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variant surface glycoprotein from trypanosoma brucei clone ytat 1.1 contains a latent calmodulin-binding domain.calmodulin affinity chromatography and chromatofocusing were used to purify calmodulin-binding proteins of 32-40-kda from homogenates of trypanosoma brucei clone ytat1.1. the trypanosome proteins associated with calmodulins from different sources and reversibly inhibited calmodulin-dependent bovine brain phosphodiesterase. purified 32-kda protein bound to calmodulin with an approximate kd of 1.3 nm. polyclonal antibodies directed against purified 32-kda protein and monoclonal antibody eca6 recog ...19911852168
comparative study on rickettsia-like organisms in the midgut epithelial cells of different glossina species.the midgut epithelium of glossina morsitans centralis, g. austeni, g. pallidipes, g. palpalis palpalis, g. p. gambiensis, g. fuscipes fuscipes, g. tachinoides and g. brevipalpis from ilrad-bred colonies was examined, by electron microscopy, for the presence and distribution of rickettsia-like organisms (rlos). rlos were present in the midgut epithelial cells of all non-teneral tsetse. in g.m. centralis, g. pallidipes and, to a much lesser extent, g. brevipalpis, rlos were numerous and were prese ...19911852486
rickettsia-like organisms, puparial temperature and susceptibility to trypanosome infection in glossina morsitans.maintaining the puparial stage of successive generations of a population of tsetse 3 degrees c lower than normal reduced the numbers of rickettsia-like organisms (rlo) carried by emerging flies. the susceptibility of these flies to midgut infection with trypanosoma congolense was also significantly reduced compared with control flies held at normal temperature. these results support the view that the relationship between rlo and susceptibility is quantitative-teneral flies with heavier rlo infec ...19911852487
diagnosis of human african trypanosomiasis and visceral leishmaniasis based on the detection of anti-parasite-enzyme antibodies.a sensitive diagnostic assay for parasitic infections based on the detection of anti-enzyme antibodies is presented. all serum antibodies produced in response to parasite antigens are immobilized via their fc domain on matrix-bound protein g. incubation of the immobilized antibodies with saturating amounts of parasite extract results in the binding of all recognized antigens, including those directed against a specific and readily measurable enzyme. the amount of bound enzyme is proportional to ...19911856492
role of phospholipids in the cytotoxic action of high density lipoprotein on trypanosomes.host range among the african trypanosomes, protozoa that cause fatal diseases both in humans and livestock, may be, in part, regulated by toxic properties associated with host high density lipoproteins (hdl). high density lipoproteins from hosts resistant (baboon, human) or susceptible (rabbit, rat) to trypanosoma brucei infection were isolated and their trypanocidal activity was determined in in vitro cell lysis assays. rabbit and rat hdl were not cytotoxic while baboon and human hdl rapidly ly ...19911856609
kinetic properties of fructose bisphosphate aldolase from trypanosoma brucei compared to aldolase from rabbit muscle and staphylococcus aureus.the kinetic properties of aldolase from trypanosoma brucei were studied in comparison with aldolase from rabbit muscle and staphylococcus aureus. the 3 enzymes displayed a similar broad ph optimum for the cleavage of fructose 1,6-bisphosphate (fru(1,6)p2) and a similar narrow ph optimum for the cleavage of fructose 1-phosphate (fru-1-p). however, small alterations in the maximal cleavage rate at more extreme ph values yielded disparities between the ph curves. the reaction catalyzed by the aldol ...19911857377
chemical modification of fructose bisphosphate aldolase from trypanosoma brucei compared to aldolase from rabbit muscle and staphylococcus aureus.chemical modifications of class i aldolases from trypanosoma brucei, rabbit muscle and staphylococcus aureus with carboxypeptidase a, glyceraldehyde 3-phosphate and cysteine-specific reagents revealed the following differences between the three homologous enzymes. aldolase from s. aureus was not affected by any of these reagents. carboxypeptidase-a treatment of rabbit-muscle and t. brucei aldolase inhibited the activity of both enzymes towards fructose-1,6-bisphosphate (fru(1,6)p2), while the ac ...19911857380
characterization of a novel developmentally regulated gene from trypanosoma brucei encoding a potential phosphoprotein.we have isolated a cdna clone corresponding to a single-copy nuclear gene that is upregulated at the mrna level during in vitro differentiation of bloodstream trypomastigotes of strains of both trypanosoma brucei brucei and trypanosoma brucei rhodesiense to procyclic forms. transcript levels begin to increase within minutes of introduction of bloodstream forms into culture and peak well before cultures exhibit a procyclic morphology. this increase in transcript levels was found to occur both in ...19911857381
characterization of pyruvate kinase of trypanosoma brucei and its role in the regulation of carbohydrate metabolism.pyruvate kinase from trypanosoma brucei is a labile enzyme, losing its activity within several hours. in mixtures containing 50 mm triethanolamine buffer, ph 7.2, 25% glycerol and 0.5 mm inorganic phosphate the enzyme remained active and could be purified to homogeneity with a specific activity of 417 units mg-1 and a yield of 65%. the enzyme has an activation energy of 31.9 kj mol-1. magnesium and potassium ions are essential for activity. cobalt or manganese ions replace mg2+ but this leads to ...19911857383
differences in glucose transport between blood stream and procyclic forms of trypanosoma brucei rhodesiense.in african trypanosomes the requirements for glucose and its metabolism vary in different stages of the life cycle. here we present evidence that cultured procyclic trypanosomes of trypanosoma brucei rhodesiense uptake glucose against a concentration gradient in a time and dose-dependent manner. moreover, glucose transport is completely inhibited by the sulphydryl inhibitor n-ethylmaleimide, suggesting the presence of a protein moiety as the carrier molecule. comparison of glucose uptake in bloo ...19911857387
sequence of a trna gene cluster in trypanosoma brucei. 19911861987
the cytoskeletal architecture of trypanosoma brucei.the cytoskeleton of trypanosoma brucei has been analyzed by the high-resolution technique of quick-freeze deep-etch rotary-shadowing electron microscopy. the study provides detailed structural information on the subpellicular array of microtubules, the flagellum, and the interaction of these 2 major structures of the trypanosomal cytoskeleton with each other. the subpellicular microtubules closely interact both with the cell membrane and with each other. at the anterior tip of the cell they conv ...19911865269
