Schistosoma mansoni: origin and expression of a tegumental surface antigen on the miracidium and primary sporocyst

A monoclonal antibody, recognizing a carbohydrate epitope associated with several tegumental surface components on Schistosoma mansoni primary sporocysts, was used to follow tegumental formation during transformation of the miracidium to sporocyst and its subsequent development in vitro and in vivo. Indirect fluorescent antibody and direct immunogold labeling methods confirm a structural connection between the intercellular ridges and a submuscular, multinucleate syncytium in the miracidium. Immunoreactive vesicles within this latter system directly contribute to elaboration of the tegumental surface membrane, through the process of membrane fusion. Lateral expansion of intercellular ridges by vesicular fusion ultimately result in fully transformed sporocysts exhibiting vesicular membrane epitopes as prominent tegumental surface components. Light microscopical and ultrastructural observations, together with Western immunoblot analyses, suggest a gradual depletion of intracellular and surface immunoreactive material of vesicular origin in primary sporocysts grown in culture for up to 12 days. In contrast, similar immunoreactive vesicles appear to be continuously synthesized throughout in vivo primary sporocyst development. Monoclonal antibody reactive epitopes appear to be uniquely expressed in the miracidium/primary sporocyst since similar molecules are absent from daughter sporocysts, cercariae, adults, and snail tissues.     

Identification of human sperm surface glycoproteins recognized by autoantisera from immune infertile men, women, and vasectomized men

To identify the surface antigens of human sperm recognized by antisera from immune infertility patients and vasectomized men, we labeled sperm surface proteins with 125I- and used patient antisera for immunoprecipitation. Sera were studied from 27 infertile males, 18 infertile females, and 4 vasectomized males, each possessing anti-sperm antibodies detected by immunobead binding. Sera from different infertile males, different infertile females, and vasectomized males were remarkably similar in their surface antigen recognition. The different sera specifically immunoprecipitated the same small group of 125I-labeled surface proteins, which included polypeptides in the region 90 kDa, 40-45 kDa, and 26 kDa. Treatment with N-glycanase showed that the proteins of 90 kDa, 40-45 kDa, and 26 kDa were glycoproteins with N-linked carbohydrate. The immunoprecipitated 125I-labeled proteins and the total extract of 125I-labeled surface proteins were compared on two-dimensional (2D) gels. The results show the 90 kDa polypeptide is a major sperm surface component, whereas 40-45 kDa and 26 kDa polypeptides are minor components. The 2D gel comparison also indicates that 90 kDa, 40-45 kDa, and 26 kDa are a small subset of the total ensemble of sperm surface proteins. Clinical data suggest antibodies to these few proteins interfere with sperm function.     

Surface membrane proteins of Biomphalaria glabrata embryonic cells bind fucosyl determinants on the tegumental surface of Schistosoma mansoni primary sporocysts

Previous observations that in vitro adherence of Biomphalaria glabrata embryonic (Bge) cells to sporocyst larval stages of Schistosoma mansoni was strongly inhibited by fucoidan, a sulfated polymer of L-fucose, suggested a role for lectinlike Bge cell receptors in sporocyst binding interactions. In the present investigation, monoclonal antibodies with specificities to 3 major glycan determinants found on schistosomes, LacdiNAc, fucosylated LacdiNAc (LDNF), and the Lewis X antigen, were used in adhesion blocking studies to further analyze the molecular interactions at the host-parasite interface. Results showed that only the anti-LDNF antibody significantly reduced snail Bge cell adhesion to the surface of sporocysts, suggesting that fucosyl determinants may be important in larval-host cell interactions. Affinity chromatographic separation of fucosyl-reactive Bge cell proteins from fucoidan-bound Sepharose 4B revealed the presence of polypeptides ranging from 6 to 200 kDa after elution with fucoidan-containing buffer. Pre-elution of the Bge protein-bound affinity column with dextran (Dex) and dextran sulfate (DexS) before introduction of the fucoidan buffer served as controls for protein binding based on nonspecific sugar or negative charge interactions. A subset of polypeptides (approximately 35-150 kDa) released by fucoidan elution was identified as Bge surface membrane proteins, representing putative fucosyl-binding proteins. Far-western blot analysis also demonstrated binding reactivity between Bge cell and sporocyst tegumental proteins. The finding that several of these parasite-binding Bge cell proteins were also fucoidan-reactive suggests the possible involvement of these molecules in mediating cellular interactions with sporocyst tegumental carbohydrates. It is concluded that Bge cells have surface protein(s) that may be playing a role in facilitating host cell adhesion to the surface of schistosome primary sporocysts through larval fucosylated glycoconjugates.     

