Chromosomal size variation in Trypanosoma cruzi is mainly progressive and is evolutionarily informative.

The evolutionary significance of chromosome size polymorphism was explored in a representative panel of 26 Trypanosoma cruzi stocks. We tested a progressive model (aCSDI) assuming that the larger the size difference between homologous chromosomes, the more divergent the parasites are. This was contrasted with a non-progressive model (Jaccard's distance), in which any chromosome size difference has the same weight. ACSDI-based dendrograms were very similar to those built-up from multilocus enzyme electrophoresis (MLEE) and random amplified polymorphic DNA (RAPD) data: structuring in 2 major lineages (T. cruzi I and T. cruz II) and 5 small subdivisions within T. cruzi II was identical, and branching was very similar. Furthermore, a significant correlation (P < 0.001) was observed between aCSDI and phenetic distances calculated from MLEE and RAPD data. In contrast, analysis of chromosome size polymorphism with Jaccard's distance generated dendrograms with relatively long branches, causing most branching points to cluster close together, which generates statistically uncertain branching points. Our results thus support a model of progressive chromosome size-variation and show that despite an extensive polymorphism, chromosomal sizes constitute valuable characters for evolutionary analyses. Furthermore, our data are consistent with the clonal evolution model previously proposed for T. cruzi.

A comparison of the stage-specific efficacy of chloroquine, artemether and dioncophylline B against the rodent malaria parasite Plasmodium chabaudi chabaudi in vivo.

Chloroquine, artemether and dioncophylline B efficacy against Plasmodium chabaudi was compared. One intraperitoneal injection (10 mg/kg body weight) was given daily over 3 consecutive days to OF1 mice when they were predominantly bearing ring, trophozoite and schizont forms. The parasitaemia was monitored every 2 h during two schizogonic cycles and daily thereafter until parasites were cleared. Chloroquine was more efficient at the trophozoite stage, while artemether was effective against all erythrocytic stages, with a marked efficacy against the trophozoite stage. Chloroquine-treated and artemether-treated parasites displayed a pigment-clumping morphology and lowered the parasitaemia faster than dioncophylline B. Dioncophylline B was effective at trophozoite and schizont stages, but completely ineffective at the ring stage. These results demonstrate that a better timing of drug administration increases the efficacy of common and new antimalarial drugs and provides a model for antimalarial-action monitoring. Drug-induced changes in infected erythrocyte morphology are presented.

Differential sensitivity of erythrocytic stages of the rodent malaria parasite Plasmodium chabaudi chabaudi to dioncophylline B, a highly active naphthylisoquinoline alkaloid.

Four-week-old OF1 mice, infected with synchronized Plasmodium chabaudi chabaudi blood forms, were intraperitoneally injected with the naphthylisoquinoline alkaloid dioncophylline B (10 mg kg(-1) day(-1)) at three consecutive days. The respective groups were treated when rings, trophozoites, and schizonts were predominant. Microscopical observations of thin blood smears were made every two hours after the start of the experiment. A clear dependency of the effectiveness of dioncophylline B treatments on the timing of drug administration was demonstrated. Based upon the evolution of total parasitaemia and the survival rates, it was concluded that ring stages are insensitive to dioncophylline B, while the drug is highly effective when given at the trophozoite stage and partially effective when given at the schizont stage. Dioncophylline B seems to act by inhibiting the haemozoin degradation, as indicated by pigment clumping, and by impairing the segmentation of schizonts.

Antimalarial and cytotoxic potential of four quassinoids from Hannoa chlorantha and Hannoa klaineana, and their structure-activity relationships.

