Showing papers on "Ultrastructure published in 2010"

Telocytes in human term placenta: morphology and phenotype.

Abstract: In the last few years, a new cell type - interstitial Cajal-like cell (ICLC) - has been described in digestive and extra-digestive organs. The name has recently been changed to telocytes (TC) and their typical thin, long processes have been named telopodes (TP). To support the hypothesis that TC may also be present in human placenta and add to the information already available, we provide evidence on the ultrastructure, immunophenotype, distribution, and interactions with the surrounding stromal cells of TC in the villous core of human term placenta. We used phase-contrast microscopy, light microscopy of semithin sections, transmission electron microscopy, immunohistochemistry, and immunofluorescence of tissue sections or cell cultures, following a pre-established diagnostic algorithm. Transmission electron microscopy showed cells resembling TC, most (∼76%) having 2-3 very thin, longprocesses (tens to hundreds of micrometers), with an uneven calibre(≤0.5 μm thick) and typical branching pattern. The dilations of processes accommodate caveolae, endoplasmic reticulum cisternae, and mitochondria. These TC have close contacts with perivascular SMC in stem villi. In situ, similar cells are positive for c-kit, CD34, vimentin, caveolin-1, vascular endothelial growth factor (VEGF), and inducible nitric oxide synathase (iNOS). The c-kit-positive cells inconsistently co-express CD34, CD44, αSMA, S100, neuron-specific enolase, and nestin. Among cells with a morphologic TC profile in cell cultures, about 13% co-express c-kit, vimentin, and caveolin-1; 70% of the c-kit-positive cells co-express CD34 and 12% co-express iNOS or VEGF. In conclusion, this study confirms the presence of TC in human term placenta and provides their ultrastructural and immunophenotypic characterization.

From dynamic live cell imaging to 3D ultrastructure: novel integrated methods for high pressure freezing and correlative light-electron microscopy.

Abstract: Background. In cell biology, the study of proteins and organelles requires the combination of 4 different imaging approaches, from live recordings with light microscopy (LM) to electron 5 microscopy (EM). 6 Methodology. To correlate dynamic events in adherent cells with both ultrastructural and 3D 7 information, we developed a method for cultured cells that combines confocal time-lapse 8 images of GFP-tagged proteins with electron microscopy. With laser micro-patterned culture 9 substrate, we created coordinates that were conserved at every step of the sample 10 preparation and visualization processes. Specifically designed for cryo-fixation, this method 11 allowed a fast freezing of dynamic events within seconds and their ultrastructural 12 characterization. We provide examples of the dynamic oligomerization of GFP-tagged 13 myotubularin (MTM1) phosphoinositides phosphatase induced by osmotic stress, and of the 14 ultrastructure of membrane tubules dependent on amphiphysin 2 (BIN1) expression. 15 Conclusion. Accessible and versatile, we show that this approach is efficient to routinely 16 correlate functional and dynamic LM with high resolution morphology by EM, with immuno- 17 EM labeling, with 3D reconstruction using serial immuno-EM or tomography, and with 18 scanning-EM. 19

Adhesive Exocrine Glands in Insects: Morphology, Ultrastructure, and Adhesive Secretion

Abstract: A literature survey is provided summarizing the available information on exocrine epidermal glands that produce adhesive secretions in insects. The focus is on both the ultrastructure of the gland cells and the identity and function of the chemical secretion produced by them. Insects employ adhesives for various functions such as tarsal attachment during locomotion, resisting external detachment forces, mating, phoresy and parasitism, egg anchorage, retreat building, self-grooming, prey capture, and active and passive defence. The available studies on the ultrastructure and the secretion of adhesive insect glands cover a broad spectrum of developmental stages and higher taxa, i.e., the Elliplura, the Ephemeroptera, the Polyneoptera, the Acercaria, the Coleoptera, the Amphiesmenoptera, the Hymenoptera, and the Diptera (Table 8.1). Based on this diversity of biological contexts and systematic groups, adhesive structures are found at various tagmata of the body, mainly at the head, the abdomen, and the legs, but also within the thorax in the form of the metapleural glands of ants. Class 1 epidermal cells are the predominant glandular cell type among the adhesive gland systems in insects. With respect to their ultrastructure, the adhesive class 1 cells show features (in terms of their provision with endoplasmic reticulum, Golgi system, free ribosomes, and secretion vesicles and granules) that are either indicative of predominant non-proteinaceous (lipid) or protein secretion. In class 1 cells that are employed in locomotion (i.e., reversible tarsal adhesion to natural substrates such as plant surfaces), lipoidal secretion seems to prevail (although these secretions often appear to be complex mixtures of lipids with proteins and carbohydrates), whereas in the contexts of more permanent body or egg anchorage and of retreat building, protein-based secretion dominates. Oenocyte-like class 2 adhesive gland cells have hitherto only been found in the defence systems of Aphidoidea and Tingidae (both Hemiptera). Adhesive class 3 glands are almost always bicellular, consisting of a terminal secretorily active cell and an adjacent canal cell that surrounds the cuticular conducting duct.

