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Peter Overath - One of the best experts on this subject based on the ideXlab platform.
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cultivation of Bloodstream Forms of trypanosoma carassii a common parasite of freshwater fish
Parasitology Research, 1998Co-Authors: Peter Overath, Jens Ruoff, Yorkdieter Stierhof, Jochen Haag, Herbert Tichy, Iva Dykova, Jiri LomAbstract:Trypanosoma carassii (syn. T. danilewskyi) is a widespread parasite of carp and other cyprinid as well as some noncyprinid freshwater fish. It lives extracellularly in the blood and tissues of its hosts, causing chronic infections. In this paper the isolation of T. carassii from fish blood and the propagation and cloning of Bloodstream Forms in vitro are described. By several criteria, cultured and fish-derived trypomastigotes are indistinguishable. The culture system should be useful for the biochemical characterization of this trypanosome and its interaction with the fish immune system.
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Invariant surface proteins in Bloodstream Forms of Trypanosoma brucei
Parasitology Today, 1994Co-Authors: Peter Overath, M. Chaudhri, Dietmar Steverding, K ZiegelbauerAbstract:Antigenic variation of the glycoprotein forming the coat of African trypanosomes has been a dominant field of investigation for many years. The extravagant potential of these parasites to change their surface coat has destroyed hopes for a vaccine based on the variant surface glycoprotein. Recently, there has been a rising interest in the characterization of surface proteins that are not subject to antigenic variation. In this review, Peter Overath, Maliha Chaudhri, Dietmar Steverding and Karl Ziegelbauer summarize the present evidence for the occurrence, cellular localization and function of invariant surface proteins in Trypanosoma brucei.
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a transferrin binding protein of trypanosoma brucei is encoded by one of the genes in the variant surface glycoprotein gene expression site
The EMBO Journal, 1991Co-Authors: Dietmar Schell, Raymond Evers, D Preis, K Ziegelbauer, H Kiefer, F Lottspeich, Albert W C A Cornelissen, Peter OverathAbstract:Abstract A transferrin-binding protein (TFBP) with an apparent molecular weight of 42 kd was purified from detergent-soluble membrane proteins of Bloodstream Forms of Trypanosoma brucei. The protein is not expressed in the insect-borne stage of the parasite's life-cycle. Purified TFBP can be converted from an amphiphilic to a hydrophilic form by cleavage with T.brucei glycosylphosphatidylinositol (GPI)-specific phospholipase C, demonstrating that the C-terminus is modified by a GPI-membrane anchor. The TFBP is encoded by an expression-site-associated gene [ESAG 6 in the nomenclature of Pays et al. (1989) Cell, 57, 835-845] which is under the control of the promoter transcribing the expressed variant surface glycoprotein gene. The possible function of TFBP as a receptor for the uptake of transferrin in Bloodstream Forms is discussed.
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a transferrin binding protein of trypanosoma brucei is encoded by one of the genes in the variant surface glycoprotein gene expression site
The EMBO Journal, 1991Co-Authors: Dietmar Schell, Raymond Evers, D Preis, K Ziegelbauer, H Kiefer, F Lottspeich, Albert W C A Cornelissen, Peter OverathAbstract:Abstract A transferrin-binding protein (TFBP) with an apparent molecular weight of 42 kd was purified from detergent-soluble membrane proteins of Bloodstream Forms of Trypanosoma brucei. The protein is not expressed in the insect-borne stage of the parasite's life-cycle. Purified TFBP can be converted from an amphiphilic to a hydrophilic form by cleavage with T.brucei glycosylphosphatidylinositol (GPI)-specific phospholipase C, demonstrating that the C-terminus is modified by a GPI-membrane anchor. The TFBP is encoded by an expression-site-associated gene [ESAG 6 in the nomenclature of Pays et al. (1989) Cell, 57, 835-845] which is under the control of the promoter transcribing the expressed variant surface glycoprotein gene. The possible function of TFBP as a receptor for the uptake of transferrin in Bloodstream Forms is discussed.
