Radiolabelling of the excretory-secretory and somatic antigens of Anisakis simplex larvae.

Anisakis simplex larvae were cultured in vitro in medium containing 35S-methionine for ten days. The medium and the larval tissues were analysed for biosynthetically labelled polypeptide by sodium dodecyl sulphate polyacrylamide gel electrophoresis and autoradiography. Immunoprecipitates with positive and negative human antisera were similarly analysed, using Staphylococcus aureus to absorb immuno-complexes. ES products of Anisakis larvae contained many polypeptides with molecular weights of less than 200 K. 180 KDa and 40 KDa polypeptides in ES products reacted with IgG in Anisakis-infected human sera. Somatic extracts also contained many polypeptides with molecular weights of less than 200 K. One of these polypeptides with a molecular weight of 130 K reacted with IgG in Anisakis-infected human sera. These polypeptides did not react with other nematode-infected human sera.     

Studies on the polypeptides of varicella-zoster (V-Z) virus. 1. Detection of varicella-zoster virus polypeptides in infected cells.

Virus-induced polypeptides in cells infected with varicella-zoster virus (VZV) were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. When human embryonic lung (HEL) cells infected with the Oka strain of VZV were labelled with 35S-methionine or 14C-glucosamine from 40 hr to 46 hr after infection, at least 18 VZV-induced polypeptides and 10 glycoproteins could be identified in the infected cells. The molecular weights of the polypeptides and glycoproteins ranged from about 145,000 to 23,000, and from about 105,000 to 48,000, respectively. Lysates of VZV-infected cells were treated with specific antisera prepared in green monkeys or guinea-pigs, and analysed by SDS-PAGE and fluorography. In all, 33 polypeptides (with molecular weight of about 145,000 to 22,000) and 13 glycoproteins (molecular weight, about 105,000 to 38,000) were found in the immunoprecipitates. None of these polypeptides and glycoproteins were detected when infected cells cultured in the presence of phosphonoacetic acid (PAA) were treated in the same way.     

Plasmodium lophurae: Antibody-induced movement and capping of surface membranes of erythrocyte-free malarial parasites

Surface antigens of the avian malarial parasite, Plasmodium lophurae, and its host cell, the duckling erythrocyte, were visualized at the ultrastructural level using rabbit antisera and ferritin-labeled goat anti-rabbit IgG. Rabbit antisera to P. lophurae caused an aggregation of parasite and parasitophorous vacuole surface membrane antigens, a phenomenon known as capping. Capping required living plasmodia and did not occur if parasites had been fixed with glutaraldehyde prior to exposure to antisera. Antisera against duckling erythrocytes did not cross-react with erythrocyte-free malarial parasites, and did not form caps on the surface of the red blood cell. Antiplasmodial sera did not react with normal or malaria-infected red cells. These results suggest that surface membrane proteins of the intracellular plasmodium are capable of lateral movement.     

Plasmodium lophurae: membrane proteins of erythrocyte-free plasmodia and malaria-infected red cells

Plasma membranes of normal duckling erythrocytes were prepared by blender homogenization and nitro-en decompression. Surface membrane vesicles of red cells infected with the avian malaria Plasmodium lophurae were produced by nitrogen decompression. Membranes of erythrocyte-free malaria parasites were removed from cytoplasmic constituents by Dounce homogenization. These membranes were collected by centrifugation in a sucrose step gradient and purified on a linear sucrose gradient. Red cell membranes had a buoyant density of 1.159 g/cm3, whereas plasmodial membranes banded at 2 densities: 1.110 g/cm3 and 1.158 g/cm3. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the isolated red cell membranes revealed 7 major protein bands with molecular weights (MW) ranging from 230, 000 to 22,000, and 3 glycoprotein bands with MW of 160,000, 88,000 and 37,000. Parasite membranes also had 7 major bands with MW ranging from 100,000 to 22,000. No glycoproteins were identifiable in these membranes. The proteins of the surface membranes from infected red cells had MW similar to those from normal red cells; however, there was some evidence of a reduction in the amount of the high MW polypeptides. The red cell membrane contained 79 nmoles sialic acid/mg membrane protein, whereas plasmodial membranes had 8 nmoles sialic acid/mg membrane protein. The sialic acid content of the surface membranes of infected red cells was significantly smaller than that of normal cells. Lactoperoxidase-glucose oxidase-catalyzed iodination of intact normal and malaria-infected erythrocytes labeled 7 surface components. Although no observable differences in iodinatable proteins were seen in these preparations, there was a striking reduction in the iodinatability of erythrocytic membranes obtained from P. lophurae-infected cells. Erythrocyte-free plasmodia bound very little radioactive iodine; the small amount of radioactivity was distributed among 3 major bands with MW of 42,000, 32,000 and 28,000. It is suggested that the alterations of the surface of the P. lophurae-infected erythrocyte do not occur by a wholesale insertion of plasmodial membrane proteins into the red cell plasma membrane, but rather that there are parasite-mediated modifications of existing membrane polypeptides.     

