Addition Of Concanavalin Research Articles

Stimuli-Responsive Microgel-Based Surface Plasmon Resonance Transducer for Glucose Detection Using a Competitive Assay with Concanavalin A

Glucose responsive microgels composed of poly(N-isopropylacrylamide-co-glycosyloxyethyl methacrylate) (p(NIPAm-co-GEMA)) were synthesized by free radical precipitation polymerization. The resultant microgels were shown to collapse upon addition of Concanavalin A (ConA) owing to its ability to bind up to four GEMA molecules. We showed that the microgel could reswell upon addition of free glucose due to the competitive binding of GEMA and glucose with ConA, effectively breaking the cross-links responsible for the collapse. Thus, by monitoring the contraction/expansion process of the p(NIPAm-co-GEMA) microgels bound to a Au surface via surface plasmon resonance (SPR), the concentration of glucose in solution can be quantified. We go on to show that there is a ninefold SPR signal enhancement compared to exposure of bare Au to glucose and a similar enhancement compared to microgels without GEMA. Finally, we showed that the p(NIPAm-co-GEMA) microgel-based SPR sensor surfaces were not responsive to other interfering species. Taken together, the results show that the competitive binding concept can be useful for detecting small biologically relevant molecules that are usually difficult to detect via SPR. Therefore, this concept can be expanded upon to detect other small molecules of interest in the future.

Macromol. Chem. Phys. 19/2014

Front Cover: Unprotected glycopolymers synthesized via RAFT polymerization are employed to stabilize gold nanoparticles by thiol chemistry through Au–S bonds. The recognition behavior of the resulting glyco-nanoparticles is assessed by studying the shift in the plasmon band with the addition of Concanavalin A (Con A). The low dissociation constant obtained indicates a strong affinity toward Con A caused by multivalent binding. Further details can be found in the article by M. Álvarez-Paino, V. Bordegé, R. Cuervo-Rodríguez, A. Muñoz-Bonilla,* and M. Fernández-García* on page 1915.

Well‐Defined Glycopolymers via RAFT Polymerization: Stabilization of Gold Nanoparticles

Well‐defined glycopolymers of poly(2‐{[(d‐glucosamin‐2‐N‐yl)carbonyl]oxy}ethyl meth(acry­late)) (PHEMAGl and PHEAGl, respectively) are first synthesized by reversible addition–fragmentation chain transfer (RAFT) polymerization in green solvents such as water and dimethyl sulfoxide (DMSO). The biomolecular recognition of the synthesized glycopolymers bearing glucose residues toward Concanavalin A lectin is confirmed by turbidimetric assays. Subsequently, HEMAGl glycopolymer is mixed with gold nanoparticles in the presence of sodium borohydride, which reduces the terminal groups to thiol and yields the preparation of synthetic carbohydrate polymer‐stabilized gold nanoparticles showing enhanced colloidal stability. The resulting glyco‐nanoparticles are characterized by dynamic light scattering (DLS) and field‐emission electron microscopy (FE‐SEM), whereas the molecular recognition abilities are investigated using UV–vis spectroscopy by analyzing the shift in the plasmon band as a consequence of the aggregation variation with the addition of Concanavalin A. image

Switchable Binding Affinity of Mannose Tethered to Collagen Peptide by Temperature-Dependent Triple-Helix Formation

Novel glycopeptides were created with a view to regulate the bindings of carbohydrates to lectins as a means of controlling biological function. We synthesized glycopeptides containing mannose (Man) tethered to a collagen peptide moiety (MPOG10: -(Pro-Hyp-Gly)10- or MGPP10: -(Gly-Pro-Pro)10-). Circular dichroism spectra showed formation of a triple helical structure for MPOG10, and the melting temperature indicates that MPOG10 forms a more stable triple helical structure than MGPP10 in phosphate buffered saline (PBS). At 25 °C, fluorescence polarization (FP) values of MPOG10 and MGPP10 increased following the addition of concanavalin A (ConA), and the addition of α-methyl-mannose (MeMan) to a mixed solution of each glycopeptide with ConA resulted in a decrease in FP values. These results confirm that the previous increase in FP values observed was caused by ConA binding to Man on MPOG10 or MGPP10. The binding affinity of MPOG10 was higher than that of MGPP10, and the dissociation constant of MPOG10 to ConA was 1.9 × 10(-5) (mol/L). The observed binding of MPOG10 to ConA at 25 °C was reduced at higher temperature (50 °C). Therefore, the enhanced binding affinity of MPOG10 to ConA could be accounted for by formation of a clustered Man moiety triggered by the formation of a more stable triple helical structure of MPOG10 compared with MGPP10.

