Viral DNA Synthesis In Vitro with the Inclusions Isolated from Adenovirus 12-Infected Cells

A fraction defined as the inclusions was isolated by banding in CsCl gradients from nuclei of adenovirus 12-infected KB cells. When examined by electron microscopy, the isolated inclusions were relatively homogeneous, finely granular materials of moderate electron density, possibly representing the disintegrated type II or IV inclusions. The conditions of endogenous DNA synthesis in vitro with the inclusions were determined. The product of DNA synthesis in vitro with the inclusions was mainly viral and scarcely cellular, as revealed by DNA-DNA hybridization and methylated albumin kieselgur column chromatography. However, viral DNA synthesized in vitro was smaller (18 S, 22 S) than viral DNA in virions (31 S, 34 S) in neutral and alkaline sucrose gradients. Effects of various treatment of the inclusions on the DNA-synthesizing activity showed that phospholipase C inhibited the activity efficiently. The in vitro DNA synthesis was stimulated by addition of the cytoplasmic extract from adenovirus 12-infected cells and not that from unifected cells. The analysis of the composition of the inclusions showed that the inclusions contained DNA, protein, phospholipid and a small amount of RNA and carbohydrate.     

Studies on Bacillus thuringiensis H-14 strains isolated in Egypt—V. Composition and toxicity of the mosquitocidal parasporal inclusions

Parasporal inclusions of Bacillus thuringlensis H-14 strains M1 and S128 were characterized by solubilization, electron microscopy, polyacrylamide gel electrophoresis, amino acid analysis and insecticidal activity. Inclusions of both strains are composed largely of protein with 8 to 9% carbohydrate. Amino acid analysis of the purlfied inclusions revealed that the two strains produce inclusions that are closely related to each other but significantly different from lepidopteran-toxic B. thuringiensis parasporal crystals. The LC₅₀ values of the purlfied inclusions of strains M1 and S128 were 3.4 and 2.9 ng/ml, respectively, for fourth instar larvae of Aedes aegypti. Inclusions from strain M1 were resolved into two inclusion bands on the basis of their densities possibly formed as a result of disruption of some envelopes during sonication. Both inclusion types contained proteins of approximately 27, 38 and 66 kDa. The heavlest and more predominant type had an envelope and was either spherical or irregular being composed of several subunits which varied in shape, size and staining densities. The LC₅₀ value was 2.2 ng/ml and the major protein was of approximately 27 kDa. The lightest inclusions type did not have an envelope and showed clear crystal lattices. They were 10 times less toxic to A. aegypti larvae, as compared to the heavy-type inclusions and contained major protein of approximately 66 kDa.     

Fibrils in marsupial enamel tubules

Summary Serial etching of cross-sectioned prisms in undecalcified adult marsupial enamel from different species, revealed distinct cylindrical acid-resistant fibrils that were demonstrable by light microscopy and by scanning electron microscopy. No fibrils were found in the enamel of Vombatus.The fibrils and the organic matrix in the remainder of the enamel stain differently. The fibrils project from the center of prisms or the borderline between prisms and interprismatic substance.It is concluded that the fibrils are chemically different from the organic matrix in the enamel, that they constitute the compact, homogenous, and morphologically well defined organic contents of the tubules in adult marsupial enamel.Since most of the material was obtained from dry museum crania, it is concluded that the fibrils are not destroyed by prolonged drying.The scanning electron micrographs were taken at the Electron Microscopical Unit for Biological Sciences, Oslo, Norway.     

Characterization of paracrystalline inclusions in chinese hamster oocytes and early embryos

Paracrystalline inclusions, readily visible with light microscopy, were found to be present in ovarian oocytes (beginning with unilaminar follicles), ovulated ova, and preimplantation embryos of the Chinese hamster. These inclusions appeared to be aggregates of finer filamentous structures visible only with electron microscopy. The use of various histochemical techniques suggested that the paracrystals were composed of protein with little or no lipid associated with them. Autoradiographic studies using ³H-uridine and enzymatic studies did not support the hypothesis that the paracrystalline inclusions were composed of RNA masked by a crystalline protein lattice.     