Paul H Voorheis - One of the best experts on this subject based on the ideXlab platform.
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factors that determine the plasma membrane potential in Bloodstream Forms of trypanosoma brucei
FEBS Journal, 2000Co-Authors: Derek P Nolan, Paul H VoorheisAbstract:The plasma-membrane potential (Δψp) in Bloodstream Forms of Trypanosoma brucei was studied using several different radiolabelled probes: 86Rb+ and [14C]SCN− were used to report Δψp directly because they distribute in easily measured quantities across the plasma membrane only, and [3H]methyltriphenylphosphonium (MePh3P+) was used to report Δψp only when Δψm had been abolished with FCCP because it reports the algebraic sum of the two potentials when used alone. The unperturbed Δψp had a value of −82 mV and was found to be essentially identical with, and determined almost completely by, the potassium diffusion potential, as evidenced by: (a) the lack of effect of valinomycin on the value obtained under appropriate conditions when any of these probes were used; (b) the close agreement of this measured value with that predicted from the measured distribution of K+ across the plasma membrane (−76 mV); (c) the large effect of changes in the extracellular K+ concentration by substitution with Na+ on Δψp together with the complete lack of effect of substitution of extracellular Na+ by the choline cation or substitution of extracellular Cl− by the gluconate anion on Δψp. The contribution to Δψp by electrogenic pumping of Na+/K+-ATPase was found to be small (of the order of 6 mV). H+ was not found to be pumped across the plasma membrane or to contribute to Δψp.
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hydrogen ion gradients across the mitochondrial endosomal and plasma membranes in Bloodstream Forms of trypanosoma brucei solving the three compartment problem
FEBS Journal, 2000Co-Authors: Derek P Nolan, Paul H VoorheisAbstract:Conditions for the use of both [14C]methylamine and 5,5-dimethyl[14C]oxa-azolidine-2,4-dione (DMO) to measure the H+ concentration of intracellular compartments of monomorphic long thin Bloodstream Forms of Trypanosoma brucei were established. Neither probe was actively transported or bound to internal components of the cell and both probes equilibrated passively with a t1/2 close to 8 min. DMO was excluded from cells, while methylamine was accumulated but not metabolized. Solution of the three-compartment problem revealed that, when cells were respiring aerobically on glucose at an external pH of 7.5, the cytoplasmic pH was in the range 6.99–7.03, the pH of the mitochondrial matrix was 7.71–7.73, and the algebraic average pH of the various endosomal compartments was 5.19–5.50. Similar values were found when cells were respiring aerobically on glycerol. However, Bloodstream Forms of T. brucei could not maintain a constant internal H+ concentration outside the external pH range 7.0–7.5, and no evidence for the presence of an H+/Na+ exchanger was found. Full motility and levels of pyruvate production were maintained as the external pH was raised as high as 9.5, suggesting that these cells tolerate significant internal alkalinisation. However, both motility and pyruvate production were severely inhibited under acidic conditions, and the cells deteriorated rapidly below an external pH of 6.5. Physiologically, the plasma membrane of T. brucei had low permeability to H+ and the internal pH was unaffected by changes in Δψp, which is dominated by the potassium diffusion potential. However, in the presence of FCCP, the internal pH fell rapidly about 0.5 pH unit and came into equilibrium with Δψp. Oligomycin abolished the mitochondrial pH gradient (ΔpHm) selectively, whereas chloroquine abolished only the endosomal pH gradient (ΔpHe). The pH gradient across the plasma membrane (ΔpHp) alone could be abolished by careful osmotic swelling of cells. The plasma membrane had an inwardly directed proton-motive force (ΔPp) of −52 mV and an inwardly directed sodium-motive force (ΔNp) of −149 mV, whereas the mitochondrial inner membrane had only an inwardly directed ΔPm of −195 mV. The pH gradient across the endosomal membranes was not accompanied by an electrical gradient. Consequently, endosomal membranes had an algebraically average outwardly directed ΔPl within the range + 89 to + 110 mV, depending on the measurement method.
