Use of electroblotting to detect and analyze phosphotyrosine containing peptides separated by two-dimensional gel electrophoresis

A technique to detect and analyze phosphotyrosine containing peptides after separation of total cellular proteins by two-dimensional gel electrophoresis is described. This is achieved by electroblotting of proteins on nylon membranes followed by alkali treatment. In comparison with direct alkali treatment of the polyacrylamide gel, this procedure is easier to perform; avoids the diffusion of proteins out of the gel during alkali treatment; allows a more precise localization of phosphotyrosine containing peptides on the untreated membrane; and is less time consuming with respect to extraction of proteins for phosphoamino acid analysis.     

Ci-FoxA-a is the earliest zygotic determinant of the ascidian anterior ectoderm and directly activates Ci-sFRP1/5

This work focuses on the anteroposterior patterning of the ectoderm in the invertebrate chordate Ciona intestinalis. Previous work indicated that, by the eight-cell stage, the anterior and posterior animal blastomeres have acquired different properties, including a differential responsiveness to inducing signals from the underlying mesendoderm. Here, we investigated the molecular basis of this distinction. For this, we studied the regulation of the earliest marker specific for the anterior ectoderm, Ci-sFRP1/5, which is activated at the 64-cell stage. We first found that the activation of this marker in the anterior ectoderm does not involve communication with other lineages. We then identified, by phylogenetic footprinting and deletion analysis, a short conserved minimal enhancer driving the onset of expression of Ci-sFRP1/5. We showed that this enhancer was a direct target of the Ci-FoxA-a gene, a FoxA/HNF3 orthologue expressed in anterior ectodermal and mesendodermal lineages from the eight-cell stage. Gain- and loss-of-function experiments revealed that Ci-FoxA-a is necessary and sufficient within the ectoderm to impose an ectodermal anterior identity, and to repress the posterior programme. Thus, Ci-FoxA-a constitutes a major early zygotic anterior determinant for the ascidian ectoderm, acting autonomously in this territory, prior to the onset of vegetal inductions. Interestingly, while vertebrate FoxA2 are also involved in the regionalization of the ectoderm, they are thought to act during gastrulation to control, in the mesendoderm, the expression of organizer signals. We discuss the evolution of chordate ectodermal patterning in light of our findings.     

DNA alters the bilayer structure of cationic lipid diC14-amidine: A spin label study

Cationic lipids-DNA complexes (lipoplexes) have been used for delivery of nucleic acids into cells in vitro and in vivo. Despite the fact that, over the last decade, significant progress in the understanding of the cellular pathways and mechanisms involved in lipoplexes-mediated gene transfection have been achieved, a convincing relationship between the structure of lipoplexes and their in vivo and in vitro transfection activity is still missing. How does DNA affect the lipid packing and what are the consequences for transfection efficiency is the point we want to address here. We investigated the bilayer organization in cationic liposomes by electron spin resonance (ESR). Phospholipids spin labeled at the 5th and 16th carbon atoms were incorporated into the DNA/diC14-amidine complex. Our data demonstrate that electrostatic interactions involved in the formation of DNA-cationic lipid complex modify the packing of the cationic lipid membrane. DNA rigidifies the amidine fluid bilayer and fluidizes the amidine rigid bilayer just below the gel-fluid transition temperature. These effects were not observed with single nucleotides and are clearly related to the repetitive charged motif present in the DNA chain and not to a charge-charge interaction. These modifications of the initial lipid packing of the cationic lipid may reorient its cellular pathway towards different routes. A better knowledge of the cationic lipid packing before and after interaction with DNA may therefore contribute to the design of lipoplexes capable to reach specific cellular targets.     

Changes in mouse whole saliva soluble proteome induced by tannin-enriched diet

Background  

Previous studies suggested that dietary tannin ingestion may induce changes in mouse salivary proteins in addition to the primarily studied proline-rich proteins (PRPs). The aim of the present study was to determine the protein expression changes induced by condensed tannin intake on the fraction of mouse whole salivary proteins that are unable to form insoluble tannin-protein complexes. Two-dimensional polyacrylamide gel electrophoresis protein separation was used, followed by protein identification by mass spectrometry.     

Allosteric Block of KCa2 Channels by Apamin

Activation of small conductance calcium-activated potassium (K_Ca2) channels can regulate neuronal firing and synaptic plasticity. They are characterized by their high sensitivity to the bee venom toxin apamin, but the mechanism of block is not understood. For example, apamin binds to both K_Ca2.2 and K_Ca2.3 with the same high affinity (K_D ∼ 5 pm for both subtypes) but requires significantly higher concentrations to block functional current (IC₅₀ values of ∼100 pm and ∼5 nm, respectively). This suggests that steps beyond binding are needed for channel block to occur. We have combined patch clamp and binding experiments on cell lines with molecular modeling and mutagenesis to gain more insight into the mechanism of action of the toxin. An outer pore histidine residue common to both subtypes was found to be critical for both binding and block by the toxin but not for block by tetraethylammonium (TEA) ions. These data indicated that apamin blocks K_Ca2 channels by binding to a site distinct from that used by TEA, supported by a finding that the onset of block by apamin was not affected by the presence of TEA. Structural modeling of ligand-channel interaction indicated that TEA binds deep within the channel pore, which contrasted with apamin being modeled to interact with the channel outer pore by utilizing the outer pore histidine residue. This multidisciplinary approach suggested that apamin does not behave as a classical pore blocker but blocks using an allosteric mechanism that is consistent with observed differences between binding affinity and potency of block.     

In vitro study of the antioxidant properties of non steroidal anti-inflammatory drugs by chemiluminescence and electron spin resonance (ESR)

Objectives. To determine the antioxidant activities of nonsteroidal anti-inflammatory drugs (NSAIDS), we examined by chemiluminescence (CL) and electron spin resonance (ESR) their scavenging properties towards lipid peroxides, hypochlorous acid and peroxynitrite.

Methods. The antioxidant properties of nimesulide (NIM), 4-hydroxynimesulide (4-HONIM), aceclofenac (ACLO), 4-hydroxyaceclofenac (4-HOA-CLO), diclofenac (DICLO) and indomethacin (INDO) were tested on four different reactive oxygen species (ROS) generating systems: (I) phorbol-myristate acetate (PMA)-activated neutrophils, (II) Fe²⁺/ascorbate-induced lipid peroxidation, (III) HOCl-induced light emission, (IV) the kinetics of ONOO^- decomposition followed by spectrophotometry. ROS production was monitored by luminol-enhanced CL or by ESR using two different spin traps.

Results. At 10 μM, ACLO, NIM, 4-HONIM, 4-HOA-CLO, and DICLO decreased luminol-enhanced CL generated by PMA-activated neutrophils. Inversely, INDO increased the luminol enhanced CL. Interestingly, hydroxylated metabolites were more potent antioxidants than the parent drugs. Furthermore, all drugs tested, excepted ACLO, lowered lipid peroxidation induced by Fe²⁺/ascorbate system. ACLO and DICLO, even at the highest concentration tested (100 μM), did not significantly lower HOCl induced CL, whereas the other drugs were potent scavengers. Finally, all the NSAIDS accelerated decomposition of ONOO^-, suggesting a potential capacity of the molecules to scavenge peroxynitrite.

Conclusion. The NSAIDs possess variable degrees of antioxidant activities, linked to their ability to react with HOCl, lipid peroxides or ONOO^-. These antioxidant activities could offer interesting targeted side-effects in the treatment of joint inflammatory diseases.