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A F Cowman

Publications and source records attributed to A F Cowman.

At least 55 records · Page 3Linked to original sources
PubMed1997

Stable transgene expression in Plasmodium falciparum.

Plasmid vectors designed to express transgenes and a selectable marker in Plasmodiumfalciparum were constructed. These consist of a selectable gene cassette comprising the Toxoplasma gondii dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene mutated to confer pyrimethamine resistance flanked by either Plasmodium chabaudi DHFR-TS or P. falciparum calmodulin promoter sequences and the P. falciparum histidine rich protein 2 3' region. Also, each vector includes a different expression cassette driven by various Plasmodium transcriptional control sequences. Initially, the chloramphenicol acetyl transferase (CAT) reporter gene was cloned into the expression site of two vectors, pCC6-CAT and pCC13-CAT, which were identical except for the orientation of the expression cassette with respect to the selectable gene cassette. Approximately 8-fold more CAT activity was detected when the direction of transcription of the expression cassettes was in a head to head, rather than a tail to head, orientation. Importantly, it was found that stable transfection could only be achieved when the gene cassettes were in the head to head direction suggesting that this orientation also has an effect on the level of expression of the selectable marker. All other plasmids were designed with the cassettes in a head to head orientation. With the exception of pCC6-CAT and a second vector pHC4-CAT, stable transfectants were obtained with each vector in which the CAT gene had been inserted into the expression cassette. This is the first time vectors for the stable expression in Plasmodium parasites of transgenes other than a selectable marker have been described.

Animals↗
PubMed1997

Targeted gene disruption shows that knobs enable malaria-infected red cells to cytoadhere under physiological shear stress.

Knobs at the surface of erythrocytes infected with Plasmodium falciparum have been proposed to be important in adherence of these cells to the vascular endothelium. This structure contains the knob-associated histidine-rich protein (KAHRP) and the adhesion receptor P. falciparum erythrocyte membrane protein 1. We have disrupted the gene encoding KAHRP and show that it is essential for knob formation. Knob-transfectants adhere to CD36 in static assays; when tested under flow conditions that mimic those of postcapillary venules, however, the binding to CD36 was dramatically reduced. These data suggest that knobs on P. falciparum-infected erythrocytes exert an important influence on adherence of parasitized-erythrocytes to microvascular endothelium, an important process in the pathogenesis of P. falciparum infections.

Animals↗
PubMed1997

The chromosomal organization of the Plasmodium falciparum var gene family is conserved.

The var gene family of Plasmodium falciparum encodes the protein PfEMP1 which is located on the surface of infected erythrocytes and is the receptor that mediates binding to ligands on endothelial cells. This family of proteins is responsible for antigenic variation and differences in binding phenotype to ligands such as CD36 and ICAM1. We have compared the organization of the var gene family in three in vitro cloned lines of P. falciparum and show that most var genes are located in the subtelomeric region of each chromosome closely linked to the repetitive sequence rep20. While most chromosomes possess var genes in the subtelomeric region, in each in vitro cloned line there are some chromosomes that have deleted subtelomeric repetitive regions which include var genes. Comparison of the location of var genes in a field isolate showed that it does not have any detectable subtelomeric deletions as all chromosomes contain var genes and rep20 sequences. We have detected three chromosomes (4, 7 and 12) that contain var gene loci in more stable central regions and the position of these genes on chromosome 4 in the cloned lines analysed is conserved. The location of most of the var gene family in the subtelomeric region of the genome of P. falciparum has important implications for the generation of antigenic diversity of the PfEMP1 protein.

Animals↗
PubMed1997

The antimalarial drug, chloroquine, interacts with lactate dehydrogenase from Plasmodium falciparum.

We have previously shown that a radioiodinated photoreactive analogue of chloroquine, [125I]N-(4-(4-diethylamino-1-methylbutylamino)quinolin-6-yl) -4-azido-2-hydroxybenzamide ([125I]ASA-Q), specifically labels two proteins in Plasmodium falciparum with apparent molecular weights (Mr) of 42 and 33 kDa (Foley M, Deady LW, Ng K, Cowman AF, Tilley L. J Biol Chem 1994:269:6955-6961). We now report the identification of the 33 kDa protein. The 33 kDa protein was purified from Plasmodium falciparum using photoaffinity labeling with [125I]ASA-Q to monitor the enrichment process. N-terminal sequence analysis of the purified protein revealed exact identity of the first 35 amino acids with P. falciparum lactate dehydrogenase (PfLDH). The plasmodial enzyme was cloned and expressed in E. coli and the recombinant protein used to produce a rabbit antiserum. Immunoprecipitation using affinity-purified anti-PfLDH antibodies confirmed the identity of the 33 kDa CQ-binding protein. The enzyme activity of purified PfLDH was not significantly affected by chloroquine indicating that PfLDH is not a direct target of CQ. PfLDH was, however, shown to be exquisitely sensitive to inhibition by free heme and chloroquine protected against this inhibitory effect.

