Plasmodium falciparum: Drug sensitivity in vitro of isolates before and after adaptation to continuous culture

Fifteen strains of Plasmodium falciparum have been cultivated since 1979 using the Trager and Jensen method of continuous culture on isolates from malaria patients. One hundred and two drug sensitivity studies have been carried out on these strains using a semimicro test. Three isolates, initially resistant to chloroquine, adapted rapidly to in vitro cultivation and maintained their high level of resistance (ED₅₀ above 660 nM). Eleven isolates, initially chloroquine sensitive (ED₅₀ under 90 nM) became resistant to this drug (ED₅₀ = 190 to 1950 nM) after the 2–15 weeks required for their adaptation to continuous culture. The resistance of these strains never decreased during the following 15 months of continuous culture. The sensitivity to quinine varied initially from one strain to another (ED₅₀= 160 to 660 nM) and fluctuated during cultivation in the ratio of 1, 3.5 for a given strain. The sensitivity of mefloquine remained high for all strains (ED₅₀ under 150 nM) but one (ED₅₀ = 560 nM). These results suggest that there might be a relationship between in vitro adaptation to culture of P. falciparum by the Trager-Jensen method and a chloroquine-resistant characteristic of the strain. There is the possibility of the emergence of a drug-resistant subpopulation or of changes in the metabolic pathways.     

Plasmodium falciparum: synchronization of asexual development with aphidicolin, a DNA synthesis inhibitor

The asexual development cycle of Plasmodium falciparum, a malarial parasite of humans, has been synchronized in culture by treating ring-stage parasites with aphidicolin, an inhibitor of DNA synthesis. Optimization of both the concentration of drug added to ring stage containing red blood cells and the duration of exposure of parasites to drug led to a reversible block of their maturation at the early trophozoite stage. Release of the aphidicolin block led to a synchronous development of parasites that was manifested by about 80% of the new ring stages being produced within a 2- to 3-hr interval.     

Plasmodium falciparum: one-step growth in a semi-defined medium and the stimulatory effect of human seric lipoproteins and liposomes

The ring stages of Plasmodium falciparum within red blood cells cultured with complete medium stop growing when transferred to a basic medium containing RPMI plus fatty acid-free bovine serum albumin and dialyzable factors from human serum. Growth and multiplication can be partially restored by the addition of lipoprotein fractions prepared from human serum. No specificity was observed with subclasses of lipoproteins. Synthetic liposomes containing lecithin, oleic acid, and cholesterol mimic the effect of lipoproteins.     

Plasmodium falciparum: loss of knobs on the infected erythrocyte surface after long-term cultivation.

After long-term in vitro cultivation in human erythrocytes, variants of three strains of the malaria parasite Plasmodium falciparum no longer produce the “knob” alterations on the host erythrocyte surface. The time in continuous culture before knobs failed to appear ranged from 18 months for the Gambian strain FCR-4 to 33 months for the Vietnamese strain FCR-1. The loss of knobs is correlated with the inability to concentrate trophozoites, schizonts, and segmenters from these variant lines by the use of gelatin-containing media. This is the first report of a change in Plasmodium falciparum or its host cell as a consequence of long-term culture.     

Plasmodium lophurae: Immunization of Pekin ducklings with different antigen preparations

Pekin ducklings were vaccinated with Freund's complete adjuvant plus free Plasmodium lophurae parasites, erythrocytes infected with P. lophurae schizonts, or parasite membrane vesicles. Approximately 50% of the vaccinated ducklings were resistant to challenge with this malarial parasite. However, little protection was afforded by immunization of ducklings with a parasite-specific histidine-rich protein.     

Plasmodium falciparum: Comparative analysis of erythrocyte stage-dependent protein antigens

Proteins of erythrocytic stages of Plasmodium falciparum were biosynthetically labeled at different times during the first cycle of in vitro synchronous cultivation after collection from patients in the Madang region of Papua New Guinea. Proteins were immunoprecipitated with a pool of hyperimmune serum collected in the region then analyzed by sodium dodecyl sulfate-gel electrophoresis. Antigens were recognized in all life cycle stages but the majority of antigens, particularly those of high molecular weight, were present in the mature forms of the parasite.     

Plasmodium falciparum: effect of time in continuous culture on binding to human endothelial cells and amelanotic melanoma cells

An in vitro correlate of the binding in vivo of Plasmodium falciparum-infected erythrocytes to capillary and venular endothelium, using cultured human endothelial cells and amelanotic melanoma cells, was previously developed. The effects of different times in continuous culture on binding of erythrocytes infected with nine different isolates of P. falciparum is now reported. Four isolates, which bound at the time they were first tested, rapidly lost the ability to bind after 26-43 days in culture. One of these, the Cameroun isolate, tested 12 h after the blood was obtained from the patient, had the highest rate of binding of all isolates (680 infected erythrocytes per 100 melanoma cells). After 37 days in culture, only 18 infected erythrocytes per 100 melanoma cells bound. Three isolates first tested after 30-62 days in culture bound poorly. In contrast, two others, the Vietnam (VI) and Brazil (It), continued to bind during the period of study. The Brazil (It) isolate studied after 43 days in culture bound 505 infected erythrocytes per 100 melanoma cells; its clone ItG2G1 continued to bind equally well after 400 days in culture. The ultrastructural morphology of knobs on the binding and nonbinding infected erythrocytes were indistinguishable. Since evidence from other studies indicates that knobs are necessary for binding to endothelium, it is proposed that some parasites in continuous culture may not express the molecules responsible for binding, although the morphologic knobs are still present.     

