MPX-004 and MPX-007: New Pharmacological Tools to Study the Physiology of NMDA Receptors Containing the GluN2A Subunit

GluN2A is the most abundant of the GluN2 NMDA receptor subunits in the mammalian CNS. Physiological and genetic evidence implicate GluN2A-containing receptors in susceptibility to autism, schizophrenia, childhood epilepsy and neurodevelopmental disorders such as Rett Syndrome. However, GluN2A-selective pharmacological probes to explore the therapeutic potential of targeting these receptors have been lacking. Here we disclose a novel series of pyrazine-containing GluN2A antagonists exemplified by MPX-004 (5-(((3-chloro-4-fluorophenyl)sulfonamido)methyl)-N-((2-methylthiazol-5-yl)methyl)pyrazine-2-carboxamide) and MPX-007 (5-(((3-fluoro-4-fluorophenyl)sulfonamido)methyl)-N-((2-methylthiazol-5-yl)methyl)methylpyrazine-2-carboxamide). MPX-004 and MPX-007 inhibit GluN2A-containing NMDA receptors expressed in HEK cells with IC₅₀s of 79 nM and 27 nM, respectively. In contrast, at concentrations that completely inhibited GluN2A activity these compounds have no inhibitory effect on GluN2B or GluN2D receptor-mediated responses in similar HEK cell-based assays. Potency and selectivity were confirmed in electrophysiology assays in Xenopus oocytes expressing GluN2A-D receptor subtypes. Maximal concentrations of MPX-004 and MPX-007 inhibited ~30% of the whole-cell current in rat pyramidal neurons in primary culture and MPX-004 inhibited ~60% of the total NMDA receptor-mediated EPSP in rat hippocampal slices. GluN2A-selectivity at native receptors was confirmed by the finding that MPX-004 had no inhibitory effect on NMDA receptor mediated synaptic currents in cortical slices from GRIN2A knock out mice. Thus, MPX-004 and MPX-007 offer highly selective pharmacological tools to probe GluN2A physiology and involvement in neuropsychiatric and developmental disorders.     

Patterns of Genetic Variability in Island Populations of the Cane Toad (Rhinella marina) from the Mouth of the Amazon

The Amazonian coast has several unique geological characteristics resulting from the interaction between drainage pattern of the Amazon River and the Atlantic Ocean. It is one of the most extensive and sedimentologically dynamic regions of the world, with a large number of continental islands mostly formed less than 10,000 years ago. The natural distribution of the cane toad (Rhinella marina), one of the world’s most successful invasive species, in this complex Amazonian system provides an intriguing model for the investigation of the effects of isolation or the combined effects of isolation and habitat dynamic changes on patterns of genetic variability and population differentiation. We used nine fast-evolving microsatellite loci to contrast patterns of genetic variability in six coastal (three mainlands and three islands) populations of the cane toad near the mouth of the Amazon River. Results from Bayesian multilocus clustering approach and Discriminant Analyses of Principal Component were congruent in showing that each island population was genetically differentiated from the mainland populations. All F_ST values obtained from all pairwise comparisons were significant, ranging from 0.048 to 0.186. Estimates of both recent and historical gene flow were not significantly different from zero across all population pairs, except the two mainland populations inhabiting continuous habitats. Patterns of population differentiation, with a high level of population substructure and absence/restricted gene flow, suggested that island populations of R. marina are likely isolated since the Holocene sea-level rise. However, considering the similar levels of genetic variability found in both island and mainland populations, it is reliable to assume that they were also isolated for longer periods. Given the genetic uniqueness of each cane toad population, together with the high natural vulnerability of the coastal regions and intense human pressures, we suggest that these populations should be treated as discrete units for conservation management purposes.     

New Insights on Wood Dimensional Stability Influenced by Secondary Metabolites: The Case of a Fast-Growing Tropical Species Bagassa guianensis Aubl.

