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Sample records for brucei puf9 regulates

  1. Regulation and spatial organization of PCNA in Trypanosoma brucei

    Energy Technology Data Exchange (ETDEWEB)

    Kaufmann, Doris; Gassen, Alwine [University of Munich (LMU), Department Biology I, Genetics, Grosshaderner Str. 2-4, 82152 Martinsried (Germany); Maiser, Andreas; Leonhardt, Heinrich [University of Munich (LMU), Department Biology II, Grosshaderner Str. 2-4, 82152 Martinsried (Germany); Janzen, Christian J., E-mail: christian.janzen@uni-wuerzburg.de [University of Munich (LMU), Department Biology I, Genetics, Grosshaderner Str. 2-4, 82152 Martinsried (Germany)

    2012-03-23

    Highlights: Black-Right-Pointing-Pointer Characterization of the proliferating cell nuclear antigen in Trypanosoma brucei (TbPCNA). Black-Right-Pointing-Pointer TbPCNA is a suitable marker to detect replication in T. brucei. Black-Right-Pointing-Pointer TbPCNA distribution and regulation is different compared to closely related parasites T. cruzi and Leishmania donovani. -- Abstract: As in most eukaryotic cells, replication is regulated by a conserved group of proteins in the early-diverged parasite Trypanosoma brucei. Only a few components of the replication machinery have been described in this parasite and regulation, sub-nuclear localization and timing of replication are not well understood. We characterized the proliferating cell nuclear antigen in T. brucei (TbPCNA) to establish a spatial and temporal marker for replication. Interestingly, PCNA distribution and regulation is different compared to the closely related parasites Trypanosoma cruzi and Leishmania donovani. TbPCNA foci are clearly detectable during S phase of the cell cycle but in contrast to T. cruzi they are not preferentially located at the nuclear periphery. Furthermore, PCNA seems to be degraded when cells enter G2 phase in T. brucei suggesting different modes of replication regulation or functions of PCNA in these closely related eukaryotes.

  2. Maf1 is a negative regulator of transcription in Trypanosoma brucei.

    Science.gov (United States)

    Romero-Meza, Gabriela; Vélez-Ramírez, Daniel E; Florencio-Martínez, Luis E; Román-Carraro, Fiordaliso C; Manning-Cela, Rebeca; Hernández-Rivas, Rosaura; Martínez-Calvillo, Santiago

    2017-02-01

    RNA polymerase III (Pol III) produces small RNA molecules that play essential roles in mRNA processing and translation. Maf1, originally described as a negative regulator of Pol III transcription, has been studied from yeast to human. Here we characterized Maf1 in the parasitic protozoa Trypanosoma brucei (TbMaf1), representing the first report to analyse Maf1 in an early-diverged eukaryote. While Maf1 is generally encoded by a single-copy gene, the T. brucei genome contains two almost identical TbMaf1 genes. The TbMaf1 protein has the three conserved sequences and is predicted to fold into a globular structure. Unlike in yeast, TbMaf1 localizes to the nucleus in procyclic forms of T. brucei under normal growth conditions. Cell lines that either downregulate or overexpress TbMaf1 were generated, and growth curve analysis with them suggested that TbMaf1 participates in the regulation of cell growth of T. brucei. Nuclear run-on and chromatin immunoprecipitation analyses demonstrated that TbMaf1 represses Pol III transcription of tRNA and U2 snRNA genes by associating with their promoters. Interestingly, 5S rRNA levels do not change after TbMaf1 ablation or overexpression. Notably, our data also revealed that TbMaf1 regulates Pol I transcription of procyclin gene and Pol II transcription of SL RNA genes. © 2016 John Wiley & Sons Ltd.

  3. NLP is a novel transcription regulator involved in VSG expression site control in Trypanosoma brucei.

    Science.gov (United States)

    Narayanan, Mani Shankar; Kushwaha, Manish; Ersfeld, Klaus; Fullbrook, Alexander; Stanne, Tara M; Rudenko, Gloria

    2011-03-01

    Trypanosoma brucei mono-allelically expresses one of approximately 1500 variant surface glycoprotein (VSG) genes while multiplying in the mammalian bloodstream. The active VSG is transcribed by RNA polymerase I in one of approximately 15 telomeric VSG expression sites (ESs). T. brucei is unusual in controlling gene expression predominantly post-transcriptionally, and how ESs are mono-allelically controlled remains a mystery. Here we identify a novel transcription regulator, which resembles a nucleoplasmin-like protein (NLP) with an AT-hook motif. NLP is key for ES control in bloodstream form T. brucei, as NLP knockdown results in 45- to 65-fold derepression of the silent VSG221 ES. NLP is also involved in repression of transcription in the inactive VSG Basic Copy arrays, minichromosomes and procyclin loci. NLP is shown to be enriched on the 177- and 50-bp simple sequence repeats, the non-transcribed regions around rDNA and procyclin, and both active and silent ESs. Blocking NLP synthesis leads to downregulation of the active ES, indicating that NLP plays a role in regulating appropriate levels of transcription of ESs in both their active and silent state. Discovery of the unusual transcription regulator NLP provides new insight into the factors that are critical for ES control.

  4. INFECTED WITH TRYPANOSOMA BRUCEI BRUCEI

    African Journals Online (AJOL)

    v brucei infection where for example tant components of extra-cellular the parasite infectivity potential and toxicological effects have been sues in animal. They are ... homeostasis (1,2, 3). With a unique ability to main- tain the cellular contents and ions stable concentrations, Trypano- soma b. brucei, to which humans.

  5. Regulation of Trypanosoma brucei Total and Polysomal mRNA during Development within Its Mammalian Host.

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    Paul Capewell

    Full Text Available The gene expression of Trypanosoma brucei has been examined extensively in the blood of mammalian hosts and in forms found in the midgut of its arthropod vector, the tsetse fly. However, trypanosomes also undergo development within the mammalian bloodstream as they progress from morphologically 'slender forms' to transmissible 'stumpy forms' through morphological intermediates. This transition is temporally progressive within the first wave of parasitaemia such that gene expression can be monitored in relatively pure slender and stumpy populations as well as during the progression between these extremes. The development also represents the progression of cells from translationally active forms adapted for proliferation in the host to translationally quiescent forms, adapted for transmission. We have used metabolic labelling to quantitate translational activity in slender forms, stumpy forms and in forms undergoing early differentiation to procyclic forms in vitro. Thereafter we have examined the cohort of total mRNAs that are enriched throughout development in the mammalian bloodstream (slender, intermediate and stumpy forms, irrespective of strain, revealing those that exhibit consistent developmental regulation rather than sample specific changes. Transcripts that cosediment with polysomes in stumpy forms and slender forms have also been enriched to identify transcripts that escape translational repression prior to transmission. Combined, the expression and polysomal association of transcripts as trypanosomes undergo development in the mammalian bloodstream have been defined, providing a resource for trypanosome researchers. This facilitates the identification of those that undergo developmental regulation in the bloodstream and therefore those likely to have a role in the survival and capacity for transmission of stumpy forms.

  6. Extracellular-signal regulated kinase 8 of Trypanosoma brucei uniquely phosphorylates its proliferating cell nuclear antigen homolog and reveals exploitable properties.

    Science.gov (United States)

    Valenciano, Ana L; Knudsen, Giselle M; Mackey, Zachary B

    2016-10-17

    The Trypanosoma brucei subspecies T. brucei gambiense and T. brucei rhodesiense are vector-borne pathogens that cause sleeping sickness also known as Human African Trypanosomiasis (HAT), which is fatal if left untreated. The drugs that treat HAT are ineffective and cause toxic side effects. One strategy for identifying safer and more effective HAT drugs is to therapeutically exploit essential gene targets in T. brucei. Genes that make up a basic mitogen-activated protein kinase (MAPK) network are present in T. brucei. Tb927.10.5140 encodes an essential MAPK that is homologous to the human extracellular-signal regulated kinase 8 (HsERK8) which forms a tight complex with the replication factor proliferating cell nuclear antigen (PCNA) to stabilize intracellular PCNA levels. Here we demonstrate that (TbPCNA) is uniquely phos-phorylated on serine (S) and threonine (T) residues in T. brucei and that TbERK8 phosphorylates TbPCNA at each of these residues. The ability of an ERK8 homolog to phosphorylate a PCNA homolog is a novel biochemical property that is first demonstrated here in T. brucei and may be unique to this pathogen. We demonstrate that the potent HsERK8 inhibitor Ro318220, has an IC50 for TbERK8 that is several hundred times higher than its reported IC50 for HsERK8. This indicated that the active sites of TbERK8 and HsERK8 can be selectively inhibited, which provides a rational basis for discovering inhibitors that specifically target this essential parasite MAPK to kill the parasite.

  7. Base J and H3.V Regulate Transcriptional Termination in Trypanosoma brucei.

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    Danae Schulz

    2016-01-01

    Full Text Available Trypanosoma brucei is a protozoan parasite that lacks many transcription factors found in other eukaryotes, such as those whose binding demarcates enhancers. T. brucei retains histone variants and modifications, however, and it is hypothesized that it relies on epigenetic marks to define transcription-related boundaries. The histone H3 variant (H3.V and an alternate nucleotide, base J (ß-D-glucosyl-hydroxymethyluracil, are two chromatin marks found at both transcription termination sites (TTSs and telomeres. Here, we report that the absence of both base J and H3.V result in transcription readthrough and the appearance of antisense transcripts near TTSs. Additionally, we find that maintaining the transcriptional silencing of pol I-transcribed telomeric Variant Surface Glycoprotein (VSG genes appears to be dependent on deposition of H3.V alone. Our study reveals that gene expression depends on different epigenetic cues depending on chromosomal location and on the transcribing polymerase. This work provides insight into how these signals may have evolved into the more nuanced and fine-tuned gene regulatory mechanisms observed in other model systems.

  8. Regulators of Trypanosoma brucei cell cycle progression and differentiation identified using a kinome-wide RNAi screen.

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    Nathaniel G Jones

    2014-01-01

    Full Text Available The African trypanosome, Trypanosoma brucei, maintains an integral link between cell cycle regulation and differentiation during its intricate life cycle. Whilst extensive changes in phosphorylation have been documented between the mammalian bloodstream form and the insect procyclic form, relatively little is known about the parasite's protein kinases (PKs involved in the control of cellular proliferation and differentiation. To address this, a T. brucei kinome-wide RNAi cell line library was generated, allowing independent inducible knockdown of each of the parasite's 190 predicted protein kinases. Screening of this library using a cell viability assay identified ≥42 PKs that are required for normal bloodstream form proliferation in culture. A secondary screen identified 24 PKs whose RNAi-mediated depletion resulted in a variety of cell cycle defects including in G1/S, kinetoplast replication/segregation, mitosis and cytokinesis, 15 of which are novel cell cycle regulators. A further screen identified for the first time two PKs, named repressor of differentiation kinase (RDK1 and RDK2, depletion of which promoted bloodstream to procyclic form differentiation. RDK1 is a membrane-associated STE11-like PK, whilst RDK2 is a NEK PK that is essential for parasite proliferation. RDK1 acts in conjunction with the PTP1/PIP39 phosphatase cascade to block uncontrolled bloodstream to procyclic form differentiation, whilst RDK2 is a PK whose depletion efficiently induces differentiation in the absence of known triggers. Thus, the RNAi kinome library provides a valuable asset for functional analysis of cell signalling pathways in African trypanosomes as well as drug target identification and validation.

  9. Digital gene expression analysis of two life cycle stages of the human-infective parasite, Trypanosoma brucei gambiense reveals differentially expressed clusters of co-regulated genes

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    Wildridge David

    2010-02-01

    Full Text Available Abstract Background The evolutionarily ancient parasite, Trypanosoma brucei, is unusual in that the majority of its genes are regulated post-transcriptionally, leading to the suggestion that transcript abundance of most genes does not vary significantly between different life cycle stages despite the fact that the parasite undergoes substantial cellular remodelling and metabolic changes throughout its complex life cycle. To investigate this in the clinically relevant sub-species, Trypanosoma brucei gambiense, which is the causative agent of the fatal human disease African sleeping sickness, we have compared the transcriptome of two different life cycle stages, the potentially human-infective bloodstream forms with the non-human-infective procyclic stage using digital gene expression (DGE analysis. Results Over eleven million unique tags were generated, producing expression data for 7360 genes, covering 81% of the genes in the genome. Compared to microarray analysis of the related T. b. brucei parasite, approximately 10 times more genes with a 2.5-fold change in expression levels were detected. The transcriptome analysis revealed the existence of several differentially expressed gene clusters within the genome, indicating that contiguous genes, presumably from the same polycistronic unit, are co-regulated either at the level of transcription or transcript stability. Conclusions DGE analysis is extremely sensitive for detecting gene expression differences, revealing firstly that a far greater number of genes are stage-regulated than had previously been identified and secondly and more importantly, this analysis has revealed the existence of several differentially expressed clusters of genes present on what appears to be the same polycistronic units, a phenomenon which had not previously been observed in microarray studies. These differentially regulated clusters of genes are in addition to the previously identified RNA polymerase I polycistronic

  10. The PARP promoter of Trypanosoma brucei is developmentally regulated in a chromosomal context

    DEFF Research Database (Denmark)

    Biebinger, S; Rettenmaier, S; Flaspohler, J

    1996-01-01

    RNA is abundant in procyclic forms and almost undetectable in blood-stream forms. Post-transcriptional mechanisms are mainly responsible for PARP mRNA regulation but results of nuclear run-on experiments suggested that transcription might also be regulated. We measured the activity of genomically-integrated PARP...... not developmentally regulated, but integration at the PARP locus reduced rRNA promoter activity in bloodstream forms. PARP promoter activity was 5-fold down-regulated in bloodstream forms when integrated at either site. Regulation was probably at the level of transcriptional initiation, but elongation through plasmid...

  11. Non-radioactive method for labelling Trypanosoma brucei brucei ...

    African Journals Online (AJOL)

    It was observed that (i) the biological activities of the T.brucei brucei like the motility, viability, and growth were not affected adversely by the biotinylation; (ii) as little as 100ug Sulfo-NHS-LC-Biotin was enough to label about 6x106 Trypanosoma Brucei Brucei; (iii) high temperature appears to have adverse effect on the ...

  12. Proximity Interactions among Basal Body Components in Trypanosoma brucei Identify Novel Regulators of Basal Body Biogenesis and Inheritance

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    Hung Quang Dang

    2017-01-01

    Full Text Available The basal body shares similar architecture with centrioles in animals and is involved in nucleating flagellar axonemal microtubules in flagellated eukaryotes. The early-branching Trypanosoma brucei possesses a motile flagellum nucleated from the basal body that consists of a mature basal body and an adjacent pro-basal body. Little is known about the basal body proteome and its roles in basal body biogenesis and flagellar axoneme assembly in T. brucei. Here, we report the identification of 14 conserved centriole/basal body protein homologs and 25 trypanosome-specific basal body proteins. These proteins localize to distinct subdomains of the basal body, and several of them form a ring-like structure surrounding the basal body barrel. Functional characterization of representative basal body proteins revealed distinct roles in basal body duplication/separation and flagellar axoneme assembly. Overall, this work identified novel proteins required for basal body duplication and separation and uncovered new functions of conserved basal body proteins in basal body duplication and separation, highlighting an unusual mechanism of basal body biogenesis and inheritance in this early divergent eukaryote.

  13. A Protein Complex Map of Trypanosoma brucei.

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    Vahid H Gazestani

    2016-03-01

    Full Text Available The functions of the majority of trypanosomatid-specific proteins are unknown, hindering our understanding of the biology and pathogenesis of Trypanosomatida. While protein-protein interactions are highly informative about protein function, a global map of protein interactions and complexes is still lacking for these important human parasites. Here, benefiting from in-depth biochemical fractionation, we systematically interrogated the co-complex interactions of more than 3354 protein groups in procyclic life stage of Trypanosoma brucei, the protozoan parasite responsible for human African trypanosomiasis. Using a rigorous methodology, our analysis led to identification of 128 high-confidence complexes encompassing 716 protein groups, including 635 protein groups that lacked experimental annotation. These complexes correlate well with known pathways as well as for proteins co-expressed across the T. brucei life cycle, and provide potential functions for a large number of previously uncharacterized proteins. We validated the functions of several novel proteins associated with the RNA-editing machinery, identifying a candidate potentially involved in the mitochondrial post-transcriptional regulation of T. brucei. Our data provide an unprecedented view of the protein complex map of T. brucei, and serve as a reliable resource for further characterization of trypanosomatid proteins. The presented results in this study are available at: www.TrypsNetDB.org.

  14. Taxonomy Icon Data: Trypanosoma brucei [Taxonomy Icon

    Lifescience Database Archive (English)

    Full Text Available Trypanosoma brucei Trypanosoma brucei Trypanosoma_brucei_L.png Trypanosoma_brucei_NL.png Trypanosoma_bruce...i_S.png Trypanosoma_brucei_NS.png http://biosciencedbc.jp/taxonomy_icon/icon.cgi?i=Trypanosoma+bruce...i&t=L http://biosciencedbc.jp/taxonomy_icon/icon.cgi?i=Trypanosoma+brucei&t=NL http://bioscie...ncedbc.jp/taxonomy_icon/icon.cgi?i=Trypanosoma+brucei&t=S http://biosciencedbc.jp.../taxonomy_icon/icon.cgi?i=Trypanosoma+brucei&t=NS http://togodb.biosciencedbc.jp/togodb/view/taxonomy_icon_comment_en?species_id=121 ...

  15. Hepatic response in Trypanosomia brucei brucei infected-rats ...

    African Journals Online (AJOL)

    Ethanol leaf extract of Tithonia diversifolia was investigated for its hepato-curative activity in Trypanosoma brucei brucei infected-rats. The phytochemical compositions of the plant extract were also evaluated. The leaf extract was administered 14 days post-infection at dose of 200 and 400 mg/kg orally once daily.

  16. SUMOylation in Trypanosoma brucei

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    Cornelia Andrea Klein

    2013-10-01

    Full Text Available Small ubiquitin like modifier (SUMO proteins are involved in many processes in eukaryotes. We here show that Trypanosoma brucei SUMO (Tb927.5.3210 modifies many proteins. The levels of SUMOylation were unaffected by temperature changes but were increased by severe oxidative stress. We obtained evidence that trypanosome homologues of the SUMO conjugating enzyme Ubc9 (Tb927.2.2460 and the SUMO-specific protease SENP (Tb927.9.2220 are involved in SUMOylation and SUMO removal, respectively.

  17. Malleable mitochondrion of Trypanosoma brucei.

    Science.gov (United States)

    Verner, Zdeněk; Basu, Somsuvro; Benz, Corinna; Dixit, Sameer; Dobáková, Eva; Faktorová, Drahomíra; Hashimi, Hassan; Horáková, Eva; Huang, Zhenqiu; Paris, Zdeněk; Peña-Diaz, Priscila; Ridlon, Lucie; Týč, Jiří; Wildridge, David; Zíková, Alena; Lukeš, Julius

    2015-01-01

    The importance of mitochondria for a typical aerobic eukaryotic cell is undeniable, as the list of necessary mitochondrial processes is steadily growing. Here, we summarize the current knowledge of mitochondrial biology of an early-branching parasitic protist, Trypanosoma brucei, a causative agent of serious human and cattle diseases. We present a comprehensive survey of its mitochondrial pathways including kinetoplast DNA replication and maintenance, gene expression, protein and metabolite import, major metabolic pathways, Fe-S cluster synthesis, ion homeostasis, organellar dynamics, and other processes. As we describe in this chapter, the single mitochondrion of T. brucei is everything but simple and as such rivals mitochondria of multicellular organisms. Copyright © 2015. Published by Elsevier Inc.

  18. Non-cytochrome mediated mitochondrial ATP production in bloodstream form Trypanosoma brucei brucei

    NARCIS (Netherlands)

    Bienen, E. J.; Maturi, R. K.; Pollakis, G.; Clarkson, A. B.

    1993-01-01

    The life cycle of Trypanosoma brucei brucei involves a series of differentiation steps characterized by marked changes in mitochondrial development and function. The bloodstream forms of this parasite completely lack cytochromes and have not been considered to have any Krebs cycle function. It has

  19. Stearoyl-CoA desaturase is an essential enzyme for the parasitic protist Trypanosoma brucei

    Energy Technology Data Exchange (ETDEWEB)

    Alloatti, Andres [Instituto de Biologia Molecular y Celular de Rosario (IBR), CONICET, Facultad de Ciencias Bioquimicas y Farmaceuticas, Universidad Nacional de Rosario, Santa Fe (Argentina); Gupta, Shreedhara; Gualdron-Lopez, Melisa; Nguewa, Paul A. [Research Unit for Tropical Diseases, de Duve Institute and Laboratory of Biochemistry, Universite Catholique de Louvain, Brussels (Belgium); Altabe, Silvia G. [Instituto de Biologia Molecular y Celular de Rosario (IBR), CONICET, Facultad de Ciencias Bioquimicas y Farmaceuticas, Universidad Nacional de Rosario, Santa Fe (Argentina); Deumer, Gladys; Wallemacq, Pierre [Department of Clinical Chemistry, Cliniques Universitaires Saint-Luc, LTAP, Universite Catholique de Louvain, Brussels (Belgium); Michels, Paul A.M. [Research Unit for Tropical Diseases, de Duve Institute and Laboratory of Biochemistry, Universite Catholique de Louvain, Brussels (Belgium); Uttaro, Antonio D., E-mail: toniuttaro@yahoo.com.ar [Instituto de Biologia Molecular y Celular de Rosario (IBR), CONICET, Facultad de Ciencias Bioquimicas y Farmaceuticas, Universidad Nacional de Rosario, Santa Fe (Argentina)

    2011-08-26

    Highlights: {yields} Inhibiting {Delta}9 desaturase drastically changes T. brucei's fatty-acid composition. {yields} Isoxyl specifically inhibits the {Delta}9 desaturase causing a growth arrest. {yields} RNA interference of desaturase expression causes a similar effect. {yields} Feeding T. brucei-infected mice with Isoxyl decreases the parasitemia. {yields} 70% of Isoxyl-treated mice survived the trypanosome infection. -- Abstract: Trypanosoma brucei, the etiologic agent of sleeping sickness, is exposed to important changes in nutrients and temperature during its life cycle. To adapt to these changes, the fluidity of its membranes plays a crucial role. This fluidity, mediated by the fatty-acid composition, is regulated by enzymes named desaturases. We have previously shown that the oleoyl desaturase is essential for Trypanosoma cruzi and T. brucei. In this work, we present experimental support for the relevance of stearoyl-CoA desaturase (SCD) for T. brucei's survival, in both its insect or procyclic-form (PCF) and bloodstream-form (BSF) stages. We evaluated this essentiality in two different ways: by generating a SCD knocked-down parasite line using RNA interference, and by chemical inhibition of the enzyme with two compounds, Isoxyl and a thiastearate with the sulfur atom at position 10 (10-TS). The effective concentration for 50% growth inhibition (EC{sub 50}) of PCF was 1.0 {+-} 0.2 {mu}M for Isoxyl and 5 {+-} 2 {mu}M for 10-TS, whereas BSF appeared more susceptible with EC{sub 50} values 0.10 {+-} 0.03 {mu}M (Isoxyl) and 1.0 {+-} 0.6 {mu}M (10-TS). RNA interference showed to be deleterious for both stages of the parasite. In addition, T. brucei-infected mice were fed with Isoxyl, causing a reduction of the parasitemia and an increase of the rodents' survival.

  20. Rab23 is a flagellar protein in Trypanosoma brucei

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    Field Mark C

    2011-06-01

    Full Text Available Abstract Background Rab small GTPases are important mediators of membrane transport, and orthologues frequently retain similar locations and functions, even between highly divergent taxa. In metazoan organisms Rab23 is an important negative regulator of Sonic hedgehog signaling and is crucial for correct development and differentiation of cellular lineages by virtue of an involvement in ciliary recycling. Previously, we reported that Trypanosoma brucei Rab23 localized to the nuclear envelope 1, which is clearly inconsistent with the mammalian location and function. As T. brucei is unicellular the potential that Rab23 has no role in cell signaling was possible. Here we sought to further investigate the role(s of Rab23 in T. brucei to determine if Rab23 was an example of a Rab protein with divergent function in distinct taxa. Methods/major findings The taxonomic distribution of Rab23 was examined and compared with the presence of flagella/cilia in representative taxa. Despite evidence for considerable secondary loss, we found a clear correlation between a conventional flagellar structure and the presence of a Rab23 orthologue in the genome. By epitope-tagging, Rab23 was localized and found to be present at the flagellum throughout the cell cycle. However, RNAi knockdown did not result in a flagellar defect, suggesting that Rab23 is not required for construction or maintenance of the flagellum. Conclusions The location of Rab23 at the flagellum is conserved between mammals and trypanosomes and the Rab23 gene is restricted to flagellated organisms. These data may suggest the presence of a Rab23-mediated signaling mechanism in trypanosomes.

  1. What happens when Trypanosoma brucei leaves Africa.

    Science.gov (United States)

    Jensen, Robert E; Simpson, Larry; Englund, Paul T

    2008-10-01

    Julius Lukes and co-workers evaluated the evolutionary origin of Trypanosoma equiperdum and Trypanosoma evansi, parasites that cause horse and camel diseases. Although similar to T. brucei, the sleeping-sickness parasite, these trypanosomes do not cycle through the tsetse fly and have been able to spread beyond Africa. Transmission occurs sexually, or via blood-sucking flies or vampire bats. They concluded that these parasites, which resemble yeast petite mutants, are T. brucei sub-species, which have evolved recently through changes in mitochondrial DNA.

  2. Interaction between Trypanosoma brucei and Haemonchus ...

    African Journals Online (AJOL)

    In order to investigate the immunomodulatory influence of concurrent T. brucei and H. contortus infection in West African Dwarf (WAD) goats, 28 infected and 7 uninfected (control) of 8-9 months old male WAD goats were studied. The infected goats were separated into resistant (Class 1) and susceptible (Class 2) Faecal ...

  3. Haematology of experimental Trypanosoma brucei rhodesiense ...

    African Journals Online (AJOL)

    Haematological aberrations associated with human infective trypanosomes were investigated in the vervet monkey model of the Rhodesian sleeping sickness. Four monkeys were infected intravenously with 104 Trypanosoma brucei rhodesiense and monitored for changes in the blood profile using a haematological ...

  4. Characterization of Trypanosoma brucei gambiense stocks isolated ...

    African Journals Online (AJOL)

    Characterization of Trypanosoma brucei gambiense stocks isolated from humans by RAPD fingerprinting in Côte d'Ivoire: another evidence for multiple infections. ... a given isoenzyme profile (zymodeme), confirming that this fingerprinting method has a higher discriminative power (faster molecular clock) than isoenzymes.

  5. Wild chimpanzees are infected by Trypanosoma brucei

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    Milan Jirků

    2015-12-01

    Finally, we demonstrated that the mandrill serum was able to efficiently lyse T. b. brucei and T. b. rhodesiense, and to some extent T. b. gambiense, while the chimpanzee serum failed to lyse any of these subspecies.

  6. Genetic control of resistance to Trypanosoma brucei brucei infection in mice.

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    Matyáš Síma

    2011-06-01

    Full Text Available Trypanosoma brucei brucei infects livestock, with severe effects in horses and dogs. Mouse strains differ greatly in susceptibility to this parasite. However, no genes controlling these differences were mapped.We studied the genetic control of survival after T. b. brucei infection using recombinant congenic (RC strains, which have a high mapping power. Each RC strain of BALB/c-c-STS/A (CcS/Dem series contains a different random subset of 12.5% genes from the parental "donor" strain STS/A and 87.5% genes from the "background" strain BALB/c. Although BALB/c and STS/A mice are similarly susceptible to T. b. brucei, the RC strain CcS-11 is more susceptible than either of them. We analyzed genetics of survival in T. b. brucei-infected F(2 hybrids between BALB/c and CcS-11. CcS-11 strain carries STS-derived segments on eight chromosomes. They were genotyped in the F(2 hybrid mice and their linkage with survival was tested by analysis of variance.We mapped four Tbbr (Trypanosoma brucei brucei response loci that influence survival after T. b. brucei infection. Tbbr1 (chromosome 3 and Tbbr2 (chromosome 12 have effects on survival independent of inter-genic interactions (main effects. Tbbr3 (chromosome 7 influences survival in interaction with Tbbr4 (chromosome 19. Tbbr2 is located on a segment 2.15 Mb short that contains only 26 genes.This study presents the first identification of chromosomal loci controlling susceptibility to T. b. brucei infection. While mapping in F(2 hybrids of inbred strains usually has a precision of 40-80 Mb, in RC strains we mapped Tbbr2 to a 2.15 Mb segment containing only 26 genes, which will enable an effective search for the candidate gene. Definition of susceptibility genes will improve the understanding of pathways and genetic diversity underlying the disease and may result in new strategies to overcome the active subversion of the immune system by T. b. brucei.

  7. Genome-wide analysis of chromatin structures in Trypanosoma brucei using high-resolution MNase-ChIP-seq.

    Science.gov (United States)

    Wedel, Carolin; Siegel, T Nicolai

    2017-09-01

    Specific DNA-protein interactions are the basis for many important cellular mechanisms like the regulation of gene expression or replication. Knowledge about the precise genomic locations of DNA-protein interactions is important because it provides insight into the regulation of these processes. Recently, we have adapted an approach that combines micrococcal nuclease (MNase) digestion of chromatin with chromatin immunoprecipitation in Trypanosoma brucei. Here, we describe in detail how this method can be used to map the genome-wide distribution of nucleosomes or other DNA-binding proteins at high resolution in T. brucei. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  8. Trypanosoma brucei: two steps to spread out from Africa.

    Science.gov (United States)

    Lun, Zhao-Rong; Lai, De-Hua; Li, Feng-Jun; Lukes, Julius; Ayala, Francisco J

    2010-09-01

    Trypanosoma brucei equiperdum and Trypanosoma brucei evansi are typically considered separate species, although a recent study suggested that these organisms can be classified as subspecies of Trypanosoma brucei, which we also favor. Here we present a scenario that attempts to explain the continuing evolution of the dyskinetoplastic and akinetoplastic strains, as a consequence of loss of selective pressure(s) leading to the loss of kinetoplast DNA. Copyright 2010 Elsevier Ltd. All rights reserved.

  9. Identification of Trypanosoma evansi, Trypanosoma equiperdum and Trypanosoma brucei brucei using repetitive DNA probes.

    Science.gov (United States)

    Zhang, Z Q; Baltz, T

    1994-06-01

    The phylogenetic relatedness of 15 stocks of Trypanosoma evansi, three stocks of Trypanosoma equiperdum and one stock of Trypanosoma brucei brucei was determined using Southern blot analysis of restriction enzyme digested DNA, probed with two repetitive DNA sequences from T. b. brucei. A dendrogram derived by cluster analysis of restriction fragment length polymorphism (RFLP) revealed three groups of related stocks. Group 1 included 14 stocks of T. evansi and one stock of T. equiperdum. Group 2 included two stocks of T. equiperdum and one stock of T. evansi. Group 3 included the one stock of T. brucei brucei. Group 2 is more closely related to Group 3 than Group 1, by analysis of the banding patterns. Further analysis of the T. evansi in Group 1 revealed that the patterns of isolates from different provinces in China were identical, but differed from T. evansi isolated from Africa, South America and the Philippines. These results provide insight into the origins of T. evansi and suggest that RFLP may be a useful means of distinguishing closely related trypanosomes.

  10. Detection of Trypanosoma brucei gambiense and T. b. rhodesiense ...

    African Journals Online (AJOL)

    Detection of Trypanosoma brucei gambiense and T. b. rhodesiense in Glossina fuscipes fuscipes ( Diptera: Glossinidae ) and Stomoxys flies using the polymerase chain reaction (PCR) technique in southern Sudan.

  11. 1H, 13C and 15N resonance assignments for a putative ADF/Cofilin from Trypanosoma brucei.

    Science.gov (United States)

    Dai, Kun; Yuan, Guangfa; Liao, Shanhui; Zhang, Jiahai; Tu, Xiaoming

    2011-10-01

    Actin-depolymerizing factor (ADF)/cofilin proteins are a family of actin-binding proteins expressed in almost all eukaryotic cells, and play a significant role in regulating actin-filament dynamics. Here we report the resonance assignments of a putative ADF/cofilin from Trypanosoma brucei for further understanding of the relationship between its structure and function.

  12. (Berenil(B)) in the Treatment of Experimental Trypanosoma brucei ...

    African Journals Online (AJOL)

    Dr Olaleye

    Animal welfare was observed in accordance with. International guidelines for the use of laboratory animals for biomedical research (EC, 1996). Trypanosomes: Trypanosoma brucei brucei (Federe strain) obtained from NITR, Vom, was used and the parasites were maintained by serial passages in rats. One millilitre of.

  13. The Effect of Garlic Extracts on Experimental Trypanosoma brucei ...

    African Journals Online (AJOL)

    The anti-trypanosomal effect of aqueous and methanolic extracts of garlic were studied in Trypanosoma brucei brucei infected rabbits. With the establishment of infection, parasitaemia, anaemia, leucopenia, neutropenia and lymphocytosis developed. There was decrease in total serum protein, albumin and increase in ...

  14. Pathogenicity of Trypanosoma brucei in African giant rats ...

    African Journals Online (AJOL)

    Pathogenicity of Trypanosoma brucei in African giant rats ( Cricetomys gambianus , Water House) ... The course of trypanosomosis was investigated over a period of two weeks in six African giant rats (Cricetomys gambianus) experimentally infected with Trypanosoma brucei. Six other rats served as uninfected control.

  15. Trypanosoma evansi is alike to Trypanosoma brucei brucei in the subcellular localisation of glycolytic enzymes

    Directory of Open Access Journals (Sweden)

    S Andrea Moreno

    2015-06-01

    Full Text Available Trypanosoma evansi, which causes surra, is descended from Trypanosoma brucei brucei, which causes nagana. Although both parasites are presumed to be metabolically similar, insufficient knowledge of T. evansi precludes a full comparison. Herein, we provide the first report on the subcellular localisation of the glycolytic enzymes in T. evansi, which is a alike to that of the bloodstream form (BSF of T. b. brucei: (i fructose-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, hexokinase, phosphofructokinase, glucose-6-phosphate isomerase, phosphoglycerate kinase, triosephosphate isomerase (glycolytic enzymes and glycerol-3-phosphate dehydrogenase (a glycolysis-auxiliary enzyme in glycosomes, (ii enolase, phosphoglycerate mutase, pyruvate kinase (glycolytic enzymes and a GAPDH isoenzyme in the cytosol, (iii malate dehydrogenase in cytosol and (iv glucose-6-phosphate dehydrogenase in both glycosomes and the cytosol. Specific enzymatic activities also suggest that T. evansi is alike to the BSF of T. b. brucei in glycolytic flux, which is much faster than the pentose phosphate pathway flux, and in the involvement of cytosolic GAPDH in the NAD+/NADH balance. These similarities were expected based on the close phylogenetic relationship of both parasites.

  16. The phosphoarginine energy-buffering system of trypanosoma brucei involves multiple arginine kinase isoforms with different subcellular locations.

    Directory of Open Access Journals (Sweden)

    Frank Voncken

    Full Text Available Phosphagen energy-buffering systems play an essential role in regulating the cellular energy homeostasis in periods of high-energy demand or energy supply fluctuations. Here we describe the phosphoarginine/arginine kinase system of the kinetoplastid parasite Trypanosoma brucei, consisting of three highly similar arginine kinase isoforms (TbAK1-3. Immunofluorescence microscopy using myc-tagged protein versions revealed that each isoform is located in a specific subcellular compartment: TbAK1 is exclusively found in the flagellum, TbAK2 in the glycosome, and TbAK3 in the cytosol of T. brucei. The flagellar location of TbAK1 is dependent on a 22 amino acid long N-terminal sequence, which is sufficient for targeting a GFP-fusion protein to the trypanosome flagellum. The glycosomal location of TbAK2 is in agreement with the presence of a conserved peroxisomal targeting signal, the C-terminal tripeptide 'SNL'. TbAK3 lacks any apparent targeting sequences and is accordingly located in the cytosol of the parasite. Northern blot analysis indicated that each TbAK isoform is differentially expressed in bloodstream and procyclic forms of T. brucei, while the total cellular arginine kinase activity was 3-fold higher in bloodstream form trypanosomes. These results suggest a substantial change in the temporal and spatial energy requirements during parasite differentiation. Increased arginine kinase activity improved growth of procyclic form T. brucei during oxidative challenges with hydrogen peroxide. Elimination of the total cellular arginine kinase activity by RNA interference significantly decreased growth (>90% of procyclic form T. brucei under standard culture conditions and was lethal for this life cycle stage in the presence of hydrogen peroxide. The putative physiological roles of the different TbAK isoforms in T. brucei are further discussed.

  17. The Phosphoarginine Energy-Buffering System of Trypanosoma brucei Involves Multiple Arginine Kinase Isoforms with Different Subcellular Locations

    Science.gov (United States)

    Wadforth, Cath; Harley, Maggie; Colasante, Claudia

    2013-01-01

    Phosphagen energy-buffering systems play an essential role in regulating the cellular energy homeostasis in periods of high-energy demand or energy supply fluctuations. Here we describe the phosphoarginine/arginine kinase system of the kinetoplastid parasite Trypanosoma brucei, consisting of three highly similar arginine kinase isoforms (TbAK1-3). Immunofluorescence microscopy using myc-tagged protein versions revealed that each isoform is located in a specific subcellular compartment: TbAK1 is exclusively found in the flagellum, TbAK2 in the glycosome, and TbAK3 in the cytosol of T. brucei. The flagellar location of TbAK1 is dependent on a 22 amino acid long N-terminal sequence, which is sufficient for targeting a GFP-fusion protein to the trypanosome flagellum. The glycosomal location of TbAK2 is in agreement with the presence of a conserved peroxisomal targeting signal, the C-terminal tripeptide ‘SNL’. TbAK3 lacks any apparent targeting sequences and is accordingly located in the cytosol of the parasite. Northern blot analysis indicated that each TbAK isoform is differentially expressed in bloodstream and procyclic forms of T. brucei, while the total cellular arginine kinase activity was 3-fold higher in bloodstream form trypanosomes. These results suggest a substantial change in the temporal and spatial energy requirements during parasite differentiation. Increased arginine kinase activity improved growth of procyclic form T. brucei during oxidative challenges with hydrogen peroxide. Elimination of the total cellular arginine kinase activity by RNA interference significantly decreased growth (>90%) of procyclic form T. brucei under standard culture conditions and was lethal for this life cycle stage in the presence of hydrogen peroxide. The putative physiological roles of the different TbAK isoforms in T. brucei are further discussed. PMID:23776565

  18. An Atypical Mitochondrial Carrier That Mediates Drug Action in Trypanosoma brucei.

    Directory of Open Access Journals (Sweden)

    Juan P de Macêdo

    2015-05-01

    Full Text Available Elucidating the mechanism of action of trypanocidal compounds is an important step in the development of more efficient drugs against Trypanosoma brucei. In a screening approach using an RNAi library in T. brucei bloodstream forms, we identified a member of the mitochondrial carrier family, TbMCP14, as a prime candidate mediating the action of a group of anti-parasitic choline analogs. Depletion of TbMCP14 by inducible RNAi in both bloodstream and procyclic forms increased resistance of parasites towards the compounds by 7-fold and 3-fold, respectively, compared to uninduced cells. In addition, down-regulation of TbMCP14 protected bloodstream form mitochondria from a drug-induced decrease in mitochondrial membrane potential. Conversely, over-expression of the carrier in procyclic forms increased parasite susceptibility more than 13-fold. Metabolomic analyses of parasites over-expressing TbMCP14 showed increased levels of the proline metabolite, pyrroline-5-carboxylate, suggesting a possible involvement of TbMCP14 in energy production. The generation of TbMCP14 knock-out parasites showed that the carrier is not essential for survival of T. brucei bloodstream forms, but reduced parasite proliferation under standard culture conditions. In contrast, depletion of TbMCP14 in procyclic forms resulted in growth arrest, followed by parasite death. The time point at which parasite proliferation stopped was dependent on the major energy source, i.e. glucose versus proline, in the culture medium. Together with our findings that proline-dependent ATP production in crude mitochondria from TbMCP14-depleted trypanosomes was reduced compared to control mitochondria, the study demonstrates that TbMCP14 is involved in energy production in T. brucei. Since TbMCP14 belongs to a trypanosomatid-specific clade of mitochondrial carrier family proteins showing very poor similarity to mitochondrial carriers of mammals, it may represent an interesting target for drug

  19. CHARACTERIZATION AND ANTIPARASITIC ACTIVITY OF BENZOPHENONE THIOSEMICARBAZONES ON Trypanosoma brucei brucei

    Directory of Open Access Journals (Sweden)

    Georges C. Accrombessi

    2011-02-01

    Full Text Available The structure of four synthesized thiosemicarbazones, substituted or not, of benzophenone has been confirmed by spectrometrical analysis IR, NMR 1H and 13C. Their anti-trypanosomal activities were evaluated on Trypanosoma brucei brucei. Among these compounds, benzophenone 4 phenyl-3-thiosemicarbazone 4 has the highest activity with the half-inhibitory concentration (IC50 = 8.48 micromolar (µM. Benzophenone 4-methyl-3-thiosemicarbazone 3 and benzophenone thiosemicarbazone 1 showed moderate anti-trypanosomal activity with IC50 values equal to 23.27 µM and 67.17 µM respectively. Benzophenone 2 methyl-3-thiosemicarbazone 2 showed no activity up to IC50 = 371.74 µM.

  20. Essential Assembly Factor Rpf2 Forms Novel Interactions within the 5S RNP in Trypanosoma brucei.

    Science.gov (United States)

    Kamina, Anyango D; Jaremko, Daniel; Christen, Linda; Williams, Noreen

    2017-01-01

    Ribosome biogenesis is a highly complex and conserved cellular process that is responsible for making ribosomes. During this process, there are several assembly steps that function as regulators to ensure proper ribosome formation. One of these steps is the assembly of the 5S ribonucleoprotein particle (5S RNP) in the central protuberance of the 60S ribosomal subunit. In eukaryotes, the 5S RNP is composed of 5S rRNA, ribosomal proteins L5 and L11, and assembly factors Rpf2 and Rrs1. Our laboratory previously showed that in Trypanosoma brucei, the 5S RNP is composed of 5S rRNA, L5, and trypanosome-specific RNA binding proteins P34 and P37. In this study, we characterize an additional component of the 5S RNP, the T. brucei homolog of Rpf2. This is the first study to functionally characterize interactions mediated by Rpf2 in an organism other than fungi. T. brucei Rpf2 (TbRpf2) was identified from tandem affinity purification using extracts prepared from protein A-tobacco etch virus (TEV)-protein C (PTP)-tagged L5, P34, and P37 cell lines, followed by mass spectrometry analysis. We characterized the binding interactions between TbRpf2 and the previously characterized members of the T. brucei 5S RNP. Our studies show that TbRpf2 mediates conserved binding interactions with 5S rRNA and L5 and that TbRpf2 also interacts with trypanosome-specific proteins P34 and P37. We performed RNA interference (RNAi) knockdown of TbRpf2 and showed that this protein is essential for the survival of the parasites and is critical for proper ribosome formation. These studies provide new insights into a critical checkpoint in the ribosome biogenesis pathway in T. brucei. IMPORTANCETrypanosoma brucei is the parasitic protozoan that causes African sleeping sickness. Ribosome assembly is essential for the survival of this parasite through the different host environments it encounters during its life cycle. The assembly of the 5S ribonucleoprotein particle (5S RNP) functions as one of the

  1. Mechanisms Mediating Antigenic Variation in Trypanosoma brucei

    Directory of Open Access Journals (Sweden)

    Rudenko Gloria

    1999-01-01

    Full Text Available Antigenic variation in Trypanosoma brucei is a highly sophisticated survival strategy involving switching between the transcription of one of an estimated thousand variant surface glycoprotein (VSG genes. Switching involves either transcriptional control, resulting in switching between different VSG expression sites; or DNA rearrangement events slotting previously inactive VSG genes into an active VSG expression site. In recent years, considerable progress has been made in techniques allowing us to genetically modify infective bloodstream form trypanosomes. This is allowing us to reengineer VSG expression sites, and look at the effect on the mechanisms subsequently used for antigenic variation. We can now begin a dissection of a highly complicated survival strategy mediated by many different mechanisms operating simultaneously.

  2. Combinations of alkaloids affecting different molecular targets with the saponin digitonin can synergistically enhance trypanocidal activity against Trypanosoma brucei brucei

    National Research Council Canada - National Science Library

    Krstin, Sonja; Peixoto, Herbenya Silva; Wink, Michael

    2015-01-01

    .... In this study, the potential synergism of mutual combinations of bioactive alkaloids and alkaloids with a membrane-active steroidal saponin, digitonin, was explored with regard to their effect on T. b. brucei...

  3. Phenolic Constituents of Medicinal Plants with Activity against Trypanosoma brucei

    Directory of Open Access Journals (Sweden)

    Ya Nan Sun

    2016-04-01

    Full Text Available Neglected tropical diseases (NTDs affect over one billion people all over the world. These diseases are classified as neglected because they impact populations in areas with poor financial conditions and hence do not attract sufficient research investment. Human African Trypanosomiasis (HAT or sleeping sickness, caused by the parasite Trypanosoma brucei, is one of the NTDs. The current therapeutic interventions for T. brucei infections often have toxic side effects or require hospitalization so that they are not available in the rural environments where HAT occurs. Furthermore, parasite resistance is increasing, so that there is an urgent need to identify novel lead compounds against this infection. Recognizing the wide structural diversity of natural products, we desired to explore and identify novel antitrypanosomal chemotypes from a collection of natural products obtained from plants. In this study, 440 pure compounds from various medicinal plants were tested against T. brucei by in a screening using whole cell in vitro assays. As the result, twenty-two phenolic compounds exhibited potent activity against cultures of T. brucei. Among them, eight compounds—4, 7, 11, 14, 15, 18, 20, and 21—showed inhibitory activity against T. brucei, with IC50 values below 5 µM, ranging from 0.52 to 4.70 μM. Based on these results, we attempt to establish some general trends with respect to structure-activity relationships, which indicate that further investigation and optimization of these derivatives might enable the preparation of potentially useful compounds for treating HAT.

  4. Evaluation of In Vitro Activity of Essential Oils against Trypanosoma brucei brucei and Trypanosoma evansi

    Directory of Open Access Journals (Sweden)

    Nathan Habila

    2010-01-01

    Full Text Available Essential oils (EOs from Cymbopogon citratus (CC, Eucalyptus citriodora (EC, Eucalyptus camaldulensis (ED, and Citrus sinensis (CS were obtained by hydrodistillation process. The EOs were evaluated in vitro for activity against Trypanosoma brucei brucei (Tbb and Trypanosoma evansi (T. evansi. The EOs were found to possess antitrypanosomal activity in vitro in a dose-dependent pattern in a short period of time. The drop in number of parasite over time was achieved doses of 0.4 g/ml, 0.2 g/mL, and 0.1 g/mL for all the EOs. The concentration of 0.4 g/mL CC was more potent at 3 minutes and 2 minutes for Tbb and T. evansi, respectively. The GC-MS analysis of the EOs revealed presence of Cyclobutane (96.09% in CS, 6-octenal (77.11% in EC, Eucalyptol (75% in ED, and Citral (38.32% in CC among several other organic compounds. The results are discussed in relation to trypanosome chemotherapy.

  5. Mechanism of Trypanosoma brucei gambiense resistance to human serum

    DEFF Research Database (Denmark)

    Uzureau, Pierrick; Uzureau, Sophie; Lecordier, Laurence

    2013-01-01

    The African parasite Trypanosoma brucei gambiense accounts for 97% of human sleeping sickness cases. T. b. gambiense resists the specific human innate immunity acting against several other tsetse-fly-transmitted trypanosome species such as T. b. brucei, the causative agent of nagana disease......GP), which prevents APOL1 toxicity and induces stiffening of membranes upon interaction with lipids. Two additional features contribute to resistance to TLFs: reduction of sensitivity to APOL1 requiring cysteine protease activity, and TbHpHbR inactivation due to a L210S substitution. According...

  6. Nucleoside analysis of DNA from Trypanosoma brucei and Trypanosoma equiperdum.

    Science.gov (United States)

    Crozatier, M; De Brij, R J; Den Engelse, L; Johnson, P J; Borst, P

    1988-11-01

    We have digested trypanosome DNA with a combination of pancreatic DNase I, nuclease P1 and bovine alkaline phosphatase and fractionated the resulting nucleosides on a Supelcosil LC-18-S column by high pressure liquid chromatography. We find less than 0.1% unusual nucleosides, both in Trypanosoma brucei and in a Trypanosoma equiperdum stock, in contrast to a previous report of an unusual nucleoside replacing dC at 1.3% of total nucleosides in T. equiperdum. Our results agree with previous suggestions that the modification of inactive telomeric expression sites for variant-specific surface glycoprotein genes in T. brucei only affects a very small fraction of the total DNA.

  7. Tail characteristics of Trypanosoma brucei mitochondrial transcripts are developmentally altered in a transcript-specific manner.

    Science.gov (United States)

    Gazestani, Vahid H; Hampton, Marshall; Shaw, Aubie K; Salavati, Reza; Zimmer, Sara L

    2018-02-01

    The intricate life cycle of Trypanosoma brucei requires extensive regulation of gene expression levels of the mtRNAs for adaptation. Post-transcriptional gene regulatory programs, including unencoded mtRNA 3' tail additions, potentially play major roles in this adaptation process. Intriguingly, T. brucei mitochondrial transcripts possess two distinct unencoded 3' tails, each with a differing functional role; i.e., while one type is implicated in RNA stability (in-tails), the other type appears associated with translation (ex-tails). We examined the degree to which tail characteristics differ among cytochrome c oxidase subunits I and III (CO1 and CO3), and NADH dehydrogenase subunit 1 (ND1) transcripts, and to what extent these characteristics differ developmentally. We found that CO1, CO3 and ND1 transcripts possess longer in-tails in the mammalian life stage. By mathematically modelling states of in-tail and ex-tail addition, we determined that the typical length at which an in-tail is extended to become an ex-tail differs by transcript and, in the case of ND1, by life stage. To the best of our knowledge, we provide the first evidence that developmental differences exist in tail length distributions of mtRNAs, underscoring the potential involvement of in-tail and ex-tail populations in mitochondrial post-transcriptional regulation mechanisms. Copyright © 2017 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.

  8. Nitroheterocyclic drug resistance mechanisms in Trypanosoma brucei.

    Science.gov (United States)

    Wyllie, Susan; Foth, Bernardo J; Kelner, Anna; Sokolova, Antoaneta Y; Berriman, Matthew; Fairlamb, Alan H

    2016-03-01

    The objective of this study was to identify the mechanisms of resistance to nifurtimox and fexinidazole in African trypanosomes. Bloodstream-form Trypanosoma brucei were selected for resistance to nifurtimox and fexinidazole by stepwise exposure to increasing drug concentrations. Clones were subjected to WGS to identify putative resistance genes. Transgenic parasites modulating expression of genes of interest were generated and drug susceptibility phenotypes determined. Nifurtimox-resistant (NfxR) and fexinidazole-resistant (FxR) parasites shared reciprocal cross-resistance suggestive of a common mechanism of action. Previously, a type I nitroreductase (NTR) has been implicated in nitro drug activation. WGS of resistant clones revealed that NfxR parasites had lost >100 kb from one copy of chromosome 7, rendering them hemizygous for NTR as well as over 30 other genes. FxR parasites retained both copies of NTR, but lost >70 kb downstream of one NTR allele, decreasing NTR transcription by half. A single knockout line of NTR displayed 1.6- and 1.9-fold resistance to nifurtimox and fexinidazole, respectively. Since NfxR and FxR parasites are ∼6- and 20-fold resistant to nifurtimox and fexinidazole, respectively, additional factors must be involved. Overexpression and knockout studies ruled out a role for a putative oxidoreductase (Tb927.7.7410) and a hypothetical gene (Tb927.1.1050), previously identified in a genome-scale RNAi screen. NTR was confirmed as a key resistance determinant, either by loss of one gene copy or loss of gene expression. Further work is required to identify which of the many dozens of SNPs identified in the drug-resistant cell lines contribute to the overall resistance phenotype. © The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.

  9. Effects of Morinda lucida leaf extract on Trypanosoma brucei brucei infection in mice.

    Science.gov (United States)

    Asuzu, I U; Chineme, C N

    1990-10-01

    The dried leaves of Morinda lucida were extracted with 50% methanol and the extract was recovered in a 9.7% w/w yield. Acute toxicity tests were performed in mice and the intraperitoneal LD50 of the extract was 2000 mg/kg. The extract induced purgation in mice from the first hour after oral administration and reached its peak between the third and fourth hour. The purgation was not dose-dependent. M. lucida leaf extract i.p. significantly suppressed the level of parasitemia after Trypanosoma brucei infection in mice. Suppression of existing parasitemia appeared dose-dependent with 1000 mg/kg i.p. producing the maximum effect. The best trypanocidal activity was obtained when treatment with M. lucida extract commenced simultaneously with trypanosome inoculation.

  10. Immunomodulatory activity of Buchholzia coriacea seed methanol extract on Trypanosoma brucei brucei infected mice.

    Science.gov (United States)

    Eze, James I; Ekelozie, Chioma F; Nweze, Nwakego E

    2017-12-01

    The seeds of Buchholzia coriacea Engler (Capparaceae) are used in Eastern Nigeria to treat feverish conditions, and to treat malaria and sleeping sickness that cause fever. The current study assesses the immunomodulatory activity of Buchholzia coriacea seed extract on Trypanosoma brucei brucei infected mice. Delayed hypersensitivity reaction, humoral antibody response and in-vivo leucocyte mobilization tests were assessed in three different experiments to determine the effect of the extract on immune response. Seventy-five (75) mice (25 mice per experiment) were used for the study and were each infected with 1.00 × 106 trypanosomes intra-peritoneally. Groups A, B and C were given 250, 500 and 1000 mg/kg of the extract, respectively, group D received 7.5 mg/kg body weight of levamisole and group E was the control. Sheep RBCs were used as antigen. The acute toxicity tests did not cause clinical signs or death within 24 h post treatment at all the doses tested. The extract inhibited delayed hypersensitivity reaction by 20.9 and 20.8% at 250 and 500 mg/kg, respectively, while at 1000 mg/kg, the paw size increased (-101.9%) when compared with the control. The extract elevated the antibody titre from 1.60 ± 0.40 for control to 8.00 ± 3.58 for 500 mg/kg group. The extract increased in total leucocytes counts. The extract has a very wide safety margin and was able to improve immune response. The results of the present study showed that Buchholzia coriacea seed methanol extract possesses immunostimulatory activity on trypanosome-infected mice.

  11. ( Nigella sativa ) oil on Trypanosoma brucei -infected rats

    African Journals Online (AJOL)

    The effect of black seed oil (Nigella sativa oil) on parasitaemia, some serum and liver enzymes as well as some haematological parameters in Trypanosoma brucei-infected rats were investigated. The results show there was low parasitaemia and extension of life span of rats from 12 days of the infected untreated (control) ...

  12. The genome of the African trypanosome Trypanosoma brucei.

    Science.gov (United States)

    Berriman, Matthew; Ghedin, Elodie; Hertz-Fowler, Christiane; Blandin, Gaëlle; Renauld, Hubert; Bartholomeu, Daniella C; Lennard, Nicola J; Caler, Elisabet; Hamlin, Nancy E; Haas, Brian; Böhme, Ulrike; Hannick, Linda; Aslett, Martin A; Shallom, Joshua; Marcello, Lucio; Hou, Lihua; Wickstead, Bill; Alsmark, U Cecilia M; Arrowsmith, Claire; Atkin, Rebecca J; Barron, Andrew J; Bringaud, Frederic; Brooks, Karen; Carrington, Mark; Cherevach, Inna; Chillingworth, Tracey-Jane; Churcher, Carol; Clark, Louise N; Corton, Craig H; Cronin, Ann; Davies, Rob M; Doggett, Jonathon; Djikeng, Appolinaire; Feldblyum, Tamara; Field, Mark C; Fraser, Audrey; Goodhead, Ian; Hance, Zahra; Harper, David; Harris, Barbara R; Hauser, Heidi; Hostetler, Jessica; Ivens, Al; Jagels, Kay; Johnson, David; Johnson, Justin; Jones, Kristine; Kerhornou, Arnaud X; Koo, Hean; Larke, Natasha; Landfear, Scott; Larkin, Christopher; Leech, Vanessa; Line, Alexandra; Lord, Angela; Macleod, Annette; Mooney, Paul J; Moule, Sharon; Martin, David M A; Morgan, Gareth W; Mungall, Karen; Norbertczak, Halina; Ormond, Doug; Pai, Grace; Peacock, Chris S; Peterson, Jeremy; Quail, Michael A; Rabbinowitsch, Ester; Rajandream, Marie-Adele; Reitter, Chris; Salzberg, Steven L; Sanders, Mandy; Schobel, Seth; Sharp, Sarah; Simmonds, Mark; Simpson, Anjana J; Tallon, Luke; Turner, C Michael R; Tait, Andrew; Tivey, Adrian R; Van Aken, Susan; Walker, Danielle; Wanless, David; Wang, Shiliang; White, Brian; White, Owen; Whitehead, Sally; Woodward, John; Wortman, Jennifer; Adams, Mark D; Embley, T Martin; Gull, Keith; Ullu, Elisabetta; Barry, J David; Fairlamb, Alan H; Opperdoes, Fred; Barrell, Barclay G; Donelson, John E; Hall, Neil; Fraser, Claire M; Melville, Sara E; El-Sayed, Najib M

    2005-07-15

    African trypanosomes cause human sleeping sickness and livestock trypanosomiasis in sub-Saharan Africa. We present the sequence and analysis of the 11 megabase-sized chromosomes of Trypanosoma brucei. The 26-megabase genome contains 9068 predicted genes, including approximately 900 pseudogenes and approximately 1700 T. brucei-specific genes. Large subtelomeric arrays contain an archive of 806 variant surface glycoprotein (VSG) genes used by the parasite to evade the mammalian immune system. Most VSG genes are pseudogenes, which may be used to generate expressed mosaic genes by ectopic recombination. Comparisons of the cytoskeleton and endocytic trafficking systems with those of humans and other eukaryotic organisms reveal major differences. A comparison of metabolic pathways encoded by the genomes of T. brucei, T. cruzi, and Leishmania major reveals the least overall metabolic capability in T. brucei and the greatest in L. major. Horizontal transfer of genes of bacterial origin has contributed to some of the metabolic differences in these parasites, and a number of novel potential drug targets have been identified.

  13. Roles of triosephosphate isomerase and aerobic metabolism in Trypanosoma brucei.

    NARCIS (Netherlands)

    Helfert, S.; Estevez, A.M.; Bakker, B.M.; Michels, P.A.M.; Clayton, C.

    2001-01-01

    Kinetoplastid protozoa compartmentalize the first seven enzymes of glycolysis and two enzymes of glycerol metabolism in a microbody, the glycosome. While in its mammalian host, Trypanosoma brucei depends entirely on glucose for ATP generation. Under aerobic conditions, most of the glucose is

  14. Population genetic structure and cladistic analysis of Trypanosoma brucei isolates

    NARCIS (Netherlands)

    Agbo, E.C.; Clausen, P.H.; Buscher, P.; Majiwa, P.A.O.; Pas, te M.F.W.; Claassen, E.

    2003-01-01

    Using a novel multilocus DNA marker analysis method, we studied the population genetic structure of Trypansoma brucei stocks and derived clones isolated from animal and rhodesiense sleeping sickness patients during a national sleeping sickness control program in Mukono district, Uganda. We then

  15. Role of cytokines in Trypanosoma brucei-induced anaemia: A ...

    African Journals Online (AJOL)

    the blood-brain barrier. The subspecies Trypanosoma brucei rhodesiense in eastern and southern Africa generally causes a more acute form of ... The persistence of anaemia beyond the initial wave of parasitaemia (after which low numbers of parasites are in circulation) indicates that anaemia may be directly induced.

  16. Trypanosoma brucei mitochondrial respiratome: Composition and organization in procyclic form

    KAUST Repository

    Acestor, Nathalie

    2011-05-24

    The mitochondrial respiratory chain is comprised of four different protein complexes (I-IV), which are responsible for electron transport and generation of proton gradient in the mitochondrial intermembrane space. This proton gradient is then used by F oF 1-ATP synthase (complex V) to produce ATP by oxidative phosphorylation. In this study, the respiratory complexes I, II, and III were affinity purified from Trypanosoma brucei procyclic form cells and their composition was determined by mass spectrometry. The results along with those that we previously reported for complexes IV and V showed that the respiratome of Trypanosoma is divergent because many of its proteins are unique to this group of organisms. The studies also identified two mitochondrial subunit proteins of respiratory complex IV that are encoded by edited RNAs. Proteomics data from analyses of complexes purified using numerous tagged component proteins in each of the five complexes were used to generate the first predicted protein-protein interaction network of the Trypanosoma brucei respiratory chain. These results provide the first comprehensive insight into the unique composition of the respiratory complexes in Trypanosoma brucei, an early diverged eukaryotic pathogen. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Crystal structure of arginine methyltransferase 6 from Trypanosoma brucei.

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    Chongyuan Wang

    Full Text Available Arginine methylation plays vital roles in the cellular functions of the protozoan Trypanosoma brucei. The T. brucei arginine methyltransferase 6 (TbPRMT6 is a type I arginine methyltransferase homologous to human PRMT6. In this study, we report the crystal structures of apo-TbPRMT6 and its complex with the reaction product S-adenosyl-homocysteine (SAH. The structure of apo-TbPRMT6 displays several features that are different from those of type I PRMTs that were structurally characterized previously, including four stretches of insertion, the absence of strand β15, and a distinct dimerization arm. The comparison of the apo-TbPRMT6 and SAH-TbPRMT6 structures revealed the fine rearrangements in the active site upon SAH binding. The isothermal titration calorimetry results demonstrated that SAH binding greatly increases the affinity of TbPRMT6 to a substrate peptide derived from bovine histone H4. The western blotting and mass spectrometry results revealed that TbPRMT6 methylates bovine histone H4 tail at arginine 3 but cannot methylate several T. brucei histone tails. In summary, our results highlight the structural differences between TbPRMT6 and other type I PRMTs and reveal that the active site rearrangement upon SAH binding is important for the substrate binding of TbPRMT6.

  18. Sec16 determines the size and functioning of the Golgi in the protist parasite, Trypanosoma brucei.

    Science.gov (United States)

    Sealey-Cardona, Marco; Schmidt, Katy; Demmel, Lars; Hirschmugl, Tatjana; Gesell, Tanja; Dong, Gang; Warren, Graham

    2014-06-01

    The Sec16 homologue in Trypanosoma brucei has been identified and characterized. TbSec16 colocalizes with COPII components at the single endoplasmic reticulum exit site (ERES), which is next to the single Golgi stack in the insect (procyclic) form of this organism. Depletion of TbSec16 reduces the size of the ERES and the Golgi, and slows growth and transport of a secretory marker to the cell surface; conversely, overexpression of TbSec16 increases the size of the ERES and Golgi but has no effect on growth or secretion. Together these data suggest that TbSec16 regulates the size of the ERES and Golgi and this size is set for optimal growth of the organism. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  19. Effect of diminazene aceturate, levamisole and vitamin C combination therapy in rats experimentally infected with Trypanosoma brucei brucei.

    Science.gov (United States)

    Chekwube, Adieme Ijeoma; Onyema, Ezeh Ikenna; Ikenna, Ugochukwu Emmanuel; Ezeokonkwo, Romanus Chukwuduruo

    2014-06-01

    To investigate the effect of diminazene aceturate (DA) alone or in combination with either levamisole and/or Vitamin C in albino rats experimentally infected with Trypanosoma brucei brucei. Thirty adult male albino rats, randomly assigned into 6 groups (A-F) of 5 rats each were used. They were either infected with 1×10(6) trypanosomes intraperitoneally (groups A-E) or uninfected (group F). The different groups were treated respectively as follows: group A-with 3.5 mg/kg DA; group B-3.5 mg/kg DA and 7.5 mg/kg levamisole; group C-3.5 mg/kg DA and 100 mg/kg vitamin C; and group D-3.5 mg/kg DA and 7.5 mg/kg levamisole and 100 mg/kg vitamin C. Group E was left untreated. Parameters assessed include: rectal temperature, body weight changes, packed cell volume (PCV), Haemoglobin concentration (Hb), total leucocyte count (TLC) differential leucocyte count (DLC), parasitaemia, clinical signs and survivability. Average pre-patent period of 5 days was recorded. Parasites in the blood were cleared in all treated groups (A-D) within 48 hours post treatment (PT). Untreated rats in group E died between 25 and 32 days post infection (PI). Relapse was not recorded in all the treated groups (A-D). The initial reduction in PCV, Hb, TLC and increases in rectal temperature following infection were reversed by the treatments. The rats that received drug combinations (groups B, C and D) showed faster and higher recovery rates than the uninfected control and group A. Levamisole and/or Vitamin C combination with DA were more effective in the treatment of rats infected with Trypanosoma brucei brucei. Copyright © 2014 Hainan Medical College. Published by Elsevier B.V. All rights reserved.

