ABSTRACT Fluoroacetyl-CoA was synthesized from sodium fluoroacetate via its anhydride. With fluor... more ABSTRACT Fluoroacetyl-CoA was synthesized from sodium fluoroacetate via its anhydride. With fluoroacetyl-CoA as the only substrate and a crude mitochondrial extract from Dichapetalum cymosum as enzyme source, CoASH release took place at a rate of 0.0295 μmoles/min/ mg protein, indicating the presence of a fluoroacetyl-CoA hydrolase enzyme in D. cymosum. This hydrolase could not use acetyl-CoA as a substrate. The presence of this enzyme in D. cymosum probably explains why this plant is not poisoned by its production of fluoroacetate.
ABSTRACT Fusarium fusarioides was found to be highly toxic to ducklings and rats. The toxic metab... more ABSTRACT Fusarium fusarioides was found to be highly toxic to ducklings and rats. The toxic metabolite, moniliformin (sodium or potassium salt of 1-hydroxycyclobut-1-ene-3,4-dione) was isolated as the sole toxin from F. fusarioides. Isolates obtained from peanuts, sorghum, millet, peaches, soil, and dried fish were all toxic to ducklings. The chronic toxicity of an isolate from millet was determined, and 10, 25, and 50% moldy meal incorporated into commercial rat mash all proved toxic. The effect of different incubation temperatures and periods was investigated. Material produced at three different temperatures was of approximately equal toxicity, although less of the material produced at higher temperature was consumed. Analysis showed that the material incubated at 31 °C contained the most moniliformin. Self-heating to a maximum of 6-8 °C took place at all incubation temperatures. The thin-layer chromatographic analytical method was applied to four randomly selected isolates, all of which produced moniliformin, ranging in quantities from 200-840 mg/kg. Methods are described for the analyses and isolation of moniliformin from yellow corn inoculated with cultures of F. fusarioides. The analytical methodology involves either thin-layer chromatography of the extracts or purification of these on Dowex-1 (Cl-) resin; 0.2 M sodium chloride elutes the moniliformin which is subjected to quantitation by UV spectroscopy.
Biochemical and Biophysical Research Communications, 1974
Cytotoxin VII4 being the feurth member of the cytotoxins isolated frem N. m. mossambioa venom was... more Cytotoxin VII4 being the feurth member of the cytotoxins isolated frem N. m. mossambioa venom was the minor component and also the least toxic (2).
Malaria remains the world's most devastating tropical infectious disease with as many as 40% of t... more Malaria remains the world's most devastating tropical infectious disease with as many as 40% of the world population living in risk areas. The widespread resistance of Plasmodium parasites to the costeffective chloroquine and antifolates has forced the introduction of more costly drug combinations, such as Coartem ® . In the absence of a vaccine in the foreseeable future, one strategy to address the growing malaria problem is to identify and characterize new and durable antimalarial drug targets, the majority of which are parasite proteins. Biochemical and structure-activity analysis of these proteins is ultimately essential in the characterization of such targets but requires large amounts of functional protein. Even though heterologous protein production has now become a relatively routine endeavour for most proteins of diverse origins, the functional expression of soluble plasmodial proteins is highly problematic and slows the progress of antimalarial drug target discovery. Here the status quo of heterologous production of plasmodial proteins is presented, constraints are highlighted and alternative strategies and hosts for functional expression and annotation of plasmodial proteins are reviewed.
The origins of tick toxicoses remain a subject of controversy because no molecular data are yet a... more The origins of tick toxicoses remain a subject of controversy because no molecular data are yet available to study the evolution of tick-derived toxins. In this study we describe the molecular structure of toxins from the soft tick, Ornithodoros savignyi. The tick salivary gland proteins (TSGPs) are four highly abundant proteins proposed to play a role in salivary gland granule biogenesis of the soft tick O. savignyi, of which the toxins TSGP2 and TSGP4 are a part. They were assigned to the lipocalin family based on sequence similarity to known tick lipocalins. Several other tick lipocalins were also identified using Smith-Waterman database searches, bringing the tick lipocalin family up to 20. Phylogenetic analysis showed that most tick lipocalins group within genus-specific clades, suggesting that gene duplication and divergence of tick lipocalin function occurred after tick speciation, most probably during the evolution of a hematophagous lifestyle. TSGP2 and TSGP3 show high sequence identity and group terminal to moubatin, an inhibitor of collagen-induced platelet aggregation from the tick, O. moubata. However, no platelet aggregation inhibitory activity is associated with the TSGPs using ADP or collagen as agonists, suggesting that TSGP2 and TSGP3 duplicated after divergence of O. savignyi and O. moubata. This timing is supported by the absence of TSGP2-4 in the salivary gland extracts of O. moubata. The absence of TSGP2 and TSGP4 in salivary gland extracts from O. moubata correlates with the nontoxicity of this tick species. The implications of this study are that the various forms of tick toxicoses do not have a common origin, but must have evolved independently in those tick species that cause pathogenesis.
