In Vitro Cultivation of Bloodstream Forms of Trypanosoma brucei, T. rhodesiense, and T. gambiense1

Brun, Reto; Jenni, L.; Schönenberger, Miriam J.; Schell, K-F.

doi:10.1111/j.1550-7408.1981.tb05322.x

Cited by 91 publications

(32 citation statements)

References 18 publications

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“…The presence of a surface coat on cultivated bloodstream forms and their infectivity for mammalian hosts as well as for the vector Glossina m. morsitans have been demonstrated earlier (BRUN et al, 1981;SCH~NI et al. 19821.…”

Section: Discussionmentioning

confidence: 92%

Comparative morphometric analysis of bloodstream and lymph forms of Trypanosoma (T.) brucei brucei grown in vitro and in vivo

Hecker

Brun

1982

Transactions of the Royal Society of Tropical Medicine and Hygi

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SummaryThe fine structure of bloodstream forms of Typanosoma brucei cultivated in vitro, and of trypanosomes from lymph and blood of mammalian hosts, was compared morphometrically. The cell volume, quantitative parameters of the mitochondrion and of glycosomes were mainly investigated. A Coulter Channelyzer was used for the first time to measure the mean cell volume of living parasites. In vitro, the monomorphic trypanosomes between the feeder layer cells showed lower values for mitochondrial parameters than the slightly pleomorphic forms from the supernatant medium. Trypanosomes in culture were very similar morphologically to forms from lymph nodes of rats. Despite some morphometric differences between cultivated blood stream forms and those grown in viva, the similarity of both populations was clear. Both populations, however, differed significantly from stages found in the vector or from procyclic culture forms.

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Section: Discussionmentioning

confidence: 92%

Comparative morphometric analysis of bloodstream and lymph forms of Trypanosoma (T.) brucei brucei grown in vitro and in vivo

Hecker

Brun

1982

Transactions of the Royal Society of Tropical Medicine and Hygi

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“…In the present case, a short-lived positive regulatory VOL. 7,1987 on protein required for VSG gene transcription would have to be postulated, because during inhibition transcription was shut off rather than stimulated. Although the postulate of such a protein is rather obvious, its half-life would have to be very short to explain the immediacy of the observed reduction in the rate of transcription (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…117) was used throughout this study. Bloodstream forms isolated from mouse blood were cultured in medium B lacking hypoxanthine with 15% inactivated horse serum over a feeder layer of mouse embryo fibroblasts at 37°C overnight (7,10,30 …”

Section: Methodsmentioning

confidence: 99%

RNA turnover in Trypanosoma brucei.

Ehlers¹,

Czichos²,

Overath³

1987

Mol. Cell. Biol.

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Regulation of variant surface glycoprotein (VSG) mRNA turnover in Trypanosoma brucei was studied in bloodstream forms, in procyclic cells, and during in vitro transformation of bloodstream forms to procycic cells by approach-to-equilibrium labeling and pulse-chase experiments. Upon initiation of transformation at 27°C in the presence of citrate-cis-aconitate, the half-life of VSG mRNA was reduced from 4.5 h in bloodstream forms to 1.2 h in transforming cells. Concomitantly, an approximately 25-fold decrease in the rate of transcription was observed, resulting in a 100-fold reduction in the steady-state level of de novo-synthesized VSG mRNA. This low level of expression was maintained for at least 7 h, finally decreasing to an undetectable level after 24 h. Transcription of the VSG gene in established procyclic cells was undetectable. For comparison, the turnover of polyadenylated and nonpolyadenylated RNA, ,-tubulin mRNA, and mini-exon-derived RNA (medRNA) was studied. For medRNA, no significant changes in the rate of transcription or stability were observed during differentiation. In contrast, while the rate of transcription of ,B-tubulin mRNA in in vitro-cultured bloodstream forms, transforming cells, and established procyclic cells was similar, the half life was four to five times longer in procyclic cells (tj/2, 7 h) than in cultured bloodstream forms (t1/2, 1.4 h) or transforming cells (t012, 1.7 h). Inhibition of protein synthesis in bloodstream forms at 37°C caused a dramatic 20-fold decrease in the rate of VSG mRNA synthesis and a 6-fold decrease in half-life to 45 min, while f8-tubulin mRNA was stabilized 2-to 3-fold and medRNA stability remained unaffected. It is postulated that triggering transformation or inhibiting protein synthesis induces changes in the abundance of the same regulatory molecules which effect the shutoff of VSG gene transcription in addition to shortening the half-life of VSG mRNA.The regulation of mRNA synthesis in the parasitic protozoan Trypanosoma brucei represents an interesting problem. First, throughout its complex life cycle in the mammalian host and the tsetse fly, this unicellular flagellate experiences dramatic changes in environment, to which it adapts by regulating the expression of numerous genes (42); second, it has the unique feature of discontinuous mRNA synthesis (4). In an as yet undefined mechanism, a 140-nucleotide (nt) transcript, designated mini-exon-derived RNA (medRNA), donates a 35-nt sequence located at its 5' end (mini-exon) to the 5' end of all mRNAs coding for proteins (8,9,12,21,26,44 (17,36,40). The mini-exon has been explicitly demonstrated at the 5' end of the VSG and tubulin mRNAs (3,17,36,41). medRNA is transcribed from mini-exon genes present in about 200 copies per nucleus. Most of these genes are clustered in the genome in tandemly linked 1.35-kilobase (kb) repeats (11,27 bloodstream forms to procyclic cells. In a second, related part, mRNA synthesis and decay in the absence of protein synthesis were investigated. MATERIALS AND METHODSTrypanos...

