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H J Allen

Publications and source records attributed to H J Allen.

At least 37 records · Page 2Linked to original sources
PubMed1991

Localization of endogenous beta-galactoside-binding lectin in human cells and tissues.

A rabbit antiserum raised against the 14.5-kilodalton (kDa) subunit of human splenic galaptin was used to probe protein blots of several tissue extracts. For all tissues examined, the only immunoreactive species detected was a 14.5-kDa polypeptide. This antiserum and a rabbit antiserum raised against native lung galaptin were used in immunohistochemical assays to determine the localization of galaptin in selected tissues and cells. In normal colon, galaptin was found prominently in the basement membrane and in the stroma. The cytoplasm of epithelial cells stained lightly for galaptin whereas mucous granules and secreted mucin were uniformly negative for galaptin. Hemagglutination inhibition assays also failed to demonstrate an interaction between galaptin and mucin. Macrophages stained conspicuously for galaptin in colonic and cutaneous tissue as did some capillary walls. In cutaneous tissue, the extracellular matrix and hair follicle cells contained abundant galaptin. Galaptin was absent in basal cell carcinoma and associated stroma. Galaptin was found throughout the cytoplasm of carcinoma cells of gynecologic origin present in effusions. Protein blot analysis of extracts of extracellular matrix synthesized in vitro by endothelial cells confirmed the presence of galaptin in matrix. The results show that: (1) galaptin is variably expressed by different cells and tissues; (2) its cellular location is not restricted to the cell surface; (3) galaptin is not associated with normal mucin; (4) the extracellular matrix is a major site of galaptin deposition, and (5) some malignant tissue may be characterized by a deficiency of galaptin.

Animals↗
PubMed1991

Synthesis and release of cell surface-derived and sulfated lactosaminoglycans by human ovarian carcinoma cells.

Ovarian carcinoma cell clusters were isolated from patient effusions. Cell-surface glycoconjugates were radiolabelled by a galactose oxidase-borotritide method. The surface-labelled glycoconjugates and metabolically labelled glycoconjugates released to culture medium were characterized. The surface-derived glycoconjugates were highly heterodisperse and had the same molecular weight distribution as the metabolically labelled components. Lectin precipitation assays showed that both classes of glycoconjugates contained N-linked oligosaccharides bearing N-acetyllactosamine moieties. A121 ovarian carcinoma cells also synthesized and released a heterodisperse array of glycoconjugates to culture medium. Ricinus communis agglutinin I (RCAI) precipitated glycoconjugates of MW greater than 100 kDa for both A121 cells and cells from effusions. Cells of different ovarian carcinoma histology yielded similar results. Metabolic labelling experiments with 35SO4 showed that the RACI-bound glycoconjugates released by A121 cells were sulfated. The RCAI-bound sulfated lactosaminoglycans may be associated with malignant transformation and/or metastasis since similar components were not produced by mesothelial cells isolated from effusions [Allen, H.J., M. Gamarra M.S. Piver and E.A.Z. Johnson (1989). Cancer Biochem. Biophys. 10, 219-226].

Amino Sugars↗
PubMed1991

Characterization of glycoconjugates released in vitro by human ovarian carcinoma cells isolated from effusions.

Ovarian carcinoma cell clusters were isolated from patient effusions. The glycoconjugates released to culture medium in vitro were characterized by electrophoretic, immunoassay and gel filtration procedures. Metabolically radiolabelled glycoconjugates were heterodisperse with respect to molecular weight and this heterodispersity was independent of incubation time in vitro. This heterodispersity was also characteristic of mixed Mullerian tumor cells of endometrial origin whereas mesothelial cells released a discrete glycoconjugate of MW 65-70 kDa. Multiple Coomassie blue-stained polypeptides were released by the carcinoma cells. These polypeptides were not adsorbed serum components as assessed by immunodiffusion analyses. Periodic acid-Schiff-reactive macromolecules appeared only at the top of electrophoresis gels. The high molecular weight glycoconjugates synthesized by ovarian carcinoma cells precipitated with an effusion globulin fraction at low ionic strength, but the low molecular weight components (40-70 kDa) were soluble. Immunoprecipitation with anti-Ig failed to precipitate carcinoma glycoconjugates. Antisera raised against the released carcinoma macromolecules precipitated carcinoma glycoconjugates and normal ovarian polypeptides. Antisera raised against normal ovarian macromolecules precipitated ovarian polypeptides but reacted only slightly with carcinoma glycoconjugates. Immunodiffusion analyses showed the presence of alpha 1-acid glycoprotein and carcinoembryonic antigen (CEA)-like components in the carcinoma glycoconjugates. The presence of CEA-like glycoconjugates was confirmed by immunoprecipitation. The antigens and antisera for different histologic types of ovarian carcinoma were cross-reactive. The presence of beta 2-microglobulin suggested that some of the glycoconjugates were shed from the cell surface.

