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Biomedical subjects

M Aikawa

Publications and source records attributed to M Aikawa.

At least 235 records · Page 13Linked to original sources
PubMed1988

Plasmodium malariae: distribution of circumsporozoite protein in midgut oocysts and salivary gland sporozoites.

The distribution of the circumsporozoite protein within developing Plasmodium malariae oocysts and salivary gland sporozoites was examined by immunoelectron microscopy using protein A-gold and a monoclonal antibody specific for the CS protein of P. malariae. Gold particles were found along the capsule of immature oocysts but rarely within the cytoplasm. Gold label was detected on the inner surface of peripheral vacuoles during oocyst maturation and the plasma membrane of the sporoblast. Salivary gland sporozoites and budding sporozoites in mature oocysts were labeled uniformly on the outer surface of their plasma membranes. The surface of sporozoites that ruptured into midgut epithelial cells were entirely covered with gold particles. No label was seen on the surface of sporozoites which ruptured into the midgut lumen. In addition, a rabbit polyclonal antibody against repeat a region of P. brasilianum CS protein reacted with P. malariae sporozoites.

Animals↗
PubMed1988

Morphological changes in erythrocytes induced by malarial parasites.

Host cell alterations induced by Plasmodium falciparum, P. brasilianum, P. vivax and P. malariae were described by electron microscopy and post-embedding immunoelectron microscopy. P. falciparum infection induces knobs, electron-dense material and clefts in the erythrocyte. Clefts are involved in exporting P. falciparum antigen from the parasite to the erythrocyte membrane. P. falciparum antigen is present in knobs which adhere to endothelial cells causing the blockage of cerebral capillaries and ensuing pathological changes in cerebral tissues. P. brasilianum infection induces knobs, short and long clefts and electron-dense material. These structures appear to contain different P. brasilianum antigens. This indicates that each structure functions independently in trafficking P. brasilianum protein to the erythrocyte surface. P. vivax infection induces caveola-vesicle complexes and clefts in the erythrocyte. These structures are also involved in trafficking P. vivax protein from the parasite to the erythrocyte membrane. P. malariae induces caveolae, electron-dense material, vesicles, clefts and knobs in the erythrocyte. Although vesicles and caveolae are seen in the erythrocyte cytoplasm, they do not form caveola-vesicle complexes as seen in P. vivax-infected erythrocytes. They also appear to be involved in trafficking of malaria antigens. These studies, therefore, indicate that host cell changes occur in order to facilitate the transport of malarial antigens to the host cell membrane. The significance of these phenomena is still not clear.

Animals↗
PubMed1988

Inhibition of surface membrane maturation in schistosomula of Schistosoma mansoni.

The surface membrane of the multicellular parasite Schistosoma mansoni is radically reorganized during the transformation of cercariae into schistosomula. The current study investigates factors involved in maturation of the surface from a trilaminate to a multilaminate membrane. When maturation was induced in the presence of puromycin (900 microM), the acquisition of a multilaminate surface and stainability with fluorescein-conjugated Con A were similar to that of control parasites. Similarly, although organisms treated with monensin (0.1 microM) for 3 hr showed large vacuoles in the perinuclear cytoplasm of the subtegumental cells, the surface membrane became multilaminate. In contrast, microtubule-active drugs interfered with maturation: the surface remained largely trilaminate and the percentage of organisms binding Con A to their surface was significantly reduced. Furthermore, large accumulations of multilaminate bodies were found in the subtegumental cells of colchicine-treated parasites, whereas few were seen in the controls. Colchicine-treated schistosomula failed to mature to adult worms upon injection into mice and, like cercariae, they were water tolerant. We therefore conclude that the components that constitute the schistosomula surface preexist in cercariae and suggest that they are stored in multilaminate bodies before being transported to the surface with the help of microtubules. The acquisition of the multilaminate membrane may be essential for survival of the parasites in vivo and in vitro.

Animals↗
PubMed1988

In vitro and in vivo studies of the effects of halogenated histidine analogs on Plasmodium falciparum.