Ticks control their host's hemostatic system by secretion of bioactive components during feeding ... more Ticks control their host's hemostatic system by secretion of bioactive components during feeding that inhibit blood coagulation and platelet aggregation. Dissolution of platelets that have already aggregated can enhance control over the hemostatic system. It has been shown that disaggregation of aggregated platelets by the enzyme apyrase was accompanied by a shape change from the aggregated spherical form back to the discoid form associated with un-activated platelets. The present study concerns the disaggregation effect of the α IIb β 3 antagonist, savignygrin. Aggregated platelets that were disaggregated by savignygrin and platelets pre-incubated with savignygrin before activation with ADP, retained a spherical form similar to platelets disaggregated by the fibrinogenolytic enzyme plasmin. The number of pseudopods were however, markedly reduced suggesting a disruption of the focal adhesion points that act as a localization point of α IIb β 3 . These results are concurrent with targeting of α IIb β 3 and dissociation of fibrinogen from its receptor, once aggregation has taken place. This is the second mediator of platelet disaggregation found in soft ticks and suggests that disaggregation of aggregated platelets might play an important part in the anti-hemostatic strategy of ticks.
The ornithine decarboxylase (ODC) component of the bifunctional S-adenosylmethionine decarboxylas... more The ornithine decarboxylase (ODC) component of the bifunctional S-adenosylmethionine decarboxylase/ornithine decarboxylase enzyme (PfAdoMetDC-ODC) of Plasmodium falciparum was modeled on the crystal structure of the Trypanosoma brucei enzyme. The homology model predicts a doughnut-shaped active homodimer that associates in a head-to-tail manner. The monomers contain two distinct domains, an N-terminal ␣/barrel and a C-terminal modified Greek-key domain. These domains are structurally conserved between eukaryotic ODC enzymes and are preserved in distant analogs such as alanine racemase and triosephosphate isomerase-like proteins. Superimposition of the PfODC model on the crystal structure of the human enzyme indicates a significant degree of deviation in the carbon ␣-backbone of the solvent accessible loops. The surface locality of the ab initio modeled 38 amino acid parasite-specific insert suggests a role in the stabilization of the large bifunctional protein complex. The active site pockets of PfODC at the interface between the monomers appear to be conserved regarding the binding sites of the cofactor and substrate, but each contains five additional malaria-specific residues. The predicted PfODC homology model is consistent with mutagenesis results and biochemical studies concerning the active site residues and areas involved in stabilizing the dimeric form of the protein. Two competitive inhibitors of PfODC could be shown to interact with several parasite-specific residues in comparison with their interaction with the human ODC. The PfODC homology model contributes toward a structurebased approach for the design of novel malariaspecific inhibitors. Proteins 2003;50:464 -473.
The organization and mining of malaria genomic and post-genomic data is important to significantl... more The organization and mining of malaria genomic and post-genomic data is important to significantly increase the knowledge of the biology of its causative agents, and is motivated, on a longer term, by the necessity to predict and characterize new biological targets and new drugs. Biological targets are sought in a biological space designed from the genomic data from Plasmodium falciparum, but using also the millions of genomic data from other species. Drug candidates are sought in a chemical space containing the millions of small molecules stored in public and private chemolibraries. Data management should, therefore, be as reliable and versatile as possible. In this context, five aspects of the organization and mining of malaria genomic and post-genomic data were examined: 1) the comparison of protein sequences including compositionally atypical malaria sequences, 2) the high throughput reconstruction of molecular phylogenies, 3) the representation of biological processes, particularly metabolic pathways, 4) the versatile methods to integrate genomic data, biological representations and functional profiling obtained from Xomic experiments after drug treatments and 5) the determination and prediction of protein structures and their molecular docking with drug candidate structures. Recent progress towards a grid-enabled chemogenomic knowledge space is discussed.
