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PubMed1999

Stimulation of Plasmodium falciparum gametocytogenesis by conditioned medium from parasite cultures.

Plasmodium falciparum gametocyte development was examined in erythrocyte monolayer cultures prepared with Cell-Tak, a cell and tissue adhesive. The monolayers, which were stable for up to 10 days in culture, supported multiple cycles of asexual growth and the development of clusters of stage IV gametocytes. Small numbers of chicken erythrocytes incorporated into the monolayers served as internal reference standards for parasite counts. This permitted quantitative assessment of gametocyte formation under different culture conditions. Gametocyte formation was limited in monolayers grown in standard culture medium but it increased slightly in monolayers cocultured with suspensions of parasitized erythrocytes. The number of gametocytes increased significantly in monolayers grown in parasite-conditioned medium. In both cases the changes resulted from increased numbers of stage II and III gametocytes in the monolayers. These results suggest that parasite conditioned medium contains a factor(s) that stimulates sexual development.

Animals↗
PubMed1983

Stage-dependent effects of chloroquine on Plasmodium falciparum in vitro.

The erythrocytic developmental cycle of Plasmodium falciparum can be conveniently divided into the ring, trophozoite, and schizont stages based on morphology and metabolism. Using highly synchronous cultures of P. falciparum, considerable variation was demonstrated among these stages in sensitivity to chloroquine. The effects of timed, sequential exposure to several clinically relevant concentrations of chloroquine were monitored by three techniques: morphological analysis, changes in the rate of glucose consumption, and changes in the incorporation of 3H-hypoxanthine into parasite nucleic acids. All three techniques gave essentially identical results. The trophozoite and schizont stages were considerably more sensitive to the drug than ring-stage parasites. Chloroquine sensitivity decreased as nuclear division neared completion. The increase in chloroquine sensitivity was coincident with a marked rise in the rate of glucose consumption and nucleic acid synthesis. The rate of nucleic acid synthesis decreased as schizogony progressed while glucose consumption continued at high rates during this process. The degree of chloroquine sensitivity was not highly correlated with either metabolic activity.

Animals↗
PubMed1989

Characterization of epitopes on the 25 kD protein of the macrogametes/zygotes of Plasmodium falciparum.

A sexual stage-specific protein of Plasmodium falciparum with a Mr of 25,000 is one of the target antigens of transmission-blocking antibodies. The contributions of tertiary structure and post-translational modifications (glycosylation and acylation) to the structure of the epitopes on this protein were the subject of detailed investigations. After modification of the three-dimensional structure and modification or cleavage of carbohydrate groups and linked fatty acids, the immunological reactivity was investigated by three different techniques: (i) immunoprecipitation of radiolabelled proteins, (ii) enzyme-linked immunosorbent assay (ELISA), and (iii) Western blotting. The results of the experiments indicate that the immunological reactivity of the major epitopes on the 25 kD protein, including the epitope involved in transmission-blocking immunity, are dependent on the tertiary structure of the protein and on the presence of linked fatty acids, but not on the presence or absence of carbohydrate groups.

Acylation↗
PubMed1993

Glutamate rich Plasmodium falciparum antigen (GLURP).

We have characterized a glutamate rich Plasmodium falciparum antigen, GLURP, which by immunoassays appears to be present in both the pre-erythrocytic and erythrocytic stages of the vertebrate life cycle. The gene, which is located on chromosome 10, encodes a polypeptide of 1271 residues with a predicted molecular mass of 145 kDa. Rabbit antiserum against a fusion protein expressing the C-terminal end of the molecule detects a protein with a molecular mass of 220 kDa. The sequence includes two hydrophobic regions: one consisting of 23 residues and located by the N-terminus which may act as signal peptide, and the second located at the C-terminus consisting of 33 predominantly hydrophobic residues. Except for these hydrophobic regions the protein is hydrophilic and highly charged. The sequence has two tandem repeats designated as R1 and R2. These regions were found to be conserved in isolates from different geographical areas. High levels of anti-GLURP antibodies have been shown to correlate with low parasite density. The indication of GLURP being present in all stages of the parasite in the human host raises significantly the prospects of the potential of this molecule.

Adult↗
PubMed1987

Characterization of gp195 processed products purified from Plasmodium falciparum culture supernates.