The effects of four halogenated analogs of histidine on in vitro growth of Plasmodium falciparum malaria parasites were monitored by measurement of the incorporation of 3H-labeled amino acids into parasite proteins and by light and electron microscopy. The uptake of [3H]isoleucine was reduced to 50% of the control value by addition of 70 microM 2-fluoro-L-histidine (2-F-HIS) or 420 microM 2-iodo-L-histidine (2-I-HIS). [3H]histidine uptake into acid-insoluble material was affected equally by these two compounds, 50% inhibition resulting at 200 microM concentration. Morphological analysis of parasite development proved a sensitive assay, since development of mature trophozoites was inhibited 50% by 25 microM 2-F-HIS or 100 2-I-HIS. Electron microscopy studies suggested different mechanisms of action of 2-F-HIS and 2-I-HIS on P. falciparum. 2-F-HIS produced a decrease in knob number at the erythrocyte surface and accumulation of electron-dense material under the parasite membrane. 2-I-HIS had no obvious effect on knobs or electron-dense material but affected parasite morphology. Surprisingly, 2-chloro-L-histidine and 2-bromo-L-histidine did not inhibit P. falciparum in vitro, even though their halogen atom substituents are intermediate in size between F and I atoms. 2-F-HIS and 2-I-HIS were tested in vivo against P. falciparum in owl monkeys (Aotus sp.) but were ineffective at doses that were nontoxic.

Amino Acids↗
PubMed1988

Membrane-associated antigens of blood stages of Plasmodium, brasilianum, a quartan malaria parasite.

The localization of Plasmodium brasilianum-derived antigens in short and long clefts within the cytoplasm of infected erythrocytes and in association with knobs of the host cell membrane was demonstrated by immunoelectron microscopy with monoclonal antibodies. Our results document that malaria-induced short and long clefts, previously distinguishable only by morphology, differ also in antigenic composition. Another parasite-derived antigen was found to be associated with the parasitophorous vacuole space in schizonts. In segmenters, this antigen was present in large amounts between merozoites and in the cytoplasm of infected cells. These antigens were characterized by biosynthetic labeling and gel electrophoresis.

Animals↗
PubMed1988

A malarial cysteine proteinase is necessary for hemoglobin degradation by Plasmodium falciparum.

To obtain free amino acids for protein synthesis, trophozoite stage malaria parasites feed on the cytoplasm of host erythrocytes and degrade hemoglobin within an acid food vacuole. The food vacuole appears to be analogous to the secondary lysosomes of mammalian cells. To determine the enzymatic mechanism of hemoglobin degradation, we incubated trophozoite-infected erythrocytes with peptide inhibitors of different classes of proteinases. Leupeptin and L-transepoxy-succinyl-leucyl-amido-(4-guanidino)-butane (E-64), two peptide inhibitors of cysteine proteinases, inhibited the proteolysis of globin and caused the accumulation of undegraded erythrocyte cytoplasm in parasite food vacuoles, suggesting that a food vacuole cysteine proteinase is necessary for hemoglobin degradation. Proteinase assays of trophozoites demonstrated cysteine proteinase activity with a pH optimum similar to that of the food vacuole and the substrate specificity of lysosomal cathepsin L. We also identified an Mr 28,000 proteinase that was trophozoite stage-specific and was inhibited by leupeptin and E-64. We conclude that the Mr 28,000 cysteine proteinase has a critical, perhaps rate-limiting, role in hemoglobin degradation within the food vacuole of Plasmodium falciparum. Specific inhibitors of this enzyme might provide new means of antimalarial chemotherapy.

Animals↗
PubMed1988

Morphological effects of pyronaridine on malarial parasites.

The ultrastructural changes caused by a new antimalarial drug, pyronaridine, were investigated using mice infected with erythrocytic forms of Plasmodium berghei and P. falciparum cultivated in vitro in human erythrocytes. The first changes observed in both parasites after exposure to pyronaridine occurred in the food vacuoles. This suggests that the target organelle of this drug may be the food vacuole of malarial parasites. In addition, rapid alterations were also noted within the pellicular complex of both plasmodia.

Animals↗
PubMed1988

Glomerulopathy in squirrel monkeys with acute Plasmodium falciparum infection.

