Labeled Wheat Germ Agglutinin Research Articles

Differences in the connectivity of rat pudendal motor nuclei as revealed by retrograde transneuronal transport of wheat germ agglutinin.

Bilateral coordinated activation of pudendal motoneurons is an essential component of penile reflexes in male rats. However, little is known about the intraspinal organization of these reflexes. In the present study, retrograde transneuronal transport of wheat germ agglutinin (WGA) was used to examine the organization of spinal motoneurons and putative interneurons mediating penile reflexes in adult male rats. Injection of WGA into the ventral bulbospongiosus muscle resulted in direct retrograde labeling of motoneurons in the ipsilateral dorsomedial (DM) nucleus and transneuronal labeling of ipsilateral and contralateral DM motoneurons. Motoneurons in the ipsilateral and contralateral dorsolateral (DL) nuclei were not labeled. WGA-labeled putative interneurons were observed bilaterally, primarily in the ventromedial spinal gray matter extending dorsally to the central canal and the dorsal gray commissure. The number of transneuronally labeled putative interneurons increased with longer survival times. Injection of WGA into the ischiocavernosus muscle resulted in direct retrograde labeling of motoneurons in the medial subdivision of the ipsilateral DL nucleus. However, no WGA labeling was detected in motoneurons in the lateral subdivision of the ipsilateral DL nucleus, the contralateral DL nucleus, or the DM nuclei at any of the survival times studied (1-7 days). Only a small number of transneuronally labeled putative interneurons was observed in the ventrolateral gray matter at longer survival times (3-7 days). Thus, marked differences were observed between the DM and DL nuclei with respect to the transneuronal transport of WGA. These results are discussed with respect to the organization of the spinal circuits that mediate pudendal motor reflexes.

Confocal laser scanning microscopy of small-intestinal mucosa in celiac disease.

Light microscopy of jejunal biopsy specimens is routinely used to diagnose celiac disease. Confocal laser scanning microscopy offers research advantages, since thin optical sectioning can be performed without mechanical damage to the specimen. Fixed as well as non-fixed specimens can be studied. Confocal laser scanning microscopy was used to study the stanining of wheat germ agglutinin, which binds to glycoconjugates of the enterocyte, and rhodamine phalloidin, which binds to the F-actin of the cells. In healthy mucosa the wheat germ agglutinin labeling showed a strong and punctate staining of microvilli, which outlined a convoluted surface. Phalloidin-labeled actin formed a three-dimensional cage at the cell membrane. In the crypt-hyperplastic mucosa, both staining patterns were irregular, and the cytoskeleton was disorganized. Confocal laser scanning microscopy offers the possibility to study the distribution of surface and cytoskeleton markers in thick, structurally intact specimens.

Focal and regional variations in the composition of the glycocalyx of large vessel endothelium.

The glycocalyx of the endothelium of the systemic arteries and vena cava of the rabbit was visualised by in situ perfusion fixation with glutaraldehyde containing Alcian blue. The thickness of the layer ranged from 45 +/- 1 nm in the coronary artery to 81 +/- 2 nm in the carotid. The glycocalyx was 20 +/- 1.5 nm thicker on the downstream side of intercostal ostia than on the upstream side. Changes in the staining pattern with increasing concentrations of MgCl2 indicated that carboxyl groups made the major contribution to the surface charge, though sulphate groups were also present, particularly in the aortic arch and carotid artery. Segments of the thoracic aorta and carotid artery were also stained in vitro with fluorescence labelled wheat germ agglutinin, and fluorescence intensity in histological sections was quantified using a video microscope equipped with a microcomputer-based image analysis system. The fluorescence intensity in the carotid was 1.65 +/- 0.15 times that in the aorta. Pretreatment with neuraminidase reduced fluorescence intensity by 60 +/- 4% in the carotid and 53 +/- 2% on the upstream side of intercostal ostia, but only by 37 +/- 3% on the downstream side. Chondroitinase and heparanase both reduced binding and when used together their effect was additive, reducing fluorescence by 27 +/- 3, 51 +/- 4, and 32 +/- 3% at the three sites, respectively. Though the interpretation of the lectin binding experiments is complicated by a number of factors, these results support previous reports that sialyl groups are abundant in the endothelial glycocalyx. Glycosaminoglycans are also present, however, in significant amounts.

