Topological characterization of the essential Escherichia coli cell division protein FtsW

The membrane topology of Escherichia coli FtsW, a 46-kDa essential protein, was analyzed using a set of 28 ftsW-alkaline phosphatase (ftsW-phoA) and nine ftsW-beta-lactamase (ftsW-bla) gene fusions obtained by in vivo and in vitro methods. The alkaline phosphatase activities or resistance pattern of cells expressing the FtsW-PhoA or FtsW-Bla fusions confirmed only eight out of 10 transmembrane segments predicted by computational methods. After comparison with the recent topology of Streptococcus pneumoniae FtsW, we could identify all the fusions in absolute agreement with the predicted model: N-terminal and C-terminal ends in the cytoplasm, 10 transmembrane segments and one large loop of 67 amino acids (E240-E306) located in the periplasm.     

Specific and Reversible Immobilization of Proteins Tagged to the Affinity Polypeptide C-LytA on Functionalized Graphite Electrodes

We have developed a general method for the specific and reversible immobilization of proteins fused to the choline-binding module C-LytA on functionalized graphite electrodes. Graphite electrode surfaces were modified by diazonium chemistry to introduce carboxylic groups that were subsequently used to anchor mixed self-assembled monolayers consisting of N,N-diethylethylenediamine groups, acting as choline analogs, and ethanolamine groups as spacers. The ability of the prepared electrodes to specifically bind C-LytA-tagged recombinant proteins was tested with a C-LytA-β-galactosidase fusion protein. The binding, activity and stability of the immobilized protein was evaluated by electrochemically monitoring the formation of an electroactive product in the enzymatic hydrolysis of the synthetic substrate 4-aminophenyl β-D-galactopyranoside. The hybrid protein was immobilized in an specific and reversible way, while retaining the catalytic activity. Moreover, these functionalized electrodes were shown to be highly stable and reusable. The method developed here can be envisaged as a general, immobilization procedure on the protein biosensor field.     

Viability of the Endangered Little Bustard <Emphasis Type="Italic">Tetrax tetrax</Emphasis> Population of Western France

Stochastic computer simulations are used to evaluate the sensitivity of Little bustard population parameters, estimating the survival probabilities of the seven endangered Little bustard populations of central-western France for which conservation actions are currentlybeing or have been implemented. Different scenarios of parameter compensation for those nuclei to establish parameter levels assuring population viability are discussed. Adult survival, productivity per female, initial population size and carrying capacity were the most sensitive parameters in a hypothetical, isolated population. Juvenile survival also affected population survival, although its sensitivity was lower. Sex ratio did not have a linear effect on population survival, but probability of extinction increased for extreme values. Productivity per female and initial population size, varied strongly among the populations studied, determining their average time of extinction and growth rate. When a metapopulation scenario was simulated, the survival probabilities of each population and the metapopulations stayed close to 1.0 if no mortality was associated to migration. When mortality during migration was included in the simulations, the metapopulation's probability of survival significantly decreased under 90%. This approach may help managers to correctly address conservation measures and design effective strategies, which should be directed mainly to improve productivity, enhance female survival, and minimise mortality during migration (e.g. promoting insect-rich nesting substrates, avoiding female killing and nest destruction at harvesting, reducing the risk of collision with powerlines, or controlling poaching).     

Population genomic analysis of a bacterial plant pathogen: novel insight into the origin of Pierce's disease of grapevine in the U.S

Invasive diseases present an increasing problem worldwide; however, genomic techniques are now available to investigate the timing and geographical origin of such introductions. We employed genomic techniques to demonstrate that the bacterial pathogen causing Pierce's disease of grapevine (PD) is not native to the US as previously assumed, but descended from a single genotype introduced from Central America. PD has posed a serious threat to the US wine industry ever since its first outbreak in Anaheim, California in the 1880s and continues to inhibit grape cultivation in a large area of the country. It is caused by infection of xylem vessels by the bacterium Xylella fastidiosa subsp. fastidiosa, a genetically distinct subspecies at least 15,000 years old. We present five independent kinds of evidence that strongly support our invasion hypothesis: 1) a genome-wide lack of genetic variability in X. fastidiosa subsp. fastidiosa found in the US, consistent with a recent common ancestor; 2) evidence for historical allopatry of the North American subspecies X. fastidiosa subsp. multiplex and X. fastidiosa subsp. fastidiosa; 3) evidence that X. fastidiosa subsp. fastidiosa evolved in a more tropical climate than X. fastidiosa subsp. multiplex; 4) much greater genetic variability in the proposed source population in Central America, variation within which the US genotypes are phylogenetically nested; and 5) the circumstantial evidence of importation of known hosts (coffee plants) from Central America directly into southern California just prior to the first known outbreak of the disease. The lack of genetic variation in X. fastidiosa subsp. fastidiosa in the US suggests that preventing additional introductions is important since new genetic variation may undermine PD control measures, or may lead to infection of other crop plants through the creation of novel genotypes via inter-subspecific recombination. In general, geographically mixing of previously isolated subspecies should be avoided.     

