Pollination of Euphorbia dendroides by lizards and insects: Spatio-temporal variation in patterns of flower visitation

The patterns of flower visitation by lizards (Podarcis lilfordi, Lacertidae) and insects (mainly flies, bees and wasps) on the shrub Euphorbia dendroides, were studied in the island of Cabrera (Balearic Islands) during the flowering seasons of 1995 and 1996. Lizards act as true pollinators of the plant, moving large quantities of pollen within and among shrubs. To our knowledge, this is the first time that pollination by lizards has been empirically demonstrated. Variation in the quantitative component of pollination (frequency of visits × flower visitation rate) by the two groups of pollinators (lizards and insects) is documented at both spatial (within a plant population) and temporal scales (throughout the flowering season and between seasons). Variation in lizard density on a small spatial scale (within c. 200 m), presumably due to differences in vegetation cover, strongly affected their frequency of flower visitation. Insects were rather scarce, mainly because the plant flowers at a time (mid-March) when temperatures are still low. At the site where lizards were abundant, their frequency of flower visits was more than 3 times that of insects, they stayed on the shrubs about 3 times longer and visited about 8 times more cyathia per minute than did insects. Fruit and seed set were greater at this site, and this is attributed to the different frequency of flower visits by lizards, as shrubs are similar in size and produce similar amounts of cyathia in the two sites compared. Both, lizards and insects went more frequently to plants with large flower crops. However, flower crop was not associated with seed viability. We found no evidence for pollinator-mediated selection on plant traits related to fitness. Received: 28 August 1996 / Accepted: 26 February 1997     

Joint ecological,geographical and cultural approach to identify territories of opportunity for large vertebrates conservation in Mexico

The objective of the present paper is to provide a holistic framework to delineate “territories of opportunity” where agrarian communities can manage areas to enhance the conservation of large vertebrates. The study was conducted Mexico, which is sociologically, culturally and ecologically complex, similar to other “megadiverse” countries. We conducted ensemble niche modeling of endangered top predators to define a set of large vertebrate species. Environmental attributes were used to perform three distance-based and two artificial intelligence-based algorithms. Socio-cultural attributes were included to depict agricultural communities with strong social government schemes and clear evidence of well-managed natural resources. Other socio-economic attributes such as land acquisition cost, human agglomeration and anthropogenic land use were included in the analysis. Scenarios were computed and displayed cartographically with the aid of a geographic information system. Results showed that the largest concentration of biodiversity converges on regions with large land cover persistence and high local governance, defined as potential willingness to engage in conservation actions. The cartographic areas identified overlapped with current Mexican protected areas in only 2.7% of the country. Thus, conservation law enforcement in most of the country seems to be ineffective. Here we show that, in a number of territories, agrarian communities that have coexisted for millennia with umbrella species can be regarded as allies in biodiversity conservation. Results are discussed in the light of their relevance for future niche modeling, environmental policy design and implications for climate change.     

Analysis of rhizobial endosymbionts of Vicia,Lathyrus and Trifolium species used to maintain mountain firewalls in Sierra Nevada National Park (South Spain)

Forest fires lead to the annual disappearance of many natural formations that require the creation of firewall areas. They can be maintained by enriching their pastures with attractive plants for grazing livestock, mainly legumes, which have a high protein content and low dependence on N fertilizers due to their ability to establish nitrogen-fixing symbiosis with rhizobia. In this study, the rhizobia isolated from the nodules of six legumes from the genera Vicia, Lathyrus and Trifolium were analysed in a firewall zone established in Lanjarón (Granada) close to the Sierra Nevada National Park (Spain). The results showed a high genetic diversity of the isolated strains that had 3, 16, 14 and 13 different types of rrs, recA, atpD and glnII genes, respectively. All strains were phylogenetically close to the species from the Rhizobium leguminosarum group, although they were not identified as any of them. The isolated strains belonged to the symbiovars viciae and trifolii but high phylogenetic diversity was found within both symbiovars, since there were 16 and 14 nodC gene types, respectively. Some of these strains clustered with strains isolated in other countries and continents, but others formed atpD, recA, glnII and nodC clusters and lineages only found to date in this study.     