Nuclear export of recombinant baculovirus nucleocapsids through small pore or nuclear-pore-like structure in Sf9 cells.

Translocation of baculovirus nucleocapsids (45 nm in diameter, approximate length of 280-300 nm) from nucleoplasm to cytoplasm was studied morphologically using cryofixation and gold labeled wheat germ agglutinin (WGA-gold) during recombinant Autographa californica nuclear polyhedrosis virus infection in Sf9 cells. Nucleocapsids formed in the nucleoplasm migrated into protrusions of the nuclear envelope, but not into nuclear pore complexes. We found cross-like membranous structures. Small pores seemed to be in the protruding nuclear double membranes. The middle piece of a nucleocapsid was located within the small pore whereas the upper part was in the cytoplasm. Other nucleocapsids were situated within pores without colocalization of WGA-gold in the nuclear envelope. These results suggest that baculovirus nucleocapsids use small pores in the nuclear-derived membranes or incomplete nuclear pores in the nuclear envelope to migrate from the nucleoplasm to the cytoplasm, but not complete nuclear pore complexes proper.

Chitin synthesis in Spodoptera frugiperda wing imaginal discs. II. Selective action of chlorfluazuron on wheat germ agglutinin binding and cuticle ultrastructure

AbstractThe action of the chitin synthesis inhibitor, chlorfluazuron, was investigated in Spodoptera frugiperda wing imaginal discs cultured in vitro. Electron microscopy and cytochemical labeling with a lectin, wheat germ agglutinin (WGA), were used to monitor morphogenesis, as well as the presence and localization of chitin and non‐polymerized N‐acetyl‐D‐glucosamine (GlcNAc). Chlorfluazuron (CFA) selectively inhibited 20‐ hydroxyecdysone–stimulated chitin synthesis and procuticle deposition in imaginal discs, without otherwise affecting their morphogenesis. Tracheole migration, evagination, exocytosis, and endocytosis in the epithelial cells, and the presence of non‐polymerized GlcNAc in the extracellular matrix, were observed in both CFA‐treated and control wing discs. On the other hand, CFA prevented the appearance of WGA‐labeled chitin in newly formed procuticle, while the deposition of proteinaceous cuticulin and epicuticle was unaffected. A brief treatment with CFA resulted in WGA labeling of non‐polymerized GlcNAc, but not chitin in the procuticle region. The lack of chitin in CFA treated wing discs was correlated with the appearance of an amorphous, non‐lamellar procuticle region. © 1994 Wiley‐Liss, Inc.This article is a US Government work and, as such, is in the public domain in the United States of America.

Characteristics of Extracellular Adhesions Produced DuringSaprolegnia FeraxSecondary Zoospore Encystment and Cystospore Germination

Fluorescence microscopy of lectin binding and electron microscopy of surface features were used in an investigation of adhesion characteristics of germinating secondary cystospores of Saprolegniaferax. Adhesive material at two locations bound S. ferax germlings to glass coverslips or to boiled onion epidermis. As secondary cystospores germinated, they released along the germ tube extracellular ad? hesive material to which fluorescently labeled wheat germ agglutinin bound. Similarly, this lectin also bound to portions of the adhesion pad between the secondary cystospore and its substrate. Scanning electron microscopy revealed that although material of the adhesion pad extended a short distance along the germ tube, the material at the germ tube tip was not continuous with this previously released adhesive material. Changes in the structure of the adhesion pad material indicated that it had fluidity when first released, but later its fluidity became constrained, possibly by progressive polymerization. Evidence suggests that adhesives found along the germ tube were released at the tip during elongation. The site of germ tube initiation appeared dependent on more cues than mere prealignment of the cystospore when a secondary zoospore adhered to a substrate along its ventral surface. It is concluded that S. ferax is well adapted to its aquatic environment because both secondary cystospores and colonizing germ tubes attached to substrates with adhesives.

