Investigation of the function of delta-cadinene synthase with aza-analogues and site directedmutagenesis

Terpenes are one of the most structurally varied families of natural products with extraordinary chemical properties that have been exploited for numerous applications. Sesquiterpene synthases are a family of metal-dependent enzymes that catalyse the cyclisation of farnesyl diphosphate (FDP) into a myriad of complex C15-isoprenoid hydrocarbons, the sesquiterpenes. δ-Cadinene synthase (DCS) from Gossypium arboreum (cotton tree) catalyses the formation of δ-cadinene (DCN), a bicyclic intermediate in the biosynthesis of important phytoalexins such us gossypol. Two mechanistic proposals have been made for the formation of δ-cadinene: a 1,10-ring closure mechanism leading to the key intermediate germacradienyl cation, or a 1,6-ring closure leading to thealpha-bisabolyl carbocation. Previous investigation with fluorinated FDP analogues were in partial agreement with both scenarios and hence it was not possible to distinguish unambiguously between the two possible cyclisation reactions. To investigate the catalytic mechanism of DCS, enantiopure samples of the azaanalogues of alpha-bisabolyl cation and germacradienyl cation were needed. These compounds are designed as stable structural and electrostatic mimics of the putative short-lived carbocationic intermediates generated by terpene synthases, and hence often act as potent reversible competitive inhibitors (low Ki) of these enzymes. Here, the enantioselective total synthesis of R- and S- aza-analogues of the alpha-bisabolyl cation are described as well as the partial racemic synthesis of azagermacradienyl cation. Both enantiomers of aza-bisabolyl cation were goodmimics of α-bisabolene. They were competitive inhibitors of DCS, providing evidence for a 1,6-cyclisation closure. The second part of the project involved the investigation of the role of tryptophan 279 for the desolvation of the active site of DCS and therefore for the formation of DCN. Seven mutants of W279 were created. The data obtained showed that W279 is essential to prevent water from entering the active site and form the hydroxylate terpenoid germacradien-4-ol (GD4ol). Mutagenesis studies yielded a mutant, W279A, capable of making GD4ol as the sole product

Polyphenolic and Methylxanthine Bioaccessibility of Cocoa Bean Shell Functional Biscuits: Metabolomics Approach and Intestinal Permeability through Caco-2 Cell Models

Cocoa bean shell (CBS), a by-product with considerable concentrations of bioactive compounds and proven biofunctional potential, has been demonstrated to be a suitable ingredient for high-fiber functional biscuits adapted to diabetic consumers. In this work, the in vitro bioaccessibility and intestinal absorption of polyphenols and methylxanthines contained in these biscuits were evaluated, and the effect of the food matrix was studied. Biscuits containing CBS and the CBS alone underwent in vitro digestion followed by an intestinal permeability study. The results confirmed that compounds were less bioavailable in the presence of a food matrix, although the digestion contributed to their release from this matrix, increasing the concentrations available at the intestinal level and making them capable of promoting antioxidant and antidiabetic activities. After digestion, CBS biscuits were shown to possess α-glucosidase inhibition capacity comparable to that of acarbose. Moreover, the presence of the food matrix improved the stability of polyphenols throughout the digestion process. Intestinal absorption of flavan-3-ols seemed to be limited to a maximum threshold and was therefore independent of the sample, while procyanidin was not absorbed. Methylxanthine absorption was high and was boosted by the presence of the food matrix. The results confirmed the biofunctional potential of CBS-based biscuitsThe present work was supported by COVALFOOD “Valorisation of high added-value compounds from cocoa industry by-products as food ingredients and additives”. This project received funding from the European Union’s Seventh Framework Programme for research and innovation under the Marie Skłodowska-Curie grant agreement no. 609402—2020 researchers: Train to Move (T2M)S

Pharmacological evidence for the stimulation of NADPH oxidase by P2X7 receptors in mouse submandibular glands

ATP in the 100 μM-1 mM concentration range provoked a calcium-independent increase of the oxidation of dichlorodihydrofluorescein (DCFH) to dichlorofluorescein (DCF) by mouse submandibular cells. 3′-O-(4-benzoyl)benzoyl adenosine 5′-triphosphate (BzATP), a P2X7 agonist, but not a muscarinic or an adrenergic agonist, reproduced the effect of ATP. The inhibition of phospholipase C by U73122 or the potentiation of P2X4 receptor activation with ivermectin did not modify the response to ATP. ATP did not increase the oxidation of DCFH in cells isolated from submandibular glands of P2X7 knockout mice or in cells pretreated with a P2X7 antagonist. The inhibition of protein kinase C or of mitogen-activated protein kinase (MAP kinase) or of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase blocked the oxidation of DCFH without affecting the increase of the intracellular concentration of calcium or the uptake of ethidium bromide in response to extracellular ATP. From these results it is concluded that the activation of the P2X7 receptors from submandibular glands triggers an intracellular signalling cascade involving protein kinase C and MAP kinase leading to the stimulation of NADPH oxidase and the subsequent generation of reactive oxygen species