diminazene aceturate residues in the tissues of healthy, trypanosoma congolense and trypanosoma brucei brucei infected dogs.the tissue distribution and residue profile of diminazene aceturate was investigated in healthy dogs and in dogs infected with trypanosoma congolense and trypanosoma brucei brucei. the drug was administered at 3.5 mg/kg i.m. and tissue samples were taken post mortem from the animals at 48, 72, 120, 168 and 240 h after injection. the drug was distributed to various organs and tissues of the body with the highest concentrations occurring in liver and kidney. higher drug levels were obtained in the ...19911868320
the glycosomes of trypanosomes: number and distribution as revealed by electron spectroscopic imaging and 3-d reconstruction.computer-aided 3-d reconstruction of trypanosomes from 0.35-micron-thick sections imaged on the zeiss 902 electron microscope are being used to study the dynamics of cell organization. segregation of glycolytic enzymes into glycosomes raises questions concerning the distribution and biogenesis of these organelles. direct counts of glycosomes from trypanosoma evansi indicate 30-40 per cell and for the closely related t. brucei, 65 per cell. these figures contrast with the estimates of others who ...19911870115
comparative histochemical studies of rats infected with a pathogenic and nonpathogenic trypanosome.histochemical variations in tissues from rats inoculated with trypanosoma lewisi and trypanosoma rhodesiense were investigated. during peak parasitemia, the liver of rats inoculated with t lewisi showed increased glycogen distribution. however, glycogen depletion was noted in the liver and spleen of animals inoculated with living cells of t rhodesiense. depletion was very apparent from day 4 to day 10. throughout the period of observation, only a small amount of lipid infiltration occurred in ti ...19911875425
basal body movements as a mechanism for mitochondrial genome segregation in the trypanosome cell cycle.the mitochondrial genome of trypanosoma brucei is organized in the form of a complex catenated network of circular dna molecules. this mass of dna, known as the kinetoplast, is present at a unique site in the single mitochondrion, and is replicated in a discrete, periodic s phase of the cell cycle. the single-copy nature of the kinetoplast suggests that there is a mechanism ensuring segregation fidelity of replicated copies to each daughter cell. historically, speculation regarding the nature of ...19911876188
molecular cloning and analysis of two tandemly linked genes for pyruvate kinase of trypanosoma brucei.in trypanosoma brucei (stock 427) genes encoding the glycolytic enzyme pyruvate kinase are present on two homologous chromosomes. we have cloned and characterized one of the alleles. two large, tandemly arranged open reading frames were found, each coding for a pyruvate kinase polypeptide of 498 amino acids. the gene sequences differ at 15 positions, resulting in five amino acid substitutions. the calculated molecular masses of the polypeptides are 54,378 da and 54,363 da. these values are somew ...19911879424
the effects of the methylating agent 1,2-bis(methylsulfonyl)-1-methylhydrazine on morphology, dna content and mitochondrial function of trypanosoma brucei subspecies.repeated exposure of trypanosomes in vitro or in vivo to low concentrations of the methylating agent 1,2-bis(methylsulfonyl)-1-methylhydrazine induces a series of moderately synchronous morphological and biochemical changes. cell division halts and the long-slender bloodstream forms transform to short-stumpy forms via larger intermediate-stage cells which contain approximately double the normal g2 content of dna. in common with naturally occurring short-stumpy trypanosomes, drug-induced short-st ...19911880758
refined 1.83 a structure of trypanosomal triosephosphate isomerase crystallized in the presence of 2.4 m-ammonium sulphate. a comparison with the structure of the trypanosomal triosephosphate isomerase-glycerol-3-phosphate complex.triosephosphate isomerase (tim) is a dimeric glycolytic enzyme. tim from trypanosoma brucei brucei has been crystallized at ph 7.0 in 2.4 m-ammonium sulphate. the well-diffracting crystals have one dimer per asymmetric unit. the structure has been refined at 1.83 a resolution with an r-factor of 18.3% for all data between 6 a and 1.83 a (37,568 reflections). the model consists of 3778 protein atoms and 297 solvent atoms. subunit 1 is involved in considerably more crystal contacts than subunit 2. ...19911880808
elusive trypanosomes.professor kershaw's encouragement of the development of anion-exchange separation of african trypanosomes from blood led to two decades of activity when, for the first time, considerable progress was made in the intrinsic characterization of these parasites. such characterization depended on establishing high infections in laboratory rodents. however, the collection of samples from the field was restricted by the failure of certain trypanosomes either to infect, or to multiply adequately in, rod ...19911888216
lipid peroxidation as a component in trypanocidal drug action.a representative range of trypanocides was examined for ability to induce lipid peroxidation in trypanosoma rhodesiense in vitro. because of drug interference, thiobarbituric acid (tba) chromogen production and diene conjugation u.v. absorbance assay methods were less satisfactory that glc analysis of polyenoic fatty acid loss. docosohexanoic acid (22:6) content was specifically reduced to a highly significant degree by treatment with ethidium or the lipid-soluble 4-demethoxydaunorubicin.19911888218
suppression by trypanosoma brucei rhodesiense of the capacities of human t lymphocytes to express interleukin-2 receptors and proliferate after mitogenic stimulation.we studied the suppressive effects induced in phytohemagglutinin (pha)-stimulated human peripheral blood mononuclear cells (pbmc) by purified blood forms of trypanosoma brucei rhodesiense. the parasite was found to markedly impair lymphocyte proliferation (measured in terms of [3h]thymidine incorporation). the extent of this effect increased with parasite concentration and was not due to mitogen absorption, depletion of medium nutrients, or pbmc killing by the parasite. significant reductions in ...19911894358
