Articoli di riviste: "Production de mutants de trypanosomes"

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Villafraz, Oriana, Marc Biran, Erika Pineda, Nicolas Plazolles, Edern Cahoreau, Rodolpho Ornitz Oliveira Souza, Magali Thonnus et al. "Procyclic trypanosomes recycle glucose catabolites and TCA cycle intermediates to stimulate growth in the presence of physiological amounts of proline". PLOS Pathogens 17, n. 3 (1 marzo 2021): e1009204. http://dx.doi.org/10.1371/journal.ppat.1009204.

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Trypanosoma brucei, a protist responsible for human African trypanosomiasis (sleeping sickness), is transmitted by the tsetse fly where the procyclic forms of the parasite develop in the proline-rich (1–2 mM) and glucose-depleted digestive tract. Proline is essential for the midgut colonization of the parasite in the insect vector, however other carbon sources could be available and used to feed its central metabolism. Here we show that procyclic trypanosomes can consume and metabolize metabolic intermediates, including those excreted from glucose catabolism (succinate, alanine and pyruvate), with the exception of acetate, which is the ultimate end-product excreted by the parasite. Among the tested metabolites, tricarboxylic acid (TCA) cycle intermediates (succinate, malate and α-ketoglutarate) stimulated growth of the parasite in the presence of 2 mM proline. The pathways used for their metabolism were mapped by proton-NMR metabolic profiling and phenotypic analyses of thirteen RNAi and/or null mutants affecting central carbon metabolism. We showed that (i) malate is converted to succinate by both the reducing and oxidative branches of the TCA cycle, which demonstrates that procyclic trypanosomes can use the full TCA cycle, (ii) the enormous rate of α-ketoglutarate consumption (15-times higher than glucose) is possible thanks to the balanced production and consumption of NADH at the substrate level and (iii) α-ketoglutarate is toxic for trypanosomes if not appropriately metabolized as observed for an α-ketoglutarate dehydrogenase null mutant. In addition, epimastigotes produced from procyclics upon overexpression of RBP6 showed a growth defect in the presence of 2 mM proline, which is rescued by α-ketoglutarate, suggesting that physiological amounts of proline are not sufficient per se for the development of trypanosomes in the fly. In conclusion, these data show that trypanosomes can metabolize multiple metabolites, in addition to proline, which allows them to confront challenging environments in the fly.

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Olayinka-Adefemi, Folayemi, Chukwunonso Onyilagha, Nipun Jayachandran, Sen Hou, Ping Jia, Jude E. Uzonna e Aaron J. Marshall. "The Function of Phosphatidylinositol 3-Kinase delta (PI3Kδ) Enzyme in Protective Immunity to Trypanosoma congolense Infection in Mice: The Role of Regulatory B cells". Journal of Immunology 204, n. 1_Supplement (1 maggio 2020): 82.39. http://dx.doi.org/10.4049/jimmunol.204.supp.82.39.

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Abstract The delta isoform of PI 3-kinase (PI3Kδ) has important functions in B cell activation. Trypanosoma parasites utilize several mechanisms to exploit and evade host B cell and antibody responses critical for immunity. We sought to determine the impact of PI3Kδ in immunity to Trypanosoma congolense infection. We found that PI3KδD910A mutant mice show a surprisingly enhanced control of parasitemia in early infection (7–9 days) when compared to wild-type (WT) C57BL/6 mice. Drug treatment with Idelalisib to acutely inhibit PI3Kδ during infection in WT mice led to improved early control of parasitemia, consistent with results from genetically deficient mice. Both mutant mice and Idelalisib-treated mice showed a delay in polyclonal B cell activation and CD80/86 expression. Analysis of cytokine levels in the blood and peritoneal cavity showed higher IFNg and lower IL-10 levels in the drug-treated group at early time points, indicating a more pro-inflammatory environment. B1 cells were identified as the major IL-10 producing cells in the peritoneal cavity in early infection, and B1 cell production of IL-10 was significantly impaired by PI3Kδ-inhibitor treatment. We further observed increased Nitric oxide production in drug-treated mice, which correlated with increased parasite killing in early infection. Despite the improved early parasite control, there was a 100% mortality in PI3KδD910A mutants and 25% mortality in Idelalisib treated mice, presumably due to compromised generation of parasite-specific antibodies required to clear the first wave of blood infection. Our findings suggest that PI3Kδ inhibition impacts both regulatory B cell functions, affecting the early innate response, and generation of critical protective antibodies.

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Beneke, Tom, Ross Madden, Laura Makin, Jessica Valli, Jack Sunter e Eva Gluenz. "A CRISPR Cas9 high-throughput genome editing toolkit for kinetoplastids". Royal Society Open Science 4, n. 5 (maggio 2017): 170095. http://dx.doi.org/10.1098/rsos.170095.

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Clustered regularly interspaced short palindromic repeats (CRISPR), CRISPR-associated gene 9 (Cas9) genome editing is set to revolutionize genetic manipulation of pathogens, including kinetoplastids. CRISPR technology provides the opportunity to develop scalable methods for high-throughput production of mutant phenotypes. Here, we report development of a CRISPR-Cas9 toolkit that allows rapid tagging and gene knockout in diverse kinetoplastid species without requiring the user to perform any DNA cloning. We developed a new protocol for single-guide RNA (sgRNA) delivery using PCR-generated DNA templates which are transcribed in vivo by T7 RNA polymerase and an online resource (LeishGEdit.net) for automated primer design. We produced a set of plasmids that allows easy and scalable generation of DNA constructs for transfections in just a few hours. We show how these tools allow knock-in of fluorescent protein tags, modified biotin ligase BirA*, luciferase, HaloTag and small epitope tags, which can be fused to proteins at the N- or C-terminus, for functional studies of proteins and localization screening. These tools enabled generation of null mutants in a single round of transfection in promastigote form Leishmania major , Leishmania mexicana and bloodstream form Trypanosoma brucei ; deleted genes were undetectable in non-clonal populations, enabling for the first time rapid and large-scale knockout screens.

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Sass, Gabriele, Laura C. Miller Conrad, Terrence-Thang H. Nguyen e David A. Stevens. "The Pseudomonas aeruginosa product pyochelin interferes with Trypanosoma cruzi infection and multiplication in vitro". Transactions of The Royal Society of Tropical Medicine and Hygiene 114, n. 7 (20 marzo 2020): 492–98. http://dx.doi.org/10.1093/trstmh/trz136.

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Abstract Background Bacteria are sources of numerous molecules used in treatment of infectious diseases. We investigated effects of molecules produced by 26 Pseudomonas aeruginosa strains against infection of mammalian cell cultures with Trypanosoma cruzi, the aetiological agent of Chagas disease. Methods Vero cells were infected with T. cruzi in the presence of wild-type P. aeruginosa supernatants or supernatants of mutants with defects in the production of various virulence, quorum sensing and iron acquisition factors. Quantification of T. cruzi infection (percentage of infected cells) and multiplication (number of amastigotes per infected cell) was performed and cell viability was determined. Results Wild-type P. aeruginosa products negatively affected T. cruzi infection and multiplication in a dose-dependent manner, without evident toxicity for mammalian cells. PvdD/pchE mutation (loss of the P. aeruginosa siderophores pyoverdine and pyochelin) had the greatest impact on anti–T. cruzi activity. Negative effects on T. cruzi infection by pure pyochelin, but not pyoverdine, or other P. aeruginosa exoproducts studied, were quantitatively similar to the effects of benznidazole, the current standard therapy against T. cruzi. Conclusions The P. aeruginosa product pyochelin showed promising activity against T. cruzi and might become a new lead molecule for therapy development.

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Vassella, Erik, Peter Bütikofer, Markus Engstler, Jennifer Jelk e Isabel Roditi. "Procyclin Null Mutants ofTrypanosoma bruceiExpress Free Glycosylphosphatidylinositols on Their Surface". Molecular Biology of the Cell 14, n. 4 (aprile 2003): 1308–18. http://dx.doi.org/10.1091/mbc.e02-10-0694.

