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At least 883 records · Page 49Linked to original sources
PubMed1999

Molecular epidemiology of malaria in Yaounde, Cameroon V. analysis of the omega repetitive region of the plasmodium falciparum CG2 gene and chloroquine resistance.

A novel Plasmodium falciparum gene, denoted cg2 gene, has been recently discovered, and a distinct genotype, characterized by 12 point mutations and 3 size polymorphisms, has been shown to be associated with chloroquine resistance in laboratory-adapted parasite strains. One of the polymorphic regions, denoted the omega region, consists of 16 tandem repeat units in chloroquine-resistant strains, while the chloroquine-sensitive strains have either < or = 15 or > or = 17 repeat units. In this study, the in vivo and in vitro responses were compared with the number of repeat units in the omega region of the cg2 gene for 75 Cameroonian isolates determined either by DNA sequencing or agarose gel electrophoresis. The 16-repeat units that characterize the resistant strains were found in 10 chloroquine-sensitive isolates (50% inhibitory concentration [IC50] < 100 nM) and 30 chloroquine-resistant isolates (IC50 > or = 100 nM). Thirty-five isolates (28 chloroquine-sensitive isolates and 7 chloroquine-resistant isolates) displayed < or = 15 or > or = 17 repeat units. Of the 18 patients responding with treatment failure, 15 were infected with parasites carrying 16 repeat units. Twenty-eight patients (11 with isolates carrying 16 repeat units and 17 with isolates carrying < or = 15 or > or = 17 repeat units) showed an adequate clinical response. The sensitivity, specificity, and predictive value were 81% (83%), 74% (61%), and 75% (58%), respectively compared with in vitro (or in vivo) responses. Neither the level of IC50 nor the key P. falciparum multidrug resistance gene 1 (pfmdr 1) allele at position 86 was associated with the number of omega repeat units. Although in vitro and in vivo resistance to chloroquine was statistically associated with the presence of 16 repeat units in the omega region (P < 0.05), the number of omega repeat units did not adequately discriminate patients infected with chloroquine-resistant parasites from those infected with chloroquine-sensitive parasites. Further studies on the cg2 gene are needed to determine whether cg2 gene is a reliable genetic marker for chloroquine resistance.

Adolescent↗
PubMed2002

Thioredoxin reductase is essential for the survival of Plasmodium falciparum erythrocytic stages.

The human malaria parasite Plasmodium falciparum poses an increasing threat to human health in the tropical regions of the world, and the validation and assessment of possible drug targets is required for the development of new antimalarials. It has been shown that the erythrocytic stages of the parasites, which are responsible for the pathology of the disease in humans, are under enhanced oxidative stress and are particularly vulnerable to exogenous challenges by reactive oxygen species. Therefore it is postulated that the disruption of the antioxidant and/or redox systems of the parasite is a feasible way to interfere with their development during erythrocytic schizogony. In order to test this suggestion thioredoxin reductase (TrxR), an enzyme heavily involved in maintenance of redox homeostasis and antioxidant defense, was knocked out in P. falciparum. It was impossible to generate parasites with a disrupted trxR gene suggesting that TrxR is essential for P. falciparum erythrocytic stages. Technical problems were excluded by transfecting a 3' replacement construct, which recombined correctly and transfectants did not show any phenotypic alterations. In order to prove that the trxR knockout was responsible for the lethal phenotype of the null mutants, a co-transfection with both the knockout construct and a construct containing the trxR coding region under the control of the calmodulin promoter was conducted. Despite the disruption of the trxR gene, parasites were viable. In a Southern blot analysis a complicated restriction pattern was obtained, but it was shown by pulse field gel electrophoresis and field inverse gel electrophoreses that only the trxR gene locus on chromosome 9 was targeted by the constructs. It was found that the co-transfected constructs form concatemeric structures prior to integration into the trxR gene locus, which is further supported by plasmid rescue followed by restriction analyses of the plasmids. Northern and Western blot analyses proved that the co-transfectants highly overexpress TrxR from the introduced gene. Our results demonstrate that TrxR is essential for the survival of the erythrocytic stages of P. falciparum.

Animals↗
PubMed1985

In vivo assessment of the sensitivity of Plasmodium falciparum to sulphadoxine/pyrimethamine combination (Fansidar) in six localities in Tanzania where chloroquine-resistant P. falciparum has been detected.

