CTF/NF1 transcription factors act as potent genetic insulators for integrating gene transfer vectors.

Gene transfer-based therapeutic approaches have greatly benefited from the ability of some viral vectors to efficiently integrate within the cell genome and ensure persistent transmission of newly acquired transgenes to the target cell progeny. However, integration of provirus has been associated with epigenetic repercussions that may influence the expression of both the transgene and cellular genes close to vector integration loci. The exploitation of genetic insulator elements may overcome both issues through their ability to act as barriers that limit transgene silencing and/or as enhancer-blockers preventing the activation of endogenous genes by the vector enhancer. We established quantitative plasmid-based assay systems to screen enhancer-blocker and barrier genetic elements. Short synthetic insulators that bind to nuclear factor-I protein family transcription factors were identified to exert both enhancer-blocker and barrier functions, and were compared to binding sites for the insulator protein CTCF (CCCTC-binding factor). Gamma-retroviral vectors enclosing these insulator elements were produced at titers similar to their non-insulated counterparts and proved to be less genotoxic in an in vitro immortalization assay, yielding lower activation of Evi1 oncogene expression and reduced clonal expansion of bone marrow cells.

Lipid binding by the unique and SH3 domains of c-Src suggests a new regulation mechanism

c-Src is a non-receptor tyrosine kinase involved in numerous signal transduction pathways. The kinase,SH3 and SH2 domains of c-Src are attached to the membrane-anchoring SH4 domain through the flexible Unique domain. Here we show intra- and intermolecular interactions involving the Unique and SH3 domains suggesting the presence of a previously unrecognized additional regulation layer in c-Src. We have characterized lipid binding by the Unique and SH3 domains, their intramolecular interaction and its allosteric modulation by a SH3-binding peptide or by Calcium-loaded calmodulin binding to the Unique domain. We also show reduced lipid binding following phosphorylation at conserved sites of the Unique domain. Finally, we show that injection of full-length c-Src with mutations that abolish lipid binding by the Unique domain causes a strong in vivo phenotype distinct from that of wild-type c-Src in a Xenopus oocyte model system, confirming the functional role of the Unique domain in c-Src regulation.

Activation of peroxisome proliferator-activated receptors (PPARs) by their ligands and protein kinase A activators.

The nuclear peroxisome proliferator-activated receptors (PPARs) alpha, beta, and gamma activate the transcription of multiple genes involved in lipid metabolism. Several natural and synthetic ligands have been identified for each PPAR isotype but little is known about the phosphorylation state of these receptors. We show here that activators of protein kinase A (PKA) can enhance mouse PPAR activity in the absence and the presence of exogenous ligands in transient transfection experiments. Activation function 1 (AF-1) of PPARs was dispensable for transcriptional enhancement, whereas activation function 2 (AF-2) was required for this effect. We also show that several domains of PPAR can be phosphorylated by PKA in vitro. Moreover, gel retardation experiments suggest that PKA stabilizes binding of the liganded PPAR to DNA. PKA inhibitors decreased not only the kinase-dependent induction of PPARs but also their ligand-dependent induction, suggesting an interaction between both pathways that leads to maximal transcriptional induction by PPARs. Moreover, comparing PPAR alpha knockout (KO) with PPAR alpha WT mice, we show that the expression of the acyl CoA oxidase (ACO) gene can be regulated by PKA-activated PPAR alpha in liver. These data demonstrate that the PKA pathway is an important modulator of PPAR activity, and we propose a model associating this pathway in the control of fatty acid beta-oxidation under conditions of fasting, stress, and exercise.

Remorin, a solanaceae protein resident in membrane rafts and plasmodesmata, impairs potato virus X movement.

Remorins (REMs) are proteins of unknown function specific to vascular plants. We have used imaging and biochemical approaches and in situ labeling to demonstrate that REM clusters at plasmodesmata and in approximately 70-nm membrane domains, similar to lipid rafts, in the cytosolic leaflet of the plasma membrane. From a manipulation of REM levels in transgenic tomato (Solanum lycopersicum) plants, we show that Potato virus X (PVX) movement is inversely related to REM accumulation. We show that REM can interact physically with the movement protein TRIPLE GENE BLOCK PROTEIN1 from PVX. Based on the localization of REM and its impact on virus macromolecular trafficking, we discuss the potential for lipid rafts to act as functional components in plasmodesmata and the plasma membrane.

