Induction of angiogenic response by chemically stable prostacyclin analogs

Angiogenic activities of several chemically stable prostacyclin analogs (isocarbacyclins and 7-fluoro prostacyclin) were evaluated by the chick embryo chorioallantoic membrane assay. These compounds showed potent angiogenic activity at very low concentration (0.1 micrograms/egg 1.0 micrograms/egg), whereas naturally occurring prostaglandins such as prostacyclin and PGE1 were almost ineffective up to 1 microgram/egg. Pretreatment of chorioallantoic membranes with dexamethasone or indomethacin inhibited the angiogenic response induced by these chemically stable prostacyclin analogs. These results indicate that these prostacyclin analogs induce the angiogenic response of chick chorioallantoic membranes via a mechanism involving activation of inflammatory cells, as well as through their direct angiogenic activity.     

Glycoproteins in the whorls of membrane produced by oligodendroglia in culture

Two glycoproteins of 99 kDa and 77 kDa which exhibit intense binding to wheat germ agglutinin have been purified from the whorls of membrane produced by oligodendroglia in culture. The whorls of membrane were isolated by gradient centrifugation from purified bovine oligodendroglia maintained in culture. The two glycoproteins were solubilized from the membranes using a non-ionic detergent and purified by Sephadex LH-60 chromatography, wheat germ agglutinin affinity chromatography, and SDS-polyacrylamide pore gradient gel electrophoresis. HPLC peptide mapping of the 99-kDa and 77-kDa glycoproteins revealed structural differences between the two proteins. Peptide mapping suggested that the 99-kDa glycoprotein from the whorls of membrane may be homologous to that from the plasma membranes. The 77-kDa glycoproteins from both sets of membrane may also be structurally related. Lectin binding studies showed that both glycoproteins from the whorls of membrane bound to wheat germ agglutinin, succinylated wheat germ agglutinin, concanavalin A, and lentil lectin, indicating the presence of high mannose and hybrid type oligosaccharide side-chains.     

Drosophila cell fusion induced by wheat germ agglutinin.

The effects of wheat germ agglutinin on Drosophila embryonic cell lines growing on cover-glasses was examined by scanning electron microscopy. At low concentrations of the lectin (5-10 mug/ml), cells spread against the glass surface and fused to form syncytia. At high concentration, damage to the cell surface was evidenced as extensive membrane shrivelling and loss of surface microfilaments. Fusion also occurred under these conditions. There was some indication that the morphology of cells in division remains undisturbed by wheat germ agglutinin. The coalescence of cells and morphologic disotrtion induced by wheat germ agglutinin were not inhibited by N-acetylglucosamine, the hapten inhibitor of the lectin, under the conditions utilized in this study.     

Identification of P-glycoprotein in renal brush border membranes

A monoclonal antibody (C219) that recognizes the P-glycoprotein (Mr = 170,000) in plasma membranes of multidrug-resistant Chinese hamster ovary (CHO) cell lines was used to assay renal brush border membrane (BBM) and basolateral membrane (BLM) fractions for the presence of a cross-reactive polypeptide. The C219 antibody bound to a 155,000 dalton protein in immunoblots of rat BBM but not BLM proteins resolved by sodium dodecyl sulfate gel electrophoresis. The corresponding human kidney BBM and dog kidney BBM proteins had molecular weights of 170,000 and 160,000 respectively. The glycoprotein nature of the renal protein was shown by its sensitivity to N-glycanase treatment which reduced the apparent molecular weight of the dog protein to 120,000. In addition, dog P-glycoprotein could be bound to and eluted from immobilized wheat germ agglutinin. The molecular weight, antibody crossreactivity, glycosidase sensitivity and lectin binding show that this protein is a normal kidney analogue of the P-glycoprotein induced in multidrug resistant cell lines.     