Ascitic Fluid↗
PubMed1990

Metabolic correction of fucosidosis lymphoid cells by galaptin-alpha-L-fucosidase conjugates.

To determine if isolated galaptin, an endogenous galactoside-binding lectin, could serve as a transport vehicle of therapeutic agents to cells, galaptin and alpha-L-fucosidase were coupled using glutaraldehyde. The conjugates were incubated with alpha-L-fucosidase-deficient, EBV-immortalized lymphoid cells from a fucosidosis patient. Conjugates were effectively bound and internalized by the cells in a lactose inhibitable manner. Internalization of conjugate resulted in the reduced accumulation of alpha-L-fucosyl-N-acetylglucosaminylasparagine, a glycopeptide that accumulates in cells of fucosidosis patients, to levels found in lymphoid cells from a healthy individual. Thus, galaptin-alpha-L-fucosidase conjugates may be useful for enzyme replacement therapy of fucosidosis. The concept of using galaptin as a transport vehicle may be applied to the delivery of other compounds to cells bearing galaptin receptors.

B-Lymphocytes↗
PubMed1990

Human splenic galaptin: physicochemical characterization.

Mammalian spleens were previously reported to contain beta-galactoside-binding lectins [Allen, H. J., Cywinski, M., Palmberg, R., & DiCioccio, R. (1987) Arch. Biochem. Biophys. 256, 523-533]. The aim of the present investigation was to determine the relationship of human splenic galaptin to other beta-galactoside-binding lectins identified in other human and animal tissues. Galaptin of subunit molecular mass 14.5 kDa was the only lectin of this type found in human spleen as assessed by SDS-PAGE, RP-HPLC, and Western blot analyses. Three polypeptides of pI 4.60, 4.80, and 4.85 were detected by isoelectric focusing of purified galaptin, with the major band having pI 4.85. UV spectral analysis indicated the absence of prosthetic groups and gave A1%(1cm), 280 = 5.5. Circular dichroic analysis suggested the presence of 40% beta structure, considerable random coil, and 10% alpha helix structure. The amino acid composition was very similar to that for human placental galaptin. Amino acid sequence analyses were carried out on V8 protease, CNBr, and iodosobenzoic acid digestion fragments. A total of 94 residues were identified. All sequences determined could be aligned with placental galaptin sequences. We conclude that human splenic galaptin is identical with human placental galaptin. A related polypeptide of molecular mass approximately 14.5 kDa was found to be present in several different mammalian spleens as assessed by Western blot analysis using a monospecific polyclonal anti-human splenic galaptin antiserum.

Amino Acid Sequence↗
PubMed1990

Human splenic galaptin: carbohydrate-binding specificity and characterization of the combining site.

A galactose-binding lectin (galaptin) from human spleen has been purified to homogeneity by affinity chromatography on asialofetuin-Sepharose. The carbohydrate-binding specificity of galaptin has been investigated by analyzing the binding of galaptin to asialofetuin in the presence of putative inhibitors. An enzyme-linked immunosorbent assay (ELISA) was developed that involved adsorption of asialofetuin to microtiter plates. Galaptin bound to asialofetuin was detected with polyclonal rabbit anti-galaptin serum followed by goat anti-rabbit IgG-peroxidase conjugate. The concentrations of inhibitors giving 50% inhibition of galaptin binding relative to controls were graphically determined and normalized relative to galactose or lactose. These analyses revealed that galaptin has a combining site at least as large as a disaccharide. The disaccharides having non-reducing-terminal beta-galactosyl residues linked (1,3), (1,4), and (1,6) to Glc or GlcNAc are better inhibitors than free Gal. GalNAc, either free or glycosidically linked, appears to have no affinity for the lectin. The nitrophenyl galactosides are better inhibitors than methyl galactosides, indicating the occurrence of hydrophobic interactions. The data indicate that OH groups at C-4 and C-6 of Gal and the OH at C-3 of GlcNAc in Gal beta(1,4)GlcNAc are important for lectin sugar interaction. Our data support the hypothesis that endogenous receptors for galaptin are most likely lactosaminoglycan moieties.

