Effect of the 8-aminoquinoline primaquine on culture-derived gametocytes of the malaria parasitePlasmodium falciparum

Gametocytes from the Honduras I/CDC clone HB-3 ofPlasmodium falciparum were exposed to different concentrations of primaquine diphosphate. It could be shown that the drug acts specifically on the mitochondria of the cells by destroying their internal structure and causing these organelles to swell.     

Endotoxin-induced serum factor kills malarial parasites in vitro.

We investigated the possibility that malarial parasites may be killed by nonspecific soluble mediators, such as those in tumor necrosis serum, that are obtained from mice given macrophage-activating agents like Corynebacterium parvum or Mycobacterium bovis BCG, followed by endotoxin. Such sera killed parasites in vitro after overnight incubation; killing was measured directly by using an in vivo infectivity assay. Parasite infectivity was not decreased by incubation in sera from mice given C. parvum or BCG alone (no endotoxin) or by incubation in sera from normal mice given endotoxin. Plasmodium yoelii, its lethal variant, and Plasmodium berghei were equally susceptible to inactivation. Sera obtained from mice given endotoxin during the course of infection with these parasites also contained parasite-killing factor. The activity of this factor appeared to be proportional to parasitemia in that it was higher in the sera from mice infected with the lethal parasites than in the sera from mice with infections which resolved either spontaneously or after vaccination.     

Ultrastructural assessment of Plasmodium falciparum in age-fractionated thalassaemic erythrocytes

Culture of Plasmodium falciparum in age-fractionated thalassaemic red blood cells (RBC) has shown evidence of parasite damage on light microscopy in older cells during the third culture cycle (96–l44 h). In this report, parasites growing in thalassaemic trait and normal RBC were examined ultrastructurally from 96 to l44 h. All parasite stages in old thalassaemic RBC showed evidence of damage worsening with culture duration. There were cytoplasmic alterations with ribosomal damage, and parasite cytoplasm became increasingly loose and grainy, with multiple fissures. Discontinuity of the nuclear membrane with an abnormal nucleolus was seen at l20 h. Cytosomes remained normal, but damage to the food vacuole and shrunken disintegrating parasites were observed at 144 h. These changes are compatible with cellular degeneration and developmental retardation and would account for the schizont maturation arrest and reduced reinvasion rates previously reported. Increased free radicals associated with thalassaemic erythrocytes would explain these changes, further supporting the role for oxidant stress in the protective mechanism.     

Thioredoxin networks in the malarial parasite Plasmodium falciparum

The intraerythrocytic protozoan parasite Plasmodium falciparum is responsible for more than 500 million clinical cases of tropical malaria annually. Although exposed to high fluxes of reactive oxygen species, Plasmodium lacks the antioxidant enzymes catalase and glutathione peroxidase. Thus, the parasite depends on the antioxidant capacity of its host cell and its own peroxidases. These are fuelled by the thioredoxin system and are considered to represent the major defense line against peroxides. Five peroxidases that act in different compartments have been described in P. falciparum. They include two typical 2-Cys peroxiredoxins (Prx), a 1-Cys Prx, the so-called antioxidant protein (AOP), which is a further Prx acting on the basis of a 1-Cys mechanism, and a glutathione peroxidase-like thioredoxin peroxidase. Because of their central function in redox regulation and antioxidant defense, some of these proteins might represent highly interesting targets for structure-based drug development. In this article we summarize the present knowledge on the thioredoxin and peroxiredoxin metabolism in malaria parasitized red blood cells. We furthermore report novel data on the biochemical and kinetic characterization of different thioredoxins, of AOP, and of the classic 1-Cys peroxiredoxin of P. falciparum.     

The effect of ascaridole on the in vitro development ofPlasmodium falciparum

Ascaridole is a terpene isolated from the plantChenopodium ambrosioides (American wormseed); it is one of the few naturally occurring endoperoxidases. Artemisinin, which also belongs to this group, is a potent antimalarial. We therefore undertook a study to determine the effect of ascaridole, a known anthelmintic, on the in vitro development ofPlasmodium falciparum. Ascaridole was found to be a potent inhibitor of plasmodial growth; after 3 days, development was arrested by a drug concentration of 0.05 M, and at 0.1 M no parasites were visible in the culture. At lower concentrations the effect was observed mainly at the trophozoite stage, whereas the ring stage was marginally affected. However, even at these lower concentrations, the ring culture could not continue normal development and ceased to grow at a later stage. The peroxide group is essential for the antimalarial activity of ascaridole, as judged from the fact that cineol, which bears an epoxide group instead of the peroxide group found in ascaridole, was totally inactive at identical concentrations.This paper is dedicated to the late Prof. R. Segal     

