Functional analysis of AtHKT1 in Arabidopsis shows that Na(+) recirculation by the phloem is crucial for salt tolerance

Two allelic recessive mutations of Arabidopsis, sas2-1 and sas2-2, were identified as inducing sodium overaccumulation in shoots. The sas2 locus was found (by positional cloning) to correspond to the AtHKT1 gene. Expression in Xenopus oocytes revealed that the sas2-1 mutation did not affect the ionic selectivity of the transporter but strongly reduced the macro scopic (whole oocyte current) transport activity. In Arabidopsis, expression of AtHKT1 was shown to be restricted to the phloem tissues in all organs. The sas2-1 mutation strongly decreased Na(+) concentration in the phloem sap. It led to Na(+) overaccumulation in every aerial organ (except the stem), but to Na(+) underaccumulation in roots. The sas2 plants displayed increased sensitivity to NaCl, with reduced growth and even death under moderate salinity. The whole set of data indicates that AtHKT1 is involved in Na(+) recirculation from shoots to roots, probably by mediating Na(+) loading into the phloem sap in shoots and unloading in roots, this recirculation removing large amounts of Na(+) from the shoot and playing a crucial role in plant tolerance to salt.     

Increased circulating AC133+ CD34+ endothelial progenitor cells in children with hemangioma

Hemangioma is the most common soft-tissue tumor of infancy. Despite the frequency of these vascular tumors, the origin of hemangioma-endothelial cells is unknown. Circulating endothelial progenitor cells (EPCs) have recently been identified as vascular stem cells with the capacity to contribute to postnatal vascular development. We have attempted to determine whether circulating EPCs are increased in hemangioma patients and thereby provide insight into the role of EPCs in hemangioma growth. METHODS AND RESULTS: Peripheral blood mononuclear cells (PBMCs) were isolated from hemangioma patients undergoing surgical resection (N = 5) and from age-matched controls (N = 5) undergoing strabismus correction surgery. PBMCs were stained with fluorescent-labeled antibodies for AC133, CD34, and VEGFR2/KDR. Fluorescent-labeled isotype antibodies served as negative controls. Histologic sections of surgical specimens were stained with the specific hemangioma markers Glut1, CD32, and merosin, to confirm the diagnosis of common hemangioma of infancy. EPCs harvested from healthy adult volunteers were stained with Glut1, CD32, and merosin, to assess whether cultured EPCs express known hemangioma markers. Hemangioma patients had a 15-fold increase in the number of circulating CD34 AC133 dual-staining cells relative to controls (0.78+/-0.14% vs.0.052+/-0.017%, respectively). Similarly, the number of PBMCs that stained positively for both CD34 and KDR was also increased in hemangioma patients (0.49+/-0.074% vs. 0.19+/-0.041% in controls). Cultured EPCs stained positively for the known hemangioma markers Glut1, CD32, merosin. CONCLUSIONS: This is the first study to suggest a role for EPCs in the pathogenesis of hemangioma. Our results imply that increased levels of circulating EPCs may contribute to the formation of this vascular tumor.     

High-density genetic linkage maps of Phytophthora infestans reveal trisomic progeny and chromosomal rearrangements

Detailed analysis of the inheritance of molecular markers was performed in the oomycete plant pathogen Phytophthora infestans. Linkage analysis in the sexual progeny of two Dutch field isolates (cross 71) resulted in a high-density map containing 508 markers on 13 major and 10 minor linkage groups. The map showed strong clustering of markers, particularly of markers originating from one parent, and dissimilarity between the parental isolates on linkage group III in the vicinity of the mating-type locus, indicating a chromosomal translocation. A second genetic map, constructed by linkage analysis in sexual progeny of two Mexican isolates (cross 68), contained 363 markers and is thus less dense than the cross 71 map. For some linkage groups the two independent linkage maps could be aligned, but sometimes markers appeared to be in a different order, or not linked at all, indicating chromosomal rearrangements between genotypes. Graphical genotyping showed that some progeny contained three copies of a homologous linkage group. This trisomy was found for several linkage groups in both crosses. Together, these analyses suggest a genome with a high degree of flexibility, which may have implications for evolution of new races and resistance development to crop protection agents.     

