Ralstonia syzygii, the Blood Disease Bacterium and some Asian R. solanacearum strains form a single genomic species despite divergent lifestyles

The Ralstonia solanacearum species complex includes R. solanacearum, R. syzygii, and the Blood Disease Bacterium (BDB). All colonize plant xylem vessels and cause wilt diseases, but with significant biological differences. R. solanacearum is a soilborne bacterium that infects the roots of a broad range of plants. R. syzygii causes Sumatra disease of clove trees and is actively transmitted by cercopoid insects. BDB is also pathogenic to a single host, banana, and is transmitted by pollinating insects. Sequencing and DNA-DNA hybridization studies indicated that despite their phenotypic differences, these three plant pathogens are actually very closely related, falling into the Phylotype IV subgroup of the R. solanacearum species complex. To better understand the relationships among these bacteria, we sequenced and annotated the genomes of R. syzygii strain R24 and BDB strain R229. These genomes were compared to strain PSI07, a closely related Phylotype IV tomato isolate of R. solanacearum, and to five additional R. solanacearum genomes. Whole-genome comparisons confirmed previous phylogenetic results: the three phylotype IV strains share more and larger syntenic regions with each other than with other R. solanacearum strains. Furthermore, the genetic distances between strains, assessed by an in-silico equivalent of DNA-DNA hybridization, unambiguously showed that phylotype IV strains of BDB, R. syzygii and R. solanacearum form one genomic species. Based on these comprehensive data we propose a revision of the taxonomy of the R. solanacearum species complex. The BDB and R. syzygii genomes encoded no obvious unique metabolic capacities and contained no evidence of horizontal gene transfer from bacteria occupying similar niches. Genes specific to R. syzygii and BDB were almost all of unknown function or extrachromosomal origin. Thus, the pathogenic life-styles of these organisms are more probably due to ecological adaptation and genomic convergence during vertical evolution than to the acquisition of DNA by horizontal transfer.     

Induction of P‐glycoprotein and Bcrp at the rat blood–brain barrier following a subchronic morphine treatment is mediated through NMDA/COX‐2 activation

Subchronic morphine treatment induces P‐glycoprotein (P‐gp) up‐regulation at the blood–brain barrier. This study investigates the rate and extent to which P‐gp and breast cancer‐resistance protein (Bcrp) increase at the rat blood–brain barrier following subchronic morphine treatment. Rats were given increasing doses of morphine (10–40 mg/kg) or saline i.p. twice daily for 5 days. The brain cortex large vessels and microvessels were then mechanical isolated 6, 9, 12, 24, and 36 h after the last injection. The gene and protein expression of P‐gp and Bcrp in morphine‐treated and control rats were compared by qRT‐PCR and western blotting. The levels of Mdr1a and Bcrp mRNAs were not significantly modified 6 h post morphine, but the Mdr1a mRNA increased 1.4‐fold and Bcrp mRNA 2.4‐fold at 24 h. P‐gp and Bcrp protein expression in brain microvessels was unchanged 6 h post morphine and increased 1.5‐fold at 24 h. This effect was more pronounced in large vessels than in microvessels. However, extracellular morphine concentrations of 0.01–10 μM did not modify the expressions of the MDR1 and BCRP genes in hCMEC/D3 human endothelial brain cells in vitro. MK‐801 (NMDA antagonist) and meloxicam (cyclo‐oxygenase‐2 inhibitor) given after morphine treatment completely blocked P‐gp and Bcrp up‐regulation. Interestingly, misoprostol and iloprost, two well‐known agonists of prostaglandin E2 receptors induced both MDR1 and BCRP mRNA levels in hCMEC/D3. Thus, morphine does not directly stimulate P‐gp and Bcrp expression by the brain endothelium, but glutamate released during morphine withdrawal may do so by activating the NMDA/cyclo‐oxygenase‐2 cascade.     

Curcumin modulates drug metabolizing enzymes in the female Swiss Webster mouse

Curcumin, the yellow pigment found in turmeric, exhibits potent chemopreventative properties in both in vivo and in vitro cancer models. We hypothesized that this effect may occur via curcumin-mediated changes in enzymes involved in both carcinogen bioactivation and estrogen metabolism. Female Swiss Webster mice were treated with either curcumin (200 mg/kg or 400 mg/kg, p.o.) or vehicle control for 1 or 2 weeks. The results demonstrated that curcumin had no effect on the catalytic activities of ovarian aromatase, hepatic catechol-O-methyltransferase or hepatic UDP-glucuronosyltransferase. However, both doses of curcumin caused a 25% decrease in CYP1A catalytic activity, but not polypeptide levels, following 2 weeks of treatment. Additionally, following 2 weeks of curcumin at 400 mg/kg, there was a 20% decrease in the catalytic activity and a 28% decrease in polypeptide levels of CYP3A. While 2 weeks of curcumin treatment (400 mg/kg) caused a 20% increase in glutathione S-transferase activity, there was no parallel increase in hepatic stores of the co-factor glutathione. In conclusion small changes in CYP1A, CYP3A and GST following long term treatment (2 weeks) suggest that the combination of all three metabolic pathways may play a small role in curcumin's chemopreventative action.     

