Luminal adenosine stimulates chloride secretion through A1 receptor in mouse jejunum

Adenosine is known to stimulate chloride secretion by mouse jejunum. Whereas the receptor on the basolateral side is believed to be A2B, the receptor involved in the luminal effect of adenosine has not been identified. We found that jejuna expressed mRNA for all adenosine receptor subtypes. In this study, we investigated the stimulation of chloride secretion by adenosine in jejuna derived from mice lacking the adenosine receptors of A1 (A1R) and A2A (A(2A)R) or control littermates. The jejunal epithelium was mounted in a Ussing chamber, and a new method on the basis of impedance analysis was used to calculate the short-circuit current (I(sc)) values. Chloride secretion was assessed by the I(sc) after inhibition of the sodium-glucose cotransporter by adding phloridzin to the apical bathing solution. The effect of apical adenosine on chloride secretion was lost in jejuna from mice lacking the A1R. There was no difference in the response to basolaterally applied adenosine or to apical forskolin. Furthermore, in jejuna from control mice, the effect of apical adenosine was also abolished in the presence of 8-cyclopentyl-1,3-dipropylxanthine, a specific A1R antagonist. Responses to adenosine were identical in jejuna from control and A(2A)R knockout mice. This study demonstrates that A1R (and not A(2A)R) mediates the enhancement of chloride secretion induced by luminal adenosine in mice jejunum.     

Activation of the lysosome-associated p61Hck isoform triggers the biogenesis of podosomes

Haematopoietic cell kinase (Hck) is a protein tyrosine kinase of the Src family specifically expressed in phagocytes as two isoforms, p59Hck and p61Hck, present at the plasma membrane and lysosomes, respectively. We report that ectopic expression of a constitutively active mutant of p61Hck (p61Hck(ca)) triggered the de novo formation of actin-rich rings at the ventral face of the cells that we characterized as bona fide podosome rosettes, structures involved in cell migration. Their formation required the adaptor domains and the kinase activity of p61Hck, the integrity of microfilament and microtubule networks and concerted action of Cdc42, Rac and Rho. Podosome rosette formation was either abolished when p61Hck(ca) was readdressed from lysosomes to the cytosol or triggered when p59Hck(ca) was relocalized to lysosomes. Lysosomal markers were present at podosome rosettes. By stimulating exocytosis of p61Hck(ca) lysosomes with a calcium ionophore, the formation of podosome rosettes was enhanced. Interestingly, we confirm that, in human macrophages, Hck and lysosomal markers were present at podosomes which were spatially reorganized as clusters, a foregoing step to form rosettes, upon expression of p61Hck(ca). We propose that lysosomes, under the control of p61Hck, are involved in the biogenesis of podosomes, a key phenomenon in the migration of phagocytes.     

Caudal and even-skipped in the annelid Platynereis dumerilii and the ancestry of posterior growth

In order to address the question of the conservation of posterior growth mechanisms in bilaterians, we have studied the expression patterns of the orthologues of the genes caudal, even-skipped, and brachyury in the annelid Platynereis dumerilii. Annelids belong to the still poorly studied third large branch of the bilaterians, the lophotrochozoans, and have anatomic and developmental characteristics, such as a segmented body plan, indirect development through a microscopic ciliated larva, and building of the trunk through posterior addition, which are all hypothesized by some authors (including us) to be present already in Urbilateria, the last common ancestor of bilaterians. All three genes are shown to be likely involved in the building of the anteroposterior axis around the slit-like amphistomous blastopore as well as in the patterning of the terminal anus-bearing piece of the body (the pygidium). In addition, caudal and even-skipped are likely involved in the posterior addition of segments. Together with the emerging results on the conservation of segmentation genes, these results reinforce the hypothesis that Urbilateria had a segmented trunk developing through posterior addition.     

Identification of six novel allosteric effectors of Arabidopsis thaliana aspartate kinase-homoserine dehydrogenase isoforms. Physiological context sets the specificity

The Arabidopsis genome contains two genes predicted to code for bifunctional aspartate kinase-homoserine dehydrogenase enzymes (isoforms I and II). These two activities catalyze the first and the third steps toward the synthesis of the essential amino acids threonine, isoleucine, and methionine. We first characterized the kinetic and regulatory properties of the recombinant enzymes, showing that they mainly differ with respect to the inhibition of the homoserine dehydrogenase activity by threonine. A systematic search for other allosteric effectors allowed us to identify an additional inhibitor (leucine) and 5 activators (alanine, cysteine, isoleucine, serine, and valine) equally efficient on aspartate kinase I activity (4-fold activation). The six effectors of aspartate kinase I were all activators of aspartate kinase II activity (13-fold activation) and displayed a similar specificity for the enzyme. No synergy between different effectors could be observed. The activation, which resulted from a decrease in the Km values for the substrates, was detected using low substrates concentrations. Amino acid quantification revealed that alanine and threonine were much more abundant than the other effectors in Arabidopsis leaf chloroplasts. In vitro kinetics in the presence of physiological concentrations of the seven allosteric effectors confirmed that aspartate kinase I and II activities were highly sensitive to changes in alanine and threonine concentrations. Thus, physiological context rather than enzyme structure sets the specificity of the allosteric control. Stimulation by alanine may play the role of a feed forward activation of the aspartate-derived amino acid pathway in plant.     

