Trypanosoma rangeli: characterization of a Mg-dependent ecto ATP-diphosphohydrolase activity

In this work we describe the ability of living Trypanosoma rangeli to hydrolyze extracellular ATP. In these intact parasites whose viability was assessed before and after the reactions by motility and by Trypan blue dye exclusion, there was a low level of ATP hydrolysis in the absence of any divalent metal (1.53+/-0.12 nmol P(i)/h x 10(7) cells). The ATP hydrolysis was stimulated by MgCl(2) and the Mg-dependent ecto-ATPase activity was 5.24+/-0.64 nmol P(i)/h x 10(7) cells. The Mg-dependent ecto-ATPase activity was linear with cell density and with time for at least 60 min. This stimulatory effect on the ATP hydrolysis was also observed when MgCl(2) was replaced by MnCl(2), but not by CaCl(2), SrCl(2), and ZnCl(2). The apparent K(m) for Mg-ATP2- was 0.53+/-0.11 mM. The optimum pH for the T. rangeli Mg-dependent ecto-ATPase activity lies in the alkaline range. This ecto-ATPase activity was insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A1, ouabain, furosemide, vanadate, molybdate, sodium fluoride, tartrate, and levamizole. To confirm that this Mg-dependent ATPase was an ecto-ATPase, we used an impermeant inhibitor, DIDS (4, 4'-diisothiocyanostylbene 2'-2'-disulfonic acid) as well as suramin, an antagonist of P2 purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg(2+)-dependent ATPase activity in a dose-dependent manner. This ecto-ATPase activity was stimulated by carbohydrates involved in the attachment/invasion of salivary glands of Rhodnius prolixus and by lipophorin, an insect lipoprotein circulating in the hemolymph.     

A sialidase mutant displaying trans-sialidase activity

Trypanosoma cruzi, the agent of Chagas disease, expresses a modified sialidase, the trans-sialidase, which transfers sialic acid from host glycoconjugates to beta-galactose present in parasite mucins. Another American trypanosome, Trypanosoma rangeli, expresses a homologous protein that has sialidase activity but is devoid of transglycosidase activity. Based on the recently determined structures of T.rangeli sialidase (TrSA) and T.cruzi trans-sialidase (TcTS), we have now constructed mutants of TrSA with the aim of studying the relevant residues in transfer activity. Five mutations, Met96-Val, Ala98-Pro, Ser120-Tyr, Gly249-Tyr and Gln284-Pro, were enough to obtain a sialidase mutant (TrSA(5mut)) with trans-sialidase activity; and a sixth mutation increased the activity to about 10% that of wild-type TcTS. The crystal structure of TrSA(5mut) revealed the formation of a trans-sialidase-like binding site for the acceptor galactose, primarily defined by the phenol group of Tyr120 and the indole ring of Trp313, which adopts a new conformation, similar to that in TcTS, induced by the Gln284-Pro mutation. The transition state analogue 2,3-didehydro-2-deoxy-N-acetylneuraminic acid (DANA), which inhibits sialidases but is a poor inhibitor of trans-sialidase, was used to probe the active site conformation of mutant enzymes. The results show that the presence of a sugar acceptor binding-site, the fine-tuning of protein-substrate interactions and the flexibility of crucial active site residues are all important to achieve transglycosidase activity from the TrSA sialidase scaffold.     

Trypanosoma rangeli uptakes the main lipoprotein from the hemolymph of its invertebrate host

During its life cycle Trypanosoma rangeli crosses the hemolymph of its invertebrate host. In the present study, we demonstrate for the first time the uptake of lipophorin (Lp), the main lipid-transporting particle of insect hemolymph. We observed that living T. rangeli parasites uptake lipids from both 32P- and 3H-, or 125I-labeled Lp. However, the parasites do not uptake any other hemolymphatic protein such as 32P-labeled vitellogenin. The presence of a specific receptor to Lp in the parasite surface is suggested based on experiments using 125I-Lp. We also investigated the intracellular fate of lipids using Texas Red-labeled phosphatidylethanolamine-Lp. Parasites were observed under confocal microscope and displayed fluorescent-labeled lipids close to the flagellar pocket and in vesicles at the posterior region. In conclusion, this study raises a novel set of molecular events which takes place during vector-parasite interaction.     

