Sequence of Plasmodium falciparum chromosomes 2, 10, 11 and 14

The mosquito-borne malaria parasite Plasmodium falciparum kills an estimated 0.7-2.7 million people every year, primarily children in sub-Saharan Africa. Without effective interventions, a variety of factors-including the spread of parasites resistant to antimalarial drugs and the increasing insecticide resistance of mosquitoes-may cause the number of malaria cases to double over the next two decades. To stimulate basic research and facilitate the development of new drugs and vaccines, the genome of Plasmodium falciparum clone 3D7 has been sequenced using a chromosome-by-chromosome shotgun strategy. We report here the nucleotide sequences of chromosomes 10, 11 and 14, and a re-analysis of the chromosome 2 sequence. These chromosomes represent about 35% of the 23-megabase P. falciparum genome.     

Sequence of Plasmodium falciparum chromosome 12

The human malaria parasite Plasmodium falciparum is responsible for the death of more than a million people every year. To stimulate basic research on the disease, and to promote the development of effective drugs and vaccines against the parasite, the complete genome of P. falciparum clone 3D7 has been sequenced, using a chromosome-by-chromosome shotgun strategy. Here we report the nucleotide sequence of the third largest of the parasite's 14 chromosomes, chromosome 12, which comprises about 10% of the 23-megabase genome. As the most (A + T)-rich (80.6%) genome sequenced to date, the P. falciparum genome presented severe problems during the assembly of primary sequence reads. We discuss the methodology that yielded a finished and fully contiguous sequence for chromosome 12. The biological implications of the sequence data are more thoroughly discussed in an accompanying Article (ref. 3).     

Size variation in chromosomes from independent cultured isolates of Plasmodium falciparum

The complexity of the life cycle of the protozoan malaria parasite Plasmodium falciparum has hindered genetic analysis; even the number of chromosomes in P. falciparum is uncertain. The blood stages of rodent malaria parasites are haploid and hybridization with cloned complementary DNAs similarly suggests a haploid genome in P. falciparum blood stages (ref. 4 and our unpublished results). A novel approach to karyoptic and linkage analysis in P. falciparum has been provided recently by the technique of pulsed-field gradient (PFG) gel electrophoresis, which allows the fractionation of DNA molecules of 30-3,000 kilobases (kb), a range including the sizes of intact chromosomal DNA molecules from eukaryotes such as yeast and trypanosomatids. We describe here the fractionation by PFG electrophoresis of chromosomal DNA molecules from P. falciparum into at least seven discrete species which vary in size by up to 20% between different isolates. Several genes for P. faciparum antigens which contain repetitive sequences are located on different chromosomes. Surprisingly, two of the chromosomes seem to contain the same sequences.     

The physical maps for sequencing human chromosomes 1, 6, 9, 10, 13, 20 and X

We constructed maps for eight chromosomes (1, 6, 9, 10, 13, 20, X and (previously) 22), representing one-third of the genome, by building landmark maps, isolating bacterial clones and assembling contigs. By this approach, we could establish the long-range organization of the maps early in the project, and all contig extension, gap closure and problem-solving was simplified by containment within local regions. The maps currently represent more than 94% of the euchromatic (gene-containing) regions of these chromosomes in 176 contigs, and contain 96% of the chromosome-specific markers in the human gene map. By measuring the remaining gaps, we can assess chromosome length and coverage in sequenced clones.     

The complete nucleotide sequence of chromosome 3 of Plasmodium falciparum.

Analysis of Plasmodium falciparum chromosome 3, and comparison with chromosome 2, highlights novel features of chromosome organization and gene structure. The sub-telomeric regions of chromosome 3 show a conserved order of features, including repetitive DNA sequences, members of multigene families involved in pathogenesis and antigenic variation, a number of conserved pseudogenes, and several genes of unknown function. A putative centromere has been identified that has a core region of about 2 kilobases with an extremely high (adenine + thymidine) composition and arrays of tandem repeats. We have predicted 215 protein-coding genes and two transfer RNA genes in the 1,060,106-base-pair chromosome sequence. The predicted protein-coding genes can be divided into three main classes: 52.6% are not spliced, 45.1% have a large exon with short additional 5' or 3' exons, and 2.3% have a multiple exon structure more typical of higher eukaryotes.     