The renal pathology of 9 squirrel monkeys (Saimiri sciureus) with acute Plasmodium falciparum infection was studied by light and electron microscopy. Endocapillary proliferative glomerulonephritis was the major pathological change observed. The peroxidase anti-peroxidase method demonstrated the presence of IgG, IgM, and P. falciparum antigens in the mesangium and basement membrane. These findings were consistent with those seen in humans with acute P. falciparum infection and indicates that squirrel monkeys are likely to be a good model for the study of renal pathology in malaria research.

Acute Disease↗
PubMed1988

Ultrastructural study of the effects of chloroquine and verapamil on Plasmodium falciparum.

Verapamil, a calcium antagonist, has recently been shown to reverse chloroquine resistance in malarial parasites in vitro. We report the first ultrastructural morphological changes associated with this phenomenon using chloroquine-sensitive and -resistant clones of Plasmodium falciparum. While the administration of 6.3 x 10(-8) M chloroquine had little morphological effect on the chloroquine-resistant strain, the combination of chloroquine and verapamil resulted in typical chloroquine-related food vacuolar swelling with increased amounts of granular matrix. Secondary morphological changes included degeneration of nuclei, mitochondria, and other organelles. These effects appeared similar to those in the chloroquine-sensitive strain of P. falciparum treated with chloroquine alone or with the chloroquine/verapamil combination. Furthermore mild food vacuolar changes were seen in a small number of parasites (from both chloroquine-sensitive and -resistant groups) exposed to high concentrations (1 x 10(-4) M) of verapamil alone.

Animals↗
PubMed1988

Human cerebral malaria.

Possible factors contributing to the development of cerebral malaria were discussed based on pathological changes in Burmese patients who died of cerebral malaria. Blockage of cerebral capillaries by Plasmodium falciparum infected erythrocytes appeared to be the principal cause of cerebral malaria. From electron microscopic results, it was concluded that knobs on infected erythrocytes acted as focal junctions which mediated adhesion to endothelial cells. The knobs are, therefore, important contributors to the blockage of the capillary lumen and ensuing pathological changes in cerebral tissues. Host cell molecules such as OKM5 and thrombospondin may function as endothelial cell surface receptors for the attachment of knobs of P. falciparum infected erythrocytes. Immunological events might also play a role in the pathogenesis of cerebral malaria. This was suggested by the presence of IgG, IgM, P. falciparum antigens, and knob proteins in the cerebral capillaries of the people with cerebral malaria. It will be important to assess the candidate malaria vaccines now in development not only for their efficacy in reducing parasitemia but for effects they may have on the sequestration of infected erythrocytes in the brain.

Adolescent↗
PubMed1988

Immunoelectron microscopic localization of vivax malaria antigens to the clefts and caveola-vesicle complexes of infected erythrocytes.

Erythrocytes infected with Plasmodium vivax show unique ultrastructural changes which include membranous structures in the host cell cytosol, called clefts, and caveola-vesicle complexes (CVC) in the infected erythrocyte membrane. It has been suggested that the latter structures correspond with the Schuffner's dots observed on Giemsastained thin films. The subcellular localization of a 28 kDa and a 95 kDa antigen of the erythrocytic stages of P. vivax was determined by post-embedding immunoelectron microscopy. Four monoclonal antibodies (MAbs) (2H12.B4,2H8.E10, 1H4.B6, and 4C12.G4) against the 95 kDa protein reacted with the vesicles of CVC and vesicles scattered in the cytoplasm of the infected erythrocytes. Two other MAbs (4C12.B10 and 4D7.B1) against a 28 kDa protein reacted with the cytoplasmic clefts and were also reactive with the vesicles and electron dense materials in parasitophorous vacuole. These parasite-induced structures make a contribution to the movement of some malaria proteins from the parasite to the erythrocyte surface.

Animals↗
PubMed1988

Ultrastructural localization of erythrocyte cytoskeletal and integral membrane proteins in Plasmodium falciparum-infected erythrocytes.

