Stage-specific inhibitory effect of cyclic AMP on asexual maturation and gametocyte formation of Plasmodium falciparum

The effect of cyclic AMP on asexual maturation and gametocyte formation of Plasmodium falciparum grown in vitro was examined over a wide range of concentrations. Cyclic AMP inhibited both processes in a stage-specific manner. Asexual maturation was inhibited from shortly after parasite entry into the red cell through the ring stage. However, trophozoites and schizonts matured normally in the presence of cyclic AMP and produced infectious merozoites. Gametocyte formation was inhibited by 95% when 1.0 mM cyclic AMP was added to synchronously growing parasites in the ring stage of development but was only inhibited by 15% when added in the trophozoite or schizont stages. Cyclic AMP was not found to increase gametocyte formation over a wide range of concentrations.     

Blood schizontocidal activity of methylene blue in combination with antimalarials against Plasmodium falciparum

Methylene blue (MB) is the oldest synthetic antimalarial. It is not used anymore as antimalarial but should be reconsidered. For this purpose we have measured its impact on both chloroquine sensitive and resistant Plasmodium strains. We showed that around 5 nM of MB were able to inhibit 50% of the parasite growth in vitro and that late rings and early trophozoites were the most sensitive stages; while early rings, late trophozoites and schizonts were less sensitive. Drug interaction study following fractional inhibitory concentrations (FIC) method showed antagonism with amodiaquine, atovaquone, doxycycline, pyrimethamine; additivity with artemether, chloroquine, mefloquine, primaquine and synergy with quinine. These results confirmed the interest of MB that could be integrated in a new low cost antimalarial combination therapy.     

Stage-specific sensitivity of Plasmodium falciparum to antifolates

Highly synchronous cultures of Plasmodium falciparum were exposed to therapeutic concentrations of sulfadoxine or pyrimethamine at different developmental stages to investigate the effect on subsequent growth. Morphological observations showed that schizont formation from uninuclear trophozoites was the only process inhibited by the drugs. Segmentation of mature schizonts, merozoite invasion and development of the ring stage remained unaffected. These results support earlier reports suggesting that DNA synthesis is most pronounced in 32-42 h old trophozoites. The possible relevance of our results to the metabolism of P. falciparum is discussed.     

Protein tyrosine kinase activity in human malaria parasite Plasmodium falciparum.

Protein tyrosine kinases (PTKs) are believed to be implicated in the parasite growth, maturation and differentiation functions. Protein tyrosine kinase activity was found to be distributed in all the stages of P. falciparum parasite maturation. Membrane bound PTK activity was found to be increased during maturation process (ring stage to trophozoite stage) in chloroquine sensitive strains. In vivo conversion of the schizont stage to ring stage via release of merozoites was associated with a decrease in PTK activity. Chloroquine inhibited the membrane bound PTK activity in a dose dependent manner (IC50 = 45 microM). Kinetic studies show that chloroquine is a competitive inhibitor of PTK with respect to peptide substrate and noncompetitive with respect to ATP indicating that chloroquine inhibits PTK activity by binding with protein substrate binding site. The results suggest that maturation of malaria parasite is related to PTK and inhibition of this activity by chloroquine could provide a hypothesis to explain the mechanism of action of chloroquine.     

Stage-dependent effects of chloroquine on Plasmodium falciparum in vitro

The erythrocytic developmental cycle of Plasmodium falciparum can be conveniently divided into the ring, trophozoite, and schizont stages based on morphology and metabolism. Using highly synchronous cultures of P. falciparum, considerable variation was demonstrated among these stages in sensitivity to chloroquine. The effects of timed, sequential exposure to several clinically relevant concentrations of chloroquine were monitored by three techniques: morphological analysis, changes in the rate of glucose consumption, and changes in the incorporation of 3H-hypoxanthine into parasite nucleic acids. All three techniques gave essentially identical results. The trophozoite and schizont stages were considerably more sensitive to the drug than ring-stage parasites. Chloroquine sensitivity decreased as nuclear division neared completion. The increase in chloroquine sensitivity was coincident with a marked rise in the rate of glucose consumption and nucleic acid synthesis. The rate of nucleic acid synthesis decreased as schizogony progressed while glucose consumption continued at high rates during this process. The degree of chloroquine sensitivity was not highly correlated with either metabolic activity.     