  20. Trypanosoma brucei RAP1 maintains telomere and subtelomere integrity by suppressing TERRA and telomeric RNA:DNA hybrids.

    Science.gov (United States)

    Nanavaty, Vishal; Sandhu, Ranjodh; Jehi, Sanaa E; Pandya, Unnati M; Li, Bibo

    2017-06-02

    Trypanosoma brucei causes human African trypanosomiasis and regularly switches its major surface antigen, VSG, thereby evading the host's immune response. VSGs are monoallelically expressed from subtelomeric expression sites (ESs), and VSG switching exploits subtelomere plasticity. However, subtelomere integrity is essential for T. brucei viability. The telomeric transcript, TERRA, was detected in T. brucei previously. We now show that the active ES-adjacent telomere is transcribed. We find that TbRAP1, a telomere protein essential for VSG silencing, suppresses VSG gene conversion-mediated switching. Importantly, TbRAP1 depletion increases the TERRA level, which appears to result from longer read-through into the telomere downstream of the active ES. Depletion of TbRAP1 also results in more telomeric RNA:DNA hybrids and more double strand breaks (DSBs) at telomeres and subtelomeres. In TbRAP1-depleted cells, expression of excessive TbRNaseH1, which cleaves the RNA strand of the RNA:DNA hybrid, brought telomeric RNA:DNA hybrids, telomeric/subtelomeric DSBs and VSG switching frequency back to WT levels. Therefore, TbRAP1-regulated appropriate levels of TERRA and telomeric RNA:DNA hybrid are fundamental to subtelomere/telomere integrity. Our study revealed for the first time an important role of a long, non-coding RNA in antigenic variation and demonstrated a link between telomeric silencing and subtelomere/telomere integrity through TbRAP1-regulated telomere transcription. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  1. Adaptations of Trypanosoma brucei to gradual loss of kinetoplast DNA: Trypanosoma equiperdum and Trypanosoma evansi are petite mutants of T. brucei.

    Science.gov (United States)

    Lai, De-Hua; Hashimi, Hassan; Lun, Zhao-Rong; Ayala, Francisco J; Lukes, Julius

    2008-02-12

    Trypanosoma brucei is a kinetoplastid flagellate, the agent of human sleeping sickness and ruminant nagana in Africa. Kinetoplastid flagellates contain their eponym kinetoplast DNA (kDNA), consisting of two types of interlocked circular DNA molecules: scores of maxicircles and thousands of minicircles. Maxicircles have typical mitochondrial genes, most of which are translatable only after RNA editing. Minicircles encode guide RNAs, required for decrypting the maxicircle transcripts. The life cycle of T. brucei involves a bloodstream stage (BS) in vertebrates and a procyclic stage (PS) in the tsetse fly vector. Partial [dyskinetoplastidy (Dk)] or total [akinetoplastidy (Ak)] loss of kDNA locks the trypanosome in the BS form. Transmission between vertebrates becomes mechanical without PS and tsetse mediation, allowing the parasite to spread outside the African tsetse belt. Trypanosoma equiperdum and Trypanosoma evansi are agents of dourine and surra, diseases of horses, camels, and water buffaloes. We have characterized representative strains of T. equiperdum and T. evansi by numerous molecular and classical parasitological approaches. We show that both species are actually strains of T. brucei, which lost part (Dk) or all (Ak) of their kDNA. These trypanosomes are not monophyletic clades and do not qualify for species status. They should be considered two subspecies, respectively T. brucei equiperdum and T. brucei evansi, which spontaneously arose recently. Dk/Ak trypanosomes may potentially emerge repeatedly from T. brucei.

  2. Trypanosoma brucei TbIF1 inhibits the essential F1-ATPase in the infectious form of the parasite.

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    Brian Panicucci

    2017-04-01

    Full Text Available The mitochondrial (mt FoF1-ATP synthase of the digenetic parasite, Trypanosoma brucei, generates ATP during the insect procyclic form (PF, but becomes a perpetual consumer of ATP in the mammalian bloodstream form (BF, which lacks a canonical respiratory chain. This unconventional dependence on FoF1-ATPase is required to maintain the essential mt membrane potential (Δψm. Normally, ATP hydrolysis by this rotary molecular motor is restricted to when eukaryotic cells experience sporadic hypoxic conditions, during which this compulsory function quickly depletes the cellular ATP pool. To protect against this cellular treason, the highly conserved inhibitory factor 1 (IF1 binds the enzyme in a manner that solely inhibits the hydrolytic activity. Intriguingly, we were able to identify the IF1 homolog in T. brucei (TbIF1, but determined that its expression in the mitochondrion is tightly regulated throughout the life cycle as it is only detected in PF cells. TbIF1 appears to primarily function as an emergency brake in PF cells, where it prevented the restoration of the Δψm by FoF1-ATPase when respiration was chemically inhibited. In vitro, TbIF1 overexpression specifically inhibits the hydrolytic activity but not the synthetic capability of the FoF1-ATP synthase in PF mitochondria. Furthermore, low μM amounts of recombinant TbIF1 achieve the same inhibition of total mt ATPase activity as the FoF1-ATPase specific inhibitors, azide and oligomycin. Therefore, even minimal ectopic expression of TbIF1 in BF cells proved lethal as the indispensable Δψm collapsed due to inhibited FoF1-ATPase. In summary, we provide evidence that T. brucei harbors a natural and potent unidirectional inhibitor of the vital FoF1-ATPase activity that can be exploited for future structure-based drug design.

  3. Minimum Information Loss Based Multi-kernel Learning for Flagellar Protein Recognition in Trypanosoma Brucei

    KAUST Repository

    Wang, Jim Jing-Yan

    2014-12-01

    Trypanosma brucei (T. Brucei) is an important pathogen agent of African trypanosomiasis. The flagellum is an essential and multifunctional organelle of T. Brucei, thus it is very important to recognize the flagellar proteins from T. Brucei proteins for the purposes of both biological research and drug design. In this paper, we investigate computationally recognizing flagellar proteins in T. Brucei by pattern recognition methods. It is argued that an optimal decision function can be obtained as the difference of probability functions of flagella protein and the non-flagellar protein for the purpose of flagella protein recognition. We propose to learn a multi-kernel classification function to approximate this optimal decision function, by minimizing the information loss of such approximation which is measured by the Kull back-Leibler (KL) divergence. An iterative multi-kernel classifier learning algorithm is developed to minimize the KL divergence for the problem of T. Brucei flagella protein recognition, experiments show its advantage over other T. Brucei flagellar protein recognition and multi-kernel learning methods. © 2014 IEEE.

  4. Telomeric expression sites are highly conserved in Trypanosoma brucei.

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    Christiane Hertz-Fowler

    Full Text Available Subtelomeric regions are often under-represented in genome sequences of eukaryotes. One of the best known examples of the use of telomere proximity for adaptive purposes are the bloodstream expression sites (BESs of the African trypanosome Trypanosoma brucei. To enhance our understanding of BES structure and function in host adaptation and immune evasion, the BES repertoire from the Lister 427 strain of T. brucei were independently tagged and sequenced. BESs are polymorphic in size and structure but reveal a surprisingly conserved architecture in the context of extensive recombination. Very small BESs do exist and many functioning BESs do not contain the full complement of expression site associated genes (ESAGs. The consequences of duplicated or missing ESAGs, including ESAG9, a newly named ESAG12, and additional variant surface glycoprotein genes (VSGs were evaluated by functional assays after BESs were tagged with a drug-resistance gene. Phylogenetic analysis of constituent ESAG families suggests that BESs are sequence mosaics and that extensive recombination has shaped the evolution of the BES repertoire. This work opens important perspectives in understanding the molecular mechanisms of antigenic variation, a widely used strategy for immune evasion in pathogens, and telomere biology.

  5. Antitrypanosomal effect of methanolic extract of Zingiber officinale (ginger on Trypanosoma brucei brucei-infected Wistar mice

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    P. I. Kobo

    2014-10-01

    Full Text Available Aim: The study was carried out to determine the in vivo antitrypanosomal effect of methanolic extract of Zingiber officinale (ginger in Trypanosoma brucei brucei-infected mice. Materials and Methods: Twenty-five mice were randomly allocated into five groups of five animals each. Group I and II were given Tween 80 (1 ml/kg and diminazene aceturate (3.5 mg/kg to serve as untreated and treated controls, respectively. Groups III-V received the extract at 200, 400 and 800 mg/kg body weight, respectively. All treatments were given for 6 consecutive days and through the oral route. The mean body weight, mean survival period and daily level of parasitaemia were evaluated. Results: Acute toxicity showed the extract to be relatively safe. There was an insignificant increase in body weight and survival rate of mice treated with the extract. The level of parasitaemia in the extract treated groups was decreased. Conclusion: This study shows the in vivo potential of methanolic extract of Z. officinale in the treatment of trypanosomiasis.

  6. In or out? On the tightness of glycosomal compartmentalizatin of metabolites and enzymes in Trypanosoma brucei.

    NARCIS (Netherlands)

    Haanstra, J.R.; Bakker, B.M.; Michels, P.A.M.

    2014-01-01

    Trypanosomatids sequester large parts of glucose metabolism inside specialised peroxisomes, called glycosomes. Many studies have shown that correct glycosomal compartmentalization of glycolytic enzymes is essential for bloodstream-form Trypanosoma brucei. The recent finding of pore-forming

  7. 1H, 13C and 15N resonance assignment of Urm1 from Trypanosoma brucei.

    Science.gov (United States)

    Zhang, Wen; Ding, Husheng; Zhang, Jiahai; Tu, Xiaoming

    2008-06-01

    Urm1 (ubiquitin-related modifier), involved in diverse biological processes in yeast, is proved to be a "molecular fossil" in ubiquitin superfamily. Here we report the resonance assignment of Urm1 from Trypanosoma brucei.

  8. Spliced leader trapping reveals widespread alternative splicing patterns in the highly dynamic transcriptome of Trypanosoma brucei.

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    Daniel Nilsson

    2010-08-01

    Full Text Available Trans-splicing of leader sequences onto the 5'ends of mRNAs is a widespread phenomenon in protozoa, nematodes and some chordates. Using parallel sequencing we have developed a method to simultaneously map 5'splice sites and analyze the corresponding gene expression profile, that we term spliced leader trapping (SLT. The method can be applied to any organism with a sequenced genome and trans-splicing of a conserved leader sequence. We analyzed the expression profiles and splicing patterns of bloodstream and insect forms of the parasite Trypanosoma brucei. We detected the 5' splice sites of 85% of the annotated protein-coding genes and, contrary to previous reports, found up to 40% of transcripts to be differentially expressed. Furthermore, we discovered more than 2500 alternative splicing events, many of which appear to be stage-regulated. Based on our findings we hypothesize that alternatively spliced transcripts present a new means of regulating gene expression and could potentially contribute to protein diversity in the parasite. The entire dataset can be accessed online at TriTrypDB or through: http://splicer.unibe.ch/.

  9. A Flagellar Pocket Membrane Fraction from Trypanosoma brucei rhodesiense: Immunogold Localization and Nonvariant Immunoprotection

    Science.gov (United States)

    1988-01-01

    membrane components. specific glycoproteins from Trypanosoma equiperdum variants. Exp. Parasitol. 64:1-11. FEBS Lett. 82:93-96. 20. Melton, D. W., D. S...Fraction from Trypanosoma brucei rhodesiense: Immunogold Localization and Nonvariant Immunoprotection JOHN G. OLENICK,* RUTH WOLFF,’ ROBERT K. NAUMAN...obtained for each of three distinct variable antigenic types (VATs) of a serodeme of Trypanosoma brucei rhodesiense Wellcome strain. Products of

  10. The Phosphoproteome of Bloodstream Form Trypanosoma brucei, Causative Agent of African Sleeping Sickness

    OpenAIRE

    Nett, Isabelle R. E.; Martin, David M. A.; Miranda-Saavedra, Diego; Lamont, Douglas; Barber, Jonathan D.; Mehlert, Angela; Ferguson, Michael A. J.

    2009-01-01

    The protozoan parasite Trypanosoma brucei is the causative agent of human African sleeping sickness and related animal diseases, and it has over 170 predicted protein kinases. Protein phosphorylation is a key regulatory mechanism for cellular function that, thus far, has been studied in T.brucei principally through putative kinase mRNA knockdown and observation of the resulting phenotype. However, despite the relatively large kinome of this organism and the demonstrated essentiality of severa...

  11. Steroid Alkaloids from Holarrhena africana with Strong Activity against Trypanosoma brucei rhodesiense

    OpenAIRE

    Charles Okeke Nnadi; Ngozi Justina Nwodo; Marcel Kaiser; Reto Brun; Thomas J. Schmidt

    2017-01-01

    In our continued search for natural compounds with activity against Trypanosoma brucei, causative agent of human African trypanosomiasis (HAT, “sleeping sickness”), we have investigated extracts from the leaves and bark of the West African Holarrhena africana (syn. Holarrhena floribunda; Apocynaceae). The extracts and their alkaloid-enriched fractions displayed promising in vitro activity against bloodstream forms of T. brucei rhodesiense (Tbr; East African HAT). Bioactivity-guided chromatogr...

  12. Characterization of Trypanosoma brucei dihydroorotate dehydrogenase as a possible drug target; structural, kinetic and RNAi studies.

    Science.gov (United States)

    Arakaki, Tracy L; Buckner, Frederick S; Gillespie, J Robert; Malmquist, Nicholas A; Phillips, Margaret A; Kalyuzhniy, Oleksandr; Luft, Joseph R; Detitta, George T; Verlinde, Christophe L M J; Van Voorhis, Wesley C; Hol, Wim G J; Merritt, Ethan A

    2008-04-01

    Nucleotide biosynthesis pathways have been reported to be essential in some protozoan pathogens. Hence, we evaluated the essentiality of one enzyme in the pyrimidine biosynthetic pathway, dihydroorotate dehydrogenase (DHODH) from the eukaryotic parasite Trypanosoma brucei through gene knockdown studies. RNAi knockdown of DHODH expression in bloodstream form T. brucei did not inhibit growth in normal medium, but profoundly retarded growth in pyrimidine-depleted media or in the presence of the known pyrimidine uptake antagonist 5-fluorouracil (5-FU). These results have significant implications for the development of therapeutics to combat T. brucei infection. Specifically, a combination therapy including a T. brucei-specific DHODH inhibitor plus 5-FU may prove to be an effective therapeutic strategy. We also show that this trypanosomal enzyme is inhibited by known inhibitors of bacterial Class 1A DHODH, in distinction to the sensitivity of DHODH from human and other higher eukaryotes. This selectivity is supported by the crystal structure of the T. brucei enzyme, which is reported here at a resolution of 1.95 A. Additional research, guided by the crystal structure described herein, is needed to identify potent inhibitors of T. brucei DHODH.

  13. A transcription-independent epigenetic mechanism is associated with antigenic switching in Trypanosoma brucei.

    Science.gov (United States)

    Aresta-Branco, Francisco; Pimenta, Silvia; Figueiredo, Luisa M

    2016-04-20

    Antigenic variation in Trypanosoma brucei relies on periodic switching of variant surface glycoproteins (VSGs), which are transcribed monoallelically by RNA polymerase I from one of about 15 bloodstream expression sites (BES). Chromatin of the actively transcribed BES is depleted of nucleosomes, but it is unclear if this open conformation is a mere consequence of a high rate of transcription, or whether it is maintained by a transcription-independent mechanism. Using an inducible BES-silencing reporter strain, we observed that chromatin of the active BES remains open for at least 24 hours after blocking transcription. This conformation is independent of the cell-cycle stage, but dependent upon TDP1, a high mobility group box protein. For two days after BES silencing, we detected a transient and reversible derepression of several silent BESs within the population, suggesting that cells probe other BESs before commitment to one, which is complete by 48 hours. FACS sorting and subsequent subcloning confirmed that probing cells are switching intermediates capable of returning to the original BES, switch to the probed BES or to a different BES. We propose that regulation of BES chromatin structure is an epigenetic mechanism important for successful antigenic switching. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  14. Role of centrins 2 and 3 in organelle segregation and cytokinesis in Trypanosoma brucei.

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    Angamuthu Selvapandiyan

    Full Text Available Centrins are calcium binding proteins involved in cell division in eukaryotes. Previously, we have shown that depletion of centrin1 in Trypanosoma brucei (T. brucei displayed arrested organelle segregation resulting in loss of cytokinesis. In this study we analyzed the role of T. brucei centrin2 (TbCen2 and T. brucei 3 (TbCen3 in the early events of T. brucei procyclic cell cycle. Both the immunofluorescence assay and electron microscopy showed that TbCen2 and 3-deficient cells were enlarged in size with duplicated basal bodies, multinuclei and new flagella that are detached along the length of the cell body. In both TbCen2 and TbCen3 depleted cells segregation of the organelles i.e. basal bodies, kinetoplast and nucleus was disrupted. Further analysis of the cells with defective organelle segregation identified three different sub configurations of organelle mis-segregations (Type 1-3. In addition, in majority of the TbCen2 depleted cells and in nearly half of the TbCen3 depleted cells, the kinetoplasts were enlarged and undivided. The abnormal segregations ultimately led to aborted cytokinesis and hence affected growth in these cells. Therefore, both centrin2 and 3 are involved in organelle segregation similar to centrin1 as was previously observed. In addition, we identified their role in kinetoplast division which may be also linked to overall mis-segregation.

  15. Trypanosoma brucei CYP51: Essentiality and Targeting Therapy in an Experimental Model.

    Science.gov (United States)

    Dauchy, Frédéric-Antoine; Bonhivers, Mélanie; Landrein, Nicolas; Dacheux, Denis; Courtois, Pierrette; Lauruol, Florian; Daulouède, Sylvie; Vincendeau, Philippe; Robinson, Derrick R

    2016-11-01

    Trypanosoma brucei gambiense is the main causative agent of Human African Trypanosomiasis (HAT), also known as sleeping sickness. Because of limited alternatives and treatment toxicities, new therapeutic options are urgently needed for patients with HAT. Sterol 14alpha-demethylase (CYP51) is a potential drug target but its essentiality has not been determined in T. brucei. We used a tetracycline-inducible RNAi system to assess the essentiality of CYP51 in T. brucei bloodstream form (BSF) cells and we evaluated the effect of posaconazole, a well-tolerated triazole drug, within a panel of virulent strains in vitro and in a murine model. Expression of CYP51 in several T. brucei cell lines was demonstrated by western blot and its essentiality was demonstrated by RNA interference (CYP51RNAi) in vitro. Following reduction of TbCYP51 expression by RNAi, cell growth was reduced and eventually stopped compared to WT or non-induced cells, showing the requirement of CYP51 in T. brucei. These phenotypes were rescued by addition of ergosterol. Additionally, CYP51RNAi induction caused morphological defects with multiflagellated cells (pnifurtimox-eflornithine combinations showed similar improvement in mice survival (p≤0.001). Our results provide support for a CYP51 targeting based treatment in HAT. Thus posaconazole used in combination may represent a therapeutic alternative for trypanosomiasis.

  16. High-throughput Gene Tagging in Trypanosoma brucei.

    Science.gov (United States)

    Dyer, Philip; Dean, Samuel; Sunter, Jack

    2016-08-12

    Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins can give insights into their function by determining their localization within the cell and enabling interaction partner identification. We recently published a fast and scalable method to generate Trypanosoma brucei cell lines that express a tagged protein from the endogenous locus. The method was based on a plasmid we generated that, when coupled with long primer PCR, can be used to modify a gene to encode a protein tagged at either terminus. This allows the tagging of dozens of trypanosome proteins in parallel, facilitating the large-scale validation of candidate genes of interest. This system can be used to tag proteins for localization (using a fluorescent protein, epitope tag or electron microscopy tag) or biochemistry (using tags for purification, such as the TAP (tandem affinity purification) tag). Here, we describe a protocol to perform the long primer PCR and the electroporation in 96-well plates, with the recovery and selection of transgenic trypanosomes occurring in 24-well plates. With this workflow, hundreds of proteins can be tagged in parallel; this is an order of magnitude improvement to our previous protocol and genome scale tagging is now possible.

  17. Spliced leader RNA silencing (SLS - a programmed cell death pathway in Trypanosoma brucei that is induced upon ER stress

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    Michaeli Shulamit

    2012-05-01

    Full Text Available Abstract Trypanosoma brucei is the causative agent of African sleeping sickness. The parasite cycles between its insect (procyclic form and mammalian hosts (bloodstream form. Trypanosomes lack conventional transcription regulation, and their genes are transcribed in polycistronic units that are processed by trans-splicing and polyadenylation. In trans-splicing, which is essential for processing of each mRNA, an exon, the spliced leader (SL is added to all mRNAs from a small RNA, the SL RNA. Trypanosomes lack the machinery for the unfolded protein response (UPR, which in other eukaryotes is induced under endoplasmic reticulum (ER stress. Trypanosomes respond to such stress by changing the stability of mRNAs, which are essential for coping with the stress. However, under severe ER stress that is induced by blocking translocation of proteins to the ER, treatment of cells with chemicals that induce misfolding in the ER, or extreme pH, trypanosomes elicit the spliced leader silencing (SLS pathway. In SLS, the transcription of the SL RNA gene is extinguished, and tSNAP42, a specific SL RNA transcription factor, fails to bind to its cognate promoter. SLS leads to complete shut-off of trans-splicing. In this review, I discuss the UPR in mammals and compare it to the ER stress response in T. brucei leading to SLS. I summarize the evidence supporting the notion that SLS is a programmed cell death (PCD pathway that is utilized by the parasites to substitute for the apoptosis observed in higher eukaryotes under prolonged ER stress. I present the hypothesis that SLS evolved to expedite the death process, and rapidly remove from the population unfit parasites that, by elimination via SLS, cause minimal damage to the parasite population.

  18. Protective effect of humus extract against Trypanosoma brucei infection in mice.

    Science.gov (United States)

    Kodama, Hiroshi; Denso; Okazaki, Fumi; Ishida, Saeko

    2008-11-01

    Humic substances are formed during the decomposition of organic matter in humus, and are found in many natural environments in which organic materials and microorganisms are present. Oral administration of humus extract to mice successfully induced effective protection against experimental challenge by the two subspecies, Trypanosoma brucei brucei and T. brucei gambiense. Mortality was most reduced among mice who received a 3% humus extract for 21 days in drinking water ad libitum. Spleen cells from humus-administered mice exhibited significant non-specific cytotoxic activity against L1210 mouse leukemia target cells. Also, spleen cells produced significantly higher amounts of Interferon-gamma when stimulated in vitro with Concanavalin A than cells from normal controls. These results clearly show that administration to mice of humus extract induced effective resistance against Trypanosoma infection. Enhancement of the innate immune system may be involved in host defense against trypanosomiasis.

  19. Ligand tunnels in T. brucei and human CYP51: Insights for parasite-specific drug design.

    Science.gov (United States)

    Yu, Xiaofeng; Nandekar, Prajwal; Mustafa, Ghulam; Cojocaru, Vlad; Lepesheva, Galina I; Wade, Rebecca C

    2016-01-01

    Cytochrome P450 sterol 14α-demethylase (CYP51) is an essential enzyme for sterol biosynthesis and a target for anti-parasitic drug design. However, the design of parasite-specific drugs that inhibit parasitic CYP51 without severe side effects remains challenging. The active site of CYP51 is situated in the interior of the protein. Here, we characterize the potential ligand egress routes and mechanisms in Trypanosoma brucei and human CYP51 enzymes. We performed Random Acceleration Molecular Dynamics simulations of the egress of four different ligands from the active site of models of soluble and membrane-bound T. brucei CYP51 and of soluble human CYP51. In the simulations, tunnel 2f, which leads to the membrane, was found to be the predominant ligand egress tunnel for all the ligands studied. Tunnels S, 1 and W, which lead to the cytosol, were also used in T. brucei CYP51, whereas tunnel 1 was the only other tunnel used significantly in human CYP51. The common tunnels found previously in other CYPs were barely used. The ligand egress times were shorter for human than T. brucei CYP51, suggesting lower barriers to ligand passage. Two gating residues, F105 and M460, in T. brucei CYP51 that modulate the opening of tunnels 2f and S were identified. Although the main egress tunnel was the same, differences in the tunnel-lining residues, ligand passage and tunnel usage were found between T. brucei and human CYP51s. The results provide a basis for the design of selective anti-parasitic agents targeting the ligand tunnels. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Isolation and analysis of the genetic diversity of repertoires of VSG expression site containing telomeres from Trypanosoma brucei gambiense, T. b. brucei and T. equiperdum

    Directory of Open Access Journals (Sweden)

    Becker Marion

    2008-08-01

    Full Text Available Abstract Background African trypanosomes (including Trypanosoma brucei are unicellular parasites which multiply in the mammalian bloodstream. T. brucei has about twenty telomeric bloodstream form Variant Surface Glycoprotein (VSG expression sites (BESs, of which one is expressed at a time in a mutually exclusive fashion. BESs are polycistronic transcription units, containing a variety of families of expression site associated genes (ESAGs in addition to the telomeric VSG. These polymorphic ESAG families are thought to play a role in parasite-host adaptation, and it has been proposed that ESAG diversity might be related to host range. Analysis of the genetic diversity of these telomeric gene families has been confounded by the underrepresentation of telomeric sequences in standard libraries. We have previously developed a method to selectively isolate sets of trypanosome BES containing telomeres using Transformation associated recombination (TAR cloning in yeast. Results Here we describe the isolation of repertoires of BES containing telomeres from three trypanosome subspecies: Trypanosoma brucei gambiense DAL 972 (causative agent of West-African trypanosomiasis, T. b. brucei EATRO 2340 (a nonhuman infective strain and T. equiperdum STIB 818 (which causes a sexually transmitted disease in equines. We have sequenced and analysed the genetic diversity at four BES loci (BES promoter region, ESAG6, ESAG5 and ESAG2 from these three trypanosome BES repertoires. Conclusion With the exception of ESAG2, the BES sequence repertoires derived from T. b. gambiense are both less diverse than and nearly reciprocally monophyletic relative to those from T. b. brucei and T. equiperdum. Furthermore, although we find evidence for adaptive evolution in all three ESAG repertoires in T. b. brucei and T. equiperdum, only ESAG2 appears to be under diversifying selection in T. b. gambiense. This low level of variation in the T. b. gambiense BES sequence repertoires is

  1. Isolation and analysis of the genetic diversity of repertoires of VSG expression site containing telomeres from Trypanosoma brucei gambiense, T. b. brucei and T. equiperdum.

    Science.gov (United States)

    Young, Rosanna; Taylor, Jesse E; Kurioka, Ayako; Becker, Marion; Louis, Edward J; Rudenko, Gloria

    2008-08-12

    African trypanosomes (including Trypanosoma brucei) are unicellular parasites which multiply in the mammalian bloodstream. T. brucei has about twenty telomeric bloodstream form Variant Surface Glycoprotein (VSG) expression sites (BESs), of which one is expressed at a time in a mutually exclusive fashion. BESs are polycistronic transcription units, containing a variety of families of expression site associated genes (ESAGs) in addition to the telomeric VSG. These polymorphic ESAG families are thought to play a role in parasite-host adaptation, and it has been proposed that ESAG diversity might be related to host range. Analysis of the genetic diversity of these telomeric gene families has been confounded by the underrepresentation of telomeric sequences in standard libraries. We have previously developed a method to selectively isolate sets of trypanosome BES containing telomeres using Transformation associated recombination (TAR) cloning in yeast. Here we describe the isolation of repertoires of BES containing telomeres from three trypanosome subspecies: Trypanosoma brucei gambiense DAL 972 (causative agent of West-African trypanosomiasis), T. b. brucei EATRO 2340 (a nonhuman infective strain) and T. equiperdum STIB 818 (which causes a sexually transmitted disease in equines). We have sequenced and analysed the genetic diversity at four BES loci (BES promoter region, ESAG6, ESAG5 and ESAG2) from these three trypanosome BES repertoires. With the exception of ESAG2, the BES sequence repertoires derived from T. b. gambiense are both less diverse than and nearly reciprocally monophyletic relative to those from T. b. brucei and T. equiperdum. Furthermore, although we find evidence for adaptive evolution in all three ESAG repertoires in T. b. brucei and T. equiperdum, only ESAG2 appears to be under diversifying selection in T. b. gambiense. This low level of variation in the T. b. gambiense BES sequence repertoires is consistent both with the relatively narrow host range

  2. Trypanosoma brucei: analysis of cytoplasmic Ca2+ during differentiation of bloodstream stages in vitro.

    Science.gov (United States)

    Stojdl, D F; Clarke, M W

    1996-06-01

    The highly regulated intracellular concentration of calcium (Ca2+) is a well-described regulator of diverse cellular events, including cell cycle control. In the present study we have addressed the regulation of cytosolic Ca2+ in differentiation events in the life cycle of the protozoan parasite Trypanosoma brucei. Bloodstream form (BSF) trypanosomes include the mitotically active long slender forms (LS) which differentiate to two nondividing stages--intermediate (INT) which transform into short stumpy (SS) forms. An axenic in vitro culture system was used to cultivate LS to a density greater than 1.0 x 10(6) cells/ml/day. Populations of the intermediate BSF (INT) and SS were derived from cultured LS by treatment with difluoromethyl ornithine (DFMO, 100 microM) for 2 and 4 days, respectively. A semiquantitative reverse transcriptase-coupled polymerase chain reaction protocol (SQ-RT-PCR) was developed to objectively distinguish the three BSF by monitoring the relative levels of stage-specific mRNAs--cytochrome oxidase II (COXII), variant surface glycoprotein, and procyclin during the differentiation of LS to SS, showing an increase in COXII and procyclin mRNA expression during this process of differentiation. Basal cytosolic Ca2+ levels [Ca2+]i of populations of LS, INT, and SS were studied using Indo-1 dual emission fluorometry. [Ca2+]i was maximal in dividing LS cells and was shown to decrease coincidentally with early events in the process of differentiation to INT and SS. Thapsigargin (1 microM), reported to cause the release of Ca2+ from the endoplasmic reticulum, elevated [Ca2+]i by about 30-60 nM in all BSF; however, the total thapsigargin-releasable stores decreased in parallel with the decrease in basal [Ca2+]i. Control treatments verified that elevations in [Ca2+]i in response to thapsigargin were intracellular in origin. These results may reflect the cessation of cytosolic Ca2+ transients involved in the regulation of mitosis as the parasite exits from

  3. Arterial blood pressure changes in acute T. brucei infection of dogs ...

    African Journals Online (AJOL)

    The aim of this study is to find out the usefulness of serial arterial blood pressure measurements in predicting severity and outcome of acute Trypanosoma brucei infection in dogs. Twenty adult dogs of mixed sexes and aged between 2 and 5 years were used for this study. The dogs were of good cardiac health and were ...

  4. Biosynthesis of SUMOylated Proteins in Bacteria Using the Trypanosoma brucei Enzymatic System

    Science.gov (United States)

    Iribarren, Paula Ana; Berazategui, María Agustina; Cazzulo, Juan José; Alvarez, Vanina Eder

    2015-01-01

    Post-translational modification with the Small Ubiquitin-like Modifier (SUMO) is conserved in eukaryotic organisms and plays important regulatory roles in proteins affecting diverse cellular processes. In Trypanosoma brucei, member of one of the earliest branches in eukaryotic evolution, SUMO is essential for normal cell cycle progression and is likely to be involved in the epigenetic control of genes crucial for parasite survival, such as those encoding the variant surface glycoproteins. Molecular pathways modulated by SUMO have started to be discovered by proteomic studies; however, characterization of functional consequences is limited to a reduced number of targets. Here we present a bacterial strain engineered to produce SUMOylated proteins, by transferring SUMO from T. brucei together with the enzymes essential for its activation and conjugation. Due to the lack of background in E. coli, this system is useful to express and identify SUMOylated proteins directly in cell lysates by immunoblotting, and SUMOylated targets can be eventually purified for biochemical or structural studies. We applied this strategy to describe the ability of TbSUMO to form chains in vitro and to detect SUMOylation of a model substrate, PCNA both from Saccharomyces cerevisiae and from T. brucei. To further validate targets, we applied an in vitro deconjugation assay using the T. brucei SUMO-specific protease capable to revert the pattern of modification. This system represents a valuable tool for target validation, mutant generation and functional studies of SUMOylated proteins in trypanosomatids. PMID:26258470

  5. Phosphatidylinositol 4-kinase III-beta is required for Golgi maintenance and cytokinesis in Trypanosoma brucei.

    Science.gov (United States)

    Rodgers, Melissa J; Albanesi, Joseph P; Phillips, Margaret A

    2007-07-01

    The parasitic protozoan Trypanosoma brucei contains two type III phosphatidylinositol 4-kinases (alpha and beta). We have cloned the gene encoding the T. brucei type III phosphatidylinositol 4-kinase beta (TbPI4KIII-beta), expressed the protein in COS-7 cells, and confirmed that the protein catalyzes the phosphorylation of phosphatidylinositol. Depletion of TbPI4KIII-beta in procyclic T. brucei by RNA interference (RNAi) resulted in inhibition of cell growth and a distorted cellular morphology. RNAi cells had a distorted Golgi apparatus, and lysosomal and flagellar pocket proteins were mislocalized. Ultrastructural analysis revealed the internal accumulation of a heterogeneous population of vesicles, abnormal positioning of organelles, and a loss of cell polarity. Scanning electron microcopy revealed a twisted phenotype, and dividing cells often exhibited a detached daughter flagellum and lacked a cleavage furrow. Cell cycle analysis confirmed that cells depleted of TbPI4KIII-beta have a postmitotic cytokinesis block that occurs after a single round of mitosis, suggestive of a specific cell cycle block. In summary, TbPI4KIII-beta is an essential protein in procyclic T. brucei, required for maintenance of Golgi structure, protein trafficking, normal cellular shape, and cytokinesis.

  6. The Flagellar Arginine Kinase in Trypanosoma brucei Is Important for Infection in Tsetse Flies.

    Directory of Open Access Journals (Sweden)

    Cher-Pheng Ooi

    Full Text Available African trypanosomes are flagellated parasites that cause sleeping sickness. Parasites are transmitted from one mammalian host to another by the bite of a tsetse fly. Trypanosoma brucei possesses three different genes for arginine kinase (AK including one (AK3 that encodes a protein localised to the flagellum. AK3 is characterised by the presence of a unique amino-terminal insertion that specifies flagellar targeting. We show here a phylogenetic analysis revealing that flagellar AK arose in two independent duplication events in T. brucei and T. congolense, the two species of African trypanosomes that infect the tsetse midgut. In T. brucei, AK3 is detected in all stages of parasite development in the fly (in the midgut and in the salivary glands as well as in bloodstream cells, but with predominance at insect stages. Genetic knockout leads to a slight reduction in motility and impairs parasite infectivity towards tsetse flies in single and competition experiments, both phenotypes being reverted upon expression of an epitope-tagged version of AK3. We speculate that this flagellar arginine kinase is important for T. brucei infection of tsetse, especially in the context of mixed infections and that its flagellar targeting relies on a system equivalent to that discovered for calflagins, a family of trypanosome flagellum calcium binding proteins.

  7. The Flagellar Arginine Kinase in Trypanosoma brucei Is Important for Infection in Tsetse Flies.

    Science.gov (United States)

    Ooi, Cher-Pheng; Rotureau, Brice; Gribaldo, Simonetta; Georgikou, Christina; Julkowska, Daria; Blisnick, Thierry; Perrot, Sylvie; Subota, Ines; Bastin, Philippe

    2015-01-01

    African trypanosomes are flagellated parasites that cause sleeping sickness. Parasites are transmitted from one mammalian host to another by the bite of a tsetse fly. Trypanosoma brucei possesses three different genes for arginine kinase (AK) including one (AK3) that encodes a protein localised to the flagellum. AK3 is characterised by the presence of a unique amino-terminal insertion that specifies flagellar targeting. We show here a phylogenetic analysis revealing that flagellar AK arose in two independent duplication events in T. brucei and T. congolense, the two species of African trypanosomes that infect the tsetse midgut. In T. brucei, AK3 is detected in all stages of parasite development in the fly (in the midgut and in the salivary glands) as well as in bloodstream cells, but with predominance at insect stages. Genetic knockout leads to a slight reduction in motility and impairs parasite infectivity towards tsetse flies in single and competition experiments, both phenotypes being reverted upon expression of an epitope-tagged version of AK3. We speculate that this flagellar arginine kinase is important for T. brucei infection of tsetse, especially in the context of mixed infections and that its flagellar targeting relies on a system equivalent to that discovered for calflagins, a family of trypanosome flagellum calcium binding proteins.

  8. Particle-bound enzymes in the bloodstream form of Trypanosoma brucei

    NARCIS (Netherlands)

    Opperdoes, F. R.; Borst, P.; Spits, H.

    1977-01-01

    We have screened the bloodstream form of Trypanosoma brucei for the presence of enzymes that could serve as markers for the microbodies and the highly repressed mitochondrion of this organism. None of seven known microbody enzymes were detected at all, but glycerol-3-phosphate oxidase, ATPase,

  9. Procyclic Trypanosoma brucei do not use Krebs cycle activity for energy generation

    NARCIS (Netherlands)

    Weelden, van S.W.H.; Fast, B.; Vogt, A.; Meer, van der P.; Saas, J.; Hellemond, van J.J.; Tielens, A.G.M.; Boshart, M.

    2003-01-01

    The importance of a functional Krebs cycle for energy generation in the procyclic stage of Trypanosoma brucei was investigated under physiological conditions during logarithmic phase growth of a pleomorphic parasite strain. Wild type procyclic cells and mutants with targeted deletion of the gene

  10. Contribution of glucose transport to the control of the glycolytic flux in Trypanosoma brucei.

    NARCIS (Netherlands)

    Bakker, B.M.; Walsh, M.C.; ter Kuile, B.; Mensonides, F.I.C.; Michels, P.A.M.; Opperdoes, F.R.; Westerhoff, H.V.

    1999-01-01

    The rate of glucose transport across the plasma membrane of the bloodstream form of Trypanosoma brucei was modulated by titration of the hexose transporter with the inhibitor phloretin, and the effect on the glycolytic flux was measured. A rapid glucose uptake assay was developed to measure the

  11. The promoter for a variant surface glycoprotein gene expression site in Trypanosoma brucei

    NARCIS (Netherlands)

    Zomerdijk, J. C.; Ouellette, M.; ten Asbroek, A. L.; Kieft, R.; Bommer, A. M.; Clayton, C. E.; Borst, P.

    1990-01-01

    The variant-specific surface glycoprotein (VSG) gene 221 of Trypanosoma brucei is transcribed as part of a 60 kb expression site (ES). We have identified the promoter controlling this multigene transcription unit by the use of 221 chromosome-enriched DNA libraries and VSG gene 221 expression site

  12. Isolation and characterization of kinetoplast DNA from the bloodstream form of Trypanosoma brucei.

    NARCIS (Netherlands)

    A.H. Fairlamb; P.O. Weislogel; J.H.J. Hoeijmakers (Jan); P. Borst (Piet)

    1978-01-01

    textabstractWe have used restriction endonucleases PstI, EcoRI, HapII, HhaI, and S1 nuclease to demonstrate the presence of a large complex component, the maxi-circle, in addition to the major mini-circle component in kinetoplast DNA (kDNA) networks of Trypanosoma brucei (East African

  13. Deletion of the Trypanosoma brucei Superoxide Dismutase Gene sodb1 Increases Sensitivity to Nifurtimox and Benznidazole▿

    Science.gov (United States)

    Prathalingham, S. Radhika; Wilkinson, Shane R.; Horn, David; Kelly, John M.

    2007-01-01

    It has been more than 25 years since it was first reported that nifurtimox and benznidazole promote superoxide production in trypanosomes. However, there has been no direct evidence of an association between the drug-induced free radicals and trypanocidal activity. Here, we identify a superoxide dismutase required to protect Trypanosoma brucei from drug-generated superoxide. PMID:17145786

  14. Deletion of the Trypanosoma brucei superoxide dismutase gene sodb1 increases sensitivity to nifurtimox and benznidazole.

    Science.gov (United States)

    Prathalingham, S Radhika; Wilkinson, Shane R; Horn, David; Kelly, John M

    2007-02-01

    It has been more than 25 years since it was first reported that nifurtimox and benznidazole promote superoxide production in trypanosomes. However, there has been no direct evidence of an association between the drug-induced free radicals and trypanocidal activity. Here, we identify a superoxide dismutase required to protect Trypanosoma brucei from drug-generated superoxide.

  15. Trypanosoma brucei Infection in a herd of sedentary cattle in Danja ...

    African Journals Online (AJOL)

    Trypanosoma brucei Infection in a herd of sedentary cattle in Danja Local Government Area, Katsina State, Northern Nigeria– A possible resurgence of Tsetse flies ... resulted in advocacy for pastoralists to settle down into productive cattle production compared to the nomadic behavior which does not enhance productivity.

  16. Novel molecular mechanism for targeting the parasite Trypanosoma brucei with snake venom toxins

    DEFF Research Database (Denmark)

    Martos Esteban, Andrea; Laustsen, Andreas Hougaard; Carrington, Mark

    Trypanosoma brucei is a parasitic protozoan species capable to infecting insect vectors whose bite further produces African sleeping sickness inhuman beings. During parasites’extracellular lives in the mammalian host, its outer coat, mainly composedof Variable surface glycoproteins (VSGs)[2], und...

  17. Aquaglyceroporins Are the Entry Pathway of Boric Acid in Trypanosoma brucei.

    Science.gov (United States)

    Marsiccobetre, Sabrina; Rodríguez-Acosta, Alexis; Lang, Florian; Figarella, Katherine; Uzcátegui, Néstor L

    2017-05-01

    The boron element possesses a range of different effects on living beings. It is essential to beneficial at low concentrations, but toxic at excessive concentrations. Recently, some boron-based compounds have been identified as promising molecules against Trypanosoma brucei, the causative agent of sleeping sickness. However, until now, the boron metabolism and its access route into the parasite remained elusive. The present study addressed the permeability of T. brucei aquaglyceroporins (TbAQPs) for boric acid, the main natural boron species. To this end, the three TbAQPs were expressed in Saccharomyces cerevisiae and Xenopus laevis oocytes. Our findings in both expression systems showed that all three TbAQPs are permeable for boric acid. Especially TbAQP2 is highly permeable for this compound, displaying one of the highest conductances reported for a solute in these channels. Additionally, T. brucei aquaglyceroporin activities were sensitive to pH. Taken together, these results establish that TbAQPs are channels for boric acid and are highly efficient entry pathways for boron into the parasite. Our findings stress the importance of studying the physiological functions of boron and their derivatives in T. brucei, as well as the pharmacological implications of their uptake by trypanosome aquaglyceroporins. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Biosynthesis of SUMOylated Proteins in Bacteria Using the Trypanosoma brucei Enzymatic System.

    Directory of Open Access Journals (Sweden)

    Paula Ana Iribarren

    Full Text Available Post-translational modification with the Small Ubiquitin-like Modifier (SUMO is conserved in eukaryotic organisms and plays important regulatory roles in proteins affecting diverse cellular processes. In Trypanosoma brucei, member of one of the earliest branches in eukaryotic evolution, SUMO is essential for normal cell cycle progression and is likely to be involved in the epigenetic control of genes crucial for parasite survival, such as those encoding the variant surface glycoproteins. Molecular pathways modulated by SUMO have started to be discovered by proteomic studies; however, characterization of functional consequences is limited to a reduced number of targets. Here we present a bacterial strain engineered to produce SUMOylated proteins, by transferring SUMO from T. brucei together with the enzymes essential for its activation and conjugation. Due to the lack of background in E. coli, this system is useful to express and identify SUMOylated proteins directly in cell lysates by immunoblotting, and SUMOylated targets can be eventually purified for biochemical or structural studies. We applied this strategy to describe the ability of TbSUMO to form chains in vitro and to detect SUMOylation of a model substrate, PCNA both from Saccharomyces cerevisiae and from T. brucei. To further validate targets, we applied an in vitro deconjugation assay using the T. brucei SUMO-specific protease capable to revert the pattern of modification. This system represents a valuable tool for target validation, mutant generation and functional studies of SUMOylated proteins in trypanosomatids.

  19. BAPTA-AM decreases cellular pH, inhibits acidocalcisome acidification and autophagy in amino acid-starved T. brucei.

    Science.gov (United States)

    Li, Feng-Jun; Tan, Kevin S W; He, Cynthia Y

    2017-04-01

    To investigate the role of Ca2+ signaling in starvation-induced autophagy in Trypanosoma brucei, the causative agent of human African trypanosomiasis, we used cell-permeant Ca2+ chelator BAPTA-AM and cell impermeant chelator EGTA, and examined the potential involvement of several intracellular Ca2+ signaling pathways in T. brucei autophagy. The results showed an unexpected effect of BAPTA-AM in decreasing cellular pH and inhibiting acidocalcisome acidification in starved cells. The implication of these results in the role of Ca2+ signaling and cellular/organellar pH in T. brucei autophagy is discussed. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Trypanosoma brucei CYP51: Essentiality and Targeting Therapy in an Experimental Model.

    Directory of Open Access Journals (Sweden)

    Frédéric-Antoine Dauchy

    2016-11-01

    Full Text Available Trypanosoma brucei gambiense is the main causative agent of Human African Trypanosomiasis (HAT, also known as sleeping sickness. Because of limited alternatives and treatment toxicities, new therapeutic options are urgently needed for patients with HAT. Sterol 14alpha-demethylase (CYP51 is a potential drug target but its essentiality has not been determined in T. brucei. We used a tetracycline-inducible RNAi system to assess the essentiality of CYP51 in T. brucei bloodstream form (BSF cells and we evaluated the effect of posaconazole, a well-tolerated triazole drug, within a panel of virulent strains in vitro and in a murine model. Expression of CYP51 in several T. brucei cell lines was demonstrated by western blot and its essentiality was demonstrated by RNA interference (CYP51RNAi in vitro. Following reduction of TbCYP51 expression by RNAi, cell growth was reduced and eventually stopped compared to WT or non-induced cells, showing the requirement of CYP51 in T. brucei. These phenotypes were rescued by addition of ergosterol. Additionally, CYP51RNAi induction caused morphological defects with multiflagellated cells (p<0.05, suggesting cytokinesis dysfunction. The survival of CYP51RNAi Doxycycline-treated mice (p = 0.053 and of CYP51RNAi 5-day pre-induced Doxycycline-treated mice (p = 0.008 were improved compared to WT showing a CYP51 RNAi effect on trypanosomal virulence in mice. The posaconazole concentrations that inhibited parasite growth by 50% (IC50 were 8.5, 2.7, 1.6 and 0.12 μM for T. b. brucei 427 90-13, T. b. brucei Antat 1.1, T. b. gambiense Feo (Feo/ITMAP/1893 and T. b. gambiense Biyamina (MHOM/SD/82, respectively. During infection with these last three virulent strains, posaconazole-eflornithine and nifurtimox-eflornithine combinations showed similar improvement in mice survival (p≤0.001. Our results provide support for a CYP51 targeting based treatment in HAT. Thus posaconazole used in combination may represent a therapeutic

  1. Diverse patterns of expression of the cytochrome c oxidase subunit I gene and unassigned reading frames 4 and 5 during the life cycle of Trypanosoma brucei.

    Science.gov (United States)

    Jasmer, D P; Feagin, J E; Stuart, K

    1985-01-01

    Transcription of a maxicircle segment from Trypanosoma brucei 164 that contains nucleotide (nt) sequences corresponding to cytochrome c oxidase subunit I (COI) and unassigned reading frames (URFs) 4 and 5 of other mitochondrial systems was investigated. Two major transcripts that differ in size by ca. 200 nt map to each of the COI and URF4 genes, while a single major transcript maps to URF5. In total RNA, the larger COI transcript is more abundant in procyclic forms (PFs) than in bloodstream forms (BFs), the smaller COI and both URF4 transcripts have similar abundances in both forms, and the single URF5 transcript is more abundant in BF than PF. These patterns of expression differ in poly(A)+ RNA as a result of a higher proportion of poly(A)+ mitochondrial transcripts in PFs than in BFs. In addition, small (300- to 500-nt) RNAs that are transcribed from C-rich sequences located between putative protein-coding genes also exhibit diverse patterns of expression between life cycle stages and differences in polyadenylation in PFs compared with BFs. These observations suggest that multiple processes regulate the differential expression of mitochondrial genes in T. brucei. Images PMID:2427925

  2. The phosphoproteome of bloodstream form Trypanosoma brucei, causative agent of African sleeping sickness.

    Science.gov (United States)

    Nett, Isabelle R E; Martin, David M A; Miranda-Saavedra, Diego; Lamont, Douglas; Barber, Jonathan D; Mehlert, Angela; Ferguson, Michael A J

    2009-07-01

    The protozoan parasite Trypanosoma brucei is the causative agent of human African sleeping sickness and related animal diseases, and it has over 170 predicted protein kinases. Protein phosphorylation is a key regulatory mechanism for cellular function that, thus far, has been studied in T.brucei principally through putative kinase mRNA knockdown and observation of the resulting phenotype. However, despite the relatively large kinome of this organism and the demonstrated essentiality of several T. brucei kinases, very few specific phosphorylation sites have been determined in this organism. Using a gel-free, phosphopeptide enrichment-based proteomics approach we performed the first large scale phosphorylation site analyses for T.brucei. Serine, threonine, and tyrosine phosphorylation sites were determined for a cytosolic protein fraction of the bloodstream form of the parasite, resulting in the identification of 491 phosphoproteins based on the identification of 852 unique phosphopeptides and 1204 phosphorylation sites. The phosphoproteins detected in this study are predicted from their genome annotations to participate in a wide variety of biological processes, including signal transduction, processing of DNA and RNA, protein synthesis, and degradation and to a minor extent in metabolic pathways. The analysis of phosphopeptides and phosphorylation sites was facilitated by in-house developed software, and this automated approach was validated by manual annotation of spectra of the kinase subset of proteins. Analysis of the cytosolic bloodstream form T. brucei kinome revealed the presence of 44 phosphorylated protein kinases in our data set that could be classified into the major eukaryotic protein kinase groups by applying a multilevel hidden Markov model library of the kinase catalytic domain. Identification of the kinase phosphorylation sites showed conserved phosphorylation sequence motifs in several kinase activation segments, supporting the view that

  3. Alkaloids Induce Programmed Cell Death in Bloodstream Forms of Trypanosomes (Trypanosoma b. brucei

    Directory of Open Access Journals (Sweden)

    Michael Wink

    2008-10-01

    Full Text Available The potential induction of a programmed cell death (PCD in Trypanosoma b. brucei by 55 alkaloids of the quinoline, quinolizidine, isoquinoline, indole, terpene, tropane, steroid, and piperidine type was studied by measuring DNA fragmentation and changes in mitochondrial membrane potential. For comparison, the induction of apoptosis by the same alkaloids in human leukemia cells (Jurkat APO-S was tested. Several alkaloids of the isoquinoline, quinoline, indole and steroidal type (berberine, chelerythrine, emetine, sanguinarine, quinine, ajmalicine, ergotamine, harmine, vinblastine, vincristine, colchicine, chaconine, demissidine and veratridine induced programmed cell death, whereas quinolizidine, tropane, terpene and piperidine alkaloids were mostly inactive. Effective PCD induction (EC50 below 10 µM was caused in T. brucei by chelerythrine, emetine, sanguinarine, and chaconine. The active alkaloids can be characterized by their general property to inhibit protein biosynthesis, to intercalate DNA, to disturb membrane fluidity or to inhibit microtubule formation.

  4. Aparasitemic serological suspects in Trypanosoma brucei gambiense human African trypanosomiasis : a potential human reservoir of parasites ?

    OpenAIRE

    Koffi, Mathurin; Solano, Philippe; Denizot, M.; Courtin, D.; Garcia, André; Lejon, V.; Buscher, P.; Cuny, Gérard; Jamonneau, Vincent

    2006-01-01

    The serological and parasitological tests used for Trypanosoma brucei gambiense human African trypanosomiasis (HAT) diagnosis have low specificity and sensitivity, respectively, and in the field, control program teams are faced with subjects with positive serology but negative parasitology who remain untreated. The aim of this work was to explore, using PCR tool, the significance of these aparasitemic serological suspects. Since discordant PCR results have been observed earlier with different...

  5. Differential virulence and tsetse fly transmissibility of Trypanosoma congolense and Trypanosoma brucei strains

    Directory of Open Access Journals (Sweden)

    Purity K. Gitonga

    2017-01-01

    Full Text Available African animal trypanosomiasis causes significant economic losses in sub-Saharan African countries because of livestock mortalities and reduced productivity. Trypanosomes, the causative agents, are transmitted by tsetse flies (Glossina spp.. In the current study, we compared and contrasted the virulence characteristics of five Trypanosoma congolense and Trypanosoma brucei isolates using groups of Swiss white mice (n = 6. We further determined the vectorial capacity of Glossina pallidipes, for each of the trypanosome isolates. Results showed that the overall pre-patent (PP periods were 8.4 ± 0.9 (range, 4–11 and 4.5 ± 0.2 (range, 4–6 for T. congolense and T. brucei isolates, respectively (p < 0.01. Despite the longer mean PP, T. congolense–infected mice exhibited a significantly (p < 0.05 shorter survival time than T. brucei–infected mice, indicating greater virulence. Differences were also noted among the individual isolates with T. congolense KETRI 2909 causing the most acute infection of the entire group with a mean ± standard error survival time of 9 ± 2.1 days. Survival time of infected tsetse flies and the proportion with mature infections at 30 days post-exposure to the infective blood meals varied among isolates, with subacute infection–causing T. congolense EATRO 1829 and chronic infection–causing T. brucei EATRO 2267 isolates showing the highest mature infection rates of 38.5% and 23.1%, respectively. Therefore, our study provides further evidence of occurrence of differences in virulence and transmissibility of eastern African trypanosome strains and has identified two, T. congolense EATRO 1829 and T. brucei EATRO 2267, as suitable for tsetse infectivity and transmissibility experiments.

  6. Procyclic Trypanosoma brucei do not use Krebs cycle activity for energy generation.

    Science.gov (United States)

    van Weelden, Susanne W H; Fast, Beate; Vogt, Achim; van der Meer, Pieter; Saas, Joachim; van Hellemond, Jaap J; Tielens, Aloysius G M; Boshart, Michael

    2003-04-11

    The importance of a functional Krebs cycle for energy generation in the procyclic stage of Trypanosoma brucei was investigated under physiological conditions during logarithmic phase growth of a pleomorphic parasite strain. Wild type procyclic cells and mutants with targeted deletion of the gene coding for aconitase were derived by synchronous in vitro differentiation from wild type and mutant (Delta aco::NEO/Delta aco::HYG) bloodstream stage parasites, respectively, where aconitase is not expressed and is dispensable. No differences in intracellular levels of glycolytic and Krebs cycle intermediates were found in procyclic wild type and mutant cells, except for citrate that accumulated up to 90-fold in the mutants, confirming the absence of aconitase activity. Surprisingly, deletion of aconitase did not change differentiation nor the growth rate or the intracellular ATP/ADP ratio in those cells. Metabolic studies using radioactively labeled substrates and NMR analysis demonstrated that glucose and proline were not degraded via the Krebs cycle to CO(2). Instead, glucose was degraded to acetate, succinate, and alanine, whereas proline was degraded to succinate. Importantly, there was absolutely no difference in the metabolic products released by wild type and aconitase knockout parasites, and both were for survival strictly dependent on respiration via the mitochondrial electron transport chain. Hence, although the Krebs cycle enzymes are present, procyclic T. brucei do not use Krebs cycle activity for energy generation, but the mitochondrial respiratory chain is essential for survival and growth. We therefore propose a revised model of the energy metabolism of procyclic T. brucei.

  7. Mapping of branch sites in trans-spliced pre-mRNAs of Trypanosoma brucei.

    Science.gov (United States)

    Patzelt, E; Perry, K L; Agabian, N

    1989-01-01

    The process of trans splicing is essential to the maturation of all mRNAs in the Trypanosomatidae, a family of protozoan parasites, and to specific mRNAs in several species of nematode. In Trypanosoma brucei, a 39-nucleotide (nt) leader sequence originating from a small, 139-nt donor RNA (the spliced leader [SL] RNA) is spliced to the 5' end of mRNAs. An intermediate in this trans-splicing process is a Y structure which contains the 3' 100 nt of the SL RNA covalently linked to the pre-mRNA via a 2'-5' phosphodiester bond at the branch point residue. We mapped the branch points in T. brucei alpha- and beta-tubulin pre-mRNAs. The primary branch acceptors for the alpha- and beta-tubulins are 44 and 56 nt upstream of the 3' splice sites, respectively, and are A residues. Minor branch acceptors were detected 42 and 49 nt upstream of the alpha-tubulin splice site and 58 nt upstream of the splice site in beta-tubulin. The regions surrounding these branch points lack homology to the consensus sequences determined for mammalian cells and yeasts; there is also no conservation among the sequences themselves. Thus, the identified sequences suggest that the mechanism of branch point recognition in T. brucei differs from the mechanism of recognition by U2 RNA that has been proposed for other eucaryotes. Images PMID:2479824

  8. The inositol pyrophosphate synthesis pathway in Trypanosoma brucei is linked to polyphosphate synthesis in acidocalcisomes.

    Science.gov (United States)

    Cordeiro, Ciro D; Saiardi, Adolfo; Docampo, Roberto

    2017-10-01

    Inositol pyrophosphates are novel signaling molecules possessing high-energy pyrophosphate bonds and involved in a number of biological functions. Here, we report the correct identification and characterization of the kinases involved in the inositol pyrophosphate biosynthetic pathway in Trypanosoma brucei: inositol polyphosphate multikinase (TbIPMK), inositol pentakisphosphate 2-kinase (TbIP5K) and inositol hexakisphosphate kinase (TbIP6K). TbIP5K and TbIP6K were not identifiable by sequence alone and their activities were validated by enzymatic assays with the recombinant proteins or by their complementation of yeast mutants. We also analyzed T. brucei extracts for the presence of inositol phosphates using polyacrylamide gel electrophoresis and high-performance liquid chromatography. Interestingly, we could detect inositol phosphate (IP), inositol 4,5-bisphosphate (IP2 ), inositol 1,4,5-trisphosphate (IP3 ), and inositol hexakisphosphate (IP6 ) in T. brucei different stages. Bloodstream forms unable to produce inositol pyrophosphates, due to downregulation of TbIPMK expression by conditional knockout, have reduced levels of polyphosphate and altered acidocalcisomes. Our study links the inositol pyrophosphate pathway to the synthesis of polyphosphate in acidocalcisomes, and may lead to better understanding of these organisms and provide new targets for drug discovery. © 2017 John Wiley & Sons Ltd.

  9. Molecular variation of Trypanosoma brucei subspecies as revealed by AFLP fingerprinting.

    Science.gov (United States)

    Agbo, E E C; Majiwa, P A O; Claassen, H J H M; te Pas, M F W

    2002-04-01

    Genetic analysis of Trypanosoma spp. depends on the detection of variation between strains. We have used the amplified fragment length polymorphism (AFLP) technique to develop a convenient and reliable method for genetic characterization of Trypanosome (sub)species. AFLP accesses multiple independent sites within the genome and would allow a better definition of the relatedness of different Trypanosome (sub)species. Nine isolates (3 from each T. brucei subspecies) were tested with 40 AFLP primer combinations to identify the most appropriate pairs of restriction endonucleases and selective primers. Primers based on the recognition sequences of EcoRI and BglII were chosen and used to analyse 31 T. brucei isolates. Similarity levels calculated with the Pearson correlation coefficient ranged from 15 to 98%, and clusters were determined using the unweighted pair-group method using arithmetic averages (UPGMA). At the intraspecific level, AFLP fingerprints were grouped by numerical analysis in 2 main clusters, allowing a clear separation of T. b. gambiense (cluster I) from T. b. brucei and T. b. rhodesiense isolates (cluster II). Interspecies evaluation of this customized approach produced heterogeneous AFLP patterns, with unique genetic markers, except for T. evansi and T. equiperdum, which showed identical patterns and clustered together.

  10. NIMA-related kinase TbNRKC is involved in basal body separation in Trypanosoma brucei.

    Science.gov (United States)

    Pradel, Lydie C; Bonhivers, Mélanie; Landrein, Nicolas; Robinson, Derrick R

    2006-05-01

    The NIMA-related kinase 2 (NEK 2) has important cell cycle functions related to centriole integrity and splitting. Trypanosoma brucei does not possess centrioles, however, cytokinesis is coupled to basal body separation events. Here we report the first functional characterisation of a T. brucei basal body-cytoskeletal NIMA-related kinase (NRK) protein, TbNRKC. The TbNRKC kinase domain has high amino acid identity with the human NEK1 kinase domain (50%) but also shares 42% identity with human NEK2. TbNRKC is expressed in bloodstream and procyclic cells and functions as a bona fide kinase in vitro. Remarkably, RNAi knockdown of TbNRKC and overexpression of kinase-dead TbNRKC in procyclic forms induces the accumulation of cells with four basal bodies, whereas overexpression of active protein produces supernumary basal bodies and blocks cytokinesis. TbNRKC is located on mature and immature basal bodies and is the first T. brucei NRK to be found associated with the basal body cytokinesis pathway.

  11. The role of the PI(3,5)P2 kinase TbFab1 in endo/lysosomal trafficking in Trypanosoma brucei.

    Science.gov (United States)

    Gilden, Julia K; Umaer, Khan; Kruzel, Emilia K; Hecht, Oliver; Correa, Renan O; Mansfield, John M; Bangs, James D

    2017-06-01

    Protein trafficking through endo/lysosomal compartments is critically important to the biology of the protozoan parasite Trypanosoma brucei, but the routes material may take to the lysosome, as well as the molecular factors regulating those routes, remain incompletely understood. Phosphoinositides are signaling phospholipids that regulate many trafficking events by recruiting specific effector proteins to discrete membrane subdomains. In this study, we investigate the role of one phosphoinositide, PI(3,5)P2 in T. brucei. We find a low steady state level of PI(3,5)P2 in bloodstream form parasites comparable to that of other organisms. RNAi knockdown of the putative PI(3)P-5 kinase TbFab1 decreases the PI(3,5)P2 pool leading to rapid cell death. TbFab1 and PI(3,5)P2 both localize strongly to late endo/lysosomes. While most trafficking functions were intact in TbFab1 deficient cells, including both endocytic and biosynthetic trafficking to the lysosome, lysosomal turnover of an endogenous ubiquitinylated membrane protein, ISG65, was completely blocked suggesting that TbFab1 plays a role in the ESCRT-mediated late endosomal/multivesicular body degradative pathways. Knockdown of a second component of PI(3,5)P2 metabolism, the PI(3,5)P2 phosphatase TbFig4, also resulted in delayed turnover of ISG65. Together, these results demonstrate an essential role for PI(3,5)P2 in the turnover of ubiquitinylated membrane proteins and in trypanosome endomembrane biology. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. A Pre-clinical Animal Model of Trypanosoma brucei Infection Demonstrating Cardiac Dysfunction.

