Schistosoma mansoni: identification and characterization of schistosomula polypeptides

Schistosomula proteins separated by a two-dimensional (NEPHGE) gel system identify 94 major silver-stained polypeptides. When compared to polypeptides similarly separated from cercariae and adult worms; cercariae share the same polypeptides as schistosomula, adult worms share ca. 60% of the polypeptides. A group of five schistosomula polypeptides 15-31 kDa (apparent pI 8.2-8.9) was not found in adult worm extracts. To identify which polypeptides were immunogens, Western blots of the NEPHGE gels were probed with sera either from humans with chronic schistosomiasis or from mice vaccinated with irradiated cercariae. For characterization studies, polyclonal antibodies were made against the five schistosomula-specific and selected immunogenic polypeptides by immunizing mice with silver-stained spots removed from NEPHGE gels. We show that the polyclonal serum against a polypeptide of 12.5 kDa and an apparent pI of 6.70 mediated complement and eosinophil-dependent killing of schistosomula in an in vitro assay. Epitopes recognized by antibody against the 12.5-kDa polypeptide show a diffuse distribution and are found on flame cells of the excretory system of the schistosomula.     

Lipoprotein-binding proteins in the human platelet plasma membrane

The binding of homologous plasma lipoproteins to specific receptor proteins in the plasma membrane of human blood platelets was studied by ligand blotting techniques. HDL3, HDL2 and LDL showed saturable binding to three bands of 156, 130 and 115 kDa, respectively. This binding was not markedly affected by the presence or absence of Ca2+ nor by covalent modification of lysine and arginine residues of the apoprotein moieties. However, it can be almost completely reversed by the addition of heparin or suramin.     

Comparison of amylase-binding proteins in oral streptococci

Abstract Certain species of oral streptococci bind salivary amylase to their cell surface. The patterns of amylase-binding proteins produced by a range of streptococci have been compared by ligand blotting and several characteristics of the binding proteins investigated. Streptococcus gordonii was the most homogeneous species and almost all strains produced proteins migrating with molecular mass 82 kDa and 20 kDa. Other species were more heterogeneous, releasing proteins that resolved at 87 or 82 kDa and/or between 20 and 36 kDa. Binding of amylase to the 82/87-kDa proteins on ligand blots was prevented by amylase inhibitors, amylase substrates and periodate treatment but these had limited or no effect on amylase binding to 20–36 kDa proteins. Also, the 20 kDa protein of S. gordonii Challis was released into culture medium before the 82-kDa protein. These data suggest that there is significant variation in amylase-binding proteins among streptococci and that the high and low molecular mass proteins differ in the way they interact with salivary amylase.     

Three major surface antigens of Schistosoma mansoni are linked to the membrane by glycosylphosphatidylinositol

Schistosomula of Schistosoma mansoni were examined for the presence of glycosylphosphatidylinositol (GPI) anchored surface membrane Ag. Parasites were surface iodinated and cultured in the presence or absence of a crude phospholipase C (PLC) preparation or phosphatidylinositol-specific PLC (PIPLC). Culture supernatants were then analyzed: 1) by centrifugation to ascertain which molecules released from the surface were soluble or contained in membrane vesicles; 2) by immunoprecipitation with antibodies specific for the "cross-reacting determinant," an epitope revealed on some GPI-anchored proteins only after cleavage of the diacylglycerol from the protein by PIPLC, and 3) by immunoprecipitation with immune mouse sera to establish co-identity with previously described, immunologically relevant surface Ag. By using these techniques, schistosomula were shown to possess three GPI-anchored surface Ag of m.w. 38,000, 32,000 and 18,000 which are spontaneously released from the surface of schistosomula in association with membrane, but remain insoluble until cleaved by PIPLC. All three molecules were recognized by antibodies from mice vaccinated with irradiated cercariae and/or chronically infected mice. Moreover, the m.w. 38,000 component was recognized by a previously described protective mAb (E.1). A major developmental modification appears to occur in the expression of these molecules because, by the same techniques, no GPI-anchored surface Ag were detectable on 7-day-old lung stage parasites. The finding that these important parasite immunogens are GPI-anchored and released from the surface of the parasite in membrane vesicles may, in part, explain why they elicit strong immune responses capable of damaging the schistosomulum tegument.     