Trypanosoma cruzi: defined medium for continuous cultivation of virulent parasites.

A liquid medium was developed for the continuous cultivation of Trypanosoma cruzi. Among the several highly purified macromolecules tested only bovine liver catalase, horseradish peroxidase, lactoperoxidase, and bovine hemoglobin supported the continuous growth, at high yield, of mice-virulent Trypanosoma cruzi; other hemoproteins were inactive. Bovine liver catalase showed optimal Trypanosoma cruzi growth-promoting activity, parasites reaching 20 × 10⁶ parasites/ml (95% epimastigotes) at about 10 days in most of the 45 subpassages to date. Furthermore, this protein in the incubation medium provided all the amino acid requirements of actively growing parasites, thus eliminating the need for exogeneous free amino acids. Additional experiments revealed that the hemoprotein's growth-promoting activity was independent of any enzymatic activity and that reconstituting the exact protein composition by means of exogeneous amino acids did not support parasite multiplication, suggesting the importance of the primary structure of the active proteins for growth-promoting activity. These active macromolecules supported the multiplication of five different strains of Trypanosoma cruzi, but did not support Leishmania brasiliensis or Leishmania mexicana proliferation, suggesting species specificity.     

Plasmodium berghei: Architectural analysis by freeze-fracturing of the intraoocyst sporozoite's pellicular system

The technique of freeze-fracturing has been used to study the architecture of the pellicular complex of the intraoocyst sporozoite of Plasmodium berghei. The sporozoite is surrounded by three plasma membranes and a layer of subpellicular microtubules. During freeze-fracturing, each of the three membranes can split along its hydrophobic interior to yield a total of six fracture faces. The most obvious feature of each fracture face is the presence of globular intramembranous particles on the surface. The six fracture faces differ from one another in arrangement, size, and density of these intramembranous particles. Two of the fracture faces exhibit a unique arrangement of particles in well-organized parallel rows along the long axis of the sporozoite. This arrangement has not been reported in either the erythrocytic or the exoerythrocytic forms of Plasmodium spp. Another unique feature in the sporozoite revealed through freeze-fracturing is a single suture line that traverses the long axis of the inner two membranes of the parasite.     

Plasmodium berghei: diet and drug dosage regimens influencing selection of drug-resistant parasites in mice

Two different diets for the host and three drug dosage regimens were used to select lines resistant to sulfadoxine and pyrimethamine from the parent strain of the rodent malaria parasite Plasmodium berghei [the N (K173) strain]. A higher yield of resistance was obtained when a high parasitemia was present at the beginning of the drug pressure schedule. The development of resistance to the association of sulfadoxine plus pyrimethamine was accelerated by a relatively high para-aminobenzoic acid (PABA) content diet. Reproducibility was satisfactory when one of the dosage regimens was applied independently by two different technicians at different times.     

Trypanosoma cruzi: Isolation and characterization of a proteinase

Lysates of Trypanosoma cruzi epimastigotes were able to hydrolyze casein (K_m = 2.5 mg/ml) as well as bovine and human hemoglobins (K_m = 12.2 mg/ml); there was optimum activity was around pH 7.0. The proteinase activity detected with these substrates was enhanced by sodium diaminotetraacetate (EDTA) and reducing agents (SO^2?₃, mercaptoethanol, cysteine) and was inhibited by sulfhydryl reagents, thus suggesting an SH-dependent enzyme. Purification (60×) of the proteinase was carried out as follows: (1) precipitation at ?20 C, pH 4.5, with 80% acetone, (2) gel filtration on Sephadex G-200, (3) affinity chromatography on Sepharose 4B covalently linked to p-aminophenyl mercuric acetate. Only a single component (with an estimated molecular weight of 60,000) was detected in purified preparations by polyacrylamide gel electrophoresis. However, in addition to the major component identified as a proteinase, crossed immunoelectrophoresis experiments indicated the presence of at least three other antigens that apparently were devoid of proteinase activity. Optimum pH activity of the purified preparations was around pH 6.0 for casein and pH 3.0 for hemoglobins, but these activities probably are due to the one enzyme since they were altered identically by the same agents.     