Affinity Labels↗
PubMed1996

Photoaffinity labeling of mefloquine-binding proteins in human serum, uninfected erythrocytes and Plasmodium falciparum-infected erythrocytes.

A photoreactive quinolinemethanol analog, N-[4-[1-hydroxy-2-(dibutylamino)ethyl]quinolin-8yl]-4- azido-2-salicylamide (ASA-MQ) has been synthesized which closely mimics the action of mefloquine. ASA-MQ possesses potent antimalarial activity against a mefloquine-sensitive strain of Plasmodium falciparum and shows decreased activity against a mefloquine-resistant parasite strain. Radioiodinated ASA-MQ has been used in photoaffinity labeling studies to identify mefloquine-interacting proteins in serum, uninfected erythrocytes and Plasmodium falciparum-infected erythrocytes. We have shown that mefloquine interacts specifically with apo-A1, the major protein of serum high density lipoproteins. In addition, mefloquine was shown to interact specifically with the erythrocyte membrane protein, band 7.2b (stomatin). A further two high affinity mefloquine-binding proteins with apparent molecular masses of 22 and 36 kDa were identified in three different strains of Plasmodium falciparum. We suggest that these two mefloquine-binding parasite proteins may be involved in the uptake of mefloquine or may represent macromolecular targets of mefloquine action in malaria parasites.

Affinity Labels↗
PubMed1996

Modulation of the function of human MDR1 P-glycoprotein by the antimalarial drug mefloquine.

MDR1 P-glycoprotein in membranes of human tumor cells of the CEM/VBL100 line was selectively labelled using photoreactive analogs of verapamil, N-(p-azido-3-[125I]salicyl)amino-verapamil ([125I]ASA-V) and prazosin, 2-[4-(4-azido-3-[125I]iodobenzoyl)piperazin-1-yl]4 -amino-6,7-dimethoxyyquinazoline ([125I]ASA-P). Mefloquine, a quinolinemethanol antimalarial drug, was shown to inhibit the labelling of P-glycoprotein with an efficiency similar to that for verapamil, a known chemosensitizer. By contrast, chloroquine competed poorly for the binding site on P-glycoprotein. Mefloquine also inhibited the functional activity of P-glycoprotein. It decreased the rates of extrusion of [3H]vinblastine and the fluorescent dyes, fluo-3 acetomethoxy ester and rhodamine 123, from drug-resistant cells and decreased the level of resistance of these cells to vinblastine. The ability of mefloquine to inhibit P-glycoprotein function may be involved in the neurotoxic side-effects occasionally associated with the use of mefloquine as an antimalarial drug.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Cloning and sequence analysis of a novel member of the ATP-binding cassette (ABC) protein gene family from Plasmodium falciparum.

We have employed oligonucleotide primers directed against the Walker A and B ATP-binding consensus motifs in a PCR-approach to clone a novel member of the eukaryotic ABC protein family of genes from Plasmodium falciparum. The novel gene is predicted to encode a 95.5-kDa protein with two ATP-binding folds each containing a Walker A and B consensus motif and an ABC protein signature sequence. The predicted protein is highly hydrophilic and contains numerous phosphorylation consensus sites but does not contain any potential membrane spanning domains. The gene is present on chromosome 11 and is expressed as a 3.3-kb transcript. The closest homologue with known function to the plasmodial gene is the yeast GCN20 gene which is part of the translation initiation pathway in amino acid starved yeast cells. We have therefore tentatively named the gene Plasmodium falciparum GCN20 homologue (pfgcn20). The pfgcn20 encoded Pfgcn20 protein is also highly homologous to a number of ATP-binding subunits of prokaryotic ABC transporters. We speculate that Pfgcn20 may be an example of a eukaryotic ATP-binding cytosolic subunit of a multipeptide ABC transporter.

ATP-Binding Cassette Transporters↗
PubMed1996

The var genes of Plasmodium falciparum are located in the subtelomeric region of most chromosomes.

PfEMP1, a Plasmodium falciparum-encoded protein on the surface of infected erythrocytes is a ligand that mediates binding to receptors on endothelial cells. The PfEMP1 protein, which is encoded by the large var gene family, shows antigenic variation and changes in binding phenotype associated with alterations in antigenicity. We have constructed a yeast artificial chromosome contig of chromosome 12 from P. falciparum and show that var genes are arranged in four clusters; two lie amongst repetitive subtelomeric sequences and two occur in the more conserved central region. Analysis of parasite chromosomes by pulsed field gel electrophoresis (PFGE) demonstrates that most contain var genes and two-dimensional PFGE has shown that var genes are located at chromosome ends interspersed amongst repetitive sequences present in the subtelomeric complex. Analysis of a var gene located in the subtelomeric region of chromosome 12 has shown that it has close homologues at the opposite end of the chromosome and in the subtelomeric region of two other chromosomes. This suggests that recombination between heterologous chromosomes has occurred in the subtelomeric regions of these chromosomes. The subtelomeric location of var genes dispersed amongst repetitive sequences has important implications for generation of antigenic variants and novel cytoadherent specificities of this protein.