Cytoarchitecture of the desiccation-tolerant green alga Zygogonium ericetorum

Abstract: In this study, the filamentous green alga Zygogonium ericetorum (Zygnematales, Chlorophyta), collected at its natural habitat in the high alps, was investigated by light, scanning, and transmission electron microscopy. The field samples were separated into a moist fraction when wetted by splattering water of a nearby spring or a desiccated one when visually dried out. Light microscopy demonstrated a purple pigmentation of the sun-exposed upper layers, the central position of the nucleus, and the starch content in the pyrenoids. The smooth surface of the cells occasionally covered with fungal hyphae was shown by scanning electron microscopy. The cytoarchitecture of moist cells revealed many vacuoles and only a thin cytoplasmic area surrounding the two chloroplasts. The secondary cell walls of older cells were up to 4 µm thick. Organelle membranes as well as thylakoid membranes occasionally showed an inversion of contrast. In the chloroplasts, distinct areas with granular content surrounding the pyrenoids were detected. Within the cytoplasm, electron-dense particles with electron-translucent crystalloid structures were observed. When desiccated samples were investigated, the vacuoles and cytoplasmatic portions appeared destroyed, whereas nucleus and chloroplasts generally remained intact. The thylakoid membranes of desiccated samples showed lumen dilatations and numerous plastoglobules. Water-soluble extracts were separated by high-pressure liquid chromatography that revealed two major compounds with UV-absorbing capacities.

Analysis of the Ultrastructure of Archaea by Electron Microscopy

Abstract: The ultrastructural characterization of archaeal cells is done with both types of electron microscopy, transmission electron microscopy, and scanning electron microscopy. Depending on the scientific question, different preparation methods have to be employed and need to be optimized, according to the special cultivation conditions of these—in many cases extreme—microorganisms. Recent results using various electron microscopy techniques show that archaeal cells have a variety of cell appendages, used for motility as well as for establishing cell–cell and cell–surface contacts. Cryo-preparation methods, in particular high-pressure freezing and freeze-substitution, are crucial for obtaining results: (1) showing the cells in ultrathin sections in a good structural preservation, often with unusual shapes and subcellular complexity, and (2) enabling us to perform immunolocalization studies. This is an important tool to make a link between biochemical and ultrastructural studies.

The morphometrical analysis on the ultrastructure of A549 cells.

Abstract: Objective: To report the morphometric characteristics of ultrastructure inside A549 cells. Methods: A549 cells were processed for inverted microscopy and transmission electron microscopy (TEM). Cell images were obtained randomly using inverted microscopy and TEM. The morphometric parameters of ultrastructure were tested using precise morphometric techniques by Image-Pro Plus analysis software. Results: (1) The diameter of A549 cells from inverted microscopy and TEM images was 14.93 µm and 10.59 µm. (2) By defining cell as reference space the volume densities (VV) of nucleus and cytoplasm were about 0.28 and 0.72; the surface densities (SV) of nucleus were 0.19 µm -1 . By defining cell nucleus as reference space the V V of nucleoli, euchromatin and heterochromatin were 0.076, 0.72 and 0.20 respectively; the SV of nucleoli was 0.15 µm-1. By defining cytoplasm as reference space the VV of mitochondria, lamellar bodies and lysosomes were 0.046, 0.025 and 0.014; the SV of mitochondria, lamellar bodies and lysosomes were 0.60 µm-1, 0.36 µm-1, and 0.18 µm -1 . (3) In individual A549 cell total volume and surface of mitochondria were 61.91 µm 3 and 1001.67 µm 2 ; Total volume and surface area of lamellar bodies were 76.82 µm3 and 428.68 µm2; Total volume and surface area of lysosomes were 21.69 µm3 and 212.04 µm2. Conclusions: The morphometric parameters of some ultrastructures within A549 cells were established using precise morphometric techniques by Image-Pro Plus analysis software.