Leishmania tropica: characterization of a lipophosphoglycan-like antigen recognized by species-specific monoclonal antibodies.

Species-specific monoclonal antibodies to Leishmania tropica, T11 and T13-15, recognize membranal and secreted antigens. The membrane form of the antigen migrates on sodium dodecyl sulfate-polyacrylamide gels with a diffuse molecular weight from 15 to 50 kDa and can be labeled with palmitic acid, myoinositol, galactose, glucosamine, and inorganic phosphate. Both phosphate and sugar-labeled material were isolated from metabolically labeled promastigotes by affinity chromatography on antibodies coupled to Sepharose 4B. No binding to Ricinus communis agglutinin was observed. This material behaves like lipophosphoglycans from other Leishmania but contains unique species-specific epitopes. It is susceptible to cleavage by phospholipase C and after digestion no longer partitions into the detergent phase following a Triton X-114 extraction. All four monoclonal antibodies appear to recognize a carbohydrate epitope on the lipophosphoglycan since periodate treatment of this material bound to nitrocellulose essentially eliminated antibody binding. In addition, T15 binding could be blocked by 5 mM mannose-6-PO4 and fructose-1- or 6-PO4, but not by mannose, glucose, fructose, or the additional PO4 derivatives examined. The antibodies recognize a similar but not identical epitope, as demonstrated by a competitive radioimmunoassay using 125I-labeled T11, T13, and T15. Expression of surface antigen is elevated during the promastigote stationary phase.     

Differential binding of hemolymph proteins from schistosome-resistant and -susceptible Biomphalaria glabrata to Schistosoma mansoni sporocysts

Humoral factors have been associated with resistance of Biomphalaria glabrata to infection by Schistosoma mansoni. The goal of this study was to determine which serum (cell-free hemolymph) proteins bind to the surface of S. mansoni sporocysts. For this, 125I-labeled serum from schistosome-resistant (10-R2) and -susceptible (M-line) B. glabrata was incubated with sporocysts, washed, and then subjected to SDS-PAGE and autoradiography. Other samples examined included radiolabeled 10-R2 and M-line serum, sporocysts incubated with unlabeled serum followed by incubation with radiolabeled serum, and radiolabeled sporocysts. Results indicated that many polypeptides in the serum from both strains of B. glabrata were radiolabeled. Dominating both profiles were bands in the 90-210-kDa range. However, some differences between the serum of the 2 snail strains were observed with M-line serum having several radiolabeled polypeptides in the 31-40- and 66-85-kDa range that were absent in serum from 10-R2 B. glabrata. When sporocysts were incubated with radiolabeled serum, 3 polypeptides (116, 180, 210 kDa) from both snail strains bound to the surface of the parasite. Further, a 55-kDa polypeptide bound to sporocysts incubated with 10-R2 serum but did not bind to those parasites incubated with M-line serum. Preincubation of sporocysts with unlabeled serum prior to incubation with radiolabeled serum significantly inhibited the uptake of radiolabeled proteins. This differential binding of serum polypeptides from different strains of B. glabrata may be important in determining resistance or susceptibility of the snail to larval schistosome infection.     

The human lymphocyte function-associated (HLFA) antigen and a related macrophage differentiation antigen (HMac-1): functional effects of subunit-specific monoclonal antibodies

