concanavalin-a and Ascites

Intraperitoneal administration of concanavalin A (ConA, 25 mg/kg b.w.), a cell-binding plant lectin was used for inducing inflammatory ascites, and potential inhibitors were tested in 1 h and 2.5 h experiments, i.e. still before the major influx of leucocytes. At the end of the experiment the peritoneal fluid was collected and measured. The ConA-induced ascites was significantly (p<0.01) and dose-dependently inhibited by icatibant (HOE-140), a synthetic polypeptide antagonist of bradykinin receptors. Aprotinin, a kallikrein inhibitor protein also had significant (p<0.01), but less marked inhibitory effect. L-NAME, an inhibitor of NO synthesis, and atropine methylnitrate, an anticholinergic compound, were ineffective. It is concluded, that the kallikrein/kinin system contributes to the mediation of the ConA-induced ascites by increasing subperitoneal vascular permeability, independent of the eventual vasodilation produced by NO. It is known, that membrane glycoproteins are aggregated by the tetravalent ConA and the resulting distortion of membrane structure may explain the activation of the labile prekallikrein. Complete inhibition of the ConA-induced ascites could not be achieved by aprotinin or icatibant, which indicates the involvement of additional mediators.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Ascites; Bradykinin; Concanavalin A; Female; Humans; Kallikreins

The early phase of the ConA-induced inflammatory ascites was studied, with special reference to histamine. Concanavalin A (ConA), a cell-surface binding lectin was injected i.p. (25 mg/kg bw) to mice. After 1 h the animals were killed, the ascitic fluid collected and measured. Other agents were injected s.c., 10 min before the ConA-challenge. Exogenous histamine markedly inhibited the ConA-induced ascites. Release of endogenous vasoactive agents from the mast cells by Compound 48/80 had a similar, but slight effect. Cromolyn, a mast cell stabilizing agent, and chloropyramine, a histamine H1 receptor antagonist was ineffective. Although histamine increases endothelial permeability, it did not enhance the formation of ascitic fluid, on the contrary, it inhibited the ConA-induced ascites, presumably due to its known hypotonic effect. It is concluded that ConA-induced ascites is not mediated by mast cell histamine.

Topics: Adult; Animals; Ascites; Ascitic Fluid; Concanavalin A; Cromolyn Sodium; Ethylenediamines; Female; Histamine; Histamine H1 Antagonists; Humans; Mast Cells; Mice; p-Methoxy-N-methylphenethylamine

The effect of pre-treatment with Escherichia coli O83 lipopolysaccharide (LPS) on concanavalin A-induced ascites was examined. The LPS was injected intraperitoneally (i.p.) in different doses to mice, and then ascites was induced by i.p. administration of concanavalin A (ConA) (25 mg/kg b.w.). After 2.5 h the mice were killed and the ascitic fluid was collected and measured. The LPS produced a marked and dose-dependent inhibition of ConA-induced ascites and the effect of pre-treatment lasted up to almost a week. Complete inhibition could not be achieved. If administered alone, LPS did not produce ascites.It is well known that LPS enhances vascular permeability in several tissues, but the present work shows that peritoneal permeability is not enhanced by this agent. Suppression of ConA-induces ascites may be explained by the hypotonic effect of LPS.

Topics: Animals; Ascites; Concanavalin A; Down-Regulation; Female; Humans; Inflammation; Injections, Intraperitoneal; Lipopolysaccharides; Mice

