Biomedical subjects

M Aikawa

Publications and source records attributed to M Aikawa.

At least 253 records · Page 14Linked to original sources

PubMed1987

Transport of an Mr approximately 300,000 Plasmodium falciparum protein (Pf EMP 2) from the intraerythrocytic asexual parasite to the cytoplasmic face of the host cell membrane.

The profound changes in the morphology, antigenicity, and functional properties of the host erythrocyte membrane induced by intraerythrocytic parasites of the human malaria Plasmodium falciparum are poorly understood at the molecular level. We have used mouse mAbs to identify a very large malarial protein (Mr approximately 300,000) that is exported from the parasite and deposited on the cytoplasmic face of the erythrocyte membrane. This protein is denoted P. falciparum erythrocyte membrane protein 2 (Pf EMP 2). The mAbs did not react with the surface of intact infected erythrocytes, nor was Pf EMP 2 accessible to exogenous proteases or lactoperoxidase-catalyzed radioiodination of intact cells. The mAbs also had no effect on in vitro cytoadherence of infected cells to the C32 amelanotic melanoma cell line. These properties distinguish Pf EMP 2 from Pf EMP 1, the cell surface malarial protein of similar size that is associated with the cytoadherent property of P. falciparum-infected erythrocytes. The mAbs did not react with Pf EMP 1. In one strain of parasite there was a significant difference in relative mobility of the 125I-surface-labeled Pf EMP 1 and the biosynthetically labeled Pf EMP 2, further distinguishing these proteins. By cryo-thin-section immunoelectron microscopy we identified organelles involved in the transit of Pf EMP through the erythrocyte cytoplasm to the internal face of the erythrocyte membrane where the protein is associated with electron-dense material under knobs. These results show that the intraerythrocytic malaria parasite has evolved a novel system for transporting malarial proteins beyond its own plasma membrane, through a vacuolar membrane and the host erythrocyte cytoplasm to the erythrocyte membrane, where they become membrane bound and presumably alter the properties of this membrane to the parasite's advantage.

Animals↗

PubMed1987

Human cerebral malaria: a pathological study.

The following report using light and electron microscopic and immunological techniques is based on a series of 19 Burmese patients who died of cerebral malaria. The principal change was blockage of cerebral capillaries by Plasmodium falciparum-infected erythrocytes. Ring hemorrhages and segmental necrosis of cerebral capillaries were common. Cerebral edema was variable in these cases. Electron-dense knobs, 40 X 80 nm in size, which protruded from the membrane of infected erythrocytes, formed focal junctions between endothelial cells and erythrocytes. These junctions resulted in the entrapment of erythrocytes and caused blockage in the capillary lumen. Immunoperoxidase study revealed that P. falciparum antigens and IgG deposits in the capillary basement membrane. This implies that damage to the cerebral capillary could be related to immune mechanisms.

Adolescent↗

PubMed1987

Ultrastructure of the erythrocytic stages of Plasmodium malariae.

This report describes the fine structure of the erythrocytic stages of Plasmodium malariae. Erythrocytic parasites from a naturally acquired human infection and an experimentally infected chimpanzee were morphologically indistinguishable and structurally similar to other primate malarias. New findings included observations of highly structured arrays of merozoite surface coat proteins in the cytoplasm of early schizonts and on the surface of budding merozoites and the presence of knobs in the membranes of Maurer's clefts. Morphological evidence is presented suggesting that proteins are transported between the erythrocyte surface and intracellular parasites via two routes: one associated with Maurer's clefts for transport of membrane-associated knob material and a second associated with caveolae in the host cell membrane for the import or export of host- or parasite-derived substances through the erythrocyte cytoplasm.

Adolescent↗

PubMed1987

Localization of circumsporozoite protein of Plasmodium ovale in midgut oocysts.

Circumsporozoite (CS) proteins are the major proteins found on the surface of salivary gland sporozoites and are the protective antigens of several species of malaria parasites. Little is known about the distribution of CS proteins in developing oocysts, however. Immunoelectron microscopy with protein A-gold and a monoclonal antibody specific for the CS protein of Plasmodium ovale was performed to investigate the distribution of CS protein within developing P. ovale oocysts. There was an almost complete absence of label in immature oocysts prior to the development of sporoblasts. In contrast, sporoblasts and budding and free sporozoites in mature oocysts were labeled uniformly on the outer surfaces of their plasma membranes, indicating a uniform distribution of CS protein on these membranes. Gold particles were frequently associated with the cytoplasm of sporoblasts and sporozoites, as well as with the inner surface of the oocyst capsule. This is the first evidence that CS protein is present in oocyst sporozoites and sporoblasts of P. ovale.

Animals↗

PubMed1987

Ultrastructural localization of the 150/130 Kd antigens in sexual and asexual blood stages of Plasmodium falciparum-infected human erythrocytes.

