Impact of selective logging on inbreeding and gene dispersal in an Amazonian tree population of Carapa guianensis Aubl

Selective logging may impact patterns of genetic diversity within populations of harvested forest tree species by increasing distances separating conspecific trees, and modifying physical and biotic features of the forest habitat. We measured levels of gene diversity, inbreeding, pollen dispersal and spatial genetic structure (SGS) of an Amazonian insect-pollinated Carapa guianensis population before and after commercial selective logging. Similar levels of gene diversity and allelic richness were found before and after logging in both the adult and the seed generations. Pre- and post-harvest outcrossing rates were high, and not significantly different from one another. We found no significant levels of biparental inbreeding either before or after logging. Low levels of pollen pool differentiation were found, and the pre- vs. post-harvest difference was not significant. Pollen dispersal distance estimates averaged between 75 m and 265 m before logging, and between 76 m and 268 m after logging, depending on the value of tree density and the dispersal model used. There were weak and similar levels of differentiation of allele frequencies in the adults and in the pollen pool, before and after logging occurred, as well as weak and similar pre- and post-harvest levels of SGS among adult trees. The large neighbourhood sizes estimated suggest high historical levels of gene flow. Overall our results indicate that there is no clear short-term genetic impact of selective logging on this population of C. guianensis.     

Proteomic profiling of a parasitic wasp exposed to constant and fluctuating cold exposure

When insects are exposed to fluctuating thermal regimes (FTRs) (i.e., cold exposure alternating with periodic short pulses to high temperature), in contrast to constant low temperature (CLT), mortality due to accumulation of chill injuries is markedly reduced. To investigate the physiological processes behind the positive impact of FTR, based on a holistic approach, two-dimensional electrophoresis (2-DE) analysis were performed with the parasitic wasp Aphidius colemani. Parasitoid proteomes revealed 369 well-distinguishable protein spots, where the overall response to cold exposure was clearly specific to treatments (CLT versus FTR). The reduced mortality under FTR was associated with up-regulation of several proteins playing key roles in energy metabolism (glycolysis, TCA cycle, synthesis and conversion of ATP), protein chaperoning (Hsp70/Hsp90), and protein degradation (proteasome). Our results also support the idea that cytoskeleton components, particularly actin arrangement, could play a role in the higher survival rates of insects under FTR.     

Members of the plant NIMA-related kinases are involved in organ development and vascularization in poplar, Arabidopsis and rice

NIMA-related kinases (Neks) are a family of serine/threonine kinases that have been linked to cell-cycle regulation in fungi and mammals. Information regarding the function of Neks in plants is very limited. We screened the three plant species that have had their genomes sequenced in an attempt to improve our understanding of their role in plants. We retrieved seven members in Arabidopsis thaliana, nine in Populus trichocarpa and six in Oryza sativa. Phylogenetic analysis showed that plant Neks are closely related to each other and contain paralogous genes. Moreover, their chromosome distribution and their exon-intron structure revealed that the actual plant Nek family was derived from a single representative followed by large segmental duplication events. Functional expression analyses in the three species relied on RTqPCR in poplar and publicly available microarray data for Arabidopsis and rice. Although plant Neks are present in every organ analyzed, their expression profiles suggest their involvement in plant development processes. Furthermore, we showed that PNek1, a member of the poplar family, is expressed at sites of free auxin synthesis and is specifically involved during the vascularization process.     

Synthesis and biological evaluation of thiazolidine-2-one 1,1-dioxide as inhibitors of Escherichia coli beta-ketoacyl-ACP-synthase III (FabH)

A series of cyclic sulfones has been synthesized and their activity against beta-ketoacyl-ACP-synthase III (FabH) has been investigated. The compounds are selectively active against Escherichia coli FabH (ecFabH), but not Mycobacterium tuberculosis FabH (mtFabH) or Plasmodium falciparum KASIII (PfKASIII). The activity against ecFabH ranges from 0.9 to >100microM and follows a consistent general SAR trend. Many of the compounds were shown to have antimalarial activity against chloroquine (CQ)-sensitive (D6) P. falciparum (IC(50)=5.3microM for the most potent inhibitor) and some were active against E. coli (MIC=6.6microg/ml for the most potent inhibitor).     

Lambing rate using vitrified blastocysts is improved by culture with BSA and hyaluronan

Fatty acid-free bovine serum albumin (BSA(FAF)) can be added to supplement medium used in the culture of sheep embryos. BSA(FAF) was able to support blastocyst and subsequent embryo development at rates equivalent to that of fetal calf serum (FCS)-supplemented medium when fresh embryos were transferred. Furthermore, culture with BSA(FAF) significantly increased development of vitrified blastocysts transferred into synchronized sheep. The addition of the glycosaminoglycan, hyaluronan (HA) to the culture medium in the third and fifth day also increased cryo-tolerance of blastocysts and in turn lambing rate was improved.     

hnRNP I/PTB can antagonize the splicing repressor activity of SRp30c

The control of alternative pre-mRNA splicing often requires the participation of factors displaying synergistic or antagonistic activities. In the hnRNP A1 pre-mRNA, three elements promote the exclusion of alternative exon 7B, while a fourth intron element (CE9) represses splicing of exon 7B to the downstream exon. We have shown previously that the 5' portion of the 38-nucleotide-long CE9 element is bound by SRp30c, and that this interaction is important for repression in vitro. To determine whether SRp30c alone can impose repression, we tested a high-affinity SRp30c binding site that we identified using the SELEX protocol. We find that multiple high-affinity SRp30c sites are required to replicate the level of repression obtained with CE9, and that both the 5' and the 3' portions of CE9 contribute to SRp30c binding. Performing RNA affinity chromatography with the complete CE9 element recovered hnRNP I/PTB. Surprisingly however, His-tagged PTB reduced the binding of SRp30c to CE9 in a nuclear extract, stimulated splicing to a downstream 3' splice site, and relieved the CE9-mediated splicing repression in vitro. Our in vivo results are consistent with the notion that increasing PTB levels alleviates the repression imposed by CE9 to a downstream 3' splice site. Thus, PTB can function as an anti-repressor molecule to counteract the splicing inhibitory activity of SRp30c.     