Animals↗
PubMed1996

Characterization of promoters and stable transfection by homologous and nonhomologous recombination in Plasmodium falciparum.

Genetic studies of the protozoan parasite Plasmodium falciparum have been severely limited by the inability to introduce or modify genes. In this paper we describe a system of stable transfection of P. falciparum using a Toxoplasma gondii dihydrofolate reductase-thymidylate synthase gene, modified to confer resistance to pyrimethamine, as a selectable marker. This gene was placed under the transcriptional control of the P. falciparum calmodulin gene flanking sequences. Transfected parasites generally maintained plasmids episomally while under selection; however, parasite clones containing integrated forms of the plasmid were obtained. Integration occurred by both homologous and nonhomologous recombination. In addition to the flanking sequence of the P. falciparum calmodulin gene, the 5' sequences of the P. falciparum and P. chabaudi dihydrofolate reductase-thymidylate synthase genes were also shown to be transcriptionally active in P. falciparum. The minimal 5' sequence that possessed significant transcriptional activity was determined for each gene and short sequences containing important transcriptional control elements were identified. These sequences will provide considerable flexibility in the future construction of plasmid vectors to be used for the expression of foreign genes or for the deletion or modification of P. falciparum genes of interest.

Animals↗
PubMed1996

Plasmodium falciparum: chloroquine selection of a cloned line and DNA rearrangements.

The chloroquine-sensitive Plasmodium falciparum clone, HB3, was selected for growth in increasing amounts of chloroquine. Increases in the size of chromosome 3 were observed with increased chloroquine selection, with the less chloroquine-sensitive parasites possessing the larger forms of the chromosome. Removal of chloroquine selection resulted in a decrease in size of the larger form to the original size and reexposure of these parasites to chloroquine selected again for parasites with a larger chromosome 3. These results suggest that the increase in size of chromosome 3 was associated with the decreased drug sensitivity in the chloroquine selected parasites. A macrorestriction map of chromosome 3 was constructed using the parent HB3 and drug selected lines. The chromosomal size increase was found to have resulted from DNA amplification occurring in 100-kb amplicons. Other isolates of P. falciparum were screened for large size polymorphism in chromosome 3 to determine if chromosome amplification events in this area of the genome also occurred in field isolates. No size changes that correlated with the amplified region in the chloroquine selected lines were observed.

ATP-Binding Cassette Transporters↗
PubMed1996

Plasmodium falciparum: amplification and overexpression of pfmdr1 is not necessary for increased mefloquine resistance.

Amplification and overexpression of the pfmdr1 gene has been associated with mefloquine resistance in Plasmodium falciparum. We have selected for parasites more resistant to mefloquine from P. falciparum FAC8, a clone that has three copies of pfmdr1. The parasite lines derived from this selection were up to threefold more resistant to mefloquine. The mefloquine-selected parasites were also more resistant to quinine and halofantrine, suggesting a multidrug resistance phenotype. In contrast to previous findings, the selection of P. falciparum FAC8 on mefloquine did not alter the copy number or the level of expression of pfmdr1. Sequence analysis of pfmdr1 from the selected lines showed no change in the amino acids. These results show that alterations in pfmdr1 are not involved in mediating the increased mefloquine resistance observed in this clone.

ATP-Binding Cassette Transporters↗
PubMed1996

Current status of the Plasmodium falciparum genome project.

The Plasmodium falciparum Genome Project is a collaborative effort by many laboratories that will provide detailed molecular information about the parasite, which may be used for developing practical control measures. Initial goals are to prepare an electronically indexed clone bank containing partially sequenced clones representing up to 80% of the parasite's genes and to prepare an ordered set of overlapping clones spanning each of the parasite's 14 chromosomes. Currently, clones of genomic DNA, prepared as yeast artificial chromosomes, are arranged into contigs covering approximately 70% of the genome of parasite clone 3D7, gene sequence tags are available from more than contigs covering approximately 70% of the genome of parasite clone 3D7, gene sequence tags are available from more than 20% of the parasite's genes, and approximately 5% of the parasite's genes are tentatively identified from similarity searches of entries in the international sequence databases. A total of > 0.5 Mb of P. falciparum sequence tag data is available. The gene sequence tags are presently being used to complete YAC contig assembly and localize the cloned genes to positions on the physical map in preparation for sequencing the genome. Routes of access to project information and services are described.