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Procyclins are abundant, glycosylphosphatidylinositol (GPI)-anchored proteins on the surface of procyclic (insect) form trypanosomes. To investigate whether trypanosomes are able to survive without a procyclin coat, all four procyclin genes were deleted sequentially. Bloodstream forms of the null mutant exhibited no detectable phenotype and were able to differentiate to procyclic forms. Initially, differentiated null mutant cells were barely able to grow, but after an adaptation period of 2 mo in culture they proliferated at the same rate as wild-type trypanosomes. Analysis of these culture-adapted null mutants revealed that they were covered by free GPIs. These were closely related to the mature procyclin anchor in structure and were expressed on the surface in numbers comparable with that of procyclin in wild-type cells. However, free GPIs were smaller than the procyclin anchor, indicative of a lower number of poly-N-acetyllactosamine repeats, and a proportion contained diacylphosphatidic acid. Free GPIs are also expressed by wild-type cells, although to a lesser extent. These have been overlooked in the past because they partition in a solvent fraction (chloroform/water/methanol) that is normally discarded when GPI-anchored proteins are purified.

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Lüscher, Alexandra, Pinar Önal, Anne-Marie Schweingruber e Pascal Mäser. "Adenosine Kinase of Trypanosoma brucei and Its Role in Susceptibility to Adenosine Antimetabolites". Antimicrobial Agents and Chemotherapy 51, n. 11 (13 agosto 2007): 3895–901. http://dx.doi.org/10.1128/aac.00458-07.

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ABSTRACT Trypanosoma brucei cannot synthesize purines de novo and relies on purine salvage from its hosts to build nucleic acids. With adenosine being a preferred purine source of bloodstream-form trypanosomes, adenosine kinase (AK; EC 2.7.1.20) is likely to be a key player in purine salvage. Adenosine kinase is also of high pharmacological interest, since for many adenosine antimetabolites, phosphorylation is a prerequisite for activity. Here, we cloned and functionally characterized adenosine kinase from T. brucei (TbAK). TbAK is a tandem gene, expressed in both procyclic- and bloodstream-form trypanosomes, whose product localized to the cytosol of the parasites. The RNA interference-mediated silencing of TbAK suggested that the gene is nonessential under standard growth conditions. Inhibition or downregulation of TbAK rendered the trypanosomes resistant to cordycepin (3′-deoxyadenosine), demonstrating a role for TbAK in the activation of adenosine antimetabolites. The expression of TbAK in Saccharomyces cerevisiae complemented a null mutation in the adenosine kinase gene ado1. The concomitant expression of TbAK with the T. brucei adenosine transporter gene TbAT1 allowed S. cerevisiae ado1 ade2 double mutants to grow on adenosine as the sole purine source and, at the same time, sensitized them to adenosine antimetabolites. The coexpression of TbAK and TbAT1 in S. cerevisiae ado1 ade2 double mutants proved to be a convenient tool for testing nucleoside analogues for uptake and activation by T. brucei adenosine salvage enzymes.

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Mensa-Wilmot, K., JH LeBowitz, KP Chang, A. al-Qahtani, BS McGwire, S. Tucker e JC Morris. "A glycosylphosphatidylinositol (GPI)-negative phenotype produced in Leishmania major by GPI phospholipase C from Trypanosoma brucei: topography of two GPI pathways". Journal of Cell Biology 124, n. 6 (15 marzo 1994): 935–47. http://dx.doi.org/10.1083/jcb.124.6.935.

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The major surface macromolecules of the protozoan parasite Leishmania major, gp63 (a metalloprotease), and lipophosphoglycan (a polysaccharide), are glycosylphosphatidylinositol (GPI) anchored. We expressed a cytoplasmic glycosylphosphatidylinositol phospholipase C (GPI-PLC) in L. major in order to examine the topography of the protein-GPI and polysaccharide-GPI pathways. In L. major cells expressing GPI-PLC, cell-associated gp63 could not be detected in immunoblots. Pulse-chase analysis revealed that gp63 was secreted into the culture medium with a half-time of 5.5 h. Secreted gp63 lacked anti-cross reacting determinant epitopes, and was not metabolically labeled with [3H]ethanolamine, indicating that it never received a GPI anchor. Further, the quantity of putative protein-GPI intermediates decreased approximately 10-fold. In striking contrast, lipophosphoglycan levels were unaltered. However, GPI-PLC cleaved polysaccharide-GPI intermediates (glycoinositol phospholipids) in vitro. Thus, reactions specific to the polysaccharide-GPI pathway are compartmentalized in vivo within the endoplasmic reticulum, thereby sequestering polysaccharide-GPI intermediates from GPI-PLC cleavage. On the contrary, protein-GPI synthesis at least up to production of Man(1 alpha 6)Man(1 alpha 4)GlcN-(1 alpha 6)-myo-inositol-1-phospholipid is cytosolic. To our knowledge this represents the first use of a catabolic enzyme in vivo to elucidate the topography of biosynthetic pathways. GPI-PLC causes a protein-GPI-negative phenotype in L. major, even when genes for GPI biosynthesis are functional. This phenotype is remarkably similar to that of some GPI mutants of mammalian cells: implications for paroxysmal nocturnal hemoglobinuria and Thy-1-negative T-lymphoma are discussed.

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Guerra-Giraldez, Cristina, Luis Quijada e Christine E. Clayton. "Compartmentation of enzymes in a microbody, the glycosome, is essential in Trypanosoma brucei". Journal of Cell Science 115, n. 13 (1 luglio 2002): 2651–58. http://dx.doi.org/10.1242/jcs.115.13.2651.

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All kinetoplastids contain membrane-bound microbodies known as glycosomes,in which several metabolic pathways including part of glycolysis are compartmentalized. Peroxin 2 is essential for the import of many proteins into the microbodies of yeasts and mammals. The PEX2 gene of Trypanosoma brucei was identified and its expression was silenced by means of tetracycline-inducible RNA interference. Bloodstream-form trypanosomes, which rely exclusively on glycolysis for ATP generation, died rapidly upon PEX2 depletion. Insect-form (procyclic) trypanosomes do not rely solely on glycolysis for ATP synthesis. PEX2 depletion in procyclic forms resulted in relocation of most tested matrix proteins to the cytosol, and these mutants also died. Compartmentation of microbody enzymes is therefore essential for survival of bloodstream and procyclic T. brucei. In contrast, yeasts and cultured mammalian cells grow normally in the absence of peroxisomal membranes unless placed on selective media.

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Lillico, Simon, Mark C. Field, Pat Blundell, Graham H. Coombs e Jeremy C. Mottram. "Essential Roles for GPI-anchored Proteins in African Trypanosomes Revealed Using Mutants Deficient in GPI8". Molecular Biology of the Cell 14, n. 3 (marzo 2003): 1182–94. http://dx.doi.org/10.1091/mbc.e02-03-0167.

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The survival of Trypanosoma brucei, the causative agent of Sleeping Sickness and Nagana, is facilitated by the expression of a dense surface coat of glycosylphosphatidylinositol (GPI)-anchored proteins in both its mammalian and tsetse fly hosts. We have characterized T. brucei GPI8, the gene encoding the catalytic subunit of the GPI:protein transamidase complex that adds preformed GPI anchors onto nascent polypeptides. Deletion ofGPI8 (to give Δgpi8) resulted in the absence of GPI-anchored proteins from the cell surface of procyclic form trypanosomes and accumulation of a pool of non–protein-linked GPI molecules, some of which are surface located. Procyclic Δgpi8, while viable in culture, were unable to establish infections in the tsetse midgut, confirming that GPI-anchored proteins are essential for insect-parasite interactions. Applying specific inducible GPI8 RNAi with bloodstream form parasites resulted in accumulation of unanchored variant surface glycoprotein and cell death with a defined multinuclear, multikinetoplast, and multiflagellar phenotype indicative of a block in cytokinesis. These data show that GPI-anchored proteins are essential for the viability of bloodstream form trypanosomes even in the absence of immune challenge and imply that GPI8 is important for proper cell cycle progression.