The subcellular localization of the 150/130 Kd antigen in Plasmodium falciparum-infected erythrocytes was determined by electron microscopy using monoclonal antibody 9B11 and immuno-gold labeling. We now find that this antigen may be associated with the membrane of newly-infected human erythrocytes and the cytoplasm of ring stage parasites. During differentiation of the parasite to the trophozoite stage, the antigens are no longer detectable on the erythrocyte membrane, while gold particles become more numerous within the parasite and in the erythrocyte cytoplasm adjacent to the parasite. As the parasites develop into schizonts, more antigen appears within the parasites, and some of it appears in the erythrocyte cytoplasm. At the segmented schizont stage, many intraparasitic gold particles are associated with rhoptries and micronemes of developing merozoites. Likewise, gold particles are associated with elements of the rhoptry-microneme complex in free merozoites. No gold particles are detected on the surface of merozoites. These antigens are found most abundantly in erythrocytes infected with gametocytes, revealing a localization pattern similar to that of mature trophozoite-infected erythrocytes. These subcellular localization patterns are similar to those described for the ring-infected erythrocyte surface antigen.

Animals↗

PubMed1987

An ultrastructural study of the effects of mefloquine on malaria parasites.

The ultrastructural changes induced by the administration of a recently developed antimalarial drug, mefloquine, were studied in mice infected with Plasmodium berghei and human erythrocytes infected with P. falciparum in vitro. Pronounced changes which occurred in both experiments comprised swelling of the parasites' food vacuoles with gradual loss of pigment granules, which did not form clumps as occurs with chloroquine. These findings suggest that the malarial parasites' food vacuole is the target of this drug.

Animals↗

PubMed1987

Knob antigen deposition in cerebral malaria.

Plasmodium falciparum-infected erythrocytes attach to the endothelial cells via electron-dense knobs and this attachment has been suggested as one of the contributing factors in the development of cerebral malaria. Monoclonal antibodies against an 80-95 Kd knob protein were prepared and applied to brain tissue from cerebral malaria patients. The deposition of the 80-95 Kd knob protein antibodies was observed in the basement membrane of cerebral capillaries by the peroxidase anti-peroxidase method. This result indicates involvement of knob protein deposition in the pathogenesis of cerebral malaria.

Adult↗

PubMed1987

Ultrastructural localization of Plasmodium falciparum circumsporozoite protein in newly invaded hepatoma cells.

The fate and disposition of the circumsporozoite (CS) protein of Plasmodium falciparum was investigated during hepatoma cell invasion with several sera raised against defined CS peptides, including both repeat and nonrepeat regions spanning approximately 60% of the P. falciparum CS gene product. Distribution of the protein, as revealed by immunoelectron microscopy, was limited to the surface of the sporozoite both before and after invasion. In particular, no CS protein antigen was detected in association with either the parasitophorous vacuole membrane or the host cell surface.

Animals↗

PubMed1987

Role of calmodulin in Plasmodium falciparum: implications for erythrocyte invasion by the merozoite.

Calmodulin, a calcium-dependent modulator protein, was shown to be indispensable for in vitro growth of erythrocytic stages of the human malaria parasite, Plasmodium falciparum. When the potent calmodulin antagonists, W7, trifluoperazine (TFP) and R24571, were added to cultures of P. falciparum they inhibited invasion of erythrocytes by merozoites, as well as maturation of schizonts. W5, a chlorine-deficient analogue of W7, was a much weaker inhibitor than W7. The concentrations of W5, W7, TFP and R24571 needed to produce 50% inhibition of schizont maturation were 63.5, 19, 18 and 8.5 microM, respectively, while concentrations needed to inhibit 50% the appearance of ring forms were only 19.5, 7, 8.4 and 4.5 microM, respectively. All the antagonists were more effective at inhibiting the invasion of erythrocytes by merozoites than maturation of schizonts. Ca2+ depletion by EGTA also inhibited merozoite invasion of erythrocytes. Unlike W5, W7, TFP and R24571, cyclosporin A (CsA) showed marked inhibition of schizont maturation at concentrations that reduce ring form production. Immunoelectron microscopy showed that calmodulin was concentrated at the apical end of both free and intraerythrocytic merozoites. No anticalmodulin immunoreactivity was observed in merozoites grown in the presence of 10 microM TFP, although the other calmodulin antagonists and EGTA did not significantly affect the calmodulin location in merozoites. These results suggest that the accumulation of calmodulin at the apical end of merozoites plays an important role during their attachment to and/or invasion of the host erythrocyte, possibly through activation of Ca2+ dependent processes.

Animals↗

PubMed1987

Ultrastructural localization of protective and nonprotective Plasmodium falciparum proteins using serum samples from vaccinated Aotus monkeys.