Different ligands with high molecular masses are immobilized on compact, porous separation units and used for affinity chromatography. In subsequent experiments different enzymes are immobilized and used for converting substrates with low and high molecular masses. Disk or tube with immobilized concanavalin A (ConA) are used as model systems for lectin affinity chromatography. The enzyme glucose oxidase is used as a standard protein to test the ConA units. Subsequently glycoproteins from plasma membranes of rat liver are separated, using units with immobilized ConA. The enzyme dipeptidyl peptidase i.v., which is used as a model protein in the experiments, is enriched about 40-fold in a single step, with a yield of over 90%. The results are only slightly better than those obtained with ConA when it is immobilized on bulk supports. The important improvement lies in the reduction of separation time to only 1 h. Experiments concerning the isolation of monoclonal antibodies against clotting factor VIII (FVIII) are carried out on disks, combining anion-exchange chromatography and protein A affinity chromatography as a model for multidimensional chromatography. Both IgG (bound to the protein A disk) and accompanying proteins (bound to the anion-exchange disk) from mouse ascites fluid are retarded and eluted separately. With the immobilized enzymes invertase and glucose oxidase (GOX) the corresponding substrates with low molecular masses, saccharose and glucose, are converted. It is shown that the amount of immobilized enzyme and the concentration of the substrate are responsible for the extent of the conversion, whereas the flow-rates used in the experiments have no effect at all. The influence of immobilization chemistry was investigated with GOX. Indirect immobilization with ConA as spacer proved to be the best alternative. With trypsin, immobilized on a disk, substrates with high molecular masses are digested in flow-through. For optimal digestion the proteins have to be denatured in the buffer for sodium dodecyl sulfate-polyacrlyamide gel electrophoresis prior to application. In contrast to the conversion of substrates with low molecular masses, flow-rates play an important part in conversion of substrates with high molecular masses. With lower flow-rates a higher degree of digestion is achieved.

Topics: Animals; Ascites; beta-Fructofuranosidase; Chromatography, Affinity; Concanavalin A; Dipeptidyl Peptidase 4; Electrophoresis, Polyacrylamide Gel; Enzymes, Immobilized; Glucose Oxidase; Glycoside Hydrolases; Immunoglobulin G; Ligands; Liver; Mice; Molecular Weight; Rats; Staphylococcal Protein A; Sucrose; Transferrin; Trypsin

Erythrocytes from the circulation of rats bearing Yoshida ascites sarcoma exhibit higher concanavalin A (ConA)-mediated agglutinability than those from normal animals. A tetrameric glycoprotein of subunit molecular mass 170 kDa, purified from the cell-free ascites fluid, was found to confer higher ConA-mediated agglutinability on erythrocytes in vitro. An antiserum to this tumour-derived protein failed to detect any cross-reactive component in normal rat plasma or in any of the normal tissues examined. An immunoreactive protein was, however, detected in blood plasma when the acute-phase reaction was stimulated by injection of turpentine. The cross-reactive acute-phase protein was purified by ConA-affinity, gel-filtration and ion-exchange chromatography, and identified as alpha2-macroglobulin. The acute-phase protein and the protein obtained from the ascites fluid have identical or very similar native and subunit molecular masses, subunit arrangement and pI. They both are able to inhibit trypsin and, as a consequence, acquire greater mobility in native PAGE. In addition, the two proteins bind to rat erythrocytes non-specifically, and in similar amounts. However, despite these similarities, the acute-phase protein is unable to enhance the agglutinability of erythrocytes. The two proteins differ in their carbohydrate content, but this differential glycosylation is not the cause of the difference in their surface modification activity. The chemically deglycosylated proteins show a small but consistent difference in the size of their polypeptides. Their tryptic peptide maps, although largely similar, show some differences, as do their amino acid compositions. It is probable that the proteins are independent members of the same (alpha-macroglobulin) family. The rat embryo is also found to express a soluble protein consisting of a 170 kDa polypeptide that cross-reacts with the antibody to the tumour-derived protein. The purified embryo protein is able to alter the ConA-mediated agglutinability of erythrocytes in vitro, and also yields a tryptic peptide map that is identical to that of the tumour-derived protein. The modification of the host cell surface in the tumour-bearing rats is thus caused by what appears to be a tumour (oncofetal?) variant of alpha2-macroglobulin.

Topics: Agglutination; alpha-Macroglobulins; Amino Sugars; Animals; Ascites; Concanavalin A; Dose-Response Relationship, Drug; Embryo, Mammalian; Erythrocyte Membrane; Erythrocytes; Genetic Variation; Monosaccharides; N-Acetylneuraminic Acid; Peptide Mapping; Protein Binding; Rats; Rats, Wistar; Sarcoma, Yoshida; Tissue Distribution