High molecular weight plant heteropolysaccharides stimulate fibroblasts but inhibit keratinocytes

Konjac glucomannan (KGM) is a natural polysaccharide of β(1-4)-d-glucomannopyranosyl backbone of d-mannose and d-glucose derived from the tuber of Amorphophallus konjac C. Koch. KGM has been reported to have a wide range of activities including wound healing. In this study we examined KGM extracts prepared from five plant species, (Amorphophallus konjac Koch, Amorphophallus oncophyllus, Amorphophallus prainii, Amorphophallus paeoniifolius and Amorphophallus elegans) for their effects on cultured human keratinocytes and fibroblasts. Extracts from A. konjac Koch, A. oncophyllus and A. prainii (but not from A. paeoniifolius or A. elegans) stimulated fibroblast proliferation both in the absence and presence of serum. However, these materials inhibited keratinocyte proliferation. The fibroblast stimulatory activity was associated with high molecular weight fractions of KGM and was lost following ethanol extraction or enzyme digestion with β-mannanase. It was also reduced by the addition of concanavalin A but not mannose suggesting that these heteropolysaccharides are acting on lectins but not via receptors specific to mannose. The most dramatic effect of KGM was seen in its ability to support fibroblasts for 3weeks under conditions of deliberate media starvation. This effect did not extend to supporting keratinocytes under conditions of media starvation but KGM did significantly help support adipose derived stem cells under media starvation conditions.

Dextran-Coated Gold Nanoparticles for the Assessment of Antimicrobial Susceptibility

Bacteria rapidly evolve mechanisms to become resistant to antibiotics. Therefore, identifying an effective antibiotic or antibacterial agent and administering it at concentrations that will successfully prevent bacterial growth (antimicrobial susceptibility) is critical for health care decision making and vital for the battle against multi-drug-resistant bacteria. Currently, the determination of antimicrobial susceptibility requires at least 24 h. Herein, we describe a nanoparticle-based antimicrobial susceptibility assay based on the concanavalin A-induced clustering of dextran-coated gold nanoparticles, which sense the presence of available complex carbohydrates in bacterial suspension. Under conditions of bacterial growth inhibition, addition of concanavalin A results in the formation of extensive dextran gold nanoassemblies, which are facilitated by the presence of free carbohydrates in solution and result in large shifts in the surface plasmon band of the nanoparticles. Meanwhile, at conditions of increased bacterial growth, a decrease in the amount of free carbohydrates in solution will occur due to an increased carbohydrate uptake by the proliferating bacteria. This will result in a decrease in the size of the gold nanoparticle clusters and an increase in the number of nanoparticles that bind to bacterial surface carbohydrates, causing lower shifts in the plasmonic band. The gold nanoparticle-based assessment of antimicrobial susceptibility yields results within 3 h and can be used for the high-throughput screening of samples during epidemics and identification of potential antimicrobial agents to expedite clinical decision-making in point-of-care diagnostics.

Studies on the Cytological Function of the Biomembrane and the Neurons

AbstractFor Abstract see ChemInform Abstract in Full Text.

