Excretory-secretory products of Echinostoma paraensei sporocysts mediate interference with Biomphalaria glabrata hemocyte functions.

Miracidia of Echinostoma paraensei were cultured in medium containing 14C-labeled amino acids, allowed to transform into sporocysts, and their excretory/secretory products (E-S) were collected and characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. Effects of E-S on hemocytes of Biomphalaria glabrata were also assessed. E-S collected during day 1 of culture (E-S1) contained several polypeptides, none of which were labeled, suggesting that E-S1 are largely preformed. E-S1 significantly depressed the ability of hemocytes to phagocytose sheep red blood cells (SRBC), but otherwise had little effect on hemocyte structure or behavior. E-S released by sporocysts in day-2 cultures (E-S2) and in older cultures generally were similar and also contained several polypeptides, many of which were labeled, indicating active synthesis of E-S in vitro. E-S2 strongly inhibited hemocyte uptake of SRBC. Also, hemocytes pretreated with E-S2 assumed a spherical shape and failed to spread normally. E-S obtained through 10 days of culture mediated this effect. Active components of E-S2 were greater than 100 kDa in their native configuration, were heat- and trypsin-labile, and were bound by anti-E-S antibodies. Both greater than 200- and 80-kDa bands were prominent in anti-E-S immunoprecipitates. Hemocytes derived from snails of the 13-16-R1 strain of B. glabrata (a strain resistant to infection with Schistosoma mansoni), when pretreated with E-S2, bound to sporocysts of S. mansoni but lost their ability to damage such sporocysts. E-S2 interfered with hemocyte functions in ways inferred from earlier classic in vivo studies of trematode-snail interactions.     

Schistosoma japonicum: excretory-secretory products of the eggs during miracidial development.

The release pattern of excretory-secretory (E-S) products of Schistosoma japonicum eggs was investigated using eggs cultured in a chemically defined medium (MEMSE-J) for 16 days. The amount of protein released in culture supernatants was greater in 0- to 4-day and 12- to 16-day cultures than in 4- to 12-day cultures. The protein composition of E-S products and soluble extracts of newly laid eggs (N-SEA) and in vitro matured eggs (M-SEA) was analyzed by SDS-PAGE. Silver staining patterns of N-SEA and M-SEA were found to be similar except for the band at approximately 66 kDa, which appeared in highest concentrations in N-SEA. Western blot analysis with human infected sera showed antibody recognition of a 140- to 160-kDa antigen present in E-S products from mature eggs, while E-S products from immature eggs were unreactive. When either [35S]methionine or [3H]glucosamine was added to the culture medium, newly synthesized proteins or glycoproteins of the SEA and E-S products were labeled. Incorporation of both isotopes into SEA appears to correlate with developmental activity of the eggs. In contrast, release of E-S proteins and glycoproteins is more apparent as the miracidium matures. These results suggested that the source of E-S products from immature eggs is likely to be the collapsing vitelline cells and that of E-S products from mature eggs to be mainly miracidial secretions.     

Products of Cultured Neuroglial Cells. III. Release of an 85,000 Molecular Weight Glycoprotein by C6 Glioma Cells In Vitro

Abstract: With [³H]fucose as a marker, C6 glioma cells in culture released an 85,000 molecular weight molecule into the medium as the major extracellular glycoprotein. The quantity and extracellularkytoplasmic ratio of this glycoprotein suggest that its cellular processing is different from that of five other released glycoproteins of molecular weights 55,000, 115,000, 130,000, 150,000, and 170,000. Nearly 40% of newly synthesized glycoproteins in the cells was released into the culture medium. Major glycoproteins retained by the cells migrated electrophoretically to molecular weight positions of 82,000, 110,000, 120,000, 140,000, and 160,000, and approximately one-third of these retained glycoproteins were labile to trypsinization. Both synthesis and release of these macromolecules were inhibited more than 95% with cycloheximide treatment, demonstrating that nearly all fucosylation was linked to protein synthesis. Since 40% of all glycoproteins was released under conditions of more than 99% cellular viability, it is likely that these extracellular glycoproteins are physiological products of membrane turnover and secretion, but not of cell lysis. The results provide a basis for the further study of glial differentiation and of shed glioma antigens.     