The lymphocyte signal transduction, as determined by intracellular free Ca2+ mobilization of concanavalin A-stimulated T lymphocytes and of anti-immunoglobulin mu chain antibody-stimulated B lymphocytes, was suppressed in spleen cells from mice injected with murine P1.HTR mastocytoma-induced ascites and in spleen cells treated with the ascites in vitro. The suppression was observed both at the peak level and in the reactive pattern of Ca2+ influx. In the suppression, the ascites were replaceable with tumor culture supernatants or tumor homogenates. Correspondingly, primary and secondary cytotoxic T lymphocyte (CTL) responses of DBA/2 mice to allogeneic antigen were also significantly suppressed by injection of the syngeneic P1.HTR tumor-derived ascites. This new finding suggested that the mechanism of the tumorous ascites or of the tumor-derived factor-mediated immunosuppression involves at least in part the suppression of the early event of the signal transduction for lymphocyte activation.

Topics: Animals; Ascites; B-Lymphocytes; Calcium; Concanavalin A; Immunoglobulin mu-Chains; Immunosuppression Therapy; Lymphocyte Activation; Lymphocytes; Mast-Cell Sarcoma; Mice; Mice, Inbred Strains; Neoplasms, Experimental; Signal Transduction; Suppressor Factors, Immunologic; T-Lymphocytes

[14C]Glucosamine metabolic labeling and concanavalin A blots were used to identify four major glycoprotein species associated with ascites tumor cell microvillar microfilament cores and with a transmembrane complex containing actin. Phalloidin shift analysis of glucosamine-labeled microvilli showed that glycoproteins of 110-120, 80, 65, and 55 kDa are stably associated with the microfilament cores. Analysis of large (greater than 10(6) kDa) transmembrane complexes from microvillar membranes made under microfilament-depolymerizing conditions (Carraway, C. A. C., Jung, G., and Carraway, K. L. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 430-434) revealed glycoproteins of the same Mr values, showing the same relative staining or labeling patterns as those observed with the microfilament cores. Gel filtration of high salt, high pH extracts of intact microvilli, microfilament cores, or transmembrane complexes showed that in all of these fractions the glycoproteins are associated in a very large, stable complex. The glycoprotein multimer was isolated essentially free of actin and other components by Sephacryl S-1000 chromatography of microvilli, microvillar membranes prepared at pH 11, microfilament cores, or transmembrane complex fractions in Triton X-100, 1 M KCl, glycine, pH 9.5. Purified glycoprotein complex bound actin when incubated under polymerizing conditions. The presence of the glycoprotein heteromultimer in both microfilament cores and transmembrane complex from isolated membranes and the association of the purified glycoprotein complex with actin are consistent with our hypothesis that the glycoprotein-containing transmembrane complex is an association site for microfilaments at the plasma membrane.

Topics: Actins; Adenocarcinoma; Animals; Ascites; Blotting, Western; Chromatography, Gel; Concanavalin A; Electrophoresis, Polyacrylamide Gel; Glucosamine; Membrane Glycoproteins; Microfilament Proteins; Microvilli; Phalloidine; Rats; Tumor Cells, Cultured

Hepatoma-associated abnormal (des-gamma-carboxy)prothrombin (HAPT) is a newly described tumor marker for hepatocellular carcinoma. HAPT has been measured in the blood of patients with hepatoma by immunoassay but has not been isolated or characterized. This paper describes the quantitative isolation and structural characterization of HAPT. Purified HAPT has the same molecular weight, amino-terminal sequence, and amino acid analysis (exclusive of gamma-carboxyglutamic acid) as native prothrombin and abnormal prothrombin isolated from the blood of patients taking sodium warfarin. HAPT is heterogeneous in gamma-carboxyglutamic acid (Gla) content with an average of 5 Gla residues/molecule compared to 10 Gla residues for native prothrombin and 2 Gla residues for abnormal prothrombin. HAPT is glycosylated in a manner equivalent to that for native prothrombin when evaluated by a concanavalin A-binding assay. These studies find structural identity between HAPT and abnormal prothrombin. Therefore the findings support the hypothesis that HAPT results from an acquired defect in the posttranslational vitamin K-dependent carboxylation of the prothrombin precursor and not an intrinsic defect in the prothrombin precursor molecule.