Studies on the Cytological Function of the Biomembrane and the Neurons

Na(+)-dependent and -independent transport sites were elucidated for glycine and L-leucine, respectively, in Chang liver cells, a human culture cell line. Findings of acceleration of the L-leucine uptake by the cells in the acidic medium and synchronized acidification within the cell membrane vesicles with the uptake by them all suggested contransport of L-leucine and proton and the uptake of L-leucine dependent on the inward proton gradient in Chang liver cells. Cotransport of L-leucine and proton was also demonstrated in human peripheral lymphocytes and accelerated by the addition of concanavalin A, probably accompanied by membrane hyperpolarization. It was shown that the Na(+)-gradient-dependent uptake of glycine can be regulated by insulin and 17 beta-estradiol in the rat uterus and by Ca(2+)-calmodulin and membrane potential in Chang liver cells. D-Aspartate uptake as a model of glutamate transport was characterized in rat hippocampal slices and found to consist of Na(+)-dependent (higher-affinity) and -independent (lower-affinity) components. The vulnerability of hippocampal neurons to the Alzheimer beta-amyloid protein was confirmed in vitro with primary cultured rat hippocampal neurons in the presence of the amyloid protein beta 1-42 or its core fragments. The toxicity of the amyloid protein could be blocked by the addition of insulin and several other growth factors to the medium. The addition of genipin, a plant-derived iridoid, was demonstrated to prevent the toxicity of a synthetic fragment of beta 1-42, beta 25-35. Genipin had a neuritogenic activity in PC12h cells, a rat pheochromocytoma cell line, an activity extremely sensitive to inhibitors of the nitrogen oxide (NO) synthase and soluble guanylate cyclase and an NO scavenger. It was also demonstrated in PC12h cells that the activation of the MAP kinase cascade was essential for the neuritogenesis of genipin. These properties of genipin are very comparable to those of nerve growth factor in the cells. It is considered likely that various useful, neurotrophic substances and their extracts will be found in plants in future.

レクチンの認識機能を有するハイブリッド型リポソームの調製

Hybrid liposomes that have a targeting effect were prepared by introducing a sugar type surfactant. The hybrid liposomes consist of dimyristoylphosphatidylcholine, polyoxyethylene glycol surfactant, and a long-chain alkyl sugar ester. The targeting effect was evaluated on the basis of the binding behavior between the hybrid liposomes and a lectine. When the hybrid liposomes were prepared with a glucose type of surfactant, aggregation was observed by the addition of concanavalin A (ConA) to the liposome solution. The result indicates that bridging should occur between the sugar moiety on the surface of liposomes and the recognition site of lectine. The aggregation behavior was also confirmed by the AFM photograph. The interaction between the hybrid liposomes and lectine was found to be reversible because the aggregates disappeared by the addition of excess glucose. Hybrid liposomes containing a galactose type of surfactant exhibited a high affinity toward the peanut lectine (PNA). The alkyl-chain length of sugar surfactants also affected the aggregation behavior. It was suggested that longer alkyl chain surfactants show a higher recognition effect.

Insulin delivery governed by covalently modified lectin-glycogen gels sensitive to glucose.

A glucose-sensitive gel formulation containing concanavalin A and glycogen has been reported previously. Precipitation resulting from the addition of concanavalin A to glycogen has been documented, but the formation of glucose-sensitive gels based on lectin-glycogen interactions is novel and used here in our studies. An improved in-vitro self-regulating drug-delivery system, using covalently modified glucose-sensitive gels based on concanavalin A and a polysaccharide displacement mechanism, is described. The successful use of the covalently modified gels addresses a problem identified previously where significant leaching of the mitogenic lectin from the gel membranes of non-coupled gels was encountered. Concanavalin A was covalently coupled to glycogen by use of derivatives of Schiff's bases. The resulting gels, like the non-coupled gels, were shown to undergo a gel-sol transformation in response to glucose. Insulin delivery was demonstrated using this covalently modified system in conditions of repeated glucose triggering at 20 degrees C and 37 degrees C. The magnitude of the response was less variable than for the dextran-based gels studied previously. The performance of this system has been improved in terms of concanavalin A leaching. This could, therefore, be used as the basis of the design of a self-regulating drug-delivery device for therapeutic agents used to treat diabetes mellitus.