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hydrogen ion gradients across the mitochondrial endosomal and plasma membranes in Bloodstream Forms of trypanosoma brucei solving the three compartment problem
FEBS Journal, 2000Co-Authors: Derek P Nolan, Paul H VoorheisAbstract:Conditions for the use of both [14C]methylamine and 5, 5-dimethyl[14C]oxa-azolidine-2,4-dione (DMO) to measure the H+ concentration of intracellular compartments of monomorphic long thin Bloodstream Forms of Trypanosoma brucei were established. Neither probe was actively transported or bound to internal components of the cell and both probes equilibrated passively with a t1/2 close to 8 min. DMO was excluded from cells, while methylamine was accumulated but not metabolized. Solution of the three-compartment problem revealed that, when cells were respiring aerobically on glucose at an external pH of 7.5, the cytoplasmic pH was in the range 6.99-7.03, the pH of the mitochondrial matrix was 7.71-7.73, and the algebraic average pH of the various endosomal compartments was 5.19-5.50. Similar values were found when cells were respiring aerobically on glycerol. However, Bloodstream Forms of T. brucei could not maintain a constant internal H+ concentration outside the external pH range 7.0-7.5, and no evidence for the presence of an H+/Na+ exchanger was found. Full motility and levels of pyruvate production were maintained as the external pH was raised as high as 9.5, suggesting that these cells tolerate significant internal alkalinisation. However, both motility and pyruvate production were severely inhibited under acidic conditions, and the cells deteriorated rapidly below an external pH of 6.5. Physiologically, the plasma membrane of T. brucei had low permeability to H+ and the internal pH was unaffected by changes in Deltapsip, which is dominated by the potassium diffusion potential. However, in the presence of FCCP, the internal pH fell rapidly about 0.5 pH unit and came into equilibrium with Deltapsip. Oligomycin abolished the mitochondrial pH gradient (DeltapHm) selectively, whereas chloroquine abolished only the endosomal pH gradient (DeltapHe). The pH gradient across the plasma membrane (DeltapHp) alone could be abolished by careful osmotic swelling of cells. The plasma membrane had an inwardly directed proton-motive force (DeltaPp) of -52 mV and an inwardly directed sodium-motive force (DeltaNp) of -149 mV, whereas the mitochondrial inner membrane had only an inwardly directed DeltaPm of -195 mV. The pH gradient across the endosomal membranes was not accompanied by an electrical gradient. Consequently, endosomal membranes had an algebraically average outwardly directed DeltaPl within the range + 89 to +110 mV, depending on the measurement method.
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the mitochondrion in Bloodstream Forms of trypanosoma brucei is energized by the electrogenic pumping of protons catalysed by the f1f0 atpase
FEBS Journal, 1992Co-Authors: Derek P Nolan, Paul H VoorheisAbstract:Bloodstream Forms of Trypanosoma brucei were found to maintain a significant membrane potential across their mitochondrial inner membrane (delta psi m) in addition to a plasma membrane potential (delta psi p). Significantly, the delta psi m was selectively abolished by low concentrations of specific inhibitors of the F1F0-ATPase, such as oligomycin, whereas inhibition of mitochondrial respiration with salicylhydroxamic acid was without effect. Thus, the mitochondrial membrane potential is generated and maintained exclusively by the electrogenic translocation of H+, catalysed by the mitochondrial F1F0-ATPase at the expense of ATP rather than by the mitochondrial electron-transport chain present in T. brucei. Consequently, Bloodstream Forms of T. brucei cannot engage in oxidative phosphorylation. The mitochondrial membrane potential generated by the mitochondrial F1F0-ATPase in intact trypanosomes was calculated after solving the two-compartment problem for the uptake of the lipophilic cation, methyltriphenylphosphonium (MePh3P+) and was shown to have a value of approximately 150 mV. When the value for the delta psi m is combined with that for the mitochondrial pH gradient (Nolan and Voorheis, 1990), the mitochondrial proton-motive force was calculated to be greater than 190 mV. It seems likely that this mitochondrial proton-motive force serves a role in the directional transport of ions and metabolites across the promitochondrial inner membrane during the Bloodstream stage of the life cycle, as well as promoting the import of nuclear-encoded protein into the promitochondrion during the transformation of Bloodstream Forms into the next stage of the life cycle of T. brucei.