Two novel types of O-glycans on the mugwort pollen allergen Art v 1 and their role in antibody binding

Art v 1, the major allergen of mugwort (Artemisia vulgaris) pollen contains galactose and arabinose. As the sera of some allergic patients react with natural but not with recombinant Art v 1 produced in bacteria, the glycosylation of Art v 1 may play a role in IgE binding and human allergic reactions. Chemical and enzymatic degradation, mass spectrometry, and 800 MHz (1)H and (13)C nuclear magnetic resonance spectroscopy indicated the proline-rich domain to be glycosylated in two ways. We found a large hydroxyproline-linked arabinogalactan composed of a short beta1,6-galactan core, which is substituted by a variable number (5-28) of alpha-arabinofuranose residues, which form branched side chains with 5-, 2,5-, 3,5-, and 2,3,5-substituted arabinoses. Thus, the design of the Art v 1 polysaccharide differs from that of the well known type II arabinogalactans, and we suggest it be named type III arabinogalactan. The other type of glycosylation was formed by single (but adjacent) beta-arabinofuranoses linked to hydroxyproline. In contrast to the arabinosylation of Ser-Hyp(4) motifs in other hydroxyproline-rich glycoproteins, such as extensins or solanaceous lectins, no oligo-arabinosides were found in Art v 1. Art v 1 and parts thereof produced by alkaline degradation, chemical deglycosylation, proteolytic degradation, and/or digestion with alpha-arabinofuranosidase were used in enzyme-linked immunosorbent assay and immunoblot experiments with rabbit serum and with the sera of patients. Although we could not observe antibody binding by the polysaccharide, the single hydroxyproline-linked beta-arabinose residues appeared to react with the antibodies. Mono-beta-arabinosylated hydroxyproline residues thus constitute a new, potentially cross-reactive, carbohydrate determinant in plant proteins.     

Automated expression and solubility screening of His-tagged proteins in 96-well format

A growing need for sensitive and high-throughput methods for screening the expression and solubility of recombinant proteins exists in structural genomics. Originally, the emergency solution was to use immediately available techniques such as manual lysis of expression cells followed by analysis of protein expression by gel electrophoresis. However, these handmade methods quickly proved to be unfit for the high-throughput demand of postgenomics, and it is now generally accepted that the long-term solution to this problem will be based on automation, on industrial standard-formatted experiments, and on downsizing samples and consumables. In agreement with this consensus, we have set up a fully automated method based on a dot-blot technology and using 96-well format consumables for assessing by immunodetection the amount of total and soluble recombinant histidine (His)-tagged proteins expressed in Escherichia coli. The method starts with the harvest of expression cells and ends with the display of solubility/expression results in milligrams of recombinant protein per liter of culture using a three-color code to assist analysis. The program autonomously processes 160 independent cultures at a time.     

Biophysical and biological studies of end-group-modified derivatives of Pep-1

Pep-1 is a tryptophane-rich cell-penetrating peptide (CPP) that has been previously proposed to bind protein cargoes by hydrophobic assembly and translocate them across cellular membranes. To date, however, the molecular mechanisms responsible for cargo binding and translocation have not been clearly identified. This study was conducted to gain insight into the interaction between Pep-1 with its cargo and the biological membrane to identify the thereby involved structural elements crucial for translocation. We studied three peptides differing in their N- and C-termini: (i) Pep-1, carrying an acetylated N-terminus and a C-terminal cysteamine elongation, (ii) AcPepWAmide, with an acetylated N-terminus and an amidated C-terminus, and (iii) PepW, with two free termini. Thioredoxin (TRX) and beta-galactosidase were used as protein cargoes. To study CPP-membrane interactions, we performed biophysical as well as biological assays. To mimic biological membranes, we used phospholipid liposomes in a dye leakage assay and surfactant micelles for high-resolution NMR studies. In addition, membrane integrity, cell viability, and translocation efficiency were analyzed in HeLa cells. An alpha-helical structure was found for all peptides in the hydrophobic N-terminal region encompassing residues 4-13, whereas the hydrophilic region remained unstructured in the presence of micelles. Our results show that the investigated peptides interacted with the micelles as well as with the protein cargo via their tryptophan-rich domain. All peptides displayed an orientation parallel to the micelle surface. The C-terminal cysteamine group formed an additional membrane anchor, leading to more efficient translocation properties in cells. No membrane permeabilization was observed, and our data were largely compatible with an endocytic pathway for cellular uptake.     

