Tuesday, September 15, 2009

What's new for 'Trypanosomatids' in PubMed

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Search kinetoplastids OR kinetoplastid OR Kinetoplastida OR "trypanosoma brucei" OR leishmania OR brucei OR leishmaniasis OR "African trypanosomiasis"
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PubMed Results
Items 1 -10 of 18

1: PLoS Pathog. 2009 Sep;5(9):e1000575. Epub 2009 Sep 11.

The Aurora Kinase in Trypanosoma brucei Plays Distinctive Roles in Metaphase-Anaphase Transition and Cytokinetic Initiation.

Li Z, Umeyama T, Wang CC.

Department of Pharmaceutical Chemistry, University of California, San Francisco, California, United States of America.

Aurora B kinase is an essential regulator of chromosome segregation with the action well characterized in eukaryotes. It is also implicated in cytokinesis, but the detailed mechanism remains less clear, partly due to the difficulty in separating the latter from the former function in a growing cell. A chemical genetic approach with an inhibitor of the enzyme added to a synchronized cell population at different stages of the cell cycle would probably solve this problem. In the deeply branched parasitic protozoan Trypanosoma brucei, an Aurora B homolog, TbAUK1, was found to control both chromosome segregation and cytokinetic initiation by evidence from RNAi and dominant negative mutation. To clearly separate these two functions, VX-680, an inhibitor of TbAUK1, was added to a synchronized T. brucei procyclic cell population at different cell cycle stages. The unique trans-localization pattern of the chromosomal passenger complex (CPC), consisting of TbAUK1 and two novel proteins TbCPC1 and TbCPC2, was monitored during mitosis and cytokinesis by following the migration of the proteins tagged with enhanced yellow fluorescence protein in live cells with time-lapse video microscopy. Inhibition of TbAUK1 function in S-phase, prophase or metaphase invariably arrests the cells in the metaphase, suggesting an action of TbAUK1 in promoting metaphase-anaphase transition. TbAUK1 inhibition in anaphase does not affect mitotic exit, but prevents trans-localization of the CPC from the spindle midzone to the anterior tip of the new flagellum attachment zone for cytokinetic initiation. The CPC in the midzone is dispersed back to the two segregated nuclei, while cytokinesis is inhibited. In and beyond telophase, TbAUK1 inhibition has no effect on the progression of cytokinesis or the subsequent G1, S and G2 phases until a new metaphase is attained. There are thus two clearly distinct points of TbAUK1 action in T. brucei: the metaphase-anaphase transition and cytokinetic initiation. This is the first time to our knowledge that the dual functions of an Aurora B homolog is dissected and separated into two clearly distinct time frames in a cell cycle.

PMID: 19750216 [PubMed - in process]

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2: J Antimicrob Chemother. 2009 Sep 12. [Epub ahead of print]

Membrane sterol depletion impairs miltefosine action in wild-type and miltefosine-resistant Leishmania donovani promastigotes.

Saint-Pierre-Chazalet M, Ben Brahim M, Le Moyec L, Bories C, Rakotomanga M, Loiseau PM.

Université Paris-Sud, UMR 8076, Chimiothérapie Antiparasitaire, Faculté de Pharmacie, 5 rue Jean-Baptiste Clément, Chatenay-Malabry, F-92296, France.

Objectives This study focuses on the importance of sterols in the action of miltefosine (hexadecylphosphocholine, HePC) against Leishmania donovani. Methods Plasma membranes of L. donovani promastigotes were depleted of sterol using methyl-beta-cyclodextrin (MCD) and cholesterol oxidase (CH-OX). Sterols were quantified and HePC susceptibility was assessed using the MTT test. A biomimetic model of the outer leaflet of a Leishmania plasma membrane was used to decipher the HePC-lipid interactions. Results CH-OX, which is known to act more specifically on condensed membranes, therefore at the level of lipid rafts, gave a better extraction yield in HePC-resistant parasites, confirming the more rigid structure of their membranes than those of wild-type parasites. Sterol depletion was responsible for a 40% decrease in HePC susceptibility in both wild-type and HePC-resistant parasites. Sterol repletion of the sterol-depleted parasites restored HePC susceptibility. The biomimetic model of the outer leaflet of a Leishmania plasma membrane confirmed that condensed microdomains were able to incorporate higher quantities of HePC than fluid ones and this result was amplified when the sterol concentration was increased. Conclusions Sterol and lipid rafts probably play a significant role as an HePC reservoir providing a constant supply to the previously described transporter. In addition, (1)H NMR experiments suggested that HePC stimulated lipid trafficking in parasites.

