Optimal assay design for determining the in vitro sensitivity of ring stage Plasmodium falciparum to artemisinins

Recent reports demonstrate that failure of artemisinin-based antimalarial therapies is associated with an altered response of early blood stage Plasmodium falciparum. This has led to increased interest in the use of pulse assays that mimic clinical drug exposure for analysing artemisinin sensitivity of highly synchronised ring stage parasites. We report a methodology for the reliable execution of drug pulse assays and detail a synchronisation strategy that produces well-defined tightly synchronised ring stage cultures in a convenient time-frame.     

Entamoeba invadens: inhibition of excystation and metacystic development by aphidicolin

The effect of aphidicolin, a specific inhibitor of the replicative DNA polymerases, on the excystation and metacystic development of Entamoeba invadens was examined. The protein profile of metacystic amoebae and their immunogenicity in the presence and absence of aphidicolin were also examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. Excystation, which was assessed by counting the number of metacystic amoebae after the induction of excystation, was inhibited by aphidicolin in a concentration-dependent manner during incubation compared to the controls. Metacystic development, when determined by the number of nuclei in amoeba, was also inhibited by aphidicolin, because the percentage of 4-nucleate amoebae in cultures with aphidicolin during incubation was higher than that in cultures without the drug. The addition of aphidicolin to cultures at day 1 of incubation reduced the number of metacystic amoebae thereafter compared to cultures without the drug. The inhibitory effect of aphidicolin on excystation and metacystic development was reversed by removal of the drug. Pretreatment of cysts with aphidicolin before transfer to a growth medium containing the drug had no further effect on the excystation and metacystic development. Cellular proteins of metacystic amoebae with 4 nuclei, which were predominant even at day 3 in the cultures with aphidicolin, reacted strongly with rabbit anticyst serum absorbed with trophozoite proteins. In contrast, those of metacystic amoebae with 1 nucleus, which were predominant at day 3 in cultures without aphidicolin, no longer reacted with the absorbed anticyst serum, suggesting change in the expression of proteins during metacystic development.     

Establishment and characterisation of a rapidly dividing diploid cell suspension culture of Arabidopsis thaliana suitable for cell cycle synchronisation

Polyploid plants often have altered gene expression, biochemistry, and metabolism compared to their diploid predecessors. Therefore cultured diploid cells have distinct benefits over cultured polyploid cells for the study of gene regulation and metabolism of the parent plant. Here we report methods for establishing and maintaining a rapidly dividing diploid Arabidopsis thaliana cell suspension culture, and subsequent cell cycle synchronisation. Rapid growth of homogeneous cell populations was achieved after 3 months of initiation of cultures from leaf calluses. The cells were grown in the dark on an orbital shaker (110 rpm, 50 mm orbit) at 24 °C. Continued maintenance of the culture required the use of late-exponential stage cells for subculture at weekly intervals using careful subculturing techniques to achieve accurate biomass transfer. Cell cycle synchronisation was achieved following sucrose starvation, phosphate starvation, hydroxyurea treatment, aphidicolin treatment, and a combination of phosphate starvation and aphidicolin treatment. Inhibition of the cell cycle and accumulation of cells in specific phases was monitored by microscopy to determine the metaphase/anaphase index, and by flow cytometry. The cell cycle was partially and reversibly blocked by sucrose or phosphate starvation and by hydroxyurea (2.5 mM) treatment. A complete block at G1/S interphase was achieved after aphidicolin treatment or phosphate starvation combined with aphidicolin treatment. Release from the aphidicolin block achieved ca. 78% cell cycle synchronisation in the cell population. Endoreduplication was evident after release from the block in all treatments but after one cycle (24 h) the cells returned to the diploid state. This diploid culture is currently being used in our laboratory for the genetic analysis of cell death.     

