Freeze-Fracture Study of Malaria Sporozoites: Antibody-Induced Changes of the Pellicular Membrane*

Plasmodium cynomolgi, Plasmodium knowlesi, and Plasmodium berghei sporozoites, before and after incubation with immune serum, were studied after freeze-fracture by electron microscopy. There were evenly distributed numerous intramembranous particles (IMP) on the P face of the outer membrane. The E face of the plasma membrane had fewer IMP than its P face. The E face of the intermediate membrane had few IMP and also linear arrays of slightly raised ridges running the length of the parasite. The P face of the intermediate membrane had many IMP aligned along the long axis of the sporozoite. On the P face of the inner membrane. IMP were arranged in very distinct rows conforming to the long axis of the parasite; the E face of this membrane had a few randomly distributed IMP. A prominent change in the sporozoite incubated in immune serum was the appearance of a layer of aggregated particles around the parasite. The P face of the plasma membrane had several clear areas devoid of IMP and IMP aggregates. No changes were seen in the other fractured faces of the pellicle. These observations suggest that immune serum acts only on the P face of the plasma membrane.     

An Electron Microscope-Cytochemical Method for Differentiating Membranes of Host Red Cells and Malaria Parasites*

Limiting membranes of malaria parasites and host red cells stain differently when exposed to positively charged iron colloid. Negatively charged red cell membranes avidly bind colloid, whereas parasite membranes do not. This selectivity in colloidal iron uptake by the 2 types of membranes can be utilized as an aid in discerning the amounts of contaminating host cell membranes in “free” malaria parasite preparations and in related cell-free membrane extracts.     

A Comparative Study of Gametocyte Ultrastructure in Avian Haemosporidia*

The fine structure of gametocytes of 3 avian haemosporidian parasites Plasmodium gallinaceum, Haemoproteus columbae, and Leucocytozoon simondi has been studied and compared by electron microscopy. The gametocytes of all 3 species are bounded by a 3-layered limiting membrane system, possess a cytostome during some portion of their residence within host cells, and their sex can be distinguished by both nuclear and cytoplasmic characteristics. L. simondi differs most significantly from P. gallinaceum and H. columbae in possessing large intranuclear granules, mitochondria associated with pocket infoldings of the nuclear envelope near the atypical centriole complex and compartmentalization of the cytoplasm by segments of closely aligned unit membranes. Further, the cytostome of L. simondi does not appear to be a persistent structure as in the other 2 species and pigment is not present within food vacuoles. L. simondi also is capable of infecting a wider variety of host cells and within leukocytes produces striations of the host nucleus and an apparent spiral banding of the host cell surface. The comparison of P. gallinaceum, H. columbae, and L. simondi gametocytes by electron microscopy leads to the conclusion that Plasmodium and Haemoproteus are more closely related to each other than either of them is on Leucocytozoon. The terminology used to describe certain organelles within the gametocyte's cytoplasm has been reexamined and the relationship of the nucleolus to parasite maturation also is described.     

Plasmodium falciparum Rhoptry Proteins of 140/130/110 kd (Rhop-H) Are Located in an Electron Lucent Compartment in the Neck of the Rhoptries

ABSTRACT. To investigate in more detail the structure of the high molecular weight rhoptry protein complex of Plasmodium falciparum, Rhop-H (140/130/110 kd), the complex was affinity purified from parasite extracts using rhoptry protein specific antisera prepared against Rhop-H proteins bound to and eluted from Balb/c mouse erythrocytes, using 0.5 M NaCl. The individual proteins (140 kd/Rhop-1, 130 kd/Rhop-2, and 110 kd/Rhop-3) were separated, electroeluted, and monospecific polyclonal antisera prepared against the individual proteins, and against the affinity purified complex. Immunofluorescence assays and immunoelectron microscopic studies were performed to verify the subcellular localization of the Rhop-H epitopes. Immunoblotting and immunoprecipitation assays were also performed. We report novel findings regarding the localization of the rhoptry proteins to an electron lucent compartment in the neck of the rhoptries. Analysis of the amino acid composition of the individually purified Rhop-H proteins demonstrated a predominance of negatively charged (E, D) as well as hydrophobic residues (L, A, P, S) in the three proteins. The percentage of negatively charged residues was high for all three proteins. Similarities in amino acid composition for the three proteins supports the previous data demonstrating shared properties such as erythrocyte and liposome binding, for the three proteins. Results of antibody characterizations using rhoptry protein specific antisera demonstrate the immunodominance of the Rhop-H complex.     