Stage-Dependent Effects of Chloroquine on Plasmodium falciparum In Vitro1

The erythrocytic developmental cycle of Plasmodium falciparum can be conveniently divided into the ring, trophozoite, and schizont stages based on morphology and metabolism. Using highly synchronous cultures of P. falciparum, considerable variation was demonstrated among these stages in sensitivity to chloroquine. The effects of timed, sequential exposure to several clinically relevant concentrations of chloroquine were monitored by three techniques: morphological analysis, changes in the rate of glucose consumption, and changes in the incorporation of ³H-hypoxanthine into parasite nucleic acids. All three techniques gave essentially identical results. The trophozoite and schizont stages were considerably more sensitive to the drug than ring-stage parasites. Chloroquine sensitivity decreased as nuclear division neared completion. The increase in chloroquine sensitivity was coincident with a marked rise in the rate of glucose consumption and nucleic acid synthesis. The rate of nucleic acid synthesis decreased as schizogony progressed while glucose consumption continued at high rates during this process. The degree of chloroquine sensitivity was not highly correlated with either metabolic activity.     

Inhibition of a protein tyrosine kinase activity in Plasmodium falciparum by chloroquine

The aim of the present study was to establish the importance of phosphorylation events for parasite growth and maturation. Investigations into the cytosolic Plasmodium falciparum protein tyrosine kinase (PTK) activity revealed that there is a stage specific increase in the activity, in the order ring < trophozoite < schizont in both chloroquine sensitive (CQ-S) and chloroquine resistant (CQ-R) strains (p < 0.05). Our data also show that in vivo conversion of the schizont stage to ring stage via release of merozoites is associated with a decrease in PTK activity. Piceatannol, a specific inhibitor of PTK inhibited the activity in both the CQ-S and CQ-R strains of the parasites. The presence of low levels of chloroquine (CQ) inhibited the cytosolic PTK activity in a dose dependent manner (IC50 = 45 mumoles or 23 micrograms/ml) in CQ-S strains. The effect of varying concentration of CQ on the kinetics of peptide phosphorylation reveal that CQ was a competitive inhibitor of PTK with respect to peptide substrate and non-competitive with respect to ATP indicating that CQ inhibits PTK activity by binding with protein substrate binding site. These data thus suggests that maturation of malaria parasite may be due to this cellular PTK and its inhibition by CQ could provide a hypothesis to explain its antimalarial activity and efficacy.     

Plasmodium falciparum: synchronization of cultures with DL-alpha-difluoromethylornithine, an inhibitor of polyamine biosynthesis

DL-alpha-Difluoromethylornithine, an inhibitor of polyamine biosynthesis, was tested for its ability to synchronize Plasmodium falciparum. Asynchronous cultures were pretreated with sorbitol and incubated for 28-30 hr. Then, when cultures consisted of mainly schizont stage parasites, DL-alpha-difluoromethylornithine was added to the growth medium for another 38-47 hr of incubation. Putrescine was added to parasites arrested at the early trophozoite stage. This resulted in a synchronous resumption of growth. After 19 hr, 83% of parasites were at the schizont stage. After 30 hr, more than 98% of the parasites were in the ring form stage. Furthermore, the transformation of early trophozoites to schizonts occurred within 3 hr, with a slight reduction in parasitemia. Synchrony was maintained for 4-5 biological cycles as confirmed also by flow fluorimetry. It appears that this new approach to synchronize P. falciparum cultures is simple, reproducible, and effective.     