The structural and functional relations between the alpha- and beta-subunits of the human lymphocyte function-associated antigen (HLFA) and the human Mac-1 antigen (HMac-1) have been analyzed with the use of five monoclonal antibodies that react with these proteins. The specificities of these antibodies were examined by immunoprecipitation of proteins from 125I-labeled cells and purified HLFA and HMac-1 antigens. Three antibodies reacted with the Mr 95,000 common beta-subunit of the proteins, and also co-precipitated the Mr 175,000 HLFA alpha-subunit, the Mr 165,000 HMac-1 alpha-subunit, and a third polypeptide alpha-subunit of Mr 150,000. The other antibodies were specific to noncross-reactive epitopes present on the alpha-subunits of HLFA or HMac-1. These specificities were confirmed in sequential immunoprecipitation studies. Peptide mapping showed that the beta-subunits of HLFA and HMac-1 were identical, whereas the two alpha-subunits differed considerably. The HLFA alpha-subunit-specific monoclonal antibody inhibited phytohemagglutinin stimulation, the mixed lymphocytes reaction, cytolytic T lymphocyte-mediated killing, and tetanus toxoid stimulation, but did not affect natural killer cell-mediated killing or complement receptor type 3 function. The HMac-1 alpha-subunit-specific monoclonal antibody inhibited complement receptor type 3 function but had no effect on T cell or natural killer cell functions. Three monoclonal antibodies to the beta-subunit inhibited all functions tested, including T cell, natural killer cell, and complement receptor type 3 activities. The results suggest that the functions of the HLFA and HMac-1 molecules may be determined by the alpha-subunit, and that the common beta-subunit also bears functionally important epitopes.     

Schistosoma mansoni: two-dimensional gel electrophoretic analysis of antigens uniquely immunoreactive with protective rat serum

Candidate vaccine antigens are defined by their differential immunoreactivity with antisera which are distinguishable by their capacity to confer passive resistance to infection. This "contrasting antisera" immunoassay has been successfully used in previous analyses of 4-week-old worm biosynthetically radiolabeled Schistosoma mansoni proteins to identify potentially protective antigens. Twice-infected Fischer (F-2x) and Wistar-Furth (W-2x) rat sera were the sources of protective and non-protective antibody, respectively. We have extended our original analysis by applying two-dimensional gel electrophoresis to resolve total and immunoreactive soluble proteins of the 4-week worms. Total proteins were characterized by silver staining and autoradiography. Radiolabeled protein antigens immunoprecipitated by F-2x and W-2x antisera were compared, and several were shown to be uniquely reactive with the protective immune serum. In a companion molecular approach to clone the candidate vaccine antigens, screening of a lambda gt11 adult S. mansoni cDNA expression library by the contrasting antisera assay has identified a clone (lambda 40) producing a fusion protein with epitopes uniquely reactive with F-2x. A rabbit antiserum to the lambda 40 fusion protein (anti-FP40) reacted with radiolabeled worm proteins in the 20-kDa size range. By 2D gel electrophoretic analysis, we can now demonstrate that anti-FP40 specifically immunoprecipitates most of the members of a multicomponent protein antigen subset 18-22 kDa in Mr, focusing over a pI range of 5.3-5.8, and recognized uniquely by F-2x.     

Identification of the outer membrane proteins of Campylobacter pyloridis and antigenic cross-reactivity between C. pyloridis and C. jejuni

The outer membrane and surface exposed proteins of four strains of the gastric Campylobacter-like organism Campylobacter pyloridis were identified by SDS-PAGE of Sarkosyl-insoluble membranous material and 125I-surface-labelled whole bacteria. Although constant outer membrane proteins (molecular mass 61, 54 and 31 kDa) were observed in these strains, several variable 125I-labelled surface proteins were detected. C. pyloridis does not appear to express a single surface-exposed major outer membrane protein like that of C. jejuni and C. coli. Putative flagella proteins were identified from isolated flagella and acid-extractable surface material and by immunoblotting with anti-flagella antibodies. Several major protein antigens were observed by immunoblotting with anti-C. pyloridis antisera. At least two of these antigens cross-reacted with anti-C. jejuni antiserum. This cross-reaction appears to be caused primarily by flagellar antigens. However, one major protein antigen (61 kDa) was not cross-reactive with C. jejuni and may, therefore, be useful in serological tests for the specific diagnosis of C. pyloridis infections.     