In vitro killing of schistosomula of Schistosoma mansoni by BCG and C. parvum-activated macrophages.

Resistance to Schistosoma mansoni infection in the mouse has been induced either specifically by a primary infection with this parasite or nonspecifically by a variety of immunostimulants such as BCG. In the present study we developed an in vitro system to examine the effector mechanism of nonspecifically induced resistance. Activated macrophage monolayers obtained from BCG- or Corynebacterium parvum treated mice killed a respective mean 32 +/- 6% and 48 +/- 5% of schistosomula after 24 hr incubation. The killing of the parasites was verified by their inability to mature to adult worms upon injection into normal mice. The activated macrophage-mediated killing was related to cell:parasite ratio, and was partially lost if the macrophage monolayers were kept in cultures for 24 hr before incubation with the organism. Supernatants of macrophages cultured in the presence of schistosomula killed a mean of 51 +/- 3% of the organisms whereas those from cells cultured alone resulted in a mean killing of 25 +/- 3%. Furthermore, toxic supernatants could be generated equally well on incubation with S. mansoni schistosomula or Trichinella spiralis larvae. Our data show that activated macrophage monolayers through soluble mediators destroy a significant proportion of the multicellular parasite S. mansoni schistosomula in vitro.     

Identification of lipoprotein-binding proteins in rat liver Golgi apparatus membranes

The low density lipoprotein (LDL) receptor has been shown to be a plasma membrane glycoprotein responsible for the cellular binding and endocytosis of plasma lipoproteins. Inasmuch as the Golgi apparatus has been shown to participate in glycoprotein processing and in the assembly of plasma lipoproteins by hepatic and intestinal epithelial cells, the present studies were designed to test the hypothesis that lipoprotein receptors are present within Golgi membranes. Utilizing ligand blotting with a variety of iodinated lipoproteins, several lipoprotein-binding proteins were identified in rat liver Golgi membranes at apparent molecular weights (Mr) 200,000, 160,000, 130,000, 120,000, 100,000, 80,000, and 70,000. The 130,000 protein was the most prominent and was identified as the mature LDL receptor by its binding characteristics and an Mr characteristic of the plasma membrane receptor. Enzymatic deglycosylation studies suggested that the 120,000 and 100,000 proteins were LDL receptor precursors lacking sialic acid. Antibody to the LDL receptor recognized all the bands on immunoblots except the 70,000 protein, with the 130,000 protein being the most prominent. Isolation of the Golgi fractions in the presence of protease inhibitors did not eliminate any of the proteins recognized by the antibody but did result in sharper bands on the blots. Additionally, we investigated the hypothesis that conditions that regulate plasma membrane LDL receptors also cause detectable changes in receptors in Golgi membranes. All the binding proteins were increased in Golgi membranes from rats treated with 17-alpha-ethynylestradiol. Colchicine caused an accumulation of 120,000 Mr protein, suggesting blockage of final sialylation in the trans Golgi. When protein synthesis was inhibited by cycloheximide, there was no reduction of mature LDL receptors in Golgi membranes, consistent with recycling of receptors through this organelle.     

Schistosoma mansoni: detection by monoclonal antibody of a 22,000-dalton surface membrane antigen which may be blocked by host molecules on lung stage parasites

Monoclonal antibody M.2 binds to the surface membranes of cercariae and developing schistosomula. This antibody was generated from mice immunized with membrane-enriched extracts of mechanically transformed schistosomula. The antigen detected by M.2 was shown to persist on developing schistosomula for at least 96 hr post-transformation. M.2 also bound to the surface of living, cultured lung worms but not to freshly harvested lung worms. The ability of M.2 to bind to cultured lung worms coincided with the loss of host H-2 from the parasite surface. The apparent m.w. of the antigen was 22,000; an antigen with the same apparent m.w. was immunoprecipitated from cercariae, schistosomula, lung worms, and adult worms.     

