Polyglucose particles histochemically synthesized by glycogen synthetase activity in the living chick retina

Summary Electron histochemical techniques for glycogen synthetase has been applied to the living retina of the chick and the polyglucose particles synthesized from UDPG in the paraboloid of the accessory cone were compared with those synthesized by the conventional histochemical techniques.In the retinal incubated in the medium for glycogen synthetase in vivo, synthesized polyglucose particles were located in the cytoplasmic matrices and most of the particles were less than 200 Å in diameter. These particles were rather well stainable with lead citrate and filled the cytoplasmic matrices. However, the tubular structures were not flattened, but slightly dilated. Compared with polyglucose particles synthesized in vitro by glycogen synthetase, those demonstrated by the in vivo histochemical techniques showed closer resemblance to native glycogen particles in size and stainability with lead citrate.The polyglucose particles synthesized from UDPG by glycogen synthetase were apparently different from those synthesized from glucose-1-phosphate by phosphorylase and branching glycosyltransferase.     

Application of histochemistry to a living organ

Summary Histochemical application in a living animal was tested on the paraboloid of the accessory cone of the chick retina.After the anterior part of the eye had been cut with a Graefe's knife under ether anesthesia, the posterior part was filled with the medium for phosphorylase under exposure to light. The specimens were embedded for routine electron microscopy and the paraboloid of the accessory cone was observed by electron microscopy.Polyglucose particles were synthesized from glucose-1-phosphate in the paraboloid by the activities of phosphorylase and branching glycosyl transferase and found to be in the cytoplasmic matrices. These particles were larger in size and better stainable with lead citrate than those found in the paraboloid of the retina incubated in the medium in vitro by the conventional histochemical method. Overproduction of polyglucose particles was not found in the paraboloid of the retina incubated in the medium in vivo. These findings suggest that polyglucose particles synthesized in vivo have a close resemblance to native glycogen particles and that glycogen metabolism is regulated by the living cell. Glycolysis may not be related to the membranous structures.Therefore, application of enzyme histochemical techniques to the living organ can demonstrate more accurate morphological aspects of metabolism in the cell.     

In vivo synthesis of glycogen by phosphorylase system

Summary Application of conventional histochemical techniques to the living chick retina demonstrates that phosphorylase can synthesize glycogen (polyglucose) in vivo, in the paraboloid of the accessory cone. Natural in vivo glycogen synthesis may therefore be due to glycogen synthetase and phosphorylase systems, although phosphorylase is normally regarded as a glycolytic enzyme.     

Macromolecular particles of polyglucose synthesized under histochemical conditions in the endothelial cells of rabbit blood vessels

Synopsis New polyglucose was synthesized artificially under histochemical conditions from glucose 1-phosphate by phosphorylase and branching glycosyltransferase in the endothelial cells of rabbit blood vessels. In electron micrographs it appeared as a large macromolecular structure of spheroidal branching bodies. The polyglucose particles were much larger in size than those observed previously. They were synthesized in intracellular cytoplasmic matrices and also within nuclei.     

Electron microscopical observation of polyglucose synthesized from glucose-1-phosphate by the phosphorylase activity in rat skeletal muscles

Summary An improved demonstration of polyglucose synthesized histochemically from glucose-1-phosphate by the phosphorylase activity in rat skeletal muscle was made by use of electron histochemical techniques.New polyglucose was found in the sarcoplasmic matrix among various organelles in muscle fibers embedded with epoxy resin. This polyglucose stained less densely with lead, forming amorphous aggregates or finer particulates with 100 to 150 Å in diameter, different from native glycogen which was separately observed in the same section.     