Scanning electron microscopic and X-ray diffraction studies of otoconia in the lizard Podarcis s. sicula

Summary The otoliths of embryos and young animals of the lizard Podarcis s. sicula were studied by X-ray diffraction and scanning electron microscopy. Two types of crystal that give different X-ray diffraction patterns were found in the membranous labyrinth of Podarcis. The crystals consist of calcite or aragonite and are easily distinguished by scanning electron microscopy because of their different morphology. The two calcium carbonate crystal forms are not mixed at random but are present in the embryo from the very beginning in specific sites. The endolymphatic sac contains aragonite crystals while the saccule contains calcite crystals adjacent to the wall, in addition to a preponderance of aragonite crystals. The utricle and lagena contain only calcite crystals. The presence of two crystal forms of calcium carbonate in the membranous labyrinth are discussed in terms of differing genetic and functional significance.     

Ultrastructure of the corpus luteum of antarctic seals during pregnancy

Summary The fine structure of granulosa lutein cells from three crabeater seals, Lobodon carcinophagus, and two leopard seals, Hydrurga leptonyx, has been studied from early through mid-pregnancy. Analysis of the arrangement and modifications of the cytoplasmic organelles and inclusions has revealed three types of lutein cells throughout the corpus. Type I cell typically possesses a central nucleus and cytoplasm containing very large amounts of smooth and/or fenestrated endoplasmic cisternae which frequently extend from the juxta-nuclear to the periphery of the cell. Type II cell contains a central or eccentric nucleus, moderate amounts of peripheral, smooth and fenestrated cisternae which often form large and concentric membranous whorls, numerous mitochondria and small lipid droplets. Frequently these cells show polarity in the arrangement of the cytoplasmic organelles and inclusions. Type III cell contains predominant large lipid droplets, many mitochondria, and small amounts of smooth and fenestrated cisternae. In light microscopy the type I cell is evenly granular, while the type III cell is highly vacuolated. Type II cells have both granular and vacuolated conditions. Ultrastructural features of type I and II cells suggest that they probably secrete most of the steroids, whereas the primary role of the type III cells appear to be lipid storage.This research was supported by National Science Foundation, Grant No. 1325 from the Office of Antarctic Biology.     

Histochemistry and ultrastructure of the mycetome and its 'symbiotes' in the pear psylla, Psylla pyricola Foerster (Homoptera)

The orange-coloured mycetome of the pear psylla, which comprises a cellular covering membrane, a number of mycetocytes and a central syncytium, contains two kinds of inclusions (micro-organisms-in the literature). As DNA could not be detected in them, their status as micro-organisms is questioned. Both kinds contain protein; one kind contains RNA, and (by electron microscopy) tubule-like peripheral maculae. In spite of their anomalous reactions, the inclusions are probably unusual, degenerate or aberrant microorganisms. The biosome theory is invoked in explanation.     

Ultrastructure and histochemistry of the supportive structures associated with the radula of the slug,Limax maximus