Membrane association, electrostatic sequestration, and cytotoxicity of Gly-Leu-rich peptide orthologs with differing functions

The skins of closely related frog species produce Gly-Leu-rich peptide orthologs that have very similar sequences, hydrophobicities, and amphipathicities but differ markedly in their net charge and membrane-damaging properties. Cationic Gly-Leu-rich peptides are hemolytic and very potent against microorganisms. Peptides with no net charge have only hemolytic activity. We have used ancestral protein reconstruction and peptide analogue design to examine the roles of electrostatic and hydrophobic interactions in the biological activity and mode of action of functionally divergent Gly-Leu-rich peptides. The structure and interaction of the peptides with anionic and zwitterionic model membranes were investigated by circular dichroism with 2-dimyristoyl-sn-glycero-3-phosphatidylcholine or 1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol vesicles and surface plasmon resonance with immobilized bilayers. The results, combined with antimicrobial assays, the kinetics of bacterial killing, and membrane permeabilization assays, reveal that Gly, Val, Thr, and Ile can all be accommodated in an amphipathic alpha helix when the helix is in a membrane environment. Binding to anionic and zwitterionic membranes fitted to a 2-stage interaction model (adsorption to the membrane followed by membrane insertion). The first step is governed by hydrophobic interactions between the nonpolar surface of the peptide helix and the membranes. The strong binding of Gly-Leu-rich cationic peptides to anionic membranes is due to the second binding step and involves short-range Coulombic interactions that prolong the residence time of the membrane-inserted peptide. The data demonstrate that evolution has positively selected charge-altering nucleotide substitutions to generate an orthologous cationic variant of neutral hemolytic peptides that bind to and permeate bacterial cell membranes.     

Peptidomic and proteomic analyses of the systemic immune response of Drosophila

Insects have developed an efficient host defense against microorganisms, which involves humoral and cellular mechanisms. Numerous data highlight similarities between defense responses of insects and innate immunity of mammals. The fruit fly, Drosophila melanogaster, is a favorable model system for the analysis of the first line defense against microorganisms. Taking advantages of improvements in mass spectrometry (MS), two-dimensional (2D) gel electrophoresis and bioinformatics, differential analyses of blood content (hemolymph) from immune-challenged versus control Drosophila were performed. Two strategies were developed: (i) peptidomic analyses through matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS and high performance liquid chromatography for molecules below 15 kDa, and (ii) proteomic studies based on 2D gel electrophoresis, MALDI-TOF fingerprinting and database searches, for compounds of greater molecular masses. The peptidomic strategy led to the detection of a large number of peptides induced in the hemolymph of challenged flies as compared to controls. Of these, 28 were characterized, amongst which were antimicrobial peptides. The 2D gel electrophoresis strategy led to the detection of 70 spots differentially regulated by at least fivefold after microbial infection. This approach yielded the identity of a series of proteins that were related to the Drosophila immune response, such as proteases, protease inhibitors, prophenoloxydase-activating enzymes, serpins and a Gram-negative binding protein-like protein. This strategy also brought to light new candidates with a potential function in the immune response (odorant-binding protein, peptidylglycine alpha-hydroxylating monooxygenase and transferrin). Interestingly, several molecules resulting from the cleavage of proteins were detected after a fungal infection. Together, peptidomic and proteomic analyses represent new tools to characterize molecules involved in the innate immune reactions of Drosophila.     

