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Biomedical subjects

R J Wilson

Publications and source records attributed to R J Wilson.

At least 127 records · Page 7Linked to original sources
PubMed1985

Selective stage-specific changes in the permeability to small hydrophilic solutes of human erythrocytes infected with Plasmodium falciparum.

The permeability characteristics of Plasmodium falciparum-infected human erythrocytes to various 3H-labelled solutes were measured during the maturation of the parasites in sorbitol-synchronised cultures. Using [14C]inulin as the extracellular marker, estimates were made of the influx kinetics of [3H]amino acids into trichloroacetic acid (TCA)-soluble pools within the erythrocyte and concomitant incorporation into TCA-precipitable material. These measurements provided values of the rates of protein synthesis by the parasite and the initial influx rates for the transport of precursor amino acids into the erythrocyte. For about 12-15 h after parasitisation, the influx of L-[3H]glutamine remained at a low level comparable to that in the uninfected cell (2-9 nmol g-1 cells min-1). As pigment appeared in the trophozoite, the initial rate of influx of L-glutamine increased to a value up to 100-fold higher than in the uninfected erythrocyte. The increase in permeability affected only the parasitised cells in a culture of partially infected erythrocytes, and was selective with respect to substrate since the influx kinetics for both [3H]isoleucine and [3H]arginine were not affected by parasitisation. The permeability changes occurred mainly over a 4-8 h period in the development of the young trophozoite, during which time [3H]glycine influx was enhanced by a factor of 3-10, with a comparable increase in the uptake of myo-[3H]inositol. L-[3H]glutamate, which did not penetrate significantly into uninfected erythrocytes, entered red cells infected with mature trophozoites at a rate which was much less than 1% of the parasite-induced-L-glutamine influx. At the stages when the permeability to L-glutamine was markedly enhanced, parasitised cells remained impermeable to [3H]sucrose. An analysis of the relative 3H activities in glutathione and free amino acid pools indicated that, if L-glutamine permeation did not increase during parasite maturation beyond the ring stage, or was blocked by a potential antimalarial compound, an insufficient supply of L-glutamine would be available for the increased rates of parasite protein synthesis and glutathione turnover within the red cell.

Animals↗
PubMed1985

Relation of red cell membrane properties to invasion by Plasmodium falciparum.

The effects of changes in red cell membrane properties on invasion by Plasmodium falciparum have been studied by varying the cholesterol content and the intracellular concentration of polyamines. Increased cholesterol content is known to cause large reductions in the internal fluidity of the phospholipid bilayer and a change in its preferred direction of bending, but does not cause changes in gross mechanical rigidity. Polyamines, on the other hand, are thought to increase the cohesion of the membrane cytoskeleton and impede translational diffusion of transmembrane particles, as well as increase the mechanical rigidity of the membrane. Cells with membranes augmented by 50% in cholesterol show no reduction in their susceptibility to parasitic invasion, whereas an increase in cytosolic polyamine (especially spermine) concentration leads to strong inhibition of invasion. In neither case is the development of the intracellular parasite affected. We conclude that it is the macroscopic, rather than the microscopic rheoelastic properties of the membrane that influence the invasion process. Depletion of membrane cholesterol leads to a substantial reduction in parasitaemia; it is suggested that this is linked to the reduced phosphorus incorporation into spectrin in these cells. Polyamines may exert a significant effect at physiological concentrations and the possibility must be considered that the elevated polyamine levels found in red cells in sickle cell disease may account for the protection against P. falciparum.

Adult↗
PubMed1985

Identifying impaired drivers among the general driving population.

The primary purpose of this study was to identify predictors of impaired driving among a random sample of Canadian drivers who consume beverage alcohol. A second purpose was to determine if impaired drivers in the general population have characteristics similar to those reported among convicted and accident-involved impaired drivers. Amount of alcohol consumed over the past seven days was found to be the single most powerful predictor of both frequency of drinking-driving and of impaired driving. Impaired drivers differed from moderate-drinking drivers on a number of measures and were characterized by more irresponsible attitudes and higher risk behavior, consistent with findings of other impaired-driver subgroups. It is concluded that impaired driving may be just one behavior which is part of a deviant behavioral syndrome typified by high-risk behaviors.

Accidents, Traffic↗
PubMed1984

A simple method for isolating viable mature parasites of Plasmodium falciparum from cultures.

Mature asexual parasites from cultures of knobby or knobless clones of P. falciparum containing 5 to 10% late forms were harvested by layering up to 10(9) erythrocytes on 3 ml Percoll. The density of the Percoll was adjusted to between 1.081 and 1.091 g cm-3, depending on the maturity of the parasites. Centrifugation at 1500 g for 10 min produced a sharp band at the interface containing mature parasites with a purity averaging 86%. Washed parasites derived from the layer or pellet showed good viability in vitro.

Animals↗
PubMed1984

Metabolic labelling and characterisation of S-antigens, the heat-stable, strain-specific antigens of Plasmodium falciparum.