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the distribution of permeant ions demonstrates the presence of at least two distinct electrical gradients in Bloodstream Forms of trypanosoma brucei
FEBS Journal, 1991Co-Authors: Derek P Nolan, Paul H VoorheisAbstract:The distribution of 86Rb+ and the radiolabelled lipophilic cation [3H]methyltriphenylphosphonium (MePh3P+) was used to investigate the membrane potentials that exist in Bloodstream Forms of Trypanosoma brucei. Even after correction for binding to cellular constituents, the accumulation of MePh3P+ was approximately tenfold greater than the accumulation of Rb+ under resting conditions. The addition of low concentrations of carbonylcyanide p-trifluoromethoxyphenylhydrazone or valinomycin reduced the accumulation of MePh3P+ tenfold without perturbing the accumulation of Rb+. Although selective permeabilization of the plasma membrane abolished the accumulation of Rb+ and caused a substantial decrease in the accumulation of MePh3P+, a significant carbonylcyanide-p-trifluoromethoxyphenylhydrazone-sensitive accumulation of MePh3P+ persisted under these conditions. These data were consistent with the presence of at least two distinct membrane potentials (delta psi) in Bloodstream Forms of T. brucei; a potential across the plasma membrane (delta psi p) and an additional delta psi, generated by the electrogenic movement of H+, across the membrane of an intracellular organelle that possesses no electrical permeability to Rb+ or K+.
Derek P Nolan - One of the best experts on this subject based on the ideXlab platform.
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characterization of two protein disulfide isomerases from the endocytic pathway of Bloodstream Forms of trypanosoma brucei
Journal of Biological Chemistry, 2005Co-Authors: Joyce Rubotham, Etienne Pays, Katherine Woods, Jose A Garciasalcedo, Derek P NolanAbstract:Proteins from the endocytic pathway in Bloodstream Forms of Trypanosome brucei are modified by the addition of linear poly-N-acetyllactosamine side chains, which permits their isolation by tomato lectin affinity chromatography. Antibodies against this tomato lectin binding fraction were employed to screen a cDNA expression library from Bloodstream Forms of T. brucei. Two cDNAs were prominent among those selected. These cDNAs coded for two putative protein disulfide isomerases (PDIs) that respectively contained one and two double-cysteine redox-active sites and corresponded to a single domain PDI and a class 1 PDI. Assays of the purified recombinant proteins demonstrated that both proteins possess isomerase activity, but only the single domain PDI had a reducing activity. These PDIs possess a number of unusual features that distinguish them from previously characterized PDIs. The expression of both is developmentally regulated, they both co-localize with markers of the endocytic pathway, and both are modified by N-glycosylation. The larger PDI possesses N-glycans containing poly-N-acetyllactosamine, a modification that is indicative of processing in the Golgi and suggests the presence of a novel trafficking pathway for PDIs in trypanosomes. Although generally PDIs are considered essential, neither activity appeared to be essential for the growth of trypanosomes, at least in vitro.
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factors that determine the plasma membrane potential in Bloodstream Forms of trypanosoma brucei
FEBS Journal, 2000Co-Authors: Derek P Nolan, Paul H VoorheisAbstract:The plasma-membrane potential (Δψp) in Bloodstream Forms of Trypanosoma brucei was studied using several different radiolabelled probes: 86Rb+ and [14C]SCN− were used to report Δψp directly because they distribute in easily measured quantities across the plasma membrane only, and [3H]methyltriphenylphosphonium (MePh3P+) was used to report Δψp only when Δψm had been abolished with FCCP because it reports the algebraic sum of the two potentials when used alone. The unperturbed Δψp had a value of −82 mV and was found to be essentially identical with, and determined almost completely by, the potassium diffusion potential, as evidenced by: (a) the lack of effect of valinomycin on the value obtained under appropriate conditions when any of these probes were used; (b) the close agreement of this measured value with that predicted from the measured distribution of K+ across the plasma membrane (−76 mV); (c) the large effect of changes in the extracellular K+ concentration by substitution with Na+ on Δψp together with the complete lack of effect of substitution of extracellular Na+ by the choline cation or substitution of extracellular Cl− by the gluconate anion on Δψp. The contribution to Δψp by electrogenic pumping of Na+/K+-ATPase was found to be small (of the order of 6 mV). H+ was not found to be pumped across the plasma membrane or to contribute to Δψp.