Dichloromethane and methanolic extracts of each plant were tested for their antiplasmodial activi... more Dichloromethane and methanolic extracts of each plant were tested for their antiplasmodial activity on chloroquineresistant strain of Plasmodium falciparum (FCB strain), based on lactate dehydrogenase activity. Cytotoxicity was assessed with the MTT test on MRC-5 human diploid embryonic lung cells. Most extracts of ten selected plants used in Malay traditional medicine in Malaysia had activity in vitro. This supports continued investigations of traditional medicine in the search for new antimalarial agent. The compounds responsible for the observed antiplasmodial effects are under investigation.
International Journal For Parasitology, Sep 2, 2012
Polyamines and the enzymes involved in their biosynthesis are present at high levels in rapidly p... more Polyamines and the enzymes involved in their biosynthesis are present at high levels in rapidly proliferating cells, including cancer cells and protozoan parasites. Inhibition of polyamine biosynthesis in asexual blood-stage malaria parasites causes cytostatic arrest of parasite development under in vitro conditions, but does not cure infections in vivo. This may be due to replenishment of the parasite's intracellular polyamine pool via salvage of exogenous polyamines from the host. However the mechanism(s) of polyamine uptake by the intra-erythrocytic parasite are not well understood. In this study, the uptake of the polyamines putrescine and spermidine into Plasmodium falciparum parasites functionally isolated from their host erythrocyte were investigated using radioisotope flux techniques. Both putrescine and spermidine were taken up into isolated parasites via a temperature-dependent process that showed cross-competition between different polyamines. There was also some inhibition of polyamine uptake by basic amino acids. Inhibition of polyamine biosynthesis led to an increase in the total amount of putrescine and spermidine taken up from the extracellular medium. The uptake of putrescine and spermidine by isolated parasites was independent of extracellular Na + but increased with increasing external pH. Uptake also showed a marked dependence on the parasite's membrane potential, decreasing with membrane depolarization and increasing with membrane hyperpolarization. The data are consistent with polyamines being taken up into the parasite via an electrogenic uptake process, energized by the parasite's inwardly negative membrane potential.
Insect Biochemistry and Molecular Biology, Jun 30, 2003
Ticks are obligate blood-feeding parasites that secrete anti-hemostatic components during feeding... more Ticks are obligate blood-feeding parasites that secrete anti-hemostatic components during feeding to enable control of the hemostatic system of the host. Complex interactions at the tick-host interface are an indication of the important role that the host played during tick evolution. The question is to what extent interaction with the host and the environment influences tick evolution. Previously, two isoforms (97% sequence identity) of savignygrin, an α IIb β 3 antagonist, have been described. The presence of both isoforms within 20 random individuals confirmed that these isoforms must be recent gene duplicates. Analysis of the sequence differences between the isoforms shows a Kn/Ks ratio of 1, which indicates neutral selection for the isoforms. However, the biased localization of differences within the 3Ј end of the genes suggests that concerted evolution acts on the isoforms. Calculation of the divergence date between the isoforms (1.6-5.2 MYA) also indicates purifying selection, as ample time had passed after duplication, for inactivation of one gene copy. We conclude that concerted evolution has functioned to maintain a high copy number of the savignygrins in order for Ornithodoros savignyi to parasitize a wide host range. This contrasts with O. moubata that expresses the savignygrin homolog, disagregin, as a single copy at lower concentration levels and correlates with the confined habitat and consequently narrow host range of O. moubata. Recent "domestication" of O. savignyi due to animal husbandry practices could however, have reduced the selection constraints acting to maintain the gene copies as evidenced by the structural instability of one of the isoforms. Our results suggest that environmental factors and host associations do play an important role in the evolution of antihemostatic components in ticks.
Two-dimensional gel electrophoresis (2-DE) is one of the most commonly used technologies to obtai... more Two-dimensional gel electrophoresis (2-DE) is one of the most commonly used technologies to obtain a snapshot of the proteome at any specific time. However, its application to study the Plasmodial (malaria parasite) proteome is still limited due to inefficient extraction and detection methods and the extraordinarily large size of some proteins. Here, we report an optimized protein extraction method, the most appropriate methods for Plasmodial protein quantification and 2-DE detection, and finally protein identification by mass spectrometry (MS). Linear detection of Plasmodial proteins in a optimized lysis buffer was only possible with the 2-D Quant kit, and of the four stains investigated, Flamingo Pink was superior regarding sensitivity, linearity, and excellent MS-compatibility. 2-DE analyses of the Plasmodial proteome using this methodology resulted in the reliable detection of 349 spots and a 95% success rate in MS/MS identification. Subsequent application to the analyses of the Plasmodial ring and trophozoite proteomes ultimately resulted in the identification of 125 protein spots, which constituted 57 and 49 proteins from the Plasmodial ring and trophozoite stages, respectively. This study additionally highlights the presence of various isoforms within the Plasmodial proteome, which is of significant biological importance within the Plasmodial parasite during development in the intraerythrocytic developmental cycle.
