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Endocrine disruption is considered a highly relevant hazard for environmental risk assessment of chemicals, plant protection products, biocides and pharmaceuticals. Therefore, screening tests with a focus on interference with estrogen, androgen, and thyroid hormone pathways in fish and amphibians have been developed. However, they use a large number of animals and short-term alternatives to animal tests would be advantageous. Therefore, the status of alternative assays for endocrine disruption in fish and frogs was assessed by a detailed literature analysis. The aim was to (i) determine the strengths and limitations of alternative assays and (ii) present conclusions regarding chemical specificity, sensitivity, and correlation with in vivo data. Data from 1995 to present were collected related to the detection/testing of estrogen-, androgen-, and thyroid-active chemicals in the following test systems: cell lines, primary cells, fish/frog embryos, yeast and cell-free systems. The review shows that the majority of alternative assays measure effects directly mediated by receptor binding or resulting from interference with hormone synthesis. Other mechanisms were rarely analysed. A database was established and used for a quantitative and comparative analysis. For example, a high correlation was observed between cell-free ligand binding and cell-based reporter cell assays, between fish and frog estrogenic data and between fish embryo tests and in vivo reproductive effects. It was concluded that there is a need for a more systematic study of the predictive capacity of alternative tests and ways to reduce inter- and intra-assay variability. [ABSTRACT FROM AUTHOR]

Generally, cationic vector-based intravenous delivery of DNA is hindered by interactions of positively charged complexes with serum proteins. However, if optimally formulated, cationic vectors can provide reasonable levels of transfection in the lung either by intravenous or intrapulmonary routes. We investigated the in vivo transfection capacity of a cationic polymer: linear, 22 kDa polyethylenimine. PEI/DNA complexes were formulated in 5% glucose and delivered into adult mice through the tail vein. Two marker genes were used, β-galactosidase and luciferase. High levels of luciferase expression (107 RLU/mg protein) were found in the lung when DNA was complexed with PEI at a ratio of 4 nitrogen equivalents per DNA phosphate. Lower levels of transfection were found in the heart, spleen, liver and kidney. Expression was dose- and time-dependent in all tissues examined. In the lung, β-galactosidase staining showed transgene expression in clusters of 10 or more pulmonary cells including the alveolar endothelium, squamous and great alveolar epithelial cells (type I and II pneumocytes) and septal cells. These findings indicate that the complexes pass the capillary barrier in the lung. Although the delivery mechanism requires elucidation, linear PEI has promise as a vector for intravenous transfer of therapeutic genes. [ABSTRACT FROM AUTHOR]

Currently in vivo gene delivery by synthetic vectors is hindered by the limited diffusibility of complexes in extracellular fluids and matrices. Here we show that certain formulations of plasmid DNA with linear polyethylenimine (22 kDa PEI, ExGene 500) can produce complexes that are sufficiently small and stable in physiological fluids so as to provide high diffusibility. When plasmid DNA was formulated with 22 kDa PEI in 5% glucose, it produced a homogeneous population of complexes with mean diameters ranging from 30 to 100 nm according to the amount of PEI used. In contrast, formulation in physiological saline produced complexes an order of magnitude greater (1 μm). Intraventricular injection of complexes formulated in glu-cose showed the complexes to be highly diffusible in the cerebrospinal fluid of newborn and adult mice, diffusing from a single site of injection throughout the entire brain ventricular spaces. Transfection efficiency was followed by histochemistry of β-galactosidase activity and double immunocytochemistry was used to identify the cells transfected. Transgene expression was found in both neurons and glia adjacent to ventricular spaces. Thus, this method of formulation is promising for in vivo work and may well be adaptable to other vectors and physiological models. [ABSTRACT FROM AUTHOR]

Efficient gene transfer is a prerequisite for analysing regulation of transfected promoters. We combined the DNA binding property of the cationic polymer polyethylenimine (PEI) and the potent endocytic activity of adenovirus in a PEI–DNA–adenovirus complex which provided efficient plasmid delivery in differentiated cultured cells. We transfected 3T3-F442A adipocytes, C2.7 myocytes and FAO hepatoma cells with a construct containing the simian virus 40 promoter fused to the chloramphenicol acetyltransferase (CAT) gene, using a combination of PEI and 200 p.f.u. per cell of replication-deficient type 5 adenovirus. Resulting CAT activities varied according to the cell type reaching about 0.6, 8 and 38 units/mg protein for respectively 3T3-F442A, FAO and C2.7 cells. Increases in transfection efficiencies were 140- to 300-fold when compared with those obtained with PEI alone. Then we tested physiologically regulated promoters: the phosphoenolpyruvate carboxykinase gene promoter in 3T3-F442A or FAO cells and the hexokinase II gene promoter in C2.7 myocytes. Gene expression was appropriately increased by clofibrate, dexamethasone and insulin for 3T3-F442A, FAO and C2.7 cells, respectively. Thus, the combination of PEI and adenovirus is a simple, efficient, inexpensive and versatile method of gene transfer which is applicable to several differentiated cells and provides a physiologically coherent transgene regulation. We name this method PEI- adenofection. [ABSTRACT FROM AUTHOR]

Presents a study which examined the role of thyroid hormone and its alpha receptor in adult neural stem cell (NSC) cycling. Information on NSC; Effect of hypothyroidism on NSC proliferation; Conclusion and findings of the study.

