Wheat Germ Agglutinin Receptors Research Articles

Targeted delivery of intranasally administered nanoparticles-mediated neuroprotective peptide NR2B9c to brain and neuron for treatment of ischemic stroke

The lack of effective therapies mandates the development of new treatment strategies for ischemic stroke. The NR2B9c peptide can prevent N-Methyl-D-aspartate receptor (NMDAR)-mediated neurotoxicity induced by ischemia without affecting essential NMDAR activity and brings hope for stroke therapy. However, it is very difficult for NR2B9c to cross by itself the blood–brain barrier (BBB) and the neuron membrane. To provide a suitable delivery for unleashing the therapeutic potential of NR2B9c, in consideration of a high affinity of wheat germ agglutinin (WGA) for WGA receptors abundantly present on olfactory epithelium and neuronal surface, we developed WGA-modified nanoparticles carrying NR2B9c (NR2B9c-WGA-NPs). Following intranasal administration, NR2B9c-WGA-NPs are able to bypass the BBB and effectively transport NR2B9c into the brain and neuron, and therefore can protect neurons against excitotoxicity, reduce ischemic brain injury in rats and ameliorate their neurological function deficits. The intranasal administration of NR2B9c-WGA-NPs may serve as a practical stroke therapy.

Lectinhistochemical staining of granuloma induced by bacillus Calmette-Guerin in Piaractus mesopotamicus

ABSTRACTObjetive. This study was conducted to evaluate, by means of lectinhistochemistry (LHC), the expression of carbohydrates in granulomas induced by the bacillus Calmette-Guerin (BCG) in muscle tissue of Piaractus mesopotamicus after 33 days. Material and methods. Histological sections with 3 μm thick were incubated with the following lectins :WGA (Wheat germ agglutinin), DBA (Dolichos biflorus agglutinin) and HPA (Helix pomatia agglutinin), and the results were evaluated by light microscopy. Results. Acid fast bacilli were stained by Ziehl Neelsen (ZN) and strong labeled by WGA in the cytoplasm of macrophages. Labeling with DBA was intense in fibroblasts and weak in macrophages. On the other hand, HPA binding was stronger in macrophages, especially in those that were in close contact with epithelioid cells, without evidence of binding to fibroblasts. The epithelioid cells were not labeled by the used lectins, but they were identified by Hematoxilin-Eosin (HE). The lectins labeled specific type saccharides in glycoproteins, as N-acetylglucosamine present in bacilli and macrophages, as well as N-acetyl-galactosamine in macrophages. The control group showed no inflammation or lectin binding. Conclusions. This technique may be useful in identifying receptors for WGA, DBA and the HPA lectins in epithelioid granuloma induced by BCG in P. mesopotamicus

QD as a bifunctional cell-surface marker for both fluorescence and atomic force microscopy

Fluorescent quantum dots (QDs) are a new class of fluorescent label and have been extensively used in cell imaging. Streptavidin-conjugated QDs have a diameter of ca. 10–15 nm; therefore when used as probes to label cell-surface biomolecules, they can provide contrast enhancement under atomic force microscopy (AFM) and allow specific proteins to be distinguished from the background. In addition, the size and fluorescent properties potentially make them as probes in correlative fluorescence microscopy (FM) and AFM. In this study, we tested the feasibility of using QD-streptavidin conjugates as probes to label wheat germ agglutinin (WGA) receptors on the membrane of human red blood cells (RBCs) and simultaneously obtain fluorescence and AFM images. The results show that the distribution of QDs labeled on human RBCs was non-uniform and that the number of labeled QDs on different erythrocytes varied significantly, which perhaps indicates different ages of the erythrocytes. Thus, QDs may be employed as bifunctional cell-surface markers for both FM and AFM to quantitatively investigate the distribution and expression of membrane proteins or receptors on cell surface.

Human alveolar macrophages: wheat germ agglutinin-dependent tumor cell killing.

