Dual pathways for ribonucleic acid turnover in WI-38 but not in I-cell human diploid fibroblasts.

The turnover rates of 3H-labeled 18S ribosomal ribonucleic acid (RNA), 28S ribosomal RNA, transfer RNA, and total cytoplasmic RNA were very similar in growing WI-38 diploid fibroblasts. The rate of turnover was at least twofold greater when cell growth stopped due to cell confluence, 3H irradiation, or treatment with 20 mM NaN3 or 2 mM NaF. In contrast, the rate of total 3H-protein turnover was the same in growing and nongrowing cells. Both RNA and protein turnovers were accelerated at least twofold in WI-38 cells deprived of serum, and this increase in turnover was inhibited by NH4Cl. These results are consistent with two pathways for RNA turnover, one of them being nonlysosomal and the other being lysosome mediated (NH4Cl sensitive), as has been suggested for protein turnover. Also consistent with the notion of two pathways for RNA turnover were findings with I-cells, which are deficient for many lysosomal enzymes, and in which all RNA turnover was nonlysosomal (NH4Cl resistant).

Properties and synthesis of multiple components in native small ribosomal subunits of mouse ascites tumor cells.

Native small ribosomal subunits in mouse ascites tumor cells prepared by a sucrose zone sedimentation and analyzed by CsCl isopycnic centrifugation, consisted of four kinds of particles which could be distinguished reproducibly by their ultraviolet absorption. These particles had buoyant densities at 1.40, 1.42, 1.46 and 1.49 g/cm3 and were designated as S1, S2, S3 and S4 particles respectively. Studies on labeling kinetics with radioactive RNA precursors and the pulse label--actinomycin D chase experiment, demonstrated that radioactivity was incorporated first in precursor particles with a density of 1.45 g/cm3 and then appeared in S3, S1 and/or S2 and S4 particles. Results on labeling after administration of actinomycin D and EDTA treatment of the labeled small subunits revealed that S1 and S2 particles contained a messenger-like ribonucleoprotein particle and this particle plus tRNA, respectively. Since appearance of newly formed S4 particle was remarkably delayed and the amount of it was reduced after incubation of the cells in vitro with 10 mM sodium fluoride, this particle originated from dissociation of the small subunit.

Monomer arrangement in HSP90 dimer as determined by decoration with N and C-terminal region specific antibodies.

Electron microscopy using the low-angle rotary shadowing replica method showed that the HSP90 dimer consists of four globular domains aligning in a tandem fashion. When decorated with two monoclonal antibodies against epitopes mapped on the N-terminal region of HSP90, these antibodies bound to both ends of the HSP90 dimer. A C-terminal region specific antibody was shown to bind to the side of HSP90. These results support a model for HSP90 dimer whereby two HSP90 monomers are arranged in an antiparallel fashion and dimerize through the C-terminal domain. Treatment of HSP90 at elevated temperatures or with ATP at room temperature, though not with ADP, induces molecular transformation of the linear HSP90 dimer into an O-ring-shaped structure.

S-Adenosyl-L-homocysteine hydrolase is sequestered into actin rods in Dictyostelium discoideum spores.

Here we show evidence that S-adenosyl-L-homocysteine hydrolase (SAHH) is linked to the actin cytoskeleton. Actin rods formed in Dictyostelium discoideum spores during the final stage of development are structurally composed of novel bundles of actin filaments. SAHH only accumulates with actin at this stage of development in the life cycle of D. discoideum. Recently SAHH is believed to be a target for antiviral chemotherapy and the suppression of T cells. Our finding may contribute to designing novel antiviral and immunosuppressive drugs.

Ultrastructural changes associated with accumulation of inclusion bodies in rat retinal pigment epithelium.

PURPOSE: To determine the structural changes in the retinal pigment epithelium (RPE) and neighboring structures induced by intravitreal injection of a lysosomal protease inhibitor. METHODS: Eleven-week-old Sprague-Dawley rats were injected with 5 microliter of a lysosomal protease inhibitor, E-64 (2.22 microM), intravitreally once and killed at 24 hours, 48 hours, or 7 days later. Others received two or three injections at 48-hour intervals or three daily injections, and killed at 1, 4, and 7 days after the last injection. Eyes were enucleated and retinal tissues were processed for light and electron microscopy. RESULTS: A single injection of E-64 caused only a transient accumulation of phagosome-like and phagolysosome-like inclusion bodies in the RPE. By contrast, repeated injection caused progressive accumulation of these inclusions followed by altered RPE cell conformation, and changes in organelles such as loss of smooth endoplasmic reticulum (SER). This was accompanied by shortening and loss of photoreceptor outer segments without prior dysmorphic changes, alteration of choroidal capillaries, and invasion of Bruch's membrane by fibroblasts and pericytes. Intravitreal injection of vehicle as control induced no structural changes. CONCLUSIONS: E-64 treatment induced structural changes in the outer retina. The causal relationship between accumulation of inclusions in RPE and changes in other subcellular organelles and neighboring cells systems is not clear. However, there are possible explanations: physical disturbance of organelles, particularly SER by inclusions; cellular damage by consequent upon accumulation of A2-E; or, shortage of recycled material due to reduced degradation of phagosomes.

