Bacterial translocation motors investigated by single molecule techniques

Translocation of DNA and protein fibers through narrow constrictions is a ubiquitous and crucial activity of bacterial cells. Bacteria use specialized machines to support macromolecular movement. A very important step toward a mechanistic understanding of these translocation machines is the characterization of their physical properties at the single molecule level. Recently, four bacterial transport processes have been characterized by nanomanipulation at the single molecule level, DNA translocation by FtsK and SpoIIIE, DNA import during transformation, and the related process of a type IV pilus retraction. With all four processes, the translocation rates, processivity, and stalling forces were remarkably high as compared with single molecule experiments with other molecular motors. Although substrates of all four processes proceed along a preferential direction of translocation, directionality has been shown to be controlled by distinct mechanisms.     

Disruption of the mouse mTOR gene leads to early postimplantation lethality and prohibits embryonic stem cell development

The mammalian target of rapamycin (mTOR) is a key component of a signaling pathway which integrates inputs from nutrients and growth factors to regulate cell growth. Recent studies demonstrated that mice harboring an ethylnitrosourea-induced mutation in the gene encoding mTOR die at embryonic day 12.5 (E12.5). However, others have shown that the treatment of E4.5 blastocysts with rapamycin blocks trophoblast outgrowth, suggesting that the absence of mTOR should lead to embryonic lethality at an earlier stage. To resolve this discrepancy, we set out to disrupt the mTOR gene and analyze the outcome in both heterozygous and homozygous settings. Heterozygous mTOR (mTOR(+/-)) mice do not display any overt phenotype, although mouse embryonic fibroblasts derived from these mice show a 50% reduction in mTOR protein levels and phosphorylation of S6 kinase 1 T389, a site whose phosphorylation is directly mediated by mTOR. However, S6 phosphorylation, raptor levels, cell size, and cell cycle transit times are not diminished in these cells. In contrast to the situation in mTOR(+/-) mice, embryonic development of homozygous mTOR(-/-) mice appears to be arrested at E5.5; such embryos are severely runted and display an aberrant developmental phenotype. The ability of these embryos to implant corresponds to a limited level of trophoblast outgrowth in vitro, reflecting a maternal mRNA contribution, which has been shown to persist during preimplantation development. Moreover, mTOR(-/-) embryos display a lesion in inner cell mass proliferation, consistent with the inability to establish embryonic stem cells from mTOR(-/-) embryos.     

An international reference consensus genetic map with 897 marker loci based on 11 mapping populations for tetraploid groundnut (Arachis hypogaea L.)

Only a few genetic maps based on recombinant inbred line (RIL) and backcross (BC) populations have been developed for tetraploid groundnut. The marker density, however, is not very satisfactory especially in the context of large genome size (2800 Mb/1C) and 20 linkage groups (LGs). Therefore, using marker segregation data for 10 RILs and one BC population from the international groundnut community, with the help of common markers across different populations, a reference consensus genetic map has been developed. This map is comprised of 897 marker loci including 895 simple sequence repeat (SSR) and 2 cleaved amplified polymorphic sequence (CAPS) loci distributed on 20 LGs (a01-a10 and b01-b10) spanning a map distance of 3, 863.6 cM with an average map density of 4.4 cM. The highest numbers of markers (70) were integrated on a01 and the least number of markers (21) on b09. The marker density, however, was lowest (6.4 cM) on a08 and highest (2.5 cM) on a01. The reference consensus map has been divided into 20 cM long 203 BINs. These BINs carry 1 (a10_02, a10_08 and a10_09) to 20 (a10_04) loci with an average of 4 marker loci per BIN. Although the polymorphism information content (PIC) value was available for 526 markers in 190 BINs, 36 and 111 BINs have at least one marker with >0.70 and >0.50 PIC values, respectively. This information will be useful for selecting highly informative and uniformly distributed markers for developing new genetic maps, background selection and diversity analysis. Most importantly, this reference consensus map will serve as a reliable reference for aligning new genetic and physical maps, performing QTL analysis in a multi-populations design, evaluating the genetic background effect on QTL expression, and serving other genetic and molecular breeding activities in groundnut.     

