Trimeric structure of OprN and OprM efflux proteins from Pseudomonas aeruginosa, by 2D electron crystallography

OprM and OprN belong to the outer membrane factor family of multidrug efflux proteins from Pseudomonas aeruginosa, a bacterium responsible of nosocomial infections. We report here the two-dimensional (2D) crystallization of OprN and OprM into lipid bilayers and the determination of their 2D projected structure by cryo-electron crystallography, at 1 and 1.4 nm, respectively. Both proteins present a dense ring of protein density, of approximately 7 nm diameter. An additional thin peripheral ring is resolved in OprN structure. Both proteins are assembled as trimers. The results presented here indicate a high structural homology between OprN (and OprM) and TolC, a multidrug efflux protein from Escherichia coli.     

Femoral intra-arterial injection: a tool to deliver and assess recombinant AAV constructs in rodents whole hind limb

With the aim of simplifying recombinant-adeno-associated virus (rAAV) delivery in muscle, a new femoral intra-arterial technique was designed and tested in rodents (rats and mice). Two serotypes, several promoters and transgenes (reporter or therapeutic) were tested using this administration route. The new route is both easy to perform and efficient. Its usefulness as a tool to assess gene delivery constructs in the muscle was established in the context of recombinant AAV serotypes 1 and 2, and with the ubiquitous CMV and two muscle-specific (C5-12 and CK6) promoters. Both serum monitoring of a secreted protein (murine alkaline phosphatase: muSEAP) and slide staining were used to compare the different constructs. Significantly different patterns of expression in kinetics of expression (muSEAP) and homogeneity of fiber transduction (staining) were evidenced with the different promoters tested, and compared with intra-muscular expression patterns. Detailed studies of differential transduction in leg and thigh muscles showed equivalent efficacy, except in rectus femoris, and to a lesser extent in soleus. In light of these results and prior data, intra-arterially mediated gene transfer mechanism is discussed.     

Immobilization induces alterations in the outer membrane protein pattern of Yersinia ruckeri

We compared the outer membrane protein (OMP) pattern of 2-day-old immobilized Yersinia ruckericells (IC) with that of early (FC24) and late (FC48) stationary-phase planktonic counterparts. Fifty-five OMPs were identified. Principal component analysis discriminated between the protein maps of FC and IC. Some OMPs involved in bacterial adaptation were accumulated by both FC48 and IC but the expression of other proteins was controlled by the sessile mode of growth.     

Genetic structure of the feral cat (Felis catus L.) introduced 50 years ago to a sub-Antarctic island

Information about the invasion dynamics and demographic status of invasive species is essential to choose the optimal control options of population numbers. While long-term direct demographic and historical records are generally lacking, the analysis of the genetic variability of a current population might supply information about past and current demographic processes. In this study, we analysed the genetic variability of the cat population living on the main island of the Kerguelen archipelago. Genetic diversity was consistent with the introduction of a very small number of individuals followed by a demographic explosion of the cat population. Significant genetic structure among sites (F_st=0.06 ±0.005) and absence of isolation by distance could indicate that the initial phase of fast colonisation is now over. Estimates of individual relatedness indicated a significant kin structure. Overall data suggested that the cat population of the main island has probably reached carrying capacity.     

Site-specific incorporation of N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF) into oligonucleotides using modified 'ultra-mild' DNA synthesis

Aromatic amino and nitro compounds are potent carcinogens found in the environment that exert their toxic effects by reacting with DNA following metabolic activation. One important adduct is N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF), which has been extensively used in studies of the mechanisms of DNA repair and mutagenesis. Despite the importance of dG-AAF adducts in DNA, an efficient method for its incorporation into DNA using solid-phase synthesis is still missing. We report the development of a modified ‘ultra-mild’ DNA synthesis protocol that allows the incorporation of dG-AAF into oligonucleotides of any length accessible by solid-phase DNA synthesis with high efficiency and independent of sequence context. Key to this endeavor was the development of improved deprotection conditions (10% diisopropylamine in methanol supplemented with 0.25 M of β-mercaptoethanol) designed to remove protecting groups of commercially available ‘ultra-mild’ phosphoramidite building blocks without compromising the integrity of the exquisitely base-labile acetyl group at N8 of dG-AAF. We demonstrate the suitability of these oligonucleotides in the nucleotide excision repair reaction. Our synthetic approach should facilitate comprehensive studies of the mechanisms of repair and mutagenesis induced by dG-AAF adducts in DNA and should be of general use for the incorporation of base-labile functionalities into DNA.     