Iodination and identification of surface membrane antigens in procyclic Trypanosoma rhodesiense

Surface membrane proteins and glycoproteins of procyclic Trypanosoma rhodesiense were labeled with 125I by the use of the insoluble catalyst Iodo-Gen. Autoradiography of whole solubilized procyclic trypanosomes after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed a minimum of 25 surface components to have incorporated radioactivity. These components ranged in m.w. from approximately 10,000 to approximately 285,000. Immunoprecipitation with rabbit antisera of Triton X-100 extracts of radiolabeled trypanosomes revealed a subset of at least 14 surface antigens. Two of these antigens (m.w. of 63,000 and 96,000) showed heavy incorporation of label and may be major proteins of the procyclic membrane. Sera from trypanosome-infected mice recognized an overlapping but different subset of surface antigens, including a doublet of very high m.w. Lectin precipitation using antilectin conjugates or bead-bound lectins indicated that many of the labeled surface components are glycoproteins including the two major proteins precipitated by rabbit antisera. Radiolabeled glycoproteins identified by these methods bear alpha-methyl-mannopyranoside and/or galactose residues but not N-acetyl glucosamine or fucose residues in quantity. The use of these methods in identifying potentially pathogenic trypanosomal antigens is suggested.     

Plasmodium falciparum antigens synthesized by schizonts and stabilized at the merozoite surface by antibodies when schizonts mature in the presence of growth inhibitory immune serum

Some immune sera that inhibit erythrocyte invasion by merozoites also agglutinate the merozoites as they emerge from rupturing schizonts. These immune clusters of merozoites (ICM) possess a surface coat that is cross-linked by antibody and is thicker than the surface coat associated with normal merozoites (NM) obtained from cultures containing preimmune serum. Analysis of metabolically labeled ICM and NM performed by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that washed ICM possessed immune complexes containing antigens representative of schizonts and merozoites. Characteristics of the immune complexes included: a) they were not soluble in pH 8 Triton X-100, b) they were soluble at an acid pH, and c) after pH neutralization they were precipitated by using staphylococcal protein A. Merozoite antigens having Mr of 83, 73, and 45 kDa were associated with immune complexes in ICM. The 83 and 73 kDa antigens were recovered in considerably larger quantities from ICM than from NM. Schizont antigens having Mr of 230, 173 (triplet), 152 (doublet), and 31 kDa were associated with immune complexes in ICM, and a 195 kDa antigen(s) from schizonts and merozoites was also present in the immune complexes. In addition, other antigens of Mr 113, 101, 65, and 51 kDa may have been immune complexed. These 15 antigens accounted for less than 30% of the schizont and merozoite antigens recognized by the immune serum. Immune complexes probably formed between antibodies and a) surface antigens of schizont-infected erythrocytes exposed to antibody before schizont rupture, b) surface antigens of merozoites and schizonts exposed during schizont rupture, and c) soluble antigens normally released during schizont rupture. The antibody components of the immune complexes may have prevented rapid degradation or shedding of some antigens from the merozoite surface. Allowing schizonts to rupture in the presence of inhibitory antibodies (to form ICM) is a useful approach to identifying exposed targets of protective immunity against malaria.     

Characterization of a developmentally regulated oocyst protein from Eimeria tenella

Changes in proteins during sporulation of Eimeria tenella oocysts were investigated. Unsporulated E. tenella oocysts collected from cecal tissue at 7 days postinoculation were sporulated in aerated media at 28 C for 0-48 hr. Gel analysis of soluble protein extracts prepared from oocysts from their respective time points indicated the presence of 2 prominent bands with relative molecular weight (Mr) in the range of 30 kDa and making up 20% of the total protein. These 2 bands, designated as major oocyst proteins (MOPs), were absent or barely detectable by 21 hr of sporulation. MOP bands were weakly reactive with glycoprotein stain but showed no mobility shift on deglycosylation. By gel analysis it was shown that the purified MOPs consisted of 2 bands of Mr 28.7 and 30.1 kDa. However, by matrix-assisted laser deabsorption-time of flight analysis it was shown that masses were about 17% lower. Internal sequence analysis of the 28.7-kDa protein generated 2 peptides of 17 and 14 amino acids in length, consistent with a recently described protein coded by the gam56 gene and expressed in E. maxima gametocytes. Rabbit antibodies made against MOPs were localized to outer portions of sporocysts before excystment and to the apical end of in vitro-derived sporozoites. These same antibodies were found to react with bands of Mr 101 and 65 kDa by Western blot but did not recognize MOPs in soluble or insoluble sporozoite extracts. The data suggest that the MOPs are derived from part of a gametocyte protein similar to that coded by gam56 and are processed during sporulation into sporocyst and sporozoite proteins. Alternatively, the binding of anti-MOP to 101- and 65-kDa proteins may result from alternatively spliced genes as the development of parasite proceeds.     