Challenging evaluation of tropical forest biodiversity requires the reporting of taxonomic diversity but also the systematic characterization of wood properties in order to discover new promising species for timber industry. Among wood properties, the dimensional stability is regarded as a major technological characteristic to validate whether a wood species is adapted to commercial uses. Cell structure and organization are known to influence the drying shrinkage making wood density and microfibrils angle markers of choice to predict wood dimensional stability. On the contrary the role of wood extractive content remains unclear. This work focuses on the fast-growing tropical species Bagassa guianensis and we report herein a correlation between heartwood drying shrinkage and extractive content. Chemical extractions and shrinkage experiments were performed on separate wood twin samples to better evaluate correctly how secondary metabolites influence the wood shrinkage behaviour. Extractive content were qualitatively and quantitatively analysed using HPLC and NMR spectroscopy. We found that B guianensis heartwood has a homogeneous low shrinkage along its radius that could not be explained only by its basic density. In fact the low drying shrinkage is correlated to the high extractive content and a corrected model to improve the prediction of wood dimensional stability is presented. Additionally NMR experiments conducted on sapwood and heartwood extracts demonstrate that secondary metabolites biosynthesis occurs in sapwood thus revealing B. guianensis as a Juglans-Type heartwood formation. This work demonstrates that B. guianensis, a fast-growing species associated with high durability and high dimensional stability, is a good candidate for lumber production and commercial purposes.     

Mammalian prion amyloid formation in bacteria

Mammalian prion proteins (PrPs) that cause transmissible spongiform encephalopathies are misfolded conformations of the host cellular PrP. The misfolded form, the scrapie PrP (PrP^Sc), can aggregate into amyloid fibrils that progressively accumulate in the brain, evolving to a pathological phenotype. A particular characteristic of PrP^Sc is to be found as different strains, related to the diversity of conformational states it can adopt. Prion strains are responsible for the multiple phenotypes observed in prion diseases, presenting different incubation times and diverse deposition profiles in the brain. PrP biochemical properties are also strain-dependent, such as different digestion pattern after proteolysis and different stability. Although they have long been studied, strain formation is still a major unsolved issue in prion biology. The recreation of strain-specific conformational features is of fundamental importance to study this unique pathogenic phenomenon. In our recent paper, we described that murine PrP, when expressed in bacteria, forms amyloid inclusion bodies that possess different strain-like characteristics, depending on the PrP construct. Here, we present an extra-view of these data and propose that bacteria might become a successful model to generate preparative amounts of prion strain-specific assemblies for high-resolution structural analysis as well as for addressing the determinants of infectivity and transmissibility.     

Life With or Without Names

The terms ‘life’, ‘species’ and ‘individuals’ are key concepts in biology. However, theoretical and practical concerns are directly associated with definitions of these terms and their use in researchers’ work. Although the practical implications of employing definition of ‘species’ and ‘individuals’ are often clear, it is surprising how most biologists work in their field of study without adhering to a specific definition of life. In everyday scientific practice, biologists rarely define life. This is somewhat understandable: the majority of biologists accept the standard definition of life without exploring it, but this represents a bad attitude. In this essay, we update the concepts of life, species, and individuals in the light of the new techniques for massive DNA sequencing collectively known as high throughput DNA sequencing (HTS). A re-evaluation of the newest approaches and traditional concepts is required, because in many scientific publications, HTS users apply concepts ambiguously (in particular that of species). However, the absence of clarity is understandable. For most of the last 250 years, from Linnaeus to the most recent researches, identification and classification have been performed applying the same process. On the contrary, through HTS, biologists have become simply identifiers, who construct boundaries around the biological entities and do not examine the taxa at length, resulting in uncertainty in most readers and displeasure in traditional taxonomists. We organised our essay to answer a basic question: can we develop new means to observe living organisms?     

A constitutive expression system for <Emphasis Type="Italic">Pichia pastoris</Emphasis> based on the <Emphasis Type="Italic">PGK1</Emphasis> promoter

Objectives

To develop a new vector for constitutive expression in Pichia pastoris based on the endogenous glycolytic PGK1 promoter.

Results

P. pastoris plasmids bearing at least 415 bp of PGK1 promoter sequences can be used to drive plasmid integration by addition at this locus without affecting cell growth. Based on this result, a new P. pastoris integrative vector, pPICK2, was constructed bearing some features that facilitate protein production in this yeast: a ~620 bp PGK1 promoter fragment with three options of restriction sites for plasmid linearization prior to yeast transformation: a codon-optimized α-factor secretion signal, a new polylinker, and the kan marker for vector propagation in bacteria and selection of yeast transformants.

Conclusions

A new constitutive vector for P. pastoris represents an alternative platform for recombinant protein production and metabolic engineering purposes.