Detection of G Proteins in Purified Bovine Brain Myelin

Following a previous report on detection of muscarinic receptors in myelin with the implied presence of G proteins, we now demonstrate by more direct means the presence of such proteins and their quantification. Using [35S]guanosine 5'-O-(3-thiotriphosphate) ([35S]GTP gamma S) as the binding ligand, purified myelin from bovine brain was found to contain approximately half the binding activity of whole white matter (138 +/- 9 vs. 271 +/- 18 pmol/mg of protein). Scatchard analysis of saturation binding data revealed two slopes, a result suggesting at least two binding populations. This binding was inhibited by GTP and its analog but not by 5'-adenylylimidodiphosphate [App(NH)p], GMP, or UTP. Following sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) of myelin proteins and blotting on nitrocellulose, [alpha-32P]GTP bound to three bands in the 21-27-kDa range in a manner inhibited by GTP and GTP gamma S but not App(NH)p. ADP-ribosylation of myelin with [32P]NAD+ and cholera toxin labeled a protein of 43 kDa, whereas reaction with pertussis toxin labeled two components of 40 kDa. Cholate extract of myelin subjected to chromatography on a column of phenyl-Sepharose gave at least three major peaks of [35S]GTP gamma S binding activity. SDS-PAGE and immunoblot analyses of peak I indicated the presence of Go alpha, Gi alpha, and Gs alpha. Further fractionation of peak II by diethyl-aminoethyl-Sephacel chromatography gave one [35S]GTP gamma S binding peak with the low-molecular-mass (21-27 kDa) proteins and a second showing two major protein bands of 36 and 40 kDa on SDS-PAGE.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ligand blot analysis of juvenile hormone esterase binding proteins in Manduca sexta L

Biotinylated recombinant juvenile hormone esterase (JHE) was used for ligand blotting of proteins from fat body tissue and pericardial athrocytes of Manduca sexta. Proteins were separated by SDS-polyacrylamide gel electrophoresis or by two-dimensional electrophoresis. Eight putative JHE binding proteins were detected in fat body tissue and in pericardial athrocytes of both M. sexta and Heliothis virescens. The predominant bands were 29, 72, 75, 125 and 240kDa, with minor bands at 50, 80 and 205kDa. All putative JHE binding proteins were present from the second through to the fifth instar larvae of M. sexta. On wide-range isoelectric focusing, the 29kDa JHE binding protein separated into three species with isoelectric points of 6.5, 6.6 and 6.8. Biotinylated-JHE did not bind recombinant M. sexta-derived juvenile hormone binding protein. The mutant JHE with mutations K29R and K524R binds weakly to the JHE binding protein P29, relative to binding of wild-type JHE [Shanmugavelu et al., J. Biol. Chem., 275 (2000) 1802-1806]. A similar reduction in binding was not seen for the 29kDa binding protein identified here in pericardial athrocytes by ligand blot. This result is discussed.     

Schistosoma mansoni host-exposed surface antigens characterized by sera and recombinant antibodies from schistosomiasis-resistant rats

Antibodies from Schistosoma mansoni-infected rats, unlike mice, show a higher titer for schistosome apical tegumental antigens compared with non-apical membrane antigens. These antibodies bind to the surface of living lung-stage worms and to formaldehyde-fixed adult worms. We produced a single-chain antibody Fv domain (scFv) phage library displaying the antibody repertoire of rats highly immune to schistosome infection and we selected for scFvs that recognize the host-exposed surface of worms. Five unique rat scFvs (Teg1, Teg4, Teg5, Teg20 and Teg37) were obtained which recognize schistosome surface epitopes. Each of the scFvs recognizes the surface of living schistosomula and lung-stage schistosomules and/or the surface of formaldehyde-fixed adult worms. None of these scFvs reproducibly stained living adult worms. This suggests that a change occurs during the transition from lung schistosomules to 4-week adults such that at least some surface antigens, although remaining on the surface in living adult worms, can no longer be immunologically stained. Teg1 and Teg4 scFvs both recognize specific bands on Western blots. No bands were observed for the other three scFvs, suggesting that these scFvs may recognize non-protein or conformationally-dependent epitopes. Teg1 was unambiguously identified as recognizing the S. mansoni tetraspanin antigen, SmTSP-2, within the large extracellular domain. Teg4 recognizes a 35 kDa band tentatively identified as Sm29 by proteomic analysis. These scFvs can now be used to characterize schistosome epitopes at the host-parasite interface, to target worms in vivo, and to study the mechanisms by which these worms naturally evade immune damage to the tegument within permissive hosts.     