Rat liver type I iodothyronine deiodinase is not identical to protein disulfide isomerase

This study was done to test the recent hypothesis (Boado et al. (1988) Biochem. Biophys. Res. Commun. 155, 1297-1304) that type I iodothyronine deiodinase (ID-I) is identical to protein disulfide isomerase (PDI). Autoradiograms of rat liver microsomal proteins, labeled with N-bromoacetyl-[125I]triiodothyronine (BrAc[125I]T3) and separated by SDS-PAGE, show predominantly 2 radioactive bands of Mr 27 and 56 kDa. Substrates and inhibitors of ID-I inhibited labeling of the 27 kDa band but not that of the 56 kDa band. Treatment of microsomes with trypsin abolished labeling of the 27 kDa protein and destroyed the activity of ID-I but did not prevent labeling of the 56 kDa protein. Following treatment of microsomes at pH 8.0-9.5 or with 0.05% deoxycholate (DOC) PDI content and labeling of the 56 kDa protein were strongly diminished but ID-I activity and labeling of the 27 kDa protein were not affected. The latter decreased in parallel after treatment at pH greater than or equal to 10. Rat pancreas microsomes contain high amounts of PDI but show no ID-I activity. Reaction of these microsomes with BrAc[125I]T3 results in extensive labeling of a 56 kDa protein but no labeling of a 27 kDa protein. Pure PDI (Mr 56 kDa) was readily labeled by BrAc[125I]T3 but showed no deiodinase activity. These results strongly suggest that the 27 kDa band represents (a subunit of) ID-I while the 56 kDa band represents PDI. From these and other data it is concluded that PDI and ID-I are not identical proteins.     

Monoclonal antibodies that inhibit IgE binding

Four monoclonal antibodies were produced that inhibit IgE binding to the high affinity IgE receptor (Fc epsilon R) on rat basophilic leukemia cells. The four monoclonal antibodies (mAb) fall into two groups. The first group was comprised of 3 antibodies (mAb BC4, mAb CD3, and mAb CA5) that reacted with the Fc epsilon R at epitopes close or identical to the IgE-binding site. With 125I-labeled antibodies there was reciprocal cross-inhibition between the antibodies and IgE. The antibodies activated both RBL-2H3 cells and normal rat mast cells for histamine release. The 3 antibodies immunoprecipitated the previously described alpha, beta, and gamma components of the receptor. The number of radiolabeled Fab fragments of 2 of these antibodies bound per cell was similar or equal to the number of IgE receptors. In contrast, the mAb BC4 Fab bound to 2.1 +/- 0.4 times the number of IgE receptor sites. Therefore, the portion of the Fc epsilon R exposed on the cell surface must have two identical epitopes and an axis of symmetry. These 3 monoclonal antibodies recognize different but closely related epitopes in the IgE-binding region of the Fc epsilon R. The fourth monoclonal antibody (mAb AA4) had different characteristics. In cross-inhibition studies, IgE and the other 3 monoclonals did not inhibit the binding of this 125I-labeled monoclonal antibody. The number of molecules of this antibody bound per cell was approximately 14-fold greater than the Fc epsilon R number. This monoclonal antibody caused the inhibition of histamine release and it appears to bind to several cell components.     

Structural inferences for the native skeletal muscle sodium channel as derived from patterns of endogenous proteolysis