The separation of proteins by hydrophobic-interaction HPLC and reversed-phase HPLC depends upon d... more The separation of proteins by hydrophobic-interaction HPLC and reversed-phase HPLC depends upon differences in the hydrophobicity of accessible surface groups. The elution order of a group of snake venom cardiotoxins was found to vary between these two HPLC methods. Circular dichroism spectroscopy showed that the eluant acetonitrile-trifluoroacetic acid used for reversed-phase HPLC altered the conformation of the toxins, whereas the salt-buffer eluting medium used for hydrophobic-interaction HPLC did not affect toxin conformation. The retention times of cardiotoxins on reversed-phase HPLC are therefore influenced by their conformational instability in the eluting medium which causes partial or complete unfolding. Hydrophobic interaction is clearly the preferred method with which to correlate the "surface hydrophobicity" of cardiotoxins and their biological effects.
The increasing emergence of Plasmodium falciparum parasites resistant to most of the cost-effecti... more The increasing emergence of Plasmodium falciparum parasites resistant to most of the cost-effective drugs has necessitated the identification of novel leads and drug targets. Parasitespecific inserts in enzymes that are essential for the differentiation and proliferation of malarial parasites have received considerable interest since it distinguishes these proteins from their human counterparts. The functions of these inserts, which include mediations of protein activities or protein-protein interactions, are being investigated by several strategies including deletion mutagenesis. A comparative study of five widely used PCR-based mutagenesis methods identified a modified inverse PCR method as particularly suitable for the deletion of large areas (>100 bp) in malaria parasite genes.
Biochimica Et Biophysica Acta General Subjects, Jun 1, 1977
Hemolysis of guinea pig erythrocytes by snake venom cardiotoxins was investigated with a semi-aut... more Hemolysis of guinea pig erythrocytes by snake venom cardiotoxins was investigated with a semi-automatic method based on light-scattering changes of erythrocyte suspensions at 700 nm which are directly related to hemoglobin release. Small amounts of phospholipase-free cardiotoxin (<100 pg) could-be conveniently and rapidly assayed with high reproducibility in a recording spectrophotometer, and reliable kinetic data were accumulated.
Malaria remains the world's most devastating tropical infectious disease with as many as 40% of t... more Malaria remains the world's most devastating tropical infectious disease with as many as 40% of the world population living in risk areas. The widespread resistance of Plasmodium parasites to the costeffective chloroquine and antifolates has forced the introduction of more costly drug combinations, such as Coartem ® . In the absence of a vaccine in the foreseeable future, one strategy to address the growing malaria problem is to identify and characterize new and durable antimalarial drug targets, the majority of which are parasite proteins. Biochemical and structure-activity analysis of these proteins is ultimately essential in the characterization of such targets but requires large amounts of functional protein. Even though heterologous protein production has now become a relatively routine endeavour for most proteins of diverse origins, the functional expression of soluble plasmodial proteins is highly problematic and slows the progress of antimalarial drug target discovery. Here the status quo of heterologous production of plasmodial proteins is presented, constraints are highlighted and alternative strategies and hosts for functional expression and annotation of plasmodial proteins are reviewed.
Biochimica et Biophysica Acta (BBA) - Protein Structure, 1978
... OF METHIONINE AND ITS EFFECT ON THE PROPERTIES OF CARDIOTOXIN VI11 FROM NAJA MELANOLEUCA VENO... more ... OF METHIONINE AND ITS EFFECT ON THE PROPERTIES OF CARDIOTOXIN VI11 FROM NAJA MELANOLEUCA VENOM FRITZ HH CARLSSON * and ABRAHAM I. LOUW ... Phospholipase A2 (Naja mossambica mos mbica fraction CM-II, [12]) was a kind gift from Dr FJ Joubert ...