The sensitivity of Plasmodium falciparum strains to Fansidar (500 mg sulphadoxine/25 mg pyrimethamine) was tested in vivo in six localities in the United Republic of Tanzania where chloroquine-resistant P. falciparum strains have been demonstrated by both in vivo and in vitro tests. Single doses as recommended by the manufacturers achieved 100% clearance of parasitaemia in five localities with mean clearance period of between 2.2 and 2.9 days. In one locality (Gonja) the recommended dose failed to clear parasitaemia in two of the 38 cases (5.3%) within seven days. The possibility of using this drug combination for the treatment of chloroquine-resistant P. falciparum strains in the United Republic of Tanzania is discussed.

Adolescent↗
PubMed1991

Clinical correlates of in vitro Plasmodium falciparum cytoadherence.

To determine whether isolates of Plasmodium falciparum have intrinsically different cytoadherent properties and whether these differences contribute to the clinical severity of human falciparum malaria, we studied the cytoadherence to C32 melanoma cells in vitro of 59 parasite isolates from patients with naturally acquired infections in Thailand. Parasitized erythrocytes adhere to these melanoma cells principally via the glycoprotein CD36, which is also expressed on most vascular endothelium. In vitro cytoadherence was significantly greater for isolates from patients with biochemical evidence of severe malaria. The cytoadherent properties of P. falciparum parasites may thus be a virulence factor in human falciparum malaria. However, there was no correlation between the degree of in vitro cytoadherence and cerebral symptoms, which suggests that other receptors and/or host factors may be important in the adherence of malaria parasites to cerebral vascular endothelium. The cytokines tumor necrosis factor, interleukin-1, and gamma interferon, which have been implicated in the pathogenesis of cerebral malaria and are known to promote intercellular adhesion in other systems, did not enhance the cytoadherence of P. falciparum isolates to C32 melanoma cells.

Adult↗
PubMed2006

Development of cellular immune responses to Plasmodium falciparum blood stage antigens from birth to 36 months of age in Cameroon.

Naturally acquired immunity to Plasmodium falciparum is related to immune system that changes during normal development and ageing. The effects of repeated infections during the early life on the maturation of the immune system are still unknown. Elucidation of these effects is of considerable interest given that malaria originates high mortality, especially during the first years of life. We conducted a cohort study to identify naturally acquired immune responses to P. falciparum. Cellular responses of Cameroonian neonates from birth to 36 months of age were evaluated every 6 months by cell proliferation and cytokines (IFN-gamma, IL-2 and IL-4) production after in vitro culture in the presence of schizont extract and Pf155/RESA peptides. Data were analyzed by a multiple correspondence analysis (MCA) exhibiting three main findings. Firstly, the lack of time-dependant evolution of specific immune pathways recruitment in the response to a given antigen, no antigen inducing a specific mode of response at a given time-point. Secondly, most of the data variability was expressed by IFN-gamma and IL-4 productions, and the major variation of the immune response with age involved this change in IFN-gamma production. Thirdly, the age-related immune response evolution is characterized by the acquisition of the capacity to mount a IFN-gamma response, a transient phase during which children produce a high IL-4 response, and the fast vanishing of the dominance of the IL-2 response. These results suggest that P. falciparum specific immune responses are first oriented towards a Th2-type of response, and later switch to Th1-type of response.

Aging↗
PubMed1999

Complexity of Plasmodium falciparum infections is consistent over time and protects against clinical disease in Tanzanian children.

The complexity of Plasmodium falciparum populations in 21 children was studied in repetitive samples over 4 years in an area of Tanzania where the organism is holoendemic. Genotyping was done by a polymerase chain reaction method that targets three highly polymorphic regions of the merozoite surface protein (MSP) 1 block 2, MSP 2, and the glutamine-rich protein. Eight children were repeatedly parasitemic, 5 had scanty parasitemias, and 8 were consistently nonparasitemic. Varying numbers of genotypes were detected in the parasitemic children, but the multiplicity of infection was significantly constant within each child. The children with frequent parasitemias experienced fewer clinical episodes during the study period than those without parasitemias. There was also a tendency for children with more complex infections to experience fewer episodes. The children had consistent parasitologic profiles over the 4 years. Although few subjects were studied and the results will require confirmation, the results suggest that asymptomatic (especially polyclonal) P. falciparum infection protects against clinical disease from new infections.

