Quantitation of the P2Y1 Receptor with a High Affinity Radiolabeled Antagonist

2-Chloro-N6-methyl-(N)-methanocarba-2′-deoxyadenosine-3′,5′-bisphosphate (MRS2279) was developed previously as a selective high-affinity, non-nucleotide P2Y1 receptor (P2Y1-R) antagonist (J Med Chem 43:829–842, 2002; Br J Pharmacol 135:2004–2010, 2002). We have taken advantage of the N6-methyl substitution in the adenine base to incorporate [3H]methylamine into the synthesis of [3H]MRS2279 to high (89 Ci/mmol) specific radioactivity and have used this molecule as a radioligand for the P2Y1-R. [3H]MRS2279 bound to membranes from Sf9 insect cells expressing recombinant human P2Y1-R but not to membranes from wild-type Sf9 cells or Sf9 cells expressing high levels of recombinant P2Y2 or P2Y12 receptors. Equilibrium binding of [3H]MRS2279 to P2Y1-R expressed in Sf9 membranes was with a high affinity (Kd = 8 nM) essentially identical to the apparent affinity of MRS2279 determined previously in studies of P2Y1-R–promoted inositol phosphate accumulation or platelet aggregation. A kinetically derived Kd calculated from independent determinations of the rate constants of association (7.15 × 107 M−1 min−1) and dissociation (0.72 min−1) of [3H]MRS2279 also was in good agreement with the Kd derived from equilibrium binding studies. Competition binding assays with [3H]MRS2279 and P2Y1-R expressing Sf9 cell membranes revealed Ki values for the P2Y1-R antagonists MRS2279 (Ki = 13 nM), N6-methyl-2′-deoxyadenosine-3′,5′-bisphosphate (MRS2179; Ki = 84 nM), adenosine-3′, 5′-bisphosphate (Ki = 900 nM), and pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid (Ki = 6 µM) that were in good agreement with antagonist activities of these molecules previously determined at the P2Y1-R in intact tissues. Moreover, [3H]MRS2279 also bound with high affinity (Kd = 4–8 nM) to Chinese hamster ovary (CHO) or 1321N1 human astrocytoma cells stably expressing the human P2Y1-R, but specific binding was not observed in wild-type CHO or 1321N1 cells. [3H]MRS2279 bound with high affinity (Kd = 16 nM) to a binding site on out-dated human platelets (5–35 receptors/platelet) and rat brain membranes (210 fmol/mg protein) that fit the expected drug selectivity of a P2Y1-R. Taken together, these results indicate that [3H]MRS2279 is the first broadly applicable antagonist radioligand for a P2Y receptor

Characterization of the unfolding of a weak focus and modulus of analytic classification