ABSTRACT Fluoroacetyl-CoA was synthesized from sodium fluoroacetate via its anhydride. With fluor... more ABSTRACT Fluoroacetyl-CoA was synthesized from sodium fluoroacetate via its anhydride. With fluoroacetyl-CoA as the only substrate and a crude mitochondrial extract from Dichapetalum cymosum as enzyme source, CoASH release took place at a rate of 0.0295 μmoles/min/ mg protein, indicating the presence of a fluoroacetyl-CoA hydrolase enzyme in D. cymosum. This hydrolase could not use acetyl-CoA as a substrate. The presence of this enzyme in D. cymosum probably explains why this plant is not poisoned by its production of fluoroacetate.
ABSTRACT Fusarium fusarioides was found to be highly toxic to ducklings and rats. The toxic metab... more ABSTRACT Fusarium fusarioides was found to be highly toxic to ducklings and rats. The toxic metabolite, moniliformin (sodium or potassium salt of 1-hydroxycyclobut-1-ene-3,4-dione) was isolated as the sole toxin from F. fusarioides. Isolates obtained from peanuts, sorghum, millet, peaches, soil, and dried fish were all toxic to ducklings. The chronic toxicity of an isolate from millet was determined, and 10, 25, and 50% moldy meal incorporated into commercial rat mash all proved toxic. The effect of different incubation temperatures and periods was investigated. Material produced at three different temperatures was of approximately equal toxicity, although less of the material produced at higher temperature was consumed. Analysis showed that the material incubated at 31 °C contained the most moniliformin. Self-heating to a maximum of 6-8 °C took place at all incubation temperatures. The thin-layer chromatographic analytical method was applied to four randomly selected isolates, all of which produced moniliformin, ranging in quantities from 200-840 mg/kg. Methods are described for the analyses and isolation of moniliformin from yellow corn inoculated with cultures of F. fusarioides. The analytical methodology involves either thin-layer chromatography of the extracts or purification of these on Dowex-1 (Cl-) resin; 0.2 M sodium chloride elutes the moniliformin which is subjected to quantitation by UV spectroscopy.
Biochemical and Biophysical Research Communications, 1974
Cytotoxin VII4 being the feurth member of the cytotoxins isolated frem N. m. mossambioa venom was... more Cytotoxin VII4 being the feurth member of the cytotoxins isolated frem N. m. mossambioa venom was the minor component and also the least toxic (2).
Malaria remains the world's most devastating tropical infectious disease with as many as 40% of t... more Malaria remains the world's most devastating tropical infectious disease with as many as 40% of the world population living in risk areas. The widespread resistance of Plasmodium parasites to the costeffective chloroquine and antifolates has forced the introduction of more costly drug combinations, such as Coartem ® . In the absence of a vaccine in the foreseeable future, one strategy to address the growing malaria problem is to identify and characterize new and durable antimalarial drug targets, the majority of which are parasite proteins. Biochemical and structure-activity analysis of these proteins is ultimately essential in the characterization of such targets but requires large amounts of functional protein. Even though heterologous protein production has now become a relatively routine endeavour for most proteins of diverse origins, the functional expression of soluble plasmodial proteins is highly problematic and slows the progress of antimalarial drug target discovery. Here the status quo of heterologous production of plasmodial proteins is presented, constraints are highlighted and alternative strategies and hosts for functional expression and annotation of plasmodial proteins are reviewed.
The origins of tick toxicoses remain a subject of controversy because no molecular data are yet a... more The origins of tick toxicoses remain a subject of controversy because no molecular data are yet available to study the evolution of tick-derived toxins. In this study we describe the molecular structure of toxins from the soft tick, Ornithodoros savignyi. The tick salivary gland proteins (TSGPs) are four highly abundant proteins proposed to play a role in salivary gland granule biogenesis of the soft tick O. savignyi, of which the toxins TSGP2 and TSGP4 are a part. They were assigned to the lipocalin family based on sequence similarity to known tick lipocalins. Several other tick lipocalins were also identified using Smith-Waterman database searches, bringing the tick lipocalin family up to 20. Phylogenetic analysis showed that most tick lipocalins group within genus-specific clades, suggesting that gene duplication and divergence of tick lipocalin function occurred after tick speciation, most probably during the evolution of a hematophagous lifestyle. TSGP2 and TSGP3 show high sequence identity and group terminal to moubatin, an inhibitor of collagen-induced platelet aggregation from the tick, O. moubata. However, no platelet aggregation inhibitory activity is associated with the TSGPs using ADP or collagen as agonists, suggesting that TSGP2 and TSGP3 duplicated after divergence of O. savignyi and O. moubata. This timing is supported by the absence of TSGP2-4 in the salivary gland extracts of O. moubata. The absence of TSGP2 and TSGP4 in salivary gland extracts from O. moubata correlates with the nontoxicity of this tick species. The implications of this study are that the various forms of tick toxicoses do not have a common origin, but must have evolved independently in those tick species that cause pathogenesis.