Hemocyte surface changes in the migratory grasshopper, Melanoplus sanguinipes in response to wounding and infection with Beauveria bassiana

AbstractSurface changes of hemocytes from the migratory grasshopper, Melanoplus sanguinipes (Fab.), following wounding and/or infection with Beauveria bassiana (Bals.) Vuillemin were assessed using fluorescein labelled wheat germ agglutinin (WGA) and concanavalin A (Con A). Binding was observed on outer wall of hemocytes when either Con A or WGA conjugates were tested. After infection there was an increase in the percentage of hemocytes which bound to WGA and which remained so for 24 h. However, after wounding this increase in binding subsided after only 1 h. Furthermore, it was found that the increase in WGA‐binding occurred within the first 15 min of wounding or infection. In addition, ConA binding in males only increased 3‐fold in fungus‐injected insects within 1 h of treatment and remained high for 24 h. These results indicated the presence of mannose (and/or glucose) residues (Con A specificity) and N‐acetyl‐D‐glucosamine residues (wheat germ agglutinin; WGA specificity) on outer wall surfaces of the hemocytes. The implications of these results in the immune response of M. sanguinipes to infection with B. bassiana are discussed.

In vivo effects of tunicamycin on chondrocytes of rat mandibular condyles as revealed by lectin cytochemistry.

The in vivo effects of tunicamycin on the glycosylation of proteoglycans and link protein in rat mandibular condylar chondrocytes were studied by ultrastructural lectin histochemistry. The binding of wheat-germ agglutinin was shown by using anti-lectin antibody followed by protein A-gold complex. In normal rats, wheat-germ agglutinin labeling was restricted to trans cisternae and vacuoles of the Golgi complex, whereas it was observed in neither the cis region of the Golgi complex nor in the rough endoplasmic reticulum. By 3 h after the drug administration, wheat-germ agglutinin binding sites on the disorganized Golgi vacuoles were dramatically reduced in number. At 6 h after the drug administration, the lectin binding sites on the Golgi vacuoles were restored. These results demonstrate that the in vivo use of tunicamycin in combination with histochemical analysis using lectin probes is of significant value for the study of protein glycosylation in chondrocytes of the rat mandibular condyle.

Ultrastructural localization of chitin in cell walls of Rigidoporus lignosus, the white-rot fungus of rubber tree roots

The distribution of N-acetylglucosamine residues in the cell wall of the white-rot pathogenic fungus, Rigidoporus lignosus, was studied by using gold labelled wheatgerm agglutinin bound to ovomucoid-colloidal gold. Ultrastructural investigation of R. lignosus-infected root tissues of Hevea brasiliensis showed a modification of the fungal cell wall throughout the infection process. Gold particles were found to occur on both thick- and thin-walled hyphae of R. lignosus rhizomorphs at the root surface. Walls of hyphae that had penetrated the roots were only labelled when they were out of the host cell, suggesting that modification of chitin molecules may be related to the excretion of host cell wall degrading enzymes. Variation in the distribution of gold particles was observed over hyphal walls of both colonized phellem and xylem cells. The observation that N-acetylglucosamine residues were released in the host cell cytoplasm suggests that lytic enzymes alter the fungal cell walls. Released chitin oligosaccharides may play a role in the induction of the root's defence system against fungal attack.

Plasma-membrane glycoproteins during spermiogenesis and in the spermatozoa of some Orthoptera

Orthoptera spermatids and spermatozoa from two species of Tettigoniidae and from one of Acrididae were analysed by means of the fluorescent lectins, concanavalin A or wheat-germ agglutinin, with the aim of finding α-D mannose· α-D glucose· β N-acetylglucosamine and sialic acid sugar residues in their plasmamembrane glycoproteins. Labelling with lectins shows remarkable changes occurring in the plasma membrane during spermiogenesis. In early spermatids, the whole cell surface is labelled, but in mature spermatids and spermatozoa, a noticeable fluorescence is restricted to the membrane that covers the acrosome. The end piece of the tail of Acrididae spermatids and spermatozoa fluoresces after wheat-germ agglutinin labelling. The intense labelling of acrosomal area is independent of acrosomal size and shape, as shown by the marked differences observed in the acrosomes of Tettigoniidae compared with Acrididae: in the former, the acrosome is a well-developed structure with an arrow-like shape, but in Acrididae, the acrosome resembles a small vesicle in the anterior tip of the cell. The large amount of some sugar residues in the plasma membrane covering the acrosome is discussed in relation to the features observed in other species, and also in connection with the physiology of the male gamete prior or during fertilization.

