Receptor-mediated delivery of siRNAs by tethered nucleic acid base-paired interactions

We report a new strategy for cell-type-specific delivery of functional siRNAs into cells. The method involves the noncovalent attachment of siRNAs to ligand-conjugated oligodeoxynucleotides via nucleic acid base-paired interactions. The resulting complexes can be directly applied to cells, leading to specific cellular uptake and gene silencing. The method is simple, economical, and can be easily adapted for other cell surface receptors. Here we show the application of this method for the delivery of siRNAs to folate receptor-expressing cells.     

Strategies for antiviral resistance in transgenic plants

Genetic engineering offers a means of incorporating new virus resistance traits into existing desirable plant cultivars. The initial attempts to create transgenes conferring virus resistance were based on the pathogen-derived resistance concept. The expression of the viral coat protein gene in transgenic plants was shown to induce protective effects similar to classical cross protection, and was therefore distinguished as 'coat-protein-mediated' protection. Since then, a large variety of viral sequences encoding structural and non-structural proteins were shown to confer resistance. Subsequently, non-coding viral RNA was shown to be a potential trigger for virus resistance in transgenic plants, which led to the discovery of a novel innate resistance in plants, RNA silencing. Apart from the majority of pathogen-derived resistance strategies, alternative strategies involving virus-specific antibodies have been successfully applied. In a separate section, efforts to combat viroids in transgenic plants are highlighted. In a final summarizing section, the potential risks involved in the introduction of transgenic crops and the specifics of the approaches used will be discussed.     

Proteomic profiling during atherosclerosis progression: Effect of nebivolol treatment

There is a great need for the identification of biomarkers for the early diagnosis of atherosclerosis and the agents to prevent its progression. The aim of this study was to explore the effect of 24 week of nebivolol (a third-generation vasodilatory beta-blocker) treatment on serum protein profiles in Apo E^?/? mice during atherosclerosis progression. Nebivolol treated and non-treated (the control group) groups consisted of 10 genetically modified homozygous Apo E^?/? mice. Proteomic analyses were performed using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) in the serum samples from the nebivolol treated and non-treated Apo E^?/? mice. The protein profiles obtained using three different chips, CM10 (weak cation-exchange), H50 (reverse phase), and IMAC30-Cu²⁺ (immobilized metal affinity capture) were statistically analyzed using the ProteinChip data manager 3.0 program. At the end of 24 week of nebivolol-treatment period, a total of 662 protein/peptide clustering peaks were detected using 12 different conditions and reading with high and low intensity laser energy. The highest total number of protein/peptide clusters was found on H50 chip array. The peak intensities of 95 of the 662 protein/peptide clusters were significantly different in the nebivolol-treated atherosclerotic group in comparison to the non-treated control mice groups (P < 0.05). Forty-three protein/peptides were up-regulated (high signal intensity) while 52 protein/peptides had lower signal intensity (down-regulated) in the nebivolol-treated atherosclerotic group. The proteomic profiles of nebivolol-treated Apo E^?/? mice were different than the control group indicating a potential role of nebivolol in atherosclerosis. Our study contributes to understand the efficacy of nebivolol on serum protein/peptide profiles during atherosclerosis development.     

Pure MnTBAP selectively scavenges peroxynitrite over superoxide: Comparison of pure and commercial MnTBAP samples to MnTE-2-PyP in two models of oxidative stress injury,an SOD-specific Escherichia coli model and carrageenan-induced pleurisy