Schizonts of the malaria parasite Plasmodium falciparum synthesize a 195 kDa surface glycoprotein (gp195) that is processed into several smaller products including one of 83 kDa, which, in the case of the Camp strain, is sequentially processed into 73 and 67 kDa products. gp195 and its processing intermediates larger than 83 kDa were not precipitated from culture supernates, but the 83 and 73 kDa products were precipitated by three monoclonal antibodies (McAbs). The 83 and 73 kDa products were affinity purified from culture supernates by adsorbing to McAb 7B2 coupled to Affigel 10 and eluting either with 0.2 N acetic acid, pH 2.8, or with 3 M potassium isothiocyanate (KSCN). The epitope recognized by McAb 7B2 was denatured by acid elution but could be regenerated by treating with 8 M urea followed by dialysis. The implications of renaturing antigens to regenerate epitopes should be considered in studies on the purification, function and immunogenicity of malaria antigens.

Animals↗
PubMed1988

An antigenic complex in the rhoptries of Plasmodium falciparum.

A previously identified putative rhoptry antigen of Plasmodium falciparum is composed of two major components, one of 80 kDa and a doublet at 42/40 kDa. An inhibitory monoclonal antibody immunoprecipitated both the 80 kDa protein and the 42/40 kDa doublet, but immunoblotted only the 80 kDa component. A second monoclonal antibody, raised against the affinity purified complex, immunoblotted only the 42 kDa band under non-reducing conditions. Electron microscopic examination of thin sections of parasites immunolabeled with these monoclonal antibodies and colloidal gold anti-mouse conjugate has confirmed that this antigen is localised in the rhoptry organelles of mature schizonts and free merozoites. The antigen is associated with apparent membranous structures released from free merozoites. Immunoblotting and immunoprecipitation with two different monoclonal antibodies, and protease digestion experiments, have clearly demonstrated that this antigen is a complex composed of two separate and distinct proteins, and does not represent a monomer/dimer pair. The 80 kDa protein is synthesised as an 84 kDa precursor.

Animals↗
PubMed2003

Expression of a recombinant IRP-like Plasmodium falciparum protein that specifically binds putative plasmodial IREs.

Plasmodium falciparum iron regulatory-like protein (PfIRPa, accession AJ012289) has homology to a family of iron-responsive element (IRE)-binding proteins (IRPs) found in different species. We have previously demonstrated that erythrocyte P. falciparum PfIRPa binds a mammalian consensus IRE and that the binding activity is regulated by iron status. In the work we now report, we have cloned a C-terminus histidine-tagged PfIRPa and overexpressed it in a bacterial expression system in soluble form capable of binding IREs. To overexpress PfIRPa, we used the T7 promoter-driven vector, pET28a(+), in conjunction with the Rosetta(DE3)pLysS strain of E. coli, which carries extra copies of tRNA genes usually found in organisms such as P. falciparum whose genome is (A+T)-rich. The histidine-tagged recombinant protein (rPfIRPa) in soluble form was partially purified using His-bind resin. We searched the plasmodial database, plasmoDB, to identify sequences capable of forming IRE loops using a specially developed algorithm, and found three plasmodial sequences matching the search criteria. In gel retardation assays, rPfIRPa bound three 32P-labeled putative plasmodial IREs with affinity exceeding the affinity for the mammalian consensus IRE. The binding was concentration-dependent and was not inhibited by heparin, an inhibitor of non-specific binding. Immunodepletion of rPfIRPa resulted in substantial inhibition of the signal intensity in the gel retardation assays and in Western blot-determinations of rPfIRPa protein levels. Endogenous PfIRPa retained all three putative 32P-IREs at the same position on the gel as the recombinant PfIRPa.

Animals↗
PubMed1993

Frequency of cross-fertilization in the human malaria parasite Plasmodium falciparum.

Two clones of the human malaria parasite Plasmodium falciparum, denoted 3D7 and HB3, were grown in vitro under conditions permitting the development of gametocytes. The two clones differ in their allelic forms of two antigen genes MSP1 and MSP2. The alleles can be distinguished as size differences of polymerase chain reaction (PCR) amplified fragments of repetitive regions of each gene. Mosquitoes (Anopheles stephensi) were fed on a mixture of these gametocytes. A total of 128 oocysts was isolated from the midguts of infected mosquitoes from 9 crossing experiments between the clones. DNA extracted from these oocysts was amplified by PCR. Oocysts which contained both alleles of each gene (MSP1 and MSP2) had developed from heterozygotes produced by cross-fertilization events between 3D7 and HB3 gametes. The remaining oocysts contained single alleles of each gene, in parent clone combinations, and these had developed from homozygotes formed by self-fertilizations. The results suggest that gametes in the original mixture fed to mosquitoes had undergone random mating.

