African Trypanosomes: Cultivation of Animal-Infective
            <i>Trypanosoma brucei</i>
            in Vitro

Hirumi, H.; Doyle, J.J.; Hirumi, K.

doi:10.1126/science.558652

Cited by 135 publications

(49 citation statements)

References 13 publications

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“…Trypanosomes were grown in 300-g male Wistar rats and isolated as described previously [6,11]. In some experiments, trypanosomes were separated from blood by density centrifugation as described by Hirumi et al [12], followed by separation from contaminating blood cells on a small DEAE-cellulose column [13]. Cells were washed either with phosphate/ saline/glucose buffer [13] or, if cellular extracts had to be prepared, with balanced-salt solution pH 7.6, whichcontained 15.5mMTris, 226mM NaC1,0.54mM KC1, 98 pM MgC12, 5 pM CaC12, 50 mM glucose.…”

Section: Growth and Isolation Of Trypanosomesmentioning

confidence: 99%

Glycolysis in Trypanosoma brucei

Visser

Opperdoes

1980

European Journal of Biochemistry

122

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The possibility that the glycosomes present in the bloodstream form of Trypanosoma brucei FEBS Lett. 80, 360-3641 constitute a separate pool of glycolytic intermediates within the cell was investigated.In titrations of intact cells with digitonin, a differential activation of glycolytic enzymes was observed. Enolase, pyruvate kinase and the cell-sap marker alanine aminotransferase were activated at 0.05 mg digitonin per mg protein. The nine glycosomal enzymes involved in the conversion of glucose and glycerol into 3-phosphoglycerate were activated only at digitonin concentrations between 0.7 and 9.8 mg/mg protein. In subcellular fractions the activities or the latter enzymes were all latent between 70 and 92 %. Latency was abolished by addition of 0.1 % Triton X-100 or partly by five cycles of freezing and thawing. We conclude that the glycosomal enzymes are surrounded by a membrane, which forms a permeability barrier to intermediates and co-factors of glycolysis.The concentrations of glycolytic intermediates and of adenine nucleotides were measured under aerobic conditions as well as in the presence of 1 mM salicylhydroxamic acid, a respiratory inhibitor. Addition of salicylhydroxamic acid caused the following changes : (a) The levels of almost all glycolytic intermediates measured decreased. Glycerol-3-phosphate, however, increased fourfold. From the high levels of metabolite concentrations found and from comparison of the apparent mass-action ratios calculated for the separate glycolytic reactions with those for other organisms, we conclude that in bloodstream form T. brucei the glycolytic intermediates are present in the glycosomes as well as in the cytosol and that the two pools of intermediates equilibrate with each other, despite the presence of the glycosomal membrane.Bloodstream forms of the African trypanosomes belonging to the brucei group, like Trypanosoma brucei, Trypanosoma rhodesiense and Trypanosoma gambiense, are the causative agents of nagana in domestic animals Abbreviations. Dihydroxyacetone-P, dihydroxyacetone phosphate ; glycerol-3-P, sn-glycerol-3-phosphate; glycerate-3-P, 3-phosphoglycerate; glucose-6-P, glucose-6-phosphate; fructose-6-P, fructose-6-phosphate; fructose-l,h-Pz, fructose-1,6-bisphosphate; glycerate-2-P, 2-phosphoglycerate; glyceraldehyde-3-P, D-glyceraldehyde-3-phosphate.Enzymes. Hexokinase (EC 2.7.1.1); phosphoglucose isomerase (EC 5.3.1.9); 6-phosphofructokinase (EC 2.7.1.11); fructose-bisphosphate aldolase (EC 4.1.2.13); triosephosphate isomerase (EC 5.3.1 .l): sn-glycerol-3-phosphate dehydrogenase (EC 1.1.1.8); glycerol kinase (EC 2.7.1.30); glyceraldehyde-phosphate dehydrogenase (EC 1.2.1.12); phosphoglycerate kinase (EC 2.7.2.3); phosphoglyceromutase (EC 2.7.5.3); enolase (EC 4.2.1.11); pyruvate kinase (EC 2.7.1.40); alanine aminotransferase (EC 2.6.1.2). and live-stock and of the acute and chronic forms of sleeping sickness in man. For their energy needs, these organisms are entirely dependent on glycolysis which proceeds at an extremely high rate and differs in its...

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Section: Growth and Isolation Of Trypanosomesmentioning

confidence: 99%

Glycolysis in Trypanosoma brucei

Visser

Opperdoes

1980

European Journal of Biochemistry

122

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“…Both bloodstream and procyclic forms are highly motile by virtue of a single flagellum, which emerges near the posterior end of the cell and is fused to the cell membrane along its length. Interest in investigating trypanosomes under controlled environmental conditions has spawned several studies describing in vitro culture of both forms of the parasite in semi-defined liquid medium (7)(8)(9)(10)(11)(12)(13)(14)(15). Procyclics have traditionally been easier to maintain in in vitro culture and thus are occasionally referred to as the "culture form."…”

mentioning

confidence: 99%

High-efficiency clonal growth of bloodstream- and insect-form Trypanosoma brucei on agarose plates.

Carruthers

Cross

1992

Proc. Natl. Acad. Sci. U.S.A.

