IDENTIFICATION OF GLYCOGEN IN ELECTRON MICROGRAPHS OF THIN TISSUE SECTIONS

The electron microscopic appearance of glycogen has been studied in the organs of several animal species. Glycogen almost always appears as roughly circular granules from 150 to 400 A in diameter. The intrinsic electron density of glycogen varies from tissue to tissue; however, treatment with lead hydroxide as described by Watson deeply stains the granules. Glycogen pellets were isolated from some of the tissues studied by centrifugation. Such pellets were shown to be glycogen by chemical and histochemical criteria. When thin sections of the pellet are examined under the electron microscope they can be seen to consist of densely packed granules similar to those found in the intact tissues. Such pellets are also stained for electron microscopy by short exposure to lead hydroxide.     

Association of AMP-activated Protein Kinase Subunits With Glycogen Particles as Revealed In Situ by Immunoelectron Microscopy

Immunogold cytochemistry was applied to reveal the intracellular location of AMP-activated protein kinase (AMPK) subunits in liver tissue of normal rats fed ad libitum. AMPK α and β subunits were located both in the cytosol and in close association with rosettes of glycogen particles (α particles). To reveal their true in situ association with glycogen, particular tissue processing conditions that retain glycogen in the cells were required. These included fixation with a combination of glutaraldehyde and paraformaldehyde, followed by postfixation with osmium tetroxide and lead citrate and embedding in Epon. Processing by less-stringent fixation conditions and embedding in Lowicryl led to the extraction of the glycogen deposits, which in turn resulted in the absence of any labeling. This indicates that the loss of glycogen deposits leads to the loss of closely associated proteins. Labeling for the α₁ and α₂ subunits of AMPK was found to be about 2-fold greater over glycogen than over cytosol, whereas labeling for β₁ was 8-fold higher over the glycogen particles than over the cytosol. Immunogold combined with morphometric analysis demonstrated that the β₁ subunits are located at the periphery of the glycogen rosettes, consistent with a recent hypothesis developed via biochemical approaches. (J Histochem Cytochem 57:963–971, 2009)     

The structure of the ventricle of Elliptio complanatus, a fresh-water lamellibranch

The morphology of the ventricle of the fresh-water lamellibranch, Elliptio complanatus, was investigated. Contrary to the condition reported previously in Tritogonia verrucosa, the two atria in Elliptio communicate with the ventricular lumen through separate openings, each guarded by an atrio-ventricular valve. Fixation of ventricle for electron microscopy with 2.5% buffered glutaraldehyde did not appear to shrink the tissue, in spite of the low blood osmolarity to which the muscle is adapted. Ventricle tissue is composed of smooth muscle fibers, containing a central nucleus, glycogen, mitochondria, paramyosin, dense bodies and “attachment plaques,” much like the ventricle of the salt-water clam, Venus (Mercenaria) mercenaria.     

Fat distribution in the skin of bottlenose dolphins (Tursiops truncatus and Tursiops gilli)

Frozen sections stained with Oil-red-O and semithin (0.5 μm) plastic sections stained with toluidine blue revealed an abundance of fat globules of various sizes in all strata of the epidermis of bottlenose dolphins (Tursiops truncatus and T. gilli). The fat was rather evenly distributed but sometimes appeared as circumscribed areas of heavier concentration involving hundreds of cells (as seen in a single plane). Occasionally, there were smaller groups of epidermal cells heavily loaded with lipid. The dermis presented a unique phenomenon in the presence of abundant extracellular fat distributed among the collagen bundles as droplets of various sizes or as larger, irregularly shaped lipid particles that seemed to conform to the spaces between collagen bundles. These lipid particles were sometimes seen to be closely applied to the dermal surface of the stratum basale. Equally unusual was the presence of lipid particles of various sizes and shapes in the lumen of some of the vessels of the dermal papillae. Granular cells resembling mast cells were commonly seen in the papillary dermis and some were closely associated with lipid particles. Blood vessels of the reticular dermis tended to have collections of lipid droplets in the loose connective tissue often found adjacent to the tunica adventitia. It is postulated that the extracellular dermal lipids (probably mainly triglycerides) are broken down to free fatty acids that diffuse into the basal layer of the epidermis and are there resynthesized into triglycerides. Possible uses for the epidermal lipids are discussed.     

