Role of calcium and erythrocyte cytoskeleton phosphorylation in the invasion of Plasmodium falciparum.

The role of calcium in the invasion of the human erythrocyte by the parasite Plasmodium falciparum was studied. The intraerythrocytic and intraparasitic concentrations of Ca2+ were modified using calcium-ionophore A23187 and the chelator EGTA. The Ca2+ inside the parasite appeared to be necessary for the normal completion of invasion. We determined that in recently invaded erythrocytes (2 h), the Ca2+ concentration increased about 10 times. Merozoite invasion produced a decrease in beta-spectrin phosphorylation and an increase in the phosphorylation of a protein with band 4.1 mobility. These changes were similar to those produced by an ionophore-mediated Ca2+ influx in uninfected erythrocytes. These facts support the idea that a calcium influx into erythrocytes might precede or accompany merozoite invasion, triggering a series of molecular events, including phosphorylation and dephosphorylation of cystoskeletal proteins.

A simple technique for entrapping rifampicin and isoniazid into liposomes.

A method for the preparation of liposomes loaded with rifampicin and isoniazid is described. Optimal conditions were established; the lipid suspension was mixed with the aqueous solution of the drugs and was sonicated in a bath for 30 min at 50 degrees C. The optimum composition tested was phosphatidyl choline, cholesterol and cardiolipin in a molar ratio of 7:2:1. The separation of unloaded drug was performed by centrifugation through three successive Sephadex G-25 columns. The liposomes were multilamellar vesicles with a size ranging from 100-300 nm. The drugs were trapped in concentrations from 6.5-9.5 mg/ml. This method is suitable for preparation of liposomes in small laboratories.

Effects of Ca++ depletion on the asexual cell cycle of Plasmodium falciparum.

Ca++ was shown to be indispensable for the normal growth of cultures of Plasmodium falciparum. Inclusion of ethyleneglycolbis (beta-amino-ethylether) N,N'-tetra-acetic acid (EGTA) caused blocking of the asexual cell cycle of the parasite in two sites, the first blockage occurring between 20 and 26 hours after invasion of the erythrocyte. It proved to be irreversible by additions of Mg++ or Ca++, and to lead to morphologically abnormal parasites arrested in the mature trophozoite stage of the cycle. The second site of inhibition was probably one of the steps in the process of invasion of the erythrocyte by the merozoite. When 1 mM EGTA was added 24--30 hours after the culture was synchronized the cell cycle of the parasite continued without any interference in the normal maturation until the development of schizonts and release of merozoites into the medium. However, reinvasion of fresh erythrocytes by these merozoites was impeded. The inhibition of reinvasion caused by EGTA was overcome by the addition of an excess of Ca++ but not by an excess of Mg++. After the addition of Ca++ to cultures blocked just before the invasion phase as schizonts, the merozoites were again rendered fully infective and the rate of invasion was similar to that in an untreated control culture. Implications of the effects of Ca++ depletion on the asexual cell cycle and possible applications of EGTA as a reversible inhibitor of the invasion process are discussed.