MnTBAP is often referred to as an SOD mimic in numerous models of oxidative stress. We have recently reported that pure MnTBAP does not dismute superoxide, but commercial or poorly purified samples are able to perform O₂^·?dismutation with low-to-moderate efficacy via non-innocent Mn-containing impurities. Herein, we show that neither commercial nor pure MnTBAP could substitute for SOD enzyme in a SOD-deficient Escherichia coli model, whereas MnTE-2-PyP-treated SOD-deficient E. coli grew as well as a wild-type strain. This SOD-specific system indicates that MnTBAP does not act as an SOD mimic in vivo. In another model, carrageenan-induced pleurisy in mice, inflammation was evidenced by increased pleural fluid exudate and neutrophil infiltration and activation: these events were blocked by 0.3 mg/kg MnTE-2-PyP and, to a slightly lesser extent, by 10 mg/kg of either MnTBAP. Also, 3-nitrotyrosine formation, an indication of peroxynitrite existence in vivo, was blocked by both compounds; again MnTE-2-PyP was 33-fold more effective. Pleurisy model data indicate that MnTBAP exerts some protective actions in common with MnTE-2-PyP, which are not O₂^·? related and can be fully rationalized if one considers that the common biological role shared by MnTBAP and MnTE-2-PyP is related to their reduction of peroxynitrite and carbonate radical, the latter arising from ONOOCO₂ adduct. The log k_cat (O₂^·?) value for MnTBAP is estimated to be about 3.16, which is ~ 5 and ~ 6 orders of magnitude smaller than the SOD activities of the potent SOD mimic MnTE-2-PyP and Cu,Zn-SOD, respectively. This very low value indicates that MnTBAP is too inefficient at dismuting superoxide to be of any biological impact, which was confirmed in the SOD-deficient E. coli model. The peroxynitrite scavenging ability of MnTBAP, however, is only ~ 2.5 orders of magnitude smaller than that of MnTE-2-PyP and is not significantly affected by the presence of the SOD-active impurities in the commercial MnTBAP sample (log k_red (ONOO^?) = 5.06 for pure and 4.97 for commercial sample). The reduction of carbonate radical is equally fast with MnTBAP and MnTE-2-PyP. The dose of MnTBAP required to yield oxidative stress protection and block nitrotyrosine formation in the pleurisy model is > 1.5 orders of magnitude higher than that of MnTE-2-PyP, which could be related to the lower ability of MnTBAP to scavenge peroxynitrite. The slightly better protection observed with the commercial MnTBAP sample (relative to the pure MnTBAP) could arise from its impurities, which, by scavenging O₂^·?, reduce consequently the overall peroxynitrite and secondary ROS/RNS levels. These observations have profound biological repercussions as they may suggest that the effect of MnTBAP observed in numerous studies may conceivably relate to peroxynitrite scavenging. Moreover, provided that pure MnTBAP is unable to dismute superoxide at any significant extent, but is able to partially scavenge peroxynitrite and carbonate radical, this compound may prove valuable in distinguishing ONOO^?/CO₃^·? from O₂^·? pathways.     

New prostaglandin derivative for glaucoma treatment

A hydrogen sulphide-releasing derivative of latanoprost acid (ACS 67) was synthesized and tested in vivo to evaluate its activity on reduction of intraocular pressure and tolerability. Glutathione (GSH) and cGMP content were also measured in the aqueous humour. The increased reduction of intraocular pressure, with a marked increase of GSH and cGMP and the related potential neuroprotective properties, make this compound interesting for the treatment of glaucoma. This is the first time that an application of a hydrogen sulphide-releasing molecule is reported for the treatment of ocular diseases.     

Tumor Suppressor Density-enhanced Phosphatase-1 (DEP-1) Inhibits the RAS Pathway by Direct Dephosphorylation of ERK1/2 Kinases

