Transfer and expression of the cryIVB and cryIVD genes of Bacillus thuringiensis subsp. israelensis in Bacillus sphaericus 2297

Abstract The genes encoding the CryIVB and CryIVD crystal polypeptides of B. thuringiensis subsp. israelensis were cloned indepently on a stable shuttle vector, and transfered into B. sphaericus 2297. Recombinant cells expressed the B. thuringiensis toxins during sporulation and were shown to be toxic to Aedes aegypti fourth instar larvae, whereas the parental strain was not.     

Evolutionary History of the Plant Pathogenic Bacterium Xanthomonas axonopodis

Deciphering mechanisms shaping bacterial diversity should help to build tools to predict the emergence of infectious diseases. Xanthomonads are plant pathogenic bacteria found worldwide. Xanthomonas axonopodis is a genetically heterogeneous species clustering, into six groups, strains that are collectively pathogenic on a large number of plants. However, each strain displays a narrow host range. We address the question of the nature of the evolutionary processes – geographical and ecological speciation – that shaped this diversity. We assembled a large collection of X. axonopodis strains that were isolated over a long period, over continents, and from various hosts. Based on the sequence analysis of seven housekeeping genes, we found that recombination occurred as frequently as point mutation in the evolutionary history of X. axonopodis. However, the impact of recombination was about three times greater than the impact of mutation on the diversity observed in the whole dataset. We then reconstructed the clonal genealogy of the strains using coalescent and genealogy approaches and we studied the diversification of the pathogen using a model of divergence with migration. The suggested scenario involves a first step of generalist diversification that spanned over the last 25 000 years. A second step of ecology-driven specialization occurred during the past two centuries. Eventually, secondary contacts between host-specialized strains probably occurred as a result of agricultural development and intensification, allowing genetic exchanges of virulence-associated genes. These transfers may have favored the emergence of novel pathotypes. Finally, we argue that the largest ecological entity within X. axonopodis is the pathovar.     

Chelate-assisted phytoextraction of lead using Fagopyrum esculentum: laboratory vs. field experiments

Abstract

The development of more sustainable remediation techniques has been receiving greater attention, as an alternative to soil excavation plan in urban gardens. An in situ phytoextraction experiment with buckwheat (Fagopyrum esculentum) was performed with a 5?mmol kg^?1 citric acid (CA) application. Joint experiments under laboratory conditions were conducted using various cultivars of F. esculentum in two soils with a Pb contamination of either geogenic or anthropogenic origin and various chelate concentrations. Results show that a minimum dose of 50?mmol kg^?1 of CA is required to lower soil pH and raise the concentration of mobile Pb–CaCl₂ for both soils. Consequently, Pb shoot uptake is increased from 6.3 to 8.9 times depending on soil type. Phytoextraction efficiency is found to be 1.3 to 2.0 times higher in the anthropogenic contaminated soil than in the soil with geogenic Pb. A scale effect has also been identified since Pb root accumulation under laboratory conditions was 2.4 times higher than in the field experiment. Despite an increase in the Pb extraction rate with CA, buckwheat appears to lack the efficiency needed to remove Pb in moderately contaminated soils. The calculated remediation period would last 166?years to remove the mobile Pb fraction.     

The methylerythritol phosphate pathway for isoprenoid biosynthesis in coccidia: presence and sensitivity to fosmidomycin

The apicoplast is a recently discovered, plastid-like organelle present in most apicomplexa. The methylerythritol phosphate (MEP) pathway involved in isoprenoid biosynthesis is one of the metabolic pathways associated with the apicoplast, and is a new promising therapeutic target in Plasmodium falciparum. Here, we check the presence of isoprenoid genes in four coccidian parasites according to genome database searches. Cryptosporidium parvum and C. hominis, which have no plastid genome, lack the MEP pathway. In contrast, gene expression studies suggest that this metabolic pathway is present in several development stages of Eimeria tenella and in tachyzoites of Toxoplasma gondii. We studied the potential of fosmidomycin, an antimalarial drug blocking the MEP pathway, to inhibit E. tenella and T. gondii growth in vitro. The drug was poorly effective even at high concentrations. Thus, both fosmidomycin sensitivity and isoprenoid metabolism differs substantially between apicomplexan species.     

The Role of Tryptophan Catabolism along the Kynurenine Pathway in Acute Ischemic Stroke

Post-stroke inflammation may induce upregulation of the kynurenine (KYN) pathway for tryptophan (TRP) oxidation, resulting in neuroprotective (kynurenic acid, KA) and neurotoxic metabolites (3-hydroxyanthranillic acid, 3-HAA). We investigated whether activity of the kynurenine pathway in acute ischemic stroke is related to initial stroke severity, long-term stroke outcome and the ischemia-induced inflammatory response. Plasma concentrations of TRP and its metabolites were measured in 149 stroke patients at admission, at 24 h, at 72 h and at day 7 after stroke onset. We evaluated the relation between the KYN/TRP ratio, the KA/3-HAA ratio and stroke severity, outcome and inflammatory parameters (C-reactive protein (CRP), erythrocyte sedimentation rate (ESR) and neutrophil/lymphocyte ratio (NLR)). KYN/TRP but not KA/3-HAA correlated with the NIHSS score and with the infarct volume. Patients with poor outcome had higher mean KYN/TRP ratios than patients with more favourable outcome. The KYN/TRP ratio at admission correlated with CRP levels, ESR and NLR. The activity of the kynurenine pathway for tryptophan degradation in acute ischemic stroke correlates with stroke severity and long-term stroke outcome. Tryptophan oxidation is related to the stroke-induced inflammatory response.     