Hannoa chlorantha and Hannoa klaineana (Simaroubaceae) are used in traditional medicine of Central African countries against fevers and malaria. Four stem bark extracts from H. klaineana and four quassinoids from H. chlorantha were examined in vitro against Plasmodium falciparum NF 54. The extracts displayed good activities, while the quassinoids were highly active, with IC50 values well below 1 microgram ml-1, those of chaparrinone and 15-desacetylundulatone being much lower than 0.1 microgram ml-1 (0.037 and 0.047 microgram ml-1, respectively). Chaparrinone is five times more active than 14-hydroxychaparrinone against P. falciparum, indicating that the hydroxyl function at C-14 is unfavourable for antiplasmodial activity. As 14-hydroxychaparrinone has a seven-times higher cytotoxic activity against P-388 cells than chaparrinone, the latter compound has the better antiplasmodial therapeutic index. All four quassinoids were evaluated in vivo in a standard 4-day test as well. 15-Desacetylundulatone was proven to be the most active compound, almost totally suppressing the parasitaemias of OF1 mice for at least 7 days, while both chaparrinone and 14-hydroxychaparrinone were active for at least 4 days. Quassinoids have ED50 values much lower than 50 mg kg-1 body weight day-1 and none of them caused obvious side effects. The keto function at C-2 in 15-desacetylundulatone is apparently of crucial importance for its high activity. 6-alpha-Tigloyloxyglaucarubol was not active at all. Chaparrinone is considered the most interesting of the investigated quassinoids and its in-vivo antimalarial potential will be examined further.

Naphthylisoquinoline alkaloids against malaria: evaluation of the curative potentials of dioncophylline C and dioncopeltine A against Plasmodium berghei in vivo.

Naphthylisoquinoline alkaloid-containing extracts from species of the families Dioncophyllaceae and Ancistrocladaceae and purified alkaloids derived therefrom were shown to exhibit antiparasitic activity in Plasmodium berghei-infected mice. Several extracts and alkaloids, especially dioncophylline C and dioncopeltine A, isolated from Triphyophyllum peltatum (Dioncophyllaceae), displayed high levels of activity. Dioncopeltine A was able to suppress parasitemia almost totally, while dioncophylline C cured infected mice completely after oral treatment with 50 mg kg of body weight(-1) day(-1) for 4 days without noticeable toxic effects. Analysis of the dose-response relationship of dioncophylline C revealed a 50% effective dosage (ED50) of 10.71 mg kg(-1) day(-1) under these conditions. Although four daily treatments with 50 mg kg(-1) day(-1) are needed to achieve radical cure, one oral dose is sufficient to kill 99.6% of the parasites. Intravenous application of dioncophylline C is even more effective, with an ED50 of 1.90 mg kg(-1) day(-1) and no noticeable toxic effects. The compound also suppressed more established P. berghei infections when orally applied at day 3 after infection. Both dioncopeltine A and dioncophylline C are active against the chloroquine-resistant P. berghei Anka CRS parasites. Sustained release of these compounds at 20 mg kg(-1) day(-1) by implanted miniosmotic pumps exhibited curative effects. The naphthylisoquinoline alkaloids are therefore promising new antimalarial agents.

In vitro inhibition of liver forms of the rodent malaria parasite Plasmodium berghei by naphthylisoquinoline alkaloids--structure-activity relationships of dioncophyllines A and C and ancistrocladine.

Naphthylisoquinoline alkaloids are derived from Dioncophyllaceae and Ancistrocladaceae species and comprise a new class of promising antimalarials with a demonstrated potential against asexual erythrocytic Plasmodium falciparum and P. berghei stages in vitro. We report herein the pronounced activity of pure naphthylisoquinoline alkaloids against exoerythrocytic malaria parasites. P. berghei-infected human hepatoma cells (Hep G2) were incubated with culture medium containing selected alkaloids at 10 micrograms/ml. The most active compounds, showing inhibitory activity of more than 40%, were dioncophylline A (compound 1), dioncophyllacine A (compound 6), and ancistrobarterine A (compound 12). For structure-activity investigations of dioncophyllines A (compound 1) and C (compound 3) and ancistrocladine (compound 7) a selection of their analogs from natural or synthetic sources was examined. Dioncophylline A (compound 16), 5'-O-demethyl-8-O-methyl-7-epi-dioncophylline A (compound 17), N-formyl-8-O-methyl-dioncophylline C (compound 21), and N-formyl-8-O-benzoyldioncophylline C (compound 24) were found to display high levels of activity as well, although the former two compounds caused damage to the host-cell monolayers. As naphthylisoquinoline alkaloids are also highly active against blood forms of Plasmodium spp., they should be regarded as lead compounds for further development as drugs against erythrocytic and exoerythrocytic stages of Plasmodium spp.