Plasmodium knowlesi: glycolytic intermediate levels of enzyme activities of infected rhesus monkey erythrocytes

In vitro glycolytic enzyme activities and in vivo glycolytic intermediate concentrations were assayed in Plasmodium knowlesi-infected rhesus monkey erythrocytes and control erythrocytes. The enzyme activities of infected erythrocytes were greater than controls indicating that P. knowlesi had its own glycolytic system and that parasite glycolysis was the source of the increased rate of glucose consumption by infected erythrocytes. The P. knowlesi glycolytic enzymes phosphofructokinase and hexokinase were less sensitive to acid inhibition than uninfected red cells.P. knowlesi-infected monkey erythrocytes and Plasmodium berghei-infected mouse erythrocytes had similar in vivo glycolytic profiles and in vitro enzyme activity increases.     

Synthetic propeptide inhibits mosquito midgut chitinase and blocks sporogonic development of malaria parasite

Incessant transmission of the parasite by mosquitoes makes most attempts to control malaria fail. Blocking of parasite transmission by mosquitoes therefore is a rational strategy to combat the disease. Upon ingestion of blood meal mosquitoes secrete chitinase into the midgut. This mosquito chitinase is a zymogen which is activated by the removal of a propeptide from the N-terminal. Since the midgut peritrophic matrix acts as a physical barrier, the activated chitinase is likely to contribute to the further development of the malaria parasite in the mosquito. Earlier it has been shown that inhibiting chitinase activity in the mosquito midgut blocked sporogonic development of the malaria parasite. Since synthetic propeptides of several zymogens have been found to be potent inhibitors of their respective enzymes, we tested propeptide of mosquito midgut chitinase as an inhibitor and found that the propeptide almost completely inhibited the recombinant or purified native Anopheles gambiae chitinase. We also examined the effect of the inhibitory peptide on malaria parasite development. The result showed that the synthetic propeptide blocked the development of human malaria parasite Plasmodium falciparum in the African malaria vector An. gambiae and avian malaria parasite Plasmodium gallinaceum in Aedes aegypti mosquitoes. This study implies that the expression of inhibitory mosquito midgut chitinase propeptide in response to blood meal may alter the mosquito's vectorial capacity. This may lead to developing novel strategies for controlling the spread of malaria.     

Studies on the in vitro cultivation of ciliate protozoa from the kangaroo forestomach

The methods used for culturing rumen protozoa were found to be unsatisfactory for growth of ciliate protozoa from the kangaroo forestomach. Based on published measurements of physical parameters in the marsupial forestomach, several modifications were incorporated into the procedure, i.e., an increase in % hydrogen in the gas phase, adjustment of initial pH of the medium to 6.9–7.0 range, feed only forage as a substrate and incubate at a lower temperature (33–36 °C). Only incubation at the lower temperature increased survival time of the kangaroo protozoa. Two species of Bitricha were still viable after 28 d in culture. Cultures had to be terminated at that time. One of the species differed considerably in size and shape from previously described species and based on 18S rRNA data, may represent a new species of Bitricha. The second species, present in low numbers was identified as Bitricha oblata. In a separate trial, Macropodinium yalanbense survived for 11 d, at which time these cultures also had to be terminated.     

Plasmodium lophurae: composition and properties of hemozoin, the malarial pigment.

Plasmodium lophurae hemozoin (malarial pigment) is composed of proteinaceous macromolecules bonded to iron III protoporphyrin IX by coordination bonding, van der Waals forces, and hydrophobic interactions but not by covalent bonding. Hemozoin is not composed of partially degraded globin peptides coordinated to heme, since fragments of molecular size less than that of globin monomers were not observed by SDS-PAGE. Two major polypeptides constituted the macromolecular portion of hemozoin; these had molecular weights of 21,000 and 15,000. The 21,000-molecular-weight protein is probably of parasite origin. The 15,000-molecular-weight polypeptide is believed to consist of globin monomers, and indicates the presence of irreversibly denatured hemoglobin (hemiglobin), as a constituent of hemozoin. The formation of hemozoin is hypothesized to play the following roles: protection of the parasite against molecular oxygen and compartmentation of the iron porphyrin which is a product of hemoglobin digestion by the plasmodium.     

Synthesis of tetra- and pentasaccharides corresponding to the capsular polysaccharide of Streptococcus pneumoniae type 9A&L, 9N and 9A

Two tetrasaccharides, alpha-D-GlcAp-(1-->3)-alpha-D-Galp-(1-->3)-beta-D-ManpNAc-(1-->4)-beta-D-Glcp and alpha-D-GlcAp-(1-->3)-alpha-D-Glcp-(1-->3)-beta-D-ManpNAc-(1-->4)-beta-D-Glcp (protected form), and a pentasaccharide, alpha-D-Glcp-(1-->4)-alpha-D-GlcAp-(1-->3)-alpha-D-Galp-(1-->3)-beta-D-ManpNAc-(1-->4)-beta-D-Glcp have been synthesised from 2-aminoethyl glycoside trisaccharide acceptors in a linear approach via consecutive alpha-glycosylations. Ethyl thioglycosides were used as glycosyl donors and DMTST in Et(2)O or NIS/TfOH in CH(2)Cl(2) were employed as promoters.