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Microglia are the resident macrophage population of the central nervous system (CNS). Adequate microglia function is crucial for the homeostasis of the CNS in health and disease, as they represent the first line of defence against pathogens, contributing to immune responses, but are also involved in tissue repair and remodeling. It is therefore crucial to better understand microglia origin and homeostasis. Much controversy remains regarding the nature of microglial progenitors, as the exact contribution and persistence of embryonic and post-natal hematopoietic progenitors to the adult microglial pool in the steady state remained unclear. In this study, we show that post-natal hematopoietic progenitors do not significantly contribute to microglia homeostasis in the adult brain in mice. In vivo lineage tracing studies established that adult microglia derives from primitive hematopoietic progenitors that arise before embryonic day 8. These results identify microglia as an ontogenically distinct population in the mononuclear phagocyte system and have implications for the use of embryonically-derived microglial progenitors for the treatment of various brain disorders.

Proteomic approaches on lipid bodies have led to the identification of proteins associated with this compartment, showing that, rather than the inert fat depot, lipid droplets appear as complex dynamic organelles with roles in metabolism control and cell signaling. We focused our investigations on caleosin [Arabidopsis thaliana caleosin 1 (AtClo1)], a minor protein of the Arabidopsis thaliana seed lipid body. AtClo1 shares an original triblock structure, which confers to the protein the capacity to insert at the lipid body surface. In addition, AtClo1 possesses a calcium-binding domain. The study of plants deficient in caleosin revealed its involvement in storage lipid degradation during seed germination. Using Saccharomyces cerevisiae as a heterologous expression system, we investigated the potential role of AtClo1 in lipid body biogenesis and filling. The green fluorescent protein-tagged protein was correctly targeted to lipid bodies. We observed an increase in the number and size of lipid bodies. Moreover, transformed yeasts accumulated more fatty acids (+46.6%). We confirmed that this excess of fatty acids was due to overaccumulation of lipid body neutral lipids, triacylglycerols and steryl esters. We showed that the original intrinsic properties of AtClo1 protein were sufficient to generate a functional lipid body membrane and to promote overaccumulation of storage lipids in yeast oil bodies.

The attention paid to endocriniens modulators for purpose micropolluants (endocrine disrupters) has been increasingly studied these last years particularly on animals. The results of this study raised big concerns from Doctors and Biologists on the eventual risks human health can face. Indeed, endocrine systems of the body play an essential and pervasive role in both the short- and long-term regulation of metabolic processes. Nutritional, behavioural, and reproductive processes are intricately regulated by endocrine systems, as are growth (including bone growth/remodelling), gut, cardiovascular, and kidney function and responses to all forms of stress. Disorders of any of the endocrine system, involving both over- and under-active hormone secretion, result inevitably in disease, the effects of which may extend to many different organs and functions and are often debilitating or life-threatening. Viewed from this general perspective, the threat posed from environmental chemicals with endocrine activity (either agonist or antagonistic) is potentially serious. However, the fact that humans and wildlife are exposed to such chemicals does not necessarily mean that clinically manifest disturbance of the relevant endocrine system will result, because much depends on the level and duration of exposure and on the timing of exposure. Indeed, a large numbers of environmental estrogens are suspected of altering human health as well as the marine ecosystem balance. The objective of this review is to study the different molecular mechanisms of these xenoestrogenes micropolluants, in order to emphasize their potential risk and to present some of the different experimental methods for their detection.

The biotechnology of recombinant protein production is now entering its most advanced stage, and the growth of industrial protein pharmaceuticals provides solid proof of this evolution. However, the systematic conversion of genetic information into a biologically active protein is constantly confronted by the fundamental problem of protein folding in cells, and many recombinant proteins are not produced in their native state. Instead, they aggregate into a biologically inactive state. Although this aggregation reaction has some practical advantages, in vitro renaturation of recombinant proteins, after solubilization of cellular aggregates, is still an empiric and random process. Thus, it is better to control cellular expression conditions to minimize this problem inside the cells. The most attractive approach is certainly the development of high throughput genetic screens to monitor efficient protein folding.