Mitochondrial DNA of the human malarial parasite Plasmodium falciparum

Covalently closed circular DNA molecules were isolated from Plasmodium falciparum total DNA by isopycnic centrifugation in CsCl gradients containing either ethidium bromide or 2',6-diamidino-2-phenylindole. The circular molecules had an average contour length of 11.1 +/- 0.5 micron, similar to the analogous molecules previously isolated from the simian malaria parasite P. knowlesi. Both circular molecules shared considerable sequence homology and conserved restriction sites. The nucleotide sequence of one 936 bp fragment of the P. falciparum molecule was determined and identified, by a data base homology search, as part of a mitochondrial small rRNA subunit, thus confirming the mitochondrial origin of the circular DNAs of both malarial species.     

Human serum haptoglobin is toxic to Plasmodium falciparum in vitro

Innate immune responses are important in the control of malaria, particularly in those who have not yet mounted an effective adaptive response. Here we report that the human serum acute phase protein, haptoglobin, is toxic to Plasmodium falciparum cultured in vitro. This effect is phenotype dependent and occurs during the trophozoite phase of the asexual life cycle. We propose that the increased levels of haptoglobin seen in the acute phase response may be protective against malaria in humans.     

Effect of drugs inhibiting spermidine biosynthesis and metabolism on the in vitro development of Plasmodium falciparum

Treatment of Plasmodium falciparum with the potent inhibitor dicyclohexylamine completely arrests in vitro cell proliferation of the chloroquine-susceptible P. falciparum strain NF54 and the R strain, which shows less sensivity to chloroquine. The average inhibitory concentration (IC50) values determined for both strains revealed different inhibition profiles. The IC50 value for the chloroquine-sensitive NF54 strain was 97 microM and 501 microM for the R strain. Monitoring polyamine pools after treatment with dicyclohexylamine leads to a significant decrease in the intracellular spermidine content, which was nearly reversed by supplementation with spermidine. Since spermidine is an important precursor for the biosynthesis of hypusine and homospermidine in eukaryotes, we studied the developmental effect on both P. falciparum strains of 1,7-diaminoheptane as an inhibitor of deoxyhypusine synthase (EC 1.1.1.249) in mammalian cells, and agmatine as a moderate inhibitor of homospermidine synthase (EC 2.5.1.44). Inhibition profiles with 1,7-diaminoheptane resulted in an IC50 value of 466 microM for the NF54 strain and 319 microM for the R strain. Spermidine pools changed significantly. Inhibition with agmatine caused a strong decrease in parasitemia for the chloroquine-susceptible NF54 strain, with a determined IC50 value of 431 microM and an IC50 value of 340 microM for the less chloroquine-susceptible R strain. Spermidine was not detectable after inhibition. The uncommon triamine homospermidine occurred in both P. falciparum strains. To our knowledge this is the first evidence of homospermidine in P. falciparum. The use of specific inhibitors of spermidine metabolism might be a novel strategy for the design of new antimalarials, and suggests the occurrence of both enzymes in the parasite.     

Antisense oligonucleotides targeting malarial aldolase inhibit the asexual erythrocytic stages of Plasmodium falciparum

A major obstacle in the global effort to control malaria is the paucity of anti-malarial drugs. This is compounded by the continuing emergence and spread of resistance to old and new anti-malarial drugs in the malarial parasites. Here we describe the anti-malarial effect of phosphorothioate antisense (AS) oligodeoxynucleotides (ODNs) targeting the aldolase enzyme of Plasmodium falciparum, using the asexual blood stages of the parasite grown in vitro. The blood stages of P. falciparum depend almost entirely on the energy produced by their own glycolysis. Aldolase, the fourth enzyme of the glycolytic pathway, is highly upregulated during the malarial 48-h life cycle. We found that the mRNA of this enzyme can be inhibited, in a sequence specific manner, using AS-ODN to the splice sites on the pre-mRNA of malarial aldolase. At the enzyme level, both specific AS-ODNs for the splice sites, as well as for the translation initiation site on mature mRNA, can inhibit aldolase enzyme activity within the trophozoites of P. falciparum. Furthermore, this downregulation of the malarial aldolase results in a reduction in the production of ATP within the parasite. Finally, the treatment reduces parasitemia. In summary, AS-ODNs targeting the aldolase gene of P. falciparum can interfere with the blood-stage life cycle of this parasite in vitro by inhibiting the expression of the enzyme aldolase which results in decreased malarial glycolysis and energy production. Thus, we conclude that blockade of the expression of malarial glycolytic enzymes using specific AS-ODNs has the potential of a new anti-malarial strategy.     