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    Charlotte S McCarroll

    2015-05-01

    Full Text Available African trypanosomiasis (AT, caused by Trypanosoma brucei species, results in both neurological and cardiac dysfunction and can be fatal if untreated. Research on the pathogenesis and treatment of the disease has centred to date on the characteristic neurological symptoms, whereas cardiac dysfunction (e.g. ventricular arrhythmias in AT remains largely unstudied. Animal models of AT demonstrating cardiac dysfunction similar to that described in field cases of AT are critically required to transform our understanding of AT-induced cardiac pathophysiology and identify future treatment strategies. We have previously shown that T. brucei can interact with heart muscle cells (cardiomyocytes to induce ventricular arrhythmias in ex vivo adult rat hearts. However, it is unknown whether the arrhythmias observed ex vivo are also present during in vivo infection in experimental animal models. Here we show for the first time the characterisation of ventricular arrhythmias in vivo in two animal models of AT infection using electrocardiographic (ECG monitoring. The first model utilised a commonly used monomorphic laboratory strain, Trypanosoma brucei brucei Lister 427, whilst the second model used a pleomorphic laboratory strain, T. b. brucei TREU 927, which demonstrates a similar chronic infection profile to clinical cases. The frequency of ventricular arrhythmias and heart rate (HR was significantly increased at the endpoint of infection in the TREU 927 infection model, but not in the Lister 427 infection model. At the end of infection, hearts from both models were isolated and Langendorff perfused ex vivo with increasing concentrations of the β-adrenergic agonist isoproterenol (ISO. Interestingly, the increased frequency of arrhythmias observed in vivo in the TREU 927 infection model was lost upon isolation of the heart ex vivo, but re-emerged with the addition of ISO. Our results demonstrate that TREU 927 infection modifies the substrate of the myocardium

  13. Immunization with recombinant actin from Trypanosoma evansi induces protective immunity against T. evansi, T. equiperdum and T. b. brucei infection.

    Science.gov (United States)

    Li, San-Qiang; Yang, Wu-Biao; Lun, Zhao-Rong; Ma, Ling-Jun; Xi, Shou-Min; Chen, Qun-Li; Song, Xiao-Wei; Kang, Jian; Yang, Lan-Ze

    2009-01-01

    Actin gene of Trypanosoma evansi (STIB 806) was cloned and expressed in Escherichia coli. The predicted amino acid sequence of T. evansi actin shows 100%, 98.7%, and 93.1%, homology with Trypanosoma equiperdum, Trypanosoma brucei brucei, and Trypanosoma cruzi. Recombinant actin was expressed as inclusion bodies in E. coli. It was purified and renatured for immunological studies. Mice immunized with the renatured recombinant actin were protected from lethal challenge with T. evansi STIB 806, T. equiperdum STIB 818, and T. b. brucei STIB 940, showing 63.3%, 56.7%, and 53.3% protection, respectively. Serum collected from the rabbit immunized with recombinant actin inhibited the growth of T. evansi, T. equiperdum, and T. b. brucei in vitro cultivation. Serum from mice and rabbits immunized with recombinant actin only recognized T. evansi actin but not mouse actin. The results of this study suggest that the recombinant T. evansi actin induces protective immunity against T. evansi, T. equiperdum, and T. b. brucei infection and may be useful in the development of a vaccine with other cytoskeletal proteins to prevent animal trypanosomiasis caused by these three trypanosome species.

  14. Chemopreventive effect of methanolic extract of Azadirachta indica on experimental Trypanosoma brucei induced oxidative stress in dogs.

    Science.gov (United States)

    Omobowale, Temidayo O; Oyagbemi, Ademola A; Oyewunmi, Oyefunbi A; Adejumobi, Olumuyiwa A

    2015-01-01

    The medicinal properties of Azadirachta indica have been harnessed for many years in the treatment of many diseases in both humans and animals. Twenty-five apparently healthy dogs weighing between 3 and 8 kg were randomly divided into five groups with five dogs in each group. Ameliorative effect of A. indica on erythrocyte antioxidant status and markers of oxidative stress were assessed. Liver and kidney function tests were also performed. Pre-treatment with methanolic extract of Azadirachta indica (MEAI) at different doses did not significantly alter the values of alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase activity in Trypanosoma brucei infection. Although, serum creatinine significantly (P indica, after 2 weeks of T. brucei infection. However, the reduced glutathione (GSH) content of the erythrocyte increased significantly in animals pre-treated with 50 mg/kg and 200 mg/kg of A. indica respectively. Markers of oxidative stress such as malondialdehyde and hydrogen peroxide generated were higher in animals infected with T. brucei with no significant (P >0.05) difference compared to the values obtained in pre-treated animals. Pre-treatment with 100 mg/kg and 200 mg/kg of A. indica significantly (P < 0.05) decreased serum myeloperoxidase activity at 2 weeks post-infection with T. brucei. From this study, MEAI showed significant ability to attenuate oxidative stress and inflammation during experimental T. brucei infection.

  15. The nucleotide sequence of the variable region in Trypanosoma brucei completes the sequence analysis of the maxicircle component of mitochondrial kinetoplast DNA

    NARCIS (Netherlands)

    Sloof, P.; de Haan, A.; Eier, W.; van Iersel, M.; Boel, E.; van Steeg, H.; Benne, R.

    1992-01-01

    The nucleotide sequence of two non-contiguous DNA fragments of 4.0 and 2.2 kb, respectively, of the kinetoplast maxicircle of Trypanosoma brucei brucei EATRO strain 427 has been determined, completing the sequence analysis of the so-called variable region (see also de Vries et al., 1988, Mol.

  16. PCR approach for the detection of Trypanosoma brucei and T. equiperdum and their differentiation from T. evansi based on maxicircle kinetoplast DNA.

    Science.gov (United States)

    Li, Feng-Jun; Gasser, Robin B; Lai, De-Hua; Claes, Filip; Zhu, Xing-Quan; Lun, Zhao-Rong

    2007-02-01

    The goal of this study was to develop a PCR approach based on the sequence of maxicircle kinetoplast DNA (kDNA) of Trypanosoma brucei to distinguish T. brucei/T. equiperdum from T. evansi and to evaluate its diagnostic use for their detection in blood samples. Primers derived from the sequence of the maxicircle kDNA of T. brucei, encoding the NADH dehydrogenase subunit 5 (nad5) gene, were used to test the PCR-amplification from T. brucei (including T. b. brucei and T. b. rhodesiense), T. equiperdum, T. evansi, T. vivax and T. congolense. A primer pair to a nuclear DNA region incorporated into a separate PCR was employed to control for the presence of amplifiable genomic DNA (representing the subgenus Trypanozoon) in each sample subjected to the PCR. Products of approximately 395bp were amplified from all T. brucei and T. equiperdum samples tested using the nad5-PCR, but not from T. evansi DNA samples or any of the control samples representing T. vivax, T. congolense, or host. The current PCR approach allows the rapid differentiation of T. brucei/T.equiperdum from T. evansi and can detect the equivalent of 20-25 cells of T. brucei or T. equiperdum in purified genomic DNA or infected blood samples.

  17. The response of trypanosomes and other eukaryotes to ER stress and the spliced leader RNA silencing (SLS) pathway in Trypanosoma brucei.

    Science.gov (United States)

    Michaeli, Shulamit

    2015-01-01

    The unfolded protein response (UPR) is induced when the quality control machinery of the cell is overloaded with unfolded proteins or when one of the functions of the endoplasmic reticulum (ER) is perturbed. Here, I describe UPR in yeast and mammals, and compare it to what we know about pathogenic fungi and the parasitic protozoans from the order kinetoplastida, focusing on the novel pathway the spliced leader silencing (SLS) in Trypanosoma brucei. Trypanosomes lack conventional transcription regulation, and thus, lack most of the UPR machinery present in other eukaryotes. Trypanosome genes are transcribed in polycistronic units that are processed by trans-splicing and polyadenylation. In trans-splicing, which is essential for processing of each mRNA, an exon known as the spliced leader (SL) is added to all mRNAs from a small RNA, the SL RNA. Under severe ER stress, T. brucei elicits the SLS pathway. In SLS, the transcription of the SL RNA gene is extinguished, and the entire transcription complex dissociates from the SL RNA promoter. Induction of SLS is mediated by an ER-associated kinase (PK3) that migrates to the nucleus, where it phosphorylates the TATA-binding protein (TRF4), leading shut-off of SL RNA transcription. As a result, trans-splicing is inhibited and the parasites activate a programmed cell death (PCD) pathway. Despite the ability to sense the ER stress, the different eukaryotes, especially unicellular parasites and pathogenic fungi, developed a variety of unique and different ways to sense and adjust to this stress in a manner different from their host.

  18. Genome and phylogenetic analyses of Trypanosoma evansi reveal extensive similarity to T. brucei and multiple independent origins for dyskinetoplasty.

    Directory of Open Access Journals (Sweden)

    Jason Carnes

    2015-01-01

    Full Text Available Two key biological features distinguish Trypanosoma evansi from the T. brucei group: independence from the tsetse fly as obligatory vector, and independence from the need for functional mitochondrial DNA (kinetoplast or kDNA. In an effort to better understand the molecular causes and consequences of these differences, we sequenced the genome of an akinetoplastic T. evansi strain from China and compared it to the T. b. brucei reference strain. The annotated T. evansi genome shows extensive similarity to the reference, with 94.9% of the predicted T. b. brucei coding sequences (CDS having an ortholog in T. evansi, and 94.6% of the non-repetitive orthologs having a nucleotide identity of 95% or greater. Interestingly, several procyclin-associated genes (PAGs were disrupted or not found in this T. evansi strain, suggesting a selective loss of function in the absence of the insect life-cycle stage. Surprisingly, orthologous sequences were found in T. evansi for all 978 nuclear CDS predicted to represent the mitochondrial proteome in T. brucei, although a small number of these may have lost functionality. Consistent with previous results, the F1FO-ATP synthase γ subunit was found to have an A281 deletion, which is involved in generation of a mitochondrial membrane potential in the absence of kDNA. Candidates for CDS that are absent from the reference genome were identified in supplementary de novo assemblies of T. evansi reads. Phylogenetic analyses show that the sequenced strain belongs to a dominant group of clonal T. evansi strains with worldwide distribution that also includes isolates classified as T. equiperdum. At least three other types of T. evansi or T. equiperdum have emerged independently. Overall, the elucidation of the T. evansi genome sequence reveals extensive similarity of T. brucei and supports the contention that T. evansi should be classified as a subspecies of T. brucei.

  19. Genome and phylogenetic analyses of Trypanosoma evansi reveal extensive similarity to T. brucei and multiple independent origins for dyskinetoplasty.

    Science.gov (United States)

    Carnes, Jason; Anupama, Atashi; Balmer, Oliver; Jackson, Andrew; Lewis, Michael; Brown, Rob; Cestari, Igor; Desquesnes, Marc; Gendrin, Claire; Hertz-Fowler, Christiane; Imamura, Hideo; Ivens, Alasdair; Kořený, Luděk; Lai, De-Hua; MacLeod, Annette; McDermott, Suzanne M; Merritt, Chris; Monnerat, Severine; Moon, Wonjong; Myler, Peter; Phan, Isabelle; Ramasamy, Gowthaman; Sivam, Dhileep; Lun, Zhao-Rong; Lukeš, Julius; Stuart, Ken; Schnaufer, Achim

    2015-01-01

    Two key biological features distinguish Trypanosoma evansi from the T. brucei group: independence from the tsetse fly as obligatory vector, and independence from the need for functional mitochondrial DNA (kinetoplast or kDNA). In an effort to better understand the molecular causes and consequences of these differences, we sequenced the genome of an akinetoplastic T. evansi strain from China and compared it to the T. b. brucei reference strain. The annotated T. evansi genome shows extensive similarity to the reference, with 94.9% of the predicted T. b. brucei coding sequences (CDS) having an ortholog in T. evansi, and 94.6% of the non-repetitive orthologs having a nucleotide identity of 95% or greater. Interestingly, several procyclin-associated genes (PAGs) were disrupted or not found in this T. evansi strain, suggesting a selective loss of function in the absence of the insect life-cycle stage. Surprisingly, orthologous sequences were found in T. evansi for all 978 nuclear CDS predicted to represent the mitochondrial proteome in T. brucei, although a small number of these may have lost functionality. Consistent with previous results, the F1FO-ATP synthase γ subunit was found to have an A281 deletion, which is involved in generation of a mitochondrial membrane potential in the absence of kDNA. Candidates for CDS that are absent from the reference genome were identified in supplementary de novo assemblies of T. evansi reads. Phylogenetic analyses show that the sequenced strain belongs to a dominant group of clonal T. evansi strains with worldwide distribution that also includes isolates classified as T. equiperdum. At least three other types of T. evansi or T. equiperdum have emerged independently. Overall, the elucidation of the T. evansi genome sequence reveals extensive similarity of T. brucei and supports the contention that T. evansi should be classified as a subspecies of T. brucei.

  20. Pyrimidine Salvage Enzymes Are Essential for De Novo Biosynthesis of Deoxypyrimidine Nucleotides in Trypanosoma brucei.

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    Christopher Leija

    2016-11-01

    Full Text Available The human pathogenic parasite Trypanosoma brucei possess both de novo and salvage routes for the biosynthesis of pyrimidine nucleotides. Consequently, they do not require salvageable pyrimidines for growth. Thymidine kinase (TK catalyzes the formation of dTMP and dUMP and is one of several salvage enzymes that appear redundant to the de novo pathway. Surprisingly, we show through analysis of TK conditional null and RNAi cells that TK is essential for growth and for infectivity in a mouse model, and that a catalytically active enzyme is required for its function. Unlike humans, T. brucei and all other kinetoplastids lack dCMP deaminase (DCTD, which provides an alternative route to dUMP formation. Ectopic expression of human DCTD resulted in full rescue of the RNAi growth phenotype and allowed for selection of viable TK null cells. Metabolite profiling by LC-MS/MS revealed a buildup of deoxypyrimidine nucleosides in TK depleted cells. Knockout of cytidine deaminase (CDA, which converts deoxycytidine to deoxyuridine led to thymidine/deoxyuridine auxotrophy. These unexpected results suggested that T. brucei encodes an unidentified 5'-nucleotidase that converts deoxypyrimidine nucleotides to their corresponding nucleosides, leading to their dead-end buildup in TK depleted cells at the expense of dTTP pools. Bioinformatics analysis identified several potential candidate genes that could encode 5'-nucleotidase activity including an HD-domain protein that we show catalyzes dephosphorylation of deoxyribonucleotide 5'-monophosphates. We conclude that TK is essential for synthesis of thymine nucleotides regardless of whether the nucleoside precursors originate from the de novo pathway or through salvage. Reliance on TK in the absence of DCTD may be a shared vulnerability among trypanosomatids and may provide a unique opportunity to selectively target a diverse group of pathogenic single-celled eukaryotes with a single drug.

  1. Knockdown of asparagine synthetase A renders Trypanosoma brucei auxotrophic to asparagine.

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    Inês Loureiro

    Full Text Available Asparagine synthetase (AS catalyzes the ATP-dependent conversion of aspartate into asparagine using ammonia or glutamine as nitrogen source. There are two distinct types of AS, asparagine synthetase A (AS-A, known as strictly ammonia-dependent, and asparagine synthetase B (AS-B, which can use either ammonia or glutamine. The absence of AS-A in humans, and its presence in trypanosomes, suggested AS-A as a potential drug target that deserved further investigation. We report the presence of functional AS-A in Trypanosoma cruzi (TcAS-A and Trypanosoma brucei (TbAS-A: the purified enzymes convert L-aspartate into L-asparagine in the presence of ATP, ammonia and Mg(2+. TcAS-A and TbAS-A use preferentially ammonia as a nitrogen donor, but surprisingly, can also use glutamine, a characteristic so far never described for any AS-A. TbAS-A knockdown by RNAi didn't affect in vitro growth of bloodstream forms of the parasite. However, growth was significantly impaired when TbAS-A knockdown parasites were cultured in medium with reduced levels of asparagine. As expected, mice infections with induced and non-induced T. brucei RNAi clones were similar to those from wild-type parasites. However, when induced T. brucei RNAi clones were injected in mice undergoing asparaginase treatment, which depletes blood asparagine, the mice exhibited lower parasitemia and a prolonged survival in comparison to similarly-treated mice infected with control parasites. Our results show that TbAS-A can be important under in vivo conditions when asparagine is limiting, but is unlikely to be suitable as a drug target.

  2. Chemical characterisation of Nigerian red propolis and its biological activity against Trypanosoma Brucei.

    Science.gov (United States)

    Omar, Ruwida M K; Igoli, John; Gray, Alexander I; Ebiloma, Godwin Unekwuojo; Clements, Carol; Fearnley, James; Ebel, Ru Angeli Edrada; Zhang, Tong; De Koning, Harry P; Watson, David G

    2016-01-01

    A previous study showed the unique character of Nigerian red propolis from Rivers State, Nigeria (RSN), with regards to chemical composition and activity against Trypanosoma brucei in comparison with other African propolis. To carry out fractionation and biological testing of Nigerian propolis in order to isolate compounds with anti-trypanosomal activity. To compare the composition of the RSN propolis with the composition of Brazilian red propolis. Profiling was carried out using HPLC-UV-ELSD and HPLC-Orbitrap-FTMS on extracts of two samples collected from RSN with data extraction using MZmine software. Isolation was carried out by normal phase and reversed phase MPLC. Elucidation of the compounds with a purity > 95% was performed by 1D/2D NMR HRMS and HRLC-MS(n) . Ten phenolic compounds were isolated or in the case of liquiritigenin partially purified. Data for nine of these correlated with literature reports of known compounds i.e. one isoflavanone, calycosin (1); two flavanones, liquiritigenin (2) and pinocembrin (5); an isoflavan, vestitol (3); a pterocarpan, medicarpin (4); two prenylflavanones, 8-prenylnaringenin (7) and 6-prenylnaringenin (8); and two geranyl flavonoids, propolin D (9) and macarangin (10). The tenth was elucidated as a previously undescribed dihydrobenzofuran (6). The isolated compounds were tested against Trypanosoma brucei and displayed moderate to high activity. Some of the compounds tested had similar activity against wild type T. brucei and two strains displaying pentamidine resistance. Nigerian propolis from RSN has some similarities with Brazilian red propolis. The propolis displayed anti-trypanosomal activity at a potentially useful level. Copyright © 2015 John Wiley & Sons, Ltd.

  3. The transcriptome of the human pathogen Trypanosoma brucei at single-nucleotide resolution.

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    Nikolay G Kolev

    2010-09-01

    Full Text Available The genome of Trypanosoma brucei, the causative agent of African trypanosomiasis, was published five years ago, yet identification of all genes and their transcripts remains to be accomplished. Annotation is challenged by the organization of genes transcribed by RNA polymerase II (Pol II into long unidirectional gene clusters with no knowledge of how transcription is initiated. Here we report a single-nucleotide resolution genomic map of the T. brucei transcriptome, adding 1,114 new transcripts, including 103 non-coding RNAs, confirming and correcting many of the annotated features and revealing an extensive heterogeneity of 5' and 3' ends. Some of the new transcripts encode polypeptides that are either conserved in T. cruzi and Leishmania major or were previously detected in mass spectrometry analyses. High-throughput RNA sequencing (RNA-Seq was sensitive enough to detect transcripts at putative Pol II transcription initiation sites. Our results, as well as recent data from the literature, indicate that transcription initiation is not solely restricted to regions at the beginning of gene clusters, but may occur at internal sites. We also provide evidence that transcription at all putative initiation sites in T. brucei is bidirectional, a recently recognized fundamental property of eukaryotic promoters. Our results have implications for gene expression patterns in other important human pathogens with similar genome organization (Trypanosoma cruzi, Leishmania sp. and revealed heterogeneity in pre-mRNA processing that could potentially contribute to the survival and success of the parasite population in the insect vector and the mammalian host.

  4. Trypanosoma brucei Co-opts NK Cells to Kill Splenic B2 B Cells.

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    Deborah Frenkel

    2016-07-01

    Full Text Available After infection with T. brucei AnTat 1.1, C57BL/6 mice lost splenic B2 B cells and lymphoid follicles, developed poor parasite-specific antibody responses, lost weight, became anemic and died with fulminating parasitemia within 35 days. In contrast, infected C57BL/6 mice lacking the cytotoxic granule pore-forming protein perforin (Prf1-/- retained splenic B2 B cells and lymphoid follicles, developed high-titer antibody responses against many trypanosome polypeptides, rapidly suppressed parasitemia and did not develop anemia or lose weight for at least 60 days. Several lines of evidence show that T. brucei infection-induced splenic B cell depletion results from natural killer (NK cell-mediated cytotoxicity: i B2 B cells were depleted from the spleens of infected intact, T cell deficient (TCR-/- and FcγRIIIa deficient (CD16-/- C57BL/6 mice excluding a requirement for T cells, NKT cell, or antibody-dependent cell-mediated cytotoxicity; ii administration of NK1.1 specific IgG2a (mAb PK136 but not irrelevant IgG2a (myeloma M9144 prevented infection-induced B cell depletion consistent with a requirement for NK cells; iii splenic NK cells but not T cells or NKT cells degranulated in infected C57BL/6 mice co-incident with B cell depletion evidenced by increased surface expression of CD107a; iv purified NK cells from naïve C57BL/6 mice killed purified splenic B cells from T. brucei infected but not uninfected mice in vitro indicating acquisition of an NK cell activating phenotype by the post-infection B cells; v adoptively transferred C57BL/6 NK cells prevented infection-induced B cell population growth in infected Prf1-/- mice consistent with in vivo B cell killing; vi degranulated NK cells in infected mice had altered gene and differentiation antigen expression and lost cytotoxic activity consistent with functional exhaustion, but increased in number as infection progressed indicating continued generation. We conclude that NK cells in T. brucei

  5. Variation of G-rich mitochondrial transcripts among stocks of Trypanosoma brucei.

    Science.gov (United States)

    Jasmer, D P; Feagin, J E; Payne, M; Stuart, K

    1987-01-15

    We have compared maxicircle transcripts from eight stocks of subspecies of Trypanosoma brucei. Transcripts from the rRNA and protein genes have a constant size among stocks and exhibit only minor variation in abundance. In contrast, four of the G+C rich sequences encode multiple transcripts that very markedly in size or abundance. Maxicircle nucleotide sequence comparison of three stocks shows very limited sequence divergence suggesting that sequence divergence may not explain the transcript variability. These results suggest that the G-rich transcripts do not encode proteins and that their variability among stocks may result from posttranscriptional processing events.

  6. Structural and functional insight into ADF/cofilin from Trypanosoma brucei.

    Science.gov (United States)

    Dai, Kun; Liao, Shanhui; Zhang, Jiahai; Zhang, Xuecheng; Tu, Xiaoming

    2013-01-01

    The ADF/cofilin family has been characterized as a group of actin-binding proteins critical for controlling the assembly of actin within the cells. In this study, the solution structure of the ADF/cofilin from Trypanosoma brucei (TbCof) was determined by NMR spectroscopy. TbCof adopts the conserved ADF/cofilin fold with a central β-sheet composed of six β-strands surrounded by five α-helices. Isothermal titration calorimetry experiments denoted a submicromolar affinity between TbCof and G-actin, and the affinity between TbCof and ADP-G-actin was five times higher than that between TbCof and ATP-G-actin at low ionic strength. The results obtained from electron microscopy and actin filament sedimentation assays showed that TbCof depolymerized but did not co-sediment with actin filaments and its ability of F-actin depolymerization was pH independent. Similar to actin, TbCof was distributed throughout the cytoplasm. All our data indicate a structurally and functionally conserved ADF/cofilin from Trypanosoma brucei.

  7. Structural and Functional Association of Trypanosoma brucei MIX Protein with Cytochrome c Oxidase Complex ▿ †

    Science.gov (United States)

    Zíková, Alena; Panigrahi, Aswini K.; Uboldi, Alessandro D.; Dalley, Rachel A.; Handman, Emanuela; Stuart, Kenneth

    2008-01-01

    A mitochondrial inner membrane protein, designated MIX, seems to be essential for cell viability. The deletion of both alleles was not possible, and the deletion of a single allele led to a loss of virulence and aberrant mitochondrial segregation and cell division in Leishmania major. However, the mechanism by which MIX exerts its effect has not been determined. We show here that MIX is also expressed in the mitochondrion of Trypanosoma brucei, and using RNA interference, we found that its loss leads to a phenotype that is similar to that described for Leishmania. The loss of MIX also had a major effect on cytochrome c oxidase activity, on the mitochondrial membrane potential, and on the production of mitochondrial ATP by oxidative phosphorylation. Using a tandem affinity purification tag, we found that MIX is associated with a multiprotein complex that contains subunits of the mitochondrial cytochrome c oxidase complex (respiratory complex IV), the composition of which was characterized in detail. The specific function of MIX is unknown, but it appears to be important for the function of complex IV and for mitochondrial segregation and cell division in T. brucei. PMID:18776036

  8. Investigating the Chaperone Properties of a Novel Heat Shock Protein, Hsp70.c, from Trypanosoma brucei

    Directory of Open Access Journals (Sweden)

    Adélle Burger

    2014-01-01

    Full Text Available The neglected tropical disease, African Trypanosomiasis, is fatal and has a crippling impact on economic development. Heat shock protein 70 (Hsp70 is an important molecular chaperone that is expressed in response to stress and Hsp40 acts as its co-chaperone. These proteins play a wide range of roles in the cell and they are required to assist the parasite as it moves from a cold blooded insect vector to a warm blooded mammalian host. A novel cytosolic Hsp70, from Trypanosoma brucei, TbHsp70.c, contains an acidic substrate binding domain and lacks the C-terminal EEVD motif. The ability of a cytosolic Hsp40 from Trypanosoma brucei J protein 2, Tbj2, to function as a co-chaperone of TbHsp70.c was investigated. The main objective was to functionally characterize TbHsp70.c to further expand our knowledge of parasite biology. TbHsp70.c and Tbj2 were heterologously expressed and purified and both proteins displayed the ability to suppress aggregation of thermolabile MDH and chemically denatured rhodanese. ATPase assays revealed a 2.8-fold stimulation of the ATPase activity of TbHsp70.c by Tbj2. TbHsp70.c and Tbj2 both demonstrated chaperone activity and Tbj2 functions as a co-chaperone of TbHsp70.c. In vivo heat stress experiments indicated upregulation of the expression levels of TbHsp70.c.

  9. KREX2 is not essential for either procyclic or bloodstream form Trypanosoma brucei.

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    Jason Carnes

    Full Text Available BACKGROUND: Most mitochondrial mRNAs in Trypanosoma brucei require RNA editing for maturation and translation. The edited RNAs primarily encode proteins of the oxidative phosphorylation system. These parasites undergo extensive changes in energy metabolism between the insect and bloodstream stages which are mirrored by alterations in RNA editing. Two U-specific exonucleases, KREX1 and KREX2, are both present in protein complexes (editosomes that catalyze RNA editing but the relative roles of each protein are not known. METHODOLOGY/PRINCIPAL FINDINGS: The requirement for KREX2 for RNA editing in vivo was assessed in both procyclic (insect and bloodstream form parasites by methods that use homologous recombination for gene elimination. These studies resulted in null mutant cells in which both alleles were eliminated. The viability of these cells demonstrates that KREX2 is not essential in either life cycle stage, despite certain defects in RNA editing in vivo. Furthermore, editosomes isolated from KREX2 null cells require KREX1 for in vitro U-specific exonuclease activity. CONCLUSIONS: KREX2 is a U-specific exonuclease that is dispensable for RNA editing in vivo in T. brucei BFs and PFs. This result suggests that the U deletion activity, which is required for RNA editing, is primarily mediated in vivo by KREX1 which is normally found associated with only one type of editosome. The retention of the KREX2 gene implies a non-essential role or a role that is essential in other life cycle stages or conditions.

  10. Secondary metabolites from Vietnamese marine invertebrates with activity against Trypanosoma brucei and T. cruzi.

    Science.gov (United States)

    Thao, Nguyen Phuong; No, Joo Hwan; Luyen, Bui Thi Thuy; Yang, Gyongseon; Byun, Soo Young; Goo, Junghyun; Kim, Kyung Tae; Cuong, Nguyen Xuan; Nam, Nguyen Hoai; Van Minh, Chau; Schmidt, Thomas J; Kang, Jong Seong; Kim, Young Ho

    2014-06-11

    Marine-derived natural products from invertebrates comprise an extremely diverse and promising source of the compounds from a wide variety of structural classes. This study describes the discovery of five marine natural products with activity against Trypanosoma species by natural product library screening using whole cell in vitro assays. We investigated the anti-trypanosomal activity of the extracts from the soft corals and echinoderms living in Vietnamese seas. Of the samples screened, the methanolic extracts of several marine organisms exhibited potent activities against cultures of Trypanosoma brucei and T. cruzi (EC50 activity against T. brucei with EC50 values ranging from 1.57 ± 0.14 to 14.6 ± 1.36 μM, relative to the positive control, pentamidine (EC50 = 0.015 ± 0.003 μM). Laevigatol B (1) and 5α-cholest-8(14)-ene-3β,7α-diol (5) exhibited also significant inhibitory effects on T. cruzi. The cytotoxic activity of the pure compounds on mammalian cells was also assessed and found to be insignificant in all cases. This is the first report on the inhibitory effects of marine organisms collected in Vietnamese seas against Trypanosoma species responsible for neglected tropical diseases.

  11. An Overview of Trypanosoma brucei Infections: An Intense Host-Parasite Interaction.

    Science.gov (United States)

    Ponte-Sucre, Alicia

    2016-01-01

    Trypanosoma brucei rhodesiense and T. brucei gambiense, the causative agents of Human African Trypanosomiasis, are transmitted by tsetse flies. Within the vector, the parasite undergoes through transformations that prepares it to infect the human host. Sequentially these developmental stages are the replicative procyclic (in which the parasite surface is covered by procyclins) and trypo-epimastigote forms, as well as the non-replicative, infective, metacyclic form that develops in the vector salivary glands. As a pre-adaptation to their life in humans, metacyclic parasites begin to express and be densely covered by the Variant Surface Glycoprotein (VSG). Once the metacyclic form invades the human host the parasite develops into the bloodstream form. Herein the VSG triggers a humoral immune response. To avoid this humoral response, and essential for survival while in the bloodstream, the parasite changes its cover periodically and sheds into the surroundings the expressed VSG, thus evading the consequences of the immune system activation. Additionally, tools comparable to quorum sensing are used by the parasite for the successful parasite transmission from human to insect. On the other hand, the human host promotes clearance of the parasite triggering innate and adaptive immune responses and stimulating cytokine and chemokine secretion. All in all, the host-parasite interaction is extremely active and leads to responses that need multiple control sites to develop appropriately.

  12. Variations in maxi-circle and mini-circle sequences in kinetoplast DNAs from different Trypanosoma brucei strains.

    NARCIS (Netherlands)

    P. Borst (Piet); F. Fase-Fowler; J.H.J. Hoeijmakers (Jan); A.C.C. Frasch

    1980-01-01

    textabstractWe have compared a total of 30 recognition sites for eight restriction endonucleases on the 20-kilobase-pair maxi-circle of kinetoplast DNAs from five different Trypanosoma brucei strains. In addition to three polymorphic sites were have found a 5 kilobase-pair region that is not cleaved

  13. THE CYTOSOLIC AND GLYCOSOMAL GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE FROM TRYPANOSOMA-BRUCEI - KINETIC-PROPERTIES AND COMPARISON WITH HOMOLOGOUS ENZYMES

    NARCIS (Netherlands)

    LAMBEIR, AM; LOISEAU, AM; KUNTZ, DA; VELLIEUX, FM; MICHELS, PAM; OPPERDOES, FR

    1991-01-01

    The protozoan haemoflagellate Trypanosoma brucei has two NAD-dependent glyceraldehyde-3-phosphate dehydrogenase isoenzymes, each with a different localization within the cell. One isoenzyme is found in the cytosol, as in other eukaryotes, while the other is found in the glycosome, a microbody-like

  14. A tropical tale: how Naja nigricollis venom beats Trypanosoma brucei

    DEFF Research Database (Denmark)

    Martos Esteban, Andrea; Laustsen, Andreas Hougaard; Carrington, Mark

    Trypanosoma brucei is a parasitic protozoan species capable to infecting insect vectors whose bite further produces African sleeping sickness inhuman beings [1]. During the parasite’s extracellular life in the mammalian host,its outer coat, mainly composed of Variable Surface Glycoproteins (VSGs...

  15. Haematological indices in Trypanosoma brucei brucei (Federe isolate infected Nigerian donkeys (Equus asinus treated with homidium and isometamidium chloride of ciprofloxacin in broiler chickens after single intravenous and intraingluvial administration

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    Queen Nneka Oparah

    2017-03-01

    Full Text Available The efficacy of intramuscular administration of Homidium chloride (Novidium® and Isometamidium chloride (Sécuridium® in Nigerian donkeys (Equus asinus experimentally infected with T. b. brucei (Federe isolate was investigated. Changes in haematological and serum biochemical indices were evaluated using clinical haematology and biochemistry methods. Red blood cell (RBC count for the negative control group was significantly higher than for the positive control, Novidium® and Sécuridium®-treatment groups. Haemoglobin (Hb concentration significantly reduced in the infected untreated group compared with other groups. Packed cell volume (PCV was significantly different between negative and positive controls, and also between the infected untreated and treatment groups. There was significant reduction in platelet counts post-infection and post-treatment. Mean corpuscular volume (MCV increased significantly in the treatment groups while mean corpuscular haemoglobin concentration (MCHC significantly reduced only in the Sécuridium®-treatment group. Lymphocyte count for infected untreated was non-significantly higher than for the uninfected controls, but treatment with both trypanocides recorded further increases, which were higher compared with that of the uninfected group. Post infection and treatment, aspartate aminotransferase (AST levels increased significantly. There were non-significant differences in electrolyte ion concentrations across the groups except for chloride ion which recorded a significant reduction in the Novidium®-treatment group. This experiment revealed that Nigerian donkeys infected with T. brucei brucei (Federe isolate developed symptoms of trypanosomosis; anaemia, lymphocytosis and thrombocytopenia. Treatment with the trypanocides ameliorated effects of the infection, and results suggest that immunosuppression may not be a substantial clinical manifestation of T. brucei brucei (Federe isolate trypanosomosis in Nigerian

  16. Sulfhydryl reagent susceptibility in proteins with high sequence similarity--triosephosphate isomerase from Trypanosoma brucei, Trypanosoma cruzi and Leishmania mexicana.

    Science.gov (United States)

    Garza-Ramos, G; Cabrera, N; Saavedra-Lira, E; Tuena de Gómez-Puyou, M; Ostoa-Saloma, P; Pérez-Montfort, R; Gómez-Puyou, A

    1998-05-01

    The amino acid sequence of triosephosphate isomerase from Trypanosoma brucei, Trypanosoma cruzi, and Leishmania mexicana have an identity of 68%. Using the numbering system for the T. brucei enzyme, in their aligned sequences, the T. cruzi and leishmanial enzymes have cysteine residues at positions 14, 40, 117 and 126. T. brucei triosephosphate isomerase has cysteine residues at positions 14, 40 and 126, and a valine residue at position 117. Dithionitrobenzoic acid and methylmethane thiosulfonate inhibited the three enzymes, but T. cruzi triosephosphate isomerase was more than 100-fold more sensitive. The sensitivity of wild type triosephosphate isomerase from T. cruzi and T. brucei to the reagents was equal to that of the Cys117Val and Val117Cys mutant enzymes, respectively. Triosephosphate isomerases that have cysteine residues at positions 40 and 126, but lack a cysteine residue at position 14 are insensitive to methylmethane thiosulfonate. Thus, sulfhydryl reagents act on Cys14. At stoichiometric concentrations, the reagents inhibited the three enzymes as a consequence of structural alterations as measured by binding of 8-anilino-1-napthalenesulfonic acid to previously buried hydrophobic regions. However, the times for half-maximal alterations were 10 min, 15 hours and over 30 hours for T. cruzi, T. brucei and L. mexicana triosephosphate isomerase, respectively. The effect of pH on the action of the sulfhydryl reagents and molecular modeling showed no differences in the solvent accessibility of Cys14. As Cys14 forms part of the dimer interface, the data indicate that, in the three enzymes, barriers of different magnitude hinder the interaction between the sulfhydryl reagents and Cys14. The barrier is lower in T. cruzi triosephosphate isomerase which makes its dimer interface more susceptible for perturbation.

  17. Molecular profiles of Trypanosoma brucei, T. evansi and T. equiperdum stocks revealed by the random amplified polymorphic DNA method.

    Science.gov (United States)

    Lun, Zhao-Rong; Li, An-Xing; Chen, Xiao-Guang; Lu, Li-Xin; Zhu, Xing-Quan

    2004-03-01

    A total of 20 random primers (10-mers) were used to amplify RAPD markers from the genomic DNA of four Trypanosoma brucei stocks from East and West Africa, four T. evansi stocks from Africa, Asia and South America and one T. equiperdum stock from Asia. Between 65 and 88 reproducible fragments ranging from 0.25 to 2.15 kb were generated from these stocks depending on the stock/primer combination. The similarity coefficient (SC) among the stocks of T. brucei from Kenya, Nigeria, Tanzania and Zambia ranged from 62.9% to 74.0% (average: 67.6%). The SC among the stocks of T. evansi from Kenya, China and Brazil was 76.4%-95.5% (average: 86.4%), while the SC between T. evansi stock from China and Brazil was 95.5%. For T. evansi and T. equiperdum, the SC among the stocks ranged from 81.2% to 94.4% (average: 87.6%). As for the SC among the stocks of T. brucei and T. evansi, it was found to be from 54.7% to 80.3% (average: 68.0%) and the SC among stocks of T. brucei and T. equiperdum was from 59.4% to 76.9% (average: 68.1%). Our results indicate that the stocks of T. evansi from China and from Brazil are more closely related to the stock of T. equiperdum from China than to the stocks of T. evansi isolated from Kenya and to the stocks of T. brucei. In addition, our results further support the hypothesis that T. evansi stocks from China and Brazil could have arisen from a single lineage. The possible evolution of T. evansi and T. equiperdum is also discussed.

  18. ATG24 Represses Autophagy and Differentiation and Is Essential for Homeostasy of the Flagellar Pocket in Trypanosoma brucei.

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    Ana Brennand

    Full Text Available We have previously identified homologs for nearly half of the approximately 30 known yeast Atg's in the genome database of the human sleeping sickness parasite Trypanosoma brucei. So far, only a few of these homologs have their role in autophagy experimentally confirmed. Among the candidates was the ortholog of Atg24 that is involved in pexophagy in yeast. In T. brucei, the peroxisome-like organelles named glycosomes harbor core metabolic processes, especially glycolysis. In the autotrophic yeast, autophagy is essential for adaptation to different nutritional environments by participating in the renewal of the peroxisome population. We hypothesized that autophagic turnover of the parasite's glycosomes plays a role in differentiation during its life cycle, which demands adaptation to different host environments and associated dramatic changes in nutritional conditions. We therefore characterized T. brucei ATG24, the T. brucei ortholog of yeast Atg24 and mammalian SNX4, and found it to have a regulatory role in autophagy and differentiation as well as endocytic trafficking. ATG24 partially localized on endocytic membranes where it was recruited via PI3-kinase III/VPS34. ATG24 silencing severely impaired receptor-mediated endocytosis of transferrin, but not adsorptive uptake of a lectin, and caused a major enlargement of the flagellar pocket. ATG24 silencing approximately doubled the number of autophagosomes, suggesting a role in repressing autophagy, and strongly accelerated differentiation, in accordance with a role of autophagy in parasite differentiation. Overexpression of the two isoforms of T. brucei ATG8 fused to GFP slowed down differentiation, possibly by a dominant-negative effect. This was overcome by ATG24 depletion, further supporting its regulatory role.

  19. Chimerization at the AQP2–AQP3 locus is the genetic basis of melarsoprol–pentamidine cross-resistance in clinical Trypanosoma brucei gambiense isolates

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    Fabrice E. Graf

    2015-08-01

    Full Text Available Aquaglyceroporin-2 is a known determinant of melarsoprol–pentamidine cross-resistance in Trypanosoma brucei brucei laboratory strains. Recently, chimerization at the AQP2–AQP3 tandem locus was described from melarsoprol–pentamidine cross-resistant Trypanosoma brucei gambiense isolates from sleeping sickness patients in the Democratic Republic of the Congo. Here, we demonstrate that reintroduction of wild-type AQP2 into one of these isolates fully restores drug susceptibility while expression of the chimeric AQP2/3 gene in aqp2–aqp3 null T. b. brucei does not. This proves that AQP2–AQP3 chimerization is the cause of melarsoprol–pentamidine cross-resistance in the T. b. gambiense isolates.

  20. Further evidence from SSCP and ITS DNA sequencing support Trypanosoma evansi and Trypanosoma equiperdum as subspecies or even strains of Trypanosoma brucei

    OpenAIRE

    Wen, Y; Lun, Z; Zhu, X; Hide, G; Lai, D

    2016-01-01

    The subgenus Trypanozoon includes three species Trypanosoma brucei, Trypanosoma evansi and Trypanosoma equiperdum, which are morphologically identical and indistinguishable even using some molecular methods. In this study, PCR-based single strand conformation polymorphism (PCR-SSCP) was used to analyze the ribosomal DNA of the Trypanozoon species. Data indicate different patterns of ITS2 fragments between T. brucei, T. evansi and T. equiperdum by SSCP. Furthermore, analysis of total ITS seque...

  1. Trypanosoma brucei FKBP12 differentially controls motility and cytokinesis in procyclic and bloodstream forms.

    Science.gov (United States)

    Brasseur, Anaïs; Rotureau, Brice; Vermeersch, Marjorie; Blisnick, Thierry; Salmon, Didier; Bastin, Philippe; Pays, Etienne; Vanhamme, Luc; Pérez-Morga, David

    2013-02-01

    FKBP12 proteins are able to inhibit TOR kinases or calcineurin phosphatases upon binding of rapamycin or FK506 drugs, respectively. The Trypanosoma brucei FKBP12 homologue (TbFKBP12) was found to be a cytoskeleton-associated protein with specific localization in the flagellar pocket area of the bloodstream form. In the insect procyclic form, RNA interference-mediated knockdown of TbFKBP12 affected motility. In bloodstream cells, depletion of TbFKBP12 affected cytokinesis and cytoskeleton architecture. These last effects were associated with the presence of internal translucent cavities limited by an inside-out configuration of the normal cell surface, with a luminal variant surface glycoprotein coat lined up by microtubules. These cavities, which recreated the streamlined shape of the normal trypanosome cytoskeleton, might represent unsuccessful attempts for cell abscission. We propose that TbFKBP12 differentially affects stage-specific processes through association with the cytoskeleton.

  2. Enhanced succinic acid production in Aspergillus saccharolyticus by heterologous expression of fumarate reductase from Trypanosoma brucei

    DEFF Research Database (Denmark)

    Yang, Lei; Lübeck, Mette; Ahring, Birgitte K.

    2015-01-01

    production medium as well as the complete medium, but the measured enzyme activities were different depending on the media. Furthermore, a soluble NADH-dependent fumarate reductase gene (frd) from Trypanosoma brucei was inserted and expressed in A. saccharolyticus. The expression of the frd gene led......Aspergillus saccharolyticus exhibits great potential as a cell factory for industrial production of dicarboxylic acids. In the analysis of the organic acid profile, A. saccharolyticus was cultivated in an acid production medium using two different pH conditions. The specific activities...... on the pattern and the amount of organic acids produced by A. saccharolyticus. The wild-type strain produced higher amount of malic acid and succinic acid in the pH buffered condition (pH 6.5) compared with the pH non-buffered condition. The enzyme assays showed that the rTCA branch was active in the acid...

  3. Extracellular Vesicles from Trypanosoma brucei Mediate Virulence Factor Transfer and Cause Host Anemia.

    Science.gov (United States)

    Szempruch, Anthony J; Sykes, Steven E; Kieft, Rudo; Dennison, Lauren; Becker, Allison C; Gartrell, Anzio; Martin, William J; Nakayasu, Ernesto S; Almeida, Igor C; Hajduk, Stephen L; Harrington, John M

    2016-01-14

    Intercellular communication between parasites and with host cells provides mechanisms for parasite development, immune evasion, and disease pathology. Bloodstream African trypanosomes produce membranous nanotubes that originate from the flagellar membrane and disassociate into free extracellular vesicles (EVs). Trypanosome EVs contain several flagellar proteins that contribute to virulence, and Trypanosoma brucei rhodesiense EVs contain the serum resistance-associated protein (SRA) necessary for human infectivity. T. b. rhodesiense EVs transfer SRA to non-human infectious trypanosomes, allowing evasion of human innate immunity. Trypanosome EVs can also fuse with mammalian erythrocytes, resulting in rapid erythrocyte clearance and anemia. These data indicate that trypanosome EVs are organelles mediating non-hereditary virulence factor transfer and causing host erythrocyte remodeling, inducing anemia. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Compositional compartmentalization of the nuclear genomes of Trypanosoma brucei and Trypanosoma equiperdum.

    Science.gov (United States)

    Isacchi, A; Bernardi, G; Bernardi, G

    1993-12-06

    High molecular weight DNA preparations from Trypanosoma brucei and Trypanosoma equiperdum were fractionated by preparative centrifugation in a Cs2SO4 density gradient in the presence of BAMD, bis(acetatomercurimethyl)dioxane, a sequence-specific DNA ligand. Analytical centrifugation in CsCl of the DNA fractions so obtained showed that both DNAs had a bimodal distribution with two major peaks banding at 1.702-1.703 and 1.708 g/cm3 and representing 1/3 and 2/3 of total DNA, respectively. Several minor components were also detected. These results indicate that a compositional compartmentalization is not only found in the genome of vertebrates and plants, as already described, but also in those of protozoa such as Trypanosomes.

  5. Zinc finger nuclease technology: A stable tool for high efficiency transformation in bloodstream form T. brucei.

    Science.gov (United States)

    Schumann, Gabriela; Kangussu-Marcolino, Monica M; Doiron, Nicholas; Käser, Sandro; de Assis Burle-Caldas, Gabriela; DaRocha, Wanderson D; Teixeira, Santuza M; Roditi, Isabel

    2017-04-01

    In Trypanosoma brucei, the generation of knockout mutants is relatively easy compared to other organisms as transfection methods are well established. These methods have their limitations, however, when it comes to the generation of genome-wide libraries that require a minimum of several hundred thousand transformants. Double-strand breaks with the meganuclease ISce-I dramatically increase transformation efficiency, but are not widely in use as cell lines need to be generated de novo before each transfection. Here we show that zinc finger nucleases are a robust and stable tool that can enhance transformation in bloodstream forms by more than an order of magnitude. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Population genetics of Trypanosoma brucei rhodesiense: clonality and diversity within and between foci.

    Directory of Open Access Journals (Sweden)

    Craig W Duffy

    2013-11-01

    Full Text Available African trypanosomes are unusual among pathogenic protozoa in that they can undergo their complete morphological life cycle in the tsetse fly vector with mating as a non-obligatory part of this development. Trypanosoma brucei rhodesiense, which infects humans and livestock in East and Southern Africa, has classically been described as a host-range variant of the non-human infective Trypanosoma brucei that occurs as stable clonal lineages. We have examined T. b. rhodesiense populations from East (Uganda and Southern (Malawi Africa using a panel of microsatellite markers, incorporating both spatial and temporal analyses. Our data demonstrate that Ugandan T. b. rhodesiense existed as clonal populations, with a small number of highly related genotypes and substantial linkage disequilibrium between pairs of loci. However, these populations were not stable as the dominant genotypes changed and the genetic diversity also reduced over time. Thus these populations do not conform to one of the criteria for strict clonality, namely stability of predominant genotypes over time, and our results show that, in a period in the mid 1990s, the previously predominant genotypes were not detected but were replaced by a novel clonal population with limited genetic relationship to the original population present between 1970 and 1990. In contrast, the Malawi T. b. rhodesiense population demonstrated significantly greater diversity and evidence for frequent genetic exchange. Therefore, the population genetics of T. b. rhodesiense is more complex than previously described. This has important implications for the spread of the single copy T. b. rhodesiense gene that allows human infectivity, and therefore the epidemiology of the human disease, as well as suggesting that these parasites represent an important organism to study the influence of optional recombination upon population genetic dynamics.

  7. Secondary Metabolites from Vietnamese Marine Invertebrates with Activity against Trypanosoma brucei and T. cruzi

    Directory of Open Access Journals (Sweden)

    Nguyen Phuong Thao

    2014-06-01

    Full Text Available Marine-derived natural products from invertebrates comprise an extremely diverse and promising source of the compounds from a wide variety of structural classes. This study describes the discovery of five marine natural products with activity against Trypanosoma species by natural product library screening using whole cell in vitro assays. We investigated the anti-trypanosomal activity of the extracts from the soft corals and echinoderms living in Vietnamese seas. Of the samples screened, the methanolic extracts of several marine organisms exhibited potent activities against cultures of Trypanosoma brucei and T. cruzi (EC50 < 5.0 μg/mL. Among the compounds isolated from these extracts, laevigatol B (1 from Lobophytum crassum and L. laevigatum, (24S-ergost-4-ene-3-one (2 from Sinularia dissecta, astropectenol A (3 from Astropecten polyacanthus, and cholest-8-ene-3β,5α,6β,7α-tetraol (4 from Diadema savignyi showed inhibitory activity against T. brucei with EC50 values ranging from 1.57 ± 0.14 to 14.6 ± 1.36 μM, relative to the positive control, pentamidine (EC50 = 0.015 ± 0.003 μM. Laevigatol B (1 and 5α-cholest-8(14-ene-3β,7α-diol (5 exhibited also significant inhibitory effects on T. cruzi. The cytotoxic activity of the pure compounds on mammalian cells was also assessed and found to be insignificant in all cases. This is the first report on the inhibitory effects of marine organisms collected in Vietnamese seas against Trypanosoma species responsible for neglected tropical diseases.

  8. Trypanocidal action of bisphosphonium salts through a mitochondrial target in bloodstream form Trypanosoma brucei

    Directory of Open Access Journals (Sweden)

    Abdulsalam A.M. Alkhaldi

    2016-04-01

    Full Text Available Lipophilic bisphosphonium salts are among the most promising antiprotozoal leads currently under investigation. As part of their preclinical evaluation we here report on their mode of action against African trypanosomes, the etiological agents of sleeping sickness. The bisphosphonium compounds CD38 and AHI-9 exhibited rapid inhibition of Trypanosoma brucei growth, apparently the result of cell cycle arrest that blocked the replication of mitochondrial DNA, contained in the kinetoplast, thereby preventing the initiation of S-phase. Incubation with either compound led to a rapid reduction in mitochondrial membrane potential, and ATP levels decreased by approximately 50% within 1 h. Between 4 and 8 h, cellular calcium levels increased, consistent with release from the depolarized mitochondria. Within the mitochondria, the Succinate Dehydrogenase complex (SDH was investigated as a target for bisphosphonium salts, but while its subunit 1 (SDH1 was present at low levels in the bloodstream form trypanosomes, the assembled complex was hardly detectable. RNAi knockdown of the SDH1 subunit produced no growth phenotype, either in bloodstream or in the procyclic (insect forms and we conclude that in trypanosomes SDH is not the target for bisphosphonium salts. Instead, the compounds inhibited ATP production in intact mitochondria, as well as the purified F1 ATPase, to a level that was similar to 1 mM azide. Co-incubation with azide and bisphosphonium compounds did not inhibit ATPase activity more than either product alone. The results show that, in T. brucei, bisphosphonium compounds do not principally act on succinate dehydrogenase but on the mitochondrial FoF1 ATPase.

  9. Trypanocidal action of bisphosphonium salts through a mitochondrial target in bloodstream form Trypanosoma brucei

    Science.gov (United States)

    Alkhaldi, Abdulsalam A.M.; Martinek, Jan; Panicucci, Brian; Dardonville, Christophe; Zíková, Alena; de Koning, Harry P.

    2015-01-01

    Lipophilic bisphosphonium salts are among the most promising antiprotozoal leads currently under investigation. As part of their preclinical evaluation we here report on their mode of action against African trypanosomes, the etiological agents of sleeping sickness. The bisphosphonium compounds CD38 and AHI-9 exhibited rapid inhibition of Trypanosoma brucei growth, apparently the result of cell cycle arrest that blocked the replication of mitochondrial DNA, contained in the kinetoplast, thereby preventing the initiation of S-phase. Incubation with either compound led to a rapid reduction in mitochondrial membrane potential, and ATP levels decreased by approximately 50% within 1 h. Between 4 and 8 h, cellular calcium levels increased, consistent with release from the depolarized mitochondria. Within the mitochondria, the Succinate Dehydrogenase complex (SDH) was investigated as a target for bisphosphonium salts, but while its subunit 1 (SDH1) was present at low levels in the bloodstream form trypanosomes, the assembled complex was hardly detectable. RNAi knockdown of the SDH1 subunit produced no growth phenotype, either in bloodstream or in the procyclic (insect) forms and we conclude that in trypanosomes SDH is not the target for bisphosphonium salts. Instead, the compounds inhibited ATP production in intact mitochondria, as well as the purified F1 ATPase, to a level that was similar to 1 mM azide. Co-incubation with azide and bisphosphonium compounds did not inhibit ATPase activity more than either product alone. The results show that, in T. brucei, bisphosphonium compounds do not principally act on succinate dehydrogenase but on the mitochondrial FoF1 ATPase. PMID:27054061

  10. Protein functional links in Trypanosoma brucei, identified by gene fusion analysis

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    Trimpalis Philip

    2011-07-01

    Full Text Available Abstract Background Domain or gene fusion analysis is a bioinformatics method for detecting gene fusions in one organism by comparing its genome to that of other organisms. The occurrence of gene fusions suggests that the two original genes that participated in the fusion are functionally linked, i.e. their gene products interact either as part of a multi-subunit protein complex, or in a metabolic pathway. Gene fusion analysis has been used to identify protein functional links in prokaryotes as well as in eukaryotic model organisms, such as yeast and Drosophila. Results In this study we have extended this approach to include a number of recently sequenced protists, four of which are pathogenic, to identify fusion linked proteins in Trypanosoma brucei, the causative agent of African sleeping sickness. We have also examined the evolution of the gene fusion events identified, to determine whether they can be attributed to fusion or fission, by looking at the conservation of the fused genes and of the individual component genes across the major eukaryotic and prokaryotic lineages. We find relatively limited occurrence of gene fusions/fissions within the protist lineages examined. Our results point to two trypanosome-specific gene fissions, which have recently been experimentally confirmed, one fusion involving proteins involved in the same metabolic pathway, as well as two novel putative functional links between fusion-linked protein pairs. Conclusions This is the first study of protein functional links in T. brucei identified by gene fusion analysis. We have used strict thresholds and only discuss results which are highly likely to be genuine and which either have already been or can be experimentally verified. We discuss the possible impact of the identification of these novel putative protein-protein interactions, to the development of new trypanosome therapeutic drugs.

  11. Reduced Mitochondrial Membrane Potential Is a Late Adaptation of Trypanosoma brucei brucei to Isometamidium Preceded by Mutations in the γ Subunit of the F1Fo-ATPase.

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    Anthonius A Eze

    2016-08-01

    Full Text Available Isometamidium is the main prophylactic drug used to prevent the infection of livestock with trypanosomes that cause Animal African Trypanosomiasis. As well as the animal infective trypanosome species, livestock can also harbor the closely related human infective subspecies T. b. gambiense and T. b. rhodesiense. Resistance to isometamidium is a growing concern, as is cross-resistance to the diamidine drugs diminazene and pentamidine.Two isometamidium resistant Trypanosoma brucei clones were generated (ISMR1 and ISMR15, being 7270- and 16,000-fold resistant to isometamidium, respectively, which retained their ability to grow in vitro and establish an infection in mice. Considerable cross-resistance was shown to ethidium bromide and diminazene, with minor cross-resistance to pentamidine. The mitochondrial membrane potentials of both resistant cell lines were significantly reduced compared to the wild type. The net uptake rate of isometamidium was reduced 2-3-fold but isometamidium efflux was similar in wild-type and resistant lines. Fluorescence microscopy and PCR analysis revealed that ISMR1 and ISMR15 had completely lost their kinetoplast DNA (kDNA and both lines carried a mutation in the nuclearly encoded γ subunit gene of F1 ATPase, truncating the protein by 22 amino acids. The mutation compensated for the loss of the kinetoplast in bloodstream forms, allowing near-normal growth, and conferred considerable resistance to isometamidium and ethidium as well as significant resistance to diminazene and pentamidine, when expressed in wild type trypanosomes. Subsequent exposure to either isometamidium or ethidium led to rapid loss of kDNA and a further increase in isometamidium resistance.Sub-lethal exposure to isometamidium gives rise to viable but highly resistant trypanosomes that, depending on sub-species, are infective to humans and cross-resistant to at least some diamidine drugs. The crucial mutation is in the F1 ATPase γ subunit, which

  12. No gold standard estimation of the sensitivity and specificity of two molecular diagnostic protocols for Trypanosoma brucei spp. in Western Kenya.

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    Barend Mark de Clare Bronsvoort

    2010-01-01

    Full Text Available African animal trypanosomiasis is caused by a range of tsetse transmitted protozoan parasites includingTrypanosoma vivax, Trypanosoma congolense and Trypansoma brucei. In Western Kenya and other parts of East Africa two subspecies of T. brucei, T.b. brucei and the zoonoticT.b. rhodesiense, co-circulate in livestock. A range of polymerase chain reactions (PCR have been developed as important molecular diagnostic tools for epidemiological investigations of T. brucei s.l. in the animal reservoir and of its zoonotic potential. Quantification of the relative performance of different diagnostic PCRs is essential to ensure comparability of studies. This paper describes an evaluation of two diagnostic test systems for T. brucei using a T. brucei s.l. specific PCR [1] and a single nested PCR targeting the Internal Transcribed Spacer (ITS regions of trypanosome ribosomal DNA [2]. A Bayesian formulation of the Hui-Walter latent class model was employed to estimate their test performance in the absence of a gold standard test for detecting T.brucei s.l. infections in ear-vein blood samples from cattle, pig, sheep and goat populations in Western Kenya, stored on Whatman FTA cards. The results indicate that the system employing the T. brucei s.l. specific PCR (Se1=0.760 had a higher sensitivity than the ITS-PCR (Se2=0.640; both have high specificity (Sp1=0.998; Sp2=0.997. The true prevalences for livestock populations were estimated (pcattle=0.091, ppigs=0.066, pgoats=0.005, psheep=0.006, taking into account the uncertainties in the specificity and sensitivity of the two test systems. Implications of test performance include the required survey sample size; due to its higher sensitivity and specificity, the T. brucei s.l. specific PCR requires a consistently smaller sample size than the ITS-PCR for the detection of T. brucei s.l. However the ITS-PCR is able to simultaneously screen samples for other pathogenic trypanosomes and may thus be, overall, a better

  13. Diagnosis of human trypanosomiasis, due to Trypanosoma brucei gambiense in central Africa, by the polymerase chain reaction

    OpenAIRE

    Penchenier, Laurent; Simo, G.; Grébaut, Pascal; Nkinin, S.; Laveissière, Claude; Herder, Stéphane

    2000-01-01

    During a mass screening of sleeping sickness conducted in 1998 and 1999, and involving 27,932 persons in Cameroon and the Central African Republic, we tested the polymerase chain reaction (PCR) on whole blood for the diagnosis of human African trypanosomiasis due to #Trypanosoma brucei gambiense$. The 1858 samples obtained were from 4 groups : 155 infected patients, 1432 serological suspects detected by the card agglutination test for trypanosomiasis (CATT), 222 negative controls living in th...

  14. T. brucei infection reduces B lymphopoiesis in bone marrow and truncates compensatory splenic lymphopoiesis through transitional B-cell apoptosis.

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    Viki Bockstal

    2011-06-01

    Full Text Available African trypanosomes of the Trypanosoma brucei species are extracellular protozoan parasites that cause the deadly disease African trypanosomiasis in humans and contribute to the animal counterpart, Nagana. Trypanosome clearance from the bloodstream is mediated by antibodies specific for their Variant Surface Glycoprotein (VSG coat antigens. However, T. brucei infection induces polyclonal B cell activation, B cell clonal exhaustion, sustained depletion of mature splenic Marginal Zone B (MZB and Follicular B (FoB cells, and destruction of the B-cell memory compartment. To determine how trypanosome infection compromises the humoral immune defense system we used a C57BL/6 T. brucei AnTat 1.1 mouse model and multicolor flow cytometry to document B cell development and maturation during infection. Our results show a more than 95% reduction in B cell precursor numbers from the CLP, pre-pro-B, pro-B, pre-B and immature B cell stages in the bone marrow. In the spleen, T. brucei induces extramedullary B lymphopoiesis as evidenced by significant increases in HSC-LMPP, CLP, pre-pro-B, pro-B and pre-B cell populations. However, final B cell maturation is abrogated by infection-induced apoptosis of transitional B cells of both the T1 and T2 populations which is not uniquely dependent on TNF-, Fas-, or prostaglandin-dependent death pathways. Results obtained from ex vivo co-cultures of living bloodstream form trypanosomes and splenocytes demonstrate that trypanosome surface coat-dependent contact with T1/2 B cells triggers their deletion. We conclude that infection-induced and possibly parasite-contact dependent deletion of transitional B cells prevents replenishment of mature B cell compartments during infection thus contributing to a loss of the host's capacity to sustain antibody responses against recurring parasitemic waves.

  15. Apocytochrome b and other mitochondrial DNA sequences are differentially expressed during the life cycle of Trypanosoma brucei.

    OpenAIRE

    Feagin, J E; Jasmer, D P; Stuart, K

    1985-01-01

    Cytochromes and Krebs cycle enzymes are not detected in bloodstream forms of Trypanosoma brucei but are present in procyclic forms. We have analyzed transcription of mitochondrial sequences which contain the apocytochrome b gene and several other open reading frames (ORFs). Multiple transcripts map to individual DNA sequences located on both DNA strands. Larger low abundance transcripts map to multiple ORFs and may be precursor RNAs. Small abundant transcripts map to G + C rich sequences that...

  16. A target-based high throughput screen yields Trypanosoma brucei hexokinase small molecule inhibitors with antiparasitic activity.

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    Elizabeth R Sharlow

    2010-04-01

    Full Text Available The parasitic protozoan Trypanosoma brucei utilizes glycolysis exclusively for ATP production during infection of the mammalian host. The first step in this metabolic pathway is mediated by hexokinase (TbHK, an enzyme essential to the parasite that transfers the gamma-phospho of ATP to a hexose. Here we describe the identification and confirmation of novel small molecule inhibitors of bacterially expressed TbHK1, one of two TbHKs expressed by T. brucei, using a high throughput screening assay.Exploiting optimized high throughput screening assay procedures, we interrogated 220,233 unique compounds and identified 239 active compounds from which ten small molecules were further characterized. Computation chemical cluster analyses indicated that six compounds were structurally related while the remaining four compounds were classified as unrelated or singletons. All ten compounds were approximately 20-17,000-fold more potent than lonidamine, a previously identified TbHK1 inhibitor. Seven compounds inhibited T. brucei blood stage form parasite growth (0.03brucei parasites, Leishmania promastigotes, and mammalian cell lines. Analysis of two structurally related compounds, ebselen and SID 17387000, revealed that both were mixed inhibitors of TbHK1 with respect to ATP. Additionally, both compounds inhibited parasite lysate-derived HK activity. None of the compounds displayed structural similarity to known hexokinase inhibitors or human African trypanosomiasis therapeutics.The novel chemotypes identified here could represent leads for future therapeutic development against the African trypanosome.

  17. Identification of a bacterial-like HslVU protease in the mitochondria of Trypanosoma brucei and its role in mitochondrial DNA replication.

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    Ziyin Li

    2008-04-01

    Full Text Available ATP-dependent protease complexes are present in all living organisms, including the 26S proteasome in eukaryotes, Archaea, and Actinomycetales, and the HslVU protease in eubacteria. The structure of HslVU protease resembles that of the 26S proteasome, and the simultaneous presence of both proteases in one organism was deemed unlikely. However, HslVU homologs have been identified recently in some primordial eukaryotes, though their potential function remains elusive. We characterized the HslVU homolog from Trypanosoma brucei, a eukaryotic protozoan parasite and the causative agent of human sleeping sickness. TbHslVU has ATP-dependent peptidase activity and, like its bacterial counterpart, has essential lysine and N-terminal threonines in the catalytic subunit. By epitope tagging, TbHslVU localizes to mitochondria and is associated with the mitochondrial genome, kinetoplast DNA (kDNA. RNAi of TbHslVU dramatically affects the kDNA by causing over-replication of the minicircle DNA. This leads to defects in kDNA segregation and, subsequently, to continuous network growth to an enormous size. Multiple discrete foci of nicked/gapped minicircles are formed on the periphery of kDNA disc, suggesting a failure in repairing the gaps in the minicircles for kDNA segregation. TbHslVU is a eubacterial protease identified in the mitochondria of a eukaryote. It has a novel function in regulating mitochondrial DNA replication that has never been observed in other organisms.

