A postsynaptic density (PSD) fraction, including some adherent subsynaptic web material, has been isolated from dog cerebral cortex by a short-procedure modification of the methods of Davis and Bloom (21,22) and Cotman and Taylor (20), using Triton X-100. The fraction has been visualized by thin-section, replica, and negative (phosphotungstic acid) staining electron microscopy and its proteins separated by high-resolution SDS gel electrophoresis. Morphologically, the preparation seems to be quite pure, with very little membrane contamination. The density is composed of protein, no nucleic acids, and very little phospholipid being detectable. The fraction had no ATPase or GTPase activity, but it did have a very small amount of cytochrome c oxidase activity (of a specific activity less than 0.5 % that of a mitochondrial fraction) and a small amount of 5'-nucleotidase activity (of a specific activity between 6 and 7% that of a synaptic membrane fraction). Electron micrographs reveal cup-shaped structures -400 nm long and -40 nm wide, made up of apparent particles 13-28 nm in diameter. However, en face views, and particularly micrographs of replicas and PTA-stained preparations, reveal a disk-shaped structure, outside diameter -400 nm, in which filaments are seen to extend from the central part of the density. High resolution gel electrophoresis studies indicated some 15 major proteins and perhaps 10 more minor ones; the predominant protein had a mol wt of 51,000, followed by ones at 45,000, 40,000, 31,000, 26,000, and several at 100,000. A comparison by gel electrophoresis of density fraction proteins with those of a lysed synaptosomal membrane fraction containing some adherent densities indicated some comigrating proteins, but the major membrane fraction protein, mol wt 52,000, was not found in the density fraction. Antibodies raised against the density fraction reacted with a preparation of solubilized synaptic membrane proteins. By both these criteria, it was considered that the density and the synaptic membrane have some proteins in common. By separately mixing 125I-labeled myelin, synaptic vesicle, and mitochondrial fraction protel'~S with synaptosomes, and then isolating the density

Monolayers of human erythrocytes (E) infected with Plasmodium falciparum were briefly fixed with 1% glutaraldehyde and air dried. They were then exposed to sera from patients with P. falciparum malaria or from donors immune to this parasite and tested in an indirect immunofluorescence assay (IFA). Parasites in infected E were made visible by counterstaining with ethidium bromide. Immunofluorescence (IF) was restricted to the surface of infected E. No antibody binding was detected unless the E were dried, suggesting that the relevant antigens were not available on the outer layers of the E surface. Staining over large parts of the E surface was seen already when the merozoite penetrated noninfected cells and was strong in E containing early stages of the parasite (rings, trophozoites). It was weak or absent from E containing schizonts. Antibodies in sera from different parts of Africa, Colombia, or Sweden reacted similarly with E infected with a Tanzanian P. falciparum strain kept in culture for many years and with parasitized E freshly drawn from African, Swedish, or Colombian patients. All sera from residents of a holoendemic area (Liberia) were IFA positive. In contrast, some sera from Colombian or Swedish patients with primary infection gave negative results. The results of the IFA and of an enzyme-linked immunosorbent assay in which fixed and dried E were the targets were well-correlated, suggesting that the same antibodies were detected by these assays. The antigens involved in the IFA were susceptible to pronase but not to trypsin or neuraminidase. E surface IF was inhibited by lysates of infected E, merozoite extracts, or soluble antigens present in P. falciparum culture supernatants but not by lysates of normal E or ghost extracts. The inhibitory antigens were heat stable (100 degrees C, 5 min). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting of either antigen-enriched preparations from culture supernatants or merozoite extracts showed that antibodies eluted from monolayers of infected E reacted consistently with a predominant polypeptide of Mr 155,000 and two to four minor polypeptides of lower molecular weights. Metabolic labeling of the parasites with 75Se-methionine indicated that these antigens were parasite derived. We conclude that the antigens involved in these reactions are released from bursting schizonts or merozoites and are deposited in the E membrane in the course of invasion.(ABSTRACT TRUNCATED AT 400 WORDS)