Characterization of cobalamin-dependent methionine synthase purified from the human malarial parasite,Plasmodium falciparum

Methionine synthase, which catalyzes the reaction, 5-methyltetrahydrofolate (5–CH₃–H₄PteGlu)+homocysteinemethionine+tetrahydrofolate, was detected and partially purified from the human malarial parasite,Plasmodium falciparum (K₁ isolate). Partial purification was achieved using high-performance size-exclusion and anion-exchange chromatography. The apparent relative molecular weight of the enzyme was estimated as 105000 daltons, and the apparent K_m for 5–CH₃–H₄PteGlu was 24.2 M. The enzyme was dependent on adenosylcobalamin or methylcobalamin but not on cobalamin, cyanocobalamin, or hydroxocobalamin in either the absence or presence ofS-adenosylmethionine. Preincubation with nitrous oxide markedly inhibited the enzyme. Methionine synthase inP. falciparum may play a role in the supply of methionine and in folate salvage using exogenous 5–CH₃–H₄PteGlu for tetrahydrofolate metabolism.     

Use of hydroethidine and flow cytometry to assess the effects of leukocytes on the malarial parasite Plasmodium falciparum.

Flow cytometry was evaluated as a method of assessing in vitro the effects of leukocytes on blood-stage Plasmodium falciparum. Hydroethidine is converted by metabolizing cells to ethidium, a nucleic acid fluorochrome. After incubation with hydroethidine, viable and dead leukocytes and parasitized and uninfected erthrocytes could all be identified on the basis of fluorescence intensity and size. Leukocytes can therefore be eliminated from further analysis; this allows assessment, at any parasite developmental stage, of the level of parasitemia within erythrocytes in the presence of any of several types of leukocytes. Whether leukocytes actually kill intraerythrocytic parasites can therefore be determined and the level of cytotoxicity can be assessed. The ability of leukocytes to prevent merozoites from invading new erythrocytes, i.e., inhibition of parasite invasion, can also be assessed by this method. When erythrocytes containing schizont-stage parasites were cocultured with different leukocyte populations and the level of parasitemia was determined after merozoite release and invasion, only cultures containing gamma delta T cells inhibited parasite invasion. The different blood-stage forms of the parasite vary in nucleic acid content, which allows each of the developmental stages to be distinguished by flow cytometry; this permits assessment of changes in parasite development in the presence of leukocytes. Monocyte-derived macrophages (MDMs) appeared to have an effect on parasite development. In this instance, when erythrocytes containing ring-form parasites were cocultured with MDMs and harvested 24 h later, the parasites in cultures containing MDMs were at the late schizont stage, whereas parasites in control cultures were early trophozoites; this finding suggests that MDMs accelerate parasite development. Together, these results indicate that flow cytometry is potentially useful for measuring the following effects mediated by leukocytes: (i) level of cytotoxicity, (ii) changes in parasite development, and (iii) inhibition of parasite invasion.     

Effects of taurine on human embryo development in vitro.

Glutamine and taurine are reported to be beneficial for mouse embryo development in vitro, and we have recently shown that glutamine improves human blastocyst formation in vitro. This randomized study compared the development of supernumerary human embryos in the presence of 1 mmol/l glutamine and/or 5 mmol/l taurine from the 2-4-cell stage to the blastocyst stage. Blastocyst development and cell numbers were similar in the presence of glutamine or taurine: 52.6% and 58.3% of the embryos reached the blastocyst stage, respectively. Pyruvate uptake was similar in the presence of glutamine or taurine throughout development, as was lactate production after the 8-cell stage. Before this stage, lactate production was 4-fold higher in the presence of taurine (P < 0.001). The proportion of embryos reaching the blastocyst stage was similar with glutamine alone or with glutamine and taurine (62.5% and 47.2% respectively), as were the blastocyst cell numbers (63.0 +/- 4.6 and 61.0 +/- 5.1 respectively). In conclusion, taurine supports development of 2-4-cell human embryos to the blastocyst stage, although it does not further augment the beneficial effects of glutamine.     