Ticks control their host's hemostatic system by secretion of bioactive components during feeding ... more Ticks control their host's hemostatic system by secretion of bioactive components during feeding that inhibit blood coagulation and platelet aggregation. Dissolution of platelets that have already aggregated can enhance control over the hemostatic system. It has been shown that disaggregation of aggregated platelets by the enzyme apyrase was accompanied by a shape change from the aggregated spherical form back to the discoid form associated with un-activated platelets. The present study concerns the disaggregation effect of the α IIb β 3 antagonist, savignygrin. Aggregated platelets that were disaggregated by savignygrin and platelets pre-incubated with savignygrin before activation with ADP, retained a spherical form similar to platelets disaggregated by the fibrinogenolytic enzyme plasmin. The number of pseudopods were however, markedly reduced suggesting a disruption of the focal adhesion points that act as a localization point of α IIb β 3 . These results are concurrent with targeting of α IIb β 3 and dissociation of fibrinogen from its receptor, once aggregation has taken place. This is the second mediator of platelet disaggregation found in soft ticks and suggests that disaggregation of aggregated platelets might play an important part in the anti-hemostatic strategy of ticks.
The ornithine decarboxylase (ODC) component of the bifunctional S-adenosylmethionine decarboxylas... more The ornithine decarboxylase (ODC) component of the bifunctional S-adenosylmethionine decarboxylase/ornithine decarboxylase enzyme (PfAdoMetDC-ODC) of Plasmodium falciparum was modeled on the crystal structure of the Trypanosoma brucei enzyme. The homology model predicts a doughnut-shaped active homodimer that associates in a head-to-tail manner. The monomers contain two distinct domains, an N-terminal ␣/barrel and a C-terminal modified Greek-key domain. These domains are structurally conserved between eukaryotic ODC enzymes and are preserved in distant analogs such as alanine racemase and triosephosphate isomerase-like proteins. Superimposition of the PfODC model on the crystal structure of the human enzyme indicates a significant degree of deviation in the carbon ␣-backbone of the solvent accessible loops. The surface locality of the ab initio modeled 38 amino acid parasite-specific insert suggests a role in the stabilization of the large bifunctional protein complex. The active site pockets of PfODC at the interface between the monomers appear to be conserved regarding the binding sites of the cofactor and substrate, but each contains five additional malaria-specific residues. The predicted PfODC homology model is consistent with mutagenesis results and biochemical studies concerning the active site residues and areas involved in stabilizing the dimeric form of the protein. Two competitive inhibitors of PfODC could be shown to interact with several parasite-specific residues in comparison with their interaction with the human ODC. The PfODC homology model contributes toward a structurebased approach for the design of novel malariaspecific inhibitors. Proteins 2003;50:464 -473.
The organization and mining of malaria genomic and post-genomic data is important to significantl... more The organization and mining of malaria genomic and post-genomic data is important to significantly increase the knowledge of the biology of its causative agents, and is motivated, on a longer term, by the necessity to predict and characterize new biological targets and new drugs. Biological targets are sought in a biological space designed from the genomic data from Plasmodium falciparum, but using also the millions of genomic data from other species. Drug candidates are sought in a chemical space containing the millions of small molecules stored in public and private chemolibraries. Data management should, therefore, be as reliable and versatile as possible. In this context, five aspects of the organization and mining of malaria genomic and post-genomic data were examined: 1) the comparison of protein sequences including compositionally atypical malaria sequences, 2) the high throughput reconstruction of molecular phylogenies, 3) the representation of biological processes, particularly metabolic pathways, 4) the versatile methods to integrate genomic data, biological representations and functional profiling obtained from Xomic experiments after drug treatments and 5) the determination and prediction of protein structures and their molecular docking with drug candidate structures. Recent progress towards a grid-enabled chemogenomic knowledge space is discussed.