The odontophore and connective tissue-filled portion of the radular sac (called the “collostyle”) of the slug, Limax maximus, were examined by light and electron microscopy. While both of these structures grossly resemble vertebrate cartilage, neither is composed of a type of tissue with the microscopic appearance and histochemical properties of cartilage. The roughly U-shaped odontophore possesses a thin capsule composed of connective tissue. The parenchyma of the odontophore consists of modified muscle cells which are organized into irregular groups by incomplete trabeculae composed of conventional muscle cells. The odontophoral cells are variable in size; they contain glycogen-filled “cores” as well as bundles of peripherally located filaments resembling myofilaments; and they are innervated like muscle cells. The nuclei of the cells are located eccentrically in the glycogen-filled portions of the cells and typically contain prominent nucleoli. The nuclei are surrounded by multiple small Golgi complexes and pleomorphic dense bodies resembling lysosomes. The extracellular matrix of the odontophore is very sparse and contains glycogen and fibrillar material but no histochemically demonstrable acidic mucosubstances. The collostyle consists of a gelatinous type of tissue somewhat like vertebrate mucoid connective tissue. The abundant extracellular matrix contains cross banded filaments, a flocculent material disposed in wavy indefinite strands, and small electron-dense particles. The matrix contains histochemically demonstrable neutral and weakly acidic mucosubstances. The cell population of the collostyle includes solitary muscle cells and fibrocytes containing large quantities of glycogen.     

ELECTRON MICROSCOPIC OBSERVATIONS ON TUBULAR STRUCTURES INVOLVED IN CRYSTAL FORMATION IN THE RED ALGA PLEONOSPORIUM SQUARRULOSUM (HARV.) ABBOTT1

Hexagonal or angular crystalline inclusions in Pleonosporium (Naeg.) Hauck vegetative cells were examined using electron microscopy. Ultrastructural analysis reveals that the inclusions initially contain tubular elements resembling microtubules but, with continued differentiation, are transformed into rod containing crystals. The tubular structures initially measure 25 nm in diameter. Scattered tubules become arranged in a parallel and alternate pattern and undergo subsequent enlargement to approximately 29 nm. Following enlargement, each tubule apparently disaggregates into rods that form a crystal having hexagonally arranged rod-like subunits. It is suggested that these tubules may represent microtubules and the resultant crystals are composed of tubulin.     

The nature and role of the basal pinacoderm of Corvomeyenia carolinensis Harrison (Porifera: Spongillidae) A histochemical and developmental study

Summary The nature and role of the basal pinacoderm in the freshwater sponge Corvomeyenia carolinensis Harrison was examined using phase contrast microscopy, cytochemistry, and morphogenic analysis of regeneration. The pinacoderm is a nonsyncytial simple squamous epithelium. Pinacocytes, normally nucleolate, contain little histochemically demonstrable RNA, suggesting low levels of protein synthesis. The lack of a major role in food storage is indicated by the slight amount of PAS-positive carbohydrate and the absence of glycogen and acid mucopolysaccharides. Marginal pinacocytes are active in feeding. High levels of acid phosphatase in these cells indicate digestion is following ingestion. Pinacocytes contain numerous contractile vacuoles and play an active role in osmoregulation. These cells play the dominant role in wound healing, becoming actively ameboid to initiate the regenerative process.

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Zusammenfassung Die vorliegende Arbeit is das Resultat von Untersuchungen des Charakters und der Rolle des basalen Pinakoderms im Süsswasserschwamme Corvomeyenia carolinensis Harrison mit Hilfe von Phasenkontrast-Mikroskopie, Cytochemie, und morphogenetischer Analyse der Regenerationsvorgänge. Das Pinakoderm ist ein nicht-syncytielles, einfaches Plattenepithel. Dessen Zellen, die normalerweise nucleoliert sind, enthalten sehr weinig nachweisbares RNA, was eine tiefe Stufe von Proteinsynthese andeutet. Das Fehlen einer bemerkenswerten Rolle dieser Zellen in Hinblick auf Nahrungseinlagerung ist angezeigt durch die kleinen Mengen von POS-positiven Kohlehydraten, als auch durch die Abwesenheit von Glycogen und saueren Mucopolysachariden. Randständige Pinacocyten sind äusserst aktiv in Nahrungsaufnahme. Ein hohes Niveau von sauerer Phosphatase in diesen Zellen beweist dass die Nahrung nach Aufnahme verdaut wird. Die Pinacocyten enthalten zahlreiche kontraktile Vakuolen und spielen eine aktive Rolle in Osmoregulationsvorgängen. Darüber hinaus sind diese Zellen in Wundheilung dominierend beteiligt, nachdem sie sich in aktive amöboide Formen verwandeln, die den Regenerationsprozess durchführen.