The effects of swimming exercise and supraphysiological doses of nandrolone decanoate on the testis in adult male rats: a transmission electron microscope study

Abstract: Anabolic-androgenic steroids (AAS) are used in high doses by athletes to improve athletic ability, physical appearance, and muscle mass. Unfortunately, the abuse of these agents has significantly increased. It has been established that exercise and high doses of AAS may influence the hypothalamic-pituitary gonadal (H-P-G) axis, which can in turn affect the ultrastructure of the testes. However, the effect of the combination of exercise and high doses of AAS on the ultrastructure of the testes is not known. This study was undertaken in order to examine the combination effects of swimming exercise and supraphysiological doses of nandrolone decanoate on the ultrastructural changes in rat testes. Five groups of male Wistar strain albino rats were treated as follows for 8 weeks: solvent of nandrolone decanoate (peanut oil) as a vehicle (sham); nandrolone decanoate (ND) (10 mg/kg/week) - ND; exercise (1 h/day, 5 days a week) - exercise; ND (10 mg/kg/week) and exercise (1 h/day, 5 days a week) - ND-EX; and sedentary control without any injection or exercise - control. Ultrastructural changes in the rat testes were characterised by transmission electron microscopy. The number and size of Leydig cells were considerably decreased in the interstitial space in the experimental rats. The increased thickness and irregular wavy multilaminar appearance of basement membrane in the treated animals, especially in the ND-EX group, are associated with well developed myoid cells. Cytoplasm vacuolisation, vesicular-like crista of the mitochondria, numerous lipid droplets, and lysosome and phagolysosome in Sertoli cells were significantly observed in the experimental groups. Several apoptotic germ cells were considerably observed in the experimental rats (p ≤ 0.05). Exercise training seems to increase the extent of ultrastructural changes caused by supraphysiological doses of ND in rats, which in turn may affect fertility.

Secondary Cell Wall Deposition in Developing Secondary Xylem of Poplar

Abstract: Although poplar is widely used for genomic and biotechnological manipulations of wood, the cellular basis of wood development in poplar has not been accurately documented at an ultrastructural level. Developing secondary xylem cells from hybrid poplar (Populus deltoides x P. trichocarpa), which were actively making secondary cell walls, were preserved with high pressure freezing/freeze substitution for light and electron microscopy. The distribution of xylans and mannans in the different cell types of developing secondary xylem were detected with immunofluorescence and immuno-gold labeling. While xylans, detected with the monoclonal antibody LM10, had a general distribution across the secondary xylem, mannans were enriched in the S2 secondary cell wall layer of fibers. To observe the cellular structures associated with secondary wall production, cryofixed fibers were examined with transmission electron microscopy during differentiation. There were abundant cortical microtubules and endomembrane activity in cells during the intense phase of secondary cell wall synthesis. Microtubule-associated small membrane compartments were commonly observed, as well as Golgi and secretory vesicles fusing with the plasma membrane.

Ultrastructure and large subunit rDNA-based phylogeny of Sphaerodinium cracoviense, an unusual freshwater dinoflagellate with a novel type of eyespot.

Abstract: Sphaerodinium cracoviense was collected near Cracow, Poland, and analysed by light microscopy, scanning electron microscopy, and serial-section transmission electron microscopy. Thecae showed a peridinioid type of plate arrangement with unusual numbers in the anterior intercalary and postcingular plate series: 4 and 6, respectively. The apical pore of S. cracoviense differed from the typical arrangement seen in many thecate forms and included a furrow with knob-like protuberances reminiscent of the apical area of some woloszynskioids. The flagellar apparatus included the three microtubular roots that extend to the left of the basal bodies and a striated root connective between the transverse striated root and the longitudinal microtubular root. Both the single-stranded root that associates with the right side of the longitudinal basal body in peridinioids and gonyaulacoids, and the layered connective typical of peridinioids were absent. The eyespot was formed by a layer of vesicle-contained crystal-like units underlain by layers of variably fused globules not bounded by membranes, and represents a novel type. The pusular system included a long canal with a dilated inner portion with radiating tubules. Bayesian and maximum likelihood analyses based on large subunit rDNA placed Sphaerodinium as a sister taxon to a group of woloszynskioids and relatively far from Peridinium and its allies.

