Ceramide induces apoptosis in PC12 cells

AbstractThe novel lipid second messenger, ceramide, induced apoptosis in PC12 cells as determined morphologically by nuclear appearance and internucleosomal DNA fragmentation. Apoptosis was induced by exogenous C2-ceramide in a dose- and time-dependent manner. Natural ceramide and C6-ceramide had a similar effect. This response was specific since the structural analog C2-dihydroceramide and other related lipids failed to initiate apoptosis. The apoptotic effect of ceramide also depends critically on cell plating density. Furthermore, the peptide inhibitor of interleukin-1β converting enzyme (ICE)-like proteases, Z-VAD.FMK, completely prevented the nuclear changes induced by ceramide, implicating the involvement of ICE-like protease activation in ceramide-induced apoptosis in PC12 cells

The effects and interactions of GABAergic and dopaminergic agents in the prevention of form deprivation myopia by brief periods of normal vision

Intravitreal injections of GABA antagonists, dopamine agonists and brief periods of normal vision have been shown separately to inhibit form-deprivation myopia (FDM). Our study had three aims: (i) establish whether GABAergic agents modify the myopia protective effect of normal vision, (ii) investigate the receptor sub-type specificity of any observed effect, and (iii) consider an interaction with the dopamine (DA) system. Prior to the period of normal vision GABAergic agents were applied either (i) individually, (ii) in combination with other GABAergic agents (an agonist with an antagonist), or (iii) in combination with DA agonists and antagonists. Water injections were given to groups not receiving drug treatments so that all experimental eyes received intravitreal injections. As shown previously, constant form-deprivation resulted in high myopia and when diffusers were removed for 2 h per day the period of normal vision greatly reduced the FDM that developed. GABA agonists inhibited the protective effect of normal vision whereas antagonists had the opposite effect. GABAA/C agonists and D2 DA antagonists when used in combination were additive in suppressing the protective effect of normal vision. A D2 DA agonist restored some of the protective effect of normal vision that was inhibited by a GABA agonist (muscimol). The protective effect of normal vision against form-deprivation is modifiable by both the GABAergic and DAergic pathways.\u

Potent, multi-target serine protease inhibition achieved by a simplified beta-sheet motif

Engagement of an extended beta-sheet is a common substrate/inhibitor interaction at the active site of serine proteases and is an important feature of Laskowski mechanism inhibitors that present a substrate-like loop to a target protease. This loop is cleaved but subsequently relegated forming a stable inhibitor/protease complex. Laskowski inhibitors are ubiquitous in nature and are used extensively in serine protease inhibitor design. However, most studies concentrate on introducing new sidechain interactions rather than the direct contributions of the substrate-like beta-sheet to enzyme inhibition. Here we report the crystal structure of an simplified beta-sheet inhibitory motif within the Sunflower Trypsin Inhibitor (SFTI) in complex with trypsin. We show that the intramolecular hydrogen bond network of this SFTI variant (SFTI-TCTR) engages the inhibitor sidechains that would normally interact with a target protease, giving mainchain interactions a more prominent role in complex formation. Despite having reduced sidechain interactions, this SFTI variant is remarkably potent and inhibits a diverse range of serine proteases. Crystal structural analysis and molecular modelling of SFTI-TCTR complexes again indicates an interface dominated by beta-sheet interactions, highlighting the importance of this motif and the adaptability of SFTI as a scaffold for inhibitor design

A Versatile and Robust Serine Protease Inhibitor Scaffold from Actinia tenebrosa

Serine proteases play pivotal roles in normal physiology and a spectrum of patho-physiological processes. Accordingly, there is considerable interest in the discovery and design of potent serine protease inhibitors for therapeutic applications. This led to concerted efforts to discover versatile and robust molecular scaffolds for inhibitor design. This investigation is a bioprospecting study that aims to isolate and identify protease inhibitors from the cnidarian Actinia tenebrosa. The study isolated two Kunitz-type protease inhibitors with very similar sequences but quite divergent inhibitory potencies when assayed against bovine trypsin, chymostrypsin, and a selection of human sequence-related peptidases. Homology modeling and molecular dynamics simulations of these inhibitors in complex with their targets were carried out and, collectively, these methodologies enabled the definition of a versatile scaffold for inhibitor design. Thermal denaturation studies showed that the inhibitors were remarkably robust. To gain a fine-grained map of the residues responsible for this stability, we conducted in silico alanine scanning and quantified individual residue contributions to the inhibitor’s stability. Sequences of these inhibitors were then used to search for Kunitz homologs in an A. tenebrosa transcriptome library, resulting in the discovery of a further 14 related sequences. Consensus analysis of these variants identified a rich molecular diversity of Kunitz domains and expanded the palette of potential residue substitutions for rational inhibitor design using this domain

Determinants of genetic diversity

International audienceGenetic polymorphism varies among species and within genomes, and has important implications for the evolution and conservation of species. The determinants of this variation have been poorly understood, but population genomic data from a wide range of organisms now make it possible to delineate the underlying evolutionary processes, notably how variation in the effective population size (Ne) governs genetic diversity. Comparative population genomics is on its way to providing a solution to 'Lewontin's paradox' — the discrepancy between the many orders of magnitude of variation in population size and the much narrower distribution of diversity levels. It seems that linked selection plays an important part both in the overall genetic diversity of a species and in the variation in diversity within the genome. Genetic diversity also seems to be predictable from the life history of a species