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Ralston, Katherine S., Neville K. Kisalu e Kent L. Hill. "Structure-Function Analysis of Dynein Light Chain 1 Identifies Viable Motility Mutants in Bloodstream-Form Trypanosoma brucei". Eukaryotic Cell 10, n. 7 (4 marzo 2011): 884–94. http://dx.doi.org/10.1128/ec.00298-10.

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ABSTRACT The flagellum of Trypanosoma brucei is an essential and multifunctional organelle that is receiving increasing attention as a potential drug target and as a system for studying flagellum biology. RNA interference (RNAi) knockdown is widely used to test the requirement for a protein in flagellar motility and has suggested that normal flagellar motility is essential for viability in bloodstream-form trypanosomes. However, RNAi knockdown alone provides limited functional information because the consequence is often loss of a multiprotein complex. We therefore developed an inducible system that allows functional analysis of point mutations in flagellar proteins in T. brucei . Using this system, we identified point mutations in the outer dynein light chain 1 (LC1) that allow stable assembly of outer dynein motors but do not support propulsive motility. In procyclic-form trypanosomes, the phenotype of LC1 mutants with point mutations differs from the motility and structural defects of LC1 knockdowns, which lack the outer-arm dynein motor. Thus, our results distinguish LC1-specific functions from broader functions of outer-arm dynein. In bloodstream-form trypanosomes, LC1 knockdown blocks cell division and is lethal. In contrast, LC1 point mutations cause severe motility defects without affecting viability, indicating that the lethal phenotype of LC1 RNAi knockdown is not due to defective motility. Our results demonstrate for the first time that normal motility is not essential in bloodstream-form T. brucei and that the presumed connection between motility and viability is more complex than might be interpreted from knockdown studies alone. These findings open new avenues for dissecting mechanisms of flagellar protein function and provide an important step in efforts to exploit the potential of the flagellum as a therapeutic target in African sleeping sickness.

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Paul, Kimberly Sue, Barbara McCall, Brette Winston, Patrick Vigueira e Jennifer McCallister. "Fluorescence‐based RNAi screen for mutants in fatty acid uptake in African trypanosomes". FASEB Journal 22, S2 (aprile 2008): 268. http://dx.doi.org/10.1096/fasebj.22.2_supplement.268.

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Rodríguez-Bolaños, Monica, Nallely Cabrera e Ruy Perez-Montfort. "Identification of the critical residues responsible for differential reactivation of the triosephosphate isomerases of two trypanosomes". Open Biology 6, n. 10 (ottobre 2016): 160161. http://dx.doi.org/10.1098/rsob.160161.

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The reactivation of triosephosphate isomerase (TIM) from unfolded monomers induced by guanidine hydrochloride involves different amino acids of its sequence in different stages of protein refolding. We describe a systematic mutagenesis method to find critical residues for certain physico-chemical properties of a protein. The two similar TIMs of Trypanosoma brucei and Trypanosoma cruzi have different reactivation velocities and efficiencies. We used a small number of chimeric enzymes, additive mutants and planned site-directed mutants to produce an enzyme from T. brucei with 13 mutations in its sequence, which reactivates fast and efficiently like wild-type (WT) TIM from T. cruzi , and another enzyme from T. cruzi, with 13 slightly altered mutations, which reactivated slowly and inefficiently like the WT TIM of T. brucei . Our method is a shorter alternative to random mutagenesis, saturation mutagenesis or directed evolution to find multiple amino acids critical for certain properties of proteins.

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Vaidya, Tushar, Moiz Bakhiet, Kent L. Hill, Tomas Olsson, Krister Kristensson e John E. Donelson. "The Gene for a T Lymphocyte Triggering Factor from African Trypanosomes". Journal of Experimental Medicine 186, n. 3 (4 agosto 1997): 433–38. http://dx.doi.org/10.1084/jem.186.3.433.

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An early and essential event in the protective immune response against most viruses and protozoa is the production of interferon-γ (IFN-γ). In contrast, during infection with African trypanosomes, protozoan parasites that cause human sleeping sickness, the increased levels of IFN-γ do not correlate with a protective response. We showed previously that African trypanosomes express a protein called T lymphocyte triggering factor (TLTF), which triggers CD8+ T lymphocytes to proliferate and to secrete IFN-γ. Here, we isolate the gene for TLTF and demonstrate that the recombinant version of TLTF specifically induces CD8+, but not CD4+, T cells to secrete IFN-γ. Studies with TLTF fused to the green fluorescent protein show that TLTF is localized to small vesicles that are found primarily at or near the flagellar pocket, the site of secretion in trypanosomes. TLTF is likely to be only the first example of a class of proteins that we designate as trypanokines, i.e., factors secreted by trypanosomes that modulate the cytokine network of the host immune system for the benefit of the parasite.

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Hutchings, Nathan R., John E. Donelson e Kent L. Hill. "Trypanin is a cytoskeletal linker protein and is required for cell motility in African trypanosomes". Journal of Cell Biology 156, n. 5 (25 febbraio 2002): 867–77. http://dx.doi.org/10.1083/jcb.200201036.

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The cytoskeleton of eukaryotic cells is comprised of a complex network of distinct but interconnected filament systems that function in cell division, cell motility, and subcellular trafficking of proteins and organelles. A gap in our understanding of this dynamic network is the identification of proteins that connect subsets of cytoskeletal structures. We previously discovered a family of cytoskeleton-associated proteins that includes GAS11, a candidate human tumor suppressor upregulated in growth-arrested cells, and trypanin, a component of the flagellar cytoskeleton of African trypanosomes. Although these proteins are intimately associated with the cytoskeleton, their function has yet to be determined. Here we use double-stranded RNA interference to block trypanin expression in Trypanosoma brucei, and demonstrate that this protein is required for directional cell motility. Trypanin(−) mutants have an active flagellum, but are unable to coordinate flagellar beat. As a consequence, they spin and tumble uncontrollably, occasionally moving backward. Immunofluorescence experiments demonstrate that trypanin is located along the flagellum/flagellum attachment zone and electron microscopic analysis revealed that cytoskeletal connections between the flagellar apparatus and subpellicular cytoskeleton are destabilized in trypanin(−) mutants. These results indicate that trypanin functions as a cytoskeletal linker protein and offer insights into the mechanisms of flagellum-based cell motility.

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Turner, C. M. R., J. Sternberg, N. Buchanan, E. Smith, G. Hide e A. Tait. "Evidence that the mechanism of gene exchange in Trypanosoma brucei involves meiosis and syngamy". Parasitology 101, n. 3 (dicembre 1990): 377–86. http://dx.doi.org/10.1017/s0031182000060571.

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SUMMARYAll pairwise combinations of three cloned stocks of Trypanosoma brucei (STIB 247L, STIB 386AA and TREU 927/4) were co-transmitted through tsetse flies (Glossina morsitans) and screened for the production of hybrid trypanosomes. Clones of metacyclic and bloodstream trypanosomes from flies harbouring mature infections containing hybrid trypanosomes were established and screened for several isoenzyme and restriction fragment length polymorphisms. For each of the three combinations of parents, some progeny clones were observed to be of a phenotype and genotype indicating that genetic exchange had occurred during development of the trypanosomes in flies. These hybrid clones shared three salient features: (1) where the parents were homozygous variants the progeny were heterozygous, (2) where one of the parents was heterozygous, allelic segregation was observed and (3) the progeny clones were shown to be recombinant when two or more markers for which one of the parents was heterozygous were examined. These results are consistent with the progeny being an F1 in a diploid mendelian genetic system involving meiosis and syngamy. Our observations show that all possible combinations of the three stocks may undergo genetic exchange. A marker analysis of a series of clones each derived from single metacyclic trypanosomes showed that individual flies transmit a mixture of trypanosome genotypes corresponding to F1 progeny and to parental types, indicating that genetic exchange was a non-obligatory event in the life-cycle of the trypanosome. In addition, a preliminary analysis of the phenotype of procyclic stage trypanosomes derived from flies infected with two stocks, indicates that genetic exchange is unlikely to occur at this stage.