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Part of this investigation is based on a portion of a dissertation submitted to the Graduate School of the University of South Carolina in partial fulfillment of the requirements for the degree of Doctor of Philosophy.     

Light and electron microscopy of virus inclusions inAmaranthus lividus cells

Summary Amaranthus plants infected with a virus of rod-shaped particles showed under the light microscope intracytoplasmic amorphous and crystalline inclusions.The submicroscopic organization of mesophyll cells from infectedAmaranthus leaves by electron microscopy is described. Besides big crystalline inclusions, long dark inclusions correspondent to needle-like inclusions observed by light microscopy are definable in the cytoplasm. The amorphous inclusion bodies were formed by an overgrown protrusion of vacuolate cytoplasm containing virus particles, long very dark stained inclusions forming dense bands and rings, normal elements of the cytoplasm such as mitochondria, endoplasmic reticulum and ribosomes, and some spherosomes. Inclusions and virus particles were not found in chloroplasts, mitochondria or nuclei of infected cells.     

Unusual polyphosphate inclusions observed in a marine Beggiatoa strain

Sulfide-oxidizing bacteria of the genus Beggiatoa are known to accumulate phosphate intracellularly as polyphosphate but little is known about the structure and properties of these inclusions. Application of different staining techniques revealed the presence of unusually large polyphosphate inclusions in the marine Beggiatoa strain 35Flor. The inclusions showed a co-occurrence of polyphosphate, calcium and magnesium when analyzed by scanning electron microscopy and energy dispersive X-ray analysis. Similar to polyphosphate-enriched acidocalcisomes of prokaryotes and eukaryotes, the polyphosphate inclusions in Beggiatoa strain 35Flor are enclosed by a lipid layer and store cations. However, they are not notably acidic. 16S rRNA gene sequence-based phylogenetic reconstruction showed an affiliation of Beggiatoa strain 35Flor to a monophyletic branch, comprising other narrow vacuolated and non-vacuolated Beggiatoa species. The polyphosphate inclusions represent a new type of membrane surrounded storage compartment within the genus Beggiatoa, distinct from the mostly nitrate-storing vacuoles known from other marine sulfide-oxidizing bacteria of the family Beggiatoaceae.     

Location of the crystal toxin gene ofBacillus thuringiensis var.aizawai

Mutants of two different isolates ofBacillus thuringiensis var.aizawai that are different from the parental strains in their plasmid profile were generated by plasmid curing experiments. Examination of these mutants by microscopy resulted in the identification of derivatives containing only the small crystal inclusions as well as those containing both the small and the large crystal inclusions. Acrystalliferous variants were also identified. Analysis of the plasmid profiles of these mutants along with the results from the bioassays seem to indicate that the spodopteran-active -endotoxin (i.e., small crystal inclusions) gene of theB. thuringiensis var.aizawai isolates used in this study is most likely located on the chromosome. The determinants of the large crystal inclusions (nontoxic toSpodopera exigua), however, appear to be located on either a plasmid (of 46 mega-Daltons in size) of one isolate or the chromosome of the other.     

Sperm transfer and storage in the lizard,Anolis carolinensis

The relationship of the hemipenis to the cloaca in copula and sperm storage and transport in the female oviduct were studied in Anolis carolinensis using light and scanning electron microscopy. During copulation, the hemipenis does not penetrate beyond the cloaca, but the two apical openings of the bifurcate sulcus spermaticus appose the openings of the oviducts from the cloaca. Sperm enter the sperm storage tubules between 2 and 6 hr after insemination and small amounts of sperm reach the infundibulum 6 to 24 hr following mating. Sperm storage tubules are embedded in the wall of the utero-vaginal transition, and are formed by the folding and fusion of the oviducal epithelium. The importance of the hemipenile-cloacal relationship and the role of sperm storage in the life history of A. carolinensis are discussed.     