Involvement of the S100B in cAMP-Induced Cytoskeleton Remodeling in Astrocytes: A Study Using TRTK-12 in Digitonin-Permeabilized Cells

1. Stellation of astrocytes in culture involves a complex rearrangement of microfilaments, intermediate filaments, and microtubules, which reflects in part the plasticity of these cells observed during development or after injury.2. An astrocytic calcium-binding protein, S100B, has been implicated in the regulation of plasticity due to its ability to interact with cytoskeletal proteins.3. We used digitonin-permeabilized astrocytes to introduce TRTK-12, a peptide that binds to the C-terminal of S100B and blocks its interaction with cytoskeletal proteins.4. TRTK-12 was able to block cAMP-induced astrocyte stellation and this effect was dependent on the concentration of the peptide. These results support the idea that S100B has a modulatory role on astrocyte morphology.     

S100B-Mediated Inhibition of the Phosphorylation of GFAP Is Prevented by TRTK-12

S100B belongs to a family of calcium-binding proteins involved in cell cycle and cytoskeleton regulation. We observed an inhibitory effect of S100B on glial fibrillary acidic protein (GFAP) phosphorylation, when stimulated by cAMP or Ca2+/calmodulin, in a cytoskeletal fraction from primary astrocyte cultures. We found that S100B has no direct effect on CaM KII activity, the major kinase in this cytoskeletal fraction able to phosphorylate GFAP. The inhibition of GFAP phosphorylation is most likely due to the binding of S100B to the phosphorylation sites on this protein and blocking the access of these sites to the protein kinases. This inhibition was dependent on Ca2+. However, Zn2+ could substitute for Ca2+. The inhibitory effect of S100B was prevented by TRTK-12, a peptide that blocks S100B interaction with several target proteins including glial fibrillary acidic protein. These data suggest a role for S100B in the assembly of intermediate filaments in astrocytes.     

Characterization of AtCHX17, a member of the cation/H+ exchangers, CHX family, from Arabidopsis thaliana suggests a role in K+ homeostasis

The Arabidopsis genome contains many sequences annotated as encoding H(+)-coupled cotransporters. Among those are the members of the cation:proton antiporter-2 (CPA2) family (or CHX family), predicted to encode Na(+),K(+)/H(+) antiporters. AtCHX17, a member of the CPA2 family, was selected for expression studies, and phenotypic analysis of knockout mutants was performed. AtCHX17 expression was only detected in roots. The gene was strongly induced by salt stress, potassium starvation, abscisic acid (ABA) and external acidic pH. Using the beta-glucuronidase reporter gene strategy and in situ RT-PCR experiments, we have found that AtCHX17 was expressed preferentially in epidermal and cortical cells of the mature root zones. Knockout mutants accumulated less K(+) in roots in response to salt stress and potassium starvation compared with the wild type. These data support the hypothesis that AtCHX17 is involved in K(+) acquisition and homeostasis.     

Cotyledon vascular pattern2-mediated inositol (1,4,5) triphosphate signal transduction is essential for closed venation patterns of Arabidopsis foliar organs

Vein patterns in leaves and cotyledons form in a spatially regulated manner through the progressive recruitment of ground cells into vascular cell fate. To gain insight into venation patterning mechanisms, we have characterized the cotyledon vascular pattern2 (cvp2) mutants, which exhibit an increase in free vein endings and a resulting open vein network. We cloned CVP2 by a map-based cloning strategy and found that it encodes an inositol polyphosphate 5' phosphatase (5PTase). 5PTases regulate inositol (1,4,5) triphosphate (IP(3)) signal transduction by hydrolyzing IP(3) and thus terminate IP(3) signaling. CVP2 gene expression is initially broad and then gradually restricted to incipient vascular cells in several developing organs. Consistent with the inferred enzymatic activity of CVP2, IP(3) levels are elevated in cvp2 mutants. In addition, cvp2 mutants exhibit hypersensitivity to the plant hormone abscisic acid. We propose that elevated IP(3) levels in cvp2 mutants reduce ground cell recruitment into vascular cell fate, resulting in premature vein termination and, thus, in an open reticulum.