Pgh1 modulates sensitivity and resistance to multiple antimalarials in Plasmodium falciparum

Throughout the latter half of this century, the development and spread of resistance to most front-line antimalarial compounds used in the prevention and treatment of the most severe form of human malaria has given cause for grave clinical concern. Polymorphisms in pfmdr1, the gene encoding the P-glycoprotein homologue 1 (Pgh1) protein of Plasmodium falciparum, have been linked to chloroquine resistance; Pgh1 has also been implicated in resistance to mefloquine and halofantrine. However, conclusive evidence of a direct causal association between pfmdr1 and resistance to these antimalarials has remained elusive, and a single genetic cross has suggested that Pgh1 is not involved in resistance to chloroquine and mefloquine. Here we provide direct proof that mutations in Pgh1 can confer resistance to mefloquine, quinine and halofantrine. The same mutations influence parasite resistance towards chloroquine in a strain-specific manner and the level of sensitivity to the structurally unrelated compound, artemisinin. This has important implications for the development and efficacy of future antimalarial agents.     

Artemisinins target the SERCA of Plasmodium falciparum

Artemisinins are extracted from sweet wormwood (Artemisia annua) and are the most potent antimalarials available, rapidly killing all asexual stages of Plasmodium falciparum. Artemisinins are sesquiterpene lactones widely used to treat multidrug-resistant malaria, a disease that annually claims 1 million lives. Despite extensive clinical and laboratory experience their molecular target is not yet identified. Activated artemisinins form adducts with a variety of biological macromolecules, including haem, translationally controlled tumour protein (TCTP) and other higher-molecular-weight proteins. Here we show that artemisinins, but not quinine or chloroquine, inhibit the SERCA orthologue (PfATP6) of Plasmodium falciparum in Xenopus oocytes with similar potency to thapsigargin (another sesquiterpene lactone and highly specific SERCA inhibitor). As predicted, thapsigargin also antagonizes the parasiticidal activity of artemisinin. Desoxyartemisinin lacks an endoperoxide bridge and is ineffective both as an inhibitor of PfATP6 and as an antimalarial. Chelation of iron by desferrioxamine abrogates the antiparasitic activity of artemisinins and correspondingly attenuates inhibition of PfATP6. Imaging of parasites with BODIPY-thapsigargin labels the cytosolic compartment and is competed by artemisinin. Fluorescent artemisinin labels parasites similarly and irreversibly in an Fe2+-dependent manner. These data provide compelling evidence that artemisinins act by inhibiting PfATP6 outside the food vacuole after activation by iron.     

Intercellular adhesion molecule-1 is an endothelial cell adhesion receptor for Plasmodium falciparum

The primary event in the pathogenesis of severe malaria in Plasmodium falciparum infection is thought to be adherence of trophozoite- and schizont-infected erythrocytes to capillary endothelium, a process called sequestration. Identifying the endothelial molecules used as receptors is an essential step in understanding this disease process. Recent work implicates the membrane glycoprotein CD36 (platelet glycoprotein IV; refs 2-5) and the multi-functional glycoprotein thrombospondin as receptors. Although CD36 has a widespread distribution on microvascular endothelium, it may not be expressed on all capillary beds where sequestration occurs, especially in the brain. The role of thrombospondin in cell adhesion, in vitro or in vivo, is less certain. We have noticed that some parasites bind to human umbilical-vein endothelial cells independently of CD36 or thrombospondin. To screen for alternative receptors, we have developed a novel cell-adhesion assay using transfected COS cells, which confirms that CD36 is a cell-adhesion receptor. In addition, we find that an endothelial-binding line of P. falciparum binds to COS cells transfected with a complementary DNA encoding intercellular adhesion molecule-1. As this molecule is widely distributed on capillaries and is inducible, this finding may be relevant to the pathogenesis of severe malaria.     