Developmental stage-specific biosynthesis of glycosylphosphatidylinositol anchors in intraerythrocytic Plasmodium falciparum and its inhibition in a novel manner by mannosamine

Glycosylphosphatidylinositols (GPIs) are the major glycoconjugates in intraerythrocytic stage Plasmodium falciparum. Several functional proteins including merozoite surface protein 1 are anchored to the cell surface by GPI modification, and GPIs are vital to the parasite. Here, we studied the developmental stage-specific biosynthesis of GPIs by intraerythrocytic P. falciparum. The parasite synthesizes GPIs exclusively during the maturation of early trophozoites to late trophozoites but not during the development of rings to early trophozoites or late trophozoites to schizonts and merozoites. Mannosamine, an inhibitor of GPI biosynthesis, inhibits the growth of the parasite specifically at the trophozoite stage, preventing further development to schizonts and causing death. Mannosamine has no effect on the development of either rings to early trophozoites or late trophozoites to schizonts and merozoites. The analysis of GPIs and proteins synthesized by the parasite in the presence of mannosamine demonstrates that the effect is because of the inhibition of GPI biosynthesis. The data also show that mannosamine inhibits GPI biosynthesis by interfering with the addition of mannose to an inositol-acylated GlcN-phosphatidylinositol (PI) intermediate, which is distinctively different from the pattern seen in other organisms. In other systems, mannosamine inhibits GPI biosynthesis by interfering with either the transfer of a mannose residue to the Manalpha1-6Manalpha1-4GlcN-PI intermediate or the formation of ManN-Man-GlcN-PI, an aberrant GPI intermediate, which cannot be a substrate for further addition of mannose. Thus, the parasite GPI biosynthetic pathway could be a specific target for antimalarial drug development.     

Plasmodium falciparum: Attenuation by irradiation

The effect of irradiation on the in vitro growth of Plasmodium falciparum was investigated. The cultured malarial parasites at selected stages of development were exposed to gamma rays and the sensitivity of each stage was determined. The stages most sensitive to irradiation were the ring forms and the early trophozoites; late trophozoites were relatively insensitive. The greatest resistance was shown when parasites were irradiated at a time of transition from the late trophozoite and schizont stages to young ring forms. The characteristics of radiosensitive variation in the parasite cycle resembled that of mammalian cells. Growth curves of parasites exposed to doses of irradiation upto 150 gray had the same slope as nonirradiated controls but parasites which were exposed to 200 gray exhibited a growth curve which was less steep than that for parasites in other groups. Less than 10 organisms survived from the 10⁶ parasites exposed to this high dose of irradiation; the possibility exists of obtaining radiation-attenuated P. falciparum.     

Plasmodium falciparum: differential sensitivity in vitro to E-64 (cysteine protease inhibitor) and Pepstatin A (aspartyl protease inhibitor).

We investigated the effect of a cysteine proteinase inhibitor (E-64) and an aspartyl proteinase inhibitor (Pepstatin A) on asexual erythrocytic stages of Plasmodium falciparum in culture. These two protease inhibitors showed different patterns of activity. E-64 acted preferentially against trophozoite and schizont stages. After 48 h incubation at high concentrations of E-64 (28, 140, 280 microM), growth was totally abolished and the parasites presented characteristic enlarged food vacuoles. Morphological alterations were also seen after shorter incubation periods (6 h at 28 microM) or 12 h at the inhibitory concentration 50% (12 microM), but an additional culture period (24 h) in inhibitor-free medium allowed normal parasite development, demonstrating a parasitostatic effect. E-64 acts on parasite multiplication; the normal merozoite maturation was altered and the normal reinvasion process partially impaired. Pepstatin A used at the inhibitory concentration 50% (4 microM) killed the parasites before trophozoite development and had a major effect on schizonts maturation. No altered parasite development occurred during an additional culture period without Pepstatin A, demonstrating a parasiticidal effect. E-64 and Pepstatin A used in combination inhibit the parasite growth with a strong synergistic effect.     