Immunohistochemical localization of vimentin in the gerbil inner ear

Cells containing immunoreactive vimentin-type intermediate filaments (IF) were identified in paraffin sections and whole-mount preparations of the gerbil inner ear. Most connective tissue cells showed positive immunostaining, although one unusual class of stromal cell lacked vimentin. Several different types of epithelial cells contained high levels of vimentin. In the cochlea, Deiters' cells, inner phalangeal cells, Boettcher's cells, some outer sulcus cells, and the intermediate cells of the stria vascularis showed strong immunoreactivity. Strial basal cells exhibited weaker and less consistent staining. Neither inner nor outer hair cells were stained. In the vestibular system, hair cells with a morphology and location more characteristic of type I than of type II cells showed strong immunostaining for vimentin. Supporting cells in vestibular neurosensory epithelium stained with less intensity. These results were surprising because epithelial cells in vivo only rarely express vimentin-type IF. Although the functional significance of vimentin remains to be established, its presence in some but not other highly specialized cell types provides an excellent marker for investigating the lineage and morphogenesis of the complex inner ear tissues.     

RABBIT SKELETAL MUSCLE GLYCOGEN : A Morphological and Biochemical Study of Glycogen β-Particles Isolated by the Precipitation-Centrifugation Method

Glycogen in its particulate β-form is localized in the sarcoplasm close to the sarcoplasmic reticulum. Some particles are in close contact with the membranes, on the outer side of the vesicles. The mild technique of differential precipitation-centrifugation has been adapted to the preparation of glycogen from adult skeletal muscle. A preliminary low-speed centrifugation which eliminates the contractile protein structures and the cell debris is followed by a high-speed centrifugation which produces pellets containing glycogen mixed with smooth-walled vesicles, the glycogen-sarcovesicular fraction. The glycogen obtained after treatment of this fraction with deoxycholate and two washings contains 3% protein. A similar protein content contaminates glycogen banded in a linear sucrose gradient. The glycogen-sarcovesicular fraction and the purified glycogen have been examined, under the electron microscope, in sections of fixed and embedded material or with the negative staining technique. The glycogen β-particles in negatively stained preparations have an average diameter of 39.4 mµ. The largest particles present irregular outlines, suggesting the presence of conglomerated subunits, about 20 mµ in diameter. These subunits seem to fall apart under the influence of concentrated potassium hydroxide. The mean sedimentation coefficients calculated for infinite dilution vary from 115 to 135S. The spectrophotometric analysis of the glycogen-iodine complex indicates the presence of long end-chains in the molecule.     

Hormonal modulation of glycogen reserves In the fat body of castor semilooperAchaea janata Linn (Lepidoptera,Noctuidea)

Histochemical details of the fat body in the fifth instar larval stage, pupa and adult moth of the castor semilooperAchaea janata were elucidated in detail using light and electron microscopy in conjunction with glycogen storage patterns using polyacrylamide gel electrophoresis. The periodic-acid Schiff staining for glycogen in fat body was maximum in the spinning stage of the larva, when compared to the feeding stage and prepupal stages, and higher in the pupa than in the larva and the adult moth. In insulin injected and juvenile hormone treated fat body, glycogen deposition was more than in glucagon injected tissues. The periodic-acid Schiff stained bands in PAGE had electrophoretic mobility similar to the corresponding protein band numbers, indicating their glycoprotein nature.     

Fine structure and distribution of three types of virus-like particles in the sheep blowfly,Lucilia cuprina and associated cytopathic effects

The ultrastructure is described for three types of virus-like particles (VLP 1–3) found in thin sections of the sheep blowfly Lucilia cuprina and, in the case of one type (VLP 1), in negatively stained preparations. VLP 1 appears to be morphologically similar to the particles of chronic bee paralysis virus and RS virus of Drosophila and causes a highly characteristic vesiculation of mitochondria. VLP 2 has two forms, spherical and filamentous; these are seen mainly in the gut where the filaments proliferate in the apical parts of the cells and resemble reovirus-like particles found in the gut of Musca domestica. The third type of particle (VLP 3) is intranuclear and seen only rarely. It is arrayed in quasi-crystalline inclusions which resemble inclusions reported in cells and tissues of Drosophila.     

