Proof of concept for microarray-based detection of DNA-binding oncogenes in cell extracts

The function of DNA-binding proteins is controlled not just by their abundance, but mainly at the level of their activity in terms of their interactions with DNA and protein targets. Moreover, the affinity of such transcription factors to their target sequences is often controlled by co-factors and/or modifications that are not easily assessed from biological samples. Here, we describe a scalable method for monitoring protein-DNA interactions on a microarray surface. This approach was designed to determine the DNA-binding activity of proteins in crude cell extracts, complementing conventional expression profiling arrays. Enzymatic labeling of DNA enables direct normalization of the protein binding to the microarray, allowing the estimation of relative binding affinities. Using DNA sequences covering a range of affinities, we show that the new microarray-based method yields binding strength estimates similar to low-throughput gel mobility-shift assays. The microarray is also of high sensitivity, as it allows the detection of a rare DNA-binding protein from breast cancer cells, the human tumor suppressor AP-2. This approach thus mediates precise and robust assessment of the activity of DNA-binding proteins and takes present DNA-binding assays to a high throughput leve

Proof of concept for microarray-based detection of DNA-binding oncogenes in cell extracts

The function of DNA-binding proteins is controlled not just by their abundance, but mainly at the level of their activity in terms of their interactions with DNA and protein targets. Moreover, the affinity of such transcription factors to their target sequences is often controlled by co-factors and/or modifications that are not easily assessed from biological samples. Here, we describe a scalable method for monitoring protein–DNA interactions on a microarray surface. This approach was designed to determine the DNA-binding activity of proteins in crude cell extracts, complementing conventional expression profiling arrays. Enzymatic labeling of DNA enables direct normalization of the protein binding to the microarray, allowing the estimation of relative binding affinities. Using DNA sequences covering a range of affinities, we show that the new microarray-based method yields binding strength estimates similar to low-throughput gel mobility-shift assays. The microarray is also of high sensitivity, as it allows the detection of a rare DNA-binding protein from breast cancer cells, the human tumor suppressor AP-2. This approach thus mediates precise and robust assessment of the activity of DNA-binding proteins and takes present DNA-binding assays to a high throughput level

Nav1.4 Deregulation in Dystrophic Skeletal Muscle Leads to Na+ Overload and Enhanced Cell Death

Duchenne muscular dystrophy (DMD) is a hereditary degenerative disease manifested by the absence of dystrophin, a structural, cytoskeletal protein, leading to muscle degeneration and early death through respiratory and cardiac muscle failure. Whereas the rise of cytosolic Ca2+ concentrations in muscles of mdx mouse, an animal model of DMD, has been extensively documented, little is known about the mechanisms causing alterations in Na+ concentrations. Here we show that the skeletal muscle isoform of the voltage-gated sodium channel, Nav1.4, which represents over 90% of voltage-gated sodium channels in muscle, plays an important role in development of abnormally high Na+ concentrations found in muscle from mdx mice. The absence of dystrophin modifies the expression level and gating properties of Nav1.4, leading to an increased Na+ concentration under the sarcolemma. Moreover, the distribution of Nav1.4 is altered in mdx muscle while maintaining the colocalization with one of the dystrophin-associated proteins, syntrophin α-1, thus suggesting that syntrophin is an important linker between dystrophin and Nav1.4. Additionally, we show that these modifications of Nav1.4 gating properties and increased Na+ concentrations are strongly correlated with increased cell death in mdx fibers and that both cell death and Na+ overload can be reversed by 3 nM tetrodotoxin, a specific Nav1.4 blocker

Preferential Binding of Bacteriophage Mu Repressor to Supercoiled Mu DNA

It was shown, using a relatively simple assay, that Mu repressor, cI, binds specifically to a region which spans the leftmost HindIII cleavage site on the phage genome. This extends the observations of Kwoh and Zipser [Nature (London) 277, 489–491 (1979)], who were able to define a binding region to the left of this site. These results provide support for the idea that the eight blocks of repeated DNA sequences, which also span the HindIII cleavage site, are involved in repressor binding. These results also indicate that cI repressor has a marked preference for supercoiled DNA

