These observations prompt a modification of the current paradigms of the pathogenesis of malaria and clear the way to investigate the pathophysiology of P. vivax infections.

Malaria is one of the leading causes of morbidity and mortality in the tropics, with 300 to 500 million clinical cases and 1.5 to 2.7 million deaths per year. Nearly all fatal cases are caused by Plasmodium falciparum. The resistance of this parasite to conventional antimalarial drugs such as chloroquine is growing at an alarming rate and therefore new efficient drugs are urgently needed (1-3).In all organisms studied so far, the biosynthesis of isoprenoids such as dolichol, cholesterol, and ubiquinones depends on the condensation of the different numbers of isopentenyl diphosphate (IPP) 1 and dimethylallyl diphosphate units. In mammals and fungi, these units are derived from the classical mevalonate pathway (4). However, in higher plants, in several algae, in some eubacteria, and in P. falciparum the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway was described as the alternative non-mevalonate pathway for the synthesis of IPP (for reviews, see Refs. 5-10). This pathway starts with the condensation of pyruvate and glyceraldehyde 3-phosphate, which yields 1-deoxy-D-xylulose-5-phosphate (DOXP) as a key metabolite (11-17). The DOXP reductoisomerase then catalyzes the simultaneous intramolecular rearrangement and reduction of DOXP to form MEP (18 -22). The activity of this enzyme is specifically inhibited by fosmidomycin (23). Several reaction steps are necessary for the conversion of MEP to IPP. The downstream intermediates of MEP for this pathway are: 4-(cytidine-5-diphospho)-2-C-methyl-D-erythritol (CDP-ME) (24), 4-(cytidine-5-diphospho)-2-C-methyl-D-erythritol-2-phosphate (CDP-MEP), 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (ME-2,4-cPP) (25, 26), and 4-hydroxy-3-methylbut-2-enyl pyrophosphate (27-31). IPP and dimethylallyl diphosphate are synthesized through independent routes in the late steps of the non-mevalonate pathway (32). These units are used for the biosynthesis of ubiquinones and dolichols, and for the prenylation of proteins and other products (33)(34)(35).Based on the sequence data provided by the malaria genome project (plasmodb.org), Jomaa and co-workers (21) identified two genes in P. falciparum that encode key enzymes of the MEP pathway: 1-deoxy-D-xylulose-5-phosphate synthase and 1-deoxy-D-xylulose-5-phosphate reductoisomerase. They also demonstrated that an amino-terminal signal sequence in 1-de- 1 The abbreviations and trivial names used are: IPP, isopentenyl diphosphate; MEP, 2-C-methyl-D-erythritol-4-phosphate; DOXP, 1-deoxy-D-xylulose-5-phosphate; DOX, 1-deoxy-D-xylulose; CDP-ME, 4-(cytidine-5-diphospho)-2-C-methyl-D-erythritol; CDP-MEP, 4-(cytidine-5-diphospho)-2-C-methyl-D-erythritol-2-phosphate; ME-2,4-cPP, 2-Cmethyl-D-erythritol-2,4-cyclodiphosphate; HPLC, high-performance liquid chromatography; ESI-QTOF-MS, ESI-quadrupole time-of-flight mass spectrometry; Q n , Coenzyme Q n .

Carotenoids are widespread lipophilic pigments synthesized by all photosynthetic organisms and some nonphotosynthetic fungi and bacteria. All carotenoids are derived from the C40 isoprenoid precursor geranylgeranyl pyrophosphate, and their chemical and physical properties are associated with light absorption, free radical scavenging, and antioxidant activity. Carotenoids are generally synthesized in well defined subcellular organelles, the plastids, which are also present in the phylum Apicomplexa, which comprises a number of important human parasites, such as Plasmodium and Toxoplasma. Recently, it was demonstrated that Toxoplasma gondii synthesizes abscisic acid. We therefore asked if Plasmodium falciparum is also capable of synthesizing carotenoids. Herein, biochemical findings demonstrated the presence of carotenoid biosynthesis in the intraerythrocytic stages of the apicomplexan parasite P. falciparum. Using metabolic labeling with radioisotopes, in vitro inhibition tests with norflurazon, a specific inhibitor of plant carotenoid biosynthesis, the results showed that intraerythrocytic stages of P. falciparum synthesize carotenoid compounds. A plasmodial enzyme that presented phytoene synthase activity was also identified and characterized. These findings not only contribute to the current understanding of P. falciparum evolution but shed light on a pathway that could serve as a chemotherapeutic target.