The effect of schistosome excretory-secretory products on Biomphalaria glabrata hemocyte motility

Excretory-secretory (E-S) products contained in supernatants from in vitro cultured Schistosoma mansoni primary sporocysts were assayed for their effects on the in vitro motility of Biomphalaria glabrata hemocytes. Both whole (unfractionated) and fractionated E-S products were tested in modified Boyden chemotaxis chambers. E-S product fractionation was accomplished using both membrane ultrafiltration (MF) and high-pressure liquid chromatography (HPLC). Transformation (Tr) products, but not those products released by 8-day sporocysts, significantly inhibited the random motility of hemocytes from an S. mansoni susceptible strain (M-Line) of B. glabrata. This activity was found in both high and low MF fractions of Tr but not in an intermediate MF fraction. In an effort to isolate the active component(s) of the high MF fraction, HPLC was used to separate components based on size exclusion. Although each of four HPLC fractions displayed some inhibitory activity, the greatest consistent activity was found in fraction 3, which was composed, predominantly, of a 108-kDa protein. In contrast to the response of M-Line cells to Tr E-S products, the motility of hemocytes from an S. mansoni-resistant strain (10-R2-OK) of B. glabrata was not significantly reduced from controls. The high MF fraction, however, elicited a slight positive chemokinetic response, while the low MF fraction reduced 10-R2-OK hemocyte motility slightly but not significantly. While three HPLC fractions significantly reduced 10-R2-OK hemocyte motility, this effect was significantly less than that produced by the same HPLC fractions on M-Line hemocyte motility. These data suggest that S. mansoni sporocyst Tr E-S products differentially affect the random motility of M-Line and 10-R2-OK snail hemocytes. Although the significance of this differential effect on the in vivo defenses of B. glabrata is not known, it could be important in the host-parasite interaction which leads to either resistance or susceptibility.     

Effects of excretory-secretory products of Echinostoma paraensei larvae on the hematopoietic organ of M-line Biomphalaria glabrata snails.

Responses of the hematopoietic organ (HO) in Biomphalaria glabrata snails to extracts and excretory-secretory (E-S) products of Echinostoma paraensei larvae were studied to understand the HO-activating mechanism. M-line B. glabrata snails were injected with materials from E. paraensei larvae, and the size of the HO was ascertained in histological sections. The size of HO in snails injected with extracts and E-S products from sporocysts and rediae was significantly larger than that in snails injected with culture medium. E-S products of sporocysts were fractionated using ultrafiltration membranes, polyacrylamide gel electrophoresis, and electrophoretic elution. Examination of fractionated E-S products of sporocysts revealed that specific components of E-S products were responsible for HO-stimulating activity.     

In vitro effect of larval Schistosoma mansoni excretory-secretory products on phagocytosis-stimulated superoxide production in hemocytes from Biomphalaria glabrata

The in vitro production of the reactive oxygen metabolite superoxide (O2-) was confirmed in hemocytes from the schistosome intermediate host Biomphalaria glabrata. Active forms of the enzyme superoxide dismutase (SOD) inhibited reduction of nitroblue tetrazolium (NBT) to formazan in cells that had phagocytozed zymosan particles, whereas an inactivated form of SOD did not. Moreover, based on the prevalence of O2(-)-positive hemocytes and the relative intensity of NBT staining reactions, hemocytes from the Schistosoma mansoni-resistant 10-R2 strain of B. glabrata possessed an overall greater capacity for generating superoxide than did those from S. mansoni-susceptible M-line snails. Schistosoma mansoni excretory-secretory (E-S) products, released during in vitro transformation of miracidia to sporocysts, inhibited phagocytosis of zymosan particles and superoxide activity in hemocytes from both snail strains, but 10-R2 hemocytes maintained higher levels of phagocytosis and superoxide production than did M-line hemocytes. The dose-dependent decreases in phagocytosis observed in both snail strains in the presence of E-S products could not account fully for the concomitant decrease in superoxide levels detected, indicating that either a single E-S factor differentially affects phagocytosis and superoxide production, or that different E-S factors are involved in the specific interference of each of these hemocyte functions.     