Alleles↗
PubMed1996

The Plasmodium falciparum 6 kb element is polycistronically transcribed.

The Plasmodium falciparum 6 kb element encodes three protein coding genes and highly fragmented large and small subunit rRNAs; its gene content makes it the probable mitochondrial genome. Many of the genes are encoded so close to each other that there is insufficient room for specific promoters upstream of each gene. RNase protection analysis of two rRNA fragments whose genes are adjacent provided evidence for a polycistronic transcript containing sequences from both, as well as separate small RNAs. To evaluate the possibility of further polycistronic transcription, several sets of oligonucleotide primers located in different regions of the 6 kb element were employed to amplify cDNAs. These analyses have revealed the existence of 6 kb element transcripts as long as 5.9 kb. Both mRNA and rRNA sequences are included on these putative precursor transcripts. Since these types of RNA are known to have different patterns of abundance changes during the erythrocytic portion of the parasite life cycle, RNA stability is presumably an important feature in regulating mitochondrial transcript abundance.

Animals↗
PubMed1994

Order and specificity of the Plasmodium falciparum hemoglobin degradation pathway.

The human malaria parasite, Plasmodium falciparum, degrades nearly all its host cell hemoglobin during a short segment of its intraerythrocytic development. This massive catabolic process occurs in an acidic organelle, the digestive vacuole. Aspartic and cysteine proteases have been implicated in this pathway. We have isolated three vacuolar proteases that account for most of the globin-degrading activity of the digestive vacuole. One is the previously described aspartic hemoglobinase that initiates hemoglobin degradation. A second aspartic protease is capable of cleaving hemoglobin with an overlapping specificity, but seems to prefer acid-denatured globin. The third is a cysteine protease that does not recognize native hemoglobin but readily cleaves denatured globin. It is synergistic with the aspartic hemoglobinase, both by in vitro assay of hemoglobin degradation, and by isobologram analysis of protease inhibitor-treated parasites in culture. The cysteine protease is highly sensitive to chloroquine-heme complex, suggesting a possible mechanism of 4-aminoquinoline antimalarial action. The data suggest an ordered pathway of hemoglobin catabolism that presents an excellent target for chemotherapy.

Amino Acid Sequence↗
PubMed2002

Molecular characterisation of drug-resistant Plasmodium falciparum from Thailand.

BACKGROUND: The increasing levels of Plasmodium falciparum resistance to chloroquine (CQ) in Thailand have led to the use of alternative antimalarials, which are at present also becoming ineffective. In this context, any strategies that help improve the surveillance of drug resistance, become crucial in overcoming the problem. METHODS: In the present study, we have established the in vitro sensitivity to CQ, mefloquine (MF), quinine (QUIN) and amodiaquine (AMQ) of 52 P. falciparum isolates collected in Thailand, and assessed the prevalence of four putative genetic polymorphisms of drug resistance, pfcrt K76T, pfmdr1 N86Y, pfmdr1 D1042N and pfmdr1 Y1246D, by PCR-RFLP. RESULTS: The percentage of isolates resistant to CQ, MF, and AMQ was 96% (50/52), 62% (32/52), and 58% (18/31), respectively, while all parasites were found to be sensitive to QUIN. In addition, 41 (79%) of the isolates assayed were resistant simultaneously to more than one drug; 25 to CQ and MF, 9 to CQ and AMQ, and 7 to all three drugs, CQ, MF and AMQ. There were two significant associations between drug sensitivity and presence of particular molecular markers, i) CQ resistance / pfcrt 76T (P = 0.001), and ii) MF resistance / pfmdr1 86N (P < 0.001) CONCLUSIONS: i) In Thailand, the high levels of CQ pressure have led to strong selection of the pfcrt 76T polymorphism and ii) pfmdr1 86N appears to be a good predictor of in vitro MF resistance.

ATP-Binding Cassette Transporters↗
PubMed1998

Antigenicity of recombinant proteins derived from rhoptry-associated protein 1 of Plasmodium falciparum.