Inhibition of the Prostaglandin Transporter PGT Lowers Blood Pressure in Hypertensive Rats and Mice

Inhibiting the synthesis of endogenous prostaglandins with nonsteroidal anti-inflammatory drugs exacerbates arterial hypertension. We hypothesized that the converse, i.e., raising the level of endogenous prostaglandins, might have anti-hypertensive effects. To accomplish this, we focused on inhibiting the prostaglandin transporter PGT (SLCO2A1), which is the obligatory first step in the inactivation of several common PGs. We first examined the role of PGT in controlling arterial blood pressure blood pressure using anesthetized rats. The high-affinity PGT inhibitor T26A sensitized the ability of exogenous PGE₂ to lower blood pressure, confirming both inhibition of PGT by T26A and the vasodepressor action of PGE₂ T26A administered alone to anesthetized rats dose-dependently lowered blood pressure, and did so to a greater degree in spontaneously hypertensive rats than in Wistar-Kyoto control rats. In mice, T26A added chronically to the drinking water increased the urinary excretion and plasma concentration of PGE₂ over several days, confirming that T26A is orally active in antagonizing PGT. T26A given orally to hypertensive mice normalized blood pressure. T26A increased urinary sodium excretion in mice and, when added to the medium bathing isolated mouse aortas, T26A increased the net release of PGE₂ induced by arachidonic acid, inhibited serotonin-induced vasoconstriction, and potentiated vasodilation induced by exogenous PGE₂. We conclude that pharmacologically inhibiting PGT-mediated prostaglandin metabolism lowers blood pressure, probably by prostaglandin-induced natriuresis and vasodilation. PGT is a novel therapeutic target for treating hypertension.     

Chronic bronchial allergic inflammation increases alveolar liquid clearance by TNF-alpha -dependent mechanism

Bronchial inflammation in allergic asthma is associated with active exudation from the bronchial tree into the interstitial space of both mucosa and submucosa. The aim of this study was to evaluate epithelial and endothelial permeability as well as alveolar fluid movement in a model of chronic allergic inflammation in Brown-Norway rats sensitized and challenged with ovalbumin (OA). Control groups were challenged with saline solution (C), and rats were immunized by OA but not challenged (Se). Lung sections showed a marked inflammatory infiltrate associated with perivascular and peribronchiolar edema in OA. To measure alveolar liquid clearance, a 5% bovine albumin solution with 1 microCi of (125)I-labeled human albumin was instilled into the air spaces. Alveolar-capillary barrier permeability was evaluated by intravascular injection of 1 microCi of (131)I-labeled albumin. Endothelial permeability was significantly increased in OA, from 0.08 +/- 0.01 in the C group to 0.19 +/- 0.03 in OA group (P < 0.05). Final-to-initial protein ratio was also statistically higher in OA (1.6 +/- 0.05) compared with C (1.38 +/- 0.03, P = 0.01) and Se groups (1.42 +/- 0.03, P = 0.04). Administration of anti-tumor necrosis factor-alpha antibodies within the instillate significantly decreased this ratio (1.32 +/- 0.08, P = 0.003 vs. OA). To conclude, we demonstrated a tumor necrosis factor-alpha-dependent increase in alveolar fluid movement in a model of severe bronchial allergic inflammation associated with endothelial and epithelial leakage.     

Malaria and urbanization in sub-Saharan Africa

There are already 40 cities in Africa with over 1 million inhabitants and the United Nations Environmental Programme estimates that by 2025 over 800 million people will live in urban areas. Recognizing that malaria control can improve the health of the vulnerable and remove a major obstacle to their economic development, the Malaria Knowledge Programme of the Liverpool School of Tropical Medicine and the Systemwide Initiative on Malaria and Agriculture convened a multi-sectoral technical consultation on urban malaria in Pretoria, South Africa from 2nd to 4th December, 2004. The aim of the meeting was to identify strategies for the assessment and control of urban malaria. This commentary reflects the discussions held during the meeting and aims to inform researchers and policy makers of the potential for containing and reversing the emerging problem of urban malaria.     