Topics: Ascites; Biomarkers; Biomarkers, Tumor; Carcinoma, Hepatocellular; Concanavalin A; Electrophoresis, Polyacrylamide Gel; Liver Neoplasms; Protein Precursors; Prothrombin

A macrophage migration inhibition factor (OC-MIF) has been isolated from ascites fluid of ovarian cancer patients by affinity chromatography on L-fucose-Sepharose 6B, and characterized biochemically. OC-MIF activity was purified approximately 10 000-fold as compared to the starting material. It exhibits molecular heterogeneity with respect to net charge and molecular weights. Compared to it, purified and radioiodinated OC-MIF is fairly homogeneous and contains a major protein component with a molecular mass of about 45 kD, and two isoelectric points of 3.0-4.0 and about 5.0. Rabbits were immunized with the highly purified MIF material and an antiserum was prepared and was used to prepare immunoadsorbent beads. Beads made with anti-OC-MIF antiserum, but not with rabbit control serum, could remove specifically OC-MIF activity and showed weak reactivity towards Con A induced MIF. Using a radioimmunoassay (RIA) anti-OC-MIF antiserum reacts with OC-MIF and also with Con A induced MIF. This antigenic relationship between conventional MIF and OC-MIF and common biochemical properties suggest that the two mediator substances are very similar and may, perhaps, be identical. Furthermore, the possibility to determine various MIF activities by means of RIA was investigated.

Topics: Antibodies; Ascites; Chromatography, Affinity; Chromatography, Gel; Concanavalin A; Electrophoresis, Polyacrylamide Gel; Female; Humans; Immunosorbent Techniques; Isoelectric Focusing; Macrophage Migration-Inhibitory Factors; Macrophages; Ovarian Neoplasms; Radioimmunoassay

We have studied some cellular characteristics and the transplantability of a newly induced squamous cell carcinoma, Sq1-SC, in comparison with the ascites-transformed subline of the same tumor, Sq1-AA. We could demonstrate that the AA tumor differed from the SC tumor in the pattern of intravenously induced 'experimental metastases'. The SC tumor preferentially gave rise to extrapulmonary tumor colonies ('metastases'), while the AA tumor exclusively gave rise to lung colonies. Comparison with the ascites tumor growing in solid form subcutaneously (AS tumor) shows that the enzymatic treatment, which is necessary to bring solid tumors into viable and dissociated suspensions, can have a decisive influence on tumor cell lodgement in vessels and metastasis.

Topics: Animals; Ascites; Blood Coagulation; Carcinoma, Squamous Cell; Cell Separation; Concanavalin A; Electrophoresis; Female; Lectins; Methylcholanthrene; Mice; Mice, Inbred CBA; Neoplasm Metastasis; Neoplasm Transplantation; Trypsin; Wheat Germ Agglutinins

Ascitic fluid haptoglobins 1-1, 2-1 and 2-2 and their tryptic glycopeptides were fractionated by affinity chromatography on Con A-Sepharose. Three peaks were obtained, corresponding to non-binding, weakly binding and strongly binding fractions. Concanavalin A-non-binding and concanavalin A-binding fractions of haptoglobin and of glycopeptide III 2-2 consisted of a series of polymers with increasing molecular mass, except for the non-binding fraction of glycopeptide III 1-1. After reduction there was no difference between the subunit composition of the glycopeptides and their concanavalin A fraction. Concanavalin A-non-binding fractions from haptoglobin 2-1 and glycopeptides III 1-1 and III 2-2 did not form an active complex with hemoglobin and, in crossed immunodiffusion, showed a reaction of partial identity with haptoglobin 2-1, glycopeptides III 1-1, III 2-2 and their concanavalin A-binding fractions. Concanavalin A-binding fractions of the above preparations exhibited with hemoglobin higher peroxidase activity than before their separation on Con A-Sepharose and immunodiffusion gave a reaction of identity among themselves and with unfractionated preparations. The concanavalin A-binding glycopeptide III is the biologically active part of the haptoglobin beta-chain.