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hydrogen ion gradients across the mitochondrial endosomal and plasma membranes in Bloodstream Forms of trypanosoma brucei solving the three compartment problem
FEBS Journal, 2000Co-Authors: Derek P Nolan, Paul H VoorheisAbstract:Conditions for the use of both [14C]methylamine and 5,5-dimethyl[14C]oxa-azolidine-2,4-dione (DMO) to measure the H+ concentration of intracellular compartments of monomorphic long thin Bloodstream Forms of Trypanosoma brucei were established. Neither probe was actively transported or bound to internal components of the cell and both probes equilibrated passively with a t1/2 close to 8 min. DMO was excluded from cells, while methylamine was accumulated but not metabolized. Solution of the three-compartment problem revealed that, when cells were respiring aerobically on glucose at an external pH of 7.5, the cytoplasmic pH was in the range 6.99–7.03, the pH of the mitochondrial matrix was 7.71–7.73, and the algebraic average pH of the various endosomal compartments was 5.19–5.50. Similar values were found when cells were respiring aerobically on glycerol. However, Bloodstream Forms of T. brucei could not maintain a constant internal H+ concentration outside the external pH range 7.0–7.5, and no evidence for the presence of an H+/Na+ exchanger was found. Full motility and levels of pyruvate production were maintained as the external pH was raised as high as 9.5, suggesting that these cells tolerate significant internal alkalinisation. However, both motility and pyruvate production were severely inhibited under acidic conditions, and the cells deteriorated rapidly below an external pH of 6.5. Physiologically, the plasma membrane of T. brucei had low permeability to H+ and the internal pH was unaffected by changes in Δψp, which is dominated by the potassium diffusion potential. However, in the presence of FCCP, the internal pH fell rapidly about 0.5 pH unit and came into equilibrium with Δψp. Oligomycin abolished the mitochondrial pH gradient (ΔpHm) selectively, whereas chloroquine abolished only the endosomal pH gradient (ΔpHe). The pH gradient across the plasma membrane (ΔpHp) alone could be abolished by careful osmotic swelling of cells. The plasma membrane had an inwardly directed proton-motive force (ΔPp) of −52 mV and an inwardly directed sodium-motive force (ΔNp) of −149 mV, whereas the mitochondrial inner membrane had only an inwardly directed ΔPm of −195 mV. The pH gradient across the endosomal membranes was not accompanied by an electrical gradient. Consequently, endosomal membranes had an algebraically average outwardly directed ΔPl within the range + 89 to + 110 mV, depending on the measurement method.
