Seed dispersal networks in tropical forest fragments: Area effects,remnant species,and interaction diversity

Seed dispersal interactions involve key ecological processes in tropical forests that help to maintain ecosystem functioning. Yet this functionality may be threatened by increasing habitat loss, defaunation, and fragmentation. However, generalist species, and their interactions, can benefit from the habitat change caused by human disturbance while more specialized interactions mostly disappear. Therefore, changes in the structure of the local, within fragment, networks can be expected. Here we investigated how the structure of seed dispersal networks changes along a gradient of increasing habitat fragmentation. We analyzed 16 bird seed dispersal assemblages from forest fragments of a biodiversity-rich ecosystem. We found significant species–, interaction–, and network–area relationships, yet the later was determined by the number of species remaining in each community. The number of frugivorous bird and plant species, their interactions, and the number of links per species decreases as area is lost in the fragmented landscape. In contrast, network nestedness has a negative relationship with fragment area, suggesting an increasing generalization of the network structure in the gradient of fragmentation. Network specialization was not significantly affected by area, indicating that some network properties may be invariant to disturbance. Still, the local extinction of partner species, paralleled by a loss of interactions and specialist–specialist bird–plant seed dispersal associations, suggests the functional homogenization of the system as area is lost. Our study provides empirical evidence for network–area relationships driven by the presence/absence of remnant species and the interactions they perform.     

The genome sequence of the gram-positive sugarcane pathogen Leifsonia xyli subsp. xyli

The genome sequence of Leifsonia xyli subsp. xyli, which causes ratoon stunting disease and affects sugarcane worldwide, was determined. The single circular chromosome of Leifsonia xyli subsp. xyli CTCB07 was 2.6 Mb in length with a GC content of 68% and 2,044 predicted open reading frames. The analysis also revealed 307 predicted pseudogenes, which is more than any bacterial plant pathogen sequenced to date. Many of these pseudogenes, if functional, would likely be involved in the degradation of plant heteropolysaccharides, uptake of free sugars, and synthesis of amino acids. Although L. xyli subsp. xyli has only been identified colonizing the xylem vessels of sugarcane, the numbers of predicted regulatory genes and sugar transporters are similar to those in free-living organisms. Some of the predicted pathogenicity genes appear to have been acquired by lateral transfer and include genes for cellulase, pectinase, wilt-inducing protein, lysozyme, and desaturase. The presence of the latter may contribute to stunting, since it is likely involved in the synthesis of abscisic acid, a hormone that arrests growth. Our findings are consistent with the nutritionally fastidious behavior exhibited by L. xyli subsp. xyli and suggest an ongoing adaptation to the restricted ecological niche it inhabits.     

Exploiting the potential of insects for in vivo pathogenicity testing of microbial pathogens

Conventional assays for quantifying the virulence of microbial pathogens and mutants have traditionally relied upon the use of a range of mammalian species. A number of workers have demonstrated that insects can be used for evaluating microbial pathogenicity and provide results comparable to those that can be obtained with mammals since one component of the vertebrate immune system, the innate immune response, remains similar to that found in insects. Larvae of the Greater Wax Moth Galleria mellonella have been used to evaluate the virulence of a range of bacterial and fungal pathogens and a correlation with the virulence of these microbes in mice has been established. This review highlights the similarities of the vertebrate and insect innate immune responses to infection and identifies the potential use of insects for the in vivo evaluation of the microbial pathogenicity.     

Novel natural peptide substrates for endopeptidase 24.15, neurolysin,and angiotensin-converting enzyme

Endopeptidase 24.15 (EC; ep24.15), neurolysin (EC; ep24.16), and angiotensin-converting enzyme (EC; ACE) are metallopeptidases involved in neuropeptide metabolism in vertebrates. Using catalytically inactive forms of ep24.15 and ep24.16, we have identified new peptide substrates for these enzymes. The enzymatic activity of ep24.15 and ep24.16 was inactivated by site-directed mutagenesis of amino acid residues within their conserved HEXXH motifs, without disturbing their secondary structure or peptide binding ability, as shown by circular dichroism and binding assays. Fifteen of the peptides isolated were sequenced by electrospray ionization tandem mass spectrometry and shared homology with fragments of intracellular proteins such as hemoglobin. Three of these peptides (PVNFKFLSH, VVYPWTQRY, and LVVYPWTQRY) were synthesized and shown to interact with ep24.15, ep24.16, and ACE, with K(i) values ranging from 1.86 to 27.76 microm. The hemoglobin alpha-chain fragment PVNFKFLSH, which we have named hemopressin, produced dose-dependent hypotension in anesthetized rats, starting at 0.001 microg/kg. The hypotensive effect of the peptide was potentiated by enalapril only at the lowest peptide dose. These results suggest a role for hemopressin as a vasoactive substance in vivo. The identification of these putative intracellular substrates for ep24.15 and ep24.16 is an important step toward the elucidation of the role of these enzymes within cells.     

