Characterization of intracytoplasmic hydrocarbon inclusions from the hydrocarbon-oxidizing Acinetobacter species HO1-N.

The ultrastructure of Acinetobacter sp. strain HO1-N grown on hydrocarbon and nonhydrocarbon substrates was compared using thin sections and freeze-etching. Hydrocarbon-grown cells were characterized by the presence of intracytoplasmic membrane-bound hexadecane inclusions. This membrane did not exhibit a typical unit membrane structure but appeared as a monolayer. The freeze-etch technique revealed the internal structure of the hexadecane inclusions and provided evidence for the presence of a smooth-surfaced limiting membrane. Freeze-etching also revealed intracytoplasmic membranes in the hexadecane-grown cells. These ultrastructural modifications were not present in nonhydrocarbon-grown cells. The hexadecane inclusions were isolated from Acinetobacter. Negative-staining of the inclusions revealed electron-transparent vesicles approximating the size of the inclusions seen in whole cells. Freeze-etching of the purified inclusions revealed membrane-bound vesicles. The purified inclusions exhibited a relatively high value of lipid phosphorus to protein. The lipid composition and the electrophoretic banding pattern of the inclusions on sodium dodecyl sulfate-polyacrylamide gels were determined and compared with other membrane fractions (outer membrane and cytoplasmic membrane) previously isolated from this organism.     

Ultrastructural organization of Alicyclobacillus tolerans strain K1T cells

Electron microscopy examinations of thin sections and freeze-fracture replicas revealed the specific ultrastructural features of Alicyclobacillus tolerans strain K1(T). In particular, the cell wall displayed an ultrastructure typical of gram-positive bacteria and consisted of a thin murein layer (50-60 A in thickness); cells exhibited a surface S-layer constituted by large hexagonally packed (p6-symmetry) rod-shaped subunits of 150-160 A in diameter and 200 A in height. In the cytoplasmic membrane, there were intramembrane vesicular structures that sometimes appeared as large leaflets in the central part. The cytoplasm contained numerous vesicular inclusions covered with a monolayered wall, dissimilar to bilamellar lipid membranes. Endospore coats displayed an intricate structure and consisted of three thick layers; the outer layer had an unusual fine structure; the exosporium was also found.     

Comparative characteristics of the ultrastructure of the causative agents of glanders and melioidosis]

The authors examined the ultrastructure of the causative agents of glanders and melio idosis. It was revealed that the structure of their cell wall and of the cytoplasmic membrane was characteristic of Gram negative bacteria. The cytoplasm of both types of the causative agents showed the presence of ribosomes, membrane structure, nucleoid, and also osmiophilic and osmiophobic inclusions.     

Cytopathology of Lettuce Mosaic Virus-infected Lettuce Seeds and Seedlings

The ultrastructure of lettuce mosaic potyvirus (LMV)-infected lettuce seeds and seedlings was studied by transmission electron microscopy. Conventional thin-section electron microscopy and immunogold cytochemistry were both successfully employed to study the location of LMV in embryonic and non-embryonic seed parts. LMV particle aggregates and cytoplasmic “pinwheel” inclusions characteristic of potyviruses were observed throughout the embryonic tissues (radicle, hypocotyl and cotyledon) of infected lettuce seeds and seedlings, and also in the non-embryonic endosperm layer. LMV particles, but not inclusions, were also located in the non-embryonic pericarp layer.     

Histology, ultrastructure, and morphogenesis of a rickettsia-like organism causing disease in the oyster, Crassostrea ariakensis Gould

Moribund specimens of the oyster, Crassostrea ariakensis Gould, aged 2-3 years were collected from Hailing Bay in Yangxi County of Guangdong Province from February to May and November to December in the years 2001, 2002, and 2003. A massive infection by an obligate intracellular prokaryote, specifically a rickettsia-like organism (RLO), was found. Here we report investigations of this RLO in the tissues of the oyster C. ariakensis Gould and describe the histology, ultrastructure, and morphogenesis of this pathogen in C. ariakensis Gould. Light microscopic observations of stained tissues revealed cytoplasmic inclusion bodies typical of prokaryote infection in about 87% (26/30) of the oysters. Most inclusions were observed in epithelial cells and connective tissues of the gill, mantle, and digestive gland of most of the infected oysters. The shape, size, and color of inclusions from different tissues were polymorphic. Electron microscopic examination of digestive gland, gill, and mantle tissues showed that the RLOs were intracytoplasmic. RLOs were often round, dumb-bell-shaped (undergoing binary fission), or occasionally rod-shaped and ranged from approximately 0.58 to 1.20microm in size. The organisms exhibited an ultrastructure characteristic of prokaryotic bacteria-like cells, including a trilaminar cell wall, electron-dense periplasmic ribosome zone, and a DNA nucleoid. Reproductive stages, including transverse binary fission, were observed by TEM. These stages were frequently observed within membrane-bound cytoplasmic vacuoles. Hexagonal phage-like particles in the cytoplasm of RLOs were also observed.     

