Functional consequences of perturbing polyamine metabolism in the malaria parasite, Plasmodium falciparum.

Inhibition of polyamine biosynthesis and/or the perturbation of polyamine functionality have been exploited with success against parasitic diseases such as Trypanosoma infections. However, when the classical polyamine biosynthesis inhibitor, alpha-difluoromethylornithine, is used against the human malaria parasite, Plasmodium falciparum, it results in only a cytostatic growth arrest. Polyamine metabolism in this parasite has unique properties not shared by any other organism. These include the bifunctional arrangement of the catalytic decarboxylases and an apparent absence of the typical polyamine interconversion pathways implying different mechanisms for the regulation of polyamine homeostasis that includes the uptake of exogenous polyamines at least in vitro. These properties make polyamine metabolism an enticing drug target in P. falciparum provided that the physiological and functional consequences of polyamine metabolism perturbation are understood. This review highlights our current understanding of the biological consequences of inhibition of the biosynthetic enzymes in the polyamine pathway in P. falciparum as revealed by several global analytical approaches. Ultimately, the evidence suggests that polyamine metabolism in P. falciparum is a validated drug target worth exploiting.

Exploring functional genomics for drug target and therapeutics discovery in Plasmodia.

Functional genomics approaches are indispensable tools in the drug discovery arena and have recently attained increased attention in antibacterial drug discovery research. However, the application of functional genomics to post-genomics research of Plasmodia is still in comparatively early stages. Nonetheless, with this genus having the most species sequenced of any eukaryotic organism so far, the Plasmodia could provide unique opportunities for the study of intracellular eukaryotic pathogens. This review presents the status quo of functional genomics of the malaria parasite including descriptions of the transcriptome, proteome and interactome. We provide examples for the in silico mining of the X-ome data sets and illustrate how X-omic data from drug challenged parasites might be used in elucidating amongst others, the mode-of-action of inhibitory compounds, validate potential targets and discover novel targets/therapeutics.

In vitro inhibition of Plasmodium falciparum early and late stage gametocyte viability by extracts from eight traditionally used South African plant species.

ETHNOPHARMACOLOGICAL RELEVANCE: Extracts of plant species, used traditionally to treat malaria, have been extensively investigated for their activity against Plasmodium intraerythrocytic asexual parasites in search of new antimalarial drugs. However, less effort has been directed towards examining their efficacy in blocking transmission. Here, we report the results of the in vitro screening of extracts from eight selected plant species used traditionally to treat malaria in South Africa for activity against Plasmodium falciparum NF54 early and late stage gametocytes. The species used were Khaya anthotheca, Trichilia emetica, Turraea floribunda, Leonotis leonurus, Leonotis leonurus ex Hort, Olea europaea subsp. Africana, Catha edulis and Artemisia afra. AIM OF THE STUDY: To investigate the activities of extracts from plant species traditionally used for malaria treatment against P. falciparum gametocytes. MATERIAL AND METHODS: Air-dried and ground plant leaves were extracted using acetone. Primary two point in vitro phenotypic screens against both early and late stage gametocytes were done at 10 and 20µg/ml followed by full IC50 determination of the most active extracts. Inhibition of gametocyte viability in vitro was assessed using the parasite lactate dehydrogenase (pLDH) assay. RESULTS: Of the eight crude acetone extracts from plant species screened in vitro, four had good activity with over 50-70% inhibition of early and late stage gametocytes' viability at 10 and 20µg/ml, respectively. Artemisia afra (Asteraceae), Trichilia emetica (Meliaceae) and Turraea floribunda (Meliaceae) were additionally highly active against both gametocyte stages with IC50 values of less than 10µg/ml while Leonotis leonurus ex Hort (Lamiaceae) was moderately active (IC50<20µg/ml). The activity of these three highly active plant species was significantly more pronounced on late stage gametocytes compared to early stages. CONCLUSION: This study shows the potential transmission blocking activity of extracts from selected South African medicinal plants and substantiates their traditional use in malaria control that broadly encompasses prevention, treatment and transmission blocking. Further studies are needed to isolate and identify the active principles from the crude extracts of A. afra, T. emetica and T. floribunda, as well as to examine their efficacy towards blocking parasite transmission to mosquitoes.

Polyamine uptake by the intraerythrocytic malaria parasite, Plasmodium falciparum.

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 intraerythrocytic 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 was 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, energised by the parasite's inwardly negative membrane potential.

Radurization of Prime Beef Cuts.

The bacteriology, organoleptic quality and shelf-life extension of radurized beef cuts were investigated. Application of doses of 2 kGy to vacuum-packed meat caused a considerable change in the bacterial population by elimination of the pseudomonads, Enterobacteriaceae and enterococci. The LLP group of lactic acid bacteria was relatively resistant to radiation, and after radurization was mostly comprised of Lactobacillus species. The lactobacilli multiplied rapidly on the meat during storage at 4 C, and reached relatively high numbers toward the end of the storage period. Odor and appearance evaluations were carried out at regular intervals and were used together with organoleptic testing and bacteriological analyses to determine the shelf-life extension of radurized beef cuts. A doubling in the shelf-life of samples irradiated to 2 kGy was attained when compared to non-irradiated (control) samples.

Radiation resistance of lactobacilli isolated from radurized meat relative to growth and environment.

Of 113 lactobacilli isolated from radurized (5 kGy) minced meat, 7 Lactobacillus sake strains, 1 L. curvatus strain, and 1 L. farciminis strain were used for radiation resistance studies in a semisynthetic substrate (i.e., modified MRS broth). Five reference Lactobacillus spp., one Staphylococcus aureus strain, and one Salmonella typhimurium strain were used for comparative purposes. All L. sake isolates exhibited the phenomenon of being more resistant to gamma-irradiation in the exponential (log) phase than in the stationary phase of their growth cycles by a factor of 28%. Four references strains also exhibited this phenomenon, with L. sake (DSM 20017) showing a 68% increase in resistance in the log phase over the stationary phase. This phenomenon was not common to all bacteria tested and is not common to all strains with high radiation resistance. Four L. sake isolates and three reference strains were used in radiation sensitivity testing in a natural food system (i.e., meat). The bacteria were irradiated in minced meat and packaged under four different conditions (air, vacuum, CO2, and N2). Organisms exhibited the highest death rate (lowest D10 values [doses required to reduce the logarithm of the bacterial population by 1] ) under CO2 packaging conditions, but resistance to irradiation was increased under N2. The D10 values of the isolates were generally greater than those of the reference strains. The D10 values were also higher (approximately two times) in meat than in semisynthetic growth medium.

Shelf-Life Extension of Minced Beef Through Combined Treatments Involving Radurization.

The effect of addition of glucose, lactic acid (LA) as well as radurization on several bacterial groups in refrigerated beef was investigated. Glucose added to meat in concentrations of 2 to 10% (w/w) had little influence on the bacteria monitored. Addition of LA to a pH of 5 had a marked effect on several bacteria groups in meat; the effect became more pronounced during storage. An increased shelf life was obtained but the appearance of LA-treated samples was undesirable. Radurization (2.5 kGy) had a far greater effect on shelf life than any of the other treatments, although an overwhelming population of lactic acid bacteria developed toward the end of the storage period. Radurization also caused a significant increase in the shelf life in comparison to control, glucose-treated and LA-treated meat samples. A combiend treatment of radurization and LA had an even greater effect on the bacterial population and the shelf life of meat than that of the two separate treatments.