Topics: Ascites; Chromatography, Affinity; Concanavalin A; Electrophoresis, Polyacrylamide Gel; Haptoglobins; Hemoglobins; Humans; Immunodiffusion; Molecular Weight; Peroxidases; Receptors, Concanavalin A; Sepharose; Trypsin

In view of the reported disagreement in the physicochemical properties of ovarian carcinoembryonic antigen (CEA), this study was undertaken to compare the properties of CEA obtained from extracts of ovarian tumour tissue, ascitic fluid and cyst fluid. On the basis of molecular weight estimation and binding properties with Concanavalin A and wheat germ lectin, ovarian CEA from these three sources appeared similar, and also possessed similar properties to those of colonic CEA. On isoelectric focusing, however, it was found that the isoelectric point of CEA from tumour tissue and cyst fluid differed from that from ascitic fluid. It is most likely that this is due to a loss of sialic acid from the CEA released into ascitic fluid.

Topics: Ascites; Carcinoembryonic Antigen; Concanavalin A; Cystadenocarcinoma; Female; Humans; Ovarian Cysts; Ovarian Neoplasms; Radioimmunoassay

Fusion of cells of the mouse myeloma line, P3/X63-Ag8 with spleen cells from AKR/J mice immunized against C3H thymocytes or from (BALB/c x BALB.K)F1 mice immunized against AKR/J thymocytes gave rise to hybrid cell lines that continuously secrete antibodies specific for the Thy-1.2 and Thy-1.1 antigens, respectively. Monoclonal antibodies from four such cell lines were analyzed in detail. All were 19S IgM, and, in the presence of complement (C), had high lytic titers on T cells of the appropriate antigenicity. Their specificity was shown by lysis of thymocytes from Thy-1 congenic mouse strains, A/J(Thy-1.2) and A. Thy 1.1. Furthermore, they lyse only 60 to 70% of lymph node cells, suggesting cytotoxicity for mature T cells and not B cells. Treatment of peripheral lymphocyte populations with monoclonal antibody plus C eliminated effector cytotoxic T lymphocytes, their precursors, and the mitogenic response to Con A, but did not affect the response to LPS. Purified, fluorescein-labeled monoclonal anti-Thy-1 antibody could be used to distinguish T and B cells. Purified antibody coupled to Sepharose 6MB was used to separate viable T and B cells. Two independently isolated anti-Thy-1.2 hybridomas are indistinguishable and bind the same determinant whereas a third is unique and may bind a separate site.

Topics: Animals; Antibody Specificity; Antilymphocyte Serum; Ascites; Binding Sites, Antibody; Cell Fusion; Cell Separation; Chromosome Mapping; Clone Cells; Concanavalin A; Epitopes; Isoantibodies; Mice; Mice, Inbred A; Mice, Inbred AKR; Mice, Inbred BALB C; Mice, Inbred C3H; Mice, Inbred C57BL; Thymus Gland

Two ascites tumors in syngeneic CBA mice are described, viz., MCB 21-AA and MCB 31-AA, with their solid progenitors: A sarcoma (MCB 21-SS) and a squamous cell carcinoma (MCB 31-SC), induced by gastric feeding of 20-methylcholanthrene. The ascites tumor cells have certain characteristics in common, which they do not share with either cells from the solid tumors or even with cells from solid ascites tumors (-21-AS and -31-AS=ascites tumor transplanted s.c.). Presumably some of these differences, for instance, in PAS stainability, electrophoretic mobility and lectin agglutinability, are due to enzyme treatment required to bring solid tumors into suspension. Between the two ascites tumors there are certain differences in cell size, aggregability, and growth rate. They are similar, however, in requiring large cell doses for transplantation in syngeneic animals, which is also true for the solid (SS and SC) tumors. MCB 21 and -AA even required fewer cells for transplantation in allogeneic A mice than in syngeneic CBA mice. MCB 31-AA is also allotransplantable. The pattern of spread, after i.v. cell injection, is almost exclusively to the lungs for all tumor lines.

Topics: Animals; Ascites; Carcinoma, Squamous Cell; Cell Nucleus; Chromosomes; Concanavalin A; Electrophoresis; Fibrosarcoma; Lectins; Methylcholanthrene; Mice; Mice, Inbred CBA; Neoplasm Transplantation; Neoplasms, Experimental; Surface Properties; Transplantation, Homologous; Transplantation, Isogeneic

Topics: Adenocarcinoma; Agglutination; Animals; Antigens, Neoplasm; Anura; Ascites; Cell Line; Cell Membrane; Cell Nucleus; Concanavalin A; Inclusion Bodies; Kidney Neoplasms; Rana pipiens