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G H Mitchell

Publications and source records attributed to G H Mitchell.

At least 19 recordsLinked to original sources
PubMed2004

Correlation of structural development and differential expression of invasion-related molecules in schizonts of Plasmodium falciparum.

During asexual development Plasmodium schizonts undergo a series of complex biochemical and structural changes. Using tightly synchronized cultures of 2 P. falciparum lines (clone C10 and strain ITO4) for light microscopy and fluorescence imaging we monitored the timing and sequence of expression of proteins associated with invasion-related organelles. Antibodies to rhoptry, micronemal and dense granule proteins (Rhoptry Associated Protein 1, Apical Membrane Antigen 1, Erythrocyte Binding Antigen 175, Ring-infected Erythrocyte Surface Antigen) and to pellicle-associated proteins (Merozoite Surface Protein 1, PfMyosin-A) were used. Clone C10 developed faster than ITO4; this difference was also found in the timing of protein expression seen by immunofluorescence. Light microscopic data were combined with transmission electron microscopic analysis using serial sectioning of ITO4 schizonts to determine nuclear number and organellar development. Thus a timetable of schizont structural maturation was established. Generally, the timing of organelle-specific antigen expression correlates well with the ultrastructural data. Rhoptries are formed mainly between second and fourth nuclear divisions, micronemes between the end of the fourth nuclear division and merozoite separation from the residual body, while dense granules are generated mainly after the micronemes. PfAMA-1 appears in micronemes before EBA-175, suggesting micronemal heterogeneity.

Animals↗
PubMed2004

Apical membrane antigen 1, a major malaria vaccine candidate, mediates the close attachment of invasive merozoites to host red blood cells.

Apical membrane antigen 1 (AMA-1) of Plasmodium merozoites is established as a candidate molecule for inclusion in a human malaria vaccine and is strongly conserved in the genus. We have investigated its function in merozoite invasion by incubating Plasmodium knowlesi merozoites with red cells in the presence of a previously described rat monoclonal antibody (MAb R31C2) raised against an invasion-inhibitory epitope of P. knowlesi AMA-1 and then fixing the material for ultrastructural analysis. We have found that the random, initial, long-range (12 nm) contact between merozoites and red cells occurs normally in the presence of the antibody, showing that AMA-1 plays no part in this stage of attachment. Instead, inhibited merozoites fail to reorientate, so they do not bring their apices to bear on the red cell surface and do not make close junctional apical contact. We conclude that AMA-1 may be directly responsible for reorientation or that the molecule may initiate the junctional contact, which is then presumably dependent on Duffy binding proteins for its completion.

Animals↗
PubMed2001

Microtubule associated motor proteins of Plasmodium falciparum merozoites.

We have studied the occurrence, stage specificity and cellular location of key molecules associated with microtubules in Plasmodium falciparum merozoites. Antibodies to gamma tubulin, conventional kinesin and cytoplasmic dynein were used to determine the polarity of merozoite microtubules (mt), the stage specificity of the motor proteins and their location during merozoite development. We conclude that the minus ends of the mts are located at their apical pole. Kinesin was present throughout the lifecycle, appearing as a distinct crescent at the apex of developing merozoites. The vast majority of cytoplasmic dynein reactivity occurred in late merogony, also appearing at the merozoite apex. Destruction of mt with dinitroanilines did not affect the cellular location of kinesin or dynein. In invasion assays, dynein inhibitors reduced the number of ring stage parasites. Our results show that both conventional kinesin and cytoplasmic dynein are abundant, located at the negative pole of the merozoite mt and, intriguingly, appear there only in very late merogony, prior to merozoite release and invasion.

Animals↗
PubMed2000

Motile systems in malaria merozoites: how is the red blood cell invaded?

The ability of the malaria parasite to invade erythrocytes is central to the disease process, but is not thoroughly understood. In particular, little attention has been paid to the motor systems driving invasion. Here, Jennifer Pinder, Ruth Fowler and colleagues review motility in the merozoite. The components of an actomyosin motor are present, including a novel unconventional class XIV myosin, now called Pfmyo-A, which, because of its time of synthesis and location, is likely to generate the force required for invasion. In addition, there is a subpellicular microtubule assemblage in falciparum merozoites, the f-MAST, the integrity of which is necessary for invasion.

Animals↗
PubMed2000

A brief illustrated guide to the ultrastructure of Plasmodium falciparum asexual blood stages.

Interpretation of the new information arising from the Plasmodium falciparum Genome Project requires a good working knowledge of the ultrastructure of the parasite; however many aspects of the morphology of this species remain obscure. Lawrence Bannister, John Hopkins and colleagues here give an illustrated overview of the three-dimensional (3-D) organization of the merozoite, ring, trophozoite and schizont stages of the parasite, based on available data that include 3-D reconstruc-tion from serial electron microscope sections. The review describes the chief organelles present in these stages, emphasizing the continuity of structure in addition to specialized, stage-specific features developed during the asexual erythrocytic cycle.