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hydrogen ion gradients across the mitochondrial endosomal and plasma membranes in Bloodstream Forms of trypanosoma brucei solving the three compartment problem
FEBS Journal, 2000Co-Authors: Derek P Nolan, Paul H VoorheisAbstract:Conditions for the use of both [14C]methylamine and 5, 5-dimethyl[14C]oxa-azolidine-2,4-dione (DMO) to measure the H+ concentration of intracellular compartments of monomorphic long thin Bloodstream Forms of Trypanosoma brucei were established. Neither probe was actively transported or bound to internal components of the cell and both probes equilibrated passively with a t1/2 close to 8 min. DMO was excluded from cells, while methylamine was accumulated but not metabolized. Solution of the three-compartment problem revealed that, when cells were respiring aerobically on glucose at an external pH of 7.5, the cytoplasmic pH was in the range 6.99-7.03, the pH of the mitochondrial matrix was 7.71-7.73, and the algebraic average pH of the various endosomal compartments was 5.19-5.50. Similar values were found when cells were respiring aerobically on glycerol. However, Bloodstream Forms of T. brucei could not maintain a constant internal H+ concentration outside the external pH range 7.0-7.5, and no evidence for the presence of an H+/Na+ exchanger was found. Full motility and levels of pyruvate production were maintained as the external pH was raised as high as 9.5, suggesting that these cells tolerate significant internal alkalinisation. However, both motility and pyruvate production were severely inhibited under acidic conditions, and the cells deteriorated rapidly below an external pH of 6.5. Physiologically, the plasma membrane of T. brucei had low permeability to H+ and the internal pH was unaffected by changes in Deltapsip, which is dominated by the potassium diffusion potential. However, in the presence of FCCP, the internal pH fell rapidly about 0.5 pH unit and came into equilibrium with Deltapsip. Oligomycin abolished the mitochondrial pH gradient (DeltapHm) selectively, whereas chloroquine abolished only the endosomal pH gradient (DeltapHe). The pH gradient across the plasma membrane (DeltapHp) alone could be abolished by careful osmotic swelling of cells. The plasma membrane had an inwardly directed proton-motive force (DeltaPp) of -52 mV and an inwardly directed sodium-motive force (DeltaNp) of -149 mV, whereas the mitochondrial inner membrane had only an inwardly directed DeltaPm of -195 mV. The pH gradient across the endosomal membranes was not accompanied by an electrical gradient. Consequently, endosomal membranes had an algebraically average outwardly directed DeltaPl within the range + 89 to +110 mV, depending on the measurement method.
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abrupt rna changes precede the first cell division during the differentiation of trypanosoma brucei Bloodstream Forms into procyclic Forms in vitro
Molecular and Biochemical Parasitology, 1993Co-Authors: Etienne Pays, J Hanocqquertier, Francoise Hanocq, Suzanne Van Assel, Derek P Nolan, Sylvie RolinAbstract:We have monitored the timing of DNA and RNA synthesis during the synchronous differentiation of Trypanosoma brucei Bloodstream Forms into procyclic Forms in vitro. Both are triggered after a lag period of 4 h and reach a first peak after 9 h. The division of the kinetoplast precedes that of the nucleus by about 4 h. The first cell divisions are observed after 10 h, and the cell number is doubled after 20 h. The total RNA content per cell increases sharply between 4 and 10 h, then progressively decreases as cell division progresses. The increase in RNA content cannot be due solely to accumulation of rRNA since it is also observed for mRNAs such as actin. The VSG mRNA has almost disappeared within 2 h, while the procyclin mRNA accumulates soon after the triggering of differentiation, with a strong peak between 4 and 6 h. At this moment, the amount of procyclin mRNA per cell is at least 20-fold higher than in established procyclic culture Forms. The loss of the VSG and the appearance of procyclic-specific proteins essentially occur before the first cell division. These observations contrast with the progressive transition observed when monomorphic slender Forms are induced to transform under the same conditions.
Dietmar Schell - One of the best experts on this subject based on the ideXlab platform.
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a transferrin binding protein of trypanosoma brucei is encoded by one of the genes in the variant surface glycoprotein gene expression site
The EMBO Journal, 1991Co-Authors: Dietmar Schell, Raymond Evers, D Preis, K Ziegelbauer, H Kiefer, F Lottspeich, Albert W C A Cornelissen, Peter OverathAbstract:Abstract A transferrin-binding protein (TFBP) with an apparent molecular weight of 42 kd was purified from detergent-soluble membrane proteins of Bloodstream Forms of Trypanosoma brucei. The protein is not expressed in the insect-borne stage of the parasite's life-cycle. Purified TFBP can be converted from an amphiphilic to a hydrophilic form by cleavage with T.brucei glycosylphosphatidylinositol (GPI)-specific phospholipase C, demonstrating that the C-terminus is modified by a GPI-membrane anchor. The TFBP is encoded by an expression-site-associated gene [ESAG 6 in the nomenclature of Pays et al. (1989) Cell, 57, 835-845] which is under the control of the promoter transcribing the expressed variant surface glycoprotein gene. The possible function of TFBP as a receptor for the uptake of transferrin in Bloodstream Forms is discussed.
