New synchronization method for <i>Plasmodium falciparum</i>

&lt;b&gt;Background&lt;/b&gt;: Plasmodium falciparum is usually asynchronous during in vitro culture. Although various synchronization methods are available, they are not able to narrow the range of ages of parasites. A newly developed method is described that allows synchronization of parasites to produce cultures with an age range as low as 30 minutes. &lt;b&gt;Methods&lt;/b&gt;: Trophozoites and schizonts are enriched using Plasmion. The enriched late stage parasites are immobilized as a monolayer onto plastic Petri dishes using concanavalin A. Uninfected erythrocytes are placed onto the monolayer for a limited time period, during which time schizonts on the monolayer rupture and the released merozoites invade the fresh erythrocytes. The overlay is then taken off into a culture flask, resulting in a highly synchronized population of parasites. &lt;b&gt;Results&lt;/b&gt;: Plasmion treatment results in a 10- to 13-fold enrichment of late stage parasites. The monolayer method results in highly synchronized cultures of parasites where invasion has occurred within a very limited time window, which can be as low as 30 minutes. The method is simple, requiring no specialized equipment and relatively cheap reagents. &lt;b&gt;Conclusions&lt;/b&gt;: The new method for parasite synchronization results in highly synchronized populations of parasites, which will be useful for studies of the parasite asexual cell cycle

Genetic polymorphism of the serine rich antigen N-terminal region in Plasmodium falciparum field isolates from Brazil

In this work we investigated the frequency of polymorphism in exon II of the gene encoding most of the amino-terminal region of the serine rich antigen (SERA) in Plasmodium falciparum field samples. The blood samples were colleted from P. falciparum infected individuals in three areas of the Brazilian Amazon. Two fragments have been characterized by polymerase chain reaction: one of 175 bp corresponding to the repeat region with 5 octamer units and one other of 199 bp related to the 6 repeat octamer units of SERA protein. The 199 bp fragment was the predominant one in all the studied areas. The higher frequency of this fragment has not been described before and could be explained by an immunological selection of the plasmodial population in the infected individuals under study. Since repeat motifs in the amino-terminal region of SERA contain epitopes recognized by parasite-inhibitor antibodies, data reported here suggest that the analysis of the polymorphism of P. falciparum isolates in different geographical areas is a preliminary stage before the final drawing of an universal vaccine against malaria can be reached

Clues to Evolution of the SERA Multigene Family in 18 Plasmodium Species

SERA gene sequences were newly determined from 11 primate Plasmodium species including two human parasites, P. ovale and P. malariae, and the evolutionary history of SERA genes was analyzed together with 7 known species. All have one each of Group I to III cysteine-type SERA genes and varying number of Group IV serine-type SERA genes in tandem cluster. Notably, Group IV SERA genes were ascertained in all mammalian parasite lineages; and in two primate parasite lineages gene events such as duplication, truncation, fragmentation and gene loss occurred at high frequency in a manner that mimics the birth-and-death evolution model. Transcription profile of individual SERA genes varied greatly among rodent and monkey parasites. Results support the lineage-specific evolution of the Plasmodium SERA gene family. These findings provide further impetus for studies that could clarify/provide proof-of-concept that duplications of SERA genes were associated with the parasites' expansion of host range and the evolutionary conundrums of multigene families in Plasmodium

Optimized high gradient magnetic separation for isolation of Plasmodium-infected red blood cells

<p>Abstract</p> <p>Background</p> <p>Highly purified infected red blood cells (irbc), or highly synchronized parasite cultures, are regularly required in malaria research. Conventional isolation and synchronization rely on density and osmotic fragility of irbc, respectively. High gradient magnetic separation (HGMS) offers an alternative based on intrinsic magnetic properties of irbc, avoiding exposure to chemicals and osmotic stress. Successful HGMS concentration in malaria research was previously reported using polymer coated columns, while HGMS depletion has not been described yet. This study presents a new approach to both HGMS concentration and depletion in malaria research, rendering polymer coating unnecessary.</p> <p>Methods</p> <p>A dipole magnet generating a strong homogenous field was custom assembled. Polypropylene syringes were fitted with one-way stopcocks and filled with stainless steel wool. Rbc from <it>Plasmodium falciparum </it>cultures were resuspended in density and viscosity optimized HGMS buffers and HGMS processed. Purification and depletion results were analysed by flow cytometer and light microscopy. Viability was evaluated by calculating the infection rate after re-culturing of isolates.</p> <p>Results</p> <p>In HGMS concentration, purity of irbc isolates from asynchronous cultures consistently ranged from 94.8% to 98.4% (mean 95.7%). With further optimization, over 90% of isolated irbc contained segmented schizonts. Processing time was less than 45 min. Reinfection rates ranged from 21.0% to 56.4%. In HGMS depletion, results were comparable to treatment with sorbitol, as demonstrated by essentially identical development of cultures.</p> <p>Conclusion</p> <p>The novel HGMS concentration procedure achieves high purities of segmented stage irbc from standard asynchronous cultures, and is the first HGMS depletion alternative to sorbitol lysis. It represents a simple and highly efficient alternative to conventional irbc concentration and synchronization methods.</p