Animals↗
PubMed2000

Ultrastructure of rhoptry development in Plasmodium falciparum erythrocytic schizonts.

Prior to the separation of merozoites from the Plasmodium falciparum schizont, various stage-specific organelles are synthesized and assembled within each merozoite bud. The apical ends of the merozoites are initiated close to the ends of endomitotic spindles. At each of these sites, the nuclear membrane forms coated vesicles, and a single discoidal or cup-like Golgi cisterna appears. Reconstruction from serial sections indicates that this structure receives vesicles from the nuclear envelope and in turn gives off coated vesicles to generate the apical secretory organelles. Rhoptries first form as spheroidal structures and grow by progressive fusion of small vesicles around their margins. As each rhoptry develops, 2 distinctive regions separate within it, an apical reticular zone with electron-lucent areas separated by cords of granular material, and a more homogenously granular basal region. The apical part elongates into the duct, with evidence for further vesicular fusion at the duct apex. The rounded rhoptry base becomes progressively more densely packed to form a spheroidal mass, and compaction also occurs in the duct. Typically, one rhoptry matures before the other. Cryofractured rhoptry membranes show asymmetry in the sizes and numbers of intramembranous particles at the internally- and externally-directed fracture faces.

Animals↗
PubMed1998

Microtubules in Plasmodium falciparum merozoites and their importance for invasion of erythrocytes.

Plasmodium falciparum merozoites have an array of 2-3 subpellicular microtubules, designated f-MAST. We have previously shown that colchicine inhibits merozoite invasion of erythrocytes, indicating a microtubular involvement in this process. Colchicine inhibition of invasion was reduced by the Taxol-stabilization of merozoite microtubules prior to colchicine exposure. Immunofluorescence assays showed that the number and length of f-MASTs were reduced in colchicine-treated merozoites, confirming that microtubules were the target of colchicine inhibition. The dinitroaniline drugs, trifluralin and pendimethalin, were shown by immunofluorescence to depolymerize the f-MAST and both drugs were inhibitory in invasion assays. These results demonstrate that the integrity of the f-MAST is important for successful invasion. Fluorescence imaging demonstrated the alignment of mitochondria to f-MAST, suggesting that mitochondrial transport might be perturbed in merozoites with disorganized f-MAST. Depolymerizing mt in late-stage schizonts did not affect the allocation of mitochondria to merozoites.

Aniline Compounds↗
PubMed1998

Actomyosin motor in the merozoite of the malaria parasite, Plasmodium falciparum: implications for red cell invasion.

The genome of the malaria parasite, Plasmodium falciparum, contains a myosin gene sequence, which bears a close homology to one of the myosin genes found in another apicomplexan parasite, Toxoplasma gondii. A polyclonal antibody was generated against an expressed polypeptide of molecular mass 27,000, based on part of the deduced sequence of this myosin. The antibody reacted with the cognate antigen and with a component of the total parasite protein on immunoblots, but not with vertebrate striated or smooth muscle myosins. It did, however, recognise two components in the cellular protein of Toxoplasma gondii. The antibody was used to investigate stage-specificity of expression of the myosin (here designated Pf-myo1) in P. falciparum. The results showed that the protein is synthesised in mature schizonts and is present in merozoites, but vanishes after the parasite enters the red cell. Pf-myo1 was found to be largely, though not entirely, associated with the particulate parasite cell fraction and is thus presumably mainly membrane bound. It was not solubilised by media that would be expected to dissociate actomyosin or myosin filaments, or by non-ionic detergent. Immunofluorescence revealed that in the merozoite and mature schizont Pf-myo1 is predominantly located around the periphery of the cell. Immuno-gold electron microscopy also showed the presence of the myosin around almost the entire parasite periphery, and especially in the region surrounding the apical prominence. Labelling was concentrated under the plasma membrane but was not seen in the apical prominence itself. This suggests that Pf-myo1 is associated with the plasma membrane or with the outer membrane of the subplasmalemmal cisterna, which forms a lining to the plasma membrane, with a gap at the apical prominence. The results lead to a conjectural model of the invasion mechanism.

Actomyosin↗
PubMed1997

A role for microtubules in Plasmodium falciparum merozoite invasion.

Colchicine, a drug which poisons the polymerization of microtubules, was assayed for effects on the invasion of Plasmodium falciparum merozoites into red cells in order to investigate if merozoite microtubules have a function in invasion. Culture conditions and concentrations of colchicine were established where the maturation and rupture of schizonts was unaffected by the drug. This was judged first by light microscopy, including morphology and counts of nuclear particle numbers, then by ultrastructural studies which excluded deranged organellogenesis as a cause of merozoite failure, and finally by diachronic cultures in which both recruitment and loss of schizonts could be counted. Specific invasion inhibition was seen when 10 microM-1 mM colchicine was present. Red cells pre-incubated in colchicine and then washed showed no reduction in their extent of invasion, and neither red cell lysis, sphering nor blebbing were apparent. We conclude that intact microtubules are necessary for successful merozoite function.