PMID: 19749205 [PubMed - as supplied by publisher]

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3: Eukaryot Cell. 2009 Sep 11. [Epub ahead of print]

Overexpression Of Mitochondrial Leishmania Major Ascorbate Peroxidase Shows Enhanced Tolerance To Oxidative Stress-Induced Programmed Cell Death And Protein Damage.

Dolai S, Yadav RK, Pal S, Adak S.

Division of Structural Biology & Bio-informatics, Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata - 700 032, India.

Ascorbate peroxidase from L. major (LmAPX) is one of the key enzymes for scavenging of reactive oxygen species generated from the mitochondrial respiratory chain. We have investigated whether mitochondrial LmAPX has any role in oxidative stress induced apoptosis. The measurement of reduced glutathione (GSH) and protein carbonyl content in cellular homogenates indicates that overexpression of LmAPX protects Leishmania cells against depletion of GSH and oxidative damage of proteins by H2O2 or camptothecin (CPT) treatment. Confocal microscopy and fluorescence spectroscopy data have revealed that the intracellular elevation of Ca(2+) attained by the LmAPX -overexpressing cells was always below that one attained in control cells. The flowcytometry assay and confocal microscopy observation strongly suggest that LmAPX overexpression protects cells from H2O2-induced mitochondrial membrane depolarization as well as ATP fall. Western blot data suggest that overexpression of LmAPX shields against H2O2 or CPT induced cytochrome c and endonuclease G release from mitochondria and subsequently its accumulation into cytoplasm. Caspase activity assay by flowcytometry shows lower level of caspase like protease activity in LmAPX overexpressing cells under apoptotic stimuli. The phosphatidylserine exposed on the cell surface data and DNA fragmentation results show that overexpression of LmAPX renders the Leishmania cells more resistant to apoptosis provoked by H2O2 or CPT treatment. Taken together, these results indicate that constitutive overexpression of LmAPX in the mitochondria of L. major prevents cells from the deleterious effects of oxidative stress that is mitochondrial dysfunction and cellular death.

PMID: 19749178 [PubMed - as supplied by publisher]

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4: J Immunol. 2009 Sep 11. [Epub ahead of print]

IL-27 Regulates IL-10 and IL-17 from CD4+ Cells in Nonhealing Leishmania major Infection.

Anderson CF, Stumhofer JS, Hunter CA, Sacks D.

*Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892.

Control of infection caused by Leishmania major requires the development of IFN-gamma(+)CD4(+) lymphocytes for the induction of microbicidal activity in host macrophages. We recently reported on the inability of conventionally resistant C57BL/6 mice to successfully resolve infection by an isolate of L. major, despite a strong IFN-gamma response by the host. Susceptibility was caused by Ag-specific IL-10 from CD4(+) cells that were also producing IFN-gamma. In the present studies, we have explored the role for IL-27 in the regulation of IL-10 from Th1 cells in leishmaniasis. Cytokine analysis of CD4(+) cells in the lesions and draining lymph nodes of infected IL-27R-deficient (WSX-1(-/-)) mice revealed diminished IL-10 from IFN-gamma(+) CD4(+) cells, which was accompanied by a reduction in total IFN-gamma(+)CD4(+) cells and an increase in IL-4. Despite the inhibition of IL-10 from CD4(+) cells, no significant change in parasite numbers was observed, due both to the shift in the Th1/Th2 balance and to residual levels of IL-10. Strikingly, infected WSX-1(-/-) mice developed more severe lesions that were associated with the appearance of IL-17(+) CD4(+) cells, demonstrating a function for IL-27 in blocking the development of inappropriate Th17 cells during L. major infection. The results demonstrate the pleiotropic effects that IL-27 has on L. major-driven Th1, Th2, and Th17 development, and reinforce its function as a key regulatory cytokine that controls the balance between immunity and pathology.