Postembedding immunoelectron microscopy, using pooled serum samples from a recent vaccination experiment involving Aotus monkeys, was used to localize immune targets in Plasmodium falciparum-infected erythrocytes and free merozoites. Serum samples from Aotus monkeys, protected completely by immunization with the P. falciparum merozoite surface coat precursor protein, identified immune targets on the surface of free and intracellular merozoites as well as the cytoplasm, plasma membrane, and parasitophorous vacuole membrane of immature schizonts. Serum samples from unprotected monkeys, which had been immunized with a complex of 143-kDa, 132-kDa, and 102-kDa polypeptides reacted specifically with the rhoptries of immature schizonts and mature merozoites.

Animals↗

PubMed1987

Phagocytosis by hemolymph cells of the land slug, Incilaria fruhstorferi Collinge (Gastropoda: Pulmonata).

Phagocytosis by hemolymph cells of the land slug, Incilaria fruhstorferi Collinge, were studied with the scanning electron microscopy (SEM) and the transmission electron microscopy (TEM). The fate of foreign materials, i.e., sheep red blood cells (SRBC) and latex beads, introduced into the hemocoel of the slug were followed. Certain hemocytes named as Type I cell were involved in spontaneous cyto-adherence while both Type II and III cells were not observed to adhere to foreign materials. SRBC and latex beads (luminal diameter 0.79 micron) were phagocytosed and latex beads (luminal diameter 15.8 micron) were engulfed by Type I cells. In vitro phagocytosis experiments showed that SRBC formed the rosette structure surrounding a Type I cell and both SRBC and latex beads (luminal diameter 0.79 micron) were swallowed by Type I cells in vivo. The plate-like structures with long fine fibers attached the latex beads. The latex beads were often seen deep in the aggregates of plate-like structures.

Animals↗

PubMed1986

Secretion of a malarial histidine-rich protein (Pf HRP II) from Plasmodium falciparum-infected erythrocytes.

Plasmodium falciparum-infected erythrocytes (IRBCs) synthesize several histidine-rich proteins (HRPs) that accumulate high levels of [3H]histidine but very low levels of amino acids such as [3H]isoleucine or [35S]methionine. We prepared a monoclonal antibody which reacts specifically with one of these HRPs (Pf HRP II) and studied the location and synthesis of this protein during the parasite's intracellular growth. With the knob-positive Malayan Camp strain of P. falciparum, the monoclonal antibody identified a multiplet of protein bands with major species at Mr 72,000 and 69,000. Pf HRP II synthesis began with immature parasites (rings) and continued through the trophozoite stage. The Mr 72,000 band of Pf HRP II, but not the faster moving bands of the multiplet, was recovered as a water-soluble protein from the culture supernatant of intact IRBCs. Approximately 50% of the total [3H]histidine radioactivity incorporated into the Mr 72,000 band was extracellular between 2 and 24 h of culture. Immunofluorescence and cryothin-section immunoelectron microscopy localized Pf HRP II to several cell compartments including the parasite cytoplasm, as concentrated "packets" in the host erythrocyte cytoplasm and at the IRBC membrane. Our results provide evidence for an intracellular route of transport for a secreted malarial protein from the parasite through several membranes and the host cell cytoplasm.

Animals↗

PubMed1986

Membrane-associated electron-dense material of the asexual stages of Plasmodium falciparum: evidence for movement from the intracellular parasite to the erythrocyte membrane.

Electron-dense material (EDM) appears at the parasite plasma membrane with trophozoites of several strains of Plasmodium falciparum cultured in vitro. The EDM is also seen associated with unit membrane-bounded Maurer's clefts in K+ P. falciparum-infected erythrocytes. The cytoplasmic clefts lack the EDM with K- parasites. Some EDM have the same density and appearance as the material located under knobs at the erythrocyte membrane. The EDM at the parasite plasma membrane is absent with schizonts when expression of new knobs at the erythrocyte membrane appears to have ceased. This electron microscopic study suggests that the parasite-derived EDM is transported from the parasite plasmalemma to the erythrocyte membrane via Maurer's clefts in the erythrocyte cytoplasm.

Animals↗

PubMed1986

Localization of protective 143/140 kDa antigens of Plasmodium knowlesi by the use of antibodies and ultracryomicrotomy.