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FAST, Beate, Katrin KREMP, Michael BOSHART e Dietmar STEVERDING. "Iron-dependent regulation of transferrin receptor expression in Trypanosoma brucei". Biochemical Journal 342, n. 3 (5 settembre 1999): 691–96. http://dx.doi.org/10.1042/bj3420691.

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Transferrin is an essential growth factor for African trypanosomes. Here we show that expression of the trypanosomal transferrin receptor, which bears no structural similarity with mammalian transferrin receptors, is regulated by iron availability. Iron depletion of bloodstream forms of Trypanosoma brucei with the iron chelator deferoxamine resulted in a 3-fold up-regulation of the transferrin receptor and a 3-fold increase of the transferrin uptake rate. The abundance of expression site associated gene product 6 (ESAG6) mRNA, which encodes one of the two subunits of the trypanosome transferrin receptor, is regulated 5-fold by a post-transcriptional mechanism. In mammalian cells the stability of transferrin receptor mRNA is controlled by iron regulatory proteins (IRPs) binding to iron-responsive elements (IREs) in the 3′-untranslated region (UTR). Therefore, the role of a T. brucei cytoplasmic aconitase (TbACO) that is highly related to mammalian IRP-1 was investigated. Iron regulation of the transferrin receptor was found to be unaffected in δaco::NEO/δaco::HYG null mutants generated by targeted disruption of the TbACO gene. Thus, the mechanism of post-transcriptional transferrin receptor regulation in trypanosomes appears to be distinct from the IRE/IRP paradigm. The transferrin uptake rate was also increased when trypanosomes were transferred from medium supplemented with foetal bovine serum to medium supplemented with sera from other vertebrates. Due to varying binding affinities of the trypanosomal transferrin receptor for transferrins of different species, serum change can result in iron starvation. Thus, regulation of transferrin receptor expression may be a fast compensatory mechanism upon transmission of the parasite to a new host species.

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Stuart, K., A. K. Panigrahi, A. Schnaufer, M. Drozdz, C. Clayton e R. Salavati. "Composition of the editing complex of Trypanosoma brucei". Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences 357, n. 1417 (29 gennaio 2002): 71–79. http://dx.doi.org/10.1098/rstb.2001.0994.

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The RNA editing that produces most functional mRNAs in trypanosomes is catalysed by a multiprotein complex. This complex catalyses the endoribonucleolytic cleavage, uridylate addition and removal, and RNA ligation steps of the editing process. Enzymatic and in vitro editing analyses reveal that each catalytic step contributes to the specificity of the editing and, together with the interaction between gRNA and the mRNA, results in precisely edited mRNAs. Tandem mass spectrometric analysis was used to identify the genes for several components of biochemically purified editing complexes. Their identity and presence in the editing complex were confirmed using immunochemical analyses utilizing mAbs specific to the editing complex components. The genes for two RNA ligases were identified. Genetic studies show that some, but not all, of the components of the complex are essential for editing. The TbMP52 RNA ligase is essential for editing while the TbMP48 RNA ligase is not. Editing was found to be essential in bloodstream form trypanosomes. This is surprising because mutants devoid of genes encoding RNAs that become edited survive as bloodstream forms but encouraging since editing complex components may be targets for chemotherapy.

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Lepesheva, G. I., T. Y. Hargrove, R. D. Ott, W. D. Nes e M. R. Waterman. "Biodiversity of CYP51 in trypanosomes". Biochemical Society Transactions 34, n. 6 (25 ottobre 2006): 1161–64. http://dx.doi.org/10.1042/bst0341161.

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Sterol 14α-demethylases (CYP51) are metabolic cytochromes P450, found in each biological kingdom. They catalyse a single three-step reaction included in all sterol biosynthetic pathways. Plant CYP51s have strict preference towards their physiological substrate O (obtusifoliol), which is C-4-monomethylated. Natural substrates of animal/fungal CYP51 (lanosterol, 24,25-dihydrolanosterol or 24-methylenelanosterol) are C-4-dimethylated. CYP51 from the pathogenic protozoa TB (Trypanosoma brucei) is the first example of O-specific sterol 14α-demethylase in non-photosynthetic organisms. Surprisingly, at 83% amino acid identity to the TB orthologue, CYP51 from TC (Trypanosoma cruzi) clearly prefers C-4-dimethylated sterols. Replacement of animal/fungi-like Ile105 in the B′ helix of TC CYP51 with phenylalanine, the residue found in this position in all plant and other trypanosome CYP51s, dramatically increases the ability of the enzyme to metabolize O, converting it into a more plant-like sterol 14α-demethylase. A more than 100-fold increase in binding and turnover is observed for the 24-desmethyl analogue of O [N (norlanosterol)], which is found in vivo in procyclic forms of TB and is a good TB CYP51 substrate in vitro. We believe that (i) N is a non-conventional CYP51 substrate, preferred in TB and perhaps other Trypanosomatidae and (ii) functional similarity of TC CYP51 to animal/fungal orthologues is a result of evolutionary convergence (including F105I mutation), leading to different pathways for sterol production in TC versus TB.

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Roger, M., e P. Viens. "Trypanosoma musculi: influence of uric acid on in vitro formation of metacyclic trypomastigotes". Parasitology 95, n. 3 (dicembre 1987): 531–35. http://dx.doi.org/10.1017/s0031182000057954.

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SUMMARYUric acid is the most important constituent in the urine of insects. Infective metacyclic forms of the stercorarian trypanosomes are produced in the rectum of their insect vector and are thus in contact with uric acid. Using a culture system which permitted the growth of the insect stages of Trypanosoma musculi, we studied the influence of uric acid on the metacyclogenesis of this parasite. When added to the culture, uric acid enhanced the production of metacyclic forms. Furthermore, it rendered these trypanosomes highly infective by the oral route. It is suggested that uric acid may play an important role on the metacyclogenesis of T. musculi.

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de Koning, Harry P., Laura F. Anderson, Mhairi Stewart, Richard J. S. Burchmore, Lynsey J. M. Wallace e Michael P. Barrett. "The Trypanocide Diminazene Aceturate Is Accumulated Predominantly through the TbAT1 Purine Transporter: Additional Insights on Diamidine Resistance in African Trypanosomes". Antimicrobial Agents and Chemotherapy 48, n. 5 (maggio 2004): 1515–19. http://dx.doi.org/10.1128/aac.48.5.1515-1519.2004.

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ABSTRACT Resistance to diminazene aceturate (Berenil) is a severe problem in the control of African trypanosomiasis in domestic animals. It has been speculated that resistance may be the result of reduced diminazene uptake by the parasite. We describe here the mechanisms by which [3H]diminazene is transported by Trypanosoma brucei brucei bloodstream forms. Diminazene was rapidly accumulated through a single transporter, with a Km of 0.45 ± 0.11 μM, which was dose dependently inhibited by pentamidine and adenosine. The Ki values for these inhibitors were consistent with this transporter being the P2/TbAT1 adenosine transporter. Yeast expressing TbAT1 acquired the ability to take up [3H]diminazene and [3H]pentamidine. TbAT1-null mutants had lost almost all capacity for [3H]diminazene transport. However, this cell line still displayed a small but detectable rate of [3H]diminazene accumulation, in a nonsaturable manner. We conclude that TbAT1 mediates [3H]diminazene transport almost exclusively and that this explains the observed diminazene resistance phenotypes of TbAT1-null mutants and field isolates.

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21

Vincendeau, Philippe, e Bernard Bouteille. "Immunology and immunopathology of African trypanosomiasis". Anais da Academia Brasileira de Ciências 78, n. 4 (dicembre 2006): 645–65. http://dx.doi.org/10.1590/s0001-37652006000400004.

