Steroid substrate-induced epimerase mechanism in the active site of the human 11β-hydroxysteroid dehydrogenase type 1

Cytochrome P4507B1 7[alpha]-hydroxylates dehydroepiandrosterone (DHEA), epiandrosterone (EpiA) and 5[alpha]-androstane-3[beta],17[beta]-diol (Adiol). 11[beta]-Hydroxysteroid dehydrogenase type 1 (11[beta]-HSD1) interconverts 7[alpha]- and 7[beta]- forms. Whether the inter-conversion proceeds through oxido-reductive steps or epimerase activity is investigated. Experiments using ^3^H-labeled 7[beta]-hydroxy-DHEA, 7[beta]-hydroxy-EpiA and 7[beta]-hydroxy-Adiol show the ^3^H-label to accumulate in 7-oxo-DHEA trap but neither in 7-oxo-EpiA nor 7-oxo-Adiol traps. Computed models of 7-oxygenated steroids dock in the active site of 11[beta]-HSD1 either in a flipped or turned form relative to cortisone and cortisol. 7-Oxo-steroid reduction in 7[alpha]- or 7[beta]-hydroxylated derivatives results from either turned or flipped forms. 11[beta]-HSD1 incubation in H~2~^18^O medium with each 7-hydroxysteroid did not incorporate ^18^O in 7-hydroxylated derivatives of EpiA and Adiol independently of the cofactor used. Thus oxido-reductive steps apply for the interconversion of 7[alpha]- and 7[beta]-hydroxy-DHEA through 7-oxo-DHEA. Epimerisation may proceed on the 7-hydroxylated derivatives of EpiA and Adiol through a mechanism involving the cofactor and Ser170

Operation Of The NuMi Beam Monitoring System

The NuMI (Neutrinos at the Main Injector) facility produces an intense neutrino beam for experiments. The NuMI Beam Monitoring system consists of four arrays of ion chambers that measure the intensity and distribution of the remnant hadron and tertiary muon beams produced in association with the neutrinos. The ion chambers operate in an environment of high particle fluxes and high radiation.Physic

The Long-Baseline Neutrino Experiment: Exploring Fundamental Symmetries of the Universe

The preponderance of matter over antimatter in the early Universe, the dynamics of the supernova bursts that produced the heavy elements necessary for life and whether protons eventually decay --- these mysteries at the forefront of particle physics and astrophysics are key to understanding the early evolution of our Universe, its current state and its eventual fate. The Long-Baseline Neutrino Experiment (LBNE) represents an extensively developed plan for a world-class experiment dedicated to addressing these questions. LBNE is conceived around three central components: (1) a new, high-intensity neutrino source generated from a megawatt-class proton accelerator at Fermi National Accelerator Laboratory, (2) a near neutrino detector just downstream of the source, and (3) a massive liquid argon time-projection chamber deployed as a far detector deep underground at the Sanford Underground Research Facility. This facility, located at the site of the former Homestake Mine in Lead, South Dakota, is approximately 1,300 km from the neutrino source at Fermilab -- a distance (baseline) that delivers optimal sensitivity to neutrino charge-parity symmetry violation and mass ordering effects. This ambitious yet cost-effective design incorporates scalability and flexibility and can accommodate a variety of upgrades and contributions. With its exceptional combination of experimental configuration, technical capabilities, and potential for transformative discoveries, LBNE promises to be a vital facility for the field of particle physics worldwide, providing physicists from around the globe with opportunities to collaborate in a twenty to thirty year program of exciting science. In this document we provide a comprehensive overview of LBNE's scientific objectives, its place in the landscape of neutrino physics worldwide, the technologies it will incorporate and the capabilities it will possess.Comment: Major update of previous version. This is the reference document for LBNE science program and current status. Chapters 1, 3, and 9 provide a comprehensive overview of LBNE's scientific objectives, its place in the landscape of neutrino physics worldwide, the technologies it will incorporate and the capabilities it will possess. 288 pages, 116 figure

Neurostéroïdes neuroprotecteurs (métabolisme, dosage et effets dans les maladies neurogégénératives)