Spatio-temporal variation in cat population density in a sub-Antarctic environment

We used the walked-line transect method for estimating the density of cats and coefficients of variation of density estimates in 4 contrasted sites on the main island of Îles Kerguelen between 1998 and 2000. Density estimates varied from 0.44ǂ.15 cats per km² to 2.42ǂ.23 cats per km² according to site and period. Coefficient of variation of density estimates ranged from 11.92% to 34.76%. The line transect method was, therefore, an efficient method for monitoring the density of the cat population in a sub-Antarctic environment characterised by short vegetation. Our results suggest that cat population size at the main island of Îles Kerguelen (the total number of cats expected is around 7,000) is much lower than previously thought.     

Raspberry vesicles

Cell swelling following hypoosmotic stress leads to the activation of volume-sensitive ion channels that allow a K+ and Cl- efflux accompanied by water loss. A Ca2+-insensitive K+ channel (I(K,vol)) has been described in Ehrlich cells that can be activated by hypotonicity and leukotriene D4 and is inhibited by clofilium. We have studied the activation and deactivation by osmotic stimuli of this channel. A G-protein appears to be involved in these processes since GTP-gamma-S accelerates deactivation, while GDP-beta-S blocks the channel in the open state, a result mimicked by pertussis toxin (PTX). In addition, PTX accelerates the onset of I(K,vol). We propose that I(K,vol) is tonically inhibited by a PTX-sensitive G-protein.     

Tree and shrub diversity and abundance in fragmented littoral forest of southeastern Madagascar

In the coastal littoral forest of extreme southeastern Madagascar, westudied tree diameter at breast height (DBH) 10 cm in 20, 50× 50 m plots in each of four forest fragments, andunderstorywoody vegetation (DBH < 10 cm, 1 m tall) in60,10 × 10 m plots in three of the fragments. Oneforestfragment was located in the highly degraded Lokaro region, and three in the nearbySainte-Luce forest. Atotal of 3476 trees, representing 169 species in 55 families, were recorded inthe50 × 50 m plots, and 10282 understory stems, representing195 species in 54 families, were found in the 10 × 10m plots. For each tree, DBH was recorded. Mean tree diameter andpatterns of tree size class distribution did not differ among the four forestfragments. However, the fragments differed significantly in both tree andunderstory stem densities, species richness and diversity values, and familyrichness values, with the Lokaro fragment having the lowest values for allmeasures. Furthermore, floristic patterns, family importance values, and communitysimilarity measures revealed that the species composition at theLokaro fragment was very different from the Sainte-Luce fragments. Anthropogenicdisturbance appears most pronounced in the isolated Lokaro forest, where bioticresources are limited to this single fragment.     

Use of HepG2 cell line for direct or indirect mutagens screening: comparative investigation between comet and micronucleus assays

In the present study, DNA-damage and clastogenic or aneugenic effects of genotoxic compounds were examined in a metabolically competent human cell line (HepG2 cells) using the micronucleus and the comet assays. Compounds with various action mechanisms were tested: direct mutagens such as 4-nitroquinoline-N-oxide (4-NQO) and methyl methanesulfonate (MMS) and indirect mutagens requiring biotransformation to be active such as N-nitrosodimethylamine (NDMA), benzo[a]pyrene (B[a]P) and 2-acetylaminofluorene (2-AAF). The compounds were first tested for cytotoxicity by measuring their effects on RNA synthesis inhibition in HepG2 cells. 4-NQO, B[a]P and 2-AAF were the most potent compounds; their IC(50) values were, respectively, 1.9 micro M (4h contact), 3.4 and 112 micro M after 20 h. MMS was mildly cytotoxic (IC(50)=0.9 mM) and NDMA had a weak effect (IC(50)=110 mM) after 4h contact. In the micronucleus and comet assays, concentrations required to obtain a significant genotoxic effect in HepG2 cells varied over a broad range, NDMA being active only at very high concentrations. To compare the sensitivity of the two assays, we measured the so-called FIC(2)-the concentration necessary to induce a 2-fold increase of the measured genotoxicity parameter. The data show that genotoxic effects were consistently observed at lower concentrations in the micronucleus test, except in the case of MMS. The measured FIC(2) values were 0.12 micro M (4-NQO), 0.17 micro M (2-AAF), 0.26 micro M (B[a]P) and 6.4mM (NDMA). MMS had such a weak effect in the HepG2 cells that we could not calculate its FIC(2) value. In the comet assay, FIC(2) values were observed, respectively, at 1.48 micro M (4-NQO), 3.67 micro M (B[a]P), 13.42 micro M (MMS) and 27 mM (NDMA). 2-AAF failed to induce DNA-damage in this assay. The present study shows that HepG2 cells could be a suitable tool for assessing the genotoxicity of direct and indirect mutagens and for establishing the lowest genotoxic concentration.