Density-enhanced phosphatase-1 (DEP-1) is a trans-membrane receptor protein-tyrosine phosphatase that plays a recognized prominent role as a tumor suppressor. However, the mechanistic details underlying its function are poorly understood because its primary physiological substrate(s) have not been firmly established. To shed light on the mechanisms underlying the anti-proliferative role of this phosphatase, we set out to identify new DEP-1 substrates by a novel approach based on screening of high density peptide arrays. The results of the array experiment were combined with a bioinformatics filter to identify eight potential DEP-1 targets among the proteins annotated in the MAPK pathway. In this study we show that one of these potential targets, the ERK1/2, is indeed a direct DEP-1 substrate in vivo. Pulldown and in vitro dephosphorylation assays confirmed our prediction and demonstrated an overall specificity of DEP-1 in targeting the phosphorylated tyrosine 204 of ERK1/2. After epidermal growth factor stimulation, the phosphorylation of the activation loop of ERK1/2 can be modulated by changing the concentration of DEP-1, without affecting the activity of the upstream kinase MEK. In addition, we show that DEP-1 contains a KIM-like motif to recruit ERK1/2 proteins by a docking mechanism mediated by the common docking domain in ERK1/2. ERK proteins that are mutated in the conserved docking domain become insensitive to DEP-1 de-phosphorylation. Overall this study provides novel insights into the anti-proliferative role of this phosphatase and proposes a new mechanism that may also be relevant for the regulation of density-dependent growth inhibition.DEP-1⁴ (also known as CD148, HPTPη, and PTPRJ) is a class III receptor protein-tyrosine phosphatase, characterized by eight fibronectin type III repeats within the extracellular domain, a trans-membrane region, and a single cytosolic catalytic domain (1, 2). DEP-1 is expressed in all human hematopoietic cell lineages and was shown to negatively regulate T cell activation. In addition, several epithelial cell types display DEP-1 on their cell membranes (3). Homozygous DEP-1 mutant mice die before embryonic day 11.5, displaying severe defects in vascular organization (4). Interestingly, DEP-1 expression levels were found to augment with increased cell density (2), suggesting a role for this tyrosine phosphatase in sensing cell-cell contacts and in density-dependent growth inhibition (5). Moreover, accumulating evidence supports a prominent role for DEP-1 as a tumor suppressor as it negatively regulates cell proliferation and is poorly expressed in many cancer cell lines (6–10). The observed anti-proliferative effect may be accounted for by the ability of DEP-1 to down-regulate growth factor signaling through the dephosphorylation of various receptor tyrosine kinases, such as PDGFR, VEGFR2, and MET (11–13), resulting in quenching of the downstream RAS-MAPK pathway. However, given the complex pleiotropic functions of DEP-1, it is also possible that additional regulatory circuits mediated by yet unknown DEP-1 substrates may play a functional role in contact inhibition and control of cell proliferation.A variety of in vivo and in vitro approaches has led us to propose a number of DEP-1 substrates as mediators of its function. These include PDGFR, p120 catenin (CTND1), hepatocyte growth factor receptor, SRC kinase, VEGFR2, phosphatidylinositol 3-kinase regulatory subunit α (P85A), and RET receptor kinase (5, 11–16).Here we report a novel, unbiased strategy based on the screening of high density phosphopeptide arrays for their ability to bind phosphatase trapping mutants. A large portion of the phosphoproteome could be explored by this approach, thus unveiling a long list of potential substrates. A selected list of potentially relevant substrates has been obtained by applying a bioinformatics context filter. In this study we report the detailed characterization of one of these substrates, and we propose that DEP-1 modulates the RAS pathway by directly dephosphorylating Tyr-204 of ERK1/2. In addition, we show that the efficient removal of the phosphate group from Tyr-204 requires the integrity of a docking site on the ERK1/2 proteins.     

Plant-based strategies aimed at expressing HIV antigens and neutralizing antibodies at high levels. Nef as a case study

The first evidence that plants represent a valid, safe and cost-effective alternative to traditional expression systems for large-scale production of antigens and antibodies was described more than 10 years ago. Since then, considerable improvements have been made to increase the yield of plant-produced proteins. These include the use of signal sequences to target proteins to different cellular compartments, plastid transformation to achieve high transgene dosage, codon usage optimization to boost gene expression, and protein fusions to improve recombinant protein stability and accumulation. Thus, several HIV/SIV antigens and neutralizing anti-HIV antibodies have recently been successfully expressed in plants by stable nuclear or plastid transformation, and by transient expression systems based on plant virus vectors or Agrobacterium-mediated infection. The current article gives an overview of plant expressed HIV antigens and antibodies and provides an account of the use of different strategies aimed at increasing the expression of the accessory multifunctional HIV-1 Nef protein in transgenic plants.     

Application of an integrated physical and functional screening approach to identify inhibitors of the Wnt pathway

Large‐scale proteomic approaches have been used to study signaling pathways. However, identification of biologically relevant hits from a single screen remains challenging due to limitations inherent in each individual approach. To overcome these limitations, we implemented an integrated, multi‐dimensional approach and used it to identify Wnt pathway modulators. The LUMIER protein–protein interaction mapping method was used in conjunction with two functional screens that examined the effect of overexpression and siRNA‐mediated gene knockdown on Wnt signaling. Meta‐analysis of the three data sets yielded a combined pathway score (CPS) for each tested component, a value reflecting the likelihood that an individual protein is a Wnt pathway regulator. We characterized the role of two proteins with high CPSs, Ube2m and Nkd1. We show that Ube2m interacts with and modulates β‐catenin stability, and that the antagonistic effect of Nkd1 on Wnt signaling requires interaction with Axin, itself a negative pathway regulator. Thus, integrated physical and functional mapping in mammalian cells can identify signaling components with high confidence and provides unanticipated insights into pathway regulators.     

HAC stability in murine cells is influenced by nuclear localization and chromatin organization

Background  

Human artificial chromosomes (HAC) are small functional extrachromosomal elements, which segregate correctly during each cell division. In human cells, they are mitotically stable, however when the HAC are transferred to murine cells they show an increased and variable rate of loss. In some cell lines the HAC are lost over a short period of time, while in others the HAC become stable without acquiring murine DNA.