Immunohistochemical localization of iodopsin in the retina of the chicken and Japanese quail

Summary Localization of iodopsin in the retina of the chicken and Japanese quail was investigated immunohistochemically with the use of monoclonal antibodies (R1-R4) highly specific for R-photopsin (protein moiety of iodopsin). In paraffin sections of the retina, the outer segments of double cones (principal and accessory cones) and of one particular type of single cones were labeled with the antibodies. In addition, reticular cytoplasmic structures, probably representing the Golgi apparatus in a position close to the vitreous pole of the paraboloid and to the outer limiting membrane were intensely stained in the cone cells bearing an immunoreactive outer segment. In whole-mount preparations, 5 types of cone cells were identified according to the color of oil droplets, i.e., red, yellow, pale-green (principal member of double cones), pale-blue and clear, in addition to a sixth type devoid of an oil droplet (accessory member of double cones). The immunohistochemical analysis of the preparations revealed that R-photopsin (suggesting the presence of iodopsin) is localized in the outer segments of both the principal and accessory members of double cones, and the population of single cones displaying a red oil droplet. Other cones endowed with a yellow, blue or clear oil droplet were not labeled with the antibodies used. Similar results were obtained in the retina of the Japanese quail.     

Particulate glycogen of mammalian liver: specificity in binding phosphorylase and glycogen synthase.

The glycogen particle - glycogen metabolizing enzyme complex was investigated to gain some understanding of its physiological significance. Fractionations of populations of particles from mouse liver were carried out utilising open column and high performance liquid chromatography, and based either on the molecular weight of the particles or the hydrophobic interactions of the glycogen-associated proteins. The activities of glycogen phosphorylase and glycogen synthase were measured in these fractions. Fractionations were of tissue in different stages of glycogen deposition or mobilization. In animals fed ad libitum, glycogen synthase was associated with the whole spectrum of molecular weights, while the glycogen phosphorylase distribution was skewed in favour of the lower molecular weight species. Under conditions of glycogen mobilization, the phosphorylase distribution changed to include all molecular weights. The hydrophobic interaction separations demonstrated that glycogen synthase binds to a specific subpopulation of particles that is a minor proportion of the total. In general, there was a direct relationship of the total amount of phosphorylase and synthase bound during periods of mobilization and deposition, respectively. Two notable exceptions were the large amounts of glucose-6-P dependent synthase present during the early period of glycogen mobilization and the high amounts of active phosphorylase appearing shortly after food withdrawal, in spite of interim glycogen deposition from presumably already ingested food.     

Evidence for the presence of glycogen in rat thymus

Chemical and biochemical analysis of the polysaccharide, present in rat thymus, indicate that it consists of glucose units alpha-1,4 and alpha-1,6 linked. Electron microscopy reveals the presence of a polysaccharide, similar to the beta-glycogen particles observed in liver and muscle with an average diameter of 20-30 nm. They are located in the cytoplasmic area of T-cells from the cortical region of the thymus. Enzymatic analysis indicates that the beta-particles contain a highly branched glucan with short external chains. Some of the enzymes of glycogen metabolism: synthase, phosphorylase and branching were for the first time partially purified from rat thymus and some of their properties were studied. Therefore, glycogen appeared to be synthesized in rat thymus.     

Intranuclear synthesized and native glycogen particles in human gastric cancer: Ultrastructure and histochemistry

Summary Ultrastructural and histochemical studies on human gastric cancer cells disclosed the presence of native and synthesized glycogen particles. The glycogen particles were investigated in the histochemical synthesis of glycogen particles from glucose 1-phosphate by the phosphorylase-branching glycosyltransferase system and non-incubated native glycogen in human gastric adenocarcinoma tubulare.It was observed that focal synthesis localized in the intracytoplasmic matrix and intranucleus. Intranuclear synthesized glycogen appeared as a rosette form ranging from 1100 to 1300 Å in diameter and free particles ranging from 325 to 900 Å in diameter. The synthesis of glycogen appeared in the nucleus as well as in the cytoplasm of the human gastric cancer cells, and the synthesized glycogen was observed as a group of particles. Newly formed glycogen particles appeared occasionally in the interchromatin area as a large macromolecular structure of rosette form.Native glycogen appeared as a free-particle (250–333 Å, medium=300 Å) and aggregated rosette from (694–1050 Å, medium=917 Å) in the autophagosome of gastric cancer cells. There was not, however, a native glycogen particle in the nuclei of gastric cancer cells.Under certain conditions the nuclei of gastric cancer cells can acquire the capacity to synthesize glycogen.     