A new green fluorescent protein-based bacterial biosensor for analysing phenanthrene fluxes.

The polycyclic aromatic hydrocarbon (PAH)-degrading strain Burkholderia sp. RP007 served as host strain for the design of a bacterial biosensor for the detection of phenanthrene. RP007 was transformed with a reporter plasmid containing a transcriptional fusion between the phnS putative promoter/operator region and the gene encoding the enhanced green fluorescent protein (GFP). The resulting bacterial biosensor--Burkholderia sp. strain RP037--produced significant amounts of GFP after batch incubation in the presence of phenanthrene crystals. Co-incubation with acetate did not disturb the phenanthrene-specific response but resulted in a homogenously responding population of cells. Active metabolism was required for induction with phenanthrene. The magnitude of GFP induction was influenced by physical parameters affecting the phenanthrene flux to the cells, such as the contact surface area between solid phenanthrene and the aqueous phase, addition of surfactant, and slow phenanthrene release from Model Polymer Release System beads or from a water-immiscible oil. These results strongly suggest that the bacterial biosensor can sense different phenanthrene fluxes while maintaining phenanthrene metabolism, thus acting as a genuine sensor for phenanthrene bioavailability. A relationship between GFP production and phenanthrene mass transfer is proposed.

Body lipid deposition in Nile tilapia fed on rations containing tannin

The aim of this work was to evaluate the effect of tannin sources and levels in rations, on the productive performance and body lipid deposition of Nile tilapias (Oreochromis niloticus) during the finishing phase. Three hundred and forty-two fishes were distributed in 18 tanks. Rations were prepared using corn, sorghum varieties, with low and high tannin content, and tannic acid at 0.08, 0.34, and 0.60%. Weight gain, apparent feed conversion and protein efficiency rate were not influenced by the treatments. The highest body lipid deposition was observed for the tannic acid treatment (14.39%), while the diet containing sorghum with high tannin content yielded leaner body (12.01%) than that of sorghum with low tannin content (13.31%). Diets containing sorghum provided lower levels of visceral fat. Rations with tannin contents did not harm the productive performance of Nile tilapia.

Efficient lentiviral gene transfer into corneal stroma cells using a femtosecond laser.

We investigated a new procedure for gene transfer into the stroma of pig cornea for the delivery of therapeutic factors. A delimited space was created at 110 mum depth with a LDV femtosecond laser in pig corneas, and a HIV1-derived lentiviral vector expressing green fluorescent protein (GFP) (LV-CMV-GFP) was injected into the pocket. Corneas were subsequently dissected and kept in culture as explants. After 5 days, histological analysis of the explants revealed that the corneal pockets had closed and that the gene transfer procedure was efficient over the whole pocket area. Almost all the keratocytes were transduced in this area. Vector diffusion at right angles to the pocket's plane encompasses four (endothelium side) to 10 (epithelium side) layers of keratocytes. After 21 days, the level of transduction was similar to the results obtained after 5 days. The femtosecond laser technique allows a reliable injection and diffusion of lentiviral vectors to efficiently transduce stromal cells in a delimited area. Showing the efficacy of this procedure in vivo could represent an important step toward treatment or prevention of recurrent angiogenesis of the corneal stroma.

Lipoproteins and mitogen-activated protein kinase signaling: a role in atherogenesis?