Further studies of the sieve-element plastids of theCaryophyllales includingBarbeuia,Corrigiola, Lyallia,Microtea, Sarcobatus,andTelephium

The sieve-element plastids of 69 species of theCaryophyllales were investigated by transmission electron microscopy. All contained the specific subtype-P3 plastids characterized by a peripheral ring of protein filaments. The presence or absence of an additional central protein crystal and their shape being either polygonal or globular as well as the average sizes of the sieve-element plastids are useful features in the characterization of some families.—Barbeuia contains sieve-element plastids that confirm its placement within thePhytolaccaceae. Lyallia differs fromHectorella by including small starch grains in their sieve-element plastids, which otherwise by their globular crystals negate a closer connection to theCaryophyllaceae. The lack of a central protein crystal in its form-P3fs plastids placesMicrotea best within theChenopodiaceae. Sarcobatus, a so far uncontested member of theChenopodiaceae, contains form-P3cf plastids, i.e., including a central crystal not found elsewhere in this family.Telephium andCorrigiola, shifted back and forth betweenMolluginaceae andCaryophyllaceae, have form-P3cf(s) plastids with a polygonal crystal which favor their placement within theCaryophyllaceae.     

Correlation of Activity and Amount of Ribulose 1,5-bisphosphate Carboxylase with Chloroplast Stroma Crystals in Water-Stressed Willow Leaves

Changes in the activity and amount of ribulose 1,5-bisphosphate(RuBP)carboxylase (E.C. 4.1.1.39 [EC] ) were studied in well-watered plantsof Salix ‘aquatica gigantea’ and in similar plantsduring three different water stress treatments and after rewatering.The chloroplast ultrastructure of these plants was examinedby electron microscopy. The amounts of crystallized proteinin the chloroplast stroma were assessed according to the areaof crystal structure seen in the thin sections. RuBP carboxylase activity decreased with decreasing leaf waterpotentials but recovered upon rewatering, except when leaveshad been exposed to severe water stress. The percentage of totalchloroplast area made up of crystal inclusions decreased withdecreasing leaf water potentials. After rewatering, the crystalseither disappeared or the amount decreased markedly. Both RuBPcarboxylase activity and the area of crystal inclusions increasedinitially with increased extractable RuBP carboxylase proteinbut decreased with further increases above 6700–7000 µgRuBP carboxylase protein mg^–1 chlorophyll. In well-wateredand water-stressed plants the activity of RuBP carboxylase,based on amount of chlorophyll, increased with an increasingamount of crystal inclusions in the chloroplast stroma. In rewateredplants no such correlation was observed, and the low percentageof crystal inclusions in the chloroplast area was independentof RuBP carboxylase activity. Key words: Chloroplast stroma crystals, ribulose 1,5-bisphosphate carboxylase, Salix, water stress     

Structure, Cytochemistry, and Locomotion of Haemogregarina sp. from Rana berlandieri

Intra- and extracellular gametocytes of Haemogregarina sp. from Rana berlandieri were studied by light and electron microscopy. Locomotion in free gametocytes appears to be related to series of horizontal “peristaltic” waves of constriction, passing from anterior to posterior along the body. Intracellular gametocytes lie within a vacuole in the erythrocyte cytoplasm. The pellicle of the parasite consists of a trilaminar plasmalemma and an inner electron dense layer, beneath which lies a ring of 80 microtubules. The inner dense layer becomes thickened and modified in the apical region, to form a cap-like structure. The gametocytes contain a prominent nucleus, several mitochondria, and many granular inclusions. One type of inclusion consists of elliptical, electron-dense, profeinaceous bodies scattered throughout the cytoplasm, while other inclusions are larger and electron-opaque, polysaccharide in nature, and occur predominantly in the pre- and post-nuclear regions. In the electron microscope, pronounced pellicular folds were observed in longitudinally sectioned extracellular gametocytes. These folds are thought to represent the waves of constriction seen in motile specimens by light microscopy. The mechanism of movement of the parasite is discussed and compared with that in haemosporidian ookinetes, as well as in gregarines.     