Stress induced biosynthesis of a 31 kd-glycoprotein in goldfish brain

The de novo protein synthesis in goldfish brain has been studied under defined stress conditions and after training in an active swimming task. One- and two-dimensional gel electrophoresis of the brain proteins previously labelled in vivo with 35S-methionine indicated that the synthesis of one distinct protein increases after stress but not after successful training. This protein is a glycoprotein with an apparent molecular weight of 31 kd and an isoelectric point of pH 5. It is discussed that the protein may be the ACTH precursor "pro-opiomelanocortin".     

The Infection of Duck and Goose Erythrocytes by the Mammalian Malaria Parasite, Plasmodium berghei

The infection of mice and baby rats by both Plasmodium lophurae , an avian parasite, and Plasmodium berghei , a mammalian malaria parasite, prompted investigation of the likelihood of P. berghei infecting avian erythrocytes. Though erythrocytes of chick embryos were not infected, those of the goose and duck embryos were. In both these cells the morphology of the parasite was markedly different from that seen in mammalian erythrocytes. Infections were transitory and it was impossible to find parasites after 4 days. Examination of the hosts of both species of parasites showed a rather wide range and examination of the susceptibility of the duck erythrocyte indicated that this cell was peculiarly receptive to infection by a variety of plasmodia.     

Radiolabelling of DNA/polypeptide complexes in isolated bulk DNA and in residual nuclear matrix DNA by nick-translation

Conditions are described that allow 32P-radiolabelling and detection of tight complexes between DNA and polypeptides by nick-translation. Prolonged nick-translation of purified bulk DNA results in radiolabelled complexes migrating on SDS-polyacrylamide gels with apparent molecular weights of 68 kd and 54 kd respectively. Residual nuclear matrix DNA which is not accessible to DNase I on the nuclear level becomes accessible to radiolabelling by nick-translation on the nuclear matrix level. In this case the in situ radiolabelled complexes migrate on SDS-polyacrylamide gels with apparent molecular weights of 68 kd and 100 kd. The DNA/polypeptide complexes are stable during treatments with SDS, beta-mercapto ethanol and alkali which points to covalent bonds between the polypeptides and DNA strands.     

Translation products of mRNA from infective larvae of Trichinella spiralis include an antigenic polypeptide

Total RNA was extracted from packed infective larvae of Trichinella spiralis by centrifugation through a 5.7 M caesium chloride cushion. Polyadenylated messenger RNA was separated from total RNA in an oligothymidylic acid-cellulose gel column. The in vitro translation of the mRNA, isolated from infective larvae of T. spiralis, was carried out using the rabbit reticulocyte cell-free translation system. Incorporation of 35S-methionine into the trichloroacetic acid precipitates in the lysate containing mRNA was 5 times greater than that in control. The translation products were analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography. Many polypeptides with molecular weights of less than 100,000 were synthesized in the lysate. A T. spiralis positive mouse serum was mixed with translation products to form antigen-antibody complexes, which were then absorbed by Staphylococcus aureus Cowan 1 strain and analysed by autoradiography of SDS-PAGE. An antigenic polypeptide with a molecular weight of 48,000 was demonstrated to react specifically with IgG antibody in T. spiralis positive mouse serum. T. spiralis larvae were cultured in methionine-free medium containing 35S-methionine, and antigenic polypeptides in somatic extracts and ES products were compared with those in translation products by autoradiography of SDS-PAGE. Several polypeptides in ES products and somatic extracts reacted specifically with IgG antibodies in positive serum. Especially the polypeptide with a molecular weight of 48,000 in ES products strongly reacted with IgG antibody in positive serum.     

Isolation and partial characterization of haemagglutinins from plasmodia of Physarum polycephalum.