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Major modifications of immune system have been observed in African trypanosomiasis. These immune reactions do not lead to protection and are also involved in immunopathology disorders. The major surface component (variable surface glycoprotein,VSG) is associated with escape to immune reactions, cytokine network dysfunctions and autoantibody production. Most of our knowledge result from experimental trypanosomiasis. Innate resistance elements have been characterised. In infected mice, VSG preferentially stimulates a Th 1-cell subset. A response of <FONT FACE=Symbol>gd</FONT> and CD8 T cells to trypanosome antigens was observed in trypanotolerant cattle. An increase in CD5 B cells, responsible for most serum IgM and production of autoantibodies has been noted in infected cattle. Macrophages play important roles in trypanosomiasis, in synergy with antibodies (phagocytosis) and by secreting various molecules (radicals, cytokines, prostaglandins,...). Trypanosomes are highly sensitive to TNF-alpha, reactive oxygen and nitrogen intermediates. TNF-alpha is also involved in cachexia. IFN-gamma acts as a parasite growth factor. These various elements contribute to immunosuppression. Trypanosomes have learnt to use immune mechanisms to its own profit. Recent data show the importance of alternative macrophage activation, including arginase induction. L-ornithine produced by host arginase is essential to parasite growth. All these data reflect the deep insight into the immune system realised by trypanosomes and might suggest interference therapeutic approaches.

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22

Leung, Ka Fai, Fay S. Riley, Mark Carrington e Mark C. Field. "Ubiquitylation and Developmental Regulation of Invariant Surface Protein Expression in Trypanosomes". Eukaryotic Cell 10, n. 7 (13 maggio 2011): 916–31. http://dx.doi.org/10.1128/ec.05012-11.

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ABSTRACTThe cell surface ofTrypanosoma bruceiis dominated by the glycosylphosphatidylinositol-anchored variant surface glycoprotein (VSG), which is essential for immune evasion. VSG biosynthesis, trafficking, and turnover are well documented, buttrans-membrane domain (TMD) proteins, including the invariant surface glycoproteins (ISGs), are less well characterized. Internalization and degradation of ISG65 depend on ubiquitylation of conserved cytoplasmic lysines. Using epitope-tagged ISG75 and reporter chimeric proteins bearing the cytoplasmic andtrans-membrane regions of ISG75, together with multiple mutants with lysine-to-arginine mutations, we demonstrate that the cytoplasmic tail of ISG75 is both sufficient and necessary for endosomal targeting and degradation. The ISG75 chimeric reporter protein localized to endocytic organelles, while lysine-null versions were significantly stabilized at the cell surface. Importantly, ISG75 cytoplasmic lysines are modified by extensive oligoubiquitin chains and ubiquitylation is abolished in the lysine-null version. Furthermore, we find evidence for differential modes of turnover of ISG65 and ISG75. Full-length lysine-null ISG65 localization and protein turnover are significantly perturbed, but ISG75 localization and protein turnover are not, while ubiquitin conjugates can be detected for full-length lysine-null ISG75 but not ISG65. We find that the ISG75 ectodomain has a predicted coiled-coil, suggesting that ISG75 could be part of a complex, while ISG65 behaves independently. We also demonstrate a developmental stage-specific mechanism for exclusion of surface ISG expression in insect-stage cells by a ubiquitin-independent mechanism. We suggest that ubiquitylation may be a general mechanism for regulatingtrans-membrane domain surface proteins in trypanosomes.

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23

Philip G, Penketh, e Patton Curtis L. "The activity of crude bromoacetyl-L-carnitine preparations against Trypanosoma brucei and the roles of threonine/pyruvate in non-hexose/glycerol ATP production". Annals of Systems Biology 6, n. 1 (29 aprile 2023): 001–3. http://dx.doi.org/10.17352/asb.000020.

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The Trypanosoma brucei group trypanosomes (Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense) cause an invariably fatal disease in humans, and Trypanosoma brucei brucei a fatal disease in cattle, if left untreated [1].

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24

Duncan, Samuel M., Carla Gilabert Carbajo, Rupa Nagar, Qi Zhong, Conor Breen, Michael A. J. Ferguson e Calvin Tiengwe. "Generation of a bloodstream form Trypanosoma brucei double glycosyltransferase null mutant competent in receptor-mediated endocytosis of transferrin". PLOS Pathogens 20, n. 6 (27 giugno 2024): e1012333. http://dx.doi.org/10.1371/journal.ppat.1012333.

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The bloodstream form of Trypanosoma brucei expresses large poly-N-acetyllactosamine (pNAL) chains on complex N-glycans of a subset of glycoproteins. It has been hypothesised that pNAL may be required for receptor-mediated endocytosis. African trypanosomes contain a unique family of glycosyltransferases, the GT67 family. Two of these, TbGT10 and TbGT8, have been shown to be involved in pNAL biosynthesis in bloodstream form Trypanosoma brucei, raising the possibility that deleting both enzymes simultaneously might abolish pNAL biosynthesis and provide clues to pNAL function and/or essentiality. In this paper, we describe the creation of a TbGT10 null mutant containing a single TbGT8 allele that can be excised upon the addition of rapamycin and, from that, a TbGT10 and TbGT8 double null mutant. These mutants were analysed by lectin blotting, glycopeptide methylation linkage analysis and flow cytometry. The data show that the mutants are defective, but not abrogated, in pNAL synthesis, suggesting that other GT67 family members can compensate to some degree for loss of TbGT10 and TbGT8. Despite there being residual pNAL synthesis in these mutants, certain glycoproteins appear to be particularly affected. These include the lysosomal CBP1B serine carboxypeptidase, cell surface ESAG2 and the ESAG6 subunit of the essential parasite transferrin receptor (TfR). The pNAL deficient TfR in the mutants continued to function normally with respect to protein stability, transferrin binding, receptor mediated endocytosis of transferrin and subcellular localisation. Further the pNAL deficient mutants were as viable as wild type parasites in vitro and in in vivo mouse infection experiments. Although we were able to reproduce the inhibition of transferrin uptake with high concentrations of pNAL structural analogues (N-acetylchito-oligosaccharides), this effect disappeared at lower concentrations that still inhibited tomato lectin uptake, i.e., at concentrations able to outcompete lectin-pNAL binding. Based on these findings, we recommend revision of the pNAL-dependent receptor mediated endocytosis hypothesis.

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25

Asahi, Satoru, Yutaka Tsunemi, Motowo Izawa e Muneharu Doi. "Cytidine Production by Mutants ofBacillus subtilis". Bioscience, Biotechnology, and Biochemistry 58, n. 8 (gennaio 1994): 1399–402. http://dx.doi.org/10.1271/bbb.58.1399.

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26

Barab�s, Zolt�n. "Hybrid seed production using nutritional mutants". Euphytica 53, n. 1 (febbraio 1991): 67–72. http://dx.doi.org/10.1007/bf00032035.

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27

Matovu, Enock, Mhairi L. Stewart, Federico Geiser, Reto Brun, Pascal Mäser, Lynsey J. M. Wallace, Richard J. Burchmore et al. "Mechanisms of Arsenical and Diamidine Uptake and Resistance in Trypanosoma brucei". Eukaryotic Cell 2, n. 5 (ottobre 2003): 1003–8. http://dx.doi.org/10.1128/ec.2.5.1003-1008.2003.