Screening of murine monoclonal antibodies against living schistosomula of Schistosoma mansoni by radioimmunoassay

A radioimmunoassay was developed to screen supernatants of murine monoclonal antibodies against surface antigens of living schistosomula of Schistosoma mansoni. Of 196 clones screened, 10% bound schistosomula. Of these, 74% bound only schistosomula. The remaining molecules also reacted with soluble adult worm antigens and soluble egg antigens as determined by enzyme-linked immunosorbent assay. Immunoblot analysis demonstrated that monoclonal antibody 204-3E4 reacted with a 68 kDa protein, a glycoprotein that induces substantial resistance against S. mansoni infection. Recognition of an 18 kDa antigen by 204-3F1 antibody was stage-specific with the antigen being expressed in cercariae, 3- and 24-h-old parasites but not 4-day, lung stage or adult worms. Monoclonal antibody 204-4E3 reacted with purified S. mansoni paramyosin. These data indicate that radioimmunoassay using living schistosomula is a rapid alternative method to identify murine hybridomas that secrete antibodies which react with surface antigens of S. mansoni.     

Inhibition of collagen biosynthesis and increases in low molecular weight IGF-I binding proteins in the skin of fasted rats

Insulin-like growth factor-I (IGF-I) is an important stimulator of collagen biosynthesis and prolidase activity in connective tissue cells. The disturbances in skin collagen metabolism (reflected by significant decrease in skin collagen content, collagen biosynthesis and prolidase activity) in fasted rats were accompanied by decrease in serum IGF-I level. Fasted rat serum was found to contain about 58% of IGF-I (101.6 +/- 15.4 ng/ml) as compared to control rat serum (175.7 +/- 19.8 ng/ml), while the skin of control and fasted rats contained similar concentrations of IGF-I (about 77 ng/g tissue). The insulin-like growth factor binding proteins (IGFBPs) of sera and tissue extracts (known to regulate IGF-I activity) were analysed by ligand blotting. In the serum of control rats one IGFBP band of about 46 kDa (corresponding to the acid-dissociated IGFBP-3) was detected. In the serum of fasted rats the 46 kDa IGFBP was not observed, however, an other IGFBP of about 30 kDa (corresponding to low molecular weight IGFBPs, e.g. IGFBP-1 or IGFBP-2) was found. The intensity of IGF-I binding to the 30 kDa IGFBP was much higher than that of IGFBP-3, found in control rat serum. Control and fasted rat skin contained similar IGFBPs, however their IGF-I binding abilities were much lower, compared to their serum counterparts. It was found that 46 kDa and 30 kDa proteins, observed in ligand blotting represent IGFBP-3 and IGFBP-1 or IGFBP-2. respectively as demonstrated by western immunoblot analysis. An increase in IGF-binding to 30 kDa IGFBP-1 and/or IGFBP-2 (known as an inhibitors of IGF-dependent functions) in the skin of fasted rats may explain the mechanism of reduced collagen biosynthesis and deposition in tissues during fasting.     

The enhanced cellular uptake of very-low-density lipoprotein enriched in apolipoprotein E.

We have recently reported an increased clearance of plasma very-low-density lipoprotein (VLDL) after intravenous injection of apolipoprotein (apo) E in Watanabe heritable hyperlipidemic (WHHL) rabbits. In the present study, we have investigated the cellular uptake of VLDL enriched in apo E (VLDL-E) which had been incubated with purified rabbit apo E. VLDL-E was taken up approx. 2-fold more than VLDL in human skin fibroblast, human monocyte-derived macrophage and Hep G2 cell and its degradation was least in macrophage. To characterize the binding of VLDL-E, we performed a binding assay using hepatic endosome isolated from estradiol-treated rats and we observed both increased EDTA-sensitive and -resistant binding of VLDL-E on endosome. Ligand blotting of hepatic endosome demonstrated two major bands of LDL receptor (130 and 260 kDa protein) and a minor band of LDL receptor-related protein (580 kDa protein) with a ligand of VLDL-E. These results suggested that VLDL-E was endocytosed in liver through a similar pathway among three cell types, and enrichment of apo E in VLDL enhanced the uptake of VLDL not only via an EDTA-sensitive binding site (classical LDL receptor) but also via other binding sites including an EDTA-resistant binding site and an LDL receptor-related protein.     