Expression of cone-like properties by chick embryo neural retina cells in glial-free monolayer cultures

We report here that cells present in embryonic chick retinal monolayer cultures express differentiated properties characteristic of chick cones developing in vivo. Cell suspensions from 8-d chick embryo retina (a stage when photoreceptor differentiation has not yet started) were cultured for up to 7 d in low density, glial-free monolayers. Under these conditions, monopolar cells represent approximately 40% of the total number of process-bearing neurons. After 6 d in vitro, most of these monopolar cells showed morphological features reminiscent of developing chick cones. These features could be detected with phase-contrast microscopy, lectin cytochemistry, and transmission and scanning electron microscopy. Characteristic cone traits expressed by cultured monopolar cells included the following: (a) a highly polarized organization; (b) a single, short, usually unbranched neurite; (c) the polarized position of the nucleus close to the origin of the neurite; (d) characteristic cone inner segment features such as abundant free ribosomes, a polarized Golgi apparatus, a cluster of mitochondria distal to the nucleus, a big, membrane-bound, pigment-containing vacuole reminiscent of the "lipid droplet" characteristic of chick cones, and at least in some cases, a well-developed paraboloid; (e) the presence of a complex of apical differentiations including abundant microvilli and in some cases also a cilium-like process; and (f) the staining of the apical region of the cell with peanut lectin, which has been shown to be selective for chick embryo cones (Blanks, J.C., and L.V. Johnson, 1983, J. Comp. Neurol., 221:31-41; and Blanks, J.C., and L.V. Johnson, 1984, Invest. Ophthalmol. Visual Sci., 25:546-557). This pattern of differentiation achieved by 8-d chick retina cells after 6 d in vitro is similar to that shown by 14-d-old chick embryo cones in vivo. Outer segments are not present at this stage of development either in vivo or in vitro. This experimental system is now being used to search for cellular and molecular signals controlling survival and differentiation of cone cells.     

Correlation of rhodopsin biogenesis with ultrastructural morphogenesis in the chick retina

The developing chick retina from stages 39-45 has been examined by biochemical and electron microscope techniques. The levels of rhodopsin contained in the maturing chick retina were evaluated by detergent extraction and correlated with rod outer segment formation. It was found that the appearance of rhodopsin in significant levels preceded outer segment formation by at least 2 days, thus implying that rhodopsin is synthesized in the receptor cell inner segment and translocated to the outer limb when disk membrane biogenesis occurs. The level of rhodopsin continues to rise as the rod outer segment develops. Development of both rods and cones originates and proceeds most rapidly in the fundus or central region and proceeds toward the periphery. In general, rod outer segments were noted to develop far more rapidly than cone outer segments.     

Electron microscopical study of a cytosolic enzyme in unfixed cryostat sections: demonstration of glycogen phosphorylase activity in rat liver and heart tissue

Summary Glycogen phosphorylase activity has been demonstrated at the ultrastructural level in liver and heart tissue of fasted rats. Unfixed cryostat sections were incubated by mounting them on a semipermeable membrane stretched over a gelled incubation medium. The medium contained a high concentration of glucose 1-phosphate which enables indirect detection of glycogen phosphorylase activity on the basis of the synthesis of glycogen. Tissue fixation, dehydration and embedding for electron microscopical study were performed after the incubation had been completed. The ultrastructure of both liver and heart tissue was rather well preserved. Glycogen granules resulting from glycogen phosphorylase activity were found in the cytoplasmic matrix of both hepatocytes and cardiomyocytes; no relationship with membranous structures could be detected. It is concluded that the semipermeable membrane method is well suited for localizing cytosolic enzyme activities at the ultrastructural level without prior tissue fixation; this opens further perspectives for correlations between histochemical and biochemical data.     

