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Dive into the research topics where W. B. Gratzer is active.

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Featured researches published by W. B. Gratzer.


Experimental Parasitology | 1986

Plasmodium falciparum: Protease inhibitors and inhibition of erythrocyte invasion

A. R. Dluzewski; K. Rangachari; R.J.M. Wilson; W. B. Gratzer

Invasion of human red blood cells by Plasmodium falciparum is inhibited by the protease inhibitors, leupeptin and chymostatin. The efficacy of chymostatin was reduced if the cells were first treated with chymotrypsin. On the other hand, exposure of fresh cells to the supernatant from a synchronous culture at the reinvasion stage showed no such effect. This suggests that a proteolytic step occurs in the course of invasion and may be confined to the region of contact between the invading parasite and the erythrocyte. To test this, leupeptin or chymostatin was introduced into lysed cells, which were then resealed. The intracellular inhibitor strongly reduced invasion. Leupeptin also caused a striking effect on the development of the trophozoite stage of the parasites: a massive vacuole, apparently containing undigested haemoglobin, developed within the parasite. This did not totally stop development and the vacuolated parasites could be recovered in relatively pure form by lysis of the parasitised host cells with saponin.


Journal of Molecular Biology | 1992

Basis of unique red cell membrane properties in hereditary ovalocytosis

Ann E. Schofield; Michael J. A. Tanner; Jennifer C. Pinder; Barbara Clough; Peter M. Bayley; Gerard B. Nash; A.R. Dluzewski; David M. Reardon; T.M. Cox; R.J.M. Wilson; W. B. Gratzer

Hereditary ovalocytes from a Mauritian subject are extremely rigid, with a shear elastic modulus about three times that of normal cells, and have increased resistance to invasion by the malaria parasite Plasmodium falciparum in vitro. The genetic anomaly resides in band 3; the protein gives rise to chymotryptic fragments with reduced mobility in SDS/polyacrylamide gel electrophoresis, but this is a result of anomalous binding of SDS and not a higher molecular weight. Analysis of the band 3 gene reveals (1) a point mutation (Lys56----Glu), which also occurs in a common asymptomatic band 3 (Memphis) variant and governs the electrophoretic properties, and (2) a deletion of nine amino acid residues, including a proline residue, encompassing the interface between the membrane-associated and the N-terminal cytoplasmic domains. The interaction of the mutant band 3 with ankyrin appears unperturbed. The fraction of band 3 capable of undergoing translation diffusion in the membrane is greatly reduced in the ovalocytes. Cells containing the asymptomatic band 3 variant were normal with respect to all the properties that we have studied. Possible mechanisms by which a structural change in band 3 at the membrane interface could regulate rigidity are examined.


Nature | 1977

Control of interaction of spectrin and actin by phosphorylation.

Jennifer C. Pinder; D. Bray; W. B. Gratzer

SPECTRIN is a high molecular weight protein located on the cytoplasmic surface of the mammalian erythrocyte membrane, from which it may be readily liberated by extraction with solutions of low ionic strength. It is thought to be a major structural element of the cell and to play a critical part in maintaining its discoid shape and characteristic viscoelastic properties. Birchmeier and Singer1 have shown that changes in the shape of red cell ghosts are associated with the phosphorylation of a single serine residue on one of the two spectrin subunits, and have suggested that this might provide a basis for the well-known control of red cell shape by ATP2. The erythrocyte membrane also contains actin, which is present in approximately equimolar proportions to the spectrin dimer (molecular weight 500,000 (ref. 3)). We have shown previously4 that there is a specific interaction between these two proteins, which reveals itself in the ability of spectrin to provoke the polymerisation of muscle actin in vitro. We show here that this effect depends on the phosphorylation of spectrin. Furthermore, in an undis-persed mixture of spectrin and its cognate actin such phosphorylation causes the formation of a gel. The striking parallel between these results and the previously demonstrated effects of phosphorylation in situ suggests that the shape of the cell is controlled primarily by the actin–spectrin complex.


Nature | 1986

Control of malarial invasion by phosphorylation of the host cell membrane cytoskeleton

K. Rangachari; A. Dluzewski; R. J. M. Wilson; W. B. Gratzer

It has been shown that the entry of the malaria parasite into the red blood cell requires the presence of ATP in the host cell cytoplasm1,2. In red blood cell ghosts that contain no ATP the receptor on the extracellular surface remains in place and parasites will bind to the membrane, but will not enter3. ATP is thus necessary for one of the steps4 in the invasion sequence that follows recognition and attachment. The process of entry appears to involve the active participation of the host cell membrane cytoskeleton5. We have suggested2 that the function of the intracellular ATP may be to regulate phosphorylation of the cytoskeleton. We now present evidence that the activity of the membrane-associated cyclic AMP-independent kinase of the red blood cell is inseparable from invasion; the active substrate may be spectrin.


Molecular and Biochemical Parasitology | 1983

A cytoplasmic requirement of red cells for invasion by malarial parasites

Anton R. Dluzewski; K. Rangachari; R.J.M. Wilson; W. B. Gratzer

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.


