Wednesday, April 13, 2011

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 23

1. PLoS One. 2011 Mar 31;6(3):e18425.

The Cell Cycle Regulated Transcriptome of Trypanosoma brucei.

Archer SK, Inchaustegui D, Queiroz R, Clayton C.

Zentrum für Molekulare Biologie Heidelberg, DKFZ-ZMBH Allianz, Heidelberg, Germany.

Abstract

Progression of the eukaryotic cell cycle requires the regulation of hundreds of genes to ensure that they are expressed at the required times. Integral to cell cycle progression in yeast and animal cells are temporally controlled, progressive waves of transcription mediated by cell cycle-regulated transcription factors. However, in the kinetoplastids, a group of early-branching eukaryotes including many important pathogens, transcriptional regulation is almost completely absent, raising questions about the extent of cell-cycle regulation in these organisms and the mechanisms whereby regulation is achieved. Here, we analyse gene expression over the Trypanosoma brucei cell cycle, measuring changes in mRNA abundance on a transcriptome-wide scale. We developed a "double-cut" elutriation procedure to select unperturbed, highly synchronous cell populations from log-phase cultures, and compared this to synchronization by starvation. Transcriptome profiling over the cell cycle revealed the regulation of at least 430 genes. While only a minority were homologous to known cell cycle regulated transcripts in yeast or human, their functions correlated with the cellular processes occurring at the time of peak expression. We searched for potential target sites of RNA-binding proteins in these transcripts, which might earmark them for selective degradation or stabilization. Over-represented sequence motifs were found in several co-regulated transcript groups and were conserved in other kinetoplastids. Furthermore, we found evidence for cell-cycle regulation of a flagellar protein regulon with a highly conserved sequence motif, bearing similarity to consensus PUF-protein binding motifs. RNA sequence motifs that are functional in cell-cycle regulation were more widespread than previously expected and conserved within kinetoplastids. These findings highlight the central importance of post-transcriptional regulation in the proliferation of parasitic kinetoplastids.

PMID: 21483801 [PubMed - in process]
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2. PLoS Negl Trop Dis. 2011 Apr 5;5(4):e1033.

High Throughput Screens Yield Small Molecule Inhibitors of Leishmania CRK3:CYC6 Cyclin-Dependent Kinase.

Walker RG, Thomson G, Malone K, Nowicki MW, Brown E, Blake DG, Turner NJ, Walkinshaw MD, Grant KM, Mottram JC.

Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom.

Abstract

BACKGROUND: Leishmania species are parasitic protozoa that have a tightly controlled cell cycle, regulated by cyclin-dependent kinases (CDKs). Cdc2-related kinase 3 (CRK3), an essential CDK in Leishmania and functional orthologue of human CDK1, can form an active protein kinase complex with Leishmania cyclins CYCA and CYC6. Here we describe the identification and synthesis of specific small molecule inhibitors of bacterially expressed Leishmania CRK3:CYC6 using a high throughput screening assay and iterative chemistry. We also describe the biological activity of the molecules against Leishmania parasites.

METHODOLOGY/PRINCIPAL FINDINGS: In order to obtain an active Leishmania CRK3:CYC6 protein kinase complex, we developed a co-expression and co-purification system for Leishmania CRK3 and CYC6 proteins. This active enzyme was used in a high throughput screening (HTS) platform, utilising an IMAP fluorescence polarisation assay. We carried out two chemical library screens and identified specific inhibitors of CRK3:CYC6 that were inactive against the human cyclin-dependent kinase CDK2:CycA. Subsequently, the best inhibitors were tested against 11 other mammalian protein kinases. Twelve of the most potent hits had an azapurine core with structure activity relationship (SAR) analysis identifying the functional groups on the 2 and 9 positions as essential for CRK3:CYC6 inhibition and specificity against CDK2:CycA. Iterative chemistry allowed synthesis of a number of azapurine derivatives with one, compound 17, demonstrating anti-parasitic activity against both promastigote and amastigote forms of L. major. Following the second HTS, 11 compounds with a thiazole core (active towards CRK3:CYC6 and inactive against CDK2:CycA) were tested. Ten of these hits demonstrated anti-parasitic activity against promastigote L. major.