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ABSTRACT Sleeping sickness, caused by Trypanosoma brucei spp., has become resurgent in sub-Saharan Africa. Moreover, there is an alarming increase in treatment failures with melarsoprol, the principal agent used against late-stage sleeping sickness. In T. brucei, the uptake of melarsoprol as well as diamidines is thought to be mediated by the P2 aminopurine transporter, and loss of P2 function has been implicated in resistance to these agents. The trypanosomal gene TbAT1 has been found to encode a P2-type transporter when expressed in yeast. Here we investigate the role of TbAT1 in drug uptake and drug resistance in T. brucei by genetic knockout of TbAT1. Tbat1-null trypanosomes were deficient in P2-type adenosine transport and lacked adenosine-sensitive transport of pentamidine and melaminophenyl arsenicals. However, the null mutants were only slightly resistant to melaminophenyl arsenicals and pentamidine, while resistance to other diamidines such as diminazene was more pronounced. Nevertheless, the reduction in drug sensitivity might be of clinical significance, since mice infected with tbat1-null trypanosomes could not be cured with 2 mg of melarsoprol/kg of body weight for four consecutive days, whereas mice infected with the parental line were all cured by using this protocol. Two additional pentamidine transporters, HAPT1 and LAPT1, were still present in the null mutant, and evidence is presented that HAPT1 may be responsible for the residual uptake of melaminophenyl arsenicals. High-level arsenical resistance therefore appears to involve the loss of more than one transporter.

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28

Zoltner, Martin, Gustavo D. Campagnaro, Gergana Taleva, Alana Burrell, Michela Cerone, Ka-Fai Leung, Fiona Achcar et al. "Suramin exposure alters cellular metabolism and mitochondrial energy production in African trypanosomes". Journal of Biological Chemistry 295, n. 24 (30 aprile 2020): 8331–47. http://dx.doi.org/10.1074/jbc.ra120.012355.

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Introduced about a century ago, suramin remains a frontline drug for the management of early-stage East African trypanosomiasis (sleeping sickness). Cellular entry into the causative agent, the protozoan parasite Trypanosoma brucei, occurs through receptor-mediated endocytosis involving the parasite's invariant surface glycoprotein 75 (ISG75), followed by transport into the cytosol via a lysosomal transporter. The molecular basis of the trypanocidal activity of suramin remains unclear, but some evidence suggests broad, but specific, impacts on trypanosome metabolism (i.e. polypharmacology). Here we observed that suramin is rapidly accumulated in trypanosome cells proportionally to ISG75 abundance. Although we found little evidence that suramin disrupts glycolytic or glycosomal pathways, we noted increased mitochondrial ATP production, but a net decrease in cellular ATP levels. Metabolomics highlighted additional impacts on mitochondrial metabolism, including partial Krebs' cycle activation and significant accumulation of pyruvate, corroborated by increased expression of mitochondrial enzymes and transporters. Significantly, the vast majority of suramin-induced proteins were normally more abundant in the insect forms compared with the blood stage of the parasite, including several proteins associated with differentiation. We conclude that suramin has multiple and complex effects on trypanosomes, but unexpectedly partially activates mitochondrial ATP-generating activity. We propose that despite apparent compensatory mechanisms in drug-challenged cells, the suramin-induced collapse of cellular ATP ultimately leads to trypanosome cell death.

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Prathalingham, S. Radhika, Shane R. Wilkinson, David Horn e John M. Kelly. "Deletion of the Trypanosoma brucei Superoxide Dismutase Gene sodb1 Increases Sensitivity to Nifurtimox and Benznidazole". Antimicrobial Agents and Chemotherapy 51, n. 2 (4 dicembre 2006): 755–58. http://dx.doi.org/10.1128/aac.01360-06.

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ABSTRACT 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.

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30

Kava - Cordeiro, V., E. A. Luna - Alves - Lima e J. L. Azevedo. "Survival and mutant production induced by mutagenic agents in Metarhizium anisopliae". Scientia Agricola 52, n. 3 (dicembre 1995): 548–54. http://dx.doi.org/10.1590/s0103-90161995000300023.

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A wild strain of Metarhizium anisopliae, an entomopathogenic fungus, was submitted to three mutagenic agents: gamma radiation, ultraviolet light and nitrous acid. Survival curves were obtained and mutants were selected using different mutagenic doses which gave 1 to 5% survival. Morphological and auxotrophic mutants were isolated. Morphological mutants were grouped in a class with yellow conidia and other with pale vinaceous conidia as opposed to the green wild type conidia. Auxotrophic mutants had requirements for vitamin and aminoacid biosynthesis. More than 58% of the total auxotrophk mutants required proline/aipnine. Gamma radiation showed to be the most efficient mutagenic agent giving 0.2% of auxotrophk mutants followed by ultraviolet light (0.12%) and nitrous acid (0.06%).The conidial colour and auxotrophk mutants isolated until now from M. anisopliae were reviewed.

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31

AJAKAIYE, A. A., M. A. ABDULLAHI e T. O. OLANREWAJU. "REPRODUCTIVE ORGAN DAMAGE OF DOMESTIC RUMINANTS IN AFRICAN ANIMAL TRYPANOSOMIASIS: A REVIEW". FUDMA Journal of Agriculture and Agricultural Technology 7, n. 2 (6 giugno 2022): 47–52. http://dx.doi.org/10.33003/jaat.2021.0702.047.

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Animal African trypanosomiasis affects both production and reproduction in domestic ruminants. The reproductive damages caused by trypanosomes have direct or indirect link to some specific organs like endocrine and pituitary gland which secrets follicle stimulating hormone (FSH), interstitial-cell stimulating hormone (ICSH) and growth hormone (GH). These hormones play an important role in the spermatogenic cycle in males and oestrus cycle in females. However, the pathogenesis of these damages have not been clearly elucidated. We believe that this aspect deserves closer study especially in livestock where reproductive performance is the pillar upon which productivity is built. It is hoped that this review which is centred on the chronological report of works that have been carried out on some reproductive organs damage in domestic ruminants infected with or affected by trypanosomes will add more light to the pathophysiology of the affected organs

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32

Richardson, J. P., L. Jenni, R. P. Beecroft e T. W. Pearson. "Procyclic tsetse fly midgut forms and culture forms of African trypanosomes share stage- and species-specific surface antigens identified by monoclonal antibodies." Journal of Immunology 136, n. 6 (15 marzo 1986): 2259–64. http://dx.doi.org/10.4049/jimmunol.136.6.2259.

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Abstract Procyclic culture form (PCF) trypanosomes were established from a bloodstream form population of cloned Trypanosoma brucei rhodesiense and were used to immunize mice for hybridoma production. Indirect immunofluorescence was used to select 10 hybridomas which secreted antibodies that bound to the surface of homologous living PCF. The antibodies reacted with PCF of several clones of T.b. brucei, T.b. gambiense, and T.b. rhodesiense, but not with PCF of T. congolense or T. vivax, or with promastigotes of several species of Leishmania parasites. The antigens were not detectable in ethanol/acetic acid-fixed bloodstream forms or in lysates of bloodstream forms of any of the T. brucei subspecies, and are thus species-specific and stage-specific markers. Selected monoclonal antibodies bound to procyclic trypanosomes taken directly from the midgut of infected tsetse flies, and to immature epimastigote forms in salivary probes, and may therefore be useful in epidemiologic investigations.

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BRINGAUD, F., C. EBIKEME e M. BOSHART. "Acetate and succinate production in amoebae, helminths, diplomonads, trichomonads and trypanosomatids: common and diverse metabolic strategies used by parasitic lower eukaryotes". Parasitology 137, n. 9 (23 dicembre 2009): 1315–31. http://dx.doi.org/10.1017/s0031182009991843.

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SUMMARYParasites that often grow anaerobically in their hosts have adopted a fermentative strategy relying on the production of partially oxidized end products, including lactate, glycerol, ethanol, succinate and acetate. This review focuses on recent progress in understanding acetate production in protist parasites, such as amoebae, diplomonads, trichomonads, trypanosomatids and in the metazoan parasites helminths, as well as the succinate production pathway(s) present in some of them. We also describe the unconventional organisation of the tricarboxylic acid cycle associated with the fermentative strategy adopted by the procyclic trypanosomes, which may resemble the probable structure of the primordial TCA cycle in prokaryotes.

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Awukew, Addisu, Getachew Terefe, Tilaye Demisie e Eyob Eshetu. "Immune Responses to Animal Trypanosomosis: A Review". Journal of Scientific and Innovative Research 6, n. 4 (30 dicembre 2017): 142–50. http://dx.doi.org/10.31254/jsir.2017.6406.