Genetic diversity and chloroquine selective sweeps in Plasmodium falciparum

Widespread use of antimalarial agents can profoundly influence the evolution of the human malaria parasite Plasmodium falciparum. Recent selective sweeps for drug-resistant genotypes may have restricted the genetic diversity of this parasite, resembling effects attributed in current debates to a historic population bottleneck. Chloroquine-resistant (CQR) parasites were initially reported about 45 years ago from two foci in southeast Asia and South America, but the number of CQR founder mutations and the impact of chlorquine on parasite genomes worldwide have been difficult to evaluate. Using 342 highly polymorphic microsatellite markers from a genetic map, here we show that the level of genetic diversity varies substantially among different regions of the parasite genome, revealing extensive linkage disequilibrium surrounding the key CQR gene pfcrt and at least four CQR founder events. This disequilibrium and its decay rate in the pfcrt-flanking region are consistent with strong directional selective sweeps occurring over only approximately 20-80 sexual generations, especially a single resistant pfcrt haplotype spreading to very high frequencies throughout most of Asia and Africa. The presence of linkage disequilibrium provides a basis for mapping genes under drug selection in P. falciparum.     

1-(6-乙酰基-9-正丁基咔唑-3-基)-3-苯丙基-1,3-二酮的合成及其光谱性能

β-二酮化合物被广泛应用于有机稀土配合物的配体,配体的最低三重态能级与稀土的最低激发态能级的匹配程度直接影响有机稀土配合物的荧光强度,为了提高有机稀土配合物的荧光强度,设计合成新的β-二酮化合物变的尤为重要。本文以咔唑为原料,经N-烷基化、乙酰化和缩合反应得到1-(6-乙酰基-9-正丁基-9H-咔唑基)-3-苯丙基-1,3-二酮。经氢核磁共振谱(1 H-NMR)、质谱(MS-ESI)和红外光谱(IR)确认,目标化合物结构正确,并测定了化合物的紫外吸收光谱、热重(TG)、示差扫描量热曲线(DSC),结果表明该化合物适合用于有机稀土配合物的配体。     

A proteomic view of the Plasmodium falciparum life cycle

The completion of the Plasmodium falciparum clone 3D7 genome provides a basis on which to conduct comparative proteomics studies of this human pathogen. Here, we applied a high-throughput proteomics approach to identify new potential drug and vaccine targets and to better understand the biology of this complex protozoan parasite. We characterized four stages of the parasite life cycle (sporozoites, merozoites, trophozoites and gametocytes) by multidimensional protein identification technology. Functional profiling of over 2,400 proteins agreed with the physiology of each stage. Unexpectedly, the antigenically variant proteins of var and rif genes, defined as molecules on the surface of infected erythrocytes, were also largely expressed in sporozoites. The detection of chromosomal clusters encoding co-expressed proteins suggested a potential mechanism for controlling gene expression.     

Genome sequence of the human malaria parasite Plasmodium falciparum

The parasite Plasmodium falciparum is responsible for hundreds of millions of cases of malaria, and kills more than one million African children annually. Here we report an analysis of the genome sequence of P. falciparum clone 3D7. The 23-megabase nuclear genome consists of 14 chromosomes, encodes about 5,300 genes, and is the most (A + T)-rich genome sequenced to date. Genes involved in antigenic variation are concentrated in the subtelomeric regions of the chromosomes. Compared to the genomes of free-living eukaryotic microbes, the genome of this intracellular parasite encodes fewer enzymes and transporters, but a large proportion of genes are devoted to immune evasion and host-parasite interactions. Many nuclear-encoded proteins are targeted to the apicoplast, an organelle involved in fatty-acid and isoprenoid metabolism. The genome sequence provides the foundation for future studies of this organism, and is being exploited in the search for new drugs and vaccines to fight malaria.     

关于无5-圈,8-圈和9-圈平面图的3-选色

图G的选色数,记为xl(G),定义为最小的自然数k,使得满足对任一顶点给定k种颜色的列表,且染色时每个顶点的颜色只能从自身的列表中选择时,总存在图G的一个顶点的正常着色.证明了每个围长至少为4且不含5-圈,8-圈和9-圈的平面图是3-选色的.     