Age Factors↗
PubMed2005

Analysis of the genetic diversity of the Plasmodium falciparum multidrug resistance gene 5' upstream region.

Recent findings indicating a low level of polymorphism in the Plasmodium falciparum genome have led to the hypothesis that existent polymorphisms are likely to have functional significance. We tested this hypothesis by developing a map of the polymorphism in the P. falciparum multidrug resistance 1 (pfmdr1) gene 5' upstream region and assaying its correlation with drug resistance in a sample of field isolates from Dakar, Senegal. A comparison of six geographically diverse laboratory strains showed that the 1.94-kb 5'-untranslated region is highly monomorphic, with a total of four unique single nucleotide polymorphisms (SNPs) being identified. All of the mutations were localized to a 462-basepair region proximal to the transcription start point. Analysis of this region in field isolates shows the prevalence of one SNP throughout the entire population of parasites, irrespective of drug resistance status. The SNP frequency of the pfmdr1 upstream region is lower than that found in the noncoding region of other genes.

ATP-Binding Cassette Transporters↗
PubMed2005

Molecular surveillance of mutations in dihydrofolate reductase and dihydropteroate synthase genes of Plasmodium falciparum in Ethiopia.

Point mutations in the genes for dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) of Plasmodium falciparum isolates are associated with sulfadoxine/pyrimethamine (SP) treatment failure, respectively. This study was conducted to assess the prevalence of SP resistance in P. falciparum isolates collected at the Jimma Health Center in southwestern Ethiopia. In this study, the genetic profile of P. falciparum isolates with respect to DHFR and DHPS genes was assessed in 124 individuals. The prevalence of single, double, and multiple mutations in these genes was calculated. The sequence profile showed that all samples carried a double mutation at the positions 51 and 108 (I51N108) in the DHFR gene. Sixty-seven (54.03%) of the isolates had an additional third mutation at position 59, resulting in the triple mutant I51R59N108. All isolates carried mutations G437 and E540 in the DHPS gene. Two isolates (1.61%) had additional mutations at codon 581 (A581).

Animals↗
PubMed1998

[Dynamics of Plasmodium falciparum gametocytemia as a function of therapeutic response to chloroquine in a mesoendemic malaria zone].

Patients with Plasmodium falciparum infections were selected with an in vivo chloroquine sensitivity assay in a mesoendemic area of Senegal. Gametocytemia was studied in relation to asexual parasite responses classified as drug-sensitive or showing RI or RII resistance. Gametocyte prevalence and density appeared significantly higher in resistant infections than in sensitive ones. These observations were made on children as well as on adults. The period between first clinical symptom appearance and treatment, varying from 0 to 5 days, was not linked to asexual parasite responses, but was significantly and positively correlated to gametocyte prevalence and density observed seven days after beginning the treatment. The comparison between these results and those previously observed in a hypoendemic area suggests that the dynamic of gametocytemia is independent of the endemic level. These observations illustrate that the dissemination of chimioresistant malaria can be reinforced--via the gametocyte stage--by selective chloroquine pressure.

Adolescent↗
PubMed1993

Expression of Plasmodium falciparum lactate dehydrogenase in Escherichia coli.

A Plasmodium falciparum gene is described which encodes lactate dehydrogenase activity (P. falciparum LDH). The P. falciparum LDH gene contains no introns and is present in a single copy on chromosome 13. P. falciparum LDH was expressed in all asexual blood stages as a 1.6-kb mRNA. The predicted 316 amino acid protein coding region of P. falciparum LDH was inserted into the prokaryotic expression vector pKK223-3 and a 33-kDa protein having LDH activity was synthesized in Escherichia coli. P. falciparum LDH primary structure displays high amino acid similarity (50-57%) to vertebrate and bacterial LDH, but lacks the amino terminal extension observed in all vertebrate LDH. The majority of amino acid residues implicated in substrate and coenzyme binding and catalysis of other LDH are well conserved in P. falciparum LDH. However, several notable differences in amino acid composition were observed. P. falciparum LDH contained several distinctive single amino acid insertions and deletions compared to other LDH enzymes, and most remarkably, it contained a novel insertion of 5 amino acids within the conserved mobile loop region near arginine residue 109, a residue which is known to make contact with pyruvate in the ternary complex of other LDH. These results suggest that novel features of P. falciparum LDH primary structure may be correlated with previously characterized and distinctive kinetic, biochemical, immunochemical, and electrophoretic properties of P. falciparum LDH.