Rhoptry-associated protein 1 (RAP1) of Plasmodium falciparum is a potential component of a malaria vaccine. We have expressed in Escherichia coli eight recombinant RAP1 proteins representing almost the entire sequence of the mature protein and assessed the antigenicity of the proteins by immunization of mice. Antisera to six of the recombinant proteins reacted specifically with parasite-derived RAP1 (PfRAP1), as determined by indirect immunofluorescence and by immunoblotting. These proteins were then used in enzyme-linked immunosorbent assays to evaluate human antibody responses to RAP1 during naturally transmitted infections in The Gambia. Immunoglobulin G (IgG) antibodies specifically reactive with the recombinant RAP1 proteins are directed mostly towards fragments containing the N-terminal sequences of mature PfRAP1. The most N-terminal segment (residues 23 to 175) contains only minor epitopes, while major epitopes are outside this region. Antibodies from malaria patients do not compete for a linear epitope recognized by an inhibitory anti-RAP1 monoclonal antibody. Analysis of IgG subclass distribution shows that human anti-RAP1 antibodies are predominantly IgG1.

Adolescent↗
PubMed1987

Putative glycophorin-binding protein is secreted from schizonts of Plasmodium falciparum.

A cDNA clone expressing an antigen of Plasmodium falciparum, selected by screening an expression library cloned in Escherichia coli, encodes a portion of the protein identified as a glycophorin-binding protein [Kochan et al. (1986) Cell 44, 689-696]. Human antibodies affinity-purified on extracts from this clone were used to characterize the antigen by immunoblotting. This protein was present in all isolates tested, restricted to mature trophozoites and schizonts. It was abundant in culture supernatants at the time of merozoite release but present in minor amounts if at all in merozoites. The pattern of antigen distribution over schizont-infected cells observed by immunoelectron microscopy differed from that of the precursor of the major merozoite surface antigens in that most of the antigen appeared to be located over the erythrocyte cytoplasm without any obvious association with organelles. It thus appears unlikely that this antigen is present on the merozoite surface prior to schizont rupture.

Amino Acid Sequence↗
PubMed1988

Variation in the precursor to the major merozoite surface antigens of Plasmodium falciparum.

The major antigens on the surface of Plasmodium falciparum merozoites are derived from a single high molecular weight polypeptide. The precursor to the major merozoite surface antigen (PMMSA) has conserved and variable antigenic determinants and varies in size in different isolates. Since the protein is a candidate for a malaria vaccine, it is important to understand the molecular basis of this variation. We present the complete sequence of the PMMSA of the Papua New Guinea isolate FC27 and the partial sequence of the West African isolate NF7. The gene consists of blocks of sequence which are either conserved or variable between different isolates. Variable sequences fall into two distinct types, indicating that the PMMSA is encoded by dimorphic alleles that undergo recombination within conserved blocks at the 5' end of the gene. Genetic exchange is not apparent within the other two-thirds of the gene in 12 isolates, suggesting strong selection against such recombinants. The most variable block located near the 5' end contains repeats that are different in independent cloned isolates. This variation presumably accounts for much of the size and antigenic variability.

Alleles↗
PubMed1992

An examination of the mitomycin C induction of chromosome polymorphisms in cultures of Plasmodium falciparum.

Cloned parasites of the FCR3 strain of Plasmodium falciparum that survived treatment with either mitomycin C or ethidium bromide were grown and subcloned. The chromosomes of 10 cloned isolates from each population were analysed by contour-clamped homogenous electric field gel electrophoresis. Eight of 10 isolates from the mitomycin C-treated population contained a detectable polymorphic chromosome change while none of the isolates from the ethidium bromide-treated population did. One of the polymorphic changes involved chromosome #4. A pyrimethamine resistant derivative of FCR3, FCR3-D5, that had previously been shown to contain a single nucleotide change in the dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene but no detectable chromosome polymorphisms, was sequentially treated with mitomycin C and a higher concentration of pyrimethamine than the strain had previously been shown to be resistant to in order to determine if there was a correlation between the selection of chromosome #4 polymorphisms and the applied selective pressure. Most of the cloned survivors of this treatment were found to contain chromosome polymorphisms that involved chromosome #4, the chromosome on which the DHFR-TS gene is located. The polymorphic changes were usually different in the different isolates. The selection of mitomycin C-treated, pyrimethamine resistant strains with chromosome #4 polymorphisms will be discussed.

Animals↗
PubMed2002

Targeting and sequestration of truncated Pfs230 in an intraerythrocytic compartment during Plasmodium falciparum gametocytogenesis.