Enzyme activity during the metabolism of glycogen. I. Demonstration of phosphorylases in the sensory cells of the tuberous organ of Gnathonemus petersii (Mormyridae). Histochemical and ultrastructural methods.

Phosphorylase activities were investigated by histochemical and ultrastructural procedures in the electroreceptive sensory cells of the tuberous organ of Gnathonemus petersii. Ater incubation in G1P, G1P activated by AMP (Takeuchi and Kuriaki medium) or in G1P activated by ATP+MgSO4 (Guha and Wegman medium) newly formed polysaccharides were analysed with the iodine and P.A.S. reactions under light microscopy and, under electron microscopy, with the periodic acid thiocarbohydrazide (TCH) silver proteinate (PATAg reaction, Thiery), The newly formed polysaccharides proved the presence of glycogen phosphorylase (2.4.1.1) activities and of their branching enzymes (2.4.1.18). When G1P was activated by ATP+MgSO4, they appeared as glycogen particles with the same constitution as native glycogen. After incubation in G1P and in G1P activated by AMP they appeared as glycogen and polyglucose filaments too. In the latter case they were high concentrated. The results show that the phosphorylases are principally present in this sensory cell in their inactive form.     

Effect of Diabetes on Glycogen Metabolism in Rat Retina

Glucose is the main fuel for energy metabolism in retina. The regulatory mechanisms that maintain glucose homeostasis in retina could include hormonal action. Retinopathy is one of the chemical manifestations of long-standing diabetes mellitus. In order to better understand the effect of hyperglycemia in retina, we studied glycogen content as well as glycogen synthase and phosphorylase activities in both normal and streptozotocin-induced diabetic rat retina and compared them with other tissues. Glycogen levels in normal rat retina are low (46 +/- 4.0 nmol glucosyl residues/mg protein). However, high specific activity of glycogen synthase was found in retina, indicating a substantial capacity for glycogen synthesis. In diabetic rats, glycogen synthase activity increased between 50% and 100% in retina, brain cortex and liver of diabetic rats, but only retina exhibited an increase in glycogen content. Although, total and phosphorylated glycogen synthase levels were similar in normal and diabetic retina, activation of glycogen synthase by glucose-6-P was remarkable increased. Glycogen phosphorylase activity decreased 50% in the liver of diabetic animals; it was not modified in the other tissues examined. We conclude that the increase in glycogen levels in diabetic retina was due to alterations in glycogen synthase regulation.     

Enzyme activity during the metabolism of glycogen

Summary Phosphorylase activities were investigated by histochemical and ultrastructural procedures in the electroreceptive sensory cells of the tuberous organ of Gnathonemus petersii.After incubation in G1P, G1P activated by AMP (Takeuchi and Kuriaki medium) or in G1P activated by ATP+MgSO₄ (Guha and Wegman medium) newly formed polysaccharides were analysed with the iodine and P.A.S. reactions under light microscopy and, under electron microscopy, with the periodic acid thiocarbohydrazide (TCH) silver proteinate (PATAg reaction, Thiery), The newly formed polysaccharides proved the presence of glycogen phosphorylase (2.4.1.1) activities and of their branching enzymes (2.4.1.18). When G1P was activated by ATP+MgSO₄, they appeared as glycogen particles with the same constitution as native glycogen. After incubation in G1P and in G1P activated by AMP they appeared as glycogen and polyglucose filaments too. In the latter case they were high concentrated. The results show that the phosphorylases are principally present in this sensory cell in their inactive form.     

Association of Shank 1A Scaffolding Protein with Cone Photoreceptor Terminals in the Mammalian Retina

