Fluorescence polarization studies of saccharide binding to Wheat germ agglutinin and lysozyme

N-acetyl-D-glucosamine causes only slight increases in the polarization of Wheat germ agglutinin fluorescence at saturating levels whereas the disaccharide and trisaccharide produce increases in the polarization value from 0.116 to 0.151 and 0.154 respectively. These increases suggest that rotational motions of the tryptophan residue at the binding sites are being restricted by an interaction between these tryptophans and the bound sugars. A model of the nature and location of these interactions is discussed.Comparable results are obtained with lysozyme, which shows a larger effect upon binding of N-acetyl-D-glucosamine, but a maximal increase in polarization upon binding the corresponding disaccharide or trisaccharide.     

Axotomy-induced apoptosis in adult rat primary sensory neurons

Neuronal death following unilateral axotomy of a sensory nerve has long been inferred from neuronal counts of dorsal root ganglion neurons, using the contralateral ganglia as a control. The counting methods used usually involved the counting of neuronal nucleoli and made assumptions about them which could conceivably be flawed. Very few studies have used direct observations of dying or degenerating neurons to address questions concerning the duration of the period of neuronal death or the mechanisms involved in this process. Here we describe a morphological, morphometric and histochemical study into the nature and duration of sensory neuron death following transection and ligation of the sciatic nerve at mid-thigh level in the adult rat. We show that at least some of this neuronal loss occurs by apoptosis as defined by morphological criteria and in situ end-labelling of damaged DNA. Absolute numbers of apoptotic neurons were counted from serial paraffin sections of ganglia and estimates of neuronal numbers obtained by disector analysis at 1, 2, 3 and 6 months after axotomy. Using this approach we show that axotomy-induced apoptosis begins at around 1 week and continues up to at least 6 months after axotomy.     

Surface localization of wheat germ agglutinin and concanavalin A binding sites on normal human blood cells: an ultrastructural histochemical study

In order to determine the extent and variations in surface concavanalin A (CON A) and wheat germ agglutinin (WGA) labeling of different varieties of normal blood cells, gluraraldehyde-fixed human blood cells were exposed to CON A-gold labeled horseradish peroxidase (CON A-HRP-G) and WGA-gold labeled ovomucoid (WGA-OVO-G) histochemical methods. The resultant particulate reaction product permitted assessment of binding and number of gold particles per micrometer of cell surface. Particle counts and data were subjected to statistical analysis. Six subjects (three female and three male) were used and compared in this study. In spite of moderate variations in surface labeling of the various types of leukocytes, erythrocytes and platelets within a given subject, determinations of mean labeling values for similar cell varieties proved quite similar between subjects with the given lectin. WGA and CON A had substantially different labeling densities on the various hemic cells. WGA surface labeling of all types of hemic cells, with the exception of platelets, showed far more labeling than was found with CON A. WGA mean labeling of the grouped subjects was significantly higher for each variety of leukocyte than for either erythrocytes or platelets although this distinction was not always evident in an individual subject. With CON A, mean labeling density for each of hemic cell types showed significant differences between each of the hemic cell varieties. Erythrocytes had only minimal CON A binding while monocyte and platelet populations represented the most reactive of the hemic cells. No difference was noted between corresponding cell varieties in the female vs. male subjects.     

Spike-timing in primary sensory neurons: a model of somatosensory transduction in the rat

In previous work, we constructed a simple electro-mechanical model of transduction in the rat mystacial follicle that was able to replicate primary afferent response profiles to a variety of whisker deflection stimuli. Here, we update that model to fit newly available spike-timing response data, and demonstrate that the new model produces appropriate responses to richer stimuli, including pseudo white noise and natural textures, at a spike-timing level of detail. Additionally, we demonstrate reliable distributed encoding of multi-component oscillatory signals. No modifications were necessary to the mechanical model of the physical components of the follicle–sinus complex, supporting its generality. We conclude that this model, and its continued development, will aid the understanding both of somatosensory systems in general, and of physiological results from higher (e.g. thalamocortical) systems by accurately characterising the signals on which they operate.     

Wheat germ agglutinin blocks the acrosome reaction in Strongylocentrotus purpuratus sperm by binding a 210,000-mol-wt membrane protein

Wheat germ agglutinin (WGA) binds to the entire surface of Strongylocentrotus purpuratus sperm, and inhibits the egg jelly-induced acrosome reaction. The binding was found to be species dependent and was completely inhibited by 5 mM N-acetyl-D-glucosamine. Blockage of the acrosome reaction by WGA was bypassed by a combination of the ionophores A23187 and monensin, although neither ionophore was effective individually. These experiments suggest that WGA blocks both Ca2+ uptake and Na+/H+ exchange in these sperm, which was confirmed by direct measurements of 45Ca2+ uptake and H+ efflux. The target of WGA in S. purpuratus sperm appears to be a membrane glycoprotein of Mr = 210,000. Treatment of this protein with neuraminidase or endo-beta-N-acetylglucosaminidase F abolished WGA binding.     