Axenic culture of Schistosoma mansoni sporocysts in low O2 environments.

Recent successes in culturing intramolluscan larval stages of Schistosoma mansoni have relied on synxenic culture with a cell line (Bge) developed from embryos of a molluscan host Biomphalaria glabrata. To further facilitate progress toward control of schistosomiasis, a system for axenic in vitro culture of the parasite has now been developed. When culture media were preconditioned by Bge cells, sporocysts lived longer in vitro and produced more offspring. Because Bge-derived components could be protecting sporocysts from oxidative stress, axenic sporocysts were cultured at lowered O2 levels. In an hypoxic environment, S. mansoni sporocysts grew well and produced daughter sporocysts continuously under axenic conditions and in a medium completely lacking host molecules. Sporocyst production occurs independently of host influence.     

Identification of species-specific and gender-specific proteins and glycoproteins of three human schistosomes

Species-specific and gender-specific polypeptides of Schistosoma haematobium, Schistosoma japonicum, and Schistosoma mansoni have been identified. Proteins of these schistosomes were metabolically labeled in vitro with 35S-methionine and their total proteins, concanavalin-A binding glycoproteins, released (shed or secreted) proteins, and released glycoproteins compared by two-dimensional polyacrylamide electrophoresis. Many of the released proteins were glycosylated, and most of the synthesized glycoproteins were released. The most striking gender-specific and species-specific differences were observed in the released glycoproteins. These results provide a basis for investigating the molecular evolution of schistosomes, the occurrence of dioecy in the schistosomatidae , and for the development of improved serodiagnostic reagents.     

Participation of N-acetyl-D-glucosamine carbohydrate moieties in the recognition of Schistosoma mansoni sporocysts by haemocytes of Biomphalaria tenagophila

Lectin-carbohydrate binding may be involved in the recognition of Schistosoma mansoni sporocysts by haemocytes of Biomphalaria; therefore, we tested if this interaction is associated with snail resistance against Schistosoma infection. In vitro data showed that most of the S. mansoni sporocysts cultured with haemocytes from Biomphalaria glabrata BH, a highly susceptible snail strain, had a low number of cells that adhered to their tegument and a low mortality rate. Moreover, the addition of N-acetyl-D-glucosamine (GlcNAc) did not alter this pattern of adherence and mortality. Using haemocytes and haemolymph of Biomphalaria tenagophila Cabo Frio, we observed a high percentage of sporocysts with adherent cells, but complete encapsulation was not detected. Low concentrations of GlcNAc increased haemocyte binding to the sporocysts and mortality, which returned to basal levels with high concentrations of the carbohydrate. In contrast, haemocytes plus haemolymph from B. tenagophila Taim encapsulated cellular adhesion index of level 3 and destroyed over 30% of the S. mansoni sporocysts in culture. Interestingly, the addition of GlcNAc, but not mannose, to the culture medium resulted in the significant inhibition of cellular adhesion to the parasite tegument and the reduction of parasite mortality, suggesting that GlcNAc carbohydrate moieties are important to the recognition of S. mansoni by B. tenagophila Taim.     

Characterization of proteins produced in vitro by periattachment bovine conceptuses

Bovine conceptuses from Days 16 (n = 4), 19 (n = 6), 22 (n = 3), and 24 (n = 4), and chorion from Day 69 (estrus/mating = Day 0) were cultured for 24 h in modified minimum essential medium (MEM) in the presence of radioactive L-leucine [( 3H] leucine) to characterize de novo synthesis and release of proteins. Proteins released into MEM were identified by two-dimensional polyacrylamide gel electrophoresis, fluorography, and gel and ion exchange chromatography. Major polypeptides identified in MEM were different from those identified in conceptus and chorionic tissues. Both uptake of [3H] leucine and quality of polypeptides produced de novo and released into MEM were related to stage of conceptus development. Percent retention of [3H] leucine in MEM was lowest (P less than 0.01) in Day 16 cultures (1.2 +/- 4.1%), increased in Days 19 (16.8 +/- 3.7%) and 22 cultures (20.9 +/- 5.8%), and decreased (P less than 0.07) in Day 24 cultures (6.9 +/- 4.1%). Complexity of polypeptides increased after Day 16. Days 16, 19, 22 and 24 conceptus culture MEM was enriched in low-Mr, acidic polypeptides (Mr/isoelectric point ranges: 22K-26K/6.5-5.6, 20K-26K/5.5-5.4, and 16K-20K/5.0-4.5), which were not prominent products of Day 29 and 69 tissues. A high-Mr (Mr +/- SEM; 735K +/- 22K) glycoprotein was produced by all conceptus and chorionic tissues. The transient nature of production of low-Mr polypeptides suggests that they may be required during the periattachment period.     