Animals↗
PubMed1996

Contractile protein system in the asexual stages of the malaria parasite Plasmodium falciparum.

F-actin was detected in asexual-stage Plasmodium falciparum parasites by fluorescence microscopy of blood films stained with fluorescent phalloidin derivatives. F-actin was present at all stages of development and appeared diffusely distributed in trophic parasites, but merozoites stained strongly at the poles and peripheries. No filament bundles could be discerned. A similar distribution was obtained by immunofluorescence with 2 polyclonal anti-actin antibodies, one of which was directed against a peptide sequence present only in parasite actin (as inferred from the DNA sequence of the gene). A monoclonal anti-actin antibody stained very mature or rupturing schizonts but not immature parasites. Myosin was identified in immunoblots of parasite protein extracts by several monoclonal anti-skeletal muscle myosin antibodies, as well as by a polyclonal antiserum directed against a consensus conserved myosin sequence (IQ motif). The identity of the polypeptides recognised by these antibodies was confirmed by overlaying blots with biotinylated F-actin. The antiserum and one of the monoclonal antibodies were used in immunofluorescence studies and were found to stain all blood-stage parasites, with maximal intensity towards the poles of merozoites. Our results are consistent with the presence of an actomyosin motor system in the blood-stage malaria parasite.

Actins↗
PubMed1995

The role of the cytoskeleton in Plasmodium falciparum merozoite biology: an electron-microscopic view.

The biochemical, ultrastructural and experimental data concerning the organization and biological roles of the merozoite cytoskeleton are briefly reviewed. Actin is known to be expressed in the asexual erythrocytic stages, and has also been demonstrated in Plasmodium falciparum merozoites biochemically and visualized by fluorescence microscopy after appropriate labelling. Experimental evidence indicates that actin-myosin-based motility is important in merozoite locomotion during red-cell invasion. Microtubules also occur in P. falciparum merozoites in the form of a small longitudinal band of subpellicular microtubules, and experiments with anti-microtubule drugs indicate that microtubules are involved in some aspect of invasion. In the late-stage schizont, microtubules are also important in merozoite morphogenesis. The numbers and positions of the merozoite apices within the schizont are spatially related to the spindle poles of the final mitotic division, and extranuclear microtubules are probably responsible for the trafficking of vesicles from a single Golgi cisterna to form the apical organelles. In addition to these cytoskeletal structures, numerous short cytoskeletal filaments of unknown composition attach the merozoite plasma membrane to the underlying pellicular cisterna, and this process may drive the budding of merozoites from the parent schizont.

Actins↗
PubMed1994

Aspects of immunity for the AMA-1 family of molecules in humans and non-human primates malarias.

The apical membrane antigen (AMA-1) family of malaria merozoite proteins is characterised by a high degree of inter-species conservation. Evidence that the protein (PK66/AMA-1) from the simian parasite Plasmodium knowlesi was protective in rhesus monkeys suggested that the 83kDa P. falciparum equivalent (PF83/AMA-1) should be investigated for protective effects in humans. Here we briefly review pertinent comparative data, and describe the use of an eukaryotic full length recombinant PF83/AMA-1 molecule to develop a sensitive ELISA for the determination of serological responses in endemic populations. The assay has revealed surprisingly high levels of humoral response to this quantitatively minor antigen. We also show that PK66/AMA-1 inhibitory mAb's are active against merozoites subsequent to release from schizont-infected red cells, further implicating AMA-1 molecules in red cell invasion.

Animals↗
PubMed1992

Origins of the parasitophorous vacuole membrane of the malaria parasite, Plasmodium falciparum, in human red blood cells.

We have attempted to determine whether the parasitophorous vacuole membrane, in which the malaria parasite (merozoite) encapsulates itself when it enters a red blood cell, is derived from the host cell plasma membrane, as the appearance of the invasion process in the electron microscope has been taken to suggest, or from lipid material stored in the merozoite. We have incorporated into the red cell membrane a haptenic phospholipid, phosphatidylethanolamine, containing an NBD (N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)) group, substituted in the acyl chain, and allowed it to translocate into the inner bilayer leaflet. After invasion of these labelled cells by the parasite, Plasmodium falciparum, immuno-gold electron microscopy was used to follow the distribution of the labelled lipid; this was found to be overwhelmingly in favour of the host cell membrane relative to the parasitophorous vacuole. Merozoites of P. knowlesi were allowed to attach irreversibly to red cells without invasion, using the method of pretreatment with cytochalasin. The region of contact between the merozoite and the host cell membrane was in all cases devoid of the labelled phosphatidylethanolamine. These results lead us to infer that the parasitophorous vacuole membrane is derived wholly or partly from lipid preexisting in the merozoite.

Animals↗