Wheat germ agglutinin evidence for a genetic basis of multiple forms

Germ from hexaploid wheat (Triticum aestivum L.) contained three forms of agglutinin separable by ion-excahnge column chromatography at pH 3.8, while germ from tetraploid wheat (Triticum turgidum L. (durum group)) contained only two such forms. The different number of forms, not due to protein modification occuring during the purification process, was demonstrable in commercial germ and in bran fractions containing germ from single wheat varieties. This evidence for a genetic basis of lectin multiple forms in wheat indicates the advisability of using genetically identified plant varieties in lectin research.     

Wheat germ agglutinin regulates cell division in wheat seedling roots

The mitogenic activity of wheat germ agglutinin (WGA) has been studied in roots of 4-day-old wheat seedlings. WGA had a more pronounced stimulating effect on cell division than the known mitogens concanavalin A and phytohemagglutinin whereas gliadin had no effect. Treatment of wheat seedling roots with exogenous WGA led to the accumulation of indoleacetic acid and cytokinins, hormones that play an important role in the activation of plant cell growth. The data on the combined effect of 24-epibrassinolide and WGA on cell division and accumulation of phytohormones in seedling roots support a possible link between the endogenous WGA level and hormonal regulation of cell division in the root meristem of wheat plants.     

Wheat germ agglutinin in wheat seedling roots: induction by elicitors and fungi

Summary Treatment of wheat (Triticum aestivum L.) seedlings with elicitors originating from either plant or fungal cell walls induces about a 2-fold increase of wheat germ agglutinin (WGA) in the roots. While the WGA content in roots of healthy plants normally decreases as a function of germination time, a transient accumulation of WGA could be observed in plants challenged with different fungi, including Rhizoctonia solani, Fusarium culmorum, Pythium ultimum and Neurospora crassa. Peak levels in challenged roots were 2 to 5 times as high as in control plants. Most of this induced WGA could be released from the roots by soaking them in a solution of the hapten N-acetylglucosamine. On the basis of the results obtained it is postulated that WGA may be involved in the defence of wheat against fungal attack.     

Heterogeneity in wheat germ agglutinin binding by mouse peritoneal macrophages

The binding of the lectin wheat germ agglutinin (WGA) to the cell surface of monocytes and macrophages obtained from the stimulated peritoneal cavity of mice was investigated electron microscopically, using ovomucoid-gold as an indirect marker. Resident (tissue) macrophages, identified by the presence of PO activity in the rough endoplasmic reticulum as well as in the nuclear envelope, showed low WGA binding, whereas monocytes and monocyte-derived macrophages with PO activity in the granules showed high WGA binding. Since cells devoid of PO activity showed variable WGA binding, the value of this gold-WGA-binding technique for discrimination on a quantitative basis between resident macrophages and monocytes or monocyte-derived macrophages, is discussed.     

Microcalorimetric study of wheat germ agglutinin binding to N-acetylglucosamine and its oligomers.

The energetics of association of wheat germ agglutinin (WGA) with N-acetylglucosamine (GlcNAc) and its beta(1,4) oligomers have been measured using isothermal titration calorimetry. Association constants of 0.4, 5.3, 11.1, 12.3, and 19.1 mM-1 and enthalpies of binding of -6.1, -15.6, -19.4, -19.3, and -18.2 kcal mol-1 were obtained at 26 degrees C for the titration of WGA with GlcNAc, (GlcNAc)2, (GlcNAc)3, (GlcNAc)4, and (GlcNAc)5, respectively. The term T delta S was always of negative value, indicating that the binding process is enthalpically driven. Titrations of WGA performed at pH 4.5 did not differ significantly from those performed at pH 7.0, suggesting that no groups with a pKa in this range are directly involved in the binding event. Also, performing the titration in a buffer system with a higher enthalpy of protonation did not change the enthalpy of binding confirming that there is no net protonation or deprotonation when WGA binds GlcNAc residues at pH 7. A model of four independent binding sites was found to adequately describe the binding curves, except in the case of (GlcNAc)4 which exhibited positive cooperativity. The energetic values are discussed within the context of the structure of the WGA-(GlcNAc)2 complex.     

Wheat germ agglutinin is selectively transported to multivesicular bodies.