Retarded development of exoerythrocytic stages of the rodent malaria parasite Plasmodium berghei in human hepatoma cells by extracts from Dioncophyllaceae and Ancistrocladaceae species.

Retarded development of exoerythrocytic stages of the rodent malaria parasite Plasmodium berghei in human hepatoma cells by extracts from Dioncophyllaceae and Ancistrocladaceae species. International Journal for Parasitology 27: 29-32. Naphthylisoquinoline alkaloid-containing extracts (10 micrograms ml-1) of species belonging to the Dioncophyllaceae and the Ancistrocladaceae, 2 small tropical plant families, display pronounced in vitro activities against exoerythrocytic stages of Plasmodium berghei (Anka), developing in human hepatoma cells (Hep G2). The highest activities were obtained with CH2Cl2 root and bark extracts, and a CH2Cl2/NH3 leaf extract from Triphyophyllum peltatum, a CH2Cl2/NH3 root extract from Ancistrocladus abbreviatus, and a CH2Cl2 leaf extract from A. tectorius. The degrees of growth inhibition ranged within 27.7-70.0%. The commercially available drug primaquine diphosphate (25 micrograms ml-1) caused a comparable effect (62.1%) in the same test system.

Chronobiology of Plasmodium chabaudi chabaudi: analysis of hourly recorded total and differential parasitaemia during a schizogonic cycle.

Detailed total and differential parasitaemia curves of asexual Plasmodium chabaudi chabaudi erythrocytic stages were recorded and analysed. Female, inbred, CBA/Ca mice were infected with the virulent IP-PC1 strain after in vitro synchronization of the parasites. Thin blood smears were made on an hourly basis, and the total and differential parasitaemia of ring forms, trophozoites and schizonts were counted after Giemsa staining. These curves reveal information that remains hidden when less detailed curves are examined: the duration and periodicity of the schizogonic cycle, the existence of a plateau, indications of a schizont withdrawal from the peripheral blood, the timing of the rise of the parasitaemia at each schizogony, and the invasion rate of the merozoites. In the perspective of developing a rational and efficient strategy for chronotherapy of malaria, such information should be taken into account.

Larvicidal activity of the naphthylisoquinoline alkaloid dioncophylline A against the malaria vector Anopheles stephensi.

The larvicidal activity of dionocophylline A, a naphthylisoquinoline alkaloid derived from the tropical vine Triphyophyllum peltatum (Dioncophyllaceae), was investigated against the malaria vector Anopheles stephensi. In direct and indirect inhibition assays it was demonstrated that the younger larval stages were very sensitive towards this natural product, with LC50 values below 1 mg/l. Pronounced effects were observed within 4 h of exposure. Aging larvae, however, were less sensitive, while pupae were totally insensitive to the action of dioncophylline A. The transformations from larvae to pupae and from pupae to adult mosquitoes remained unaffected. Therefore, dioncophylline A can be regarded as a promising specific larvicide.

Naphthylisoquinoline alkaloids exhibit strong growth-inhibiting activities against Plasmodium falciparum and P. berghei in vitro--structure-activity relationships of dioncophylline C.

The growth-inhibiting activities of naturally occurring naphthylisoquinoline alkaloids against asexual blood stages of Plasmodium falciparum (NF 54, clone A1A9) and P. berghei (Anka) were studied in vitro. Three of the alkaloids [7-epi-dioncophylline A (8b), dioncolactone A (4), and 5'-O-demethyl-8-O-methyl-7-epidioncophylline A (11)] displayed good activities against both parasites, with median inhibitory concentrations (IC50) of 1-5 micrograms/ml. Dioncophylline C (2), however, was even better, with IC50 of 0.014 microgram/ml (P. falciparum) and 0.015 microgram/ml (P. berghei) and therefore regarded as a promising lead for studies of structure-activity relationships. The free N- and 8-OH-functions were shown to be prerequisites for the outstanding activity of this molecule against P. falciparum, the presence of at least one free phenolic OH-function appearing to be essential for any activity. Initial experiments with derivatives of ancistrocladine (1) show that, in contrast to 2, N-derivatization of this alkaloid leads to increased activity against P. falciparum.