For malaria to be transmitted, the Plasmodium falciparum parasite must invade an erythrocyte and undergo gametocytogenesis. When mature intraerythrocytic gametocytes are taken up in a blood meal by a mosquito they emerge as gametes and, once fertilized, continue to differentiate into infectious sporozoites. One of the major proteins associated with the surface of the parasite during gamete differentiation is Pfs230, a 360 kDa member of a family of P. falciparum proteins that contains a repeated cysteine motif domain. To characterize the role of different regions of Pfs230, the gene was disrupted by targeted integration and clones isolated that expressed distinct sections of Pfs230. Independent clones D1.356 a and b express the first 452 amino acids (aa) of Pfs230 and do not contain a cysteine motif domain, whereas clones D2.850 a and b express the first 950 aa, including the first cysteine motif domain. Although both sets of clones undergo gametogenesis and produce morphologically normal gametes, neither truncated Pfs230 is located on the surface of the gamete. In clones D1.356 a and b, the 452 aa Pfs230 is secreted into the parasitophorous vacuole and released as a soluble protein when the parasite emerges from the erythrocyte as a gamete. In marked contrast, the 950 aa form of Pfs230 expressed by clones D2.850 a and b is sequestered in a novel tubular compartment in the erythrocyte cytoplasm. This sexual-stage tubular intraerythrocytic compartment (STIC) is not recognized by antibodies specific for proteins associated with the parasitophorous vacuole membrane (Pfs16 or Exp-1) or Maurer's clefts (Pfsbp 1 or mAb LWL1) or intraerythrocytic asexual parasite proteins (PfEMP2 or HRP II).

Animals↗
PubMed1984

Effect of salinomycin-Na on malaria parasites (Plasmodium falciparum and P. berghei).

In vitro exposure of Plasmodium falciparum and P. berghei to salinomycin-Na showed that 10 min incubation in RPMI 1640 medium containing 100 micrograms/ml of the polyether antibiotic led to complete destruction of most parasites; in media containing 10 or 1 microgram/ml salinomycin-Na some young developmental stages seemed to survive, apparently due to the protection of the mostly intact host cell. In vitro treatment of rats infected with P. berghei revealed that a single subcutaneous (oral) dose of 20 mg/kg (80 mg/kg salinomycin-Na caused complete destruction of parasites. Incipient degeneration of the parasites could be already observed 1 h after treatment. After 22 h parasites had disappeared from blood smears. Repeated subcutaneous doses of the polyether as low as 5 mg/kg (X3) completely destroyed the asexual stages of P. berghei. During the ultrastructural investigation of the action of salinomycin-Na it was found that initially the inner lacunes (such as endoplasmic reticulum and perinuclear space) and the mitochondrion were markedly swollen. This was followed by mitochondrial disruption with rupture of the parasites' pellicle. Since the infected host cells also ruptured, mainly extracellular parasites were seen in blood smears beginning 6 h after treatment. Salinomycin-Na seems to act similarly on the malarial parasites and on the free merozoites of chicken intestinal coccidia.

Animals↗
PubMed1985

Studies of resistance to chloroquine, quinine, amodiaquine and mefloquine among Philippine strains of Plasmodium falciparum.

One hundred cases of slide-confirmed Plasmodium falciparum malaria admitted to the San Lazaro Hospital, Manila, Philippines were screened for in vitro resistance to chloroquine, quinine, amodiaquine and mefloquine using the microtechnique. 59 of the 100 primary parasite isolates produced schizonts, whereas the remaining 41 isolates did not. 51 of the 59 isolates tested were resistant in vitro to chloroquine and eight were sensitive. In contrast, three of the primary isolates were resistant to quinine, three showed resistance to amodiaquine and four were mefloquine-resistant. 43 of the strains judged chloroquine-resistant in vitro were fully in vitro sensitive to amodiaquine, quinine and mefloquine. One chloroquine-resistant isolate was also resistant to quinine alone. Three isolates that were resistant to chloroquine were also resistant to amodiaquine. An additional three were cross-resistant to chloroquine and mefloquine. A single isolate was found to be resistant to chloroquine, quinine and mefloquine and another was cross-resistant to chloroquine, quinine and amodiaquine. All strains demonstrating in vitro resistance to amodiaquine, quinine or mefloquine also showed in vitro resistance to chloroquine. The parasites in 22 patients showed in vivo resistance to chloroquine therapy. 86% were of the R1 type, 9% were R2 and 5% R3. All 22 patients demonstrating in vivo resistance to chloroquine showed in vitro resistance.

Amodiaquine↗