Trypanosoma cruzi epimastigotes are able to store and mobilize high amounts of cholesterol in reservosome lipid inclusions

Background

Reservosomes are lysosome-related organelles found in Trypanosoma cruzi epimastigotes. They represent the last step in epimastigote endocytic route, accumulating a set of proteins and enzymes related to protein digestion and lipid metabolism. The reservosome matrix contains planar membranes, vesicles and lipid inclusions. Some of the latter may assume rectangular or sword-shaped crystalloid forms surrounded by a phospholipid monolayer, resembling the cholesterol crystals in foam cells.

Methodology/Principal Findings

Using Nile Red fluorimetry and fluorescence microscopy, as well as electron microscopy, we have established a direct correlation between serum concentration in culture medium and the presence of crystalloid lipid inclusions. Starting from a reservosome purified fraction, we have developed a fractionation protocol to isolate lipid inclusions. Gas-chromatography mass-spectrometry (GC-MS) analysis revealed that lipid inclusions are composed mainly by cholesterol and cholesterol esters. Moreover, when the parasites with crystalloid lipid-loaded reservosomes were maintained in serum free medium for 48 hours the inclusions disappeared almost completely, including the sword shaped ones.

Conclusions/Significance

Taken together, our results suggest that epimastigote forms of T. cruzi store high amounts of neutral lipids from extracellular medium, mostly cholesterol or cholesterol esters inside reservosomes. Interestingly, the parasites are able to disassemble the reservosome cholesterol crystalloid inclusions when submitted to serum starvation.     

Ultrastructural features of the retina and optic nerve of Strombus luhuanus,a marine gastropod

The retina and optic nerve of Strombus luhuanus were examined by transmission electron microscopy in order to provide an ultrastructural basis for their electrophysiological responses, described elsewhere. The retina exhibits a distinct rhabdomeric layer and layers of cell nuclei and neuropile. These layers are comprised predominantly of three cell types that can be readily distinguished on the basis of their shape, their nuclei and cytoplasmic inclusions such as vesicles and filaments. One type of cell, apparently a photoreceptor that depolarizes in response to photic stimulation, possesses a long distal segment with microvilli; such distal segments comprise the bulk of the rhabdomeric layer. A second cell type, which appears to be supportive in function, contains a bundle of tightly packed tonofilaments that extend across the retina from the capsule to the vitreous body; this cell is quite narrow except in the region near the rhabdomeric layer, where it is expanded and wraps around the other cell types. A third type of cell possesses many short microvilli that project from its apical end into the rhabdomeric layer; it may be a second type of photoreceptor or another type of neuron. The retina also contains bundles of cilia that appear to project from a possible fourth type of cell. The layer of neuropile contains numerous processes that exhibit a variety of vesicle types and structures generally associated with synapses; these appear to play a role in mediating inhibitory and excitatory interactions between the retinal neurons. The optic nerve exhibits two populations of fiber distinguishable on the basis of mean diameter. Fibers in these two populations apparently yield “on” and “off” discharges in response to photic stimulation of the eye.     

INCLUSIONS OF THE PROPLASTIDS AND VACUOLES IN THE SHOOT APICES OF BRYOPHYLLUM AND KALANCHOË

Spherical, membrane-bound inclusions occur in the proplastids and vacuoles of cells of Bryophyllum and Kalanchoë shoot apices. Evidence is presented indicating that the inclusions arise by the accumulation of material within the cisternae of certain tubular proplastid membranes and are then transferred to vacuoles. The results obtained from electron microscopy and from histochemical studies indicate that the contents of the inclusions are predominantly lipid.