Clinicopathological correlation of polypoidal choroidal vasculopathy revealed by ultrastructural study.

AIMS: To describe the clinical and histopathological findings in a patient with polypoidal choroidal vasculopathy. METHODS: A 76 year old Japanese man had a discrete, orange-red lesion of 1 disc diameter in the macula, with the fluorescein and indocyanine green angiographic and optical coherence tomographic findings compatible with polypoidal choroidal vasculopathy. He underwent a surgical removal of the macular lesion, followed by light and electron microscopic examinations. RESULTS: The histopathological examination revealed that the specimen consisted of degenerated retinal pigment epithelium-Bruch's membrane-choriocapillaris complex and inner choroid. A tortuous, unusually dilated venule was present adjacent to an arteriole with marked sclerotic changes, appearing to form arteriovenous crossing. These vessels seemed to represent native inner choroidal vessels, and had haemorrhage per diapedesis. Blood cells and fibrin filled the lumina of the vessels and accumulated in the extravascular spaces, indicating vascular stasis. CONCLUSION: Hyperpermeability and haemorrhage due to stasis of a dilated venule and an arteriole involved by sclerosis at the site where they cross in the inner choroid might cause oedema and degeneration of the tissue. Voluminous accumulation of blood cells and fibrin might generate elevation of tissue pressure sufficient to displace the weakened lesion anteriorly. The result suggests that the polypoidal vessels in this case represent abnormality in the inner choroidal vasculature.

Leber's hereditary optic neuropathy: mitochondrial and biochemical studies on muscle biopsies.

Two patients with Leber's hereditary optic neuropathy underwent morphological and biochemical investigations of muscle biopsy samples from the biceps brachii. Although clinically there was no muscle weakness or atrophy, specific histochemical and electron microscopic examinations showed mild but distinct myopathic changes, including abnormal oxidative enzyme activities, aggregates of enlarged mitochondria in the subsarcolemmal regions, and disruptions of myofilaments. Biochemical analyses of mitochondria isolated from muscle samples did not show and deficiency in respiratory-chain enzyme complexes or defect in content of cytochromes. Leber's hereditary optic neuropathy is assumed to be a multisystem disorder involving skeletal muscle also.

Preoperative localization of brain lesions by magnetic resonance imaging with a marking device. Technical note.

A new technique for preoperative localization of brain lesions using magnetic resonance imaging and a marking device is described. The projection of lesions, especially superficial brain tumors, is demonstrated on the scalp, with an acceptable tolerance for open procedures. This simple and inexpensive method, which was use in a group of 34 patients, can assist surgeons in planning an operative approach to intracranial lesions.

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.

Orbital rhabdomyosarcoma: case report with immunohistochemical detection of muscle cell type intermediate filament proteins.

A 4 1/2-year-old girl had a rapidly growing tumor in the orbit. An extirpated mass consisted of small round or spindle neoplastic cells containing hyperchromatic mitotic nuclei. The cytoplasm had many lipids and glycogens with few organelles. A light and electron microscopic examination revealed an exceptional tumor cell with distinct cross striations compatible with typical rhabdomyoblast; occasional cells exhibited a few bundles of thin filaments scattered in the cytoplasm. Immunohistochemical studies for muscle cell-related proteins by avidin-biotin-peroxidase complex methods revealed positive reactions of the tumor cells to the antiserum against myoglobin, myosin, desmin and vimentin, providing evidence for muscle cell derivation of the neoplastic cells.

Complications of hypermature cataract: spontaneous absorption of lens material and phacolytic glaucoma-associated retinal perivasculitis.