Colloidal iron dextran particles bearing wheat germ agglutinin (WGA/FeDex) were bound by glycoconjugates expressed at the surface of HepG2 cells. Bound WGA/FeDex was internalized when cells were incubated at 37 degrees C and accumulated in intracellular structures which have the same buoyant density as the plasma membrane when examined on Percoll density gradients. The intracellular structures containing WGA/FeDex were identified as multivesicular bodies (MVB) by transmission electron microscopy. WGA/FeDex was not transported to lysosomes nor did it interfere with uptake and transport of GalBSA to lysosomes by the asialoglycoprotein receptor. WGA/FeDex was seen predominantly in non-coated invaginations at the cell surface, suggesting it may enter cells at a different site than GalBSA/FeDex. Highly enriched plasma membranes and MVBs containing superparamagnetic [125I]WGA/FeDex particles were prepared by high gradient magnetic affinity chromatography (HIMAC). Plasma membranes prepared by HIMAC were enriched 30-fold for [125I]WGA/FeDex, 15-fold for alkaline phosphodiesterase I, and 9-fold for galactosyltransferase relative to the crude post-nuclear homogenate and consisted entirely of plasmalemmal sheets. Intracellular structures containing WGA/FeDex were enriched 35-fold for [125I]WGA/FeDex, 10-fold for alkaline phosphodiesterase I, and 10-fold for galactosyltransferase but did not contain lysosomal beta-galactosidase. WGA/FeDex has a different ultimate destination in HepG2 cells than ligands internalized by the asialoglycoprotein receptor and can be used to obtain highly enriched plasma membranes and MVBs from cultured cells.     

Heterogeneity in wheat germ agglutinin binding by mouse peritoneal macrophages

Summary The binding of the lectin wheat germ agglutinin (WGA) to the cell surface of monocytes and macrophages obtained from the stimulated peritoneal cavity of mice was investigated electron microscopically, using ovomucoid-gold as an indirect marker. Resident (tissue) macrophages, identified by the presence of PO activity in the rough endoplasmic reticulum as well as in the nuclear envelope, showed low WGA binding, whereas monocytes and monocyte-derived macrophages with PO activity in the granules showed high WGA binding. Since cells devoid of PO activity showed variable WGA binding, the value of this gold-WGA-binding technique for discrimination on a quantitative basis between resident macrophages and monocytes or monocyte-derived macrophages, is discussed.     

Wheat germ agglutinin functionalized complexation hydrogels for oral insulin delivery

Insulin was loaded into hydrogel microparticles after two hours with loading efficiencies greater than 70% for both poly(methacrylic acid-grafted-ethylene glycol) (P(MAA-g-EG)) and poly(methacrylic acid-grafted-ethylene glycol) functionalized with wheat germ agglutinin (P(MAA-g-EG) WGA). The pH-responsive release results demonstrated that the pH shift from the stomach to the small intestine can be used as a physiologic trigger to release insulin from P(MAA-g-EG) and P(MAA-g-EG) WGA microparticles, thus limiting release of insulin into the acidic environment of the stomach. Microplates were successfully treated with PGM to create a surface that allowed for specific binding between mucins and lectins. The 1% PGM treatment followed by a 2 h BSA blocking step gave the most consistent results when incubated with F-WGA. In addition, the PGM-treated microplates were shown to create specific interactions between F-WGA and the PGM by use of a competitive carbohydrate. The 1% PGM treated microplates were also used to show that adhesion was improved in the P(MAA-g-EG) WGA microparticles over the P(MAA-g-EG) microparticles. The interaction between the PGM-treated microplate and P(MAA-g-EG) WGA was again shown to be specific by adding a competitive carbohydrate, while the interaction between P(MAA-g-EG) and the PGM-treated microplate was nonspecific. Cellular monolayers were used as another method for demonstrating that the functionalized microparticles increase adhesion over the nonfunctionalized microparticles. This work has focused on improving the mucoadhesive nature of P(MAA-g-EG) by functionalizing these hydrogel carriers with wheat germ agglutinin (WGA) to create a specific mucosal interaction and then evaluating the potential of these carriers as oral insulin delivery systems by in vitro methods. From these studies, it is concluded that the addition of the WGA on the microparticles produces a specific adhesion to carbohydrate-containing surfaces and that P(MAA-g-EG) WGA shows great promise as an oral insulin delivery system.     