Abrogation of the effects of aphidicolin on NIH3T3 and V79 cells by caffeine

In this communication I show that caffeine (1,3,7-trimethylxanthine) stimulates [³H]thymidine incorporation in aphidicolin-treated V79 and NIH3T3 cells. Flow microfluorometric analysis showed that caffeine, partially or fully, abrogates the cell cycle progression block produced by aphidicolin. Increased cell growth is also observed in cultures treated with both aphidicolin and caffeine compared to cultures treated with aphidicolin only. Microscopic examination of V79 cultures treated with aphidicolin for 8 h showed a marked reduction in the freqeuncy of round mitotic cells, as is expected from a drug which inhibits progression through the cell cycle by inhibiting DNA replication; this effect of aphidicolin was also reduced by caffeine. Biochemical analysis showed that caffeine did not directly interfere with the inhibition of DNA polymerase-α by aphidicolin. Analysis of dNTP pools indicated that caffeine increased the level of dCTP in V79 cells. In aphidicolin-treated V79 cells, the increase in the dCTP level due to exogenous cytidine was almost completely blocked; caffeine also substantially overcame this effect of aphidicolin. These results indicate that caffeine produces its effects on aphidicolin-treated cells by altering the dCTP metabolism.     

3-bromohomofascaplysin A, a fascaplysin analogue from a Fijian Didemnum sp. ascidian

A new fascaplysin analogue, 3-bromohomofascaplysin A (1), along with two known analogues, homofascaplysin A (2) and fascaplysin (3), were isolated from a Fijian Didemnum sp. ascidian. The absolute configurations of 3-bromohomofascaplysin A (1) and homofascaplysin A (2) were determined via experimental and theoretically calculated ECD spectra. The differential activities of 1-3 against different blood-borne life stages of the malaria pathogen Plasmodium falciparum were assessed. Homofascaplysin A (2) displayed an IC(50) of 0.55±0.11 nM against ring stage parasites and 105±38 nM against all live parasites. Given the stronger resistance of ring stage parasites against most current antimalarials relative to the other blood stages, homofascaplysin A (2) represents a promising agent for treatment of drug resistant malaria.     

Inhibition of DNA replication in preimplantation mouse embryos by aphidicolin

The regulation of trophectoderm differentiation in mouse embryos was studied by inhibiting DNA synthesis with aphidicolin, a specific inhibitor of DNA polymerase alpha. Embryos were exposed to aphidicolin (0.5 micrograms/ml) for 16 h at various preimplantation stages and scored for their ability to form a blastocyst and develop beyond the blastocyst stage. Embryos were most sensitive to aphidicolin at the late 4-cell stage and became progressively less sensitive as they developed. Aphidicolin inhibited blastocyst formation by 70%, 100%, 77%, and 24% after treatment at the 2-cell, 4-cell, noncompacted 8-cell, and compacted 8-cell stages, respectively. Although the inhibitory effect of aphidicolin on blastocyst formation decreased markedly as 8-cell embryos underwent compaction, developmental capacity beyond the blastocyst stage was poor after treatment of either noncompacted or compacted 8-cell embryos. Treatment at the morula and early blastocyst stages was less harmful to embryos than treatment at earlier stages but reduced the number of trophoblast outgrowths by interfering with hatching. Autoradiographic analysis showed that during aphidicolin treatment, incorporation of 3H-thymidine was inhibited over 90% at all stages examined, indicating an inhibition of DNA synthesis. Because inhibition of blastocyst formation by aphidicolin decreased at the compacted 8-cell stage, we suggest that approximately the first half of the fourth DNA replication cycle is critical for subsequent blastocyst formation. Furthermore, the poor further development of blastocysts formed after aphidicolin treatment of compacted 8-cell embryos suggests that the DNA replication requirements for initial trophectoderm differentiation are distinct from requirements for further development of blastocysts in vitro.     

Inhibition by aphidicolin of cell cycle progression and DNA replication in sea urchin embryos.

We have recently found that aphidicolin, a tetracyclic diterpene-tetraol produced by several fungi, blocks DNA synthesis of sea urchin embryos by interfering with the activity of DNA polyermase alpha. These cells fail to proliferate in the presence of aphidicolin. In continuation of these studies, we determined the drug-sensitive stage in the first cell cycle of the sea urchin Clypeaster japonicus embryo. In continuous exposure to aphidicolin (2 micrograms/ml) from five minutes after fertilization, mitotic division of the embryo was completely suppressed. Embryos were exposed to the drug at progressively later intervals and their capability for cytokinesis was examined. Evidence was thereby obtained that aphidicolin acts at the S-period to inhibit DNA synthesis resulting in developmental arrest of the embryo.     

