Identification of inhibitors of the E. coli cyclopropane fatty acid synthase from the screening of a chemical library: In vitro and in vivo studies

Using an automated coupled colorimetric assay for the Escherichia coli cyclopropane fatty acid synthase (CFAS), we have screened an academic chemical library of 3040 compounds, to identify new inhibitors of this enzyme. We identified 8 compounds as potent inhibitors of this enzyme, with IC(50) ranging from 1 to 10 microM, in the presence of 750 microM S-adenosyl-l-methionine and 1 mg/mL phospholipids. We conducted kinetic analyses of the inhibition of the CFAS using dioctylamine and three inhibitors identified in this report: sinefungin, 1, a synthetic S-adenosyl-l-homocysteine analog, 2, and an indoloquinolizine derivative, 3. The inhibition patterns observed were interpreted assuming that the E. coli CFAS operated via an ordered Bi Bi mechanism with binding of S-adenosyl-l-methionine first. Dioctylamine was the most potent inhibitor with a competitive inhibition constant of 130 nM with respect to the phospholipids. Compound 2 bound to the two substrate-binding sites of the enzyme suggesting that it acted as a bisubstrate analog (apparent inhibition constant, K(I)=6 microM). Compound 2 was also found to completely inhibit cyclopropanation of the phospholipids in growing E. coli cells, at 150 microM. This molecule is thus the first inhibitor of a cyclopropane synthase that is active in vivo, contrary to sinefungin and other analogs that are only active on the isolated enzyme.     

Root to leaf electrical signaling in avocado in response to light and soil water content

Phytomonitoring techniques for irrigation of avocado orchards indicate that plants respond very rapidly to fluctuations in soil water content. Root to leaf abscicic acid transport cannot fully explain the almost immediate response of stomata to either irrigation and/or sudden changes in climatic conditions. Therefore, we studied the existence of a fast conducting signal between roots and leaves, and the possible involvement of such a signal in the regulation of stomatal behavior. Two-year-old avocado trees were subjected to drying and re-watering cycles or changes in incident radiation (light or darkness). The difference in extracellular electrical potential between the leaf petiole and the base of stem (DeltaV(L-S)) was continuously recorded. Stomatal conductance (gs) was also recorded for the same leaves that were used for voltage difference measurements. A sudden change in soil water content induced by root drying and re-watering was accompanied by a slow, significant change in the recorded DeltaV(L-S) signal, which was fully developed at 52 and 32min for root drying and re-watering, respectively. We found an inverse correlation (r=-0.56) between the change of DeltaV(L-S) and the gs difference measured before and after each soil-drying treatment. Plants that were girdled to disrupt the phloem and then irrigated tended to have lower DeltaV(L-S) differences over time than non-girdled irrigated plants, suggesting that the electrical signal was transmitted in the phloem. The existence of a fast signal transmitted from the root to the leaf that can be measured and correlated with stomatal control opens the possibility of developing a new phytomonitoring technique and/or artificially modifying plant responses by imposing agronomic management strategies aimed at rapid stomatal adaptation to changes in soil water content.     

Floral integration,phenotypic covariance structure and pollinator variation in bumblebee‐pollinated Helleborus foetidus

By analysing patterns of phenotypic integration and multivariate covariance structure of five metric floral traits in nine Iberian populations of bumblebee‐pollinated Helleborus foetidus (Ranunculaceae), this paper attempts to test the general hypothesis that pollinators enhance floral integration and selectively modify phenotypic correlations between functionally linked floral traits. The five floral traits examined exhibited significant phenotypic integration at all populations, and both the magnitude and the pattern of integration differed widely among populations. Variation in extent and pattern of integration was neither distance‐dependent nor significantly related to between‐population variation in taxonomical composition and morphological diversity of the pollinator assemblage. Patterns of floral integration were closer to expectations derived from consideration of developmental affinities between floral whorls than to expectations based on a pollinator‐mediated adaptive hypothesis. Taken together, results of this study suggest that between‐population differences in magnitude and pattern of floral integration in H. foetidus are probably best explained as a consequence of random genetic sampling in the characteristically small and ephemeral populations of this species, rather than reflecting the selective action of current pollinators.     