The fine structure ofCristispira from the lamellibranchCryptomya californica conrad

The fine structure ofCristispira from the lamellibranchCryptomya californica Conrad was examined by Nomarski differential interference, Hoffman modulation contrast, phase contrast, polarizing, and darkfield optics, which were useful for observing the motility, morphology, and organelles of livingCristispira within the crystalline style. Transmission electron microscopy studies of double-stained sections and negatively stained whole cells revealed details of the outer sheath, axial filament, and the protoplasmic cylinder. Hundreds of periplasmic flagella were observed, as well as membrane-bound vesicles and lipid accumulations in the protoplasm, which may help to explain the mutualistic relationship which seems to exist betweenCristispira spp. and their molluscan hosts. Also described are a dialysis method for producing concentrated populations of viableCristispira from the clam, and a minimal-salts medium for their short-term maintenance.     

Organization of intermediate filaments and their association with collagen-containing phagosomes in mouse decidual cells

We have analyzed the distribution of intermediate filaments (IF) in the cytoplasm of mature decidual cells of mice. IF were scattered throughout the cytoplasm of these cells although there was a preferential accumulation around the nuclei. In many cells a large area of the cytoplasm was occupied by a rich network of IF that extended from the perinuclear region toward the cell surface. Thin bundles of IF crossed the cytoplasm without a preferential orientation. IF were also seen in close association with nuclear pore complexes, gap junctions, mitochondria, and lysosomes. A very developed network of IF surrounded phagosomes that contained collagen fibrils. Longitudinal and cross sections of these phagosomes showed a very close association of IF with the phagosome membrane.     

Ultrastructural and Cytochemical Observations on Sporogenesis of Myxobolus sp. (Myxosporida: Myxobolidae) from the Common Shiner Notropis cornutus*

SYNOPSIS. The structure and cytochemistry of spores of Myxobolus sp. from plasmodia which occur in the gill filaments of the common shiner Notropis cornutus were studied by light microscopy and by scanning and transmission electron microscopy. The thin-walled valves of the pyriform spores are thickened in the lateral sutural and apical regions. Mucous material is associated predominantly with the posterior end of many spores. The plasmodium is surrounded by a syncytial wall bounded by 2 membranes. Pinocytotic channels are formed by the inner membrane and numerous dense vesicles are pinched off at the distal ends of the channels. Sporogenesis is initiated by the envelopment of one vegetative cell by another. The larger, enveloped cell divides to form a disporous pansporoblast, which contains 2 pairs of capsulogenic and valvogenic cells and 2 binucleate sporoplasm cells. Each capsular primordium and connecting external tubule gives rise to a polar capsule which houses a helically coiled polar tubule. The apical end of each polar capsule is plugged by a stopper. The valvogenic cells surround the capsulogenic and posteriorly situated sporoplasm cells to form the spore valves. Iodinophilic (glycogen) inclusions were not seen in spores stained with iodine or Best's carmine. A darkly stained band was observed around the posterior region of most spores stained with Best's carmine. In the electron microscope large aggregates of β glycogen particles were seen in the cytoplasm of sporoplasm cells in mature spores.     

Glycogen in the neurohypophysis of toads

Summary Studies with the electron microscope revealed the presence of glycogen granules in the neurohypophysis of toads (Bufo arenarum Hensel). The glycogen appeared as scattered particles or as clusters formed by these particles within the nerve fibers. The scattered granules were intermingled with the neurosecretory granules and with the microvesicles. Chemical assays showed the presence of glycogen in the neurointermediate lobe of the toad's hypophysis.This work was supported by research grants from the Consejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina and The Rockefeller Foundation.The authors wish to express their appreciation to Prof. Bernardo A. Houssay and to Dr. Luis F. Leloir for their encouragement during the preparation of this paper; to Miss Susana Camocardi for her technical assistance and to Mr. Luis Millara for the photographic work.     

Presence of an α-galactolipid on the cell surfaces of endothelial cells of human kidney