The C-10 clone of Plasmodium falciparum was metabolically labelled with [3H]glycine. Analysis by sodium dodecylsulphate/polyacrylamide gel electrophoresis (SDS/PAGE) revealed that S-antigens were the only significantly labelled products released into culture supernatants by the end of the asexual cycle. This result indicates conclusively that heat-stable, strain-specific antigens (the 'S-antigens') emanate from the parasite and not the host. The S-antigen of clone C-10 was resolved on SDS/PAGE as two labelled products with molecular weights of 156000 +/- 5000 and 130000 +/- 2000. The two components were differentially susceptible to proteolysis with trypsin. In other experiments, boiled plasma from Aotus monkeys infected with a different isolate of P. falciparum was partially purified by isoelectric focusing and the fraction containing S-antigen was iodinated by the Iodogen procedure. Analysis by SDS/PAGE of immunoprecipitated material revealed an iodinated component corresponding to the lower molecular weight band of the metabolically labelled S-antigen.

Animals↗
PubMed1984

A possible molecular basis for strain specific immunity to malaria.

A 250 kDa antigen implicated in the induction of protective immunity to Plasmodium chabaudi was examined with a panel of 11 monoclonal antibodies in cloned parasite lines. 2 antibodies cross-reacted with the different parasite lines while 9 were specific for one line. This antigenic diversity was correlated with major differences in one dimensional peptide maps between the purified antigen from different lines of parasites. The peptide maps also revealed some apparently conserved structure which may have been responsible for the antigenic cross reactivity. Using cloned lines of P. falciparum and a second series of monoclonal antibodies, similar antigenic and structural diversity was evident in the equivalent antigen from the important human pathogen. These findings are discussed with relationship to the induction of protective immunity to malaria.

Animals↗
PubMed1983

Intraerythrocytic development and antigenicity of Plasmodium falciparum and comparison with simian and rodent malaria parasites.

Using sorbitol-synchronised cultures and metabolic labelling with [35S]methionine, the stage specificity of polypeptides synthesised by the intraerythrocytic stages of Plasmodium falciparum was studied. We confirmed that the synthesis of many polypeptides is restricted to defined morphological stages of parasite development, while other polypeptides are synthesised more or less throughout the cycle. The synthesis of at least 6 polypeptides was confined to the period of differentiation of mature trophozoites to schizonts and merozoites. Polypeptides synthesised by a cloned long-term passage isolate were very similar to those of a recently cultured uncloned isolate. Comparison of polypeptides synthesized during differentiation of mature trophozoites to schizonts and merozoites by P. falciparum with those of P. chabaudi and P. knowlesi showed that while P. chabaudi and P. knowlesi synthesised a 250 000 molecular weight polypeptide at this stage the apparently equivalent polypeptide of P. falciparum was of significantly lower molecular weight being 200 000. Using a surface immunoprecipitation technique, it was shown that this 200 000 mol. wt. polypeptide was accessible to antibodies on the surface of erythrocytes infected with mature trophozoites and schizonts. A 150 000 mol. wt. polypeptide was also accessible to antibodies. By comparing polypeptides synthesised during the differentiation of mature trophozoites to schizonts and merozoites with those recovered in the ring stage parasites after schizogony and erythrocyte invasion, it was shown that this 200 000 mol. wt. polypeptide and 140 000 and 120 000 mol. wt. polypeptides were not taken into the erythrocyte by the invading merozoite. The importance of these polypeptides in terms of the parasite biology and in the induction and expression of immunity to malaria is discussed.

Animals↗
PubMed1983

A cytoplasmic requirement of red cells for invasion by malarial parasites.

Human red cells, when lysed by dialysis at high haematocrit against a medium of low ionic strength and then dialysed back to physiological saline at 37 degrees C, give rise to resealed ghosts that are invaded with high efficiency by Plasmodium falciparum parasites. When the haematocrit is reduced, a critical concentration is reached, such that the resealed ghosts no longer support invasion. This indicates that a constituent of the cytoplasm becomes diluted to a concentration below a critical level. This constituent is evidently ATP, for when extraneous ATP is added to the diluent and the dialysate, the susceptibility to invasion is fully restored. This does not occur when the non-hydrolysable analogue, adenylyl-imidodiphosphate (AMP-PNP) is substituted for ATP, whereas the hydrolysable ATP analogue, adenosine-5'-O-(3-thiotriphosphate) (ATP-gamma-S), which can be utilised by kinases, can partly replace ATP. Stimulation of invasion by the addition of 2,3-diphosphoglycerate was also associated with a perceptible rise in ATP concentration. The invasion process does not appear to involve intracellular calcium, for EGTA introduced into the resealed ghost has no detectable effect. Moreover, vanadate in the medium does not appreciably inhibit invasion, and it is thus unlikely that the requirement for ATP is linked to the activity of membrane ion-pump enzymes. An inhibitor of phosphorylation, adenosine, introduced into the cells at high concentration, causes significant inhibition of invasion. The results suggest that ATP is required for maintaining the turnover of phosphoryl groups of membrane-associated proteins, such as spectrin. A basic scheme for the mechanism of the invasion process is suggested. In addition to the effect of ATP, it is also shown that with greater dilution, and in the presence of ATP, there is an abrupt loss of susceptibility to invasion. It is inferred that this is due to the dilution of another essential cytoplasmic constituent to below a critical concentration. This second constituent has not yet been identified.

