Fumiyuki Uehara

Localization of fluorescence-labeled lectin binding sites on photoreceptor cells of the monkey retina

The binding of eight fluorescence-labeled lectins to the photoreceptors of the monkey retina was investigated using a post-embedding staining method. Concanavalin A (specific for mannosyl and glucosyl residues) bound to the outer and inner segments of both rods and cones, while the degree of staining was more intense in the rods. The rod outer segments showed patchy fluorescence and the proximal portions of the inner segments were diffusely stained. Wheat germ agglutinin (specific for sialyl and N-acetylglucosaminyl residues) bound preferentially to the surface of the outer and inner segments of both rods and cones. Ricinum communis agglutinin-1 (specific for galactosyl residues) stained the rod outer segments in patches, particularly strongly in the region dividing the outer and inner segments. The cones were also stained, although faintly, in the same pattern as the rods. The distal halves of the rods and cones showed diffuse weak staining and their proximal halves stained spotty. Peanut agglutinin (specific for Gal beta 1 leads to 3GalNAc sequence) bound preferentially to the cones and only scarcely to the rods. The external surface of both outer and inner segments of cones were uniformly stained; the interior of the cone outer segments was also stained, while the interior of the inner segments was not. Two lectins specific for fucosyl residues, namely, Ulexeuropaeus agglutinin-1 and Lotustetragonolobus agglutinin, bound diffusely to the distal halves of the inner segments of both rods and cones. Lectins reacting with N-acetyl-galactosamine residue, i.e. Dolichos biflorus agglutinin and soybean agglutinin, bound weakly to the distal portions of rods and cones.

Specialization of the interphotoreceptor matrices around cone and rod photoreceptor cells in the monkey retina, as revealed by lectin cytochemistry

The binding sites of two lectins, peanut agglutinin (PNA) and wheat germ agglutinin (WGA), in the interphotoreceptor matrix (IPM) and photoreceptor plasma membranes of the Japanese monkey (Macaca fuscata) retina were localized using a pre-embedding staining method with ferritin-conjugated (Fer) lectins as well as a postembedding staining method with fluorescence-labeled (FITC) lectins. FITC-PNA, but not WGA, stained cylindrical domains of the IPM around cone outer and inner segments, while the IPM around rods stained with FITC-WGA but not PNA. When the intact (not detached) retinal tissues were incubated with Fer-lectin, the lectin generally labeled neither the IPM nor photoreceptor plasma membranes, but labeled only those structures in detached portions occurring at the edges of occasional retinal tissue blocks. Thus, the neural retinas physically isolated from the retinal pigment epithelium (RPE) were utilized principally here. Ultrastructurally, the IPM in the intact retina consisted of granular and filamentous materials; the IPM in the isolated neutral retina also retained those components, although somewhat loosely organized, and the IPM around cones appeared to be preserved better than did the IPM around rods. Fer-PNA bound to the IPM associated with cones, but not rods; Fer-WGA bound to the rod- but not cone-associated IPM. The ferritin particles were found to lie close to the granular and filamentous materials. Those photoreceptor-associated IPMs extended to the apical surface of the RPE in detached portions or to the apical villi of the RPE which were frequently found in the isolated neural retinas. Also, Fer-PNA labeled the cone, but not rod, plasma membranes; Fer-WGA bound heavily to the plasma membranes of rod and cone outer segments, but sparsely to those of their inner segments. These results suggest that the IPM comprises chemically and physically differential domains specialized for cone and rod photoreceptor cells, and that these specialized IPM are structurally so stable that may be involved in isolating photoreceptor cells physicochemically from each other and in the interactions between the photoreceptors and the RPE, such as retinal adhesion.

Application of lectins for detection of goblet cell carbohydrates of the human conjunctiva

Paraffin-embedded and frozen biopsies from the human conjunctival epithelium were examined by fluorescence microscopy after labeling with eight fluorescein-conjugated lectins: wheat germ agglutinin; soybean agglutinin; peanut agglutinin; Ricinus communis agglutinin-1; Limulus polyphemus agglutinin; Ulex europaeus agglutinin-1; Dolichos biflorus agglutinin; concanavalin A. The lectins were used as specific molecular probes to detect carbohydrate composition of glycoproteins secreted from the conjunctival goblet cells. The labeling pattern of goblet cells and conjunctival epithelial surfaces with various lectins suggested that N-acetyl-glucosamine, galactose, N-acetyl-galactosamine and sialic acid are contained in goblet cells and contribute to the formation of tear mucus glycoprotein. Fucose and mannose, which are present in the tear mucus, were not detectable in goblet cells.