Lectin-Reactive Components in White Matter Membranes from Normal and Multiple Sclerosis Brains

Abstract: Polypeptides derived from human white matter membranes reacted with the radioiodinated lectins concanavalin A, Lens culinaris phytohemagglutinin, Ricinus communis agglutinin and wheat germ agglutinin after electrophoresis in polyacrylamide pore gradient gels. The molecular weights of these lectin-reactive bands were estimated by comparison with radioiodinated protein standards by using the linear relationship between log of the molecular weight and log of the gel concentration reached by the protein after electrophoresis in a polyacrylamide gradient gel. The molecular weight estimates for components reactive with concanavalin A were 176,800, 141,200, 72,800, 52,800, 44,700, 40,000, 24,800 and 23,900. The molecular weights of the bands reactive with both wheat germ agglutinin and Lens culinaris phytohemagglutinin were 138,000, 113,500, 92,100, 52,800, 44,700, 24,800 and 23,900. Wheat germ agglutinin was bound also to a band with a molecular weight of 72,800. Ricinus communis agglutinin bound to bands with estimated molecular weights of 138,000, 72,800, 52,800, 44,700, 24,800 and 23,900. The electrophoretic pattern of lectin-reactive polypeptides derived from normal-appearing white matter of multiple sclerosis brains was not qualitatively different from the lectin-binding pattern of control brain membrane polypeptides.     

Tumor angiogenesis factors produced by cancer cells.

Tumor angiogenic activity from tumor angiogenesis factors (TAFs) produced by 25 cell lines was assayed onto chorioallantoic membranes (CAMs). Neovascularization occurred prominently in such cell lines, as HTBOA (poorly differentiated ovarian carcinoma), HUOCA-II (poorly differentiated clear cell adenocarcinoma), HWUA (poorly differentiated endometrial adenocarcinoma), HKUS (uterine cervical small cell carcinoma), and in HOTHC (anaplastic thyroid carcinoma). The cell lines which secreted TAF showed high heterotransplantability in nude mice and produced rapidly growing tumors which were rich in blood vessels. The TAFs polypeptides of 14,000 and 78,000 molecular weight, were extracted and purified from the conditioned medium of HUOCA-II or W3UF (sub-line of HUOCA-II) lines, respectively. TAFs at concentrations of 10 ng/ml and 100 ng/ml promoted proliferation of the endothelial cells and induced tube formation. Microsequencing analysis revealed that TAF of 78,000 molecular weight has sequence identity with human hepatocyte growth factor (hHGF).     

Wheat germ agglutinin inhibits basal- and stimulated-adenylate cyclase activity as well as the binding of [3H] caerulein to rat pancreatic plasma membranes

Wheat germ agglutinin, but not concanavalin A or soybean lectin, inhibited the basal-and stimulated-adenylate cyclase activity which was present in a plasma membrane preparation from the rat pancreas. The inhibition by wheat germ agglutinin was rapid and sustained. It was of the non-competitive type and never exceeded 20% for Gpp (NH) p- and NaF-stimulated adenylate cyclase activity. The inhibition of secretin-stimulated activity was also non-competitive but more pronounced (57% inhibition at a wheat germ agglutinin concentration of 20 microgram/ml). For the C-terminal octapeptide of cholecystokinin-pancreozymin (OC-PZ)-stimulated cyclase, the inhibition amounted to 68% and was of a mixed type (both competitive and non-competitive). This last observation might be explained by the competitive inhibition exerted by wheat germ agglutinin on the binding of peptides of the OC-PZ family to their membrane specific receptors. The various inhibitory effects of wheat germ agglutinin were completely suppressed by incubating the membranes in the presence of ovomucoid, a N-acetyl-D-glucosamine rich glycoprotein. The possible functional implication of these results is discussed.     

Labelling of regenerating retinal pigment epithelium by colloidal iron oxide and ferritin conjugated to wheat germ agglutinin

Summary In order to determine if there are biochemical changes in plasma-membrane oligosaccharides of regenerating retinal pigment epithelium, the binding of colloidal iron oxide at low pH and ferritin-conjugated wheat germ agglutinin — probes of sialic acid and N-acetylglucosamine on the cell surface — was examined electron-microscopically. An animal model of retinal pigment epithelium regeneration — rabbits with sodium iodate induced retinopathy — was used. In this model, large expanses of regenerating pigment epithelium are present for comparison with zones of spared pgiment epithelium in the same animals. In thin sections examined by transmission electron microscopy, ferritin-conjugated wheat germ agglutinin appeared to bind more intensely to the exposed plasma membrane of regenerating retinal pigment epithelium than to spared pigment epithelium, or that of normal rabbits. Morphometry verified this. Colloidal iron oxide intensely labelled the plasma membranes of regenerating, spared, and normal pigment epithelium, and was visibly reduced after exposure of tissue to neuraminidase. The observations indicate that the plasma membrane of regenerating retinal pigment epithelium bears sialic acid and N-acetylglucosamine residues as in normal retinal pigment epithelium. However, the amount of plasma membrane bearing exposed N-acetylglucosamine increases during regeneration.     

