Paper analytical devices for dynamic evaluation of cell surface N-glycan expression via a bimodal biosensor based on multibranched hybridization chain reaction amplification.

A novel colorimetric/fluorescence bimodal lab-on-paper cyto-device was fabricated based on concanavalin A (Con A)-integrating multibranched hybridization chain reaction (mHCR). The product of mHCR was modified PtCu nanochains (colorimetric signal label) and graphene quantum dot (fluorescence signal label) for in situ and dynamically evaluating cell surface N-glycan expression. In this strategy, preliminary detection was carried out through colorimetric method, if needed, then the fluorescence method was applied for a precise determination. Au-Ag-paper devices increased the surface areas and active sites for immobilizing larger amount of aptamers, and then specifically and efficiently captured more cancer cells. Moreover, it could effectively reduce the paper background fluorescence. Due to the specific recognition of Con A with mannose and the effective signal amplification of mHCR, the proposed strategy exhibited excellent high sensitivity for the cytosensing of MCF-7 cells ranging from 100 to 1.0×10(7) and 80-5.0×10(7) cellsmL(-1) with the detection limit of 33 and 26 cellsmL(-1) for colorimetric and fluorescence, respectively. More importantly, this strategy was successfully applied to dynamically monitor cell-surface multi-glycans expression on living cells under external stimuli of inhibitors as well as for N-glycan expression inhibitor screening. These results implied that this biosensor has potential in studying complex native glycan-related biological processes and elucidating the N-glycan-related diseases in biological and physiological processes.

Functional roles for C5a and C5aR but not C5L2 in the pathogenesis of human and experimental cerebral malaria.

The host immune response plays an important role in the onset and progression of cerebral malaria (CM). The complement system is an essential component of the innate immune response to malaria, and its activation generates the anaphylatoxin C5a. To test the hypothesis that C5a signaling contributes to the pathogenesis of CM, we investigated a causal role for the C5a receptors C5aR and C5L2 in a mouse model of experimental CM (ECM) induced by Plasmodium berghei ANKA infection, and using a case-control design, we examined levels of C5a in plasma samples from Ugandan children presenting with CM or uncomplicated malaria (UM). In the ECM model, C5aR(-/-) mice displayed significantly improved survival compared to their wild-type (WT) counterparts (P = 0.004), whereas C5L2(-/-) mice showed no difference in survival from WT mice. Improved survival in C5aR(-/-) mice was associated with reduced levels of the proinflammatory cytokines tumor necrosis factor (TNF) and gamma interferon (IFN-γ) and the chemokine, monocyte chemoattractant protein 1 (MCP-1) (CCL2). Furthermore, endothelial integrity was enhanced, as demonstrated by increased levels of angiopoietin-1, decreased levels of angiopoietin-2 and soluble ICAM-1, and decreased Evans blue extravasation into brain parenchyma. In the case-control study, the median levels of C5a at presentation were significantly higher in children with CM versus those in children with UM (43.7 versus 22.4 ng/ml; P < 0.001). These findings demonstrate that C5a is dysregulated in human CM and contributes to the pathogenesis of ECM via C5aR-dependent inflammation and endothelial dysfunction.

Fluorescence assay for glycan expression on living cancer cells based on competitive strategy coupled with dual-functionalized nanobiocomposites.

Cell surface glycans are a class of sophisticated biomolecules related to cancer development and progression, and their analysis is of great significance for early cancer diagnosis and treatment. In this paper, we proposed a fluorescence assay to evaluate glycan expression on living cancer cells based on a competitive strategy coupled with dual-functionalized nanobiocomposites. The competitive assay was conducted between living cancer cells and thiomannosyl derivatives using concanavalin A (Con A)-modified electrode as the interaction platform. To impart fluorescence signaling ability to competitive derivatives, quantum dots (QDs) were anchored on BSA-protected Au nanoparticles, and thiomannosyl derivatives were further immobilized on the nanoparticle surface through Au-S binding. Due to the spacing between QDs and Au nanoparticles by BSA, the {QDs-Au-BSA-mannose} nanobiocomposites maintained the fluorescence of QDs and showed binding ability with the Con A-modified electrode. Au nanorods (AuNRs)-modified electrode was used as an effective substrate to immobilize Con A. This assay was successfully applied to the analysis of two cancer cells lines (A549 and QGY-7701). The method is simple and shows promise for the study of glycan expression on living cancer cells.