Substrate specificities of a bacterial sialidase and rat liver ganglioside GM3 sialyltransferase

Sialidases cleave off sialic acid residues from the oligosaccharide chain of gangliosides in their catabolic pathway while sialyltransferases transfer sialic acid to the growing oligosaccharide moiety in ganglioside biosynthesis. Ganglioside G_M3 is a common substrate for both types of enzymes, for sialidase acting on ganglioside G_M3 as well as for ganglioside G_D3 synthase. Therefore, it is possible that both enzymes recognize similar structural features of the sialic acid moiety of their common substrate, ganglioside G_M3. Based on this idea we used a variety of G_M3 derivatives as glycolipid substrates for a bacterial sialidase (Clostridium perfringens) and for G_D3 synthase (of rat liver Golgi vesicles). This study revealed that those G_M3 derivatives that were poorly degraded by sialidase also were hardly recognized by sialyltransferase (G_D3 synthase). This may indicate similarities in the substrate binding sites of these enzymes.     

Identification of a developmentally regulated sialidase inEimeria tenella that is immunologically related to theTrypanosoma cruzi enzyme

Sporozoites and merozoites of three species ofEimeria, E. tenella, E. maxima, andE. necatrix, that cause diarrhea in chickens worldwide, were examined for their expression of sialidase (SA) activity. The enzyme was found in three species, and the activity of merozoites was 10–20 times higher than that of sporozoites. The enzyme was resistant to degradation by proteases that are normally present in the intestine, a site inhabited by theEimeria parasites, and it was relatively resistant to heat, with optimum activity being at 40°C, which is within the range of temperature in the chicken intestine (40–43°C).E. tenella SA was immuniprecipitated by monoclonal and polyclonal antibodies raised against theTrypanosoma cruzi SA (TCSA), and enzyme activity was neutralized by these antibodies.E. tenella SA was identified by immunoblots as a doublet of molecular weight 190 000 and 180 000 using, as a probe, anti-TCSA antibodies and antibodies against a synthetic peptide (TR) derived from the long tandem repeat domain of TCSA. Binding of the monoclonal and polyclonal antibodies toE. tenella was completely blocked by TR, but not by an irrelevant peptide (BR). Therefore,E. tenella expresses a developmentally regulated SA that is structurally related to theT. cruzi counterpart. Because of the high SA activity in merozoites, and by analogy with other SA-producing microbes that inhabit mucin-rich epithelia, we suggest that theEimeria SA plays a role in desialylating intestinal mucins to reduce viscosity of the local environment and thereby facilitate parasite migration. The enzyme could also play a role in host cell-parasite interaction.Abbreviations SA sialidase (neuraminidase) - Neu5Ac N-acetylneuraminic acid - 4-MU-Neu5Ac 2-(4-methylumbelliferyl)--N-acetyl-d-neuraminic acid - BSA bovine serum albumin - PBS phosphate buffered saline - PMSF phenylmethylsulfonyl fluoride - PNA peanut agglutinin - Ab antibody - TCN-2 monoclonal antibody toT. cruzi sialidase, anti-Ars, monoclonal antibody top-azophenylarsonate - TCSA Trypanosoma cruzi sialidase     

WEB 2086, a platelet-activating factor antagonist, inhibits prophenoloxidase-activating system and hemocyte microaggregation reactions induced by Trypanosoma rangeli infection in Rhodnius prolixus hemolymph

The effects of the triazolodiazepine WEB 2086, a platelet-activating factor (PAF) antagonist, on hemocyte microaggregation and prophenoloxidase (proPO)-activating system in the hemolymph, hemocoelic infection and mortality in fifth-instar larvae of Rhodnius prolixus inoculated with Trypanosoma rangeli were investigated. Hemocoelic injection of short T. rangeli epimastigotes (1x10(4) parasites/insect) in R. prolixus that were previously fed with blood containing 1muM of WEB 2086 resulted in (i) reduced hemocyte microaggregations as well as an attenuated proPO system in the hemolymph and (ii) greater parasitemia and mortality among the insects. In vitro assays using hemolymph from insects previously fed with blood containing WEB 2086 exhibited attenuated hemocyte microaggregations when T. rangeli was employed as the inducer of the reaction, and this effect was not counteracted by PAF treatment. In vitro assays using hemolymph from insects previously fed with blood, regardless of WEB 2086 presence increased the PO activity when incubated with the parasites. However, the PO activity was drastically inhibited when hemolymph from insects fed with blood, whether or not it contained WEB 2086, was incubated with fat body homogenates from insects fed with blood containing WEB 2086. The addition of PAF did not enhance the PO activity. These analyses did not reveal any PAF influence on WEB 2086 effects in the two defense reactions.     