Ficus carica L. leaf anatomy: Trichomes and solid inclusions

Ficus carica L., a typical plant of the Mediterranean environment, presents leaves covered by an extensive indumentum, and a mesophyll full of solid inclusions. The morphology and ultrastructure of the trichomes, calcium carbonate cystoliths and silicified structures of leaves of F. carica cv Dottato were investigated with light, confocal, scanning electron microscopy, and energy-dispersive X-ray spectroscopy. At the same time, histochemical reactions were also employed to analyse the indumentum composed by glandular and non-glandular trichomes by applying chemical reagents and fluorescence microscopy. Non-glandular and glandular trichomes, capitate, are described. Non-glandular trichomes are unicellular simple, spine-like and present different morphology and sizes. The capitate glandular trichomes are present on leaf adaxial and abaxial surface and consist of one-celled stalk and 3/4 cells spherical head. Histochemical characterisation of leaf hairs revealed the presence of flavonoids, while glandular trichome head cells showed a complex mixture of alkaloids, essential oil and flavonoids. Cu and Al were found in the constitutive structures, spike and dome, of the cystoliths. Several epidermal cells and non-glandular trichomes were silicified. Leaf hairs, trichomes secretions, solid inclusions and silicification of F. carica leaf have significant roles to play in relation to leaf protection from external factors, including high-intensity radiation, herbivores or pathogens.     

An ultrastructural study of oogenesis in the planarian Schmidtea mediterranea (Platyhelminthe, Paludicola)

The ovary of the freshwater planarian Schmidtea mediterranea has been studied for the first time using both light and electron microscopy methods. The ultrastructure of the ovary revealed two types of cells: accessory cells and germinal cells at various stages of differentiation, distributed along a maturation axis. Initially, oogonia underwent cytoplasm growth due to the development of organelles, such as endoplasmic reticulum, Golgi complex, and mitochondria, which are all involved in the production of cytoplasmic inclusions or yolk globules. It is shown that the chromatoid body and fibrogranular aggregates may participate in the synthesis of vitelline inclusions. When completely mature, the oocytes have become larger, due to the accumulation of nutritive inclusions, which are round in shape and have a paracrystalline structure. These inclusions are interpreted as being yolk globules and may represent a kind of nutritive material for the developing embryo. These ultrastructural features of the ovary agree with the available phylogenetic tree, based on morphological and karyological characters that considers Schmidtea group as a genus and not a subgenus. The presence of sperm between the oocytes suggests that fertilization may occur within the ovary, representing an uncommon condition within the Triclads, in which fertilization usually takes places outside of the ovaries.     

On the morphology of the human sertoli cell

Summary As revealed by light microscopical investigations the human Sertoli cell presents different appearances according to the pattern of infranuclear cytoplasmic inclusions. Although two or three stages of spermatogenesis are seen in a single cross section of a seminiferous tubule the Sertoli cells all show virtually the same features in such a cross sectioned tubule.The different appearances are also evident under the electron microscope. Although no obvious correlation was found with the stages of spermatogenesis in the seminiferous epithelium, the Sertoli cell appearances described here may be assumed to represent different metabolic situations.Other features of Sertoli cell ultrastructure are discussed such as the presence of residual bodies in the apical cytoplasm, glycogen-rich areas protruding towards the tubular lumen or the extracellular space, and membrane bound, round structures, found between the membranes of the smooth endoplasmic reticulum and resembling the microbodies of steroid producing cells.Presented in part at the 69^th Versammlung der Anatomischen Gesellschaft, Kiel, 1974.     

Cytochemical localization of glycogen in Chlamydia trachomatis inclusions.