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The aim of this document is to review the current knowledge on Trypanosome parasites of animals with emphasis on the immunological response and points to the wealth of information available for trypanosomiasis, in contrast to the numerous gaps in our understanding of immune responses to trypanosomal infections. African trypanosomes are pathogens for humans and livestock. They are single-cell, extra-cellular parasites that cause persistent infections of the blood and induce profound immune-suppression. The specific immune response to the infecting parasites is complex and involves both the humoral and cellular branches of immune systems. In trypanosomosis, parasite growth is primarily controlled through T-cell dependent antibody responses to the variable surface glycoproteins and possibly to other molecules embedded on the surface of the parasites. Cellular immune responses also occur but at the level of immune-suppression directed against B cells. Additionally, a variety of immune-modulatory cytokines like TNF-α, IFN-γ, IL-10, IL-4, IL-6, IL-12 and etc are produced during the course of infection. The center of the immune-pathology is the T-cell-independent production of antibodies to the variant surface glycoprotein of trypanosomes, the anti-VSG antibody-mediated phagocytosis of trypanosomes by macrophages, and the subsequent profound dysregulation of the macrophage system. It has, however, been demonstrated that a T-cell subset is critical in protective immunity.

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Matthews, Keith R., Richard McCulloch e Liam J. Morrison. "The within-host dynamics of African trypanosome infections". Philosophical Transactions of the Royal Society B: Biological Sciences 370, n. 1675 (19 agosto 2015): 20140288. http://dx.doi.org/10.1098/rstb.2014.0288.

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African trypanosomes are single-celled protozoan parasites that are capable of long-term survival while living extracellularly in the bloodstream and tissues of mammalian hosts. Prolonged infections are possible because trypanosomes undergo antigenic variation—the expression of a large repertoire of antigenically distinct surface coats, which allows the parasite population to evade antibody-mediated elimination. The mechanisms by which antigen genes become activated influence their order of expression, most likely by influencing the frequency of productive antigen switching, which in turn is likely to contribute to infection chronicity. Superimposed upon antigen switching as a contributor to trypanosome infection dynamics is the density-dependent production of cell-cycle arrested parasite transmission stages, which limit the infection while ensuring parasite spread to new hosts via the bite of blood-feeding tsetse flies. Neither antigen switching nor developmental progression to transmission stages is driven by the host. However, the host can contribute to the infection dynamic through the selection of distinct antigen types, the influence of genetic susceptibility or trypanotolerance and the potential influence of host-dependent effects on parasite virulence, development of transmission stages and pathogenicity. In a zoonotic infection cycle where trypanosomes circulate within a range of host animal populations, and in some cases humans, there is considerable scope for a complex interplay between parasite immune evasion, transmission potential and host factors to govern the profile and outcome of infection.

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Vergara-Meza, José Gabriel, Andreia Fernandes Brilhante, Vera da Costa Valente, Evaristo Villalba-Alemán, Paola Andrea Ortiz, Sueli Cosmiro de Oliveira, Maxdelles Rodrigues Cavalcante et al. "Trypanosoma cruzi and Trypanosoma rangeli in Acre, Brazilian Amazonia: Coinfection and Notable Genetic Diversity in an Outbreak of Orally Acquired Acute Chagas Disease in a Forest Community, Wild Reservoirs, and Vectors". Parasitologia 2, n. 4 (2 dicembre 2022): 350–65. http://dx.doi.org/10.3390/parasitologia2040029.

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Acute Chagas disease (ACD) caused by Trypanosoma cruzi has emerged as a major food-borne disease in Brazilian Amazonia. For the first time, we characterized an outbreak of orally acquired ACD in Acre, in the forest community of Seringal Miraflores, affecting 13 individuals who shared the pulp of açai palm berries: 11 adults and two children (one newborn), all diagnosed by thick-drop blood smears. The fluorescent fragment length barcoding method, which simultaneously identifies species/genotypes of trypanosomes in blood samples, uncovered an unprecedented genetic diversity in patients from a single outbreak of ACD: T. cruzi TcI in all patients, mostly concomitantly with the non-pathogenic Trypanosoma rangeli of genotypes TrA or TrB, and TcI, TcIV, and TrB in the child. The patients presented persistent fever, asthenia, myalgia, edema of the face and lower limbs, hepatosplenomegaly and, rarely, cardiac arrhythmia. The clinical symptoms were not correlated to gender, age, or to trypanosome species and genotypes. The inferred SSU rRNA phylogenetic analyses of trypanosomes from humans, triatomines and sylvatic hosts included the first sequences of T. cruzi and T. rangeli from humans in southwestern (Acre and Rondônia) Amazonia, and the first TcI/TcIV sequences from Rhodnius spp. from Acre. The sylvatic transmission cycles of genetically different trypanosomes in landscapes changed by deforestation for human settlements and increasing açai production is a novel scenario favoring trypanosome transmission to humans in Acre.

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Krykbayev, Yerkin, Askar Kondybayev, Symbat Dzhunisbayeva e Moldir Akhmetzhanova. "UTILIZATION OF GORYAEV CHAMBER TO DETERMINE THE CONCENTRATION OF TRYPANOSOMA EQUIPERDUM IN LABORATORY ANIMALS". 3i intellect idea innovation - интеллект идея инновация 2 (2024): 19–28. http://dx.doi.org/10.52269/22266070_2024_2_19.

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This article reflects the results of studies on the possibility of using the Goryaev chamber to determine concentration of parasitemia of the Trypanosoma equiperdum strain in laboratory animals, as one of the quick and convenient methods for determining the accumulation kinetics. The research findings revealed that the Goryaev camera is recommended to be used with a minimum dilution of biological samples (blood) of 1:100, and the use of 1x40 microscope magnification can be used as a method for determining the accumulation dynamics of the Trypanosoma equiperdum strain in laboratory animals, allowing one to determine the concentration of trypanosomes at various stages of parasitemia, starting from the first days of infection and the detection of single trypanosomes, and ending with the last days of infection at high concentrations and dilutions of 1:200 or more. The study of the kinetics of trypanosome accumulation is a pressing issue, allowing assessing the rate of parasitemia in laboratory and industrial conditions, as well as organizing the production of diagnostic test systems and kits. The study used parasitological methods of infection and assessment of the kinetics of trypanosome accumulation. The practical significance of the study is associated with the possibility of using the presented method in industrial conditions of accumulation of trypanosomes both in laboratory animals (mice, rats) and in target animals (horses, donkeys).

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Gashururu S., Richard, Ndichu Maingi, Samuel M. Githigia, Methode N. Gasana, Peter O. Odhiambo, Dennis O. Getange, Richard Habimana et al. "Occurrence, diversity and distribution of Trypanosoma infections in cattle around the Akagera National Park, Rwanda". PLOS Neglected Tropical Diseases 15, n. 12 (15 dicembre 2021): e0009929. http://dx.doi.org/10.1371/journal.pntd.0009929.

