Heparin modulates integrin function in human platelets.

PURPOSE: Heparin binds to human platelets and can cause activation and aggregation, although the mechanisms are unknown. To determine how heparin alters platelet function, we identified platelet-binding sites for heparin and measured heparin's influence on the function of platelet integrin alpha(IIb)beta(3) (glycoprotein IIb/IIIa). METHODS: Photoaffinity cross-linking and affinity chromatography experiments were performed to identify platelet membrane proteins that bind heparin. Heparin's effect on fibrinogen binding to platelets was measured with a radioligand-binding assay. The translocation to the cytoskeleton of Rap2, a guanosine triphosphate-binding protein, was measured from platelets aggregating in response to heparin and other agonists. RESULTS: Cross-linking and affinity chromatographic experiments positively identified the integrin alpha(IIb)beta(3) as a heparin-binding site. Heparin aggregation was calcium dependent. Low concentrations of unfractionated porcine mucosal heparin (2-5 U/mL) significantly increased fibrinogen I 125 binding to activated platelets, whereas higher doses did not. Heparin-mediated platelet aggregation was completely blocked by GRGDS peptide (5 mmol/L), a competitive inhibitor of fibrinogen binding, and was blocked by EDTA (2 mmol/L), which dissociates the functional integrin complex. Aggregation was associated with Rap2 translocation to the cytoskeleton, a sign of outside-in signaling. CONCLUSIONS: Heparin binds to the alpha(IIb)beta(3) integrin in vitro and ex vivo, and heparin increases fibrinogen binding to the integrin. Heparin-mediated aggregation requires an intact integrin and ligand and leads to Rap2 translocation to the cytoskeleton-an outside-in signal of ligand engagement. Heparin may directly modulate platelet integrin function, most likely through direct binding and modulation of integrin function.

Primary structure and partial characterization of a life-cycle-regulated cysteine protease from Trypanosoma (Nannomonas) congolense.

Trypanosoma (Nannomonas) congolense is an important pathogenic parasite of domestic livestock in Africa. We have cloned a cDNA encoding a prepro-cysteine protease of this protozoan, the sequence of which indicates it is an early mRNA processing intermediate. Northern analysis demonstrates a life-cycle-stage specificity similar to previously described enzymatic data. The deduced amino-acid sequence shows extensive similarity to cysteine proteases of other parasitic protozoa, as well as papain and cathepsin L. As with other African trypanosomes, a poly-proline tract connects the catalytic domain with an unusual C-terminal extension.

Three genes and two isozymes: gene conversion and the compartmentalization and expression of the phosphoglycerate kinases of Trypanosoma (Nannomonas) congolense.

The glycosome, a microbody organelle found only in kinetoplastid protozoa, compartmentalizes the first six enzymes of glycolysis. In order to better understand the regulation and targeting of glycolytic enzymes in trypanosomes, we have cloned and analyzed the three genes of the phosphoglycerate kinase (PGK) complex of Trypanosoma (Nannomonas) congolense. The organization of the genes within the complex is similar to that of Trypanosoma brucei brucei. The nucleotide and amino-acid sequences, including those of the novel high-molecular-weight 56PGK, show substantial cross-species similarity. However, the two downstream genes, c1PGK and c2PGK, encode identical isozymes in T. congolense, while they encode distinct glycosomal and cytoplasmic isozymes in T. brucei. Western analysis also indicated that there are only two isozymes in T. congolense and that these are constitutively expressed. Differential digitonin solubilization of the trypanosomes indicated that 56PGK is primarily localized to the glycosome, as expected, and that c1/c2PGK is cytoplasmic. Northern analysis demonstrates that while 56PGK is constitutively expressed, c1PGK and c2PGK mRNAs are differentially expressed in the T. congolense developmental stages. This work demonstrates that T. congolense has only one PGK isozyme, 56PGK, that is predominantly localized in glycosomes.

Developmental regulation of RNA editing and polyadenylation in four life cycle stages of Trypanosoma congolense.