Human alveolar macrophages (AM) obtained by bronchoalveolar lavage from healthy donors were examined for ability to cause lectin-dependent tumor cell killing. Of five plant and two animal lectins tested, only one lectin, wheat germ agglutinin (WGA), induced significant and reproducible lectin-dependent macrophage-mediated cytotoxicity (LDMC) against human bladder cancer (T-24) cells. There was no significant difference between the LDMCs of AM and blood monocytes. All 6 tumor cell lines tested were sensitive to various extents to LDMC induced by WGA. Quantitative analysis with WGA-FITC conjugate showed the presence of various levels of receptors for WGA on the surface of AM, monocytes and tumors. A relatively good correlation was found between the sensitivities of the tumor cells to LDMC mediated by AM and the numbers of receptors for WGA. Pretreatment of AM or monocytes with LPS did not affect their LDMC. These results indicate that a plant lectin, WGA which binds to both human AM and tumor cells, renders human AM cytotoxic to allogeneic tumor cells by a different mechanism(s) from that involved in the nonspecific tumor cytotoxicity of activated macrophages.

Heterogeneous distribution of plasma membrane glycoconjugates in pancreatic acinar cells

Flow-cytometric studies of lectin binding to individual acinar cells have been carried out in order to analyse the distribution of membrane glycoconjugates in cells from different areas of the pancreas: duodenal lobule (head) and splenic lobule (body and tail). The following fluoresceinated lectins were used: wheat germ agglutinin (WGA), Tetragonolobus purpureus agglutinin (TP) and concanavalin A (Con A), which specifically bind to N-acetyl d-glucosamine and sialic acid, l-fucose and d-mannose, respectively. In both pancreatic areas, two cell populations (R1 and R2) were identified according to the forward scatter (size). On the basis of their glycoconjugate pattern, R1 cells displayed higher density of WGA and TP receptors than R2 cells throughout the pancreas. Although no difference in size was found between the cells from duodenal and splenic lobules, N-acetyl d-glucosamine and/or sialic acid and l-fucose residues were more abundant in plasma membrane cell glycoconjugates from the duodenal lobule. The results provide evidence for biochemical heterogeneity among individual pancreatic cells according to the distribution of plasma membrane glycoconjugates.

Effect of cholecystokinin blockade on the recovery of alterations induced by acute pancreatitis in glycoconjugates of rat zymogen granules.

Lectin-binding studies have been performed on rat zymogen granules to investigate alterations in the carbohydrate membrane composition that occur in acute pancreatitis induced by caerulein. The influence of treatment with hydrocortisone for seven days before inducing pancreatitis was also studied. Lectin labeling on zymogen granules was also analyzed seven days after inducing pancreatitis in rats that had previously received a hydrocortisone treatment. During this period L 364,718 (0.1 mg/kg)--specific cholecystokinin (CCK) receptor antagonist--was administered daily to some of the rats, and no treatment was applied to others. Using fluorescein-labelled T. purpureus (TP)lectin, a significant decrease in the amount of L-fucose in the granule membrane was observed in rats with caerulein-induced pancreatitis. This effect was directly caused by the pancreatitis and was not influenced by previous hydrocortisone treatment. Seven days later, the density of TP receptors in the granule membrane was similar to the controls both in L-364,718-treated and untreated rats. Therefore, we suggest that endogenous CCK is not an essential factor in the recovery of L-fucose containing glycoconjugates the granule membrane after pancreatitis. Acute pancreatitis did not alter the expression of wheat germ agglutinin (WGA) receptors in the zymogen granule membrane. WGA specifically binds N-acetyl glucosamine and sialic acids. L 364,718 administered for seven days after inducing pancreatitis significantly reduced WGA binding, untreated rats showed a normal zymogen granule membrane. Therefore, the blockade of CCK-induced alterations in membrane glycoconjugates enriched in N-acetyl glucosamine and sialic acid of newly formed granules after pancreatitis, a finding that could explain the delay in the regression of the disease.