Binding Sites↗
PubMed1990

Binding characteristics of galactoside-binding lectin (galaptin) from human spleen.

Binding characteristics of human spleen soluble galactoside-binding protein (galaptin) were studied using simple galactosides, galactose-terminated disaccharides, cluster glycosides containing up to 6 terminal lactosyl residues, bovine serum albumin derivatives containing 7 to 40 lactosyl residues, desialylated serum glycoproteins, and glycopeptides derived thereof as inhibitors in a newly developed binding assay. In this assay, aminohexyl lactoside was attached to divinyl sulfone-activated Sepharose, which was then used to bind 125I-galaptin. Similarly derivatized Sepharose containing mannoside served as a control. The assay is sensitive, maintains linearity in the concentration range of 125I-galaptin tested, and has very low nonspecific binding. The following new findings were made. 1) All the alpha-D-galactopyranosides with non-sugar aglycon were better inhibitors than the corresponding beta-D-galactopyranoside. 2) The S-galactosides were better inhibitors than the corresponding O-galactosides, regardless of the anomeric configuration. 3) Many Gal beta 1-4- and Gal beta 1-3-linked disaccharides were tested. Although the galaptin did not appear to recognize N-acetylglucosamine as a monosaccharide, the presence of this sugar penultimate to galactose increased the binding affinity by as much as 500-fold, as was the case for N-acetyllactosamine. Of a particular importance is the presence of an equatorial 3-OH group on this sugar. We synthesized the 3-deoxy derivative of N-acetyllactosamine and found that it had 50-fold lower binding affinity compared to N-acetyllactosamine. 4) The binding sites of this lectin do not seem to be operating in a cooperative fashion, since synthetic lactose-containing divalent ligands with various inter-galactose distances did not increase the binding affinity significantly.

Binding Sites↗
PubMed1990

Role of galaptin in ovarian carcinoma adhesion to extracellular matrix in vitro.

Immunohistochemical studies indicated that galaptin is a major protein of ovarian carcinoma cells present in patient effusions and it is distributed throughout the cytoplasm. Enzyme-linked immunoadsorbent assay (ELISA) and immunoprecipitation experiments demonstrated that galaptin is also a major protein of the A121 ovarian carcinoma cell line, constituting less than or equal to 1% of extractable protein bound by DEAE Sephacel. Western blot analyses revealed that the galaptin present in ovarian carcinoma consists of a 14.5 KD subunit. Ovarian carcinoma and mesothelial cells isolated from patient effusions display surface receptors for galaptin with an apparently greater density of receptors present on the carcinoma cells. A121 cells also display surface receptors for galaptin: binding sites/cell = 3 X 10(8) and Ka = 1.2 X 10(9) M-1. The presence of galaptin in bovine corneal endothelial cells (BCEC) and BCEC-derived extracellular matrix (ECM) was demonstrated by ELISA. Of the total ECM-bound galaptin, about 75% appears to be insoluble in phosphate-buffered saline (PBS) lactose. ECM was also found to contain abudnant receptors for galaptin. Treatment of ECM with lactose increased the apparent galaptin receptor density:binding sites/cm2 = 7 X 10(13) and Ka = 2.6 X 10(9) M-1. Pretreatment of A121 cells with galaptin inhibited adhesion to ECM. The addition of exogenous galaptin to ECM had variable effect on cell adhesion. The data presented here suggest that early adhesion events may be carbohydrate-specific involving interaction between ECM-bound galaptin and cell surface galaptin receptors.