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a transferrin binding protein of trypanosoma brucei is encoded by one of the genes in the variant surface glycoprotein gene expression site
The EMBO Journal, 1991Co-Authors: Dietmar Schell, Raymond Evers, D Preis, K Ziegelbauer, H Kiefer, F Lottspeich, Albert W C A Cornelissen, Peter OverathAbstract:Abstract A transferrin-binding protein (TFBP) with an apparent molecular weight of 42 kd was purified from detergent-soluble membrane proteins of Bloodstream Forms of Trypanosoma brucei. The protein is not expressed in the insect-borne stage of the parasite's life-cycle. Purified TFBP can be converted from an amphiphilic to a hydrophilic form by cleavage with T.brucei glycosylphosphatidylinositol (GPI)-specific phospholipase C, demonstrating that the C-terminus is modified by a GPI-membrane anchor. The TFBP is encoded by an expression-site-associated gene [ESAG 6 in the nomenclature of Pays et al. (1989) Cell, 57, 835-845] which is under the control of the promoter transcribing the expressed variant surface glycoprotein gene. The possible function of TFBP as a receptor for the uptake of transferrin in Bloodstream Forms is discussed.
Dietmar Steverding - One of the best experts on this subject based on the ideXlab platform.
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a cultivation method for growing Bloodstream Forms of trypanosoma brucei to higher cell density and for longer time
Parasitology Research, 2015Co-Authors: Chinenye Ajoko, Dietmar SteverdingAbstract:A novel cultivation technique using transwells allowing the growth of Bloodstream Forms of Trypanosoma brucei for longer periods of time and to higher cell densities is described. Trypanosomes cultured in transwells placed in cups of tissue culture plates containing sixfold excess of medium grew within 4 days to maximum cell densities of 2 × 107 parasites per milliliter. Compared to control cultures, the exponential growth of trypanosomes was 1 day longer and the maximum cell concentration was increased fourfold. The new culture system may be useful in drug screening assays and analysing the T. brucei secretome.
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bitter melon extract inhibits proliferation of trypanosoma brucei Bloodstream Forms in vitro
Experimental Parasitology, 2013Co-Authors: Elizabeth A Phillips, Darren W Sexton, Dietmar SteverdingAbstract:Trypanosoma brucei is the causative agent of sleeping sickness, a fatal disease prevalent in sub-Saharan Africa. The few currently available drug treatments are dated and face problems with toxicity and resistance. For these reasons, there is an urgent need for the development of new chemotherapies for the treatment of sleeping sickness. In this study, we investigated the trypanocidal activity of bitter melon extract. Recently, it has been shown that bitter melon extracts display cytotoxic activity towards different cancer cell lines. However, agents exhibiting anti-tumour activity are usually also inhibiting the growth of T. brucei. Treatment of Bloodstream Forms of T. brucei with extracts prepared from Chinese and Indian bitter melon varieties resulted in a decrease in cell proliferation. In contrast, human myeloid leukaemia HL-60 cells were 3-6 times less sensitive to the extracts than trypanosomes. Initial fractionation of bitter melon extracts indicated that the trypanocidal activity of the extract is associated with at least two different classes of substances: one class of larger molecular weight compounds (>3 kDa) causing rapid lysis of trypanosomes and one class of smaller molecular weight compounds (<3 kDa) inducing accumulation of the parasites in the G(2)-M phase of the cell cycle. Together, the results suggest that bitter melon is a promising source for trypanocidal agents which could be used as lead compounds for the development of novel anti-sleeping sickness drugs.