Use of the fluorescence-activated cell sorter (FACS) for in vitro assays of developmental toxicity

Our objective is to predict embryotoxicity with reliable in vitro techniques. In several experimental systems, differentiation is accompanied by changes in the glycosylation pattern of cell-surface glycoconjugates. This is also the case with embryonal carcinoma cells. We have monitored the expression of receptors for wheat germ agglutinin (WGA). Murine embryonal carcinoma cells (P19 and F9) were exposed in vitro to xenobiotics for 1–3 days, then incubated successively with WGA-biotin (15 μg/ml) and streptavidin-phycoerythrin (SA-PE) (20 μg/ml), each for 30 min at room temperature. Cell-surface fluorescence was then analysed using a fluorescence-activated cell sorter (FACS). Exposure to 1 μm retinoic acid, a known inducer of differentiation, altered glycosylation as indicated by changes in WGA binding. Clear-cut effects were also observed after exposure to salts of arsenic (20 μM), or nickel (50 μm), and to methotrexate (1 μg/ml), fluorouracil (1.3 μg/ml) or actinomycin D (0.04 μg/ml). These compounds affected the percentage of positive cells the intensity of labelling, or both. Two non-teratogenic compounds (metronidazole and sulfonilamide) have also been tested and had no effect. Lectin histochemistry of embryonal carcinoma cells exposed to potentially toxic agents holds promise as a method for predicting embryotoxicity. FACS analysis allows rapid quantification.

A lectin histochemical study of intrahepatic ducts in patients with hepatolithiasis

A panel of 12 biotinylated lectins was used to investigate the diversity of glycoconjugate on the epithelium of stone-containing intrahepatic bile ducts and compared to controls. Among the 12 lectins, only WGA (wheat germ agglutinin) and Con A (concanavalin agglutinin) stained the epithelium of stone-containing intrahepatic ducts. Con A, a glucose/mannose-specific lectin, bound weakly on the epithelium of the stone-bearing intrahepatic duct in 10 of the 25 specimens, but none of the controls. All stone-containing intrahepatic bile ducts were stained heavily and homogenously by WGA, the N-acetylglucosamine-specific lectin. The high columnar epithelia of both intramural and extramural glands were stained in the supranuclear region, while the serous acini of extramural glands were stained in whole cytoplasm. The epithelium of intrahepatic ducts from the controls was stained weakly by WGA only. The WGA receptors were not abolished by pretreatment of neuraminidase. This led us to conclude that the stone-containing intrahepatic ducts were rich in N-acetylglucosamine and the heavy and homogenous staining with WGA will be indicative of hypersecretion of mucus from stone-bearing intrahepatic bile ducts.

Duck lymphocytes. VII. Selection of subpopulations using lectin-coated magnetic beads

The optimum conditions (time, concentration of duck serum, ratio of cells:beads) for binding of duck lymphocytes to magnetic beads (Dynabeads) coated with the lectins phytohaemagglutinin (PHA), wheat germ agglutinin (WGA), lentil lectin (LL) and concanavalin A (Con A) were determined. In blood and lymphoid organs, the largest population was cells binding PHA (55–80%) and the smallest was cells binding Con A (0–15%) with cells binding WGA and LL occurring at 15–58%. Most blood lymphocytes had receptors for more than one lectin, though cells with receptors for WGA were mostly a subpopulation within those possessing receptors for PHA. It is possible that, as in some other animals, lectins identify functionally important duck lymphocyte populations. Notably, PHA deserves further investigation as a marker for duck T lymphocytes, while receptors for Con A appear to be associated with duck macrophages.

Localization of wheat germ agglutinin lectin receptors on human sperm by fluorescence microscopy: utilization of different fixatives.