Animals↗
PubMed1989

Synthesis and release of glycoconjugates bearing N-linked oligosaccharides by ovarian carcinoma cells isolated from effusions.

Ovarian carcinoma cell clusters were isolated from patient effusions. The cell isolates were incubated in vitro with radioactive glycoconjugate precursors. Radiolabelled glycoconjugates released to culture media were analyzed for molecular mass heterogeneity and lectin binding activity. From 10 to 50% of the released glycoconjugates were present as a heterogeneous array of glycoconjugates of molecular mass greater than 250 kilodaltons. The remaining glycoconjugates were dispersed in a molecular mass range extending down to approximately 15 kilodaltons. Concanavalin A-Sepharose affinity chromatography revealed the presence of N-linked oligosaccharides. Interaction of glycoconjugates with lentil and pea lectins indicated the presence of L-fucose residues linked to asparagine-bound N-acetylglucosamine. Precipitation of glycoconjugates with ricinus communis agglutinin I showed the presence of nonreducing terminal N-acetyllactosamine residues. Collectively, the data indicate that ovarian carcinoma cells release to culture medium fucosylated glycoconjugates bearing complex-type oligosaccharides. The synthesis and release of these glycoconjugates showed no significant differences among different histologic types of ovarian carcinoma; however, modulation as a function of tumor progression may occur.

Cell Separation↗
PubMed1989

Synthesis and release of glycoconjugates in vitro by human ovarian carcinoma cells isolated from effusions.

Human ovarian carcinoma cells were isolated as multicellular aggregates from patient effusions. Freshly isolated cells were incubated with radioactive glycoconjugate precursors and the glycoconjugates released to culture medium were analyzed. The glycoconjugates appeared to contain both O- and N-linked oligosaccharides. Both fucosylation and sialylation of glycoconjugates occurred. The results of SDS-PAGE analysis showed the presence of a very heterogeneous array of glycoconjugates with no discrete components resolved. The results of glycoconjugate analyses were independent of carcinoma histology, differentiation, progression, and patient chemotherapy. In contrast, mesothelial cells synthesized a major glycoconjugate of molecular mass 65 KDa. The heterogeneous array of carcinoma-derived glycoconjugates may be transformation-related and may have utility in tumor diagnosis and prognosis.

Carbon Radioisotopes↗
PubMed1987

Comparative analysis of galactoside-binding lectins isolated from mammalian spleens.

Galactoside-inhibitable lectins have been isolated from rabbit, rat, mouse, pig, lamb, calf, and human spleens. Native molecular mass, subunit structure, pI, and hemagglutinating activity have been compared for these lectins. The yields of lectin varied from 1.8 mg/kg for rabbit spleen to 79 mg/kg for lamb spleen. Pig, lamb, calf, and human spleen lectins yielded single protein peaks when subjected to Superose 12 fast-protein liquid chromatography. The apparent molecular mass for these lectins was 33-34 kDa. In contrast, rat and mouse spleen lectin preparations were separated into three components ranging from 8.4 to 34 kDa. Superose 12 chromatography of rabbit spleen lectin revealed the presence of at least six components. Gradient slab gel sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the presence of single polypeptides for pig, calf, lamb, and human lectins corresponding to a molecular mass of 14-14.5 kDa. Multiple polypeptides were detected for the mouse, rat, and rabbit lectins. The molecular mass of the major polypeptides were 15, 15, and 17 kDa for rat, mouse, and rabbit, respectively. The presence of isolectins in all preparations was shown by isoelectric focusing. The major isolectins were acidic proteins with pI 4.38-4.80. Hemagglutination and hemagglutination inhibition assays demonstrated similarities as well as differences among the lectin preparations. Hemagglutinating activity could not be demonstrated in rabbit spleen extracts nor for isolated putative lectin. Human buffy coat cells were reversibly agglutinated by calf and human spleen lectins, demonstrating the presence of leucocyte cell surface lectin receptors.

Animals↗
PubMed1987

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.

Adenocarcinoma↗
PubMed1987

Galactoside-binding lectin in human tissues.

