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

In silico and in vivo combinatorial design of Octarellin VI, an artificial protein modeled on the (B/A)8 fold

One way to gain insight into the sequence-structure-function relationship in proteins is to perform de novo design of artificial proteins. The applications of such a study are varied. For example, in medicine and industry, it would give us the ability to precisely engineer proteins to perform a specific function under a wider range of conditions. Despite impressive successes in the de novo protein design, designing a folded protein of more than 100 amino acids remains a challenge. In our lab, four generations of Octarellins, de novo polypeptides of more than two hundred amino acids modelled on the (beta/alpha)8 barrel fold, have been built and structurally characterized using biophysical and spectroscopic methods. The last generation of Octarellins was designed following a hierarchical method combining the specificity of rational design and the power of computational design. The resulting artificial protein, named Octarellin VI, was expressed in E. coli and purified from inclusion bodies. The biophysical characterization showed a monomeric protein, with a secondary structure level similar to the computationally designed model and thermostability. However, the poor solubility in bacteria and low stability of the protein at long term make impossible determine its structure to criticize the model. To improve these negative features, we performed a directed evolution process over the Octarellin, following the improvement at solubility level in the bacteria, thanks to the fusion of Octarellin to the fluorescent folding reporter GFP. After 8 cycles of directed evolution by Error Prone PCR technique, we obtained a most soluble protein, with a 92% of sequence identity with the original protein. This soluble variant is under study to characterize its structural features. The combination between in silico design and directed evolution process emerges as a powerful tool for protein engineering, showing be complementaries techniques and the information obtained by the whole process of design and posterior comparison between 3D structure of Octarellin with the computational model will allow to improve the algorithms for protein design

Development of mesoporous sol-gel film for anti-fingerprint applications on glass

Mesoporous sol-gel film can be used as smart coatings due their capability to embed active species in applications such as drug delivery, catalysis, sensors, active corrosion protection layer, ... These materials were synthesized by specific sol-gel route including the use of surfactant molecules acting as templating agent through the evaporation induced self-assembly (EISA) process. Controlled mesoporosity of the film is obtained by the thermal removal of the templating agent. After this treatment, the mesopores can be employed to host active molecules. In this work, the mesostructure of the sol-gel layer is used as reservoir for active enzymatic molecules providing anti-fingerprint coating. The mesopore size and their configuration were designed to assure a good impregnation by lipases. The coatings were applied on glass. The influence of the deposition process and the composition of the sol-gel on the structure of the mesoporous film were characterized. The morphologies of films were characterized by scanning electron microscopy (SEM) and water adsorption/desorption isotherms recorded with a quartz crystal microbalance (QCM). The stability of the sol-gel solution was checked by rheological measurements. The efficiency of the combination of mesoporous film and the enzymatic solution was evaluated in terms of anti-fingerprint properties using an artificial sebum

Le biomimétisme moléculaire : du concept universitaire à l'application industrielle

Le biomimétisme moléculaire est une science transdisciplinaire qui permet aux différents points de vue de se rencontrer et de faire émerger, grâce à leur complémentarité, des projets de recherches variés et de multiples applications potentielles. A notre tribune ce midi, pour nous en parler, nous accueillions Cécile Van de Weerdt (Group leader au GIGA, laboratoire de Biomimétique moléculaire) et Carine Bebrone (Chef de projet chez Symbiose Biomaterials). C'est avec le projet Biocoat que la recherche a démarré au sein du GIGA-R. L'objectif était d'apporter de nouvelles fonctionnalités sur les matériaux, dans une logique de développement durable et avec des procédés verts. Un projet qui rassemblait une équipe multidisciplinaire (biologie, chimie et matériaux) et dont le succès a mené à la création de Symbiose Biomaterials afin d'accélérer la mise sur le marché des résultats de recherche. Lors de cette conférence, nous nous sommes plus particulièrement attardés sur la technologie GEPI qui repose sur des protéines capables de reconnaître des matières organiques ou inorganiques dans la nature. Une technologie qui fonctionne notamment grâce aux outils du génie génétique. Cécile Van de Weerdt nous a présenté ses caractéristiques ainsi que la technique dite du "phage display" permettant la sélection des peptides d'intérêt. Carine Bebrone nous a quant a elle détaillé le projet Adekit, né d'une rencontre "brainstorming" avec le CSTC, d'où est ressorti le souhait de développer un kit de détection in situ de l'amiante dans les matériaux. Un projet qui est aujourd'hui à la fin de sa phase de développement laboratoire et qui a déjà entamé ses phases de prototypage et de marketing. Un projet ambitieux qui offre diverses applications potentielles et qui dispose de nombreux facteurs-clés pour un succès qu'on leur souhaite... Au terme de la séance, Symbiose Biomaterials a sondé les participants afin de connaître leurs préférences quant aux kits en développement