Fas/Fas Ligand System in Prolactin-Induced Apoptosis in Rat Corpus Luteum: Possible Role of Luteal Immune Cells

The prolactin (PRL) surge in cycling rats during the proestrous afternoon is an inducer of apoptotic cell death in luteal cells. This luteolytic action of PRL is peculiar, because PRL may be categorized as a survival factor, if other known physiological functions of PRL are taken into account. Here we analyzed the underlying molecular/cellular mechanisms of this PRL-induced apoptosis. Corpora lutea (CL) were prepared from the ovary on the proestrous day and cultured with or without PRL (2 μg/ml). An addition of PRL to the culture medium induced DNA breakdown in the nuclei of cells mostly identified as steroidogenic by 3β-HSD activity staining, and the number of 3β-HSD-positive cells were significantly decreased, indicating the induction of apoptotic cell death by PRL among luteal cells in culture. Next, the expression of membrane form-Fas ligand (mFasL) in the luteal cell lysate was quantified, because Fas receptor is known to have an exact physiological role in luteolysis. An addition of PRL increased the expression of mFasL. Immunostaining and TUNEL assay on regressing CL revealed that both CD3-positive cells and FasL-positive cells were co-localized in the regions where apoptosis convergently occurred. Moreover, an addition of concanavalin A (ConA), a T-cell specific activator, to the culture mimicked the PRL action by inducing apoptosis in luteal cells and enhancing the expression of mFasL. These data suggest that the CD3-positive T lymphocyte in the CL is at least one of the PRL-effector cell species during the process of luteolysis in rats, and that FasL expression of these cells is upregulated by PRL.

Effects of reducing, oxidizing and alkylating agents on early steps of Junin virus multiplication.

The action of reducing, oxidizing and thiol-alkylating agents on early steps of Junin virus (JV) multiplication in Vero cells was investigated. The presence of reducing agents during virus adsorption as well as incubation of viral particles with these compounds before infection enhanced JV infectivity. On the contrary, the thiol-alkylating agent 5,5' dithiobis (2-nitrobenzoic acid) and the oxidizing compound potassium periodate showed an inhibitory effect, suggesting that sulfhydryl groups, and certain sugar moieties of viral glycoproteins play an important role in the first steps of JV infection. Also enzymatic treatment of cell monolayers and addition of concanavalin A to cultures prior to infection suggest that cellular glycoproteins are involved in virus attachment.

Lymphocyte apoptosis during classical swine fever: implication of activation-induced cell death.

Infection of pigs with classical swine fever virus (CSFV), a member of the Flaviviridae family, causes a severe leukopenia, particularly notable with the lymphocytes. The goal of this study was to analyze mechanisms behind this CSFV-induced lymphopenia. To this end, the kinetics of leukocyte depletion, the appearance of apoptotic cells, and virus infection of leukocytes after infection of pigs with the virulent CSFV strain Brescia were analyzed. Depletion of B and T lymphocytes was noted as early as 1 day postinfection (p.i.). Circulating viable lymphocytes with reduced mitochondrial transmembrane potential--a particular early marker for apoptosis--were also detectable as early as 1 day p.i. When isolated peripheral blood mononuclear cells were cultured for 6 h, significantly more sub-G1 cells with reduced DNA content were detected among the lymphocytes from CSFV-infected animals, again as early as 1 to 3 days p.i. The first time virus was first found in the plasma, as well as infection of leukocytes, was 3 days p.i. However, throughout the observation time of 1 week, <3% of the circulating leukocytes and no lymphocytes contained virus or viral antigen. Further analysis of the T lymphocytes from infected animals demonstrated an increase in CD49d, major histocompatibility complex class II, and Fas expression. An increased susceptibility to apoptosis in vitro was also observed, particularly after addition of concanavalin A as well as apoptosis-inducing anti-Fas antibody to the cultures. Taken together, these results imply that activation-induced programmed cell death was the mechanism behind lymphopenia during classical swine fever.

In vitro replication of epizootic hemorrhagic disease and bluetongue viruses in white-tailed deer peripheral blood mononuclear cells and virus-cell association during in vivo infections.