Secondary heterologous dengue infection risk: Disequilibrium between immune regulation and inflammation?

Increased serum levels of cytokines released by cells of the immune response have been detected in patients suffering from dengue disease. Likewise, secondary infections by a different dengue virus serotype result in a highest risk of development of the severe dengue disease. Both findings suggest that the memory immune response is one of the key players in the pathogenesis of this disease. Here we take advantage of the particular Cuban epidemiological situation in dengue to analyze a broad spectrum of cell-mediated immune response mediators at mRNA and protein level. Evidences for a regulatory immune pattern in homologous (TGF-β, IL-10) vs. pro-inflammatory pattern (IFN-γ, TNF-α) in heterologous dengue virus re-challenge were found, suggesting a possible association with the higher incidence of severe dengue cases in the latter case.     

Soybean seed damage by different species of stink bugs

Abstract

• 1 Damage caused by the three main species of stink bugs occurring on soybean Nezara viridula (Linnaeus), Piezodorus guildinii (Westwood) and Euschistus heros (Fabricius) was compared in field cages and in greenhouses. Infestation levels of 4 stink bugs/m row of plants (field cages) and 2 stink bug/plant (greenhouse) for 15 days during the pod filling stage are reported. At harvest, the yield and seed quality were evaluated.
• 2 In the field, there was no difference in yield between infested and insect‐free plants, but damage to seed quality varied with stink bug species. Plants damaged by P. guildinii had the lowest quality seeds. From 50 g seed samples harvested in the field, the mean weight of seeds classified as ‘good’ was 37.3 g in plants infested with P. guildinii, compared to 41.8, 44.2 and 46.6 g in plants infested with E. heros, N. viridula and the control, respectively.
• 3 Plants infested with P. guildinii showed the highest number of seeds damaged by stink bugs, whereas those infested with E. heros showed the lowest damage.
• 4 Plants infested with P. guildinii had 18.5% damaged seeds, higher than the 3.6% and 3.4% damaged seeds from plants infested with the two other species and 0.1% in control plants. The percentage of non‐viable seeds due to stink bug damage was 5.7% for P. guildinii but lower for the other two species.

     

The Highly Conserved Layer-3 Component of the HIV-1 gp120 Inner Domain Is Critical for CD4-Required Conformational Transitions

The trimeric envelope glycoprotein (Env) of human immunodeficiency virus type 1 (HIV-1) mediates virus entry into host cells. CD4 engagement with the gp120 exterior envelope glycoprotein subunit represents the first step during HIV-1 entry. CD4-induced conformational changes in the gp120 inner domain involve three potentially flexible topological layers (layers 1, 2, and 3). Structural rearrangements between layer 1 and layer 2 have been shown to facilitate the transition of the envelope glycoprotein trimer from the unliganded to the CD4-bound state and to stabilize gp120-CD4 interaction. However, our understanding of CD4-induced conformational changes in the gp120 inner domain remains incomplete. Here, we report that a highly conserved element of the gp120 inner domain, layer 3, plays a pivot-like role in these allosteric changes. In the unliganded state, layer 3 modulates the association of gp120 with the Env trimer, probably by influencing the relationship of the gp120 inner and outer domains. Importantly, layer 3 governs the efficiency of the initial gp120 interaction with CD4, a function that can also be fulfilled by filling the Phe43 cavity. This work defines the functional importance of layer 3 and completes a picture detailing the role of the gp120 inner domain in CD4-induced conformational transitions in the HIV-1 Env trimer.     

Plasmodium circumsporozoite protein promotes the development of the liver stages of the parasite

The liver stages of malaria are clinically silent but have a central role in the Plasmodium life cycle. Liver stages of the parasite containing thousands of merozoites grow inside hepatocytes for several days without triggering an inflammatory response. We show here that Plasmodium uses a PEXEL/VTS motif to introduce the circumsporozoite (CS) protein into the hepatocyte cytoplasm and a nuclear localization signal (NLS) to enter its nucleus. CS outcompetes NFkappaB nuclear import, thus downregulating the expression of many genes controlled by NFkappaB, including those involved in inflammation. CS also influences the expression of over one thousand host genes involved in diverse metabolic processes to create a favorable niche for the parasite growth. The presence of CS in the hepatocyte enhances parasite growth of the liver stages in vitro and in vivo. These findings have far reaching implications for drug and vaccine development against the liver stages of the malaria parasite.