Photoreceptor terminals contain post-synaptic density (PSD) proteins e.g., PSD-95/PSD-93, but their role at photoreceptor synapses is not known. PSDs are generally restricted to post-synaptic boutons in central neurons and form scaffolding with multiple proteins that have structural and functional roles in neuronal signaling. The Shank family of proteins (Shank 1–3) functions as putative anchoring proteins for PSDs and is involved in the organization of cytoskeletal/signaling complexes in neurons. Specifically, Shank 1 is restricted to neurons and interacts with both receptors and signaling molecules at central neurons to regulate plasticity. However, it is not known whether Shank 1 is expressed at photoreceptor terminals. In this study we have investigated Shank 1A localization in the outer retina at photoreceptor terminals. We find that Shank 1A is expressed presynaptically in cone pedicles, but not rod spherules, and it is absent from mice in which the Shank 1 gene is deleted. Shank 1A co-localizes with PSD-95, peanut agglutinin, a marker of cone terminals, and glycogen phosphorylase, a cone specific marker. These findings provide convincing evidence for Shank 1A expression in both the inner and outer plexiform layers, and indicate a potential role for PSD-95/Shank 1 complexes at cone synapses in the outer retina.     

Immunohistochemical Localization of Glycogen Synthase and GSK3β: Control of Glycogen Content in Retina

Glycogen has an important role in energy handling in several brain regions. In the brain, glycogen is localized in astrocytes and its role in several normal and pathological processes has been described, whereas in the retina, glycogen metabolism has been scarcely investigated. The enzyme glycogen phosphorylase has been located in retinal Müller cells; however the cellular location of glycogen synthase (GS) and its regulatory partner, glycogen synthase kinase 3β (GSK3β), has not been investigated. Our aim was to localize these enzymes in the rat retina by immunofluorescence techniques. We found both GS and GSK3β in Müller cells in the synaptic layers, and within the inner segments of photoreceptor cells. The presence of these enzymes in Müller cells suggests that glycogen could be regulated within the retina as in other tissues. Indeed, we showed that glycogen content in the whole retina in vitro was increased by high glucose concentrations, glutamate, and insulin. In contrast, retina glycogen levels were not modified by norepinephrine nor by depolarization with high KCl concentrations. Insulin also induced an increase in glycogen content in cultured Müller cells. The effect of insulin in both, whole retina and cultured Müller cells was blocked by inhibitors of phosphatidyl-inositol 3-kinase, strongly suggesting that glycogen content in retina is modulated by the insulin signaling pathway. The expression of GS and GSK3β in the synaptic layers and photoreceptor cells suggests an important role of GSK3β regulating glycogen synthase in neurons, which opens multiple feasible roles of insulin within the retina.     

THE FINE STRUCTURE OF ASTROCYTES IN THE CEREBRAL CORTEX AND THEIR RESPONSE TO FOCAL INJURY PRODUCED BY HEAVY IONIZING PARTICLES

Normal and reactive astrocytes in the cerebral cortex of the rat have been studied with the electron microscope following focal alpha particle irradiation. The presence of glycogen and approximately 60-A fibrils identify astrocyte cytoplasm in formalin-perfused tissue. The glycogen particles facilitate the identification of small processes and subpial and perivascular end-feet. Both protoplasmic and fibrous astrocytes contain cytoplasmic fibrils and should be distinguished on the basis of the configuration of their processes and their distribution. Acutely reactive astrocytes are characterized by a marked increase in the number of glycogen granules and mitochondria from the first day after irradiation. These cells later hypertrophy and accumulate lipid bodies and increased numbers of cytoplasmic fibrils. The glial "scar" consists of a greatly expanded volume of astrocyte cytoplasm filled with fibrils and displays no signs of astrocyte death, reversion to primitive forms, or extensive multiplication.     

Restoration of glycogenesis in hepatocytes from starved rats.

Hepatocytes isolated from the liver of rats starved for two days synthesized glycogen only when incubated in the presence of both glucose and glucogenic precursors (combinations of alanine, glycerol, pyruvate, lactate or fructose). Unlabeled glucogenic precursors facilitated the incorporation of [U-14C]glucose into glycogen. Unlabeled glucose likewise greatly enhanced glycogen synthesis from isotopically labeled lactate and other glucogenic precursors.In those systems which contained no added endocrines glucose dampened glycogen phosphorylase activity in a cAMP-independent fashion. Fructose is unable to mimic the effects of glucose on glycogen deposition and on glycogen phosphorylase activity.