Lectin-cytochemical study on epithelial mucus glycoprotein of conjunctiva and pterygium

The epithelium of pterygium and conjunctiva was studied with reference to cytochemical reactivity to six fluorescein-labeled lectins that recognize a certain carbohydrate residue(s) of cellular membrane-bound or secretory glycoprotein: Ulex europaeus agglutinin-1 (UEA-1, specific for fucose); Dolichos biflorus agglutinin (DBA, specific for N-acetylgalactosamine); peanut agglutinin (PNA, specific for galactose-beta 1-3N-acetylgalactosamine): wheat germ agglutinin (WGA, specific for N-acetylglucosamine and N-acetylneuraminic acid); Concanavalia ensiformis (Con A, specific for mannose); Ricinus communis agglutinin-1 (RCA-1, specific for galactose). Non-goblet epithelial cells of pterygium were labeled with UEA-1, DBA and PNA, while those of conjunctiva were not. Distribution density of goblet cells was larger in pterygium than in conjunctiva, but there was no distinct difference in lectin reactivity between the two tissues, with marked label with WGA, PNA and RCA-1. Con A did not bind to either pterygium or conjunctiva. The observations suggest the presence of anomalous mucus glycoproteins secreted from pterygium.

Diagnostic imaging in patients with orbital cellulitis and inflammatory pseudotumor.

We have had the opportunity, at an eye clinic, to examine and treat a patient with orbital inflammatory disease of which the main symptom was severe eyelid swelling. When a patient has general symptoms of an infection, and a pathogenic organism is isolated and detected in an orbital lesion or blood culture, she or he is aggressively treated for orbital cellulitis with antibiotics and surgical drainage. Staphylococcus and Streptococcus species are the most frequent pathogens in adulthood. With respect to the microbiological spectrum in childhood, it was recently reported that Streptococcus species are the predominant cause of orbital cellulitis now that Haemophilus influenzae bacteremia has decreased in the United States, owing to the advent of an H. influenzae bacteremia vaccine. However, we sometimes encounter a patient who has eyelid swelling without general symptoms or bacteremia. In such a case, it is not easy to differentiate orbital cellulitis from inflammatory pseudotumor, which comprises nongranulomatous idiopathic inflammation within the orbit. The treatment strategies are different for these inflammatory disorders: Orbital cellulitis is basically treated with antibiotics, whereas steroid therapy is effective for inflammatory pseudotumor. Therefore, a differential diagnosis is important for the treatment of these orbital disorders. Recently, magnetic resonance imaging (MRI) has been widely used for the diagnosis of orbital inflammatory disorders. The findings on MRI or computed tomography (CT) have usually been included in recent case reports of orbital cellulitis and inflammatory pseudotumor. However, little literature has described how to distinguish orbital cellulitis from inflammatory pseudotumor on the basis of MRI findings. In the present study, therefore, we retrospectively examined our cases at Kagoshima University Hospital to characterize the MRI features of orbital inflammatory diseases for the differential diagnosis between orbital cellu-

Two-dimensional gel electrophoretic analysis of lectin receptors in the bovine interphotoreceptor matrix.

Proteins and glycoproteins of the bovine interphotoreceptor matrix (IPM) with or without neuraminidase treatment was analysed by two-dimensional gel electrophoresis combined with Western blotting and staining with seven horseradish peroxidase-labeled lectins. More than 80 spots of proteins and glycoproteins were revealed on the gel. Nineteen spots (or groups of spots) were revealed by staining with five lectins [concanavalin A, wheat germ agglutinin (WGA), peanut agglutinin (PNA), Ricinus communis agglutinin-1 (RCA-1) and soybean agglutinin (SBA)]; some of those spots were specific for one lectin and others reacted with several lectins. We could not detect distinct spots reacting with Dolichos biflorus agglutinin or Ulex europaeus agglutinin-1. Neuraminidase digestions of the IPM increased and unmasked the binding spots for PNA, RCA-1 and SBA. The spots of WGA-receptors without neuraminidase treatment were mostly identical to the receptors for PNA, RCA-1 and SBA, which became prominent after the digestion. Spots reacting with RCA-1 were mostly identical to the spots of SBA-receptors. The spots reacting with PNA coincided only partially with the spots reacting with RCA-1 and SBA.