Isolation and identification of Concanavalin A binding glycoproteins from human seminal plasma: a step towards identification of male infertility marker proteins.

Human seminal plasma contains a large array of proteins of clinical importance which are essentially needed to maintain the reproductive physiology of spermatozoa and for successful fertilization. Thus, isolation and identification of seminal plasma proteins is of paramount significance for their biophysical characterization and functional analysis in reproductive physiological processes. In this study, we have isolated Concanavalin-A binding glycoproteins from human seminal plasma and subsequently identified them by MALDI-TOF/MS analysis. The major proteins, as identified in this study, are Aminopeptidase N, lactoferrin, prostatic acid phosphatase, zinc-alpha-2-glycoprotein, prostate specific antigen, progestagen-associated endometrial protein, Izumo sperm-egg fusion protein and prolactin inducible protein. This paper also reports preliminary studies to identify altered expression of these proteins in oligospermia and azoospermia in comparison to normospermia. In oligospermia, five proteins were found to be downregulated while in azoospermia, four proteins were downregulated and two proteins were upregulated. Thus, this study is of immense biomedical interest towards identification of potential male infertility marker proteins in seminal plasma.

A Con A- purified hydatid glycoprotein fraction effectively diagnoses human hydatidosis.

Diagnosis and quantification of Echinococcus granulosus infection in man and animal hosts are centralized to feasible control. This study included 93 serum samples, 25 sure positive hydatid cases confirmed surgically, 7 suspected cases diagnosed by indirect haemagglutination IHA and 41 cases other parasitic infections (15 S. mansoni, 8 Fasciola, 7 Ascaris, 5 H. nana & 6 Ancylostoma) diagnosed by microscopic examination and were negative by ELISA and/or IHA for anti-hydatid antibody. Twenty negative serum samples served as healthy controls. Six types of hydatid fluid antigens (crude, host-free & Con-A purified) of human and camel origin were subjected to electrophoretic separation (SDS-PAGE) and immunoblotting (EITB). The anti-hydatid IgG was detected in sera of the different groups for evaluation of sensitivity, specificity and diagnostic efficacy of each type of antigens. Detection of circulating hydatid antigen (CAg) was performed using anti rabbit hyperimmune sera raised against Con-A purified either human or camel hydatid antigen. SDS-PAGE revealed several bands ranging from 55-185 kDa with 10 kDa band shared by all antigens. The specific bands revealed by EITB for Con-A purified camel and human antigens were at 80, 110 & 55, 110 kDa respectively. ELISA highest sensitivity (96.9%) was by using host-free Con-A purified glycoprotein fraction of human hydatid antigen. Highest specificity (98.4%) was recorded upon use of either Con-A purified camel or human antigen with 94.5% & 97.7% diagnostic efficacy respectively. Detection of circulating antigen by polyclonal antibodies against Con-A purified human hydatid antigen revealed 91.8% specificity.

Mosaicism in the organization of Con A binding sites on the membrane surface of female cells of Nicotiana tabacum.

The presence of mosaicism in the organization of concanavalin agglutinin (Con A) binding sites on murine egg cells was first reported 30 year ago. This discovery has triggered extensive studies into the roles of glycoproteins in gamete interactions in animals. This report comprises the first account of the existence of the mosaicism in higher plants. The distribution of Con A binding sites on both egg cells and central cells of tobacco (Nicotiana tabacum) was found to be polar and apparently determined by the location of the nucleus of the cell. On central cells, Con A binding sites were distributed on the section of the plasma membrane surface near the nucleus. By contrast, the binding sites on egg cells were concentrated away from the nucleus. Therefore, polarity of the plasma membrane component of female cells was confirmed for the first time. It is proposed that such polarized ConA binding sites could be involved in sperm recognition.