The accumulation of many edited mRNAs is developmentally regulated in a transcript-specific fashion in Trypanosoma brucei. In addition, these transcripts are frequently present in two size classes which differ substantially in the lengths of their poly(A) tails, and poly(A) tail length is also developmentally regulated. Previously, these phenomena have only been studied in the mammalian bloodstream and insect procyclic forms (BF and PF, respectively) of T. brucei. In this paper, we examine developmental regulation of edited RNA abundance and poly(A) tail length of 3 mitochondrially encoded RNAs in mammalian BF and 3 insect stages (PF, epimastigotes, and metacyclics) of T. congolense. T. congolense BF and PF are similar, but not identical, to these stages of T. brucei with regard to edited RNA accumulation and poly(A) tail length. At the level of edited RNA, both epimastigotes and metacyclic stage parasites appear to be pre-adapted for the respiratory mechanisms of BF but not yet down-regulated from the cytochrome-based respiration of PF since edited RNAs encoding NADH dehydrogenase components are up-regulated and edited CYb RNA is abundant in these stages. Poly(A) tail lengths of mitochondrial mRNAs appear to be regulated independently of edited RNA abundance. These results indicate that multiple mechanisms for regulation of mitochondrial gene expression are active throughout the trypanosome life cycle.

A colorimetric assay for trypanosome viability and metabolic function.

We have adapted a tetrazolium salt (MTT) colorimetric cytotoxicity assay to the assessment of viability and metabolic function in cultured African trypanosomes. Trypomastigotes of Trypanosoma congolense and T. brucei rhodesianse were harvested from the blood of parasitemic rats and cultured under axenic conditions that support trypanosome viability and growth. Analysis of serial dilutions of these bloodstream forms indicated that the assay could detect 10(4) parasites. To assess the effect of lymphoid cytokines on trypomastigote viability, 10(5) freshly harvested parasites were cultured with a wide range of dilutions of human recombinant IL-1, IL-3, IL-6, interferon-gamma (IFN gamma) or tumor necrosis factor-alpha (TNF alpha), or bovine recombinant IFN gamma or TNF alpha for 24, 48 or 96 h. These cytokines had no apparent growth enhancing or inhibitory effect on the trypomastigotes compared with growth in supplemented medium alone. This assay has several advantages over traditional counting methods, including increased sensitivity and rapid, repeatable quantitation. This adaptation of the MTT colorimetric assay should be useful in screening drugs and host-derived factors for growth-modulating effects on trypanosomes and other extracellular protozoan parasites.

The transferrin receptor in African trypanosomes: identification, partial characterization and subcellular localization.

All eukaryotic cells, including African trypanosomes, require iron for growth and division, and this iron is acquired by the receptor-mediated endocytosis of iron-loaded transferrin (diFe(3+)-transferrin). In trypanosomes transferrin (Tf) has been shown to be delivered into lysosomes and may not recycle back to the cell surface as it does in mammalian cells (Grab, D. J., et al., Eur. J. Cell Biol. 59, 398-404 (1992)). Here, we describe for the first time, the characteristics of a Tf-binding protein with receptor-like properties in Trypanosoma brucei brucei. Bloodstream forms of rodent-adapted T. brucei were incubated with [35S]methionine and detergent lysates chromatographed on a Sephacryl S-300 column. Fractions were incubated with anti-Tf serum to immunoprecipitate Tf/Tf-binding protein complexes. On sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) the molecular mass of the major protein in the immunoprecipitate was 88 to 92 kDa. Tf-binding proteins could also be isolated using diferric Tf-Sepharose. The molecular mass of the major Tf-binding protein, as estimated from Sephacryl S-300 column chromatography, in the presence of detergent, was approximately 90 to 100 kDa and 90 kDa with SDS-PAGE. Each 90 kDa Tf-binding protein was able to bind one molecule of diferric Tf. Since monoclonal antibodies to human and bovine Tf receptors failed to react with any trypanosome proteins, antisera were raised against the T. brucei Tf-binding proteins eluted from Tf-Sepharose at low pH. These antibodies recognized a 90 kDa protein on Western blots of a T. brucei lysate and inhibited the growth of T. brucei in vitro. Immunolocalization studies, using this antiserum showed that the Tf-binding protein was localized in the flagellar pocket and within the early endosomal compartments. In the presence of protease inhibitors there was additional localization in lysosome-like organelles. The Tf-binding characteristics and localization of this 90 kDa protein suggest that this molecule is a strong candidate as a physiological receptor for Tf in these parasites.

