Cécile Van de Weerdt

Antibacterial Polyelectrolyte Micelles for Coating Stainless Steel

In this study, we report on the original synthesis and characterization of novel antimicrobial coatings for stainless steel by alternating the deposition of aqueous solutions of positively charged polyelectrolyte micelles doped with silver-based nanoparticles with a polyanion. The micelles are formed by electrostatic interaction between two oppositely charged polymers: a polycation bearing 3,4-dihydroxyphenylalanine units (DOPA, a major component of natural adhesives) and a polyanion (poly(styrene sulfonate), PSS) without using any block copolymer. DOPA units are exploited for their well-known ability to anchor to stainless steel and to form and stabilize biocidal silver nanoparticles (Ag(0)). The chlorine counteranion of the polycation forms and stabilizes biocidal silver chloride nanoparticles (AgCl). We demonstrate that two layers of micelles (alternated by PSS) doped with silver particles are enough to impart to the surface strong antibacterial activity against gram-negative E. coli. Moreover, micelles that are reservoirs of biocidal Ag(+) can be easily reactivated after depletion. This novel water-based approach is convenient, simple, and attractive for industrial applications.

Thyroid Hormone Inhibits the Human Prolactin Gene Promoter by Interfering with Activating Protein-1 and Estrogen Stimulations

Transcription of the human PRL (hPRL) gene in the pituitary is subject to tissue-specific and multihormonal regulation involving two main regulatory regions, a proximal promoter and a distal enhancer. In this report we show that thyroid hormone inhibits the expression of the hPRL gene in rat pituitary cells. Transient expression experiments show that thyroid hormone regulation involves a strong inhibitory element, located in the proximal (-164/-35) promoter, which is modulated by a more distal stimulatory response control region. Gel retardation experiments reveal that the thyroid hormone receptor does not bind to the proximal negative element. We show the existence of an activating protein-1 (AP-1) response element located at positions -61 to -54 of the proximal promoter, conferring AP-1 stimulation to the hPRL promoter. This AP-1 induction is abolished when hormone-bound thyroid hormone receptor is present, indicating that there is an interference between the thyroid hormone receptor and AP-1 regulatory pathways. Furthermore, using the complete hPRL upstream region, we show that estrogen induction is abolished by simultaneous thyroid hormone treatment.

Detection of illegal growth promoters in biological samples using receptor binding assays

Abstract In the European Union (EU), the use of growth-promoting substances in meat production is banned. The control of growth promoters, especially steroid hormones, is presently based on expensive and time-consuming chromatographic methods of analysis or, sometimes, for screening purposes, on radio- or enzyme-immunoassays, all of which are often too specific to allow effective multi-analyte control. In order to develop rapid and inexpensive multi-analyte detection tests, we proposed the use of hormonal receptors as detection tools. The system described here (radio-receptor assays) is based on a direct binding assay of steroid hormones to their respective receptors. Human receptors to estrogens (hERα), androgens (hAR), progestagens (hPR) and glucocorticoids (hGR) have been produced by genetic engineering in bacteria or in eucaryotic cells. Binding analyses revealed that the obtained receptor proteins retained a high affinity for their corresponding native ligand. In addition, competition studies confirmed that each of the four receptors displays a specificity profile for a series of analogs in agreement with the literature. Finally, the stability of these recombinant receptors is sufficient to allow their use in test kits.

Crystallization of ornithine acetyltransferase from yeast by counter-diffusion and preliminary X-ray study

A study is presented on the crystallization of ornithine acetyltransferase from yeast, which catalyzes the fifth step in microbial arginine synthesis. The use of the counter-diffusion technique removes the disorder present in one dimension in crystals grown by either the batch or hanging-drop techniques. This makes the difference between useless crystals and crystals that allow successful determination of the structure of the protein. The crystals belong to space group P4, with unit-cell parameters a = b = 66.98, c = 427.09 A, and a data set was collected to 2.76 A.

Clay and DOPA Containing Polyelectrolyte Multilayer Film for Imparting Anticorrosion Properties to Galvanized Steel

A facile and green approach is developed to impart remarkable protection against corrosion to galvanized steel. A protecting multilayer film is formed by alternating the deposition of a polycation bearing catechol groups, used as corrosion inhibitors, with clay that induces barrier properties. This coating does not affect the esthetical aspect of the surface and does not release any toxic molecules in the environment.