Wheat germ agglutinin treatment of follicle cell-free mouse oocytes inhibits fertilization

Controversy exists whether treatment of follicle cell-free oocytes with wheat germ agglutinin (WGA) prevents fertilization. Lack of inhibition in one case has led to the suggestion that acrosin may not be a zona lysin. To re-examine the effect of the WGA, the zona pellucida of follicle cell-free mouse oocytes was made more resistant to proteinase digestion by treatment with 10 or 50 μg/ml WGA. Such WGA-treated oocytes showed decreased fertilizability when washed to remove excess WGA and incubated with capacitated spermatozoa. Oocyte cleavage was used as an end point, because a large number of spermatozoa adhered to the eggs after WGA treatment, making observation of sperm penetration and pronucleus formation unreliable. Resistance to proteinase digestion increased, and the fertilizability decreased with the higher amount of WGA. The action of WGA was most likely not mediated by a direct effect on sperm motility, sperm acrosin activity, sperm binding to the zona pellucida, or oocyte cleavage. WGA did not affect the acrosome reaction of guinea pig spermatozoa. These data show that WGA treatment of follicle cell-free mouse oocytes results in decreased fertilizability, possibly by rendering the zona pellucida more resistant to sperm proteinase digestion.     

Wheat germ agglutinin blocks the biological effects of nerve growth factor

The binding of nerve growth factor (NGF) to specific cell surface receptors initiates a variety of effects that lead to the morphological and biochemical differentiation of clonal pheochromocytoma, PC12, cells. The lectin wheat germ agglutinin (WGA) alters the characteristics of NGF-receptor interaction. We have found that treatment of PC12 cells with WGA dramatically and reversibly inhibits the ability of NGF to elicit three distinct biological effects characteristic of NGF action. Two of these events, the rapid ruffling of cell-surface membranes and the stimulation of the phosphorylation of a 250-kD cytoskeletal protein in situ, occur rapidly and are an immediate consequence of receptor occupancy. Both of these effects are blocked by pretreatment of the cells with WGA. WGA was also found to inhibit the NGF-stimulated regeneration of neurites that occurs over 1- 2 d. Both the WGA inhibition of neurite outgrowth and the phosphorylation of the 250-kD cytoskeletal protein were reversed upon addition of the specific sugar N-acetylglucosamine. These data demonstrate that the WGA-induced changes in the NGF-receptor interaction reflect important alterations in the ability of the receptor to transmit biological signals, resulting in the abrogation of the biological effects of NGF on these cells.     

Identification of sensory neurons supplying receptors in lingual muscles of the rat: histochemical and retrograde labeling study with horseradish peroxidase.

Sensory innervation of lingual musculature was studied in young adult Wistar rats using retrograde labeling by horseradish peroxidase (HRP) and combined silver impregnation and acetylcholinesterase (AchE) methods. Intra-lingual injection of HRP resulted in labeling of neuronal somata in the trigeminal, superior vagal, and second cervical spinal (C2) ganglia. When HRP was directly applied to the proximal stump of severed hypoglossal nerve, labeling occurred only in the cervical and superior vagal ganglia. Morphometric analysis revealed that the labeled neurons were of the small-sized category in all ganglia. However, in the trigeminal and C2 ganglia, labeling occurred also among the medium-sized neurons. Combined silver and AchE preparations from lingual muscles revealed the absence of typical muscle spindles. Instead, there were free and spiral nerve terminals in the interstitium, and epilemmal knob-like or bouton-like endings surrounding non-encapsulated muscle fibers. These terminals showed AchE -ve reaction in contrast to the motor ones. Few ganglionic cells were scattered along the hypoglossal nerve with uniform AchE +ve reaction in their perikarya. This indicates that medium-sized neurons in the trigeminal and C2 ganglia, and probably sensory neurons along the hypoglossal nerve mediate lingual muscle sensibility perceived by atypical sensory terminals.     

Fluorescence analysis of hormone binding activities of wheat germ agglutinin

Wheat germ agglutinin (WGA) from embryos of the monocotyledonous plant Triticum vulgaris (Graminaceae) is a carbohydrate binding protein characterized by high specificity to N-acetyl-d-glucosamine and N-acetyl-d-neuraminic acid. In this study we show that parallel to its carbohydrate binding activities, WGA binds with several orders of magnitude higher affinity adenine, adenine-related cytokinins: kinetin, zeatin and isopentenyl-adenine as well as abscisic and gibberellic acids (K(d) 0.43-0.65 microM). Its interactions with these ligands cause conformational rearrangements in the protein molecules and significant enhancement of the protein tryptophan fluorescence (up to 60%) allowing characterization of the protein-hormone complexes. Dimeric WGA molecules possess two different classes of binding sites for the fluorescent hydrophobic probe 2-(p-toluidinyl) naphthalene sulfonic acid (TNS) as suggested by the sigmoid shape of the fluorescence titration curve and the value of the Hill coefficient (n(H) 1.6+/-0.3). The plant hormones displace part of the bound TNS probe and share the higher affinity TNS binding sites. These results characterize WGA as a hormone-binding protein.