Novel retinoid-binding proteins from filarial parasites.

The present study deals with the discovery and partial characterization of specific binding proteins for retinol and retinoic acid from filarial parasites (worms of the superfamily Filarioidea), including those from two species of Onchocerca. These binding proteins, which are distinct in their physicochemical properties and in the mode of ligand interactions from the host-tissue retinoid-binding proteins, may be involved in the mediation of the putative biological roles of retinoids in the control of parasitic growth, differentiation and reproduction. Parasite retinol-binding protein and retinoic acid-binding protein exhibited specificity for binding retinol and retinoic acid respectively. Both the binding proteins showed an s20,w value of 2.0 S. On gel filtration, both proteins were retarded to a position corresponding to the same molecular size (19.0 kDa). On preparative columns, the parasite binding proteins exhibited isoelectric points at pH 5.7 and 5.75. Unlike the retinoid-binding proteins of mammalian and avian origin, the parasite retinoid-binding proteins showed a lack of mercurial sensitivity in ligand binding. The comparative amounts of retinoic acid-binding protein in five parasites, Onchocerca volvulus, Onchocerca gibsoni, Dipetalonema viteae, Brugia pahangi and Dirofilaria immitis, were between 2.7 and 3.1 pmol of retinoic acid bound/mg of extractable protein. However, the levels of parasite retinol-binding protein were between 4.8 and 5.8 pmol/mg, which is considerably higher than the corresponding levels of cellular retinol-binding protein of mammalian and avian origin. Both retinol- and retinoic acid-binding-protein levels in O. volvulus-infected human nodules and O. gibsoni-infected bovine nodules were similar to their levels in mammalian tissues. Also, these nodular binding proteins, like the host-binding proteins, exhibited mercurial sensitivity to ligand interactions.     

Schistosomula of Schistosoma mansoni clear concanavalin A from their surface by sloughing

The lectin concanavalin A (Con A) was used as a model probe to study the behavior of molecules bound to the surface of recently transformed schistosomula of Schistosoma mansoni. Con A binding was saturable (150- 180 pg/organism) and specifically competed by alpha-methyl mannoside. Both FITC-Con A and 125-I-Con A were lost from the surface of schistosomula with a halftime of 8-10 h in culture in defined medium. A comparable decrease in the binding of Con A to schistosomula cultured and then labeled with the lectin indicated that the labeling procedure itself was not inducing the observed change. Internalization of Con A was not seen by either fluorescence microscopy or electron microscope radioautography. In addition, 70-80% of the radioactivity lost from the parasite was recoverable by TCA precipitation from the culture medium as intact Con A (27,000 mol wt on SDS PAGE). Thus, the mechanism of clearance of bound Con A from the surface of cultured schistosomula is apparently by sloughing of Con A molecules intact into the culture media and not by endocytosis and degradation. Con A binding sites, visualized with hemocyanin by scanning electron microscopy, appeared homogeneously distributed over the surface of schistosomula when organisms were labeled at 4 degree C or after fixation with glutaraldehyde. However, Con A and hemocyanin formed aggregates on the surface of schistosomula when labeling was performed at 37 degrees C, which suggests that lectin binding sites have lateral mobility within the plane of the membrane. These aggregates are likely independent of metabolism by the parasite because aggregation also occurs on the surface of organisms killed with azide.     

