Exploiting differences in caspase-2 and -3 S₂ subsites for selectivity: structure-based design, solid-phase synthesis and in vitro activity of novel substrate-based caspase-2 inhibitors

Several caspases have been implicated in the pathogenesis of Huntington's disease (HD); however, existing caspase inhibitors lack the selectivity required to investigate the specific involvement of individual caspases in the neuronal cell death associated with HD. In order to explore the potential role played by caspase-2, the potent but non-selective canonical Ac-VDVAD-CHO caspase-2 inhibitor 1 was rationally modified at the P(2) residue in an attempt to decrease its activity against caspase-3. With the aid of structural information on the caspase-2, and -3 active sites and molecular modeling, a 3-(S)-substituted-l-proline along with four additional scaffold variants were selected as P(2) elements for their predicted ability to clash sterically with a residue of the caspase-3 S(2) pocket. These elements were then incorporated by solid-phase synthesis into pentapeptide aldehydes 33a-v. Proline-based compound 33h bearing a bulky 3-(S)-substituent displayed advantageous characteristics in biochemical and cellular assays with 20- to 60-fold increased selectivity for caspase-2 and ～200-fold decreased caspase-3 potency compared to the reference inhibitor 1. Further optimization of this prototype compound may lead to the discovery of valuable pharmacological tools for the study of caspase-2 mediated cell death, particularly as it relates to HD.     

Viral genome segmentation can result from a trade-off between genetic content and particle stability

The evolutionary benefit of viral genome segmentation is a classical, yet unsolved question in evolutionary biology and RNA genetics. Theoretical studies anticipated that replication of shorter RNA segments could provide a replicative advantage over standard size genomes. However, this question has remained elusive to experimentalists because of the lack of a proper viral model system. Here we present a study with a stable segmented bipartite RNA virus and its ancestor non-segmented counterpart, in an identical genomic nucleotide sequence context. Results of RNA replication, protein expression, competition experiments, and inactivation of infectious particles point to a non-replicative trait, the particle stability, as the main driver of fitness gain of segmented genomes. Accordingly, measurements of the volume occupation of the genome inside viral capsids indicate that packaging shorter genomes involves a relaxation of the packaging density that is energetically favourable. The empirical observations are used to design a computational model that predicts the existence of a critical multiplicity of infection for domination of segmented over standard types. Our experiments suggest that viral segmented genomes may have arisen as a molecular solution for the trade-off between genome length and particle stability. Genome segmentation allows maximizing the genetic content without the detrimental effect in stability derived from incresing genome length.     

Microtriches of tetraphyllidean metacestodes from Western Mediterranean striped dolphins (Stenella coeruleoalba)

The tegumental structures of two types of tetraphyllidean plerocercoids and two types of merocercoids (Phyllobothrium delphini and Monorygma grimaldii) from Mediterranean striped dolphins, Stenella coeruleoalba, are described for the first time using scanning electron microscopy. The tegument of all of the specimens was fully covered with microtriches. Four basic types were found: filitriches, blade-like spinitriches of different sizes and shapes, cone-shaped spinitriches (with two parallel small projections of equal length at the apex), and crowned cylindrical spinitriches (with 6-7 small papillae forming a crown at the apex); the two latter types are newly described. The two types of plerocercoids had a similar morphology and distribution of microtriches; in addition, cilium-like projections appeared interspersed among the microtriches on the apical sucker, accessory suckers, and distal bothridial loculus. Merocercoids exhibited a greater variety of tegumental structures, especially on the distal bothridial loculus. Both merocercoid types had regularly spaced papillae or "buttons" on the accessory suckers and the distal bothridial loculus that were composed of a central cilium-like projection surrounded by numerous filitriches. However, crowned cylindrical spinitriches were specific to P. delphini and cone-shaped spinitriches were specific to M. grimaldii. Differences in the morphology and distribution of scolex microtriches of adult cestodes have been considered useful for species identification. A previous molecular study has suggested that P. delphini and M. grimaldii are actually different congeneric species. Our study has shown that significant differences in the morphology and distribution of microtriches occur between these species at the merocercoid stage.     

Food and forensic molecular identification: update and challenges

The need for accurate and reliable methods for animal species identification has steadily increased during past decades, particularly with the recent food scares and the overall crisis of biodiversity primarily resulting from the huge ongoing illegal traffic of endangered species. A relatively new biotechnological field, known as species molecular identification, based on the amplification and analysis of DNA, offers promising solutions. Indeed, despite the fact that retrieval and analysis of DNA in processed products is a real challenge, numerous technically consistent methods are now available and allow the detection of animal species in almost any organic substrate. However, this field is currently facing a turning point and should rely more on knowledge primarily from three fundamental fields--paleogenetics, molecular evolution and systematics.     

