Radioimmuno-thin-layer chromatographic detection of Forssman antigen in human carcinoma cell lines

Glycolipid antigen was examined by radioimmuno-thin-layer chromatography (RITLC), which is a combination of a thin-layer chromatography and radioimmunoassay. In this way Forssman antigen was studied in seven carcinoma cell lines. The usual Forssman antigen with a ceramide pentasaccharide structure was detected in cell lines of a gastric cancer and a breast cancer. In addition another glycolipid with slower mobility on thin-layer chromatography and with Forssman reactivity was found in cell lines of three gastric cancers and one lung cancer.     

Isolation and partial characterization of a 700 kilodalton human colon carcinoma associated antigen

An antigen of high molecular weight (CA-3) was isolated from the cytosol fraction of GW-39 human colon tumor cells by antibody affinity chromatography. CA-3 was characterized by an acidic pI value of 4.5-4.9, a molecular weight of 700 kilodaltons and a sedimentation coefficient of 13S. It contained all of the commonly occurring amino acids and had an acidic to basic amino acid ratio of 1.4. CA-3 was resistant to dissociation by reducing agents as well as by sodium dodecylsulfate. Quantitation of CA-3 by a radioimmunoassay employing rabbit anti-CA-3 antiserum revealed a marked elevation of CA-3 in the cytosol extracts of human primary colon carcinoma in comparison to normal colon. The molecular properties of CA-3 are compared to those of carcinoembryonic antigen, high molecular weight colon specific antigen CSAp and two other high molecular weight proteins, fibronectin and conglutinin. Colon antigen CA-3 appears to be different from these other molecules in terms of its molecular weight, sedimentation value, isoelectric point and amino acid composition.     

Isolation and characterization of angiogenin, an angiogenic protein from human carcinoma cells

The first human tumor derived protein with in vivo angiogenic activity to be obtained in pure form has been isolated from serum-free supernatants of an established human adenocarcinoma cell line (HT-29) and named angiogenin. It was purified by cation-exchange and reversed-phase high-performance liquid chromatography; the yield was approximately 0.5 microgram/L of medium. Biological activity of angiogenin was monitored throughout purification by using the chick embryo chorioallantoic membrane assay. Statistical evaluation demonstrates that it displays activity in this system with as little as 35 fmol per egg. Moreover, only 3.5 pmol is required to induce extensive blood vessel growth in the rabbit cornea. The amino acid composition of this basic (isoelectric point greater than 9.5), single-chain protein of molecular weight approximately 14 400 has been determined. The amino terminus is blocked, and the carboxyl-terminal residue is proline.     

Isolation and characterization of an unique kidney antigen of relevance in human renal disease

Recently we described a monoclonal antibody, designated 7-5Q, with specificity for a human non-glomerular basement membrane capillary wall antigen. In order to purify and characterize the corresponding antigen for the development of sensitive ELISA techniques, applicable to the diagnosis and monitoring of renal disease, human kidney cortices were extracted with a variety of detergents. A double band of 98/105 kD was evident by immunoblotting in all preparations most notably with a Triton X-114 extract. The caprylic acid- and ammonium sulfate-purified monoclonal antibody 7-5Q was covalently bound to CNBr-activated Sepharose and detergent extracts were applied to this affinity material. A pure protein of 98/105 kD (double band on SDS-polyacrylamide gels) was eluted. Glycan typing with lectins revealed N-acetyl glucosamine-residues and amino acid analysis a relatively high content of acidic (31%) and hydrophobic (30%) amino acids, indicating that the antigen is an acidic, membrane-bound glycoprotein.     

Isolation and characterization of a carcinoma-associated antigen

GA733 is a murine IgG2a monoclonal antibody (MAb) against human gastric carcinoma and is highly tumoricidal in nude mice. The GA733 antigen is a cell surface protein with two subunits of 30,000 and 40,000 daltons. The antigen isolated by immunoaffinity chromatography consists mainly of the 30,000-dalton subunit which bears the GA733 epitope. This subunit displayed several isoelectric points between 6.9 and 7.7. Anti-colon carcinoma MAb 17-1A also detects this antigen, but probably binds to a different epitope.     