Cell aggregation by scaffolded receptor clusters.

The aggregation of cells by lectins or antibodies is important for biotechnological and therapeutic applications. One strategy to augment the avidity and aggregating properties of these mediators is to maximize the number of their ligand binding sites. The valency of lectins and antibodies, however, is limited by their quaternary structures. To overcome this limitation, we explored the use of polymers generated by ring-opening metathesis polymerization (ROMP) as scaffolds to noncovalently assemble multiple copies of a lectin, the tetravalent protein concanavalin A (Con A). We demonstrate that complexes between Con A and multivalent scaffolds aggregate cells of a T cell leukemia line (Jurkat) more effectively than Con A alone. We anticipate that synthetic scaffolds will offer a new means of facilitating processes that rely on cell aggregation, such as pathogen clearance and immune recognition.

The distribution of MUC1, an apical membrane glycoprotein, in mammary epithelial cells at the resolution of the electron microscope: implications for the mechanism of milk secretion.

The distribution of the glycoprotein, mucin 1 (MUC1), was determined in lactating guinea-pig mammary tissue at the resolution of the electron microscope. MUC1 was detected on the apical plasma membrane of secretory epithelial cells, the surface of secreted milk-fat globules, the limiting membranes of secretory vesicles containing casein micelles and in small vesicles and tubules in the apical cytoplasm. Some of the small MUC1-containing vesicles were associated with the surfaces of secretory vesicles and fat droplets in the cytoplasm. MUC1 was detected in much lower amounts on basal and lateral plasma membranes. By quantitative immunocytochemistry, the ratio of MUC1 on apical membranes and milk-fat globules to that on secretory vesicle membranes was estimated to be 9.2:1 (density of colloidal gold particles/microm membrane length). The ratio of MUC1 on apical membranes compared with basal/lateral membranes was approximately 99:1. The data are consistent with a mechanism for milk-fat secretion in which lipid globules acquire an envelope of membrane from the apical surface and possibly from small vesicles containing MUC1 in the cytoplasm. During established lactation, secretory vesicle membrane does not appear to contribute substantially to the milk-fat globule membrane, or to give rise in toto to the apical plasma membrane.

Presence of aberrant tumor-reactive immunoglobulins in the circulation of patients with ovarian cancer.

OBJECTIVES: Cancer patients generally exhibit circulating tumor-reactive immunoglobulins; however, these antibodies fail to eradicate tumors or prevent their progression. This study identifies and characterizes an aberrant tumor-reactive IgG population present in women with ovarian cancer. METHODS: In this pilot study, IgG was isolated from the sera of women with advanced-stage ovarian cancer (stages III and IV, n = 62) and age-matched female volunteers (n = 50) by affinity chromatography. These IgGs were characterized on the basis on their aberrant binding to concanavalin A affinity columns. Subsequently, the concanavalin A-binding moiety was localized following IgG fragmentation, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and characterized by oligosaccharide profiling. RESULTS: The level of concanavalin A-binding IgG in our control population was 8.9 +/- 2.9%, whereas in ovarian cancer patients, the level of concanavalin A-binding IgG was 38.8 +/- 7.4%. In the patients with ovarian cancer, 87.5 +/- 5.7% of the tumor-reactive IgG was demonstrated to be concanavalin A-binding. Based on oligosaccharide profiling of the fragmented concanavalin A-binding IgG, the aberrant lectin binding appeared to be the consequence of altered glycosylation of one of the two Fc chains. CONCLUSIONS: While our previous studies have identified the presence of circulating IgG reactive with specific tumor-associated antigens and its association with poor prognosis, this report demonstrated the presence of an aberrantly glycosylated IgG population in cancer patients. This altered IgG appeared to be the primary class of tumor-reactive antibodies in these women.