CONCLUSIONS/SIGNIFICANCE: The pharmacophores identified from the high throughput screens, and the derivatives synthesised, selectively target the parasite enzyme and represent compounds for future hit-to-lead synthesis programs to develop therapeutics against Leishmania species. Challenges remain in identifying specific CDK inhibitors with both target selectivity and potency against the parasite.

PMID: 21483720 [PubMed - in process]
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3. PLoS Negl Trop Dis. 2011 Apr 5;5(4):e1017.

Trypanosoma bruc ei Glycogen Synthase Kinase-3, A Target for Anti-Trypanosomal Drug Development: A Public-Private Partnership to Identify Novel Leads.

Oduor RO, Ojo KK, Williams GP, Bertelli F, Mills J, Maes L, Pryde DC, Parkinson T, Van Voorhis WC, Holler TP.

Opportunities for Partnering in Medicine, Pfizer Global Research and Development, Sandwich, Kent, United Kingdom.

Abstract

BACKGROUND: Trypanosoma brucei, the causative agent of Human African Trypanosomiasis (HAT), expresses two proteins with homology to human glycogen synthase kinase 3β (HsGSK-3) designated TbruGSK-3 short and TbruGSK-3 long. TbruGSK-3 short has previously been validated as a potential drug target and since this enzyme has also been pursued as a human drug target, a large number of inhibitors are available for screening against the parasite enzyme. A collaborative industrial/academic partnership facilitated by the World Health Organisation Tropical Diseases Research division (WHO TDR) was initiated to stimulate research aimed at identifying new drugs for treating HAT.

METHODOLOGY/PRINCIPAL FINDINGS: A subset of over 16,000 inhibitors of HsGSK-3 β from the Pfizer compound collection was screened against the shorter of two orthologues of TbruGSK-3. The resulting active compounds were tested for selectivity versus HsGSK-3β and a panel of human kinases, as well as in vitro anti-trypanosomal activity. Structural analysis of the human and trypanosomal enzymes was also performed.

CONCLUSIONS/SIGNIFICANCE: We identified potent and selective compounds representing potential attractive starting points for a drug discovery program. Structural analysis of the human and trypanosomal enzymes also revealed hypotheses for further improving selectivity of the compounds.

PMID: 21483717 [PubMed - in process]
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4. PLoS One. 2011 Apr 5;6(4):e18467.

Berberine Chloride Mediates Its Anti-Leishmanial Activity via Differential Regulation of the Mitogen Activated Protein Kinase Pathway in Macrophages.

Saha P, Bhattacharjee S, Sarkar A, Manna A, Majumder S, Chatterjee M.

Department of Pharmacology, Institute of Post Graduate Medical Education and Research, Kolkata, India.

Abstract

BACKGROUND: A complex interplay between Leishmania and macrophages influences parasite survival and necessitates disruption of signaling molecules, eventually resulting in impairment of macrophage function. In this study, we demonstrate the immunomodulatory activity of Berberine chloride in Leishmania infected macrophages.

PRINCIPAL FINDINGS: The IC(50) of Berberine chloride, a quaternary isoquinoline alkaloid was tested in an amastigote macrophage model and its safety index measured by a cell viability assay. It eliminated intracellular amastigotes, the IC(50) being 2.8 fold lower than its IC(50) in promastigotes (7.10 µM vs. 2.54 µM) and showed a safety index >16. Levels of intracellular and extracellular nitric oxide (NO) as measured by flow cytometry and Griess assay respectively showed that Berberine chloride in Leishmania infected macrophages increased production of NO. Measurement of the mRNA expression of iNOS, IL-12 and IL-10 by RT-PCR along with levels of IL-12p40 and IL-10 by ELISA showed that in infected macrophages, Berberine chloride enhanced expression of iNOS and IL-12p40, concomitant with a downregulation of IL-10. The phosphorylation status of extracellular signal related kinase (ERK1/2) and p38 mitogen activated protein kinase (p38 MAPK) was studied by western blotting. In infected macrophages, Berberine chloride caused a time dependent activation of p38 MAPK along with deactivation of ERK1/2; addition of a p38 MAPK inhibitor SB203580 inhibited the increased generation of NO and IL-12p40 by Berberine chloride as also prevented its decrease of IL-10.