Our laboratory has been using various bioluminescent imaging systems for more than 20 years to visualize and quantify bioluminescent and chemiluminescent reactions. This equipment allowed us to establish numerous cell lines expressing bioluminescent reporter genes to study the mechanism of action of nuclear receptors. Cells expressing the luciferase gene under the control of a constitutive promoter were used to follow in vivo proliferation of cancer cells. Intensities of in vitro and in vivo bioluminescent signals were compared and the conditions of bioluminescent reaction measurements were determined. These bioluminescent models are new tools for evaluating cancer treatment efficiencies and the role of hormone receptors in invasion. Cells expressing the luciferase gene under the control of hormones are used as in vivo biosensors for studying analog bioavailabilities and in vivo response kinetics. They are complementary models to in vitro models that have been developed in our laboratory for several years. In the future, targeting reporter gene (luciferase and GFP) expression to specific tissues should allow the detailed localisation of the action of nuclear receptor ligands.

Because the GnRH receptor plays a paramount role within the reproductive axis, the understanding of the molecular apparatus that governs the tissue-specific expression and regulation of this gene must lead to a better knowledge of the physiology and the physiopathology of the gonadotrope function. To elucidate these mechanisms, we have used two complementary in vivo and in vitro approaches. Firstly, we have isolated the pituitary promoter of the rat GnRH receptor gene and investigated its activity using transient transfection into two gonadotrope-derived cell lines, the alphaT3-1 and the LbetaT2 cell lines. We have thus defined a primary set of transcription factors involved in the tissue-specific expression of the GnRH receptor gene. These include the steroidogenic factor-1 (SF-1) which plays a decisive role while functionally interacting with proteins related to the GATA and LIM homeodomain families of transcription factors. In addition, we highlighted the critical implication of SF-1 and its functional interaction with a CREB-related factor in the stimulatory action of PACAP (Pituitary Adenylate Cyclase Activating Polypeptide) on promoter activity. These results have led us to analyze the activity of this promoter by transgenesis in the mouse using human placental alkaline phosphatase as a reporter gene. In agreement with the in vitro data, the pituitary promoter was found to confer gonadotrope-specific activity in the pituitary. It was also able to direct transgene expression in several areas of the central nervous system known to express the endogenous GnRH receptor, in particular in the hippocampo-septal complex. Some of these tissue do not express SF-1, suggesting that, in vivo, its role would not be as decisive as suggested by the in vitro experiments. Surprisingly, during pituitary ontogenesis, the transgene is expressed as early as E 13.5 whereas SF-1 is not yet present in the pituitary. Thus, in vivo, SF-1 would not be necessary for the activation of the GnRH receptor gene during the early developmental stages in the pituitary. These results are consistent with data obtained following general or pituitary-specific knockout of the gene encoding SF-1, suggesting that the GnRH receptor is expressed despite the absence of this factor. Identifying the factors responsible for the activation of the GnRH receptor gene at these early developmental stages should make it possible to refine the role of SF-1, not only in gene regulation but more generally, in the physiology and the physiopathology of the gonadotrope function.

18F-FDG is currently the only fluorinated tracer used in routine clinical positron emission tomography (PET). Fluorine 18 is considered as the ideal radioisotope for PET, thanks to a low positron energy, which not only limits the dose rate to the patients but also provides high-resolution images. Furthermore, the 110 min. physical half-life allows for high-yield radiosynthesis, transport from the production site to the imaging site, and imaging protocols that could span hours, which permits dynamic studies and assessing metabolic processes that may be fairly slow. Recently, synthesis of fluorinated tracers from prosthetic group precursors, which allows easier radiolabeling of biomolecules, has given a boost to the development of numerous fluorinated tracers. Given the wide availability of fluorine 18, such tracers may well develop into important routine tracers. This article is a review of the literature concerning fluorinated analogs of nucleosides and fluorinated radiotracers of gene expression recently developed and under investigation.