Cloning and expression of the phage Mu <i>A</i> gene

We have cloned the phage Mu A gene, with and without the gene ner, under the control of the PL promoter of phage λ in a multicopy plasmid vector. We demonstrate that plasmid-carrying cells are able to support growth of superinfecting Mu A am phages in a temperature-dependent fashion in a host strain carrying a defective λ prophage which specifies the cI857-coded λ repressor. In addition, we show that the presence of the ner gene reduces the efficiency of plating of the superinfecting phage. Analysis of proteins specified by the cloned Mu fragments indicates that two proteins, 70 and 33 kDal, are synthesized. The level of synthesis, compared to that of the vector-encoded ß-lactamase, was found to increase with temperature. This indicates that their transcription is driven by the PL promoter. The MPr of the 70-kDal protein is identical to that previously observed for pA

Na1.4 Deregulation in Dystrophic Skeletal Muscle Leads to Na Overload and Enhanced Cell Death-1

F Na1.4 channels from muscle extracts of control C57BL/6J and mice. Each slot represents the extract from one muscle of one 12–16-wk-old mouse. (B) Quantification of Na1.4 expression levels (four mice/group). Protein concentrations were measured in triplicate to guarantee equal loading, and Na1.4 density is expressed in arbitrary units using SERCA1 as an internal control (**, P < 0.01; Student's unpaired test). Values are reported as means ± SEM.<p><b>Copyright information:</b></p><p>Taken from "Na1.4 Deregulation in Dystrophic Skeletal Muscle Leads to Na Overload and Enhanced Cell Death"</p><p></p><p>The Journal of General Physiology 2008;132(2):199-208.</p><p>Published online Jan 2008</p><p>PMCID:PMC2483333.</p><p></p

Aerosols released during the laser cutting of a Fukushima Daiichi debris simulant

International audienceOne of the important challenges for the decommissioning of the damaged reactors of the Fukushima Daiichi Nuclear Power Plant is the safe retrieval of the fuel debris or corium. It is especially primordial to investigate the cutting conditions for air configuration and for underwater configuration at different water levels. Concerning the cutting techniques, the laser technique is well adapted to the cutting of expected material such as corium that has an irregular shape and heterogeneous composition. A French consortium (ONET Technologies, CEA and IRSN) is being subsidized by the Japanese government to implement R&D related to the laser cutting of Fukushima Daiichi fuel debris and related to dust collection technology. Debris simulant have been manufactured in the PLINIUS platform to represent Molten Core Concrete Interaction as estimated from Fukushima Daiichi calculations. In this simulant, uranium is replaced by hafnium and the major fission products have been replaced by their natural isotopes. During laser cutting experiments in the DELIA facility, aerosols have been collected thanks to filters and impactors. The collected aerosols have been analyzed. Both chemical analysis (dissolution + ICP MS and ICP AES) and microscopic analyses (SEM EDS) will be presented and discussed. These data provide insights on the expected dust releases during cutting and can be converted to provide radioactivity estimates. They have also been successfully compared to thermodynamic calculations with the NUCLEA database

Na1.4 Deregulation in Dystrophic Skeletal Muscle Leads to Na Overload and Enhanced Cell Death-4

Zation. Isolated fibers were immunolabeled with mouse antibodies to Na1.4. A fluorescent signal was observed primarily at the sarcolemma. The luminescence distribution plot (inset) shows nonuniform density corresponding to apparent clusters of Na channels. (B) Distribution of Na1.4 expression levels in cell-attached patches (model parameter in fits with the Hodgkin-Huxley model) displays a similar non-Gaussian shape corresponding to that observed in confocal microscopy. The continuous curve represents the distribution density of amplitudes calculated using a Gaussian kernel density estimate with the bandwidth of 40 pA from the data points plotted below the histogram as thin vertical lines.<p><b>Copyright information:</b></p><p>Taken from "Na1.4 Deregulation in Dystrophic Skeletal Muscle Leads to Na Overload and Enhanced Cell Death"</p><p></p><p>The Journal of General Physiology 2008;132(2):199-208.</p><p>Published online Jan 2008</p><p>PMCID:PMC2483333.</p><p></p