Cytokine biosynthesis by tumor-infiltrating T lymphocytes from human non-small-cell lung carcinoma

The purpose of this work was to assess the capacity of tumor-infiltrating leukocytes (TIL) from human non-small-cell lung carcinoma (NSCLC) specimens to synthesize type-1 and type-2 cytokines. Methods: TIL were isolated from tumors following digestion with collagenase/DNase and further enriched by ficoll-hypaque gradient centrifugation. Membrane phenotypes and intracellular cytokine protein expression of TIL were assessed by flow cytometry. Results: The majority of TIL expressed the CD3 antigen with a CD4:CD8 ratio of approximately 2:1. Other leukocytes such as macrophages (CD14), B lymphocytes (CD20), and natural killer (NK) cells (CD56) were also found to infiltrate the tumors, but in significantly lower numbers. Owing to the limited recovery of non-CD3⁺ leukocytes, our analysis of cytokine biosynthesis has focused on T lymphocytes. In the absence of activation, a small percentage of CD3⁺ TIL synthesized cytokines ( <4%). Following activation with anti-CD3+interleukin-2 (IL-2), CD3⁺ TIL synthesized predominantly a type-1 cytokine profile; however, the type-2 cytokines, IL-6 and IL-10, were also detected in a small percentage of infiltrating cells. Following activation with phorbol 12-myristate 13-acetate + ionomycin, CD3⁺ TIL also expressed more type-1 than type-2 cytokines and in significantly greater numbers of cells. The CD3⁺CD8⁺ component of the TIL synthesized only type-1 cytokines, whereas the CD3⁺CD4⁺ component synthesized both type-1 and type-2 cytokines. Conclusion: These results show that the majority of the TIL isolated from NSCLC specimens are T lymphocytes with the capacity to synthesize type-1 cytokines. Received: 24 March 1999 / Accepted: 9 September 1999     

Isolation, characterization, and biosynthesis of a phosphorylated glycoprotein from rat bone

A phosphorylated glycoprotein was purified from the mixture of proteins extracted by demineralization of rat bone with 0.5 M EDTA in 4 M guanidinium chloride. A high level of purity for the preparation was indicated by a single band on sodium dodecyl sulfate (SDS)-gradient gel electrophoresis, sedimentation equilibrium ultracentrifugal data, and by automated Edman degradation results. The molecular weight of the phosphoprotein was shown to be about 44,000 by sedimentation equilibrium analyses in 4 M guanidinium chloride, even though an Mr of 75,000 was obtained by 5-15% SDS-polyacrylamide gel electrophoresis. Subsequent analysis by 15% SDS-polyacrylamide gel electrophoresis gave an Mr of 45,000. Analytical data showed that the protein contained 16.6% carbohydrate, possibly including 1 N-linked oligosaccharide and 5-6 O-linked oligosaccharides. The aspartic acid- and glutamic acid-rich protein contained about 300 amino acid residues including 1 phosphothreonine and 12 phosphoserine residues. Alkaline beta-elimination/NaBH4 reduction data showed that the phosphate obtained by complete acid hydrolysis prior to amino acid analysis was equivalent to the phosphate subject to alkaline beta-elimination. In this experiment, the losses of serine plus threonine exceeded the amount of phosphate liberated by 5-6 residues/protein. These serine and threonine residues probably represent O-linked oligosaccharides, since the protein contained about this number of N-acetyl-galactosamine residues. That the phosphoprotein is synthesized and secreted by osteoblast-like cells was shown with cultures of clonal rat osteosarcoma cells. After pulsing with 32PO4 the proteins secreted into the medium were precipitated with trichloroacetic acid and the radiolabeled proteins were immunoadsorbed. A protein migrating in the same position, on 5-15% SDS-polyacrylamide gel electrophoresis (i.e. with an Mr = 75,000) and on 15% gels (Mr = 45,000), as the phosphoprotein obtained from bone could be specifically immunoprecipitated.     

Isolation and characterization of a novel Forssman active acidic glycosphingolipid with branched isoglobo-, ganglio-, and neolacto-series hybrid sugar chains

