the nuclear association of internalized FGF-2 is essential for its mitogenic activity and that Translokin is important in this translocation pathway.     

Splanchnic free fatty acid kinetics

These studies were conducted to assess the relationship between visceral adipose tissue free fatty acid (FFA) release and splanchnic FFA release. Steady-state splanchnic bed palmitate ([9,10-(3)H]palmitate) kinetics were determined from 14 sampling intervals from eight dogs with chronic indwelling arterial, portal vein, and hepatic vein catheters. We tested a model designed to predict the proportion of FFAs delivered to the liver from visceral fat by use of hepatic vein data. The model predicted that 15 +/- 2% of hepatic palmitate delivery originated from visceral lipolysis, which was greater (P = 0.004) than the 11 +/- 2% actually observed. There was a good relationship (r(2) = 0.63) between the predicted and observed hepatic palmitate delivery values, but the model overestimated visceral FFA release more at lower than at higher palmitate concentrations. The discrepancy could be due to differential uptake of FFAs arriving from the arterial vs. the portal vein or to release of FFAs in the hepatic circulatory bed. Splanchnic FFA release measured using hepatic vein samples was strongly related to visceral adipose tissue FFA release into the portal vein. This finding suggests that splanchnic FFA release is a good indicator of visceral adipose tissue lipolysis.     

Effects of overstory and understory vegetation on the understory light environment in mixed boreal forests

Abstract. The percentage of above-canopy Photosynthetic Photon Flux Density (%PPFD) was measured at 0, 50 and 100 cm above the forest floor and above the main understory vegetation in stands of (1) pure Betula papyrifera (White birch), (2) pure Populus tremuloides (Trembling aspen), (3) mixed broad-leaf-conifer, (4) shade-tolerant conifer and (5) pure Pinus banksiana (Jack pine) occurring on both clay and till soil types. %PPFD was measured instantaneously under overcast sky conditions (nine locations within each of 29 stands) and continuously for a full day under clear sky conditions (five locations within each of eight stands). The percentage cover of the understory layer was estimated at the same locations as light measurements. Mean %PPFD varied from 2% at the forest floor under Populus forests to 15% above the understory vegetation cover under Betula forests. Percent PPFD above the understory vegetation cover was significantly higher under shade intolerant tree species such as Populus, Betula and Pinus than under shade tolerant conifers. No significant differences were found in %PPFD above the understory vegetation cover under similar tree species between clay and till soil types. The coefficient of variation in %PPFD measured in the nine locations within each stand was significantly lower under deciduous dominated forests (mean of 19%) than under coniferous dominated forests (mean of 40%). %PPFD measured at the forest floor was positively correlated with %PPFD measured above the understory vegetation and negatively correlated with cumulative total percent cover of the understory vegetation (R² = 0.852). The proportion of sunflecks above 250 and 500 mmol m^–2 s^–1 was much lower and %PPFD in shade much higher under Populus and Betula forests than under the other forests. Differences in the mean, variability and nature of the light environment found among forest and soil types are discussed in relation to their possible influences on tree succession.     

The Syk Protein Tyrosine Kinase Is Essential for Fcγ Receptor Signaling in Macrophages and Neutrophils

The cytoplasmic protein tyrosine kinase Syk has two amino-terminal SH2 domains that engage phosphorylated immunoreceptor tyrosine-based activation motifs in the signaling subunits of immunoreceptors. Syk, in conjunction with Src family kinases, has been implicated in immunoreceptor signaling in both lymphoid and myeloid cells. We have investigated the role of Syk in Fcγ receptor (FcγR)-dependent and -independent responses in bone marrow-derived macrophages and neutrophils by using mouse radiation chimeras reconstituted with fetal liver cells from Syk^−/− embryos. Chimeric mice developed an abdominal hemorrhage starting 2 to 3 months after transplantation that was ultimately lethal. Syk-deficient neutrophils derived from the bone marrow were incapable of generating reactive oxygen intermediates in response to FcγR engagement but responded normally to tetradecanoyl phorbol acetate stimulation. Syk-deficient macrophages were defective in phagocytosis induced by FcγR but showed normal phagocytosis in response to complement. The tyrosine phosphorylation of multiple cellular polypeptides, including the FcγR γ chain, as well as Erk2 activation, was compromised in Syk^−/− macrophages after FcγR stimulation. In contrast, the induction of nitric oxide synthase in macrophages stimulated with lipopolysaccharide and gamma interferon was not dependent on Syk. Surprisingly, Syk-deficient macrophages were impaired in the ability to survive or proliferate on plastic petri dishes. Taken together, these results suggest that Syk has specific physiological roles in signaling from FcγRs in neutrophils and macrophages and raise the possibility that in vivo, Syk is involved in signaling events other than those mediated by immunoreceptors.     

Synthesis and in vitro antiplasmodial evaluation of 4-anilino-2-trichloromethylquinazolines

To identify a new safe antiplasmodial molecular scaffold, an original series of 2-trichloromethylquinazolines, functionalized in position 4 by an alkyl- or arylamino substituent, was synthesized from 4-chloro-2-trichloromethylquinazoline 1, via a cheap, fast and efficient solvent-free operating procedure. Among the 40 molecules prepared, several exhibit a good profile with both a significant antiplasmodial activity on the W2 Plasmodium falciparum strain (IC₅₀ values: 0.4–2.2 μM) and a promising toxicological behavior regarding human cells (HepG2/W2 selectivity indexes: 40–83), compared to the antimalarial drug compounds chloroquine and doxycycline. The in vitro antitoxoplasmic and antileishmanial evaluations were conducted in parallel on the most active molecules, showing that these ones specifically display antiplasmodial properties.     