Immunosuppressive acidic protein (IAP) level used to monitor adjuvant arthritis in rats.

To determine if immunosuppressive acidic protein (IAP) responds to the degree of inflammation in adjuvant arthritis in rats (AA), we measured the serum IAP level of the rats by using the single immunodiffusion method, and the level of concanavalin A (Con A) binding protein by using nepherometry. Pharmacologic treatment with 0.3mg/kg of methotrexate (MTX) was done from day 5 to 14 postimmunization in rats with AA. The measured serum IAP level on day 21 indicated the most severe inflamed phase. IAP levels reflected the degree of primary and secondary inflammatory phases in AA, whereas ConA binding protein (CBP) levels did not. Our results suggest that IAP can be used to monitor the symptoms of inflammation and the efficacy of anti-inflammatory drugs.

Preliminary validation of tumor cell attachment inhibition assay for developmental toxicants with mouse S180 cells.

This study was designed to explore the possibility of using ascitic mouse sarcoma cell line (S180) to validate the mouse tumor cell attachment assay for developmental toxicants, and to test the inhibitory effects of various developmental toxicants. The results showed that 2 of 3 developmental toxicants under consideration, sodium pentobarbital and ethanol, significantly inhibited S180 cells attachment to Concanavalin A-coated surfaces. Inhibition was dependent on concentration, and the IC50 (the concentration that reduced attachment by 50%), of these 2 chemicals was 1.2 x 10(-3) mol/L and 1.0 mol/L, respectively. Another developmental toxicant, hydrocortisone, did not show inhibitory activity. Two non-developmental toxicants, sodium chloride and glycine were also tested and these did not decrease attachment rates. The main results reported here were generally similar to those obtained with ascitic mouse ovarian tumor cells as a model. Therefore, this study added further evidence to the conclusion that cell specificity does not limit attachment inhibition to Con A-coated surfaces, so S180 cell may serve as an alternative cell model, especially when other cell lines are unavailable. Furthermore, after optimal validation, it can be suggested that an S180 cell attachment assay may be a candidate for a series of assays to detect developmental toxicants.

Dictyostelium myosin II null mutant can still cap Con A receptors.

Cross-linked antigens on the surface of a motile cell cap at the trailing end of the cell. In Dictyostelium discoideum, myosin II null mutants have previously been reported to be unable to cap Con A receptors, although they are able to locomote. This finding implicated myosin II as an essential component of the capping mechanism, although not of the machinery for locomotion. Here we show that myosin II null mutants do cap Con A receptors, albeit less efficiently than does wild type. This shows that cap formation is not absolutely dependent on myosin II and that a close mechanistic relationship between capping, particle movement, and cell migration may still exist.

[The actions of nucleating proteins in vesicle aggregation and fusion: a preliminary study].

After investigation of the effects of Con A-binding proteins, the major biliary pronucleating effectors system on the figures and lipid composition of model biliary vesicles, the authors found that Con A-binding proteins accelerate aggregation and fusion of vesicles as a result of increased vesicular cholesterol and decreased vesicular phospholipids as well as cholesterol-saturated vesicles. We also found that vesicular proteins though in small quantity are potent in pronucleating effects. The distribution of several known nucleating proteins between the vesicles and micelles is quite different, and there are higher content and more potent pronucleating effectors in patients with cholesterol gallstone than those with pigment stone. By affinity staining method, con A-binding proteins were shown in native biliary vesicles as lipid-protein complex. These results suggest that the existence and changes in their quantity and quality of vesicular nucleating proteins play an important role in vesicle aggregation and fusion.

Cross-linking of concanavalin A receptors on cortical neurons induces programmed cell death.

