Basal and luminal are two molecular subtypes of breast cancer with opposite histoclinical features. We report a combined, high-resolution analysis of genome copy number and gene expression in primary basal and luminal breast cancers. First, we identified and compared genomic alterations in 45 basal and 48 luminal tumors by using 244K oligonucleotide array comparative genomic hybridization (aCGH). We found various genome gains and losses and rare high-level gene amplifications that may provide therapeutic targets. We show that gain of 10p is a new alteration in basal breast cancer and that a subregion of the 8p12 amplification is specific of luminal tumors. Rare high-level amplifications contained BCL2L2, CCNE, EGFR, FGFR2, IGF1R, NOTCH2, and PIK3CA. Potential gene breaks involved ETV6 and FLT3. Second, we analyzed both aCGH and gene expression profiles for 42 basal and 32 luminal breast cancers. The results support the existence of specific oncogenic pathways in basal and luminal breast cancers, involving several potential oncogenes and tumor suppressor genes (TSG). In basal tumors, 73 candidate oncogenes were identified in chromosome regions 1q21-23, 10p14, and 12p13 and 28 candidate TSG in regions 4q32-34 and 5q11-23. In luminal breast cancers, 33 potential oncogenes were identified in 1q21-23, 8p12-q21, 11q13, and 16p12-13 and 61 candidate TSG in 16q12-13, 16q22-24, and 17p13. HORMAD1 (P = 6.5 Â 10 À5 ) and ZNF703 (P = 7 Â 10 À4

No abstract

Background: Parasite concentration methods facilitate molecular, biochemical and immunological research on the erythrocytic stages of Plasmodium. In this paper, an adaptation of magnetic MACS ® columns for the purification of human Plasmodium species is presented. This method was useful for the concentration/purification of either schizonts or gametocytes.

The cDNA encoding Pfmap-2, an enzyme of the human malaria parasite Plasmodium falciparum, was cloned, sequenced, and expressed in Escherichia coli. The open reading frame carried by the Pfmap-2 cDNA encodes a 508-amino acid polypeptide of 59.2 kDa with maximal homology to mitogen-activated protein kinases (MAPKs) from various organisms. The purified recombinant enzyme displayed functional characteristics of MAPKs such as (i) ability to undergo autophosphorylation, (ii) ability to phosphorylate myelin basic protein, a classical MAPK substrate, (iii) regulation of kinase activity by a MAPK-specific phosphatase, and (iv) ability to be activated by component(s) present in cell extracts. Mutational analysis of the recombinant protein allowed the identification of residues that are important for enzymatic activity. Northern blot analysis and immunofluorescence assays indicated that Pfmap-2 is expressed specifically in gametocytes, the form that is responsible for transmission of the parasite to the mosquito vector. Gametocyte extracts activated recombinant Pfmap-2 more efficiently than extracts from asexual parasites, which is consistent with this stage specificity. Despite its overall high level of homology to MAPKs, Pfmap-2 presents the peculiarity of not possessing the conserved threonine-X-tyrosine activation motif usually found in enzymes of this family; instead, it has a threonine-serine-histidine at the same location. This atypical feature formed the basis for a detailed analysis of the primary structure of MAPKs, allowing us to define an operational MAPK signature, which is shared by Pfmap-2. The fact that no MAPK from vertebrates diverge in the activation motif suggests that the fine mechanisms of Pfmap-2 regulation may offer an opportunity for antimalarial drug targeting.The spread of drug resistance in Plasmodium falciparum, the parasite responsible for the lethal form of human malaria, represents one of the most pressing public health problems in many parts of the world (1, 2). Parasites that are resistant to anti-malarials are selected under drug pressure in treated patients, develop into male and female gametocytes that are infective to the mosquito vector, and hence can be transmitted to new human hosts. One possible way to limit the spread of P. falciparum resistance might consist in interfering with sexual development of the parasite, thereby preventing transmission. A rational approach to this goal requires a detailed knowledge of the molecular mechanisms of Plasmodium sexual development.After invasion of a red blood cell, a merozoite can either embark on a new cycle of asexual multiplication leading to the formation of a schizont ultimately releasing 8 -32 new merozoites or undergo sexual differentiation (gametocytogenesis), a process characterized by cell cycle arrest, a shift in the transcriptional repertoire, and morphological changes (reviewed in Refs. 3-4). Mature gametocytes maintain their cell cycle arrested while in the blood of the human host, but this block is relieved immediately after th...