Dichloromethane and methanolic extracts of each plant were tested for their antiplasmodial activi... more Dichloromethane and methanolic extracts of each plant were tested for their antiplasmodial activity on chloroquineresistant strain of Plasmodium falciparum (FCB strain), based on lactate dehydrogenase activity. Cytotoxicity was assessed with the MTT test on MRC-5 human diploid embryonic lung cells. Most extracts of ten selected plants used in Malay traditional medicine in Malaysia had activity in vitro. This supports continued investigations of traditional medicine in the search for new antimalarial agent. The compounds responsible for the observed antiplasmodial effects are under investigation.
International Journal For Parasitology, Sep 2, 2012
Polyamines and the enzymes involved in their biosynthesis are present at high levels in rapidly p... more Polyamines and the enzymes involved in their biosynthesis are present at high levels in rapidly proliferating cells, including cancer cells and protozoan parasites. Inhibition of polyamine biosynthesis in asexual blood-stage malaria parasites causes cytostatic arrest of parasite development under in vitro conditions, but does not cure infections in vivo. This may be due to replenishment of the parasite's intracellular polyamine pool via salvage of exogenous polyamines from the host. However the mechanism(s) of polyamine uptake by the intra-erythrocytic parasite are not well understood. In this study, the uptake of the polyamines putrescine and spermidine into Plasmodium falciparum parasites functionally isolated from their host erythrocyte were investigated using radioisotope flux techniques. Both putrescine and spermidine were taken up into isolated parasites via a temperature-dependent process that showed cross-competition between different polyamines. There was also some inhibition of polyamine uptake by basic amino acids. Inhibition of polyamine biosynthesis led to an increase in the total amount of putrescine and spermidine taken up from the extracellular medium. The uptake of putrescine and spermidine by isolated parasites was independent of extracellular Na + but increased with increasing external pH. Uptake also showed a marked dependence on the parasite's membrane potential, decreasing with membrane depolarization and increasing with membrane hyperpolarization. The data are consistent with polyamines being taken up into the parasite via an electrogenic uptake process, energized by the parasite's inwardly negative membrane potential.
Insect Biochemistry and Molecular Biology, Jun 30, 2003
Ticks are obligate blood-feeding parasites that secrete anti-hemostatic components during feeding... more Ticks are obligate blood-feeding parasites that secrete anti-hemostatic components during feeding to enable control of the hemostatic system of the host. Complex interactions at the tick-host interface are an indication of the important role that the host played during tick evolution. The question is to what extent interaction with the host and the environment influences tick evolution. Previously, two isoforms (97% sequence identity) of savignygrin, an α IIb β 3 antagonist, have been described. The presence of both isoforms within 20 random individuals confirmed that these isoforms must be recent gene duplicates. Analysis of the sequence differences between the isoforms shows a Kn/Ks ratio of 1, which indicates neutral selection for the isoforms. However, the biased localization of differences within the 3Ј end of the genes suggests that concerted evolution acts on the isoforms. Calculation of the divergence date between the isoforms (1.6-5.2 MYA) also indicates purifying selection, as ample time had passed after duplication, for inactivation of one gene copy. We conclude that concerted evolution has functioned to maintain a high copy number of the savignygrins in order for Ornithodoros savignyi to parasitize a wide host range. This contrasts with O. moubata that expresses the savignygrin homolog, disagregin, as a single copy at lower concentration levels and correlates with the confined habitat and consequently narrow host range of O. moubata. Recent "domestication" of O. savignyi due to animal husbandry practices could however, have reduced the selection constraints acting to maintain the gene copies as evidenced by the structural instability of one of the isoforms. Our results suggest that environmental factors and host associations do play an important role in the evolution of antihemostatic components in ticks.
Two-dimensional gel electrophoresis (2-DE) is one of the most commonly used technologies to obtai... more Two-dimensional gel electrophoresis (2-DE) is one of the most commonly used technologies to obtain a snapshot of the proteome at any specific time. However, its application to study the Plasmodial (malaria parasite) proteome is still limited due to inefficient extraction and detection methods and the extraordinarily large size of some proteins. Here, we report an optimized protein extraction method, the most appropriate methods for Plasmodial protein quantification and 2-DE detection, and finally protein identification by mass spectrometry (MS). Linear detection of Plasmodial proteins in a optimized lysis buffer was only possible with the 2-D Quant kit, and of the four stains investigated, Flamingo Pink was superior regarding sensitivity, linearity, and excellent MS-compatibility. 2-DE analyses of the Plasmodial proteome using this methodology resulted in the reliable detection of 349 spots and a 95% success rate in MS/MS identification. Subsequent application to the analyses of the Plasmodial ring and trophozoite proteomes ultimately resulted in the identification of 125 protein spots, which constituted 57 and 49 proteins from the Plasmodial ring and trophozoite stages, respectively. This study additionally highlights the presence of various isoforms within the Plasmodial proteome, which is of significant biological importance within the Plasmodial parasite during development in the intraerythrocytic developmental cycle.
