Relative activity of abdominal muscles during commonly prescribed strengthening exercises.

The purpose of this study was to determine the relative electromyographic (EMG) activity of the upper and lower rectus abdominis and the external oblique muscles during 5 commonly performed abdominal strengthening exercises. Twenty-five healthy subjects participated in the study. EMG data were collected under isometric and dynamic conditions. The reverse curl resulted in the greatest amount of lower rectus activity, the v-sit and reverse curl exercises resulted in the greatest amount of external oblique activity, and the trunk curl, reverse curl, trunk curl with a twist, and v-sit all resulted in similar amounts of upper rectus EMG activity. The vacuum exercise resulted in moderate levels of external oblique EMG activity but very low levels of activity in the rectus abdominis. Our findings support the concept that abdominal strengthening exercises can differentially activate various abdominal muscle groups, but contradict some traditionally held assumptions regarding the effects of specific exercises.

Characterization of three genes encoding enzymes of the folate biosynthetic pathway in Plasmodium falciparum.

Although the folate metabolic pathway in malaria parasites is a major chemotherapeutic target, resistance to currently available antifolate drugs is an increasing problem. This pathway, however, includes a number of enzymes that, to date, have not been characterized despite their potential for clinical exploitation. As a step towards evaluation of additional targets in this pathway, we report the isolation and characterization of 3 new genes that encode homologues of GTP cyclohydrolase I (GTP-CH), dihydrofolate synthase/folylpolyglutamate synthase (DHFS/FPGS) and serine hydroxymethyltransferase (SHMT). The genes encoding GTP-CH and SHMT are unambiguously assigned to chromosome 12, while that for DHFS/FPGS is tentatively assigned to chromosome 13. All 3 genes are expressed in blood-stage parasites, yielding transcripts of which only ca 60-70% is accounted for by coding sequence. All 3 of the proteins predicted to be encoded by these genes display sequence differences compared to the human host homologues that may be of functional significance. These data bring the complement of cloned genes that encode activities in the pathway to seven, leaving only the gene encoding dihydroneopterin aldolase (DHNA) to be identified in the route from GTP to folate synthesis and folate turnover in the thymidylate cycle.

A bifunctional dihydrofolate synthetase--folylpolyglutamate synthetase in Plasmodium falciparum identified by functional complementation in yeast and bacteria.

Folate metabolism in the human malaria parasite Plasmodium falciparum is an essential activity for cell growth and replication, and the target of an important class of therapeutic agents in widespread use. However, resistance to antifolate drugs is a major health problem in the developing world. To date, only two activities in this complex pathway have been targeted by antimalarials. To more fully understand the mechanisms of antifolate resistance and to identify promising targets for new chemotherapies, we have cloned genes encoding as yet uncharacterised enzymes in this pathway. By means of complementation experiments using 1-carbon metabolism mutants of both Escherichia coli and Saccharomyces cerevisiae, we demonstrate here that one of these parasite genes encodes both dihydrofolate synthetase (DHFS) and folylpolyglutamate synthetase (FPGS) activities, which catalyse the synthesis and polyglutamation of folate derivatives, respectively. The malaria parasite is the first known example of a eukaryote encoding both DHFS and FPGS activities in a single gene. DNA sequencing of this gene in antifolate-resistant strains of P. falciparum, as well as drug-inhibition assays performed on yeast and bacteria expressing PfDHFS--FPGS, indicate that current antifolate regimes do not target this enzyme. As PfDHFS--FPGS harbours two activities critical to folate metabolism, one of which has no human counterpart, this gene product offers a novel chemotherapeutic target with the potential to deliver a powerful blockage to parasite growth.

Pyrimethamine-sulfadoxine resistance in Plasmodium falciparum: what next?

Chemotherapy remains the only practicable tool to control falciparum malaria in sub-Saharan Africa, where >90% of the world's burden of malaria mortality and morbidity occurs. Resistance is rapidly eroding the efficacy of chloroquine, and the combination pyrimethamine-sulfadoxine is the most commonly chosen alternative. Resistant populations of Plasmodium falciparum were selected extremely rapidly in Southeast Asia and South America. If this happens in sub-Saharan Africa, it will be a public health disaster because no inexpensive alternative is currently available. This article reviews the molecular mechanisms of this resistance and discusses how to extend the therapeutic life of antifolate drugs.

Utilization of exogenous folate in the human malaria parasite Plasmodium falciparum and its critical role in antifolate drug synergy.