La déhydroépiandrostérone (DHEA) est produite dans le cerveau humain et métabolisée en 7[alpha]- et 7[bêta]-hydroxy-DHEA par le cytochrome P4507B1 (CYP7B1). Le taux de la DHEA chez l'homme diminue avec l'âge, et on considère donc qu'il existe une relation entre la DHEA et la neuroprotection. Cependant, le taux de la DHEA mesuré dans le cerveau de sujets atteints de la maladie d'Alzheimer est plus élevé que chez des contrôles du même âge. On sait aussi que la 7-hydroxylation est la voie principale du métabolisme de la DHEA dans le cerveau. Nous avons donc étudié les propriétés enzymatiques du CYP7B1 humain. Pour cela, nous avons utilisé le CYP7B1 humain recombinant et plusieurs 3[bêta]-hydroxystéroïdes marqués au 14C, dont la prégnénolone, la DHEA, l'épiandrostérone, le 5[alpha]-androstane-3[bêta], 17[bêta]-diol et l'estrone. Les métabolites ont été identifiés par la GC/MS. Les dérivés 7[alpha]-hydroxylés sont produits en quantité majoritaire, et les dérivés 7[bêta]-hydroxylés sont également produits en quantité minoritaire. Les KM de leurs productions sont identiques, la 7[bêta]-hydroxylation est une réaction accessoire de la 7[alpha]-hydroxylation. La 7[alpha]-hydroxylation de la DHEA est inhibée par l'estrone et l'estradiol (inhibition mixte) et par des peptides amyloïdes [bêta] (inhibition non-compétitive). Nous avons également mesuré et comparé les taux de la DHEA et ses dérivés (la DHEAS, la 7[alpha]-hydroxy-DHEA, la 7[bêta]-hydroxy-DHEA et la 16[alpha]-hydroxy-DHEA) dans le liquide céphalo-rachidien (LCR) de patients atteints de la maladie d'Alzheimer (MA) ou de la démence vasculaire (DV) et les contrôles du même âge. La DHEA est augmentée et la DHEAS est diminuée dans les deux pathologies. Les rapports de DHEA/7-hydroxy-DHEA, 7[bêta]-hydroxy-DHEA/DHEA et DHEAS/DHEA montrent une différence significative entre les deux pathologies et les contrôles. Nous avons également noté que le rapport 7[alpha]-hydroxy-DHEA/7[bêta]-hydroxy-DHEA est plus élevé chez les patients de MA et que chez les DV. Les résultats indiquent que la DHEA augmentée dans les cerveaux des patients n'est pas neuroprotective, et suggèrent que la sulfotransférase et l'enzyme responsable pour la 7-hydroxylation de la DHEA soient moins présentes chez les malades ou qu'elles soient formées par le polymorphisme en enzymes moins efficaces.Dehydroepiandrosterone (DHEA) is produced in the human brain and metabolised to 7[alpha]- and 7[bêta]-hydroxy-DHEA by the cytochrome P4507B1 (CYP7B1). Since the DHEA levels in humans decrease with aging, it was considered that a relation between DHEA and neuroprotection was possible. However, the DHEA levels were reported much higher in the brain of patients with Alzheimer's disease (AD) than in age matched controls. It is also known that the 7-hydroxylation of DHEA is the major metabolic route in the brain. We studied the enzymatic properties of the human CYP7B1. To that end, we used recombinant human CYP7B1 and several C-labelled 2[bêta]-hydroxysteroid substrates such as pregnenolone, DHEA, epiandrosterone, 5[alpha]-androstane-3[bêta], 17[bêta]-diol and estrone. The metabolites are identified by GC/MS. The major product was the 7[alpha]-hydroxy derivatives and the small amounts of the 7[bêta]-hydroxy derivative were also characterized. Identical Km for 7[alpha]- and 7[bêta]-hydroxylation indicated that 7[bêta]-hydroxylation was a minor side reaction catalysed by th enzyme. The DHEA 7[alpha]-hydroxylation was inhibited by estrone and estradiol (mixed type inhibition) and by the [bêta]-amyloid peptides (non-competitive inhibition). We have also measured and compared the levels of DHEA and its metabolites such as DHEAS, 7[alpha]-hydroxy-DHEA, 7[bêta]-hydroxy-DHEA and 16[alpha]-hydroxy-DHEA in the cerebrospinal fluid (CSF) of patients with AD, vascular dementia (VD) and the age matched controls. DHEA was increased and DHEAS was decreased in both diseased patients. The ratios of DHEA/7-hydroxy-DHEA, 7[bêta]-hydroxy-DHEA/DHEA and DHEAS/DHEA were significantly different between the patients and the controls. We also observed that the 7[alpha]-hydroxy-DHEA/7[bêta]-hydroxy-DHEA ratio differed significantly between the AD and the VD. Our results indicate that the increased DHEA in neurodegenerative diseases is not neuroprotective and suggest that the sulfotransferase and the enzyme responsible for the 7-hydroxylation of DHEA are either present in lower levels or transfered by polymorphism into less efficient enzymes.PARIS-CNAM (751032301) / SudocSudocFranceF

La 7b-hydroxy-épiandrostérone dans des modèles in vitro de cancer du sein (effets anti-estrogéniques et rôle des récepteurs des estrogènes)

