Etude de l'interaction entre les domaines transmembranaires du récepteur du polypeptide insulinotrope glucose-dépendant (GIP) et la région amino terminale du peptide

Le récepteur du polypeptide insulinotrope glucose-dependant (GIP-R), membre de la sous-famille B des récepteurs couplés aux protéines G (RCPG), module la régulation des fonctions physiologiques et métaboliques de l'organisme telles que l'homéostasie glucidique et lipidique. Ces propriétés font du récepteur du GIP une cible thérapeutique potentielle pour le traitement du diabète sucré et de l'obésité. Le développement de nouveaux agents pharmacologiques, notamment des ligands non peptidiques du récepteur du GIP, constitue un enjeu important. Les mécanismes moléculaires responsables de l'activation des récepteurs de la famille B ne sont cependant pas bien compris, même si un mécanisme d'activation général en deux étapes a été postulé pour ces récepteurs. Cependant, les acides aminés qui participent au processus d'activation restent encore à définir. L'objectif majeur du projet de recherche était d'identifier le site de liaison du GIP-R humain en caractérisant précisément les principaux résidus du récepteur en interaction fonctionnelle avec les acides aminés du domaine N-terminal du GIP, connu pour correspondre au domaine d'activation du GIP. Par modélisation moléculaire, des modèles de complexes GIP.GIP-R ont été construits, sur la base des alignements multiples de séquences des récepteurs et des ligands. La partie contenant les domaines transmembranaires du récepteur du GIP a été modélisée par homologie sur la base des coordonnées du cristal du récepteur adénosine A2A. Puis, le modèle correspondant au cristal du complexe comprenant le GIP lié au domaine extracellulaire du récepteur GIP a été " docké " sur la partie transmembranaire du récepteur du GIP. Les acides aminés du récepteur identifiés comme étant en interaction avec le N-terminal du GIP ont été mutés. L'analyse pharmacologique des mutants a démontré que l'Arg183 et l'Arg190 de l'hélice transmembranaire II (TMH-II), l'Arg300 du TMH-V et la Phe357 du TMH-VI étaient importants pour l'activation du récepteur. En effet la mutation de ces acides aminés a entrainé une diminution significative de la capacité du récepteur à induire la formation d'AMPc. Une caractérisation plus poussée de ces mutants, ainsi que des tests utilisant des analogues du GIP dont certains résidus ont été substitués par une alanine, ont démontré l'interaction du Glu3 du GIP avec l'Arg183 du GIP-R. Nous avons également préparé du GIP (1-30)-Alexa-F-647 et vérifié sa capacité à stimuler le récepteur avec une puissance équivalente au GIP(1-30). Le peptide fluorescent a été ensuite utilisé pour démontrer que tous les récepteurs mutés étaient exprimés à la surface cellulaire HEK293 à un niveau similaire de celui du WT-GIP-R, ceci grâce à des expériences de cytométrie en flux et de microscopie confocale. Nous présentons donc un modèle du complexe GIP.GIP-R identifiant les résidus et les hélices du récepteur qui sont impliqués dans le processus d'activation, ainsi que leurs interactions avec les acides aminés de l'extrémité N-terminale du peptide. En outre, la région N-terminale du GIP se place à l'intérieur dune poche formée par les hélices transmembranaires 2, 3, 5 et 6 du récepteur, notamment du fait de l'interaction de Glu3 avec l'Arg183 du récepteur et de la Tyr1 biologiquement cruciale du GIP avec la Gln224 (TMH-3), l'Arg300 (TMH-5) et la Phe357 (TMH-6) du récepteur. Ce modèle validé expérimentalement représente une étape importante vers la compréhension du mécanisme d'activation du GIP-R qui devrait faciliter la conception rationnelle d'agents thérapeutiques.Glucose-dependent insulinotropic polypeptide receptor (GIP-R), a member of subfamily B of G-protein coupled receptor (GPCR), modulates the regulation of important physiologic and metabolic functions in the body such as glucose and lipid homeostasis that make it a potentially attractive therapeutic target for new pharmacological agents such as non-peptide ligands for the treatment of diabetes mellitus and obesity. However, the molecular mechanisms responsible for receptor activation are poorly understood in receptors belonging to family B. Although a general two step activation mechanism has been postulated for family B receptors yet the precise residues of the receptor that participate in the process remain to be delineated. One of the principal objectives of my research project was to identify the binding site of human GIP-R by precisely recognizing the residues that are implicated in interactions with the amino acids of the critically important N-terminal bioactive domain of the hormone that has been shown to be responsible for receptor activation. GIP.GIP-R complex models were constructed, based on multiple sequence alignments of both receptors and the ligands and the in-silico docking of the peptide using Adenosine A2a receptor as template for transmembrane domain of receptor while also taking X-ray structural data on the interaction of GIP and GIP-R ECD into consideration. Residues of the receptor identified to be involved in interaction with the ligand were subjected to site-directed mutagenesis. The pharmacological activity assays of the mutants demonstrated that Arg183 and Arg190 in TMH2, Arg300 in TMH5 and Phe357 in TMH6 were important determinants for receptor activation as demonstrated by their significant decrease in potency to induce cAMP formation, a measure of ligand induce receptor activation. Further characterization of these mutants, including tests with alanine substituted GIP analogues, demonstrated interaction of Glu3 in GIP with Arg183 in GIP-R. Furthermore, they strongly supported a binding mode of GIP to GIP-R in which the N-terminal moiety of GIP was sited within transmembrane helices 2, 3, 5, and 6 with biologically crucial Tyr1 interacting with Gln224 (TMH3), Arg300 (TMH5) and Phe357 (TMH6) of the receptor. We also prepared and ascertained the ability of GIP (1-30)-Alexa- F-647 to stimulate the receptor in comparison to GIP (1-30) and found that both activated the receptor with equal potency. The fluorescent peptide was then used to demonstrate that all the mutated receptors were expressed at HEK293 cell surface at levels similar to that of the WT-GIP-R by performing Flow cytometery and Confocal microscopy. We therefore present a model for GIP.GIP-R pin pointing the exact residues and the helixes involved of the receptor involved in activation process and the interactions with their partner amino acids in the N-terminal of the peptide. This experimentally validated model represents an important step towards understanding activation mechanism of GIP-R which should facilitate the rational design of therapeutic agents