Acinetobacter strains IH9 and OCI1, two rhizospheric phosphate solubilizing isolates able to promote plant growth,constitute a new genomovar of Acinetobacter calcoaceticus

During a screening of phosphate solubilizing bacteria (PSB) in agricultural soils, two strains, IH9 and OCI1, were isolated from the rhizosphere of grasses in Spain, and they showed a high ability to solubilize phosphate in vitro. Inoculation experiments in chickpea and barley were conducted with both strains and the results demonstrated their ability to promote plant growth. The 16S rRNA gene sequences of these strains were nearly identical to each other and to those of Acinetobacter calcoaceticus DSM 30006^T, as well as the strain CIP 70.29 representing genomospecies 3. Their phenotypic characteristics also coincided with those of strains forming the A. calcoaceticus–baumannii complex. They differed from A. calcoaceticus in the utilization of l-tartrate as a carbon source and from genomospecies 3 in the use of d-asparagine as a carbon source. The 16S–23S intergenic spacer (ITS) sequences of the two isolates showed nearly 98% identities to those of A. calcoaceticus, confirming that they belong to this phylogenetic group. However, the isolates appeared as a separate branch from the A. calcoaceticus sequences, indicating their molecular separation from other A. calcoaceticus strains. The analysis of three housekeeping genes, recA, rpoD and gyrB, confirmed that IH9 and OCI1 form a distinct lineage within A. calcoaceticus. These results were congruent with those from DNA–DNA hybridization, indicating that strains IH9 and OCI1 constitute a new genomovar for which we propose the name A. calcoaceticus genomovar rhizosphaerae.     

Thermodynamics of zinc binding to hepatitis C virus NS3 protease: A folding by binding event

The hepatitis C virus (HCV) nonstructural protein 3 (NS3) protease is responsible for the processing of the non‐structural region of the viral precursor polyprotein in infected hepatic cells. HCV NS3 is a zinc‐dependent serine protease. The zinc ion, which is bound far away from the active site and considered to have a structural role, is essential for the structural integrity of the protein; furthermore, the ion is required for the hydrolytic activity. Consequently, the NS3 zinc binding site has been considered for a long time as a possible target for drug discovery. As a first step towards this goal, the energetics of the NS3‐zinc interaction and its effect on the NS3 conformation must be established and discussed. The thermodynamic characterization of zinc binding to NS3 protease by isothermal titration calorimetry and spectroscopy is presented here. Spectroscopic and calorimetric results suggest that a considerable conformational change in the protein is coupled to zinc binding. The energetics of the conformational change is comparable to that of the folding of a protein of similar size. Therefore, zinc binding to NS3 protease can be considered as a “folding by binding” event. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Piezoelectric biosensors assisted with electroacoustic impedance spectroscopy: a tool for accurate quantitative molecular recognition analysis

In this work, electroacoustic impedance analysis based on a modified Butterworth-Van Dyke (BVD) model is used to complement resonance frequency measurements of piezoelectric crystal sensors for the identification and removal of interfering signals. This approach enables the accurate use of the Sauerbrey correlation to establish a direct relationship between mass deposited at the sensor surface and measured frequency variations. Kinetic models can thus be evaluated and binding constants estimated directly from the measured data. We further demonstrate the usefulness of this approach by applying it to the study of the formation of 11-hydroxy-1-undecanothiol self-assembled monolayers (SAM) as well as to the binding of streptavidin to immobilized biotin. Kinetic and equilibrium parameters were estimated from transient analysis, adsorption isotherms, Scatchard and Hill plots obtained from the frequency data for both the alkanethiol and streptavidin films. This strategy based on electroacoustic impedance assisted quartz-crystal microbalance (QCM) biosensors is expected to be a major contribution for the use of these piezoelectric devices as a reliable and cheap detection system that can easily be integrated into analytical techniques.     

Cyanochromes Are Blue/Green Light Photoreversible Photoreceptors Defined by a Stable Double Cysteine Linkage to a Phycoviolobilin-type Chromophore