X-ray sensitivity and DNA synthesis in synchronous culture of Plasmodium falciparum

The relationship between parasite development and sensitivity to irradiation with X-rays was investigated during a single synchronous cycle of Plasmodium falciparum in culture. The sensitivity of the parasites to irradiation was closely correlated with the phases of DNA synthesis. Their sensitivity was greatest at the ring stage in development, but decreased at the trophozoite stage when DNA synthesis begins. Lowest sensitivity was found when DNA synthesis was most rapid as the parasites were transforming from late trophozoite to schizont forms. These findings suggest that DNA is the target of the lethal radiation damage in the parasites.     

Synchronization of cell division in eight-cell bovine embryos produced in vitro: effects of aphidicolin

To date, methods for synchronizing the cell division of ungulate embryos without reducing their developmental potential have not been reliable or simple. The overall objective of this study was to determine the reliability of aphidicolin, a powerful inhibitor of eukaryotic DNA synthesis, to arrest and synchronize blastomere division in cleavage-stage bovine embryos and to assess its reversibility and toxicity in vitro. Eight-cell stage embryos obtained at 58 h post insemination were treated with several concentrations of aphidicolin for 12 h. Treated embryos were assessed for cleavage arrest, chromatin morphology and DNA synthesis; scored for blastocyst formation and hatching rate; and fixed for determination of the number of nuclei. Complete arrest of cell division was observed at aphidicolin concentrations of 1.4 microM and above. At these concentrations, no morphological alteration to interphase chromatin was observed in treated embryos compared with the controls. Removal of aphidicolin led to at least a 4-h delay before resumption of DNA synthesis and cleavage. The ability of treated embryos to reach the blastocyst stage in vitro, the hatching rate and the number of cells per blastocyst were significantly reduced compared with the control group. Since the ability of treated embryos to develop to the blastocyst stage was significantly reduced even at the minimal effective dosage, it is concluded that aphidicolin is unlikely to provide suitable cell cycle synchronization without damage to the embryos.     

DNA replication is required for tissue-specific enzyme development in ascidian embryos

Tyrosinase which is a tissue-specific enzyme in the pigment cells of the brain of the ascidian embryo, is thought to be synthesized with activation of appropriate genes, and the enzyme synthesis begins at the early tailbud stage. If embryos at early cleavage stages up to the 64-cell stage are continuously treated with aphidicolin (a specific inhibitor of DNA synthesis), cleavage of the embryos is arrested and they do not differentiate the enzyme. However, the early gastrulae and embryos at later stage that have been permanently arrested with aphidicolin do produce the enzyme. Alkaline phosphatase, a tissue-specific enzyme of the endodermal cells, has been shown to be synthesized by a preformed maternal mRNA and is first detected histochemically at the late gastrula stage. If embryos at early cleavage stages up to the 16-cell stage are prevented from undergoing further divisions with aphidicolin, the arrested embryos do not form the enzyme. However, embryos at the 32-cell and later stages that have been permanently arrested with aphidicolin are able to differentiate the enzyme activity. These results suggest that several DNA replications are required for the histospecific enzyme development in ascidian embryos.     

Inhibition of hexose transport and abrogation of pH homeostasis in the intraerythrocytic malaria parasite by an O-3-hexose derivative

An O-3-hexose derivative, shown previously to inhibit a malaria parasite hexose transporter expressed in Xenopus oocytes as well as to suppress the multiplication of parasites, both in vitro and in vivo, was shown here to block the uptake of hexose sugars into isolated blood-stage parasites. This led to a decline in ATP levels and the loss of intracellular pH control. The results are consistent with those obtained with the cloned transporter. They support the notion that the transporter mediates uptake of glucose into the intraerythrocytic parasite and provide further support for the view that it is a suitable antimalarial drug target.     