Isoprenylation is an essential protein modification in eukaryotic cells. Herein, we report that in Plasmodium falciparum, a number of proteins were labeled upon incubation of intraerythrocytic forms with either [ 3 H]farnesyl pyrophosphate or [ 3 H]geranylgeranyl pyrophosphate. By thin-layer chromatography, we showed that attached isoprenoids are partially modified to dolichol and other, uncharacterized, residues, confirming active isoprenoid metabolism in this parasite. Incubation of blood-stage P. falciparum treated with the isoprenylation inhibitor limonene significantly decreased the parasites' progression from the ring stage to the trophozoite stage and at 1.22 mM, 50% of the parasites died after the first cycle. Using Ras-and Rap-specific monoclonal antibodies, putative Rap and Ras proteins of P. falciparum were immunoprecipitated. Upon treatment with 0.5 mM limonene, isoprenylation of these proteins was significantly decreased, possibly explaining the observed arrest of parasite development.Malaria, a major tropical disease caused by protozoa of the genus Plasmodium, affects 300 to 500 million people and causes the deaths of over 1 million individuals per year, mostly African children under 10 years of age. Plasmodium falciparum, the most virulent of the four species which infect humans, is associated with potentially fatal disease (37). The major clinical symptoms of the disease stem from the destruction of red blood cells during the multiplication of asexual parasites, leading to anemia, or cytoadherence of parasitized red blood cells to endothelial receptors, resulting in severe forms of malaria (26). Because of the expanding resistance of these parasites to virtually all of the reagents used in malaria therapy, new approaches to drug design are urgently needed. The identification of potential targets that are essential to the parasite's life cycle is a prerequisite for rational drug development.Protein prenylation is a general phenomenon in eukaryotic cells and was recently detected in parasites like Giardia lamblia (22), Trypanosoma brucei (12), and Schistosoma mansoni (6). In P. falciparum, Chakrabarti et al. detected protein prenyl transferase activities (5). Additionally, Rab GTP-binding proteins (Rab6) cloned from P. falciparum (21, 34) have the carboxyl-terminal motif Cys-AAX (where the letter A initially signified an aliphatic amino acid and the letter X denoted an undefined amino acid) and Cys-Cys residues, which suggests that they may be prenylated. Finally, Jomaa et al. demonstrated an alternative isopentenyl synthesis pathway so far described only in algae or cyanobacteria, which could be efficiently inhibited by fosmidomycin (17).The aims of this work were to characterize protein geranylgeranylation and farnesylation in the protozoan parasite P. falciparum and to test whether the monoterpene limonene, a nontoxic inhibitor of the prenyl protein transferase enzyme and initially used against tumor cells (1, 36), is also active against the fast-growing malaria parasite P. falciparum. MATERIALS...

These results suggest that a possible interaction between human and simian malaria coming from a zoonotic cycle cannot be discarded because simians that live in the areas of the Atlantic Forest could play a role as a reservoir for Plasmodium.

We have recently reported on our experience with C-X-C-motif chemokine receptor 4- (CXCR4-) directed radioligand therapy (RLT) in multiple myeloma and acute leukemia. Six patients with heavily pre-treated relapsed diffuse large B cell lymphoma (DLBCL) (3 males, 3 females; aged, 54±8 years) underwent CXCR4-directed RLT in combination with conditioning chemotherapy and allogeneic stem cell transplantation (SCT). In 2 patients, radioimmunotherapy (RIT) targeting CD20 or CD66 was added to enhance anti-lymphoma activity. Endpoints were incidence and severity of adverse events, progression-free and overall survival. RLT as well as additional RIT were well-tolerated without any acute adverse events or changes in vital signs. Successful engraftment was recorded after a median of 11 days (range, 9-13 d). In the 4 patients who were available for follow-up (one patient died of CNS aspergillosis 29 days, another of sepsis in aplasia 34 days after after RLT), CXCR4-directed RLT resulted in partial response in 2/4 cases (both treated with additional RIT) and mixed response in the remaining 2 subjects. Response duration was rather short-lived with median progression-free survival of 62 days (range, 29-110 d) and median overall survival of 76 days (range, 29-313 d). CXCR4-directed RLT (in combination with additional RIT) as a conditioning regimen prior to allogeneic SCT in DLBCL is feasible.