1-(9-菲基)-1-苯基乙烯的合成与表征

由于1-(9-菲基)-1-苯基乙烯本身具有荧光的特点，把此荧光单体接到嵌段共聚物上，从而研究嵌段共聚物的相行为具有重要意义。以9-溴菲、苯乙酮等为原料，成功地合成了1-(9-菲基)-1-苯基乙烯。并通过UV，NMR，MS，FTIR和PL对合成样品进行表征。     

The global distribution of clinical episodes of Plasmodium falciparum malaria

Interest in mapping the global distribution of malaria is motivated by a need to define populations at risk for appropriate resource allocation and to provide a robust framework for evaluating its global economic impact. Comparison of older and more recent malaria maps shows how the disease has been geographically restricted, but it remains entrenched in poor areas of the world with climates suitable for transmission. Here we provide an empirical approach to estimating the number of clinical events caused by Plasmodium falciparum worldwide, by using a combination of epidemiological, geographical and demographic data. We estimate that there were 515 (range 300-660) million episodes of clinical P. falciparum malaria in 2002. These global estimates are up to 50% higher than those reported by the World Health Organization (WHO) and 200% higher for areas outside Africa, reflecting the WHO's reliance upon passive national reporting for these countries. Without an informed understanding of the cartography of malaria risk, the global extent of clinical disease caused by P. falciparum will continue to be underestimated.     

3-(4-氯苯基)-1,3-2H-3-苯基恶唑[3,4-a]吲哚合成

以溴化亚铜作催化剂,DMF作溶剂,用N-(o-alkynylphenyl)imines亚胺在温和条件下环合-加成反应,一步合成吲哚衍生物,结果发现恶唑类吲哚在不同催化剂、不同底物种类及反应条件下的反应速度和产率具有较大差异性.     

Analysis of the Plasmodium falciparum proteome by high-accuracy mass spectrometry

The annotated genomes of organisms define a 'blueprint' of their possible gene products. Post-genome analyses attempt to confirm and modify the annotation and impose a sense of the spatial, temporal and developmental usage of genetic information by the organism. Here we describe a large-scale, high-accuracy (average deviation less than 0.02 Da at 1,000 Da) mass spectrometric proteome analysis of selected stages of the human malaria parasite Plasmodium falciparum. The analysis revealed 1,289 proteins of which 714 proteins were identified in asexual blood stages, 931 in gametocytes and 645 in gametes. The last two groups provide insights into the biology of the sexual stages of the parasite, and include conserved, stage-specific, secreted and membrane-associated proteins. A subset of these proteins contain domains that indicate a role in cell-cell interactions, and therefore can be evaluated as potential components of a malaria vaccine formulation. We also report a set of peptides with significant matches in the parasite genome but not in the protein set predicted by computational methods.     

Specific role of mitochondrial electron transport in blood-stage Plasmodium falciparum

The origin of all mitochondria can be traced to the symbiotic arrangement that resulted in the emergence of eukaryotes in a world that was exclusively populated by prokaryotes. This arrangement, however, has been in continuous genetic flux: the varying degrees of gene loss and transfer from the mitochondrial genome in different eukaryotic lineages seem to signify an ongoing 'conflict' between the host and the symbiont. Eukaryotic parasites belonging to the phylum Apicomplexa provide an excellent example to support this view. These organisms contain the smallest mitochondrial genomes known, with an organization that differs among various genera; one genus, Cryptosporidium, seems to have lost the entire mitochondrial genome. Here we show that erythrocytic stages of the human malaria parasite Plasmodium falciparum seem to maintain an active mitochondrial electron transport chain to serve just one metabolic function: regeneration of ubiquinone required as the electron acceptor for dihydroorotate dehydrogenase, an essential enzyme for pyrimidine biosynthesis. Transgenic P. falciparum parasites expressing Saccharomyces cerevisiae dihydroorotate dehydrogenase, which does not require ubiquinone as an electron acceptor, were completely resistant to inhibitors of mitochondrial electron transport. Maintenance of mitochondrial membrane potential, however, was essential in these parasites, as indicated by their hypersensitivity to proguanil, a drug that collapsed the membrane potential in the presence of electron transport inhibitors. Thus, acquisition of just one enzyme can render mitochondrial electron transport nonessential in erythrocytic stages of P. falciparum.