Maxicircle DNA and edited mRNA sequences of closely related trypanosome species: implications of kRNA editing for evolution of maxicircle genomes.

kRNA editing produces functional mRNAs by uridine insertion and deletion. We analyzed portions of the apocytochrome b and NADH dehydrogenase subunits 7 and 8 (ND7 and 8) genes and their edited mRNAs in Trypanosoma congolense and compared these to the corresponding sequences in T.brucei. We find that these genes are highly diverged between the two species, especially in the positions of thymidines and in nucleotide transitions. Editing eliminates differences in encoded uridines producing edited mRNAs that are identical except for the nucleotide substitutions. The resulting predicted proteins are identical since all nucleotide substitutions are silent. A T.congolense minicircle-encoded gRNA which can specify editing of ND8 mRNA was identified. This gRNA can basepair with both T.congolense and T.brucei ND8 mRNA despite nucleotide transitions due to the flexibility of G:U base-pairing. These results illustrate how editing affects the characteristics of maxicircle sequence divergence and allows protein sequence conservation despite a level of DNA sequence divergence which would be predicted to be intolerable in the absence of editing.

Metacyclic form-specific variable surface glycoprotein-encoding genes of Trypanosoma (Nannomonas) congolense.

A complementary DNA expression library in phage lambda gt11 was synthesized using mRNA from in vitro-produced metacyclic forms of a clone of Trypanosoma (Nannomonas) congolense. The unamplified library was screened with antiserum from a goat immune to infection with metacyclic (m)-forms of T. congolense ILRAD Nannomonas antigen repertoire 2(ILNaR2). Of the 100 antiserum-reactive phage clones identified, 22 were analyzed further: 21 of the clones contained overlapping portions of a single transcript, while one other contained a different transcript. Northern blot analyses indicated that the sequences contained in the clones were transcribed only by m-forms of ILNaR2. Immunological and sequence analyses indicated that the two different cloned sequences encode m-form-specific variable surface glycoproteins.

Trypanosoma (Nannomonas) congolense: changes in respiratory metabolism during the life cycle.

All four life cycle stages (bloodstream, procyclic, epimastigote, and metacyclic) of Trypanosoma congolense IL 3000 were assayed with an oxygen electrode (polarograph) for the presence of terminal oxidases and carbon-source preference. In addition, these stages were used for histochemical analysis of mitochondrial activity using rhodamine 123, nitroblue tetrazolium, and diaminobenzidine. Morphometry was used to compare mitochondrial volumes and surface area among the different life cycle stages. It was found that in contrast to epimastigote forms, which were metabolically almost identical to procyclic forms, metacyclic forms showed characteristics of, and seemed preadapted to, differentiation into the bloodstream stage. While mitochondrial NAD+ diaphorase activity and an electrochemical potential were detected in all life cycle stages, metacyclic metabolism was glucose-based and terminal oxidase activity was primarily dependent upon the trypanosome alternative oxidase with the contribution of cyanide-sensitive respiration accounting for only 20-30% of the total respiratory capacity.

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 observed in lysates of both insect and mammalian infective forms of T. brucei and T. congolense. Compared to their other life-cycle stages, procyclic forms of T. brucei and epimastigote forms of T. congolense exhibited enhanced hydrolysis of these substrates. Low levels of hydrolysis of Z-Arg-Arg-NHMec were observed in the bloodstream and epimastigote forms of T. vivax. The hydrolysis of Z-Gly-Gly-Arg-NHMec in each of the life-cycle stages of T. vivax was generally below detectable levels. In lysates of T. congolense, proteolytic and Z-Phe-Arg-NHMec-hydrolytic activity in bloodstream forms greater than metacyclic greater than epimastigote greater than procyclic forms. In T. vivax Z-Phe-Arg-NHMec-hydrolytic activity differed slightly according to the origin of the parasite but, in general, followed the same pattern (i.e. bloodstream forms greater than epimastigote forms, with metacyclic forms usually intermediate between these two). In T. brucei, Z-Phe-Arg-NHMec-hydrolytic activity in bloodstream forms greater than procyclic forms. Upon differentiation of the long, slender bloodstream forms into short, stumpy forms the Z-Phe-Arg-NHMec-hydrolytic activity was elevated even further. Thus, during their life cycle, each of these African trypanosomes exhibits complex changes of endopeptidase activity, suggestive of an induction of lysosomal activity between the insect and mammalian forms.

Endocytosis by African trypanosomes. II. Occurrence in different life-cycle stages and intracellular sorting.