The Impact of the Invasive Alien Plant,Impatiens glandulifera,on Pollen Transfer Networks

Biological invasions are a threat to the maintenance of ecological processes, including pollination. Plant-flower visitor networks are traditionally used as a surrogated for pollination at the community level, despite they do not represent the pollination process, which takes place at the stigma of plants where pollen grains are deposited. Here we investigated whether the invasion of the alien plant Impatiens glandulifera (Balsaminaceae) affects pollen transfer at the community level. We asked whether more alien pollen is deposited on the stigmas of plants on invaded sites, whether deposition is affected by stigma type (dry, semidry and wet) and whether the invasion of I. glandulifera changes the structure of the resulting pollen transfer networks. We sampled stigmas of plants on 10 sites invaded by I. glandulifera (hereafter, balsam) and 10 non-invaded control sites. All 20 networks had interactions with balsam pollen, although significantly more balsam pollen was found on plants with dry stigmas in invaded areas. Balsam pollen deposition was restricted to a small subset of plant species, which is surprising because pollinators are known to carry high loads of balsam pollen. Balsam invasion did not affect the loading of native pollen, nor did it affect pollen transfer network properties; networks were modular and poorly nested, both of which are likely to be related to the specificity of pollen transfer interactions. Our results indicate that pollination networks become more specialized when moving from the flower visitation to the level of pollen transfer networks. Therefore, caution is needed when inferring pollination from patterns of insect visitation or insect pollen loads as the relationship between these and pollen deposition is not straightforward.     

Catalytic properties of thimet oligopeptidase H600A mutant

Thimet oligopeptidase (EC 3.4.24.15, TOP) is a metallo-oligopeptidase that participates in the intracellular metabolism of peptides. Predictions based on structurally analogous peptidases (Dcp and ACE-2) show that TOP can present a hinge-bend movement during substrate hydrolysis, what brings some residues closer to the substrate. One of these residues that in TOP crystallographic structure are far from the catalytic residues, but, moves toward the substrate considering this possible structural reorganization is His⁶⁰⁰. In the present work, the role of His⁶⁰⁰ of TOP was investigated by site-directed mutagenesis. TOP H600A mutant was characterized through analysis of S₁ and S₁′ specificity, pH-activity profile and inhibition by JA-2. Results showed that TOP His⁶⁰⁰ residue makes important interactions with the substrate, supporting the prediction that His⁶⁰⁰ moves toward the substrate due to a hinge movement similar to the Dcp and ACE-2. Furthermore, the mutation H600A affected both K_m and k_cat, showing the importance of His⁶⁰⁰ for both substrate binding and/or product release from active site. Changes in the pH-profile may indicate also the participation of His⁶⁰⁰ in TOP catalysis, transferring a proton to the newly generated NH₂-terminus or helping Tyr⁶⁰⁵ and/or Tyr⁶¹² in the intermediate oxyanion stabilization.     

The role of Tyr605 and Ala607 of thimet oligopeptidase and Tyr606 and Gly608 of neurolysin in substrate hydrolysis and inhibitor binding

The physicochemical properties of TOP (thimet oligopeptidase) and NEL (neurolysin) and their hydrolytic activities towards the FRET (fluorescence resonance energy transfer) peptide series Abz-GFSXFRQ-EDDnp [where Abz is o-aminobenzoyl; X=Ala, Ile, Leu, Phe, Tyr, Trp, Ser, Gln, Glu, His, Arg or Pro; and EDDnp is N-(2,4-dinitrophenyl)-ethylenediamine] were compared with those of site-mutated analogues. Mutations at Tyr605 and Ala607 in TOP and at Tyr606 and Gly608 in NEL did not affect the overall folding of the two peptidases, as indicated by their thermal stability, CD analysis and the pH-dependence of the intrinsic fluorescence of the protein. The kinetic parameters for the hydrolysis of substrates with systematic variations at position P1 showed that Tyr605 and Tyr606 of TOP and NEL respectively, played a role in subsite S1. Ala607 of TOP and Gly608 of NEL contributed to the flexibility of the loops formed by residues 600-612 (GHLAGGYDGQYYG; one-letter amino acid codes used) in NEL and 599-611 (GHLAGGYDAQYYG; one-letter amino acid codes used) in TOP contributing to the distinct substrate specificities, particularly with an isoleucine residue at P1. TOP Y605A was inhibited less efficiently by JA-2 {N-[1-(R,S)-carboxy-3-phenylpropyl]Ala-Aib-Tyr-p-aminobenzoate}, which suggested that the aromatic ring of Tyr605 was an important anchor for its interaction with wild-type TOP. The hydroxy groups of Tyr605 and Tyr606 did not contribute to the pH-activity profiles, since the pKs obtained in the assays of mutants TOP Y605F and NEL Y606F were similar to those of wild-type peptidases. However, the pH-kcat/Km dependence curve of TOP Y605A differed from that of wild-type TOP and from TOP Y606F. These results provide insights into the residues involved in the substrate specificities of TOP and NEL and how they select cytosolic peptides for hydrolysis.     

Heterodimerization of the alpha and beta isoforms of the human thromboxane receptor enhances isoprostane signaling

Isoprostanes are free radical catalyzed products of arachidonic acid that are elevated in pro-oxidant disease states. Two isoprostanes, 8-isoprostaglandin F(2alpha) (iPF(2alpha)III) and 8-isoprostaglandin E2 (iPE2III), act at the receptor for thromboxane A2 (the TP) to mediate pro-atherogenic effects in vivo. We confirmed dimerization of the human TP isoforms, TPalpha and TPbeta, and determined the impact on isoprostane signaling. No overt changes in ligand binding at the TP were observed as a result of TPalpha/TPbeta coexpression. The response to iPF(2alpha)III or iPE2III was enhanced in HEK293 cells stably coexpressing TPalpha and TPbeta, as measured by inositol phosphate generation or intracellular calcium mobilization, relative to cells expressing TPalpha or TPbeta individually. In contrast, the response to traditional thromboxane analogs was unaltered. Augmented isoprostane signaling was similarly observed in HEK 293 cell transiently transfected with TPalpha and TPbeta. These results indicate that TPalpha/TPbeta dimerization enhances isoprostane-mediated signal transduction.