PURPOSE OF REVIEW: Lipoproteins play a critical role in the development of atherosclerosis, which might result partly from their capacity to induce specific intracellular signaling pathways. The goal of this review is to summarize the signaling properties of lipoproteins, in particular, their capacity to induce activation of mitogen-activated protein kinase pathways and the resulting modulation of cellular responses in blood vessel cells. RECENT FINDINGS: Lipoproteins activate the extracellular signal-regulated kinase and p38 mitogen-activated protein kinase pathways in all blood vessel cell types. This may require lipoprotein docking to scavenger receptor B1, allowing transfer of cholesterol and sphingosine-1-phosphate to plasma membranes. Subsequent propagation of the signals probably requires the stimulation of G protein-coupled receptors, followed by the transactivation of receptor tyrosine kinases. Lipoprotein-induced extracellular signal-regulated kinase activity favors cell proliferation, whereas lipoprotein-induced p38 mitogen-activated protein kinase activity leads to cell hyperplasia and promotes cell migration. Some signaling pathways and cellular effects induced by lipoproteins have been observed in atherosclerotic plaques and therefore represent potential targets for the development of anti-atherosclerotic drugs. SUMMARY: The main blood vessel cell types have the capacity to activate protein kinase pathways in the presence of lipoproteins. This induces cell proliferation, hyperplasia and migration, known to be dysregulated in atherosclerotic lesions.

Growth performance and body composition of giant trahira fingerlings fed diets with different protein and energy levels

The objective of this work was to determine the proper levels of protein and energy in diets of Hoplias lacerdae fingerlings. The dietary crude protein (CP) and gross energy (GE) levels for fingerlings of giant trahira were evaluated in a completely randomized 4x3 factorial design with 35, 39, 43 and 47% CP and 4,100, 4,300 and 4,500 kcal kg-1 of GE, and four replicates. The survival rate was 99.22%, and a linear improvement on the performance parameters was detected after increasing diet crude protein levels. Feed conversion ratio decreased with increasing levels of dietary protein and energy in the diets. A significant interaction between crude protein and gross energy was observed over body protein and mineral matter. Body lipid has increased linearly as gross energy in the diet increased. The retention of crude protein and energy showed a linear increasing with rising of crude protein levels in the diet. Crude protein level at 47% provides the best performance and energy retention, independently of the gross energy levels in the diet.

Watermelon transformation with Zucchini yellow mosaic virus coat protein gene and comparison with parental cultivar

The objective of this work was to transfer Zucchini yellow mosaic virus coat protein (ZYMV-CP) and neomycin phosphotransferase II (NPT II) genes to the watermelon 'Crimson Sweet'(CS) genome, and to compare the transgenic progenies T1 and T2 with the nontransformed parental cultivar for morphological, pomological, growth and yield characteristics. The ZYMV-CP gene was transferred by Agrobacterium tumefaciens. The presence of the gene in transgenic T0, T1 and T2 plants was determined by polymerase chain reaction, and the results were confirmed by Southern blot. Two experiments were performed, one in the winter-spring and the other in the summer-autumn. In both experiments, the hypocotyl length of transgenic seedlings was significantly higher than that of nontransgenic parental ones. In the second experiment, the differences between transgenic and nontransgenic individuals were significant concerning fruit rind thickness, flesh firmness, fruit peduncle length, size of pistil scar, and a* values for fruit stripe or flesh color. Transferring ZYMV-CP gene to CS genome affected only a few characteristics from the 80 evaluated ones. The changes in rind thickness, flesh firmness and flesh color a* values are favorable, while the increase in the size of pistil scar is undesirable. The transgenic watermelon line having ZYMV-CP gene and the parental cultivar CS are very similar.

Membrane insertion and antibody recognition of a glycosylphosphatidylinositol-anchored protein: an optical study.

The kinetics of binding of a glycolipid-anchored protein (the promastigote surface protease, PSP) to planar lecithin bilayers is studied by an integrated optics technique, in which the bilayer membrane is supported on an optical wave guide and the phase velocities of guided light modes in the wave guide are measured. From these velocities, the optical parameters of the membrane and PSP layers deposited on the waveguide are determined, yielding in particular the mass of PSP bound to the membrane, which is followed in real time. From a comparison of the binding rates of PSP and PSP from which the lipid moiety has been removed, it is shown that the lipid moiety plays a key role in anchoring the protein to the membrane. Specific and nonspecific binding of antibodies to membrane-anchored PSP is also investigated. As little as a fifth of a monolayer of PSP is sufficient to suppress the appreciable nonspecific binding of antibodies to the membrane.

Amphipathic alpha-helix AH2 is a major determinant for the oligomerization of hepatitis C virus nonstructural protein 4B.