Characterization of the maize xylem sap proteome

The xylem in plants has mainly been described as a conduit for water and minerals, but emerging evidence also indicates that the xylem contains protein. To study the proteins in xylem sap, we characterized the identity and composition of the maize xylem sap proteome. The composition of the xylem sap proteome in maize revealed proteins related to different phases of xylem differentiation including cell wall metabolism, secondary cell wall synthesis, and programmed cell death. Many proteins were found to be present as multiple isoforms and some of these isoforms are glycosylated. Proteins involved in defense mechanisms were also present in xylem sap and the sap proteins were shown to have antifungal activity in bioassays.     

Construction of a Highly Active Xylanase Displaying Oleaginous Yeast: Comparison of Anchoring Systems

Three Yarrowia lipolytica cell wall proteins (YlPir, YlCWP1 and YlCBM) were evaluated for their ability to display the xylanase TxXYN from Thermobacillus xylanilyticus on the cell surface of Y. lipolytica. The fusion proteins were produced in Y. lipolytica JMY1212, a strain engineered for mono-copy chromosomal insertion, and enabling accurate comparison of anchoring systems. The construction using YlPir enabled cell bound xylanase activity to be maximised (71.6 U/g). Although 48% of the activity was released in the supernatant, probably due to proteolysis at the fusion zone, this system is three times more efficient for the anchoring of TxXYN than the YlCWP1 system formerly developed for Y. lipolytica. As far as we know it represents the best displayed xylanase activity ever published. It could be an attractive alternative anchoring system to display enzymes in Y. lipolytica.     

Lipid free apolipoprotein E binds to the class B Type I scavenger receptor I (SR-BI) and enhances cholesteryl ester uptake from lipoproteins

The Class B type I scavenger receptor I (SR-BI) is a physiologically relevant high density lipoprotein (HDL) receptor that can mediate selective cholesteryl ester (CE) uptake by cells. Direct interaction of apolipoprotein E (apoE) with this receptor has never been demonstrated, and its implication in CE uptake is still controversial. By using a human adrenal cell line (NCI-H295R), we have addressed the role of apoE in binding to SR-BI and in selective CE uptake from lipoproteins to cells. This cell line does not secrete apoE and SR-BI is its major HDL-binding protein. We can now provide evidence that 1) free apoE is a ligand for SR-BI, 2) apoE associated to lipids or in lipoproteins does not modulate binding or CE-selective uptake by the SR-BI pathway, and 3) the direct interaction of free apoE to SR-BI leads to an increase in CE uptake from lipoproteins of both low and high densities. We propose that this direct interaction could modify SR-BI structure in cell membranes and potentiate CE uptake.     

Elemental composition and food intake of Phialidium hydromedusae in the laboratory

Elemental composition and feeding rate of hydromedusae Phialidium sp. on copepods were studied in the laboratory. Regression equations for both mature and immature medusae allowed the estimation of their dry weight (DW), total C and N content as a function of their diameter. The mean C content as percentage of the DW varied from 13.13% ( ) for the immature to 19.38% (5.68) for the mature individuals. The mean N content is 4.03% (2.49) of DW of immatures and 5.85% (2.70) of the matures. Ingestion rate of Phialidium sp. fed on copepods (200–500 μm) increased with prey density but reached a maximum at high prey concentrations. A maximum ingestion rate of 8.55 (1.6) copepods · medusa ⁻¹ · h⁻¹ was reached for prey concentrations of > 140 copepods · 1 ⁻¹ for both immature and mature medusae. Maximum daily consumption of prey weight varied from 1.41 to 978% C body weight for mature medusae and from 2.90 to 975% for the immature individuals.     

Combined Approaches for Drug Design Points the Way to Novel Proline Racemase Inhibitor Candidates to Fight Chagas’ Disease

Chagas’ disease is caused by Trypanosoma cruzi, a protozoan transmitted to humans by blood-feeding insects, blood transfusion or congenitally. Previous research led us to discover a parasite proline racemase (TcPRAC) and to establish its validity as a target for the design of new chemotherapies against the disease, including its chronic form. A known inhibitor of proline racemases, 2-pyrrolecarboxylic acid (PYC), is water-insoluble. We synthesized soluble pyrazole derivatives, but they proved weak or inactive TcPRAC inhibitors. TcPRAC catalytic site is too small and constrained when bound to PYC to allow efficient search for new inhibitors by virtual screening. Forty-nine intermediate conformations between the opened enzyme structure and the closed liganded one were built by calculating a transition path with a method we developed. A wider range of chemical compounds could dock in the partially opened intermediate active site models in silico. Four models were selected for known substrates and weak inhibitors could dock in them and were used to screen chemical libraries. Two identified soluble compounds, (E)-4-oxopent-2-enoic acid (OxoPA) and its derivative (E)-5-bromo-4-oxopent-2-enoic acid (Br-OxoPA), are irreversible competitive inhibitors that presented stronger activity than PYC on TcPRAC. We show here that increasing doses of OxoPA and Br-OxoPA hamper T. cruzi intracellular differentiation and fate in mammalian host cells. Our data confirm that through to their binding mode, these molecules are interesting and promising as lead compounds for the development of chemotherapies against diseases where active proline racemases play essential roles.