Immune sera from mice immunized with the 143/140 kDa protein have been shown to partially block erythrocyte invasion by P. knowlesi merozoites. Therefore, immunoelectron microscopy utilizing ultracryomicrotomy, antibody to 143/140 kDa protein, and protein A gold particles were used to determine the precise localization of this protein in malarial parasites. Gold particles were not seen associated with young trophozoites but appeared in the parasite cytoplasm as the parasites grew to multi-nucleate schizonts. In presegmenter-schizonts, gold particles were associated with the well-developed endoplasmic reticulum, the parasite plasma membrane, and the parasitophorous vacuole membrane. The surface of merozoites was covered with gold particles. Maurer's clefts, which appeared in Plasmodium infected erythrocytes, were also associated with gold particles. These observations suggest that 143/140 kDa protective malarial proteins may be synthesized in the endoplasmic reticulum of P. knowlesi schizonts before being transported to the surface of the schizonts and merozoites. Shedding of the merozoite surface coat may be responsible for the presence of the 143/140 kDa proteins in the parasitophorous vacuole and Maurer's clefts.

Animals↗

PubMed1985

Plasmodium gallinaceum: critical role for microtubules in the transformation of zygotes into Ookinetes.

The role of microtubules and microfilaments in the transformation of spherical zygotes of Plasmodium gallinaceum (avian malaria parasite) into vermiform ookinetes has been studied by using specific drugs (taxol, colchicine, and cytochalasin-B). Both taxol and colchicine completely abolished the transformation of zygotes into ookinetes. The inhibitory effect was seen only if the drugs were added during the initial 6 hr of total time (20-24 hr) required for complete transformation; the addition of drugs after 6-8 hr of initiation of transformation had no effect. Electron microscopy revealed that microtubules were depolymerized by colchicine treatment, whereas in taxol-treated cells there was an extensive array of cytoplasmic and nuclear microtubules which appeared to be clumped in bundles. In contrast to the effects of taxol and colchicine, cytochalasin-B, which affects the microfilament system, had no effect on the transformation. Protein synthesis and expression of two ookinete-specific surface proteins were not affected in the drug-inhibited parasites. Zygotes treated with taxol for 4 hr at room temperature failed to develop into oocysts when they were subsequently fed to mosquitoes. These studies demonstrate a critical role for microtubules in the initial stages of transformation of zygotes into ookinetes.

Alkaloids↗

PubMed1985

Isolation of a Plasmodium falciparum rhoptry protein.

A monoclonal antibody raised against the malaria parasite Plasmodium falciparum recognised a protein of 140000 molecular weight which was synthesized during schizogony. The protein has been purified by monoclonal antibody affinity chromatography from extracts of parasitized red cells. Antibodies against the protein have been used to determine its subcellular location. The protein is not expressed on the merozoite surface and has been located in the rhoptries, the apical organelles of the merozoite.

Animals↗

PubMed1985

An in vitro assay for sequestration: binding of Plasmodium falciparum-infected erythrocytes to formalin-fixed endothelial cells and amelanotic melanoma cells.

Erythrocytes infected with Plasmodium falciparum bind specifically to cultured endothelial cells and to a line of amelanotic melanoma cells. We have fixed endothelial cells and amelanotic melanoma cells in various ways and determined whether the fixed cells were still able to bind infected erythrocytes. Only cells fixed with 1.0-2.5% formalin in phosphate-buffered saline continued to bind infected erythrocytes as well as unfixed cells. The mechanism of binding to fixed and unfixed cells appeared to be identical for the following reasons. First, erythrocytes infected by parasite strains that bound to unfixed cells also bound to fixed cells while those that did not bind to unfixed cells did not bind to fixed cells. Second, immune serum that inhibited binding to unfixed cells also inhibited binding to fixed cells. Third, electron microscopy showed that knobs were the points of attachment between infected erythrocytes and both fixed and unfixed melanoma cells. Fixed cells gave reproducible results over at least 2 months. Thus, we have developed a simplified, reproducible assay for measuring binding of P. falciparum-infected erythrocytes to target cells.

Animals↗

PubMed1985

Structural alteration of the membrane of erythrocytes infected with Plasmodium falciparum.

Human erythrocytes infected with five strains of Plasmodium falciparum and Aotus erythrocytes infected with three strains of P. falciparum were studied by thin-section and freeze-fracture electron microscopy. All strains of P. falciparum we studied induced electron-dense conical knobs, measuring 30-40 nm in height and 90-100 nm in diameter on erythrocyte membranes. Freeze-fracture demonstrated that the knobs were distributed over the membrane of both human and Aotus erythrocytes. A distinct difference was seen between the intramembrane particle (IMP) distribution over the knobs of human and Aotus erythrocyte membranes. There was no change in IMP distribution in infected human erythrocyte membranes, but infected Aotus erythrocytes showed an aggregation of IMP over the P face of the knobs with a clear zone at the base. This difference in IMP distribution was related only to the host species and not to parasite strains. Biochemical analysis demonstrated that a higher proportion of band 3 was bound to the cytoskeleton of uninfected Aotus erythrocytes than uninfected human erythrocytes after Triton X-100 extraction. This may account for the different effects of P. falciparum infection on IMP distribution in the two different cell types.

Animals↗