The chick embryo chorioallantoic membrane as a model for tumor biology

Among the in vivo models, the chick embryo chorioallantoic membrane (CAM) has been used to implant several tumor types as well as malignant cell lines to study their growth rate, angiogenic potential and metastatic capability. This review article is focused on the major compelling literature data on the use of the CAM to investigate tumor growth and the metastatic process.     

The lectin binding sites on the plasma membrane components of human lymphoblastoid cell lines.

The plasma membrane components of five human B-cell lines and three human T-cell lines were separated by dodecyl sulfate polyacrylamide gel electrophoresis, incubated with the radioactive labeled lectins from lentil, castor bean, wheat germ, Phaseolus bean, peanut, gorse and the Roman snail and the molecular weights of the binding sites determined. The lentil, castor bean and wheat germ lectin bound to multiple components from molecular weights (Mr) 20 000 to 200 000 within the plasma membranes, whereas peanut lectin bound preferentially to glycoproteins of Mr 150 000 and 83 000 in B-cells, and 150 000 and 130 000 in T-cells. The gorse lectin bound to a 220 000 component in B-cells which was not labeled in T-cells.     

Effect of polyene antibiotics on the lectin-induced agglutination of transformed and untransformed cell lines.

Treatment of transformed Py3T3, SV101-3T3, and L1210 cells, as well as mitotic and Pronase-treated untransformed 3T3 cells, with the polyene antibiotics filipin, nystatin, and amphotericin B inhibited agglutination by wheat germ agglutinin. The effect of polyene antibiotic treatment was lectin and cell specific. Concanavalin A induced agglutination was not inhibited, wheat germ agglutination induced agglutination of untransformed 3T3 interphase cells was not influenced, and other aggregation phenomena, including those of erythrocytes with blood group specific antibodies or divalent cations, were unaffected by polyene treatments. This suggests that the formation of polyene-cholesterol complexes in transformed and erythrocyte cell membranes may specifically affect wheat germ agglutinin receptors and/or secondary events necessary for wheat germ agglutinin induced agglutination. Fluorescence studies of membrane filipin-cholesterol complexes showed that pretreating the cells with wheat germ agglutinin, but not concanavalin A, perturbed the fluorescence properties of filipin. Electron spin resonance studies with spin-labeled fatty acids revealed at best only a slight decrease in fatty acyl chain flexibility following filipin treatment. These studies indicate that there are not only quantitative differences between the agglutinability of transformed and untransformed cells with wheat germ agglutinin but that qualitative differences exist as well.     

Reduction of spontaneous metastases through induction of carbohydrate cross-reactive apoptotic antibodies

The selective targeting of tumor-associated carbohydrate Ags by the induction of serum Abs that trigger apoptosis of tumor cells as a means to reduce circulating tumor cells and micrometastases would be an advantage in cancer vaccine development. Some plant lectins like Griffonia simplicifolia lectin I and wheat germ agglutinin mediate the apoptosis of tumor cells. We investigated the possibility of using these lectins as templates to select peptide mimotopes of tumor-associated carbohydrate Ags as immunogens to generate cross-reactive Abs capable of mediating apoptosis of tumor cells. In this study, we show that immunization with a mimotope selected based on its reactivity with Griffonia simplicifolia lectin I and wheat germ agglutinin induced serum IgM Abs in mice that mediated the apoptosis of murine 4T1 and human MCF7 cell lines in vitro, paralleling the apoptotic activity of the lectins. Vaccine-induced anti-carbohydrate Abs reduced the outgrowth of micrometastases in the 4T1 spontaneous tumor model, significantly increasing survival time of tumor-bearing animals. This finding parallels suggestions that carbohydrate-reactive IgM with apoptotic activity may have merit in the adjuvant setting if the right carbohydrate-associated targets are identified.     