Adenosine↗
PubMed1983

Properties of red cell ghost preparations susceptible to invasion by malaria parasites.

Experimental procedures are described that lead to the formation of sealed red cell ghosts capable of efficient invasion by malaria parasites. Both human and monkey cells have been studied with respect to invasion by Plasmodium falciparum and P. knowlesi respectively. Resealed ghosts containing about half the normal concentration of haemoglobin are prepared by osmotic lysis and resealing at a haematocrit of 70%. When examined in the scanning electron microscope, populations of these ghosts contain few echinocytes, but have an abundance of stomatocytic forms. When undiluted haemolysate is substituted for the aqueous saline diluent, in which the cells are suspended for lysis and resealing, discocytes result, with a morphology very similar to that of the original cells. Invasion is somewhat, but not dramatically higher for this material. An investigation of the effect of intracellular potassium concentration revealed that this had no major effect on invasion. A small increase in invasion resulted from addition of 50 or 100 mg/ml of albumin to the medium in which the cells were resealed, but intra-erythrocytic protein content per se is evidently not a major factor in determining the efficiency of invasion. Augmentation of the cytoplasmic ATP concentration during lysis and resealing increased the level of parasitaemia significantly, and the parasites in these ghosts developed normally and gave rise to viable merozoites. It was found that albumin at certain concentrations passed freely into the lysed cells and reached equilibrium with that in the external medium. By contrast, the high molecular weight solute Blue Dextran 2000 was completely excluded from the lysed cells.

Adenosine Triphosphate↗
PubMed1983

Inhibition of malarial invasion of red cells by chemical and immunochemical linking of spectrin molecules.

The invasion of resealed human red cell ghosts by Plasmodium falciparum, and those from monkey cells by P. knowlesi, was strongly inhibited by anti-spectrin antibodies introduced into their cytoplasm. Univalent F(ab)1 fragments gave no such effect, but a combination of these fragments and goat anti-rabbit immunoglobulin, to restore bifunctionality, caused perceptible inhibition of invasion. Disulphide cross-links introduced between spectrin molecules in intact red cells by the membrane-permeant oxidizing agent, diamide, again led to inhibition of invasion. This effect was largely reversed by reduction of the cross-links. Gel electrophoresis was used to confirm that cross-linking was essentially confined to spectrin, and that extended covalent networks were not formed. It follows that local formation of bridges, whether by antibodies or oxidation of thiol groups, functions by inhibiting a local rearrangement of the cytoskeleton that forms a step in the invasion process.

Animals↗
PubMed1981

A compact semi-automated continuous cultivation system for Plasmodium falciparum.

A compact inexpensive semi-automated continuous cultivation system for Plasmodium falciparum is described. The system is simple to operate and allows cultures to proceed unattended for several days. A particularly useful feature is a portable cultivation module that is freely transferable from the incubator to a tissue culture cabinet for manual operations such as sampling or addition of red cells. Each cultivation module holds plastic flasks which can give a combined culture surface area of up to 500 cm2. Parasitaemias of 12% are attained routinely at red cell densities of 1 to 7 x 10(8) per ml, if the medium is changed daily.

Animals↗
PubMed1981

S-antigens and isozymes in strains of Plasmodium falciparum.

Evidence was found for the independent reassortment of parasite genes for S-antigens and isozymes. The Lagos and Palo Alto strains of Plasmodium falciparum had the same isozyme forms of PGD and LDH but different S-antigens. In contrast, the BW (Gambia) and Palo Alto strains had different isozyme forms of GDH but shared some S-antigens. From artificial mixtures of parasites with different isozymes of GDH and different S-antigens, clones were derived in vitro which had a single isozyme type of GDH and a single type of S-antigen.

Animals↗
PubMed1981

Detection of glucose-6-phosphate dehydrogenase in malarial parasites.

The ability to carry out the initial reaction of the pentose phosphate pathway was investigated with extracts of mouse erythrocytes infected with Plasmodium chabaudi, purified merozoites of P. knowlesi, and schizonts of P. falciparum grown in vitro in human erythrocytes. Glucose-6-phosphate dehydrogenase activity (G-6pd) was detected in extracts of all the cells after electrophoresis on polyacrylamide gels. Separate host cell and parasite glucose-6-phosphate dehydrogenase activities were demonstrated with extracts of P. knowlesi and P. falciparum but not with P. chabaudi.

Animals↗
PubMed1981

Entry of malaria parasites into resealed ghosts of human and simian erythrocytes.

Whereas "white' ghosts, conventionally prepared by hypotonic lysis and washing are resistant to invasion by malaria parasites, cells lysed at high haematocrit and resealed without separating the membranes from the cytosol are invaded with high efficiency (not less than one-third the frequency of intact cells). Similar results were obtained with human and monkey cells, exposed respectively to Plasmodium falciparum and P. knowlesi. Microspectrophotometry of individual cells was used to ascertain the the susceptibility to invasion of lysed, resealed cells was not related to its haemoglobin content.

Animals↗