Uncommon complications of hypermature cataract in two aged patients are described. One patients, a 58-year-old woman, with unilateral hypermature cataract secondary to Fuchs' heterochromic cyclitis developed spontaneous absorption of the lens material, followed by its dislocation into the anterior chamber. The other patient, a 63-year-old woman, showed phacolytic glaucoma associated with extensive retinal perivasculitis which concurred with leakage of hypermature cataractous lens material and subsided with its complete absorption. Histopathology of the membraneous cataract revealed degenerative changes of the capsule and extinction of the epithelial cells and cortical fibers.

Binding sites of peanut agglutinin in mammalian retina.

Histochemical studies were carried out of the binding sites of a lectin, peanut agglutinin (PNA), in the mammalian retina, using lectin labeled with either fluorescein or horseradish peroxidase. In all mammalian species examined, monkey, pig, cat and rabbit, the cones were intensely labeled with the lectin but the rods were unlabeled. The cone synaptic pedicles, the inner synaptic layer, the internal limiting membrane and the retinal vessels were also labeled. It is suggested from the specificity of the PNA binding that galactosyl and/or galactosaminyl residues are present in the mammalian cones, but not in the rods.

The ultrastructural study of ribosomes in photoreceptor inner segments of the pcd cerebellar mutant mouse.

Ultrastructural changes in the distribution of free ribosomes in photoreceptor inner segments in relation to photoreceptor degeneration in Purkinje cell degeneration (pcd) were studied in mutant mice reared under cyclic light and sacrificed at 30 and 120 postnatal days. On postnatal day 30, some photoreceptors appeared to be normal whereas others had degenerated to varying degrees with numerous spherules in the extracellular space surrounding degenerating inner segments. By postnatal day 120, retinal degeneration had progressed with a marked loss of photoreceptors. The distribution of free ribosomes in the mutant inner segments was random, clustered, or uniform. This was distinct from normal control retinas in which random distribution predominated. Markedly degenerated inner segments contained sparse ribosomes which coalesced in small aggregates. These changes in ribosome distribution seemed to be associated with the extent of photoreceptor degeneration. The significance of these changes in ribosome distribution, in particular clustered distribution, is discussed together with our previous findings.

Lectin-histochemical study of O-linked glycoconjugates in dysplastic retina of Norrie disease.

The carbohydrate chains of O-linked glycoconjugates in dysplastic retina from a Japanese female infant with Norrie disease were examined by lectin histochemistry. The avidin-biotinylated peroxidase method was used. The retina was highly dysplastic and composed of undifferentiated embryonic tissues containing a number of rosettes of varying sizes. The lumina of the rosettes were stained by peanut agglutinin, which recognizes the Gal beta 1,3GalNAc sequence of O-linked glycans. However, the lumina were not labeled by wheat germ agglutinin, which reacts with sialic acid and/or N-acetylglucosamine. These observations suggest that the O-linked glycoconjugates in the lumina of rosettes were not sialylated in the present case. Their lack of terminal sialic acids may be related to the rosette formation.

Purification of antibody against peanut agglutinin-receptors of bovine interphotoreceptor matrix.

From rabbit antiserum against peanut agglutinin (PNA)-receptors of the bovine interphotoreceptor matrix, an antibody was isolated by immunoaffinity chromatography on immobilized PNA-receptors. The antibody reacted with the bovine interphotoreceptor matrix at the molecular weight of 130,000 daltons. Immunohistochemical studies revealed that the antibody was bound not only to the surfaces of the cone photoreceptor cells but also to the surfaces of the rods, suggesting that the antibody purified here may have been formed by the major structure of glycoprotein that is common in the interphotoreceptor matrix around the cones and rods.

Effects of neuraminidase on lectin binding sites in photoreceptor cells of monkey retina.

Binding of sugar-specific lectins to the monkey retina was investigated with particular reference to the effects of pretreatment with neuraminidase on the photoreceptor cells. Neuraminidase-treated or untreated retinal sections were examined with a fluorescence microscope after incubation with the following fluorescein-labeled lectins: peanut agglutinin (PNA), wheat germ agglutinin (WGA), Ricinus communis agglutinin-1 (RCA-1) and Dolichos biflorus agglutinin (DBA). In the neuraminidase-untreated tissue, PNA, which is specific for D-galactose beta 1----3 N-acetyl-D-galactosamine (Gal beta 1----3GalNAc), was bound preferentially to the cones, and after treatment of sections with neuraminidase, PNA could bind to both cones and rods. WGA, which is specific for sialic acid and N-acetyl-D-glucosamine, was strongly bound to the rods but moderately to the cones without neuraminidase pretreatment, whereas neuraminidase-treated sections showed a weak binding. RCA-1, which is specific for D-galactose, showed patchy fluorescence on the basal and distal portions of the outer segments of the cones and rods, whereas neuraminidase-treated sections had uniform fluorescence throughout the tissues. DBA, which is specific for N-acetyl-D-galactosamine, did not bind to either cones or rods before and after neuraminidase treatment. These results suggest that there is a difference in the carbohydrate chains of glycoconjugates in the cones and rods: in the cones Gal beta 1----3GalNAc may be the terminal sugar as recognized by PNA, whereas in the rods this sugar is contained in the carbohydrate chain but masked by terminal sialyl residues so that those glycoconjugates are inaccessible to PNA without neuraminidase treatment.