A model to explain differentiation of appressoria by germlings of Metarhizium anisopliae

The roles of the cell wall and cytoskeleton in cell differentiation of Metarhizium anisopliae were examined by treating germlings with antagonists of wall formation or with drugs which bind to microtubules and filamentous actin. Evidence for the greater plasticity of the appressorial wall compared with nongrowing regions comes from differential staining with fluorescent brighteners (Uvitex and Calcofluor) and fluorescently labeled wheat germ agglutinin; agents which detect chitin. Sorbose, which interferes with primary wall formation, was a potent inhibitor of differentiation which indicates that formation of appressoria involves addition of materials to the wall which matches the wall extension. Treatment with the anti-microfilament agent, cytochalasin A, disrupted polar growth and produced “ballooning” of the hyphal tip, indicating that actin stabilizes the tip against turgor pressure and may be involved in the transition to nonpolar growth of appressoria. Flooding differentiated germlings with 0.5% acetic acid produced bursting of appressoria and extrusion of the cytoplasm, indicating that growing appressoria are also turgescent and that internal hydrostatic pressure is a component in their growth. The effects of anti-microtubule agents are consistent with the well-known role for microtubules in the transport of vesicles. Taken together, our studies indicate that the microtubule-microfilament network is involved in the mediation of extracellular signals but not in initial reception. Inhibition of differentiation by vanadate (an inhibitor of the plasma membrane ATPase) and gadilonium (a blocker of stretch-activated ion channels) implies that changes in membrane potential may play a role in signal reception. We propose a model for the sequence of events during differentiation in which an induction signal, perhaps involving a localized change in transmembrane potential produced by deformation of the plastic hyphal tip, disrupts the apical Ca 2+ gradient required for maintenance of polar growth and the actin cytoskeleton, the consequent differential redistribution of Ca 2+ in the cell enlargement zone initiates germ tube swelling and appressorium formation by turgor pressure against an expanded area of the cell wall produced by disruption of exocytosis at the original apex and randomly dispersed intussusception of new wall material over the entire cell surface. A possible role for the Spitzenkörper (vesicle generating apparatus) is discussed.

Flow cytometric analysis of human erythrocytes: II. Possible identification of senescent RBC with fluorescently labelled wheat germ agglutinin

In the first paper of a series (Gutowski, et al., 1991) we discussed the use of flow cytometry to follow at the cellular level the aging of red blood cells (RBC) in circulation, using fluorescently labelled lectins and goat anti-human-IgG and -IgM. The Coulter Epics 541 was used for those studies. In this report we describe more extensive experiments using the Becton-Dickinson FACScan flow cytometer, and compare the results with those obtained with the Coulter Epics 541. By changing sample conditions from isotonic to hypotonic, compensation for differences of the two instruments was accomplished. We confirmed our previous observations that RBC react very strongly with fluorescein isothiocyanate labelled wheat germ agglutinin (FITC-WGA) and that there is little change in the intensity of fluorescence given by RBC of all sizes with the exception of the smallest. Reactivity with FITC-WGA is markedly decreased in the presence of competitive inhibitors of sialic acid or upon enzymatic removal of sialic acid from RBC. Removal of sialic acid is accompanied by increased reaction with peanut agglutinin (FITC-PNA). Flow cytometry was also used to monitor the enrichment of a population of smallest RBC (less than 0.05%), isolated from both counterflow centrifugation and the interface obtained from Histopaque separation. These smallest RBC showed low reactivity with FITC-WGA and higher binding of FITC-goat-anti-human-IgG, and -IgM, and therefore represent the most senescent RBC, just prior to their clearance from circulation by the reticuloendothelial system. These observations are in compliance with the hypothesis that physiological desialylation of glycophorin is responsible for clearance of senescent RBC from circulation (Aminoff, 1988).

