Anopheles hervyi in Niger: no evidence for a role in Plasmodium falciparum transmission

Anopheles hervyi is an endemic mosquito species with a very limited spatial distribution in the south east of Niger. No new captures have been reported since the 1960s and its role in malaria transmission has not been studied. In the present study, the use of CDC light traps showed it to be much more abundant than previously found but there was no evidence to suggest it was a malaria vector in this region. The larval habitats have not been identified but the potential role of a saline lake in determining the distribution of this species is discussed.     

Dynamics of DNA methylation pattern

Methylation patterns are the result of de novo methylation, demethylation, and the maintenance of existing methylation. Although the existence and identity of an active demethylase remain in doubt, recent evidence suggests that protein binding can specify sites of demethylation through a replication-dependent pathway. By using a stable episomal system in human cells, plus the Drosophila system, and mouse embryonic stem cells, we are beginning to understand the function and targets of de novo methyltransferases in murine and human cells.     

Glyoxalase I of the malarial parasite Plasmodium falciparum: evidence for subunit fusion

Recombinant Plasmodium falciparum glyoxalase I (PfGlx I) was characterized as monomeric Zn(2+)-containing enzyme of 44 kDa. The K(M) value of the methylglyoxal-glutathione adduct is 77+/-15 microM, the k(cat) value being 4000 min(-1) at 25 degrees C and pH 7.0. PfGlx I consists of two halves, each of which is homologous to the small 2-domain glyoxalase I of man. Both parts of the pfglx I gene were overexpressed; the C-terminal half of PfGlx I was found to be a stable protein and formed an enzymatically active dimer. These results support the hypothesis of domain-swapping and subunit fusion as mechanisms in glyoxalase I evolution.     

Plasmodium falciparum: DNA probe diagnosis of malaria in Kenya

We previously reported isolation of DNA probe which specifically recognizes Plasmodium falciparum and developed a simple method for its use. The sensitivity and specificity of this DNA probe method have now been extensively field tested in comparison with those of conventional microscopic examination of blood films in two separate studies in Malindi, Kenya, involving a total of 1179 patients. In the second study, which used improved techniques, sensitivity of the DNA probe was 89% when compared to microscopy. We conclude that the DNA probe method compares favorably with conventional microscopy in detecting parasite densities as low as 25 parasites per microliter of blood. A significant advantage of the DNA probe method is that it utilizes a standardized procedure which can simultaneously and reproducibly analyze a large number of samples without opportunity for significant reader bias.     

The evolutionary origin of the 35 kb circular DNA of Plasmodium falciparum: new evidence supports a possible rhodophyte ancestry

In common with other Apicomplexan parasites, Plasmodium falciparum carries two extrachromosomal DNAs, one of which, the 6 kb element, is undoubtedly mitochondrial. The second, generally referred to as the 35 kb circle, is of unknown provenance, but the nature and organization of its genetic content makes a mitochondrial association unlikely and the molecule has features reminiscent of plastid genomes. We now report the occurrence on the circle of an open reading frame specifying a predicted 470 amino acid protein that shares more than 50% identity with a gene currently known only on the plastome of red algae. This high degree of conservation confirms the 35 kb circle's plastid ancestry, and we speculate that it may have originated from the rhodoplast of an ancient red algal endosymbiont in the progenitor of the Apicomplexa.     

Organization of Plasmodium falciparum genome: 1. Evidence for a highly repeated DNA sequence.

Plasmodium falciparum DNA, isolated from the merozoite stage, was cleaved with HindIII and cloned in pBR322 and lambda L47.1 vectors. Plasmid clones containing 13.4, 7.0, 4.3, 4.1 and 1.5 kb inserts were characterized in some detail. The inserts contain several repeating units of smaller size. Nucleic acid hybridization studies showed that the repeat element is present in the Plasmodium DNA at a very high copy number and appears to be distributed widely throughout the genome.     

Detection of Plasmodium falciparum DNA in a microtitration plate-based hybridization test

A rapid DNA-test, depending on the affinity based hybrid collection principle, was developed for the detection of Plasmodium falciparum DNA from clinical specimens. In this method, hybridization takes place in solution and the hybrids are collected onto a solid phase for measurement. Two probes are used, one labelled with an affinity tag (biotin) and the other with a detectable label (32P). In the present test a single oligonucleotide complementary to a 21-base pair sequence which is highly repeated in the parasite genome served both as capture and detector probe. The test is a 2-h hybridization performed in streptavidin coated microtitration plate wells, onto which the labelled hybrids simultaneously bind. The sensitivity of the assay with a crude erythrocyte lysate specimen was 1.6 x 10(9) repeat units corresponding to about 160 parasites in one microliter blood. The results allowed quantification of the repeat sequences and thus estimation of the degree of parasitemia in clinical specimens.     

Plasmodium falciparum: DNA sequence specificity of cisplatin and cisplatin analogues

In this paper, we provided evidence that cisplatin is able to form adducts with cellular DNA in Plasmodium falciparum. The DNA sequence specificity of cisplatin adduct formation was determined in trophozoite-enriched P. falciparum cells and this paper represents the first occasion that the sequence specificity of cisplatin DNA damage has been observed in malaria cells. Utilising a sub-telomeric, 692 bp repeat sequence in the P. falciparum genome, we were able to investigate the DNA adducts formed by cisplatin and five analogues. A run of eight consecutive guanines was the most prominent site of DNA damage in the malarial cells. This study suggests that the mechanism of P. falciparum cell death caused by cisplatin involves damage to DNA and hence inhibition of DNA replication and cell division.     