The distributions of ankyrin, spectrin, band 3, and glycophorin A were examined in Plasmodium falciparum-infected erythrocytes by immunoelectron microscopy to determine whether movement of parasite proteins and membrane vesicles between the parasitophorous vacuole membrane and erythrocyte surface membrane involves internalization of host membrane skeleton proteins. Monospecific rabbit antisera to spectrin, band 3 and ankyrin and a mouse monoclonal antibody to glycophorin A reacted with these erythrocyte proteins in infected and uninfected human erythrocytes by immunoblotting. Cross-reacting malarial proteins were not detected. The rabbit sera also failed to immunoprecipitate [3H]isoleucine labeled malarial proteins from Triton X-100 and sodium dodecyl sulfate (SDS) extracts of infected erythrocytes. These three antibodies as well as the monoclonal antibody to glycophorin A bound to the membrane skeleton of infected and uninfected erythrocytes. The parasitophorous vacuole membrane was devoid of bound antibody, a result indicating that this membrane contains little, if any, of these host membrane proteins. With ring-, trophozoite- and schizont-infected erythrocytes, spectrin, band 3 and glycophorin A were absent from intracellular membranes including Maurer's clefts and other vesicles in the erythrocyte cytoplasm. In contrast, Maurer's clefts were specifically labeled by anti-ankyrin antibody. There was a slight, corresponding decrease in labeling of the membrane skeleton of infected erythrocytes. A second, morphologically distinct population of circular, vesicle-like membranes in the erythrocyte cytoplasm was not labeled with anti-ankyrin antibody. We conclude that membrane movement between the host erythrocyte surface membrane and parasitophorous vacuole membrane involves preferential sorting of ankyrin into a subpopulation of cytoplasmic membranes.

Animals↗
PubMed1988

Immunoelectron microscopy of Schüffner's dots in Plasmodium vivax-infected human erythrocytes.

Plasmodium vivax induces morphologic alterations in infected host erythrocytes that are visible by light microscopy in Romanovsky-stained blood smears as multiple brick-red dots. These morphologic changes, referred to as Schüffner's dots, are important in the identification of this species of malarial parasite and have been associated by electron microscopy with caveolavesicle complexes along the erythrocyte plasmalemma. We have produced a monoclonal antibody (MAb A 20) that identifies an antigen in Plasmodium vivax-infected erythrocytes that is associated with the caveola-vesicle complexes of the parasitized host cell. This monoclonal antibody reacts with air-dried P vivax-infected erythrocytes to produce a pattern by the indirect immunofluorescence test (IFT) that is evocative of Schüffner's dots. Immunoelectron microscopy of P vivax-infected human erythrocytes using MAb A 20 confirmed the location of this antigen within vesicles of caveola-vesicle complexes. On Western blots MAb A 20 recognized four polypeptides of 54, 64, 72, and 86 kd. MAb A 20 reacted by IFT with 90% of Sri Lankan isolates and with a Brazilian P vivax isolate, which indicates that the epitope identified by this monoclonal is conserved.

Animals↗
PubMed1987

Monoclonal antibody characterization of Plasmodium falciparum antigens in immune complexes formed when schizonts rupture in the presence of immune serum.

When Plasmodium falciparum parasites are cultured with some immune sera, merozoites are agglutinated by antibodies to form immune clusters of merozoites and prevent their invasion into erythrocytes. Within these immune clusters of merozoites, several antigens that are normally found in the soluble fraction after detergent extraction accumulate in relatively insoluble immune complexes. From mice immunized with these immune complexes, we obtained hybridomas secreting monoclonal antibodies (mAb) that react with various immune clusters of merozoites antigens, including mAb 3D5, which recognizes a 101-kDa antigen (p101) and mAb, 5E3, which recognizes a 113-kDa antigen (p113). Both mAb reacted with antigens at the surface of schizonts, in the vacuolar space, and at the surface of merozoites before their release from schizont-infected cells. Both p101 and p113 were synthesized by mature trophozoites and young schizonts. In pulse-chase experiments, p113 was processed to 100-, 70-, 55-, and 50-kDa products. Both p101 and p113 appeared in the culture medium when schizont rupture occurred in normal culture medium but were found in immune complexes when schizont rupture occurred in the presence of immune serum. Antibodies in immune complexes, when dissociated with acid and used to probe immunoblots, reacted with affinity-purified p101 and p113. Antigens such as these, which are accessible at the parasite surface and react with antibodies present in immune serum that inhibits parasite invasion, are logical candidates to study in the search for a vaccine against the erythrocytic stages of malaria.

