Abstract. Cultivated Plasmodium falciparum gametocytes reach maturity in vitro in approximately 14-16 days, during which they pass through five morphologically distinct developmental stages. Purification of the earlier developmental stages has not been previously reported. We have modified the standard discontinuous Percoll gradient method for the separation of stage IV and V gametocytes to obtain enriched preparations of those and the earlier P. falciparum gametocyte stages. In contrast to the stages II, III, and IV, the mature stage V gametocytes from our gradient readily transformed into gametes. Such preparations may be useful in research studies on the mechanisms that underlie gametocytogenesis.Several previously reported techniques for the purification of mature Plasmodium falciparum gametocytes exploit the differences in sedimentation rates and buoyant densities of uninfected erythrocytes and erythrocytes infected with the various stages of the parasite. 1,2 The major limitation of these techniques is their inability to maintain physiologic osmolality throughout the purification procedure and avoid cell damage. 3 Percoll, a colloidal solution of polyvinylpyrrolidone-coated silica particles, can be used to overcome this problem of osmotically induced cell damage because of its low osmolality, 10 mOsm at 1.13 g/ml. 4 A Percoll step gradient centrifugation method has been successfully used to obtain highly purified preparations of viable mature P. falciparum and P. yoelii gametocytes. 5 The addition of fresh erythrocytes to P. falciparum cultures leads to loss of synchrony. 6,7 Synchrony can be restored by the addition of DNA inhibitors during gametocytogenesis. 8 However, inhibitors such as mitomycin C and chloroquine are toxic to stages I and II gametocytes. Mature gametocytes that have been synchronized by the addition of mitomycin C cannot be used immediately for research studies and membrane feeds because gametogenesis is inhibited for 24-48 hr after removal of the drug. 8 To avoid this transient drug toxicity, Ponnudurai and others synchronized gametocytes in an automated culture system by using gelatin flotation and N-acetyl glucosamine treatment. 9 Despite the fact that methods for synchronization and purification of the mature sexual stages of P. falciparum cultures have been separately described, these two techniques have not been combined to obtain purified fractions of the early gametocyte stages. This is a crucial requirement for research because the events that regulate gametocytogenesis are still in question. Our ability to study the early sexual stages, soon after morphologic differentiation, may provide insight into this intriguing aspect of cell cycle regulation. We have modified the Percoll step gradient technique to obtain enriched fractions of almost all the stages of P. falciparum gametocytes from in vitro culture. MATERIALS AND METHODS Culture of gametocytes.Plasmodium falciparum strain NF54 was cultured in vitro according to the Ifediba and Vandeberg 10 modification of the Trager and ...

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Puf proteins are a family of evolutionarily conserved translational regulators in eukaryotes. The malaria parasite has two Puf proteins (PfPuf1 and PfPuf2) that share 25% homology in the RNA binding domain. Here we confirmed the preferential expression of PfPuf2 in gametocyte stages using Northern analysis. The transcriptional initiation site of this gene, mapped using RNA ligase-mediated rapid amplification of cDNA end and primer extension, is located approximately 300 bp upstream from the translational start codon. The 3' end of PfPuf2 is located approximately 250 bp downstream from the stop codon. The total length of the RNA is approximately 2.1 kb, consistent with the mRNA size determined by Northern analysis. Recombinant PfPuf2 proteins expressed in bacteria were purified and used to produce polyclonal antibodies. Western blot further established the preferential synthesis of PfPuf2 in gametocyte stages. Using the Nanos-responsive elements (NRE) in the Hunchback mRNA of Drosophila melanogaster as an artificial target sequence, we tested the binding of PfPuf2 Puf domain to this sequence using the yeast three-hybrid system. The results showed that PfPuf2 Puf domain bound specifically to NRE, suggesting that PfPuf2 may be involved in translational regulation of target genes using a conserved mechanism of the Puf family proteins.

BackgroundBrugia malayi and B. pahangi are two closely related nematodes that cause filariasis in humans and animals. However, B. pahangi microfilariae are able to develop in and be transmitted by the mosquito, Armigeres subalbatus, whereas most B. malayi are rapidly melanized and destroyed within the mosquito hemocoel. A cross-species microarray analysis employing the B. malayi V2 array was carried out to determine the transcriptional differences between B. malayi and B. pahangi microfilariae with similar age distribution.ResultsFollowing microarray data analysis, a list of preferentially expressed genes in both microfilariae species was generated with a false discovery rate estimate of 5% and a signal intensity ratio of 2 or higher in either species. A total of 308 probes were preferentially expressed in both species with 149 probes, representing 123 genes, in B. pahangi microfilariae and 159 probes, representing 107 genes, in B. malayi microfilariae. In B. pahangi, there were 76 (62%) up-regulated transcripts that coded for known proteins that mapped into the KEGG pathway compared to 61 (57%) transcripts in B. malayi microfilariae. The remaining 47 (38%) transcripts in B. pahangi and 46 (43%) transcripts in B. malayi microfilariae were comprised almost entirely of hypothetical genes of unknown function. Twenty-seven of the transcripts in B. pahangi microfilariae coded for proteins that associate with the secretory pathway compared to thirty-nine in B. malayi microfilariae. The data obtained from real-time PCR analysis of ten genes selected from the microarray list of preferentially expressed genes showed good concordance with the microarray data, indicating that the microarray data were reproducible.ConclusionIn this study, we identified gene transcripts that were preferentially expressed in the microfilariae of B. pahangi and B. malayi, some of which coded for known immunomodulatory proteins. These comparative transcriptome data will be of interest to researchers keen on understanding the inherent differences, at the molecular level, between B. malayi and B. pahangi microfilariae especially because these microfilariae are capable of surviving in the same vertebrate host but elicit different immune response outcomes in the mosquito, Ar. subalbatus.