Phytochromes are a collection of bilin-containing photoreceptors that regulate a diverse array of processes in microorganisms and plants through photoconversion between two stable states, a red light-absorbing Pr form, and a far red light-absorbing Pfr form. Recently, a novel set of phytochrome-like chromoproteins was discovered in cyanobacteria, designated here as cyanochromes, that instead photoconvert between stable blue and green light-absorbing forms Pb and Pg, respectively. Here, we show that the distinctive absorption properties of cyanochromes are facilitated through the binding of phycocyanobilin via two stable cysteine-based thioether linkages within the cGMP phosphodiesterase/adenyl cyclase/FhlA domain. Absorption, resonance Raman and infrared spectroscopy, and molecular modeling of the Te-PixJ GAF (cGMP phosphodiesterase/adenyl cyclase/FhlA) domain assembled with phycocyanobilin are consistent with attachments to the C3¹ carbon of the ethylidene side chain and the C4 or C5 carbons in the A–B methine bridge to generate a double thioether-linked phycoviolobilin-type chromophore. These spectroscopic methods combined with NMR data show that the bilin is fully protonated in the Pb and Pg states and that numerous conformation changes occur during Pb → Pg photoconversion. Also identified were a number of photochromically inactive mutants with strong yellow or red fluorescence that may be useful for fluorescence-based cell biological assays. Phylogenetic analyses detected cyanochromes capable of different signaling outputs in a wide range of cyanobacterial species. One unusual case is the Synechocystis cyanochrome Etr1 that also binds ethylene, suggesting that it works as a hybrid receptor to simultaneously integrate light and hormone signals.Phytochromes (Phys)³ comprise a large and diverse superfamily of photoreceptors that regulate a wide range of physiological responses in plants, fungi, bacteria, and cyanobacteria (1–3). They are unique among photoreceptors by being able to photoconvert between two stable states, a red light-absorbing Pr form that is typically the dark-adapted and biologically inactive conformer and a far-red light-absorbing Pfr form that requires light for its production and is typically the biologically active conformer. By interconverting between Pr and Pfr, Phys act as light-regulated switches in controlling processes ranging from phototaxis and pigmentation in bacteria to seed germination, photomorphogenesis, and flowering time in higher plants.Light absorption by Phys is directed by a bilin (or linear tetrapyrrole) chromophore produced by the oxidative cleavage of heme. Although bacterial and fungal Phys use the immediate cleavage product biliverdin (BV), cyanobacterial and higher plant Phys use phycocyanobilin (PCB) and phytochromobilin, respectively, produced by enzymatic reduction of BV (1, 2). The bilin is then covalently bound autocatalytically to the photosensory unit of the apoprotein, which typically contains a sequence of Per/Arndt/Sim (PAS), cGMP phosphodiesterase/adenyl cyclase/FhlA (GAF), and Phy-associated (PHY) domains. Intimate contact between the bilin and surrounding protein residues then generates the unique photochromic properties of Phys. Recent three-dimensional structures of the Pr form of several bacterial Phys (BphPs) and two cyanobacterial Phys (Cphs) have shown that the bilin is deeply buried within the GAF domain in a ZZZssa configuration and that the connection between the GAF and PAS domains is stabilized by a rare figure-of-eight knot involving the region upstream of the PAS domain being lassoed by a conserved loop within the GAF domain (4–9). Although the structure of Pfr remains unsolved, various physicochemical studies have proposed that photoconversion involves a rotation of one of the three methine bridges between the pyrrole rings (1, 10–14). This rotation then induces much slower thermally driven movements of the protein to initiate signal transduction.In microorganisms, Pfr can activate a variety of signaling systems using output motifs directly appended to the C-terminal end of the photosensory region. The most prevalent are histidine kinase domains that then begin specific two-component phosphorelays (3, 15, 16). Although the output of plant Phys remains unclear, the presence of a C-terminal HK-related domain suggests that they also work as light-regulated protein kinases (17).In addition to the canonical Phys, it has become apparent through phylogenetic and biochemical studies that a heterogeneous collection of Phy-like photoreceptors exists (e.g. Refs. 3 and 18). These include Phys that prefer Pfr as the dark-adapted state (7, 19, 20), Phys that photoconvert from Pr to shorter wavelength-absorbing “near red” or Pnr forms (6, 21), and Phy-like photoreceptors that bind bilins but instead photoconvert between forms with maximal absorption other than red and far-red light (22–25). Often these Phy-like sequences are missing key residues or domains common among canonical Phys, suggesting that they employ novel bilins as chromophores, bind the bilin in different architectures, and/or use distinct photochemistries.One subclass of novel Phy-like photoreceptors present in a number of cyanobacteria, which we have designated cyanochromes (or Cycs) to better distinguish them from Cphs, is exemplified by Synechocystis sp. PCC6803 (Syn) PixJ (or TaxD1, locus sll0041) and its relatives. Syn-PixJ was discovered based on its involvement in blue light-mediated phototaxis in this mesophilic cyanobacterium (26, 27) with its close homolog Te-PixJ (locus tll0569) then found in the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 by sequence similarity (28). Like Cphs, the cyanochromes tested thus far covalently bind PCB but then generate photoreceptors that convert between blue and green light-absorbing forms designated Pb and Pg, respectively (22, 24, 29). Subsequent studies proposed that PCB is converted to phycoviolobilin (PVB) upon attachment to the apoprotein (30). PVB differs from PCB by having a methylene instead of a methine bridge between the A and B pyrrole rings, which blue-shifts the absorption of the chromophore by shortening the π-conjugation system. Phototransformation of Pb to Pg could then occur by a mechanism similar to Phys.How Te-PixJ and related cyanochromes bind PCB to generate more blue-shifted PVB-type chromophores remains unclear. Like Cphs, two cyanochromes examples link PCB via a thioether linkage between a cysteine in the Cyc-GAF domain and the C3¹ carbon of the ethylidene side chain of ring A (24, 28). Additionally, loss of the C4C5 double bond is necessary to generate PVB. One model by Ishizuka et al. (30) from studies with Te-PixJ proposed that the double bond moves from the C4-C5 position to the C2-C3 position by an autoisomerase activity intrinsic to the GAF domain. A more recent model by Rockwell et al. (24) using another Syn-PixJ relative in T. elongatus, Tlr0924, invoked the possibility of a second cysteine that also participates in PCB ligation. This cysteine was proposed to bind the bilin at the C10 position via a reversible thioether linkage. In the dark-adapted Pb state, the second linkage would then be formed to generate a rubin-like chromophore attached to the bridge between the B and C pyrrole rings. This bond would then break upon photoconversion to generate the more π-conjugated green light-absorbing photoproduct Pg.In this report, we employed a number of physicochemical approaches to help resolve the unique chromophore architecture and photochemical properties of cyanochromes, using Te-PixJ as the example. By independently mutagenizing the cysteine that binds the A ring ethylidene (Cys-522 (22)) and that proposed by Rockwell et al. (24) to reversibly bind the bilin at a second site (Cys-494), we demonstrate that both residues form light-stable covalent adducts with a PVB-type chromophore. In addition, we employed various spectroscopic methods to show that the bound PVB is fully protonated as both Pb and Pg, that only one pyrrole ring is active during photoconversion, and that the polypeptide may undergo extensive remodeling as Pb converts to Pg. We identified a set of conserved amino acids in Te-PixJ important for cyanochrome photochemistry, including several that when substituted generate yellow or red fluorescent chromoproteins potentially useful for cell biological applications. Phylogenetic analyses show that cyanochromes are widespread among cyanobacteria with their closest relatives being members of the red/far-red light-absorbing Phy subfamily defined by the absence of the N-terminal PAS domain (31).     