CONCLUSIONS: Berberine chloride modulated macrophage effector responses via the mitogen activated protein kinase (MAPK) pathway, highlighting the importance of MAPKs as an antiparasite target.

PMID: 21483684 [PubMed - in process]
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5. Cell Immunol. 2011 Apr 8. [Epub ahead of print]

IL-2 limits IL-12 enhanced lymphocyte proliferation during Leishmania amazonensis infection.

Ramer-Tait AE, Petersen CA, Jones DE.

Immunobiology Program and Department of Veterinary Pathology, College of Veterinary Medicine, Iowa State University, Ames, IA 50011-1250, USA.

Abstract

C3H mice infected with Leishmania amazonensis develop persistent, localized lesions with high parasite loads. During infection, memory/effector CD44(hi)CD4(+) T cells proliferate and produce IL-2, but do not polarize to a known effector phenotype. Previous studies have demonstrated IL-12 is insufficient to skew these antigen-responsive T cells to a functional Th1 response. To determine the mechanism of this IL-12 unresponsiveness, we used an in vitro assay of repeated antigen activation. Memory/effector CD44(hi)CD4(+) T cells did not increase proliferation in response to either IL-2 or IL-12, although these cytokines upregulated CD25 expression. Neutralization of IL-2 enhanced CD4(+) T cell proliferation in response to IL-12. This cross-regulation of IL-12 responsiveness by IL-2 was confirmed in vivo by treatment with anti-IL-2 antibodies and IL-12 during antigen challenge of previously infected mice. These results suggest that during chronic infection with L. amazonensis, IL-2 plays a dominant, immunosuppressive role independent of identifiable conventional T(reg) cells.

Copyright © 2011 Elsevier Inc. All rights reserved.
PMID: 21481338 [PubMed - as supplied by publisher]
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6. Parasite Immunol. 2011 Apr 11. doi: 10.1111/j.1365-3024.2011.01293.x. [Epub ahead of print]

Mouse models for pathogenic African trypanosomes: unravelling the immunology of host-parasite-vector intera ctions.

Magez S, Caljon G.

VIB Department of Molecular and Cellular Interactions, Laboratory for Cellular and Molecular Immunology. Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium Department of Animal Health, Unit of Veterinary Protozoology, Institute of Tropical Medicine Antwerp, Nationalestraat 155, B-2000 Antwerp, Belgium.

Abstract

African trypanosomiasis is a parasitic disease that affects a variety of mammals, including humans, on the sub-Saharan African continent. In order to understand the diverse parameters that govern the host-parasite-vector interactions, mouse models for the disease have proven to be a corner stone. Despite the fact that most trypanosomes cannot be considered as natural pathogens for rodents, experimental infections in mice have shed a tremendous amount of light on the general biology of these parasites and their interaction with and evasion of the mammalian immune system. Different aspects including inflammation, vaccine failure, antigenic variation, resistance/sensitivity to normal human serum and the influence of tsetse compounds on parasite transmission have all been addressed using mouse models. In more recent years, the introduction of various 'knock-out' mouse strains has allowed to analyse the implication of various cytokines, particularly TNF, IFNγ and IL-10 in the regulation of parasitemia and induction of pathological conditions during infection.

Copyright © 2011 Blackwell Publishing Ltd.
PMID: 21480934 [PubMed - as supplied by publisher]
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7. Biochem Biophys Res Commun. 2011 Feb 25;405(4):604-9. Epub 2011 Jan 24.

Phagocytosis is inhibited by autophagic induction in murine macrophages.

