The malaria merozoite, forty years on.

The invasive blood stage of malaria parasites, merozoites, are complex entities specialized for the capture and entry of red blood cells. Their potential for vaccination and other anti-malaria strategies have attracted much research attention over the last 40 years, and there is now a considerable body of data relating to their biology. In this article some of the major advances over this period and remaining challenges are reviewed.

Studies on the receptors to 5alpha-androst-16-en-3-one and 5alpha-androst-16-en-3alpha-ol in sow nasal mucosa.

The presence of receptors to the "boar taint" pheromones 5alpha-androst-16-en-3-one and 5alpha-androst-16-en-3alpha-ol has been demonstrated in sow olfactory mucosa. Binding studies indicated that a sufficiently low concentration of olfactory tissue homogenate exhibited saturation of binding of 5alpha-androst-16-en-3-one, and this was of high affinity compared with control tissues of non-olfactory and heated olfactory tissues. Analysis of receptor binding of 5alpha-androst-16-en-3-one gave a value for the affinity constant (Ka) of approx. 8.3-10(8) M-1 and the value for the molar concentration of binding sites (n[M]) was approx. 3.3 pmol/mg protein. Almost identical values of Ka and n [M] were obtained when receptor binding of 5alpha-[5alpha-3H]androst-16-en-3alpha-ol was investigated (Ka 8.4-10(8) M-1; n [M] 3.7 pmol/mg protein). This suggests that the same receptor binds both 5alpha-androst-16-en-3-one and 5alpha-androst-16-en-3alpha-ol with equally high affinity. In a preliminary investigation to establish the specificity of the receptor, the binding of 17beta-hydroxy-5alpha-androstan-3-one was assayed; this steroid is odourless but has a similar structure except in ring D to 5alpha-androst-16-en-3-one. Binding was of the low affinity, non-specific type only, indicating that the sow olfactory receptors are not sensitive to this androgen.

Malaria parasite invasion: interactions with the red cell membrane.

The capacity to invade red cells is central to the biology of malaria parasites; both asexual multiplication and reinfection of the definitive mosquito host depend upon intraerythrocytic stages. The invasion process is complex. The briefly free merozoite specifically recognizes and adheres to ligands on the red cell surface, then alters the red cell membrane to produce an invagination into which it moves, and so becomes enclosed in a membrane-bound parasitophorous vacuole. Here we assess new evidence that bears on our understanding of this process. This has come from sources including biochemical and ultrastructural studies of the specialized surface and organelles of merozoites, from in vitro invasion studies using naturally refractory or artificially modified red cells, and from structural, chemical, and immunological analyses of the newly parasitized cell.

Apical organelles of Apicomplexa: biology and isolation by subcellular fractionation.

The apical organelles are characteristic secretory vesicles of Plasmodium, Toxoplasma, Cryptosporidium and other apicomplexan organisms. They consist of rhoptries, micronemes and dense granules. Recent research has provided much new data concerning their structure, contents, functions and development. All of these organelles contain complex mixtures of proteins, with broad homologies as well as differences in molecular structure between species and genera. Many of the proteins interact with host cell membranes, and are thought to mediate selective adhesion to host cells as well as membrane modification during intracellular invasion. Micronemal proteins are important in the initial selection of host cells, and in enabling gliding motility of the parasites, while rhoptries appear to be more important in parasitophorous vacuole formation. Dense granules are involved predominantly in modifying the host cell after invasion. Research into apical organellar composition and function depends on accurate assignment of molecular identity. This requires the simultaneous application of several complementary approaches including immunolocalisation by light- and electron-microscopy, subcellular fractionation, and transgene expression. The merits and limitations of these different types of approach are discussed, and the importance of cell fractionation methods in characterising apical organelle proteins is stressed.

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.

Endocytic pathways in the olfactory and vomeronasal epithelia of the mouse: ultrastructure and uptake of tracers.