Animals↗
PubMed1987

cDNA sequence encoding a Plasmodium falciparum protein associated with knobs and localization of the protein to electron-dense regions in membranes of infected erythrocytes.

Plasmodium falciparum modifies the host erythrocyte's plasma membrane by the formation of electron-dense structures called knobs. We have produced monoclonal antibodies (McAbs) which specifically bind to the knobs in immunoelectron microscopic experiments with thin sections of parasitized erythrocytes. However, the McAbs fail to bind to the surface of live parasitized erythrocytes. Immunoblotting experiments with these McAbs show the antigen is localized to the erythrocyte plasma membrane. The antigen with which the McAbs react varies in mol. wt from 80 to 95 kd in different knob-producing isolates of P. falciparum and is absent in knobless variants. The McAbs react with the expressed product of a P. falciparum cDNA clone, thus demonstrating that the clone encodes part of this knob-associated protein. The sequence of the cDNA fragment partially overlaps a published cDNA sequence reported to encode the amino-terminal portion of the knob protein, and extends the predicted open reading frame by 190 amino acids. The carboxyl-terminal portion of the predicted amino acid sequence contains a highly charged stretch of approximately 100 amino acid residues. We suggest that this unusual, highly charged region participates in intermolecular salt bridging leading to dense packing of these molecules. This would create the electron-dense regions observed by electron microscopy and might also explain the insolubility of the knob-associated protein in the absence of strong ionic detergents or chaotropic agents.

Amino Acid Sequence↗
PubMed1987

Localization of Plasmodium falciparum histidine-rich protein 1 in the erythrocyte skeleton under knobs.

Plasmodium falciparum parasites that induce knobs in the host erythrocyte membrane (K+ phenotype) synthesize a 90 kDa histidine-rich protein (PfHRP-1), whereas knobless variants do not. A monoclonal antibody (mAb 89) to PfHRP-1, in combination with cryo-thin section immunoelectron microscopy, localized the antigen in the parasitophorous vacuolar space and vesicles within the erythrocyte cytosol. Additional immunoelectron microscopic studies showed that PfHRP-1 was also associated with submembranous electron-dense material under knobs and with microfilaments of the host erythrocyte skeletal network. Immunofluorescence and immunoelectron microscopy of intact, non-fixed K+ infected erythrocytes using mAb 89 and a rabbit antiserum raised against purified PfHRP-1, failed to identify any surface exposed epitopes. These antibodies also failed to block cytoadherence of infected erythrocytes to C32 melanoma cells or to affect macrophage phagocytosis of infected erythrocytes.

Animals↗
PubMed1987

Calcium and calmodulin antagonists inhibit human malaria parasites (Plasmodium falciparum): implications for drug design.

The malaria parasite has an obligate calcium requirement for normal intracellular growth and invasion of host erythrocytes. Calmodulin (CaM) is a vital calcium-dependent protein present in eukaryotes. We found by radioimmunoassay that free parasites contain CaM. Schizont-infected erythrocytes had CaM levels of 23.3 +/- 2.7 ng per 10(6) cells compared to normals (11.2 +/- 1.5 ng per 10(6) cells). CaM levels were proportional to parasite maturity. Immunoelectron microscopy identified CaM diffusely within the cytoplasm of mature parasites and at the apical end of merozoites within the ductule of rhoptries, which may explain the calcium requirement for invasion. Cyclosporin A (CsA) was also found by electron microscopic autoradiography to concentrate in the food vacuole, as do chloroquine and mefloquine, and to distribute within the cytoplasm of mature parasites. The binding of dansylated CsA to schizont-infected erythrocytes was higher than to normal erythrocytes as analyzed by flow cytometry. Kinetic analysis revealed that binding was saturable for normal and infected erythrocytes and possibly free parasites. Competition for binding existed between dansylated CsA and native CsA as well as the CaM inhibitor W-7 and the classic antimalarial chloroquine. The in vitro growth of Plasmodium falciparum was sensitive to CaM antagonists, and in large part inhibition of the parasite was proportional to known anti-CaM potency. Antagonism existed between combinations of these drugs in multi-drug-resistant strains of P. falciparum, suggesting possible competition for the same binding site. In addition, the malaria parasite was also susceptible to calcium antagonists.

Animals↗