Sensitive and specific DNA probe for detection of Plasmodium falciparum.

The isolation and some characteristics of a very sensitive DNA probe for the detection of Plasmodium falciparum are described. The probe is species specific and represents a large, albeit variable, fraction of the genome in all the strains tested. In addition to its immediate practical uses for the detection and quantitation of parasites, the probe defines an interesting family of repeated sequences.     

Plasmodium falciparum: synchronization of asexual development with aphidicolin, a DNA synthesis inhibitor

The asexual development cycle of Plasmodium falciparum, a malarial parasite of humans, has been synchronized in culture by treating ring-stage parasites with aphidicolin, an inhibitor of DNA synthesis. Optimization of both the concentration of drug added to ring stage containing red blood cells and the duration of exposure of parasites to drug led to a reversible block of their maturation at the early trophozoite stage. Release of the aphidicolin block led to a synchronous development of parasites that was manifested by about 80% of the new ring stages being produced within a 2- to 3-hr interval.     

Specific detection of Plasmodium falciparum malaria by a molecularly cloned DNA probe

A highly repeated DNA sequence from Plasmodium falciparum was cloned and used as a probe in molecular hybridization to detect malaria. Our results indicate that the probe is specific to P. falciparum but not to other species of Plasmodium and is extremely sensitive. As little as a 20 pg parasite DNA, which is equivalent to about 1000 parasites can be detected. The cloned DNA can be used as a diagnostic tool to follow the course of infection of falciparum malaria.     

Mesodermal determination genes: evidence from DNA methylation studies

Mouse embryo cells, primed to differentiate with the hypomethylating agent 5-azacytidine (5-aza-CR), provide an excellent model system in which cellular differentiation can be studied at the molecular level. An inherent advantage of this system is the availability of clonal populations of cells representative of the non-differentiated precursor, those whose determinative state is that of a specific lineage, and the end stage, phenotypically mature cell. Analysis of these cultures at the cellular and molecular level will advance our understanding of requirements for and the mechanism of action of growth modulators as well as the necessity for controlled expression of certain proto-oncogenes. At a more fundamental level, the system can be used to identify, isolate and characterize genes whose expression alters the phenotypic fate of cells.     

Serological evidence of discrete spatial clusters of Plasmodium falciparum parasites

Background

Malaria transmission may be considered to be homogenous with well-mixed parasite populations (as in the classic Ross/Macdonald models). Marked fine-scale heterogeneity of transmission has been observed in the field (i.e., over a few kilometres), but there are relatively few data on the degree of mixing. Since the Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) is highly polymorphic, the host''s serological responses may be used to infer exposure to parasite sub-populations.

Methods and Findings

We measured the antibody responses to 46 individual PfEMP1 domains at four time points among 450 children in Kenya, and identified distinct spatial clusters of antibody responses to individual domains. 35 domains showed strongly significant sero-clusters at p = 0.001. Individuals within the high transmission hotspot showed the greatest diversity of anti-PfEMP1 responses. Individuals outside the hotspot had a less diverse range of responses, even if as individuals they were at relatively intense exposure.

Conclusions

We infer that antigenically distinct sub-populations of parasites exist on a fine spatial scale in a study area of rural Kenya. Further studies should examine antigenic variation over longer periods of time and in different study areas.     

Plasmodium falciparum: cytoadherence of a knobless clone

Sequestration of Plasmodium falciparum-infected erythrocytes is crucial to parasite survival as it prevents destruction in the liver and spleen. Knobs have been considered necessary but not sufficient for cytoadherence to vascular endothelial cells in vivo and to melanoma or umbilical vein endothelial cells in vitro. We describe here a knobless clone that cytoadheres strongly to C32 melanoma cells. This clone cannot express the knob-associated histidine-rich protein (KAHRP) due to the deletion of the KAHRP gene. Our results raise the possibility of an alternative mechanism for in vitro cytoadherence and suggest that the use of long term cultured isolates and melanoma cells as a model for cytoadherence in vivo may be misleading.     

Plasmodium falciparum: S-adenosyl-L-methionine decarboxylase

Putrescine-dependent S-adenosyl-L-methionine decarboxylase has been detected in the malaria parasite Plasmodium falciparum. Mg2+ did not affect the enzyme activity. The apparent Km value of the plasmodial enzyme for adenosyl-methionine was found to be 33 microM. Methylglyoxal bis(guanylhydrazone) competitively inhibited the enzyme activity with respect to adenosylmethionine. The inhibition constant for methylglyoxal bis(guanylhydrazone) was determined to be 0.46 microM. Spermidine was the main polyamine detected in the parasite. There was significant decrease in the S-adenosyl-L-methionine decarboxylase activity when the infected erythrocytes were incubated with chloroquine and mefloquine for 2 hr at 1 and 10 microM, respectively. Since at similar concentrations these drugs did not directly affect the plasmodial enzyme activity, the interaction of these drugs with the polyamine biosynthesis remains unclear.     

The genome of Plasmodium falciparum. I: DNA base composition.

Some structural properties of the DNA of Plasmodium falciparum were studied thoroughly using several techniques. Its G+C content was found to be extremely low (17-19%), the lowest reported for a living organism. The DNA seems to be composed only of the four major bases as no methylated bases were detected. This DNA had a Tm value of 62.5 degrees C and its denaturation profile showed no marked intramolecular heterogeneity.