  18. Channel-forming activities in the glycosomal fraction from the bloodstream form of Trypanosoma brucei.

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    Melisa Gualdron-López

    Full Text Available BACKGROUND: Glycosomes are a specialized form of peroxisomes (microbodies present in unicellular eukaryotes that belong to the Kinetoplastea order, such as Trypanosoma and Leishmania species, parasitic protists causing severe diseases of livestock and humans in subtropical and tropical countries. The organelles harbour most enzymes of the glycolytic pathway that is responsible for substrate-level ATP production in the cell. Glycolysis is essential for bloodstream-form Trypanosoma brucei and enzymes comprising this pathway have been validated as drug targets. Glycosomes are surrounded by a single membrane. How glycolytic metabolites are transported across the glycosomal membrane is unclear. METHODS/PRINCIPAL FINDINGS: We hypothesized that glycosomal membrane, similarly to membranes of yeast and mammalian peroxisomes, contains channel-forming proteins involved in the selective transfer of metabolites. To verify this prediction, we isolated a glycosomal fraction from bloodstream-form T. brucei and reconstituted solubilized membrane proteins into planar lipid bilayers. The electrophysiological characteristics of the channels were studied using multiple channel recording and single channel analysis. Three main channel-forming activities were detected with current amplitudes 70-80 pA, 20-25 pA, and 8-11 pA, respectively (holding potential +10 mV and 3.0 M KCl as an electrolyte. All channels were in fully open state in a range of voltages ±150 mV and showed no sub-conductance transitions. The channel with current amplitude 20-25 pA is anion-selective (P(K+/P(Cl-∼0.31, while the other two types of channels are slightly selective for cations (P(K+/P(Cl- ratios ∼1.15 and ∼1.27 for the high- and low-conductance channels, respectively. The anion-selective channel showed an intrinsic current rectification that may suggest a functional asymmetry of the channel's pore. CONCLUSIONS/SIGNIFICANCE: These results indicate that the membrane of glycosomes

  19. Steroid Alkaloids from Holarrhena africana with Strong Activity against Trypanosoma brucei rhodesiense.

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    Nnadi, Charles Okeke; Nwodo, Ngozi Justina; Kaiser, Marcel; Brun, Reto; Schmidt, Thomas J

    2017-07-06

    In our continued search for natural compounds with activity against Trypanosoma brucei, causative agent of human African trypanosomiasis (HAT, "sleeping sickness"), we have investigated extracts from the leaves and bark of the West African Holarrhenaafricana (syn. Holarrhena floribunda; Apocynaceae). The extracts and their alkaloid-enriched fractions displayed promising in vitro activity against bloodstream forms of T. brucei rhodesiense (Tbr; East African HAT). Bioactivity-guided chromatographic fractionation of the alkaloid-rich fractions resulted in the isolation of 17 steroid alkaloids, one nitrogen-free steroid and one alkaloid-like non-steroid. Impressive activities (IC50 in µM) against Tbr were recorded for 3β-holaphyllamine (0.40 ± 0.28), 3α-holaphyllamine (0.37 ± 0.16), 3β-dihydroholaphyllamine (0.67 ± 0.03), N-methylholaphyllamine (0.08 ± 0.01), conessimine (0.17 ± 0.08), conessine (0.42 ± 0.09), isoconessimine (0.17 ± 0.11) and holarrhesine (0.12 ± 0.08) with selectivity indices ranging from 13 to 302. Based on comparison of the structures of this congeneric series of steroid alkaloids and their activities, structure-activity relationships (SARs) could be established. It was found that a basic amino group at position C-3 of the pregnane or pregn-5-ene steroid nucleus is required for a significant anti-trypanosomal activity. The mono-methylated amino group at C-3 represents an optimum for activity. ∆(5,6) unsaturation slightly increased the activity while hydrolysis of C-12β ester derivatives led to a loss of activity. An additional amino group at C-20 engaged in a pyrrolidine ring closed towards C-18 significantly increased the selectivity index of the compounds. Our findings provide useful empirical data for further development of steroid alkaloids as a novel class of anti-trypanosomal compounds which represent a promising starting point towards new drugs to combat human African trypanosomiasis.

  20. Steroid Alkaloids from Holarrhena africana with Strong Activity against Trypanosoma brucei rhodesiense

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    Charles Okeke Nnadi

    2017-07-01

    Full Text Available In our continued search for natural compounds with activity against Trypanosoma brucei, causative agent of human African trypanosomiasis (HAT, “sleeping sickness”, we have investigated extracts from the leaves and bark of the West African Holarrhena africana (syn. Holarrhena floribunda; Apocynaceae. The extracts and their alkaloid-enriched fractions displayed promising in vitro activity against bloodstream forms of T. brucei rhodesiense (Tbr; East African HAT. Bioactivity-guided chromatographic fractionation of the alkaloid-rich fractions resulted in the isolation of 17 steroid alkaloids, one nitrogen-free steroid and one alkaloid-like non-steroid. Impressive activities (IC50 in µM against Tbr were recorded for 3β-holaphyllamine (0.40 ± 0.28, 3α-holaphyllamine (0.37 ± 0.16, 3β-dihydroholaphyllamine (0.67 ± 0.03, N-methylholaphyllamine (0.08 ± 0.01, conessimine (0.17 ± 0.08, conessine (0.42 ± 0.09, isoconessimine (0.17 ± 0.11 and holarrhesine (0.12 ± 0.08 with selectivity indices ranging from 13 to 302. Based on comparison of the structures of this congeneric series of steroid alkaloids and their activities, structure-activity relationships (SARs could be established. It was found that a basic amino group at position C-3 of the pregnane or pregn-5-ene steroid nucleus is required for a significant anti-trypanosomal activity. The mono-methylated amino group at C-3 represents an optimum for activity. ∆5,6 unsaturation slightly increased the activity while hydrolysis of C-12β ester derivatives led to a loss of activity. An additional amino group at C-20 engaged in a pyrrolidine ring closed towards C-18 significantly increased the selectivity index of the compounds. Our findings provide useful empirical data for further development of steroid alkaloids as a novel class of anti-trypanosomal compounds which represent a promising starting point towards new drugs to combat human African trypanosomiasis.

  1. Polysomes of Trypanosoma brucei: Association with Initiation Factors and RNA-Binding Proteins.

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    Cornelia Klein

    Full Text Available We report here the results of experiments designed to identify RNA-binding proteins that might be associated with Trypanosoma brucei polysomes. After some preliminary mass spectrometry of polysomal fractions, we investigated the distributions of selected tagged proteins using sucrose gradients and immunofluorescence. As expected, the polysomal fractions contained nearly all annotated ribosomal proteins, the translation-associated protein folding complex, and many translation factors, but also many other abundant proteins. Results suggested that cap-binding proteins EIF4E3 and EIF4E4 were associated with both free and membrane-bound polysomes. The EIF4E binding partners EIF4G4 and EIF4G3 were present but the other EIF4E and EIF4G paralogues were not detected. The dominant EIF4E in the polysomal fraction is EIF4E4 and very few polysomal mRNAs are associated with EIF4G. Thirteen potential mRNA-binding proteins were detected in the polysomes, including the known polysome-associated protein RBP42. The locations of two of the other proteins were tested after epitope tagging: RBP29 was in the nucleus and ZC3H29 was in the cytoplasm. Quantitative analyses showed that specific association of an RNA-binding protein with the polysome fraction in sucrose gradients will not be detected if the protein is in more than 25-fold molar excess over its target binding sites.

  2. Dynamic modelling under uncertainty: the case of Trypanosoma brucei energy metabolism.

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    Fiona Achcar

    2012-01-01

    Full Text Available Kinetic models of metabolism require detailed knowledge of kinetic parameters. However, due to measurement errors or lack of data this knowledge is often uncertain. The model of glycolysis in the parasitic protozoan Trypanosoma brucei is a particularly well analysed example of a quantitative metabolic model, but so far it has been studied with a fixed set of parameters only. Here we evaluate the effect of parameter uncertainty. In order to define probability distributions for each parameter, information about the experimental sources and confidence intervals for all parameters were collected. We created a wiki-based website dedicated to the detailed documentation of this information: the SilicoTryp wiki (http://silicotryp.ibls.gla.ac.uk/wiki/Glycolysis. Using information collected in the wiki, we then assigned probability distributions to all parameters of the model. This allowed us to sample sets of alternative models, accurately representing our degree of uncertainty. Some properties of the model, such as the repartition of the glycolytic flux between the glycerol and pyruvate producing branches, are robust to these uncertainties. However, our analysis also allowed us to identify fragilities of the model leading to the accumulation of 3-phosphoglycerate and/or pyruvate. The analysis of the control coefficients revealed the importance of taking into account the uncertainties about the parameters, as the ranking of the reactions can be greatly affected. This work will now form the basis for a comprehensive Bayesian analysis and extension of the model considering alternative topologies.

  3. Functional analysis of TOEFAZ1 uncovers protein domains essential for cytokinesis in Trypanosoma brucei.

    Science.gov (United States)

    Sinclair-Davis, Amy N; McAllaster, Michael R; de Graffenried, Christopher L

    2017-10-09

    The parasite Trypanosoma brucei is highly polarized, including a flagellum that is attached along the cell surface by the flagellum attachment zone (FAZ). During cell division, the new FAZ positions the cleavage furrow, which ingresses from the anterior tip of the cell towards the posterior. We recently identified Tip Of the Extending FAZ protein 1 (TOEFAZ1) as an essential protein in trypanosome cytokinesis. We analyzed the localization and function of TOEFAZ1 domains using overexpression and RNAi complementation. TOEFAZ1 comprises three domains with separable functions: an N-terminal α-helical domain that may be involved in FAZ recruitment, a central intrinsically disordered domain that retains the morphogenic kinase TbPLK at the new FAZ tip, and a C-terminal zinc finger domain necessary for TOEFAZ1 oligomerization. Both the N-terminal and C-terminal domains are essential for TOEFAZ1 function, but TbPLK retention at the FAZ is not necessary for cytokinesis. The feasibility of alternate cytokinetic pathways that do not employ TOEFAZ1 are also assessed. Our results show that TOEFAZ1 is a multimeric scaffold for recruiting proteins that control the timing and location of cleavage furrow ingression. © 2017. Published by The Company of Biologists Ltd.

  4. Isothermal microcalorimetry, a new tool to monitor drug action against Trypanosoma brucei and Plasmodium falciparum.

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    Tanja Wenzler

    Full Text Available Isothermal microcalorimetry is an established tool to measure heat flow of physical, chemical or biological processes. The metabolism of viable cells produces heat, and if sufficient cells are present, their heat production can be assessed by this method. In this study, we investigated the heat flow of two medically important protozoans, Trypanosoma brucei rhodesiense and Plasmodium falciparum. Heat flow signals obtained for these pathogens allowed us to monitor parasite growth on a real-time basis as the signals correlated with the number of viable cells. To showcase the potential of microcalorimetry for measuring drug action on pathogenic organisms, we tested the method with three antitrypanosomal drugs, melarsoprol, suramin and pentamidine and three antiplasmodial drugs, chloroquine, artemether and dihydroartemisinin, each at two concentrations on the respective parasite. With the real time measurement, inhibition was observed immediately by a reduced heat flow compared to that in untreated control samples. The onset of drug action, the degree of inhibition and the time to death of the parasite culture could conveniently be monitored over several days. Microcalorimetry is a valuable element to be added to the toolbox for drug discovery for protozoal diseases such as human African trypanosomiasis and malaria. The method could probably be adapted to other protozoan parasites, especially those growing extracellularly.

  5. Flux Analysis of the Trypanosoma brucei Glycolysis Based on a Multiobjective-Criteria Bioinformatic Approach

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    Amine Ghozlane

    2012-01-01

    Full Text Available Trypanosoma brucei is a protozoan parasite of major of interest in discovering new genes for drug targets. This parasite alternates its life cycle between the mammal host(s (bloodstream form and the insect vector (procyclic form, with two divergent glucose metabolism amenable to in vitro culture. While the metabolic network of the bloodstream forms has been well characterized, the flux distribution between the different branches of the glucose metabolic network in the procyclic form has not been addressed so far. We present a computational analysis (called Metaboflux that exploits the metabolic topology of the procyclic form, and allows the incorporation of multipurpose experimental data to increase the biological relevance of the model. The alternatives resulting from the structural complexity of networks are formulated as an optimization problem solved by a metaheuristic where experimental data are modeled in a multiobjective function. Our results show that the current metabolic model is in agreement with experimental data and confirms the observed high metabolic flexibility of glucose metabolism. In addition, Metaboflux offers a rational explanation for the high flexibility in the ratio between final products from glucose metabolism, thsat is, flux redistribution through the malic enzyme steps.

  6. Duplicated paralogous genes subject to positive selection in the genome of Trypanosoma brucei.

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    Richard D Emes

    2008-05-01

    Full Text Available Whole genome studies have highlighted duplicated genes as important substrates for adaptive evolution. We have investigated adaptive evolution in this class of genes in the human parasite Trypanosoma brucei, as indicated by the ratio of non-synonymous (amino-acid changing to synonymous (amino acid retaining nucleotide substitution rates.We have identified duplicated genes that are most rapidly evolving in this important human parasite. This is the first attempt to investigate adaptive evolution in this species at the codon level. We identify 109 genes within 23 clusters of paralogous gene expansions to be subject to positive selection.Genes identified include surface antigens in both the mammalian and insect host life cycle stage suggesting that competitive interaction is not solely with the adaptive immune system of the mammalian host. Also surface transporters related to drug resistance and genes related to developmental progression are detected. We discuss how adaptive evolution of these genes may highlight lineage specific processes essential for parasite survival. We also discuss the implications of adaptive evolution of these targets for parasite biology and control.

  7. Cultivation in a semi-defined medium of animal infective forms of Trypanosoma brucei, T. equiperdum, T. evansi, T. rhodesiense and T. gambiense.

    Science.gov (United States)

    Baltz, T; Baltz, D; Giroud, C; Crockett, J

    1985-05-01

    A semi-defined medium for the cultivation of bloodstream forms of the African trypanosome brucei subgroup was developed. Out of 14 different strains tested, 10 could be cultured including Trypanosoma brucei, T. equiperdum, T. evansi, T. rhodesiense and T. gambiense. The presence of a reducing agent (2-mercaptoethanol or thioglycerol) was found to be essential for growth. The standard medium consisted of Hepes buffered minimum essential medium with Earle's salts supplemented with 0.2 mM 2-mercaptoethanol, 2 mM pyruvate and 10% inactivated serum either from rabbit (T. brucei, T. equiperdum, T. evansi and T. rhodesiense) or human (T. gambiense). Although a general medium could be defined for the long-term maintenance of trypanosome cultures, the initiation to culture nevertheless required particular conditions for the different strains. The cultured trypanosomes had all the characteristics of the in vivo bloodstream forms including: morphology, infectivity, antigenic variation and glucose metabolism.

  8. Further evidence from SSCP and ITS DNA sequencing support Trypanosoma evansi and Trypanosoma equiperdum as subspecies or even strains of Trypanosoma brucei.

    Science.gov (United States)

    Wen, Yan-Zi; Lun, Zhao-Rong; Zhu, Xing-Quan; Hide, Geoff; Lai, De-Hua

    2016-07-01

    The subgenus Trypanozoon includes three species Trypanosoma brucei, Trypanosoma evansi and Trypanosoma equiperdum, which are morphologically identical and indistinguishable even using some molecular methods. In this study, PCR-based single strand conformation polymorphism (PCR-SSCP) was used to analyze the ribosomal DNA of the Trypanozoon species. Data indicate different patterns of ITS2 fragments between T. brucei, T. evansi and T. equiperdum by SSCP. Furthermore, analysis of total ITS sequences within these three members of the subgenus Trypanozoon showed a high degree of homology using phylogenetic analysis but were polyphyletic in haplotype networks. These data provide novel nuclear evidence to further support the notion that T. evansi and T. equiperdum should be subspecies or even strains of T. brucei. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Diagnostic accuracy of loopamp Trypanosoma brucei detection kit for diagnosis of human African trypanosomiasis in clinical samples.

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    Patrick Mitashi

    Full Text Available BACKGROUND: Molecular methods have great potential for sensitive parasite detection in the diagnosis of human African trypanosomiasis (HAT, but the requirements in terms of laboratory infrastructure limit their use to reference centres. A recently developed assay detects the Trypanozoon repetitive insertion mobile element (RIME DNA under isothermal amplification conditions and has been transformed into a ready-to-use kit format, the Loopamp Trypanosoma brucei. In this study, we have evaluated the diagnostic performance of the Loopamp Trypanosoma brucei assay (hereafter called LAMP in confirmed T.b. gambiense HAT patients, HAT suspects and healthy endemic controls from the Democratic Republic of the Congo (DRC. METHODOLOGY/PRINCIPAL FINDINGS: 142 T.b. gambiense HAT patients, 111 healthy endemic controls and 97 HAT suspects with unconfirmed status were included in this retrospective evaluation. Reference standard tests were parasite detection in blood, lymph or cerebrospinal fluid. Archived DNA from blood of all study participants was analysed in duplicate with LAMP. Sensitivity of LAMP in parasitologically confirmed cases was 87.3% (95% CI 80.9-91.8% in the first run and 93.0% (95% CI 87.5-96.1% in the second run. Specificity in healthy controls was 92.8% (95% CI 86.4-96.3% in the first run and 96.4% (95% CI 91.1-98.6% in the second run. Reproducibility was excellent with a kappa value of 0.81. CONCLUSIONS/SIGNIFICANCE: In this laboratory-based study, the Loopamp Trypanosoma brucei Detection Kit showed good diagnostic accuracy and excellent reproducibility. Further studies are needed to assess the feasibility of its routine use for diagnosis of HAT under field conditions.

  10. New functions for parts of the Krebs cycle in procyclic Trypanosoma brucei, a cycle not operating as a cycle.

    Science.gov (United States)

    van Weelden, Susanne W H; van Hellemond, Jaap J; Opperdoes, Fred R; Tielens, Aloysius G M

    2005-04-01

    We investigated whether substrate availability influences the type of energy metabolism in procyclic Trypanosoma brucei. We show that absence of glycolytic substrates (glucose and glycerol) does not induce a shift from a fermentative metabolism to complete oxidation of substrates. We also show that glucose (and even glycolysis) is not essential for normal functioning and proliferation of pleomorphic procyclic T. brucei cells. Furthermore, absence of glucose did not result in increased degradation of amino acids. Variations in availability of glucose and glycerol did result, however, in adaptations in metabolism in such a way that the glycosome was always in redox balance. We argue that it is likely that, in procyclic cells, phosphoglycerate kinase is located not only in the cytosol, but also inside glycosomes, as otherwise an ATP deficit would occur in this organelle. We demonstrate that procyclic T. brucei uses parts of the Krebs cycle for purposes other than complete degradation of mitochondrial substrates. We suggest that citrate synthase plus pyruvate dehydrogenase and malate dehydrogenase are used to transport acetyl-CoA units from the mitochondrion to the cytosol for the biosynthesis of fatty acids, a process we show to occur in proliferating procyclic cells. The part of the Krebs cycle consisting of alpha-ketoglutarate dehydrogenase and succinyl-CoA synthetase was used for the degradation of proline and glutamate to succinate. We also demonstrate that the subsequent enzymes of the Krebs cycle, succinate dehydrogenase and fumarase, are most likely used for conversion of succinate into malate, which can then be used in gluconeogenesis.

  11. 3D Architecture of the Trypanosoma brucei Flagella Connector, a Mobile Transmembrane Junction.

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    Johanna L Höög

    2016-01-01

    Full Text Available Cellular junctions are crucial for the formation of multicellular organisms, where they anchor cells to each other and/or supportive tissue and enable cell-to-cell communication. Some unicellular organisms, such as the parasitic protist Trypanosoma brucei, also have complex cellular junctions. The flagella connector (FC is a three-layered transmembrane junction that moves with the growing tip of a new flagellum and attaches it to the side of the old flagellum. The FC moves via an unknown molecular mechanism, independent of new flagellum growth. Here we describe the detailed 3D architecture of the FC suggesting explanations for how it functions and its mechanism of motility.We have used a combination of electron tomography and cryo-electron tomography to reveal the 3D architecture of the FC. Cryo-electron tomography revealed layers of repetitive filamentous electron densities between the two flagella in the interstitial zone. Though the FC does not change in length and width during the growth of the new flagellum, the interstitial zone thickness decreases as the FC matures. This investigation also shows interactions between the FC layers and the axonemes of the new and old flagellum, sufficiently strong to displace the axoneme in the old flagellum. We describe a novel filament, the flagella connector fibre, found between the FC and the axoneme in the old flagellum.The FC is similar to other cellular junctions in that filamentous proteins bridge the extracellular space and are anchored to underlying cytoskeletal structures; however, it is built between different portions of the same cell and is unique because of its intrinsic motility. The detailed description of its structure will be an important tool to use in attributing structure / function relationships as its molecular components are discovered in the future. The FC is involved in the inheritance of cell shape, which is important for the life cycle of this human parasite.

  12. Glucose uptake occurs by facilitated diffusion in procyclic forms of Trypanosoma brucei.

    Science.gov (United States)

    Wille, U; Seyfang, A; Duszenko, M

    1996-02-15

    The glucose transporter of Trypanosoma brucei procyclic forms was characterized and compared with its bloodstream form counterpart. Measuring the glucose consumption enzymatically, we determined a saturable uptake process of relatively high affinity (Km = 80 microM, Vmax = 4 nmol min-1 10(-8) cells), which showed substrate inhibition at glucose concentrations above 1.5 mM (Ki = 21 mM). Control experiments measuring deoxy-D-[3H]Glc uptake under zero-trans conditions indicated that substrate inhibition occurred on the level of glycolysis. Temperature-dependent kinetics revealed a temperature quotient of Q10 = 2.33 and an activation energy of Ea = 64 kJ mol-1. As shown by trans-stimulation experiments, glucose uptake was stereospecific for the D isomer, whereas L-glucose was not recognized. Inhibitor studies using either the uncoupler carbonylcyanide-4-(trifluoromethoxy)phenylhydrazone (5 microM), the H+/ATPase inhibitor N,N'-dicyclohexylcarbodiimide (20 microM), the ionophor monensin (1 microM), or the Na+/K+-ATPase inhibitor ouabain (1 mM) showed insignificant effects on transport efficiency. The procyclic glucose transporter was subsequently enriched in a plasma-membrane fraction and functionally reconstituted into proteoliposomes. Using Na+-free conditions in the absence of a proton gradient, the specific activity of D-[14C]glucose transport was determined as 2.9 nmol min-1 (mg protein)-1 at 0.2 mM glucose. From these cumulative results, we conclude that glucose uptake by the procyclic insect form of the parasite occurs by facilitated diffusion, similar to the hexose-transport system expressed in bloodstream forms. However, the markedly higher substrate affinity indicates a differential expression of different transporter isoforms throughout the lifecycle.

  13. 3D Architecture of the Trypanosoma brucei Flagella Connector, a Mobile Transmembrane Junction

    Science.gov (United States)

    Höög, Johanna L.; Lacomble, Sylvain; Bouchet-Marquis, Cedric; Briggs, Laura; Park, Kristin; Hoenger, Andreas; Gull, Keith

    2016-01-01

    Background Cellular junctions are crucial for the formation of multicellular organisms, where they anchor cells to each other and/or supportive tissue and enable cell-to-cell communication. Some unicellular organisms, such as the parasitic protist Trypanosoma brucei, also have complex cellular junctions. The flagella connector (FC) is a three-layered transmembrane junction that moves with the growing tip of a new flagellum and attaches it to the side of the old flagellum. The FC moves via an unknown molecular mechanism, independent of new flagellum growth. Here we describe the detailed 3D architecture of the FC suggesting explanations for how it functions and its mechanism of motility. Methodology/Principal Findings We have used a combination of electron tomography and cryo-electron tomography to reveal the 3D architecture of the FC. Cryo-electron tomography revealed layers of repetitive filamentous electron densities between the two flagella in the interstitial zone. Though the FC does not change in length and width during the growth of the new flagellum, the interstitial zone thickness decreases as the FC matures. This investigation also shows interactions between the FC layers and the axonemes of the new and old flagellum, sufficiently strong to displace the axoneme in the old flagellum. We describe a novel filament, the flagella connector fibre, found between the FC and the axoneme in the old flagellum. Conclusions/Significance The FC is similar to other cellular junctions in that filamentous proteins bridge the extracellular space and are anchored to underlying cytoskeletal structures; however, it is built between different portions of the same cell and is unique because of its intrinsic motility. The detailed description of its structure will be an important tool to use in attributing structure / function relationships as its molecular components are discovered in the future. The FC is involved in the inheritance of cell shape, which is important for the life

  14. Virulence and pathogenicity of three Trypanosoma brucei rhodesiense stabilates in a Swiss white mouse model

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    Christopher Kariuki

    2015-05-01

    Full Text Available Background: A key objective in basic research on human African trypanosomiasis (HAT is developing a cheap and reliable experimental model of the disease for use in pathogenesis and drug studies.Objective: With a view to improving current models, a study was undertaken to characterise the virulence and pathogenicity of three Trypanosoma brucei rhodesiense stabilates, labelled as International Livestock Research Institute (ILRI-2918, ILRI-3953, and Institute of Primate Research (IPR-001, infected into Swiss white mice.Methods: Swiss white mice were infected intraperitoneally with trypanosomes and observedfor parasitaemia using wet blood smears obtained by tail snipping. Induction of late-stagedisease was undertaken using diminazene aceturate (40 mg/kg, Berenil with curativetreatment done using melarsoprol (3.6 mg/kg, Arsobal.Results: The prepatent period for the stabilates ranged from three to four days with mean peak parasitaemia ranging from Log10 6.40 to 8.36. First peak parasitaemia for all stabilates varied between six and seven days post infection (DPI followed by secondary latency in ILRI-2918 (15–17 DPI and IPR-001 (17–19 DPI. Survival times ranged from six DPI (ILRI-3953 to 86 DPI (IPR-001. Hindleg paresis was observed in both ILRI-3953 (at peak parasitaemia and ILRI-2918 (after relapse parasitaemia. Mice infected with IPR-001 survived until 54 DPI when curative treatment was undertaken.Conclusions: This study demonstrated that the stabilates ILRI-2918 and ILRI-3953 were unsuitable for modelling late-stage HAT in mice. The stabilate IPR-001 demonstrated the potential to induce chronic trypanosomiasis in Swiss white mice for use in development of a late-stage model of HAT.

  15. Interaction between Gallotannin and a Recombinant Form of Arginine Kinase of Trypanosoma brucei: Thermodynamic and Spectrofluorimetric Evaluation

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    O. S. Adeyemi

    2014-01-01

    Full Text Available Current chemotherapies against trypanosomiasis are beset with diverse challenges, a situation which underscores the numerous research efforts aimed at finding newer and effective treatments. Arginine kinase of trypanosome has been validated as target for drug development against trypanosomiasis. The present study investigated the interaction between a recombinant form of the arginine kinase (rTbAK of trypanosome and gallotannin. The interaction between gallotannin and recombinant arginine kinase of Trypanosoma brucei caused significant decrease of enzyme activity. Kinetic analysis revealed the interaction to be of noncompetitive inhibition. Further thermodynamic analysis showed that the interaction between gallotannin and the recombinant arginine kinase was nonspontaneous and involved hydrophobic forces. The Ksv values and the FRET analysis suggest that static quenching of fluorescence intensity by gallotannin was static. Data revealed inhibitory interactions between gallotannin and rTbAK of trypanosome. Although the mechanism of inhibition is not clear yet, molecular docking studies are ongoing to clearly define the inhibitory interactions between the gallotannin and rTbAK. The knowledge of such binding properties would enrich development of selective inhibitors for the arginine kinase of Trypanosoma brucei.

  16. In Silico Identification and in Vitro Activity of Novel Natural Inhibitors of Trypanosoma brucei Glyceraldehyde-3-phosphate-dehydrogenase

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    Fabian C. Herrmann

    2015-09-01

    Full Text Available As part of our ongoing efforts to identify natural products with activity against pathogens causing neglected tropical diseases, we are currently performing an extensive screening of natural product (NP databases against a multitude of protozoan parasite proteins. Within this project, we screened a database of NPs from a commercial supplier, AnalytiCon Discovery (Potsdam, Germany, against Trypanosoma brucei glyceraldehyde-3-phosphate dehydrogenase (TbGAPDH, a glycolytic enzyme whose inhibition deprives the parasite of energy supply. NPs acting as potential inhibitors of the mentioned enzyme were identified using a pharmacophore-based virtual screening and subsequent docking of the identified hits into the active site of interest. In a set of 700 structures chosen for the screening, 13 (1.9% were predicted to possess significant affinity towards the enzyme and were therefore tested in an in vitro enzyme assay using recombinant TbGAPDH. Nine of these in silico hits (69% showed significant inhibitory activity at 50 µM, of which two geranylated benzophenone derivatives proved to be particularly active with IC50 values below 10 µM. These compounds also showed moderate in vitro activity against T. brucei rhodesiense and may thus represent interesting starting points for further optimization.

  17. In vitro investigation of Brazilian Cerrado plant extract activity against Plasmodium falciparum, Trypanosoma cruzi and T. brucei gambiense.

    Science.gov (United States)

    Charneau, Sébastien; de Mesquita, Mariana Laundry; Bastos, Izabela Marques Dourado; Santana, Jaime Martins; de Paula, José Elias; Grellier, Philippe; Espindola, Laila Salmen

    2016-06-01

    The threatened Brazilian Cerrado biome is an important biodiversity hotspot but still few explored that constitutes a potential reservoir of molecules to treat infectious diseases. We selected eight Cerrado plant species for screening against the erythrocytic stages of Plasmodium falciparum, human intracellular stages of Trypanosoma cruzi and bloodstream forms of T. brucei gambiense, and for their cytotoxicity upon the rat L6-myoblast cell line. Bioassays were performed with 37 hexane, ethyl acetate and ethanol extracts prepared from different plant organs. Activities against parasites were observed for 24 extracts: 9 with anti-P. falciparum, 4 with anti-T. cruzi and 11 with anti-T. brucei gambiense activities. High anti-protozoal activity (IC50 values < 10 μg/mL) without obvious cytotoxicity to L6 cells was observed for eight extracts from plants: Connarus suberosus, Blepharocalyx salicifolius, Psidium laruotteanum and Myrsine guianensis. Overall, studies of plant extracts will contribute to increase the biodiversity knowledge essential for Cerrado conservation and sustainable development.

  18. In Silico Prediction and Experimental Evaluation of Furanoheliangolide Sesquiterpene Lactones as Potent Agents against Trypanosoma brucei rhodesiense

    Science.gov (United States)

    Da Costa, Fernando B.; Lopes, Norberto P.; Kaiser, Marcel; Brun, Reto

    2014-01-01

    As a continuation of our earlier study on the in vitro antiprotozoal activity of 40 natural sesquiterpene lactones (STLs), we extended the set of tested compounds from our laboratories to 59. On the basis of this extended data set, further enriched by literature data for 10 compounds tested under the same conditions, our quantitative structure-activity relationship (QSAR) analyses for activity against T. brucei rhodesiense (etiologic agent of human African trypanosomiasis, or sleeping sickness) were continued, and the QSAR model thus obtained with 69 structures was used to predict the activity of a virtual library of 1,750 STL structures. As a major result from these calculations, furanoheliangolide-type compounds, a subclass of STLs hitherto untested against T. brucei rhodesiense, were predicted to have an exceptionally high level of in vitro activity. Four representative compounds of this type, goyazensolide, 4,5-dihydro-2′,3′-epoxy-15-deoxygoyazensolide, budlein A, and 4,15-isoatriplicolide tiglate, were therefore tested. They displayed 50% inhibitory concentrations (IC50s) of 0.07, 0.20, 0.07, and 0.015 μM, respectively, so that the in silico prediction was experimentally confirmed. 4,15-Isoatriplicolide tiglate is the most potent STL against T. b. rhodesiense found. Furanoheliangolide STLs were thus identified as interesting leads against this parasite which deserve more detailed investigations. PMID:24165182

  19. Analysis of cosmid clones of nuclear DNA from Trypanosome brucei shows that the genes for variant surface glycoproteins are clustered in the genome.

    NARCIS (Netherlands)

    D. Valerio (Dinko); T. de Lange; P. Borst (Piet); F.G. Grosveld (Frank); L.H.T. van der Ploeg

    1982-01-01

    textabstractTrypanosoma brucei contains more than a hundred genes coding for the different variant surface glycoproteins (VSGs). Activation of some of these genes involves the duplication of the gene (the basic copy or BC) and transposition of the duplicate to an expression site (yielding the

  20. The mitochondrial FAD-dependent glycerol-3-phosphate dehydrogenase of Trypanosomatidae and the glycocomal redox balance of insect stages of Trypanosoma brucei and Leishmania spp.

    NARCIS (Netherlands)

    Guerra, D.G.; Decottignies, A.; Bakker, B.M.; Michels, P.A.M.

    2006-01-01

    The genes for the mitochondrial FAD-dependent glycerol-3-phosphate dehydrogenase were identified in Trypanosoma brucei and Leishmania major genomes. We have expressed the L. major gene in Saccharomyces cerevisiae and confirmed the subcellular localization and activity of the produced enzyme. Using

  1. Trypanosoma brucei TBRGG1, a mitochondrial oligo(U)-binding protein that co-localizes with an in vitro RNA editing activity

    NARCIS (Netherlands)

    Vanhamme, L.; Perez-Morga, D.; Marchal, C.; Speijer, D.; Lambert, L.; Geuskens, M.; Alexandre, S.; Ismaïli, N.; Göringer, U.; Benne, R.; Pays, E.

    1998-01-01

    We report the characterization of a Trypanosoma brucei 75-kDa protein of the RGG (Arg-Gly-Gly) type, termed TBRGG1. Dicistronic and monocistronic transcripts of the TBRGG1 gene were produced by both alternative splicing and polyadenylation. TBRGG1 was found in two or three forms that differ in their

  2. Discovery of novel Trypanosoma brucei phosphodiesterase B1 inhibitors by virtual screening against the unliganded TbrPDEB1 crystal structure

    NARCIS (Netherlands)

    Jansen, C.; Wang, H.; Kooistra, A.J.; de Graaf, C.; Orrling, K.M.; Tenor, H.; Seebeck, T.; de Esch, I.J.P.; Ke, H.; Leurs, R.

    2013-01-01

    Trypanosoma brucei cyclic nucleotide phosphodiesterase B1 (TbrPDEB1) and TbrPDEB2 have recently been validated as new therapeutic targets for human African trypanosomiasis by both genetic and pharmacological means. In this study we report the crystal structure of the catalytic domain of the

  3. Trypanosoma brucei Plimmer & Bradford, 1899 is a synonym of T. evansi (Steel, 1885) according to current knowledge and by application of nomenclature rules.

    Science.gov (United States)

    Molinari, Jesús; Moreno, S Andrea

    2018-02-06

    Proper application of the principles of biological nomenclature is fundamental for scientific and technical communication about organisms. As other scientific disciplines, taxonomy inherently is open to change, thus species names cannot be final and immutable. Nevertheless, altering the names of organisms of high economical, medical, or veterinary importance can become a complex challenge between the scientific need to have correct classifications, and the practical ideal of having fixed classifications. Trypanosoma evansi (Steel, 1885), T. brucei Plimmer & Bradford, 1899 and T. equiperdum Doflein, 1901 are important parasites of mammals. According to current knowledge, the three names are synonyms of a single trypanosome species, the valid name of which should be T. evansi by the mandatory application of the Principle of Priority of zoological nomenclature. Subspecies known as T. brucei brucei Plimmer & Bradford, 1899, T. b. gambiense Dutton, 1902 and T. b. rhodesiense Stephens & Fantham, 1910 should be referred to respectively as T. evansi evansi (Steel, 1885), T. e. gambiense and T. e. rhodesiense. The polyphyletic groupings so far known as T. evansi and T. equiperdum should be referred respectively to as surra- and dourine-causing strains of T. e. evansi. Likewise, trypanosomes so far known as T. b. brucei should be referred to as nagana-causing strains of T. e. evansi. Though it modifies the scientific names of flagship human and animal parasites, the amended nomenclature proposed herein should be adopted because it reflects phylogenetic and biological advancements, fixes errors, and is simpler than the existing classificatory system.

  4. Immunospecific immunoglobulins and IL-10 as markers for Trypanosoma brucei rhodesiense late stage disease in experimentally infected vervet monkeys

    DEFF Research Database (Denmark)

    Ngotho, Maina; Kagira, J.M.; Jensen, Henrik Michael Elvang

    2009-01-01

    OBJECTIVE: To determine the usefulness of IL-10 and immunoglobulin M (IgM) as biomarkers for staging HAT in vervet monkeys, a useful pathogenesis model for humans. METHODS: Vervet monkeys were infected with Trypanosoma brucei rhodesiense and subsequently given sub-curative and curative treatment 28...

  5. The use of yellow fluorescent hybrids to indicate mating in Trypanosoma brucei

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    Ferris Vanessa

    2008-02-01

    Full Text Available Abstract Background Trypanosoma brucei undergoes genetic exchange in its insect vector, the tsetse fly, by an unknown mechanism. The difficulties of working with this experimental system of genetic exchange have hampered investigation, particularly because the trypanosome life cycle stages involved cannot be cultured in vitro and therefore must be examined in the insect. Searching for small numbers of hybrid trypanosomes directly in the fly has become possible through the incorporation of fluorescent reporter genes, and we have previously carried out a successful cross using a reporter-repressor strategy. However, we could not be certain that all fluorescent trypanosomes observed in that cross were hybrids, due to mutations of the repressor leading to spontaneous fluorescence, and we have therefore developed an alternative strategy. Results To visualize the production of hybrids in the fly, parental trypanosome clones were transfected with a gene encoding Green Fluorescent Protein (GFP or Red Fluorescent Protein (RFP. Co-infection of flies with red and green fluorescent parental trypanosomes produced yellow fluorescent hybrids, which were easily visualized in the fly salivary glands. Yellow trypanosomes were not seen in midgut or proventricular samples and first appeared in the glands as epimastigotes as early as 13 days after fly infection. Cloned progeny originating from individual salivary glands had yellow, red, green or no fluorescence and were confirmed as hybrids by microsatellite, molecular karyotype and kinetoplast (mitochondrial DNA analyses. Hybrid clones showed biparental inheritance of both nuclear and kinetoplast genomes. While segregation and reassortment of the reporter genes and microsatellite alleles were consistent with Mendelian inheritance, flow cytometry measurement of DNA content revealed both diploid and polyploid trypanosomes among the hybrid progeny clones. Conclusion The strategy of using production of yellow hybrids

  6. Aquaporin 2 mutations in Trypanosoma brucei gambiense field isolates correlate with decreased susceptibility to pentamidine and melarsoprol.

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    Fabrice E Graf

    Full Text Available The predominant mechanism of drug resistance in African trypanosomes is decreased drug uptake due to loss-of-function mutations in the genes for the transporters that mediate drug import. The role of transporters as determinants of drug susceptibility is well documented from laboratory-selected Trypanosoma brucei mutants. But clinical isolates, especially of T. b. gambiense, are less amenable to experimental investigation since they do not readily grow in culture without prior adaptation. Here we analyze a selected panel of 16 T. brucei ssp. field isolates that (i have been adapted to axenic in vitro cultivation and (ii mostly stem from treatment-refractory cases. For each isolate, we quantify the sensitivity to melarsoprol, pentamidine, and diminazene, and sequence the genomic loci of the transporter genes TbAT1 and TbAQP2. The former encodes the well-characterized aminopurine permease P2 which transports several trypanocides including melarsoprol, pentamidine, and diminazene. We find that diminazene-resistant field isolates of T. b. brucei and T. b. rhodesiense carry the same set of point mutations in TbAT1 that was previously described from lab mutants. Aquaglyceroporin 2 has only recently been identified as a second transporter involved in melarsoprol/pentamidine cross-resistance. Here we describe two different kinds of TbAQP2 mutations found in T. b. gambiense field isolates: simple loss of TbAQP2, or loss of wild-type TbAQP2 allele combined with the formation of a novel type of TbAQP2/3 chimera. The identified mutant T. b. gambiense are 40- to 50-fold less sensitive to pentamidine and 3- to 5-times less sensitive to melarsoprol than the reference isolates. We thus demonstrate for the first time that rearrangements of the TbAQP2/TbAQP3 locus accompanied by TbAQP2 gene loss also occur in the field, and that the T. b. gambiense carrying such mutations correlate with a significantly reduced susceptibility to pentamidine and melarsoprol.

  7. Programmed Cell Death in Procyclic Form Trypanosoma brucei rhodesiense - Identification of Differentially Expressed Genes during Con A Induced Death

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    Welburn Susan C

    1999-01-01

    Full Text Available Trypanosoma brucei rhodesiense can be induced to undergo apoptosis after stimulation with Con A. As cell death in these parasites is associated with de novo gene expression we have applied a differential display technique, Randomly Amplified Differential Expressed Sequence-Polymerase Chain Reaction (RADES-PCR to the study of gene expression during Con A induced cell death in these organisms. Twenty-two differentially displayed products have been cloned and sequenced. These represent the first endogenous genes to be identified as implicated in cellular death in trypanosomatids (the most primitive eukaryote in which apoptosis has been described. Evidence for an ancestral death machinery, `proto-apoptosis' in single celled organisms is discussed.

  8. High-confidence glycosome proteome for procyclic form Trypanosoma brucei by epitope-tag organelle enrichment and SILAC proteomics.

    Science.gov (United States)

    Güther, Maria Lucia S; Urbaniak, Michael D; Tavendale, Amy; Prescott, Alan; Ferguson, Michael A J

    2014-06-06

    The glycosome of the pathogenic African trypanosome Trypanosoma brucei is a specialized peroxisome that contains most of the enzymes of glycolysis and several other metabolic and catabolic pathways. The contents and transporters of this membrane-bounded organelle are of considerable interest as potential drug targets. Here we use epitope tagging, magnetic bead enrichment, and SILAC quantitative proteomics to determine a high-confidence glycosome proteome for the procyclic life cycle stage of the parasite using isotope ratios to discriminate glycosomal from mitochondrial and other contaminating proteins. The data confirm the presence of several previously demonstrated and suggested pathways in the organelle and identify previously unanticipated activities, such as protein phosphatases. The implications of the findings are discussed.

  9. Crystal Structures of TbCatB and rhodesain, potential chemotherapeutic targets and major cysteine proteases of Trypanosoma brucei.

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    Iain D Kerr

    2010-06-01

    Full Text Available Trypanosoma brucei is the etiological agent of Human African Trypanosomiasis, an endemic parasitic disease of sub-Saharan Africa. TbCatB and rhodesain are the sole Clan CA papain-like cysteine proteases produced by the parasite during infection of the mammalian host and are implicated in the progression of disease. Of considerable interest is the exploration of these two enzymes as targets for cysteine protease inhibitors that are effective against T. brucei.We have determined, by X-ray crystallography, the first reported structure of TbCatB in complex with the cathepsin B selective inhibitor CA074. In addition we report the structure of rhodesain in complex with the vinyl-sulfone K11002.The mature domain of our TbCat*CA074 structure contains unique features for a cathepsin B-like enzyme including an elongated N-terminus extending 16 residues past the predicted maturation cleavage site. N-terminal Edman sequencing reveals an even longer extension than is observed amongst the ordered portions of the crystal structure. The TbCat*CA074 structure confirms that the occluding loop, which is an essential part of the substrate-binding site, creates a larger prime side pocket in the active site cleft than is found in mammalian cathepsin B-small molecule structures. Our data further highlight enhanced flexibility in the occluding loop main chain and structural deviations from mammalian cathepsin B enzymes that may affect activity and inhibitor design. Comparisons with the rhodesain*K11002 structure highlight key differences that may impact the design of cysteine protease inhibitors as anti-trypanosomal drugs.

  10. Structural Characterization of CYP51 from Trypanosoma cruzi and Trypanosoma brucei Bound to the Antifungal Drugs Posaconazole and Fluconazole

    Science.gov (United States)

    Chen, Chiung-Kuang; Leung, Siegfried S. F.; Guilbert, Christophe; Jacobson, Matthew P.; McKerrow, James H.; Podust, Larissa M.

    2010-01-01

    Background Chagas Disease is the leading cause of heart failure in Latin America. Current drug therapy is limited by issues of both efficacy and severe side effects. Trypansoma cruzi, the protozoan agent of Chagas Disease, is closely related to two other major global pathogens, Leishmania spp., responsible for leishmaniasis, and Trypansoma brucei, the causative agent of African Sleeping Sickness. Both T. cruzi and Leishmania parasites have an essential requirement for ergosterol, and are thus vulnerable to inhibitors of sterol 14α-demethylase (CYP51), which catalyzes the conversion of lanosterol to ergosterol. Clinically employed anti-fungal azoles inhibit ergosterol biosynthesis in fungi, and specific azoles are also effective against both Trypanosoma and Leishmania parasites. However, modification of azoles to enhance efficacy and circumvent potential drug resistance has been problematic for both parasitic and fungal infections due to the lack of structural insights into drug binding. Methodology/Principal Findings We have determined the crystal structures for CYP51 from T. cruzi (resolutions of 2.35 Å and 2.27 Å), and from the related pathogen T. brucei (resolutions of 2.7 Å and 2.6 Å), co-crystallized with the antifungal drugs fluconazole and posaconazole. Remarkably, both drugs adopt multiple conformations when binding the target. The fluconazole 2,4-difluorophenyl ring flips 180° depending on the H-bonding interactions with the BC-loop. The terminus of the long functional tail group of posaconazole is bound loosely in the mouth of the hydrophobic substrate binding tunnel, suggesting that the major contribution of the tail to drug efficacy is for pharmacokinetics rather than in interactions with the target. Conclusions/Significance The structures provide new insights into binding of azoles to CYP51 and mechanisms of potential drug resistance. Our studies define in structural detail the CYP51 therapeutic target in T. cruzi, and offer a starting point for

  11. The acidocalcisome vacuolar transporter chaperone 4 catalyzes the synthesis of polyphosphate in insect-stages of Trypanosoma brucei and T. cruzi.

    Science.gov (United States)

    Ulrich, Paul N; Lander, Noelia; Kurup, Samarchith P; Reiss, Laura; Brewer, Jessica; Soares Medeiros, Lia C; Miranda, Kildare; Docampo, Roberto

    2014-01-01

    Polyphosphate is a polymer of inorganic phosphate found in both prokaryotes and eukaryotes. Polyphosphate typically accumulates in acidic, calcium-rich organelles known as acidocalcisomes, and recent research demonstrated that vacuolar transporter chaperone 4 catalyzes its synthesis in yeast. The human pathogens Trypanosoma brucei and T. cruzi possess vacuolar transporter chaperone 4 homologs. We demonstrate that T. cruzi vacuolar transporter chaperone 4 localizes to acidocalcisomes of epimastigotes by immunofluorescence and immuno-electron microscopy and that the recombinant catalytic region of the T. cruzi enzyme is a polyphosphate kinase. RNA interference of the T. brucei enzyme in procyclic form parasites reduced short chain polyphosphate levels and resulted in accumulation of pyrophosphate. These results suggest that this trypanosome enzyme is an important component of a polyphosphate synthase complex that utilizes ATP to synthesize and translocate polyphosphate to acidocalcisomes in insect stages of these parasites. © 2013 The Author(s) Journal of Eukaryotic Microbiology © 2013 International Society of Protistologists.

  12. Trypanosoma brucei aquaglyceroporin 2 is a high-affinity transporter for pentamidine and melaminophenyl arsenic drugs and the main genetic determinant of resistance to these drugs

    Science.gov (United States)

    Munday, Jane C.; Eze, Anthonius A.; Baker, Nicola; Glover, Lucy; Clucas, Caroline; Aguinaga Andrés, David; Natto, Manal J.; Teka, Ibrahim A.; McDonald, Jennifer; Lee, Rebecca S.; Graf, Fabrice E.; Ludin, Philipp; Burchmore, Richard J. S.; Turner, C. Michael R.; Tait, Andy; MacLeod, Annette; Mäser, Pascal; Barrett, Michael P.; Horn, David; De Koning, Harry P.

    2014-01-01

    Objectives Trypanosoma brucei drug transporters include the TbAT1/P2 aminopurine transporter and the high-affinity pentamidine transporter (HAPT1), but the genetic identity of HAPT1 is unknown. We recently reported that loss of T. brucei aquaglyceroporin 2 (TbAQP2) caused melarsoprol/pentamidine cross-resistance (MPXR) in these parasites and the current study aims to delineate the mechanism by which this occurs. Methods The TbAQP2 loci of isogenic pairs of drug-susceptible and MPXR strains of T. brucei subspecies were sequenced. Drug susceptibility profiles of trypanosome strains were correlated with expression of mutated TbAQP2 alleles. Pentamidine transport was studied in T. brucei subspecies expressing TbAQP2 variants. Results All MPXR strains examined contained TbAQP2 deletions or rearrangements, regardless of whether the strains were originally adapted in vitro or in vivo to arsenicals or to pentamidine. The MPXR strains and AQP2 knockout strains had lost HAPT1 activity. Reintroduction of TbAQP2 in MPXR trypanosomes restored susceptibility to the drugs and reinstated HAPT1 activity, but did not change the activity of TbAT1/P2. Expression of TbAQP2 sensitized Leishmania mexicana promastigotes 40-fold to pentamidine and >1000-fold to melaminophenyl arsenicals and induced a high-affinity pentamidine transport activity indistinguishable from HAPT1 by Km and inhibitor profile. Grafting the TbAQP2 selectivity filter amino acid residues onto a chimeric allele of AQP2 and AQP3 partly restored susceptibility to pentamidine and an arsenical. Conclusions TbAQP2 mediates high-affinity uptake of pentamidine and melaminophenyl arsenicals in trypanosomes and TbAQP2 encodes the previously reported HAPT1 activity. This finding establishes TbAQP2 as an important drug transporter. PMID:24235095

  13. Trypanosoma brucei TbIF1 inhibits the essential Finf1/inf-ATPase in the infectious form of the parasite

    Czech Academy of Sciences Publication Activity Database

    Panicucci, Brian; Gahura, Ondřej; Zíková, Alena

    2017-01-01

    Roč. 11, č. 4 (2017), č. článku e0005552. ISSN 1935-2735 R&D Projects: GA MŠk(CZ) EE2.3.30.0032; GA ČR GA17-22248S Institutional support: RVO:60077344 Keywords : mt * TblF1 * Trypanosoma brucei Subject RIV: EE - Microbiology, Virology Impact factor: 3.834, year: 2016

  14. Identification and Functional Characterisation of CRK12:CYC9, a Novel Cyclin-Dependent Kinase (CDK-Cyclin Complex in Trypanosoma brucei.

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    Séverine Monnerat

    Full Text Available The protozoan parasite, Trypanosoma brucei, is spread by the tsetse fly and causes trypanosomiasis in humans and animals. Both the life cycle and cell cycle of the parasite are complex. Trypanosomes have eleven cdc2-related kinases (CRKs and ten cyclins, an unusually large number for a single celled organism. To date, relatively little is known about the function of many of the CRKs and cyclins, and only CRK3 has previously been shown to be cyclin-dependent in vivo. Here we report the identification of a previously uncharacterised CRK:cyclin complex between CRK12 and the putative transcriptional cyclin, CYC9. CRK12:CYC9 interact to form an active protein kinase complex in procyclic and bloodstream T. brucei. Both CRK12 and CYC9 are essential for the proliferation of bloodstream trypanosomes in vitro, and we show that CRK12 is also essential for survival of T. brucei in a mouse model, providing genetic validation of CRK12:CYC9 as a novel drug target for trypanosomiasis. Further, functional characterisation of CRK12 and CYC9 using RNA interference reveals roles for these proteins in endocytosis and cytokinesis, respectively.

  15. Detection of Trypanosoma brucei in field-captured tsetse flies and identification of host species fed on by the infected flies.

    Science.gov (United States)

    Konnai, Satoru; Mekata, Hirohisa; Odbileg, Raadan; Simuunza, Martin; Chembensof, Mwelwa; Witola, William Harold; Tembo, Mwase Enala; Chitambo, Harrison; Inoue, Noboru; Onuma, Misao; Ohashi, Kazuhiko

    2008-08-01

    The prevalence of trypanosome infections in tsetse flies in the Chiawa area of Lower Zambezi in Zambia, with endemic trypanosomosis, was determined by a polymerase chain reaction (PCR) method that allowed the detection of trypanosome DNA and determination of the type of animal host fed on by the tsetse fly Glossina pallidipes, using tsetse-derived DNA extracts as templates. Ninety G. pallidipes (82 females and 8 males; 18.3%) of the 492 flies captured by baited biconical traps tested positive for the presence of Trypanosoma brucei species genomic DNA. Of the 90 T. brucei-positive flies, 47 (52.2%) also tested positive for vertebrate mitochondrial DNA. Sequence analysis of the vertebrate mitochondrial DNA amplicons established that they originated from 8 different vertebrate species, namely, human (Homo sapiens), African elephant (Loxodonta cyclotis), African buffalo (Syncerus caffer), waterbuck (Kobus ellipsiprymnus), roan antelope (Hippotragus equinus), greater kudu (Tragelaphus strepsiceros), warthog (Phacochoerus africanus), and goat (Capra hircus). Furthermore, to investigate the prevalence of trypanosome infections in domestic goats in the same area where trypanosomes had been detected in tsetse files, a total of 86 goats were randomly selected from 6 different herds. Among the selected goats, 36 (41.9%) were found to be positive for T. brucei species. This combined detection method would be an ideal approach not only for mass screening for infection prevalence in tsetse populations, but also for the prediction of natural reservoirs in areas endemic for trypanosomosis.

  16. Immunization with recombinant beta-tubulin from Trypanosoma evansi induced protection against T. evansi, T. equiperdum and T. b. brucei infection in mice.

    Science.gov (United States)

    Li, S-Q; Fung, M-C; Reid, S A; Inoue, N; Lun, Z-R

    2007-04-01

    The beta-tubulin gene of Trypanosoma evansi (STIB 806) was cloned and expressed in Escherichia coli. The predicted amino acid sequence of T. evansi beta-tubulin shows 100%, 99.8%, 99.1%, and 98.6% homology with T. equiperdum, T. b. brucei, T. cruzi and T. danilewskyi, respectively, but is diverse from that of T. cyclops, showing only 51.6% of homology. Recombinant beta-tubulin was expressed as inclusion bodies in E. coli. It was purified and renatured for immunological studies. Mice immunized with the renatured recombinant beta-tubulin were protected from lethal challenge with T. evansi STIB 806, T. equiperdum STIB 818 and T. b. brucei STIB 940, showing 83.3%, 70% and 76.7% protection, respectively. Serum collected from the rabbit immunized with recombinant beta-tubulin inhibited the growth of T. evansi, T. equiperdum and T. b. brucei in vitro. Serum from mice and rabbits immunized with recombinant beta-tubulin recognized only T. evansi beta-tubulin and not mouse beta-tubulin. The results of this study demonstrated that the recombinant T. evansi beta-tubulin is a potential candidate for the development of a vaccine to prevent animal trypanosomiasis caused by these three trypanosome species.

  17. Antitrypanosomal compounds from the essential oil and extracts of Keetia leucantha leaves with inhibitor activity on Trypanosoma brucei glyceraldehyde-3-phosphate dehydrogenase.

    Science.gov (United States)

    Bero, J; Beaufay, C; Hannaert, V; Hérent, M-F; Michels, P A; Quetin-Leclercq, J

    2013-02-15

    Keetia leucantha is a West African tree used in traditional medicine to treat several diseases among which parasitic infections. The dichloromethane extract of leaves was previously shown to possess growth-inhibitory activities on Plasmodium falciparum, Trypanosoma brucei brucei and Leishmania mexicana mexicana with low or no cytotoxicity (>100 μg/ml on human normal fibroblasts) (Bero et al. 2009, 2011). In continuation of our investigations on the antitrypanosomal compounds from this dichloromethane extract, we analyzed by GC-FID and GC-MS the essential oil of its leaves obtained by hydrodistillation and the major triterpenic acids in this extract by LC-MS. Twenty-seven compounds were identified in the oil whose percentages were calculated using the normalization method. The essential oil, seven of its constituents and the three triterpenic acids were evaluated for their antitrypanosomal activity on Trypanosoma brucei brucei bloodstream forms (Tbb BSF) and procyclic forms (Tbb PF) to identify an activity on the glycolytic process of trypanosomes. The oil showed an IC(50) of 20.9 μg/ml on Tbb BSF and no activity was observed on Tbb PF. The best antitrypanosomal activity was observed for ursolic acid with IC(50) of 2.5 and 6.5 μg/ml respectively on Tbb BSF and Tbb PF. The inhibitory activity on a glycolytic enzyme of T. brucei, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was also evaluated for betulinic acid, olenaolic acid, ursolic acid, phytol, α-ionone and β-ionone. The three triterpenic acids and β-ionone showed inhibitory activities on GAPDH with oleanolic acid being the most active with an inhibition of 72.63% at 20 μg/ml. This paper reports for the first time the composition and antitrypanosomal activity of the essential oil of Keetia leucantha. Several of its constituents and three triterpenic acids present in the dichloromethane leaves extract showed a higher antitrypanosomal activity on bloodstream forms of Tbb as compared to procyclic forms

  18. Probing the metabolic network in bloodstream-form Trypanosoma brucei using untargeted metabolomics with stable isotope labelled glucose.

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    Darren J Creek

    2015-03-01

    Full Text Available Metabolomics coupled with heavy-atom isotope-labelled glucose has been used to probe the metabolic pathways active in cultured bloodstream form trypomastigotes of Trypanosoma brucei, a parasite responsible for human African trypanosomiasis. Glucose enters many branches of metabolism beyond glycolysis, which has been widely held to be the sole route of glucose metabolism. Whilst pyruvate is the major end-product of glucose catabolism, its transamination product, alanine, is also produced in significant quantities. The oxidative branch of the pentose phosphate pathway is operative, although the non-oxidative branch is not. Ribose 5-phosphate generated through this pathway distributes widely into nucleotide synthesis and other branches of metabolism. Acetate, derived from glucose, is found associated with a range of acetylated amino acids and, to a lesser extent, fatty acids; while labelled glycerol is found in many glycerophospholipids. Glucose also enters inositol and several sugar nucleotides that serve as precursors to macromolecule biosynthesis. Although a Krebs cycle is not operative, malate, fumarate and succinate, primarily labelled in three carbons, were present, indicating an origin from phosphoenolpyruvate via oxaloacetate. Interestingly, the enzyme responsible for conversion of phosphoenolpyruvate to oxaloacetate, phosphoenolpyruvate carboxykinase, was shown to be essential to the bloodstream form trypanosomes, as demonstrated by the lethal phenotype induced by RNAi-mediated downregulation of its expression. In addition, glucose derivatives enter pyrimidine biosynthesis via oxaloacetate as a precursor to aspartate and orotate.

  19. Flagellar adhesion in Trypanosoma brucei relies on interactions between different skeletal structures in the flagellum and cell body.

    Science.gov (United States)

    Rotureau, Brice; Blisnick, Thierry; Subota, Ines; Julkowska, Daria; Cayet, Nadège; Perrot, Sylvie; Bastin, Philippe

    2014-01-01

    The Trypanosoma brucei flagellum is an essential organelle anchored along the surface of the cell body through a specialized structure called the flagellum attachment zone (FAZ). Adhesion relies on the interaction of the extracellular portion of two transmembrane proteins, FLA1 and FLA1BP. Here, we identify FLAM3 as a novel large protein associated with the flagellum skeleton whose ablation inhibits flagellum attachment. FLAM3 does not contain transmembrane domains and its flagellar localization matches closely, but not exactly, that of the paraflagellar rod, an extra-axonemal structure present in the flagellum. Knockdown of FLA1 or FLAM3 triggers similar defects in motility and morphogenesis, characterized by the assembly of a drastically reduced FAZ filament. FLAM3 remains associated with the flagellum skeleton even in the absence of adhesion or a normal paraflagellar rod. However, the protein is dispersed in the cytoplasm when flagellum formation is inhibited. By contrast, FLA1 remains tightly associated with the FAZ filament even in the absence of a flagellum. In these conditions, the extracellular domain of FLA1 points to the cell surface. FLAM3 is essential for proper distribution of FLA1BP, which is restricted to the most proximal portion of the flagellum upon knockdown of FLAM3. We propose that FLAM3 is a key component of the FAZ connectors that link the axoneme to the adhesion zone, hence it acts in an equivalent manner to the FAZ filament complex, but on the side of the flagellum.

  20. A global comparison of the human and T. brucei degradomes gives insights about possible parasite drug targets.

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    Susan T Mashiyama

    Full Text Available We performed a genome-level computational study of sequence and structure similarity, the latter using crystal structures and models, of the proteases of Homo sapiens and the human parasite Trypanosoma brucei. Using sequence and structure similarity networks to summarize the results, we constructed global views that show visually the relative abundance and variety of proteases in the degradome landscapes of these two species, and provide insights into evolutionary relationships between proteases. The results also indicate how broadly these sequence sets are covered by three-dimensional structures. These views facilitate cross-species comparisons and offer clues for drug design from knowledge about the sequences and structures of potential drug targets and their homologs. Two protease groups ("M32" and "C51" that are very different in sequence from human proteases are examined in structural detail, illustrating the application of this global approach in mining new pathogen genomes for potential drug targets. Based on our analyses, a human ACE2 inhibitor was selected for experimental testing on one of these parasite proteases, TbM32, and was shown to inhibit it. These sequence and structure data, along with interactive versions of the protein similarity networks generated in this study, are available at http://babbittlab.ucsf.edu/resources.html.

  1. Loop Mediated Isothermal Amplification for Detection of Trypanosoma brucei gambiense in Urine and Saliva Samples in Nonhuman Primate Model

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    Maina Ngotho

    2015-01-01

    Full Text Available Human African trypanosomiasis (HAT is a vector-borne parasitic zoonotic disease. The disease caused by Trypanosoma brucei gambiense is the most prevalent in Africa. Early diagnosis is hampered by lack of sensitive diagnostic techniques. This study explored the potential of loop mediated isothermal amplification (LAMP and polymerase chain reaction (PCR in the detection of T. b. gambiense infection in a vervet monkey HAT model. Six vervet monkeys were experimentally infected with T. b. gambiense IL3253 and monitored for 180 days after infection. Parasitaemia was scored daily. Blood, cerebrospinal fluid (CSF, saliva, and urine samples were collected weekly. PCR and LAMP were performed on serum, CSF, saliva, and urine samples. The detection by LAMP was significantly higher than that of parasitological methods and PCR in all the samples. The performance of LAMP varied between the samples and was better in serum followed by saliva and then urine samples. In the saliva samples, LAMP had 100% detection between 21 and 77 dpi, whereas in urine the detection it was slightly lower, but there was over 80% detection between 28 and 91 dpi. However, LAMP could not detect trypanosomes in either saliva or urine after 140 and 126 dpi, respectively. The findings of this study emphasize the importance of LAMP in diagnosis of HAT using saliva and urine samples.

  2. Loop Mediated Isothermal Amplification for Detection of Trypanosoma brucei gambiense in Urine and Saliva Samples in Nonhuman Primate Model.

    Science.gov (United States)

    Ngotho, Maina; Kagira, John Maina; Gachie, Beatrice Muthoni; Karanja, Simon Muturi; Waema, Maxwell Wambua; Maranga, Dawn Nyawira; Maina, Naomi Wangari

    2015-01-01

    Human African trypanosomiasis (HAT) is a vector-borne parasitic zoonotic disease. The disease caused by Trypanosoma brucei gambiense is the most prevalent in Africa. Early diagnosis is hampered by lack of sensitive diagnostic techniques. This study explored the potential of loop mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR) in the detection of T. b. gambiense infection in a vervet monkey HAT model. Six vervet monkeys were experimentally infected with T. b. gambiense IL3253 and monitored for 180 days after infection. Parasitaemia was scored daily. Blood, cerebrospinal fluid (CSF), saliva, and urine samples were collected weekly. PCR and LAMP were performed on serum, CSF, saliva, and urine samples. The detection by LAMP was significantly higher than that of parasitological methods and PCR in all the samples. The performance of LAMP varied between the samples and was better in serum followed by saliva and then urine samples. In the saliva samples, LAMP had 100% detection between 21 and 77 dpi, whereas in urine the detection it was slightly lower, but there was over 80% detection between 28 and 91 dpi. However, LAMP could not detect trypanosomes in either saliva or urine after 140 and 126 dpi, respectively. The findings of this study emphasize the importance of LAMP in diagnosis of HAT using saliva and urine samples.