Spinal neurons in the zebrafish labeled with Fluoro-gold and wheat-germ agglutinin

This study concerns the identification and location of interneurons in the spinal cord of the zebrafish by way of retrograde tracing from the body musculature. To distinguish motoneurons from interneurons, two tracers were applied: the fluorochrome Fluoro-Gold to label motoneurons, and the trans-synaptically transported compound wheat-germ agglutinin to label motoneurons and the associated interneurons that are considered to be premotoneurons. Because the tracer accumulated mainly in cell bodies (not in neurites), premotoneurons labeled directly from motoneurons could not be distinguished from those labeled via interjacent cells. Both tracers were unilaterally injected into the myotomal muscle at the anal level of the animal. The number of labeled cell bodies in the spinal cord was examined 6, 10 and 16 days after injection. Several technique-oriented experiments were performed in order to map all pathways along which the tracers were incorporated in the neurons. The following observations were made. (1) All Fluoro-Gold-positive cells contained wheat-germ agglutinin, but yet more cells contained only wheat-germ agglutinin; the number of wheat-germ agglutinin-labeled cells was about tenfold higher than the number of Fluoro-Gold-labeled cells. (2) Fluoro-Gold labeling was restricted to cells within one to two spinal cord segments corresponding to the injection site, whereas wheat-germ agglutinin labeling was more diffuse. (3) The position and size of Fluoro-Gold-labeled cells corresponded to those of motoneurons described in previous horse-radish peroxidase experiments. (4) Statistical analysis of the group of wheat-germ agglutinin-labeled cells showed two subpopulations, one with a mean cell size and position corresponding to motoneurons and one with a smaller mean cell size, also positioned within the motor column. The smaller cells were considered to be premotoneurons. The ratio motoneuron: premotoneuron was lowest in the ventrolateral area of the motor column.

Ultrastructural localization of lectin binding sites in human melanocytes

The ultrastructural localization of carbohydrate residues in human melanocytes of normal epidermis and of one compound naevus was studied. The following lectins were used in a post-embedding technique: 1. peanut agglutinin (PNA), which reacts specifically with N-acetylgalactosamine; 2. Concanavalia ensiformis (Con A) indicating alpha-D-glucose and alpha-D-mannose binding sites; 3. Ulex europaeus agglutinin (UEA I) specific for alpha-L-fucose; 4. Wheat germ agglutinin (WGA), reacting specifically with N-acetyl-glucosamine and neuraminic acid (sialic acid); and 5. Limax flavus agglutinin (LFA), also specific for sialic acid (Neu5Ac-alpha-2,3-Gal and Neu5Ac-alpha-2,6-Gal). When incubated with WGA, Con A and LFA strong labelling was seen within the cytoplasm and in the plasma membrane of melanocytes, whereas incubations with PNA and UEA I revealed an occasional gold particle only. The determination of the distribution of carbohydrate residues in normal melanocytes is a prerequisite for future studies of abnormal melanocytes.

Comparison of photoreceptor-specific matrix domains in the cat and monkey retinas

Two lectins, wheat germ agglutinin (WGA) and peanut agglutinin (PNA), were used to compare domains within the interphotoreceptor matrices (IPM) of the cat and monkey, two species where the morphological relationship between the retinal pigment epithelium (RPE) and photoreceptors is distinctly different. In the monkey, PNA labeling was heaviest over the cone outer segments and a discrete region of the interphotoreceptor matrix bordering the cone inner and outer segments—a region which has been termed the cone matrix sheath. Near the apical border of the RPE, the outer margin of the PNA-labeled matrix is surrounded by a circular array of apical microvilli. In the cat retina, PNA labeling was highest over a region of the IPM lying between the outer margin of the cone sheath processes and surrounding rod matrix. In contrast, intracellular labeling of cone inner and outer segments was sparse. The RPE apical processes forming the cone sheath were not labeled. In the monkey retina, WGA preferentially labeled rod outer segments and the region of the IPM around rod inner and outer segments. The cone matrix sheath was not preferentially labeled using this lectin. Rod inner segments and cone inner and outer segments were labeled moderately. In the cat retina, WGA labeling was dense over both rod outer segments and cone outer segments as well as the cone sheath. Rod and cone inner segments, as well as the IPM around both rods and cones, were moderately labeled. These observations suggest that the specialized processes arising from the apical surface of retinal pigment epithelial cells, together with photoreceptor-specific extracellular matrix domains, contribute to the formation of specific micro-environments around rod and cone photoreceptor cells.