1-(2-Oxoindolin-3-yl­idene)-4-[2-(tri­fluoro­meth­yl)phen­yl]thio­semicarbazide

In the title compound, C16H11F3N4OS, the dihedral angle between the aromatic ring systems is 69.15 (10)°. Intra­molecular N—H⋯N and N—H⋯O hydrogen bonds generate S(5) and S(6) rings, respectively. A short N—H⋯F contact also occurs. In the crystal, inversion dimers linked by pairs of N—H⋯O hydrogen bonds generate R 2 2(8) loops. The dimers are linked by N—H⋯F hydrogen bonds, forming polymeric chains propagating in [100]. π–π inter­actions also exist between the centroids of the benzene rings of the 2-oxoindoline group at a distance of 3.543 (3) Å and a short C=O⋯π contact occurs. Two F atoms of the trifluoro­methyl group are disordered over two sets of sites in a 0.517 (8):0.483 (8) ratio

(±)-4,12,15,18,26-Penta­hydroxy-13,17-dioxahepta­cyclo­[14.10.0.03,14.04,12.06,11.018,26.019,24]hexa­cosa-1,3(14),6(11),7,9,15,19,21,23-nona­ene-5,25-dione monohydrate

The title compound, C24H14O9·H2O, displays a cup-shaped form. The water mol­ecule is disordered over two set of sites with an occupancy ratio of 0.78:0.22. The mol­ecule of the compound has four stereocenters and corresponds to the SSRR/RRSS diastereoisomer. In the mol­ecule, the maximum dihedral angle between the planar benzene rings is 80.40 (4)°. The H atoms of the hy­droxy groups are engaged in hydrogen bonding, forming infinite chains parallel to the a axis. These chains are inter­linked through water mol­ecules, resulting in the formation of a two-dimensional network parallel to the (001) plane. Futhermore C—H⋯O, C—H⋯π and slipped π–π inter­actions result in the formation of a three-dimensional network

A Value Chain Approach to Characterize the Chicken Sub-sector in Pakistan

The chicken industry of Pakistan is a major livestock sub-sector, playing a pivotal role in economic growth and rural development. This study aimed to characterize and map the structure of broiler and layer production systems, associated value chains, and chicken disease management in Pakistan. Qualitative data were collected in 23 key informant interviews and one focus group discussion on the types of production systems, inputs, outputs, value addition, market dynamics, and disease management. Quantitative data on proportions of commodity flows were also obtained. Value chain maps were generated to illustrate stakeholder groups and their linkages, as well as flows of birds and products. Thematic analysis was conducted to explain the functionality of the processes, governance, and disease management. Major chicken production systems were: (1) Environmentally controlled production (97–98%) and (2) Open-sided house production (2–3%). Broiler management systems were classified as (I) Independent broiler production; (II) Partially integrated broiler production; and (III) Fully integrated broiler production, accounting for 65–75, 15–20, and 10–15% of commercial broiler meat supply, respectively. The management systems for layers were classified as (I) Partially integrated layer production and (II) Independent layer production, accounting for 10 and 80–85% in the egg production, respectively. The share of backyard birds for meat and eggs was 10–15%. Independent, and integrated systems for chicken production could be categorized in terms of value chain management, dominance of actors, type of finished product and target customers involved. Integrated systems predominantly targeted high-income customers and used formal infrastructure. Numerous informal chains were identified in independent and some partially integrated systems, with middlemen playing a key role in the distribution of finished birds and eggs. Structural deficiencies in terms of poor farm management, lack of regulations for ensuring good farming practices and price fixing of products were key themes identified. Both private and public stakeholders were found to have essential roles in passive disease surveillance, strategy development and provision of health consultancies. This study provides a foundation for policy-makers and stakeholders to investigate disease transmission, its impact and control and the structural deficiencies identified could inform interventions to improve performance of the poultry sector in Pakistan