chemotherapy of cns-trypanosomiasis: the combined use of the arsenicals and nitro-compounds.in trypanosomiasis chemotherapy the main mode of action of the nitro-compounds, including the 5-nitroimidazoles or nitrofurans (nifurtimox) is to increase the oxidative stress on the organism either directly by the production of peroxides or indirectly by "futile redox cycling". if this is the case, then these nitro-compounds and the arsenicals should act in combination on the trypanothione oxidation-reduction reaction of the trypanosome. in this paper this has been demonstrated to occur with al ...19911896771
efficacy of combination of dfmo and diminazene aceturate in the treatment of late stage trypanosoma brucei brucei infection in rats.the therapeutic activity of difluoromethylornithine and diminazene aceturate alone and in combination was investigated in albino rats with late stage trypanosoma brucei brucei infection. the criteria used in the assessment of the trypanocidal effect of the compounds include the degree of parasitaemia, and frequency of relapse and death. dfmo (4 percent administered in drinking water for 6 days) and diminazene aceturate (10 mg per kg intraperitoneal) produced an intermittent aparasitaemia with re ...19911896772
therapeutic effect of berenil and samorin in mice infected with four trypanosome populations isolated from zambian cattle.four populations of trypanosoma congolense and trypanosoma brucei brucei were isolated from cattle under different management practices and environments in zambia. all four isolates had varied responses to both diminazene aceturate (berenil) and isometamidium chloride (samorin) as curative drugs in infected mice. trypanosomes from a traditionally managed herd in a high-tsetse-challenge area had the strains most resistant to berenil, with maximum curative dose of 45 mg kg-1 body weight. another i ...19911897118
trypanosoma brucei: cytotoxicity of host high-density lipoprotein is not mediated by apolipoprotein a-i. 19911901271
alterations in krebs cycle enzyme activities and carbohydrate catabolism in two strains of trypanosoma brucei during in vitro differentiation of their bloodstream to procyclic stages.a rapid switch from a fermentative to a primarily oxidative type of glucose utilization was observed during in vitro differentiation of trypanosoma brucei stib348 and eatro1244 bloodstream to procyclic trypomastigotes. in accordance with previously published reports bloodstream populations produced pyruvate as the major end product of glucose catabolism, together with very small amounts of co2, succinate and glycerol. during differentiation pyruvate excretion decreased within 48 h to the low lev ...19911904988
rna polymerase i can mediate expression of cat and neo protein-coding genes in trypanosoma brucei.we show that the ribosomal rna (rrna) promoter can efficiently direct expression of protein-coding genes in the parasitic protozoan trypanosoma brucei. the rrna promoter was characterized by: (i) point mutations at the rrna transcription initiation site which completely abolished its promoter function in transient cat transformation assays; (ii) the alpha-amanitin resistance of transcription of rrna promoter-neomycin phosphotransferase (neo) genes in stably transformed trypanosomes; and (iii) th ...19911915299
bidirectional activating signals between trypanosoma brucei and cd8+ t cells: a trypanosome-released factor triggers interferon-gamma production that stimulates parasite growth.the hemoflagellate trypanosoma brucei (t.b.) is the cause of african sleeping sickness. t. b. brucei which is pathogenic for rodents but nonpathogenic for humans was used to examine the interactions between the parasite and mononuclear cells (mnc). co-cultivation in vitro of rat or human mnc and t.b. brucei resulted in a rapid non-antigen-specific release of interferon-gamma (ifn-gamma) which was dependent on cd8+ lymphoid cells. the parasites triggered mnc proliferation if ifn-gamma was blocked ...19911915554
the mevalonate pathway in the bloodstream form of trypanosoma brucei. identification of dolichols containing 11 and 12 isoprene residues.the major surface antigen of the bloodstream form of trypanosoma brucei, the variant surface glycoprotein, is attached to the plasma membrane via a glycosylphosphatidylinositol anchor. the biosynthesis of the glycosylphosphatidylinositol anchor, as well as the assembly of the asparagine-linked oligosaccharide chains found on the variant surface glycoproteins, involves polyisoprenoid lipids that act as sugar carriers. preliminary observations (menon, a.k., schwarz, r.t., mayor, and cross, g.a.m. ...19911918042
an mrna-dependent in vitro translation system from trypanosoma brucei.we have demonstrated efficient protein synthesis in a cell-free system from the bloodstream form of trypanosoma brucei. this system was able to translate endogenous mrna, added mrna, or (apparently at much lower efficiency) three synthetic rna transcripts lacking 5' mini-exon and 3' poly(a) sequences. translation was resistant to chloramphenicol and greater than 95% inhibited by low concentrations of anisomycin and puromycin, but only partially inhibited by cycloheximide. variant surface glycopr ...19911922198
alpha-amanitin-resistant transcription units in trypanosomes: a comparison of promoter sequences for a vsg gene expression site and for the ribosomal rna genes.transcription of the predominant surface antigen genes in trypanosoma brucei is unusual in its resistance to the rna polymerase inhibitor alpha-amanitin, a property typical for rdna transcription in eukaryotes. transcription of most other protein-coding genes in trypanosomes is sensitive to alpha-amanitin. to investigate whether rna polymerase i, the polymerase that transcribes rrna genes, can give rise to functional mrnas in trypanosomes, we have fused the putative promoter of the t.brucei rrna ...19911923801
5'-alkyl-substituted analogs of 5'-methylthioadenosine as trypanocides.5'-deoxy-5'-(methylthio)adenosine (mta) is a by-product of polyamine metabolism and is phosphoryolytically cleaved to adenine and 5-deoxy-5-(methylthio)ribose-1-phosphate (mtr-1-p) by mta phosphorylase. in eukaryotes, adenine is subsequently salvaged and converted to nucleotides, while mtr-1-p is converted to methionine. we examined 5'-deoxy-5'-substituted analogs of mta for trypanocidal activity in vitro and in vivo. 5'-deoxy-5'-(hydroxyethyl)thioadenosine (heta) and its 5'-bromo,5'-chloro and ...19911929287
trypanosoma brucei glycosomal glyceraldehyde-3-phosphate dehydrogenase genes are stage-regulated at the transcriptional level.regions 5' of the glycosomal glyceraldehyde-3-phosphate dehydrogenase (ggapdh) gene from trypanosoma brucei were tested for their ability to promote chloramphenicol acetyl-transferase (cat) expression on reintroduction by electroporation into the parasite. deletion analysis mapped the ggapdh promoter to within 403 nts of the start of translation. a transcription initiation site was mapped at around -190 nts from the atg start codon by rnase protection and by primer extension. the higher expressi ...19911935905