The separation of proteins by hydrophobic-interaction HPLC and reversed-phase HPLC depends upon d... more The separation of proteins by hydrophobic-interaction HPLC and reversed-phase HPLC depends upon differences in the hydrophobicity of accessible surface groups. The elution order of a group of snake venom cardiotoxins was found to vary between these two HPLC methods. Circular dichroism spectroscopy showed that the eluant acetonitrile-trifluoroacetic acid used for reversed-phase HPLC altered the conformation of the toxins, whereas the salt-buffer eluting medium used for hydrophobic-interaction HPLC did not affect toxin conformation. The retention times of cardiotoxins on reversed-phase HPLC are therefore influenced by their conformational instability in the eluting medium which causes partial or complete unfolding. Hydrophobic interaction is clearly the preferred method with which to correlate the &quot;surface hydrophobicity&quot; of cardiotoxins and their biological effects.
The increasing emergence of Plasmodium falciparum parasites resistant to most of the cost-effecti... more The increasing emergence of Plasmodium falciparum parasites resistant to most of the cost-effective drugs has necessitated the identification of novel leads and drug targets. Parasitespecific inserts in enzymes that are essential for the differentiation and proliferation of malarial parasites have received considerable interest since it distinguishes these proteins from their human counterparts. The functions of these inserts, which include mediations of protein activities or protein-protein interactions, are being investigated by several strategies including deletion mutagenesis. A comparative study of five widely used PCR-based mutagenesis methods identified a modified inverse PCR method as particularly suitable for the deletion of large areas (>100 bp) in malaria parasite genes.
Biochimica Et Biophysica Acta General Subjects, Jun 1, 1977
Hemolysis of guinea pig erythrocytes by snake venom cardiotoxins was investigated with a semi-aut... more Hemolysis of guinea pig erythrocytes by snake venom cardiotoxins was investigated with a semi-automatic method based on light-scattering changes of erythrocyte suspensions at 700 nm which are directly related to hemoglobin release. Small amounts of phospholipase-free cardiotoxin (<100 pg) could-be conveniently and rapidly assayed with high reproducibility in a recording spectrophotometer, and reliable kinetic data were accumulated.
Malaria remains the world's most devastating tropical infectious disease with as many as 40% of t... more Malaria remains the world's most devastating tropical infectious disease with as many as 40% of the world population living in risk areas. The widespread resistance of Plasmodium parasites to the costeffective chloroquine and antifolates has forced the introduction of more costly drug combinations, such as Coartem ® . In the absence of a vaccine in the foreseeable future, one strategy to address the growing malaria problem is to identify and characterize new and durable antimalarial drug targets, the majority of which are parasite proteins. Biochemical and structure-activity analysis of these proteins is ultimately essential in the characterization of such targets but requires large amounts of functional protein. Even though heterologous protein production has now become a relatively routine endeavour for most proteins of diverse origins, the functional expression of soluble plasmodial proteins is highly problematic and slows the progress of antimalarial drug target discovery. Here the status quo of heterologous production of plasmodial proteins is presented, constraints are highlighted and alternative strategies and hosts for functional expression and annotation of plasmodial proteins are reviewed.
Biochimica et Biophysica Acta (BBA) - Protein Structure, 1978
... OF METHIONINE AND ITS EFFECT ON THE PROPERTIES OF CARDIOTOXIN VI11 FROM NAJA MELANOLEUCA VENO... more ... OF METHIONINE AND ITS EFFECT ON THE PROPERTIES OF CARDIOTOXIN VI11 FROM NAJA MELANOLEUCA VENOM FRITZ HH CARLSSON * and ABRAHAM I. LOUW ... Phospholipase A2 (Naja mossambica mos mbica fraction CM-II, [12]) was a kind gift from Dr FJ Joubert ...
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Papers by Abraham (Braam) Louw