Solubilization, identification, and localization of vitellogenin-binding sites in follicles of the cockroach, Nauphoeta cinerea

Binding sites for vitellogenin were solubilized and analyzed either with a filter assay or with ligand blotting. We tested sodium dodecylsulfate (SDS), Chaps, octyl-beta-D-glucoside, and sodium deoxycholate and found SDS and sodium deoxycholate to be most effective in solubilizing both high and low molecular weight binding sites. In the filter assay the sodium deoxycholate extracts, but not the SDS extracts, maintained binding activity after dilution of the solubilizer below its critical micellar concentration. In ligand blotting we consistently observed, in vitellogenic follicles, binding sites with an apparent Mr of approximately 200,000, 35,000, and three closely spaced bands between 14,000 and 20,000. Binding of vitellogenin to all binding sites was suppressed in the presence of the drug suramin. Analysis of corpora lutea and oothecae as well as of ovariole sheath, follicle cell/basal lamina, and oocyte plasma membrane preparations showed that the 35 and 14-20 kDa binding sites are located in the outer follicle compartments, and the 200 kDa binding site in the oocyte plasma membrane. In the latter we occasionally also observed binding sites with an apparent Mr of approximately 150,000, 95,000, 67,000 and 30,000, particularly at stages after ovulation. The 35 and 14-20 kDa binding sites, as visualized in stained gels and in ligand blotting, are rather abundant and were also seen in several other male and female tissues of Nauphoeta and even in other species. They also bound other 14C-labeled hemolymph proteins and thus appear to be rather unspecific. As binding analysis with nonsolubilized and sodium deoxycholate-solubilized membranes revealed that the quantity of vitellogenin bound by binding sites of the outer follicle compartments was low, it is conceivable that the abundance of the 14-20 kDa and 35 kDa binding sites in ligand blotting is merely an effect of SDS and does not reflect the in vivo situation. We suppose that the 200 kDa binding site of the oocyte plasma membrane represents the vitellogenin receptor involved in endocytosis.     

Endothelial albumin binding proteins are membrane-associated components exposed on the cell surface

The heterobifunctional, photoactivatable, thiol-cleavable cross-linker sulfosuccinimidyl 2-(p-azido-salicylamido)ethyl-1,3'-dithiopropionate (SASD) was radioiodinated and used to determine whether endothelial albumin binding proteins (ABP) recently identified (Ghinea, N., Fixman, A., Alexandru, D., Popov, D., Hasu, M., Ghitescu, L., Eskenasy, M., Simionescu, M., and Simionescu, N. (1988) J. Cell Biol. 107, 231-239) are plasma membrane-associated components exposed on the cell surface. Microvascular endothelial cells (MEC) freshly isolated from rat epididymal fat were incubated with 125I-2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (ASD)-albumin conjugate which upon photolysis by UV light was cross-linked to the receptor proteins. By cleaving the disulfide linkages of the cross-linker with 5% beta-mercaptoethanol and the ligand-receptor interactions with 0.1% sodium dodecyl sulfate, the radioiodinated ASD moiety remained attached to the receptor peptides which were further detected by 5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography. In parallel, samples were examined by ligand blotting with albumin-gold complex. The results showed that in these experimental conditions ABP are represented by two major peptides of 31 and 18 kDa and two minor bands of 73 and 56 kDa. Densitometric scanning showed that the two major bands constitute more than 70% of the total ABP. The four peptides were not apparent if the samples were not UV-irradiated. The binding of the radioiodinated ligand to ABPs was reduced by approximately 82% in the presence of excess competitive unlabeled albumin. When MEC were incubated with unlabeled SASD and exposed to UV light, the autoradiographic banding pattern obtained was similar to that of either radioiodinated receptor proteins or MEC not treated with SASD. This indicated that the four albumin binding peptides are distinct proteins of the endothelial cell plasmalemma.     