La 7b-hydroxy-épiandrostérone, stéroïde endogène dérivant de la DHEA, présente des propriétés anti-inflammatoires. En effet, elle module la voie des prostaglandines (PGs) en inhibant la production de la PGE2 pro-inflammatoires et en augmentant la production de la 15-Deoxy- 12,14-PGJ2 cyto-protectrice in vivo et in vitro. Les faibles doses de 7b-hydroxy-épiandrostérone (1nM, 10nM, 100nM) pour lesquelles ces effets sont observés, suggèrent une liaison à un récepteur spécifique. L inflammation et la production des PGs jouent un rôle important dans le développement et la prolifération des tumeurs mammaires estrogéno-dépendantes. Le 17b-estradiol (E2), en se fixant sur les récepteurs des estrogènes (REs), induit la production de PGE2 et la prolifération cellulaire dans ces cellules tumorales. De ce fait, notre objectif était de tester les effets de la 7b-hydroxy-épiandrostérone sur la prolifération (comptage avec exclusion au bleu trypan), le cycle cellulaire et l apoptose (cytométrie de flux) dans les lignées cellulaires de cancer du sein MCF-7 (REa+, REb+, GPR30+) et MDA-MB-231 (REa-, REb+, GPR30+) et d identifier une(des) cible(s) potentielle(s) dans ces cellules (transactivation) et dans des cellules négatives pour les REs nucléaires SKBr3 (GPR30+) (études de prolifération). Cette étude a montré que la 7b-hydroxy-épiandrostérone exerce des effets anti-estrogéniques dans les cellules MCF-7 et MDA-MB-231 associés à une inhibition de la prolifération et un arrêt du cycle cellulaire. Les études de transactivation et de prolifération avec les agonistes spécifiques des REs ont montré une interaction avec le REb. De plus, les résultats des études de proliférations sur les trois lignées cellulaires suggèrent que la 7b-hydroxy-épiandrostérone pourrait également interagir avec le GPR30. Ces résultats indiquent que ce stéroïde androgène agit comme un anti-estrogène. De plus, c est la première fois qu un stéroïde androgène à faible dose montre une action anti-proliférative dans des lignées de cancers du sein. Des études ultérieures restent à réaliser afin de mieux comprendre ces effets observés.7b-hydroxy-epiandrosterone, an endogenous androgenic derivative of DHEA, has previously been shown to exert anti-inflammatory action in vitro and in vivo via a shift from prostaglandin E2 (PGE2) to 15-deoxy- 12,14-PGJ2 production. This modulation in prostaglandin production was obtained with low concentrations of 7b-hydroxy-epiandrosterone (1 100 nM) and suggested that it might act through a specific receptor. Inflammation and prostaglandin synthesis is important in the development and survival of estrogen-dependent mammary cancers. Estrogen induced PGE2 production and cell proliferation via its binding to estrogen receptors (ERs) in these tumors. Our objective was to test the effects of 7b-hydroxy-epiandrosterone on the proliferation (by counting with trypan blue exclusion), cell cycle and cell apoptosis (by flow cytometry) of breast cancer cell lines MCF-7 (ERa+, ERb +, G-protein coupled receptor 30 : GPR30+) and MDA-MB-231 (ERa-, ERb +, GPR30+) and to identify a potential target of this steroid in these cell lineages (by transactivations) and in the nuclear ER-negative SKBr3 cells (GPR30+) (by proliferation assays). 7b-hydroxy-epiandrosterone exerted anti-estrogenic effects in MCF-7 and MDA-MB-231 cells associated with cell proliferation inhibition and cell cycle arrest. Moreover, transactivation and proliferation with ER agonists assays indicated that 7b-hydroxy-epiandrosterone interacted with ERb. Data from proliferation assays on the MCF-7, MDA-MB-231 and SKBr3 cell lines suggested that 7b-hydroxy-epiandrosterone may also act through the membrane GPR30 receptor.These results support that this androgenic steroid acts as an anti-estrogenic compound. Moreover, this is the first evidence that low doses of androgenic steroid exert antiproliferative effects in these mammary cancer cells. Further investigations are needed to improve understanding of the observed actions of endogenous 7b-hydroxy-epiandrosterone.PARIS-CNAM (751032301) / SudocSudocFranceF

Reducing discards of demersal species using a 100 mm square mesh cylinder: Size selectivity and catch comparison analysis

We assessed the impact of an additional 100 mm square mesh cylinder (SMC) on the selective property of the demersal whitefish trawl in the Celtic Sea. Sea trials were conducted on board a French trawler using twin trawl rigging. We tested the effects of the position of the SMC (in front of and behind the mandatory 100 mm square mesh panel) and of the insertion of a dispersive float. Selectivity analysis revealed the 50% retention length () to be greater than the Minimum Conservation Reference Sizes regardless of species (haddock, whiting or megrim) or rigging configuration. Results did not reveal a clear effect of the SMC position. However, the insertion of the float led to a decrease in selection ranges suggesting enhanced contact probabilities with the square meshes. With the largest values, the SMC placed in the front position (and without the float) proved to be the most selective rigging configuration and was then tested under commercial conditions. Catch comparisons revealed that the test gear retained less fish across all size classes than the commercial gear. For haddock, the test gear retained less fish below 50 cm in length. Catch comparisons also indicated significant discards of fish above the MCRS, i.e. “high-grading” practices. Under the requirements of the landing obligation (LO), unwanted catches must be stored on-board and landed regardless of economic value. The SMC is thus a valuable tool to mitigate the impact of the LO on the demersal whitefish fishing fleets operating in the Celtic Sea