In vitro and in vivo infections were conducted to determine if the epizootic hemorrhagic disease (EHD) and bluetongue (BT) viruses would replicate in peripheral blood mononuclear (PBM) cells of white-tailed deer (Odocoileus virginianus). All of the North American EHD and BT viruses (EHD virus serotypes 1 and 2, and BT virus serotypes 2, 10, 11, 13, and 17) replicated in vitro in cultures of white-tailed deer PBM cells. However, this replication appeared to be monocyte-dependent and was not enhanced by lymphocyte blastogenesis induced by the addition of concanavalin A. In white-tailed deer infected with either EHD virus serotype 2 or BT virus serotype 10, virus could be isolated consistently from PBM cells only from post-infection day 4 through 8, although they remained viremic through post-infection day 21. In deer, highest viral titers were associated with the erythrocyte fraction, and in no cases did viral titers detected in the platelet, PBM cell or polymorphonuclear cell fractions approach titers observed in whole blood. In the in vitro infections of white-tailed deer erythrocytes, the EHD and BT viruses were associated with pits in the erythrocyte membrane. This association may be important in the long-term viremia observed in deer.

Immunosuppression by breast cancer associated p43-effect of immunomodulators.

It has been previously shown that p43- a breast cancer associated antigen-has immunosuppressive properties. The present study was carried out in order to elucidate the pathomechanisms of immunosuppression in breast cancer patients influenced by the expression of p43. Lymphocytes were cultured from blood of 29 women with benign lesions in the breast as well as from 41 female patients with breast cancer. Lymphocyte stimulation was performed by addition of Concanavalin (Con A) in cultures with lymphocytes alone (CONLYM) or in lymphocytes incubated with p43 (CONAg). In other series immunomodulation was tried by addition of indomethacin (INDLYM, INDAg), levamisole (LEVLYM, LEVAg), or interleukin-2 (ILLYM, ILAG). In breast cancer patients, addition of p43 significantly inhibited the activation of lymphocyte proliferation by Con A compared to women with benign tumors. The addition of indomethacin or levamisole did not influence this inhibitory effect of p43 in breast cancer patients. Contrary to these observations, addition of IL-2 resulted in increased proliferation of lymphocytes from patients with benign as well as malignant tumors, which was inhibited after addition of p43. Analysis of the correlation of the immunosuppressive properties of p43 in correlation with prognostic factors for breast cancer showed evidence for a stronger activity of p43 in early stage tumors (i.e. smaller than 2 cm, lymph node negative, histologic grading GI), confirming previous observations of a higher expression of p43 in early stages of breast cancer.

Isolation and Characterization of a Glycosylation Mutant fromSchizosaccharomyces pombe

N-Linked oligosaccharides were elongated by glycosylation with mannose and galactose residues in the secretory pathway of Schizosaccharomyces pombe. The wild-type S. pombe cells were agglutinated by the additions of not only concanavalin A lectin, which is specific for mannose residues, but also PNA (from Arachis hypogaea) and RCA (Ricinus communis) lectins, which are specific for terminal galactose residues. By PNA-binding selection, we isolated an S. pombe mutant defective in protein glycosylation. The mutant cells, named gms1, were not agglutinated by PNA or RCA. In contrast, agglutination of the gms1 cells by the addition of concanavalin A was markedly increased. Structural studies on N-linked oligosaccharides from gms1 mutant cells showed that the number of alpha-1,2-linked galactose residues wes markedly reduced, and unsubstituted alpha-1,6-linked polymannose outer chains were attached to the core oligosaccharides.

Downregulation of lymphocyte mitogenesis by breast cancer-associated p43

Placental isoferritin-associated p43 has proven to induce immune suppression during pregnancy in order to avoid rejection of the fetus' alloantigens by maternal lymphocytes. It has been demonstrated previously that p43 is also synthesized by breast cancer cells and can also be found on the surface of a subpopulation of T cells in women with this disease. Therefore, it was the aim of the present study to investigate if breast cancer-associated p43 has immunosuppressive properties. In 40 women undergoing surgical excision of a suspicious lump in their breast, blood was withdrawn and lymphocytes were isolated. Lymphocyte cultures were incubated with p43 antigen and anti-p43 antibody (CM-H-9). In a second series, lymphocyte mitogenesis was activated by addition of concanavalin A (Con A), Con A + p43 and Con A + anti-p43, respectively. While lymphocytes of breast cancer patients ( n = 21) and women with benign breast disease ( n = 19) incubated with p43 as well as with anti-p43 antibody did not show any difference in terms of incorporation of [ 3H]thymidine, activation of lymphocytes by addition of Con A was significantly inhibited after addition of p43 antigen in breast cancer patients compared to women with benign breast disease ( P = 0.0178). Analysis of prognostic factors for breast cancer showed that inhibition of lymphocyte mitogenesis was dependent on the degree of tumor differentiation and was significantly higher in well differentiated tumors (GI) compared with more dedifferentiated tumors (GIII). The present study shows that breast cancer-associated antigen p43 is able to induce immune suppression in breast cancer patients but not in women with benign breast disease.