The alpha subunit (Mr approximately 260,000) of the rat skeletal muscle sodium channel is sensitive to cleavage by endogenous proteases during the isolation of muscle surface membrane. Antisera against synthetic oligopeptides were used to map the resultant fragments in order to identify protease-sensitive regions of the channel's structure in its native membrane environment. Antibodies to the amino terminus labeled major fragments of Mr approximately 130,000 and 90,000 and lesser amounts of other peptides as small as Mr approximately 12,000. Antisera to epitopes within the carboxyl-terminal half of the primary sequence recognized two fragments of Mr approximately 110,000 and 78,000. Individual antisera also selectively labeled smaller polypeptides in the most extensively cleaved preparations. The immunoreactivity patterns of monoclonal antibodies previously raised against the purified channel were then surveyed. The binding sites for one group of monoclonals, including several that recognize subtype-specific epitopes in the channel structure, were localized within a 12-kDa fragment near the amino terminus. The distribution of carbohydrate along the primary structure of the channel was also assessed by quantitating 125I-wheat germ agglutinin and 125I-concanavalin A binding to the proteolytic peptides. Most of the carbohydrate detected by these lectins was located between 22 and 90 kDa from the amino terminus of the protein. No lectin binding was detected to fragments arising from carboxyl-terminal half of the protein. These results were analyzed in terms of current models of sodium channel tertiary structure. In its normal membrane environment, the skeletal muscle sodium channel appears sensitive to cleavage by endogenous proteases in regions predicted to link the four repeat domains on the cytoplasmic side of the membrane while the repeat domains themselves are resistant to proteolysis.     

Characterization of the Target Antigens of Phytomonas-specific Monoclonal Antibodies

ABSTRACT. Seven Phytomonas -specific monoclonal antibodies produced against Phytomonas serpens and Phytomonas françai were further characterised in order to identify and localise their target antigens. Four monoclonal antibodies recognized carbohydrate surface epitopes, in three of the cases associated with surface glycoproteins with apparent molecular weight of 80 kDa. One monoclonal antibody apparently bound to a surface/internal protein epitope, whereas the two others recognized intra-cellular proteins. The cell surface epitopes recognized by monoclonal antibodies were detected specifically in the genus Phytomonas. These epitopes, which are detected in culture, plant and insect forms, may be useful as targets for Phytomonas identification.     

Plasmodium vivax: malarial proteins associated with the membrane-bound caveola-vesicle complexes and cytoplasmic cleft structures of infected erythrocytes

The identification of antigens of parasite origin associated with the altered membrane of Plasmodium vivax-infected erythrocytes was undertaken in this study. The 125I-lactoperoxidase catalyzed surface radiolabeling of trophozoite-infected erythrocytes revealed new bands of 95 and 70 kDa not labeled in normal erythrocytes. Erythrocyte membrane-enriched preparations from [35S]methionine biosynthetically labeled-infected erythrocytes also indicated that in addition to bands at 95 and 70 kDa, several other parasite proteins were possibly membrane associated. Five monoclonal antibodies (Mabs) reactive with P. vivax produced an immunofluorescent pattern of numerous small dots scattered over the entire infected erythrocyte. This pattern mimics that of Schuffner's stippling; small red dots seen in Giemsa-stained P. vivax-infected erythrocytes, which represent accumulations of dye in caveola-vesicle complexes (CVC). Four of the monoclonal antibodies immunoprecipitated a Triton X-100 detergent-insoluble 95-kDa parasite protein which was localized by immunofluorescent assay and immunoelectron microscopy exclusively to the CVC. Two of these Mabs were immunofluorescence reactive with the surface of intact infected erythrocytes in suspension. The fifth Mab, which also localized exclusively to the CVC structures, immunoprecipitated a Triton X-100 extractable protein of 70 kDa. Two other monoclonal antibodies reacted exclusively with the numerous membranous cleft structures found in the cytoplasm of infected erythrocytes. This cleft-associated parasite antigen was 28 kDa in size. Some of these Mabs recognize epitopes and produce similar IFA patterns on erythrocytes infected with P. cynomolgi, P. knowlesi, and P. ovale parasites, but not with P. falciparum- or P. brasilianum-infected erythrocytes.     