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Background African Trypanosomiases threaten the life of both humans and animals. Trypanosomes are transmitted by tsetse and other biting flies. In Rwanda, the African Animal Trypanosomiasis (AAT) endemic area is mainly around the tsetse-infested Akagera National Park (NP). The study aimed to identify Trypanosoma species circulating in cattle, their genetic diversity and distribution around the Akagera NP. Methodology A cross-sectional study was carried out in four districts, where 1,037 cattle blood samples were collected. The presence of trypanosomes was determined by microscopy, immunological rapid test VerY Diag and PCR coupled with High-Resolution Melt (HRM) analysis. A parametric test (ANOVA) was used to compare the mean Packed cell Volume (PCV) and trypanosomes occurrence. The Cohen Kappa test was used to compare the level of agreement between the diagnostic methods. Findings The overall prevalence of trypanosome infections was 5.6%, 7.1% and 18.7% by thin smear, Buffy coat technique and PCR/HRM respectively. Microscopy showed a low sensitivity while a low specificity was shown by the rapid test (VerY Diag). Trypanosoma (T.) congolense was found at a prevalence of 10.7%, T. vivax 5.2%, T. brucei brucei 2% and T. evansi 0.7% by PCR/HRM. This is the first report of T.evansi in cattle in Rwanda. The non-pathogenic T. theileri was also detected. Lower trypanosome infections were observed in Ankole x Friesian breeds than indigenous Ankole. No human-infective T. brucei rhodesiense was detected. There was no significant difference between the mean PCV of infected and non-infected animals (p>0.162). Conclusions Our study sheds light on the species of animal infective trypanosomes around the Akagera NP, including both pathogenic and non-pathogenic trypanosomes. The PCV estimation is not always an indication of trypanosome infection and the mechanical transmission should not be overlooked. The study confirms that the area around the Akagera NP is affected by AAT, and should, therefore, be targeted by the control activities. AAT impact assessment on cattle production and information on the use of trypanocides are needed to help policymakers prioritise target areas and optimize intervention strategies. Ultimately, these studies will allow Rwanda to advance in the Progressive Control Pathway (PCP) to reduce or eliminate the burden of AAT.

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Masuda, N., N. Gotoh, S. Ohya e T. Nishino. "Quantitative correlation between susceptibility and OprJ production in NfxB mutants of Pseudomonas aeruginosa." Antimicrobial Agents and Chemotherapy 40, n. 4 (aprile 1996): 909–13. http://dx.doi.org/10.1128/aac.40.4.909.

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Various Pseudomonas aeruginosa PAO1 NfxB mutants were isolated on agar plates containing cefpirome and ofloxacin. They were classified into type A and type B, based on the degrees of changes in their susceptibilities. Type A mutants were four to eight times more resistant to ofloxacin, erythromycin, and new zwitterionic cephems, i.e., cefpirome, cefclidin, cefozopran, and cefoselis, than was the parent strain, PAO1. In contrast, type B mutants were more resistant to tetracycline and chloramphenicol, as well as ofloxacin, erythromycin, and the new zwitterionic cephems, than was PAO1, and they were four to eight times more susceptible to carbenicillin, sulbenicillin, imipenem, panipenem, biapenem, moxalactam, aztreonam, gentamicin, and kanamycin that was PAO1. The changes in susceptibilities of type B mutants were greater than those of type A mutants. The susceptibilities of both type A and type B mutants were restored to the level of PAO1 by transformation with plasmid pNF111, which contained the wild-type nfxB gene, demonstrating that they are NfxB mutants. Immunoblot analysis with a monoclonal antibody to OprJ revealed that type B mutants produced larger amounts of outer membrane protein OprJ than did type A mutants and that PAO1 produced an undetectable amount of it. Moreover, transconjugants obtained with the different types of NfxB mutants as the donor strains showed almost the same phenotypes as the corresponding donor strains. These results suggest that there are at least two nfxB mutations that show different phenotypes and that production of OprJ is associated with changes in susceptibilities of NfxB mutants.

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Bakhiet, Moiz, Maha Hamadien, Annelie Tjernlund, Alyaa Mousa e Åke Seiger. "African trypanosomes activate human fetal brain cells to proliferation and IFN-γ production". Neuroreport 13, n. 1 (gennaio 2002): 53–56. http://dx.doi.org/10.1097/00001756-200201210-00015.

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41

Kickhoefer, Valerie A. "A new role for vault RNA–TEP1 complexes in mRNA production in trypanosomes". Journal of Biological Chemistry 294, n. 43 (25 ottobre 2019): 15575–76. http://dx.doi.org/10.1074/jbc.h119.011130.

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42

Nwanta, John, Shodeinde Shoyinka, Kennedy Chah, Joseph Onunkwo, Ifediora Onyenwe, James Eze, Chijioke Iheagwam et al. "Production characteristics, disease prevalence, and herd-health management of pigs in Southeast Nigeria". Journal of Swine Health and Production 19, n. 6 (1 novembre 2011): 331–39. http://dx.doi.org/10.54846/jshap/640.

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Abstract (sommario):

Objectives: To assess the management practices of swine production and herd health and disease prevalence in Southeast Nigeria. Materials and methods: Fifty-four farms were conveniently selected from three states of Southeast Nigeria. Information on socio-economic characteristics of farmers (sex, occupation, educational status, and farming experience), management practices, and disease prevalence were collected. Samples were screened for ectoparasites (skin scrapings), trypanosomes and Brucella antibodies (blood samples), and helminth and cestode ova and coccidia oocysts (fecal samples). Results: Of 54 farm owners surveyed, 43% were exclusively farmers, 32% were in the buying and selling business, 17% were civil servants, and 6% were students. More men (89%) than women (11%) kept pigs, with the majority having a herd size of < 100 pigs. Most pigs were crosses between native and European breeds. Management was predominantly intensive (96%), with most of the barns built of cement blocks, with concrete floors and galvanized roofing sheets. Prevalences of 47%, 25%, 20%, and 0.95% were recorded for infection with coccidia, helminths, ectoparasites, and trypanosomes, respectively; 0.6% of pigs tested were positive for Brucella antibodies. Significant associations were noted between disease prevalence and litter size and management system, and between productivity and farmer’s educational level. Implications: In spite of the good productivity recorded in this study (farms having ≥ 6 pigs marketed per litter), efforts should be made to encourage better management practices to significantly reduce disease prevalence for better performance. Public-health risks associated with Brucella and trypanosome infections recorded in this study should not be neglected.

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Deryto, Mieczyslawa, Anna Skorupska, Jozef Bednara e Zbigniew Lorkiewicz. "Rhizobium trifolii mutants deficient in exopolysaccharide production". Physiologia Plantarum 66, n. 4 (aprile 1986): 699–704. http://dx.doi.org/10.1111/j.1399-3054.1986.tb05602.x.

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44

Ikeda, Masato, e Ryoichi Katsumata. "Tryptophan Production by Transport Mutants ofCorynebacterium glutamicum". Bioscience, Biotechnology, and Biochemistry 59, n. 8 (gennaio 1995): 1600–1602. http://dx.doi.org/10.1271/bbb.59.1600.

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45

Yoshida, Hajime, Kazumi Araki e Masanobu Kawai. "Production of Lactase by Mutants ofKluyveromyces lactis". Agricultural and Biological Chemistry 52, n. 4 (aprile 1988): 951–55. http://dx.doi.org/10.1080/00021369.1988.10868792.

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46

Mehta, B. J., e E. Cerdá-Olmedo. "Mutants of carotene production in Blakeslea trispora". Applied Microbiology and Biotechnology 42, n. 6 (marzo 1995): 836–38. http://dx.doi.org/10.1007/bf00191178.

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47

Mehta, B. J., e E. Cerd�-Olmedo. "Mutants of carotene production in Blakeslea trispora". Applied Microbiology and Biotechnology 42, n. 6 (1 marzo 1995): 836–38. http://dx.doi.org/10.1007/s002530050339.

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48

Hutter, A., e S. G. Oliver. "Ethanol production using nuclear petite yeast mutants". Applied Microbiology and Biotechnology 49, n. 5 (25 maggio 1998): 511–16. http://dx.doi.org/10.1007/s002530051206.

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49

Doostmohammadi, Mohsen, Mohammad Ali Asadollahi, Iraj Nahvi, Davoud Biria, Gholam Reza Ghezelbash e Maryam Kheyrandish. "L-phenylacetylcarbinol production by yeast petite mutants". Annals of Microbiology 66, n. 3 (20 gennaio 2016): 1049–55. http://dx.doi.org/10.1007/s13213-015-1190-2.

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50

Egorova, Olga V., Seraphima A. Gulevskaya, Irina F. Puntus, Andrey E. Filonov e Marina V. Donova. "Production of androstenedione using mutants ofMycobacterium sp". Journal of Chemical Technology & Biotechnology 77, n. 2 (2002): 141–47. http://dx.doi.org/10.1002/jctb.536.

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