specificity of glucose transport in trypanosoma brucei. effective inhibition by phloretin and cytochalasin b.glucose transport in the bloodstream form of the protozoan parasite trypanosoma brucei was characterized by enzymatically measuring the d-glucose uptake. uptake kinetics showed a concentration-dependent saturable process, typical for a carrier-mediated transport system, with an apparent km = 0.49 +/- 0.14 mm and vmax = 252 +/- 43 nmol.min-1.mg cell protein-1 (equal to 2.25 x 10(8) trypanosomes). the specificity of glucose transport was investigated by inhibitor studies. glucose uptake was shown ...19911935976
homologous recombination in the tandem calmodulin genes of trypanosoma brucei yields multiple products: compensation for deleterious deletions by gene amplification.homologous recombination between a calmodulin-neomycin-resistance fusion gene and the trypanosoma brucei chromosome takes place not only in the large 5'- and 3'-flanking segments of the calmodulin locus but also in any of the four tandem genomic calmodulin genes. this results in a recombined locus consisting of the chimeric neor gene and four, three, two, one, or zero functional calmodulin genes. cells bearing this latter event have half of their normal number of intact calmodulin genes and an a ...19911936992
variant specific glycoprotein of trypanosoma brucei consists of two domains each having an independently conserved pattern of cysteine residues.the complete amino acid sequences for nine variant specific glycoproteins (vsgs) of trypanosoma brucei are presented. these have more than doubled the size of the vsg sequence data base and have enabled a new and more rigorous comparison to be made between amino acid sequences of different vsgs. each vsg can be defined as a combination of an n-terminal domain type and a c-terminal domain type, based on the distribution of cysteine residues within the molecule. this identifies three n-terminal do ...19911942032
the actin gene promoter of trypanosoma brucei.the actin genes of trypanosoma brucei are transcribed constitutively during the parasite life-cycle, by a polymerase sensitive to alpha-amanitin. the start region of the actin gene transcription unit was mapped by virtue of the accumulation of promoter-proximal transcripts which occurs following moderate uv irradiation. this region, located about 4 kilobases upstream from the genes, was able to direct transient expression of the bacterial chloramphenicol acetyl transferase (cat) gene in both blo ...19911945875
growing and in-situ processing of cells on beem capsule caps for scanning electron microscopy.a simple technique for processing cells grown on beem capsule caps for scanning electron microscopic (sem) observation is described. the cells adhere to a substratum on the inner surface of caps coated with either an egg white-polyvinylpyrrolidone solution or a polylysine solution in the case of eukaryotic cells and an agar solution in the case of bacteria. the adhering cells are cultured and then processed in situ for sem observation. electron micrographs revealed details of the shape of the ce ...19911947942
intermolecular interactions involved in the association of the variant surface glycoprotein of trypanosoma brucei.trypanosomes in their mammalian host are covered by the densely packed variant surface glycoprotein (vsg). depending on the presence or absence of a glycosyl-phosphatidyl inositol anchor. vsg is accessible as soluble globular protein (svsg), or as insoluble membrane form (mfvsg). in order to get insight into the two-dimensional association of vsg within the surface layer, protein-protein interactions were investigated in a wide range of protein concentrations. no self-assembly of svsg could be d ...19911958317
genetics of antigenic variation in african trypanosomes.the major surface antigens of trypanosoma brucei are the vsg (variant surface glycoprotein) at the bloodstream stage, and procyclin at the procyclic stage. variation in the vsg allows the parasite to escape the antibody response of its mammalian host. this occurs through either dna rearrangement in the telomeric vsg gene expression site, or alternate activation, without dna rearrangement, of different telomeric expression sites. the vsg and procyclin genes each belong to large, polycistronic tra ...19911961983
purification of the membrane-form variant surface glycoprotein of trypanosoma brucei.the membrane-form variant surface glycoprotein (mfvsg) is anchored in the plasma membrane of african trypanosomes by a diacylglycerol residue. on cell rupture the anchor is rapidly cleaved by an endogenous phospholipase c. a purification procedure is described which results in native mfvsg devoid of lipase activity. a total membrane fraction is prepared in the presence of the sh-inhibitor p-chloromercuribenzenesulphonic acid (pcmbs). membrane proteins are solubilized in the presence of pcmbs and ...19901962787
multiple roles of inositol in trypanosomes. 19901964647
the effect of temperature and storage on the infectivity and motility of african animal trypanosomes in the blood of different hosts.blood from mice, rats, goats or cattle infected with trypanosoma congolense, t. vivax or t. brucei was stored at 0-4 degrees c, 20-25 degrees c, 30-35 degrees c or 36-40 degrees c. each sample was examined after set intervals to determine the maximum period the trypanosomes could remain motile and infective. t. brucei in blood remained motile for 96 h at 0-4 degrees c, being the longest period that was observed, but remained infective for only 8 h. t. vivax survived poorly in rodent blood, but d ...19901967509
the identification of trypanosoma brucei subspecies using repetitive dna sequences.we describe the use of repetitive dna probes to characterise the relationships between different stocks of african trypanosomes representing the subspecies of trypanosoma brucei. probes derived from the ribosomal rna genes (coding region and nontranscribed spacer) and another repetitive dna sequence were used to characterise trypanosome stocks by southern blotting. numerical taxonomy methods applied to the resulting restriction enzyme patterns were used to derive a dendrogram depicting the relat ...19901969612
influence of fever and flurbiprofen on trypanosome growth.the administration of flurbiprofen, a potent non-steroidal anti-inflammatory drug (nsaid), to goats infected with trypanosomes resulted in high elevated parasitaemia and suppression of fever. in contrast to goats, rats infected with trypanosomes do not show febrile reactions. therefore, the role of body temperature was investigated with yeast-induced fever in trypanosoma evansi and t. brucei infected rats. these investigations did not support the hypothesis that a high body temperature causes a ...19901969700