Summary The presence of an -galactolipid was investigated with a peroxidase-labelled lectin fromGriffonia simplicifolia (GSA-I) with specific binding for terminal -d-galactose residues. Normal kidney tissue was obtained from patients undergoing nephrectomy for renal neoplasms. For light microscopy, tissue was snap-frozen; 4 µm-thick sections were briefly fixed in paraformaldehyde and incubated with GSA (0.025 mg ml^–1). The peroxidase activity was developed with 3-amino-9-ethylcarbazole. Adjacent sections were stained at the same time after lipid extraction with 3:1 (v/v) chloroform/methanol. For electron microscopy, 0.2–0.5 mm-thick paraformaldehyde-fixed blocks, with or without lipid extraction, were stained with peroxidase-labelled GSA. The label was developed with diaminobenzidine and osmium tetroxide. Some structures, such as tubular epithelia, stained both in lipid-extracted and non-extracted tissues, suggesting that glycoproteins were most likely involved. In addition, tissue stained immediately after fixation showed GSA reactivity on endothelial cell surfaces of intertubular capillaries and larger vessels. In lipid-extracted tissues, however, tubular epithelium was still positive for GSA but endothelial cells failed to stain. These findings suggest that a glycolipid, bearing a terminal -galactose residue, is present on the endothelial cells in human kidney and possibly on tubular epithelia. Our data may explain the preferential storage of -galactolipid in endothelial cells of patients with Fabry's disease and other biological phenomena such asEscherichia coli adhesion.     

Fine structural details of human muscle fibres after fibre type specific glycogen depletion

Summary Type 1 and Type 2 fibres of skeletal muscle (human m. vastus lateralis), selectively depleted of glycogen by sustained submaximal muscular exercise (running 30 km), were identified at light and electron microscopical level by examination of thin and ultra-thin serial sections treated particularly for visualization of glycogen. Averaged images, obtained by lateral smearing of depleted fibres (Type 1) exhibited five clearly visible cross-bridges in the M-band and had broad Z-bands. Nondepleted fibres (Type 2) showed either three central strong and two weak outer lines in the M-band and intermediate Z-bands (Type 2A), or only three central strong lines in the M-band and narrow Z-bands (Type 2B). The depleted fibres had no subsarcolemmal accumulation of glycogen particles and practically no intermyofibrillar particles. The remaining particles were small in size and seemed almost rudimentary. In nonexercised individuals, a peculiar distribution of individual glycogen particles in the I-band and A-band was found. This distribution was accounted by the structural arrangement of the myofibrillar material.     

An ultrastructural and cytochemical study of the interaction between latex particles and the haemocytes of the wax moth Galleria mellonella in vitro

Summary The plasmatocytes are the major phagocytic blood-cell type in the haemolymph of the wax-moth, Galleria mellonella. In the present study, these cells were allowed to attach to tissue culture dishes for 1 h, rinsed and then incubated with latex beads for up to 72 h. These cells were then fixed for routine transmission electron microscopy and acid phosphatase cytochemistry. Intracellular latex particles were found in tight, ill-defined phagosomes, which were often clearly associated with the Golgi complexes of the plasmatocytes. Fusion of both primary lysosomes and multivesicular bodies with the phagosomes occasionally occurred and this resulted in the accumulation of an acid phosphatase positive reaction product around the test particles. Subsequent experiments showed that this acid phosphatase activity was mainly associated with the primary lysosomes. The results of the lysosome/latex interactions are compared with those obtained from similar studies on the digestive mechanisms in other phagocytes.We are grateful to Professor E.W. Knight-Jones in whose department this work was carried out and to Mrs. M. Colley for help in insect rearing. This work was supported by grants from the Royal Society and the Science Research Council (B/73/0176. and B/RG/2286.0)     

Electron microscopic demonstration of rat liver glycogen phosphorylase activity with iron (Fe++)

Summary In this study a new electron microscopic method for the demonstration of liver glycogen phosphorylase activity has been presented.Prior to incubation the liver samples were shortly fixed in cold paraformaldehyde. Inorganic phosphate, liberated in the reaction catalyzed by the enzyme, were precipitated with iron (Fe⁺⁺) present in the incubating medium. Postfixation was performed in glutaraldehyde and osmium tetroxide.The ferrous phosphate precipitate was detected electron microscopically in unstained sections.The precipitate was mainly localized to endoplasmic membranes but also in glycogen particles. The method is imperfect in demonstrating phosphorylase activity bound to glycogen particles because of poor preservation of glycogen during treatment.     