  3. Molecular Evidence of a Trypanosoma brucei gambiense Sylvatic Cycle in the Human African Trypanosomiasis Foci of Equatorial Guinea

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    Carlos eCordon-Obras

    2015-07-01

    Full Text Available Gambiense trypanosomiasis is considered an anthroponotic disease. Consequently, control programs are generally aimed at stopping transmission of Trypanosoma brucei gambiense (T. b. gambiense by detecting and treating human cases. However, the persistence of numerous foci despite efforts to eliminate this disease questions this strategy as unique tool to pursue the eradication. The role of animals as a reservoir of T. b. gambiense is still controversial, but could partly explain maintenance of the infection at hypo-endemic levels. In the present study, we evaluated the presence of T. b. gambiense in wild animals in Equatorial Guinea. The infection rate ranged from 0.8% in the insular focus of Luba to more than 12% in Mbini, a focus with a constant trickle of human cases. The parasite was detected in a wide range of animal species including four species never described previously as putative reservoirs. Our study comes to reinforce the hypothesis that animals may play a role in the persistence of T. b. gambiense transmission, being particularly relevant in low transmission settings. Under these conditions the integration of sustained vector control and medical interventions should be considered to achieve the elimination of Gambiense trypanosomiasis.

  4. IL-6 is Upregulated in Late-Stage Disease in Monkeys Experimentally Infected with Trypanosoma brucei rhodesiense

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    Dawn Nyawira Maranga

    2013-01-01

    Full Text Available The management of human African trypanosomiasis (HAT is constrained by lack of simple-to-use diagnostic, staging, and treatment tools. The search for novel biomarkers is, therefore, essential in the fight against HAT. The current study aimed at investigating the potential of IL-6 as an adjunct parameter for HAT stage determination in vervet monkey model. Four adult vervet monkeys (Chlorocebus aethiops were experimentally infected with Trypanosoma brucei rhodesiense and treated subcuratively at 28 days after infection (dpi to induce late stage disease. Three noninfected monkeys formed the control group. Cerebrospinal fluid (CSF and blood samples were obtained at weekly intervals and assessed for various biological parameters. A typical HAT-like infection was observed. The late stage was characterized by significant (P<0.05 elevation of CSF IL-6, white blood cell count, and total protein starting 35 dpi with peak levels of these parameters coinciding with relapse parasitaemia. Brain immunohistochemical staining revealed an increase in brain glial fibrillary acidic protein expression indicative of reactive astrogliosis in infected animals which were euthanized in late-stage disease. The elevation of IL-6 in CSF which accompanied other HAT biomarkers indicates onset of parasite neuroinvasion and show potential for use as an adjunct late-stage disease biomarker in the Rhodesian sleeping sickness.

  5. IL-6 is Upregulated in Late-Stage Disease in Monkeys Experimentally Infected with Trypanosoma brucei rhodesiense

    Science.gov (United States)

    Nyawira Maranga, Dawn; Kagira, John Maina; Kinyanjui, Christopher Kariuki; Muturi Karanja, Simon; Wangari Maina, Naomi; Ngotho, Maina

    2013-01-01

    The management of human African trypanosomiasis (HAT) is constrained by lack of simple-to-use diagnostic, staging, and treatment tools. The search for novel biomarkers is, therefore, essential in the fight against HAT. The current study aimed at investigating the potential of IL-6 as an adjunct parameter for HAT stage determination in vervet monkey model. Four adult vervet monkeys (Chlorocebus aethiops) were experimentally infected with Trypanosoma brucei rhodesiense and treated subcuratively at 28 days after infection (dpi) to induce late stage disease. Three noninfected monkeys formed the control group. Cerebrospinal fluid (CSF) and blood samples were obtained at weekly intervals and assessed for various biological parameters. A typical HAT-like infection was observed. The late stage was characterized by significant (P < 0.05) elevation of CSF IL-6, white blood cell count, and total protein starting 35 dpi with peak levels of these parameters coinciding with relapse parasitaemia. Brain immunohistochemical staining revealed an increase in brain glial fibrillary acidic protein expression indicative of reactive astrogliosis in infected animals which were euthanized in late-stage disease. The elevation of IL-6 in CSF which accompanied other HAT biomarkers indicates onset of parasite neuroinvasion and show potential for use as an adjunct late-stage disease biomarker in the Rhodesian sleeping sickness. PMID:24194772

  6. A single amino acid substitution in the group 1 Trypanosoma brucei gambiense haptoglobin-hemoglobin receptor abolishes TLF-1 binding.

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    E DeJesus

    Full Text Available Critical to human innate immunity against African trypanosomes is a minor subclass of human high-density lipoproteins, termed Trypanosome Lytic Factor-1 (TLF-1. This primate-specific molecule binds to a haptoglobin-hemoglobin receptor (HpHbR on the surface of susceptible trypanosomes, initiating a lytic pathway. Group 1 Trypanosoma brucei gambiense causes human African Trypanosomiasis (HAT, escaping TLF-1 killing due to reduced uptake. Previously, we found that group 1 T. b. gambiense HpHbR (TbgHpHbR mRNA levels were greatly reduced and the gene contained substitutions within the open reading frame. Here we show that a single, highly conserved amino acid in the TbgHpHbR ablates high affinity TLF-1 binding and subsequent endocytosis, thus evading TLF-1 killing. In addition, we show that over-expression of TbgHpHbR failed to rescue TLF-1 susceptibility. These findings suggest that the single substitution present in the TbgHpHbR directly contributes to the reduced uptake and resistance to TLF-1 seen in these important human pathogens.

  7. Similarity in variable antigen type composition of Trypanosoma brucei rhodesiense populations in different sites within the mouse host.

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    Turner, C M; Hunter, C A; Barry, J D; Vickerman, K

    1986-01-01

    Trypanosoma brucei rhodesiense subpopulations in different sites within the body of infected mice were isolated and enumerated on day 6 of cyclically transmitted infections. Most trypanosomes were in the blood vasculature and spleen but approximately 6% occurred in lymph nodes and about 9% were extravascular. Most of the extravascular trypanosomes were in the peritoneal and pleural cavities; significant numbers also occurred in the brain and kidneys. Six major variable antigen types (VATs) were detected by immunofluorescence using specific antisera and monoclonal antibodies. The prevalence of each VAT was essentially the same in subpopulations in the blood, mesenteric and inguinal lymph nodes, brain, kidneys and peritoneal and pleural cavities. This similarity of VAT composition in different subpopulations is probably caused by high rates of dynamic interchange of trypanosomes between sites. Extravascular trypanosomes, therefore, form a significant proportion of the total population in acute infections of mice but they do not appear to play any special role in the population biology of antigenic variation at this stage of infection.

  8. Trypanosoma brucei Invasion and T-Cell Infiltration of the Brain Parenchyma in Experimental Sleeping Sickness: Timing and Correlation with Functional Changes.

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    Claudia Laperchia

    2016-12-01

    Full Text Available The timing of Trypanosoma brucei entry into the brain parenchyma to initiate the second, meningoencephalitic stage of human African trypanosomiasis or sleeping sickness is currently debated and even parasite invasion of the neuropil has been recently questioned. Furthermore, the relationship between neurological features and disease stage are unclear, despite the important diagnostic and therapeutic implications.Using a rat model of chronic Trypanosoma brucei brucei infection we determined the timing of parasite and T-cell neuropil infiltration and its correlation with functional changes. Parasite DNA was detected using trypanosome-specific PCR. Body weight and sleep structure alterations represented by sleep-onset rapid eye movement (SOREM periods, reported in human and experimental African trypanosomiasis, were monitored. The presence of parasites, as well as CD4+ and CD8+ T-cells in the neuropil was assessed over time in the brain of the same animals by immunocytochemistry and quantitative analyses.Trypanosome DNA was present in the brain at day 6 post-infection and increased more than 15-fold by day 21. Parasites and T-cells were observed in the parenchyma from day 9 onwards. Parasites traversing blood vessel walls were observed in the hypothalamus and other brain regions. Body weight gain was reduced from day 7 onwards. SOREM episodes started in most cases early after infection, with an increase in number and duration after parasite neuroinvasion.These findings demonstrate invasion of the neuropil over time, after an initial interval, by parasites and lymphocytes crossing the blood-brain barrier, and show that neurological features can precede this event. The data thus challenge the current clinical and cerebrospinal fluid criteria of disease staging.

  9. Trypanosoma brucei gambiense group 1 is distinguished by a unique amino acid substitution in the HpHb receptor implicated in human serum resistance.

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    Rebecca E Symula

    Full Text Available Trypanosoma brucei rhodesiense (Tbr and T. b. gambiense (Tbg, causative agents of Human African Trypanosomiasis (sleeping sickness in Africa, have evolved alternative mechanisms of resisting the activity of trypanosome lytic factors (TLFs, components of innate immunity in human serum that protect against infection by other African trypanosomes. In Tbr, lytic activity is suppressed by the Tbr-specific serum-resistance associated (SRA protein. The mechanism in Tbg is less well understood but has been hypothesized to involve altered activity and expression of haptoglobin haemoglobin receptor (HpHbR. HpHbR has been shown to facilitate internalization of TLF-1 in T.b. brucei (Tbb, a member of the T. brucei species complex that is susceptible to human serum. By evaluating the genetic variability of HpHbR in a comprehensive geographical and taxonomic context, we show that a single substitution that replaces leucine with serine at position 210 is conserved in the most widespread form of Tbg (Tbg group 1 and not found in related taxa, which are either human serum susceptible (Tbb or known to resist lysis via an alternative mechanism (Tbr and Tbg group 2. We hypothesize that this single substitution contributes to reduced uptake of TLF and thus may play a key role in conferring serum resistance to Tbg group 1. In contrast, similarity in HpHbR sequence among isolates of Tbg group 2 and Tbb/Tbr provides further evidence that human serum resistance in Tbg group 2 is likely independent of HpHbR function.

  10. Comparative biochemical and pathological changes in some laboratory animals experimentally infected with Trypanosoma brucei and their responses to diminazene diaceturate (Veriben® therapy

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    Amina Ibrahim

    2015-12-01

    Full Text Available Objective: To compare the biochemical and pathological changes of Trypanosoma brucei brucei infected mice, rats and rabbits, and study the chemotherapeutic effects according to standard criteria. Methods: A total of 20 Balb/c Albino mice, 20 Wister Albino rats and 20 New Zealand rabbits, all adults and of both sexes were used in the study. Each rodent group was divided into four groups (A, B, C and D of five animals each. Animals in Groups A and C were individually infected with 0.5 mL of blood from donor rats containing 1.5 ××106 Trypanasoma brucei brucei, while Groups B and D remained uninfected. The animals were later treated like this: Group A (infected and untreated control, Group B (uninfected and untreated control, Group C (infected and treated and Group D (uninfected and treated. The treatment was administered on Day 12 after infection. Results: The prepatent period for mice and rats was 4 days while that for rabbits was 8 days. There was a significant (P < 0.05 increase in levels of liver enzymes and serum metabolites which were more marked among the mice and rats than rabbits. These changes were modulated to their preinfection values in the infected treated animals. At necropsy, all animals showed splenomegaly, hepatomegaly and nephritis. However, cardiomegaly was exclusive to the rabbits. Histopathologically, degenerative changes were observed in both the liver and kidneys which were more severe among mice and rats and moderate in rabbits. Spleen of mice showed giant macrophages, while that of the rats showed epithelioid giant cells. However, spleen of rabbits showed haemosiderosis and eosinophilic infiltrations. Hydrophobic degeneration, necrosis and mononuclear cell infiltrations in the myocardium were observed only among rabbits. Conclusions: Diminazene diaceturate was able to ameliorate the various biochemical and pathological changes which were suggestive of severe liver and kidney dysfunctions with greater intensity occurring

  11. Interaction between the flagellar pocket collar and the hook complex via a novel microtubule-binding protein in Trypanosoma brucei.

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    Anna Albisetti

    2017-11-01

    Full Text Available Trypanosoma brucei belongs to a group of unicellular, flagellated parasites that are responsible for human African trypanosomiasis. An essential aspect of parasite pathogenicity is cytoskeleton remodelling, which occurs during the life cycle of the parasite and is accompanied by major changes in morphology and organelle positioning. The flagellum originates from the basal bodies and exits the cell body through the flagellar pocket (FP but remains attached to the cell body via the flagellum attachment zone (FAZ. The FP is an invagination of the pellicular membrane and is the sole site for endo- and exocytosis. The FAZ is a large complex of cytoskeletal proteins, plus an intracellular set of four specialised microtubules (MtQ that elongate from the basal bodies to the anterior end of the cell. At the distal end of the FP, an essential, intracellular, cytoskeletal structure called the flagellar pocket collar (FPC circumvents the flagellum. Overlapping the FPC is the hook complex (HC (a sub-structure of the previously named bilobe that is also essential and is thought to be involved in protein FP entry. BILBO1 is the only functionally characterised FPC protein and is necessary for FPC and FP biogenesis. Here, we used a combination of in vitro and in vivo approaches to identify and characterize a new BILBO1 partner protein-FPC4. We demonstrate that FPC4 localises to the FPC, the HC, and possibly to a proximal portion of the MtQ. We found that the C-terminal domain of FPC4 interacts with the BILBO1 N-terminal domain, and we identified the key amino acids required for this interaction. Interestingly, the FPC4 N-terminal domain was found to bind microtubules. Over-expression studies highlight the role of FPC4 in its association with the FPC, HC and FPC segregation. Our data suggest a tripartite association between the FPC, the HC and the MtQ.

  12. Trypanosoma brucei gambiense Infections in Mice Lead to Tropism to the Reproductive Organs, and Horizontal and Vertical Transmission.

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    Nicolas Biteau

    2016-01-01

    Full Text Available Trypanosoma brucei gambiense, transmitted by the tsetse fly, is the main causative agent of Human African trypanosomosis in West Africa and poses a significant health risk to 70 million people. Disease progression varies depending on host immunity, but usually begins with a haemo-lymphatic phase, followed by parasite invasion of the central nervous system. In the current study, the tropism of T. b. gambiense 1135, causing a low level chronic 'silent' infection, was monitored in a murine model using bioluminescence imaging and PCR. A tropism to the reproductive organs, in addition to the central nervous system, after 12-18 months of infection was observed. Bioluminescent analysis of healthy females crossed with infected males showed that 50%, 62.5% and 37.5% of the female mice were subsequently positive for parasites in their ovaries, uteri and brain respectively. Although PCR confirmed the presence of parasites in the uterus of one of these mice, the blood of all mice was negative by PCR and LAMP. Subsequently, bioluminescent imaging of the offspring of infected female mice crossed with healthy males indicated parasites were present in the reproductive organs of both male (80% and female (60% offspring. These findings imply that transmission of T. b. gambiense 1135 occurs horizontally, most probably via sexual contact, and vertically in a murine model, which raises the possibility of a similar transmission in humans. This has wide reaching implications. Firstly, the observations made in this study are likely to be valid for wild animals acting as a reservoir for T. b. gambiense. Also, the reproductive organs may act as a refuge for parasites during drug treatment in a similar manner to the central nervous system. This could leave patients at risk of a relapse, ultimately allowing them to act as a reservoir for subsequent transmission by tsetse and possibly, horizontally and vertically.

  13. Trypanosoma brucei rhodesiense transmitted by a single tsetse fly bite in vervet monkeys as a model of human African trypanosomiasis.

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    John K Thuita

    Full Text Available We have investigated the pathogenicity of tsetse (Glossina pallidipes-transmitted cloned strains of Trypanosoma brucei rhodesiense in vervet monkeys. Tsetse flies were confirmed to have mature trypanosome infections by xenodiagnosis, after which nine monkeys were infected via the bite of a single infected fly. Chancres developed in five of the nine (55.6% monkeys within 4 to 8 days post infection (dpi. All nine individuals were successfully infected, with a median pre-patent period of 4 (range = 4-10 days, indicating that trypanosomes migrated from the site of fly bite to the systemic circulation rapidly and independently of the development of the chancre. The time lag to detection of parasites in cerebrospinal fluid (CSF was a median 16 (range = 8-40 days, marking the onset of central nervous system (CNS, late stage disease. Subsequently, CSF white cell numbers increased above the pre-infection median count of 2 (range = 0-9 cells/microl, with a positive linear association between their numbers and that of CSF trypanosomes. Haematological changes showed that the monkeys experienced an early microcytic-hypochromic anaemia and severe progressive thrombocytopaenia. Despite a 3-fold increase in granulocyte numbers by 4 dpi, leucopaenia occurred early (8 dpi in the monkey infection, determined mainly by reductions in lymphocyte numbers. Terminally, leucocytosis was observed in three of nine (33% individuals. The duration of infection was a median of 68 (range = 22-120 days. Strain and individual differences were observed in the severity of the clinical and clinical pathology findings, with two strains (KETRI 3741 and 3801 producing a more acute disease than the other two (KETRI 3804 and 3928. The study shows that the fly-transmitted model accurately mimics the human disease and is therefore a suitable gateway to understanding human African trypanosomiasis (HAT; sleeping sickness.

  14. Crystal Structures of Trypanosoma brucei Sterol 14[alpha]-Demethylase and Implications for Selective Treatment of Human Infections

    Energy Technology Data Exchange (ETDEWEB)

    Lepesheva, Galina I.; Park, Hee-Won; Hargrove, Tatiana Y.; Vanhollebeke, Benoit; Wawrzak, Zdzislaw; Harp, Joel M.; Sundaramoorthy, Munirathinam; Nes, W. David; Pays, Etienne; Chaudhuri, Minu; Villalta, Fernando; Waterman, Michael R. (ULdB); (Vanderbilt); (TTU); (Toronto); (NWU); (Meharry)

    2010-01-25

    Sterol 14{alpha}-demethylase (14DM, the CYP51 family of cytochrome P450) is an essential enzyme in sterol biosynthesis in eukaryotes. It serves as a major drug target for fungal diseases and can potentially become a target for treatment of human infections with protozoa. Here we present 1.9 {angstrom} resolution crystal structures of 14DM from the protozoan pathogen Trypanosoma brucei, ligand-free and complexed with a strong chemically selected inhibitor N-1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl-4-(5-phenyl-1,3,4-oxadi-azol-2-yl)benzamide that we previously found to produce potent antiparasitic effects in Trypanosomatidae. This is the first structure of a eukaryotic microsomal 14DM that acts on sterol biosynthesis, and it differs profoundly from that of the water-soluble CYP51 family member from Mycobacterium tuberculosis, both in organization of the active site cavity and in the substrate access channel location. Inhibitor binding does not cause large scale conformational rearrangements, yet induces unanticipated local alterations in the active site, including formation of a hydrogen bond network that connects, via the inhibitor amide group fragment, two remote functionally essential protein segments and alters the heme environment. The inhibitor binding mode provides a possible explanation for both its functionally irreversible effect on the enzyme activity and its selectivity toward the 14DM from human pathogens versus the human 14DM ortholog. The structures shed new light on 14DM functional conservation and open an excellent opportunity for directed design of novel antiparasitic drugs.

  15. Functional and structural insights revealed by molecular dynamics simulations of an essential RNA editing ligase in Trypanosoma brucei.

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    Rommie E Amaro

    2007-11-01

    Full Text Available RNA editing ligase 1 (TbREL1 is required for the survival of both the insect and bloodstream forms of Trypanosoma brucei, the parasite responsible for the devastating tropical disease African sleeping sickness. The type of RNA editing that TbREL1 is involved in is unique to the trypanosomes, and no close human homolog is known to exist. In addition, the high-resolution crystal structure revealed several unique features of the active site, making this enzyme a promising target for structure-based drug design. In this work, two 20 ns atomistic molecular dynamics (MD simulations are employed to investigate the dynamics of TbREL1, both with and without the ATP substrate present. The flexibility of the active site, dynamics of conserved residues and crystallized water molecules, and the interactions between TbREL1 and the ATP substrate are investigated and discussed in the context of TbREL1's function. Differences in local and global motion upon ATP binding suggest that two peripheral loops, unique to the trypanosomes, may be involved in interdomain signaling events. Notably, a significant structural rearrangement of the enzyme's active site occurs during the apo simulations, opening an additional cavity adjacent to the ATP binding site that could be exploited in the development of effective inhibitors directed against this protozoan parasite. Finally, ensemble averaged electrostatics calculations over the MD simulations reveal a novel putative RNA binding site, a discovery that has previously eluded scientists. Ultimately, we use the insights gained through the MD simulations to make several predictions and recommendations, which we anticipate will help direct future experimental studies and structure-based drug discovery efforts against this vital enzyme.

  16. Nifurtimox-eflornithine combination therapy for second-stage Trypanosoma brucei gambiense sleeping sickness: a randomized clinical trial in Congo.

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    Priotto, Gerardo; Kasparian, Serena; Ngouama, Daniel; Ghorashian, Sara; Arnold, Ute; Ghabri, Salah; Karunakara, Unni

    2007-12-01

    Human African trypanosomiasis caused by Trypanosoma brucei gambiense is a fatal disease. Current treatment options for patients with second-stage disease are either highly toxic or impracticable in field conditions. We compared the efficacy and safety of the nifurtimox-eflornithine drug combination with the standard eflornithine regimen for the treatment of second-stage disease. A randomized, open-label, active-control, phase III clinical trial comparing 2 arms was conducted at the Sleeping Sickness Treatment Center, which was run by Medecins Sans Frontieres, in Nkayi, Bouenza Province, Republic of Congo. Patients were screened for inclusion and randomly assigned to receive eflornithine alone (400 mg/kg per day given intravenously every 6 h for 14 days) or eflornithine (400 mg/kg per day given intravenously every 12 h for 7 days) plus nifurtimox (15 mg/kg per day given orally every 8 h for 10 days). Patients were observed for 18 months. The study's outcomes were cure and adverse events attributable to treatment. A total of 103 patients with second-stage disease were enrolled. Cure rates were 94.1% for the eflornithine group and 96.2% for the nifurtimox-eflornithine group. Drug reactions were frequent in both arms, and severe reactions affected 25.5% of patients in the eflornithine group and 9.6% of those in the nifurtimox-eflornithine group, resulting in 2 and 1 treatment suspensions, respectively. There was 1 death in the eflornithine arm and no deaths in the nifurtimox-eflornithine arm. The nifurtimox-eflornithine combination appears to be a promising first-line therapy for second-stage sleeping sickness. If our findings are corroborated by ongoing findings from additional sites (a multicenter extension of this study), the new nifurtimox-eflornithine combination therapy will mark a major and multifaceted advance over current therapies.

  17. Trypanosoma brucei Inhibition by Essential Oils from Medicinal and Aromatic Plants Traditionally Used in Cameroon (Azadirachta indica, Aframomum melegueta, Aframomum daniellii, Clausena anisata, Dichrostachys cinerea and Echinops giganteus).

    Science.gov (United States)

    Kamte, Stephane L Ngahang; Ranjbarian, Farahnaz; Campagnaro, Gustavo Daniel; Nya, Prosper C Biapa; Mbuntcha, Hélène; Woguem, Verlaine; Womeni, Hilaire Macaire; Ta, Léon Azefack; Giordani, Cristiano; Barboni, Luciano; Benelli, Giovanni; Cappellacci, Loredana; Hofer, Anders; Petrelli, Riccardo; Maggi, Filippo

    2017-07-06

    Essential oils are complex mixtures of volatile components produced by the plant secondary metabolism and consist mainly of monoterpenes and sesquiterpenes and, to a minor extent, of aromatic and aliphatic compounds. They are exploited in several fields such as perfumery, food, pharmaceutics, and cosmetics. Essential oils have long-standing uses in the treatment of infectious diseases and parasitosis in humans and animals. In this regard, their therapeutic potential against human African trypanosomiasis (HAT) has not been fully explored. In the present work, we have selected six medicinal and aromatic plants ( Azadirachta indica , Aframomum melegueta , Aframomum daniellii , Clausena anisata , Dichrostachys cinerea , and Echinops giganteus ) traditionally used in Cameroon to treat several disorders, including infections and parasitic diseases, and evaluated the activity of their essential oils against Trypanosma brucei TC221. Their selectivity was also determined with Balb/3T3 (mouse embryonic fibroblast cell line) cells as a reference. The results showed that the essential oils from A. indica , A . daniellii , and E. giganteus were the most active ones, with half maximal inhibitory concentration (IC 50 ) values of 15.21, 7.65, and 10.50 µg/mL, respectively. These essential oils were characterized by different chemical compounds such as sesquiterpene hydrocarbons, monoterpene hydrocarbons, and oxygenated sesquiterpenes. Some of their main components were assayed as well on T. brucei TC221, and their effects were linked to those of essential oils.

  18. RNA interference analyses suggest a transcript-specific regulatory role for mitochondrial RNA-binding proteins MRP1 and MRP2 in RNA editing and other RNA processing in Trypanosoma brucei

    NARCIS (Netherlands)

    Vondrusková, Eva; van den Burg, Janny; Zíková, Alena; Ernst, Nancy Lewis; Stuart, Kenneth; Benne, Rob; Lukes, Julius

    2005-01-01

    Mitochondrial RNA-binding proteins MRP1 and MRP2 occur in a heteromeric complex that appears to play a role in U-insertion/deletion editing in trypanosomes. Reduction in the levels of MRP1 (gBP21) and/or MRP2 (gBP25) mRNA by RNA interference in procyclic Trypanosoma brucei resulted in severe growth

  19. Expression of Trypanosoma brucei gambiense Antigens in Leishmania tarentolae. Potential for Use in Rapid Serodiagnostic Tests (RDTs.

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    Barrie Rooney

    2015-12-01

    Full Text Available The development of rapid serodiagnostic tests for sleeping sickness and other diseases caused by kinetoplastids relies on the affordable production of parasite-specific recombinant antigens. Here, we describe the production of recombinant antigens from Trypanosoma brucei gambiense (T.b. gambiense in the related species Leishmania tarentolae (L. tarentolae, and compare their diagnostic sensitivity and specificity to native antigens currently used in diagnostic kits against a panel of human sera. A number of T.b. gambiense protein antigen candidates were chosen for recombinant expression in L. tarentolae based on current diagnostics in field use and recent findings on immunodiagnostic antigens found by proteomic profiling. In particular, the extracellular domains of invariant surface glycoprotein 65 (ISG65, variant surface glycoproteins VSG LiTat 1.3 and VSG LiTat 1.5 were fused with C-terminal histidine tags and expressed as soluble proteins in the medium of cultured, recombinant L. tarentolae. Using affinity chromatography, on average 10 mg/L of recombinant protein was purified from cultures and subsequently tested against a panel of sera from sleeping sickness patients from controls, i.e. persons without sleeping sickness living in HAT endemic countries. The evaluation on sera from 172 T.b. gambiense human African trypanosomiasis (HAT patients and from 119 controls showed very high diagnostic potential of the two recombinant VSG and the rISG65 fragments with areas under the curve between 0.97 and 0.98 compared to 0.98 and 0.99 with native VSG LiTat 1.3 and VSG LiTat 1.5 (statistically not different. Evaluation on sera from 78 T.b. rhodesiense HAT patients and from 100 controls showed an acceptable diagnostic potential of rISG65 with an area under the curve of 0.83. These results indicate that a combination of these recombinant antigens has the potential to be used in next generation rapid serodiagnostic tests. In addition, the L. tarentolae

  20. Blocking Synthesis of the Variant Surface Glycoprotein Coat in Trypanosoma brucei Leads to an Increase in Macrophage Phagocytosis Due to Reduced Clearance of Surface Coat Antibodies.

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    Jackie L Y Cheung

    2016-11-01

    Full Text Available The extracellular bloodstream form parasite Trypanosoma brucei is supremely adapted to escape the host innate and adaptive immune system. Evasion is mediated through an antigenically variable Variant Surface Glycoprotein (VSG coat, which is recycled at extraordinarily high rates. Blocking VSG synthesis triggers a precytokinesis arrest where stalled cells persist for days in vitro with superficially intact VSG coats, but are rapidly cleared within hours in mice. We therefore investigated the role of VSG synthesis in trypanosome phagocytosis by activated mouse macrophages. T. brucei normally effectively evades macrophages, and induction of VSG RNAi resulted in little change in phagocytosis of the arrested cells. Halting VSG synthesis resulted in stalled cells which swam directionally rather than tumbling, with a significant increase in swim velocity. This is possibly a consequence of increased rigidity of the cells due to a restricted surface coat in the absence of VSG synthesis. However if VSG RNAi was induced in the presence of anti-VSG221 antibodies, phagocytosis increased significantly. Blocking VSG synthesis resulted in reduced clearance of anti-VSG antibodies from the trypanosome surface, possibly as a consequence of the changed motility. This was particularly marked in cells in the G2/ M cell cycle stage, where the half-life of anti-VSG antibody increased from 39.3 ± 4.2 seconds to 99.2 ± 15.9 seconds after induction of VSG RNAi. The rates of internalisation of bulk surface VSG, or endocytic markers like transferrin, tomato lectin or dextran were not significantly affected by the VSG synthesis block. Efficient elimination of anti-VSG-antibody complexes from the trypanosome cell surface is therefore essential for trypanosome evasion of macrophages. These experiments highlight the essentiality of high rates of VSG recycling for the rapid removal of host opsonins from the parasite surface, and identify this process as a key parasite

  1. The orthologue of Sjögren's syndrome nuclear autoantigen 1 (SSNA1 in Trypanosoma brucei is an immunogenic self-assembling molecule.

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    Helen P Price

    Full Text Available Primary Sjögren's Syndrome (PSS is a highly prevalent autoimmune disease, typically manifesting as lymphocytic infiltration of the exocrine glands leading to chronically impaired lacrimal and salivary secretion. Sjögren's Syndrome nuclear autoantigen 1 (SSNA1 or NA14 is a major specific target for autoantibodies in PSS but the precise function and clinical relevance of this protein are largely unknown. Orthologues of the gene are absent from many of the commonly used model organisms but are present in Chlamyodomonas reinhardtii (in which it has been termed DIP13 and most protozoa. We report the functional characterisation of the orthologue of SSNA1 in the kinetoplastid parasite, Trypanosoma brucei. Both TbDIP13 and human SSNA1 are small coiled-coil proteins which are predicted to be remote homologues of the actin-binding protein tropomyosin. We use comparative proteomic methods to identify potential interacting partners of TbDIP13. We also show evidence that TbDIP13 is able to self-assemble into fibril-like structures both in vitro and in vivo, a property which may contribute to its immunogenicity. Endogenous TbDIP13 partially co-localises with acetylated α-tubulin in the insect procyclic stage of the parasite. However, deletion of the DIP13 gene in cultured bloodstream and procyclic stages of T. brucei has little effect on parasite growth or morphology, indicating either a degree of functional redundancy or a function in an alternative stage of the parasite life cycle.

  2. Characterization of a Novel Class I Transcription Factor A (CITFA) Subunit That Is Indispensable for Transcription by the Multifunctional RNA Polymerase I of Trypanosoma brucei

    KAUST Repository

    Nguyen, T. N.

    2012-10-26

    Trypanosoma brucei is the only organism known to have evolved a multifunctional RNA polymerase I (pol I) system that is used to express the parasite\\'s ribosomal RNAs, as well as its major cell surface antigens, namely, the variant surface glycoprotein (VSG) and procyclin, which are vital for establishing successful infections in the mammalian host and the tsetse vector, respectively. Thus far, biochemical analyses of the T. brucei RNA pol I transcription machinery have elucidated the subunit structure of the enzyme and identified the class I transcription factor A (CITFA). CITFA binds to RNA pol I promoters, and its CITFA-2 subunit was shown to be absolutely essential for RNA pol I transcription in the parasite. Tandem affinity purification (TAP) of CITFA revealed the subunits CITFA-1 to -6, which are conserved only among kinetoplastid organisms, plus the dynein light chain DYNLL1. Here, by tagging CITFA-6 instead of CITFA-2, a complex was purified that contained all known CITFA subunits, as well as a novel proline-rich protein. Functional studies carried out in vivo and in vitro, as well as a colocalization study, unequivocally demonstrated that this protein is a bona fide CITFA subunit, essential for parasite viability and indispensable for RNA pol I transcription of ribosomal gene units and the active VSG expression site in the mammalian-infective life cycle stage of the parasite. Interestingly, CITFA-7 function appears to be species specific, because expression of an RNA interference (RNAi)-resistant CITFA-7 transgene from Trypanosoma cruzi could not rescue the lethal phenotype of silencing endogenous CITFA-7.

  3. The role of B-cells and IgM antibodies in parasitemia, anemia, and VSG switching in Trypanosoma brucei-infected mice.

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    Stefan Magez

    Full Text Available African trypanosomes are extracellular parasitic protozoa, predominantly transmitted by the bite of the haematophagic tsetse fly. The main mechanism considered to mediate parasitemia control in a mammalian host is the continuous interaction between antibodies and the parasite surface, covered by variant-specific surface glycoproteins. Early experimental studies have shown that B-cell responses can be strongly protective but are limited by their VSG-specificity. We have used B-cell (microMT and IgM-deficient (IgM(-/- mice to investigate the role of B-cells and IgM antibodies in parasitemia control and the in vivo induction of trypanosomiasis-associated anemia. These infection studies revealed that that the initial setting of peak levels of parasitemia in Trypanosoma brucei-infected microMT and IgM(-/- mice occurred independent of the presence of B-cells. However, B-cells helped to periodically reduce circulating parasites levels and were required for long term survival, while IgM antibodies played only a limited role in this process. Infection-associated anemia, hypothesized to be mediated by B-cell responses, was induced during infection in microMT mice as well as in IgM(-/- mice, and as such occurred independently from the infection-induced host antibody response. Antigenic variation, the main immune evasion mechanism of African trypanosomes, occurred independently from host antibody responses against the parasite's ever-changing antigenic glycoprotein coat. Collectively, these results demonstrated that in murine experimental T. brucei trypanosomiasis, B-cells were crucial for periodic peak parasitemia clearance, whereas parasite-induced IgM antibodies played only a limited role in the outcome of the infection.

  4. Kinetic and mutational analysis of the Trypanosoma brucei NBT1 nucleobase transporter expressed in Saccharomyces cerevisiae reveals structural similarities between ENT and MFS transporters.

    Science.gov (United States)

    Papageorgiou, I; De Koning, H P; Soteriadou, K; Diallinas, G

    2008-05-01

    Parasitic protozoa are unable to synthesise purines de novo and thus depend on the uptake of nucleosides and nucleobases across their plasma membrane through specific transporters. A number of nucleoside and nucleobase transporters from Trypanosoma brucei brucei and Leishmania major have recently been characterised and shown to belong to the equilibrative nucleoside transporter (ENT) family. A number of studies have demonstrated the functional importance of particular transmembrane segments (TMS) in nucleoside-specific ENT proteins. TbNBT1, one of only three bona fide nucleobase-selective members of the ENT family, has previously been shown to be a high-affinity transporter for purine nucleobases and guanosine. In this study, we use the Saccharomyces cerevisiae expression system to build a biochemical model of how TbNBT1 recognises nucleobases. We next performed random in vitro and site-directed mutagenesis to identify residues critical for TbNBT1 function. The identification of residues likely to contribute to permeant binding, when combined with a structural model of TbNBT1 obtained by homology threading, yield a tentative three-dimensional model of the transporter binding site that is consistent with the binding model emerging from the biochemical data. The model strongly suggests the involvement of TMS5, TMS7 and TMS8 in TbNBT1 function. This situation is very similar to that concerning transporters of the major facilitator superfamily (MFS), one of which was used as a template for the threading. This point raises the possibility that ENT and MFS carriers, despite being considered evolutionarily distinct, might in fact share similar topologies and substrate translocations pathways.

  5. Oral fexinidazole for late-stage African Trypanosoma brucei gambiense trypanosomiasis: a pivotal multicentre, randomised, non-inferiority trial.

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    Mesu, Victor Kande Betu Ku; Kalonji, Wilfried Mutombo; Bardonneau, Clélia; Mordt, Olaf Valverde; Blesson, Séverine; Simon, François; Delhomme, Sophie; Bernhard, Sonja; Kuziena, Willy; Lubaki, Jean-Pierre Fina; Vuvu, Steven Lumeya; Ngima, Pathou Nganzobo; Mbembo, Hélène Mahenzi; Ilunga, Médard; Bonama, Augustin Kasongo; Heradi, Josué Amici; Solomo, Jean Louis Lumaliza; Mandula, Guylain; Badibabi, Lewis Kaninda; Dama, Francis Regongbenga; Lukula, Papy Kavunga; Tete, Digas Ngolo; Lumbala, Crispin; Scherrer, Bruno; Strub-Wourgaft, Nathalie; Tarral, Antoine

    2017-11-04

    Few therapeutic options are available to treat the late-stage of human African trypanosomiasis, a neglected tropical disease, caused by Trypanosoma brucei gambiense (g-HAT). The firstline treatment is a combination therapy of oral nifurtimox and intravenous eflornithine that needs to be administered in a hospital setting by trained personnel, which is not optimal given that patients often live in remote areas with few health resources. Therefore, we aimed to assess the safety and efficacy of an oral regimen of fexinidazole (a 2-substituted 5-nitroimidazole with proven trypanocidal activity) versus nifurtimox eflornithine combination therapy in patients with late-stage g-HAT. In this randomised, phase 2/3, open-label, non-inferiority trial, we recruited patients aged 15 years and older with late-stage g-HAT from g-HAT treatment centres in the Democratic Republic of the Congo (n=9) and the Central African Republic (n=1). Patients were randomly assigned (2:1) to receive either fexinidazole or nifurtimox eflornithine combination therapy according to a predefined randomisation list (block size six). The funder, data management personnel, and study statisticians were masked to treatment. Oral fexinidazole was given once a day (days 1-4: 1800 mg, days 5-10: 1200 mg). Oral nifurtimox was given three times a day (days 1-10: 15 mg/kg per day) with eflornithine twice a day as 2 h infusions (days 1-7: 400 mg/kg per day). The primary endpoint was success at 18 months (ie, deemed as patients being alive, having no evidence of trypanosomes in any body fluid, not requiring rescue medication, and having a cerebrospinal fluid white blood cell count ≤20 cells per μL). Safety was assessed through routine monitoring. Primary efficacy analysis was done in the modified intention-to-treat population and safety analyses in the intention-to-treat population. The acceptable margin for the difference in success rates was defined as 13%. This study has been completed and is registered with

  6. The study of trypanosome species circulating in domestic animals in two human African trypanosomiasis foci of Côte d'Ivoire identifies pigs and cattle as potential reservoirs of Trypanosoma brucei gambiense.

    Science.gov (United States)

    N'Djetchi, Martial Kassi; Ilboudo, Hamidou; Koffi, Mathurin; Kaboré, Jacques; Kaboré, Justin Windingoudi; Kaba, Dramane; Courtin, Fabrice; Coulibaly, Bamoro; Fauret, Pierre; Kouakou, Lingué; Ravel, Sophie; Deborggraeve, Stijn; Solano, Philippe; De Meeûs, Thierry; Bucheton, Bruno; Jamonneau, Vincent

    2017-10-01

    Important control efforts have led to a significant reduction of the prevalence of human African trypanosomiasis (HAT) in Côte d'Ivoire, but the disease is still present in several foci. The existence of an animal reservoir of Trypanosoma brucei gambiense may explain disease persistence in these foci where animal breeding is an important source of income but where the prevalence of animal African trypanosomiasis (AAT) is unknown. The aim of this study was to identify the trypanosome species circulating in domestic animals in both Bonon and Sinfra HAT endemic foci. 552 domestic animals (goats, pigs, cattle and sheep) were included. Blood samples were tested for trypanosomes by microscopic observation, species-specific PCR for T. brucei sl, T. congolense, T. vivax and subspecies-specific PCR for T. b. gambiense and T. b. gambiense immune trypanolysis (TL). Infection rates varied significantly between animal species and were by far the highest in pigs (30%). T. brucei s.l was the most prevalent trypanosome species (13.7%) followed by T. congolense. No T. b. gambiense was identified by PCR while high TL positivity rates were observed using T. b. gambiense specific variants (up to 27.6% for pigs in the Bonon focus). This study shows that domestic animals are highly infected by trypanosomes in the studied foci. This was particularly true for pigs, possibly due to a higher exposure of these animals to tsetse flies. Whereas T. brucei s.l. was the most prevalent species, discordant results were obtained between PCR and TL regarding T. b. gambiense identification. It is therefore crucial to develop better tools to study the epidemiological role of potential animal reservoir for T. b. gambiense. Our study illustrates the importance of "one health" approaches to reach HAT elimination and contribute to AAT control in the studied foci.

  7. Megazol and its bioisostere 4H-1,2,4-triazole: comparing the trypanocidal, cytotoxic and genotoxic activities and their in vitro and in silico interactions with the Trypanosoma brucei nitroreductase enzyme

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    Alcione Silva de Carvalho

    2014-06-01

    Full Text Available Megazol (7 is a 5-nitroimidazole that is highly active against Trypanosoma cruzi and Trypanosoma brucei, as well as drug-resistant forms of trypanosomiasis. Compound 7 is not used clinically due to its mutagenic and genotoxic properties, but has been largely used as a lead compound. Here, we compared the activity of 7 with its 4H-1,2,4-triazole bioisostere (8 in bloodstream forms of T. brucei and T. cruzi and evaluated their activation by T. brucei type I nitroreductase (TbNTR enzyme. We also analysed the cytotoxic and genotoxic effects of these compounds in whole human blood using Comet and fluorescein diacetate/ethidium bromide assays. Although the only difference between 7 and 8 is the substitution of sulphur (in the thiadiazole in 7 for nitrogen (in the triazole in 8, the results indicated that 8 had poorer antiparasitic activity than 7 and was not genotoxic, whereas 7 presented this effect. The determination of Vmax indicated that although 8 was metabolised more rapidly than 7, it bounds to the TbNTR with better affinity, resulting in equivalent kcat/KM values. Docking assays of 7 and 8 performed within the active site of a homology model of the TbNTR indicating that 8 had greater affinity than 7.

  8. Gene conversion transfers the GAF-A domain of phosphodiesterase TbrPDEB1 to one allele of TbrPDEB2 of Trypanosoma brucei.

    Science.gov (United States)

    Kunz, Stefan; Luginbuehl, Edith; Seebeck, Thomas

    2009-06-09

    Chromosome 9 of Trypanosoma brucei contains two closely spaced, very similar open reading frames for cyclic nucleotide specific phosphodiesterases TbrPDEB1 and TbrPDEB2. They are separated by 2379 bp, and both code for phosphodiesterases with two GAF domains in their N-terminal moieties and a catalytic domain at the C-terminus. The current study reveals that in the Lister427 strain of T. brucei, these two genes have undergone gene conversion, replacing the coding region for the GAF-A domain of TbrPDEB2 by the corresponding region of the upstream gene TbrPDEB1. As a consequence, these strains express two slightly different versions of TbrPDEB2. TbrPDEB2a represents the wild-type phosphodiesterase, while TbrPDEB2b represents the product of the converted gene. Earlier work on the subcellular localization of TbrPDEB1 and TbrPDEB2 had demonstrated that TbrPDEB1 is predominantly located in the flagellum, whereas TbrPDEB2 partially locates to the flagellum but largely remains in the cell body. The current findings raised the question of whether this dual localization of TbrPDEB2 may reflect the two alleles. To resolve this, TbrPDEB2 of strain STIB247 that is homozygous for TbrPDEB2a was tagged in situ, and its intracellular localization was analyzed. The results obtained were very similar to those found earlier with Lister427, indicating that the dual localization of TbrPDEB2 reflects its true function and is not simply due to the presence of the two different alleles. Notably, the gene conversion event is unique for the Lister427 strain and all its derivatives. Based on this finding, a convenient PCR test has been developed that allows the stringent discrimination between Lister-derived strains that are common in many laboratories and other isolates. The technique is likely very useful to resolve questions about potential mix-ups of precious field isolates with the ubiquitous Lister strain.

  9. Gene conversion transfers the GAF-A domain of phosphodiesterase TbrPDEB1 to one allele of TbrPDEB2 of Trypanosoma brucei.

    Directory of Open Access Journals (Sweden)

    Stefan Kunz

    Full Text Available BACKGROUND: Chromosome 9 of Trypanosoma brucei contains two closely spaced, very similar open reading frames for cyclic nucleotide specific phosphodiesterases TbrPDEB1 and TbrPDEB2. They are separated by 2379 bp, and both code for phosphodiesterases with two GAF domains in their N-terminal moieties and a catalytic domain at the C-terminus. METHODS AND FINDINGS: The current study reveals that in the Lister427 strain of T. brucei, these two genes have undergone gene conversion, replacing the coding region for the GAF-A domain of TbrPDEB2 by the corresponding region of the upstream gene TbrPDEB1. As a consequence, these strains express two slightly different versions of TbrPDEB2. TbrPDEB2a represents the wild-type phosphodiesterase, while TbrPDEB2b represents the product of the converted gene. Earlier work on the subcellular localization of TbrPDEB1 and TbrPDEB2 had demonstrated that TbrPDEB1 is predominantly located in the flagellum, whereas TbrPDEB2 partially locates to the flagellum but largely remains in the cell body. The current findings raised the question of whether this dual localization of TbrPDEB2 may reflect the two alleles. To resolve this, TbrPDEB2 of strain STIB247 that is homozygous for TbrPDEB2a was tagged in situ, and its intracellular localization was analyzed. CONCLUSIONS: The results obtained were very similar to those found earlier with Lister427, indicating that the dual localization of TbrPDEB2 reflects its true function and is not simply due to the presence of the two different alleles. Notably, the gene conversion event is unique for the Lister427 strain and all its derivatives. Based on this finding, a convenient PCR test has been developed that allows the stringent discrimination between Lister-derived strains that are common in many laboratories and other isolates. The technique is likely very useful to resolve questions about potential mix-ups of precious field isolates with the ubiquitous Lister strain.

  10. Structural characterization reveals a novel bilobed architecture for the ectodomains of insect stage expressed Trypanosoma brucei PSSA-2 and Trypanosoma congolense ISA.

    Science.gov (United States)

    Ramaswamy, Raghavendran; Goomeshi Nobary, Sarah; Eyford, Brett A; Pearson, Terry W; Boulanger, Martin J

    2016-12-01

    African trypanosomiasis, caused by parasites of the genus Trypanosoma, is a complex of devastating vector-borne diseases of humans and livestock in sub-Saharan Africa. Central to the pathogenesis of African trypanosomes is their transmission by the arthropod vector, Glossina spp. (tsetse fly). Intriguingly, the efficiency of parasite transmission through the vector is reduced following depletion of Trypanosoma brucei Procyclic-Specific Surface Antigen-2 (TbPSSA-2). To investigate the underlying molecular mechanism of TbPSSA-2, we determined the crystal structures of its ectodomain and that of its homolog T. congolense Insect Stage Antigen (TcISA) to resolutions of 1.65 Å and 2.45 Å, respectively using single wavelength anomalous dispersion. Both proteins adopt a novel bilobed architecture with the individual lobes displaying rotational flexibility around the central tether that suggest a potential mechanism for coordinating a binding partner. In support of this hypothesis, electron density consistent with a bound peptide was observed in the inter-lob cleft of a TcISA monomer. These first reported structures of insect stage transmembrane proteins expressed by African trypanosomes provide potentially valuable insight into the interface between parasite and tsetse vector. © 2016 The Protein Society.

  11. Crassiflorone derivatives that inhibit Trypanosoma brucei glyceraldehyde-3-phosphate dehydrogenase (TbGAPDH) and Trypanosoma cruzi trypanothione reductase (TcTR) and display trypanocidal activity.

    Science.gov (United States)

    Uliassi, Elisa; Fiorani, Giulia; Krauth-Siegel, R Luise; Bergamini, Christian; Fato, Romana; Bianchini, Giulia; Carlos Menéndez, J; Molina, Maria Teresa; López-Montero, Eulogio; Falchi, Federico; Cavalli, Andrea; Gul, Sheraz; Kuzikov, Maria; Ellinger, Bernhard; Witt, Gesa; Moraes, Carolina B; Freitas-Junior, Lucio H; Borsari, Chiara; Costi, Maria Paola; Bolognesi, Maria Laura

    2017-12-01

    Crassiflorone is a natural product with anti-mycobacterial and anti-gonorrhoeal properties, isolated from the stem bark of the African ebony tree Diospyros crassiflora. We noticed that its pentacyclic core possesses structural resemblance to the quinone-coumarin hybrid 3, which we reported to exhibit a dual-targeted inhibitory profile towards Trypanosoma brucei glyceraldehyde-3-phosphate dehydrogenase (TbGAPDH) and Trypanosoma cruzi trypanothione reductase (TcTR). Following this basic idea, we synthesized a small library of crassiflorone derivatives 15-23 and investigated their potential as anti-trypanosomatid agents. 19 is the only compound of the series showing a balanced dual profile at 10 μM (% inhibition TbGAPDH  = 64% and % inhibition TcTR  = 65%). In phenotypic assay, the most active compounds were 18 and 21, which at 5 μM inhibited Tb bloodstream-form growth by 29% and 38%, respectively. Notably, all the newly synthesized compounds at 10 μM did not affect viability and the status of mitochondria in human A549 and 786-O cell lines, respectively. However, further optimization that addresses metabolic liabilities including solubility, as well as cytochromes P450 (CYP1A2, CYP2C9, CYP2C19, and CYP2D6) inhibition, is required before this class of natural product-derived compounds can be further progressed. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  12. Proteins and lipids of glycosomal membranes from Leishmania tarentolae and Trypanosoma brucei [v1; ref status: indexed, http://f1000r.es/x1

    Directory of Open Access Journals (Sweden)

    Claudia Colasante

    2013-01-01

    Full Text Available In kinetoplastid protists, several metabolic pathways, including glycolysis and purine salvage, are located in glycosomes, which are microbodies that are evolutionarily related to peroxisomes. With the exception of some potential transporters for fatty acids, and one member of the mitochondrial carrier protein family, proteins that transport metabolites across the glycosomal membrane have yet to be identified. We show here that the phosphatidylcholine species composition of Trypanosoma brucei glycosomal membranes resembles that of other cellular membranes, which means that glycosomal membranes are expected to be impermeable to small hydrophilic molecules unless transport is facilitated by specialized membrane proteins. Further, we identified 464 proteins in a glycosomal membrane preparation from Leishmania tarentolae. The proteins included approximately 40 glycosomal matrix proteins, and homologues of peroxisomal membrane proteins - PEX11, GIM5A and GIM5B; PXMP4, PEX2 and PEX16 - as well as the transporters GAT1 and GAT3. There were 27 other proteins that could not be unambiguously assigned to other compartments, and that had predicted trans-membrane domains. However, no clear candidates for transport of the major substrates and intermediates of energy metabolism were found. We suggest that, instead, these metabolites are transported via pores formed by the known glycosomal membrane proteins.

  13. A comparative proteomic analysis reveals a new bi-lobe protein required for bi-lobe duplication and cell division in Trypanosoma brucei.

    Directory of Open Access Journals (Sweden)

    Qing Zhou

    Full Text Available A Golgi-associated bi-lobed structure was previously found to be important for Golgi duplication and cell division in Trypanosoma brucei. To further understand its functions, comparative proteomics was performed on extracted flagellar complexes (including the flagellum and flagellum-associated structures such as the basal bodies and the bi-lobe and purified flagella to identify new bi-lobe proteins. A leucine-rich repeats containing protein, TbLRRP1, was characterized as a new bi-lobe component. The anterior part of the TbLRRP1-labeled bi-lobe is adjacent to the single Golgi apparatus, and the posterior side is tightly associated with the flagellar pocket collar marked by TbBILBO1. Inducible depletion of TbLRRP1 by RNA interference inhibited duplication of the bi-lobe as well as the adjacent Golgi apparatus and flagellar pocket collar. Formation of a new flagellum attachment zone and subsequent cell division were also inhibited, suggesting a central role of bi-lobe in Golgi, flagellar pocket collar and flagellum attachment zone biogenesis.

  14. 3-(3-amino-3-carboxypropyl)-5,6-Dihydrouridine is one of two novel post-transcriptional modifications in tRNALys(UUU) from Trypanosoma brucei

    DEFF Research Database (Denmark)

    Krog, Jesper Schak; Español, Yaiza; Giessing, Anders M B

    2011-01-01

    was MALDI-TOF MS of two independent digests of the tRNA, with RNase A and RNase T1, respectively. This revealed digestion products harbouring mass-changing modifications. Next, the modifications were mapped at the nucleotide level in the RNase products by tandem MS. Comparison with the sequence....... Furthermore, the tRNA has to be investigated with single-nucleotide resolution in order to ensure complete mapping of all modifications. In the present work, we characterized tRNA(Lys) (UUU) from Trypanosoma brucei, and provide a complete overview of its post-transcriptional modifications. The first step...

  15. First evidence that parasite infecting apparent aparasitemic serological suspects in human African trypanosomiasis are Trypanosoma brucei gambiense and are similar to those found in patients.

    Science.gov (United States)

    Kaboré, Jacques; Koffi, Mathurin; Bucheton, Bruno; MacLeod, Annette; Duffy, Craig; Ilboudo, Hamidou; Camara, Mamadou; De Meeûs, Thierry; Belem, Adrien Marie Gaston; Jamonneau, Vincent

    2011-08-01

    Thanks to its sensitivity and its ease of use in the field, the card agglutination test for trypanosomiasis (CATT) is widely used for serological screening of Trypanosoma brucei gambiense human African trypanosomiasis (HAT). Positive subjects are then examined by microscopy to confirm the disease. However, the CATT exhibits false-positive results raising the question of whether CATT-positive subjects who are not confirmed by microscopic detection of trypanosomes (SERO) are truly exposed to T.b. gambiense infection. For this purpose, we applied microsatellite genotyping on DNA extracted from blood of both HAT confirmed patients and SERO subjects in Guinea and Côte d'Ivoire since microsatellite genotyping has proved useful for the study of T.b. gambiense genetic diversity. Problems of amplification failures raise the question of the sensitivity of microsatellite markers when applied on biological samples especially from SERO subjects for who low blood parasitaemia are suspected. Nevertheless, we have shown that the trypanosomes from SERO individuals that have been genotyped belong to T.b. gambiense group 1 and were identical to those found in HAT patients. These results constitute the first evidences that at least some SERO are indeed infected by T.b. gambiense group 1 and that they may constitute a human reservoir of parasite in HAT foci. Whether these individuals should undergo treatment remains an open question as long as their role in HAT transmission is unknown. Our results strongly recommend the follow-up of such subjects to improve control strategies. Copyright © 2011 Elsevier B.V. All rights reserved.

  16. Structure determination of glycogen synthase kinase-3 from Leishmania major and comparative inhibitor structure-activity relationships with Trypanosoma brucei GSK-3

    Energy Technology Data Exchange (ETDEWEB)

    Ojo, Kayode K; Arakaki, Tracy L; Napuli, Alberto J; Inampudi, Krishna K; Keyloun, Katelyn R; Zhang, Li; Hol, Wim G.J.; Verlind, Christophe L.M.J.; Merritt, Ethan A; Van Voorhis, Wesley C [UWASH

    2012-04-24

    Glycogen synthase kinase-3 (GSK-3) is a drug target under intense investigation in pharmaceutical companies and constitutes an attractive piggyback target for eukaryotic pathogens. Two different GSKs are found in trypanosomatids, one about 150 residues shorter than the other. GSK-3 short (GeneDB: Tb927.10.13780) has previously been validated genetically as a drug target in Trypanosoma brucei by RNAi induced growth retardation; and chemically by correlation between enzyme and in vitro growth inhibition. Here, we report investigation of the equivalent GSK-3 short enzymes of L. major (LmjF18.0270) and L. infantum (LinJ18_V3.0270, identical in amino acid sequences to LdonGSK-3 short) and a crystal structure of LmajGSK-3 short at 2 Å resolution. The inhibitor structure-activity relationships (SARs) of L. major and L. infantum are virtually identical, suggesting that inhibitors could be useful for both cutaneous and visceral leishmaniasis. Leishmania spp. GSK-3 short has different inhibitor SARs than TbruGSK-3 short, which can be explained mostly by two variant residues in the ATP-binding pocket. Indeed, mutating these residues in the ATP-binding site of LmajGSK-3 short to the TbruGSK-3 short equivalents results in a mutant LmajGSK-3 short enzyme with SAR more similar to that of TbruGSK-3 short. The differences between human GSK-3β (HsGSK-3β) and LmajGSK-3 short SAR suggest that compounds which selectively inhibit LmajGSK-3 short may be found.

  17. Equivalence trial of melarsoprol and nifurtimox monotherapy and combination therapy for the treatment of second-stage Trypanosoma brucei gambiense sleeping sickness.

    Science.gov (United States)

    Bisser, Sylvie; N'Siesi, François-Xavier; Lejon, Veerle; Preux, Pierre-Marie; Van Nieuwenhove, Simon; Miaka Mia Bilenge, Constantin; Būscher, Philippe

    2007-02-01

    Treatment of second-stage sleeping sickness relies mainly on melarsoprol. Nifurtimox has been successfully used to cure melarsoprol-refractory sleeping sickness caused by Trypanosoma brucei gambiense infection. An open, randomized trial was conducted to test for equivalence between the standard melarsoprol regimen and 3 other regimens, as follows: standard melarsoprol therapy (3 series of 3.6 mg/kg/day intravenously [iv] for 3 days, with 7-day breaks between the series); 10-day incremental-dose melarsoprol therapy (0.6 mg/kg iv on day 1, 1.2 mg/kg iv on day 2, and 1.8 mg/kg iv on days 3-10); nifurtimox monotherapy for 14 days (5 mg/kg orally 3 times per day); and consecutive 10-day melarsoprol-nifurtimox combination therapy (0.6 mg/kg iv melarsoprol on day 1, 1.2 mg/kg iv melarsoprol on day 2, and 1.2 mg/kg/day iv melarsoprol combined with oral 7.5 mg/kg nifurtimox twice a day on days 3-10). Primary outcomes were relapse, severe adverse events, and death attributed to treatment. A total of 278 patients were randomized. The frequency of adverse events was similar between the standard melarsoprol regimen and the other regimens. Encephalopathic syndromes occurred in all groups and caused all deaths that were likely due to treatment. Relapses (n=48) were observed only with the 3 monotherapy regimens. A consecutive 10-day low-dose melarsoprol-nifurtimox combination is more effective than the standard melarsoprol regimen.

  18. Coenzyme Q10 prevented full blown splenomegaly and decreased melarsoprol-induced reactive encephalopathy in mice infected with Trypanosoma brucei rhodesiense

    Directory of Open Access Journals (Sweden)

    James Nyabuga Nyariki

    2015-03-01

    Full Text Available Objective: To establish the modulatory effects of coenzyme Q10 on experimental trypanosome infections in mice and evaluate the risk of occurrence and severity of melarsoprol-induced post treatment reactive encephalopathy (PTRE. Methods: Female Swiss white mice were orally administered with 200 mg/kg of coenzyme Q10 after which they were intraperitoneally inoculated with Trypanasoma brucei rhodesiense (T. b. rhodesiense. The resultant infection was allowed to develop and simulate all phases of human African trypanosomiasis and PTRE. Parasitaemia development, packed cell volume, haematological and pathological changes were determined. Results: A histological study in the brain tissue of T. b. rhodesiense infected mice demonstrated neuroinflammatory pathology which was highly amplified in the PTRE-induced groups. A prominent reduction in the severity of the neuroinflammatory response was detected when coenzyme-Q10 was administered. Furthermore, the mean tissue weight of spleen to body ratio in coenzyme Q10 supplemented group was significantly (P<0.05 different compared to un-supplemented groups, and clearly indicated that coenzyme Q10 prevented full blown splenomegaly pathogenesis by T. b. rhodesiense. A significant (P<0.05 increase in hemoglobin levels and red blood cells was observed in coenzyme Q10 mice compared to those infected and un-supplemented with coenzyme Q10. Conclusions: The capacity of coenzyme Q10 to alter the pathogenesis of T. b. rhodesiense infection in mice and following treatment with melarsoprol, may find application by rendering humans and animals less susceptible to deleterious effects of trypanosome infection such as splenomegaly and melarsoprol-induced PTRE and neurotoxicity.

  19. Regulating the regulators : accountability of Australian regulators

    National Research Council Canada - National Science Library

    Bird, Joanna

    2011-01-01

    Accountability of Australian regulators - Australian Securities and Investments Commission - Australian Prudential Regulation Authority - concept of 'accountability' - mechanisms for accountability...

  20. Persistent ER stress induces the spliced leader RNA silencing pathway (SLS, leading to programmed cell death in Trypanosoma brucei.

    Directory of Open Access Journals (Sweden)

    Hanoch Goldshmidt

    2010-01-01

    Full Text Available Trypanosomes are parasites that cycle between the insect host (procyclic form and mammalian host (bloodstream form. These parasites lack conventional transcription regulation, including factors that induce the unfolded protein response (UPR. However, they possess a stress response mechanism, the spliced leader RNA silencing (SLS pathway. SLS elicits shut-off of spliced leader RNA (SL RNA transcription by perturbing the binding of the transcription factor tSNAP42 to its cognate promoter, thus eliminating trans-splicing of all mRNAs. Induction of endoplasmic reticulum (ER stress in procyclic trypanosomes elicits changes in the transcriptome similar to those induced by conventional UPR found in other eukaryotes. The mechanism of up-regulation under ER stress is dependent on differential stabilization of mRNAs. The transcriptome changes are accompanied by ER dilation and elevation in the ER chaperone, BiP. Prolonged ER stress induces SLS pathway. RNAi silencing of SEC63, a factor that participates in protein translocation across the ER membrane, or SEC61, the translocation channel, also induces SLS. Silencing of these genes or prolonged ER stress led to programmed cell death (PCD, evident by exposure of phosphatidyl serine, DNA laddering, increase in reactive oxygen species (ROS production, increase in cytoplasmic Ca(2+, and decrease in mitochondrial membrane potential, as well as typical morphological changes observed by transmission electron microscopy (TEM. ER stress response is also induced in the bloodstream form and if the stress persists it leads to SLS. We propose that prolonged ER stress induces SLS, which serves as a unique death pathway, replacing the conventional caspase-mediated PCD observed in higher eukaryotes.

  1. Dual functions of α-ketoglutarate dehydrogenase E2 in the Krebs cycle and mitochondrial DNA inheritance in Trypanosoma brucei.

    Science.gov (United States)

    Sykes, Steven E; Hajduk, Stephen L

    2013-01-01

    The dihydrolipoyl succinyltransferase (E2) of the multisubunit α-ketoglutarate dehydrogenase complex (α-KD) is an essential Krebs cycle enzyme commonly found in the matrices of mitochondria. African trypanosomes developmentally regulate mitochondrial carbohydrate metabolism and lack a functional Krebs cycle in the bloodstream of mammals. We found that despite the absence of a functional α-KD, bloodstream form (BF) trypanosomes express α-KDE2, which localized to the mitochondrial matrix and inner membrane. Furthermore, α-KDE2 fractionated with the mitochondrial genome, the kinetoplast DNA (kDNA), in a complex with the flagellum. A role for α-KDE2 in kDNA maintenance was revealed in α-KDE2 RNA interference (RNAi) knockdowns. Following RNAi induction, bloodstream trypanosomes showed pronounced growth reduction and often failed to equally distribute kDNA to daughter cells, resulting in accumulation of cells devoid of kDNA (dyskinetoplastic) or containing two kinetoplasts. Dyskinetoplastic trypanosomes lacked mitochondrial membrane potential and contained mitochondria of substantially reduced volume. These results indicate that α-KDE2 is bifunctional, both as a metabolic enzyme and as a mitochondrial inheritance factor necessary for the distribution of kDNA networks to daughter cells at cytokinesis.

  2. SUMO: regulating the regulator

    Directory of Open Access Journals (Sweden)

    Bossis Guillaume

    2006-06-01

    Full Text Available Abstract Post-translational modifiers of the SUMO (Small Ubiquitin-related Modifier family have emerged as key regulators of protein function and fate. While the past few years have seen an enormous increase in knowledge on SUMO enzymes, substrates, and consequences of modification, regulation of SUMO conjugation is far from being understood. This brief review will provide an overview on recent advances concerning (i the interplay between sumoylation and other post-translational modifications at the level of individual targets and (ii global regulation of SUMO conjugation and deconjugation.

  3. in the Treatment of Experimental Trypanosoma brucei brucei ...

    African Journals Online (AJOL)

    Dr Olaleye

    single dose, group B with 1.0mg/Kg of Cotecxin(R) and group C with a combination of diminazene aceturate (3.5mg/Kg) and. Cotecxin(R) (1.0mg/Kg). Rats in group D remained untreated while group E animals served as the uninfected and untreated control. The pre-patent periods in groups A, B, C and D were 3.5 ± 0.9, 3.1 ...