Lactose-inhibitable lectin activity has been analyzed by hemagglutination assay in a variety of human tissues and cells obtained at surgery and autopsy. The lectin activity was detected in surgically removed melanoma, sarcoma, colon carcinoma, breast carcinoma, adjacent non-malignant tissues, non-malignant tissues obtained at autopsy and in cells isolated from malignant effusions. Although, on average, malignant tissue had a higher hemagglutinating titer than non-malignant tissue, similar tissues from different individuals varied widely in their apparent lectin content. The lectin was isolated from lung by affinity chromatography and was found to have a native molecular mass of 31,000 daltons and a subunit molecular mass of 14,000 daltons. Utilizing rabbit anti-lung lectin serum in an immunohistochemical assay, the lectin was found to be distributed throughout the cytoplasm of lung epithelial cells. Ouchterlony immunodiffusion analysis confirmed the presence of this lectin in a variety of tissues and in some body fluids. In vitro metabolic radiolabelling experiments showed that the presence of lectin in tissues was most likely due to endogenous synthesis rather than absorption from body fluids. Lectin isolated from several tissues was found to bind to human buffy coat cell receptors.

Electrophoresis, Polyacrylamide Gel↗
PubMed1986

Binding specificity of a human leucocyte carbohydrate-binding protein.

The binding site specificity of a carbohydrate-binding protein (CBP) synthesized in vitro by normal human peripheral leucocytes was analyzed by affinity chromatography on asialofetuin-Sepharose. Radiolabelled cell extracts were applied to affinity columns. After washing with buffer, the columns were eluted with varying concentrations of different saccharides. The results show that lactose and thiodigalactoside were two of the best inhibitors of CBP binding to asialofetuin. The weakest inhibitors were methyl-beta-D-galactoside and raffinose. Binding of galactose to the CBP was enhanced by the presence of p-NO2-phenyl aglycones. Saccharides unrelated to the D-galactosyl residue failed to inhibit the CBP. Heat-inactivated plasma appeared to have little, if any, binding inhibitors. Binding of CBP to asialofetuin appeared to be inhibited by amine-containing compounds.

Asialoglycoproteins↗
PubMed1986

In vitro synthesis of carbohydrate-binding proteins by human leucocytes.

A carbohydrate-binding protein (CBP) synthesized in vitro by normal human peripheral leucocytes was isolated by affinity chromatography on asialofetuin-Sepharose. The CBP was eluted with lactose and it had a native molecular mass of 15,500 daltons. Analysis by SDS-PAGE revealed a single polypeptide of 18,000 daltons. CBP synthesis was time dependent and cell concentration dependent. The CBP appeared to be both cell bound and secreted with the apparent amount secreted inversely proportional to cell concentration. CBP did not appear to be synthesized by T and B leukemic cell lines examined. Promyelocytic HL-60 cells, however, synthesized at least two lactose-eluted CBP's corresponding to native molecular masses of 28,000 and 19,500 daltons. SDS-PAGE analysis of radiolabelled HL-60 CBP's showed the presence of two polypeptides of MM 17,700 and 16,000 daltons suggesting that one of the CBP's was a dimer.

Carbohydrate Metabolism↗
PubMed1980

Chromatographic behavior of pea lectin receptors from the 6C3HED murine ascites tumor.

We have previously shown that pea lectin and concanavalin A bind to different receptors as well as to common receptors on the cell surface of 6C3HED murine ascites tumor cells (Allen, H.J. nd Johnson, E.A.Z. (1976) Biochim. Biophys. Acta 436, 557-566). We have characterized the metabolically labeled pea lectin receptor from trypsinates of viable tumor cells by gel filtration and affinity chromatography. The pea lectin receptor eluted as an apparent high molecular weight glycopeptice (Mr 200 000) from Sephadex G-200 columns. Other glycopeptides were present in the trypsinates but they did not bind to either concanavalin A or to pea lectin in significant amounts. The high molecular weight glycopeptide was resistant to digestion by a high concentration of trypsin but it was absent in non-specific protease digests of viable cells. Sequential lectin affinity chromatography of Nonidet P-40 extracts of cells permitted the probable identification of the native glycoprotein corresponding to the high molecular weight glycopeptide which binds to pea lectin.

Cell Membrane↗