Erythrocytes infected with late asexual stages of Plasmodiumfalciparum and P. fragile may form spontaneous rosettes with uninfected erythrocytes (1-3). Here we present a detailed study of rosette formation in P. falciparum malaria .Materials and Methods Assessment ofRosetting . P. falciparum parasites were cultured in vitro under standard conditions . Infected erythorcytes (Ei) were mixed with acridine orange, and E; that had bound two or more noninfected erythrocytes (E.) were scored as rosetting. Blood samples drawn from patients with a blood smear positive for P. falciparum were washed and cultured in vitro. The number of rosettes was counted after 35 h and expressed as a percentage of the total number of trophozoite-or schizont-containing erythrocytes.Enrichment ofRosetting and Nonrosetting Parasites. Cultures containing mature parasite-infected erythrocytes were layered on Ficoll-Isopaque (FIP) and centrifuged for 5 s at room temperature (RT). The cells that passed through the FIP layer were collected in a pellet, washed twice in RPMI 1640, and cultured as described . For enrichment of nonrosetting E;, the cultures were layered over 60% Percoll and centrifuged (500 g) at RT for 20 min. The layer at the interface was collected and washed twice in RPMI 1640 and cultured as above. Rosette-forming parasites were cloned by limiting dilution and screened for rosetting on day 10-14.EM. Erythrocytes from P. falciparum cultures were fixed with 2 17c glutaraldehyde in 0.05 M phosphate buffer containing 4% sucrose (pH 7.4) for 1 h, followed by embedding and cutting procedures as described by Aikawa et al. (4) .Effect ofEnzymes on Rosetting Parasites. Cultures containing rosetted Ei were washed three times in RPMI 1640, treated with 10 or 100 ug/ml of trypsin for 15 min at 37°C, and subsequently washed . In some experiments, trypsinized E; were cultured in complete medium for observation of reappearance of rosettes . Experiments with neuraminidase were performed as above using 0.2, 1, and 5 U/ml, respectively. Cytoadherent Parasites . The assay for cytoadherence of E; to melanoma cells (ATCC 1585, C32r; American Type Culture Collection, Rockville, MD) was performed as described elsewhere (5) . To enrich for cytoadherent parasites, P. falciparum cultures were incubated with melanoma cells grown in a tissue culture flask at RT for 1-2 h with continuous rotation . The bound erythrocytes were retrieved by flushing the melanoma cells with culture medium .

The subcellular distribution of Proteins la and lb, two proteins which serve as specific substrates for protein kinases present in mammalian brain, was studied in the dog cerebral cortex. Proteins Ia and Ib were found to be most highly enriched in synaptic vesicle fractions ; they were also present in postsynaptic density and synaptic membrane fractions in significant amounts, Proteins la and Ib present in the synaptic vesicle fraction appear to be similar, if not identical, to those present in the postsynaptic density fraction as judged by several criteria : (a) the ability to serve as substrate for cAMP-dependent protein kinase, (b) electrophoretic mobility in the presence of sodium dodecyl sulfate, (c) extractability with NH4 C1 or EGTA, and (d) fragmentation to electrophoretically similar peptides by a purified Staphylococcus aureus protease . In addition, the postsynaptic density fraction has been found to contain cAMP-dependent Protein Ia and Protein Ib kinase activity . The subcellular localization of Proteins la and Ib suggests a role for these proteins in the physiology of the synapse .

IgG from a donor clinically immune to Plasmodiumfalciparum malaria strongly inhibited reinvasion in vitro of human erythrocytes by the parasite. When added to monolayers of glutaraldehyde-fixed and air-dried erythrocytes infected with the parasite, this IgG also displayed a characteristic immunofluorescence restricted to the surface of infected erythrocytes. Elution of the IgG adsorbed to such monolayers gave an antibody fraction that was 40 times more efficient in the reinvasion inhibition assay (50% inhibition titer, <1 ,ug/ml) than the original IgG preparation. falciparum (1, 2). These antigens are released from bursting schizonts or merozoites and are deposited in the RBC membrane in the course of invasion. Antibodies recognizing these antigens are of high prevalence in sera from residents of a holoendemic area of Africa (Liberia) but are also present in many sera of acutely infected patients from different parts of the world. A polypeptide of Mr 155,000 (Pf 155) appears to be primarily responsible for this RBC membrane IF. A similar antigen of the same apparent molecular weight has also been found recently by two other groups (3, 4). To further investigate the function of these antigens and the possible significance of the immune response against them, we have now tested the antibodies reacting with Pf 155 and related minor components for their capacity to inhibit RBC invasion by merozoites in vitro. MATERIAL AND METHODSParasites. Parasites were from a Tanzanian strain of P. falciparum (F 32) isolated in 1978 (5) and cultured in vitro in blood group O RBCs (6).Immune Sera. Five sera (Kinon, IS-6 to -8, X-12) were from Liberians, >15 years old (except X-12, from a 12-yearold boy), living in a P. falciparum holoendemic area in which in adult life clinical illness is rare and parasitemia is kept at a low-grade, mostly subpatent, level despite high sporozoite inoculation rates (7). These donors, designated as clinically immune, had not taken any antimalarial drug for the last 6 weeks (X-12) or longer. The 6 remaining donors had acute P. falciparum infections. Four donors (ASF, MV, JOR, FG) were South Americans from an endemic area of Colombia. Two Swedish patients (YC, HP) were suffering from a first infection acquired in Kenya. IgG was prepared by ammonium sulfate precipitation and ion-exchange chromatography on DEAE-Sephadex (Pharmacia). Immunoglobulin concentrations were determined by ELISA.In Vitro Growth Inhibition Assay. Infected RBCs from P. falciparum cultures (5-10% parasitemia, -70% schizonts) were diluted with normal O RBCs to a parasitemia of 0.5%. They were adjusted to 2% hematocrit with Hepes-buffered (20 mM) RPMI-1640 medium (GIBCO) containing 15% normal human serum, 2 mM glutamine, 25 pg of gentamycin per ml, and 0.2% NaHCO3 [complete tissue culture medium (TC medium)]. In some experiments, synchronized parasite cultures were used. In this case, the original cultures (5-10% parasitemia) were adjusted to 10% hematocrit and layered on top of 2.5 ml of 60% Percoll (Pharmacia) diluted in com...