  4. Nifurtimox-eflornithine combination therapy for second-stage African Trypanosoma brucei gambiense trypanosomiasis: a multicentre, randomised, phase III, non-inferiority trial.

    Science.gov (United States)

    Priotto, Gerardo; Kasparian, Serena; Mutombo, Wilfried; Ngouama, Daniel; Ghorashian, Sara; Arnold, Ute; Ghabri, Salah; Baudin, Elisabeth; Buard, Vincent; Kazadi-Kyanza, Serge; Ilunga, Médard; Mutangala, Willy; Pohlig, Gabriele; Schmid, Caecilia; Karunakara, Unni; Torreele, Els; Kande, Victor

    2009-07-04

    Human African trypanosomiasis (HAT; sleeping sickness) caused by Trypanosoma brucei gambiense is a fatal disease. Current treatment options for patients with second-stage disease are toxic, ineffective, or impractical. We assessed the efficacy and safety of nifurtimox-eflornithine combination therapy (NECT) for second-stage disease compared with the standard eflornithine regimen. A multicentre, randomised, open-label, active control, phase III, non-inferiority trial was done at four HAT treatment centres in the Republic of the Congo and the Democratic Republic of the Congo. Patients aged 15 years or older with confirmed second-stage T b gambiense infection were randomly assigned by computer-generated randomisation sequence to receive intravenous eflornithine (400 mg/kg per day, every 6 h; n=144) for 14 days or intravenous eflornithine (400 mg/kg per day, every 12 h) for 7 days with oral nifurtimox (15 mg/kg per day, every 8 h) for 10 days (NECT; n=143). The primary endpoint was cure (defined as absence of trypanosomes in body fluids and a leucocyte count

  5. Market, Regulation, Market, Regulation

    DEFF Research Database (Denmark)

    Frankel, Christian; Galland, Jean-Pierre

    2015-01-01

    This paper focuses on the European Regulatory system which was settled both for opening the Single Market for products and ensuring the consumers' safety. It claims that the New Approach and Standardization, and the Global Approach to conformity assessment, which suppressed the last technical...... barriers to trade in Europe, realized the free movement of products by organizing progressively several orders of markets and regulation. Based on historical and institutional documents, on technical publications, and on interviews, this article relates how the European Commission and the Member States had...... alternatively recourse to markets and to regulations, at the three main levels of the New Approach Directives implementation. The article focuses also more specifically on the Medical Devices sector, not only because this New Approach sector has long been controversial in Europe, and has recently been concerned...

  6. AcSDKP is down-regulated in anaemia induced by Trypanosoma ...

    African Journals Online (AJOL)

    We studied the responses of a tetrapeptide, AcSDKP, and IL-10, and their association with bone marrow nucleated cells in a Trypanosoma brucei brucei GVR35 experimental infection model. Methods Mouse infection was done intraperitoneally with 1 × 103 trypanosomes/mL. Mice were either infected or left uninfected (N ...

  7. NORM regulations

    Energy Technology Data Exchange (ETDEWEB)

    Gray, P. [ed.

    1997-02-01

    The author reviews the question of regulation for naturally occuring radioactive material (NORM), and the factors that have made this a more prominent concern today. Past practices have been very relaxed, and have often involved very poor records, the involvment of contractors, and the disposition of contaminated equipment back into commercial service. The rationale behind the establishment of regulations is to provide worker protection, to exempt low risk materials, to aid in scrap recycling, to provide direction for remediation and to examine disposal options. The author reviews existing regulations at federal and state levels, impending legislation, and touches on the issue of site remediation and potential liabilities affecting the release of sites contaminated by NORM.

  8. Affect Regulation

    DEFF Research Database (Denmark)

    Pedersen, Signe Holm; Poulsen, Stig Bernt; Lunn, Susanne

    2014-01-01

    Gergely and colleagues’ state that their Social Biofeedback Theory of Parental Affect Mirroring” can be seen as a kind of operationalization of the classical psychoanalytic concepts of holding, containing and mirroring. This article examines to what extent the social biofeedback theory of parental...... affect mirroring may be understood as a specification of these concepts. It is argued that despite similarities at a descriptive level the concepts are embedded in theories with different ideas of subjectivity. Hence an understanding of the concept of affect regulation as a concretizisation...... and specification of the classical concepts dilutes the complexity of both the concept of affect regulation and of the classical concepts....

  9. Regulation Theory

    CERN Document Server

    Bouvet, F.

    2015-06-15

    This paper reviews the design of regulation loops for power converters. Power converter control being a vast domain, it does not aim to be exhaustive. The objective is to give a rapid overview of the main synthesis methods in both continuous- and discrete-time domains.

  10. RegulatING chromatin regulators

    DEFF Research Database (Denmark)

    Satpathy, Shankha; Nabbi, Arash; Riabowol, Karl

    2013-01-01

    The five human ING genes encode at least 15 splicing isoforms, most of which affect cell growth, differentiation and apoptosis through their ability to alter gene expression by epigenetic mechanisms. Since their discovery in 1996, ING proteins have been classified as type II tumour suppressors...... on the basis of reports describing their down-regulation and mislocalization in a variety of cancer types. In addition to their regulation by transcriptional mechanisms, understanding the range of PTMs (post-translational modifications) of INGs is important in understanding how ING functions are fine...

  11. Differential trypanosome surface coat regulation by a CCCH protein that co-associates with procyclin mRNA cis-elements.

    Directory of Open Access Journals (Sweden)

    Pegine Walrad

    2009-02-01

    Full Text Available The genome of Trypanosoma brucei is unusual in being regulated almost entirely at the post-transcriptional level. In terms of regulation, the best-studied genes are procyclins, which encode a family of major surface GPI-anchored glycoproteins (EP1, EP2, EP3, GPEET that show differential expression in the parasite's tsetse-fly vector. Although procyclin mRNA cis-regulatory sequences have provided the paradigm for post-transcriptional control in kinetoplastid parasites, trans-acting regulators of procyclin mRNAs are unidentified, despite intensive effort over 15 years. Here we identify the developmental regulator, TbZFP3, a CCCH-class predicted RNA binding protein, as an isoform-specific regulator of Procyclin surface coat expression in trypanosomes. We demonstrate (i that endogenous TbZFP3 shows sequence-specific co-precipitation of EP1 and GPEET, but not EP2 and EP3, procyclin mRNA isoforms, (ii that ectopic overexpression of TbZFP3 does not perturb the mRNA abundance of procyclin transcripts, but rather that (iii their protein expression is regulated in an isoform-specific manner, as evidenced by mass spectrometric analysis of the Procyclin expression signature in the transgenic cell lines. The TbZFP3 mRNA-protein complex (TbZFP3mRNP is identified as a trans-regulator of differential surface protein expression in trypanosomes. Moreover, its sequence-specific interactions with procyclin mRNAs are compatible with long-established predictions for Procyclin regulation. Combined with the known association of TbZFP3 with the translational apparatus, this study provides a long-sought missing link between surface protein cis-regulatory signals and the gene expression machinery in trypanosomes.

  12. Effect of Concurrent Trypanosoma brucei Infection on ...

    African Journals Online (AJOL)

    Reduction in the packed cell volume, increased erythrocyte sedimentation rate, persistent monocytosis and leucopenia characterised by lymphopenia and neutropenia were associated with the concurrent infection while Haemonchus infection alone resulted in persistent eosinophilia. The results of the study show that a ...

  13. Polymerase chain reaction identification of Trypanosoma brucei ...

    African Journals Online (AJOL)

    The midguts of 206 flies were positive and were individually put in eppendorf tubes containing phosphate-buffered saline (PBS buffer) for DNA extraction. Internal transcribed spacer (ITS)-PCR was first used to isolate all trypanosome species from the flies. TBR PCR was then used to isolate the Trypanozoon group.

  14. Effects of Trypanosoma brucei and Heligmosomoides bakeri ...

    African Journals Online (AJOL)

    Group D served as pregnant uninfected control while a different group (E) was used as non-pregnant and uninfected control. Packed cell volumes (PCV), faecal egg counts (FEC), worm burden and water consumption of the mice were determined. Mean live-weights of surviving offspring and their numbers were recorded.

  15. Regulating through leverage: Civil regulation in China

    NARCIS (Netherlands)

    Fürst, K.

    2016-01-01

    The overarching goal of this study is to examine the efforts of Chinese NGOs to prevent and/or control industrial pollution risks and then use the findings of this research to study the nature of civil regulation in, and beyond, China’s authoritarian setting. It first argues that 'regulation through

  16. Response of Tripanosoma brucei brucei –induced anaemia to a ...

    African Journals Online (AJOL)

    Jubi Formula® can restore the PCV and Hb concentrations in anaemic conditions and is a potential substitute for blood transfusion. However, further studies are needed to investigate the potentials of the herbal preparation in reversing anaemia. Key words: Anaemia, PCV, rabbits, haemoglobin, herbal preparation.

  17. Combinatorial Gene Regulation Using Auto-Regulation

    Science.gov (United States)

    Hermsen, Rutger; Ursem, Bas; ten Wolde, Pieter Rein

    2010-01-01

    As many as 59% of the transcription factors in Escherichia coli regulate the transcription rate of their own genes. This suggests that auto-regulation has one or more important functions. Here, one possible function is studied. Often the transcription rate of an auto-regulator is also controlled by additional transcription factors. In these cases, the way the expression of the auto-regulator responds to changes in the concentrations of the “input” regulators (the response function) is obviously affected by the auto-regulation. We suggest that, conversely, auto-regulation may be used to optimize this response function. To test this hypothesis, we use an evolutionary algorithm and a chemical–physical model of transcription regulation to design model cis-regulatory constructs with predefined response functions. In these simulations, auto-regulation can evolve if this provides a functional benefit. When selecting for a series of elementary response functions—Boolean logic gates and linear responses—the cis-regulatory regions resulting from the simulations indeed often exploit auto-regulation. Surprisingly, the resulting constructs use auto-activation rather than auto-repression. Several design principles show up repeatedly in the simulation results. They demonstrate how auto-activation can be used to generate sharp, switch-like activation and repression circuits and how linearly decreasing response functions can be obtained. Auto-repression, on the other hand, resulted only when a high response speed or a suppression of intrinsic noise was also selected for. The results suggest that, while auto-repression may primarily be valuable to improve the dynamical properties of regulatory circuits, auto-activation is likely to evolve even when selection acts on the shape of response function only. PMID:20548950

  18. Regulating fisheries under uncertainty

    DEFF Research Database (Denmark)

    Hansen, Lars Gårn; Jensen, Frank

    2017-01-01

    Regulator uncertainty is decisive for whether price or quantity regulation maximizes welfare in fisheries. In this paper, we develop a model of fisheries regulation that includes ecological uncertainly, variable economic uncertainty as well as structural economic uncertainty. We aggregate...... qualification of the pro-price regulation message dominating the fisheries economics literature. We also believe that the model of a fishery developed in this paper could be applied to the regulation of other renewable resources where regulators are subject to uncertainty either directly or with some...

  19. Untranslated regions of mRNA and their role in regulation of gene ...

    Indian Academy of Sciences (India)

    2017-01-11

    Jan 11, 2017 ... poly(A)-binding protein; RBP, RNA binding protein; SIDER, short interspersed degenerated retrotransposons; SL, spliced leader;. TbZFP3, T. brucei zinc finger protein 3; TcSMUG, T. cruzi ... growth retardation, impairment of cognitive skills and de- velopment in young children, in addition to the serious.

  20. Interpersonal instrumental emotion regulation

    NARCIS (Netherlands)

    Netzer, L.; Van Kleef, G.A.; Tamir, M.

    2015-01-01

    What motivates people to regulate the emotions of others? Prior research has shown that people are motivated to regulate the emotions of others to make others feel better. This investigation, however, was designed to test whether people are also motivated to regulate the emotions of others to

  1. General Theories of Regulation

    NARCIS (Netherlands)

    Hertog, J.A. den

    1999-01-01

    This chapter makes a distinction between three types of theories of regulation: public interest theories, the Chicago theory of regulation and the public choice theories. The Chicago theory is mainly directed at the explanation of economic regulation; public interest theories and public choice

  2. TOWARD MORE EFFECTIVE REGULATION

    Energy Technology Data Exchange (ETDEWEB)

    J. GRAF

    2000-06-01

    This paper proposes a model relationship between the operator engaged in a hazardous activity, the regulator of that activity, and the general public. The roles and responsibilities of each entity are described in a way that allows effective communication flow. The role of the regulator is developed using the steam boiler as an example of a hazard subject to regulation; however, the model applies to any regulated activity. In this model the safety analyst has the extremely important role of communicating sometimes difficult technical information to the regulator in a way that the regulator can provide credible assurance to the general public as to the adequacy of the control of the hazardous activity. The conclusion asserts that acceptance of the model, understanding of the roles and responsibilities and definition of who communicates what information to whom will mitigate frustration on the part of each of the three entities.

  3. Aviation Flight Regulations

    National Research Council Canada - National Science Library

    2006-01-01

    .... This regulation covers aircraft operations, crew requirements and flight rules. It also covers Army aviation general provisions, training, standardization, and management of aviation resources...

  4. Regulating household financial advice

    Directory of Open Access Journals (Sweden)

    Benjamin F. Cummings

    2012-08-01

    Full Text Available This paper reviews economic theory related to investment advice. This theory explains 1 why financial advisors need to be carefully regulated for the benefit of both the investment advice industry and for consumers, 2 why principles-based regulation (e.g., a fiduciary standard is more efficient than rules-based regulation, 3 why dual regulation of financial professionals providing investment or insurance advice is inefficient and inequitable policy, and 4 why the application of a universal and uniform fiduciary standard will be difficult to implement.

  5. Emotion-regulation choice

    NARCIS (Netherlands)

    Sheppes, Gal; Scheibe, Susanne; Suri, Gaurav; Gross, James J.

    2011-01-01

    Despite centuries of speculation about how to manage negative emotions, little is actually known about which emotion-regulation strategies people choose to use when confronted with negative situations of varying intensity. On the basis of a new process conception of emotion regulation, we

  6. Mortgage market regulation: Europe

    NARCIS (Netherlands)

    Aalbers, M.B.; Smith, S.J.

    2012-01-01

    Despite several European Union (EU) initiatives, there is only limited pan-European mortgage market regulation. The EU strategy can be characterised as one of parallel liberalisation and consolidation. This article highlights the key differences in regulation among European mortgage markets.

  7. Practicing reflexive regulation

    NARCIS (Netherlands)

    S.I. Rutz (Suzanne)

    2017-01-01

    textabstractReflexive regulation has been developed to provide inspectors with strategies to deal with uncertain situations in which rules and roles are unclear, and inspecting involves multiple actors and learning how to deal with the situation is crucial. Reflexive regulation offers an alternative

  8. Plant Growth Regulators.

    Science.gov (United States)

    Nickell, Louis G.

    1978-01-01

    Describes the effect of "plant growth regulators" on plants, such as controlling the flowering, fruit development, plant size, and increasing crop yields. Provides a list of plant growth regulators which includes their chemical, common, and trade names, as well as their different use(s). (GA)

  9. Regulation of SUMO Modification

    NARCIS (Netherlands)

    P.M. Knipscheer (Puck Maria)

    2007-01-01

    textabstractThe small ubiquitin related modifier SUMO is a posttranslational modifier that functions in a wide range of cellular processes like intracellular transport, cell cycle regulation, DNA repair and regulation of transcription. SUMO is an 11 kDa protein and is ligated to its target proteins

  10. The regulation of hunting

    DEFF Research Database (Denmark)

    Abildtrup, Jens; Jensen, Frank

    Within hunting, wildlife populations are estimated to be too high in many countries which is assumed to be due to the market failure, that each hunter harvests too little compared to what the regulator wants. This may be due to the existing regulation which, among other things, requires knowledge...

  11. Reconceptualizing Civil Regulation

    DEFF Research Database (Denmark)

    Galang, Roberto Martin; Castello, Itziar

    2011-01-01

    This article re-conceptualizes the notion of civil regulation, through an analysis of 775 projects by firms located in 21 Asian countries, wherein we map the state of civil regulation initiatives in the region. We challenge two established assumptions in the Corporate Social Responsibility...... and environmental standards; but also that local, small and medium companies play a key role in the development of Asian civil regulation. We call this second finding the “CSR importation trap”. Our findings are supported by evidence on the limitations in the interchangeable properties of business and governments...... literature. First, contrary to what is commonly argued, we claim that strong states in Asia promote civil regulation in what we call the “paradox of the weak state”. Second, we not only argue that civil regulation is mainly enforced by multinational enterprises willing to promote international social...

  12. Epigenetic Regulation of Adipokines

    Directory of Open Access Journals (Sweden)

    Tho X. Pham

    2017-08-01

    Full Text Available Adipose tissue expansion in obesity leads to changes in the expression of adipokines, adipocyte-specific hormones that can regulate whole body energy metabolism. Epigenetic regulation of gene expression is a mechanism by which cells can alter gene expression through the modifications of DNA and histones. Epigenetic mechanisms, such as DNA methylation and histone modifications, are intimately tied to energy metabolism due to their dependence on metabolic intermediates such as S-adenosylmethionine and acetyl-CoA. Altered expression of adipokines in obesity may be due to epigenetic changes. The goal of this review is to highlight current knowledge of epigenetic regulation of adipokines.

  13. Electrical installations and regulations

    CERN Document Server

    Whitfield, J F

    1966-01-01

    Electrical Installations and Regulations focuses on the regulations that apply to electrical installations and the reasons for them. Topics covered range from electrical science to alternating and direct current supplies, as well as equipment for providing protection against excess current. Cables, wiring systems, and final subcircuits are also considered, along with earthing, discharge lighting, and testing and inspection.Comprised of 12 chapters, this book begins with an overview of electrical installation work, traits of a good electrician, and the regulations governing installations. The r

  14. Regulation of cholesterol homeostasis

    NARCIS (Netherlands)

    van der Wulp, Mariette Y. M.; Verkade, Henkjan J.; Groen, Albert K.

    2013-01-01

    Hypercholesterolemia is an important risk factor for cardiovascular disease. It is caused by a disturbed balance between cholesterol secretion into the blood versus uptake. The pathways involved are regulated via a complex interplay of enzymes, transport proteins, transcription factors and

  15. Sport Fishing Regulations

    Data.gov (United States)

    US Fish and Wildlife Service, Department of the Interior — The regulations for sport fishing on St. Vincent National Wildlife Refuge are outlined in this document. Fishing is only permitted from sunrise to sunset, and only...

  16. Collaborative Tax Regulation

    DEFF Research Database (Denmark)

    Boll, Karen

    2016-01-01

    This article shows a new form of regulation within a tax administration where tax administrators abate tax evasion by nudging and motivating consumers to only purchase services from tax compliant businesses. This indirectly closes or forces tax evading businesses to change their practices, because...... their customer bases decline to commercially non-viable levels. The analysis is framed by public governance literature and argues that the regulation is an example of collaborative or interactive governance, because the tax administrators do not regulate non-compliance directly, but activate external...... stakeholders, i.e. the consumers, in the regulatory craft. The study is based on a qualitative methodology and draws on a unique case of regulation in the cleaning sector. This sector is at high risk of tax evasion and human exploitation of vulnerable workers operating in the informal economy. The article has...

  17. Focus on PTEN regulation

    Directory of Open Access Journals (Sweden)

    Miriam eBermudez-Brito

    2015-07-01

    Full Text Available The role of PTEN as a tumour suppressor has been for a long time attributed to its lipid phosphatase activity against PI(3,4,5P3, the phospholipid product of the class I PI3Ks. Besides its traditional role as a lipid phosphatase at the plasma membrane, a wealth of data has shown that PTEN can function independently of its phosphatase activity and that PTEN also exists and plays a role in the nucleus, in cytoplasmic organelles and extracellularly. Accumulating evidence has shed light on diverse physiological functions of PTEN which are accompanied by a complex regulation of its expression and activity. PTEN levels and function are regulated transcriptionally, post-transcriptionally and post-translationally. PTEN is also sensitive to regulation by its interacting proteins and its localization. Herein, we summarize the current knowledge on mechanisms that regulate the expression and enzymatic activity of PTEN and its role in human diseases.

  18. Regulation of Genetic Tests

    Science.gov (United States)

    Skip to main content Regulation of Genetic Tests Enter Search Term(s): Español Research Funding An Overview Bioinformatics Current Grants Education and Training Funding Extramural Research News Features Funding Divisions ...

  19. Legislation and regulation

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1998-09-01

    This document presents the fulfilling of the Brazilian obligations under the Convention on Nuclear Safety. The Chapter 3 of the document contains some details about the Brazilian legislation and regulation, the nuclear and environmental licensing, and emergency preparedness legislation.

  20. Benchmarking and Regulation

    DEFF Research Database (Denmark)

    Agrell, Per J.; Bogetoft, Peter

    . The application of benchmarking in regulation, however, requires specific steps in terms of data validation, model specification and outlier detection that are not systematically documented in open publications, leading to discussions about regulatory stability and economic feasibility of these techniques...

  1. Restructuring nuclear regulations.

    OpenAIRE

    Mossman, Kenneth L

    2003-01-01

    Nuclear regulations are a subset of social regulations (laws to control activities that may negatively impact the environment, health, and safety) that concern control of ionizing radiation from radiation-producing equipment and from radioactive materials. The impressive safety record among nuclear technologies is due, in no small part, to the work of radiation safety professionals and to a protection system that has kept pace with the rapid technologic advancements in electric power generati...

  2. Epigenetic Regulation of Adipokines

    OpenAIRE

    Pham, Tho X.; Ji-Young Lee

    2017-01-01

    Adipose tissue expansion in obesity leads to changes in the expression of adipokines, adipocyte-specific hormones that can regulate whole body energy metabolism. Epigenetic regulation of gene expression is a mechanism by which cells can alter gene expression through the modifications of DNA and histones. Epigenetic mechanisms, such as DNA methylation and histone modifications, are intimately tied to energy metabolism due to their dependence on metabolic intermediates such as S-adenosylmethion...

  3. Turning regulation into value

    OpenAIRE

    Laamanen, Tomi; Reuter, Emmanuelle; Steiger, Fabio

    2015-01-01

    As an export-based industry, the survival of Swiss private banking depends on its access to an international client base in relevant markets. Adoption of transnational regulation is especially critical as the foreign onshore business gains importance while the offshore business declines. Transnational regulation should therefore not be seen as an option. Rather, it is central to a sustainable and successful Swiss private banking model.

  4. In regulation we trust.

    Science.gov (United States)

    Wiig, Siri; Tharaldsen, Jorunn Elise

    2012-01-01

    The role of trust has been argued to play an increasingly important role in modern, complex, and ambivalent risk societies. Trust within organizational research is anticipated to have a general strategic impact on aspects such as organizational performance, communication and knowledge exchange, and learning from accidents. Trust is also an important aspect related to regulation of risk. Diverse regulatory regimes, their contexts and risks influence regulators use of trust and distrust in regulatory practice. The aim of this paper is to discuss the relationship between risk regulation and trust across diverse risk regulation regimes. By drawing from studies of risk regulation, risk perception, and trust the purpose is to discuss how regulation and trust are linked and used in practice to control risk across system levels in socio-technical systems in high risk industries. This paper provides new knowledge on 1) how functional and dysfunctional trust and distrust are grounded in the empirical realities of high risk industries, 2) how different perspectives on trust and distrust act together and bring new knowledge on how society control risk.

  5. Interpersonal emotion regulation.

    Science.gov (United States)

    Zaki, Jamil; Williams, W Craig

    2013-10-01

    Contemporary emotion regulation research emphasizes intrapersonal processes such as cognitive reappraisal and expressive suppression, but people experiencing affect commonly choose not to go it alone. Instead, individuals often turn to others for help in shaping their affective lives. How and under what circumstances does such interpersonal regulation modulate emotional experience? Although scientists have examined allied phenomena such as social sharing, empathy, social support, and prosocial behavior for decades, there have been surprisingly few attempts to integrate these data into a single conceptual framework of interpersonal regulation. Here we propose such a framework. We first map a "space" differentiating classes of interpersonal regulation according to whether an individual uses an interpersonal regulatory episode to alter their own or another person's emotion. We then identify 2 types of processes--response-dependent and response-independent--that could support interpersonal regulation. This framework classifies an array of processes through which interpersonal contact fulfills regulatory goals. More broadly, it organizes diffuse, heretofore independent data on "pieces" of interpersonal regulation, and identifies growth points for this young and exciting research domain.

  6. Worldwide regulations for mycotoxins.

    Science.gov (United States)

    van Egmond, Hans P

    2002-01-01

    Since the discovery of the aflatoxins in the 1960s, regulations have been established in many countries to protect the consumer from the harmful effects of mycotoxins that may contaminate foodstuffs. Various factors play a role in the decision-making process of setting limits for mycotoxins. These include scientific factors such as the availability of toxicological data, survey data, knowledge about the distribution of mycotoxins in commodities, and analytical methodology. Economical and political factors such as commercial interests and sufficiency of food supply have their impact as well. International enquiry's on existing mycotoxin legislation in foodstuffs and animal feedstuffs have been carried out several times in the 1980s and 1990s and details about tolerances, legal basis, responsible authorities, official protocols of analysis and sampling have been published. Recently a comprehensive update on worldwide regulations was published as FAO Food and Nutrition Paper 64. It appeared that at least 77 countries now have specific regulations for mycotoxins, 13 countries are known to have no specific regulations, whereas no data are available for about 50 countries, many of them in Africa. Over the years, a large diversity in tolerance levels for mycotoxins has remained. Some free trade zones (EU, MERCOSUR) are in the process of harmonizing the limits and regulations for mycotoxins in their respective member states, but it is not likely that worldwide harmonized limits for mycotoxins will soon be within reach.

  7. Staff rules and regulations

    CERN Multimedia

    HR Department

    2007-01-01

    The 11th edition of the Staff Rules and Regulations, dated 1 January 2007, adopted by the Council and the Finance Committee in December 2006, is currently being distributed to departmental secretariats. The Staff Rules and Regulations, together with a summary of the main modifications made, will be available, as from next week, on the Human Resources Department's intranet site: http://cern.ch/hr-web/internal/admin_services/rules/default.asp The main changes made to the Staff Rules and Regulations stem from the five-yearly review of employment conditions of members of the personnel. The changes notably relate to: the categories of members of the personnel (e.g. removal of the local staff category); the careers structure and the merit recognition system; the non-residence, installation and re-installation allowances; the definition of family, family allowances and family-related leave; recognition of partnerships; education fees. The administrative circulars, some of which are being revised following the ...

  8. Volume Regulated Channels

    DEFF Research Database (Denmark)

    Klausen, Thomas Kjær

    of volume perturbations evolution have developed system of channels and transporters to tightly control volume homeostasis. In the past decades evidence has been mounting, that the importance of these volume regulated channels and transporters are not restricted to the defense of cellular volume......- serves a multitude of functions in the mammalian cell, regulating the membrane potential (Em), cell volume, protein activity and the driving force for facilitated transporters giving Cl- and Cl- channels a major potential of regulating cellular function. These functions include control of the cell cycle....... Understanding the structure/function relationship of TRPV4 is essential for future development of specific TRPV4 agonist for treatment of diseases causes by dysfunctional TRPV4. E.g. two inherited bone dysplasias have recently been demonstrated in humans to originate from TRPV4 mutations....

  9. Volume regulation in epithelia

    DEFF Research Database (Denmark)

    Larsen, Erik Hviid; Hoffmann, Else Kay

    2016-01-01

    We review studies on regulatory volume decrease (RVD) and regulatory volume increase (RVI) of major ion and water transporting vertebrate epithelia. The rate of RVD and RVI is faster in cells of high osmotic permeability like amphibian gallbladder and mammalian proximal tubule as compared...... function of iso-osmotic fluid transport that depends on Na+ recirculation. The causative relationship is discussed for a fluid-absorbing and a fluid-secreting epithelium of which the Na+ recirculation mechanisms have been identified. A large number of transporters and ion channels involved in cell volume...... regulation are cloned. The volume-regulated anion channel (VRAC) exhibiting specific electrophysiological characteristics seems exclusive to serve cell volume regulation. This is contrary to K+ channels as well as cotransporters and exchange mechanisms that may serve both transepithelial transport and cell...

  10. Quantitative analysis of flux regulation through hierarchical regulation analysis

    NARCIS (Netherlands)

    Eunen, K. van; Rossell, S.; Bouwman, J.; Westerhoff, H.V.; Bakker, B.M.

    2011-01-01

    Regulation analysis is a methodology that quantifies to what extent a change in the flux through a metabolic pathway is regulated by either gene expression or metabolism. Two extensions to regulation analysis were developed over the past years: (i) the regulation of Vmax can be dissected into the

  11. QUANTITATIVE ANALYSIS OF FLUX REGULATION THROUGH HIERARCHICAL REGULATION ANALYSIS

    NARCIS (Netherlands)

    van Eunen, Karen; Rossell, Sergio; Bouwman, Jildau; Westerhoff, Hans V.; Bakker, Barbara M.; Jameson, D; Verma, M; Westerhoff, HV

    2011-01-01

    Regulation analysis is a methodology that quantifies to what extent a change in the flux through a metabolic pathway is regulated by either gene expression or metabolism. Two extensions to regulation analysis were developed over the past years: (i) the regulation of V(max) can be dissected into the

  12. Quantitative analysis of flux regulation through hierarchical regulation analysis.

    NARCIS (Netherlands)

    Eunen, K. van; Rossell, S.L.; Bouwman, J.; Westerhoff, H.V.; Bakker, B.M.

    2011-01-01

    Regulation analysis is a methodology that quantifies to what extent a change in the flux through a metabolic pathway is regulated by either gene expression or metabolism. Two extensions to regulation analysis were developed over the past years: (i) the regulation of V(max) can be dissected into the

  13. The Impact of Regulating Social Science Research with Biomedical Regulations

    Science.gov (United States)

    Durosinmi, Brenda Braxton

    2011-01-01

    The Impact of Regulating Social Science Research with Biomedical Regulations Since 1974 Federal regulations have governed the use of human subjects in biomedical and social science research. The regulations are known as the Federal Policy for the Protection of Human Subjects, and often referred to as the "Common Rule" because 18 Federal…

  14. Rapamycin regulates biochemical metabolites

    OpenAIRE

    Tucci, Paola; Porta, Giovanni; Agostini, Massimiliano; Antonov, Alexey; Garabadgiu, Alexander Vasilievich; Melino, Gerry; Willis, Anne E

    2013-01-01

    The mammalian target of rapamycin (mTOR) kinase is a master regulator of protein synthesis that couples nutrient sensing to cell growth, and deregulation of this pathway is associated with tumorigenesis. p53, and its less investigated family member p73, have been shown to interact closely with mTOR pathways through the transcriptional regulation of different target genes. To investigate the metabolic changes that occur upon inhibition of the mTOR pathway and the role of p73 in this response p...

  15. Nuclear regulation and safety

    Energy Technology Data Exchange (ETDEWEB)

    Hendrie, J.M.

    1982-01-01

    Nuclear regulation and safety are discussed from the standpoint of a hypothetical country that is in the process of introducing a nuclear power industry and setting up a regulatory system. The national policy is assumed to be in favor of nuclear power. The regulators will have responsibility for economic, reliable electric production as well as for safety. Reactor safety is divided into three parts: shut it down, keep it covered, take out the afterheat. Emergency plans also have to be provided. Ways of keeping the core covered with water are discussed. (DLC)

  16. Cyberplagiarism in University Regulations

    Directory of Open Access Journals (Sweden)

    Santiago Cavanillas

    2008-12-01

    Full Text Available The article examines the legal framework for plagiarism, and its twofold nature of illicit appropriation (from the author of the plagiarized work and fraud (with regard to the target audience of the plagiarism. Based on these premises, academic cyberplagiarism is analysed as a form of plagiarism carried out using electronic tools in the university setting. The question of responsibility (who can regulate the legal consequences of plagiarism? before and after the Ley Orgánica de Universidades (organic law on universities, LOU is studied, as is the disciplinary handling of cyberplagiarism with the limited regulations currently in place at universities.

  17. Regulation as Rhetoric

    DEFF Research Database (Denmark)

    Boll, Karen; Györy, Csaba

    environment, these two agencies apply strategies that appear to be strikingly similar, and these similarities are worth investigating not despite, but exactly because of the differing political and social environment. We track recent shifts in organizational practice at these two agencies and argue that both...... engage reflectively in image promotion which serves two purposes: establishing and maintaining legitimacy in a particular social and political environment and producing compliance. Further, we argue that this regulation is a form of ‘post-bureaucratic’ regulation in which compliance is achieved...

  18. Role of BRCA2 in the Expressions of IRF9-Regulated Genes in Human Breast Cells

    Science.gov (United States)

    2011-07-01

    Chem. 286, 469-479. Journal Impact factor : 5.325. 2. Hall, M. 3rd, Misra, S., Chaudhuri, M., and Chaudhuri, G. (2011) Peptide aptamer mimicking...RAD51-binding domain of BRCA2 inhibits DNA damage repair and survival in Trypanosoma brucei. Microb. Pathog. 50, 252-262. Journal Impact factor : 2.324...containing B- 15 box element at their promoters in alternatively polarized M2 macrophages. Mol. Cell. Biochem. 350, 47-57. Journal Impact factor : 1,896

  19. Regulating the Internet

    Science.gov (United States)

    Anderson, Byron

    2007-01-01

    The Internet's breakthrough to primetime usage beginning in the mid-1990s evolved in an era of openness. Unfettered access seemed key to Internet development. An important foundation for the 1996 Telecommunications Act was the theory that the telecom industry would work best if it were free of government regulation, a guiding principle that has…

  20. Adaptation with transcriptional regulation

    Science.gov (United States)

    Shi, Wenjia; Ma, Wenzhe; Xiong, Liyang; Zhang, Mingyue; Tang, Chao

    2017-02-01

    Biochemical adaptation is one of the basic functions that are widely implemented in biological systems for a variety of purposes such as signal sensing, stress response and homeostasis. The adaptation time scales span from milliseconds to days, involving different regulatory machineries in different processes. The adaptive networks with enzymatic regulation (ERNs) have been investigated in detail. But it remains unclear if and how other forms of regulation will impact the network topology and other features of the function. Here, we systematically studied three-node transcriptional regulatory networks (TRNs), with three different types of gene regulation logics. We found that the topologies of adaptive gene regulatory networks can still be grouped into two general classes: negative feedback loop (NFBL) and incoherent feed-forward loop (IFFL), but with some distinct topological features comparing to the enzymatic networks. Specifically, an auto-activation loop on the buffer node is necessary for the NFBL class. For IFFL class, the control node can be either a proportional node or an inversely-proportional node. Furthermore, the tunability of adaptive behavior differs between TRNs and ERNs. Our findings highlight the role of regulation forms in network topology, implementation and dynamics.

  1. Emotion regulation during isolation

    Czech Academy of Sciences Publication Activity Database

    Poláčková Šolcová, Iva; Šolcová, Iva

    2012-01-01

    Roč. 47, Suppl. 1 (2012) ISSN 0020-7594. [International Congress of Psychology /30./. 22.07.2012-27.07.2012, Cape Town] R&D Projects: GA ČR(CZ) GAP407/11/2226 Institutional support: RVO:68081740 Keywords : emotion regulation * isolation * Mars 500 Subject RIV: AN - Psychology

  2. Allosteric Regulation of Proteins

    Indian Academy of Sciences (India)

    ... Lecture Workshops · Refresher Courses · Symposia · Live Streaming. Home; Journals; Resonance – Journal of Science Education; Volume 22; Issue 1. Allosteric Regulation of Proteins: A Historical Perspective on the Development of Concepts and Techniques. General Article Volume 22 Issue 1 January 2017 pp 37-50 ...

  3. Federal Gasoline Regulations

    Science.gov (United States)

    The Clean Air Act requires EPA to regulate fuels and fuel additives for use in mobile sources if such fuel, fuel additive or any emission products causes or contributes to air or water pollution that may endanger the public health or welfare.

  4. Understanding medical device regulation.

    Science.gov (United States)

    Galgon, Richard E

    2016-12-01

    The purpose of this article is to provide a structural and functional understanding of the systems used for the regulation of medical devices in the USA and European Union (EU). Safe and effective anesthesia care depends heavily on medical devices, including simple, low risk devices to complex life-supporting and life-sustaining devices. In the USA and EU, the Food and Drug Administration and European Commission, respectively, provide regulatory oversight to ensure medical devices are reasonably safe and effective when used for their intended purposes. Unfortunately, practicing anesthesiologists generally have little or no understanding of how medical devices are regulated, nor do they have sufficient knowledge of available adverse event reporting systems. The US and EU medical device regulatory systems are similar in many ways, but differ in important ways too, which impacts the afforded level of safety and effectiveness assurance. In both systems, medical devices are classified and regulated on a risk basis, which fundamentally differs from drug regulation, where uniform requirements are imposed. Anesthesia providers must gain knowledge of these systems and be active players in both premarket and postmarket activities, particularly with regard to vigilance and adverse event/device failure reporting.

  5. Sink regulation of photosynthesis

    National Research Council Canada - National Science Library

    Matthew J. Paul; Christine H. Foyer

    2001-01-01

    ... in the effects of elevated CO2 on photosynthesis. Photosynthesis is one of the most highly integrated and regulated metabolic processes to maximize the use of available light, to minimize the damaging effects of excess light and to optimize the use...

  6. Situated bio-regulation

    DEFF Research Database (Denmark)

    Prainsack, Barbara; Wahlberg, Ayo

    2013-01-01

    Several years ago, both authors engaged in research into bioscience and biomedical regulation in Asian countries. One of us (BP) explored why the regulatory and discursive embedding of human embryonic stem cell in Israel was much more permissive than elsewhere. The other author (AW) sought to und...

  7. Regulating groundwater use

    NARCIS (Netherlands)

    Hoogesteger van Dijk, Jaime; Wester, Flip

    2017-01-01

    Around the world it has proven very difficult to develop policies and interventions that ensure socio-environmentally sustainable groundwater use and exploitation. In the state of Guanajuato, Central Mexico, both the national government and the decentralized state government have pursued to regulate

  8. Regulation and deregulation

    NARCIS (Netherlands)

    Brouwer, M.

    2010-01-01

    Market regulation has been induced by market failures such as natural monopoly and a-symmetric information. It has also been motivated by considerations to provide universal access and services to remote regions and to sustain farmers’ incomes. The wave of deregulations that characterized the 1980s

  9. Legislation and regulation

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2001-09-01

    This document presents the fulfilling of the Brazilian obligations under the Convention on Nuclear Safety. The Chapter 3 of the document contains some details about the Brazilian legislation and regulation, the legislative and regulatory framework, regulatory body and responsibility of the license holder.

  10. Vehicle recycling regulations

    DEFF Research Database (Denmark)

    Smink, Carla

    2007-01-01

    The number of end-of-life vehicles (ELVs) in the EU is increasing continously. Around 75 percent of an ELV are recyclable metals. The forecast growth in the number of ELVs calls for regulation that aims to minimise the environmental impact of a car. Using Denmark as an example, this article...

  11. Optimal Regulation of Lumpy Investments

    NARCIS (Netherlands)

    Zwart, G.; Broer, D.P.

    2012-01-01

    When a monopolist has discretion over the timing of infrastructure investments, regulation of post-investment prices interferes with incentivizing socially optimal investment timing. In a model of regulated lumpy investment under uncertainty, we study regulation when the regulator can condition

  12. Meat and Appetite Regulation

    DEFF Research Database (Denmark)

    Kehlet, Ursula Nana

    source (animal vs. vegetable protein patties). In a controlled cross-over design, 40 healthy men consumed four meals with similar weight, energy and macronutrient composition (13 g fiber in the three fiber meals) that consisted of meatballs/vegetable patties, bread with butter, a dipping sauce...... by the viscous properties of the fiber ingredients. Moreover, our results suggest that animal and vegetable protein-based, fiber-matched meals had similar effects on appetite regulation. Paper III compared the physico-chemical, orosensory, and microstructural properties of meatballs and sausages containing......Obesity is a significant risk factor for lifestyle related diseases. Foods capable of suppressing hunger and decreasing energy intake could be an efficient tool in obesity prevention. Numerous randomized controlled trials report a beneficial effect of diets high in protein on appetite regulation...

  13. Regulation of Meiotic Recombination

    Energy Technology Data Exchange (ETDEWEB)

    Gregory p. Copenhaver

    2011-11-09

    Meiotic recombination results in the heritable rearrangement of DNA, primarily through reciprocal exchange between homologous chromosome or gene conversion. In plants these events are critical for ensuring proper chromosome segregation, facilitating DNA repair and providing a basis for genetic diversity. Understanding this fundamental biological mechanism will directly facilitate trait mapping, conventional plant breeding, and development of genetic engineering techniques that will help support the responsible production and conversion of renewable resources for fuels, chemicals, and the conservation of energy (1-3). Substantial progress has been made in understanding the basal recombination machinery, much of which is conserved in organisms as diverse as yeast, plants and mammals (4, 5). Significantly less is known about the factors that regulate how often and where that basal machinery acts on higher eukaryotic chromosomes. One important mechanism for regulating the frequency and distribution of meiotic recombination is crossover interference - or the ability of one recombination event to influence nearby events. The MUS81 gene is thought to play an important role in regulating the influence of interference on crossing over. The immediate goals of this project are to use reverse genetics to identify mutants in two putative MUS81 homologs in the model plant Arabidopsis thaliana, characterize those mutants and initiate a novel forward genetic screen for additional regulators of meiotic recombination. The long-term goal of the project is to understand how meiotic recombination is regulated in higher eukaryotes with an emphasis on the molecular basis of crossover interference. The ability to monitor recombination in all four meiotic products (tetrad analysis) has been a powerful tool in the arsenal of yeast geneticists. Previously, the qrt mutant of Arabidopsis, which causes the four pollen products of male meiosis to remain attached, was developed as a facile system

  14. Probiotics and Appetite Regulation

    DEFF Research Database (Denmark)

    Bjerg, Anne Toksvig

    resistance and blood lipid profile among others. Probiotics which are health promoting bacteria can potentially be used to affect the GM and thereby change metabolic outcomes of the host. Animal studies have shown associations between intake of probiotics and appetite regulation, but currently no human...... studies have investigated this effect. Supplementation with different probiotic strains have been shown to have an effect on blood lipid profiles in both animals and humans and the mechanisms behind have been studied in vitro and in rodents. The aim of the present thesis was to examine in an ex vivo...... intestine, in an animal study and in two human studies the effect of the probiotic bacteria Lactobacillus paracasei subsp. paracasei L. casei W8 (W8) on appetite regulation, blood lipids and blood fatty acids. In addition, it was investigated if W8 had an effect on the fecal microbiota of the human...

  15. Markets, religion, regulation

    DEFF Research Database (Denmark)

    Fischer, Johan

    2016-01-01

    of regulation, certification and standardization on a global scale. Building on research on global kosher (a Hebrew term meaning “fit” or “proper”), halal (an Arabic word that literally means “permissible” or “lawful”) and Hindu vegetarianism this paper argues that these economies or markets to a large extent...... and consumers. Epistemologically, comparison is used as a powerful conceptual mechanism that fixes attention on kosher, halal and Hindu vegetarian similarities and differences....

  16. Cyberplagiarism in University Regulations

    OpenAIRE

    Santiago Cavanillas

    2008-01-01

    The article examines the legal framework for plagiarism, and its twofold nature of illicit appropriation (from the author of the plagiarized work) and fraud (with regard to the target audience of the plagiarism). Based on these premises, academic cyberplagiarism is analysed as a form of plagiarism carried out using electronic tools in the university setting. The question of responsibility (who can regulate the legal consequences of plagiarism?) before and after the Ley Orgánica de Universidad...

  17. Telecommunications Competition Regulation

    OpenAIRE

    Productivity Commission

    2002-01-01

    On 21 June 2000 the Commission received a reference from the Treasurer on telecommunications-specific competition regulation for inquiry and report within 12 months of receipt of the reference. In conducting the review, the Commission was to have regard to the state of competition in the telecommunications market, and the impact of new technologies and delivery platforms. In making its recommendations, the Commission would aim to improve the overall economic performance of the Australian econ...

  18. Regulation of Organelle Acidity

    OpenAIRE

    Grabe, Michael; Oster, George

    2001-01-01

    Intracellular organelles have characteristic pH ranges that are set and maintained by a balance between ion pumps, leaks, and internal ionic equilibria. Previously, a thermodynamic study by Rybak et al. (Rybak, S., F. Lanni, and R. Murphy. 1997. Biophys. J. 73:674–687) identified the key elements involved in pH regulation; however, recent experiments show that cellular compartments are not in thermodynamic equilibrium. We present here a nonequilibrium model of lumenal acidification based on t...

  19. Staff rules and regulations

    CERN Multimedia

    HR Department

    2007-01-01

    The 11th edition of the Staff Rules and Regulations, dated 1 January 2007, adopted by the Council and the Finance Committee in December 2006, is currently being distributed to departmental secretariats. The Staff Rules and Regulations, together with a summary of the main modifications made, will be available, as from next week, on the Human Resources Department's intranet site: http://cern.ch/hr-web/internal/admin_services/rules/default.asp The main changes made to the Staff Rules and Regulations stem from the five-yearly review of employment conditions of members of the personnel. The changes notably relate to: the categories of members of the personnel (e.g. removal of the local staff category); the careers structure and the merit recognition system; the non-residence, installation and re-installation allowances; the definition of family, family allowances and family-related leave; recognition of partnerships; education fees. The administrative circulars, some of which are being revised following the m...

  20. Cytokines in sleep regulation.

    Science.gov (United States)

    Krueger, J M; Takahashi, S; Kapás, L; Bredow, S; Roky, R; Fang, J; Floyd, R; Renegar, K B; Guha-Thakurta, N; Novitsky, S

    1995-01-01

    The central thesis of this essay is that the cytokine network in brain is a key element in the humoral regulation of sleep responses to infection and in the physiological regulation of sleep. We hypothesize that many cytokines, their cellular receptors, soluble receptors, and endogenous antagonists are involved in physiological sleep regulation. The expressions of some cytokines are greatly amplified by microbial challenge. This excess cytokine production during infection induces sleep responses. The excessive sleep and wakefulness that occur at different times during the course of the infectious process results from dynamic changes in various cytokines that occur during the host's response to infectious challenge. Removal of any one somnogenic cytokine inhibits normal sleep, alters the cytokine network by changing the cytokine mix, but does not completely disrupt sleep due to the redundant nature of the cytokine network. The cytokine network operates in a paracrine/autocrine fashion and is responsive to neuronal use. Finally, cytokines elicit their somnogenic actions via endocrine and neurotransmitter systems as well as having direct effects neurons and glia. Evidence in support of these postulates is reviewed in this essay.

  1. Improving CS regulations.

    Energy Technology Data Exchange (ETDEWEB)

    Nesse, R.J.; Scheer, R.M.; Marasco, A.L.; Furey, R.

    1980-10-01

    President Carter issued Executive Order 12044 (3/28/78) that required all Federal agencies to distinguish between significant and insignificant regulations, and to determine whether a regulation will result in major impacts. This study gathered information on the impact of the order and the guidelines on the Office of Conservation and Solar Energy (CS) regulatory practices, investigated problems encountered by the CS staff when implementing the order and guidelines, and recommended solutions to resolve these problems. Major tasks accomplished and discussed are: (1) legislation, Executive Orders, and DOE Memoranda concerning Federal administrative procedures relevant to the development and analysis of regulations within CS reviewed; (2) relevant DOE Orders and Memoranda analyzed and key DOE and CS staff interviewed in order to accurately describe the current CS regulatory process; (3) DOE staff from the Office of the General Counsel, the Office of Policy and Evaluation, the Office of the Environment, and the Office of the Secretary interviewed to explore issues and problems encountered with current CS regulatory practices; (4) the regulatory processes at five other Federal agencies reviewed in order to see how other agencies have approached the regulatory process, dealt with specific regulatory problems, and responded to the Executive Order; and (5) based on the results of the preceding four tasks, recommendations for potential solutions to the CS regulatory problems developed. (MCW)

  2. FDA 101: Regulating Biological Products

    Science.gov (United States)

    ... Products For Consumers Home For Consumers Consumer Updates FDA 101: Regulating Biological Products Share Tweet Linkedin Pin ... and highly important field. What biological products does FDA regulate? The Center for Biologics Evaluation and Research ( ...

  3. [Sleep: regulation and phenomenology].

    Science.gov (United States)

    Vecchierini, M-F

    2013-12-01

    This article describes the two-process model of sleep regulation. The 24-hour sleep-wake cycle is regulated by a homeostatic process and an endogenous, 2 oscillators, circadian process, under the influence of external synchronisers. These two processes are partially independent but influence each other, as shown in the two-sleep-process auto-regulation model. A reciprocal inhibition model of two interconnected neuronal groups, "SP on" and "SP off", explains the regular recurrence of paradoxical sleep. Sleep studies have primarily depended on observation of the subject and have determined the optimal conditions for sleep (position, external conditions, sleep duration and need) and have studied the consequences of sleep deprivation or modifications of sleep schedules. Then, electrophysiological recordings permitted the classification of sleep stages according to the observed EEG patterns. The course of a night's sleep is reported on a "hypnogram". The adult subject falls asleep in non-REM sleep (N1), then sleep deepens progressively to stages N2 and N3 with the appearance of spindles and slow waves (N2). Slow waves become more numerous in stage N3. Every 90minutes REM sleep recurs, with muscle atonia and rapid eye movements. These adult sleep patterns develop progressively during the 2 first years of life as total sleep duration decreases, with the reduction of diurnal sleep and of REM sleep. Around 2 to 4 months, spindles and K complexes appear on the EEG, with the differentiation of light and deep sleep with, however, a predominance of slow wave sleep. Copyright © 2013 SPLF. Published by Elsevier Masson SAS. All rights reserved.

  4. Wiring regulations in brief

    CERN Document Server

    Tricker, Ray

    2012-01-01

    Tired of trawling through the Wiring Regs?Perplexed by Part P?Confused by cables, conductors and circuits?Then look no further! This handy guide provides an on-the-job reference source for Electricians, Designers, Service Engineers, Inspectors, Builders, Students, DIY enthusiastsTopic-based chapters link areas of working practice - such as cables, installations, testing and inspection, special locations - with the specifics of the Regulations themselves. This allows quick and easy identification of the official requirements relating to the situati

  5. Regulations, 30 January 1987.

    Science.gov (United States)

    1987-01-01

    Regulations adopted by the All-Union Women's Committee give recently established women's councils greater scope to initiate legislation and provide that laws and statutory instruments affecting women's interests cannot be discussed without their participation. The Councils are also to play an active role in representing the interests of the female workforce at the enterprise level through participation in formulating and monitoring collective agreements, planning the allocation of resources on the social and day-to-day needs of the staff, and supervising the work of medical, pre-school and educational institutions, commercial and public catering enterprises, and day-to-day services. full text

  6. Epigenetic regulation in plants.

    Science.gov (United States)

    Pikaard, Craig S; Mittelsten Scheid, Ortrun

    2014-12-01

    The study of epigenetics in plants has a long and rich history, from initial descriptions of non-Mendelian gene behaviors to seminal discoveries of chromatin-modifying proteins and RNAs that mediate gene silencing in most eukaryotes, including humans. Genetic screens in the model plant Arabidopsis have been particularly rewarding, identifying more than 130 epigenetic regulators thus far. The diversity of epigenetic pathways in plants is remarkable, presumably contributing to the phenotypic plasticity of plant postembryonic development and the ability to survive and reproduce in unpredictable environments. Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reserved.

  7. [Regulation of terpene metabolism

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, R.

    1992-01-01

    This report describes accomplishments over the past year on understanding of terpene synthesis in mint plants and sage. Specifically reported are the fractionation of 4-S-limonene synthetase, the enzyme responsible for the first committed step to monoterpene synthesis, along with isolation of the corresponding RNA and DNA cloning of its gene; the localization of the enzyme within the oil glands, regulation of transcription and translation of the synthetase, the pathway to camphor biosynthesis,a nd studies on the early stages and branch points of the isoprenoid pathway.

  8. Epigenetic Regulation in Plants

    Science.gov (United States)

    Pikaard, Craig S.; Mittelsten Scheid, Ortrun

    2014-01-01

    The study of epigenetics in plants has a long and rich history, from initial descriptions of non-Mendelian gene behaviors to seminal discoveries of chromatin-modifying proteins and RNAs that mediate gene silencing in most eukaryotes, including humans. Genetic screens in the model plant Arabidopsis have been particularly rewarding, identifying more than 130 epigenetic regulators thus far. The diversity of epigenetic pathways in plants is remarkable, presumably contributing to the phenotypic plasticity of plant postembryonic development and the ability to survive and reproduce in unpredictable environments. PMID:25452385

  9. Trypanosoma brucei INFECTION IN A HERD OF SEDENTARY ...

    African Journals Online (AJOL)

    Stella

    as a result of deaths drop in calving rate, reduced market value of animals as a result of ... does not favour the survival of tsetse flies. However, with forestations, strict laws on bush burning and global warming, tsetse flies seems to be moving back into ... Sedentary cattle located within the Northern Guinea Savannah area.

  10. Some liver function indices and blood parameters in T. brucei ...

    African Journals Online (AJOL)

    We investigated whether honey has protective effect on some liver functions and blood parameters affected by trypanosome infection. The serum albumin concentration in infected untreated rats increased significantly (p<0.05) compared with control whereas treatment with honey returned this effect to normal values.

  11. Probing for primary functions of prohibitin in Trypanosoma brucei

    Czech Academy of Sciences Publication Activity Database

    Týč, Jiří; Faktorová, Drahomíra; Kriegová, Eva; Jirků, Milan; Vávrová, Zuzana; Maslov, D. A.; Lukeš, Julius

    2010-01-01

    Roč. 40, č. 1 (2010), s. 73-83 ISSN 0020-7519 R&D Projects: GA ČR GA204/09/1667; GA AV ČR IAA500960705; GA ČR(CZ) GP204/06/P423 Institutional research plan: CEZ:AV0Z60220518 Keywords : prohibitin * mitochondrion * morphology * mitochondrial translation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.822, year: 2010

  12. Serum Iron and Nitric Oxide Production in Trypanosoma brucei ...

    African Journals Online (AJOL)

    Infected rats, treated and untreated, were sacrificed daily for the serum iron levels and nitric oxide synthase activities. For haematological parameters, infected and uninfected but treated rats were sacrificed on days 7 and 12 along with untreated rats. Results showed that tetracycline brought about a significant reduction in ...

  13. Understanding chromatin dynamics during antigenic variation in trypanosoma brucei

    OpenAIRE

    Branco, Francisco Maria dos Santos e Silva Aresta, 1985-

    2016-01-01

    Tese de doutoramento, Ciências Biomédicas (Microbiologia e Parasitologia), Universidade de Lisboa, Faculdade de Medicina, 2016 African trypanosomiasis is a disease restricted to sub-Saharian Africa and it assumes two forms: Human African trypanosomiasis (HAT or sleeping sickness) or Animal African trypanosomiasis (AAT or n’gana), both being fatal if untreated. While HAT causes an estimated number of 15000 new cases each year and 70 million people are at risk, AAT causes an enormous economi...

  14. Role of Cytokines in Trypanosoma brucei -Induced Anaemia: A ...

    African Journals Online (AJOL)

    A number of studies have implicated inflammatory cytokines, but these data are limited and inconsistent. In this article, we reviewed the published literature on ... The mechanism of anaemia is multifactorial and therefore requires further, more elaborate research. Data from human subjects would also shed more light.

  15. Some liver function indices and blood parameters in T. brucei ...

    African Journals Online (AJOL)

    JTEkanem

    investigated whether honey has protective effect on some liver functions and blood parameters affected by trypanosome infection. The serum albumin concentration in infected untreated rats increased significantly (p<0.05) compared with control whereas treatment with honey returned this effect to normal values. Anaemia ...

  16. Population Size and Diet of Bush Hyrax Hetrohyrax brucei (Gray ...

    African Journals Online (AJOL)

    user

    leishmaniasis occurring in the highlands of Kenya and Ethiopia (Ashford et al., 1973; Lemma et al., 2009). During a field excursion to Romanat Michael church forest (Fig 2) located on the outskirts of. Mekelle city, capital of the Tigray Region in northern Ethiopia, we encountered a population of bush hyrax basking on trees ...

  17. Serum Iron and Nitric Oxide Production in Trypanosoma brucei ...

    African Journals Online (AJOL)

    JTEkanem

    dependent enzyme that plays a central role in cell division and proliferation6. Another importance of this therapy, which is of interest in this study, is the modulation of the activity of nitric oxide synthase- a cytokine inducible enzyme which catalyses the formation of ..... lifespan of the rats, unlike when tetracycline alone was ...

  18. Trypanosoma brucei INFECTION IN A HERD OF SEDENTARY ...

    African Journals Online (AJOL)

    Stella

    Savannah, visited the Veterinary Teaching. Hospital of the Ahmadu Bello University,. Zaria (V.T.H-A.B.U-Zaria) and presented a case of emaciation, repeated abortions, pica, and alopecia of the tips of the tails despite supposedly good appetite in a herd of 120 heads of cattle. Furthermore, a report of “swollen” liver in cattle.

  19. About Trypanosoma brucei gambiense , the causative agent of the ...

    African Journals Online (AJOL)

    This must consider it within the whole context of "most-neglected diseases" but some findings obtained through biotechnology are discussed in terms of epidemiology and control applications. Key words: Human African Trypanosomiasis, neglected disease, biotechnologies. African Journal of Biotechnology Vol. 2 (12), pp.

  20. Therapeutic properties and serum iron in T. brucei infected rats ...

    African Journals Online (AJOL)

    For prophylactic treatments of infected rat, amodiaquine extended the lifespan of the rats for 14 days while mefloquine extended it for 7 days. For early stage treatment, amodiaquine and mefloquine extended life span for 7 and 4 days respectively while late stage treatment the extensions were 2 and 1 day respectively.

  1. Tissue Lipid Peroxidation in Trypanosoma brucei Infection: Testing ...

    African Journals Online (AJOL)

    Whereas both superoxide dismutase (SOD) and catalase were significantly reduced in the liver and kidney 28 days post-inoculation; the decrease in SOD activity in the heart was accompanied by an increase in catalase activity in that tissue. Treatment with S. dulcis at a daily oral dose of 200mg/Kg body weight resulted in a ...

  2. About Trypanosoma brucei gambiense, the causative agent of the ...

    African Journals Online (AJOL)

    Admin

    Since the discovery of this pathology at the beginning of 20th century (e.g. Bruce and Nabarro, 1903),. HAT has killed several hundred thousands of individuals in Africa, mainly in Central part of the continent (Martin et al., 1909; W.H.O., 1998). Although the epidemic of 1920-. 1930's was successfully controlled thanks to Dr.

  3. Detection of Trypanosoma brucei gambiense and T. b. rhodesiense ...

    African Journals Online (AJOL)

    GREGORY

    2010-09-20

    Sep 20, 2010 ... central Sudan. J. Anim. Vet. Adv. 4(11): 945-948. Baker JR (1974). Epidemiology of African sleeping sickness. In: Trypanosomiasis and Leishaniasis with Special Reference to Chagas. Disease. CIBA Foundation Symposium, pp. 29-50. Bloss JFE (1960). The history of sleeping sickness in sudan. Proc. R.

  4. Role of cytokines in Trypanosoma brucei-induced anaemia: A ...

    African Journals Online (AJOL)

    Publications on the cytokines studied in trypanosomiasis-associated anaemia ordered by year. Publication. Infecting trypanosome. Clinical sample. Cytokines. Other factors associated with anaemia. Magez et al. 1999. T.b.b AnTat1.1E. Blood from TNF-α -/-. TNF-α. -. Namangala et al. 2001. T.b.b AnTat1.1E. T.b.b PLC-/-.

  5. The Regulation of Street Foods

    DEFF Research Database (Denmark)

    Forkour, John Boulard; Samuelsen, Helle; Yeboah, Eric Henry

    2017-01-01

    the challenges and negotiating strategies of regulators of street-vended foods in Ghana and analyses the implication for their relationship with street food vendors. The paper reveals that regulators operate in a context of limited resources, leading to a general feeling of neglect. In coping, regulators adopt...

  6. Orphan drug regulations. Final rule.

    Science.gov (United States)

    2013-06-12

    The Food and Drug Administration (FDA) is issuing final regulations amending the 1992 Orphan Drug Regulations issued to implement the Orphan Drug Act. These amendments are intended to clarify regulatory provisions and make minor improvements to address issues that have arisen since those regulations were issued.

  7. Regulations and Procedures Manual

    Energy Technology Data Exchange (ETDEWEB)

    Young, Lydia J. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)

    2011-07-25

    The purpose of the Regulations and Procedures Manual (RPM) is to provide LBNL personnel with a reference to University and Lawrence Berkeley National Laboratory (LBNL or Laboratory) policies and regulations by outlining normal practices and answering most policy questions that arise in the day-to-day operations of Laboratory organizations. Much of the information in this manual has been condensed from detail provided in LBNL procedure manuals, Department of Energy (DOE) directives, and Contract DE-AC02-05CH11231. This manual is not intended, however, to replace any of those documents. RPM sections on personnel apply only to employees who are not represented by unions. Personnel policies pertaining to employees represented by unions may be found in their labor agreements. Questions concerning policy interpretation should be directed to the LBNL organization responsible for the particular policy. A link to the Managers Responsible for RPM Sections is available on the RPM home page. If it is not clear which organization is responsible for a policy, please contact Requirements Manager Lydia Young or the RPM Editor.

  8. Transcriptional regulation of metabolism.

    Science.gov (United States)

    Desvergne, Béatrice; Michalik, Liliane; Wahli, Walter

    2006-04-01

    Our understanding of metabolism is undergoing a dramatic shift. Indeed, the efforts made towards elucidating the mechanisms controlling the major regulatory pathways are now being rewarded. At the molecular level, the crucial role of transcription factors is particularly well-illustrated by the link between alterations of their functions and the occurrence of major metabolic diseases. In addition, the possibility of manipulating the ligand-dependent activity of some of these transcription factors makes them attractive as therapeutic targets. The aim of this review is to summarize recent knowledge on the transcriptional control of metabolic homeostasis. We first review data on the transcriptional regulation of the intermediary metabolism, i.e., glucose, amino acid, lipid, and cholesterol metabolism. Then, we analyze how transcription factors integrate signals from various pathways to ensure homeostasis. One example of this coordination is the daily adaptation to the circadian fasting and feeding rhythm. This section also discusses the dysregulations causing the metabolic syndrome, which reveals the intricate nature of glucose and lipid metabolism and the role of the transcription factor PPARgamma in orchestrating this association. Finally, we discuss the molecular mechanisms underlying metabolic regulations, which provide new opportunities for treating complex metabolic disorders.

  9. Hormonal Regulation of Adipogenesis.

    Science.gov (United States)

    Lee, Mi-Jeong

    2017-09-12

    Adipose tissue includes multiple anatomical depots that serve as an energy reserve that can expand or contract to maintain metabolic homeostasis. During normal growth and in response to overnutrition, adipose tissue expands by increasing the volume of preexisting adipocytes (hypertrophy) and/or by generating new adipocytes (hyperplasia) via recruitment and differentiation of adipose progenitors. This so-called healthy expansion through hyperplasia is thought to be beneficial in that it protects against obesity associated metabolic disorders by allowing for the "safe" storage of excess energy. Remodeling adipose tissue to replace dysfunctional adipocytes that accumulate with obesity and age also requires new fat cell formation and is necessary to maintain metabolic health. Adipogenesis is the process by which adipose progenitors become committed to an adipogenic lineage and differentiate into mature adipocytes. This transition is regulated by complex array of transcriptional factors and numerous autocrine, paracrine, and endocrine signals. We will focus on hormonal factors that regulate adipocyte differentiation and their molecular mechanisms of actions on adipogenesis as studied in vitro and in vivo. Accumulating evidence indicates that adipose progenitors isolated from different adipose tissues exhibit intrinsic differences in adipogenic potential that may contribute to the depot and sex differences in adipose expansion and remodeling capacity. We will put special emphasis on the hormonal factors that are known to depot-dependently affect body fat accumulation and adipocyte development. © 2017 American Physiological Society. Compr Physiol 7:1151-1195, 2017. Copyright © 2017 John Wiley & Sons, Inc.