Isolation and properties of the natural and the recombinant sialidase fromClostridium septicum NC 0054714

The natural sialidase ofClostridium septicum was purified and characterized in parallel with the recombinant enzyme expressed byEscherichia coli. The two enzymes exhibit almost identical properties. The maximum hydrolytic activity was measured at 37 °C in 60mm sodium acetate buffer, pH 5.3. Glycoproteins like fetuin and saponified bovine submandibular gland mucin, most of them having (2-6) linked sialic acids, are preferred substrates, while sialic acids from gangliosides, sialyllactoses, or the (2-8) linked sialic acid polymer (colominic acid) are hydrolysed at lower rates. (2-3) Linkages are more rapidly hydrolysed than (2-6) bonds of sialyllactoses. The cleavage rate is markedly reduced by O-acetylation of the sialic acid moiety. These properties are similar to those of other secreted clostridial sialidases. The enzyme exists in mono-, di- and trimeric forms, the monomer exhibiting a molecular mass of 125 kDa, which is close to the protein mass of 111 kDa deduced from the nucleotide sequence of the cloned gene.Abbreviations BSM bovine submandibular gland mucine - CMM cooked meat medium - EDTA ethylenediaminetetraacetic acid - FPLC fast performance liquid chromatography - LB Luria-Bertani - MU-Neu5Ac 4-methylumbelliferyl--d-N-acetylneuraminic acid - Neu5Ac N-acetylneuraminic acid - Neu5Ac2en 2-deoxy-2,3-didehydro-N-acetylneuraminic acid - Neu4,5Ac₂ N-acetyl-4-O-acetylneuraminic acid - pI isoelectric point - SDS sodium dodecyl sulfate     

Antigenic significance of a Trypanosoma rangeli sialidase

The Trypanosoma rangeli-secreted sialidase was purified by bovine submaxillary gland mucin-sepharose affinity chromatography. In immunoblotting analysis, antibodies raised against this molecule recognized polypeptides of 73 kDa in T. rangeli medium supernatant (TrSialr) and of 70 kDa in the cell lysates of T. rangeli (TrSials) and T. cruzi (TcSialL) epimastigotes. TrSialr, TrSials, and TcSialL were subjected to proteolytic cleavage with papain; the resultant peptide pattern displayed differences in the immunoblotting profiles. TrSials was purified by immunoprecipitation, and this protein band was recognized by sera from T. cruzi-infected chronic mice and Chagas' disease patients. In contrast, TrSialr was not recognized by these sera. The antibodies from the infected mice also recognized a band of 70 kDa present in the medium. These preliminary observations imply that the released and somatic sialidases are partially different molecules, with probably different biological roles. The related proteins recognized in T. rangeli and T. cruzi epimastigotes share many antigenic characteristics but have some structural differences, probably related to their function in the parasitic cell. On the basis of the strong antigenicity of TrSials, this molecule is proposed as the antigen for the detection of antibodies arising during T. cruzi infection.     

Lipid mediators and vector infection: Trypanosoma rangeli inhibits Rhodnius prolixus hemocyte phagocytosis by modulation of phospholipase A2 and PAF-acetylhydrolase activities

In this work we investigated the effects of Trypanosoma rangeli infection through a blood meal on the hemocyte phagocytosis in experiments using the 5th instar larvae of Rhodnius prolixus. Hemocyte phagocytic activity was strongly blocked by oral infection with the parasites. In contrast, hemocyte phagocytosis inhibition caused by T. rangeli infection was rescued by exogenous arachidonic acid (20 microg/insect) or platelet activating factor (PAF; 1 microg/insect) applied by hemocelic injection. Following the oral infection with the protozoan we observed significant attenuation of phospholipase A2 (PLA2) activities in R. prolixus hemocytes (cytosolic PLA2: cPLA2, secreted PLA2: sPLA2 and Ca+2-independent PLA2: iPLA2) and enhancement of sPLA2 activities in cell-free hemolymph. At the same time, the PAF-acetyl hydrolase (PAF-AH) activity in the cell-free hemolymph increased considerably. Our results suggest that T. rangeli infection depresses eicosanoid and insect PAF analogous (iPAF) pathways giving support to the role of PLA2 in the regulation of arachidonic acid and iPAF biosynthesis and of PAF-AH by reducing the concentration of iPAF in R. prolixus. This illustrates the ability of T. rangeli to modulate the immune responses of R. prolixus to favor its own multiplication in the hemolymph.     