Horseradish peroxidase (HRP) and colloidal gold-labeled proteins enter many of the endocytic organelles of bloodstream forms of Trypanosoma brucei and T. congolense. However, the colloidal gold markers were excluded from substantial parts of the pathway that contained HRP. Morphometric studies revealed that HRP entered organelles that accounted for approximately 5% of the total cell volume while transferrin-gold entered organelles that comprised approximately 2% of the total cell volume. In addition, large colloidal gold particles were excluded from organelles that contained smaller gold particles. Antibodies, raised against the variable surface glycoprotein, when applied to thawed cryosections were found to label structures from which endocytosed colloidal gold coupled to bovine serum albumin (BSA) was excluded. Endocytosis was shown to occur in two in vitro propagated forms of trypanosomes, similar to those found in the insect vector (Glossina spp.). The mammal-infective metacyclic forms were similar to bloodstream forms in that they endocytosed HRP and colloidal gold markers but excluded colloidal gold from approximately 3% of the endocytic organelles. Estimation of the flagellar pocket volumes of bloodstream form T. brucei showed that this organelle occupied 0.5% to 1.4% of the total cell volume. The flagellar pocket volume of T. congolense varied between life-cycle stages, with a fractional volume of 4.4% for bloodstream forms, 2.3% for metacyclic forms and 1.4% for procyclic forms. Endocytosis of HRP, but not of protein-gold markers, occurred in procyclic (uncoated) forms. Endocytosis by procyclic forms has heretofore not been reported.(ABSTRACT TRUNCATED AT 250 WORDS)

Disulfide bond involvement in the maintenance of the cryptic nature of the cross-reacting determinant of metacyclic forms of Trypanosoma congolense.

The variable surface glycoprotein (VSG) of African trypanosomes possesses a 1,2-dimyristoylglycosylphosphatidylinositol at the carboxy terminus. Cleavage of the 1,2-dimyristoylglycerol (1,2-DMG) moiety from the VSG reportedly results in a higher apparent molecular mass and an increased binding of antibodies against the "cross-reacting determinant" (CRD), a cryptic epitope present on most VSGs. Using metacyclic forms of Trypanosoma congolense, we show that the processes involved are more complex than heretofore presumed and that the removal of the 1,2-DMG moiety may not be necessary for binding of anti-CRD antibodies (RxCRD). Among other findings, we observe the following: (1) in sonicated samples of trypanosomes metabolically labeled with [3H]myristate, the binding of RxCRD on Western blots is coincident with bands containing labeled (membrane form) VSGs; (2) disulfide reduction of trypanosome sonicates suffices to promote RxCRD binding in the presence or absence of inhibitors of a glycosylphosphatidylinositol-specific phospholipase C; (3) trypanosomes directly solubilized in detergents show quantitative and qualitative differences in RxCRD binding which depend upon the detergent used and the order of addition of disulfide reducing agents. We conclude that the binding of RxCRD to T. congolense metacyclic VSGs depends upon the degree of unfolding of the molecule and is clearly a complex, multistep process in which structural changes and disulfide reduction play pivotal roles.

Cell adhesion in Trypanosoma: in vitro studies of the interaction of Trypanosoma vivax with immobilized organic dyes.

Certain bloodstream forms of Trypanosoma vivax have been shown to attach to Amicon Matrex Gel Green A dye beads in a manner similar to the in vivo binding of T. vivax to the inner surface of the tsetse fly proboscis. We now report an in vitro assay for trypanosome-bead attachment and show that only the 9,10-anthraquinone portion of the dye molecule is involved in the binding of trypanosomes to beads and that bead-bound dyes with similar structures also support binding to differing degrees. The binding is dependent upon the amount of dye on the beads and this, and other evidence, suggests that an array of dye molecules, rather than individual molecules, may be the actual recognition site. Various external effectors, including temperature, soluble protein-dye complexes, and serum of mice with chronic T. vivax infections, reduce trypanosome binding, indicating that at least one immunogenic trypanosome macromolecule is involved. The trypanosome-bead interaction mimics the in vivo binding to tsetse proboscis and warrants closer examination as a model of trypanosome cell adhesion in the tsetse fly.

Subcellular localization of a variable surface glycoprotein phosphatidylinositol-specific phospholipase-C in African trypanosomes.