An Analytical Method for Assessing Stage-Specific Drug Activity in Plasmodium vivax Malaria: Implications for Ex Vivo Drug Susceptibility Testing

The emergence of highly chloroquine (CQ) resistant P. vivax in Southeast Asia has created an urgent need for an improved understanding of the mechanisms of drug resistance in these parasites, the development of robust tools for defining the spread of resistance, and the discovery of new antimalarial agents. The ex vivo Schizont Maturation Test (SMT), originally developed for the study of P. falciparum, has been modified for P. vivax. We retrospectively analysed the results from 760 parasite isolates assessed by the modified SMT to investigate the relationship between parasite growth dynamics and parasite susceptibility to antimalarial drugs. Previous observations of the stage-specific activity of CQ against P. vivax were confirmed, and shown to have profound consequences for interpretation of the assay. Using a nonlinear model we show increased duration of the assay and a higher proportion of ring stages in the initial blood sample were associated with decreased effective concentration (EC(50)) values of CQ, and identify a threshold where these associations no longer hold. Thus, starting composition of parasites in the SMT and duration of the assay can have a profound effect on the calculated EC(50) for CQ. Our findings indicate that EC(50) values from assays with a duration less than 34 hours do not truly reflect the sensitivity of the parasite to CQ, nor an assay where the proportion of ring stage parasites at the start of the assay does not exceed 66%. Application of this threshold modelling approach suggests that similar issues may occur for susceptibility testing of amodiaquine and mefloquine. The statistical methodology which has been developed also provides a novel means of detecting stage-specific drug activity for new antimalarials.     

DNA Replication is Required for Tissue-Specific Enzyme Development in Ascidian Embryos

Tyrosinase which is a tissue-specific enzyme in the pigment cells of the brain of the ascidian embryo, is thought to be synthesized with activation of appropriate genes, and the enzyme synthesis begins at the early tailbud stage. If embryos at early cleavage stages up to the 64-cell stage are continuously treated with aphidicolin (a specific inhibitor of DNA synthesis), cleavage of the embryos is arrested and they do not differentiate the enzyme. However, the early gastrulae and embryos at later stages that have been permanently arrested with aphidicolin do produce the enzyme. Alkaline phosphatase, a tissue-specific enzyme of the endodermal cells, has been shown to be synthesized by a preformed maternal mRNA and is first detected histochemically at the late gastrula stage. If embryos at early cleavage stages up to the 16-cell stage are prevented from undergoing further divisions with aphidicolin, the arrested embryos do not form the enzyme. However, embryos at the 32-cell and later stages that have been permanently arrested with aphidicolin are able to differentiate the enzyme activity. These results suggest that several DNA replications are required for the histospecific enzyme development in ascidian embryos.     

Plasmodium species: flow cytometry and microfluorometry assessments of DNA content and synthesis

Fluorescence intensities were established by flow cytometry of different erythrocytic stages of Plasmodium berghei after staining of their DNA with Hoechst-33258 or Hoechst-33342. Parasites were obtained from highly synchronized infections or in vitro cultures. Most fluorescence measurements were performed using a low cost, clinical flow cytometer, equipped with a mercury arc lamp. Cells infected with P. berghei could be readily distinguished from uninfected cells on the basis of Hoechst-DNA fluorescence and single, double, and triple ring infected cells were separated clearly. The relative fluorescence intensities of different developmental stages (merozoites, ringforms, trophozoites, schizonts, and gametocytes) corresponded closely to the relative DNA contents of these stages as measured by microfluorometry. Flow cytometry appeared to be a sensitive and rapid method to measure DNA synthesis during asexual development; a C50 value of 5 microM of aphidicolin, a specific inhibitor of DNA synthesis, was established. Vital staining of parasites in culture was possible with both Hoechst dyes. After removal of Hoechst-33258, normal in vitro development of the stained parasites was observed. After Hoechst staining, the haploid ringforms of P. vivax showed slightly less fluorescence (15%) than ringforms of P. berghei and P. falciparum. No differences in fluorescence intensity were observed, however, by direct microfluorometry after Feulgen-pararosaniline staining, indicating that all three species have the same DNA content.     