Lectin bindings and diethylstilbestrol effects on the recognition of mullerian inhibiting substance (MIS) on chick mullerian ducts by MIS-antiserum

A high intensity of lectin bindings was demonstrated on the epithelial cells and serosa cells of the regressing right Mullerian ducts (Mds) in the female chick embryos. The strong lectin bindings occurs on, or in the regressing Md cells along with marked surface MIS bindings at the age of day 13. However, at the age of days 5-7 1/2, bindings of lectins were weak. Neither Wheat-germ agglutinin (WGA) or Concanavalin A (Con-A) labelings before MIS-antiserum (MIS-Ab) incubation can block antibody recognitions to the antigens, including MIS and growth hormone at the age of day 13. Our previous studies indicated that after WGA labeling on the surfaces of Md epithelial cells prior to the incubation of MIS-Ab at day 10 did not prevent the recognition of MIS-Ab (Wang 1989). On the contrary, at day 7 1/2, the specific binding of MIS was eliminated after preincubations with lectins and prenatal diethylstilbestrol (DES) treatment at the age of day 5. It is suggested that DES provides a protection to the Mds from MIS-induced regression by preventing the MIS binding to its specific membrane receptors. An increase of extra- and intracellular glycoproteins or carbohydrates of regressing Md epithelial cells were suggested. Internalization of WGA but not MIS molecules was found in Md epithelial cells. The Golgi saccules were negative of lectin bindings.

Gold labeling with wheat germ agglutinin and RNAse on osmicated tissue embedded in epoxy resin.

Tissue of an insect, Lucilia cuprina, fixed conventionally in buffered glutaraldehyde and osmium and embedded in epoxy resin (epon or epon/araldite), provided sections which could readily be labeled with RNAse/gold and wheat germ agglutinin (WGA)/gold. This method offers labeling of tissues with improved contrast and allows the retrospective application of RNAse and WGA labeling to conventionally prepared tissues, without recourse to oxidizing/etching agents.

Wheat germ agglutinin uptake by cytotoxic lymphocytes, a quantitative model of receptor-mediated endocytosis

The binding and uptake of fluorescence labeled wheat germ agglutinin into cytotoxic T-cells was measured by single cell cytophotometric analysis. The intensity of fluorescence in these cells increased continuously over 24 hrs, indicating a permanent turnover of the ligands for WGA. Although the labeling of the cells was intense, no change in the proliferation rate of this interleukin-2 dependent cell line was observed. Therefore no interaction between the interleukin-2 receptor and other receptors regulating the cellular proliferation with the lectin is likely.

Alterations of wheat-germ agglutinin binding pattern on cell surface of blood platelets after thrombin stimulation

An attempt was made to demonstrate wheat-germ agglutinin (WGA) binding sites on platelet surfaces after thrombin stimulation, by means of a post-embedding cytochemical technique using colloidal gold as marker at an ultrastructural level. In unstimulated platelets washed with EDTA, an intense uniform labeling of WGA-gold complexes was found on the surface membrane. When washed platelets were stimulated by thrombin in the absence of Ca2+, only a release reaction was induced. WGA labeling on the surface membranes of these platelets decreased dramatically. However, the labeling intensity of WGA-gold complexes on the surface membrane of aggregated platelets induced by thrombin in the presence of Ca2+ increased significantly compared to that of thrombin-stimulated platelets in the absence of Ca2+. In contrast to the uniform labeling on the surface membranes of unstimulated platelets, clusters of gold label were often found on the surface membrane of the aggregated platelets, although there was no significant quantitative difference in the labeling intensity between these two groups. Thus, we present direct morphological evidence demonstrating qualitative and quantitative alterations of WGA labeling on the surface membrane of platelets after thrombin stimulation. The possibility is considered that WGA-binding glycoproteins in the surface membrane are involved in the aggregation response after thrombin stimulation.

Selective staining of fungal hyphae in parasitic and symbiotic plant-fungus associations

A cytochemical method for light microscopical studies is described which allows the specific detection of fungal hyphae in plant-fungus associations: e.g. lichens, mycorrhiza, or fungal infections of plant tissue. The specimens were fixed and embedded in epoxy resin by a standard protocol for electron microscopy. Semithin sections were successively incubated with fluorescein isothiocyanate labelled wheat germ agglutinin (FITC-WGA) and calcofluor white (CW). FITC-WGA stained exclusively the fungal cell walls while CW stained both the fungal and the plant cell walls. Therefore, FITC-WGA is an excellent marker for the fungal hyphae.