The origin and distribution of glycogen in inclusions of Chlamydia trachomatis were demonstrated with silver proteinate stain for electron microscopy. Glycogen particles were detected in all developmental stages of C. trachomatis, as well as free in the inclusions. Intrachlamydial glycogen was most common in elementary bodies but was also detected in intermediate forms and reticulate bodies (RBs). Abnormal divisions and breakdown of cytoplasmic membranes were common in RBs. Cytoplasmic contents, including glycogen particles, were released into the inclusions after rupture of the outer membranes of abnormal RBs and intermediate forms. From these observations, we conclude that glycogen in inclusions of C. trachomatis originates in the organisms themselves.     

Purification and Characterization of a Jordanian Isolate of Watermelon Mosaic Virus

The Jordanian isolate of watermelon mosaic virus-2 (WMV-2Jo) was purified from infected Cucurbita pepo cv. Top Capi by extraction in 0.05 M sodium citrate buffer containing 0.01 M sodium diethyl dithiocarbamate and 0.01 M cysteine hydrochloride (0.01 M D + C), clarification with chloroform and n-butanol, sedimentation by ultracentrifugation, and rate-zonal centrifugation in 10–40% sucrose gradient. The purified virus had an ultraviolet absorption spectrum typical of a nucleoprotein with a low nucleic acid content. Homologous antiserum had a titre of 1: 256, as determined by the ring interface test. Electron microscopy of negatively stained purified virus revealed flexuous particles with a normal length of 750 nm. Cytoplasmic spindle-shaped inclusions were readily detectable in infected epidermal cells under the light microscope. Thin sections of infected tissue revealed the presence of laminated aggregates, pinwheel and scroll inclusions. The virus reacted with antisera prepared to the Florida strain of WMV-2 and zucchini yellow mosaic virus in a sodium dodecyl sulfate (SDS) agar gel diffusion test. Using the Derrick-decoration combined technique of immune electron microscopy, the virus reacted strongly with the homologous antiserum and zucchini yellow mosaic virus antiserum, but less with antiserum prepared to the Florida strain of WMV-2.     

Circulating hemocytes from larvae of the paper wasp Polistes dominulus (Hymenoptera, Vespidae)

Circulating hemocytes from larval stages of the paper wasp Polistes dominulus were characterized by light and transmission electron microscopy. Three types were identified: prohemocytes, plasmatocytes and granulocytes. The first two are agranular cells while the latter present typical cytoplasmic inclusions called granules. Plasmatocytes differ from prohemocytes being larger, showing lower nucleus/cytoplasm ratio and they possess many phagolysosomes. The substantial uniformity of most subcellular features and the presence of "intermediate forms" support the "single-cell theory" i.e., there is only one cell line that originates from the prohemocyte and leads to the granular cell passing through the plasmatocyte. This hypothesis seems to be confirmed by functional tests. Indeed, most part of cells adheres to the glass and is able to phagocytize fluorescent microspheres.     

The fine structure of the midgut in the mite Anystis baccarum (L.) (Acari, Actinedida: Anystidae)

The ventriculus and the midgut caeca of the fed females of Anystis baccarum (L.) were investigated by using light and electron microscopy. In addition to the main type of polyfunctional digestive cells, special secretory cells were detected in the anterior region of the ventriculus. The shape and the ultrastructure of the digestive cells vary depending on their physiological state. Intracellular digestion, absorption or excretion processes prevail at different stages of the cell cycle. The secretory cells are characterized by the presence of extensive rough endoplasmic reticulum, filling whole space of the cell. These cells do not contain the apical network of pinocytotic canals, which are typical for the digestive cells. Three types of secretory granules were found in the cytoplasm of the secretory cells that probably correspond to three sequential stages of granulogenesis. The primary secretory granules are formed by the fusion of Golgi vesicles. The primary granules fuse to form complex vesicles with heterogeneous contents. These secondary granules aggregate to form very large inclusions of high electron density (tertiary secretory granules), which probably represent the storage of the secretory product. All types of secretory granules were observed close to the apical plasmalemma.     