regulation of b cell responses to the variant surface glycoprotein molecule in trypanosomiasis. ii. down-regulation of idiotype expression is associated with the appearance of lymphocytes expressing antiidiotypic receptors.the current study examines the idiotypic expression and regulation of variant surface glycoprotein (vsg)-specific b cell responses during african trypanosomiasis. utilizing competitive inhibition ria analysis, we detected antibodies in the serum of balb/cbyj mice infected with trypanosoma brucei rhodesiense clone loutat 1.5 that recognized the same vsg epitopes as three vsg 1.5-specific mab. these epitope-specific antibody responses were detectable by day 5 of infection, peaked by day 10, and th ...19901970594
gonadotropic dysfunction produced by trypanosoma brucei brucei in the rat.hormonal disorders have been frequently observed in humans and animals infected with tsetse-transmitted (african) trypanosomes. we studied the pituitary gonadal axis (plasma concentrations of testosterone, luteinizing hormone (lh), follicle stimulating hormone (fsh) and the pituitary gonadotropin (lh, fsh) concentrations) in rats as an experimental model infected with an acute stock of trypanosoma brucei brucei (antat 1.1a). the same investigations in vivo were carried out with rats inoculated b ...19901971495
sfii and noti polymorphisms in theileria stocks detected by pulsed field gel electrophoresis.dnas of theileria parva parva, t. p. lawrencei, t. p. bovis and theileria mutans stocks, from kenya, uganda, zanzibar and zimbabwe were digested with either sfii or noti and analysed using contour-clamped homogeneous electric field (chef) and field-inversion gel electrophoresis (fige). the sfii-digested t. parva genomic dna resolved into approximately 30 fragments while the noti digestion produced between 4-7 bands. the summation of the sizes of sfii fragments gave an estimate of 9-10 x 10(6) ba ...19901972977
cure of trypanosoma brucei brucei and trypanosoma brucei rhodesiense infections in mice with an irreversible inhibitor of s-adenosylmethionine decarboxylase.a structural analog, 5'-([(z)-4-amino-2-butenyl]methylamino)-5'-deoxy adenosine (mdl 73811), of decarboxy s-adenosyl-l-methionine, the product of the reaction catalyzed by s-adenosyl-l-methionine (adomet) decarboxylase (dc), was found to inhibit trypanosoma brucei brucei adomet dc. the inhibition was time dependent (tau 50, 0.3 min), exhibited pseudo-first-order kinetics (ki, 1.5 microm), and was apparently irreversible. the natural substrate of the reaction, adomet, protected the enzyme from in ...19901977366
the association of glycosomal enzymes and microtubules: a physiological phenomenon or an experimental artifact?subpellicular microtubules isolated from trypanosoma brucei parasites were fractionated on a phosphocellulose column, and the trypanosomal p52 microtubule-associated protein was eluted along with two other proteins of 41 and 36 kda. these proteins were found to be the glycosomal enzymes aldolase (41 kda) and glyceraldehyde-3-phosphate dehydrogenase (gapdh, 36 kda) by enzyme activity, antibody cross-reaction, and n-terminal sequencing. these enzymes were coprecipitated with tubulin in the presenc ...19901979542
trypanosoma brucei evansi: dyskinetoplasia and loss of infectivity after long-term in vitro cultivation.bloodstream forms of a stock of trypanosoma brucei evansi were propagated in vitro for more than 14 months. after that period, all organisms were dyskinetoplastic and had lost their infectivity for mice. an increase in diminazene aceturate resistance in vitro was observed whereas the susceptibility to isometamidium chloride, quinapyramine sulphate and suramin was unaltered. trypanosomes derived from the long term culture had a surface coat.19901980574
salivary gland infection: a sex-linked recessive character in tsetse?male tsetse, when infected in the laboratory with trypanosomes of the subgenus trypanozoon, usually produce greater salivary gland infection rates than females of the same species. we show that a single sex-linked gene model can be fitted to most recently published data for salivary gland infection rates in tsetse. the maturation of trypanosoma congolense infections is shown to be independent of fly sex. the possible effects of genetic control of maturation of trypanozoon infections in tsetse po ...19901980807
bioenergetic studies of bloodstream forms of trypanosoma brucei: electrical and h+ gradients. 19901982094
potential therapeutic exploitation of the pulmonary polyamine uptake system. 19901982095
inhibition of the bloodstream/procyclic transformation in trypanosoma brucei. 19901982098
role of the pentose phosphate pathway in the provision of precursors for nucleic acid biosynthesis in bloodstream trypanosoma brucei. 19901982099
evidence for kinetoplast and nuclear dna homogeneity in trypanosoma evansi isolates.the kinetoplast dna minicircles from 13 stocks of trypanosomes designated as trypanosoma evansi were digested with various restriction enzymes. we also examined the distribution of restriction site polymorphisms in the nuclear dna of 9 of these stocks, using 7 different variable surface glycoprotein (vsg) and non-vsg probes. restricted kinetoplast dna (kdna) fragments of some of these strains were cloned into m13 or puc 18 vectors and sequenced. the restriction and sequence mapping showed that m ...19901982554
differential expression of a family of putative adenylate/guanylate cyclase genes in trypanosoma brucei.the expression site for the variant surface glycoprotein (vsg) gene of trypanosoma brucei contains several genes of unknown function (esags, for expression site-associated genes). among these, esag 4 shows homology to eukaryotic adenylate/guanylate cyclase genes, in the region encoding the presumptive enzyme catalytic domain. this gene belongs to a family of related sequences, and hybridizes to the genomic dna of other trypanosomatids, such as trypanosoma congolense, trypanosoma vivax and trypan ...19901982555
evidence that the mechanism of gene exchange in trypanosoma brucei involves meiosis and syngamy.all 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 ...19901982633
glucose uptake by trypanosoma brucei. rate-limiting steps in glycolysis and regulation of the glycolytic flux.glucose uptake and metabolism in the bloodstream form of the glycosome-containing protozoan parasite trypanosoma brucei was measured using 14c-labeled glucose in combination with the silicone oil centrifugation technique in short term (5-60 s) incubations. glucose rather than glucose analogues was used to study the interrelation between the uptake process and the subsequent metabolic steps. glucose uptake over the plasma membrane occurred by facilitated diffusion, which limited the overall glyco ...19911985967
