Thiol-reactive analogues of galanthamine, codeine and morphine as potential probes to interrogate allosteric binding within nAChRs

Alkaloids including galanthamine (1) and codeine (2) are reported to be positive allosteric modulators of nicotinic acetylcholine receptors (nAChRs) but the binding sites responsible for this activity are not known with certainty. Analogues of galanthamine (1), codeine (2) and morphine (3) with reactivity towards cysteine thiols were synthesised including conjugated enone derivatives of the three alkaloids 4-6 and two chloro-alkane derivatives of codeine 7 and 8. The stability of the enones was deemed sufficient for use in buffered aqueous solutions and their reactivity towards thiols was assessed by determining the kinetics of reaction with a cysteine derivative. All three enone derivatives were of sufficient reactivity and stability to be used in covalent trapping, an extension of the substituted cysteine accessibility method (SCAM), to elucidate the allosteric binding sites of galanthamine and codeine at nAChRs

Alpha9 nicotinic acetylcholine receptors and the treatment of pain

Chronic pain is a vexing worldwide problem that causes substantial disability and consumes significant medical resources. Although there are numerous analgesic medications, these work through a small set of molecular mechanisms. Even when these medications are used in combination, substantial amounts of pain often remain. It is therefore highly desirable to develop treatments that work through distinct mechanisms of action. While agonists of nicotinic acetylcholine receptors (nAChRs) have been intensively studied, new data suggest a role for selective antagonists of nAChRs. α-Conotoxins are small peptides used offensively by carnivorous marine snails known as Conus. A subset of these peptides known as α-conotoxins RgIA and Vc1.1 produces both acute and long lasting analgesia. In addition, these peptides appear to accelerate the recovery of function after nerve injury, possibly through immune mediated mechanisms. Pharmacological analysis indicates that RgIA and Vc1.1 are selective antagonists of α9α10 nAChRs. A recent study also reported that these α9α10 antagonists are also potent GABA-B agonists. In the current study, we were unable to detect RgIA or Vc1.1 binding to or action on cloned GABA-B receptors expressed in HEK cells or Xenopus oocytes. We review the background, findings and implications of use of compounds that act on α9* nAChRs.11* indicates the possible presence of additional subunits.Fil: McIntosh, J. Michael. University of Utah; Estados UnidosFil: Absalom, Nathan. The University of Sydney; AustraliaFil: Chebib, Mary. The University of Sydney; AustraliaFil: Elgoyhen, Ana Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Farmacología; ArgentinaFil: Vincler, Michelle. Wake Forest University Health Sciences; Estados Unido

Efficiency considerations in the construction of interpolated potential energy surfaces for the calculation of quantum observables by diffusion Monte Carlo

A modified Shepard interpolation scheme is used to construct global potential energy surfaces (PES) in order to calculate quantum observables--vibrationally averaged internal coordinates, fully anharmonic zero-point energies and nuclear radial distribution functions--for a prototypical loosely bound molecular system, the water dimer. The efficiency of PES construction is examined with respect to (a) the method used to sample configurational space, (b) the method used to choose which points to add to the PES data set, and (c) the use of either a one- or two-part weight function. The most efficient method for constructing the PES is found to require a quantum sampling regime, a combination of both h-weight and rms methods for choosing data points and use of the two-part weight function in the interpolation. Using this regime, the quantum diffusion Monte Carlo zero-point energy converges to the exact result within addition of 50 data points. The vibrationally averaged O-O distance and O-O radial distribution function, however, converge more slowly and require addition of over 500 data points. The methods presented here are expected to be applicable to both other loosely bound complexes as well as tightly bound molecular species. When combined with high quality ab initio calculations, these methods should be able to accurately characterize the PES of such species.D.L.C. would like to acknowledge the financial support of an Australian Postgraduate Research Award. This work has also been supported by Large Grant No. A00104447 from the Australian Research Council and by grants of computer time from the Australian Partnership in Advanced Computing (APAC) National Merit Allocation Scheme

發作性血色素尿症ニ就テ

The α4β2 nicotinic acetylcholine receptor (nAChR) is the most abundant subtype in the brain and exists in two functional stoichiometries: (α4)3(β2)2 and (α4)2(β2)3. A distinct feature of the (α4)3(β2)2 receptor is the biphasic activation response to the endogenous agonist acetylcholine, where it is activated with high potency and low efficacy when two α4-β2 binding sites are occupied and with low potency/high efficacy when a third α4-α4 binding site is occupied. Further, exogenous ligands can bind to the third α4-α4 binding site and potentiate the activation of the receptor by ACh that is bound at the two α4-β2 sites. We propose that perturbations of the recently described pre-activation step when a third binding site is occupied are a key driver of these distinct activation properties. To investigate this, we used a combination of simple linear kinetic models and voltage clamp electrophysiology to determine whether transitions into the pre-activated state were increased when three binding sites were occupied. We separated the binding at the two different sites with ligands selective for the α4-β2 site (Sazetidine-A and TC-2559) and the α4-α4 site (NS9283) and identified that when a third binding site was occupied, changes in the concentration-response curves were best explained by an increase in transitions into a pre-activated state. We propose that perturbations of transitions into a pre-activated state are essential to explain the activation properties of the (α4)3(β2)2 receptor by acetylcholine and other ligands. Considering the widespread clinical use of benzodiazepines, this discovery of a conserved mechanism that benzodiazepines and ACh potentiate receptor activation via a third binding site can be exploited to develop therapeutics with similar properties at other cys-loop receptors

Prognostic Factors in Dedifferentiated Chondrosarcoma: A Retrospective Analysis of a Large Series Treated at a Single Institution.