壮筋续骨汤含药血清体外培养大鼠成骨细胞的增殖

背景：壮筋续骨汤具有促进胫骨骨折愈合的作用。 目的：观察壮筋续骨汤含药血清对体外培养大鼠成骨细胞增殖的影响。 方法：大鼠灌胃给予壮筋续骨汤后制备含药血清。取第2代生长状况良好出生48 h内的SD大鼠成骨细胞,对照组和壮筋续骨汤组分别加入5%,10%,20%的正常SD大鼠血清和含药血清培养72 h。 结果与结论：MTT法检测发现壮筋续骨汤含药血清对成骨细胞的增殖有明显的促进作用,以10%壮筋续骨汤组作用最明显(P < 0.05)。流式细胞仪检测各组细胞DNA合成期(S期)的比例,发现壮筋续骨汤组处于S期的细胞比例明显大于对照组 (P < 0.05)。说明壮筋续骨汤含药血清能促进成骨细胞DNA的合成,使更多的细胞进入S期,促进体外培养的成骨细胞增殖。     

Effects of streptokinase on the development of rat cerebral cortical cells in vitro

The aim of the present work was to study the effects of streptokinase (SK) on the ultrastructure of the cellular elements of the cerebral cortex of neonatal rats in vitro. Three series of cultures were used: cultures maintained in DMEM enriched with 15% fetal calf serum (control 1), some transferred to minimal nutritive medium containing only 0.5% serum (control 2), and some supplemented with SK (2000 MU/ml) (experimental). Addition of SK to the nutritive medium prevented a decrease in the viability of cells in a mature (14 days) dissociated culture from the neocortex of rat pups aged 1–2 days induced by transfer of the cultures to medium lacking serum proteins. The action of SK on 7-day cultures enhanced the loss of cell viability. Electron microscopy studies of organotypic cultures showed that at this concentration, SK prevented the development of destructive changes of astrocytes, oligodendrocytes, and neurons induced in explants by serum protein deficiency. Neurons showed an abundance of mitochondria, some of which had few cristae. Signs of destruction affected only the nuclei of neurons. After exposure to SK for 48 h, oligodendrocytes were activated (they contained an abundance of myelin-like bodies), with degradation of most astrocytes (surviving examples showing nuclear hyperchromicity). Neurons were resistant to these effects. __________ Translated from Morfologiya, Vol. 128, No. 5, pp. 33–36, September–October, 2005.     

Opsonic activity of human immune serum on in vitro phagocytosis of Plasmodium falciparum infected red blood cells by monocytes.

In vitro human monocytes from normal blood donors ingest red blood cells infected with Plasmodium falciparum more efficiently than normal red blood cells (NRBC). The phagocytic activity of human monocytes for infected red blood cells (IRBC) is greatly enhanced by the addition of immune sera obtained from individuals living in areas with endemic malaria. In contrast, the addition of sera obtained from individuals recovering from a first infection, or pooled normal sera, does not result in increased phagocytosis of IRBC. The phagocytosis enhancing activity of immune sera is associated with the IgG fraction and IgG depleted sera do not stimulate phagocytosis. Enhanced immune serum mediated phagocytosis occurs as a result of opsonization of IRBC. This was demonstrated by experiments in which monocytes or IRBC were preincubated with immune serum prior to the phagocytic assay. The opsonic activity could be absorbed by IRBC but not by NRBC. The opsonization of IRBC and subsequent phagocytosis were also dependent on the stage of development of the intracellular parasite. IRBC containing schizonts and trophozoites were preferentially phagocytosed as compared with ring forms. The role of malaria induced surface alterations and/or malaria surface antigens in the opsonization of IRBC by immune sera is discussed. These experiments suggest that phagocytosis of P. falciparum IRBC by monocytes may play a role in the immune elimination of malaria infection in humans.     

同种异体血清体外培养兔膝关节软骨细胞的增殖*◆

背景：兔关节软骨细胞体外单层培养采用胎牛血清培养基易去分化,有必要寻找合适培养基来提高兔关节软骨细胞培养质量。 目的：观察同种异体兔血清对体外培养的兔膝关节软骨细胞增殖能力的影响。 方法：以0.4%链霉蛋白酶和0.025%Ⅱ型胶原酶分离成年兔膝关节软骨细胞,将获得的软骨细胞随机分为实验组和对照组。实验组以体积分数10%异体兔血清+DMEM/F12培养;对照组以体积分数10%胎牛血清+DMEM/F12培养,传代培养至4代。 结果与结论：实验组前4代软骨细胞增殖较对照组慢,但软骨细胞形态未发生明显改变而对照组软骨细胞出现去分化现象。提示体积分数10%异体兔血清培养利于维持软骨细胞增殖和形态的稳定,是较好的获取大量优良软骨细胞的体外培养方式。