The antifolate combination pyrimethamine/sulphadoxine (PYR/SDX; Fansidar) is frequently used to combat chloroquine-resistant malaria. Its success depends upon pronounced synergy between the two components, which target dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) in the folate pathway. This synergy permits clearance of parasites resistant to either drug alone, but its molecular basis is still unexplained. Plasmodium falciparum can use exogenous folate, which is normally present in vivo, bypassing SDX inhibition of DHPS and, apparently, precluding synergy under these conditions. However, we have measured parasite inhibition by SDX/PYR combinations in assays in which folate levels are strictly controlled. In parasites that use exogenous folate efficiently, SDX inhibition can be restored by levels of PYR significantly lower than those required to inhibit DHFR. Isobolograms show that the degree of synergy between PYR and SDX is highly dependent upon prevailing folate concentrations and are indicative of PYR acting to block folate uptake and/or utilization. No significant synergy was observed at physiological drug levels when PYR/SDX acted on purified DHFR, whether wild type or mutant. We conclude that the primary basis for antifolate synergy in these organisms arises from PYR targeting a site (or sites) in addition to DHFR, which restores DHPS as a relevant target for SDX.

Allelic exchange at the endogenous genomic locus in Plasmodium falciparum proves the role of dihydropteroate synthase in sulfadoxine-resistant malaria.

We have exploited the recently developed ability to trans- fect the malaria parasite Plasmodium falciparum to investigate the role of polymorphisms in the enzyme dihydropteroate synthase (DHPS), identified in sulfadoxine-resistant field isolates. By using a truncated form of the dhps gene, specific mutations were introduced into the endogenous gene by allelic replacement such that they were under the control of the endogenous promoter. Using this approach a series of mutant dhps alleles that mirror P.falciparum variants found in field isolates were found to confer different levels of sulfadoxine resistance. This analysis shows that alteration of Ala437 to Gly (A437G) confers on the parasite a 5-fold increase in sulfadoxine resistance and addition of further mutations increases the level of resistance to 24-fold above that seen for the transfectant expressing the wild-type dhps allele. This indicates that resistance to high levels of sulfadoxine in P.falciparum has arisen by an accumulation of mutations and that Gly437 is a key residue, consistent with its occurrence in most dhps alleles from resistant isolates. These studies provide proof that the mechanism of resistance to sulfadoxine in P.falciparum involves mutations in the dhps gene and determines the relative contribution of these mutations to this phenotype.

Rational drug design approach for overcoming drug resistance: application to pyrimethamine resistance in malaria.

Pyrimethamine acts by selectively inhibiting malarial dihydrofolate reductase-thymidylate synthase (DHFR-TS). Resistance in the most important human parasite, Plasmodium falciparum, initially results from an S108N mutation in the DHFR domain, with additional mutation (most commonly C59R or N51I or both) imparting much greater resistance. From a homology model of the 3-D structure of DHFR-TS, rational drug design techniques have been used to design and subsequently synthesize inhibitors able to overcome malarial pyrimethamine resistance. Compared to pyrimethamine (Ki 1.5 nM) with purified recombinant DHFR fromP. falciparum, the Ki value of the m-methoxy analogue of pyrimethamine was 1.07 nM, but against the DHFR bearing the double mutation (C59R + S108N), the Ki values for pyrimethamine and the m-methoxy analogue were 71.7 and 14.0 nM, respectively. The m-chloro analogue of pyrimethamine was a stronger inhibitor of both wild-type DHFR (with Ki 0.30 nM) and the doubly mutant (C59R +S108N) purified enzyme (with Ki 2.40 nM). Growth of parasite cultures of P. falciparum in vitro was also strongly inhibited by these compounds with 50% inhibition of growth occurring at 3.7 microM for the m-methoxy and 0.6 microM for the m-chloro compounds with the K1 parasite line bearing the double mutation (S108N + C59R), compared to 10.2 microM for pyrimethamine. These inhibitors were also found in preliminary studies to retain antimalarial activity in vivo in P. berghei-infected mice.

Development and comparison of quantitative assays for the dihydropteroate synthetase codon 540 mutation associated with sulfadoxine resistance in Plasmodium falciparum.

A point mutation in codon 540 of the dihydropteroate synthetase (dhps) gene affecting sulfadoxine resistance has previously been found in parasites from patients with Plasmodium falciparum infection. Here, we investigated 4 methods of identifying this mutation in clinical specimens and established a reliable quantitative assay to estimate the percentage of resistant type in mixed infections. A diagnostic PCR assay based on allele-specific amplification was developed, which clearly typed the clinical specimens examined. The mutation in codon 540 introduces an additional FokI cleavage site which provided a second method to differentiate mutant from wild type, where the former gives rise to 2 characteristic fragments of 538 and 326 bp that are absent from the latter. To calibrate quantitatively the ratio of alleles in mixed samples, we constructed artificial mixes containing 2 plasmid DNAs, one carrying the mutation and the other a wild-type insert. When 32P-labelling was employed, the allele-specific PCR assay could detect the level of resistant type in a mixture down to 0.1-1%, while for the restriction enzyme/PCR analysis, the figure was approximately 10%. Furthermore, neither fluorescent dye-labelled terminator nor dye-labelled primer cycle sequencing was able to detect the mutant allele if it was present at less than 20-30%. We conclude that the allele-specific PCR assay is the most sensitive method of detecting the codon 540 mutation in P. falciparum dhps, and the method of choice for estimating the composition of mixed samples.