Wheat germ agglutinin (WGA) and Concanavalin A (Con A) seem to be the most widely studied of the lectins used to investigate sperm surface receptors. It has been reported that WGA receptors on human sperm membranes may be closely related to male fertility and that WGA receptor deficiency may cause human infertility. Although these claims may not be negated, there are many discrepancies in current literature regarding the localization and detection of human sperm WGA receptors. These discrepancies are probably the result of variations in the pretreatment of sperm before fixation and sperm-lectin interaction. This study investigated the effect of different fixation procedures on the localization of human sperm WGA receptors on normal spermatozoa. Comparative studies were performed on spermatozoa separated by wash-swim-up and fixed with 4% formaldehyde, 95% ethanol, or Bouin's solution, or nonfixed (air-dried) using fluorescein isothiocyanate-conjugated WGA. There were significant differences (p < .01) in the % WGA receptor localization following the different fixation procedures on samples receiving the same treatment previous to fixation. The different fixatives affect the plasma membrane to different degrees, resulting in an alteration of lectin receptor localization. To allow a better comparison and understanding of results, a standardized preparation procedure is recommended during studies using lectins as molecular probes. The results suggest than an aldehyde fixative may be the fixative of choice for sperm membrane studies.

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.

Calyculin A induces contractile ring-like apparatus formation and condensation of chromosomes in unfertilized sea urchin eggs.

Calyculin A, a protein phosphatase inhibitor, induced cleavage-like morphological change in unfertilized sea urchin eggs. A contractile ring-like apparatus containing both filamentous actin and myosin was formed in the cleavage furrow. Wheat germ agglutinin receptors were also found in the same region. The eggs did not develop further after constriction of the ring. No aster-like microtubular structure was found in the calyculin A-treated eggs. The cleavage was not inhibited by the antimicrotubule drug griseofulvin. Calyculin A also increased histone H1 kinase activity and induced chromosome condensation. These changes also occurred in the presence of emetine (an inhibitor of protein synthesis) and aphidicolin (an inhibitor of DNA synthesis). It is suggested that calyculin A induced these changes in the sea urchin eggs by inhibiting the activity of protein phosphatase 1.

Pregnancy-associated suppressor cells in mice: Functional characteristics of CD3 +4 −8 −45R + T cells with natural suppressor activity

Natural suppressor (NS) cells are MHC-unrestricted regulatory cells with non-specific inhibitory activity for immune responses. In adult mice, NS cells are characteristically found in bone marrow and in splenic tissue following total lymphoid irradiation and cyclophosphamide treatments. Recently, we have shown that the spleens of pregnant mice harbour a population of lymphocytes which resemble NS cells in terms of phenotype and inhibitory activity. In this study, we use positive and negative selection techniques to further characterize splenic pregnancy-associated NS (SPANS) cells as predominantly ‘double negative’ T cells (CD3 +4 −8 −) bearing receptors for the lectins wheat germ agglutinin and soybean agglutinin, as well as expressing CD45R and the heat-stable J11d.2 antigen. Taken together, these findings lead us to conclude that SPANS cells belong to an immature T cell lineage. In keeping with their T cell phenotype, SPANS cells do not express the natural killer (NK) cell-specific markers NK2.1 and asialoGM1 and do not mediate lytic activity against NK-sensitive YAC-1 cells, although natural cytotoxic activity against WEHI-164 cells was found to co-purify with SPANS cells. Suppressive activity of SPANS cells in mixed lymphocyte reactions (MLR) is abolished by treatment with mitomycin C, suggesting that natural suppression in this system is a proliferation-dependent phenomenon. Preincubation of SPANS cells with conditioned medium from Con A-stimulated T cell cultures results in augmented NS activity, indicating that SPANS cells respond to T cell signals. Our data suggest that SPANS cells mediate suppression via the elaboration of a soluble suppressor factor since SPANS cells do not require cell-cell contact to mediate suppression and supernatants from short-term cultures of SPANS cell-enriched SBA + pregnancy spleen cells inhibit MLR. We believe that SPANS cells may be important in regulating hematopoiesis and maternal anti-fetal immunity during murine pregnancy.