The soluble haemagglutinins produced by plasmodia of Physarum polycephalum were purified by chromatographic methods and resolved into haemagglutinins I and II. On SDS-PAGE, purified haemagglutinins I and II each gave a single band with an apparent molecular mass of 6 and 11 kDa, respectively. The results of gel-filtration chromatography suggested that both haemagglutinins were dimers of the respective subunits under non-denaturing conditions. Rabbit erythrocytes were preferentially agglutinated by both haemagglutinins. The human type A, B and O erythrocytes were agglutinated by haemagglutinin II to an equal degree but were not agglutinated by haemagglutinin I. Simple sugars failed to inhibit the activities of both haemagglutinins. The activities, however, were effectively inhibited by the addition of thyroglobulin. Other glycoproteins such as fetuin, orsomucoid and transferrin inhibited the activity of haemagglutinin I but not that of haemagglutinin II. These haemagglutinins were detected in a slime fraction obtained from the culture media of starved plasmodia, suggesting that they are released to the outside of the plasmalemma to become associated with the slime layer on the plasmodial surface.     

Plasmodium lophurae: Membrane Proteins of Erythrocyte-free Plasmodia and Malaria-Infected Red Cells*

SYNOPSIS. Plasma membranes of normal duckling erythrocytes were prepared by blender homogenization and nitrogen decompression. Surface membrane vesicles of red cells infected with the avian malaria Plasmodium lophurae were produced by nitrogen decompression. Membranes of erythrocyte-free malaria parasites were removed from cytoplasmic constituents by Dounce homogenization. These membranes were collected by centrifugation in a sucrose step gradient and purified on a linear sucrose gradient. Red cell membranes had a buoyant density of 1.159 g/cm³, whereas plasmodial membranes banded at 2 densities: 1.110 g/cm³ and 1.158 g/cm³. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the isolated red cell membranes revealed 7 major protein bands with molecular weights (MW) ranging from 230,000 to 22,000, and 3 glycoprotein bands with MW of 160,000, 88,000 and 37,000. Parasite membranes also had 7 major bands with MW ranging from 100,000 to 22,000. No glycoproteins were identifiable in these membranes. The proteins of the surface membranes from infected red cells had MW similar to those from normal red cells; however, there was some evidence of a reduction in the amount of the high MW polypeptides. The red cell membrane contained 79 nmoles sialic acid/mg membrane protein, whereas plasmodial membranes had 8 nmoles sialic acid/mg membrane protein. The sialic acid content of the surface membranes of infected red cells was significantly smaller than that of normal cells. Lactoperoxidase-glucose oxidase-catalyzed iodination of intact normal and malaria-infected erythrocytes labeled 7 surface components. Although no observable differences in iodinatable proteins were seen in these preparations, there was a striking reduction in the iodinatability of erythrocytic membranes obtained from P. lophurae-infected cells. Erythrocyte-free plasmodia bound very little radioactive iodine; the small amount of radioactivity was distributed among 3 major bands with MW of 42,000, 32,000 and 28,000. It is suggested that the alterations of the surface of the P. lophurae-infected erythrocyte do not occur by a wholesale insertion of plasmodial membrane proteins into the red cell plasma membrane, but rather that there are parasite-mediated modifications of existing membrane polypeptides.     

Modulation of the Host's Immune Response to Plasmodium berghei by a Parasite-Derived Immunosuppressive Factor

ABSTRACT. It is well known that Plasmodium -infected hosts are immunosuppressed, as shown by their depressed immune responsiveness to a variety of antigens. It is not known, however, whether the immune response of malaria-infected animals to the malarial parasite itself is suppressed. The availability of a noninfectious, immunosuppressive factor (ISF) derived from Plasmodium berghei -infected rat erythrocytes made it possible to investigate this question. Mice infected with P. berghei and injected with the ISF had higher levels of parasitemia and shorter survival times than control mice that were similarly infected but were treated with control material derived from noninfected rat erythrocytes or with saline solution. Conversely, mice immunized against the ISF and then infected with P. berghei had lower parasitemias and longer survival times than mice immunized with the control material or with saline solution. We conclude that immunosuppression in murine malaria affects the course of malaria infection.     

Purification of the major UsnRNPs from broad bean nuclear extracts and characterization of their protein constituents.