Role of the cholinergic pathway in mediating platelet-Activating factor-induced pulmonary dysfunctions in unsedated calves

The purpose of this study was to investigate the possible role of the cholinergic pathway in mediating platelet-activating factor (PAF)-induced pulmonary dysfunctions in unsedated calves. In a placebo group, PAF infusion challenge induced significant dysfunctions in the pattern of breathing [a significant increase in respiratory rate (RR) and a significant decrease in tidal volume (VT)], the mechanics of breathing [a significant increase in total lung resistance (RL) and a significant decrease in dynamic lung compliance (CLdyn)] and gas exchange, whereas in atropine pre-treated calves, PAF infusion challenge induced a significant increase in RR, VT and HR and a significant decrease in CLdyn. The RL increase was prevented by atropine pre-treatment. On the basis of our findings, we suggest that, in cattle, the PAF-induced pattern of breathing dysfunctions and the diffuse bronchoconstriction and microvascular leakage of small airways are not mediated through the cholinergic pathway. By contrast, our data suggest that PAF-induced bronchoconstriction of upper airways is at least partly mediated through muscarinic receptors

Multifunctional coatings

publication date: 2009-12-09; filing date: 2008-06-06New polyelectrolyte copolymer, composite material, multilayer film and substrate carrying such polyelectrolyte copolymer, composite and multilayer film wherein the polyelectrolyte copolymer comprises a) a first type of identical or different units (A) each comprising one or more dihydroxyphenyl groups such that sidechains are present along the backbone of the polyelectrolyte copolymer which contain at least one dihydroxyphenyl group each; and (b1) a second type of identical or different units (B1) each comprising a cationic moiety, or (b2) a second type of identical or different units (B2) each comprising an anionic moiety

A novel protocol for the design of artificial (β/α)8-barrel proteins

Designing de novo proteins of more than 100 amino acids is still challenging. The creation of artificial (β/α)8-barrel proteins had only one successful example in literature, thank to use of internal spatial symmetry. Here we present a protocol to design de novo (β/α)8-barrel proteins without symmetry restriction. First, the backbone was created in 4 steps: (I) Rosetta ParametricDesign produced an highly symmetric polyalanine scaffold with no loops; (II) Rosetta Fixed-Backbone Design used the previous output to substitute the alanines in all the position; (III) Loops were constructed with Modeller joining the terminus of the secondary structure elements and (IV) RosettaRelax performed relaxation, creating around 4000 different models. 28 backbone models were selected for the next steps of sequence design. To design the final proteins for experimental validation, 10 cycles of Rosetta Design and Relax were performed. In the first cycle only apolar amino acids were allowed in hydrophobic regions; in the next 6 cycles, amino acids were allowed based on the definition of 3 regions: core, boundaries and surface. All the amino acids were allowed in each position in the last 3 cycles. More than 10000 different sequences were created and analyzed in term of amino acid composition, sequence similarity with natural protein, secondary structure prediction, and molecular dynamics simulations. The 30 best candidate sequences have been selected for experimental verification.Octarellin VI

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

Inorganic Binding Peptides and quality control methods using them

publication date: 2010-01-07; filing date: 2009-07-06BIOCOA