PMID: 19748991 [PubMed - as supplied by publisher]

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5: Mol Biol Evol. 2009 Sep 11. [Epub ahead of print]

The evolution of amastin surface glycoproteins in trypanosomatid parasites.

Jackson AP.

Amastin is a transmembrane glycoprotein found on the cell surfaces of trypanosomatid parasites. Encoded by a large, diverse gene family, amastin was initially described from the intracellular, amastigote stage of Trypanosoma cruzi and Leishmania donovani. Genome sequences have subsequently shown that the amastin repertoire is much larger in Leishmania relative to Trypanosoma. However, it is not known when this expansion occurred, whether it is associated with the origins of Leishmania and vertebrate parasitism itself, or prior to this. To examine the timing of amastin diversification, as well as the evolutionary mechanisms regulating gene repertoire and sequence diversity, this study sequenced the genomic regions containing amastin loci from two related insect parasites (Leptomonas seymouri and Crithidia sp.), and estimated a phylogeny for these and other amastin sequences. The phylogeny shows that amastin includes four subfamilies with distinct genomic positions, secondary structures, and evolution, which were already differentiated in the ancestral trypanosomatid. Diversification in Leishmania was initiated from a single ancestral locus on chromosome 34, with rapid derivation of novel loci through transposition and accelerated sequence divergence. This is absent from related organisms showing that diversification occurred after the origin of Leishmania. These results describe a substantial elaboration of amastin repertoire directly associated with the origin of Leishmania, suggesting that some amastin genes evolved novel functions crucial to cell function in leishmanial parasites after the acquisition of a vertebrate host.

PMID: 19748930 [PubMed - as supplied by publisher]

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6: Bioorg Med Chem Lett. 2009 Aug 7. [Epub ahead of print]

Synthesis and biological evaluation of new heterocyclic quinolinones as anti-parasite and anti-HIV drug candidates.

Darque A, Dumètre A, Hutter S, Casano G, Robin M, Pannecouque C, Azas N.

UMR-MD3-Relations Hôte-Parasites, Pharmacologie et Thérapeutique, Faculté de Pharmacie, Université de la Méditerranée, Marseille Cedex 05, France.

We have synthesized quinolinones with potential antiparasitic and anti-HIV activities by an original two-step method involving microwave irradiation and have evaluated their activities against Plasmodium falciparum, Leishmania donovani, Trichomonas vaginalis, and HIV. None of the tested compounds had been previously described using this method of synthesis. One of the compounds had interesting antiparasitic and anti-HIV activity, which could be improved by substitution with different radicals.

PMID: 19748781 [PubMed - as supplied by publisher]

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7: Vaccine. 2009 Sep 10. [Epub ahead of print]

Recombinant outer membrane vesicles to augment antigen-specific live vaccine responses.

Schroeder J, Aebischer T.

Marie Curie Team Pathogen Habitats, Institute of Immunology and Infection Research, University of Edinburgh, UK.

Release of outer membrane vesicles is a common feature of Gram negative bacteria. There is growing interest in the use of these vesicles in the development of affordable vaccines. However, to exploit their full potential a convenient system to generate recombinant vesicles would be highly desirable. Here, we report the design of a versatile system for preparation of recombinant outer membrane vesicles based on an engineered autotransporter. Two model vaccine antigens of Leishmania were expressed as fusion proteins with the transporter domain of AIDA, the Escherichia coli adhesin involved in diffuse adhesion. Single subcutaneous injections of recombinant vesicles boosted vaccine antigen-specific antibody responses in mice primed with live recombinant salmonella vaccines by 6-40-fold. The results further show an expansion of only the vaccine antigen-specific antibody response indicating a great potential of this approach for prime boost vaccination strategies.

PMID: 19748581 [PubMed - as supplied by publisher]

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8: Mol Biochem Parasitol. 2009 Sep 10. [Epub ahead of print]

Genetic validation of aldolase and glyceraldehyde-3-phosphate dehydrogenase as drug targets in Trypanosoma brucei.