Space use patterns of mountain hare (<Emphasis Type="Italic">Lepus timidus</Emphasis>) on the Alps

Populations on the limits of species’ distribution can show different behavioral adaptations to strong ecological pressure than in the central part of the range. We investigated space use patterns of alpine mountain hare (Lepus timidus) at two areas on the southern edge of the species’ range. We monitored 34 hares between 2005 and 2008, estimating home range size, overlap, and site fidelity, and compared our results with space use in Scottish and North-European populations. Home ranges of mountain hares did not differ between two study areas with different habitat types. Subadult animals used larger ranges than adults and both age groups reduced home range size in autumn, a period that might be critical for hares due to changes in diet and/or high energy expenditure during the previous breeding season. Home ranges in these alpine populations were smaller than in Scandinavian populations but within the range of populations in different habitat types in Scotland. Seasonal home ranges overlapped considerably, but differed among the sexes: male–female overlap was higher than same sex (male–male and female–female) spatial overlap. Seasonal shifts of home ranges were small, and site fidelity remained high over the seasons, suggesting that resource distribution remained constant throughout the year and that the knowledge of an intensively frequented area is an important element of habitat quality. We concluded that habitat structure and availability of mates interact in affecting mountain hare space use in alpine habitats.     

Hormones, IgG and lactose changes around parturition in plasma, and colostrum or saliva of multiparous sows

Blood, colostrum and saliva samples were serially taken from 6 multiparous sows from day 109 of gestation until day 3 postpartum. Plasma was assayed for oestradiol-17beta (E2), progesterone (P4), prolactin (PRL), cortisol, immunoglobulin G (IgG) and lactose. Colostrum was assayed for E2, P4, IgG and lactose. Lactoserum, obtained after ultra centrifugation of colostrum, was assayed for PRL. Saliva was assayed for cortisol. Time-related variations in hormone, IgG and lactose concentrations measured in plasma were parallel to those measured in colostrum, lactoserum or saliva. However, the concentrations were higher in colostrum or lactoserum and lower in saliva than in plasma. Ratios of concentrations of cortisol in saliva and PRL in lactoserum over those in plasma did not vary with time and averaged 0.2 and 1.6, respectively. Conversely, the ratios of concentrations of E2 and P4 in colostrum over those in plasma varied with time (P < 0.05) but were quite constant before the end of parturition, averaging 2.7 and 3.6, respectively. The ratios of concentrations of IgG and lactose in colostrum over those in plasma also varied with time (P < 0.05). The concentrations of hormones in plasma on the one hand and in colostrum, lactoserum or saliva on the other hand were significantly correlated but correlations varied with time (PRL across periods: r = 0.31; cortisol across periods: r = 0.60; E2 during parturition: r = 0.83; P4 before parturition: r = 0.82; P4 during parturition: r = 0.67). The present results indicate that around parturition, assays of hormones in colostrum or saliva can be used to study the hormonal status of sows. Furthermore, variations in colostrum and plasma concentrations of IgG and lactose are good indicators of the transition from colostrum to milk synthesis.     

Internalization of Human 5-HT<Subscript>4a</Subscript> and 5-HT<Subscript>4b</Subscript> Receptors is Splice Variant Dependent

The family of 5-HT₄ receptors comprises 16 putative splice variants. We have previously shown that there are differences in signal transduction of the h5-HT_4a and h5-HT_4b receptors. In the present study, the internalization of these two splice variants following receptor stimulation was investigated with confocal microscopy on living cells. Chimeric receptors, h5-HT_4a-GFP and h5-HT_4b-GFP were generated by fusing the coding sequence of the 5-HT₄ receptor with the coding sequence of the GFP. The agonist stimulation of fluorescent receptors resulted in a time-dependent internalization of the h5-HT_4b-GFP receptor, but not of the h5-HT_4a-GFP receptor. The h5-HT_4b receptor displays a dual coupling to Gαi,o and Gαs proteins, in contrast to the h5-HT_4a receptor, which couples to Gαs proteins only. We investigated whether the difference in internalization of the two splice variant receptors was related to their differential coupling. Therefore, we performed agonist-stimulation of the receptor following inhibition of the Gαi,o protein coupling using PTX. The h5-HT_4b receptor internalization is PTX insensitive. We co-transfected the fluorescent chimeric receptors with other wild-type variants, which did not produce an alteration of the receptor trafficking. These findings provide the first evidence of differential internalization between the two splice variants, 5-HT_4a and 5-HT_4b receptors.     

Construction of replicative and integrative plasmids for setting up the in vivo expression technology in Helicobacter pylori

Some pathogenic factors of Helicobacter pylori, a bacterium involved in peptic ulcer and gastric cancer, have already been identified using either global or particular approach, but there are still some orphan genes and unidentified pathogenic factors. One of the methods used successfully for the identification of virulence genes of many pathogens is the in vivo expression technology. We describe here the construction and sequences of three different plasmids, one integrative and two replicatives, for the identification of virulence genes by using in vivo expression technology in H. pylori, and of potential use in other bacteria such as Campylobacter spp. Moreover, the use of the green fluorescent protein could allow to classify the genes according to the strength of their expression and to identify those which are repressed upon interaction with gastric mucosa.