Equine kidney and spleen contain a Forssman active glycosphingolipid, and the structure of this glycolipid has been reported to be that of a globopentaosylceramide (GalNAcalpha-1,3GalNAcbeta-1,3Galalpha-1, 4Galbeta-1,4Glcbeta-1,1'Ceramide). We found that equine kidney contains several other anti-Forssman antibody-reactive glycosphingolipids. One of these acidic Forssman active glycosphingolipids was isolated and characterized by means of NMR, mass spectrometry, permethylation studies, and TLC-immunostaining. This glycolipid contains three moles of galactose, one mole of glucose, three moles of N-acetylgalactosamine, one mole of N-acetylglucosamine, and one mole of N-acetylneuraminic acid, and is stained on TLC with anti-Forssman antibodies and anti-GM2 ganglioside antibodies. HOHAHA and ROESY experiments and permethylation studies showed this glycolipid oligosaccharide to be branched at the innermost galactose; one chain has an isoglobo structure with a terminal Forssman disaccharide and the other chain is branched through the linkage of N-acetylglucosaminebeta-1,6 to the inner galactose. The nonreducing end of the GM2 trisaccharide is linked to this glucosamine. The structure of the oligosaccharide of the glycolipid presented here is a novel type, having branched isoglobo-, ganglio-, and neolacto-series oligosaccharides. Mass spectrometric analyses indicated the ceramide moiety of the glycolipid to be composed predominantly of hydroxy fatty acids (C20:0, C22:0, C23:0, C24:0, and C25:0) and hydroxysphinganine. GalNAcalpha-1,3GalNAcbeta-1,3Galalpha-1,3[GalNAcbet a-1, 4(NeuAcalpha-2,3)Galbeta-1,4GlcNAcbeta-1,6]Galbeta+ ++-1,4Glcbeta-1, 1'Ceramide     

Isolation and morphologic characterization of human ovarian carcinoma cell clusters present in effusions

Serous, mucinous, endometrioid and clear cell human ovarian carcinoma cells were isolated as multicellular aggregates from patient effusions by filtration on nylon mesh of defined porosity and examined by light microscopy. The cell clusters ranged from compact to loosely adherent groups of cells to spheroids with a central lumen surrounded by a cell monolayer. There was considerable variation in cluster morphology between effusions from different patients as well as within effusion from the same patient. Apparent budding of clusters was observed as well as different stages of cluster growth and development. This was observed for all histologic types studied. Electron microscopy of serous, mucinous and clear cell types showed that cells forming clusters were attached to each other by desmosomes, demonstrating that cluster formation did not result from a nonspecific stickiness of cells. Irregular microvilli were present on the external periphery of the various carcinoma cells and a prominent glycocalyx was present on the surface of mucinous carcinoma cells. Extensive interdigitation of cytoplasmic extensions and extended villi was present in mucinous and serous clusters which appeared to strengthen cluster cohesiveness. Nuclei were irregular with prominent nucleoli frequently present. The cell clusters usually remained intact and viable in culture but generally did not attach to glass or plastic substrata, whereas mesothelial cells and nonactivated histiocytes rapidly attached. When carcinoma cell clusters did attach, they were resistant to detachment by trypsin-EDTA treatment, in contrast to the nonmalignant cells.     

Isolation and characterization of a multipotent clone of human embryonal carcinoma cells

Histopathological studies suggest that the stem cells of human teratomas may be classified into two major categories: nullipotent stem cells, and multipotent stem cells, capable both of self-renewal and differentiation into a wide range of somatic and extraembryonic cell types. We have isolated a multipotent stem cell clone from the human teratoma cell line GCT 27, and compared its properties to a nullipotent clone derived from the same strain. The multipotent clone GCT 27 X-1 gave rise to colonies of mixed cell morphology in vitro. Analysis of cell surface, cytostructural and extracellular matrix markers in GCT 27 X-1 cells showed that the stem cells of this line were very similar in phenotype to nullipotent cells. The two cell clones were predominantly hypotriploid, and contained several marker chromosomes in common. GCT 27 X-1 was feeder-cell-dependent for continuous growth in vitro; removal of the feeder layer resulted in differentiation of the stem cells into a variety of cell types, some with characteristics of extraembryonic endoderm, others showing neuronal properties. When transplanted into nude mice, GCT 27 X-1 cells gave rise to teratocarcinomas containing embryonal carcinoma stem cells, and many other cell types: yolk sac carcinoma cells; cells producing alphafetoprotein or human chorionic gonadotrophin; glandular, columnar, cuboidal, and squamous epithelium; primitive mesenchyme and cartilage; neuroectodermal cells. Nullipotent GCT 27 C-1 cells could form colonies in the absence of feeder layers, but multipotent GCT 27 X-1 cells could not. While a range of known growth factors and related substances failed to substitute for feeder layers in supporting the growth of GCT 27 X-1 stem cells, supernatants from yolk sac carcinoma cell line GCT 44 could partially replace the feeder cell requirement. Thus, the results revealed a basic difference in growth control between these multipotent and nullipotent human embryonal carcinoma cells, and suggested a possible paracrine regulatory pathway between multipotent stem cells and yolk sac carcinoma cells.     

Histone metabolism during amphibian metamorphosis. Isolation, characterization, and biosynthesis.

