Munefumi Sameshima

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.

Capsaicin-induced neuroparalytic keratitis-like corneal changes in the mouse

A single subcutaneous injection of 12.5-100 mg kg-1 capsaicin to newborn mice produced gross corneal changes. The changes were manifest about three weeks after capsaicin treatment and progressed dose-dependently from slight punctate vesiculations in the epithelium to diffuse edematous opacities and vascularizations in the stroma, followed by recovery in several weeks with or without residual scars. Control newborn mice with vehicle solution did not show any corneal abnormality. The most prominent histopathological feature of the affected corneas was a marked loss of nerve axons in the epithelium with associated disorganization of the epithelium. Similar corneal changes were observed with systemic capsaicin treatment to young or adult mice. The pathogenesis of the capsaicin-induced corneal changes was discussed with reference to the trophic action of the trigeminal nerve.

Synthesis and transport of various RNA species in developing embryos of Xenopus laevis

Abstract Embryonic cells of Xenopus laevis were labeled for varying lengths of time, and their nuclear and cytoplasmic RNAs were analyzed, with the following results. (1) The synthesis of small nuclear RNAs (snRNAs) is detected from blastula stage on. (2) The initiation of 4 S and 5 S RNA syntheses occurs at blastula stage. However, while the former is transported into the cytoplasm immediately after its synthesis, the latter remains within the nucleus, until its transport starts later, concomitantly with that of 28 S rRNA. (3) As soon as “blastula” cells start to synthesize 40 S rRNA precursor at 5th hr of cultivation, 18 S rRNA is transported first; the transport of 28 S rRNA begins 2 hr later. (4) On a per-cell basis, poly(A)-RNA is synthesized in blastula stage at a much higher rate than in the later stages. About one-third of the total blastula poly(A)-RNA, and about one-fifth in the case of tailbud cells, is transported quickly into the cytoplasm. Then, it appears that the RNAs which are synthesized at early embryonic stages are transported to the cytoplasm without delays, except for 5 S RNA and snRNAs.

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.

Formation of nucleus-like structure in the cytoplasm of λ-DNA-injected fertilized eggs and its partition into blastomeres during early embryogenesis in Xenopus laevis☆

The fate of bacteriophage lambda-DNA was examined after injection into the fertilized eggs of Xenopus laevis. Injection of a large amount of lambda-DNA (ca. 24 ng) into a fertilized Xenopus egg induced the formation around the injected DNA of a giant nucleus-like structure which was surrounded by an apparently normal bilayered nuclear membrane with nuclear pore complexes. Southern blot analysis revealed the persistence of injected lambda-DNA until the blastula stage. The nucleus-like structure was partitioned into blastomeres during cleavage through a process of nuclear fission, and was maintained in a group of extraordinarily large blastomeres until the blastula stage.

Plasticity of polypoidal lesions in polypoidal choroidal vasculopathy.

PurposeThe aim of this study was to describe the clinical course in a patient with polypoidal choroidal vasculopathy (PCV).MethodsA 68-year-old man with PCV in the left eye was followed up by means of routine examinations including fluorescein angiography and indocyanine green angiography for over 60 months.ResultsThroughout the follow-up period, the patient experienced repeated lesions in the macula, such as serosanguineous detachment of the retinal pigment epithelium and neurosensory retina, but retained good visual acuity. Indocyanine green angiography disclosed spontaneous regression of polypoidal vessels followed by significant changes in the choroidal circulation: a group of polypoidal structures disappeared, and after several months a small choroidal vessel became apparent that was distant from the previously observed polypoidal structure rather than representing an extension of the original lesion.ConclusionThe clinical observation suggests that in some cases of PCV the choroidal vasculature may be altered with time, in that some vessels in the inner choroid and even the choriocapillaris may close and collateral vessels and/or new vessels may develop to form complex such as that described here.

Effects of the injection of exogenous DNAs on gene expression in early embryos and coenocytic egg cells ofXenopus laevis

SummaryInXenopus laevis embryogenesis, the synthesis of heterogeneous mRNA-like RNA starts at the cleavage stage, whereas that of low-molecular-mass RNAs and rRNA occurs at the early blastula and late blastula stages, respectively. In coenocytic fertilizedXenopus egg cells, which fail to cleave, an excess of exogenously injected DNA (pBR322) induces ‘premature’ expression of previously injected exogenous genes (yeast tRNA genes). We have carried out experiments to discover whether the injection of excess exogenous DNAs of various origins modifies the expression of endogenous genes and previously injected exogenous genes inXenopus embryogenesis. We found that injection of a relatively large amount of exogenous DNA (Xenopus rDNA clone) induces the premature expression, or enhanced expression, of previously injected bacterial chloramphenicol acetyltransferase genes in coenocytic cells. In embryos, however, the injection of exogenous DNAs of various origins did not appreciably modify the expression of either endogenous or previously injected CAT genes. The DNAs injected into fertilized eggs were not degraded and were partitioned into the nuclei of most (at least 80%) of the descendant blastomeres at least during early stages of development. Therefore, we concluded that the program of gene expression in normally developing embryos cannot easily be altered by the introduction of excess exogenous DNAs.

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.

Blood Staining of the Cornea in Hansen's Disease

Blood staining of the cornea was studied by light and electron microscopy: a 55-year-old male with Hansens disease had blood staining of the cornea due to intracorneal hemorrhage; he received a partial-thickness keratoplasty following 1 year after the onset of the staining. The excised specimens revealed deposits of degraded erythrocytes in the stroma. Numerous dense granules, probably of erythrocytic breakdown products, were phagocytosed by macrophages as well as parenchymal cells. The presence of macrophages was limited to the middle part of the stroma in which newly formed vessels were remarkable.