Transfer origins in the conjugative Enterococcus faecalis plasmids pAD1 and pAM373: identification of the pAD1 nic site, a specific relaxase and a possible TraG-like protein

The Enterococcus faecalis conjugative plasmids pAD1 and pAM373 encode a mating response to the peptide sex pheromones cAD1 and cAM373 respectively. Sequence determination of both plasmids has recently been completed with strong similarity evident over many of the structural genes related to conjugation. pAD1 has two origins of transfer, with oriT1 being located within the repA determinant, whereas the more efficiently utilized oriT2 is located between orf53 and orf57 , two genes found in the present study to be essential for conjugation. We have found a similarly located oriT to be present in pAM373. oriT2 corresponds to about 285 bp based on its ability to facilitate mobilization by pAD1 when ligated to the shuttle vector pAM401; however, it was not mobilized by pAM373. In contrast, a similarly ligated fragment containing the oriT of pAM373 did not facilitate mobilization by pAD1 but was efficiently mobilized by pAM373. The oriT sites of the two plasmids each contained a homologous large inverted repeat (spanning about 140 bp) adjacent to a series of non-homologous short (6 bp) direct repeats. A hybrid construction containing the inverted repeat of pAM373 and direct repeats of pAD1 was mobilized efficiently by pAD1 but not by pAM373, indicating a significantly greater degree of specificity is associated with the direct repeats. Mutational (deletion) analyses of the pAD1 oriT2 inverted repeat structure suggested its importance in facilitating transfer or perhaps ligation of the ends of the newly transferred DNA strand. Analyses showed that Orf57 (to be called TraX) is the relaxase, which was found to induce a specific nick in the large inverted repeat inside oriT ; the protein also facilitated site-specific recombination between two oriT2 sites. Orf53 (to be called TraW) exhibits certain structural similarities to TraG-like proteins, although there is little overall homology.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72536/1/j.1365-2958.2002.03007.x.pd

Plasmid macro-evolution: selection of deletions during adaptation in a nutrient-limited environment

Under conditions where plasmid-carriage is deleterious to the cell, evolutionary changes may be expected which result in an attenuation of the deleterious effect of the plasmid. During long-term growth in glucose-limited continuous culture, initiated with a single clone of Escherichia coli containing a derivative of the plasmid pBR322, a structural change arose in the plasmid and predominated in the plasmid-containing sector of the population. This variant possessed a 2.25 kb deletion encompassing the tetracycline resistance operon as well as a region of about 1.5 kb upstream from this operon. Competition experiments involving strains carrying the plasmid with the spontancous deletion, and strains carrying plasmids with artificially constructed deletions, revealed that deletion of this region of the plasmid, involving loss of tetracycline resistance, resulted in an increment in fitness of between 10 and 20%. From the magnitude of the growth advantage, we conclude that the attenuation of the deleterious effect of the plasmid was mainly due to a reduction in the plasmid mediated interference in the metabolism of the cell caused by a deletion of the tetracycline resistance gene.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/42797/1/10709_2004_Article_BF00127247.pd

Platform for Plasmodium vivax vaccine discovery and development

Plasmodium vivax is the most prevalent malaria parasite on the American continent. It generates a global burden of 80-100 million cases annually and represents a tremendous public health problem, particularly in the American and Asian continents. A malaria vaccine would be considered the most cost-effective measure against this vector-borne disease and it would contribute to a reduction in malaria cases and to eventual eradication. Although significant progress has been achieved in the search for Plasmodium falciparum antigens that could be used in a vaccine, limited progress has been made in the search for P. vivax components that might be eligible for vaccine development. This is primarily due to the lack of in vitro cultures to serve as an antigen source and to inadequate funding. While the most advanced P. falciparum vaccine candidate is currently being tested in Phase III trials in Africa, the most advanced P. vivax candidates have only advanced to Phase I trials. Herein, we describe the overall strategy and progress in P. vivax vaccine research, from antigen discovery to preclinical and clinical development and we discuss the regional potential of Latin America to develop a comprehensive platform for vaccine development

Functional relationship between parts of the replication region of plasmid ColE1.

The inhibition of plasmid ColE1 replication caused by a deletion of the ColE1 plasmid replication origin has been previously reported (T. Hashimoto-Gotoh and J. Inselburg, J. Bacteriol. 139:597-619). Evidence is presented showing that restoration of the deleted nucleotide sequence in the precise relationship it normally has to the rest of the replication region is essential for restoration of ColE1 replication capability to the deletion mutant