Tracking the effects of one century of habitat loss and fragmentation on calcareous grassland butterfly communities

Habitat loss and fragmentation are known to reduce patch sizes and increase their isolation, consequently leading to modifications in species richness and community structure. Calcareous grasslands are among the richest ecosystems in Europe for insect species. About 10% (1,150 ha) of the total area of a calcareous ridge region (Calestienne, Belgium) and its butterfly community was analysed over a timeframe of about 100 years. Since 1905 to present day (2005), the Calestienne region has undergone both calcareous grassland loss and fragmentation: not only did calcareous grassland size decrease and isolation increase, but also, the number of calcareous grassland patches within the landscape increased until 1965, and subsequently decreased, clearly reflecting the effects of fragmentation. These processes have had a profound effect on the butterfly community: extinction and rarefaction affected significantly more often specialist species, which means that generalist species are more and more overrepresented. This ecological drift, i.e. the replacement of specialists by generalists in species assemblages is likely to be a general effect of habitat loss and fragmentation on natural communities.     

Synthesis and biological activation of an ethylene glycol-linked amino acid conjugate of cyclic cidofovir

Cidofovir (HPMPC) is a broad-spectrum anti-viral agent whose potential, particularly in biodefense scenarios, is limited by its low oral bioavailability. Two prodrugs (3 and 4) created by conjugating ethylene glycol-linked amino acids (L-Val, L-Phe) with the cyclic form of cidofovir (cHPMPC) via a P-O ester bond were synthesized and their pH-dependent stability (3 and 4), potential for in vivo reconversion to drug (3), and oral bioavailability (3) were evaluated. The prodrugs were stable in buffer between pH 3 and 5, but underwent rapid hydrolysis in liver (t(1/2) = 3.7 min), intestinal (t(1/2) = 12.5 min), and Caco-2 cell homogenates (t(1/2) = 20.2 min). In vivo (rat), prodrug 3 was >90% reconverted to cHPMPC. The prodrug was 4x more active than ganciclovir (IC50 value, 0.68 microM vs 3.0 microM) in a HCMV plaque reduction assay. However, its oral bioavailability in a rat model was similar to the parent drug. The contrast between the promising activation properties and unenhanced transport of the prodrug is briefly discussed.     

Fluorescence spectroscopic and (19)f NMR studies of human thymidylate synthase with its cognate RNA

In order to investigate the interaction between hTS protein and its cognate mRNA, a 29nt fragment of TS mRNA was synthesized. This region has been suggested as a putative stem-loop involved in translational autoregulation. The melting temperature of the 29ntRNA was 65 degrees C, suggesting that this region does indeed form a stem-loop. Fluorescence spectroscopy was used to monitor the RNA: hTS protein interaction [dissociation constant (K(d)) 3.9 +/- 0.8 nM; stoichiometry of binding 1dimeric hTS: 1RNA]. When hTS was titrated against FdUMP, this gave the expected stoichiometry of 1dimeric hTS: 1.7 FdUMP but in the presence of the 29ntRNA, the stoichiometry of binding changed to 1dimeric hTS: 1RNA: 1FdUMP. Experiments using methotrexate (MTX) gave a stoichiometry of 1dimeric hTS: 1MTX and in the presence of 29ntRNA, the stoichiometry was unchanged. (19)F-NMR spectra of human TS: FdUMP complexes were found to be strikingly similar to analogous NMR spectra of complexes formed by L.casei TS and mouse TS. In the presence of FdUMP, spectra exhibited two additional resonances (-1.50 ppm and -34.4 ppm). The resonance at -1.50 ppm represents non-covalently bound FdUMP, the peak at -34.4 ppm represents covalently bound FdUMP. The addition of methotrexate to the binary TS-FdUMP complex caused a displacement of the internal equilibrium, with only the covalently-bound form seen, and with a slightly disturbed (19)F chemical shift (-36.5 ppm). Similar results were found when MTX was replaced by folinic or folic acid. The addition of 29ntRNA caused no changes to the (19)F spectra of either the binary or ternary complexes.     

The type III secretion system needle tip complex mediates host cell sensing and translocon insertion

Type III secretion systems (T3SSs) are essential virulence determinants of many Gram-negative bacterial pathogens. The Shigella T3SS consists of a cytoplasmic bulb, a transmembrane region and a hollow 'needle' protruding from the bacterial surface. Physical contact with host cells initiates secretion and leads to assembly of a pore, formed by IpaB and IpaC, in the host cell membrane, through which proteins that facilitate host cell invasion are translocated. As the needle is implicated in host cell sensing and secretion regulation, its tip should contain components that initiate host cell contact. Through biochemical and immunological studies of wild-type and mutant Shigella T3SS needles, we reveal tip complexes of differing compositions and functional states, which appear to represent the molecular events surrounding host cell sensing and pore formation. Our studies indicate that the interaction between IpaB and IpaD at needle tips is key to host cell sensing, orchestration of IpaC secretion and its subsequent assembly at needle tips. This allows insertion into the host cell membrane of a translocation pore that is continuous with the needle.     

Intracellular survival of Burkholderia cenocepacia in macrophages is associated with a delay in the maturation of bacteria-containing vacuoles

Strains of the Burkholderia cepacia complex (Bcc) are opportunistic bacteria that can cause life-threatening infections in patients with cystic fibrosis and chronic granulomatous disease. Previous work has shown that Bcc isolates can persist in membrane-bound vacuoles within amoeba and macrophages without bacterial replication, but the detailed mechanism of bacterial persistence is unknown. In this study, we have investigated the survival of the Burkholderia cenocepacia strain J2315 within RAW264.7 murine macrophages. Strain J2315 is a prototypic isolate of the widespread and transmissible ET12 clone. Unlike heat-inactivated bacteria, which reach lysosomes shortly after internalization, vacuoles containing live B. cenocepacia J2315 accumulate the late endosome/lysosome marker LAMP-1 and start fusing with lysosomal compartments only after 6 h post internalization. Using fluorescent fluid-phase probes, we also demonstrated that B. cenocepacia-containing vacuoles continued to interact with newly formed endosomes, and maintained a luminal pH of 6.4 +/- 0.12. In contrast, vacuoles containing heat-inactivated bacteria had an average pH of 4.8 +/- 0.03 and rapidly merged with lysosomes. Additional experiments using concanamycin A, a specific inhibitor of the vacuolar H+-ATPase, revealed that vacuoles containing live bacteria did not exclude the H+-ATPase. This mode of bacterial survival did not require type III secretion, as no differences were found between wild type and a type III secretion mutant strain. Collectively, our results suggest that intracellular B. cenocepacia cause a delay in the maturation of the phagosome, which may contribute to facilitate bacterial escape from the microbicidal activities of the host cell.     

Alkyne-azide click chemistry mediated carbanucleosides synthesis

Hitherto unknown 1,4-disubstituted-[1,2,3]-triazolo-4',4'-dihydroxymethyl-3'-deoxy carbanucleosides were synthesized based on a "click approach." Various alkynes were introduced on a key azido intermediate by the "click" 1,3-dipolar Huisgen cycloaddition. Their antiviral activities and cellular toxicities were evaluated on vaccinia virus. None of the synthesized compounds exhibited a significant antiviral activity.