  10. Regulations and Procedures Manual

    Energy Technology Data Exchange (ETDEWEB)

    Young, Lydia [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)

    2010-09-30

    The purpose of the Regulations and Procedures Manual (RPM) is to provide Laboratory personnel with a reference to University and Lawrence Berkeley National Laboratory policies and regulations by outlining the normal practices and answering most policy questions that arise in the day-to-day operations of Laboratory departments. Much of the information in this manual has been condensed from detail provided in Laboratory procedure manuals, Department of Energy (DOE) directives, and Contract DE-AC02-05CH11231. This manual is not intended, however, to replace any of those documents. The sections on personnel apply only to employees who are not represented by unions. Personnel policies pertaining to employees represented by unions may be found in their labor agreements. Questions concerning policy interpretation should be directed to the department responsible for the particular policy. A link to the Managers Responsible for RPM Sections is available on the RPM home page. If it is not clear which department should be called, please contact the Associate Laboratory Director of Operations.

  11. Strategisk compliance og regulering

    DEFF Research Database (Denmark)

    Kühn Pedersen, Mogens

    2016-01-01

    Denne artikel introducerer strategisk compliance og påpeger dens samspil med klassiske og nyere former for reguleringer i digital værdiskabelse. Konteksten er den digitale økonomi, som vokser frem imellem den materielle økonomis bærepiller: Virksomheder og markeder, men består af en helt ny...... materialitet, som er det digitale univers og dets modsvarighed i nye krav til compliance. Den nye materialitet stiller nye krav, hvad angår digitale processer og transaktioner. Klassisk regulering, som aktører ikke selv kan ændre, støder på egenregulering, hvor aktørerne selv opsætter regler for at skabe...... digital værdi. Dette kalder på strategisk compliance. Med digitalisering er strategisk compliance sat på dagsordnen i reguleringsdebatten. Vi hævder, at regulering og egenregulering kan komme til at virke komplementært i det post-industrielle, digitaliserede samfund....

  12. Effective doses, guidelines & regulations.

    Science.gov (United States)

    Burch, Michael D

    2008-01-01

    A number of countries have developed regulations or guidelines for cyanotoxins and cyanobacteria in drinking water, and in some cases in water used for recreational activity and agriculture. The main focus internationally has been upon microcystin toxins, produced predominantly by Microcystis aeruginosa. This is because microcystins are widely regarded as the most significant potential source of human injury from cyanobacteria on a world-wide scale. Many international guidelines have taken their lead from the World Health Organization's (WHO) provisional guideline of 1 microg L(-1) for microcystin-LR in drinking-water released in 1998 (WHO 2004). The WHO guideline value is stated as being 'provisional', because it covers only microcystin-LR, for reasons that the toxicology is limited and new data for toxicity of cyanobacterial toxins are being generated. The derivation of this guideline is based upon data that there is reported human injury related to consumption of drinking water containing cyanobacteria, or from limited work with experimental animals. It was also recognised that at present the human evidence for microcystin tumor promotion is inadequate and animal evidence is limited. As a result the guideline is based upon the model of deriving a Tolerable Daily intake (TDI) from an animal study No Observed Adverse Effects Level (NOAEL), with the application of appropriate safety or uncertainty factors. The resultant WHO guideline by definition is the concentration of a toxin that does not result in any significant risk to health of the consumer over a lifetime of consumption. Following the release of this WHO provisional guideline many countries have either adopted it directly (e.g., Czech Republic, France, Japan, Korea, New Zealand, Norway, Poland, Brazil and Spain), or have adopted the same animal studies, TDI and derivation convention to arrive at slight variants based upon local requirements (e.g., Australia, Canada). Brazil currently has the most

  13. Translational regulation in nutrigenomics.

    Science.gov (United States)

    Liu, Botao; Qian, Shu-Bing

    2011-11-01

    The emergence of genome-wide analysis to interrogate cellular DNA, RNA, and protein content has revolutionized the study of the control network that mediates cellular homeostasis. Nutrigenomics addresses the effect of nutrients on gene expression, which provides a basis for understanding the biological activity of dietary components. Translation of mRNAs represents the last step of genetic flow and primarily defines the proteome. Translational regulation is thus critical for gene expression, in particular, under nutrient excess or deficiency. Until recently, it was unclear how the global effects of translational control are influenced by nutrient signaling. An emerging concept of translational reprogramming addresses how to maintain the expression of specific proteins during pathophysiological conditions by translation of selective mRNAs. Here we describe recent advances in our understanding of translational control, nutrient signaling, and their dysregulation in aging and cancer. The mechanistic understanding of translational regulation in response to different nutrient conditions may help identify potential dietary and therapeutic targets to improve human health.

  14. Regulation of osteoclast polarization.

    Science.gov (United States)

    Takahashi, Naoyuki; Ejiri, Sadakazu; Yanagisawa, Shigeru; Ozawa, Hidehiro

    2007-07-01

    Osteoclast function consists of several processes: recognition of mineralized tissues, development of ruffled borders and sealing zones, secretion of acids and proteolytic enzymes into the space beneath the ruffled border, and incorporation and secretion of bone degradation products using the transcytosis system. One of the most important questions concerning osteoclast function is how osteoclasts recognize bone and polarize. During the past decade, new approaches have been taken to investigate the regulation of osteoclast polarization. Attachment of osteoclasts to some proteins containing the Arg-Gly-Asp sequence motif through vitronectin receptors is the first step in inducing the polarization of osteoclasts. Physical properties of bone such as hardness or roughness are also required to induce osteoclast polarity. Osteoclasts cultured even on plastic dishes secrete protons toward the dish surface, suggesting that osteoclasts recognize plastic as a mineralized matrix and secrete protons. This notion was supported by the recent findings that bisphosphonates and reveromycin A were specifically incorporated into polarized osteoclasts cultured even on plastic dishes. On the other hand, a sealing zone, defined as a thick band of actin, is induced in osteoclasts adherent only on an apatite-containing mineralized matrix. These results suggest that osteoclasts recognize physical properties of the mineralized tissue to secrete protons, and also sense apatite itself or components of apatite to form the sealing zone. Here, we review recent findings on the regulation of osteoclast polarization. We also discuss how osteoclasts recognize mineralized tissues to form the sealing zone.

  15. Environmental regulations on chlorofluorocarbons

    Science.gov (United States)

    Hoffman, J. S.; Wells, J. B.

    1989-05-01

    In August 1988, the U.S. Environmental Protection Agency issued final regulations that implement the Montreal Protocol on Substances that Deplete the Ozone Layer. The regulations require a 50% reduction in consumption of fully halogenated chlorofluorocarbons (CFCs) within 10 years and a freeze on consumption of halons within 4 years. The Montreal Protocol provisions were designed in September 1987 based on the results of a 2-year international series of scientific, technical, and economic workshops. As would be expected, scientific investigations continued during this period. While these investigations suggested that significant global depletion had already occurred, these preliminary findings were not taken into account during negotiations or rulemaking. In March 1988, however, the international Ozone Trends Panel confirmed the findings. Depletion greater than that projected under the Montreal Protocol has already occurred. An early reassessment of the Protocol provisions appears to be inevitable. Restrictions on CFCs will affect the refrigeration and air-conditioning industries. Emerging alternatives to CFCs include newly developed refrigerants, innovative designs, and engineering controls. Key issues in evaluating these alternatives include energy efficiency, capital costs, service to consumers, and compatibility with existing designs.

  16. [Regulation of terpene metabolism

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, R.

    1991-01-01

    During the last grant period, we have completed studies on the key pathways of monoterpene biosynthesis and catabolism in sage and peppermint, and have, by several lines of evidence, deciphered the rate-limiting step of each pathway. We have at least partially purified and characterized the relevant enzymes of each pathway. We have made a strong case, based on analytical, in vivo, and in vitro studies, that terpene accumulation depends upon the balance between biosynthesis and catabolism, and provided supporting evidence that these processes are developmentally-regulated and very closely associated with senescence of the oil glands. Oil gland ontogeny has been characterized at the ultrastructural level. We have exploited foliar-applied bioregulators to delay gland senescence, and have developed tissue explant and cell culture systems to study several elusive aspects of catabolism. We have isolated pure gland cell clusters and localized monoterpene biosynthesis and catabolism within these structures, and have used these preparations as starting materials for the purification to homogeneity of target regulatory'' enzymes. We have thus developed the necessary background knowledge, based on a firm understanding of enzymology, as well as the necessary experimental tools for studying the regulation of monoterpene metabolism at the molecular level. Furthermore, we are now in a position to extend our systematic approach to other terpenoid classes (C[sub 15]-C[sub 30]) produced by oil glands.

  17. Trust-based environmental regulation.

    Science.gov (United States)

    Lange, Bettina; Gouldson, Andy

    2010-10-15

    Within this paper, we examine the contribution that trust-based relationships can make to achieving better-and particularly more effective, efficient and equitable-environmental regulation. While levels of trust in regulators, regulatory processes and outcomes are often discussed, the influence of trust on different actors and on different measures of regulatory performance is poorly understood. Within this paper, we define trust-based environmental regulation as a specific regulatory style that involves openness and cooperation in interaction between regulated, regulators and third-party stakeholders in order to achieve environmental protection objectives. We then discuss the pros and cons of trust relationships between regulators, regulated businesses and citizens for achieving behavioural change towards greater environmental protection. To illustrate the significance of these issues, we then examine three forms of contractual regulatory style where trust relationships are critically important: responsive regulation, self-regulation and environmental agreements. Based on this analysis, we highlight the importance of trust-based relationships, and we argue that one of the greatest contributions of trust-based environmental regulation is to challenge how we think about regulation. Trust is often understood as enabling existing regulatory relationships or in the case of self-regulation as a complement to regulation. However, we argue that the real potential of trust is to open up new ways for participants in regulatory regimes to engage in collective action, to go beyond a perception of regulation as driven by the competing interests of individual actors, and thus, to open up new channels of influence for behavioural change towards greater environmental protection. Our analysis therefore has great relevance for future research and for on-going debates on the future of regulation. Copyright © 2010. Published by Elsevier B.V.

  18. Regulation as delegation

    Directory of Open Access Journals (Sweden)

    Oren Bar-Gill

    2016-03-01

    Full Text Available Objective to consider the conception of reverse delegation when the government acts a principal and an individual ndash an agent from the point of view of behavioral PrincipalAgent Theory. Methods statistical method sociological polling. Results In diverse areas ndash from retirement savings to consumer credit to prescription drug use to fuel economy and energy efficiency rules to tobacco consumption to food and beverage consumption ndash government makes decisions for us or endeavors to help us make better decisions thus serving as our agent. From the point of view of PrincipalAgent Theory and behavioral PrincipalAgent Theory a great deal of modern regulation can be helpfully evaluated as a hypothetical delegation. Shifting from personal decisions to public goods problems the authors view the idea of reverse delegation with the government as principal and the individuals as agents. They show that the essence of delegation changes depending on the context. The article describes conditions under which various approaches will make sense. Scientific novelty the paper is devoted to the foreign experience of regulation through delegation by the example of a country with developed market economy the USA. It shows the prospects of such approach in solving both the public and the private tasks. Application of PrincipalAgent Theory and behavioral PrincipalAgent Theory is viewed to distinguish between such types of hypothetical delegation as information default rules incentives precommitments mandates and prohibitions. The article considers the benefits and costs of delegation and circumstances in which one or another approach makes sense. Practical significance PrincipalAgent Theory is widely used in economics and political science and can serve as a convenient tool to consider the optimal scale and essence of the assistance rendered to us by the government as our agent. The paper is of interest for the Russian legal science as the institution of

  19. Respiration of bloodstream forms of the parasite Trypanosoma brucei brucei is dependent on a plant-like alternative oxidase

    NARCIS (Netherlands)

    Clarkson, A. B.; Bienen, E. J.; Pollakis, G.; Grady, R. W.

    1989-01-01

    CoQ links the sn-glycerol-3-phosphate dehydrogenase and oxidase components of the cyanide-insensitive, non-cytochrome-mediated respiratory system of bloodstream African trypanosomes. In this and other characteristics, their respiratory system is similar to the alternative oxidase of plants. The

  20. Higher regulators, algebraic

    CERN Document Server

    Bloch, Spencer J

    2000-01-01

    This book is the long-awaited publication of the famous Irvine lectures. Delivered in 1978 at the University of California at Irvine, these lectures turned out to be an entry point to several intimately-connected new branches of arithmetic algebraic geometry, such as regulators and special values of L-functions of algebraic varieties, explicit formulas for them in terms of polylogarithms, the theory of algebraic cycles, and eventually the general theory of mixed motives which unifies and underlies all of the above (and much more). In the 20 years since, the importance of Bloch's lectures has not diminished. A lucky group of people working in the above areas had the good fortune to possess a copy of old typewritten notes of these lectures. Now everyone can have their own copy of this classic work.

  1. Regulation of Transcript Elongation

    Science.gov (United States)

    Belogurov, Georgiy A.; Artsimovitch, Irina

    2015-01-01

    Bacteria lack subcellular compartments and harbor a single RNA polymerase that synthesizes both structural and protein-coding RNAs, which are cotranscriptionally processed by distinct pathways. Nascent rRNAs fold into elaborate secondary structures and associate with ribosomal proteins, whereas nascent mRNAs are translated by ribosomes. During elongation, nucleic acid signals and regulatory proteins modulate concurrent RNA-processing events, instruct RNA polymerase where to pause and terminate transcription, or act as roadblocks to the moving enzyme. Communications among complexes that carry out transcription, translation, repair, and other cellular processes ensure timely execution of the gene expression program and survival under conditions of stress. This network is maintained by auxiliary proteins that act as bridges between RNA polymerase, ribosome, and repair enzymes, blurring boundaries between separate information-processing steps and making assignments of unique regulatory functions meaningless. Understanding the regulation of transcript elongation thus requires genome-wide approaches, which confirm known and reveal new regulatory connections. PMID:26132790

  2. Mitosis and its regulation

    Directory of Open Access Journals (Sweden)

    Frías Vázquez Sara

    2014-07-01

    Full Text Available Cell division by mitosis is essential for the development of organisms and their reproduction; it is also neces- sary that each new cell is genetically identical to that from which it comes. In eukaryotes this is achieved by the presence of complex mechanisms that ensure the integrity of genomic material and their proper segregation during mitosis. The traditional view of mitosis has been divided into different stages that were characterized by morphological studies in dividing cells; advances in molecular biology have led beyond this characterization, so that we now know a range of participant molecules. This article will discuss the process of mitosis, both at the cellular and molecular level and a brief summary of the molecular players that regulate this process.

  3. Meniscus regulator system

    Science.gov (United States)

    Thomas, Marshall K.

    1995-06-01

    A method and system is provided for regulating the meniscus of a water droplet in a high velocity water droplet apparatus in which a large pulse of energy are applied to the water droplet to propel the water droplet at high velocity. In this method the shape of the meniscus of the water droplet is observed and adjusted in accordance with the observation. The energy is applied to the water droplet substantially simultaneously with the observing of the shape of the meniscus. The shape of the meniscus is determined by observing a magnified image of the water droplet so that the adjustment may be made in accordance with the magnified image. The high energy is applied to the water droplet by means of a projectile and an energy transfer body which transfers energy from the projectile to the water droplet. A pressure adjustment device is provided for adjusting the pressure applied to the water droplet in order to adjust the meniscus.

  4. Robust regulering af destillationskolonner

    DEFF Research Database (Denmark)

    Nielsen, Kim

    1987-01-01

    Sharke og Dixons metode benyttes til minimalrealisering af Laplace-overføringsmatricer. Ud fra denne metode til minimalrealisering opnåes en tilstandsbeskrivelse med første ordens Pade'-approximation af tidsforsinkelsen i Laplace-overføringsfunktionsmatricerne. Ud fra A.N Hansens frekvensanalyser...... bør denne regulator dog undersøges med eksakt beskrivelse af tidsforsinkelsen, hvilket ikke er nået indenfor denne opgave, væsenligst på grund af problemer med MIMOFAD Konklusion. Med Sharked og Dixons metode er der opnået kendskab til en simpel metode til minimalrealisering af Laplace......-overføringsmatricer. Metoden munder ud i et simpelt program der foretager minimalrealisering af Laplace-overføringsfunktionsmatricer med reelle adskildte poler. Anvendelsen af programmet resulterer i en tilstandsbeskrivelse af Laplace-overføringsfunktioner for velbeskrevne kolonner i litteraturen. Den opnåede...

  5. Regulation of Terpene Metabolism

    Energy Technology Data Exchange (ETDEWEB)

    Rodney Croteau

    2004-03-14

    OAK-B135 Research over the last four years has progressed fairly closely along the lines initially proposed, with progress-driven expansion of Objectives 1, 2 and 3. Recent advances have developed from three research thrusts: 1. Random sequencing of an enriched peppermint oil gland cDNA library has given access to a large number of potential pathway and regulatory genes for test of function; 2. The availability of new DNA probes and antibodies has permitted investigation of developmental regulation and organization of terpenoid metabolism; and 3. The development of a transformation system for peppermint by colleagues at Purdue University has allowed direct transgenic testing of gene function and added a biotechnological component to the project. The current status of each of the original research objectives is outlined below.

  6. NCAM regulates cell motility

    DEFF Research Database (Denmark)

    Prag, Søren; Lepekhin, Eugene A; Kolkova, Kateryna

    2002-01-01

    Cell migration is required during development of the nervous system. The regulatory mechanisms for this process, however, are poorly elucidated. We show here that expression of or exposure to the neural cell adhesion molecule (NCAM) strongly affected the motile behaviour of glioma cells...... independently of homophilic NCAM interactions. Expression of the transmembrane 140 kDa isoform of NCAM (NCAM-140) caused a significant reduction in cellular motility, probably through interference with factors regulating cellular attachment, as NCAM-140-expressing cells exhibited a decreased attachment...... to a fibronectin substratum compared with NCAM-negative cells. Ectopic expression of the cytoplasmic part of NCAM-140 also inhibited cell motility, presumably via the non-receptor tyrosine kinase p59(fyn) with which NCAM-140 interacts. Furthermore, we showed that the extracellular part of NCAM acted as a paracrine...

  7. [Regulation of terpene metabolism

    Energy Technology Data Exchange (ETDEWEB)

    Croteau, R.

    1989-11-09

    Terpenoid oils, resins, and waxes from plants are important renewable resources. The objective of this project is to understand the regulation of terpenoid metabolism using the monoterpenes (C[sub 10]) as a model. The pathways of monoterpene biosynthesis and catabolism have been established, and the relevant enzymes characterized. Developmental studies relating enzyme levels to terpene accumulation within the oil gland sites of synthesis, and work with bioregulators, indicate that monoterpene production is controlled by terpene cyclases, the enzymes catalyzing the first step of the monoterpene pathway. As the leaf oil glands mature, cyclase levels decline and monoterpene biosynthesis ceases. Yield then decreases as the monoterpenes undergo catabolism by a process involving conversion to a glycoside and transport from the leaf glands to the root. At this site, the terpenoid is oxidatively degraded to acetate that is recycled into other lipid metabolites. During the transition from terpene biosynthesis to catabolism, the oil glands undergo dramatic ultrastructural modification. Degradation of the producing cells results in mixing of previously compartmentized monoterpenes with the catabolic enzymes, ultimately leading to yield decline. This regulatory model is being applied to the formation of other terpenoid classes (C[sub 15] C[sub 20], C[sub 30], C[sub 40]) within the oil glands. Preliminary investigations on the formation of sesquiterpenes (C[sub 15]) suggest that the corresponding cyclases may play a lesser role in determining yield of these products, but that compartmentation effects are important. From these studies, a comprehensive scheme for the regulation of terpene metabolism is being constructed. Results from this project wail have important consequences for the yield and composition of terpenoid natural products that can be made available for industrial exploitation.

  8. Regulation of organelle acidity.

    Science.gov (United States)

    Grabe, M; Oster, G

    2001-04-01

    Intracellular organelles have characteristic pH ranges that are set and maintained by a balance between ion pumps, leaks, and internal ionic equilibria. Previously, a thermodynamic study by Rybak et al. (Rybak, S., F. Lanni, and R. Murphy. 1997. Biophys. J. 73:674-687) identified the key elements involved in pH regulation; however, recent experiments show that cellular compartments are not in thermodynamic equilibrium. We present here a nonequilibrium model of lumenal acidification based on the interplay of ion pumps and channels, the physical properties of the lumenal matrix, and the organelle geometry. The model successfully predicts experimentally measured steady-state and transient pH values and membrane potentials. We conclude that morphological differences among organelles are insufficient to explain the wide range of pHs present in the cell. Using sensitivity analysis, we quantified the influence of pH regulatory elements on the dynamics of acidification. We found that V-ATPase proton pump and proton leak densities are the two parameters that most strongly influence resting pH. Additionally, we modeled the pH response of the Golgi complex to varying external solutions, and our findings suggest that the membrane is permeable to more than one dominant counter ion. From this data, we determined a Golgi complex proton permeability of 8.1 x 10(-6) cm/s. Furthermore, we analyzed the early-to-late transition in the endosomal pathway where Na,K-ATPases have been shown to limit acidification by an entire pH unit. Our model supports the role of the Na,K-ATPase in regulating endosomal pH by affecting the membrane potential. However, experimental data can only be reproduced by (1) positing the existence of a hypothetical voltage-gated chloride channel or (2) that newly formed vesicles have especially high potassium concentrations and small chloride conductance.

  9. Standard types of regulation loops; Chaines de regulation types

    Energy Technology Data Exchange (ETDEWEB)

    Bertrand, M. [ENSAM, Centre d`Enseignement et de Recherche de Lille, 59 - Lille (France)

    1997-12-01

    The aim of this paper is to give help in the analysis of industrial regulation problems using different types of real installations. The increasing complexity of industrial systems requires the use of a decomposition-recomposition procedure using a scheme with different blocs. Examples are given to help the non-specialist users in the mastery of essential choices and in the distinction between operational and material separations. The examples concern: the heating loop of a central heating installation, the sensors and actuators of industrial systems (the temperature regulation of a tubular furnace, the electro-hydraulic positioning systems used in machine tools, forming, aeronautics etc.., the regulation of a mixing system for hot and cold fluids, and the regulation of a fluidizing system. The usual types of regulation loops are presented with the different steps of the resolution of a regulation problem. (J.S.) 7 refs.

  10. Branded prescription drug fee. Final regulations, temporary regulations, and removal of temporary regulations.

    Science.gov (United States)

    2014-07-28

    This document contains final regulations that provide guidance on the annual fee imposed on covered entities engaged in the business of manufacturing or importing branded prescription drugs. This fee was enacted by section 9008 of the Patient Protection and Affordable Care Act, as amended by section 1404 of the Health Care and Education Reconciliation Act of 2010. This document also withdraws the Branded Prescription Drug Fee temporary regulations and contains new temporary regulations regarding the definition of controlled group that apply beginning on January 1, 2015. The final regulations and the new temporary regulations affect persons engaged in the business of manufacturing or importing certain branded prescription drugs. The text of the temporary regulations in this document also serves as the text of proposed regulations set forth in a notice of proposed rulemaking (REG-123286-14) on this subject in the Proposed Rules section in this issue of the Federal Register.

  11. Sustainable regulation of construction.

    Science.gov (United States)

    2000-11-01

    The seminar examined the role building codes and regulations can have in promoting a more sustainable approach to construction, particularly through their application to non-industrial building materials. A range of building materials such as straw, bamboo, rammed earth, adobe, and cob (a mixture of clay and chopped straw) were described and illustrated by slides to show their building potential. The current codes have a prime concern to protect the health and safety of people from the built environment. They have been developed almost exclusively for mainstream industrial materials and methods of construction, which makes them difficult to use with alternative, indigenous, or non-industrial building materials, even though those materials may be considered more sustainable. The argument was put forward that with only one-third of the world population living in modern industrial buildings today, it is not sustainable to re-house the remaining rapidly expanding population in high technology dwellings. Many of the low technology building materials and methods now used by the majority of people in the world need only incremental improvement to be equal or superior to many of their industrial replacements. Since these can be more sustainable methods of building, there needs to be an acceptance of the use of alternative materials, particularly in the developing parts of the world, where they are being rejected for less sustainable industrial methods. However, many codes make it difficult to use non-industrial materials; indeed, many of the industrial materials would not meet the demands that must be now met if they were now being introduced as new materials. Consequently, there is a need to develop codes to facilitate the use of a wider range of materials than in current use, and research is needed to assist this development. Sustainable regulation should take into account the full range of real impacts that materials and systems have in areas such as resource use and

  12. Regulation of the power sector

    CERN Document Server

    2013-01-01

    Regulation of the Power Sector is a unified, consistent and comprehensive treatment of the theories and practicalities of regulation in modern power-supply systems. The need for generation to occur at the time of use occasioned by the impracticality of large-scale electricity storage coupled with constant and often unpredictable changes in demand make electricity-supply systems large, dynamic and complex and their regulation a daunting task. Conceptually arranged in four parts, this book addresses both traditional regulatory frameworks and also liberalized and re-regulated environments. First, an introduction gives a full characterization of power supply including engineering, economic and regulatory viewpoints. The second part presents the fundamentals of regulation and the third looks at the regulation of particular components of the power sector in detail. Advanced topics and subjects still open or subject to dispute form the content of the fourth part. In a sector where regulatory design is the key driver...

  13. Epigenetic regulation in obesity.

    Science.gov (United States)

    Drummond, Elaine M; Gibney, Eileen R

    2013-07-01

    Research suggests that 65% of variation in obesity is genetic. However, much of the known genetic associations have little known function and their effect size small, thus the gene-environment interaction, including epigenetic influences on gene expression, is suggested to be an important factor in the susceptibilty to obesity. This review will explore the potential of epigenetic markers to influence expression of genes associated with obesity. Epigenetic changes in utero are known to have direct implications on the phenotype of the offspring. More recently work has focused on how such epigenetic changes continue to regulate risk of obesity from infancy through to adulthood. Work has shown that, for example, hypomethylation of the MC4 gene causes an increase in expression, and has a direct impact on appetite and intake, and thus influences risk of obesity. Similar influences are also seen in other aspects of obesity including inflammation and adiposity. Maternal diet during foetal development has many epigenetic implications, which affect the offspring's risk factors for obesity during childhood and adulthood, and even in subsequent generations. Genes associated with risk of obesity, are susceptible to epigenetic mutations, which have subsequent effects on disease mechanisms, such as appetite and impaired glucose and insulin tolerance.

  14. Regulating the sharing economy

    Directory of Open Access Journals (Sweden)

    Kristofer Erickson

    2016-06-01

    Full Text Available In this introductory essay, we explore definitions of the ‘sharing economy’, a concept indicating both social (relational, communitarian and economic (allocative, profit-seeking aspects which appear to be in tension. We suggest combining the social and economic logics of the sharing economy to focus on the central features of network enabled, aggregated membership in a pool of offers and demands (for goods, services, creative expressions. This definition of the sharing economy distinguishes it from other related peer-to-peer and collaborative forms of production. Understanding the social and economic motivations for and implications of participating in the sharing economy is important to its regulation. Each of the papers in this special issue contributes to knowledge by linking the social and economic aspects of sharing economy practices to regulatory norms and mechanisms. We conclude this essay by suggesting future research to further clarify and render intelligible the sharing economy, not as a contradiction in terms but as an empirically observable realm of socio-economic activity.

  15. Personality and Emotion Regulation Strategies

    Directory of Open Access Journals (Sweden)

    Esti Hayu Purnamaningsih

    2017-01-01

    Full Text Available The emotions has many important functions in our life such as in relation of interpersonal communication, and health. In interpersonal communicative function aimed to signal to other information about internal state. Emotions manifests in specific cognitive, behavioural, and physiological reactions, thus closely related to health. There is wide variety of ways for individuals to regulate their emotion. In this regard, there are two kinds of emotion regulation strategy; first Antecedent-focused emotion regulation consisting of situation selection, situation modification, attentional deployment, cognitive change and second, Response-focused emotion regulation consisting of suppression. The purpose of this research is to investigate personality factors relate with emotion regulation strategies. 339 students from Faculty of Psychology, Universitas Gadjah Mada were participating in this study and given The Big Five Personality Factors (Ramdhani, 2012, adaptation, and the modified version of the Emotion Regulation Scale was used, Emotion Regulation Questionnaire (John & Gross, 2004 which measure personality and emotion regulation respectively. Using multiple regression analysis, the study indicated that personality predicts emotion regulation strategies.

  16. Power-MOSFET Voltage Regulator

    Science.gov (United States)

    Miller, W. N.; Gray, O. E.

    1982-01-01

    Ninety-six parallel MOSFET devices with two-stage feedback circuit form a high-current dc voltage regulator that also acts as fully-on solid-state switch when fuel-cell out-put falls below regulated voltage. Ripple voltage is less than 20 mV, transient recovery time is less than 50 ms. Parallel MOSFET's act as high-current dc regulator and switch. Regulator can be used wherever large direct currents must be controlled. Can be applied to inverters, industrial furnaces photovoltaic solar generators, dc motors, and electric autos.

  17. Glucocorticoid Regulation of Reproduction.

    Science.gov (United States)

    Geraghty, Anna C; Kaufer, Daniela

    2015-01-01

    It is well accepted that stress, measured by increased glucocorticoid secretion, leads to profound reproductive dysfunction. In times of stress, glucocorticoids activate many parts of the fight or flight response, mobilizing energy and enhancing survival, while inhibiting metabolic processes that are not necessary for survival in the moment. This includes reproduction, an energetically costly procedure that is very finely regulated. In the short term, this is meant to be beneficial, so that the organism does not waste precious energy needed for survival. However, long-term inhibition can lead to persistent reproductive dysfunction, even if no longer stressed. This response is mediated by the increased levels of circulating glucocorticoids, which orchestrate complex inhibition of the entire reproductive axis. Stress and glucocorticoids exhibits both central and peripheral inhibition of the reproductive hormonal axis. While this has long been recognized as an issue, understanding the complex signaling mechanism behind this inhibition remains somewhat of a mystery. What makes this especially difficult is attempting to differentiate the many parts of both of these hormonal axes, and new neuropeptide discoveries in the last decade in the reproductive field have added even more complexity to an already complicated system. Glucocorticoids (GCs) and other hormones within the hypothalamic-pituitary-adrenal (HPA) axis (as well as contributors in the sympathetic system) can modulate the hypothalamic-pituitary-gonadal (HPG) axis at all levels-GCs can inhibit release of GnRH from the hypothalamus, inhibit gonadotropin synthesis and release in the pituitary, and inhibit testosterone synthesis and release from the gonads, while also influencing gametogenesis and sexual behavior. This chapter is not an exhaustive review of all the known literature, however is aimed at giving a brief look at both the central and peripheral effects of glucocorticoids on the reproductive function.

  18. Algorithms for optimal price regulations

    NARCIS (Netherlands)

    Grigoriev, Alexander; van Loon, Joyce; Uetz, Marc Jochen; Papadimitriou, C.; Zhang, S.

    2008-01-01

    Since summer 2007, mobile phone users in the European Union (EU) are protected by a ceiling on the roaming tariff when calling or receiving a call abroad. We analyze the effects of this price regulative policy, and compare it to alternative implementations of price regulations. The problem is a

  19. Regulating Pornography: A Public Dilemma.

    Science.gov (United States)

    Thompson, Margaret E.; And Others

    1990-01-01

    Examines attitudes toward sex and pornography by means of a telephone survey of Dane County, Wisconsin, adults. Describes survey questions about sexual attitudes, perceived effects of pornography, and pornography regulation. Concludes that adults who feel more strongly that pornography has negative effects are more opposed to its regulation. (SG)

  20. Teachers' Regulation of the Classroom.

    Science.gov (United States)

    Muir, William K., Jr.

    The nature of teachers' control in classrooms is explored in order: to understand the tension created when noneducators superimpose their rules on the regime of teachers at work and to learn something of a general nature about the antagonism between regulators and those they regulate. Teachers' regulatory powers are based on coercion, exchange, or…

  1. Designing Next Generation Telecom Regulation

    DEFF Research Database (Denmark)

    Henten, Anders; Samarajiva, Rohan

    – ICT convergence regulation and multisector utility regulation. Whatever structure of next generation telecom regulation is adopted, all countries will need to pay much greater attention to the need for increased coordination of policy directions and regulatory activities both across the industries......Continuously expanding applications of information and communication technologies (ICT) are transforming local, national, regional and international economies into network economies, the foundation for information societies. They are being built upon expanded and upgraded national telecom networks...... to creating an environment to foster a massive expansion in the coverage and capabilities of the information infrastructure networks, with national telecom regulators as the key implementers of the policies of reform. The first phase of reform has focused on industry specific telecom policy and regulation...

  2. RNA-guided transcriptional regulation

    Energy Technology Data Exchange (ETDEWEB)

    Church, George M.; Mali, Prashant G.; Esvelt, Kevin M.

    2016-02-23

    Methods of modulating expression of a target nucleic acid in a cell are provided including introducing into the cell a first foreign nucleic acid encoding one or more RNAs complementary to DNA, wherein the DNA includes the target nucleic acid, introducing into the cell a second foreign nucleic acid encoding a nuclease-null Cas9 protein that binds to the DNA and is guided by the one or more RNAs, introducing into the cell a third foreign nucleic acid encoding a transcriptional regulator protein or domain, wherein the one or more RNAs, the nuclease-null Cas9 protein, and the transcriptional regulator protein or domain are expressed, wherein the one or more RNAs, the nuclease-null Cas9 protein and the transcriptional regulator protein or domain co-localize to the DNA and wherein the transcriptional regulator protein or domain regulates expression of the target nucleic acid.

  3. Regulation of GMOs in China.

    Science.gov (United States)

    Liu, Yinliang

    2008-12-01

    Genetically modified organisms (GMOs) are created by biotechnology to serve people with much benefit while may impose risks to ecological environment and human health and therefore need careful regulation. During the past two decades, GMOs have been well developed in China and so has their corresponding regulation. This paper reviews and comments the multiple aspects of mainly the agricultural GMOs, including their safety assessment, control measures, trade activities, import, labels, and GM food, which have been prescribed by the corresponding laws, regulations and administrative measures. It is held that till present a framework for regulation of agricultural GMOs and GM food has been established basically in China, while a more comprehensive system for regulation of all kinds of GMOs and all kinds of related activities is still needed at present and in the future.

  4. PPARγ regulates exocrine pancreas lipase.

    Science.gov (United States)

    Danino, Hila; Naor, Ronny Peri-; Fogel, Chen; Ben-Harosh, Yael; Kadir, Rotem; Salem, Hagit; Birk, Ruth

    2016-12-01

    Pancreatic lipase (triacylglycerol lipase EC 3.1.1.3) is an essential enzyme in hydrolysis of dietary fat. Dietary fat, especially polyunsaturated fatty acids (PUFA), regulate pancreatic lipase (PNLIP); however, the molecular mechanism underlying this regulation is mostly unknown. As PUFA are known to regulate expression of proliferator-activated receptor gamma (PPARγ), and as we identified in-silico putative PPARγ binding sites within the putative PNLIP promoter sequence, we hypothesized that PUFA regulation of PNLIP might be mediated by PPARγ. We used in silico bioinformatics tools, reporter luciferase assay, PPARγ agonists and antagonists, PPARγ overexpression in exocrine pancreas AR42J and primary cells to study PPARγ regulation of PNLIP. Using in silico bioinformatics tools we mapped PPARγ binding sites (PPRE) to the putative promoter region of PNLIP. Reporter luciferase assay in AR42J rat exocrine pancreas acinar cells transfected with various constructs of the putative PNLIP promoter showed that PNLIP transcription is significantly enhanced by PPARγ dose-dependently, reaching maximal levels with multi PPRE sites. This effect was significantly augmented in the presence of PPARγ agonists and reduced by PPARγ antagonists or mutagenesis abrogating PPRE sites. Over-expression of PPARγ significantly elevated PNLIP transcript and protein levels in AR42J cells and in primary pancreas cells. Moreover, PNLIP expression was up-regulated by PPARγ agonists (pioglitazone and 15dPGJ2) and significantly down-regulated by PPARγ antagonists in non-transfected rat exocrine pancreas AR42J cell line cells. PPARγ transcriptionally regulates PNLIP gene expression. This transcript regulation resolves part of the missing link between dietary PUFA direct regulation of PNLIP. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Evidence for regulated expression of Telomeric Repeat-containing RNAs (TERRA) in parasitic trypanosomatids.

    Science.gov (United States)

    Damasceno, Jeziel D; Silva, Gabriel LA; Tschudi, Christian; Tosi, Luiz Ro

    2017-08-01

    The Telomeric Repeat-containing RNAs (TERRA) participate in the homeostasis of telomeres in higher eukaryotes. Here, we investigated the expression of TERRA in Leishmania spp. and Trypanosoma brucei and found evidences for its expression as a specific RNA class. The trypanosomatid TERRA are heterogeneous in size and partially polyadenylated. The levels of TERRA transcripts appear to be modulated through the life cycle in both trypanosomatids investigated, suggesting that TERRA play a stage-specific role in the life cycle of these early-branching eukaryotes.

  6. Mental fatigue impairs emotion regulation.

    Science.gov (United States)

    Grillon, Christian; Quispe-Escudero, David; Mathur, Ambika; Ernst, Monique

    2015-06-01

    Because healthy physical and mental functioning depends on the ability to regulate emotions, it is important to identify moderators of such regulations. Whether mental fatigue, subsequent to the depletion of cognitive resources, impairs explicit emotion regulation to negative stimuli is currently unknown. This study explored this possibility. In a within-subject design over 2 separate sessions, healthy individuals performed easy (control session) or difficult (depletion session) cognitive tasks. Subsequently, they were presented with neutral and negative pictures, with instructions to either maintain or regulate (i.e., reduce) the emotions evoked by the pictures. Emotional reactivity was probed with the startle reflex. The negative pictures evoked a similar aversive state in the control and depletion sessions as measured by startle potentiation. However, subjects were able to down-regulate their aversive state only in the control session, not in the depletion session. These results indicate that mental fatigue following performance of cognitive tasks impairs emotion regulation without affecting emotional reactivity. These findings suggest that mental fatigue needs to be incorporated into models of emotion regulation. (c) 2015 APA, all rights reserved).

  7. Emotion Regulation in Alcohol Dependence.

    Science.gov (United States)

    Petit, Géraldine; Luminet, Olivier; Maurage, François; Tecco, Juan; Lechantre, Stéphane; Ferauge, Marc; Gross, James J; de Timary, Philippe

    2015-12-01

    The main aim of this study was to investigate, in alcohol-dependent (AD) patients, the use of the 5 emotion regulation strategies specified in Gross's (1998, Rev Gen Psychol, 2, 271) process model of emotion regulation with the use of a semi-structured interview allowing a detailed and high-quality assessment of emotion regulation strategies. A secondary aim was to examine the possible influence of protracted abstinence and detoxification on emotion dysregulation. Finally, the association between the level of craving and the types of regulation strategies was investigated. Forty-four treatment-seeking AD patients with varying time spent in rehabilitation, and 26 healthy controls were interviewed using a version of the Emotion Regulation Interview (Werner et al., 2011, J Psychopathol Behav Assess, 33, 346) adapted to alcohol dependence. Compared to controls, AD patients reported significantly greater use of response modulation and attentional deployment, but lesser use of cognitive change. Among patients, (1) rehabilitation duration was positively correlated with the use of cognitive change and (2) the use of response modulation was positively associated with the level of craving. These findings clarify the specific pattern of emotion dysregulation associated with alcohol dependence. They also suggest that (1) abstinence is associated with a shift toward more adaptive emotion regulation patterns and that (2) inefficient regulation strategies may lead to craving and the maintenance of alcohol use. If these findings are confirmed through longitudinal and mediation designs, they will have important clinical implications. Copyright © 2015 by the Research Society on Alcoholism.

  8. Regulation of Drosophila metamorphosis by xenobiotic response regulators

    National Research Council Canada - National Science Library

    Deng, Huai; Kerppola, Tom K

    2013-01-01

    Mammalian Nrf2-Keap1 and the homologous Drosophila CncC-dKeap1 protein complexes regulate both transcriptional responses to xenobiotic compounds as well as native cellular and developmental processes...

  9. Designing Next Generation Telecom Regulation

    DEFF Research Database (Denmark)

    Henten, Anders; Samarajiva, Rohan; Melody, William H.

    2003-01-01

    This article critically examines the multiple rationales for telecom, IT, media convergence regulation, on the one hand, and multisector utility regulation, on the other, and the practical questions of implementation they pose, with a view to contributing to informed policy and regulatory decisions...... to the regulatory process such as scarcity of regulatory resources and safeguards for regulatory independence, are examined. It is concluded that ICT and media convergence issues are primarily about improving the efficiency of market economies, and how changes in regulation can facilitate this process. Multi...

  10. Nanometrology - challenges for health regulation

    Directory of Open Access Journals (Sweden)

    Jailton Carreteiro Damasceno

    2013-11-01

    Full Text Available The relationship between metrology, nanotechnology and nanoscience and sanitary regulation is discussed from the point of view of its importance and the interrelationship between the themes for the development of products and services involving nanotech-nology. The discussion involves the main techniques for measuring dimensional, chemical and biological properties of materials, and presents some of the challenges for the future. Issues such as processes of standardization and regulation in Europe, U.S. and Brazil are also addressed, providing an overview of how these processes are related to sanitary regulation.

  11. Electronic Code of Federal Regulations

    Data.gov (United States)

    National Archives and Records Administration — The Electronic Code of Federal Regulations (e-CFR) is the codification of the general and permanent rules published in the Federal Register by the executive...

  12. Network Regulation and Support Schemes

    DEFF Research Database (Denmark)

    Ropenus, Stephanie; Schröder, Sascha Thorsten; Jacobsen, Henrik

    2009-01-01

    -in tariffs to market-based quota systems, and network regulation approaches, comprising rate-of-return and incentive regulation. National regulation and the vertical structure of the electricity sector shape the incentives of market agents, notably of distributed generators and network operators....... This article seeks to investigate the interactions between the policy dimensions of support schemes and network regulation and how they affect the deployment of distributed generation. Firstly, a conceptual analysis examines how the incentives of the different market agents are affected. In particular......At present, there exists no explicit European policy framework on distributed generation. Various Directives encompass distributed generation; inherently, their implementation is to the discretion of the Member States. The latter have adopted different kinds of support schemes, ranging from feed...

  13. PDH regulation in skeletal muscle

    DEFF Research Database (Denmark)

    Kiilerich, Kristian

    regulation in human skeletal muscle. 2: Effect of muscle glycogen on PDH regulation in human skeletal muscle at rest and during exercise. 3: The impact of physical inactivity on PDH regulation in human skeletal muscle at rest and during exercise. 4: Elucidating the importance of PGC-1? in PDH regulation...... in mouse skeletal muscle at rest and in response to fasting and during recovery from exercise. The studies indicate that the content of PDH-E1? in human muscle follows the metabolic profile of the muscle, rather than the myosin heavy chain fiber distribution of the muscle. The larger lactate accumulation...... in human skeletal muscle. It may be noted that the increased PDK4 protein associated with elevated plasma FFA occurs already 2 hours after different dietary intake. A week of physical inactivity (bed rest), leading to whole body glucose intolerance, does not affect muscle PDH-E1? content, or the exercise...

  14. Comparison of some European regulations

    Energy Technology Data Exchange (ETDEWEB)

    Argyriadis, K. [Germanisher Lloyd, Hamburg (Germany)

    1996-09-01

    Fatigue calculations are an essential part in certification of a wind turbine. Manufacturers have to fulfill recommendations of several different regulations throughout Europe with the result that the design has often to be altered to satisfy them. In general three national (D/GL, NL, DK), and two international (GL, IEC) regulations are in use, with the IEC standard getting more importance with wind energy deploying to more in regions with no yet clearly defined national standards (India, Spain). The Germanischer Lloyd made calculations for wind turbines they are certifying and in one case we compared the resulting damages for different regulations and classes on a 600 kW, three bladed, stall regulated wind turbine. (EG) 18 refs.

  15. Firms' Compliance with Complex Regulations

    NARCIS (Netherlands)

    Mendoza Rodriguez, J.P.; Dekker, H.C.; Wielhouwer, J.L.

    2016-01-01

    This study addresses the question of what explains compliance with complex regulations, which are technical, extensive, and often subject to modifications. Based on official (anonymized) data of financial intermediaries in the Netherlands (N = 602), we examined the association between compliance

  16. Microbial regulation in gorgonian corals

    National Research Council Canada - National Science Library

    Hunt, Laura R; Smith, Stephanie M; Downum, Kelsey R; Mydlarz, Laura D

    2012-01-01

    .... Since many bacteria use quorum sensing (QS) signals to facilitate colonization of host organisms, regulation of prokaryotic cell-to-cell communication may represent an important bacterial control mechanism...

  17. How should Bitcoin be regulated ?

    OpenAIRE

    SHCHERBAK, Sergii

    2014-01-01

    The lack of clarity about Bitcoin’s legal framework has meant that none of the regulators across the EU have yet achieved sufficient clarity in the legal treatment of Bitcoin and its stakeholders. This uncertainty poses a number of substantial risks to Bitcoin stakeholders and creates challenges for regulatory authorities. Therefore, there is a need for a clear strategy for Bitcoin’s regulation aiming to ensure the maximum possible balance between the interests of Bitcoin stakeholders longing...

  18. Money Laundering and its Regulation

    OpenAIRE

    Alberto E. Chong; Florencio López-de-Silanes

    2007-01-01

    The recent wave of terrorist attacks has increased the attention paid to money laundering activities. Using several methodologies, this paper investigates empirically the determinants of money laundering and its regulation in over 80 countries by assembling a cross-country dataset on proxies for money laundering and the prevalence of feeding activities. The paper additionally constructs specific money laundering regulation indices based on available information on laws and their mechanisms of...

  19. Epigenetic regulation of persistent pain

    Science.gov (United States)

    Bai, Guang; Ren, Ke; Dubner, Ronald

    2014-01-01

    Persistent or chronic pain is tightly associated with various environmental changes and linked to abnormal gene expression within cells processing nociceptive signaling. Epigenetic regulation governs gene expression in response to environmental cues. Recent animal model and clinical studies indicate that epigenetic regulation plays an important role in the development/maintenance of persistent pain and, possibly the transition of acute pain to chronic pain, thus shedding light in a direction for development of new therapeutics for persistent pain. PMID:24948399

  20. Liquidity regulation and bank behavior

    OpenAIRE

    Bonner, C.

    2014-01-01

    In response to the 2007-08 financial crisis, the Basel Committee on Banking Supervision proposed two liquidity standards to reinforce banks’ resilience to liquidity risks. The purpose of this thesis is to analyze the impact of liquidity regulation on bank behavior. The first of four main chapters analyzes the development of global liquidity standards, their objectives as well as their interaction with capital standards. The analysis suggests that regulating capital is associated with declinin...

  1. Civilsamfundets ABC: R for Regulering

    DEFF Research Database (Denmark)

    Meyer, Gitte; Lund, Anker Brink

    2016-01-01

    Hvad er civilsamfundet? Anker Brink Lund og Gitte Meyer fra CBS Center for Civil Society Studies gennemgår civilsamfundet bogstav for bogstav. Vi er nået til R for Regulering.......Hvad er civilsamfundet? Anker Brink Lund og Gitte Meyer fra CBS Center for Civil Society Studies gennemgår civilsamfundet bogstav for bogstav. Vi er nået til R for Regulering....

  2. Regulating Power from Supermarket Refrigeration

    DEFF Research Database (Denmark)

    O'Connell, Niamh; Madsen, Henrik; Pinson, Pierre

    2014-01-01

    This paper presents an analysis of the demand response capabilities of a supermarket refrigeration system, with a particular focus on the suitability for participation in the regulating power market. An ARMAX model of a supermarket refrigeration system is identified using experimental data from...... be represented in a manner that is sufficiently simple to communicate to a market operator in the form of a bid for the provision of regulating power....

  3. Emotion regulation and sport performance.

    Science.gov (United States)

    Wagstaff, Christopher R D

    2014-08-01

    This study used a single-blind, within-participant, counterbalanced, repeated-measures design to examine the relationship between emotional self-regulation and sport performance. Twenty competitive athletes completed four laboratory-based conditions; familiarization, control, emotion suppression, and nonsuppression. In each condition participants completed a 10-km cycling time trial requiring self-regulation. In the experimental conditions participants watched an upsetting video before performing the cycle task. When participants suppressed their emotional reactions to the video (suppression condition) they completed the cycling task slower, generated lower mean power outputs, and reached a lower maximum heart rate and perceived greater physical exertion than when they were given no self-regulation instructions during the video (nonsuppression condition) and received no video treatment (control condition). The findings suggest that emotional self-regulation resource impairment affects perceived exertion, pacing and sport performance and extends previous research examining the regulation of persistence on physical tasks. The results are discussed in line with relevant psychophysiological theories of self-regulation and fatigue and pertinent potential implications for practice regarding performance and well-being are suggested.

  4. Thyroid Hormone Regulation of Metabolism

    Science.gov (United States)

    Mullur, Rashmi; Liu, Yan-Yun

    2014-01-01

    Thyroid hormone (TH) is required for normal development as well as regulating metabolism in the adult. The thyroid hormone receptor (TR) isoforms, α and β, are differentially expressed in tissues and have distinct roles in TH signaling. Local activation of thyroxine (T4), to the active form, triiodothyronine (T3), by 5′-deiodinase type 2 (D2) is a key mechanism of TH regulation of metabolism. D2 is expressed in the hypothalamus, white fat, brown adipose tissue (BAT), and skeletal muscle and is required for adaptive thermogenesis. The thyroid gland is regulated by thyrotropin releasing hormone (TRH) and thyroid stimulating hormone (TSH). In addition to TRH/TSH regulation by TH feedback, there is central modulation by nutritional signals, such as leptin, as well as peptides regulating appetite. The nutrient status of the cell provides feedback on TH signaling pathways through epigentic modification of histones. Integration of TH signaling with the adrenergic nervous system occurs peripherally, in liver, white fat, and BAT, but also centrally, in the hypothalamus. TR regulates cholesterol and carbohydrate metabolism through direct actions on gene expression as well as cross-talk with other nuclear receptors, including peroxisome proliferator-activated receptor (PPAR), liver X receptor (LXR), and bile acid signaling pathways. TH modulates hepatic insulin sensitivity, especially important for the suppression of hepatic gluconeogenesis. The role of TH in regulating metabolic pathways has led to several new therapeutic targets for metabolic disorders. Understanding the mechanisms and interactions of the various TH signaling pathways in metabolism will improve our likelihood of identifying effective and selective targets. PMID:24692351

  5. To Regulate or Not to Regulate? Views on Electronic Cigarette Regulations and Beliefs about the Reasons for and against Regulation.

    Science.gov (United States)

    Sanders-Jackson, Ashley; Tan, Andy S L; Bigman, Cabral A; Mello, Susan; Niederdeppe, Jeff

    2016-01-01

    Policies designed to restrict marketing, access to, and public use of electronic cigarettes (e-cigarettes) are increasingly under debate in various jurisdictions in the US. Little is known about public perceptions of these policies and factors that predict their support or opposition. Using a sample of US adults from Amazon Mechanical Turk in May 2015, this paper identifies beliefs about the benefits and costs of regulating e-cigarettes and identifies which of these beliefs predict support for e-cigarette restricting policies. A higher proportion of respondents agreed with 8 different reasons to regulate e-cigarettes (48.5% to 83.3% agreement) versus 7 reasons not to regulate e-cigarettes (11.5% to 18.9%). The majority of participants agreed with 7 out of 8 reasons for regulation. When all reasons to regulate or not were included in a final multivariable model, beliefs about protecting people from secondhand vapor and protecting youth from trying e-cigarettes significantly predicted stronger support for e-cigarette restricting policies, whereas concern about government intrusion into individual choices was associated with reduced support. This research identifies key beliefs that may underlie public support or opposition to policies designed to regulate the marketing and use of e-cigarettes. Advocates on both sides of the issue may find this research valuable in developing strategic campaigns related to the issue. Specific beliefs of potential benefits and costs of e-cigarette regulation (protecting youth, preventing exposure to secondhand vapor, and government intrusion into individual choices) may be effectively deployed by policy makers or health advocates in communicating with the public.

  6. To Regulate or Not to Regulate? Views on Electronic Cigarette Regulations and Beliefs about the Reasons for and against Regulation.

    Directory of Open Access Journals (Sweden)

    Ashley Sanders-Jackson

    Full Text Available Policies designed to restrict marketing, access to, and public use of electronic cigarettes (e-cigarettes are increasingly under debate in various jurisdictions in the US. Little is known about public perceptions of these policies and factors that predict their support or opposition.Using a sample of US adults from Amazon Mechanical Turk in May 2015, this paper identifies beliefs about the benefits and costs of regulating e-cigarettes and identifies which of these beliefs predict support for e-cigarette restricting policies.A higher proportion of respondents agreed with 8 different reasons to regulate e-cigarettes (48.5% to 83.3% agreement versus 7 reasons not to regulate e-cigarettes (11.5% to 18.9%. The majority of participants agreed with 7 out of 8 reasons for regulation. When all reasons to regulate or not were included in a final multivariable model, beliefs about protecting people from secondhand vapor and protecting youth from trying e-cigarettes significantly predicted stronger support for e-cigarette restricting policies, whereas concern about government intrusion into individual choices was associated with reduced support.This research identifies key beliefs that may underlie public support or opposition to policies designed to regulate the marketing and use of e-cigarettes. Advocates on both sides of the issue may find this research valuable in developing strategic campaigns related to the issue.Specific beliefs of potential benefits and costs of e-cigarette regulation (protecting youth, preventing exposure to secondhand vapor, and government intrusion into individual choices may be effectively deployed by policy makers or health advocates in communicating with the public.

  7. Balancing Public and Private Regulation

    Directory of Open Access Journals (Sweden)

    Martijn Scheltema

    2016-01-01

    Full Text Available Voluntary Sustainability Standards (VSS might develop into a viable alternative to public regulation. However, it turns on the (regulatory circumstances whether that holds true in practice. If public regulation on CSR topics is lacking, governments are unable to agree upon certain topics on a global level or diverging public regulation exists, VSS can be helpful to set global standards. Obviously, private standards will especially be helpful if they are commensurate with local public legislation (and e.g. treaties and/or are accepted by local governments. If one neglects this, numerous domestic structures might exist that frustrate VSS. Furthermore, governments have to remain vigilant as to whether these private regimes do not result in market disruption, consumer detriment or hamper trade. VSS might also compete with public arrangements which might limit the uptake of VSS. However, if public regulation exists VSS might be a viable alternative if compliance with not too compelling public norms by market participants is rather poor and the public policymaker is aiming to incentivize the better performing part of the market to embark on higher standards and thus only desires to regulate the less performing part of the market. However, of paramount importance is the effectiveness of VSS in order to be a viable alternative to public regulation. The effectiveness of VSS should be assessed using an integrated multi-disciplinary (comparative approach entailing legal, impact-assessment, legitimacy, governance and behavioural aspects. Only effective VSS in the aforementioned sense are a true alternative to public regulation.Beyond that, the legal perspective in connection with (the effectiveness of VSS is discussed, featuring FSC and UTZ Certified as an example. It is important from this perspective that VSS have a clear and sufficiently selective objective and sufficiently specific norms, are regularly evaluated, entail ‘conflict of law rules’ and

  8. 77 FR 43082 - Federal Acquisition Regulation; Information Collection; Commerce Patent Regulations

    Science.gov (United States)

    2012-07-23

    ... Regulation; Information Collection; Commerce Patent Regulations AGENCIES: Department of Defense (DOD... approved information collection requirement concerning Department of Commerce patent regulations. Public...: Submit comments identified by Information Collection 9000- 0095, Commerce Patent Regulations, by any of...

  9. Molecular basis of osmotic regulation.

    Science.gov (United States)

    Burg, M B

    1995-06-01

    Cells almost universally respond to the stress of long-term hyperosmolality by accumulating compatible organic osmolytes. This allows them to maintain normal cell volume without a deleterious increase in intracellular inorganic ion concentration. Cells in the renal inner medulla are exposed to variable concentrations of salt and urea that may reach molal levels. The organic osmolytes that they accumulate include sorbitol, betaine, inositol, taurine, and glycerophosphocholine (GPC). This review considers recent advances in understanding osmotic regulation of these substances. Sorbitol is synthesized from glucose catalyzed by aldose reductase. Hypertonicity elevates the abundance of this enzyme by increasing transcription of its gene. Betaine is taken up via a specialized transporter. Hypertonicity raises the number of transporters by increasing their transcription. Current studies demonstrate that the 5' regions flanking the aldose reductase and betaine transporter genes contain osmotic response elements that increase transcription in response to hypertonicity. Osmotic regulation of inositol and taurine uptake also involves increased expression of specific transporter genes. GPC is unique in that its level rises in response to high urea, as well as hypertonicity. GPC accumulation is mainly regulated by changes in its degradation to choline, catalyzed by GPC:choline phosphodiesterase. Numerous other genes, including those for heat shock proteins, are also induced by hypertonicity. Their regulation and their role in osmotic regulation are the subject of considerable ongoing research.

  10. Progress toward risk informed regulation

    Energy Technology Data Exchange (ETDEWEB)

    Rogers, K.C.

    1997-01-01

    For the last several years, the NRC, with encouragement from the industry, has been moving in the direction of risk informed regulation. This is consistent with the regulatory principle of efficiency, formally adopted by the Nuclear Regulatory Commission in 1991, which requires that regulatory activities be consistent with the degree of risk reduction they achieve. Probabilistic risk analysis has become the tool of choice for selecting the best of several alternatives. Closely related to risk informed regulation is the development of performance based rules. Such rules focus on the end result to be achieved. They do not specify the process, but instead establish the goals to be reached and how the achievement of those goals is to be judged. The inspection and enforcement activity is based on whether or not the goals have been met. The author goes on to offer comments on the history of the development of this process and its probable development in the future. He also addresses some issues which must be resolved or at least acknowledged. The success of risk informed regulation ultimately depends on having sufficiently reliable data to allow quantification of regulatory alternatives in terms of relative risk. Perhaps the area of human reliability and organizational performance has the greatest potential for improvement in reactor safety. The ability to model human performance is significantly less developed that the ability to model mechanical or electrical systems. The move toward risk informed, performance based regulation provides an unusual, perhaps unique, opportunity to establish a more rational, more effective basis for regulation.

  11. Regulation of Autophagy by Kinases

    Energy Technology Data Exchange (ETDEWEB)

    Sridharan, Savitha; Jain, Kirti; Basu, Alakananda, E-mail: alakananda.basu@unthsc.edu [Department of Molecular Biology and Immunology, Institute for Cancer Research, University of North Texas Health Science Center, Fort Worth, TX 76107 (United States)

    2011-06-09

    Autophagy is a process of self-degradation that maintains cellular viability during periods of metabolic stress. Although autophagy is considered a survival mechanism when faced with cellular stress, extensive autophagy can also lead to cell death. Aberrations in autophagy are associated with several diseases, including cancer. Therapeutic exploitation of this process requires a clear understanding of its regulation. Although the core molecular components involved in the execution of autophagy are well studied there is limited information on how cellular signaling pathways, particularly kinases, regulate this complex process. Protein kinases are integral to the autophagy process. Atg1, the first autophagy-related protein identified, is a serine/threonine kinase and it is regulated by another serine/threonine kinase mTOR. Emerging studies suggest the participation of many different kinases in regulating various components/steps of this catabolic process. This review focuses on the regulation of autophagy by several kinases with particular emphasis on serine/threonine protein kinases such as mTOR, AMP-activated protein kinase, Akt, mitogen-activated protein kinase (ERK, p38 and JNK) and protein kinase C that are often deregulated in cancer and are important therapeutic targets.

  12. Re-Framing Biotechnology Regulation.

    Science.gov (United States)

    Peck, Alison

    Biotechnology is about to spill the banks of federal regulation. New genetic engineering techniques like CRISPR-Cas9 promise revolutionary breakthroughs in medicine, agriculture, and public health—but those techniques would not be regulated under the terms of the Coordinated Framework for Regulation of Biotechnology. This revolutionary moment in biotechnology offers an opportunity to correct the flaws in the framework, which was hastily patched together at the advent of the technology. The framework has never captured all relevant technologies, has never satisfied the public that risk is being effectively managed, and has never been accessible to small companies and publicly-funded labs that increasingly are positioned to make radical, life-saving innovations. This Article offers a proposal for new legislation that would reshape biotechnology regulation to better meet these goals. Key reforms include tying regulation to risk rather than technology category; consolidating agency review; capturing distinct regulatory expertise through inter-agency consultations; creating a clearinghouse to help guide applicants and disseminate information; setting up more comprehensive monitoring of environmental effects; and providing federal leadership to fill key data gaps and address socio-economic impacts.

  13. Regulation of class V myosin.

    Science.gov (United States)

    Zhang, Ning; Yao, Lin-Lin; Li, Xiang-Dong

    2018-01-01

    Class V myosin (myosin-5) is a molecular motor that functions as an organelle transporter. The activation of myosin-5's motor function has long been known to be associated with a transition from the folded conformation in the off-state to the extended conformation in the on-state, but only recently have we begun to understand the underlying mechanism. The globular tail domain (GTD) of myosin-5 has been identified as the inhibitory domain and has recently been shown to function as a dimer in regulating the motor function. The folded off-state of myosin-5 is stabilized by multiple intramolecular interactions, including head-GTD interactions, GTD-GTD interactions, and interactions between the GTD and the C-terminus of the first coiled-coil segment. Any cellular factor that affects these intramolecular interactions and thus the stability of the folded conformation of myosin-5 would be expected to regulate myosin-5 motor function. Both the adaptor proteins of myosin-5 and Ca2+ are potential regulators of myosin-5 motor function, because they can destabilize its folded conformation. A combination of these regulators provides a versatile scheme in regulating myosin-5 motor function in the cell.

  14. Cosmetic Regulations: A Comparative Study.

    Science.gov (United States)

    Suhag, Jyoti; Dureja, Harish

    2015-01-01

    The regulatory framework, compliance requirement, efficacy, safety, and marketing of cosmetic products are considered the most important factors for growth of the cosmetic industry. There are different regulatory bodies across the globe that have their own insights for regulation; moreover, governments such as the United States, European Union, and Japan follow a stringent regulatory framework, whereas cosmetics are not so much strictly regulated in countries such as India, Brazil, and China. The alignment of a regulatory framework will play a significant role in the removal of barriers to trade, growth of market at an international level, innovation in the development and presentation of new products, and most importantly safety and efficacy of the marketed products. The present contribution gives insight into the important cosmetic regulations in areas of premarket approval, ingredient control, and labeling and warnings, with a special focus on the cosmetic regulatory environments in the United States, European Union, Japan, and India. Most importantly, the authors highlight the dark side of cosmetics associated with allergic reactions and even skin cancer. The importance of cosmetic regulations has been highlighted by dint of which the society can be healthier, accomplished by more stringent and harmonized regulations.

  15. Guidelines on Building Regulations 2008

    DEFF Research Database (Denmark)

    Thse guidelines clarify and intepret the provisions of the Building Regulations of 2008 (BR08). The Guidelines, which match BR08 in terms of organisation into Parts, are accompanied by the full text of the regulations and the explanatory notes issued by the Danish Enterprise and Construction...... Authority. The Guidelines refer the reader to sources such as relevant standards, instructions and other background material which provides more detailed information. The Guidelines cover the same ground as BR08, including building control regulations, layout, fitting out, structures, fire safety, indoor...... climate, energy consumotion and services. The Guidelines are aimed at all professionals involved in building projects, particularly building design consultants, contractors and municipal application officers....

  16. Digital Convergence and Content Regulation

    Directory of Open Access Journals (Sweden)

    Michael John Starks

    2014-12-01

    Full Text Available Broadcasting, Press and Internet journalism systems of distribution are converging: the same infrastructure can deliver all three historically separate services. Reception devices mirror this: the Connected TV, the tablet and the smart phone overlap in their functionality. Service overlaps are evident too, with broadcasters providing online and on-demand services and newspapers developing electronic versions. Does this mean that media regulation policies must converge too?My argument is that they should, though only where historically different communications are now fulfilling a similar function, e.g. broadcaster online services and electronic versions of newspapers. Convergence requires a degree of harmonisation and, to this end, I advocate a review of UK broadcasting's 'due impartiality' requirement and of the UK's application of the public service concept. I also argue for independent self-regulation (rather than state-based regulation of non-public-service broadcasting journalism.