transient activity assays of the trypanosoma brucei variant surface glycoprotein gene promoter: control of gene expression at the posttranscriptional level.the putative promoter of the variant surface glycoprotein (vsg) gene of trypanosoma brucei was cloned into a plasmid containing the chloramphenicol acetyltransferase (cat) gene. after electroporation into trypanosomes, this construct directed the expression of the cat reporter gene. the essential region for promoter activity was found to reside within 88 bp upstream of the putative transcription start site. transcription of the cat construct occurred at approximately the same level in both blood ...19911986230
endopeptidase variations among different life-cycle stages of african trypanosomes.lysates of different life-cycle stages of trypanosoma congolense, trypanosoma vivax and trypanosoma brucei were analysed for endopeptidase activity, using reaction conditions which permitted a distinction to be made between lysosomal and non-lysosomal activity [lonsdale-eccles, j. d. & grab, d. j. (1987) eur. j. biochem. 169, 467-475]. hydrolysis of z-arg-arg-nhmec (z = benzyloxycarbonyl, nhmec = 7-amino-4-methylcoumaryl) and z-gly-gly-arg-nhmec occurred predominantly at alkaline ph and was obse ...19911991468
a new photometric assay for testing trypanocidal activity in vitro.a new in vitro test was developed to assess the trypanocidal activity of compounds against axenically grown trypanosoma brucei bloodstream forms. trypanosomes from continuous culture were exposed to series of drug dilutions in a microtiter plate. after an incubation time of 72 h at 37 degrees c, the ph indicator of the medium had changed its colour in non-inhibited cultures due to the production of pyruvate. inhibited cultures showed less, if any, colour changes. plates could be read by eye or u ...19911994369
effect of polyclonal anti-procyclic antibodies on development of trypanosoma brucei brucei in tsetse flies.results obtained in experiments testing the efficacy of anti-procyclic-form rabbit sera on the development of homologous and heterologous stocks of trypanosoma brucei brucei in glossina morsitans morsitans indicated that this development was affected little, or not at all, by such sera. the absence of effect of anti-procyclic stage antibodies can be explained by the failure to detect by either direct or indirect fluorescent antibody methods the presence of antibodies acquired in vivo by either t ...19911994370
a similar gene is shared by both the variant surface glycoprotein and procyclin gene transcription units of trypanosoma brucei.the genes for the variant surface glycoprotein (vsg) and procyclin are expressed in a mutually exclusive manner during the life cycle of trypanosoma brucei and synthesize the most abundant mrnas specific to the bloodstream and procyclic stages of the parasite, respectively. genes belonging to the polycistronic transcription unit of the vsg gene (expression site-associated genes [esags]) are uniquely expressed in the bloodstream form, but some members of esag families (genes related to esags [gre ...19911996104
expression and deletion analysis of the trypanosoma brucei rhodesiense cysteine protease in escherichia coli.trypanosoma brucei, the cause of african sleeping sickness, differentiates in the mammalian bloodstream from a long, slender trypanosome into a short, stumpy trypanosome. this event is necessary for infection of the tsetse fly and maintenance of the life cycle. we have previously shown that the stumpy form contains 10- to 15-fold-greater cysteine protease activity than either the slender form or the insect midgut procyclic, and we have isolated a cdna encoding the protease. in order to determine ...19911997411
a novel suicide inhibitor strategy for antiparasitic drug development.dl-alpha-difluoromethylornithine (dfmo), a suicide inhibitor of eukaryotic ornithine decarboxylase (odc), has therapeutic activities against african trypanosomiasis. the ki value of dfmo for odc of trypanosoma brucei is somewhat higher than that for mouse odc. the therapeutic efficacy of dfmo cannot therefore be attributed to a preferential inhibition of the parasite enzyme. the t. brucei gene encoding odc was cloned and sequenced, and the derived amino acid sequence has 61.5% homology with that ...19912005184
stable integrative transformation of trypanosoma brucei that occurs exclusively by homologous recombination.a calmodulin-neomycin-resistance fusion gene was introduced into trypanosoma brucei by electroporation, and stably transformed cell lines were obtained. in all of the transformants, the fusion gene had integrated into the host genome at the cognate locus, evidently by homologous recombination within flanking calmodulin dna. this unusual observation distinguishes trypanosomes as the only eukaryote other than yeast known to undergo gene targeting in essentially 100% of the stable transformants. it ...19912006150
high level expression in escherichia coli of soluble, enzymatically active schistosomal hypoxanthine/guanine phosphoribosyltransferase and trypanosomal ornithine decarboxylase.the bacterial alkaline phosphatase (phoa) promoter and signal peptide have been used previously to control recombinant expression and secretion of eukaryotic proteins in escherichia coli. other reports have shown that this expression system can generate relatively modest levels of active hypoxanthine/guanine phosphoribosyltransferase (hprt; hypoxanthine phosphoribosyltransferase; imp:pyrophosphate phosphoribosyltransferase, ec 2.4.2.8), which carries part of the signal peptide but remains in the ...19912006185
inhibition of protein synthesis results in super-induction of procyclin (parp) rna levels.procyclin is an abundant surface antigen found exclusively on the procyclic forms of african trypanosomes. we are interested in the induction of procyclin gene expression during differentiation from bloodstream forms. we find that increased levels of procyclin rna are evident as early as 15 min after triggering differentiation. the increase in procyclin rna levels requires the temperature shift from 37 degrees c to 27 degrees c and is aided by addition of the tricarboxylic acid cycle intermediat ...19912011148
self-proteolysis of the cysteine proteinase, cruzipain, from trypanosoma cruzi gives a major fragment corresponding to its carboxy-terminal domain.the major cysteine proteinase (cruzipain) purified from trypanosoma cruzi epimastigotes catalyzes its own degradation in the presence of beta-mercaptoethanol, at 56 degrees c and ph 6. the reaction is affected by the same inhibitors which inhibit the azocaseinase activity, and yields a major 25-kda fragment, which contains carbohydrate, few, if any, aromatic amino acids, and presents a proline-rich n-terminus (gpgpxpep...), in addition to a number of small peptides, which can be isolated by reve ...19912011151