African trypanosomes contain a membrane-bound enzyme capable of removing dimyristylglycerol from the membrane-attached form of the variable surface glycoprotein (mfVSG; Ferguson, M. A. J., K. Halder, and G. A. M. Cross, 1985, J. Biol Chem., 260:4963-4968). Although mfVSG phospholipase-C has been implicated in the removal of the VSG from the trypanosome surface (Cardoso de Almeida, M. L., and M. J. Turner, 1983, Nature (Lond.)., 302:349-352; Ferguson, M. A. J., K. Halder, and G. A. M. Cross, 1985, J. Biol Chem., 260:4963-4968), its precise function and subcellular location have not been determined. We have developed a procedure for the separation of the cell fractions and organelles of Trypanosoma brucei brucei (and other trypanosome species) by differential sucrose and isopycnic PercollR centrifugation. These fractions were tested for mfVSG phospholipase activity using Trypanosoma brucei mfVSG labeled with 3H-myristic acid as substrate. The highest enzyme-specific activity was associated with the flagella and evidence is presented to suggest that it is localized in the flagellar pocket. Some activity was also associated with the Golgi complex. These results suggest that the mfVSG phospholipase is localized primarily in the membrane of the flagella pocket and possibly other membrane organelles derived from and associated with this structure, and may be part of the VSG-membrane recycling system in African trypanosomes. The activity of mfVSG phospholipase amongst various trypanosome species was determined. We show that, in contrast to the bloodstream forms of Trypanosoma brucei, cultured procyclic Trypanosoma brucei and bloodstream Trypanosoma vivax had little or no mfVSG phospholipase activity. The activity found in bloodstream forms of Trypanosoma congolense was intermediate between Trypanosoma vivax and Trypanosoma brucei.

Inosine analogs as chemotherapeutic agents for African trypanosomes: metabolism in trypanosomes and efficacy in tissue culture.

Certain purine analogs, the pyrazolopyrimidines, are effective chemotherapeutic agents against Leishmania spp. and Trypanosoma cruzi both in vitro and in some clinical models. Heretofore they have not been effective against the African trypanosomes; this suggested that these organisms were not comparable to the other pathogens with respect to their purine metabolism. We have studied the efficacy and metabolism of the pyrazolopyrimidine bases allopurinol and thiopurinol, their respective ribonucleosides, and the C-nucleosides formycin B and 9-deazainosine in Trypanosoma brucei subsp. gambiense and Trypanosoma brucei subsp. rhodesiense. The efficacy of these compounds was dependent on the purine content of the culture medium. The C-nucleosides were the most effective, with 90% effective doses for formycin B and 9-deazainosine of 0.01 and 2 micrograms/ml, respectively. Metabolism was the same in both the bloodstream and culture forms and identical to that reported for Leishmania spp. and T. cruzi. Both agents were phosphorylated to the ribonucleotide and then aminated to produce adenine nucleotide analogs. Growth inhibition studies were performed with three inosine analogs (allopurinol riboside, formycin B, and 9-deazainosine) on trypomastigotes grown in bone marrow tissue culture. Both C-nucleosides eradicated the infection at a concentration of 0.25 micrograms/ml. Unlike formycin B, 9-deazainosine is not known to be aminated by mammalian cells and appears to be relatively nontoxic in three different mammalian tissue culture systems. This nucleoside was very active against all pathogenic leishmaniae and trypanosomes investigated and is worthy of further study.

Biosynthesis of the novel fatty acid, 17-methyl-cis-9,10-methyleneoctadecanoic acid, by the parasitic protozoan, Herpetomonas megaseliae.

Herpetomonas megaseliae, a flagellate protozoan parasite of the gut of a dipteran, Megaselia scalaris, is shown by chromatographic, spectrometric and radiotracer methods to synthesize de novo an iso-branched chain cyclopropane fatty acid, 17-methyl-cis-9,10-methyleneoctadecanoic acid.

Purine metabolism in the bloodstream forms of Trypanosoma gambiense and Trypanosoma rhodesiense.

Bloodstream forms of Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense are incapable of de novo purine synthesis. Purine bases are converted directly to ribonucleotides and with the exception of guanine, are stable. Guanine is incorporated directly into ribonucleotides and also deaminated to xanthine. Purine ribonucleosides are hydrolyzed rapidly; these reactions may limit their incorporation since purine bases label the nucleotide pools more efficiently than do ribonucleosides. The apparent order of salvage efficiency for ribonucleosides is adenosine greater than inosine greater than guanosine greater than xanthosine for both organisms. T. b. gambiense salvages purine bases in the same order, while T. b. rhodesiense salvages purine bases in the order hypoxanthine greater than adenine greater than guanine greater than xanthine.