Régulation par l’ATP d’une phospholipase D présente dans les cellules ductales de glandes sous-maxillaires

ATP increased the PLD activity of rat submandibular ductal cells in a concentration-dependent and calcium-sensitive manner. Among ATP analogs, BZATP and ATPgammaS were the only ones able to stimulate the PLD activity. ATP and BZ-ATP also activated PLD in control mice, but not in mice lacking the P2X7 receptors. Oxidized ATP, Coomassie blue, NiCl2 and MgCl2 inhibited the response to ATP. PLD stimulation by ATP was inhibited by calphostin C and chelerythrine, two PKC inhibitors. The PLD stimulation by BZ-ATP and TPA, a phorbol ester which activates PKC, were not additive. Purinergic agonists did not increase the phosphorylation of proteins on tyrosyl residues. We can conclude that the activation of P2X7 receptors in rat submandibular glands ductal cells, is coupled to the activation of PLD. This activation is partly mediated by PKC and is not secondary to the activation of a tyrosine kinase.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Etude de l'activité phospholipase D dans les canaux de glandes sous-maxillaires :régulation par les agents purinergiques et muscariniques

Doctorat en sciences pharmaceutiquesinfo:eu-repo/semantics/nonPublishe

Effectors coupled to muscarinic receptors in rat submandibular ductal cells.

info:eu-repo/semantics/nonPublishe

Les glandes salivaires :Des glandes pleines de promesses

info:eu-repo/semantics/publishe

Development and Delphi validation of a Best Possible Medication History form

Objective: To develop and validate a standardized Best Possible Medication History (BPMH) form that could be used by clinical pharmacists. Methods: The draft version was presented to a focus group and was adapted following their comments. A three-rounds e-Delphi method was used to validate content, usability and face validity of the BPMH form. We supplemented the quantitative analysis with a qualitative analysis of comments for each Delphi round. Results: The draft BPMH form contained 23 items grouped into eight tabs. Refinement of these tabs and items by the focus group resulted in 7 tabs and 21 items, which were included in the Delphi survey. The consensus was obtained for all tabs within the second round (p=0.072). Consensus was reached on 76% (16/21) of items in the third round. 20 items were included following the qualitative analysis of the experts' comments in the third round. Conclusions: The findings of this study provide data on the content of the BPMH form. This form can be used to help clinical pharmacists to collect a complete and accurate medication list on admission. It could have an impact on inpatient safety and improve inpatient management. Studies with an international e-Delphi should be conducted for wider useSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Development and Delphi validation of a Best Possible Medication History form

BackgroundCompletion of the Best Possible Medication History (BPMH) form is the first step for medication reconciliation across the continuum of care. However, getting a complete picture of the patient’s current medication is a particularly difficult challenge.Methods The BPMH form was developed and refined by the research group. The draft was presented to a focus group and was adapted following their comments. A three-rounds e-Delphi method was used to validate content, usability and face validity of BPMH form. In the first round, items and tabs with 100% of expert rating ≥ 3 were retained for the final version of the BPMH form. At the beginning of the second round and the third round, results of previous rounds were provided to experts. Each expert confirmed or re-rated their level of agreement. Consensus was defined in the second round and the third round as 85% of experts rating items/tabs as 3 and greater. We supplemented the quantitative analysis with a qualitative analysis of comments for each Delphi round.ResultsThe draft BPMH form contained 23 items grouped into 8 tabs. Refinement of these tabs and items by the focus group resulted in 7 tabs and 21 items, which were included in the Delphi survey. The consensus was obtained for 6 tabs within the second round (p= 0,072). Consensus was reached on 76% (16/21) of items in the third round. 20 items were included following the qualitative analysis of the experts' comments in the third round.Conclusions This study provides the BPMH form, including 20 items arranged under six tabs. The systematic use of standardized BPMH form can be used to help clinical pharmacists to collect a complete and accurate medication list on admission. It could impact inpatient safety and improve inpatient management. The BPMH collecting process is the starting point for the medication reconciliation process which has the ability to reduce medication errors. An international e-Delphi should be conducted for wider use of the BPMH form.AcknowledgementsA particularly warm thank you goes to Delphi panels.info:eu-repo/semantics/publishe

Effects of sidestream cigarette smoke extract and homocysteine on vascular endothelial function

info:eu-repo/semantics/nonPublishe