Apical secretion of apolipoproteins from enterocytes

Synthesis and secretion of apolipoproteins in pig small intestine was studied by pulse-chase labeling of jejunal segments, kept in organ culture. Apo A-1 and apo B-48 were the two major proteins released, constituting 25 and 10%, respectively, of the total amount of labeled protein in the mucosal-side medium where they appeared with a t1/2 of 50-60 min. Using tissue from fasting animals, > 85% of newly synthesized apo A-1 and about one third of apo B-48 was released to the mucosal-side medium. Newly synthesized apolipoprotein that remained associated with the intestinal segment accumulated in the soluble fraction, suggesting a basolateral secretion into the intercellular space, and both this accumulation and the release to the medium was prevented by culture at 20 degrees C. The specific radioactivity of apo A-1 and apo B-48 released to the medium was significantly higher than that of the corresponding apolipoproteins remaining associated with the intestinal tissue. Furthermore, during culture periods of up to 5 h, the enterocytes and their tight junctions largely remained intact as evidenced by the inaccessibility of the nonpermeable surface marker Ruthenium red. We therefore propose that enterocytes release most of their newly made free apo A-1 and a significant portion of apo B-48 by exocytosis via the brush border membrane into the intestinal lumen. Fat absorption reduced apolipoprotein secretion to the medium and induced the formation of chylomicrons, containing apo A-1 at their surface, as evidenced by immunogold electron microscopy. The chylomicrons were localized in the Golgi complex and near the basolateral plasma membrane, but not in the apical region of the enterocytes, indicating that only free apolipoproteins are secreted to the intestinal lumen.     

The pattern of collagen degradation in cultured tadpole tissues

A characteristic pattern of selective degradation of isotopically labeled collagen in tadpole tail fin in culture was observed by measuring the amount and radioactivity of degraded collagen fragments released into the culture medium as a function of time of incubation. The changes in specific activity and total amount of hydroxyproline released with time indicated early degradation apparent at 3 hr of incubation of a small fraction of newly synthesized heavily labeled collagen followed by breakdown of the bulk of old lightly labeled fibrils. Collagenase activity rose in the culture medium with the release of collagen breakdown products and continued long afterward. Serum in the medium significantly reduced the release of collagen degradation products to the medium and greatly lowered their specific activity. Possible mechanisms of selective collagen degradation are discussed.     

Characterization of protein production by bovine chorionic and allantoic membranes

Bovine allantoic (A) and chorionic (C) membranes from Days 29, 32, 36, and 40 of pregnancy were isolated by dissection and cultured in a modified minimum essential medium in the presence of L-[35S]methionine to characterize in vitro synthesis and release of proteins. Membranes were also cultured in the presence of the glycosylation inhibitor tunicamycin. Proteins synthesized and released into the medium were analyzed by two-dimensional polyacrylamide gel electrophoresis and fluorography of dried gels. Stained gels were used to analyze protein from allantoic fluids. Percent incorporation of the radiolabeled amino acid into nondialyzable protein was higher for A than for C (A = 8.0 +/- 1.2 vs. C = 6.4 +/- 0.5 per 200 mg tissue) but not significantly different. C released significantly more total protein (nonradioactive) into the medium (57.0 +/- 3 vs. 9.6 +/- 0.6 micrograms/ml). Of the 25 proteins analyzed, 19 appeared to be produced primarily by one membrane or the other. In general, C was the source of a number of basic-to-neutral glycosylated proteins and A was the source of a number of more acidic glycosylated proteins. Many but not all proteins synthesized by A were present in allantoic fluid. The present study is the first to characterize protein production by isolated chorionic and allantoic membranes and to demonstrate that both tissues contribute to the production of secretory conceptus proteins.     