Amino Acid Sequence↗
PubMed1983

Genetic variation in the host and adaptive phenomena in Plasmodium falciparum infection.

The in vitro culture of Plasmodium falciparum, after synchronization, lends itself well to an analysis of the asexual schizogonic cycle. We have found in this system that DNA synthesis and RNA synthesis are associated mainly with the trophozoite stage, with the latter peaking slightly ahead of the former. Distinctive patterns of protein synthesis are seen at serial times along the cycle, with a number of 'stage-specific' bands identifiable on SDS gels. The fate of the infection can be influenced by the genotype of the host cell. Thus, glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes are invaded normally, but maturation of intracellular parasites is delayed and impaired. However, the parasites that do develop will then have a normal behaviour in their next rounds in G6PD-deficient cells, suggesting that an adaptive change has taken place. These results fit well with the relative protection against P. falciparum in vivo of girls heterozygous for G6PD deficiency but not of hemizygous G6PD-deficient boys.

Animals↗
PubMed2005

The human malaria parasite Plasmodium falciparum possesses two distinct dihydrolipoamide dehydrogenases.

The Plasmodium falciparum genome contains genes encoding three alpha-ketoacid dehydrogenase multienzyme complexes (KADHs) that have central metabolic functions. The parasites possess two distinct genes encoding dihydrolipoamide dehydrogenases (LipDH), which are indispensable subunits of KADHs. This situation is reminiscent of that in plants, where two distinct LipDHs are found in mitochondria and chloroplasts, respectively, that are part of the organelle-specific KADHs. In this study, we show by reverse transcription polymerase chain reaction (RT-PCR) that the genes encoding subunits of all three KADHs, including both LipDHs, are transcribed during the erythrocytic development of P. falciparum. Protein expression of mitochondrial LipDH and mitochondrial branched chain alpha-ketoacid dihydrolipoamide transacylase in these parasite stages was confirmed by Western blotting. The localization of the two LipDHs to the parasite's apicoplast and mitochondrion, respectively, was shown by expressing the LipDH N-terminal presequences fused to green fluorescent protein in erythrocytic stages of P. falciparum and by immunofluorescent colocalization with organelle-specific markers. Biochemical characterization of recombinantly expressed mitochondrial LipDH revealed that the protein has kinetic and physicochemical characteristics typical of these flavo disulphide oxidoreductases. We propose that the mitochondrial LipDH is part of the mitochondrial alpha-ketoglutarate dehydrogenase and branched chain alpha-ketoacid dehydrogenase complexes and that the apicoplast LipDH is an integral part of the pyruvate dehydrogenase complex which occurs only in the apicoplast in P. falciparum.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1999

Intra-cluster recombination and var transcription switches in the antigenic variation of Plasmodium falciparum.

Antigenic variation and immune evasion by Plasmodium falciparum parasitized erythrocytes are mediated by expression switches among members of the multicopy var gene family. Here we describe a cluster of var genes on chromosome 12 that showed spontaneous recombination and switches in the transcription of individual genes. The transcription switches were not associated with sequence changes in promoter regions. Transfected episomes containing a luciferase reporter under control of a var promoter were expressed regardless of the transcriptional status of the endogenous promoter. The results suggest epigenetic regulation of P. falciparum var gene transcription that depends upon the local structure of chromatin and its associated proteins.

Animals↗
PubMed2002

Mosaic-like transcription of var genes in single Plasmodium falciparum parasites.