Identification of Ki (Ku, p70/p80) autoantigens and analysis of anti-Ki autoantibody reactivity

Anti-Ki (Ku, p70/p80) autoantibodies, named after the prototype patient Kikuta by Tojo et al., occur in approximately 10% of patients with SLE, often in association with anti-Sm autoantibodies. The immunofluorescent staining pattern characteristic of anti-Ki antibodies is diffuse speckled nuclear, although some substrates show nucleolar staining as well. Anti-Ki sera specifically immunoprecipitated two protein antigens, Ki86 (Mr 86,000) and Ki66 (Mr 66,000), from radiolabeled cell extracts. The Ki system was found to be immunologically identical to the Ku system described by Mimori et al. and the p70/p80 system described by Reeves. The Ki primary in vitro translation products were identified and proved similar in size to the cellular antigens. The Ki antigens were purified from human spleen by immunoaffinity chromatography followed by SDS-PAGE. The purified Ki antigens proved to be closely related by amino acid composition, and did not appear to be phosphorylated, glycosylated, or associated with RNA. The Ki antigens were found to bind to DNA, in agreement with the observations on the Ku and p70/p80 antigens. They were found to be widely conserved in mammals and were coordinately expressed in all tissues tested. Anti-Ki autoantibodies were purified by antigen-affinity chromatography and were tested by immunoblotting. The antibodies were classified as class I, II, or III, depending on their reactivity with the Ki antigens in immunoblots. Class I antibodies cross-reacted with both Ki antigens, class II antibodies reacted solely with Ki66, and class III antibodies reacted solely with Ki86. These results suggest that at least three different epitopes are present on the Ki autoantigens and that patients differ in their autoantibody response to each epitope.     

Lectin and human blood group determinants of Schistosoma mansoni: alteration following in vitro transformation of miracidium to mother sporocyst.

A mixed agglutination assay method was employed to detect the presence of surface determinants for various lectins and human blood group antibodies on Schistosoma mansoni miracidia and cultured mother sporocysts. Miracidia were found to possess surface receptors for the lectins Con A (concanavalin A), anti-Heel (eel serum agglutinin), and anti-ADb (Dolichos seed extract), as well as human anti-A antibodies. Following in vitro transformation of the miracidium to mother sporocyst, anti-Heel and human anti-A receptors were no longer detectable on the sporocyst surface, while determinants for Con A and anti-ADb remained essentially unaltered. It is concluded that transition of the miracidium to the sporocyst results in the alteration of surface molecular structures on schistosome larve. Furthermore, since determinants for Con A, anti-Heel, anti ADb, and human anti-A have been found associated with macromolecules in the hemolymph of the snail Biomphalaria glabrata (Stnislawski et al., 1976), there is now evidence that miracidia and mother sporocysts of S. mansoni and their snail host share molecules with common lectin and human blood group determinants.     

Polypeptide components of the apamin receptor associated with a calcium activated potassium channel

Photoaffinity labeling of rat brain membranes with [125I]ANPAA-apamin incorporated radioactivity into polypeptides of 86 and 59 kDa and occasionally a more weakly labeled component of 45 kDa. These polypeptides were immunoprecipitated with anti-apamin antibodies and treated with glycosidases. Neither the 86 nor the 59 kDa polypeptide appeared to be N-glycosylated. Partial proteolytic mapping of affinity labeled polypeptides with chymotrypsin or V8 protease generated an identical pattern. These results suggest that the 59 and 45 kDa components are not additional subunits of an oligomeric protein but result from cleavage of the 86 kDa polypeptide.     

Schistosoma mansoni: antigen preparations which induce antibodies to schistosomula surface antigens

To determine the easiest method of raising antibodies to antigens exposed on the surface of schistosomula of Schistosoma mansoni, several crude preparations of the parasite were used to immunize mice. Schistosomula released products, whole worm homogenate, and parasite eggs all raised antibodies which bound to the surface of live schistosomula, although the anti-egg antiserum did so less strongly. Anti-schistosomula released products antiserum recognized three schistosomula surface antigens of Mr 15,000, 20,000, and 32,000, anti-whole worm homogenate recognized 20,000, 32,000, and 38,000 Mr surface antigens, and anti-egg recognized a less than 200,000 Mr surface antigen. None of these antigens was recognized when the labeled preparation was immunoprecipitated with its homologous antiserum. When these antisera were used to immunoprecipitate cell free translation products of adult worm RNA, the antischistosomula released products and anti-whole worm homogenate recognized an 11,000 Mr doublet while the anti-egg precipitated 14,000 and 44,000 Mr antigens. Other crude preparations were used to immunize rabbits; Formalin-fixed schistosomula, denuded adult worms, and purified worm tegument all induced antibodies which recognized the 20,000, 32,000, and 38,000 Mr schistosomula surface antigens.