Immunohistochemical localization of cytosolic sialidase in photoreceptor cells.

The binding sites of the antibody to cytosolic sialidase on the rat and monkey photoreceptor cells were examined immunohistochemically using the avidin-biotinylated peroxidase method. In the rat photoreceptor cells, the antibody bound diffusely to the inner segment and the outer nuclear layers which are composed chiefly of rod cells. In the monkey photoreceptor cells, the antibody bound to the rod inner segments which were clearly distinct, morphologically, from the cone inner segments. The antibody also bound to the rod cell bodies in the outer nuclear layer. These binding patterns show that the antibody bound preferentially to rod photoreceptor cells. This observation is consistent with previous lectin histochemical findings that sialoglycans are preferentially present on the surfaces of rod photoreceptors, and in the rod-associated interphotoreceptor matrix. Sialidase in rod inner segments may function by balancing with sialyltransferase, also preferentially expressed in rod inner segments, to form sialyl residues on the termini of sugar chains in the rod-associated sialoglycoconjugates.

Gray-white, spherical deposition on retinal vessel associated with acute retinal necrosis and diabetic retinopathy in HTLV-I carriers.

Tiny, gray-white, spherical deposits were found on the retinal vessels and vitreoretinal interface of the fovea in a 55-year-old woman with acute retinal necrosis due to varicella-zoster virus, and in a 47-year-old man with preproliferative diabetic retinopathy. Both patients developed massive vitreous opacities due to uveal inflammation or hemorrhages that rendered the fundus difficult to visualize. A therapeutic vitrectomy revealed gray-white, tiny spherical deposits, about blood-vessel-diameter, which were scattered on seemingly intact retinal arteries and veins in the posterior fundus and over the vitreoretinal interface overlying the fovea. The deposits were loosely adherent to vessel walls and were easily aspirated, and the residual materials resolved in the early postoperative days. These characteristic retinal vascular deposits resembled those seen in patients with HTLV-I associated uveitis. The two patients reported herein were otherwise asymptomatic carriers of HTLV-I. The findings provide additional information about the etiological role of HTLV-I in the development of characteristic retinal vascular deposition, although its pathogenesis remains to be elucidated.

Maackia amurensis lectin binding in developing rat retina.

The developmental changes in the binding of Maackia amurensis lectin, specific for sialic acid alpha 2,3 galactose sequence, to the rat retina was investigated using the avidin-biotinylated peroxidase method. The lectin bound to the surfaces of photoreceptor outer segments from postnatal day 16 (P16), whereas it had bound to the other retinal layers from P14. The intense labelings of the outer segments were interspersed with unstained portions, which may correspond to cone photoreceptors. These results confirm that the sialic acid residues on the terminus of carbohydrate chains increase at P16 and mask the beta-galactose residues around rod outer segments.

Differential expression of mRNA for alpha 2,3-sialyltransferase during development of rat retina.

Glycoconjugates, consisting of O- and N-linked types, are present on the surface of photoreceptor cells and in the interphotoreceptor matrix (IPM). This study was undertaken to facilitate understanding of the metabolic features of O-linked sialoglycoconjugates in comparison with those of N-linked glycoconjugates in the rat retina. We examined the developmental change in distribution of Gal beta 1,3GalNAc alpha 2,3-sialyltransferase mRNA in rat retinas using in situ hybridization histochemistry to detect the synthesis period of O-linked sialoglycans. Positive hybridization signal was observed in the ganglion cells throughout the postnatal days (P) examined, whereas strong signals were detected in the photoreceptor inner segments and the inner nuclear layer exclusively at P16 and P18. At P20 and older, weak or sparse signals were detected in these regions. It is likely that O-linked sialoglycoconjugates, which are detected in the mature IPM, are actively synthesized from P16 to P18 in the rat photoreceptor inner segments, suggesting that the O-linked sialoglycoconjugates in the photoreceptor layer may be stable with litter turnover in the mature retina.