Establishment of a Murine Model for Metastasis of Cytokine-Producing Tumor to the Brain

The A375 cell line, derived from human malignant melanoma, has characteristics of interleukin-6 (IL-6) production. By using this cell line, we have investigated a murine metastasis model of IL-6-producing tumors to the brain by injecting A375 cells directly into the left cardiac ventricle. Nude mice were anesthetized with intraperitoneal injection of pentobarbital sodium. Next, A375 cells suspended in phosphate-buffered saline (PBS) were injected into the left cardiac ventricle of mice. An intracardiac injection of 10⁵ cells developed tumor colonies in the brain after 4 to 6 weeks. Metastatic cells were found in every lobe of the brain. An immunocytochemical study revealed IL-6 production by A375 cells at the metastatic sites in the brain. By the transfection of genes encoding proteins into A375 cells, a novel model of protein expression in the brain in vivo could be constructed. Our system does not require great skill. Our experimental model will facilitate future studies of the local effects of proteins in the brain.     

New Observations on Gametogenesis,Fertilization, and Zygote Transformation in Plasmodium gallinaceum1

The ultrastructure of the sexual stages of Plasmodium gallinaceum during gametogenesis, fertilization, and early zygote transformation is described. New observations are made regarding the parasitophorous vacuole (PV) of gametocytes and the process of emergence in male and female gametocytes. Whereas female gametocytes readily disrupted both the PV membrane and host cell plasmalemma during emergence, male gametocytes frequently failed to break down the plasmalemma of the host cell. New observations and hypotheses are presented on the behavior of the male gamete nucleus. Following fertilization, the male nucleus appears to travel through a channel of endoplasmic reticulum in the female gamete before fusing with the female nucleus at a region in which the nuclear envelope is thrown into extensive convoluted folds. Polarization of the zygote nucleus, in association with the appearance of a perinuclear spindle of cytoplasmic microtubules, preceded all other changes in the developing zygote. After nuclear polarization becomes apparent, electron-dense material is deposited beneath the zygote pellicle, and a canopy is formed which eventually extends over the entire apical end of the developing ookinete. As the apical end begins to extend outward, polar rings, micronemes, and subpellicular microtubules become visible in this portion and a “virus-like” inclusion known as a crystalloid is formed in the posterior portion of the zygote. When female gametes are prevented from being fertilized, the cytoplasm at 24 h after gametogenesis is devoid of most of those organelles found in the developing zygote or the mature ookinete. The cell is surrounded only by a single membrane. Although at various points beneath the membrane there are deposits of electron-dense material reminiscent of those deposited in the zygote, no further development of ookinete structures takes place in the unfertilized female gamete.     

Ultrastructure of the Erythrocytic Stages of Plasmodium malariae1

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.     

油菜叶绿体被膜的制备和Toc33的检测

采用蔗糖密度离心方法分离完整叶绿体,进一步分离叶绿体被膜,借助SDS-PAGE分析了2种油菜叶绿体被膜的蛋白组分.用对拟南芥叶绿体外被膜上存在Toc33的特异抗体,检测到油菜叶绿体被膜上存在Toc33转运蛋白.Toc33在2种油菜中的相对含量不同,黄化油菜叶绿体被膜中高于野生油菜叶绿体被膜.     

Structural Alteration of the Membrane of Erythrocytes Infected with Plasmodium falciparum1

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.