Purification and characterization of sialidase fromClostridium sordellii G12

Sialidase secreted by the urease-positiveClostridium sordellii strain G12 was isolated from culture medium and purified to apparent homogeneity as estimated by Fast Protein Liquid Chromatography (FPLC) and sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE). For this purpose, ion-exchange chromatography, gel filtration, isoelectric focusing, and FPLC on ion-exchange resin and gel filtration materials were used. The sialidase was purified 159 300-fold from 5 l of culture medium, yielding 9 g of enzyme protein with a specific activity of 480 U/mg. For the denatured (SDS-PAGE) and native (FPLC) sialidase relative molecular masses of 40 000 and 38 500 Da, respectively, were estimated. The substrate specificity, kinetic data, and pH-optimum of the enzyme are similar to those of other bacterial sialidases. The influences of salt or serum proteins on enzyme activity are of interest.Abbreviations MU-Neu5Ac 4-methylumbelliferyl -d-N-acetylneuraminic acid - Ganglioside GD1a IV³NeuAc, ll³NeuAc-GgOse₄Cer - Neu5Ac2en 2-deoxy-2,3-didehydro-N-acetylneuraminic acid     

Trypanosoma rangeli: effects of physalin B on the immune reactions of the infected larvae of Rhodnius prolixus

Physalins are seco-steroids obtained from plants of the family Solanaceae. Herein, we tested Physalis angulata L purified physalin B as an immunomodulatory compound in 5th-instar larvae of Rhodnius prolixus, which were systemically infected with the H14 Trypanosoma rangeli strain protozoan. In uninfected insects, the effective concentration of physalin B, which inhibited 50% of the blood ingested (ED(50)) volume, was 15.2+/-1.6 microg/ml of the meal. Ecdysis processes and mortality in uninfected larvae, treated orally with physalin B in concentrations ranging from 1 to 10 microg/ml, was similar to that observed in insects not treated with physalin B. However, R. prolixus larvae previously fed on blood containing 1.0, 0.1, and 0.01 microg of physalin B/ml exhibited mortality rates of 78.1, 54.3, and 12.7%, respectively, 6 days after inoculation of T. rangeli (1 x 10(3) parasites/insect), whereas only 7.2% mortality was observed in the control group, injected with sterile culture medium. The insects treated with physalin B (0.1 microg/ml) and inoculated with T. rangeli did not modify the phenoloxidase (PO) activity and total hemocyte count in the hemolymph. However, physalin B treatment caused a reduction in hemocyte micro-aggregation and nitric oxide production and enhanced the parasitemia in the hemolymph. These results demonstrate that physalin B from P. angulata is a potent immunomodulatory substance for the bloodsucking insect, R. prolixus.     

Isolation and properties of a lectin from the seeds ofMimosa invisa L.

A lectin has been purified from the seeds ofMimosa invisa L. by gel filtration and preparative Polyacrylamide gel electrophoresis. The purified lectin was homogeneous as judged by analytical Polyacrylamide gel electrophoresis, immunodiffusion and Immunoelectrophoresis. The apparent molecular weight is 100,000; the protein is a tetramer with two types of subunits (molecular weight 35,000 and 15,000). The lectin is a glycoprotein with approximately 21% carbohydrate and interacts with Sephadex and concanavalin A-Sepharose. It agglutinates erthrocytes non-specifically, does not agglutinate leucocytes and is not mitogenic, agglutinates Mimosa-nodulatingRhizobium and is a panagglutinin; the agglutination is not inhibited by several simple sugars. It is thermo-stable and has no metal ions.     

Resolution of multiclonal infections of Trypanosoma cruzi from naturally infected triatomine bugs and from experimentally infected mice by direct plating on a sensitive solid medium

The isolation of biological clones of Trypanosoma cruzi by microscopically dispensing individual organisms or by serial dilution is laborious and time consuming. The inability to resolve mixed T. cruzi infections, from vectors and hosts, and to isolate clones of slow growing genotypes by efficient plating on solid media, has hindered characterisation studies and downstream applications. We have devised and validated a sensitive, solid medium plating technique for rapid in vitro isolation of clones representative of all the recognised T. cruzi lineages (TCI, TCIIa-e), including the slow growing strain CANIII (TC IIa) and Trypanosoma rangeli, with high plating efficiencies. Furthermore, the method is effective for the isolation of clones directly from silvatic triatomine bugs and from experimentally infected mice harbouring mixed infections, allowing resolution of multiclonal infections from varied sources.     