Interactions of lectins with plasma membrane glycoproteins of the Ehrlich ascites carcinoma cell.

Several aspects of the interaction of various lectins with the surface of Ehrlich ascites carcinoma cells are described. The order of agglutinating activity for various lectins is Ricinus communis greater than wheat germ greater than or equal to concanavalin A greater than or equal to soybean greater than Limulus polyphemus. No agglutination was noted for Ulex europaeus. Using 125I-labeled lectins it was determined that there are 1.6 and 7 times as many Ricinus communis lectin binding sites for concanavalin A and soybean lectins. Sodium deoxycholate-solubilized plasma membrane material was subjected to lectin affinity chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The lectin receptors of the plasma membrane appeared to be heterogeneous and some qualitative differences could be discerned among the electrophoretically analyzed material, which bound to and was specifically eluted from the various lectin affinity columns. The characteristics of elution of bound material from individual lectin columns indicated secondary hydrophobic interactions between concanavalin A or wheat germ agglutinin and their respective lectin receptor molecules.     

Interaction of sialoglycoproteins with wheat germ agglutinin-sepharose of varying ratio of lectin to Sepharose. Use for the purification of mucin glycoproteins from membrane extracts

The influence of varying the amount of wheat germ agglutinin immobilized on Sepharose beads on the binding of glycoproteins to these beads was investigated. A series of wheat germ agglutinin-Sepharose gels containing between 0.10 and 10.0 mg of lectin/ml of gel was prepared, and the actual lectin content was established by acid hydrolysis of the gel followed by analysis of glycine, a major amino acid in wheat germ agglutinin. Affinity chromatography of labeled glycoproteins indicated that glycophorin bound to all the wheat germ agglutinin-Sepharose preparations. Fetuin, ovomucoid, and alpha 1-acid glycoprotein bound not at all or very poorly to gels with a low content of wheat germ agglutinin (less than 0.95 mg/ml). The specific binding of these glycoproteins increased with increasing lectin content on the gels, and on gels of high content (greater than 3 mg/ml) the binding was virtually quantitative. On chromatographing a mixture of glycophorin, alpha 1-acid glycoprotein, fetuin, and ovomucoid on wheat germ agglutinin-Sepharose, containing 0.08 mg of lectin/ml of gel, glycophorin was selectively retained on the gel. It was possible to purify glycophorin from an extract of human erythrocyte membranes in one step by chromatography on the above gel. By using the series of gels, it was demonstrated that Morris hepatoma 7777 membranes contained at least 4-fold more sialoglycoproteins which bound to low density wheat germ agglutinin-Sepharose compared to rat liver membranes. These hepatoma sialoglycoproteins were isolated, purified, and partially characterized as having a high proportion of O-linked sialyloligosaccharides. Our studies illustrate the use of low density wheat germ agglutinin-Sepharose gels both for the detection and for easy isolation of mucin-type glycoproteins from crude extracts of cells or membranes.     

Isolation of a Zn-binding protein mediating cell adhesion from common carp

Extraordinarily high concentrations of Zn (300-500 microg/[g fresh tissue]) are often found in the digestive tract tissue of common carp Cyprinus carpio, and most of the Zn is bound to membrane protein located on plasma membranes that are attached to basal laminae. To isolate the Zn-binding protein, the basolateral plasma membranes were separated from the extracellular matrix by treating the nuclei/cell debris fraction of the tissue with collagenase type IV and Arg-Gly-Asp (RGD) peptide. The Zn-binding protein was isolated from the separated plasma membranes by immobilized metal affinity chromatography and affinity chromatography on laminin-Sepharose. A 43 kDa protein was bound by the laminin-Sepharose and specifically eluted with tirofiban (a mimic of RGD). Affinity chromatography on wheat germ agglutinin and concanavalin A-Sepharose showed that the 43 kDa protein is a glycoprotein. The 43 kDa protein was labelled with 65Zn and became incorporated into liposomes at a high efficiency. Liposomes containing this protein were bound to laminin-Sepharose or reconstituted basement membrane. We propose that the Zn-binding protein is a cell surface receptor involved in the adhesion of cells to laminin.     