Genomic analysis of tyrosine hydroxylase gene sequence variations and its association with D-9- tetrahydrocannabinol dependence in addicts

Purpose: To elucidate the genetic basis of drug addiction by conducting a genetic analysis of TH (tyrosine hydroxylase) gene and the novel polymorphisms that might help in understanding addiction and its molecular basis. Methods: Forty-two subjects were recruited into three groups for this study. DNA was isolated from the individuals. PCR amplification of TH gene was carried out and amplicons were sequenced. Genomic characterization of TH gene provided five polymorphic loci – TH 1, TH 2, TH 3, TH 4 and TH 5 which were found among all the groups. Results: According to Shannon’s diversity index, the studied population was between 0.0762 and 0.6032. Heterozygosity index depicted that TH 1 locus was less heterozygous (0.3288), followed by TH 5 (0.3152). TH 1 (0.1462) was the least heterozygous. Genotypic analysis predicted that among these five loci, TH 4 (p = 0.039898) and TH 2 (p = 0.851716) were non-significant (p > 0.05) and obeyed Hardy Weinberg Equilibrium (HWE) law. There are few genetic changes in the studied population that can statistically be associated with drug addiction. Still, their genotypic distribution in the gene pool was very low. Conclusion: On the basis of these findings, drug addiction in the studied population is more likely a social issue rather than a genetic one. Keywords: Tyrosine hydroxylase, SNP, Drug dependenc

N′-[(E)-(4-Bromo-2-thienyl)methyl­ene]isonicotinohydrazide

In title compound, C11H8BrN3OS, the dihedral angle between the two aromatic rings is 27.61 (14)° and the Br atom is disordered over two sites with an occupancy ratio of 0.804 (2):0.196 (2). In the crystal, the mol­ecules are linked by N—H⋯O, C—H⋯O and C—H⋯N inter­actions, resulting in chains

(Z)-4-Hexyl-1-(5-nitro-2-oxo-2,3-dihydro-1H-indol-3-yl­idene)thio­semicarbazide

In the title compound, C15H19N5O3S, intra­molecular N—H⋯O, N—H⋯N and C—H⋯S inter­actions occur and the three terminal C atoms of the hexyl group are disordered over two sites with an occupancy ratio of 0.664 (12):0.336 (12). In the crystal, inversion dimers linked by pairs of N—H⋯O hydrogen bonds occur and C—H⋯O bonds link the dimers into chains. A short C=O⋯π contact is also present

N′-[(E)-4-Hydr­oxy-3-methoxy­benzyl­idene]pyridine-4-carbohydrazide

In the title compound, C14H13N3O3, the two six-membered rings are oriented at a dihedral angle of 15.17 (11)° and an intra­molecular O—H⋯O hydrogen bond occurs. In the crystal, mol­ecules inter­act by way of N—H⋯O, O—H⋯N and C—H⋯O hydrogen bonds, thereby generating S(5) chain and R 2 1(7) ring motifs

N′-[(E)-4-Hydr­oxy-3-methoxy­benzyl­idene]benzohydrazide

In the title compound, C15H14N2O3, the phenyl ring is disordered over two set of sites with an occupancy ratio of 0.810 (3):0.190 (3); the dihedral angle between the two components is 72.3 (4)°. The benzene and phenyl rings are oriented at dihedral angles of 69.18 (8) and 26.0 (5)° (major and minor orientations, respectively), and an intra­molecular O—H⋯O hydrogen bond occurs. In the crystal, mol­ecules are linked by N—H⋯O, O—H⋯O and C—H⋯O inter­actions, generating a three-dimensional network

N′-[(E)-(1-Methyl-1H-pyrrol-2-yl)methyl­idene]pyridine-4-carbohydrazide. Corrigendum

Corrigendum to Acta Cryst. (2010), E66, o1881