Selective inhibition of the human tie-1 promoter with triplex-forming oligonucleotides targeted to ets binding sites

The Tie receptors (Tie-1 and Tie-2/Tek) are essential for angiogenesis and vascular remodeling/integrity. Tie receptors are up-regulated in tumor-associated endothelium, and their inhibition disrupts angiogenesis and can prevent tumor growth as a consequence. To investigate the potential of anti-gene approaches to inhibit tie gene expression for anti-angiogenic therapy, we have examined triple-helical (triplex) DNA formation at 2 tandem Ets transcription factor binding motifs (designated E-1 and E-2) in the human tie-1 promoter. Various tie-1 promoter deletion/mutation luciferase reporter constructs were generated and transfected into endothelial cells to examine the relative activities of E-1 and E-2. The binding of antiparallel and parallel (control) purine motif oligonucleotides (21-22 bp) targeted to E-1 and E-2 was assessed by plasmid DNA fragment binding and electrophoretic mobility shift assays. Triplex-forming oligonucleotides were incubated with tie-1 reporter constructs and transfected into endothelial cells to determine their activity. The Ets binding motifs in the E-1 sequence were essential for human tie-1 promoter activity in endothelial cells, whereas the deletion of E-2 had no effect. Antiparallel purine motif oligonucleotides targeted at E-1 or E-2 selectively formed strong triplex DNA (K(d) approximately 10(-7) M) at 37 degrees C. Transfection of tie-1 reporter constructs with triplex DNA at E-1, but not E-2, specifically inhibited tie-1 promoter activity by up to 75% compared with control oligonucleotides in endothelial cells. As similar multiple Ets binding sites are important for the regulation of several endothelial-restricted genes, this approach may have broad therapeutic potential for cancer and other pathologies involving endothelial proliferation/dysfunction

Magnetic and electrokinetic manipulations on a microchip device for bead-based immunosensing applications

9 páginas, 5 figuras.The combination of electrophoretic and magnetic manipulations with electrochemical detection for a versatile microfluidic and bead-based biosensing application is demonstrated. Amperometric detection is performed in an off-channel setup by means of a voltammetric cell built at the microchannel outlet and using a gold working electrode. Superparamagnetic particles are introduced and handled inside the channel by means of an external permanent magnet in combination with the electrogenerated flow which allows reproducible loading. The specific detection of phenol as electroactive alkaline phosphatase product is used in this study as proof of concept for a sensitive protein quantification. Characterizations and optimization of different parameters have been carried out in order to achieve the best detection signal. The applicability of the device has been finally demonstrated by the detection of rabbit IgG as model protein after an immunoassay performed on magnetic particles as immobilization platform. A comparison between the electrochemical detection using the developed device and the optical standard detection revealed similar performances with, however, extremely lower amount of reagent used and shorter analysis time. The developed electrophoretic- and magnetic-based chip may open the way to several other biosensing applications with interest not only for other proteins but also for DNA analysis, cell counting, and environmental control.The authors acknowledge funding from the ‘‘Ministerio de Ciencia e Innovación’’ (Madrid, Spain) for Projects MAT 2008-03079/NAN and CSD2006-00012 ‘‘NANOBIOMED’’ (Consolider-Ingenio 2010).Peer reviewe

On the influence of gas inlet configuration with respect to homogeneity in a horizontal single wafer reactor

In this paper two different gas inlet modifications for a horizontal single-wafer MOVPE reactor were studied: the conventional one ensured gas mixing of groups III and V precursors by forcibly merging both gases in a gas mixing unit, the newer one allowed mixing of groups III and V precursors solely by diffusion after the separation plate. AlAs/ GaAs Bragg reflector structures were deposited in nitrogen atmosphere for both gas inlet types and the uniformity determined by X-ray diffraction. Three-dimensional detailed numerical modeling was employed to explain the results. They establish that the observed strong non-uniformity of growth for the new inlet as well as the unusual concave instead of the common convex growth profile found is due to the incomplete mixing of precursor gases in a highly dense gas phase. By adjusting the groups III/V gas stream ratio an effect could be taken on the diffusion processes. Furthermore the total flow in the reactor was optimized for the new gas inlet. By taking advantage of changed diffusion properties in nitrogen atmosphere, however, perfect uniformity with an absolute deviation smaller than 1% was obtained. The modeling results are presented and agree very well with the experimental data. (C) 2001 Elsevier Science B.V. All rights reserved