Few studies have included subjects with the propensity to reach old age in good health, with the aim to disentangle mechanisms contributing to staying healthier for longer. The hypothalamic-pituitary-thyroid (HPT) axis maintains circulating levels of thyroid stimulating hormone (TSH) and thyroid hormone (TH) in an inverse relationship. Greater longevity has been associated with higher TSH and lower TH levels, but mechanisms underlying TSH/TH differences and longevity remain unknown. The HPT axis plays a pivotal role in growth, development and energy metabolism. We report that offspring of nonagenarians with at least one nonagenarian sibling have increased TSH secretion but similar bioactivity of TSH and similar TH levels compared to controls. Healthy offspring and spousal controls had similar resting metabolic rate and core body temperature. We propose that pleiotropic effects of the HPT axis may favour longevity without altering energy metabolism. [ABSTRACT FROM AUTHOR]

Abstract Background The western African clawed frog Xenopus tropicalis is an anuran amphibian species now used as model in vertebrate comparative genomics. It provides the same advantages as Xenopus laevis but is diploid and has a smaller genome of 1.7 Gbp. Therefore X. tropicalis is more amenable to systematic transcriptome surveys. We initiated a large-scale partial cDNA sequencing project to provide a functional genomics resource on genes expressed in the nervous system during early embryogenesis and metamorphosis in X. tropicalis. Results A gene index was defined and analysed after the collection of over 48,785 high quality sequences. These partial cDNA sequences were obtained from an embryonic head and retina library (30,272 sequences) and from a metamorphic brain and spinal cord library (27,602 sequences). These ESTs are estimated to represent 9,693 transcripts derived from an estimated 6,000 genes. Comparison of these cDNA sequences with protein databases indicates that 46% contain their start codon. Further annotation included Gene Ontology functional classification, InterPro domain analysis, alternative splicing and non-coding RNA identification. Gene expression profiles were derived from EST counts and used to define transcripts specific to metamorphic stages of development. Moreover, these ESTs allowed identification of a set of 225 polymorphic microsatellites that can be used as genetic markers. Conclusion These cDNA sequences permit in silico cloning of numerous genes and will facilitate studies aimed at deciphering the roles of cognate genes expressed in the nervous system during neural development and metamorphosis. The genomic resources developed to study X. tropicalis biology will accelerate exploration of amphibian physiology and genetics. In particular, the model will facilitate analysis of key questions related to anuran embryogenesis and metamorphosis and its associated regulatory processes.

L'Homme est exposé aux polluants ayant des propriétés de perturbateurs endocriniens (PE) dès la vie intra-utérine. Parmi eux, un mix de 15 polluants différents (composants perfluorés, bisphénol A ...) ubiquitaires et retrouvés dans le liquide amniotique de femmes enceintes, est associé à une dysfonction thyroïdienne et à des retards d'acquisition chez les enfants nés de femme exposées [1]. Des données épidémiologiques suggèrent une association entre l'exposition aux polluants et le risque de maladie articulaire [2]. Notre objectif est de déterminer l'effet de ce mix de polluant sur le risque d'arthrose et la fonction chondrocytaire. Des souris femelles C57/B6 ont été exposées au mix de 15 polluants, par l'eau de leur boisson (dose équivalente de bisphénol A [BPA] de 1,5 μg/kg/j) 15 jours avant conception et pendant toute la gestation. Les souriceaux nés ont été exposés de la même manière jusqu'à leur sacrifice à 10 mois (n = 18). Un groupe contrôle de femelles et de souriceaux (n = 18) non exposés a été élevé dans les mêmes conditions. Au sacrifice, une analyse histologique du genou a été réalisée (score OARSI). In vitro, des cultures primaires de chondrocytes murins (n = 7), ont été exposées ou non à différentes doses du mix de polluants pendant 24 h. La toxicité cellulaire a été évaluée par dosage LDH, l'expression de cytokines (interleukine [IL]–6) chimiokines (MCP1) et métalloprotéases (MMP-3) par RT-qPCR et ELISA et le dosage des espèces réactives de l'oxygène (ROS) par un test au Nitroblue tetrazolium (NBT). Une analyse transcriptomique a été réalisée pour déterminer l'ensemble des gènes différentiellement exprimés (DE) entre les chondrocytes exposés (n = 4) ou non (n = 4) au mix (eq. BPA 10 ng/mL) pendant 24 h. Enfin, à 15 minutes de stimulation, nous avons évalué la phosphorylation de la voie de signalisation MAPK p38 par Western Blot. À 10 mois, les mâles exposés aux polluants avaient des scores OARSI supérieurs à ceux des mâles non exposés (3,25 ± 0,31 vs 2,12 ± 0,31 p = 0,02). Cette différence n'a pas été retrouvée chez les femelles. Il n'y avait pas de différence entre les deux groupes concernant le phénotype (poids, taille), la synoviale et l'os sous-chondral. In vitro, le mix de polluants n'a pas induit de toxicité cellulaire direct (LDH). Les chondrocytes stimulés par le mix surexprimaient, par qPCR, des cytokines inflammatoires et métalloprotéases avec un effet-dose : IL6 : + 222 % (p < 0,05) ; MCP 1 : + 155 % (p < 0,05), MMP-3 : +222 % (p < 0,05) pour une dose eq. BPA 10 ng/mL. Des résultats similaires ont été obtenus en ELISA. L'activation pro-inflammatoire a été confirmée puisque le mix induisait une augmentation significative de la phosphorylation de p38MAPK. Parmi les 265 gènes DE entre chondrocytes exposés ou non au mix dans l'analyse transcriptomique, plusieurs voies cellulaires impliquées dans le stress oxydant (comme le métabolisme du glutathion, la voie des pentose et la ferroptose) ont été identifiées. Ceci a été confirmé par test au NBT puisque les chondrocytes exposés au mix produisaient significativement plus de ROS (p < 0,05). L'exposition aux PE induit des altérations précoces du cartilage chez les souris. Ceci pourrait être lié à un effet local pro-inflammatoire et pro-oxydant sur les chondrocytes. [ABSTRACT FROM AUTHOR]