PfCDPK1 [Plasmodium falciparum CDPK1 (calcium-dependent protein kinase 1)] is highly expressed in parasite asexual blood and mosquito stages. Its role is still poorly understood, but unsuccessful gene knockout attempts suggest that it is essential for parasite replication and/or RBC (red blood cell) invasion. In the present study, by tagging endogenous CDPK1 with GFP (green fluorescent protein), we demonstrate that CDPK1 localizes to the parasite plasma membrane of replicating and invasive forms as well as very young intracellular parasites and does not appear to be exported into RBCs. Although a knockdown of endogenous CDPK1 was achieved using a destabilization domain, parasites tolerated reduced expression without displaying a phenotype. Because of this, the PfCDPK1 auto-inhibitory J (junction) domain was explored as a means of achieving inducible and specific inhibition. Under in vitro conditions, a fusion protein comprising a J-GFP fusion specifically bound to PfCDPK1 and inhibited its activity. This fusion protein was conditionally expressed in P. falciparum asexual blood stages under the regulation of a DD (destabilization domain) (J-GFP-DD). We demonstrate that J-GFP-DD binds to CDPK1 and that this results in the arrest of parasite development late in the cell cycle during early schizogony. These data point to an early schizont function for PfCDPK1 and demonstrate that conditionally expressing auto-inhibitory regions can be an effective way to address the function of Plasmodium enzymes.

Violacein is a violet pigment extracted from the gram-negative bacterium Chromobacterium violaceum. It presents bactericidal, tumoricidal, trypanocidal, and antileishmanial activities. We show that micromolar concentrations efficiently killed chloroquine-sensitive and -resistant Plasmodium falciparum strains in vitro; inhibited parasitemia in vivo, even after parasite establishment; and protected Plasmodium chabaudi chabaudiinfected mice from a lethal challenge.Violacein is a violet pigment isolated from Chromobacterium violaceum, a gram-negative betaproteobacterium found in the Amazon River in Brazil. It has been reported to kill bacteria (4) and induces apoptosis in various types of cancer cells (1,5,7,8,10,11). Moderate activity against Trypanosoma cruzi and Leishmania amazonensis promastigotes has also been observed (3, 9). Due to the widespread presence of drug resistance in the malaria parasite, resulting in dramatically decreased efficacy of available antimalarial drugs (15), and the fact that immunoprotection achieved by the most successful malaria vaccine is only partial and short-lived (14), we evaluated the in vitro and in vivo effects of violacein on human and murine blood stage forms of Plasmodium parasites.Isolation and purification of violacein, (Fig. 1), from C. violaceum (CCT3496) were performed as previously described (12). Toxicity was measured as the concentration-dependent lysis of normal erythrocytes (NE) by counting red blood cells per milliliter with the aid of a Neubauer chamber. After 48 h of exposure to various concentrations of violacein, the percent red blood cell density (RBCD) relative to that of the control (without violacein) was monitored and calculated according to the formula (RBCD per milliliter in the presence of violacein/RBCD per milliliter without violacein) ϫ 100. As shown in Fig. 2A, a slight reduction in the RBCD percentage at violacein concentrations of Ͼ8.0 M was observed. Significant (Mann-Whitney U test, P Ͻ 0.05) toxicity to NE occurred at a concentration of 14.0 M.Next, we performed dose-response assays to obtain the 50% inhibitory concentrations (IC 50 s) of violacein against erythrocytes infected with chloroquine-sensitive or -resistant strains of P. falciparum (3D7 [16] or S20 [2], respectively) at 1% parasitemia and a 2% final hematocrit. We used [ 3 H]hypoxanthine (Amersham Biosciences, Amersham, United Kingdom) incorporation to assess parasite growth according to a protocol described elsewhere (13). Violacein was tested in triplicate at least three times with different batches and cells, and parasite growth was compared to that in nontreated infected erythrocytes (IE), which represented 100% parasite growth. Percent parasite growth inhibition was calculated according to the formula [1 Ϫ (cpm of treated IE Ϫ cpm of NE/cpm of nontreated IE Ϫ cpm of NE)] ϫ 100. After a 48-h incubation, violacein inhibited parasite development even at the lowest tested concentration of 0.06 M and completely abrogated parasite viability at concentrations of Ͼ1.0 M (Fig. 2B).The ...