Nonstructural protein 4B (NS4B) is a key organizer of hepatitis C virus (HCV) replication complex formation. It induces a specific membrane rearrangement, designated membranous web, that serves as a scaffold for the HCV replication complex. However, the mechanisms underlying membranous web formation are poorly understood. Based on fluorescence resonance energy transfer (FRET) and confirmatory coimmunoprecipitation analyses, we provide evidence for an oligomerization of NS4B in the membrane environment of intact cells. Several conserved determinants were found to be involved in NS4B oligomerization, through homotypic and heterotypic interactions. N-terminal amphipathic ?-helix AH2, comprising amino acids 42 to 66, was identified as a major determinant for NS4B oligomerization. Mutations that affected the oligomerization of NS4B disrupted membranous web formation and HCV RNA replication, implying that oligomerization of NS4B is required for the creation of a functional replication complex. These findings enhance our understanding of the functional architecture of the HCV replication complex and may provide new angles for therapeutic intervention. At the same time, they expand the list of positive-strand RNA virus replicase components acting as oligomers.

Regulation of the vitellogenin gene B1 promoter after transfer into hepatocytes in primary cultures.

The estrogen-dependent and tissue-specific regulation of the Xenopus laevis vitellogenin gene B1 promoter has been studied by lipid-mediated DNA transfer into Xenopus hepatocytes in primary culture. Hepatocytes achieve an efficient hormonal control of this promoter through a functional interaction between the estrogen responsive elements and a promoter proximal region upstream of the TATA box, which is characterized by a high density of binding sites for the transcription factors CTF/NF-1, C/EBP and HNF3. DNA accessibility to restriction enzymes within the chromosomal copy of the vitellogenin gene B1 promoter shows that the estrogen responsive unit and the promoter proximal region are sensitive to digestion in uninduced and estrogen-induced hepatocytes but not in erythrocyte nuclei. Together, these findings support the notion that chromatin configuration as well as the interplay of promoter elements mediate proper hormone-dependent and tissue-specific expression of the B1 vitellogenin gene.

Heat shock response in photosynthetic organisms: membrane and lipid connections.

The ability of photosynthetic organisms to adapt to increases in environmental temperatures is becoming more important with climate change. Heat stress is known to induce heat-shock proteins (HSPs) many of which act as chaperones. Traditionally, it has been thought that protein denaturation acts as a trigger for HSP induction. However, increasing evidence has shown that many stress events cause HSP induction without commensurate protein denaturation. This has led to the membrane sensor hypothesis where the membrane's physical and structural properties play an initiating role in the heat shock response. In this review, we discuss heat-induced modulation of the membrane's physical state and changes to these properties which can be brought about by interaction with HSPs. Heat stress also leads to changes in lipid-based signaling cascades and alterations in calcium transport and availability. Such observations emphasize the importance of membranes and their lipids in the heat shock response and provide a new perspective for guiding further studies into the mechanisms that mediate cellular and organismal responses to heat stress.

Identification of in vitro HSC fate regulators by differential lipid raft clustering.

Most hematopoietic stem cells (HSC) in the bone marrow reside in a quiescent state and occasionally enter the cell cycle upon cytokine-induced activation. Although the mechanisms regulating HSC quiescence and activation remain poorly defined, recent studies have revealed a role of lipid raft clustering (LRC) in HSC activation. Here, we tested the hypothesis that changes in lipid raft distribution could serve as an indicator of the quiescent and activated state of HSCs in response to putative niche signals. A semi-automated image analysis tool was developed to map the presence or absence of lipid raft clusters in live HSCs cultured for just one hour in serum-free medium supplemented with stem cell factor (SCF). By screening the ability of 19 protein candidates to alter lipid raft dynamics, we identified six factors that induced either a marked decrease (Wnt5a, Wnt3a and Osteopontin) or increase (IL3, IL6 and VEGF) in LRC. Cell cycle kinetics of single HSCs exposed to these factors revealed a correlation of LRC dynamics and proliferation kinetics: factors that decreased LRC slowed down cell cycle kinetics, while factors that increased LRC led to faster and more synchronous cycling. The possibility of identifying, by LRC analysis at very early time points, whether a stem cell is activated and possibly committed upon exposure to a signaling cue of interest could open up new avenues for large-scale screening efforts.