Small nuclear ribonucleoprotein particles containing the five major nucleoplasmic snRNAs U1, U2, U4, U5 and U6 as well as two smaller sized snRNAs were purified from broad bean nuclear extracts by anti-m3G, monoclonal antibody, immunoaffinity chromatography. We have so far defined 13 polypeptides of approximate mol. wts. of 11 kd, 11.5 kd, 12.5 kd, 16 kd, 17 kd, 17.5 kd, 18.5 kd, 25 kd (double band), 30 kd, 31 kd, 35 kd, 36 kd and 54 kd. Upon fractionation of the UsnRNPs by anion exchange chromatography, essentially pure U5 snRNPs were obtained, containing the 11 kd, 11.5 kd, 12.5 kd, 16 kd, 17 kd, 17.5 kd, 35 kd and 36 kd polypeptides. These may therefore represent the common snRNP polypeptides and which may also be present in the other snRNPs. By immunoblotting studies, using anti-Sm sera and mouse monoclonal antibodies we show that the 35 kd and 36 kd proteins are immunologically related to the mammalian common B/B' proteins. The broad bean 16 kd and 17 kd proteins appear to share structural elements with the mammalian D protein. The three proteins of mol. wts. 11 kd, 11.5 kd and 12.5 kd probably represent the broad bean polypeptides E, F, and G. Cross-reactivity of proteins of mol. wts of 30 kd and 31 kd with Anti-(U1/U2)RNP antibodies suggests that they may represent the broad bean A and B" polypeptides. The 54 kd protein and the 18.5 kd protein could be candidates for the U1 specific 70 k and C polypeptides. Our results demonstrate a strong similarity between the overall structure of broad bean and mammalian snRNPs.     

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.     

Isolation of messenger ribonucleoproteins from mammalian cells

EDTA-dissociated polysomes from normally growing and adenovirus-infected KB cells were fractionated by affinity chromatography on oligo(dT)-cellulose into an unadsorbed fraction containing the ribosomal subunits and a second fraction which bound to the adsorbent. The latter fraction, recovered from the oligo(dT)-cellulose by elution with formamide in a buffer containing 0.2 m-NaCl, was shown to contain messenger RNA together with protein. Evidence was obtained that a substantial part (50%) of ³⁵S-labelled protein was associated with the mRNA when eluted from the oligo(dT)-cellulose, and that up to 75% of the labelled protein was mRNA-associated after dilution of the eluted material in buffer not containing formamide. As judged from the characteristic gel electrophoresis pattern of adenovirus-specific mRNA derived from such messenger ribonucleoprotein complexes, the mRNA remained intact during the isolation procedure. Analysis of the fraction containing the majority of the messenger ribonucleoprotein complexes on sodium dodecyl sulfate-polyacrylamide gels showed a specific pattern of labelled polypeptides, which in the case of material from uninfected cells consisted of four major labelled polypeptides; approximate molecular weights 56,000, 68,000, 78,000 and 130,000. When the material from the adenovirus infected cells was analysed a set of four polypeptides, which migrated identically to those of the uninfected cells, was found. However, in addition to this apparently common set of polypeptides, the material from the infected cells, harvested late in the infectious cycle, contained one extra polypeptide with an approximate molecular weight of 110,000.     

Babesia argentina: observations on the immunogenicity of the cryofibrinogen complex.

Rabbit antisera were prepared against the soluble and insoluble fractions of a cryofibrinogen complex that formed in plasma of cattle acutely infected with Babesia argentina. Analyses of the rabbit antisera indicated that the cryofibrinogen complex contained proteins from erythrocytes and parasites as well as fibrinogen and related proteins.     

The Duffy receptor family of Plasmodium knowlesi is located within the micronemes of invasive malaria merozoites

Plasmodium vivax and Plasmodium knowlesi merozoites invade human erythrocytes that express Duffy blood group surface determinants. A soluble parasite protein of 135 kd binds specifically to a human Duffy antigen. Using antisera affinity purified on the 135 kd protein, we cloned a gene that encodes a member of a P. knowlesi family of erythrocyte binding proteins. The gene is a member of a family that includes three homologous genes located on separate chromosomes. Two genes are expressed as major membrane-bound products that give rise to soluble erythrocyte binding proteins: the 135 kd Duffy binding protein and a 138 kd protein that binds only rhesus erythrocytes. These different erythrocyte binding specificities may result from sequence divergence of the homologous genes. The Duffy receptor family is localized in micronemes, an organelle found in all organisms of the phylum Apicomplexa.     