Effects of site-specific mutations on the enzymatic properties of a sialidase fromClostridium perfringens

Three site-specific mutations were performed in two regions of a sialidase gene fromClostridium perfringens which are known to be conserved in bacterial sialidases. The mutant enzymes were expressed inEscherichia coli and, when measured with MU-Neu5Ac as substrate, exhibited variations in enzymatic properties compared with the wild-type enzyme. The conservative substitution of Arg 37 by Lys, located in a short conserved region upstream from the four repeated sequences common in bacterial sialidase genes, was of special interest, asK _M andV _max, as well asK _i measured with Neu5Ac2en, were dramatically changed. These data suggest that this residue may be involved in substrate binding. In addition to its low activity, this mutant enzyme has a lower temperature optimum and is active over a more limited pH range. This mutation also prevents the binding of an antibody able to inhibit the wild-type sialidase. The other mutations, located in one of the consensus sequences, were of lower influence on enzyme activity and recognition by antibodies.     

Trypanosoma cruzi: variability of stocks from Colombia determined by molecular karyotype and minicircle Southern blot analysis

Nineteen Trypanosoma cruzi stocks, most of them of wild origin, and four Trypanosoma rangeli stocks from Colombia were analysed by molecular karyotype analysis with cloned DNA cruzipain as the probe. Another 27 cloned stocks of T. cruzi from different geographic areas of South America were used as reference for T. cruzi lineages. Phenetic analysis of chromosome size polymorphism demonstrated a great variability of Colombian T. cruzi stocks, suggesting that most belong to lineage I, although two of them belong to lineage II. The 2 lineage II T. cruzi, 17 T. cruzi lineage I, and 3 T. rangeli stocks from Colombia were studied further by Southern blot analysis with a panel of kinetoplast DNA minicircle probes. Hybridisation results indicate that the two T. cruzi II stocks are genetically distant from each other and from T. cruzi lineages IIb, IId, and IIe from Chile. Finally, T. cruzi minicircle probes do not cross-hybridise in any stringency condition tested with T. rangeli minicircles, a clear indication that these parasites can be easily distinguished by this method.     

The interaction ofClostridium perfringens sialidase with immobilized sialic acids and sialyl-glycoconjugates

Clostridium perfringens sialidase is adsorbed by sialic acid immobilized on adipic acid dihydrazido-Sepharose 4B and/or polymethylacrylic hydrazido-Sepharose 4B, through its carboxyl group, C-7 to C-9 side chain, or its amino function asd-neuraminic acid--methyl glycoside or 2-deoxy-2,3-didehydroneuraminic acid. Sialidase binding was strongest to the amino-linked adsorbents, but purification was low and the enzyme could not be eluted with substrate or free sialic acid. Low binding of the sialidase to the non-substituted, blocked supports suggested that hydrophobic interactions were involved, and this was confirmed by adsorption of the enzyme on alkyl agaroses with approximately 80% of total sialidase adsorbed on decyl-agarose. Genuine affinity chromatography of sialidases is possible on immobilized sialyl-glycoconjugates, andC. perfringens sialidase could be purified to the same specific activity as the electrophoretically homogeneous enzyme using submandibular gland mucus glycoprotein adsorbents. Sialidases fromVibrio cholerae, Arthrobacter ureafaciens, Newcastle disease virus, Fowl plague virus and Influenza A₂ virus also bound to immobilized sialic acids and sialyl-glycocojugates.Dedicated to Prof. Dr. Hans Faillard on the occasion of his 60th birthday.     

New ganglioside analogs that inhibit influenze virus sialidase

Synthetic thioglycoside-analogs of gangliosides such as Neu5Ac)2-S-6)Glc-(1-1)Ceramide (1) and the GM3 analog Neu5Ac(2-S-6)Gal-(1–4)Glc(1-1)Ceramide (2), competitively inhibited GM3 hydrolysis by the sialidase of different subtypes of human and animal influenza viruses with an apparent K_i value of 2.8×10^–6 and 1.5×10^–5 M, respectively. The inhibitory activity of the ganglioside GM4 analog [Neu5Ac-(2-S-6)Gal-(1-1)Ceramide (3)], in which the glucose of 1 was substituted by galactose, was lower than that of 1 (K_i =1.0×10^–4 M). The thioglycoside-analogs (1, 2, 3) of the gangliosides were nonhydrolyzable substrates for influenza virus sialidase. The inhibitory activity of 1 to bacterial sialidases fromClostridium perfringens andArthrobacter ureafaciens was considerably lower than that to influenza virus sialidase, indicating that the structure of the active site in bacterial and influenza virus sialidase may be different and the analogs may be useful to determine the orientation of the substrate to the active site of sialidases, especially of influenza viruses.Abbreviations Cer ceramide - GM3 Neu5Ac(2–3)Gal(1–4)Glc(1-1)Cer - GM4 Neu5Ac(2–3)Gal(1-1)Cer Gangliosides were abbreviated according to Svennerholm [1] and the recommendation of the IUPAC-IUB Commission on Biochemical Nomenclature [2].     