La thèse présente une description géométrique d’un germe de famille générique déployant un champ de vecteurs réel analytique avec un foyer faible à l’origine et son complexifié : le feuilletage holomorphe singulier associé. On montre que deux germes de telles familles sont orbitalement analytiquement équivalents si et seulement si les germes de familles de difféomorphismes déployant la complexification de leurs fonctions de retour de Poincaré sont conjuguées par une conjugaison analytique réelle. Le “caractère réel” de la famille correspond à sa Z2-équivariance dans R^4, et cela s’exprime comme l’invariance du plan réel sous le flot du système laquelle, à son tour, entraîne que l’expansion asymptotique de la fonction de Poincaré est réelle quand le paramètre est réel. Le pullback du plan réel après éclatement par la projection monoidal standard intersecte le feuilletage en une bande de Möbius réelle. La technique d’éclatement des singularités permet aussi de donner une réponse à la question de la “réalisation” d’un germe de famille déployant un germe de difféomorphisme avec un point fixe de multiplicateur égal à −1 et de codimension un comme application de semi-monodromie d’une famille générique déployant un foyer faible d’ordre un. Afin d’étudier l’espace des orbites de l’application de Poincaré, nous utilisons le point de vue de Glutsyuk, puisque la dynamique est linéarisable auprès des points singuliers : pour les valeurs réels du paramètre, notre démarche, classique, utilise une méthode géométrique, soit un changement de coordonée (coordonée “déroulante”) dans lequel la dynamique devient beaucoup plus simple. Mais le prix à payer est que la géométrie locale du plan complexe ambiante devient une surface de Riemann, sur laquelle deux notions de translation sont définies. Après avoir pris le quotient par le relèvement de la dynamique nous obtenons l’espace des orbites, ce qui s’avère être l’union de trois tores complexes plus les points singuliers (l’espace résultant est non-Hausdorff). Les translations, le caractère réel de l’application de Poincaré et le fait que cette application est un carré relient les différentes composantes du “module de Glutsyuk”. Cette propriété implique donc le fait qu’une seule composante de l’invariant Glutsyuk est indépendante.The thesis gives a geometric description for the germ of the singular holomorphic foliation associated with the complexification of a germ of generic analytic family unfolding a real analytic vector field with a weak focus at the origin. We show that two such germs of families are orbitally analytically equivalent if and only if the germs of families of diffeomorphisms unfolding the complexified Poincaré map of the singularities are conjugate by a real analytic conjugacy. The Z2-equivariance of the family of real vector fields in R^4 is called the “real character” of the system. It is expressed by the invariance of the real plane under the flow of the system which, in turn, carries the real asymptotic expansion of the Poincaré map when the parameter is real. After blowing up the singularity, the pullback of the real plane by the standard monoidal map intersects the foliation in a real Möbius strip. The blow up technique allows to “realize” a germ of generic family unfolding a germ of diffeomorphism of codimension one and multiplier −1 at the origin as the semi-monodromy of a generic family unfolding an order one weak focus. In order to study the orbit space of the Poincaré map, we perform a trade-off between geometry and dynamics under the Glutsyuk point of view (where the dynamics is linearizable near the singular points): in the resulting “unwrapping coordinate” the dynamics becomes much simpler, but the price we pay is that the local geometry of the ambient complex plane turns into a much more involved Riemann surface. Over the latter, two notions of translations are defined. After taking the quotient by the lifted dynamics we get the orbit space, which turns out to be the union of three complex tori and the singular points (this space is non- Hausdorff). The Glutsyuk invariant is then defined over annular-like regions on the tori. The translations, the real character and the fact that the Poincaré map is the square of the semi-monodromy map, relate the different components of the Glutsyuk modulus. That property yields only one independent component of the Glutsyuk invariant

Quasi four-level Tm:LuAG laser

A quasi four-level solid-state laser is provided. A laser crystal is disposed in a laser cavity. The laser crystal has a LuAG-based host material doped to a final concentration between about 2% and about 7% thulium (Tm) ions. For the more heavily doped final concentrations, the LuAG-based host material is a LuAG seed crystal doped with a small concentration of Tm ions. Laser diode arrays are disposed transversely to the laser crystal for energizing the Tm ions

Physiological Benefits of Dietary Lysophospholipid Supplementation in a Marine Fish Model: Deep Analyses of Modes of Action

Given the hydrophilic structure of lysophospholipids (LPLs), their dietary inclusion translates into a better emulsifying capacity of the dietary components. The present study aimed to understand the mechanisms underlying the growth-promoting effect of LPL supplementation by undertaking deep analyses of the proximal intestine and liver interactomes. The Atlantic salmon (Salmo salar) was selected as the main aquaculture species model. The animals were divided into two groups: one was fed a control diet (C-diet) and the other a feed (LPL-diet) supplemented with an LPL-based digestive enhancer (0.1% AQUALYSO®, Adisseo). The LPL-diet had a positive effect on the fish by increasing the final weight by 5% and reducing total serum lipids, mainly due to a decrease in the plasma phospholipid (p < 0.05). In the intestine, the upregulated interactome suggests a more robust digestive capacity, improving vesicle-trafficking-related proteins, complex sugar hydrolysis, and lipid metabolism. In the liver, the LPL-diet promotes better nutrients, increasing several metabolic pathways. The downregulation of the responses to stress and stimuli could be related to a reduced proinflammatory state. This study on the benefits and modes of action of dietary LPLs opens a new window into fish nutrition and could be extended to other productive species.info:eu-repo/semantics/publishedVersio