Integrated gut/liver microphysiological systems elucidates inflammatory inter‐tissue crosstalk

A capability for analyzing complex cellular communication among tissues is important in drug discovery and development, and in vitro technologies for doing so are required for human applications. A prominent instance is communication between the gut and the liver, whereby perturbations of one tissue can influence behavior of the other. Here, we present a study on human gut‐liver tissue interactions under normal and inflammatory contexts, via an integrative multi‐organ platform comprising human liver (hepatocytes and Kupffer cells), and intestinal (enterocytes, goblet cells, and dendritic cells) models. Our results demonstrated long‐term (>2 weeks) maintenance of intestinal (e.g., barrier integrity) and hepatic (e.g., albumin) functions in baseline interaction. Gene expression data comparing liver in interaction with gut, versus isolation, revealed modulation of bile acid metabolism. Intestinal FGF19 secretion and associated inhibition of hepatic CYP7A1 expression provided evidence of physiologically relevant gut‐liver crosstalk. Moreover, significant non‐linear modulation of cytokine responses was observed under inflammatory gut‐liver interaction; for example, production of CXCR3 ligands (CXCL9,10,11) was synergistically enhanced. RNA‐seq analysis revealed significant upregulation of IFNα/β/γ signaling during inflammatory gut‐liver crosstalk, with these pathways implicated in the synergistic CXCR3 chemokine production. Exacerbated inflammatory response in gut‐liver interaction also negatively affected tissue‐specific functions (e.g., liver metabolism). These findings illustrate how an integrated multi‐tissue platform can generate insights useful for understanding complex pathophysiological processes such as inflammatory organ crosstalk. Biotechnol. Bioeng. 2017;114: 2648–2659. © 2017 Wiley Periodicals, Inc.     

Conformational selection in the recognition of phosphorylated substrates by the catalytic domain of human Pin1

Post-translational phosphorylation and the related conformational changes in signaling proteins are responsible for regulating a wide range of subcellular processes. Human Pin1 is central to many of these cell signaling pathways in normal and aberrant subcellular processes, catalyzing cis-trans isomerization of the peptide ω-bond in phosphorylated serine/threonine-proline motifs in many proteins. Pin1 has therefore been identified as a possible drug target in many diseases, including cancer and Alzheimer's. The effects of phosphorylation on Pin1 substrates, and the atomistic basis for Pin1 recognition and catalysis, are not well understood. Here, we determine the conformational consequences of phosphorylation on Pin1 substrate analogues and the mechanism of recognition by the catalytic domain of Pin1 using all-atom molecular dynamics simulations. We show that phosphorylation induces backbone conformational changes on the peptide substrate analogues. We also show that Pin1 recognizes specific conformations of its substrate by conformational selection. Furthermore, dynamical correlated motions in the free Pin1 enzyme are present in the enzyme of the enzyme-substrate complex when the substrate is in the transition state configuration, suggesting that these motions play significant roles during catalytic turnover. These results provide a detailed atomistic picture of the mechanism of Pin1 recognition that can be exploited for drug design purposes and further our understanding of the synergistic complexities of post-translational phosphorylation and cis-trans isomerization.