The var gene family of Plasmodium falciparum encodes the clonally variant adhesin PfEMP1 present on the surface of infected erythrocytes. A poorly understood mechanism of allelic exclusion controls the expression of PfEMP1. Transcription of var genes is developmentally and, most likely, epigenetically regulated. Here we have studied the transcriptional pattern of 28 members of this multigene family in individual parasites, early in the intraerythrocytic cycle. The results show unique patterns (type and number) of var transcripts in each individual PRBC, with 1-15 mRNA species detected per cell at 2-4 h post-invasion. When a panel of ten single PRBC was analyzed, the var gene coding for the expressed PfEMP1 was transcribed in more cells than any other, although transcripts from this gene did not give the strongest hybridization signal within each individual cell. Chromosomal mapping of transcriptionally active var genes indicated that their distribution reflects that of var loci in the genome, including a pronounced clustering in chromosome 4. These findings, taken together with existing data on var transcription at later developmental stages, suggest that the mosaic-like transcription of multiple var genes detected at the ring stage and the steady transcription of the gene encoding the expressed PfEMP1 are distinct although superimposed events, one of them random and the other taking place under some form of imprinting. With its unique features, the expression of P. falciparum var genes may reveal new principles of gene regulation.

Amino Acid Sequence↗
PubMed1994

Primary structure and expression of the dihydropteroate synthetase gene of Plasmodium falciparum.

The enzyme dihydropteroate synthetase (DHPS) from Plasmodium falciparum is involved in the mechanism of action of the sulfone/sulfonamide group of drugs. We describe the cloning and sequencing of the gene encoding the P. falciparum DHPS enzyme and show that it is a bifunctional enzyme that includes dihydro-6-hydroxymethylpterin pyrophosphokinase (PPPK) at the N terminus of DHPS. The gene encodes a putative protein of 83 kDa that contains two domains that are homologous with the DHPS and PPPK enzymes of other organisms. The PPPK-DHPS gene is encoded on chromosome 8 and has two introns. An antibody raised to the PPPK region of the protein was found to recognize a 68-kDa protein that is expressed throughout the asexual life cycle of the parasite. We have determined the sequence of the DHPS portion of the gene from sulfadoxine-sensitive and -resistant P. falciparum clones and identified sequence differences that may have a role in sulfone/sulfonamide resistance.

Amino Acid Sequence↗
PubMed2002

Properties, stage-dependent expression and localization of Plasmodium falciparum M1 family zinc-aminopeptidase.

A Plasmodium falciparum single copy gene predicting a 122 kDa protein belonging to the Ml family of zincmetallopeptidases was previously reported and related to erythrocytic schizont proteins of 96 (p96) and 68 (p68) kDa. By using protease inhibitors during parasite harvest and enzyme preparations, and polyclonal antibodies specific for 2 peptidic domains deduced from the gene, we identified the 120 kDa precursor and demonstrated its processing into p96 and p68. The N-terminal ends of p96 and p68 were mapped between glycine-123 and lysine-163, both proteins thus containing the catalytic domain. The purified enzyme, here named PfA-M1 (p96/p68), displayed strict aminopeptidase activity, optimal at pH 74, with broad substrate spectrum. Its inhibition and reactivation profiles were typical of zinc-metalloaminopeptidases. By Western blotting, PfA-M1 was detected in trophozoites, in addition to schizonts, but not in early rings. PfA-M1 was localized by indirect immunofluorescence confocal microscopy. In trophozoites, the labelling was diffuse in the parasite cytoplasm, with accumulations around the food vacuole. In schizonts, it turned progressively to a vesicle-like pattern, ending as a clear spot in released merozoites. The involvement of PfA-M1 in haemoglobin breakdown and erythrocyte reinvasion is discussed in light of the dual functions recently reported for several P. falciparum proteases.

Amino Acid Motifs↗
PubMed1995

Complete development of the liver stage of Plasmodium falciparum in a human hepatoma cell line.

Plasmodium falciparum parasites develop in the liver before being released into the bloodstream, where they exert the potentially lethal effects characteristic of malaria. Our understanding of the hepatic phase of the life cycle is limited by the parasite's requirement for fresh human liver cells in which to mature. In this work, liver parasites completed their development within a Thai human hepatoma cell line (HHS-102), and the presence of ring-form parasites in erythrocytes overlying the liver cell culture confirmed that an entire liver cycle was completed, culminating in the production of viable blood-stage parasites. The HHS-102 cell line allows investigation of the undefined liver stage of falciparum malaria previously unavailable in the laboratory.

Animals↗