Calcium dynamics of hemocytes of the gastropod Biomphalaria glabrata: effects of digenetic trematodes and selected bioactive compounds

Abstract. Two fluorescent calcium indicators, Calcium Green AM (CG) and Fura Red AM (FR), were used in conjunction with confocal microscopy to monitor hemocyte calcium dynamics following exposure to digenetic trematode larvae or relevant bioactive compounds. Changes in intracellular calcium levels, as measured by fluctuations in the CG/FR ratio, were correlated with hemocyte morphological changes. Hemocytes exposed to culture medium remained spread and had few calcium transients. However, following exposure to sporocysts, sporocyst secretory-excretory products, or small rediae of Echinostoma paraensei in culture medium, significantly more hemocytes both rounded up and exhibited calcium transients, though some hemocytes showed one response or the other but not both. Hemocytes did not respond significantly to large rediae, to sporocysts of another digenean ( Schistosoma mansoni ), or to bacterial lipopolysaccharides. Exposure to either zymosan particles or mannose BSA provoked responses similar to those seen with sporocysts of E. paraensei Caffeine caused rounding but no calcium transients, and phorbol myristate acetate provoked calcium transients but no rounding. The results show that sporocysts and small rediae of E. paraensei have pronounced effects on hemocyte rounding and calcium dynamics, and that these two events can occur independently of one another. This suggests that parasites may influence hemocytes in at least two separate ways.     

Synthesis of serum proteins by cultured aggregates from endodermal cells of the area opaca of the primitive streak chick embryo

Summary The pattern of serum protein synthesis and secretion in aggregates of extraembryonic endoderm cells (EEC) from the area opaca of primitive streak chick embryos was studied. EEC aggregates were cultured for various time intervals and serum proteins were detected using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. Serum proteins were identified based on their comigration with reference proteins from 4 day chick embryo serum and with reference proteins from egg white albumen and chicken serum. A number of serum proteins were detected in EEC aggregates including: two variants of immunoglobulin (IgG), four variants of transferrin, a protein with a molecular weight of 66 500 which may correspond to globulins, prealbumin, and a protein with a molecular weight of 38 600 (serum protein 11) which remains unidentified. These proteins were also detected in the culture medium. The banding profiles of EEC extracts and culture medium were compared over various time intervals of culture (6, 18 and 30 h). The IgGs, transforms and serum protein 11 decreased in concentration in EEC extracts over the culture interval. These proteins as well as prealbumin, were detected in the culture medium. A number of proteins were synthesized by EEC, as determined by radiolabelled amino acid incorporation. All of the labelled serum proteins were detected in the culture medium, not in EEC extracts. These results suggest that serum proteins are synthesized by EEC then rapidly released into the medium. Labelled serum proteins detected in the culture medium include prealbumin and an unidentified serum protein (serum protein 14) which migrates with the tracking dye, both synthesized early in culture (6 h), and transferrin which was synthesized later (18 h) during culture.     

Maternal recognition of pregnancy in the goat: effects of conceptus removal on interestrus intervals and characterization of conceptus protein production during early pregnancy