The loss of neurons by programmed cell death is a normal feature of the nervous system during development and has recently been implicated as a major mechanism of cell death in neurodegenerative diseases. In some cases, programmed cell death is induced by the activation of membrane receptors and is referred to as activation-induced programmed cell death. Activation-induced programmed cell death has been previously described in cells from the immune system, in which the activation of receptors by receptor clustering leads to programmed cell death. To determine whether activation-induced programmed cell death occurs in neurons, Concanavalin A was used to cross-link membrane receptors on cortical neurons. Concanavalin A-induced neuronal death was dose dependent and effective at concentrations previously shown to induce activation-induced programmed cell death in lymphocytes. Programmed cell death was attenuated when Concanavalin A-specific binding to neurons was blocked with methyl alpha-D-mannopyranoside. Succinyl Concanavalin A, which bound to Concanavalin A receptors but was ineffective at cross-linking them, did not induce programmed cell death. Concanavalin A-induced neuronal death exhibited many of the hallmarks associated with programmed cell death, such as membrane blebbing, nuclear condensation and margination, and internucleosomal DNA cleavage. In addition, neurons exposed to Concanavalin A displayed a rapid, robust, and persistent increase in the immediate early gene protein c-Jun. A similar increase in c-Jun precedes programmed cell death induced by beta-amyloid in neurons, and under some conditions an increase in c-Jun has been shown to be required for programmed cell death to occur in neurons. Increased expression of c-jun and other immediate early genes has also been correlated with activation-induced programmed cell death in lymphocytes. These observations suggest that Concanavalin A induces activation-induced programmed cell death in neurons via signals produced from the cross-linking of receptors on neuronal membranes. These results also raise the possibility that beta-amyloid induces programmed cell death in a similar manner, by causing the cross-linking of receptors on neuronal membranes. This mechanism may be relevant to neuronal programmed cell death that occurs during development and neurodegeneration.

[Metastatic potential correlates with cell surface carbohydrate profiles of nasopharyngeal carcinoma].

In order to investigate the changes in glycoprotein structure between non-metastatic and metastatic cells of nasopharyngerl carcinoma (NPC), peanut agglutinin (PNA), ulex europeaus (UEA-I) and concanavalin ensifomis agglutinin (ConA) staining were used to examine 102 nasopharyngeal carcinoma tissues (84 primary tumors and 18 metastatic lymph nodes) with the avidin-biotinperoxidase complex method. PNA reaction was positive in most cells of nasopharyngeal carcinomas regardles of in histopathologic type, the degree of differentiation and metastasis. The levels of UEA-I receptors apparently increased during nasopharyngeal carcinoma progression from non-metastatic to metastatic tumors. The binding to ConA clearly decreased in the cells of metastatic cases and metastatic lymph node. So, the increased expression of UEA-I receptors and decrease of ConA receptors on tumor cells might have been implicated in the expression of metastatic potential.

Myosin II is involved in capping and uroid formation in the human pathogen Entamoeba histolytica.

The redistribution and capping of surface receptors on the human pathogen Entamoeba histolytica was observed in the presence of concanavalin A (ConA). Capping was correlated with plasma membrane folding towards the rear of the amoeba and with uroid formation. The uroid is thought to play a role in the escape of amoebae from the host immune response. To localize myosin II during capping, amoebae were incubated in the presence of ConA and then analyzed by microscopy. Myosin II was three times more concentrated within the uroid compared with the rest of the cell, suggesting that the release of caps may depend upon mechanical contraction driven by myosin II activity. The use of drugs that disrupt cytoskeletal structure or that inhibit myosin heavy chain phosphorylation demonstrated that inhibition of capping prevents uroid formation. Biochemical analysis allowed the identification of two ConA receptors which have been previously described as major pathogenic antigens of this parasite: the 96-kDa antigen, which carries alcohol dehydrogenase 2 activity and binds extracellular matrix proteins, and the Gal-GalNAc-inhibitable surface lectin, which is involved in amoeba-cell interactions and in the degradation of complement particles attached to the parasite.