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trypanosoma brucei unexpected azide sensitivity of Bloodstream Forms
Journal of Parasitology, 2004Co-Authors: Dietmar Steverding, Stefan ScoryAbstract:Bloodstream Forms of Trypanosoma brucei lack cytochromes and are, therefore, insensitive to cyanide. Azide is a toxic anion that bears chemical and biological properties in common with cyanide and may act in a similar way by inhibition of cytochrome c oxidase. It was, therefore, surprising to find that Bloodstream Forms of T. brucei are sensitive to azide; growth is reduced by 50% with 0.1 mM azide. So far, the only enzyme known in Bloodstream Forms of T. brucei to be sensitive to azide is the iron-containing superoxide dismutase. However, because the activity of the superoxide dismutase was not affected in parasites incubated for 16 hr with 0.5 mM azide (a concentration at which no cell proliferates), the toxic action of azide cannot be due to inhibition of this enzyme. These results indicate that the general toxicity of azide is different from that of cyanide.
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trypanosoma brucei in vitro slender to stumpy differentiation of culture adapted monomorphic Bloodstream Forms
Experimental Parasitology, 2002Co-Authors: Tanja Breidbach, Dietmar Steverding, Elise NgazoaAbstract:Pleomorphic Trypanosoma brucei strains are characterized by their ability to differentiate from replicating long slender Forms into non-dividing short stumpy Forms in the mammalian host. The differentiation process can be efficiently induced in vitro by treatment with the membrane-permeable cAMP derivative 8-(4-chlorophenylthio)-cAMP (pCPTcAMP). In contrast, monomorphic T. brucei strains do not differentiate to stumpy Forms in the host. Here, we show that exposure of monomorphic, culture-adapted T. brucei Bloodstream Forms to pCPTcAMP allowed their subsequent differentiation into short stumpy Forms. The stumpy nature of pCPTcAMP-treated parasites was confirmed by (1) morphological change, (2) inhibition of growth and DNA synthesis, (3) cell cycle arrest in the G(1)/G(0) phase, (4) expression of NADH diaphorase activity and dihydrolipoamide dehydrogenase, (5) disappearance of the small subunit of ribonucleotide reductase, (6) up-regulation of the major lysosomal membrane protein, and (7) efficient transformation into replicating procyclic insect Forms after induction with citrate/cis-aconitate. Our results indicate that the inability of monomorphic T. brucei Bloodstream Forms to differentiate into short stumpy Forms in the host may be due to a failure in the signalling pathway rather than in the differentiation process itself. Treatment of monomorphic Bloodstream trypanosomes with pCPTcAMP could be a useful method for identifying the genes involved in the slender-to-stumpy differentiation process.
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growth inhibition of Bloodstream Forms of trypanosoma brucei by the iron chelator deferoxamine
International Journal for Parasitology, 2002Co-Authors: Tanja Breidbach, Stefan Scory, Luise R Krauthsiegel, Dietmar SteverdingAbstract:Treatment of Bloodstream Forms of Trypanosoma brucei with the iron chelator deferoxamine inhibits the proliferation of the parasites. Compared with mammalian cells, Bloodstream Forms of Trypanosoma brucei are 10 times more sensitive to iron depletion. The primary target of the chelator is obviously the intracellular iron as the toxicity of deferoxamine is abolished by addition of holotransferrin, the exogenous source of iron for the parasite. To identify probable target sites, the effect of deferoxamine on ribonucleotide reductase, alternative oxidase and superoxide dismutase, three iron-dependent enzymes in Bloodstream-form trypanosomes, was studied. Incubation of the parasites with the chelator leads to inhibition of DNA synthesis and lowers oxygen consumption indicating that deferoxamine may affect ribonucleotide reductase and alternative oxidase. The compound does not inhibit the holoenzymes directly but probably acts by chelating cellular iron thus preventing its incorporation into the newly synthesised apoproteins. Treatment of the parasites with deferoxamine for 24 h has no effect on the activity of superoxide dismutase. The results have implications for antitrypanosomal drug development based on specific intervention with the parasite's iron metabolism.