Effects of Boron on Proton Transport and Membrane Properties of Sunflower (Helianthus annuus L.) Cell Microsomes

Boron deficiency and toxicity inhibit ATP-dependent H+ pumping and vanadate-sensitive ATPase activity in sunflower roots and cell suspensions. The effects of boron on H+ pumping and on passive H+ conductance, as well as on fluorescence anisotropy in KI-washed microsomes isolated from sunflower (Helianthus annuus L. cv Enano) cell suspensions, have been investigated. Boron deficiency reduced the total and vanadate-sensitive ATPase activities as well as the vanadate-sensitive ATP-dependent H+ pumping without affecting the amount of antigenic ATPase protein as measured by immunoblotting with an Arabidopsis thaliana plasma membrane anti-H+-ATPase polyclonal antibody. Kinetic studies revealed that boron deficiency reduced Vmax of vanadate-sensitive ATPase activity with little change in the apparent Km for Mg2+-ATP. Proton leakage was greater in microsomal vesicles isolated from cells grown without boron and incubated in reaction medium without added boron, and this effect was reversed by addition of boron to the reaction medium. Fluorescence anisotropy indicated that diphenyl hexatriene and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene probes were immobilized to a greater extent in microsomes from cells grown without boron than in those from cells grown with 100 [mu]M H3BO3. The apparent decrease of membrane fluidity in microsomes from cells grown without boron was reversed by the addition of boron to the reaction medium. Taken together these data suggest that inhibition of H+ gradient formation in microsomes from sunflower cells grown in the absence of boron could be due to the combined effects of reduced H+-ATPase activity and increased passive conductance across the membrane, possibly resulting from increased membrane rigidity.     

Three-dimensional interdigitated electrode array as a transducer for label-free biosensors

A new transducer for biosensor applications has been developed based on a three-dimensional interdigitated electrode array (IDEA) with electrode digits separated by an insulating barrier. Binding of molecules to a chemically modified surface of the transducer induces important changes in conductivity between the electrodes. Three-dimensional sensor shows considerable improvement compared with a standard planar IDEA design. The potential of the developed device as a sensor transducer to detect immunochemical and enzymatic reactions, as well as DNA hybridization events is demonstrated. The immunosensor allows direct detection of the antibiotic sulfapyridine and shows the IC(50) parameter value of 5.6 microgL(-1) in a buffer. Immunochemical determination occurs under competitive configurations and without the use of any label. Each modified sensor is of a single use. Nevertheless, biochemical reagents can be easily cleaned off the sensor surface for its reuse. Layer-by-layer method of used to deposit polyethyleneimine and glucose oxidase showed that the sensor is also highly effective for detecting single and multilayered molecular assemblies.     

Chemokines determine local lymphoneogenesis and a reduction of circulating CXCR4+ T and CCR7 B and T lymphocytes in thyroid autoimmune diseases

Chemokines and their corresponding receptors are crucial for the recruitment of lymphocytes into the lymphoid organs and for its organization acting in a multistep process. Tissues affected by autoimmune disease often contain ectopic lymphoid follicles which, in the case of autoimmune thyroid disorders, are highly active and specific for thyroid Ags although its pathogenic role remains unclear. To understand the genesis of these lymphoid follicles, the expression of relevant cytokines and chemokines was assessed by real time PCR, immunohistochemistry and by in vitro assays in autoimmune and nonautoimmune thyroid glands. Lymphotoxin alpha, lymphotoxin beta, C-C chemokine ligand (CCL) 21, CXC chemokine ligand (CXCL) 12, CXCL13, and CCL22 were increased in thyroids from autoimmune patients, whereas CXCL12, CXCL13, and CCL22 levels were significantly higher in autoimmune glands with ectopic secondary lymphoid follicles than in those without follicles. Interestingly, thyroid epithelium produced CXCL12 in response to proinflammatory cytokines providing a possible clue for the understanding of how tissue stress may lead to ectopic follicle formation. The finding of a correlation between chemokines and thyroid autoantibodies further suggests that intrathyroidal germinal centers play a significant role in the autoimmune response. Unexpectedly, the percentage of circulating CXCR4(+) T cells and CCR7(+) B and T cells (but not of CXCR5) was significantly reduced in PBMCs of patients with autoimmune thyroid disease when they were compared with their intrathyroidal lymphocytes. This systemic effect of active intrathyroidal lymphoid tissue emerges as a possible new marker of thyroid autoimmune disease activity.     

Deciphering function and mechanism of calcium-binding proteins from their evolutionary imprints

Calcium-binding proteins regulate ion metabolism and vital signalling pathways in all living organisms. Our aim is to rationalize the molecular basis of their function by studying their evolution using computational biology techniques. Phylogenetic analysis is of primary importance for classifying cognate orthologs; profile hidden Markov models (HMM) of individual subfamilies discern functionally relevant sites by conservation probability analysis; and 3-dimensional structures display the integral protein in context. The major classifications of calcium-binding proteins, viz. EF-hand, C2 and ANX, exhibit structural diversity in their HMM fingerprints at the subfamily level, with functional consequences for protein conformation, exposure of receptor interaction sites and/or binding to membrane phospholipids. Calmodulin, S100 and annexin families were characterized in Petromyzon marinus (sea lamprey) to document genome duplication and gene creation events during the key evolutionary transition to primitive vertebrates. Novel annexins from diverse organisms revealed calcium-binding domains with accessory structural features that define their unique molecular fingerprints, protein interactivity and functional specificity. These include the first single-domain, bacterial annexin in Cytophaga hutchinsonii, the 21 tetrad annexins from the unicellular protist Giardia intestinalis, an ancestor to land plant annexins from the green alga Ostreococcus lucimarinus, invertebrate octad annexins and a critical polymorphism in human ANXA7. Receptor docking models supported the hypothesis of a potential interaction between annexin and C2 domains as a propitious mechanism for ensuring membrane translocation during signal transduction.     

Vacuolar Cation/H+ Antiporters of Saccharomyces cerevisiae

We previously demonstrated that Saccharomyces cerevisiae vnx1Δ mutant strains displayed an almost total loss of Na⁺ and K⁺/H⁺ antiporter activity in a vacuole-enriched fraction. However, using different in vitro transport conditions, we were able to reveal additional K⁺/H⁺ antiporter activity. By disrupting genes encoding transporters potentially involved in the vnx1 mutant strain, we determined that Vcx1p is responsible for this activity. This result was further confirmed by complementation of the vnx1Δvcx1Δ nhx1Δ triple mutant with Vcx1p and its inactivated mutant Vcx1p-H303A. Like the Ca²⁺/H⁺ antiporter activity catalyzed by Vcx1p, the K⁺/H⁺ antiporter activity was strongly inhibited by Cd²⁺ and to a lesser extend by Zn²⁺. Unlike as previously observed for NHX1 or VNX1, VCX1 overexpression only marginally improved the growth of yeast strain AXT3 in the presence of high concentrations of K⁺ and had no effect on hygromycin sensitivity. Subcellular localization showed that Vcx1p and Vnx1p are targeted to the vacuolar membrane, whereas Nhx1p is targeted to prevacuoles. The relative importance of Nhx1p, Vnx1p, and Vcx1p in the vacuolar accumulation of monovalent cations will be discussed.