Lima JG, de Freitas Vinhas C, Gomes IN, Azevedo CM, dos Santos RR, Vannier-Santos MA, Veras PS.

Laboratório de Patologia e Biointervenção, IGM-FIOCRUZ, Bahia, Brazil.

Abstract

Recent studies have demonstrated that communication takes place between the autophagic and phagocytic pathways, indicating that the convergence of these two pathways plays an important role in the innate immune response against intracellular microbes. The present study investigated the effect of autophagic induction on the phagocytic capacity of murine macrophages. Autophagy induced by physiological and pharmacological means was shown to reduce the phagocytic capacity of murine macrophages, regardless of cell origin or the nature of the phagocytosed particles themselves. This autophagic inhibitory effect on phagocytosis was shown to be an early and reversible event that results in no loss of cell viability. Furthermore, the data presented herein demonstrate that the induction of autophagy does not affect a macrophage's capacity to recognize and bind to particles, indicating that autophagy does not inhibit the particle recognition process, even though particle internalization is suppressed. The findings herein support the notion that phagocytosis and autophagy may be interdependent and complementary processes.

Copyright © 2011 Elsevier Inc. All rights reserved.
PMID: 21272565 [PubMed - indexed for MEDLINE]
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8. PLoS Negl Trop Dis. 2010 Nov 30;4(11):e899.

Chagas cardiomyopathy manifestations and Trypanosoma cruzi genotypes circulating in chronic Chagasic patients.

Ramírez JD, Guhl F, Rendón LM, Rosas F, Marin-Neto JA, Morillo CA.

Centro de investigaciones en Microbiología y Parasitología Tropical (CIMPAT), Facultad de Ciencias, Departamento de Ciencias Biológicas, Universidad de los Andes, Bogotá, Colombia.

Abstract

Chagas disease caused by Trypanosoma cruzi is a complex disease that is endemic and an important problem in public health in Latin America. The T. cruzi parasite is classified into six discrete taxonomic units (DTUs) based on the recently proposed nomenclature (TcI, TcII, TcIII, TcIV, TcV and TcVI). The discovery of genetic variability within TcI showed the presence of five genotypes (Ia, Ib, Ic, Id and Ie) related to the transmission cycle of Chagas disease. In Colombia, TcI is more prevalent but TcII has also been reported, as has mixed infection by both TcI and TcII in the same Chagasic patient. The objectives of this study were to determine the T. cruzi DTUs that are circulating in Colombian chronic Chagasic patients and to obtain more information about the molecular epidemiology of Chagas disease in Colombia. We also assessed the presence of electrocardiographic, radiologic and echocardiographic abnormalities with the purpose of correlating T. cruzi genetic variability and cardiac disease. Molecular characterization was performed in Colombian adult chronic Chagasic patients based on the intergenic region of the mini-exon gene, the 24Sα and 18S regions of rDNA and the variable region of satellite DNA, whereby the presence of T.cruzi I, II, III and IV was detected. In our population, mixed infections also occurred, with TcI-TcII, TcI-TcIII and TcI-TcIV, as well as the existence of the TcI genotypes showing the presence of genotypes Ia and Id. Patients infected with TcI demonstrated a higher prevalence of cardiac alterations than those infected with TcII. These results corroborate the predominance of TcI in Colombia and show the first report of TcIII and TcIV in Colombian Chagasic patients. Findings also indicate that Chagas cardiomyopathy manifestations are more correlated with TcI than with TcII in Colombia.

PMCID: PMC2994916 Free PMC Article
PMID: 21152056 [PubMed - indexed for MEDLINE]
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9. PLoS Negl Trop Dis. 2010 Nov 9;4(11):e880.

A comprehensive genetic analysis of candidate genes regulating response to Trypanosoma congolense infection in mice.

Goodhead I, Archibald A, Amwayi P, Brass A, Gibson J, Hall N, Hughes MA, Limo M, Iraqi F, Kemp SJ, Noyes HA.

Centre for Genomic Research, School of Biological Sciences, University of Liverpool, Liverpool, United Kingdom.