Electron microscopic cytochemistry on the distribution of wheat germ agglutinin receptor on the platelet plasma membrane after treatment with convulxin isolated from Crotalus durissus terrificus venom

The effects of convulxin, isolated from the venom of Crotalus durissus terrificus, on the localization and distribution of wheat germ agglutinin (WGA) binding sites on platelet surfaces have been investigated at an ultrastructural level. A post-embedding cytochemical technique using WGA-gold complexes was used and the quantitative intensity of WGA-labeling on the surface membrane of platelets after convulxin stimulation was determined. In the presence of Ca 2+, convulxin induced platelet release and aggregation, while in its absence, the platelets formed pseudopodia and showed release reaction, but without aggregation. After treatment with convulxin, WGA-labeling on the surface membrane decreased compared with intact washed (control) platelets. In the presence of Ca 2+, clusters of gold label were often found on the surface membrane. However, the WGA-labeling intensity of the membrane of the open canalicular system increased significantly compared with that of platelets stimulated by convulxin in the absence of Ca 2+. Direct morphological evidence demonstrates qualitative and quantitative alterations of WGA-labeling on the surface membrane of platelets, after convulxin stimulation. The possibility is considered that WGA-binding glycoproteins on the surface membrane are involved in the aggregation response after convulxin stimulation.

Proliferation of natural suppressor cells in long-term cultures of spleen cells from normal adult mice.

Natural suppressor cells were induced by culturing spleen cells from normal adult mice for 2 to 3 wk. The suppressor cells were large in size, nonadherent and nonspecifically suppressed the plaque-forming cells response of fresh spleen cells to SRBC in vitro. The suppressive activity of the cells was not affected by treatment with indomethacin or anti-Thy-1, anti-Ig, anti-Ia, or anti-asialoGM1 plus complement. Phenotype analysis by FACS showed that Thy-1, L3T4, Ly-2, CD3-epsilon, TCR-alpha beta, Ig, B220, Ia, and asialoGM1 Ag were all absent in the suppressor cells, although they were wheat germ agglutinin receptor positive. The suppressor cells did not demonstrate cytotoxicity against either YAC-1 or P-815 cells. Enriched large cell populations from fresh normal spleens expressed the same phenotypes and also exhibited the suppressive activity. These findings suggest that a minor population of natural suppressor cells exist in the normal adult mouse spleen and they proliferate during the in vitro culture of spleen cells.

Goat sperm membrane: lectin-binding sites of sperm surface and lectin affinity chromatography of the mature sperm membrane antigens

The cell surface glycoproteins of goat epididymal maturing spermatozoa have been investigated using lectins as surface probes that interact with specific sugars with high affinity. Concanavalin A (ConA) and wheat-germ agglutinin (WGA) showed high affinity for mature cauda epididymal sperm agglutination, whereas RCA 2, kidney beans lectin and peanut agglutinin caused much lower or little agglutination of the cells. The mature sperm exhibited markedly higher efficacy than the immature caput epididymal sperm for binding both ConA and WGA, as evidenced by sperm agglutination and the binding of the fluorescence isothiocyanate (FITC)-labelled lectins. FITC-ConA binds uniformly to the entire mature sperm surface whereas FITC-WGA binds to the acrosomal cap region of the head. The FITC-RCA 2 mainly labelled the posterior head of mature cauda sperm. However, no WGA-specific glycoprotein receptors could be detected in sperm plasma membrane (PM) by WGA-Sepharose affinity chromatography. The data implied that the epididymal sperm maturation is associated with a marked increase in the ConA/WGA receptors and that WGA receptors may be glycolipids rather that glycoproteins. Analysis of the ConA receptors of cauda sperm PM identified by ConA-Sepharose affinity chromatography and subsequent resolution in SDS-PAGE demonstrated the presence of five glycopolypeptides of different concentrations (98, 96, 43, 27 and 17 kDa) of goat sperm membrane. The immunoblot of these ConA-specific glycopeptides with anti-sperm membrane antiserum showed that 98- and 96-kDa receptors are immunoresponsive.