Cáceres AJ, Michels PA, Hannaert V.

Unidad de Bioquímica de Parásitos, Centro de Ingeniería Genética, Facultad de Ciencias, Universidad de Los Andes, Mérida 5101, Venezuela.

Aldolase (ALD) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of Trypanosoma brucei are considered promising targets for chemotherapeutic treatment of African sleeping sickness, because glycolysis is the single source of ATP for the parasite when living in the human bloodstream. Moreover, these enzymes appeared to possess distinct kinetic and structural properties that have already been exploited for the discovery of effective and selective inhibitors with trypanocidal activity. Here we present an experimental, quantitative assessment of the importance of these enzymes for the glycolytic pathway. This was achieved by decreasing the concentrations of ALD and GAPDH by RNA interference. The effects of these knockdowns on parasite growth, levels of various enzymes and transcripts, enzyme activities and glucose consumption were studied. A partial depletion of ALD and GAPDH was already sufficient to rapidly kill the trypanosomes. An effect was also observed on the activity of some other glycolytic enzymes.

PMID: 19748525 [PubMed - as supplied by publisher]

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9: Anal Biochem. 2009 Sep 10. [Epub ahead of print]

Development and validation of a cytochrome c coupled assay for pteridine reductase 1 and dihydrofolate reductase.

Shanks EJ, Ong HB, Robinson DA, Thompson S, Sienkiewicz N, Fairlamb AH, Frearson JA.

Division of Biological Chemistry & Drug Discovery, College of Life Sciences, University of Dundee, Dundee, DD1 5EH.

Activity of the pterin- and folate-salvaging enzymes pteridine reductase 1 (PTR1) and dihydrofolate reductase thymidylate synthetase (DHFR-TS) is commonly measured as a decrease in absorbance at 340 nm, corresponding to oxidation of NADPH. While this assay has been adequate to study the biology of these enzymes, it is not amenable to support any degree of routine inhibitor assessment since its restricted linearity is incompatible with enhanced throughput microtitre plate screening. In this paper, we report the development and validation of a non-enzymatically coupled screening assay, in which the product of the enzymatic reaction reduces cytochrome c, causing an increase in absorbance at 550 nm. We demonstrate this assay to be robust and accurate and describe its utility in supporting a structure-based design, small molecule inhibitor campaign against T. brucei PTR1 and DHFR-TS.

PMID: 19748480 [PubMed - as supplied by publisher]

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10: Vaccine. 2009 Sep 9. [Epub ahead of print]

Antibiotic resistance free plasmid DNA expressing LACK protein leads towards a protective Th1 response against Leishmania infantum infection.

Ramos I, Alonso A, Peris A, Marcen JM, Abengozar MA, Alcolea PJ, Castillo JA, Larraga V.

Centro de Investigaciones Biológicas, Spanish Research Council, Ramiro de Maeztu 9, 28040 Madrid, Spain.

Canine visceral leishmaniasis is a serious public health concern in the Mediterranean basin since dogs are the main Leishmania infantum reservoir. However, there is not a vaccination method in veterinary use in this area, and therefore the development of a vaccine against this parasite is essential for the possible control of the disease. Previous reports have shown the efficacy of heterologous prime-boost vaccination with the pCIneo plasmid and the poxvirus VV (both Western Reserve and MVA strains) expressing L. infantum LACK antigen against canine leishmaniasis. As pCIneo-LACK plasmid contains antibiotic resistance genes, its use as a profilactic method is not recommended. Hence, the antibiotic resistance gene free pORT-LACK plasmid is a more suitable tool for its use as a vaccine. Here we report the protective and immunostimulatory effect of the prime-boost pORT-LACK/MVA-LACK vaccination tested in a canine experimental model. Vaccination induced a reduction in clinical signs and in parasite burden in the liver, an induction of the Leishmania-specific T cell activation, as well as an increase of the expression of Th1 type cytokines in PBMC and target organs.

PMID: 19747996 [PubMed - as supplied by publisher]

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