Abstract

BACKGROUND: African trypanosomes are protozoan parasites that cause "sleeping sickness" in humans and a similar disease in livestock. Trypanosomes also infect laboratory mice and three major quantitative trait loci (QTL) that regulate survival time after infection with T. congolense have been identified in two independent crosses between susceptible A/J and BALB/c mice, and the resistant C57BL/6. These were designated Tir1, Tir2 and Tir3 for Trypanosoma infection response, and range in size from 0.9-12 cM.

PRINCIPAL FINDINGS: Mapping loci regulating survival time after T. congolense infection in an additional cross revealed that susceptible C3H/HeJ mice have alleles that reduce survival time after infection at Tir1 and Tir3 QTL, but not at Tir2. Next-generation resequencing of a 6.2 Mbp region of mouse chromosome 17, which includes Tir1, identified 1,632 common single nucleotide polymorphisms (SNP) including a probably damaging non-synonymous SNP in Pram1 (PML-RAR alpha-regulated adaptor molecule 1), which was the most plausible candidate QTL gene in Tir1. Genome-wide comparative genomic hybridisation identified 12 loci with copy number variants (CNV) that correlate with differential gene expression, including Cd244 (natural killer cell receptor 2B4), which lies close to the peak of Tir3c and has gene expression that correlates with CNV and phenotype, making it a strong candidate QTL gene at this locus.

CONCLUSIONS: By systematically combining next-generation DNA capture and sequencing, array-based comparative genomic hybridisation (aCGH), gene expression data and SNP annotation we have developed a strategy that can generate a short list of polymorphisms in candidate QTL genes that can be functionally tested.

PMCID: PMC2976683 Free PMC Article
PMID: 21085469 [PubMed - indexed for MEDLINE]
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10. PLoS Negl Trop Dis. 2010 Nov 2;4(11):e864.

Role of the gp85/trans-sialidases in Trypanosoma cruzi tissue tropism: preferential binding of a conserved peptide motif to the vasculature in vivo.

Tonelli RR, Giordano RJ, Barbu EM, Torrecilhas AC, Kobayashi GS, Langley RR, Arap W, Pasqualini R, Colli W, Alves MJ.

Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo, São Paulo, Brazil.

Abstract

BACKGROUND: Transmitted by blood-sucking insects, the unicellular parasite Trypanosoma cruzi is the causative agent of Chagas' disease, a malady manifested in a variety of symptoms from heart disease to digestive and urinary tract dysfunctions. The reasons for such organ preference have been a matter of great interest in the field, particularly because the parasite can invade nearly every cell line and it can be found in most tissues following an infection. Among the molecular factors that contribute to virulence is a large multigene family of proteins known as gp85/trans-sialidase, which participates in cell attachment and invasion. But whether these proteins also contribute to tissue homing had not yet been investigated. Here, a combination of endothelial cell immortalization and phage display techniques has been used to investigate the role of gp85/trans-sialidase in binding to the vasculature.

METHODS: Bacteriophage expressing an important peptide motif (denominated FLY) common to all gp85/trans-sialidase proteins was used as a surrogate to investigate the interaction of this motif with the endothelium compartment. For that purpose phage particles were incubated with endothelial cells obtained from different organs or injected into mice intravenously and the number of phage particles bound to cells or tissues was determined. Binding of phages to intermediate filament proteins has also been studied.

FINDINGS AND CONCLUSIONS: Our data indicate that FLY interacts with the endothelium in an organ-dependent manner with significantly higher avidity for the heart vasculature. Phage display results also show that FLY interaction with intermediate filament proteins is not limited to cytokeratin 18 (CK18), which may explain the wide variety of cells infected by the parasite. This is the first time that members of the intermediate filaments in general, constituted by a large group of ubiquitously expressed proteins, have been implicated in T. cruzi cell invasion and tissue homing.

PMCID: PMC2970537 Free PMC Article
PMID: 21072227 [PubMed - indexed for MEDLINE]
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