Ultracytochemical demonstration of glycogen in cone, but not in rod, photoreceptor cells in the rat retina

The presence of native glycogen in photoreceptor cells of the rat retina has not been identified in the literature. We have studied this ultracytochemically. After perfusion with glutaraldehyde fixative, the eyes were enucleated, and the retinal tissues, postfixed with OsO4, were embedded in epoxy resin. Some tissues were treated with saliva before postfixation. Ultrathin sections, stained by the periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) method or with uranyl acetate and lead citrate, were examined by electron microscopy. On routinely stained sections, glycogen particles seemed to be absent in the cytoplasmic matrix of the photoreceptor cells because they were indistinguishable from the numerous ribosomes. This was due to a similarity in size and electron density. After PA-TCH-SP staining, fine electron-dense reaction products appeared on small cytoplasmic particles (but not on ribosomes) in the inner segments, perikarya and synaptic terminals of a subpopulation of photoreceptor cells. These particles, 15-25 nm in diameter, were identified as beta-particles of glycogen because of their susceptibility to enzyme digestion. The glycogen-rich photoreceptor cells were thought to be cone cells by reasons of their morphological features, such as synaptic terminals, nuclei and outer segments. These results suggest that the cone, but not the rod, photoreceptor cells in the rat contain abundant glycogen.

Effect of enucleation on the expression of c-Fos protein in the supraoptic nucleus of the Japanese monkey (Macaca fuscata)

The aim of this study was to examine the effects of one eye enucleation on the expression of c-Fos protein in the hypothalamus of the Japanese monkey (Macaca fuscata). Compared with an intact monkey, significantly increased numbers of c-Fos positive neurons were observed in the supraoptic nuclei on both sides at 1 h after eye enucleation. This maximal c-Fos expression then started to decrease at 3 h after eye enucleation. Furthermore, by a dual-labeled immunocytochemical study, the c-Fos immunoreactivity was found mainly in the vasopressinergic but not in the oxytocinergic neurons within the supraoptic nucleus. These results suggest that vasopressinergic but not oxytocinergic neurons within the supraoptic nucleus may have critical roles in the stimulation of this nucleus in response to eye enucleation.

Reduced sialylation of glycoproteins in nasal glands of patients with chronic sinusitis.

This study was conducted to investigate the sialylations of glycoproteins in the nasal glands of patients with chronic sinusitis. Sialic acids were detected using lectin histochemistry, and the mRNA of sialyltransferase was evaluated by in situ hybridization histochemistry. Sambucus nigra agglutinin (SNA), which recognizes terminal sialic acids, strongly stained the glandular mucous cells of normal subjects, but not those of patients with chronic sinusitis. In situ hybridization histochemistry showed that the expression of alpha2,6 sialyltransferase mRNA was decreased in the secretory cells of patients with chronic sinusitis. Our present results suggest that a reduction in sialyltransferase activity at the mRNA level in the nasal glands may lead to the persistence of chronic sinusitis.

External Dacryocystorhinostomy Combined with Mucosal Grafting and Magnetic Resonance Imaging

PURPOSE To facilitate understanding of the use of magnetic resonance imaging (MRI) in nasolacrimal obstructive diseases and to determine the indication for external dacryocystorhinostomy (DCR) combined with mucosal grafting. METHODS We retrospectively studied the correlation between MRI images and surgical findings in 13 consecutive patients with swollen lacrimal sacs because of obstruction of the nasolacrimal duct or lacrimal sac. They were treated at the Kagoshima University Hospital between June 1999 and May 2001. RESULTS A simple procedure of external DCR was performed in 9 cases, and a procedure combined with mucosal grafting was performed in 4 cases (age range, 51-88 years). Surgical findings in the fibrous region of the lacrimal sac corresponded to the hyperintense signal on MRI T(1)-weighted (T1W) images, more remarkably after enhancement. Surgical findings in the granulomatous sac and proteinaceous contents corresponded to the isointense areas on T2W images. Cases with thin sac were treated by standard DCR, whereas cases with thick, fibrous and granulomatous sac were treated by external DCR combined with mucosal grafting. CONCLUSIONS The MRI images provide a useful preoperative determination for indications of external DCR combined with mucosal grafting.