The crystal structure of the pyoverdine outer membrane receptor FpvA from Pseudomonas aeruginosa at 3.6 angstroms resolution

The pyoverdine outer membrane receptor FpvA from Pseudomonas aeruginosa translocates ferric-pyoverdine across the outer membrane via an energy consuming mechanism that involves the inner membrane energy transducing complex of TonB-ExbB-ExbD and the proton motive force. We solved the crystal structure of FpvA loaded with iron-free pyoverdine at 3.6 angstroms resolution. The pyoverdine receptor is folded in two domains: a transmembrane 22-stranded beta-barrel domain occluded by an N-terminal domain containing a mixed four-stranded beta-sheet (the plug). The beta-strands of the barrel are connected by long extracellular loops and short periplasmic turns. The iron-free pyoverdine is bound at the surface of the receptor in a pocket lined with aromatic residues while the extracellular loops do not completely cover the pyoverdine binding site. The TonB box, which is involved in intermolecular contacts with the TonB protein of the inner membrane, is observed in an extended conformation. Comparison of this first reported structure of an iron-siderophore transporter from a bacterium other than Escherichia coli with the known structures of the E.coli TonB-dependent transporters reveals a high structural homology and suggests that a common sensing mechanism exists for the iron-loading status in all bacterial iron siderophore transporters.     

Disruption of postsynaptic GABA receptor clusters leads to decreased GABAergic innervation of pyramidal neurons

We have used RNA interference (RNAi) to knock down the expression of the gamma2 subunit of the GABA(A) receptors (GABA(A)Rs) in pyramidal neurons in culture and in the intact brain. Two hairpin small interference RNAs (shRNAs) for the gamma2 subunit, one targeting the coding region and the other one the 3'-untranslated region (UTR) of the gamma2 mRNA, when introduced into cultured rat hippocampal pyramidal neurons, efficiently inhibited the synthesis of the GABA(A) receptor gamma2 subunit and the clustering of other GABA(A)R subunits and gephyrin in these cells. More significantly, this effect was accompanied by a reduction of the GABAergic innervation that these neurons received. In contrast, the gamma2 shRNAs had no effect on the clustering of postsynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, postsynaptic density protein 95 (PSD-95) or presynaptic glutamatergic innervation. A gamma2-enhanced green fluorescent protein (EGFP) subunit construct, whose mRNA did not contain the 3'-UTR targeted by gamma2 RNAi, rescued both the postsynaptic clustering of GABA(A)Rs and the GABAergic innervation. Decreased GABA(A)R clustering and GABAergic innervation of pyramidal neurons in the post-natal rat cerebral cortex was also observed after in utero transfection of these neurons with the gamma2 shRNAs. The results indicate that the postsynaptic clustering of GABA(A)Rs in pyramidal neurons is involved in the stabilization of the presynaptic GABAergic contacts.     

First report of an intraerythrocytic small piroplasm in wild Iberian lynx (Lynx pardinus)

A wild injured Iberian lynx (Lynx pardinus) was taken from the Sierra Morena population. During the health check small intraerythrocytic piroplasms, morphologically indistinguishable from other feline piroplasms, were observed in Wright-Giemsa-stained blood films. Amplification by polymerase chain reaction of a portion of the 18S nuclear small subunit (NSS) rRNA gene and sequencing revealed similarity of the unknown organism with sequences obtained from Pallas's cat from Mongolia and from a domestic cat in Spain. In a retrospective (1993-2003) study of 50 Iberian lynx tissue samples, no amplifications of the 18S NSS rRNA gene of the organism were obtained. This is the first report of a naturally occurring erythroparasitemia in the Iberian lynx and the first documented case of naturally occurring piroplasm infection in a free-ranging felid from Europe.     

Design and synthesis of potent pyridazine inhibitors of p38 MAP kinase

Novel potent trisubstituted pyridazine inhibitors of p38 MAP (mitogen activated protein) kinase are described that have activity in both cell-based assays of cytokine release and animal models of rheumatoid arthritis. They demonstrated potent inhibition of LPS-induced TNF-alpha production in mice and exhibited good efficacy in the rat collagen induced arthritis model.