Background:Dedifferentiated chondrosarcomas (DDCSs) are highly malignant tumors with a dismal prognosis and present a significant challenge in clinical management. Methods:In an IRB approved retrospective protocol, we identified 72 patients with DDCS treated at our institution between 1993 and 2017 and reviewed clinicopathological characteristics, treatment modalities, and outcomes to analyze prognostic factors. Results:Femur (44.4%), pelvis (22.2%), and humerus (12.5%) were most commonly involved sites. Twenty-three patients (31.9%) presented with distant metastasis, and 3 (4.2%) of them also had regional lymph node involvement. The median overall survival (OS) was 13.9 months. On multivariate analysis, pathological fracture, larger tumor size, lymph node involvement, metastasis at diagnosis, extraosseous extension, and undifferentiated pleomorphic sarcoma component correlated with worse OS, whereas surgical resection and chemotherapy were associated with improved OS. For progression-free survival (PFS), pathological fracture and metastasis at diagnosis showed increased risk, while chemotherapy was associated with decreased risk. Among patients who received chemotherapy, doxorubicin and cisplatin were significantly associated with improved PFS but not OS. Among patients without metastasis at diagnosis, 17 (34.7%) developed local recurrence. Thirty-one (63.3%) developed distant metastases at a median interval of 18.1 months. On multivariate analysis, R1/R2 resection was related with local recurrence, while macroscopic dedifferentiated component was associated with distant metastasis. Conclusions:The prognosis of DDCS is poor. Complete resection remains a significant prognostic factor for local control. Chemotherapy with doxorubicin and cisplatin seems to have better PFS. More prognostic, multicenter trials are warranted to further explore the effectiveness of chemotherapy in selected DDCS patients

Characterization of different DLC and DLN electrodes for biosensor design

International audienceDiamond-Like Carbon and Carbon-Like Nanocomposite electrodes, novel materials in the field of biosensors, made with different ratio of sp3/sp2 carbon hybridization or doped with elements such as Ni, Si and W, were characterized electrochemically by cyclic voltammetry and by amperometric measurements towards hydrogen peroxide. SiCAr1 and SiCNi5% were chosen as sensitive transducers for elaboration of amperometric glucose biosensors. Immobilization of glucose oxidase was carried out by cross-linking with glutareldehyde. Measurements were made at a fixed potential + 1.0 V in 40 mM phosphate buffer pH 7.4. SiCAr1 seems to be more sensitive for glucose (0.6875 µA/mM) then SiCNi5% (0.3654 µA/mM). Detections limits were respectively 20 µM and 30 µM. Michaelis-Menten constants for the two electrodes were found around 3 mM. 48% and 79% of the original response for 0.5 mM glucose remained respectively for both electrodes after 10 days

Generation and phenotypic characterization of Pde1a mutant mice

Contains fulltext : 177029.pdf (publisher's version ) (Open Access)It has been proposed that a reduction in intracellular calcium causes an increase in intracellular cAMP and PKA activity through stimulation of calcium inhibitable adenylyl cyclase 6 and inhibition of phosphodiesterase 1 (PDE1), the main enzymes generating and degrading cAMP in the distal nephron and collecting duct, thus contributing to the development and progression of autosomal dominant polycystic kidney disease (ADPKD). In zebrafish pde1a depletion aggravates and overexpression ameliorates the cystic phenotype. To study the role of PDE1A in a mammalian system, we used a TALEN pair to Pde1a exon 7, targeting the histidine-aspartic acid dipeptide involved in ligating the active site Zn++ ion to generate two Pde1a null mouse lines. Pde1a mutants had a mild renal cystic disease and a urine concentrating defect (associated with upregulation of PDE4 activity and decreased protein kinase A dependent phosphorylation of aquaporin-2) on a wild-type genetic background and aggravated renal cystic disease on a Pkd2WS25/- background. Pde1a mutants additionally had lower aortic blood pressure and increased left ventricular (LV) ejection fraction, without a change in LV mass index, consistent with the high aortic and low cardiac expression of Pde1a in wild-type mice. These results support an important role of PDE1A in the renal pathogenesis of ADPKD and in the regulation of blood pressure

The relative impact of evolving pleiotropy and mutational correlation on trait divergence

Both pleiotropic connectivity and mutational correlations can restrict the decoupling of traits under divergent selection, but it is unknown which is more important in trait evolution. To address this question, we create a model that permits within-population variation in both pleiotropic connectivity and mutational correlation, and compare their relative importance to trait evolution. Specifically, we developed an individual-based stochastic model where mutations can affect whether a locus affects a trait and the extent of mutational correlations in a population. We find that traits can decouple whether there is evolution in pleiotropic connectivity or mutational correlation, but when both can evolve, then evolution in pleiotropic connectivity is more likely to allow for decoupling to occur. The most common genotype found in this case is characterized by having one locus that maintains connectivity to all traits and another that loses connectivity to the traits under stabilizing selection (subfunctionalization). This genotype is favored because it allows the subfunctionalized locus to accumulate greater effect size alleles, contributing to increasingly divergent trait values in the traits under divergent selection without changing the trait values of the other traits (genetic modularization). These results provide evidence that partial subfunctionalization of pleiotropic loci may be a common mechanism of trait decoupling under regimes of corridor selection.Peer reviewe