Resistance to antifolates in Plasmodium falciparum monitored by sequence analysis of dihydropteroate synthetase and dihydrofolate reductase alleles in a large number of field samples of diverse origins.

Resistance of Plasmodium falciparum to antifolate chemotherapy is a significant problem where combinations such as Fansidar (pyrimethamine-sulfadoxine; PYR-SDX) are used in the treatment of chloroquine-resistant malaria. Antifolate resistance has been associated with variant sequences of dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS), the targets of PYR and SDX respectively. However, while the nature and distribution of mutations in the dhfr gene are well established, this is not yet the case for dhps. We have thus examined by DNA sequence analysis 141 field samples from several geographical regions with differing Fansidar usage (West and East Africa, the Middle East and Viet Nam) to establish a database of the frequency and repertoire of dhps mutations, which were found in 60% of the samples. We have also simultaneously determined from all samples their dhfr sequences, to better understand the relationship of both types of mutation to Fansidar resistance. Whilst the distribution of mutations was quite different across the regions surveyed, it broadly mirrored our understanding of relative Fansidar usage. In samples taken from individual patients before and after drug treatment, we found an association between the more highly mutated forms of dhps and/or dhfr and parasites that were not cleared by antifolate therapy. We also report a novel mutation in a Pakistani sample at position 16 of DHFR (A16S) that is combined with the familiar C59R mutation, but is wild-type at position 108. This is the first observation in a field sample of a mutant dhfr allele where the 108 codon is unchanged.

Special care team: a welcome choice.

What happens when a patient isn't ready to accept hospice care? Or when curative rather than palliative care is needed? Some patients need the skills of a hospice nurse in a curative setting--which calls for a special care team.

A modified in vitro sulfadoxine susceptibility assay for Plasmodium falciparum suitable for investigating Fansidar resistance.

The combination of pyrimethamine and sulfadoxine (PSD or Fansidar) represents one of the most important chemotherapeutic agents currently used to treat falciparum malaria. To investigate the molecular basis of resistance to PSD, reliable in vitro drug assays are required to permit correlation of resistance levels with different genotypes. We describe here protocols that permit accurate evaluation of IC50 values for sulfadoxine (SDX) inhibition of Plasmodium falciparum. Historically, tests for this drug have suffered from poor reproducibility and extreme variability in reported values. We have examined a series of variables, including serum-containing versus serum-free media, erythrocyte source, pre-test growth conditions, test components and post-test processing. We define conditions which better control the levels of the drug antagonists folate and p-aminobenzoate, yielding reproducible differences between lines of P. falciparum with differing alleles of the dihydropteroatic synthetase gene, which encodes the target enzyme of SDX. We also use this assay to demonstrate a marked difference in the response of different parasite lines to antagonism of SDX inhibition by exogenous folate. The ability to measure reliable IC50 values for SDX inhibition should greatly facilitate further experiments to explore the molecular basis of Fansidar resistance.

Isolation and molecular characterization of the bifunctional hydroxymethyldihydropterin pyrophosphokinase-dihydropteroate synthase gene from Toxoplasma gondii.

Toxoplasma gondii is an important cause of AIDS-related opportunistic infection, manifest as toxoplasmic encephalitis. The clinical treatment of choice is the synergistic combination of antifolate agents, pyrimethamine and sulphadiazine, of which the latter targets the parasite's dihydropteroate synthase (DHPS) activity. Here, we describe the isolation of the gene encoding this activity in T. gondii. The nucleotide sequence contains an open reading frame interrupted by five introns, which encodes a protein of 664 amino acids with an M(r) of 72991. Sequence analysis revealed that, in addition to DHPS, the predicted protein contains a second enzyme function, hydroxymethyldihydropterin pyrophosphokinase (PPPK). This enzyme immediately precedes DHPS in the folate biosynthetic pathway. The bifunctional arrangement of the T. gondii pppk-dhps gene is the same as that observed in the related protozoan parasite, Plasmodium falciparum, and confirms previous biochemical data that these activities were inseparable. Recently, specific mutations within conserved motifs of the DHPS gene of P. falciparum have been identified which give rise to sulphonamide drug resistance. Analysis of seven clinical isolates of T. gondii did not reveal any similar mutations in this limited sample of organisms that had been subjected to drug pressure.