Purification and characterization of a sialidase fromClostridium chauvoei NC08596

The sialidase secreted byClostridium chauvoei NC08596 was purified to apparent homogeneity by ion-exchange chromatography, gel filtration, hydrophobic interaction-chromatography, FPLC ion-exchange chromatography, and FPLC gel filtration. The enzyme was enriched about 10 200-fold, reaching a final specific activity of 24.4 U mg^–1. It has a relatively high molecular mass of 300 kDa and consists of two subunits each of 150 kDa. The cations Mn²⁺, Mg²⁺, and Ca²⁺ and bovine serum albumin have a positive effect on the sialidase activity, while Hg²⁺, Cu²⁺, and Zn²⁺, chelating agents and salt decrease enzyme activity. The substrate specificity, kinetic data, and pH optimum of the enzyme are similar to those of other bacterial sialidases.Abbreviations FPLC fast protein liquid chromatography - NCTC National Collection of Type Cultures - ATCC American Type Culture Collection - MU-Neu5Ac 4-methylumbelliferyl--d-N-acetylneuraminic acid - buffer A 0.02m piperazine, 0.01m CaCl₂, pH 5.5 - buffer B 0.02m piperazine, 0.01m CaCl₂, 1.0m NaCl, pH 5.5 - buffer C 0.1m sodium acetate, 0.01m CaCl₂, pH 5.5 - SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis - Neu5Ac N-acetylneuraminic acid - BSM bovine submandibular gland mucin - GD1a IV³Neu5Ac, II³Neu5Ac-GgOse₄Cer - GM1 II³Neu5Ac-GgOse₄Cer - MU-Neu4,5Ac₂ 4-methylumbelliferyl--d-N-acetyl-4-O-acetylneuraminic acid - TLC thin-layer chromatography - HPTLC high performance thin-layer chromatography - EDTA ethylenediamine tetraacetic acid - EGTA ethylene glycol bis(2-aminoethyl-ethen)-N,N,N,N-tetraacetic acid - BSA bovine serum albumin - Neu5Ac2en 2-deoxy-2,3-didehydro-N-acetylneuraminic acid - IEF isoelectric focusing - IEP isoelectric point     

Trypanosoma rangeli: A possible role for ecto-phosphatase activity on cell proliferation

Here we demonstrate for the first time that growth of Trypanosoma rangeli, a protozoa parasite, is strongly dependent on the presence of inorganic phosphate (Pi) in the culture medium and that the replacement of the inorganic phosphate in the culture medium by β-glycerophosphate, a substrate for phosphatases lead the cells to achieve its maximal growth. The ecto-phosphatase activity present on the external surface of T. rangeli decreased during the growth phase of the parasite, suggesting that this enzyme could be important for the development. Accordingly, the inhibition of this ecto-phosphatase activity by sodium orthovanadate also inhibited the proliferation of T. rangeli. Parasites maintained in a Pi-starved culture medium (2 mM Pi) had 4-fold more ecto-phosphatase activity as compared to parasites maintained in a Pi-supplemented culture medium (50 mM Pi). Altogether, these results presented here suggest that this ecto-phosphatase activity leads to hydrolysis of phosphorylated compounds present in the extracellular medium, which could contribute to the acquisition of inorganic phosphate during the development of T. rangeli epimastigotes.     

Isolation and some properties of ciguatoxin

Ciguatoxin, the principal toxin of ciguatera produced by a toxic dinoflagellate and accumulated through the food chain in the viscera of moray eels, was purified. The probable molecular formula C₆₀H₈₆O₁₉ was obtained for the first time by high resolution mass spectrometry. ¹H NMR spectra suggested the presence of a primary hydroxyl group suitable for preparing an antigen necessary to develop an enzyme immunoassay. author for correspondence