Mammalian olfactory neurons possess a well-developed system of endocytic vesicles, endosomes, and lysosomes in their dendrites and perikarya. Vomeronasal neurons are similar and also contain much perikaryal agranular endoplasmic reticulum (AER). Olfactory supporting cells contain endocytic vesicles and endosomes associated closely with abundant fenestrated AER, and vesicles and numerous large dense vacuoles are present basally. Vomeronasal supporting cells have little AER, and few dense vacuoles occur in their bases. In olfactory neurons, ultrastructural tracers (0.08% horseradish peroxidase, thorium dioxide, ferritin) are endocytosed by olfactory receptor endings and transported to the cell body, where their movement is halted in lysosomes. Higher concentrations (1%) of horseradish peroxidase penetrate olfactory receptor plasma membranes and intercellular junctions. In olfactory supporting cells, endocytosed tracers pass through endosomes to accumulate in dense basal vacuoles. These observations indicate that olfactory sensory membranes are rapidly cycled and that endocytosed materials are trapped within the epithelium. It is proposed that in the olfactory epithelium, endocytosis presents redundant odorants to the enzymes of the supporting cell AER to prevent their accumulation, whereas in the vomeronasal epithelium the receptor cells carry out this activity.

Effects of unilateral deafening on the cochlear nucleus of the guinea pig at different ages.

Developmental changes in spherical cell sizes were measured in the ventral cochlear nucleus (VCN) in normal guinea pigs aged 2 days and 2, 7 and 12 weeks to establish the time course of postnatal neuronal growth, as a baseline for our experimental work. A continued growth of spherical cell size was observed in the VCN up to 7 weeks postnatally. Animals were unilaterally deafened by cochlear perfusion with kanamycin sulphate at ages 1 and 6 weeks. After 6 weeks survival the spherical cells were measured in the VCNs of both sides. Unilateral deafferentation at both ages caused an ipsilateral reduction in spherical cell size, neurons of the younger group being smaller than in the older. On the contralateral side these cells in the older group were larger than age-matched controls while in the younger group there was no difference compared with age-matched controls. These findings suggest that the results of deafferentation are age-dependent, and may indicate an ability of the neural circuitry to adapt to the loss of sensory input on the other side.

Structural aspects of Plasmodium relevant to vaccination against malaria.

The extracellular stages of Plasmodium which can be inactivated with antibodies include the sporozoites, merozoites and microgametes. The surface of sporozoites and merozoites are known to bind antibodies which render them unable to invade host cells. Intra-erythrocytic merozoites, trophozoites and schizonts release antigens which are conveyed to the surface of the host cell, although they do not appear to be attacked by antibodies directly. The significance of these observations is discussed.

Structural effects of short term and chronic extracochlear electrical stimulation on the guinea pig spiral organ.

To assess the effects of extracochlear electrical stimulation on cochlear structure, guinea pigs were implanted and stimulated with single middle ear electrodes either at round window or promontory sites, and their cochleae examined by transmission electron microscopy. Implanted but unstimulated, or unimplanted control animals were examined in the same way. Alternating current stimulation at the promontory for 2 h at 150 Hz, 500 microA, caused outer hair cell efferent endings to become dense and vacuolated, but no hair cells were damaged. With direct current stimulation at 500 microA for 2 h the basal regions of the stimulated cochlea were badly damaged and many outer hair cells lysed. Long term (up to 1200 h) round window stimulation at 100 or 141 Hz, 15-91 microA rms, did not cause cell death or inner hair cell damage, but basal outer hair cells and their efferent endings were badly affected in both ipsilateral and contralateral cochleae. The compound action potential of the auditory evoked response to broad band click stimuli was not altered by chronic electrical stimulation. It is concluded that chronic stimulation with the parameters used does not threaten cochlear survival, and it is proposed that the bilateral structural changes induced by chronic stimulation are caused by excessive activation of the cochlear efferent pathways.

Further studies of the effects of continuous white noise of moderate intensity (70--80 dB SPL) on the cochlea in young guinea pigs. Time course and distribution of hair cell degeneration.

Guinea pigs aged one week were exposed to white noise at a maximum of 76 dB SPL for 7 days and were then killed 3, 8 and 16 weeks later for histological examination of the cochlea by the surface preparation method. Appreciable increases in outer hair cell losses were observed in the apical turn 3/3 1/2, chiefly in the outer two rows, between the 3rd and 8th week, but not between the 8th and 16th week. No significant losses were seen in control groups corresponding to 3- and 8-week periods, although in the control group of 16 weeks' survival, small deficits, attributable to natural ageing, were seen in the apical half-turn, 3 1/2.

The effects of combined gentamicin and white noise on the spiral organ of young guinea pigs. A structural study.