Pyrimidine de novo synthesis during the life cycle of the intraerythrocytic stage of Plasmodium falciparum

The 6 enzymes involved in de novo synthesis of pyrimidines were measured in Plasmodium falciparum isolated by saponin lysis from RBC's nonsynchronized and synchronized in vitro cultures. The total activities were found to be dependent on the stage of the P. falciparum cycle. In parasites isolated from synchronized cultures, the highest activities for all enzymes were found at about 27 hr after synchronization in the late trophozoite stage, or just before schizont formation. Merozoites and ring forms contained little de novo activity. The first enzyme of the pathway, carbamyl phosphate synthetase (CPS-II) preferentially utilized glutamine. Ammonia was a poor substrate. CPS-II was unstable in the absence of the cryoprotectants, dimethylsulfoxide and glycerol. The apparent Km for MgATP--was 3.8 +/- 0.7 mM and the enzyme in all morphological forms of P. falciparum (ring, mature trophozoites and schizonts) was inhibited by UTP. The activity of the fourth enzyme of the pathway, dihydroorotate dehydrogenase, appeared to be linked to the cell's respiratory chain; inhibitors of mammalian electron transport such as cyanide, amytal, antimycin A, thenoyltrifluoroacetone and ubiquinone analogs also inhibited the P. falciparum enzyme. The demonstration of the variation of activity of the pyrimidine enzymes correlates with the increased synthesis of nucleic acids in the late trophozoite stage. These observations provide a basis for the testing of the effectiveness of pyrimidine analogs as potential antimetabolites against various forms of the parasite.     

An interplay between 2 signaling pathways: Melatonin-cAMP and IP3–Ca signaling pathways control intraerythrocytic development of the malaria parasite Plasmodium falciparum

Plasmodium falciparum spends most of its asexual life cycle within human erythrocytes, where proliferation and maturation occur. Development into the mature forms of P. falciparum causes severe symptoms due to its distinctive sequestration capability. However, the physiological roles and the molecular mechanisms of signaling pathways that govern development are poorly understood. Our previous study showed that P. falciparum exhibits stage-specific spontaneous Calcium (Ca²⁺) oscillations in ring and early trophozoites, and the latter was essential for parasite development. In this study, we show that luzindole (LZ), a selective melatonin receptor antagonist, inhibits parasite growth. Analyses of development and morphology of LZ-treated P. falciparum revealed that LZ severely disrupted intraerythrocytic maturation, resulting in parasite death. When LZ was added at ring stage, the parasite could not undergo further development, whereas LZ added at the trophozoite stage inhibited development from early into late schizonts. Live-cell Ca²⁺ imaging showed that LZ treatment completely abolished Ca²⁺ oscillation in the ring forms while having little effect on early trophozoites. Further, the melatonin-induced cAMP increase observed at ring and late trophozoite stage was attenuated by LZ treatment. These suggest that a complex interplay between IP₃–Ca²⁺ and cAMP signaling pathways is involved in intraerythrocytic development of P. falciparum.     

Plasmodium falciparum: Differential Sensitivity In Vitro to E-64 (Cysteine Protease Inhibitor) and Pepstatin A (Aspartyl Protease Inhibitor)

ABSTRACT We investigated the effect of a cysteine proteinase inhibitor (E-64) and an aspartyl proteinase inhibitor (Pepstatin A) on asexual erythrocytic stages of Plasmodium falciparum in culture. These two protease inhibitors showed different patterns of activity. E-64 acted preferentially against trophozoite and schizont stages. After 48 h incubation at high concentrations of E-64 (28, 140, 280 μM), growth was totally abolished and the parasites presented characteristic enlarged food vacuoles. Morphological alterations were also seen after shorter incubation periods (6 h at 28 μM) or 12 h at the inhibitory concentration 50% (12 μM), but an additional culture period (24 h) in inhibitor-free medium allowed normal parasite development, demonstrating a parasitostatic effect. E-64 acts on parasite multiplication; the normal merozoite maturation was altered and the normal reinvasion process partially impaired. Pepstatin A used at the inhibitory concentration 50% (4 μM) killed the parasites before trophozoite development and had a major effect on schizonts maturation. No altered parasite development occurred during an additional culture period without Pepstatin A, demonstrating a parasiticidal effect. E-64 and Pepstatin A used in combination inhibit the parasite growth with a strong synergistic effect.     