113

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This report describes a method for growing both bloodstream-and procyclic-form Trypanosoma brucei as colonies on agarose plates. Procyclic colonies, which took 2 weeks to develop, grew with approximately 17% plating efficiency on SDM-79/0.65% agarose supplemented with 20% (vol/vol) conditioned medium. Bloodstream forms were adapted to in vitro growth in liquid HM-9 medium and then spread on HMI-9/0.65% agarose plates, where they grew to visible colonies in 3-5 days. Plating efficiencies were from 3 to 80%, depending upon the trypanosome variant and experiment. Colonies were proven to be the result of growth from a single cell and contained approximately 106 cells at maturity. Tanzania (19) and was cloned as a metacyclic taken directly from an infected tsetse salivary gland (kindly provided by L. Jenni, Swiss Tropical Institute, Basel). It has since been propagated through a total of eight animals (mice or rats) with intermittent cryopreservation. Prior to use in this study, all of these cell lines were recloned by limiting dilution in vitro and checked by immunofluorescence using variant-specific antibodies to ensure expression of the correct variant surface glycoprotein (VSG; except STIB 366E, for which variant-specific antibodies are unavailable).Adaptation of Bloodstream Forms to Liquid Culture. Trypanosomes were grown in either mice or rats to subsaturation density (1-5 x 108 cells per ml of blood) and adapted to grow in HMI-9 medium essentially as described (12). Briefly, 1 ml of infected blood [collected with 0.2 vol of citrate glucose anticoagulant (0.1 M sodium citrate/0.04 M glucose, pH 7.7)] was diluted in 9 ml of HMI-9 medium and then centrifuged at 200 x g for 5 min at room temperature. The supernatant (containing trypanosomes) was transferred to a T-25 culture flask and incubated upright at 370C for 2 hr to allow the majority ofthe remaining blood cells to settle. Trypanosomes in the supernatant were then used to initiate 5-ml cultures in HMI-9 at a density of 1.2 x 106 cells per ml. Cultures were fed daily by adding 2.5 ml of fresh medium after removing an equal volume. In the initial stages, many cells died. Once the trypanosomes began to divide rapidly (when densities recovered to 106 cells per ml), cultures were routinely diluted 1:10 to 1:20 daily to maintain densities in the range of 105-106 cells per ml.Preparation of Agarose Plates and Growth of Colonies.Agarose plates were prepared using 0.65% agarose as suggested by Lee and Van der Ploeg (17). To achieve the desired final concentration ofingredients, it was necessary to prepare and then mix 2x solutions ofmedium and agarose. 2x HMI-9 medium was prepared by dissolving 5.31 g of powdered Iscove's modified Dulbecco's medium (IMDM, GIBCO/ BRL) and 0.907 g of sodium bicarbonate in 92 ml of lowconductivity water followed by the addition of 40 ml of Abbreviation: VSG, variant surface glycoprotein.

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“…The enhancement of replication which occurred in the good sera when macrophages were also present, suggested that the latter cells were secreting an additional growthpromoting substance or substances (M factor), effective only in the presence of s+ and essential for growth at 37 "C but not 28 "C. A similar essential growth-promoting factor is presumably produced by the 'fibroblast-like' cells in cultures of haematozoic T. brucei (Hirumi, Doyle & Hirumi, 1977). This interpretation was supported by the observation that extracellular trypanosomal growth in flasks without macrophages, but replenished daily with medium conditioned by prior contact with uninfected macrophages, was as good as in flasks containing macrophages.…”

Section: Discussionmentioning

confidence: 99%

Trypanosoma (Schizotrypanum) dionisii: Influence of Mouse Peritoneal Macrophages and Calf Sera on Extracellular Growth in vitro at 37 C

Baker

Selden

1978

Journal of General Microbiology

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27Macrophages and certain batches of sera were essential for extracellular multiplication of Trypanosoma dionisii in vitro in medium 199 with 20 % (v/v) calf serum at 37 "C. In mixtures of ' good ' and ' bad ' batches of sera, multiplication increased as the proportion of the former was increased. Mixtures of 'bad' and 'intermediate' sera permitted virtually no growth. Phagocytosis of parasites by macrophages was unaffected by different batches of sera, though reduced in medium 199 alone. Replacement of supernatant medium by medium containing 'bad' serum reduced the macrophage infection rate no more than did transfer to medium containing 'good' serum. Daily addition of medium conditioned by prior contact with macrophages in vitro to cultures of trypanosomes without macrophages resulted in growth at least as good as that in the presence of macrophages. Extracellular replication in medium 199 at 37 "C apparently required at least two factors:'M factor' provided by macrophages, but not required at 28 " C ; and 'S + factor' present in some batches of calf serum, essential also at 28 "C but not required by intracellular parasites. Some sera appeared to contain an inhibitory ' S -factor'.

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African Trypanosomes: Cultivation of Animal-Infective Trypanosoma brucei in Vitro

Cited by 135 publications

References 13 publications

Glycolysis in Trypanosoma brucei

Glycolysis in Trypanosoma brucei

High-efficiency clonal growth of bloodstream- and insect-form Trypanosoma brucei on agarose plates.

Trypanosoma (Schizotrypanum) dionisii: Influence of Mouse Peritoneal Macrophages and Calf Sera on Extracellular Growth in vitro at 37 C

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