Wheat germ agglutinin fracture-label of Golgi apparatus membranes in proliferating cells

The thin section fracture-label technique has been recently used to analyze the distribution and compartmentalization of fully glycosylated components on intracellular membranes. Labelling with the lectin wheat germ agglutinin over the freeze-fractured membranes of Golgi apparatus in various secretory and non-secretory cells as well as in human peripheral lymphocytes was always very weak or absent even over the trans-most cisternae. In order to investigate if the labelling density may reflect the cellular activity in membrane biogenesis, we used in this study wheat germ agglutinin fracture-label of rapidly proliferating cells and mitogen-activated lymphocytes. Labelling over the fractured cisternae of the medial and trans portions of the Golgi apparatus was intense. Treatment with cycloheximide of proliferating cells induced a drastic reduction of the labelling over the Golgi cisternae.     

The chick embryo chorioallantoic membrane as an in vivo experimental model to study human neuroblastoma

The chick embryo chorioallantoic membrane (CAM) has long been a favored system for the study of tumor angiogenesis because at the stage of development when generally tumor grafts are placed (6–10 days of incubation), the chick’s immunocompetent system is not fully developed and the conditions for rejection have not yet been established. All studies for mammalian neoplasms, including neuroblastoma, have used tumor cell lines, tumor bioptic specimens, cell suspensions derived from tumors, and mouse tumor xenografts bioptic specimens. CAM can also be used to study the effects of antiangiogenic molecules on tumor cell suspensions of tumor bioptic specimens. This review article summarizes and discusses the literature data on the use of the CAM as an in vivo experimental model to study human neuroblastoma.     

Isolation of a domain of the plasma membrane in Chinese hamster ovary cells which contains iodinatable, acidic glycoproteins of high molecular weight

A light vesicle fraction, apparently derived from the plasma membrane, was obtained following breakage of Chinese hamster ovary (CHO) cells by means of a fluid pump disrupting device. The final preparation was enriched approx. 40-fold over the homogenate in K+,Na+-stimulated ATPase and phosphodiesterase I, but only approx. 10-fold in 125I specific radioactivity after lactoperoxidase-catalyzed iodination. This preparation was compared with another plasma membrane fraction purified as large sheets via a two-phase centrifugation procedure. Two-dimensional polyacrylamide gel electrophoresis followed by Coomassie blue staining indicated that both fractions were fairly similar in polypeptide composition, although a few consistent differences were evident. However, staining of glycoproteins by the periodic acid-Schiff technique or by overlaying with 125I-labeled concanavalin A showed that the vesicle fraction was highly enriched in groups of high molecular weight, acidic glycoproteins which stain only weakly with Coomassie blue. These glycoproteins also bound 125I-labeled ricin I agglutinin and wheat germ agglutinin. They appear to be the major receptors for wheat germ agglutinin on the CHO cell surface. After surface labeling of cells by the 125I-lactoperoxidase technique, the membrane sheet fraction contained a large number of iodinated polypeptides, whereas labeling in the vesicle fraction was restricted almost entirely to the high molecular weight, acidic glycoproteins. It is proposed that the vesicle fraction constitutes a specific domain of the cell surface which is coated on its exterior by this group of glycoproteins. These components probably mask underlying proteins of the plasma membrane from external labeling.     

The surface glycoproteins of the HeLa cell. Internalization of wheat germ agglutinin-receptors.

The sensitivity of 125I-labeled sialoglycoproteins to neuraminidase digestion was used to monitor the loss of specific membrane glycoproteins from the cell surface in to the cytoplasmic compartment during lectin-mediated endocytosis. These studies demonstrated that a major portion of the surface glycoproteins had undergone internalization concurrently with wheat germ agglutinin in a time- and temperature-dependent process. The internalized 125I-labeled glycoproteins were associated with the small vesicle fraction and were present in the same relative proportion as they existed in the plasma membrane isolated from control untreated cells. Many of the 125I-labeled membrane proteins were shown to be receptors and were isolated after affinity chromatography of the solubilized plasma membranes on wheat germ agglutinin-agarose columns.