Gametocytogenesis Induction by Berenil in Cultured Plasmodium falciparum

Berenil, which is known to inhibit synthesis of nucleic acid, DNA and RNA, and polyamine, induced gametocytogenesis in cultured Plasmodium falciparum, although the mechanism by which Berenil induces gametocytogenesis is unknown. Asexual parasites of a strain of P. falciparum that seldom produce gametocytes in in vitro culture began gametocytogenesis after 2–24 hr treatment with RPMI 1640 medium containing Berenil (final concentration, 0.1–10.0 μg/ml) with horse serum. Gametocytogenesis was consistently observed from 3 days after the addition of Berenil to culture. After parasites were cultivated in RPMI 1640 medium containing Berenil (final concentration, 3.0–10.0 μg/ml) and concanavalin A with horse serum for 2 hr, Berenil and concanavalin A were removed from culture medium by centrifugation. Nevertheless, induction of gametocytogenesis was observed. The addition of concanavalin A (final concentration, 10 μg/ml) to RPMI 1640 medium containing Berenil enhanced the induction of gametocytogenesis by Berenil. However, when RPMI 1640 medium with concanavalin A was used without the addition of Berenil, no gametocytogenesis was observed as in RPMI 1640 medium alone.

Non-selective interactions of concanavalin A with phosphatidylcholine small unilamellar vesicles: implications for chemical sensor development

Addition of Concanavalin A (Con A) and succinyl—Con A (Suc—Con A) to suspensions of phosphatidylcholine small unilamellar vesicles (SUV), internally loaded with the dye, 6-carboxyfluorescein, caused an increase in fluorescence over several hours. This indicates that the presence of protein—polysaccharide aggregates was not required to alter SUV membrane permeability behaviour, in direct contrast to findings previously observed for anchored planar bilayer lipid membranes. It is proposed that suc—Con A molecules penetrated the SUV membrane, creating enough of a structural change in the bilayer matrix so as to alter permeability behaviour, but not affect membrane integrity. These results suggest that development of chemical sensors employing aggregation or any other interaction which perturbs the lipid matrix sufficiently to alter electrochemical behaviour may be hindered due to interferences resulting from non-specific adsorption of proteins to phospholipid membranes.

Effects of anti-oxidants on ornithine decar☐ylase in mitogenically-activated T lymphocytes

Reactive oxygen species formation is an early event following lymphocyte activation and may trigger other biochemical processes. Anti-oxidants (desferrioxamine (DES), nordihydroguaiaretic acid (NDGA) and potassium ferricyanide) inhibit human and murine T cell proliferation in vitro. Since an increase in ornithine decar☐ylase (ODC) activity is an essential concomitant of T lymphocyte proliferation, we have examined in vitro the effects of anti-oxidants on this activity increase. ODC activity in mouse lymph node T cells increased steadily from 6–24 h after addition of concanavalin A. The combination of phorbol myristate acetate (PMA) and ionomycin also stimulated ODC activity in these cells. The anti-oxidants DES, NDGA and ferricyanide strongly inhibited the increase in ODC activity seen in response to either concanavalin A or PMA/ionomycin. Dose-response curves for the inhibitory effects of the anti-oxidants on DNA synthesis and ODC activoty at 48 h after mitogen addition were very similar. Addition of putrescine or spermidine (10–1000 μM) could not overcome the block in DNA synthesis induced by DES. Although the anti-oxidants inhibited the ODC activity increase in mitogen-stimulated T cells, they did not decrease mRNA levels for the enzyme. These results suggest that intracellular formation of reactive oxygen species is involved in the induction of ODC activity by a mechanism exerted at a posttranscriptional stage. The anti-oxidants can be employed as tools to elucidate contigent relationships between some intracellular events that follow T cell activation.