No abstract

We have cloned Pfnek-1, a gene encoding a novel protein kinase from the human malaria parasite Plasmodium falciparum. This enzyme displays maximal homology to the never-in-mitosis/Aspergillus (NIMA)/NIMA-like kinase (Nek) family of protein kinases, whose members are involved in eukaryotic cell division processes. Similar to other P. falciparum protein kinases and many enzymes of the NIMA /Nek family, Pfnek-1 possesses a large C-terminal extension in addition to the catalytic domain. Bacterially expressed recombinant Pfnek-1 protein is able to autophosphorylate and phosphorylate a panel of protein substrates with a specificity that is similar to that displayed by other members of the NIMA /Nek family. However, the FXXT motif usually found in NIMA /Nek protein kinases is substituted in Pfnek-1 by a SMAHS motif, which is reminiscent of a MAP/ERK kinase (MEK) activation site.Mutational analysis indicates that only one of the serine residues in this motif is essential for Pfnek-1 kinase activity in vitro. We show (a) that recombinant Pfnek-1 is able to specifically phosphorylate Pfmap-2, an atypical P. falciparum MAPK homologue, in vitro, and (b) that coincubation of Pfnek-1 and Pfmap-2 results in a synergistic increase in exogenous substrate labelling. This suggests that Pfnek-1 may be involved in the modulation of MAPK pathway output in malaria parasites. Finally, we demonstrate that recombinant Pfnek-1 can be used in inhibition assays to monitor the effect of kinase inhibitors, which opens the way to the screening of chemical libraries aimed at identifying potential new antimalarials.

The in vitro activities of cyclines (tetracycline, doxycycline, minocycline, oxytetracycline, and rolitetracycline), macrolides (erythromycin, spiramycin, roxithromycin, and lincomycin), quinolones (norfloxacin and ofloxacin), rifampin, thiamphenicol, tobramycin, metronidazole, vancomycin, phosphomycin, and cephalosporins (cephalexin, cefaclor, cefamandole, cefuroxime, ceftriazone, cefotaxime, and cefoxitin) were evaluated on Plasmodium falciparum clones, using an isotopic, micro-drug susceptibility test. Only tetracyclines, macrolides, quinolones, and rifampin demonstrated in vitro activity against P. falciparum, which increased after a prolonged exposure (96 or 144 h). In the presence of iron (FeCl 3 ), only the activities of tetracyclines and norfloxacin were decreased. Their in vitro activity against intraerythrocytic stages of multidrug-resistant P. falciparum and their efficacy in vivo favor the use of antibiotics as antimalarial drugs. However, due to their slow antimalarial action and to the fact that they act better after prolonged contact, they probably need to be administered in conjunction with a rapidly acting antimalarial drug, such as a short course of chloroquine or quinine.

In Plasmodium falciparum, the causative agent of human malaria, the catalytic subunit gene of cAMP-dependent protein kinase (Pfpka-c ) exists as a single copy. Interestingly, its expression appears developmentally regulated, being at higher levels in the pathogenic asexual stages than in the sexual forms of parasite that are responsible for transmission to the mosquito vector. Within asexual parasites, PfPKA activity can be readily detected in schizonts. Similar to endogenous PKA activity of noninfected red blood cells, the parasite enzyme can be stimulated by cAMP and inhibited by protein kinase inhibitor. Importantly, ex vivo treatment of infected erythrocytes with the classical PKA-C inhibitor H89 leads to a block in parasite growth. This suggests that the PKA activities of infected red blood cells are essential for parasite multiplication. Finally, structural considerations suggest that drugs targeting the parasite, rather than the erythrocyte enzyme, might be developed that could help in the fight against malaria.Keywords: parasite; PKA; inhibition; H89.The emergence and dissemination of drug-resistant malaria parasites represents one of the most important public health problems in many parts of the world today. New antimalarials are urgently required, whose rational design and development requires the identification of potential therapeutic targets. This in turns rests on a better understanding of the molecular mechanisms controlling the progression of the complex life cycle of malaria parasites, especially Plasmodium falciparum, the species responsible for the lethal form of the disease. All Plasmodium species are intracellular parasites during infection of their vertebrate hosts. Sporozoites inoculated into a host during a bite by an infected Anopheles mosquito soon invade hepatocytes, within which intense asexual division takes place (exoerythrocytic schizogony), yielding up to 40 000 merozoites in the case of P. falciparum (http:// www.malaria.org). Upon schizont rupture, these merozoites invade red blood cells, where additional rounds of asexual replication occur (erythrocytic schizogony, the phase responsible for the pathogenesis of the disease). Some merozoites, instead of undergoing a further asexual cycle, arrest their cell cycle and differentiate into male or female gametocytes. Unlike asexual forms, these sexual forms are infective to the Anopheles vector.cAMP is involved in the regulation of development of several microorganisms, and cAMP-dependent pathways exist in most eukaryotic cells. For example, in the slime mold Dictyostelium discoideum, cAMP acts as a signal for the aggregation and differentiation of cells into a multicellular organism (reviewed in [1]). In most instances, cAMP exerts its action by binding to the regulatory subunit complexed to the catalytic subunit in an inactive holoenzyme of the protein kinase A (PKA or cAMPdependent protein kinase), thereby releasing the active catalytic subunit (PKA-C), whose substrates can include other protein kinases and transcription factors. P...