  17. Regulation of beta cell replication

    DEFF Research Database (Denmark)

    Lee, Ying C; Nielsen, Jens Høiriis

    2008-01-01

    Beta cell mass, at any given time, is governed by cell differentiation, neogenesis, increased or decreased cell size (cell hypertrophy or atrophy), cell death (apoptosis), and beta cell proliferation. Nutrients, hormones and growth factors coupled with their signalling intermediates have been...... suggested to play a role in beta cell mass regulation. In addition, genetic mouse model studies have indicated that cyclins and cyclin-dependent kinases that determine cell cycle progression are involved in beta cell replication, and more recently, menin in association with cyclin-dependent kinase...... inhibitors has been demonstrated to be important in beta cell growth. In this review, we consider and highlight some aspects of cell cycle regulation in relation to beta cell replication. The role of cell cycle regulation in beta cell replication is mostly from studies in rodent models, but whether...

  18. Calcium regulation of muscle contraction.

    Science.gov (United States)

    Szent-Györgyi, A G

    1975-07-01

    Calcium triggers contraction by reaction with regulatory proteins that in the absence of calcium prevent interaction of actin and myosin. Two different regulatory systems are found in different muscles. In actin-linked regulation troponin and tropomyosin regulate actin by blocking sites on actin required for complex formation with myosin; in myosin-linked regulation sites on myosin are blocked in the absence of calcium. The major features of actin control are as follows: there is a requirement for tropomyosin and for a troponin complex having three different subunits with different functions; the actin displays a cooperative behavior; and a movement of tropomyosin occurs controlled by the calcium binding on troponin. Myosin regulation is controlled by a regulatory subunit that can be dissociated in scallop myosin reversibly by removing divalent cations with EDTA. Myosin control can function with pure actin in the absence of tropomyosin. Calcium binding and regulation of molluscan myosins depend on the presence of regulatory light chains. It is proposed that the light chains function by sterically blocking myosin sites in the absence of calcium, and that the "off" state of myosin requires cooperation between the two myosin heads. Both myosin control and actin control are widely distributed in different organisms. Many invertebrates have muscles with both types of regulation. Actin control is absent in the muscles of molluscs and in several minor phyla that lack troponin. Myosin control is not found in striated vertebrate muscles and in the fast muscles of crustacean decapods, although regulatory light chains are present. While in vivo myosin control may not be excluded from vertebrate striated muscles, myosin control may be absent as a result of mutations of the myosin heavy chain.

  19. Autoimmune regulator, Aire, is a novel regulator of chondrocyte differentiation.

    Science.gov (United States)

    Si, Yuan; Inoue, Kazuki; Igarashi, Katsuhide; Kanno, Jun; Imai, Yuuki

    2013-08-09

    Chondrocyte differentiation is controlled by various regulators, such as Sox9 and Runx2, but the process is complex. To further understand the precise underlying molecular mechanisms of chondrocyte differentiation, we aimed to identify a novel regulatory factor of chondrocyte differentiation using gene expression profiles of micromass-cultured chondrocytes at different differentiation stages. From the results of microarray analysis, the autoimmune regulator, Aire, was identified as a novel regulator. Aire stable knockdown cells, and primary cultured chondrocytes obtained from Aire(-/-) mice, showed reduced mRNA expression levels of chondrocyte-related genes. Over-expression of Aire induced the early stages of chondrocyte differentiation by facilitating expression of Bmp2. A ChIP assay revealed that Aire was recruited on an Airebinding site (T box) in the Bmp2 promoter region in the early stages of chondrocyte differentiation and histone methylation was modified. These results suggest that Aire can facilitate early chondrocyte differentiation by expression of Bmp2 through altering the histone modification status of the promoter region of Bmp2. Taken together, Aire might play a role as an active regulator of chondrocyte differentiation, which leads to new insights into the regulatory mechanisms of chondrocyte differentiation. Copyright © 2013 Elsevier Inc. All rights reserved.

  20. Minsky and dynamic macroprudential regulation

    Directory of Open Access Journals (Sweden)

    Jan Kregel

    2014-06-01

    Full Text Available In the context of current debates about the proper form of prudential regulation and proposals for the imposition of liquidity and capital ratios, the paper examines Hyman Minsky’s work as a consultant to government agencies exploring financial regulatory reform in the 1960s. As the author explains, this often-overlooked early work, a precursor to Minsky’s “financial instability hypothesis”, serves as yet another useful guide to explaining why regulation and supervision in the lead-up to the 2008 financial crisis were flawed, and why the approach to reregulation after the crisis has been incomplete.

  1. Wave Dragon Buoyancy Regulation Study

    DEFF Research Database (Denmark)

    Jakobsen, Jens; Kofoed, Jens Peter

    Wave Dragon is a wave energy converter, which was deployed offshore at Nissum Bredning in Denmark in 2003. The experience gained from operating Wave Dragon during 2003 and 2004 has shown that the buoyancy regulation system can be improved in a number of ways. This study describes the current...... situation, and proposes a number of activities in order to improve the buoyancy regulation system. This work was performed under EU ENERGIE contract no. ENK5-CT-2002-00603, and is a contribution to WP 2.3/2.4 and D40/D41....

  2. The Organization of Regulated Production

    DEFF Research Database (Denmark)

    Jansen, Jos; Jeon, Doh-Shin; Menicucci, Domenico

    2008-01-01

    and weak positive (respectively, strong positive) correlation. Second, if the firms can collude under VS and know all costs, then VS is equivalent to VI. However, if firms collude under asymmetric information, then collusion does not affect the choice between VS and VI, since the regulator takes advantage...... of the transaction costs created by asymmetric information....

  3. Liquidity regulation and bank behavior

    NARCIS (Netherlands)

    Bonner, C.

    2014-01-01

    In response to the 2007-08 financial crisis, the Basel Committee on Banking Supervision proposed two liquidity standards to reinforce banks’ resilience to liquidity risks. The purpose of this thesis is to analyze the impact of liquidity regulation on bank behavior. The first of four main chapters

  4. Capital regulation and tail risk

    NARCIS (Netherlands)

    Perotti, E.; Ratnovski, L.; Vlahu, R.

    2011-01-01

    The paper studies risk mitigation associated with capital regulation, in a context where banks may choose tail risk assets. We show that this undermines the traditional result that higher capital reduces excess risk taking driven by limited liability. Moreover, higher capital may have an unintended

  5. Capital regulation and tail risk

    NARCIS (Netherlands)

    Perotti, E.; Ratnovski, L.; Vlahu, R.

    2011-01-01

    The paper studies risk mitigation associated with capital regulation, in a context when banks may choose tail risk assets. We show that this undermines the traditional result that higher capital reduces excess risk-taking driven by limited liability. When capital raising is costly, poorly

  6. Histaminergic regulation of prolactin secretion

    DEFF Research Database (Denmark)

    Knigge, U P

    1990-01-01

    Histamine (HA), which acts as a neurotransmitter in the central nervous system, participates in the neuroendocrine regulation of prolactin (PRL) secretion. HA has a predominant stimulatory effect which is mediated via H2-receptors following central administration and via H1-receptors following...

  7. Spatial regulation of Rap signaling

    NARCIS (Netherlands)

    Gloerich, M.

    2011-01-01

    By cycling between an inactive GDP-bound and active GTP-bound state, small G-proteins of the Rap family act as molecular switches that relay upstream signals to diverse cellular processes. This GDP/GTP-cycle and consequently downstream signaling by Rap is under tight regulation by its GEFs and GAPs.

  8. Incentives and regulation in banking

    NARCIS (Netherlands)

    Martynova, N.

    2015-01-01

    The financial crisis of 2007-2008 has unveiled the hidden flaws in the regulatory framework of the financial sector. The rules of the game established by regulators were not stringent enough and provided bankers with wrong incentives to gamble with depositors’ money. There are two major challenges

  9. The Regulation of Carcinogenic Hazards.

    Science.gov (United States)

    Gori, Gio Batta

    1980-01-01

    It is suggested that a system of relative standards be formulated which would compare utility of substances to their relative risk as carcinogens. This would define a range of use restrictions. Substances intended for specific uses would then be regulated according to these standards. (Author/RE)

  10. Lasp-1 regulates podosome function.

    Directory of Open Access Journals (Sweden)

    Miriam Stölting

    Full Text Available Eukaryotic cells form a variety of adhesive structures to connect with their environment and to regulate cell motility. In contrast to classical focal adhesions, podosomes, highly dynamic structures of different cell types, are actively engaged in matrix remodelling and degradation. Podosomes are composed of an actin-rich core region surrounded by a ring-like structure containing signalling molecules, motor proteins as well as cytoskeleton-associated proteins. Lasp-1 is a ubiquitously expressed, actin-binding protein that is known to regulate cytoskeleton architecture and cell migration. This multidomain protein is predominantely present at focal adhesions, however, a second pool of Lasp-1 molecules is also found at lamellipodia and vesicle-like microdomains in the cytosol.In this report, we show that Lasp-1 is a novel component and regulator of podosomes. Immunofluorescence studies reveal a localization of Lasp-1 in the podosome ring structure, where it colocalizes with zyxin and vinculin. Life cell imaging experiments demonstrate that Lasp-1 is recruited in early steps of podosome assembly. A siRNA-mediated Lasp-1 knockdown in human macrophages affects podosome dynamics as well as their matrix degradation capacity. In summary, our data indicate that Lasp-1 is a novel component of podosomes and is involved in the regulation of podosomal function.

  11. Peripheral chemoreceptors and cardiovascular regulation

    National Research Council Canada - National Science Library

    Marshall, J M

    1994-01-01

    ... I. INTRODUCTION Peripheral chemoreceptors are located in the carotid and aortic bodies and in clumps along the route of the abdominal vagus, although aortic and abdominal chemoreceptors are not present in all species. Their role in the regulation of the cardiovascular system cannot be understood without knowledge of the various factors that can chan...

  12. Light regulates ascorbate in plants

    NARCIS (Netherlands)

    Ntagkas, Nikolaos; Woltering, Ernst J.; Marcelis, Leo F.M.

    2017-01-01

    l-ascorbate (vitamin. C, ASC) is an antioxidant that is essential for the proper function not only of plants but also animals. Light is a major regulatory factor for ASC levels in plants. In this paper, we review the regulation of ASC by light and the involved biochemical and physiological

  13. Regulating collaboration in teacher education

    NARCIS (Netherlands)

    Dobber, M.; Akkerman, S.F.; Verloop, N.; Vermunt, J.D.

    2014-01-01

    Collaboration in teacher education can be seen as a way to prepare student teachers for future social practices at school. When people collaborate with each other, they have to regulate their collaboration. In the Dutch teacher education programme that was investigated, student teachers were members

  14. 75 FR 67912 - North Korea Sanctions Regulations

    Science.gov (United States)

    2010-11-04

    ... 31 CFR Part 510 North Korea Sanctions Regulations AGENCY: Office of Foreign Assets Control, Treasury...'') is issuing regulations with respect to North Korea to implement Executive Order 13466 of June 26... issuing the North Korea Sanctions Regulations, 31 CFR part 510 (the ``Regulations''), to implement E.O...

  15. The Legal Regulation of Cybersecurity

    Directory of Open Access Journals (Sweden)

    Darius Štitilis

    2013-08-01

    Full Text Available Cybercrime has become a global phenomenon, which is causing more harm to individual citizens, organizations, society and the state. Most countries in the world compare cybercrime with offences such as terrorism and drug trafficking due to its risks and profitability. Cybersecurity is the central category to fight cybercrime in cyberspace. Therefore, the strategic legal regulation of cybersecurity is one of the most relevant problems in EU, including Lithuania. So far cybersecurity legal regulation analysis in scientific literature has been rather limited. The European Commission, together with the High Representative of the Union for Foreign Affairs and Security Policy, has published a cybersecurity strategy alongside a Commission proposed directive on network and information security (NIS. The cybersecurity strategy – “An Open, Safe and Secure Cyberspace” - represents the EU’s comprehensive vision on how best to prevent and respond to cyber disruptions and attacks. The purpose of its is to further European values of freedom and democracy and ensure the digital economy can safely grow. Specific actions are aimed at enhancing cyber resilience of information systems, reducing cybercrime and strengthening EU international cyber-security policy and cyber defence. The main goal of the paper is to analyze and compare the EU cybersecurity strategy and experience of several foreign countries with the strategic legal regulation of cybersecurity in Lithuania. The article consists of four parts. The first part dealt with the EU cybersecurity strategy. The second part of the article examines the comparative aspect of foreign cybersecurity strategic legal regulation. The third part deals with attempts in Lithuania to draft cybersecurity law and the holistic approach of cybersecurity legal regulation. The fourth part examines Lithuanian cybersecurity strategy and comments on the main probleas related with the strategy. Several different approaches

  16. Essential infrastructure: national nuclear regulation.

    Science.gov (United States)

    Paperiello, Carl J

    2011-01-01

    In order for nuclear power to expand to many countries that do not currently have it, it will be essential for these countries to have laws, regulations, guidance and organizations that can license or permit nuclear power plants and support nuclear facilities, ensure compliance by inspection, and enforce nuclear regulations. The viability of nuclear power worldwide depends on an extremely high level of safety everywhere, and compliance with a number of international treaties is required before supplier nations will provide the material, both hardware and software, to build and operate nuclear power plants. While infrastructure support can be obtained from the IAEA and other countries, an essential core of expertise must exist in the country seeking to establish domestic nuclear power generation. While some reliance can be placed on the safety reviews of standard reactor designs by the nuclear regulators in supplier nations, the certification of fuel design, the quality of instruments, and the matching of a new reactor to a proposed site in the importing nation will require site-specific reviews. National arrangements are also needed for emergency preparedness, environmental protection, fuel transportation and the storage, transportation and disposal of radioactive waste. If foreign contractors and consultants are engaged to perform much of the technical work for the regulatory body(s) that has to be performed by the importing nation, that nation must have a core cadre of technically knowledgeable regulators and an organization to provide management and oversight of the contractors and consultants. Consistency in national nuclear regulations, the deployment of standardized nuclear power plant designs and standardized supporting material infrastructure can promote the safe and secure worldwide growth in nuclear power. Copyright © 2010 Health Physics Society

  17. miRNA regulation of cytokine genes

    OpenAIRE

    Asirvatham, Ananthi J.; Magner, William J.; Tomasi, Thomas B.

    2009-01-01

    In this review we discuss specific examples of regulation of cytokine genes and focus on a new mechanism involving post-transcriptional regulation via miRNAs. The post-transcriptional regulation of cytokine genes via the destabilizing activity of AU-rich elements [AREs] and miRNAs is a pre-requisite for regulating the half-life of many cytokines and achieving the temporal and spatial distributions required for regulation of these genes.

  18. Regulation of Drosophila metamorphosis by xenobiotic response regulators.

    Science.gov (United States)

    Deng, Huai; Kerppola, Tom K

    2013-01-01

    Mammalian Nrf2-Keap1 and the homologous Drosophila CncC-dKeap1 protein complexes regulate both transcriptional responses to xenobiotic compounds as well as native cellular and developmental processes. The relationships between the functions of these proteins in xenobiotic responses and in development were unknown. We investigated the genes regulated by CncC and dKeap1 during development and the signal transduction pathways that modulate their functions. CncC and dKeap1 were enriched within the nuclei in many tissues, in contrast to the reported cytoplasmic localization of Keap1 and Nrf2 in cultured mammalian cells. CncC and dKeap1 occupied ecdysone-regulated early puffs on polytene chromosomes. Depletion of either CncC or dKeap1 in salivary glands selectively reduced early puff gene transcription. CncC and dKeap1 depletion in the prothoracic gland as well as cncC(K6/K6) and dKeap1(EY5/EY5) loss of function mutations in embryos reduced ecdysone-biosynthetic gene transcription. In contrast, dKeap1 depletion and the dKeap1(EY5/EY5) loss of function mutation enhanced xenobiotic response gene transcription in larvae and embryos, respectively. Depletion of CncC or dKeap1 in the prothoracic gland delayed pupation by decreasing larval ecdysteroid levels. CncC depletion suppressed the premature pupation and developmental arrest caused by constitutive Ras signaling in the prothoracic gland; conversely, constitutive Ras signaling altered the loci occupied by CncC on polytene chromosomes and activated transcription of genes at these loci. The effects of CncC and dKeap1 on both ecdysone-biosynthetic and ecdysone-regulated gene transcription, and the roles of CncC in Ras signaling in the prothoracic gland, establish the functions of these proteins in the neuroendocrine axis that coordinates insect metamorphosis.

  19. Targeted genome regulation via synthetic programmable transcriptional regulators

    KAUST Repository

    Piatek, Agnieszka Anna

    2016-04-19

    Regulation of gene transcription controls cellular functions and coordinates responses to developmental, physiological and environmental cues. Precise and efficient molecular tools are needed to characterize the functions of single and multiple genes in linear and interacting pathways in a native context. Modular DNA-binding domains from zinc fingers (ZFs) and transcriptional activator-like proteins (TALE) are amenable to bioengineering to bind DNA target sequences of interest. As a result, ZF and TALE proteins were used to develop synthetic programmable transcription factors. However, these systems are limited by the requirement to re-engineer proteins for each new target sequence. The clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR associated 9 (Cas9) genome editing tool was recently repurposed for targeted transcriptional regulation by inactivation of the nuclease activity of Cas9. Due to the facile engineering, simplicity, precision and amenability to library construction, the CRISPR/Cas9 system is poised to revolutionize the functional genomics field across diverse eukaryotic species. In this review, we discuss the development of synthetic customizable transcriptional regulators and provide insights into their current and potential applications, with special emphasis on plant systems, in characterization of gene functions, elucidation of molecular mechanisms and their biotechnological applications. © 2016 Informa UK Limited, trading as Taylor & Francis Group

  20. 76 FR 35739 - Foreign Assets Control Regulations; Transaction Control Regulations (Regulations Prohibiting...

    Science.gov (United States)

    2011-06-20

    ... authorities with respect to North Korea, which were implemented by the Foreign Assets Control Regulations, 31... and procedure, Banking, Banks, Blocking of assets, Credit, Foreign trade, Imports, North Korea... practice and procedure, Banking, Banks, Blocking of assets, Credit, Foreign trade, North Korea, Penalties...

  1. 78 FR 31551 - Federal Acquisition Regulation; Submission for OMB Review; Commerce Patent Regulations

    Science.gov (United States)

    2013-05-24

    ... Regulation; Submission for OMB Review; Commerce Patent Regulations AGENCIES: Department of Defense (DOD... approved information collection requirement concerning Department of Commerce patent regulations. A notice... Collection 9000- 0095, Commerce Patent Regulations, by any of the following methods: Regulations.gov : http...

  2. Gene regulation by mechanical forces

    Science.gov (United States)

    Oluwole, B. O.; Du, W.; Mills, I.; Sumpio, B. E.

    1997-01-01

    Endothelial cells are subjected to various mechanical forces in vivo from the flow of blood across the luminal surface of the blood vessel. The purpose of this review was to examine the data available on how these mechanical forces, in particular cyclic strain, affect the expression and regulation of endothelial cell function. Studies from various investigators using models of cyclic strain in vitro have shown that various vasoactive mediators such as nitric oxide and prostacyclin are induced by the effect of mechanical deformation, and that the expression of these mediators may be regulated at the transcription level by mechanical forces. There also seems to be emerging evidence that endothelial cells may also act as mechanotransducers, whereby the transmission of external forces induces various cytoskeletal changes and second messenger cascades. Furthermore, it seems these forces may act on specific response elements of promoter genes.

  3. Molecular Mechanisms of Appetite Regulation

    Directory of Open Access Journals (Sweden)

    Ji Hee Yu

    2012-12-01

    Full Text Available The prevalence of obesity has been rapidly increasing worldwide over the last several decades and has become a major health problem in developed countries. The brain, especially the hypothalamus, plays a key role in the control of food intake by sensing metabolic signals from peripheral organs and modulating feeding behaviors. To accomplish these important roles, the hypothalamus communicates with other brain areas such as the brainstem and reward-related limbic pathways. The adipocyte-derived hormone leptin and pancreatic β-cell-derived insulin inform adiposity to the hypothalamus. Gut hormones such as cholecystokinin, peptide YY, pancreatic polypeptide, glucagon-like peptide 1, and oxyntomodulin transfer satiety signals to the brain and ghrelin relays hunger signals. The endocannabinoid system and nutrients are also involved in the physiological regulation of food intake. In this article, we briefly review physiological mechanisms of appetite regulation.

  4. Information, Interests, and Environmental Regulation

    DEFF Research Database (Denmark)

    Winter, Søren; May, Peter J.

    2002-01-01

    This study contributes to the understanding of informational approaches to bringing about compliance with environmental regulations with particular attention to differences in the influence of information provided by different information sources. Based on theorizing from a combination of informa......This study contributes to the understanding of informational approaches to bringing about compliance with environmental regulations with particular attention to differences in the influence of information provided by different information sources. Based on theorizing from a combination...... of information processing and interest group literatures, we develop hypotheses about regulatees' reliance upon and the influence of different sources of information. We test these hypotheses for Danish farmers’ compliance with agro-environmental rules. Our findings show that information plays a role in bringing...

  5. Regulated nucleocytoplasmic transport during gametogenesis.

    Science.gov (United States)

    Miyamoto, Yoichi; Boag, Peter R; Hime, Gary R; Loveland, Kate L

    2012-06-01

    Gametogenesis is the process by which sperm or ova are produced in the gonads. It is governed by a tightly controlled series of gene expression events, with some common and others distinct for males and females. Nucleocytoplasmic transport is of central importance to the fidelity of gene regulation that is required to achieve the precisely regulated germ cell differentiation essential for fertility. In this review we discuss the physiological importance for gamete formation of the molecules involved in classical nucleocytoplasmic protein transport, including importins/karyopherins, Ran and nucleoporins. To address what functions/factors are conserved or specialized for these developmental processes between species, we compare knowledge from mice, flies and worms. The present analysis provides evidence of the necessity for and specificity of each nuclear transport factor and for nucleoporins during germ cell differentiation. This article is part of a Special Issue entitled: Nuclear Transport and RNA Processing. Copyright © 2012 Elsevier B.V. All rights reserved.

  6. Lipid Regulation of Acrosome Exocytosis.

    Science.gov (United States)

    Cohen, Roy; Mukai, Chinatsu; Travis, Alexander J

    2016-01-01

    Lipids are critical regulators of mammalian sperm function, first helping prevent premature acrosome exocytosis, then enabling sperm to become competent to fertilize at the right place/time through the process of capacitation, and ultimately triggering acrosome exocytosis. Yet because they do not fit neatly into the "DNA--RNA-protein" synthetic pathway, they are understudied and poorly understood. Here, we focus on three lipids or lipid classes-cholesterol, phospholipids, and the ganglioside G(M1)--in context of the modern paradigm of acrosome exocytosis. We describe how these various- species are precisely segregated into membrane macrodomains and microdomains, simultaneously preventing premature exocytosis while acting as foci for organizing regulatory and effector molecules that will enable exocytosis. Although the mechanisms responsible for these domains are poorly defined, there is substantial evidence for their composition and functions. We present diverse ways that lipids and lipid modifications regulate capacitation and acrosome exocytosis, describing in more detail how removal of cholesterol plays a master regulatory role in enabling exocytosis through at least two complementary pathways. First, cholesterol efflux leads to proteolytic activation of phospholipase B, which cleaves both phospholipid tails. The resultant changes in membrane curvature provide a mechanism for the point fusions now known to occur far before a sperm physically interacts with the zona pellucida. Cholesterol efflux also enables G(M1) to regulate the voltage-dependent cation channel, Ca(V)2.3, triggering focal calcium transients required for acrosome exocytosis in response to subsequent whole-cell calcium rises. We close with a model integrating functions for lipids in regulating acrosome exocytosis.

  7. Regulators of Tfh cell differentiation

    Directory of Open Access Journals (Sweden)

    Gajendra Motiram Jogdand

    2016-11-01

    Full Text Available The follicular helper T (Tfh cells help is critical for activation of B cells, antibody class switching and germinal center formation. The Tfh cells are characterized by the expression of CXCR5, ICOS, PD-1, Bcl-6, and IL-21. They are involved in clearing infections and are adversely linked with autoimmune diseases and also have a role in viral replication as well as clearance. Tfh cells are generated from naïve CD4 T cells with sequential steps involving cytokine signaling (IL-21, IL-6, IL-12, activin A, migration and positioning in the germinal center by CXCR5, surface receptors (ICOS/ICOSL, SAP/SLAM as well as transcription factor (Bcl-6, c-Maf, STAT3 signaling and repressor miR155. On the other hand Tfh generation is negatively regulated at specific steps of Tfh generation by specific cytokine (IL-2, IL-7, surface receptor (PD-1, CTLA-4, transcription factors Blimp-1, STAT5, T-bet, KLF-2 signaling and repressor miR 146a. Interestingly, miR 17-92 and FOXO1 acts as a positive as well as a negative regulator of Tfh differentiation depending on the time of expression and disease specificity. Tfh cells are also generated from the conversion of other effector T cells as exemplified by Th1 cells converting into Tfh during viral infection. The mechanistic details of effector T cells conversion into Tfh are yet to be clear. To manipulate Tfh cells for therapeutic implication and or for effective vaccination strategies, it is important to know positive and negative regulators of Tfh generation. Hence, in this review we have highlighted and interlinked molecular signaling from cytokines, surface receptors, transcription factors, ubiquitin Ligase and miRNA as positive and negative regulators for Tfh differentiation.

  8. Frequency regulator for synchronous generators

    Science.gov (United States)

    Karlicek, Robert F.

    1982-01-01

    The present invention is directed to a novel frequency regulator which controls a generator output frequency for variations in both the input power to the generator and the power supplied to an uncontrolled external load. The present invention further includes over current and current balance protection devices which are relatively inexpensive to manufacture, which may be encapsulated to provide protection from the operating environment and which respond more quickly than previously known electromechanical devices.

  9. VOLTAGE REGULATORS OF SYNCHRONOUS GENERATORS

    OpenAIRE

    Grigorash O. V.; Korzenkov P. G.; Popuchieva M. A.

    2015-01-01

    Synchronous generators are the primary source of electrical power autonomous electrosupply systems, including backup systems. They are also used in a structure of rotating electricity converters and are widely used in renewable energy as part of wind power plants of small, mini and micro hydroelectric plants. Increasing the speed and the accuracy of the system of the voltage regulation of synchronous generators is possible due to the development of combined systems containing more stabilizers...

  10. Environmental regulations and labor markets

    OpenAIRE

    Deschenes, Olivier

    2014-01-01

    Environmental regulations such as air quality standards can lead to notable improvements in ambient air quality and to related health benefits. 
But they impose additional production costs on firms and may reduce productivity, earnings, and employment, especially in sectors exposed to trade and intensive in labor. The limited empirical evidence suggests that the benefits are likely to outweigh 
the costs.

  11. Regulation of Information and Advertising

    OpenAIRE

    Paul Rubin

    2008-01-01

    Deception is the manipulation of information to gain some advantage. This paper considers commercial deception through advertising. The paper first discusses the economics of information. The literature has derived four major policy conclusions. First, truthful information regarding price should not be restricted by regulatory authorities. Second, deception is most likely and most harmful for credence goods, and regulation is most useful (if it is useful at all) for these goods. Third, truthf...

  12. Energy saving statutes and regulations

    Energy Technology Data Exchange (ETDEWEB)

    Rado, L.

    1981-11-01

    The West German Federal government and the state governments are endeavouring to introduce energy saving measures with the aid of statutes, regulations, and ordinances. In his introductory remarks, the author briefly refers to the various activities since 1974 and on the basis of a 1976 report subjects the present status of statutes and ordinances on energy saving measures to a critical analysis. Special emphasis is placed on the interest of the gas supply industry.

  13. Epigenetic regulation of neuroblastoma development.

    Science.gov (United States)

    Durinck, Kaat; Speleman, Frank

    2018-01-19

    In recent years, technological advances have enabled a detailed landscaping of the epigenome and the mechanisms of epigenetic regulation that drive normal cell function, development and cancer. Rather than merely a structural entity to support genome compaction, we now look at chromatin as a very dynamic and essential constellation that is actively participating in the tight orchestration of transcriptional regulation as well as DNA replication and repair. The unique feature of chromatin flexibility enabling fast switches towards more or less restricted epigenetic cellular states is, not surprisingly, intimately connected to cancer development and treatment resistance, and the central role of epigenetic alterations in cancer is illustrated by the finding that up to 50% of all mutations across cancer entities affect proteins controlling the chromatin status. We summarize recent insights into epigenetic rewiring underlying neuroblastoma (NB) tumor formation ranging from changes in DNA methylation patterns and mutations in epigenetic regulators to global effects on transcriptional regulatory circuits that involve key players in NB oncogenesis. Insights into the disruption of the homeostatic epigenetic balance contributing to developmental arrest of sympathetic progenitor cells and subsequent NB oncogenesis are rapidly growing and will be exploited towards the development of novel therapeutic strategies to increase current survival rates of patients with high-risk NB.

  14. Nongenomic regulation of gene expression.

    Science.gov (United States)

    Iglesias-Platas, Isabel; Monk, David

    2016-08-01

    The purpose of this review is to highlight the recent advances in epigenetic regulation and chromatin biology for a better understanding of gene regulation related to human disease. Alterations to chromatin influence genomic function, including gene transcription. At its most simple level, this involves DNA methylation and posttranscriptional histone modifications. However, recent developments in biochemical and molecular techniques have revealed that transcriptional regulation is far more complex, involving combinations of histone modifications and discriminating transcription factor binding, and long-range chromatin loops with enhancers, to generate a multifaceted code. Here, we describe the most recent advances, culminating in the example of genomic imprinting, the parent-of-origin monoallelic expression that utilizes the majority of these mechanisms to attain one active and one repressed allele. It is becoming increasingly evident that epigenetic mechanisms work in unison to maintain tight control of gene expression and genome function. With the wealth of knowledge gained from recent molecular studies, future goals should focus on the application of this information in deciphering their role in developmental diseases.

  15. Substrate curvature regulates cell migration.

    Science.gov (United States)

    He, Xiuxiu; Jiang, Yi

    2017-05-23

    Cell migration is essential in many aspects of biology. Many basic migration processes, including adhesion, membrane protrusion and tension, cytoskeletal polymerization, and contraction, have to act in concert to regulate cell migration. At the same time, substrate topography modulates these processes. In this work, we study how substrate curvature at micrometer scale regulates cell motility. We have developed a 3D mechanical model of single cell migration and simulated migration on curved substrates with different curvatures. The simulation results show that cell migration is more persistent on concave surfaces than on convex surfaces. We have further calculated analytically the cell shape and protrusion force for cells on curved substrates. We have shown that while cells spread out more on convex surfaces than on concave ones, the protrusion force magnitude in the direction of migration is larger on concave surfaces than on convex ones. These results offer a novel biomechanical explanation to substrate curvature regulation of cell migration: geometric constrains bias the direction of the protrusion force and facilitates persistent migration on concave surfaces.

  16. Musical affect regulation in infancy.

    Science.gov (United States)

    Trehub, Sandra E; Ghazban, Niusha; Corbeil, Mariève

    2015-03-01

    Adolescents and adults commonly use music for various forms of affect regulation, including relaxation, revitalization, distraction, and elicitation of pleasant memories. Mothers throughout the world also sing to their infants, with affect regulation as the principal goal. To date, the study of maternal singing has focused largely on its acoustic features and its consequences for infant attention. We describe recent laboratory research that explores the consequences of singing for infant affect regulation. Such work reveals that listening to recordings of play songs can maintain 6- to 9-month-old infants in a relatively contented or neutral state considerably longer than recordings of infant-directed or adult-directed speech. When 10-month-old infants fuss or cry and are highly aroused, mothers' multimodal singing is more effective than maternal speech at inducing recovery from such distress. Moreover, play songs are more effective than lullabies at reducing arousal in Western infants. We explore the implications of these findings along with possible practical applications. © 2014 New York Academy of Sciences.

  17. ENERGY REGULATION IN YOUNG PEOPLE

    Directory of Open Access Journals (Sweden)

    Caroline J. Dodd

    2007-09-01

    Full Text Available Obesity in young people is now realised as a worldwide crisis of epidemic proportion. The aetiology of this disease suggests a disruption in regulation of energy at the population level, leading to a positive energy balance and excess adiposity. The relative contribution of food intake and physical inactivity remains to be elucidated. Treatment interventions have aimed to create a deficit in energy balance through manipulation of physical activity, behavioural components or, to a lesser extent, dietary modification. Whether such intervention is maintained in the long-term is as yet unclear, however it seems a combination of therapies is optimal. Mindful of a mismatch between energy intake and expenditure, recent work has begun to examine the acute relationship between physical activity and food intake in children. Initial findings suggest a short-term delay in compensation through energy intake for exercise- induced energy expenditure. The overarching study of energy regulation in children and adolescents is clearly multifaceted in nature and variables to be assessed or manipulated require careful consideration. The collection of paediatric physical activity, energy expenditure and food intake data is a time-consuming process, fraught with potential sources of error. Investigators should consider the validity and reliability of these and other issues, alongside the logistics of any proposed study. Despite these areas of concern, recent advances in the field should provide exciting opportunities for future research in paediatric energy regulation on a variety of levels

  18. Hate crimes and normative regulation

    Directory of Open Access Journals (Sweden)

    Kovačević Milica

    2011-01-01

    Full Text Available This paper is primarily devoted to issues related to the normative regulation of hate crimes, with special reference to the regulations of the Republic of Serbia, which are indirectly related to this matter. This kind of crimes are characterized by prejudices that perpetrators have towards injured parties, as members of certain, mostly, minority groups, due to which many hate crimes could be also called crimes of prejudice. In comparative law there are two different basic directions when it comes to regulating hate crimes: separation of hate crimes in a separate category on the one hand, and punishment of perpetrators of criminal acts with the detriment of minority groups through the usual charges of a given criminal justice system, on the other. The author finds that, regardless of the formal response forms, real life suggests that hate crimes can be essentially suppressed only by promoting values such as equality, respect for diversity and tolerance, and by continuous education of public about the danger of hate crimes.

  19. Regulation of Drosophila metamorphosis by xenobiotic response regulators.

    Directory of Open Access Journals (Sweden)

    Huai Deng

    Full Text Available Mammalian Nrf2-Keap1 and the homologous Drosophila CncC-dKeap1 protein complexes regulate both transcriptional responses to xenobiotic compounds as well as native cellular and developmental processes. The relationships between the functions of these proteins in xenobiotic responses and in development were unknown. We investigated the genes regulated by CncC and dKeap1 during development and the signal transduction pathways that modulate their functions. CncC and dKeap1 were enriched within the nuclei in many tissues, in contrast to the reported cytoplasmic localization of Keap1 and Nrf2 in cultured mammalian cells. CncC and dKeap1 occupied ecdysone-regulated early puffs on polytene chromosomes. Depletion of either CncC or dKeap1 in salivary glands selectively reduced early puff gene transcription. CncC and dKeap1 depletion in the prothoracic gland as well as cncC(K6/K6 and dKeap1(EY5/EY5 loss of function mutations in embryos reduced ecdysone-biosynthetic gene transcription. In contrast, dKeap1 depletion and the dKeap1(EY5/EY5 loss of function mutation enhanced xenobiotic response gene transcription in larvae and embryos, respectively. Depletion of CncC or dKeap1 in the prothoracic gland delayed pupation by decreasing larval ecdysteroid levels. CncC depletion suppressed the premature pupation and developmental arrest caused by constitutive Ras signaling in the prothoracic gland; conversely, constitutive Ras signaling altered the loci occupied by CncC on polytene chromosomes and activated transcription of genes at these loci. The effects of CncC and dKeap1 on both ecdysone-biosynthetic and ecdysone-regulated gene transcription, and the roles of CncC in Ras signaling in the prothoracic gland, establish the functions of these proteins in the neuroendocrine axis that coordinates insect metamorphosis.

  20. RegulatING chromatin regulators: post-translational modification of the ING family of epigenetic regulators.

    Science.gov (United States)

    Satpathy, Shankha; Nabbi, Arash; Riabowol, Karl

    2013-03-15

    The five human ING genes encode at least 15 splicing isoforms, most of which affect cell growth, differentiation and apoptosis through their ability to alter gene expression by epigenetic mechanisms. Since their discovery in 1996, ING proteins have been classified as type II tumour suppressors on the basis of reports describing their down-regulation and mislocalization in a variety of cancer types. In addition to their regulation by transcriptional mechanisms, understanding the range of PTMs (post-translational modifications) of INGs is important in understanding how ING functions are fine-tuned in the physiological setting and how they add to the repertoire of activities affected by the INGs. In the present paper we review the different PTMs that have been reported to occur on INGs. We discuss the PTMs that modulate ING function under normal conditions and in response to a variety of stresses. We also describe the ING PTMs that have been identified by several unbiased MS-based PTM enrichment techniques and subsequent proteomic analysis. Among the ING PTMs identified to date, a subset has been characterized for their biological significance and have been shown to affect processes including subcellular localization, interaction with enzymatic complexes and ING protein half-life. The present review aims to highlight the emerging role of PTMs in regulating ING function and to suggest additional pathways and functions where PTMs may effect ING function.

  1. Regulation, control, tele-management; Regulation, commande, telegestion

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2001-07-01

    In 1998, the French public authorities have started an ambitious energy mastery policy under the auspices of the Agency of environment and energy mastery (Ademe). One aspect of this policy concerns the R and D, industrial development and applications of automation and computerized control to the energy systems of buildings. This RCT 2000 seminar takes stock of the recent advances in this domain 7 years after the previous RCT issue. The first day of the seminar was devoted to new developments in regulation and control (ergonomics aspects, lighting systems, air conditioning in accommodations, solar systems etc..). Stress was put on the revolution expected with the introduction of domestic computer applications and Internet. This first day ended with a precise status of the standardization and future regulatory aspects. The second day was dealing with tele-management and technical management systems for buildings with an analysis of the impact of Internet and of the sensors and actuators capacity in information processing. The consequences and new requirements of the thermal regulation 2000 and the factors of success of a new start-up of the tele-management market in local authorities and social accommodations were analyzed too. (J.S.)

  2. 7 CFR 966.323 - Handling regulation.

    Science.gov (United States)

    2010-01-01

    ... packed must be clean and bright in appearance without marks, stains, or other evidence of previous use... Code of Federal Regulations. For Federal Register citations affecting these regulations, see the List...

  3. Emotion regulation strategies in Patients with schizophrenia

    NARCIS (Netherlands)

    van der Meer, Lisette; van't Wout, Mascha; Aleman, Andre

    2009-01-01

    Schizophrenia patients might experience difficulties in applying two widely used emotion regulation strategies, reappraisal and suppression. We investigated the relationships among emotion regulation strategies, alexithymia (i.e. inability to identify and verbalize feelings) and the role of

  4. Securities regulation and implicit penalties

    Directory of Open Access Journals (Sweden)

    Donghua Chen

    2011-06-01

    Full Text Available The extant literature offers extensive support for the significant role played by institutions in financial markets, but implicit regulation and monitoring have yet to be examined. This study fills this void in the literature by employing unique Chinese datasets to explore the implicit regulation and penalties imposed by the Chinese government in regulating the initial public offering (IPO market. Of particular interest are the economic consequences of underwriting IPO deals for client firms that violate regulatory rules in China’s capital market. We provide evidence to show that the associated underwriters’ reputations are impaired and their market share declines. We further explore whether such negative consequences result from a market disciplinary mechanism or a penalty imposed by the government. To analyze the possibility of a market disciplinary mechanism at work, we investigate (1 the market reaction to other client firms whose IPO deals were underwritten by underwriters associated with a violation at the time the violation was publicly disclosed and (2 the under-pricing of IPO deals undertaken by these underwriters after such disclosure. To analyze whether the government imposes an implicit penalty, we examine the application processing time for future IPO deals underwritten by the associated underwriters and find it to be significantly longer than for IPO deals underwritten by other underwriters. Overall, there is little evidence to suggest that the market penalizes underwriters for the rule-violating behavior of their client firms in China. Instead, the Chinese government implicitly penalizes them by imposing more stringent criteria on and lengthening the processing time of the IPO deals they subsequently underwrite.

  5. Challenges in Regulating Pesticide Mixtures

    Directory of Open Access Journals (Sweden)

    Michael Lydy

    2004-12-01

    Full Text Available This paper introduces the field of mixture toxicity and the challenges in regulating pesticide mixtures. Even though pesticides are unique chemical stressors designed to have biological activity that can affect a number of nontarget species, they are intentionally placed into the environment in large quantities. Currently, methods and terminology for evaluating mixture toxicity are poorly established. The most common approach used is the assumption of additive concentration, with the concentrations adjusted for potency to a reference toxicant. Using this approach, the joint action of pesticides that have similar chemical structures and modes of toxic action can be predicted. However, this approach and other modeling techniques often provide little insight into the observed toxicity produced by mixtures of pesticides from different classes. Particularly difficult to model are mixtures that involve a secondary toxicant that changes the toxicokinetics of a primary toxicant. This may result in increased activation or a change in the persistence of the primary toxicant within the organism and may be responsible for a several-fold increase or decrease in toxicity. At present, the ecological effects caused by mixtures of pesticides are given little consideration in the regulatory process. However, mixtures are being considered in relation to human health in the pesticide registration process, setting a precedent that could be followed for ecological protection. Additionally, pesticide mixtures may be regulated through toxicity testing of surface water under the Clean Water Act. The limits of our basic knowledge of how mixtures interact are compromising both these avenues for regulating mixtures. We face many challenges to adequately protecting the environment from mixture toxicity; these challenges include understanding the interactions of toxicants within an organism, identifying the mixtures that most commonly occur and cause adverse effects, and

  6. Regulated polyploidy in halophilic archaea.

    Directory of Open Access Journals (Sweden)

    Sebastian Breuert

    Full Text Available Polyploidy is common in higher eukaryotes, especially in plants, but it is generally assumed that most prokaryotes contain a single copy of a circular chromosome and are therefore monoploid. We have used two independent methods to determine the genome copy number in halophilic archaea, 1 cell lysis in agarose blocks and Southern blot analysis, and 2 Real-Time quantitative PCR. Fast growing H. salinarum cells contain on average about 25 copies of the chromosome in exponential phase, and their ploidy is downregulated to 15 copies in early stationary phase. The chromosome copy number is identical in cultures with a twofold lower growth rate, in contrast to the results reported for several other prokaryotic species. Of three additional replicons of H. salinarum, two have a low copy number that is not growth-phase regulated, while one replicon even shows a higher degree of growth phase-dependent regulation than the main replicon. The genome copy number of H. volcanii is similarly high during exponential phase (on average 18 copies/cell, and it is also downregulated (to 10 copies as the cells enter stationary phase. The variation of genome copy numbers in the population was addressed by fluorescence microscopy and by FACS analysis. These methods allowed us to verify the growth phase-dependent regulation of ploidy in H. salinarum, and they revealed that there is a wide variation in genome copy numbers in individual cells that is much larger in exponential than in stationary phase. Our results indicate that polyploidy might be more widespread in archaea (or even prokaryotes in general than previously assumed. Moreover, the presence of so many genome copies in a prokaryote raises questions about the evolutionary significance of this strategy.

  7. Environmental justice regulations draw fire

    Science.gov (United States)

    Showstack, Randy

    Advocates of "environmental justice" say that proposed U.S. Environmental Protection Agency (EPA) regulations are necessary to ensure that an unfair share of industrial facilities and waste plants are not sited in poor and minority communities, as they claim has occurred in the past.However, a number of state and local government agencies, business groups, and Democratic and Republican politicians argue that EPA guidelines—written to put some teeth into the Title VI clause of the Civil Rights Act that prohibits discrimination in all federally funded programs and activities—are unworkable and need to be overhauled.

  8. Mathematical Models of Gene Regulation

    Science.gov (United States)

    Mackey, Michael C.

    2004-03-01

    This talk will focus on examples of mathematical models for the regulation of repressible operons (e.g. the tryptophan operon), inducible operons (e.g. the lactose operon), and the lysis/lysogeny switch in phage λ. These ``simple" gene regulatory elements can display characteristics experimentally of rapid response to perturbations and bistability, and biologically accurate mathematical models capture these aspects of the dynamics. The models, if realistic, are always nonlinear and contain significant time delays due to transcriptional and translational delays that pose substantial problems for the analysis of the possible ranges of dynamics.

  9. Regulating renewable resources under uncertainty

    DEFF Research Database (Denmark)

    Hansen, Lars Gårn

    ) that a pro-quota result under uncertainty about prices and marginal costs is unlikely, requiring that the resource growth function is highly concave locally around the optimum and, 3) that quotas are always preferred if uncertainly about underlying structural economic parameters dominates. These results......Renewable natural resources (like water, fish and wildlife stocks, forests and grazing lands) are critical for the livelihood of millions of people and understanding how they can be managed efficiently is an important economic problem. I show how regulator uncertainty about different economic...

  10. Autonomic Regulation of Splanchnic Circulation

    Directory of Open Access Journals (Sweden)

    Kathleen A Fraser

    1991-01-01

    Full Text Available The role of the autonomic nervous system in circulatory regulation of the splanchnic organs (stomach, small intestine, colon, liver, pancreas and spleen is reviewed. In general, the sympathetic nervous system is primarily involved in vasoconstriction, while the parasympathetic contributes to vasodilation. Vasoconstriction in the splanchnic circulation appears to be mediated by alpha-2 receptors and vasodilation by activation of primary afferent nerves with subsequent release of vasodilatory peptides, or by stimulation of beta-adrenergic receptors. As well, an important function of the autonomic nervous system is to provide a mechanism by which splanchnic vascular reserve can be mobilized during stress to maintain overall cardiovascular homeostasis.

  11. Professional Disruption in Health Regulation

    DEFF Research Database (Denmark)

    Hasselbalch, Jacob

    2016-01-01

    in framing contests that draw on cognitive, normative and relational keys to signal their expectations. It is in these framing contests that professionals run the risk of disruption. Drawing on interview data with key policy actors, I investigate electronic cigarettes regulation in the European Union and its...... recent revision to the Tobacco Products Directive. Medical and public health professionals that control tobacco issues were challenged by a coalition of e-cigarette industry representatives, e-cigarette users, and liberal politicians. The challengers drew on the contending norm of harm reduction...

  12. QB1 - Stochastic Gene Regulation

    Energy Technology Data Exchange (ETDEWEB)

    Munsky, Brian [Los Alamos National Laboratory

    2012-07-23

    Summaries of this presentation are: (1) Stochastic fluctuations or 'noise' is present in the cell - Random motion and competition between reactants, Low copy, quantization of reactants, Upstream processes; (2) Fluctuations may be very important - Cell-to-cell variability, Cell fate decisions (switches), Signal amplification or damping, stochastic resonances; and (3) Some tools are available to mode these - Kinetic Monte Carlo simulations (SSA and variants), Moment approximation methods, Finite State Projection. We will see how modeling these reactions can tell us more about the underlying processes of gene regulation.

  13. Regulating Youth Access to Pornography

    OpenAIRE

    Flood, Michael; Hamilton, Clive

    2003-01-01

    This report on the regulation of pornography in Australia is meant to be read in conjunction with the earlier Australia Institute Discussion Paper Youth and Pornography in Australia: Evidence on the extent of exposure and likely effects (Discussion Paper Number 52, February 2003). Children in Australia have extensive exposure to pornography. Just under three-quarters(73 per cent) of boys and 11 per cent of girls report that they have watched an X-rated video. Eighty-four per cent of boys and ...

  14. Hard work in soft regulation

    DEFF Research Database (Denmark)

    Hohnen, Pernille; Hasle, Peter; Helbo Jespersen, Anne

    Certified occupational health and safety management (OHSM) systems have become a global instrument in the regulation of work environment. However, their actual impact on occupational health and safety – in particular on ‘softer’ psychosocial areas of the working environment – has been questioned....... This has resulted in recent British attempts to develop publically available guidelines (PAS 1010) to be used together with OHSAS 18001 focusing specifically on psychosocial risk management. The paper discusses these attempts in light of recent sociological theories on the regulatory mechanisms...

  15. Regulation of entry into gametogenesis.

    Science.gov (United States)

    van Werven, Folkert J; Amon, Angelika

    2011-12-27

    Gametogenesis is a fundamental aspect of sexual reproduction in eukaryotes. In the unicellular fungi Saccharomyces cerevisiae (budding yeast) and Schizosaccharomyces pombe (fission yeast), where this developmental programme has been extensively studied, entry into gametogenesis requires the convergence of multiple signals on the promoter of a master regulator. Starvation signals and cellular mating-type information promote the transcription of cell fate inducers, which in turn initiate a transcriptional cascade that propels a unique type of cell division, meiosis, and gamete morphogenesis. Here, we will provide an overview of how entry into gametogenesis is initiated in budding and fission yeast and discuss potential conserved features in the germ cell development of higher eukaryotes.

  16. Menstrual regulation with prostaglandin analogues.

    Science.gov (United States)

    Hagenfeldt, K; Bygdeman, M

    1981-01-01

    The development of generally applicable, simple non-surgical methods for menstrual regulation has been desired for a long time. Such an approach to fertility control depends on the availability of a suitable therapeutic agent that should be effective, reliable, simple to administer and free from disturbing side effects. The classical prostaglandins have shown the capability but are unsuitable mainly due to high incidence of side effects. Most of these drawbacks seem to be overcome when prostaglandin analogues are used; however, vaginal administration caused appreciable pain in 10-30% of women, and so severely limits self-administration of these analogues.

  17. Explaining (Missing) Regulator Paradigm Shifts

    DEFF Research Database (Denmark)

    Wigger, Angela; Buch-Hansen, Hubert

    2014-01-01

    of competition regulation is heaving into sight. It sets out to explain this from the vantage point of a critical political economy perspective, which identifies the circumstances under which a crisis can result in a regulatory paradigm shift. Contrasting the current situation with the shift in EC/EU competition...... capitalism; the social power configuration underpinning the neoliberal order remains unaltered; no clear counter-project has surfaced; the European Commission has been (and remains) in a position to oppose radical changes; and finally, there are no signs of a wider paradigm shift in the EU's regulatory...

  18. Regulating a Monopoly Offering Priority Service

    OpenAIRE

    Matsukawa, Isamu

    2006-01-01

    This paper investigates the effects of alternative forms of regulation on the market penetration and capacity, which are determined by a profit-maximizing monopolist providing priority service to consumers. For continuous priority service, a minimum reliability standard, price cap and rate of return regulation lead to larger capacity than in the absence of regulation. A minimum reliability standard reduces the market penetration while price cap and rate of return regulation increase it. T...

  19. Regulation of Motivation: Contextual and Social Aspects

    Science.gov (United States)

    Wolters, Christopher A.

    2011-01-01

    Background: Models of self-regulated learning have been used extensively as a way of understanding how students understand, monitor, and manage their own academic functioning. The regulation of motivation is a facet of self-regulated learning that describes students' efforts to control their own motivation or motivational processing. The…

  20. Self-Regulation in Online Learning

    Science.gov (United States)

    Cho, Moon-Heum; Shen, Demei

    2013-01-01

    The purpose of this study was to examine the role of goal orientation and academic self-efficacy in student achievement mediated by effort regulation, metacognitive regulation, and interaction regulation in an online course. The results show that intrinsic goal orientation and academic self-efficacy predicted students' metacognitive…

  1. 7 CFR 993.50 - Outgoing regulation.

    Science.gov (United States)

    2010-01-01

    ... specifications, or more restrictive grade regulations with respect to prunes that may be shipped or otherwise... restrictive grade regulation is established in connection with § 993.97 (Exhibit A) it shall insofar as.... The committee shall issue any such rules and regulations as may be necessary to insure such uses. (f...

  2. 76 FR 62630 - Information Security Regulations

    Science.gov (United States)

    2011-10-11

    ... 32 CFR Part 1902 Information Security Regulations AGENCY: Central Intelligence Agency. ACTION: Final rule. SUMMARY: The Central Intelligence agency is removing certain information security regulations... Information security regulations. PART 1902 Sec. 1902.13 0 Accordingly, under the authority of Executive Order...

  3. Transcriptional regulation of the cell cycle

    NARCIS (Netherlands)

    Stahl, M.

    2006-01-01

    Transcriptional regulators play an important role during cell cycle progression. A subset of these even seems to have a critical function in regulating cell cycle transitions. In this thesis, I have addressed the importance of transcriptional control in the regulation of cell cycle progression, in

  4. Identification of plant defence regulators through transcriptional

    Indian Academy of Sciences (India)

    Through bacterial resistance, callose deposition and pathogenesis-associated expression analyses, we identified four novel regulators of plant defence. Resistance levels in the mutants suggest that At2g19810 and [rom] At5g05790 are positive regulators, whereas At1g61370 and At3g42790 are negative regulators of plant ...

  5. 10 CFR 850.26 - Regulated areas.

    Science.gov (United States)

    2010-01-01

    ... 10 Energy 4 2010-01-01 2010-01-01 false Regulated areas. 850.26 Section 850.26 Energy DEPARTMENT... Regulated areas. (a) If airborne concentrations of beryllium in areas in DOE facilities are measured at or above the action level, the responsible employer must establish regulated areas for those areas. (b) The...

  6. Towards spatially differentiated regulation of nitrogen

    DEFF Research Database (Denmark)

    Lajer Højberg, Anker; Refsgaard, Jens Christian; Jørgensen, Lisbeth Flindt

    of drains and hydro-biogeochemical conditions in associated riparian lowlands. Hence, a shift of paradigm in regulation practice is needed, whit a cost-effective regulation accounting for this variability and differentiate the regulations/restrictions between resilient and vulnerable areas. However...

  7. Identification of let-7-regulated oncofetal genes

    DEFF Research Database (Denmark)

    Boyerinas, Benjamin; Park, Sun-Mi; Shomron, Noam

    2008-01-01

    -regulated at the end of embryonic development. Let-7 is often down-regulated early during cancer development, suggesting that let-7-regulated oncofetal genes (LOG) may become reexpressed in cancer cells. Using comparative bioinformatics, we have identified 12 conserved LOGs that include HMGA2 and IMP-1/CRD-BP. IMP-1...

  8. 7 CFR 983.50 - Aflatoxin regulations.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 8 2010-01-01 2010-01-01 false Aflatoxin regulations. 983.50 Section 983.50..., ARIZONA, AND NEW MEXICO Regulations § 983.50 Aflatoxin regulations. The committee shall establish, with the approval of the Secretary, such aflatoxin sampling, analysis, and inspection requirements...

  9. Emotion Regulation and Depressive Symptoms in Preadolescence

    Science.gov (United States)

    Siener, Shannon; Kerns, Kathryn A.

    2012-01-01

    This study examined associations among several measures of emotion regulation, and their links to depressive symptoms, in a sample of children ages 10-12 years old (N = 87). Both temporal features of emotion regulation and regulation processes involved in the evaluation, monitoring, and modification of emotion were assessed through parent and…

  10. Self-Regulation in Democratic Communities.

    Science.gov (United States)

    Yowell, Constance M.; Smylie, Mark A.

    1999-01-01

    Examines self-regulation as a nonacademic outcome of schooling and assesses school- and community-based programs and practices intended to promote self-regulation. Examines how self-regulation develops and is supported within and across three types of person-context interactions. Explores implications for cultural enrichment and social…

  11. Microbial regulation in gorgonian corals.

    Science.gov (United States)

    Hunt, Laura R; Smith, Stephanie M; Downum, Kelsey R; Mydlarz, Laura D

    2012-06-01

    Gorgonian corals possess many novel natural products that could potentially mediate coral-bacterial interactions. Since many bacteria use quorum sensing (QS) signals to facilitate colonization of host organisms, regulation of prokaryotic cell-to-cell communication may represent an important bacterial control mechanism. In the present study, we examined extracts of twelve species of Caribbean gorgonian corals, for mechanisms that regulate microbial colonization, such as antibacterial activity and QS regulatory activity. Ethanol extracts of gorgonians collected from Puerto Rico and the Florida Keys showed a range of both antibacterial and QS activities using a specific Pseudomonas aeruginosa QS reporter, sensitive to long chain AHLs and a short chain N-acylhomoserine lactones (AHL) biosensor, Chromobacterium violaceium. Overall, the gorgonian corals had higher antimicrobial activity against non-marine strains when compared to marine strains. Pseudopterogorgia americana, Pseusopterogorgia acerosa, and Pseudoplexuara flexuosa had the highest QS inhibitory effect. Interestingly, Pseudoplexuara porosa extracts stimulated QS activity with a striking 17-fold increase in signal. The stimulation of QS by P. porosa or other elements of the holobiont may encourage colonization or recruitment of specific microbial species. Overall, these results suggest the presence of novel stimulatory QS, inhibitory QS and bactericidal compounds in gorgonian corals. A better understanding of these compounds may reveal insight into coral-microbial ecology and whether a therapeutic potential exists.

  12. Legislation to regulate medical devices.

    Science.gov (United States)

    Harris, M

    1975-01-01

    The history of medical device regulation began with the need to rid the marketplace of bogus inventions which were either harmful in themselves or harmful because they delayed meaningful treatment of illness. Since World War II, sophistication in medical technology and development of electronic and other types of medical devices has created a new need for regulation of safety and performance of devices used to cure and mitigate disease in man. The 1938 amendments to the Food, Drug, and Cosmetic Act gave FDA authority over labeling and advertising of devices, enforceable only after devices were marketed. In 1969 a study by an HEW commission documented the need for further legislation. The commission recommended three categories of medical devices: those requiring premarket clearance or scientific review, those for which standards could be established to protect the public, and those which are generally recognized as safe and for which nor standards would be necessary. In 1974 the Senate unanimously approved Senator Kennedy's "Medical Device Amendments of 1973" legislation which fulfills the recommendations of the HEW commission report. The House of Representatives failed to pass their version of the legislation in the 93rd Congress. Senator Kennedy re-introduced the bill in the 94th Congress and it passed the Senate in April 1975. Representative Rogers re-introduced an amended bill. The bill is expected to become law in 1975.

  13. Understanding regulations affecting pet foods.

    Science.gov (United States)

    Dzanis, David A

    2008-08-01

    In the United States, pet foods are subject to regulation at both the federal and the state levels. The US Food and Drug Administration has jurisdiction over all animal feeds (including pet foods, treats, chews, supplements, and ingredients) in interstate commerce, which includes imported products. Many states adopt and enforce at least in part the Association of American Feed Control Officials Model Bill and Model Regulations for Pet Food and Specialty Pet Food. Thus, all pet foods in multi-state distribution are subject to a host of labeling requirements covering aspects such as product names, ingredient lists, nutrient content guarantees, and nutritional adequacy statements. Ingredients must be GRAS (generally recognized as safe) substances, approved food additives, or defined by Association of American Feed Control Officials for their intended use. Pet food labels may not bear claims that are false or misleading or that state or imply use for the treatment or prevention of disease. Pet foods that are found to be adulterated or misbranded may be subject to seizure or other enforcement actions.

  14. Epigenetic microRNA Regulation

    DEFF Research Database (Denmark)

    Wiklund, Erik Digman

    2011-01-01

    distinguish oral cancer patients from healthy controls; these miRNAs therefore have potential applications as clinical biomarkers. Finally, we show that the predominantly nuclear miR-671 controls CDR1 expression via a novel regulatory mechanism involving a circular antisense RNA. The nuclear function of mi......MicroRNAs (miRNAs) are small non-coding RNAs (ncRNAs) that negatively regulate gene expression post-transcriptionally by binding to complementary sequences in the 3’UTR of target mRNAs in the cytoplasm. However, recent evidence suggests that certain miRNAs are enriched in the nucleus......, and their targets do not seem restricted to mRNA 3’UTRs. Therefore, miRNAs are predicted to have a variety functions throughout mammalian cells. MiRNA genes appear to be regulated in much the same way as coding genes, but current insight into transcriptional miRNA control lacks detail, as mapping miRNA promoters...

  15. Translational Regulation in Nutrigenomics12

    Science.gov (United States)

    Liu, Botao; Qian, Shu-Bing

    2011-01-01

    The emergence of genome-wide analysis to interrogate cellular DNA, RNA, and protein content has revolutionized the study of the control network that mediates cellular homeostasis. Nutrigenomics addresses the effect of nutrients on gene expression, which provides a basis for understanding the biological activity of dietary components. Translation of mRNAs represents the last step of genetic flow and primarily defines the proteome. Translational regulation is thus critical for gene expression, in particular, under nutrient excess or deficiency. Until recently, it was unclear how the global effects of translational control are influenced by nutrient signaling. An emerging concept of translational reprogramming addresses how to maintain the expression of specific proteins during pathophysiological conditions by translation of selective mRNAs. Here we describe recent advances in our understanding of translational control, nutrient signaling, and their dysregulation in aging and cancer. The mechanistic understanding of translational regulation in response to different nutrient conditions may help identify potential dietary and therapeutic targets to improve human health. PMID:22332093

  16. Epigenetic regulation of human retinoblastoma.

    Science.gov (United States)

    Singh, Usha; Malik, Manzoor Ahmad; Goswami, Sandeep; Shukla, Swati; Kaur, Jasbir

    2016-11-01

    Retinoblastoma is a rare type of eye cancer of the retina that commonly occurs in early childhood and mostly affects the children before the age of 5. It occurs due to the mutations in the retinoblastoma gene (RB1) which inactivates both alleles of the RB1. RB1 was first identified as a tumor suppressor gene, which regulates cell cycle components and associated with retinoblastoma. Previously, genetic alteration was known as the major cause of its occurrence, but later, it is revealed that besides genetic changes, epigenetic changes also play a significant role in the disease. Initiation and progression of retinoblastoma could be due to independent or combined genetic and epigenetic events. Remarkable work has been done in understanding retinoblastoma pathogenesis in terms of genetic alterations, but not much in the context of epigenetic modification. Epigenetic modifications that silence tumor suppressor genes and activate oncogenes include DNA methylation, chromatin remodeling, histone modification and noncoding RNA-mediated gene silencing. Epigenetic changes can lead to altered gene function and transform normal cell into tumor cells. This review focuses on important epigenetic alteration which occurs in retinoblastoma and its current state of knowledge. The critical role of epigenetic regulation in retinoblastoma is now an emerging area, and better understanding of epigenetic changes in retinoblastoma will open the door for future therapy and diagnosis.

  17. Human morphology and temperature regulation

    Science.gov (United States)

    Anderson, G. S.

    For nearly a century individuals have believed that there is a link between human morphology and one's thermoregulatory response in adverse environments. Most early research was focussed on the rate of core cooling in a male adult population and the role of subcutaneous adipose tissue, surface area and the surface-area-to-mass ratio in one's ability to withstand varying degrees of cold stress. More recently research has addressed heat tolerance in various populations, exploring the role of subcutaneous adipose tissue, surface area and the surface-area-to-mass ratio in one's ability to maintain thermal equilibrium in warm and hot, dry and humid environments. Since the late 1970s an emphasis has been placed on the role of muscle and muscle perfusion in total-body thermal insulation. Yet, despite the history of research pertaining to human morphology and temperature regulation there is little consensus as to the impact of variations in human morphology on thermoregulatory responses. Individuals differing in body size, shape and composition appear to respond quantitatively differently to variations in both ambient and core temperatures but the interrelations between morphological components and temperature regulation are complex. It is the purpose of this paper to examine the literature pertaining to the impact of variations in muscularity, adipose tissue thickness and patterning, surface area and the surface-area-to-mass ratio on thermoregulation and thermal stability in response to both heat and cold stress.

  18. Corruption and optimal regulation under common agency

    Directory of Open Access Journals (Sweden)

    Pedro Hemsley

    2016-05-01

    Full Text Available I show that it is optimal to separate non-benevolent regulators when regulated projects are large. Separation prevents regulators from coordinating to appropriate all of the agent's informational rent when they know the type of the latter; therefore, there is a trade-off between saving on informational rent and efficiency, since the game between the regulators induced by separation causes further distortions when compared to the allocation under one regulator. When the informational rent at stake is large due to the size of the project, separation is the optimal institutional answer.

  19. 75 FR 75904 - Global Terrorism Sanctions Regulations; Terrorism Sanctions Regulations; Foreign Terrorist...

    Science.gov (United States)

    2010-12-07

    ... Office of Foreign Assets Control 31 CFR Parts 594, 595, and 597 Global Terrorism Sanctions Regulations; Terrorism Sanctions Regulations; Foreign Terrorist Organizations Sanctions Regulations AGENCY: Office of... (``OFAC'') of the U.S. Department of the Treasury is amending the Global Terrorism Sanctions Regulations...

  20. Private regulation in EU better regulation : Past performance and future promises

    NARCIS (Netherlands)

    Verbruggen, Paul

    The promotion of private regulation is frequently part of better regulation programmes. Also the Better Regulation programme of the European Union (EU) initiated in 2002 advocated forms of private regulation as important means to improve EU law-making activities. However, for various reasons the