Stereospecific synthesis of "para-hydroxymexiletine" and sodium channel blocking activity evaluation

Both enantiomers of "para-hydroxymexiletine" (PHM), one of the main metabolites of mexiletine, were synthesized and fully characterized. Properties of (R)- and (S)-PHM, in terms of blocking potency and stereoselectivity on frog skeletal muscle Na(+) channels, were evaluated. The presence of a hydroxy group on the aryloxy moiety in the 4-position, as in PHM, reduced potency with respect to mexiletine in reducing I(Na max). However, PHM showed clear use-dependent behavior similar to that of mexiletine and, in contrast with what is observed with the parent compound, maintained its stereoselectivity during the use-dependent block. Chirality 16:72-78, 2004.     

Glutamine Supplementation Alleviates Vasculopathy and Corrects Metabolic Profile in an In Vivo Model of Endothelial Cell Dysfunction

Endothelial Cell Dysfunction (ECD) is a recognized harbinger of a host of chronic cardiovascular diseases. Using a mouse model of ECD triggered by treatment with L-Nω-methylarginine (L-NMMA), we previously demonstrated that renal microvasculature displays a perturbed protein profile, including diminished expression of two key enzymes of the Krebs cycle associated with a Warburg-type suppression of mitochondrial metabolism. We hypothesized that supplementation with L-glutamine (GLN), that can enter the Krebs cycle downstream this enzymatic bottleneck, would normalize vascular function and alleviate mitochondrial dysfunction. To test this hypothesis, mice with chronic L-NMMA-induced ECD were co-treated with GLN at different concentrations for 2 months. Results confirmed that L-NMMA led to a defect in acetylcholine-induced relaxation of aortic rings that was dose-dependently prevented by GLN. In caveolin-1 transgenic mice characterized by eNOS inactivation, L-NMMA further impaired vasorelaxation which was partially rescued by GLN co-treatment. Pro-inflammatory profile induced by L-NMMA was blunted in mice co-treated with GLN. Using an LC/MS platform for metabolite profiling, we sought to identify metabolic perturbations associated with ECD and offset by GLN supplementation. 3453 plasma molecules could be detected with 100% frequency in mice from at least one treatment group. Among these, 37 were found to be differentially expressed in a 4-way comparison of control vs. LNMMA both with and without GLN. One of such molecules, hippuric acid, an “uremic toxin” was found to be elevated in our non-uremic mice receiving L-NMMA, but normalized by treatment with GLN. Ex vivo analysis of hippuric acid effects on vasomotion demonstrated that it significantly reduced acetylcholine-induced vasorelaxation of vascular rings. In conclusion, functional and metabolic profiling of animals with early ECD revealed macrovasculopathy and that supplementation GLN is capable of improving vascular function. Metabolomic analyses reveal elevation of hippuric acid, which may further exacerbate vasculopathy even before the development of uremia.     

Molecular signals in the trafficking of Toxoplasma gondii protein MIC3 to the micronemes

The protozoan parasite Toxoplasma gondii is equipped with a sophisticated secretory apparatus, including three distinct exocytic organelles, named micronemes, rhoptries, and dense granules. We have dissected the requirements for targeting the microneme protein MIC3, a key component of T. gondii infection. We have shown that MIC3 is processed in a post-Golgi compartment and that the MIC3 propeptide and epidermal growth factor (EGF) modules contain microneme-targeting information. The minimal requirement for microneme delivery is defined by the propeptide plus any one of the three EGF domains. We have demonstrated that the cleavage of the propeptide, the dimerization of MIC3, and the chitin binding-like sequence, which are crucial for host cell binding and virulence, are dispensable for proper targeting. Finally, we have shown that part of MIC3 is withheld in the secretory pathway in a cell cycle-dependent manner.