The amino acid incorporation rates of several classes of liver protein from Rana catesbeiana tadpoles were examined at different stages of spontaneous and thyroxine-induced metamorphosis, particular attention being given to histones. Incorporation data were corrected for the specific radioactivity of the free amino acid pools in tadpole liver. Little change was observed in the overall incorporation rates for the crude mitochondrial and total liver proteins during thyroxine treatment or at selected stages of spontaneous metamorphosis, except that the incorporation rates for these proteins were approximately twofold greater for the newly metamorphosed froglet than for the other stages. However, an increase in the ratio of the specific radioactivities of the total and crude mitochondrial liver protein within each set of animals was observed during late stages of spontaneous metamorphosis, as well as during the second through sixth days of thyroxine treatment. The amino acid incorporation rates of the histones for the late metamorphic and froglet stages of spontaneous metamorphosis were three- to fourfold higher than those of premetamorphic animals, but no significant changes were observed during thyroxine treatment. Thyroxine treatment also produced no detectable changes in the relative amounts or incorporation rates of the histone fractions or subfractions. Apparently the developmental changes induced by thyroxine do not involve a reorganization of the histone complement of chromatin at this level of analysis. Furthermore, since histone and DNA syntheses are tightly coupled, our results show that the extensive metabolic changes induced in tadpole liver by thyroxine occur in the absence of significant levels of cell division.     

Isolation and characterization of an asthma-inducing sea-squirt antigen

An antigen capable of inducing asthmatic attack in patients with sea-squirt allergy has been isolated from body fluid of sea-squirt, Styela plicata, and designated as DIIIa. The chemical composition and the behavior in anion-exchange chromatography showed that the antigen was a weakly acidic glycoprotein. The weight-average molecular weight of DIIIa was estimated to be 9,880 by the sedimentation equilibrium method. This antigen was apparently discriminated from the previously isolated sea-squirt antigens, Gi-rep and Ei-M, by its activity to induce asthmatic attack and conjunctival congestion, though it gave a strongly positive reaction in skin tests against allergic patients, similarly to the previous antigens. From the cross-reaction of DIIIa to rabbit anti-Gi-rep and anti-Ei-M sera in vitro, it was confirmed that the antigen carried essentially the same antigenic determinant as Gi-rep and Ei-M, and this was termed type alpha determinant. Furthermore, the allergenic activities detectable in vivo and the reactivity to the rabbit antisera in vitro were simultaneously absorbed by immobilized immunoglobulin from anti-Gi-rep serum. Thus, it was suggested that the antigenic determinant responsible for the allergic reactions in vivo corresponded to that specified as type alpha in vitro on the basis of the reactivity against the rabbit anti-Gi-rep serum.     

Isolation and characterization of a Forssman antigen-binding lectin from velvet bean (Mucuna derringiana) seeds

A Forssman antigen (GalNAc1-3GalNAc1-3Gal1-4Gal1-4Glc1-1Cer)-binding lectin has been purified from velvet bean (Mucuna derringiana) seeds by a combination of affinity chromatography and reversed phase HPLC. This lectin agglutinates both native and trypsin-treated sheep erythrocytes as well as trypsinized rabbit erythrocytes, but neither native rabbit nor human erythrocytes, irrespective of blood group type. SDS-PAGE and gel filtration chromatography reveal the lectin to be a homodimer consisting of two 54 kDa subunits linked by non-covalent bonds. The results obtained by quantitative precipitation, haemagglutination inhibition and TLC overlay assays indicate that theMucuna lectin specifically recognizes Forssman antigen and Forssman disaccharide (GalNAc1-3GalNAc)-related structures. Abbreviations: The abbreviations and the trivial names used are: AH, 6-aminohexyl; BSA, bovine serum albumin; Cer, ceramide; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; PBS, 10mm phosphate-buffered saline, pH 7,2, containing 0.15m NaCl; PMSF, phenyl methyl sulfonyl fluoride; SDS, sodium dodecyl sulphate; TFA, trifluoroacetic acid; TBS, 20mm tris-buffered saline, pH 7.2; TLC, thin-layer chromatography; A disaccharide, GalNAc1-3Gal; A trisaccharide, GalNAc1-3[Fuc1-2]Gal; Forssman disaccharide, GalNAc1-3GalNAc; CDH (ceramide dihexoside or lactosyl ceramide) Gal1-4Glc1-1Cer (LacCer); CTH (ceramide trihexoside or globotriosyl ceramide), Gal1-4Gal1-4Glc1-1Cer (GbOse₃Cer or Gb₃); globoside (globotetraosyl ceramide), GalNAc1-3Gal1-4Gal1-4Glc1-1Cer (GbOse₄Cer or Gb₄); Forssman antigen (globopentaosyl ceramide), GalNAc1-3GalNAc1-3Gal1-4Gal1-4Glc1-1Cer (GbOse₅Cer).     