Plasmodium berghei ookinetes bind to Anopheles gambiae and Drosophila melanogaster annexins

Using a proteomic approach we identified polypeptides from Anopheles gambiae and Drosophila melanogaster protein extracts that selectively bind purified Plasmodium berghei ookinetes in vitro; these were two and three distinct polypeptides, respectively, with an apparent molecular weight of about 36 kDa. Combining two-dimensional electrophoresis and MALDI-TOF (matrix-associated laser desorption ionization time of flight) mass spectrometry we determined that the polypeptides correspond to isomorphs of the annexin B11 protein of the fruit fly. When protein extracts derived from A. gambiae and D. melanogaster tissue culture cells were further fractionated, the binding activity matching the annexin protein could be localized in the fraction derived from cell membranes in both diptera. Antibody staining showed that annexin also binds to ookinetes during the invasion of the mosquito midgut. Finally, inclusion of antiannexin antisera in a mosquito blood meal impaired parasite development, suggesting a facilitating role for annexins in the infection of the mosquito by Plasmodium.     

Immunoelectrophoretic analysis of erythrocytic stages of Plasmodium yoelii and P. chabaudi

Triton X-100 extracts of erythrocytes infected with Plasmodium chabaudi and P. yoelii were analysed in crossed immunoelectrophoresis with rabbit antisera. The parasite origin of the antigens detected was assessed by metabolic radiolabelling of the parasites with 35S-methionine. About 12 immunoprecipitates were obtained with both extracts and their homologous antiserum. Cross-tests showed that the two parasite strains were very similar antigenically. Species-specific antigens could, however, also be demonstrated. Two antigens, present on both P. yoelii- and P. chabaudi-infected erythrocytes, were located on the surface of the host cell membrane as judged from 125I-labellings with lactoperoxidase. Experiments with phenyl-Sepharose showed that most of the antigens detected were hydrophilic and none of them reacted with concanavalin A.     

Neurofilament Proteins in Cultured Chromaffin Cells

Antibodies were raised against the 200-kd, 145-kd, and 68-kd subunits of a rat neurofilament preparation. Immunoblots showed that each antibody was specific for its antigen and that it did not cross-react with any of the two other neurofilament polypeptides. Use of the three antibody preparations to stain bovine chromaffin cells in culture by the indirect immunofluorescence technique indicated that the three neurofilament polypeptides are present in chromaffin cells maintained in culture for 3 or 7 days. The three anti-neurofilament antibodies labelled the cells in a similar pattern: very thin filaments specifically localized around the nucleus were observed whereas neurites and growth cones, developed by cultured chromaffin cells, were generally not stained. Some fibroblasts were present in our cultures but they were never stained by any of the neurofilament antibodies. This indicated that the antibodies used do not react with vimentin, the major intermediate filament protein found in fibroblasts. The three neurofilament antibodies were also used to immunoprecipitate specifically three proteins of molecular weights 210 kd, 160 kd, 70 kd from solubilized extracts of cultured chromaffin cells that were radiolabelled with [35S]methionine. These proteins correspond in molecular weight to the neurofilament triplet found in bovine brain. Finally, the presence of neurofilaments in freshly isolated chromaffin cells was tested by immunoblotting using the 68-kd antibody. A 70-kd protein was specifically stained by this antibody, suggesting that neurofilaments are not only present in cultured chromaffin cells but also in the adrenal gland in vivo. It is concluded from these results that chromaffin cells contain completely assembled neurofilaments. This additional neuronal property again illustrates that chromaffin cells are closely related to neurons and therefore represent an attractive model system for the study of functional aspects of adrenergic neurons.     

Alkali-extraction of membranes from mouse erythrocytes infected with Plasmodium berghei

Alkali-extracted membrane material from hypotonically lysed Plasmodium berghei-infected mouse erythrocytes has been analyzed by dodecyl sulphate polyacrylamide gel electrophoresis and fractionated by discontinuous sucrose gradient centrifugation. Components characteristic for the protein pattern associated with P. berghei infection have been recovered in the alkali-extracted material. Parasite components, free of host cell membrane contamination, have been obtained as a high-density fraction from sucrose gradients. This fraction is characterized by polypeptides with apparent molecular weight of 71,000 and 52,000.