L-phenylalanine binding and domain organization in human phenylalanine hydroxylase: a differential scanning calorimetry study

Human phenylalanine hydroxylase (hPAH) is a tetrameric enzyme that catalyzes the hydroxylation of L-phenylalanine (L-Phe) to L-tyrosine; a dysfunction of this enzyme causes phenylketonuria. Each subunit in hPAH contains an N-terminal regulatory domain (Ser2-Ser110), a catalytic domain (Asp112-Arg410), and an oligomerization domain (Ser411-Lys452) including dimerization and tetramerization motifs. Two partially overlapping transitions are seen in differential scanning calorimetry (DSC) thermograms for wild-type hPAH in 0.1 M Na-Hepes buffer, 0.1 M NaCl, pH 7.0. Although these transitions are irreversible, studies on their scan-rate dependence support that the equilibrium thermodynamics analysis is permissible in this case. Comparison with the DSC thermograms for truncated forms of the enzyme, studies on the protein and L-Phe concentration effects on the transitions, and structure-energetic calculations based on a modeled structure support that the thermal denaturation of hPAH occurs in three stages: (i) unfolding of the four regulatory domains, which is responsible for the low-temperature calorimetric transition; (ii) unfolding of two (out of the four) catalytic domains, which is responsible for the high-temperature transition; and (iii) irreversible protein denaturation, which is likely responsible for the observed exothermic distortion in the high-temperature side of the high-temperature transition. Stages 1 and 2 do not appear to be two-state processes. We present an approach to the analysis of ligand effects on DSC transition temperatures, which is based on the general binding polynomial formalism and is not restricted to two-state transitions. Application of this approach to the L-Phe effect on the DSC thermograms for hPAH suggests that (i) there are no binding sites for L-Phe in the regulatory domains; therefore, contrary to the common belief, the activation of PAH by L-Phe seems to be the result of its homotropic cooperative binding to the active sites. (ii) The regulatory domain appears to be involved in cooperativity through its interactions with the catalytic and oligomerization domains; thus, upon regulatory domain unfolding, the cooperativity in the binding of L-Phe to the catalytic domains seems to be lost and the value of the L-Phe concentration corresponding to half-saturation is increased. Overall, our results contribute to the understanding of the conformational stability and the substrate-induced cooperative activation of this important enzyme.     

Can current farmland landscapes feed declining steppe birds? Evaluating arthropod abundance for the endangered little bustard (Tetrax tetrax) in cereal farmland during the chick‐rearing period: Variations between habitats and localities

Agriculture intensification threatens farmland bird populations because, among other reasons, it reduces the availability of food resources required to rear their offspring. In our study, we sampled and analyzed total arthropod abundance, biomass and richness, and orthopteran and coleopteran abundance and biomass in different agricultural habitats (alfalfa fields, stubble fields, grazed fields, and field margins) across 4 study localities with different levels of agriculture abandonment–intensification, comparing between areas used and not used by one of the most threatened farmland birds in Europe, the little bustard (Tetrax tetrax), during the chick‐rearing season. Field margins were the taxonomically richest habitat, while alfalfa fields presented significantly higher total arthropod abundance and biomass than other habitats. All arthropod variables were the highest in the localities with clear conservation‐focused agrarian management, and the lowest in the most intensive one. Areas used by little bustards had higher orthopteran and coleopteran abundance and biomass than nonused areas, except for coleopteran biomass in grazed fields. These results highlight the relevance of these arthropods for the species, the importance of dry alfalfa fields as food reservoirs in this critical time of year, the food scarcity in sites where agrarian management disregards farmland bird conservation, and the role of stubbles as providers of food resources during the chick‐rearing season in areas used by the species. The adequate management of alfalfa fields and stubbles to provide those key resources seems crucial to improve little bustard breeding success.     

Molecular basis of the differential interaction with lithium of glycine transporters GLYT1 and GLYT2

Glycine synaptic levels are controlled by glycine transporters (GLYTs) catalyzing Na(+)/Cl(-)/glycine cotransport. GLYT1 displays a 2:1 :1 stoichiometry and is the main regulator of extracellular glycine concentrations. The neuronal GLYT2, with higher sodium coupling (3:1 :1), supplies glycine to the pre-synaptic terminal to refill synaptic vesicles. In this work, using structural homology modelling and molecular dynamics simulations of GLYTs, we predict the conservation of the two sodium sites present in the template (leucine transporter from Aquifex aeolicus), and confirm its use by mutagenesis and functional analysis. GLYTs Na1 and Na2 sites show differential cation selectivity, as inferred from the action of lithium, a non-transport-supporting ion, on Na(+)-site mutants. GLYTs lithium responses were unchanged in Na1-site mutants, but abolished or inverted in mutants of Na2 site, which binds lithium in the presence of low sodium concentrations and therefore, controls lithium responses. Here, we report, for the first time, that lithium exerts opposite actions on GLYTs isoforms. Glycine transport by GLYT1 is inhibited by lithium whereas GLYT2 transport is stimulated, and this effect is more evident at increased glycine concentrations. In contrast to GLYT1, high and low affinity lithium-binding processes were detected in GLYT2.     

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.