Isolation and characterization ofSerratia marcescens mutants defective in prodigiosin biosynthesis

Mutants deficient in the biosynthesis of prodigiosin have been obtained by treatingSerratia marcescens with high doses of ultraviolet radiation. Mutants were selected on the basis of the color characteristics of their colonies when grown on peptone glycerol medium. New types of mutants, with unusual blocks in the biosynthetic pathway of prodigiosin, were obtained. All the mutants were classified under a new scheme on the basis of the syntrophic pigmentation characteristic and infrared spectroscopic analysis of their pigment. By these criteria mutants could be distinguished into eight distinct classes. Classes I to III include mutants of the three classes (M1, B3, and B1) reported previously [Morrison, DA (1966) J Bacteriol 91:1599–1604] and several new ones. Mutants blocked in the methylamylpyrrole (MAP) arm of the bifurcated pathway were assigned to class I. A class II mutant was distinguished by its inability to synthesize methoxybipyrrolecarboxyaldehyde (MBC), but was able to produce norprodigiosin. Class III mutants were deficient in the synthesis of hydroxybipyrrolecarboxaldehyde (HBC). Double mutants were obtained with defects in the expression of both MBC and MAP and were assigned to class IV. Mutants of class V were unable to synthesize HBC and MAP, but could form MBC when furnished with exogenous HBC. Class VI and VII mutants were defective in the synthesis of all three precursors, but differed in their ability to perform the coupling step. Finally, a mutant of class VIII was found to produce the three intermediates, but was deficient in prodigiosin or norprodigiosin biosynthesis, indicative of a defect in the enzymatic condensation of MAP with the bipyrroles MBC and HBC. The anomalous pattern of syntrophism among certain interclass mutants suggests that the physiology of pigment formation inS. marcescens is quite complex.     

Isolation and characterization of vitamin-A-storing lung cells

Vitamin A-storing cells have been shown to be distributed among various organs and tissues, including the lungs. In order to investigate this unique type of cell, the in vitro isolation has been carried out from rat lungs. Lungs were perfused with EGTA and collagenase solution in situ, and were digested with trypsin-collagenase solution at 60-min intervals for 2 h. Then, the cell suspensions obtained were incubated at 37 degrees C in F-10 medium supplemented with 10% fetal bovine serum (FBS) for 72 h. Non-adherent cells were then removed by vigorous washing with medium, and the resultant cell monolayer was harvested with trypsin to remove the contaminating macrophages. These cell fractions were shown to contain more than 96% of vitamin A-storing cells, judged by electron and fluorescence microscopic examinations. The cells grown in vitro retained well the overall morphology characteristic of the vitamin A-storing cells found in lung tissues. The isolated cells grew well in vitro and the growth was inhibited by D-valine or cis-hydroxyproline. The progeny of the cells still contained vitamin A lipid droplets after several transfer generations. Characteristic networks of fibronectin were also demonstrated around the cells. These results have shown that vitamin A-storing cells in the lung was successfully isolated from rat lungs and the cells possessed fibroblast-like characters storing vitamin A in small lipid droplets.     

Isolation and characterization of the thymus-brain antigen (analogous to thy-1 antigen) from human brain.

1. The human thymus-brain antigen, which corresponds to the murine (mouse or rat) Thy-1 antigen complex, was isolated from brain after solubilization in deoxycholate by gel-permeation chromatography, wheat-germ-lectin affinity chromatography and ion-exchange chromatography. 2. The isolated antigen is a glycoprotein displaying an apparent molecular weight of 26 000-29 000 in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 3. No antigen activity was found with the lipid fraction from human brain. 4. The protein has a tendency for spontaneous self-association (dimerization), leading to aggregates resistant to dissociating and reducing agents on prolonged storage. 5. The antigen is microheterogeneous with respect to size, charge (approximate isoelectric points of the monomer 7.7, 7.0 and 6.5) and to lectin-binding affinity. 6. The antigen can be reconstituted to protein-lipid vesicles. The antigen activity of solubilized antigen is strongly increased by reconstitution and that of membranes decreased by solubilization with detergent.