Morpho–anatomical and ultrastructural analysis of extrafloral nectaries in Inga edulis (Vell.) Mart. (Leguminosae)

The presence of extrafloral nectaries (EFNs) between leaflets is an usual feature in Inga edulis (Vell.) Mart. (Leguminosae). Extrafloral nectaries are secretory structures involved in production of nectar and which serve in the protection of plants against herbivores through association with ants. This study aimed to characterize the EFNs of I. edulis at different developmental stages and describe their morphology, histochemistry and ultrastructure. Leaf fragments, containing secretory structures, were processed according to standard methods for light, scanning and transmission electron microscopy. The EFNs were classified into three stages based on morphology: pre‐secretory, secretory and post‐secretory. The pre‐secretory stage occurs in young leaves, whereas secretory and post‐secretory stages occur in developed and senescent leaves, respectively. The EFNs possess a concave surface and a central cleft in which nectar is accumulated and which was not observed in pre‐secretory EFNs. Histochemical tests identified the presence of sugars, proteins, phenolic compounds, mucilage and lipids at all developmental stages of the EFNs. Calcium crystals were identified in all tissues and stages of the EFNs. The secretory cells of the EFNs exhibit a granular cytoplasm, small vacuoles, prominent nuclei, smooth endoplasmic reticulum and mitochondria. Post‐secretory stage EFNs exhibited intense cytoplasmic degradation and the presence of microorganisms. The performance of EFNs of I. edulis appear to follow the leaf development.     

Reovirus Replication Protein μ2 Influences Cell Tropism by Promoting Particle Assembly within Viral Inclusions

The double-stranded RNA virus mammalian reovirus displays broad cell, tissue, and host tropism. A critical checkpoint in the reovirus replication cycle resides within viral cytoplasmic inclusions, which are biosynthetic centers of genome multiplication and new-particle assembly. Replication of strain type 3 Dearing (T3) is arrested in Madin-Darby canine kidney (MDCK) cells at a step subsequent to inclusion development and prior to formation of genomic double-stranded RNA. This phenotype is primarily regulated by viral replication protein μ2. To understand how reovirus inclusions differ in productively and abortively infected MDCK cells, we used confocal immunofluorescence and thin-section transmission electron microscopy (TEM) to probe inclusion organization and particle morphogenesis. Although no abnormalities in inclusion morphology or viral protein localization were observed in T3-infected MDCK cells using confocal microscopy, TEM revealed markedly diminished production of mature progeny virions. T3 inclusions were less frequent and smaller than those formed by T3-T1M1, a productively replicating reovirus strain, and contained decreased numbers of complete particles. T3 replication was enhanced when cells were cultivated at 31°C, and inclusion ultrastructure at low-temperature infection more closely resembled that of a productive infection. These results indicate that particle assembly in T3-infected MDCK cells is defective, possibly due to a temperature-sensitive structural or functional property of μ2. Thus, reovirus cell tropism can be governed by interactions between viral replication proteins and the unique cell environment that modulate efficiency of particle assembly.     

Membrane-bound intranuclear inclusions in the Leydig cell of the Chinese hamster (Cricetulus griseus)

Membrane-bound intranuclear inclusions have been described, for the first time, in the Leydig cell of the Chinese hamster (Cricetulus griseus). The inclusions were not found in the 1-day-old animal, rarely found prior to sexual maturity, and commonly found in the sexually mature animals. The incidence of inclusions increases with aging. Their size and content varies greatly. They are surrounded by a single membrane and completely enclosed by nucleoplasm. Their close association with nuclear invaginations of cytoplasmic material, and their content of cytoplasmic structures along with some exhibiting the presence of trimetaphosphatase reaction product, suggest a cytoplasmic origin. This phenomenon involves the migration of cytoplasmic structures into the nucleus followed by detachment on the nucleoplasmic side. The presence of the inclusions is not an indication of an abnormality of the Leydig cell. The Leydig cell of the Chinese hamster may be an excellent model to study factors that initiate inclusion formation, and to determine the functional role of membrane-bound intranuclear inclusions.     

A novel betaproteobacterial agent of gill epitheliocystis in seawater farmed Atlantic salmon (Salmo salar)