Polymerase Chain Reaction for the Detection of Chlamydia psittaci in the Feces of Budgerigars

The polymerase chain reaction (PCR) targeting the ompA gene of Chlamydia psittaci was evaluated for its ability to detect chlamydiae in fecal specimens of budgerigars as compared with isolation procedures using cell culture and embryonated egg inoculations. Several procedures for PCR template DNA preparation were compared so as to determine their detection levels for chlamydiae propagated in cell culture in the presence of fecal materials. Tween-20 and proteinase K treatments followed by centrifugation of the template DNA were found to be an appropriate procedure for DNA preparation for primary PCR. Subsequent nested PCR was shown to detect 4.8 IFU/ml or 84 particles/ml of chlamydiae. Chlamydiae in 50 fecal specimens from apparently healthy budgerigars were examined by nested PCR and several other methods. Nested PCR detected chlamydiae at a higher rate (12/50, 24%) than the isolation procedure in embryonated eggs (6/50, 12%). Primary PCR combined with the isolation procedure in cell culture gave a detection rate (5/50, 10%) similar to that of isolation from embryonated eggs. Detection rates by primary PCR (1/50, 2%) and in cell culture (0%) were inferior to the other procedures.     

Ultrastructural visualization of complex carbohydrates in epiphyseal cartilage with the tannic acid-metal salt methods

The present study has ultrastructurally applied the tannic acid-ferric chloride (TA-Fe) and the TA-uranyl acetate (TA-UA) methods to thin sections of glutaraldehyde-fixed, unosmicated embedded epiphyseal cartilage from rat tibiae to demonstrate complex carbohydrates. The strongest TA-Fe and TA-UA staining was observed after fixation of the specimens in glutaraldehyde containing TA. TA-Fe (pH 1.5) strongly stained matrix granules presumed to be proteoglycan monomers and chondrocyte secretory granules at various maturational stages but did not stain collagen fibrils and glycogen. TA-UA (pH 4.2) strongly stained matrix granules, intracellular glycogen, and chondrocyte secretory granules, and moderately stained collagen fibrils in the cartilage matrix. Ribosomes and nuclei were not stained above background staining with UA alone. In alpha-amylase-digested specimens, all TA-UA-reactive cytoplasmic glycogen was selectively removed. Testicular hyaluronidase digestion of specimens selectively removed TA-UA staining in matrix granules and all TA-Fe staining. When the pH of the UA solution was reduced to 1.5, TA-UA staining of glycogen and collagen was markedly decreased or absent, whereas staining of anionic sites was unaltered and significantly greater than with UA staining alone. Thus the TA-metal salt methods are pH dependent and allow differential intracellular and extracellular localization of complex carbohydrates in cartilage tissues at the electron microscope level.     

Identity of Holmes Positive Bodies with Electron Microscopically Demonstrable Nucleolus-Like Bodies in Neuronal Cytoplasm

By light microscopic observation of mouse brain stained by Holmes' silver method deeply stained cytoplasmic inclusion bodies were seen in almost all nerve cells of the locus coeruleus. Electron microscopy of tissue samples from floating Vibratome sections stained by Holmes' silver method demonstrated that the nucleolus-like bodies in the cytoplasm were densely impregnated with gold particles. Hence, it was confirmed that the cytoplasmic inclusion bodies of paraffin sections stained by Holmes' method are identical to the so-called nucleolus-like bodies seen in electron microscopic studies.     

On the presence of high glycogen concentration and intermediate filaments in the flat cells of the electroreceptive epidermis of mormyrid fish

The cytoplasm of the flat cells of the electroreceptive epidermis of Mormyrids was examined in the light and the electron microscope, in order to reveal the presence of glycogen and to study its distribution. In the electroreceptive epidermis, which consists of three layers, the periodic acid Schiff reaction used to stain polysaccharides is strongly positive in the superficial polyhedral cells and in the flat cells of the intermediate layer. Polysaccharides are absent in the basal polyhedral cells. Pre-incubation with alpha-amylase shows that glycogen is present only in the intermediate cell layer. In the electron microscope, after reaction with periodic acid, thiocarbohydrazide and silver proteinate, glycogen is seen in the form of rosettes of monoparticles. These rosettes occupy both the central region of the cytoplasm of these cells, and the more peripheral parts, where alignments of desmosomes are found. In the cytoplasm of certain flat cells, the rosettes are grouped to form accumulations of glycogen which cover several mu2. Observation in the electron microscope reveals that in addition to glycogen, these cells contain tonofilaments or intermediate filaments, common to epithelial cells, which may group themselves in bundles. Glycogen and the intermediate filaments are thus the principal constituents of the cytoplasm of the flat cells of the electroreceptive epidermis of Mormyrids. The possible role of the filaments, and especially of the glycogen which is a polysaccharide high in energy, in the flat cells which apparently have a low metabolic rate, is discussed.