Goat conceptuses were surgically removed from the uterus at different days during early pregnancy and cultured for 24-30 h in the presence of L-[3H]leucine to determine the effects of embryo removal on the interestrus interval and to characterize in vitro synthesis and release of conceptus proteins. Normal cyclic and animals (controls) exhibited interestrus intervals of 20.44 +/- 0.89 days. Removal of conceptuses on Days 13 and 15 did not alter interestrus intervals compared to cyclic animals. Removal of conceptuses on Day 17 and times thereafter resulted in significant (p less than 0.05) prolongation of interestrus intervals. These results demonstrate that maternal recognition of pregnancy in the goat occurs between Days 15 and 17. Proteins synthesized and released into the medium by conceptuses were first detectable at Day 16 by the analytical method employed (two-dimensional polyacrylamide gel electrophoresis followed by fluorography). The major protein synthesized at this time was acidic (pI = 5.2-5.7) and consisted of two isotypes with molecular weights of about 17,000. Although patterns of protein production became more complex with conceptus development, this protein remained as a major product through Day 21 but not afterwards. This protein, as well as two other low molecular weight acidic proteins (Mr approximately equal to 21,000, 23,000; pI = 5.7-6.0) were shown by immunoprecipitation to react with anti-ovine trophoblast protein-1 (oTP-1) serum. Hence, these products may comprise a caprine trophoblast protein-1 (cTP-1) complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Estrogen-dependent synthesis and release of a protein (CUPED) by cultured uterine explants from the domestic cat

Media from cultured cat endometrial explants were analyzed for the presence of a previously characterized high molecular weight estrogen-dependent secretory protein (CUPED) by polyacrylamide gel electrophoresis and radioimmunoassay (RIA). Both L-[(3)H]-leucine and D-[(3)H]-glucosamine were incorporated into newly synthesized CUPED during the culture of endometrial explants obtained from estradiol-treated ovariectomized cats, but not during the culture of tissue obtained from untreated ovariectomized animals or estradiol-primed ovariectomized animals treated with progesterone. The addition of tunicamycin to the culture medium inhibited the synthesis and release of the glycosylated form of CUPED from endometrial explants obtained from estradiol-treated cats. These data demonstrate that CUPED is synthesized and released in vitro from endometrial explants obtained from estradiol-treated cats as a glycoprotein possessing N-linked oligosaccharide chains.     

Release of the NILE and Other Glycoproteins from Cultured PC 12 Rat Pheochromocytoma Cells and Sympathetic Neurons

Studies were carried out on the glycoproteins (GPs) released by cultured rat sympathetic neurons and by cultured PC12 rat pheochromocytoma cells with and without nerve growth factor (NGF) treatment. Cultures were prelabeled with [3H]fucose and then incubated for 4-8 h in fresh unlabeled medium. The material released into the medium was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography. The patterns of labeled material released by all three types of cultures were similar. One of the major components released was of apparent Mr less than or equal to 230,000. Another major component of apparent Mr = 55,000 as well as minor components of apparent Mr less than or equal to 180,000, 140,000, 118,000, and 105,000 were also detected. An additional peptide of apparent Mr less than or equal to 210,000 was released only by the sympathetic neurons. The soluble released Mr less than or equal to 230,000 component appeared to be derived from a previously characterized neuronal integral membrane GP referred to as the NILE (NGF-inducible large external) GP. Evidence for this included recognition of the released component by a monospecific antiserum prepared against membrane-derived NILE GP. At least several of the other released GPs appeared to be derived from membrane-bound components with which they share immuno-crossreactivity. Since the soluble NILE and other released GPs had somewhat faster mobilities on SDS-polyacrylamide gels than their apparent membrane-bound correspondents, release could either be due to, or accompanied by, minor changes in molecular structure.     

Taenia taeniaeformis: characterization of larval metabolic products and growth of host gastric cells in vitro

Development of larvae of the cestode parasite Taenia taeniaeformis in the liver of rats induces gross hyperplasia of the gastric mucosa and excessive mucus production in the stomach without any direct contact with the stomach. Because the taeniid larvae are known to elaborate excretory-secretory (E-S) product in vivo and in vitro, the product was analyzed further, and its effects on cultured rat and dog stomach cells were investigated. In vitro E-S product contained less negatively charged glycosaminoglycan than either heparin or chondroitin sulfate, and proteins of various molecular weights. It stimulated the growth of both rat and dog stomach cells at concentrations of 3-9 micrograms protein/ml culture medium. At a concentration of 30 micrograms protein/ml culture medium, it stimulated hexosamine production in the cells up to 20 times, and multiple intracytoplasmic granules were found in both rat and dog cultured cells by light and electron microscopy. These results suggest that larval E-S product may be involved in the induction of gastric hyperplasia and hypermucus secretion.