Dynamic interactions of Fc gamma receptor IIB with filamin-bound SHIP1 amplify filamentous actin-dependent negative regulation of Fc epsilon receptor I signaling

The engagement of high affinity receptors for IgE (FcepsilonRI) generates both positive and negative signals whose integration determines the intensity of mast cell responses. FcepsilonRI-positive signals are also negatively regulated by low affinity receptors for IgG (FcgammaRIIB). Although the constitutive negative regulation of FcepsilonRI signaling was shown to depend on the submembranous F-actin skeleton, the role of this compartment in FcgammaRIIB-dependent inhibition is unknown. We show in this study that the F-actin skeleton is essential for FcgammaRIIB-dependent negative regulation. It contains SHIP1, the phosphatase responsible for inhibition, which is constitutively associated with the actin-binding protein, filamin-1. After coaggregation, FcgammaRIIB and FcepsilonRI rapidly interact with the F-actin skeleton and engage SHIP1 and filamin-1. Later, filamin-1 and F-actin dissociate from FcR complexes, whereas SHIP1 remains associated with FcgammaRIIB. Based on these results, we propose a dynamic model in which the submembranous F-actin skeleton forms an inhibitory compartment where filamin-1 functions as a donor of SHIP1 for FcgammaRIIB, which concentrate this phosphatase in the vicinity of FcepsilonRI and thereby extinguish activation signals.     

Non-Mendelian transmission of alleles at microsatellite loci: an example in Ixodes ricinus, the vector of Lyme disease

Microsatellite loci are usually considered to be neutral co-dominant and Mendelian markers. We undertook to study the inheritance of five microsatellite loci in the European Lyme disease vector, the tick Ixodes ricinus. Only two loci appeared fully Mendelian while the three others displayed non-Mendelian patterns that highly frequent null alleles could not fully explain. At one locus, IR27, some phenomenon seems to hinder the PCR amplification of one allele, depending on its origin (maternal imprinting) and/or its size (short allele dominance). DNA methylation, which appeared to be a possible explanation of this amplification bias, was rejected by a specific test comparing the amplification efficiency that did not differ between unmethylated and experimentally methylated DNA. The role of allele size in heterozygous individuals was then revealed from the data available on field collected ticks and consistent with the results of a theoretical approach. These observations highlight the need for prudence while inferring reproductive systems (selfing rates), parentage or even allelic frequencies from microsatellite markers, in particular for parasitic organisms for which molecular approaches often represent the only way for population biology inferences.     

Regulation of the cystic fibrosis transmembrane conductance regulator channel by beta-adrenergic agonists and vasoactive intestinal peptide in rat smooth muscle cells and its role in vasorelaxation

The signaling events that regulate vascular tone include voltage-dependent Ca(2+) influx and the activities of various ionic channels; which molecular entities are involved and their role are still a matter of debate. Here we show expression of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel in rat aortic smooth muscle cells. Immunoprecipitation and in vitro protein kinase A phosphorylation show the appearance of mature band C of CFTR. An immunohistochemistry study shows CFTR proteins in smooth muscles of aortic rings but not in skeletal muscles. Using the iodide efflux method, a combination of agonists and pharmacological agents was used to dissect the function of CFTR. Agonists of the cAMP pathway, the beta-adrenergic agonist isoproterenol, and the neuropeptide vasoactive intestinal peptide activate CFTR-dependent transport from cells maintained in a high but not low extracellular potassium-rich saline, suggesting that depolarization of smooth muscle is critical to CFTR activation. Smooth muscle CFTR possesses all of the pharmacological attributes of its epithelial homologues: stimulation by the CFTR pharmacological activators MPB-07 (EC(50) = 158 microm) and MPB-91 (EC(50) = 20 microm) and inhibition by glibenclamide and diphenylamine-2-carboxylic acid but not by 5,11,17,23-tetrasulfonato-25,26,27,28-tetramethoxy-calix[4]arene. Contraction measurements on isolated aortic rings were performed to study the contribution of CFTR to vascular tone. With aortic rings (without endothelium) preconstricted by high K(+) saline or by the alpha-adrenergic agonist norepinephrine, CFTR activators produced a concentration-dependent relaxation. These results identify for the first time the expression and function of CFTR in smooth muscle where it plays an unexpected but fundamental role in the autonomic and hormonal regulation of the vascular tone.