A Revised Time Tree of the Asterids: Establishing a Temporal Framework For Evolutionary Studies of the Coffee Family (Rubiaceae)

Divergence time analyses in the coffee family (Rubiaceae) have all relied on the same Gentianales crown group age estimate, reported by an earlier analysis of the asterids, for defining the upper age bound of the root node in their analyses. However, not only did the asterid analysis suffer from several analytical shortcomings, but the estimate itself has been used in highly inconsistent ways in these Rubiaceae analyses. Based on the original data, we here reanalyze the divergence times of the asterids using relaxed-clock models and 14 fossil-based minimum age constraints. We also expand the data set to include an additional 67 taxa from Rubiaceae sampled across all three subfamilies recognized in the family. Three analyses are conducted: a separate analysis of the asterids, which completely mirrors the original asterid analysis in terms of taxon sample and data; a separate analysis of the Gentianales, where the result from the first analysis is used for defining a secondary root calibration point; and a combined analysis where all taxa are analyzed simultaneously. Results are presented in the form of a time-calibrated phylogeny, and age estimates for asterid groups, Gentianales, and major groups of Rubiaceae are compared and discussed in relation to previously published estimates. Our updated age estimates for major groups of Rubiaceae provide a significant step forward towards the long term goal of establishing a robust temporal framework for the divergence of this biologically diverse and fascinating group of plants.     

Diverse Virulent Pneumophages Infect Streptococcus mitis

Streptococcus mitis has emerged as one of the leading causes of bacterial endocarditis and is related to Streptococcus pneumoniae. Antibiotic resistance has also increased among strains of S. mitis and S. pneumoniae. Phages are being reinvestigated as alternatives to antibiotics for managing infections. In this study, the two virulent phages Cp-1 (Podoviridae) and Dp-1 (Siphoviridae), previously isolated from S. pneumoniae, were found to also infect S. mitis. Microbiological assays showed that both pneumophages could not only replicate in S. mitis but also produced more visible plaques on this host. However, the burst size and phage adsorption data were lower in S. mitis as compared to S. pneumoniae. A comparison of the genomes of each phage grown on both hosts produced identical nucleotide sequences, confirming that the same phages infect both bacterial species. We also discovered that the genomic sequence of podophage Cp-1 of the Félix d’Hérelle collection is different than the previously reported sequence and thus renamed SOCP.     

Bushmeat genetics: setting up a reference framework for the DNA typing of African forest bushmeat

The bushmeat trade in tropical Africa represents illegal, unsustainable off‐takes of millions of tons of wild game – mostly mammals – per year. We sequenced four mitochondrial gene fragments (cyt b, COI, 12S, 16S) in >300 bushmeat items representing nine mammalian orders and 59 morphological species from five western and central African countries (Guinea, Ghana, Nigeria, Cameroon and Equatorial Guinea). Our objectives were to assess the efficiency of cross‐species PCR amplification and to evaluate the usefulness of our multilocus approach for reliable bushmeat species identification. We provide a straightforward amplification protocol using a single ‘universal’ primer pair per gene that generally yielded >90% PCR success rates across orders and was robust to different types of meat preprocessing and DNA extraction protocols. For taxonomic identification, we set up a decision pipeline combining similarity‐ and tree‐based approaches with an assessment of taxonomic expertise and coverage of the GENBANK database. Our multilocus approach permitted us to: (i) adjust for existing taxonomic gaps in GENBANK databases, (ii) assign to the species level 67% of the morphological species hypotheses and (iii) successfully identify samples with uncertain taxonomic attribution (preprocessed carcasses and cryptic lineages). High levels of genetic polymorphism across genes and taxa, together with the excellent resolution observed among species‐level clusters (neighbour‐joining trees and Klee diagrams) advocate the usefulness of our markers for bushmeat DNA typing. We formalize our DNA typing decision pipeline through an expert‐curated query database – DNAbushmeat – that shall permit the automated identification of African forest bushmeat items.     

In Silico Analysis of Usher Encoding Genes in Klebsiella pneumoniae and Characterization of Their Role in Adhesion and Colonization

Chaperone/usher (CU) assembly pathway is used by a wide range of Enterobacteriaceae to assemble adhesive surface structures called pili or fimbriae that play a role in bacteria-host cell interactions. In silico analysis revealed that the genome of Klebsiella pneumoniae LM21 harbors eight chromosomal CU loci belonging to γκп and ϭ clusters. Of these, only two correspond to previously described operons, namely type 1 and type 3-encoding operons. Isogenic usher deletion mutants of K. pneumoniae LM21 were constructed for each locus and their role in adhesion to animal (Intestine 407) and plant (Arabidopsis thaliana) cells, biofilm formation and murine intestinal colonization was investigated. Type 3 pili usher deleted mutant was impaired in all assays, whereas type 1 pili usher deleted mutant only showed attenuation in adhesion to plant cells and in intestinal colonization. The LM21ΔkpjC mutant was impaired in its capacity to adhere to Arabidopsis cells and to colonize the murine intestine, either alone or in co-inoculation experiments. Deletion of LM21kpgC induced a significant decrease in biofilm formation, in adhesion to animal cells and in colonization of the mice intestine. The LM21∆kpaC and LM21∆kpeC mutants were only attenuated in biofilm formation and the adhesion abilities to Arabidopsis cells, respectively. No clear in vitro or in vivo effect was observed for LM21∆kpbC and LM21∆kpdC mutants. The multiplicity of CU loci in K. pneumoniae genome and their specific adhesion pattern probably reflect the ability of the bacteria to adhere to different substrates in its diverse ecological niches.