Plant diversity,biogeography and environment in Iberia: Patterns and possible causal factors

Abstract. We associated patterns of plant diversity with possible causal factors by considering 93 local regions in the Iberian Peninsula and Balearic Islands with respect to biogeography, environmental favourability, and environmental heterogeneity, and their relationship with measured species diversity at four different scales: mean local species richness standardized at a grain of 100 m², total species richness in a community type within a region (regional community richness), mean compositional similarity, and mosaic diversity. Local regions in biogeographic transition zones to the North African and Atlantic floras had higher regional community richness and greater mosaic diversity than did non‐transitional regions, whereas no differences existed in mean local species richness or mean compositional similarity. Mean local species richness was positively related to environmental favourability as measured by actual evapotranspiration, but negatively related to total precipitation and temporal heterogeneity in precipitation. Mean local species richness was greatest in annual grassland and dwarf shrubland communities, and on calcareous bedrock types. Regional community richness was similarly related to actual evapotranspiration and total precipitation, but in addition was positively related to spatial heterogeneity in topography and soil water holding capacity. Mean compositional similarity decreased with increasing spatial heterogeneity and temperature seasonality. Mosaic diversity, a measure of complexity, increased with increasing local and regional richness. We hypothesize that these relationships can be explained by four ecological and evolutionary classes of causal factors: numbers of individuals, intermediate environments, limits to adaptation, and niche variation. These factors operate at various scales and manifest themselves in various ways. For example, at the site level, apparently processes that increase the number of individuals increase mean local species richness, but at the level of the entire region no such effects were found.     

A role for the Rho-p160 Rho coiled-coil kinase axis in the chemokine stromal cell-derived factor-1alpha-induced lymphocyte actomyosin and microtubular organization and chemotaxis.

The possible involvement of the Rho-p160ROCK (Rho coiled-coil kinase) pathway in the signaling induced by the chemokine Stromal cell-derived factor (SDF)-1alpha has been studied in human PBL. SDF-1alpha induced activation of RhoA, but not that of Rac. RhoA activation was followed by p160ROCK activation mediated by RhoA, which led to myosin light chain (MLC) phosphorylation, which was dependent on RhoA and p160ROCK activities. The kinetics of MLC activation was similar to that of RhoA and p160ROCK. The role of this cascade in overall cell morphology and functional responses to the chemokine was examined employing different chemical inhibitors. Inhibition of either RhoA or p160ROCK did not block SDF-1alpha-induced short-term actin polymerization, but induced the formation of long spikes arising from the cell body, which were found to be microtubule based. This morphological change was associated with an increase in microtubule instability, which argues for an active microtubule polymerization in the formation of these spikes. Inhibition of the Rho-p160ROCK-MLC kinase signaling cascade at different steps blocked lymphocyte migration and the chemotaxis induced by SDF-1alpha. Our results indicate that the Rho-p160ROCK axis plays a pivotal role in the control of the cell shape as a step before lymphocyte migration toward a chemotactic gradient.     

Involvement of Arabidopsis glutaredoxin S14 in the maintenance of chlorophyll content

Plant class‐II glutaredoxins (GRXs) are oxidoreductases carrying a CGFS active site signature and are able to bind iron–sulfur clusters in vitro. In order to explore the physiological functions of the 2 plastidial class‐II isoforms, GRXS14 and GRXS16, we generated knockdown and overexpression Arabidopsis thaliana lines and characterized their phenotypes using physiological and biochemical approaches. Plants deficient in one GRX did not display any growth defect, whereas the growth of plants lacking both was slowed. Plants overexpressing GRXS14 exhibited reduced chlorophyll content in control, high‐light, and high‐salt conditions. However, when exposed to prolonged darkness, plants lacking GRXS14 showed accelerated chlorophyll loss compared to wild‐type and overexpression lines. We observed that the GRXS14 abundance and the proportion of reduced form were modified in wild type upon darkness and high salt. The dark treatment also resulted in decreased abundance of proteins involved in the maturation of iron–sulfur proteins. We propose that the phenotype of GRXS14‐modified lines results from its participation in the control of chlorophyll content in relation with light and osmotic conditions, possibly through a dual action in regulating the redox status of biosynthetic enzymes and contributing to the biogenesis of iron–sulfur clusters, which are essential cofactors in chlorophyll metabolism.     