Possibility of signal transduction through microfilaments below the membrane following ligand-receptor interaction.

In this paper, we describe the changes of microfilament assembly and 3H-TdR incorporation in mouse ascites liver cancer cells under the action of concanavalin A (ConA) and laminin (LN). We have also studied the variation of 3H-TdR incorporation induced by destroying microfilaments with cytochalasin B (CB) following ConA and LN binding with their membrane receptors. It was found that ConA and LN interactions with their membrane receptors could induce the assembly of microfilaments below the membrane and promote DNA synthesis in these cells, but this effect was inhibited when microfilaments were destroyed by CB treatment. These results suggest that microfilaments might play a role in transferring signals from the membrane to the nucleus.

Analysis of lectin receptors in normal nasal mucosa, nasal polyp, inverted papilloma and papillary adenocarcinoma.

In order to investigate the changes in glycoprotein structure in the process of cellular differentiation of the nasal mucosa, formalin-fixed, paraffin-embedded biopsy specimens of normal nasal mucosae, nasal polyps, inverted papillomas and papillary adenocarcinomas were analysed by the Avidin Biotin-Peroxidase Complex technique for the demonstration of peanut agglutinin (PNA) receptors, concanavalin ensifomis agglutinin (ConA) receptors, ulex europeaus agglutinin (UEA-I) receptors, wheat germ agglutinin (WGA) receptors, carcino-embryonic antigen (CEA) and keratin. The quantity and distribution of PNA receptors, ConA receptors, UEA-I receptors and CEA were different, in relation to the varying pathological changes. The results suggest that the glycoprotein structure in the cells of the nasal mucosa will change following their differentiation and malignant transformation, which may be helpful in establishing the diagnosis.

Increase in concanavalin A cap formation on lymphoma cells following interaction with inactive influenza viruses.

Binding of the lectin Con A to its ligand on the cell surface of normal circulating lymphocytes induces capping in 28-32% of these cells. This Con A cap formation is markedly decreased in malignant cells from the human hematopoietic system. Among others, the human lymphoma Daudi cell line exhibit a cap formation with Con A in only 5-10% of the cells. In this study, we found that inactivated influenza viruses induced changes in the cell surface membrane of Daudi cells resulting in an increased percentage of Con A cap forming cells (30-40%). This phenomenon occurred independently of viral replication and was initiated by adsorption of inactivated viral particles or isolated hemagglutinin and neuraminidase viral glycoproteins. This phenomenon may be due to the binding of Con A molecules to viral receptors and to cell receptors leading to crosslinking of Con A receptors that will induce their mobility and the formation of a cap. Alternatively, experiments performed with cytochalasin B and colchicine suggest that the viral interaction with the cell membrane may have induced changes in the cytoskeleton at the level of microtubules. These changes induced increased lateral movement of the Con A receptors resulting into formation of a cap.

Differential effects of glycoprotein processing inhibition on experimental metastasis formation by T24-H-ras transformed fibroblasts.

Highly metastatic mouse 10T1/2 cell lines (Ciras 2, Ciras 3 and dGC2M5) which have been T24-H-ras transfected, are shown to have differential responses in metastatic properties when grown in the presence of the processing inhibitors, swainsonine, castanospermine and deoxymannojirimycin. Concanavalin A binding data indicated the inhibitors caused similar shifts in oligo-saccharide structures, resulting in more high mannose character for all cell lines. However, swainsonine inhibited the experimental metastasis of dGC2M5, but did not affect the metastatic properties of Ciras 2 and Ciras 3. Inversely, castanospermine reduced experimental metastasis of Ciras 2 and 3 and did not inhibit dGC2M5. These results show that closely related metastatic cell lines respond differently in their metastatic ability when changes occur in N-linked oligosaccharide content. This observation emphasizes the importance of oligosaccharide structure in the malignant phenotype and indicates that some caution should be used when generalizing about the effects of processing inhibitors on a complex process like metastasis.