Visualization and quantification of <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> intraerythrocytic merozoites

Malaria, a leading parasitic killer, is caused by Plasmodium spp. The pathology of the disease starts when Plasmodium merozoites infect erythrocytes to form rings, that matures through a large trophozoite form and develop into schizonts containing multiple merozoites. The number of intra-erythrocytic merozoites is a key-determining factor for multiplication rate of the parasite. Counting of intraerythrocytic merozoites by classical 2-D microscopy method is error prone due to insufficient representation of merozoite in one optical plane of a schizont. Here, we report an alternative 3-D microscopy based automated method for counting of intraerythrocytic merozoites in entire volume of schizont. This method offers a considerable amount of advantages in terms of both, ease and accuracy.     

贝氏隐孢子虫在北京鸭体内发育的超微结构研究

贝氏隐孢子虫各期虫体均位于宿主粘膜上皮细胞的带虫空泡中。在虫体与上皮细胞接触处,虫体表膜反复折迭形成营养器。子孢子或裂殖子与粘膜上皮细胞接触后,逐步过渡为球形的滋养体;滋养体经2—3次核分裂、产生含4或8个裂殖子的两代裂殖体,裂殖体以外出芽方式产生裂殖子;裂殖子无微孔,顶端表皮形成3—4个环嵴,裂殖子进一步发育成为配子体;大配子体含有两种类型的成囊体。小配子呈楔形,无鞭毛和顶体,有一个致密的长椭圆形细胞核,小配子表膜内侧有9根膜下微管;孢子化卵囊内含四个裸露的子孢子和一个大残体。本文是有关鸭体内隐孢子虫超微结构的首次报导。     

Activation of a PAK-MEK signalling pathway in malaria parasite-infected erythrocytes

Merozoites of malaria parasites invade red blood cells (RBCs), where they multiply by schizogony, undergoing development through ring, trophozoite and schizont stages that are responsible for malaria pathogenesis. Here, we report that a protein kinase-mediated signalling pathway involving host RBC PAK1 and MEK1, which do not have orthologues in the Plasmodium kinome, is selectively stimulated in Plasmodium falciparum-infected (versus uninfected) RBCs, as determined by the use of phospho-specific antibodies directed against the activated forms of these enzymes. Pharmacological interference with host MEK and PAK function using highly specific allosteric inhibitors in their known cellular IC50 ranges results in parasite death. Furthermore, MEK inhibitors have parasiticidal effects in vitro on hepatocyte and erythrocyte stages of the rodent malaria parasite Plasmodium berghei, indicating conservation of this subversive strategy in malaria parasites. These findings have profound implications for the development of novel strategies for antimalarial chemotherapy.     

Ultrastructure of Cryptosporidium wrairi from the Guinea Pig

SYNOPSIS. The ultrastructure of the known tissue stages of Cryptosporidium wrairi Vetterling, Jervis, Merrill, and Sprinz, 1971 parasitizing the ileum of guinea pigs is described. Young trophozoites are surrounded by 4 unit membranes, the outer 2 of host origin, the inner 2 the pellicle of the parasite. Each trophozoite contains a vesicular nucleus with a large nucleolus. Its cytoplasm contains ribosomes, but eventually fills with cisternae of the rough endoplasmic reticulum. As the trophozoite matures the area of attachment of the parasite to the host cell becomes vacuolated, with vertical membranous folds. It is apparent that the parasite acquires nourishment from the host cell thru this area of attachment. As schizonts develop, (a) multiple nuclei appear, (b) the endoplasmic reticulum enlarges, (c) the attachment zone increases in area, (d) large vacuoles, which develop as endocytotic vesicles in the attachment area, are found in the cytoplasm and (e) the inner unit membrane of the parasite pellicle is resorbed around the sides of the developing schizont. Following nuclear division, merozoites develop from the schizont by budding. Merozoites have an ultrastructure similar to that described for other coccidia except that no mitochondria, micropores, or subpellicular tubules were observed. Merozoites penetrate the epithelial cell causing invagination of the microvillar membrane and lysing it. No unit membrane is formed between the parasite and the host cell. However, the cell produces one or 2 dense bands adjacent to the parasite attachment area. The macrogamete contains a nucleus, endoplasmic reticulum, attachment zone, and large vacuoles. It also contains a variety of granules, some of which are polysaccharide. The immature microgametocyte contains multiple compact nuclei. No mature microgametocytes or zygotes were found.