The augmentative effect of a protein-bound polysaccharide (PSK) on tumoricidal activity of the soluble factor produced by a streptococcal preparation (OK-432)-activated macrophages

The enhancement of antitumor activities of the tumoricidal soluble factor (SF) from a streptococcal preparation (OK-432)-activated macrophages by the pretreatment with a protein-bound polysaccharide (PSK) was investigated in tumor-bearing mice. Two-step stimulations with OK-432 at in vivo priming and in vitro eliciting were required for the production of the tumoricidal SF by macrophages, and the tumoricidal activity of the SF apparently correlated with the uptake of OK-432 by macrophages at priming phase. Tumoricidal activity of the SF from OK-432-activated macrophages in proteose-peptone (P-P)-pretreated mice significantly decreased with the development of the tumor, whereas in PSK-pretreated mice did not. Pretreatment of tumor-bearing mice with PSK saved a decrease in the macrophages carrying Iak or asialo GM1 antigens and an increase in wheat germ agglutinin (WGA) receptors. Furthermore, the uptake of OK-432 by macrophages at priming phase was enhanced. The tumoricidal activity of the SF from OK-432-activated macrophages was augmented. Thus, PSK may restore the depressed functions of macrophages, and the combination therapy with PSK and OK-432 may be effective to enhance the production of tumoricidal SF in tumor-bearing mice.

Enrichment of natural suppressor activity in a wheat germ agglutinin positive hematopoietic progenitor-enriched fraction of monkey bone marrow

Natural suppressor (NS) activity, the capacity of unprimed cells to suppress immunologic responses, is present in mouse, rabbit, and human bone marrow (BM). In this study we characterize NS activity in bone marrow cells of the rhesus monkey. Greatest NS activity was found in low-density cells (1.0600 to 1.0655 g/mL) obtained by density centrifugation on a discontinuous Percoll gradient. NS activity was further enriched when cells were separated by affinity for wheat germ agglutinin (WGA). Cells with high affinity to WGA demonstrated potent NS activity, whereas cells with low affinity to WGA had no NS activity. A significant relationship between NS activity and hematopoietic activity was demonstrated using in vitro assays of colony formation (CFU-GM and CFU-MIX). NS activity was not affected by treatment with monoclonal antibodies (MoAbs) to human Fc gamma receptors (Leu, 11a,b,c) or treatment with MoAbs to monkey natural killer cells. These findings extend our prior observations by showing that cells with NS activity, which apparently have WGA receptors, are present not only in murine BM but also in monkey bone marrow, and suggest that such cells may be involved in immunoregulation by primitive cells of BM.

Enrichment of natural suppressor activity in a wheat germ agglutinin positive hematopoietic progenitor-enriched fraction of monkey bone marrow

Natural suppressor (NS) activity, the capacity of unprimed cells to suppress immunologic responses, is present in mouse, rabbit, and human bone marrow (BM). In this study we characterize NS activity in bone marrow cells of the rhesus monkey. Greatest NS activity was found in low-density cells (1.0600 to 1.0655 g/mL) obtained by density centrifugation on a discontinuous Percoll gradient. NS activity was further enriched when cells were separated by affinity for wheat germ agglutinin (WGA). Cells with high affinity to WGA demonstrated potent NS activity, whereas cells with low affinity to WGA had no NS activity. A significant relationship between NS activity and hematopoietic activity was demonstrated using in vitro assays of colony formation (CFU-GM and CFU-MIX). NS activity was not affected by treatment with monoclonal antibodies (MoAbs) to human Fc gamma receptors (Leu, 11a,b,c) or treatment with MoAbs to monkey natural killer cells. These findings extend our prior observations by showing that cells with NS activity, which apparently have WGA receptors, are present not only in murine BM but also in monkey bone marrow, and suggest that such cells may be involved in immunoregulation by primitive cells of BM.

Wheat Germ Agglutinin (WGA) Receptors on Human Sperm Membrane and Male Infertility

Wheat Germ Agglutinin (WGA) Receptors on Human Sperm Membrane and Male Infertility