Osteopontin (OPN), an RGD containing extracellular matrix protein, is associated with arterial smooth muscle cells (SMC) activation in vitro and in vivo. OPN has been shown to be overexpressed in vascular injury. Its expression can be induced by many factors including growth factors, cytokines, hormones and extracellular nucleotides. We are interested in understanding mechanisms regulating the OPN mRNA steady state level in SMC. We compared the effect of two G-protein coupled receptors agonists (UTP and angiotensin II [AII]) and one tyrosin kinase receptor agonist (PDGF). We explored the effect of these three agonists both on OPN transcription using gene reporter assay and on OPN mRNA stabilisation using actinomycin D. We showed that UTP 100 microM. AII 10 microM and PDGF 50 ng/microL induced OPN transcription. Whereas UTP and AII induced a 366 +/- 81% and 338 +/- 115% activation of transcription respectively, PDGF demonstrated a lower efficiency (195 +/- 59%) inducing the transcription. Moreover, we demonstrated that UTP and AII but not PDGF were able to stabilize OPN mRNA. This effect seems to be specific to G-protein coupled receptor agonists since previous studies demonstrated that intracellular receptor agonists did not stabilise OPN mRNA. Thus, the lower increase of OPN mRNA level in response to PDGF stimulation compared to AII or UTP could be explain by both, the lower activation of the OPN promoter and the effect of UTP and AII on OPN mRNA stabilisation.

Vasoactive peptides are implied in the development of renal sclerosis as evidenced by the efficiency of their antagonists in preventing glomerulosclerosis of experimental and human nephropathies. Genetically engineered models provide a new approach to investigate the mechanisms of the renal profibrotic actions of angiotensin II and endothelin. Overexpression of the human angiotensinogen and renin genes in rats induces renal sclerosis independently of changes in systemic hemodynamics. The same results are observed when the endothelin-1 gene is overexpressed in mice. Transgenic mice harboring the luciferase gene under the control of the collagen I-alpha 2 chain promoter (procol alpha 2[1]) and made hypertensive by induction of nitric oxide (NO) deficiency were used to study the renal profibrotic actions of vasoactive peptides. In this strain of mice, luciferase activity is an early index of renal fibrosis. Luciferase activity was increased in preglomerular arterioles and glomeruli when mice were deficient in NO. The pharmacological blockade of angiotensin II and endothelin prevented the development of renal sclerosis without modifying blood pressure. Moreover, when the endothelin receptor antagonist was administered after the development of renal fibrosis, preformed glomerulosclerosis partially regressed. Acute administration of vasoactive peptides and TGF-beta in transgenic procol alpha 2[1] mice showed that the angiotensin II activation of collagen I gene requires participation and/or cooperation of endothelin and TGF-beta. Recent data suggest that the profibrotic actions of vasoactive peptides also need the activation of EGF receptor, ERK and rho kinase pathways in renal and vascular cells.

The methylation of 5'CpG 3' dinucleotides within genes creates potential targets for protein complexes that bind to methylated DNA sequences and to histone deacetylases (MBD-HDAC). This can lead to transcriptional repression by modification of chromatic. To test the importance of this repression in vivo and to determine when during development these epigenetic controls are placed on genes, two novel genes have been engineered by directed mutagenesis of the CpG-rich LacZ gene that are depleted of (LagZ) or completely lacking (LagoZ) CpG sequences. We report that the expression (transcriptional and translational) of the three genes is indistinguishable in transient assays in cleaving mouse embryos. Therefore, the complete absence of CpG sequences within three kilobases of coding sequence is compatible with its maintenance in the nucleus and with its expression. These molecules can now be used to study the ontogenesis of the CpG-dependent repressive system in intact organisms.