Schistosoma mansoni: surface membrane stability in vitro and in vivo

The human complement component C3b is known to bind in vitro to the surfaces of all developmental stages of schistosomes as a consequence of complement activation by the alternative pathway. C3b bound to Schistosoma mansoni parasites has now been used in combination with fluorescent labeled antibodies against C3b to label the surfaces of living schistosomes. Binding of complement components and labeled antibodies to adult schistosomes rendered their surface membrane homogeneously fluorescent. At the ultrastructural level, the label was seen as a dense deposit lying on the tegumental membrane. Surface damage was not observed in labeled adults by electron microscopy. Fluorescent schistosomes were cultured in vitro for periods of up to 2 weeks, during which time the parasites remained fully viable and their surface membrane was still fluorescent. The electron dense deposit persisted, and tegumental damage at the electron microscope level was minimal or absent. Consequently, adult schistosomes would seem able to survive in vitro in the absence of rapid and general turnover of their surface membrane. Loss of fluorescence was observed consistently only at the anterior end of the parasite, including the suckers, a finding which indicates that membrane turnover may occur at different rates on different parts of the body. Fluorescent 3-week-old juveniles and 6-day-old lung stage parasites were cultured under the same conditions with similar results: they remained viable and fluorescent for at least 2 weeks. Results with skin schistosomula were different in the sense that many worms died during culture, and those which survived lost large parts of their fluorescent surface. A few of the surviving and fluorescent schistosomula developed the elongate shape typical of lung stage parasites. Fluorescent viable skin schistosomula were injected intravenously into mice and subsequently recovered from the lungs after varying periods. Fluorescence was lost in a patchy way within a few minutes from some individuals and within several hours from most of the worms. These data permit the following conclusions: C3b is a suitable tracer for membrane renewal in all developmental stages of schistosomes. Very slow membrane renewal in vitro and very rapid renewal in vivo are both compatible with parasite survival.     

Surface Coat of Meloidogyne incognita

The nematode surface coat is defined as an extracuticular component on the outermost layer of the nematode body wall, visualized only by electron microscopy. Surface coat proteins of Meloidogyne incognita race 3 infective juveniles were characterized by electrophoresis and Western blotting of extracts from radioiodine and biotin-labeled nematodes. Extraction of labeled nematodes with cetyltrimethylammonium bromide yielded a principal protein band larger than 250 kDa and, with water soluble biotin, several faint bands ranging from 31 kDa to 179 kDa. The pattern of labeling was similar for both labeling methods. Western blots of unlabeled proteins were probed with a panel of biotin-lectin conjugates, but only Concanavalin A bound to the principal band. Nematodes labeled with radioiodine and biotin released ¹²⁵I and biotin-labeled molecules into water after 20 hours incubation, indicating that surface coat proteins may be loosely attached to the nematode. Antiserum to the partially purified principal protein bound to the surface of live nematodes and to several proteins on Western blots. Differential patterns of antibody labeling were obtained on immuno-blots of extracts from M. incognita race 1, 2, and 3; Meloidogyne hapla race 2; and Meloidogyne arenaria cytological race B.     

Purification and partial amino acid sequence analysis of two distinct tumor necrosis factor receptors from HL60 cells

Two distinct tumor necrosis factor (TNF) receptors of 55- and 75-kDa apparent molecular masses previously identified on the cell surface by monoclonal antibodies have been solubilized with Triton X-100 from HL60 cells. A filter-based dot blot assay was developed to monitor specific 125I-TNF alpha binding during fractionation of the cell extract. By a combination of immuno- and ligand affinity chromatography and reverse phase high performance liquid chromatography both receptor proteins were purified to apparent homogeneity. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed two bands at 55 and 51 kDa for the 55-kDa TNF receptor and a major 75-kDa and a minor 65-kDa band for the 75-kDa TNF receptor. All these bands specifically bound TNF alpha and TNF beta in ligand blot experiments. The exclusive specificity of monoclonal antibodies of the utr series for the 75.65-kDa bands and of the htr series for the 55.51-kDa bands was demonstrated with the purified antigens on Western blots. Both TNF receptor types were found to contain N-linked carbohydrates. N-terminal amino acid sequence analysis of the 55- and 51-kDa bands of the 55-kDa TNF receptor revealed identical sequences suggesting a possible truncation at the C-terminal end. Two different N-terminal sequences were determined for the 65-kDa band. One corresponded to the published sequence of ubiquitin; the other was therefore assumed to be a unique sequence of the 75-kDa TNF receptor. Additional internal sequences of this receptor were determined after proteolytic cleavage.