Epitheliocystis, a disease characterised by cytoplasmic bacterial inclusions (cysts) in the gill and less commonly skin epithelial cells, has been reported in many marine and freshwater fish species and may be associated with mortality. Previously, molecular and ultrastructural analyses have exclusively associated members of the Chlamydiae with such inclusions. Here we investigated a population of farmed Atlantic salmon from the west coast of Norway displaying gill epitheliocystis. Although 'Candidatus Piscichlamydia salmonis', previously reported to be present in such cysts, was detected by PCR in most of the gill samples analysed, this bacterium was found to be a rare member of the gill microbiota, and not associated with the observed cysts as demonstrated by fluorescence in situ hybridization assays. The application of a broad range 16 S rRNA targeted PCR assay instead identified a novel betaproteobacterium as an abundant member of the gill microbiota. Fluorescence in situ hybridization demonstrated that this bacterium, tentatively classified as 'Candidatus Branchiomonas cysticola', was the cyst-forming agent in these samples. While histology and ultrastructure of 'Ca. B. cysticola' cysts revealed forms similar to the reticulate and intermediate bodies described in earlier reports from salmon in seawater, no elementary bodies typical of the chlamydial developmental cycle were observed. In conclusion, this study identified a novel agent of epitheliocystis in sea-farmed Atlantic salmon and demonstrated that these cysts can be caused by bacteria phylogenetically distinct from the Chlamydiae.     

Hepatocyte-derived cultured cells with unusual cytoplasmic keratin-rich spheroid bodies

Cytoplasmic inclusions are found in a variety of diseases that are characteristic morphological features of several hepatic, muscular and neurodegenerative disorders. They display a predominantly filamentous ultrastructure that is also observed in malignant rhabdoid tumor (MRT). A cellular clone containing an intracytoplasmic body was isolated from hepatocyte cell culture, and in the present study we examined whether this body might be related or not to Mallory-Denk body (MDB), a well characterized intracytoplasmic inclusion, or whether this cellular clone was constituted by malignant rhabdoid tumor cells. The intracytoplasmic body was observed in electron microscopy (EM), confocal immunofluorescence microscopy and several proteins involved in the formation of its structure were identified. Using light microscopy, a spheroid body (SB) described as a single regular-shaped cytoplasmic body was observed in cells. During cytokinesis, the SB was disassembled and reassembled in a way to reconstitute a unique SB in each progeny cell. EM examination revealed that the SB was not surrounded by a limiting membrane. However, cytoplasmic filaments were concentrated in a whorled array. These proteins were identified as keratins 8 and 18 (K8/K18), which formed the central core of the SB surrounded by a vimentin cage-like structure. This structure was not related to Mallory-Denk body or aggresome since no aggregated proteins were located in SB. Moreover, the structure of SB was not due to mutations in the primary sequence of K8/K18 and vimentin since no difference was observed in the mRNA sequence of their genes, isolated from Huh-7 and Huh-7w7.3 cells. These data suggested that cellular factor(s) could be responsible for the SB formation process. Aggregates of K18 were relocated in the SB when a mutant of K18 inducing disruption of K8/K18 IF network was expressed in the cellular clone. Furthermore, the INI1 protein, a remodeling-chromatin factor deficient in rhabdoid cells, which contain a spheroid perinuclear inclusion body, was found in our cellular clone. In conclusion, our data suggest that Huh-7w7.3 cells constitute an excellent model for determining the cellular factor(s) involved in the process of spheroid perinuclear body formation.     

Primary sideroblastic anemia masked by bleeding.

A patient with characteristic features of iron deficiency was unexpectedly found to have circulating siderocytes. Bone marrow iron stain at this time showed absence of both hemosiderin and ringed sideroblasts; electron microscopy revealed absence of mitochondrial iron loading but presence of cytoplasmic ferritin in normoblasts. Replenishment of iron stores led to development of typical sideroblastic anemia. These observations suggest that increased percentage of siderocytes in otherwise typical iron deficiency anemia may signify the presence of a sideroblastic process masked by iron deficiency due to bleeding.     

Intranuclear structures in pyrimethamine-resistant isolates of the malaria parasite Plasmodium falciparum

The ultrastructure of the malaria parasite Plasmodium falciparum is well known, both from natural infections and from culture material ( Aikawa , 1977, Langreth et al., 1978). It is noteworthy that all of these studies were done with pyrimethamine-sensitive strains, e.g. FCR-3/Gambia. Except for spindle microtubules during schizogony, no intranuclear structures have been described in any of the asexual erythrocytic stages. In the course of isolating clones from the pyrimethamine-resistant strain Honduras I/CDC (V.K. Bhasin and W. Trager , in print) and checking by electron microscopy for the presence or absence of knobs, we noticed intranuclear structures that might be correlated with pyrimethamine resistance. For comparison, we then examined the multi-drug-resistant strain Indochina 1. We present here a first report on these structures as a basis for further studies.