ultraviolet irradiation inhibits rna decay and modifies ribosomal rna processing in trypanosoma brucei.a known effect of ultraviolet radiation on transcription is the arrest of rna elongation. in trypanosoma brucei, we show that uv also inhibits rna decay, leading to specific accumulation of transcripts from the beginning of several transcription units. in addition, uv irradiation changes the pattern of ribosomal rna processing, probably by altering the order in which the non-coding spacers are excised. these effects are still observed on rna synthesized more than 2 h after irradiation, and do no ...19912011153
science and secrecy. 19912017252
phosphorylation differences among proteins of bloodstream developmental stages of trypanosoma brucei brucei.early in an infection the bloodstream forms of the african trypanosome trypanosoma brucei brucei are long, slender and rapidly dividing. later, non-dividing, short, stumpy forms may be found. in this report we described biochemical differences between the two parasite populations in the phosphorylation of their proteins in vitro. compared with the slender populations, the non-dividing stumpy forms of the parasites exhibit decreased phosphorylation of an 80 kda protein and enhanced phosphorylatio ...19912018486
crystallization and preliminary x-ray analysis of an intact soluble-form variant surface glycoprotein from the african trypanosome, trypanosoma brucei.the intact variant surface glycoprotein (vsg) iltat 1.24 from trypanosoma brucei has been crystallized. an amino-terminal domain of the protein comprising two thirds of the sequence had been crystallized previously after proteolytic digestion. now intact vsg crystals have been grown from 50 mm-mes (ph 6.5) containing 62% (w/v) saturated ammonium sulfate. the crystals are demonstrated to contain the intact vsg by h.p.l.c. gel filtration and reaction with an antibody to the inositol phosphate olig ...19912023242
trypanosoma brucei rhodesiense: characterisation of stocks from zambia, kenya, and uganda using repetitive dna probes.we have previously described a system for characterising the relationships between trypanosome stocks of the t.brucei group based on southern blotting with repetitive dna probes followed by cluster analysis of resultant banding patterns (g. hide et al. molec. bioch. parasitol. 39, 213-226, 1990). in this study, we extend this analysis to examine the relationships between trypanosome stocks isolated from major sleeping sickness foci in zambia, kenya, and uganda. we show that the trypanosome strai ...19912026217
galactocerebrosides are antigens for immunoglobulins in sera of an experimental model of trypanosomiasis in sheep.in an experimental model of human african trypanosomiasis in sheep inoculated with trypanosoma brucei brucei, we report an immunoglobulin reactivity towards central nervous system (cns) glycolipids. immunocharacterization of the glycolipid antigens was performed on thin-layer chromatography using peroxidase-labelled second antibody. an immunoreactivity against galactocerebroside antigens was observed in inoculated animals up to a dilution of 1:600 and was suppressed after immunoadsorption of ser ...19912027031
protein methylases in trypanosoma brucei brucei: activities and response to dl-alpha-difluoromethylornithine.protein methylases i, ii and iii were detected in extracts of trypanosoma brucei brucei, and characterized according to the specific amino substituent methylated. only protein methylase ii activity was elevated by difluoromethylornithine treatment of t. b. brucei, and hence this enzyme was characterized further. protein methylase ii transferred methyl groups from s-adenosyl-l-methionine (s-adomet) to the carboxyl residues of several protein substrates, exhibiting highest activity with histone vi ...19912033384
microtubules, tubulin, and microtubule-associated proteins of trypanosomes. 19912034124
recognition of a peroxisomal tripeptide entry signal by the glycosomes of trypanosoma brucei.trypanosomes compartmentalise the first 9 enzymes of glycolysis and glycerol metabolism in peroxisome-like microbodies known as glycosomes. the identity of the sequences that direct proteins into the glycosome has until now been uncertain. we show here that the peroxisomal tripeptide entry signal is sufficient to cause association of a bacterial enzyme with the glycosomes of trypanosoma brucei. however, it works less efficiently than the c-terminal 21 amino acids of trypanosome glycosomal phosph ...19912038359
porcine cerebral trypanosoma brucei brucei trypanosomiasis.this paper describes the investigation of a disease outbreak among 10 adult pigs in nsukka, anambra state, nigeria. prior to the investigation one sow died of the disease. trypanosomes were later detected in the blood of two of the nine pigs subsequently investigated. all the pigs were then treated with deep intramuscular injection of 8 mg/kg diminazene aceturate (berenil). thirty six days after treatment a boar and a sow relapsed with signs similar to the ones shown previously. further examinat ...19912038769
anion binding at the active site of trypanosomal triosephosphate isomerase. monohydrogen phosphate does not mimic sulphate.the three-dimensional structure of triosephosphate isomerase complexed with the competitive inhibitor so-4(2) was determined by x-ray crystallography to a resolution of 0.24 nm. a comparison with the native crystal structure, where so-4(2) is bound, revealed five changes: (a) a 0.10-nm shift of the anion-binding site; (b) a further closing of the flexible loop of the enzyme; (c) a 'swinging in' of the side chain of the catalytic glu, that is chi 1 changes from (+) to (-) synclinal; (d) an altere ...19912040290
the cytosolic and glycosomal isoenzymes of glyceraldehyde-3-phosphate dehydrogenase in trypanosoma brucei have a distant evolutionary relationship.trypanosoma brucei contains two isoenzymes for glyceraldehyde-3-phosphate dehydrogenase: one enzyme resides in a microbody-like organelle, the glycosome; the other is found in the cytosol. previously we have reported the characterization of the gene for the glycosomal enzyme [michels, p. a. m., poliszczak, a., osinga, k. a., misset, o., van beeumen, j., wierenga, r. k., borst, p. & opperdoes, f. r. (1986) embo j. 5, 1049-1056]. here we describe the cloning and analysis of the gene that codes for ...19912040303