Sulfadoxine resistance in the human malaria parasite Plasmodium falciparum is determined by mutations in dihydropteroate synthetase and an additional factor associated with folate utilization.

Sulfadoxine/pyrimethamine (Fansidar) is widely used in Africa for treating chloroquine-resistant falciparum malaria. To clarify how parasite resistance to this combination arises, various lines of Plasmodium falciparum were used to investigate the role of naturally occurring mutations in the target enzyme, dihydropteroate synthetase (DHPS), in the parasite response to sulfadoxine inhibition. An improved drug assay was employed to identify a clear correlation between sulfadoxine-resistance levels and the number of DHPS mutations. Moreover, tight linkage was observed between DHPS mutations and high-level resistance in the 16 progeny of a genetic cross between sulfadoxine-sensitive (HB3) and sulfadoxine-resistant (Dd2) parents. However, we also demonstrate a profound influence of exogenous folate on IC50 values, which, under physiological conditions, may have a major role in determining resistance levels. Importantly, this phenotype does not segregate with dhps genotypes in the cross, but shows complete linkage to the two alleles of the dihydrofolate reductase (dhfr) gene inherited from the parental lines. However, in unrelated lines, this folate effect correlates less well with DHFR sequence, indicating that the gene responsible may be closely linked to dhfr, rather than dhfr itself. These results have major implications for the acquisition of Fansidar resistance by malaria parasites.

A mutation-specific PCR system to detect sequence variation in the dihydropteroate synthetase gene of Plasmodium falciparum.

Sulphur-based antimalarial drugs targeted at dihydropteroate synthetase (DHPS) are frequently used in synergistic combination with inhibitors of dihydrofolate reductase (DHFR) to combat chloroquine-resistant malaria. We have previously shown that lines of Plasmodium falciparum resistant to the most commonly used sulpha drug, sulphadoxine, carry point mutations in the DHPS coding region, relative to the sequence of sensitive strains (Brooks et al., Eur. J. Biochem. 224 (1994) 397-405). We have now developed PCR diagnostic assays based on allele-specific amplification that are able to detect such mutations. The four tests described can reliably discriminate all of the mutations observed to alter codons 436, 581 and 613, yielding allele-specific amplification products of different sizes in each case. Moreover, by careful adjustment of primer length and the degree of mismatch to target and non-target alleles, we were able to standardise all four tests to a single set of PCR conditions, allowing all possible mutations to be monitored simultaneously on one thermocycler. These assays should prove invaluable in further assessing the contribution of specific base changes in the DHPS gene of the parasite to the sulphadoxine resistance phenotype and to the clinical failure of the sulphadoxine/pyrimethamine combination Fansidar.

Sequence variation of the hydroxymethyldihydropterin pyrophosphokinase: dihydropteroate synthase gene in lines of the human malaria parasite, Plasmodium falciparum, with differing resistance to sulfadoxine.

Dihydropteroate synthase (H2Pte synthase) is the target of the sulfur-based antimalarial drugs, which are frequently used in synergistic combination with inhibitors of dihydrofolate reductase (H2folate reductase) to combat chloroquine-resistant malaria. We have isolated the H2Pte synthase coding sequence of the most pathogenic human parasite Plasmodium falciparum. It forms part of a longer coding sequence, located on chromosome 8, that also specifies 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (CH2OH-H2pterinPP kinase) at its 5' proximal end. This domain is unusually large, with two long insertions relative to other CH2OH-H2pterinPP kinase molecules. To investigate a possible genetic basis for clinical resistance to sulfa drugs, we sequenced the complete H2Pte synthase domains from eleven isolates of P. falciparum with diverse geographical origins and levels of sulfadoxine resistance. Overall, point mutations in five positions were observed, affecting four codons. Parasite lines exhibiting high-level resistance were found to carry either a double mutation, altering both Ser436 and Ala613, or a single mutation affecting Ala581. The mutations at positions 436 and 581 have the same location relative to each of two degenerate repeated amino acid motifs that are conserved across all other known H2Pte synthase molecules. The amino acid alteration at residue 613 is identically positioned relative to a different conserved motif. The fourth amino acid residue (437) affected by mutation, though adjacent to the apparently crucial residue 436, shows no obvious correlation with resistance. Although these mutations have no exact counterparts in any other organism, that at position 581 falls within a region of three amino acids where H2Pte synthase is modified in various ways in a number of sulfonamide-resistant pathogenic bacteria. Copy-number analysis indicated that there was no amplification of the H2Pte synthase domain in resistant parasite lines of P. falciparum, compared to sensitive lines.