Functional characterisation of sexual stage specific proteins in Plasmodium falciparum

The various stages of the malaria parasites in the vertebrate host and in the mosquito vector offer numerous candidates for vaccine and drug development. However, the biological complexity of the parasites and the interaction with the immune system of the host continue to frustrate all such efforts thus far. While most of the targets for drug and vaccine design have focused on the asexual stages, the sexual stages of the parasite are critical for transmission and maintenance of parasites among susceptible vertebrate hosts. Sexual stage parasites undergo a series of morphological and biochemical changes during their development, accompanied by a co-ordinated cascade of a distinct expression pattern of sexual stage specific proteins. Mechanisms underlying the developmental switch from asexual parasite to sexual parasite still remain elusive. Methods that can break the malaria transmission cycle thus occupy a central place in the overall malaria control strategies. This paper provides a review of genes expressed in sexually differentiated Plasmodium. In the past few years, a molecular approach based on targeted gene disruption has revealed fascinating biological roles for many of the sexual stage gene products. In addition, we will briefly discuss other functional genomic approaches employed to study not only sexual but also other aspects of host-parasite biology.     

Current opinion on an emergence of drug resistant strains of Plasmodium falciparum through genetic alterations

The human malarial parasite Plasmodium falciparum is one of the world''s most devastating pathogen. Its capability to regulate its genes under various stages of its life cycle as well as under unfavourable environmental conditions has led to the development of vaccine resistant strains. Similarly, under drug pressure it develops mutations in the target genes. These mutations confer mid and high-level resistance to the antimalarial drugs. Increasing a resistance of malaria parasites to conventional antimalarial drugs is an important factor contributing to the persistence of the disease as a major health threat. This article reviews current knowledge of stage specific malarial targets, antimalarial drugs and the mutations that have led to the emergence of resistant strains.     

Effect of aphidicolin on vaccinia virus: isolation of an aphidicolin-resistant mutant.

Vaccinia virus growth in BSC-1 and HeLa cells was inhibited by aphidicolin concentrations of 20 microM or more. Virus yield, which decreased only when the drug was added early in infection, was reduced several 100-fold by 80 microM aphidicolin. Viral inhibition was reversed by the suspension of the infected cells in drug-free medium. DNA synthesis in uninfected cells was reduced about 10-fold by 1 microM aphidicolin. In infected cells, aphidicolin concentrations over 10 microM were needed to reduce DNA synthesis to the same extent as in uninfected cells. Fractionation of infected cells which were incubated with 1 microM drug showed that cytoplasmic viral DNA synthesis was resistant to this aphidicolin concentration. The radioactivity associated with crude nuclei from these cells was estimated to be from vaccinia DNA synthesis. Spontaneous virus mutants which were resistant to 80 microM aphidicolin did not appear. However, after mutagenesis, mutants were generated which formed large plaques in medium with 80 microM drug. In cells with replicating aphidicolin-resistant virus, DNA synthesis was about four times more resistant to 80 microM aphidicolin than in cells with replicating wild-type virus. Chromatographic patterns of viral DNA polymerase isolated from cells with wild-type or resistant virus were similar. However, in an in vitro assay, 50% inhibition of enzyme activity was obtained with ca. 75 and 188 microM aphidicolin for the wild-type and resistant DNA polymerases, respectively. Viral enzymes were much more resistant to the drug than were the cell polymerases.     

Tolerance is the key to understanding antimalarial drug resistance

The evolution of antimalarial drug resistance is often considered to be a single-stage process in which parasites are either fully resistant or completely sensitive to a drug. However, this does not take into account the important intermediate stage of drug tolerance. Drug-tolerant parasites are killed by the high serum concentrations of drugs that occur during direct treatment of the human host. However, these parasites can spread in the human population because many drugs persist long after treatment, and the tolerant parasites can infect people in which there are residual levels of the drugs. This intermediate stage between fully sensitive and fully resistant parasites has far-reaching implications for the evolution of drug-resistant malaria.