Distribution of virulence profiles related to new toxins and putative adhesins in Shiga toxin-producing Escherichia coli isolated from diverse sources in Brazil

The distribution of virulence markers related to cytolethal distending toxin-V (CDT-V), subtilase cytotoxin (SubAB), the enterohemorrhagic Escherichia coli factor for adherence (Efa1), the adhesin similar to IrgA (Iha), the long polar fimbriae (LpfO113), the autoagglutinating adhesin (Saa), and the protein required for full expression of adherence of O157:H7 Sakai strain (ToxB) was investigated in 121 Shiga toxin-producing E. coli (STEC) strains isolated in Brazil. STEC strains were isolated from human infections (n=49), cattle (n=68) and ground meat samples (n=4). Overall, the lpfA(O113), iha, efa1, saa, and toxB sequences were observed in 89.2%, 87.6%, 47.1%, 43%, and 13.2% of the strains, respectively. The genes efa1 (96.6%) and toxB (27%) were only identified among eae-positive strains, while saa (83.8%), cdt-V (12.9%), and subAB (48.4%) just occurred in eae-negative STEC strains. STEC strains harboring cdt-V and subAB were for the first time described in the South American subcontinent. In addition, the simultaneous presence of cdt-V and subAB has not been previously reported, nor the presence of subAB in STEC O77, O79, O105, O174, and O178 serogroups. A diversity of virulence profiles was observed among the STEC strains studied. The most prevalent profile observed among eae-positive STEC strains mainly isolated from humans was eae efa1 iha lpfA(O113), whereas iha lpfA(O113) saa ehxA subAB prevailed among eae-negative STEC strains, mostly isolated from cattle and foods.     

Implications for molecular mechanisms of glycoprotein hormone receptors using a new sequence-structure-function analysis resource

Comparison between wild-type and mutated glycoprotein hormone receptors (GPHRs), TSH receptor, FSH receptor, and LH-chorionic gonadotropin receptor is established to identify determinants involved in molecular activation mechanism. The basic aims of the current work are 1) the discrimination of receptor phenotypes according to the differences between activity states they represent, 2) the assignment of classified phenotypes to three-dimensional structural positions to reveal 3) functional-structural hot spots and 4) interrelations between determinants that are responsible for corresponding activity states. Because it is hard to survey the vast amount of pathogenic and site-directed mutations at GPHRs and to improve an almost isolated consideration of individual point mutations, we present a system for systematic and diversified sequence-structure-function analysis (http://www.fmp-berlin.de/ssfa). To combine all mutagenesis data into one set, we converted the functional data into unified scaled values. This at least enables their comparison in a rough classification manner. In this study we describe the compiled data set and a wide spectrum of functions for user-driven searches and classification of receptor functionalities such as cell surface expression, maximum of hormone binding capability, and basal as well as hormone-induced Galphas/Galphaq mediated cAMP/inositol phosphate accumulation. Complementary to known databases, our data set and bioinformatics tools allow functional and biochemical specificities to be linked with spatial features to reveal concealed structure-function relationships by a semiquantitative analysis. A comprehensive discrimination of specificities of pathogenic mutations and in vitro mutant phenotypes and their relation to signaling mechanisms of GPHRs demonstrates the utility of sequence-structure-function analysis. Moreover, new interrelations of determinants important for selective G protein-mediated activation of GPHRs are resumed.     

Presymbiotic growth and sporal morphology are affected in the arbuscular mycorrhizal fungus Gigaspora margarita cured of its endobacteria

Some arbuscular mycorrhizal fungi contain endocellular bacteria. In Gigaspora margarita BEG 34, a homogenous population of beta-Proteobacteria is hosted inside the fungal spore. The bacteria, named Candidatus Glomeribacter gigasporarum, are vertically transmitted through fungal spore generations. Here we report how a protocol based on repeated passages through single-spore inocula caused dilution of the initial bacterial population eventually leading to cured spores. Spores of this line had a distinct phenotype regarding cytoplasm organization, vacuole morphology, cell wall organization, lipid bodies and pigment granules. The absence of bacteria severely affected presymbiotic fungal growth such as hyphal elongation and branching after root exudate treatment, suggesting that Ca. Glomeribacter gigasporarum is important for optimal development of its fungal host. Under laboratory conditions, the cured fungus could be propagated, i.e. could form mycorrhizae and sporulate, and can therefore be considered as a stable variant of the wild type. The results demonstrated that - at least for the G. margarita BEG 34 isolate - the absence of endobacteria affects the spore phenotype of the fungal host, and causes delays in the growth of germinating mycelium, possibly affecting its ecological fitness. This cured line is the first manipulated and stable isolate of an arbuscular mycorrhizal fungus.