Immunity to Plasmodium berghei exoerythrocytic forms derived from irradiated sporozoites.

The nature of immunity generated by Plasmodium berghei exoerythrocytic (EE) stages developing from irradiated sporozoites was studied using in vivo parameters of host protection on immunization with irradiated sporozoites and in vitro parameters of inhibition of sporozoite invasion and EE form development by serum antibodies from immunized mice. On in vivo challenge of immunized mice by sporozoites, protection was observed in an irradiation-dose-dependent manner. This finding stresses that protection is dependent on the irradiation dose of sporozoites that allows sporozoite penetration yet controls EE form development within the liver. Using the human hepatoma line Hep G2 as host cells in vitro, we observed that serum antibodies raised in mice immunized with irradiated sporozoites reacted with sporozoite- and hepatic-stage parasites in an immunofluorescent antibody test (IFAT). No reactivity was observed with blood-stage parasites. Serum antibodies from mice immunized with 6- to 18-krad-irradiated sporozoites inhibited sporozoite invasion and caused severe inhibition of EE form development in hepatoma cells, pointing to the antigenic content of EE forms developing from irradiated sporozoites (irra EE forms) as critical immunogens. Moreover, in an enzyme-linked immunosorbent assay (ELISA), serum antibodies raised to 12-krad-irradiated sporozoites showed reactivity to synthetic peptides representing the conserved Region II sequences of the P. falciparum circumsporozoite (CS) protein as well as the P. falciparum liver-stage-specific antigen (LSA-1)-based repeat sequences, thus implicating an important role for both the sporozoite and the hepatic stage in protection.

Activities of extracts and naphthylisoquinoline alkaloids from Triphyophyllum peltatum, Ancistrocladus abbreviatus and Ancistrocladus barteri against Plasmodium berghei (Anka strain) in vitro.

Extracts from three tropical medicinal plant species belonging to the Dioncophyllaceae (Triphyophyllum peltatum) and the Ancistrocladaceae (Ancistrocladus abbreviatus and Ancistrocladus barteri), and pure naphthylisoquinoline alkaloids derived from these species have been examined for the first time for their activity against asexual blood forms of Plasmodium berghei (Anka strain) in vitro. These activities were considerable and comparable with those earlier found against erythrocytic forms of Plasmodium falciparum. The extracts and constituents of species belonging to the Dioncophyllaceae (dioncophylline B, dioncopeltine A and dioncophylline A) appear to be more promising than those from the Ancistrocladaceae.

Karyotype plasticity in neotropical Leishmania: an index for measuring genomic distance among L. (V.) peruviana and L. (V.) braziliensis populations.

A method for phenetic analysis of karyotype data has been developed for Leishmania populations. Measurement of size difference between chromosomes recognized by a given DNA probe in different isolates led to the formulation of a Chromosome Size Difference Index (CSDI). The method was applied to phenetic analysis of 4 sets of chromosomes--each set being recognized by a different probe--in 37 L. (Viannia) peruviana isolates sampled along a North-South transect through the Peruvian Andes and, in 11 L. (V.) braziliensis isolates from the Amazonian forest (Peru, Bolivia and Brazil). Karyotype variability was better accounted for by CSDI than by a method based on disjunctive encoding of karyotype data. CSDI evidenced the nature of relationships between L. braziliensis and L. peruviana and it provided a coherent picture of geographical and genomic differentiation among parasite populations. The latter did cluster according to their geographical origin. L. braziliensis was found karyotypically more homogeneous than L. peruviana. Within L. peruviana, Northern populations were closer to L. braziliensis than to Southern L. peruviana populations. The validity of karyotypic populations, or karyodemes, was sustained.