The present work describes the effects on the spiral organ of very young guinea pigs, of continuous white noise (76 dB SPL, 7 days) combined with gentamicin, using surface preparations and both transmission and scanning electron microscopy to analyse structural changes. Gentamicin only, at 80 mg/kg/day for 5 days did not cause any perceptible loss of hair cells, and only minimal structural changes. When combined with 7 days of white noise, there was a widespread effect on outer hair cells throughout the cochlea, some changes being similar to those induced by sound alone, others being seen only in the combined experiments. Outer hair cell losses with sound and gentamicin treatment were highly variable, but in addition to apical damage, also seen in sound-treated animals, a more basal area of cell loss appeared in the spiral organ.

Vesicle trafficking during sporozoite development in Plasmodium berghei: ultrastructural evidence for a novel trafficking mechanism.

Oocysts from Anopheles stephensi mosquitoes fed on murine blood infected with Plasmodium berghei berghei, were fixed for electron microscopy 6-12 days post-feeding. Ultrastructural analysis focused on Golgi-related trafficking pathways for rhoptry and microneme formation during sporogony. A small Golgi complex of 1-3 cisternae is formed close to the spindle pole body from coated vesicles budded from the nuclear envelope which is confluent with the endoplasmic reticulum. Rhoptries begin as small spheroidal bodies apparently formed by fusion of Golgi-derived vesicles, lengthening to 3-4 microm, and increasing in number to 4 per sporozoite. Ultrastructural data indicate the presence of a novel mechanism for vesicle transport between the Golgi complex and rhoptries along a longitudinal 30 nm - thick fibre (rootlet fibre or tigelle). Filamentous links between vesicles and rootlet indicate that this is a previously undescribed vesicle transport organelle. Genesis of micronemes occurs late in bud maturation and starts as spheroidal dense-cored vesicles (pro-micronemes), transforming to their mature bottle-like shape as they move apically. Filamentous links also occur between micronemes and subpellicular microtubules, indicating that as in merozoites, micronemes are trafficked actively along these structures.

The interactions of intracellular Protista and their host cells, with special reference to heterotrophic organisms.

Intracellular genera are found in all the major groups of Protista, but are particularly common among the dinoflagellates, trypanosomatid zooflagellates and suctorian ciliates; the Sporozoa are nearly all intracellular at some stage of their life, and the Microspora entirely so. Intracellular forms can dwell in the nucleus, within phagosomal or other vacuoles or may lie free in the hyaloplasm of their host cells. Organisms tend to select their hosts from a restricted taxonomic range although there are some notable exceptions. There is also great variation in the types of host cell inhabited. There are various reasons for both host and cell selectivity including recognition phenomena at the cell surfaces. Invasion of host cells is usually preceded by surface interactions with the invader. Some organisms depend upon phagocytosis for entry, but others induce host cells to engulf them by non-phagocytic means or invade by microinjection through the host plasma membrane. Protista avoid lysosomal destruction by their resistance to enzyme attack, by surrounding themselves with lysosome-inhibiting vacuoles, by escaping from the phagosomal system into the hyaloplasm and by choosing host cells which lack lysosomes. Nutrition of intracellular heterotrophic organisms involves some degree of competition with the host cell's metabolism as well as erosion of host cell cytoplasm. In Plasmodium infections, red cells are made more permeable to required nutrients by the action of the parasite on the host cell membrane. The parasite is often dependent upon the host cell for complex nutrients which it cannot synthesize for itself. Intracellular forms often profoundly modify the structure and metabolism of the host cell or interfere with its growth and multiplication. This may result in the final lysis of the host cell at the end of the intracellular phase or before the infection of other cells. Certain types of intracellular organisms may have arisen initially as forms attached to the cell surface of digestive or other organs, but the intracellular habit appears to have arisen independently in several groups of Protista.

Lipidic vacuoles in Plasmodium knowlesi erythrocytic schizonts.

Electron microscopy of schizont development in erythrocytic Plasmodium knowlesi has revealed that spheroidal vacuoles 250 nm in diameter with semi-dense contents appear at the periphery of the parasite prior to the budding of merozoites. When treated with non-polar solvents, their contents are completely extracted, and after fixation in tannic-glutaraldehyde they contain regular lamellae with a periodicity of 5.5 nm. Both of these reactions are typical of lipids. Some of these structures are associated with phagosomal vacuoles which may contribute to their lamellae. They disappear at the onset of merozoite formation, but membranous whorls of various sizes continue to be associated with the schizont surface during budding of merozoites. It is suggested that the lipidic vacuoles are a source of preformed lipid which can be utilized rapidly during the generation of merozoites.