Animals↗
PubMed1996

The ATP-binding cassette (ABC) gene family of Plasmodium falciparum.

Multidrug resistance (MDR) in mammalian tumour cells is mediated by P-glycoproteins. The apparent similarities between MDR and the chloroquine-resistance phenotype (CQR) in Plasmodium falciparum suggests that homologous proteins may be involved. In mammals, P-glycoproteins are encoded by mdr genes that are a subset of a super-family characterized by ATP-binding cassettes (ABC). Three genes, pfmdr1, pfmdr2 and pfef3-rl, have been identified in P. falciparum that have homology to the ABC transporter gene family. Each protein encoded by these genes has a distinct structure, suggesting functional differences between the three. Justin Rubio and Alan Cowman here discuss the structure and possible function of the ABC proteins from P. falciparum and evidence that the protein encoded by the pfmdr1 gene can influence quinoline-containing antimalarial drug-resistance phenotypes.

Journal Article↗
PubMed1996

Point mutations in the dihydrofolate reductase and dihydropteroate synthetase genes and in vitro susceptibility to pyrimethamine and cycloguanil of Plasmodium falciparum isolates from Papua New Guinea.

Plasmodium falciparum isolates from 24 Papua New Guinean patients with symptomatic malaria were tested for susceptibility to pyrimethamine and cycloguanil. Thirteen isolates were sensitive to both agents and the remainder exhibited varying degrees of resistance. No isolates were found to be resistant to one agent yet sensitive to the other and a positive correlation suggesting cross-resistance was found. Parasite DNA extracted from the patients' stained blood slides was amplified and sequenced to examine point mutations in the dihydrofolate reductase (DHFR) and dihydropteroate synthetase genes (DHPS) associated with antifolate resistance. All resistant isolates possessed mutations in the DHFR gene at codon 108, the majority changing from Ser to Asn, but one isolate from Ser to Thr, a change not previously reported in field isolates. A second mutation of the DHFR gene at Cys-59 to Arg was present in isolates with higher level resistance, but not exclusively so. Sequencing the DHPS gene, as a predictor of sulfadoxine resistance, revealed only one example that was different from DHPS alleles of sensitive isolates.

Animals↗
PubMed1995

Functional complementation of the ste6 gene of Saccharomyces cerevisiae with the pfmdr1 gene of Plasmodium falciparum.

The pfmdr1 gene has been associated with a drug-resistant phenotype in Plasmodium falciparum, and overexpression of pfmdr1 has been associated with mefloquine- and halofantrine-resistant parasites, but little is known about the functional role of pfmdr1 in this process. Here, we demonstrate that the pfmdr1 gene expressed in a heterologous yeast system functions as a transport molecule and complements a mutation in ste6, a gene which encodes a mating pheromone a-factor export molecule. In addition, the pfmdr1 gene containing two mutations which are associated with naturally occurring chloroquine resistance abolishes this mating phenotype, suggesting that these genetic polymorphisms alter this transport function. Our results support the functional role of pfmdr1 as a transport molecule in the mediation of drug resistance and provide an assay system to address the nature of this transport function.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Cloning of a new cation ATPase from Plasmodium falciparum: conservation of critical amino acids involved in calcium binding in mammalian organellar Ca(2+)-ATPases.

In order to study molecules that may be involved in pH gradient formation in Plasmodium, we have identified a novel cation-translocating ATPase (P-type ATPase) gene from P. falciparum (Pf). We report the full-length nucleotide and deduced amino acid (aa) sequences of this gene that we called PfATPase4. The PfATPase4 protein shares features with the different members of eukaryotic P-type ATPases, such as a similar transmembrane (TM) organization and aa identity in functionally important regions. Interestingly, the PfATPase4 protein possesses conserved aa involved in calcium binding in mammalian organellar Ca(2+)-ATPases.

Adenosine Triphosphatases↗
PubMed1995

A YAC contig map of Plasmodium falciparum chromosome 4: characterization of a DNA amplification between two recently separated isolates.

We have generated a physical map of Plasmodium falciparum chromosome 4 using yeast artificial chromosomes (YACs). The map is defined by a YAC contig spanning approximately 1.05 Mb, which has been restriction mapped to a resolution of 30 kb and is punctuated by 22 sequence-tagged sites. The physical information obtained has enabled us to compare and contrast the structure of chromosome 4 in detail between FCR3 and B8, two recently separated isolates of P. falciparum, leading to characterization of a novel chromosome polymorphism occurring in a subtelomeric region. Comparison of chromosomes 4 from 10 different isolates has shown that chromosome size polymorphisms are restricted to both subtelomeric regions. These analyses provide a high-resolution physical map that will be important to complement genetic analysis of this human pathogen.

Animals↗