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“…043-5140) in 500 ml minimal essential medium (Gibco no. 041-1090), final pH 7.5 [30]. TM medium was supplemented with 15% inactivated horse Serum (iHS) prepared from blood obtained from a local slaughterhouse.…”

Section: Tsetse Fliesmentioning

confidence: 99%

The effect of citrate/cis‐aconitate on oxidative metabolism during transformation of Trypanosoma brucei

Overath¹,

Czichos²,

Haas³

1986

European Journal of Biochemistry

140

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Monomorphic bloodstream forms of Trypanosoma brucei, grown in the mammal, are deficient in aconitase and 2-oxoglutarate dehydrogenase and they do not respire in the presence of the substrates citrate, cis-aconitate, succinate, proline or 2-oxoglutarate. When grown in vitro low levels of aconitase, succinate oxidase and proline oxidase are detected.Addition of citratelcis-aconitate at 37 "C to bloodstream forms leads to the formation of aconitase and proline oxidase. Most cells undergo an 'abortive' transformation to non-dividing procyclic-like cells while some cells adapt to the presence of the citric acid cycle intermediates and continue to multiply as bloodstream forms.At 27°C and in the presence of citratelcis-aconitate bloodstream forms transform synchronously to dividing procyclic cells. Within 72 h the rate of respiration with proline, succinate and 2-oxoglutarate becomes similar to that in established procyclic cells while the rate of glucose oxidation decreases.The possible role of citric acid cycle intermediates in determining whether a trypanosome will retain the properties of a bloodstream trypomastigote or differentiate to a procyclic trypomastigote is discussed.The parasitic protozoan, Trypanosoma brucei, undergoes a series of differentiation steps during its cyclical development in the mammalian host and the arthropod vector, the tsetse fly [l]. Differentiation of bloodstream forms to procyclic cells, a process called transformation, is initiated in the mammal by the transition of dividing slender forms to intermediate and non-dividing stumpy forms, giving rise to a pleomorphic population. After uptake with the blood meal into the midgut of the fly, cells with stumpy morphology are considered to transform most readily to dividing procyclic cells.Transformation of pleomorphic as well as monomorphic populations of rodent-adapted strains which have a uniform slender morphology [2] can be studied in various in vitro systems [3 -91. Synchronous transformation requires two external signals, a temperature change from 37°C to 27°C and the addition of cis-aconitate and/or citrate as inducers Some of the complex morphological, ultrastructural and metabolic changes which characterize transformation have been studied in detail. First, the repression of the synthesis of the variant surface glycoprotein (VSG) in the coated bloodstream forms is an early event which is followed by coat [lo-131. release to form coatless procyclic cells [7, 8, 111. Second, transformation involves profound changes in energy metabolism [14-161. In bloodstream forms glucose is degraded solely by glycolysis to form two mol pyruvate/mol glucose. Reducing equivalents are transferred to oxygen via a unique mitochondrial glycerol-3-phosphate oxidase [17,18]. The promitochondrion lacks a functional citric acid cycle as well as a cytochrome-linked respiratory chain. Stumpy forms contain some mitochondrial enzymes, such as 2-oxoglutarate dehydrogenase and proline oxidase [19]. During transformation to procyclic cells a functional respiratory ...

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In Vitro Cultivation of Bloodstream Forms of Trypanosoma brucei, T. rhodesiense, and T. gambiense¹

Cited by 91 publications

References 18 publications

Comparative morphometric analysis of bloodstream and lymph forms of Trypanosoma (T.) brucei brucei grown in vitro and in vivo

Comparative morphometric analysis of bloodstream and lymph forms of Trypanosoma (T.) brucei brucei grown in vitro and in vivo

RNA turnover in Trypanosoma brucei.

The effect of citrate/cis‐aconitate on oxidative metabolism during transformation of Trypanosoma brucei

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