Parasitology | 1985

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

A. R. Dluzewski; K. Rangachari; R.J.M. Wilson; W. B. Gratzer

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.


Molecular and Biochemical Parasitology | 1995

Mechanism of regulation of malarial invasion by extraerythrocytic ligands

Barbara Clough; Manrico Paulitschke; Gerard B. Nash; Peter M. Bayley; David J. Anstee; R.J.M. Wilson; Geoffrey Pasvol; W. B. Gratzer

Invasion of red cells by Plasmodium falciparum in vitro was inhibited by a range of extracellular ligands, none of which block the major receptors for merozoites. Most effective, in terms of dose response, were two monoclonal antibodies against the Wrb antigen on glycophorin A; wheat germ agglutinin which also binds to glycophorin, and an anti-band 3 monoclonal antibody, caused inhibition of invasion at higher levels of saturation, while concanavalin A, which binds to band 3, was without effect. All the ligands except concanavalin A, increased the rigidity of the host cell membrane. The anti-Wrb antibodies generated the highest dose response effect, but no correlation between invasion and shear elastic modulus of the membrane could be established. All ligands, with the exception of concanavalin A, caused a reduction in the translationally mobile fractions of band 3 and glycophorin, as revealed by fluorescence recovery after photobleaching (FRAP). Invasion diminished with loss of mobile band 3, engendered by bound wheat germ agglutinin or anti-band 3, falling precipitately when the mobile fraction fell below 40% of that in unperturbed membranes. Both anti-Wrb antibodies suppressed invasion completely at concentrations insufficient to affect significantly either membrane rigidity or intramembrane protein diffusion. A univalent anti-glycophorin A (Fab) fragment, the parent antibody of which was previously shown to inhibit invasion strongly, had only a modest effect on invasion and induced a correspondingly small change in the mobile fraction of band 3.(ABSTRACT TRUNCATED AT 250 WORDS)


British Journal of Haematology | 1981

Entry of Malaria Parasites into Resealed Ghosts of Human and Simian Erythrocytes

A. R. Dluzewski; K. Rangachari; R.J.M. Wilson; W. B. Gratzer

Summary. 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 that the susceptibility to invasion of lysed, resealed cells was not related to its haemoglobin content.


British Journal of Haematology | 1986

Prenatal diagnosis of hereditary red cell membrane defect

S. A. Morris; V. Ohanian; M. L. Lewis; S. B. Chahwala; C. H. Rodeck; R. S. Mibashan; W. B. Gratzer

Summary The red cells of a severely anaemic 2‐year‐old child of a white British family showed high haemolytic fragility with poikilocytosis. The cells showed markedly impaired thermal stability. The mother was phenotypically normal, but the fathers red cells showed mild elliptocytosis. The spectrin from the latter, extracted at low temperature, was 30% dimeric (cf. 5–10% in normals). Tryptic digests of the spectrin from both father and daughter showed a reduction in the fragment of 80 000 molecular weight, derived from the terminus of the α‐chain, and the elevation of a fragment of molecular weight 46 000, as well as one of 53 000. These characteristics and the autosomal recessive inheritance lead to a diagnosis of type II hereditary pyropoikilocytosis, so far reported only in two black American families (Lawler et al, 1983). The spectrin from the father was examined with respect to thermal conformational stability, and was found to be normal. The spectrin from the cells of the daughter gave evidence of the presence of oxidative (disulphide) cross‐links, as well as of extensive noncovalent aggregation. Blood was obtained from the umbilical cord vein of the 19‐week fetus of the pregnant mother: 250 μl of blood was used for preparation of red cell membranes for SDS‐gel electrophoresis and for extraction of spectrin. Analysis of the spectrin by gel electrophoresis in the native state revealed that the proportion of dimer was within the normal range, and the fetus therefore did not possess the hereditary pyropoikilocytosis phenotype. It is suggested that the procedures described could be generally applied to the prenatal identification of phenotypes associated with severe haemolytic anaemias.


Parasitology | 1986

Inhibition of malarial invasion by intracellular antibodies against intrinsic membrane proteins in the red cell.

A. R. Dluzewski; K. Rangachari; M. J. A. Tanner; D. J. Anstee; R.J.M. Wilson; W. B. Gratzer

It has previously been shown that antibodies against the transmembrane proteins, band 3 and glycophorin A, inhibit entry of the merozoite into the red cell and, in the case of band 3, it was established that attachment of the parasite to the cell is not prevented. We have found that antibodies against the cytoplasmic domains of band 3 and of glycophorin A, when present in the interior of resealed ghosts of human red cells, also inhibit invasion by P. falciparum. It is inferred that attachment of the merozoite to the red cell causes structural effects that are transduced to the membrane cytoskeleton and the antibodies against transmembrane proteins interfere with the invasion sequence at this level.

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K. Rangachari

Medical Research Council

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D. Bray

Medical Research Council

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Gerard B. Nash

University of Birmingham

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D. Z. Staynov

Bulgarian Academy of Sciences

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A. Pekrun

Medical Research Council

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A.R. Dluzewski

Medical Research Council

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