Robust bio-inspired antibacterial surfaces based on the covalent binding of peptides on functional atmospheric plasma thin films

Here, we describe a robust process aiming at conferring antibacterial properties on stainless steel through the covalent grafting of nisin, a natural antimicrobial peptide, onto a functional plasma thin film deposited by an atmospheric pressure dielectric barrier discharge process. The three different steps of the procedure, namely the deposition of a carboxyl rich thin layer, the surface activation by using a zero-length crosslinking agent and the nisin immobilisation, are reported and thoroughly characterised. A correlation between the carboxylic group surface concentration and the surface roughness onto the antibacterial properties of the layers is evidenced. Finally, IR analyses appear as a powerful analytical tool allowing us to validate the different chemical surface modifications, to confirm the relevance of the activation step to achieve a stable and homogenous peptide grafting over all the surfaces, as well as to investigate the secondary structure of immobilized peptides.

Inorganic-binding peptides as tools for surface quality control.

This paper highlights an innovative application of inorganic-binding peptides as quality control tools for detecting defects on inorganic surfaces of any shape. The approach involves attaching a fluorescent label to an inorganic-binding peptide and exploiting the peptides high binding specificity to detect, by simple fluorescence microscopy, chemical composition defects of microm size and crystallographic state defects. Proof of concept was demonstrated by monitoring binding of a previously isolated ZnO-binding peptide to galvanized steel substrates. The approach was further validated for TiO(2) coatings and stainless steel, with two new, specific inorganic-binding peptides isolated by phage display.

EGF stimulates Pit-1 independent transcription of the human prolactin pituitary promoter in human breast cancer SK-BR-3 cells through its proximal AP-1 response element

Normal and neoplastic human mammary gland cells are targets for the proliferative action of prolactin. These cells also synthesize prolactin, thereby inducing an autocrine/paracrine proliferative loop. We present the first extensive analysis of the transcriptional regulation of the human prolactin gene (hPRL) in human mammary tumor cells, SK-BR-3. We show that the pituitary promoter is functional in these cells in the absence of the pituitary-specific factor Pit-1. Expression of exogenous Pit-1 or epidermal growth factor (EGF) treatment stimulates the transfected hPRL pituitary promoter and the endogenous hPRL expression. EGF stimulation is mediated by increased synthesis of c-fos and c-jun, resulting in AP-1 binding to the proximal hPRL pituitary promoter. This regulation involves the EGF receptor, possibly ErbB2 that is highly expressed in SK-BR-3 cells, and a PI3K/JNK pathway. The stimulation of hPRL gene transcription by EGF in mammary cells may include hPRL in a complex regulatory network controlling growth of human mammary cells.

Far Upstream Sequences Regulate the Human Prolactin Promoter Transcription

The human prolactin gene is mainly expressed in pituitary lactotrope cells, but transcription from an alternative, far upstream promoter was detected in lymphoid, placental and mammary cells. We describe the transcriptional activity in rat pituitary cells of the complete region separating the two promoters, using transient transfection experiments. A far upstream activating region was only functional in combination with the prolactin promoter. DNaseI protection experiments revealed, in addition to binding sites for the pituitary-specific factor Pit-1, sites (e.g. SD1) for several ubiquitous factors and one lymphoid-specific factor (SD4). A single copy of the ubiquitous site SD1 or the lymphoid-specific site SD4 was unable to activate transcription of a heterologous promoter in pituitary cells. However, SD1 activated transcription in nonpituitary cells and SD4 was functional specifically in lymphoid cells. Five copies of a distal site (D8) activated transcription in each cell type tested. Gel retardation experiments show that this site binds the specific factor C/EBP in liver and a distinct factor in other cell types. Our results suggest that different elements within this large region direct specific expression from each promoter via a complex interplay between cell-specific and ubiquitous transcription factors.

Octarellin VI: using rosetta to design a putative artificial (β/α)8 protein.

The computational protein design protocol Rosetta has been applied successfully to a wide variety of protein engineering problems. Here the aim was to test its ability to design de novo a protein adopting the TIM-barrel fold, whose formation requires about twice as many residues as in the largest proteins successfully designed de novo to date. The designed protein, Octarellin VI, contains 216 residues. Its amino acid composition is similar to that of natural TIM-barrel proteins. When produced and purified, it showed a far-UV circular dichroism spectrum characteristic of folded proteins, with α-helical and β-sheet secondary structure. Its stable tertiary structure was confirmed by both tryptophan fluorescence and circular dichroism in the near UV. It proved heat stable up to 70°C. Dynamic light scattering experiments revealed a unique population of particles averaging 4 nm in diameter, in good agreement with our model. Although these data suggest the successful creation of an artificial α/β protein of more than 200 amino acids, Octarellin VI shows an apparent noncooperative chemical unfolding and low solubility.