Peripheral blood aspirates overexpressing IGF‐I via rAAV gene transfer undergo enhanced chondrogenic differentiation processes

Implantation of peripheral blood aspirates induced towards chondrogenic differentiation upon genetic modification in sites of articular cartilage injury may represent a powerful strategy to enhance cartilage repair. Such a single‐step approach may be less invasive than procedures based on the use of isolated or concentrated MSCs, simplifying translational protocols in patients. In this study, we provide evidence showing the feasibility of overexpressing the mitogenic and pro‐anabolic insulin‐like growth factor I (IGF‐I) in human peripheral blood aspirates via rAAV‐mediated gene transfer, leading to enhanced proliferative and chondrogenic differentiation (proteoglycans, type‐II collagen, SOX9) activities in the samples relative to control (reporter rAAV‐lacZ) treatment over extended periods of time (at least 21 days, the longest time‐point evaluated). Interestingly, IGF‐I gene transfer also triggered hypertrophic, osteo‐ and adipogenic differentiation processes in the aspirates, suggesting that careful regulation of IGF‐I expression may be necessary to contain these events in vivo. Still, the current results demonstrate the potential of targeting human peripheral blood aspirates via therapeutic rAAV transduction as a novel, convenient tool to treat articular cartilage injuries.     

Bradyrhizobium valentinum sp. nov., isolated from effective nodules of Lupinus mariae-josephae, a lupine endemic of basic-lime soils in Eastern Spain

Bacterial strains isolated from nitrogen-fixing nodules of Lupinus mariae-josephae have been characterized following genetic, phenotypic and symbiotic approaches. Analysis of 16S rRNA genes placed them in a group together with Bradyrhizobium elkanii USDA 76^T, B. pachyrhizi PAC48^T, B. jicamae PAC68^T, ‘B. retamae’ Ro19^T and B. lablabi CCBAU 23086^T with over 99.0% identity. Phylogenetic analysis of concatenated housekeeping genes, recA, atpD and glnII, suggested that L. mariae-josephae strains represent a new Bradyrhizobium species, closely related to B. lablabi CCBAU 23086^T, B. jicamae PAC68^T and ‘B. retamae’ Ro19^T with 92.1, 91.9 and 90.8% identity, respectively. These results are consistent with overall genomic identities calculated as Average Nucleotide Identity (ANIm) using draft genomic sequences obtained for relevant strains. While L. mariae-josephae strains LmjM3^T/LmjM6 exhibited a 99.2% ANIm value, they were significantly distant (<93% ANIm) from type strains of their closest species (‘B. retamae’ Ro19^T, B. lablabi CCBAU 23086^T and B. jicamae PAC68^T). Whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (WC-MALDI-TOF-MS) analysis of proteomic patterns of the same strains was consistent with these results. The symbiosis-related genes nodC, nodA and nifH genes from strains nodulating L. mariae-josephae were phylogenetically related to those from ‘B. retamae’ Ro19^T, but divergent from those of strains that nodulate other lupine species. Based on genetic, genomic, proteomic and phenotypic data presented in this study, L. mariae-josephae nodulating strains LmjM3^T, LmjM6 and LmjM2 should be grouped within a new species for which the name Bradyrhizobium valentinum sp. nov. is proposed (type strain LmjM3^T = CECT 8364^T, LMG 2761^T)