the cytosolic and glycosomal glyceraldehyde-3-phosphate dehydrogenase from trypanosoma brucei. kinetic properties and comparison with homologous enzymes.the protozoan haemoflagellate trypanosoma brucei has two nad-dependent glyceraldehyde-3-phosphate dehydrogenase isoenzymes, each with a different localization within the cell. one isoenzyme is found in the cytosol, as in other eukaryotes, while the other is found in the glycosome, a microbody-like organelle that fulfils an essential role in glycolysis. the kinetic properties of the purified glycosomal and cytosolic isoenzymes were compared with homologous enzymes from other organisms. both trypa ...19912040304
nucleotide sequences of the six very small molecules of trypanosoma cruzi ribosomal rna. 19912041786
the in vitro differentiation of pleomorphic trypanosoma brucei from bloodstream into procyclic form requires neither intermediary nor short-stumpy stage.our studies on the in vitro differentiation of a pleomorphic trypanosoma brucei strain treu667 indicate that the parasite differentiates directly from long-slender into procyclic form when incubated in cunningham's medium at 26 degrees c. the intermediary and the short-stumpy bloodstream forms harvested from infected mice can also differentiate directly into procylic form in vitro with time courses similar to that for the long-slender form. thus, none of the three bloodstream forms appear to be ...19912052026
chemostat cultures of leishmania donovani promastigotes and trypanosoma brucei procyclic trypomastigotes. 19912052036
in vitro activity of organometallic complexes of ir, pt and rh on trypanosoma b. gambiense, t. b. rhodesiense and t. b. brucei.a series of organometallic drugs were tested in a photometric in vitro assay for trypanocidal activity against the three subspecies of the trypanosoma brucei group. trypanosoma b. rhodesiense, t. b. gambiense and t. b. brucei displayed a similar sensitivity pattern. the following ic50 values (drug concentration inhibiting decrease of extinction by 50%) could be determined: ircl6 (iv)-pentamidine (1.5 ng/ml), ircl6(iv)-ro15-0216 (10 micrograms/ml), ptbr6-metamidium (30 micrograms/ml), ptbr6(iv)-b ...19912052855
proofreading genes. a molecular editor makes sensible additions to rna. 19912052934
trypanosoma brucei: analysis of codon usage and nucleotide composition of nuclear genes. 19912055297
the crystal structure of the "open" and the "closed" conformation of the flexible loop of trypanosomal triosephosphate isomerase.triosephosphate isomerase has an important loop near the active site which can exist in a "closed" and in an "open" conformation. here we describe the structural properties of this "flexible" loop observed in two different structures of trypanosomal triosephosphate isomerase. trypanosomal triosephosphate isomerase, crystallized in the presence of 2.4 m ammonium sulfate, packs as an asymmetric dimer of 54,000 da in the crystallographic asymmetric unit. due to different crystal contacts, peptide 1 ...19912062827
the adaptability of the active site of trypanosomal triosephosphate isomerase as observed in the crystal structures of three different complexes.crystals of triosephosphate isomerase from trypanosoma brucei brucei have been used in binding studies with three competitive inhibitors of the enzyme's activity. highly refined structures have been deduced for the complexes between trypanosomal triosephosphate isomerase and a substrate analogue (glycerol-3-phosphate to 2.2 a), a transition state analogue (3-phosphonopropionic acid to 2.6 a), and a compound structurally related to both (3-phosphoglycerate to 2.2 a). the active site structures of ...19912062828
a rapid method purifies a glycoprotein of mr 145,000 as the ldl receptor of trypanosoma brucei brucei.the trypanosome ldl receptor has been isolated from bloodstream form and cultured insect-stage trypanosomes as a protein of mr 145,000, using a rapid purification procedure in the presence of a cocktail of protease inhibitors, whereas previously a polypeptide of mr 86,000 was purified as the ldl receptor. both the 145,000 and the 86,000 polypeptides are glycosylated and recognized by a monospecific antibody raised against the 86,000 species. this antibody inhibits ldl binding to the intact trypa ...19912069558
molecular genetics of antigenic variation.antigenic variation is one of the most effective strategies developed by parasites to escape immune destruction. it requires a large wardrobe of surface coats and mechanisms to exchange one coat for an unrelated one. the molecular principles of antigenic variation are now largely known in the bacterial species borrelia and neisseria and in the protozoa of the african trypanosome group and these three examples are discussed here by piet borst.19912069675
a soluble factor from trypanosoma brucei rhodesiense that prevents progression of activated human t lymphocytes through the cell cycle.african sleeping sickness is accompanied by a severe immunosuppression. as part of our efforts to examine the mechanisms by which this suppressive state is induced, we studied alterations in human t-lymphocyte function caused by trypanosoma brucei rhodesiense. to this end, we used an in vitro system in which phytohaemagglutinin (pha)-stimulated human peripheral blood mononuclear cells (pbmc) were cultured in a medium containing soluble, non-dialysable parasite products. we were able to demonstra ...19912071163
chromosome structure: dna nucleotide sequence elements of a subset of the minichromosomes of the protozoan trypanosoma brucei.the genome of the protozoan trypanosoma brucei contains a set of about 100 minichromosomes of about 50 to 150 kb in size. the small size of these chromosomes, their involvement in antigenic variation, and their mitotic stability make them ideal candidates for a structural analysis of protozoan chromosomes and their telomeres. we show that a subset of the minichromosomes is composed predominantly of simple-sequence dna, with over 90% of the length of the minichromosome consisting of a tandem arra ...19912072894
molecular cloning of aldolases for synthetic applications. 19902076016
phylogenetic analysis of the rna polymerases of trypanosoma brucei, with special reference to class-specific transcription.we have sequenced the genes encoding to largest subunits of the three classes of dna-dependent rna polymerases of trypanosoma brucei. the nucleotide and deduced amino acid sequences were compared and aligned with the corresponding sequences of other eukaryotes. phylogenetic relationships were subsequently calculated with a distant matrix, a bootstrapped parsimony and a maximum-likelihood method. these independent calculations resulted in trees with very similar topologies. the analyses show that ...19902076555
changes in the pattern of cell surface proteins during transformation of bloodstream forms of trypanosoma brucei in vitro. 19902083652
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