Antigens associated with the surface of merozoites of the malaria parasite Plasmodium falciparum are directly accessible to immune attack and therefore are prime vaccine candidates. We have previously shown that one of the two known merozoite surface antigens (merozoite surface antigen 2; MSA-2) exhibits considerable sequence and antigenic diversity in different isolates. The sequences of MSA-2 from three isolates revealed a central domain composed of repeats that vary in number, length, and sequence, flanked in turn by nonrepetitive variable sequences and by conserved Nand C-terminal domains. We report here the sequences of a further four MSA-2 alleles, containing repetitive sequences that are related but not identical to each other. The seven alleles of MSA-2 can be divided into two distinct allele families on the basis of nonrepetitive sequences. Hybridization studies with repeat probes indicated that all of the 44 P. falciparum isolates examined contained repeat regions similar to those dermed in known MSA-2 sequences.

SummaryPlasmodium falciparum is a protozoan parasite responsible for the most severe forms of human malaria. All the clinical symptoms and pathological changes seen during human infection are caused by the asexual blood stages of Plasmodium. Within host red blood cells, the parasite undergoes enormous developmental changes during its maturation. In order to analyse the expression of genes during intraerythrocytic development, DNA microarrays were constructed and probed with stage-specific cDNA. Developmental upregulation of specific mRNAs was found to cluster into functional groups and revealed a co-ordinated programme of gene expression. Those involved in protein synthesis (ribosomal proteins, translation factors) peaked early in development, followed by those involved in metabolism, most dramatically glycolysis genes. Adhesion/ invasion genes were turned on later in the maturation process. At the end of intraerythrocytic development (late schizogony), there was a general shut-off of gene expression, although a small set of genes, including a number of protein kinases, were turned on at this stage. Nearly all genes showed some regulation over the course of development. A handful of genes remained constant and should be useful for normalizing mRNA levels between stages. These data will facilitate functional analysis of the P. falciparum genome and will help to identify genes with a critical role in parasite progression and multiplication in the human host.

The emergence of chloroquine resistance in Plasmodium falciparum has necessitated the development of alternate strategies for chemotherapy and chemoprophylaxis. One approach has been the identification of drugs that do not possess any intrinsic antimalarial activity when used alone but that potentiate the effect of currently available antimalarial drugs, such as chloroquine. We identified fluoxetine hydrochloride (Prozac), a commonly prescribed antidepressant, as another resistance modulator for drug-resistant P. falciparum. Studies with chloroquine-resistant clones and isolates from various geographical locations confirmed our initial observations with a chloroquine-resistant P. falciparum clone, W2. Fluoxetine concentrations of 500 nM were found to effectively modulate chloroquine resistance by 66% in clone W2. In comparison, verapamil at similar concentrations was observed to modulate chloroquine resistance in clone W2 by 61%. Neither fluoxetine nor verapamil was observed to possess any innate antimalarial activity. These data augment the current description of the chloroquine resistance phenotype and may provide additional insights into lead-directed synthesis of new antimalarial drugs.Malaria is a significant source of morbidity in the world, with an estimated annual prevalence of 270 million infections (19). It is a serious impediment to economic and agricultural growth and development, particularly in less developed countries. At present, the quinoline ring-containing drugs remain the most efficacious drugs for chemoprophylaxis and chemotherapy of Plasmodium falciparum malaria. Chloroquine, a 4-aminoquinoline, has been the drug of choice for several decades. However, the increasing prevalence and the degree of chloroquine resistance in regions endemic for P. falciparum malaria have substantially compromised its clinical utility. Although more than 350,000 compounds have been evaluated for their antimalarial activities at the Walter Reed Army Institute of Research, few new leads for drug development have been identified. Despite the development of mefloquine, halofantrine, and artemisinin as new blood schizonticides, few effective drugs with enhanced antimalarial activity, reduced cost, and low toxicity exist. In order to preserve the efficacies of current and future chemotherapeutic agents, alternative drug strategies must continually be developed, evaluated, and implemented. One approach that merits further investigation and possible implementation has been the identification of drugs that, at subinhibitory concentrations, modulate chloroquine resistance. Such drugs include verapamil (14), desipramine (3), chlorpromazine (12), ketotifen (1), tetrandrine (20, 21), and cyproheptadine (16).Our rationale for screening fluoxetine hydrochloride (Prozac) as a potential modulator was based on our previous observations that various compounds with neuroleptic activ-* Corresponding author. ity, desipramine (3) and chlorpromazine (12), are effective modulators of chloroquine resistance; in contrast, penfluridol (13,...

Chloroquine is one of the most effective antimalarials, but resistance to it is becoming widespread. However, we do not fully understand either the drug's mode of action or the mechanism of resistance. In an effort to expand our understanding of the mechanism of action and resistance associated with chloroquine, we used Saccharomyces cerevisiae as a model eukaryotic system. To aid in the discovery of potential drug targets we applied the transcriptional profiling method to identify genes transcriptionally responsive to chloroquine treatment in S. cerevisiae. Among the genes that were differentially expressed with chloroquine treatment were a number of metal transporters involved in iron acquisition (SIT1, ARN2, ARN4, and SMF2). These genes exhibit similar expression patterns, and several are known to be regulated by AFT1, a DNA binding protein, which responds to iron levels in the cell. We investigated the role of chloroquine in iron metabolism by using a variety of approaches, including pharmacological, genetic, and biochemical techniques. For these experiments, we utilized yeast lacking the major iron uptake pathways (FET3 and FET4) and yeast deficient in SIT1, encoding the major up-regulated iron siderophore transporter. Our experiments show that yeast genetically or environmentally limited in iron availability has increased sensitivity to chloroquine in pharmacological assays and that the addition of iron rescues these cells from chloroquine killing.55 FeCl 3 accumulation was inhibited in the presence of chloroquine, and kinetic analysis demonstrated that inhibition was competitive. These results are consistent with deprivation of iron as a mechanism of chloroquine killing in yeast.Chloroquine (CQ) is commonly used for treatment of malaria; however, its mode of action and the mechanism of resistance are still not fully understood. Better knowledge of CQ's mode of action may make it possible to identify new drugs that target similar pathways or to reverse existing resistant phenotypes.CQ has been shown to interact in both mammalian cells and Plasmodium spp. with a number of different pathways, including changes in vacuolar (or lysosomal) pH (9,22,39,56), binding to DNA and RNA (3,11,41,53), binding to heme and ␤-hematin in Plasmodium falciparum (1, 55; reviewed in reference 20). In the case of P. falciparum, CQ was an effective drug whose efficacy has been severely compromised by the emergence of drug-resistant parasites (8,20).To analyze the mechanism of CQ action, we used transcriptional profiling in a model eukaryotic system, Saccharomyces cerevisiae. Differential transcriptional profiling using microarray analysis is based on detecting differences in expression of mRNAs in cells treated under different conditions. No information other than the genomic sequence and the open reading frame (ORF) predictions is necessary to assay mRNA expression. Such whole-genome analysis allows the determination of expression profiles without preselection of genes. For the experiments described here, we used the Affymetrix...

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