Transmission electron microscopic observation on ultrastructural alterations in Schistosoma japonicum caused by mefloquine

Abstract: The purpose of the study was to explore the ultrastructural alterations of adult Schistosoma japonicum induced by mefloquine. Eight out of ten mice infected with 60–80 S. japonuicum cercariae for 35 days were treated orally with mefloquine at a single dose of 400 mg/kg. Four groups of two mice were killed at 8 h, 24 h, 3 days, and 7 days post-treatment, and schistosomes were collected by perfusion technique, fixed, and examined under a transmission electron microscope. Schistosomes obtained from the remaining two mice served as control. Eight hours after mefloquine 400 mg/kg was administered to the infected mice, various alterations in the tegument and subtegument tissues of both male and female worms were seen, which included focal lysis of tegmental matrix resulted in vacuole formation, decrease in rod-like and discoid-like secretary bodies, light swelling or focal lysis of musculature, extensive lysis of internal structure of sensory organelles, and appearance of vacuole or myelin-like structure in perinuclear cytoplasm of syncytium and epithelial cells. In vitelline cells of female worms, the most significant alteration was extensive lysis or fusion of vitelline balls in vitelline droplets and decrease in granular endoplasmic reticulum Twenty-four hours post-treatment, damage to the tegument and subtegument tissues had increased in severity. In male worms, the most prominent alterations were emergence of large vacuoles in the tegument, detachment of cytoplasmic process from the tegumental surface, focal collapse of internal structure of sensory organelle, and loss of definition of syncytium and gut epithelial cell. In female worms, focal lysis in tegumental matrix, musculatures, and parenchymal tissues resulted in emergence of vacuole or myelin-like structure, reduction of nucleoli, fusion of partial nuclear membrane together with cytoplasm in epithelial cell, and lysis of interstitial tissues among the vitelline cells which were universal. Three and 7 days post-treatment, besides the aforementioned alterations, the significant damage to the male worms were disrupted outer plasma membrane detached from the cytoplasmic process, swelling of individual cytoplasmic process, extensive swelling and focal lysis in the musculature, parenchymal tissues and perinuclear cytoplasm of syncytium, accompanied by emergence of swollen mitochondria, vacuoles, and myelin-like structure, and severe damage to gut epithelial cell. In female worms, apart from disruption of outer plasmic membrane in cytoplasmic process, severe swelling of tegumental matrix accompanied by emergence of vacuoles, swollen mitochondria and myelin-like structure, focal lysis of heterochromatin and nucleoli, disappearance of microvilli in gut epithelial cells, and emergence of myelin-like structures in vitelline cells were observed. The results indicate that administration of mefloquine to mice infected with adult S. japonicum exhibits an extensive damage to the ultrastructure in tegument and subtegument tissues including syncytium, gut epithelial cells, parenchymal tissues, and vitelline cells of schistosomes.

Ultrastructure of secretory and senescence phase in colleters of Bathysa gymnocarpa and B. stipulata (Rubiaceae)

Abstract: (Ultrastructure of secretory and senescence phase in colleters of Bathysa gymnocarpa and B. stipulata (Rubiaceae)). Colleters are secretory structures formed by a parenchymatic axis with vascular bundles, bound by a layer of secretory palisade-like epidermis. Some studies regarding the structure of colleters have focused on secretory cells structure, but not distinguished the secretory and senescent phases. Generally, in mucilage-secreting cells such as colleters, the endoplasmic reticulum and Golgi apparatus are involved in secretion production and transport. In these study, colleters structure of Bathysa gymnocarpa K. Schum. and B. stipulata (Vell.) C. Presl. (Rubiaceae) were determined in two phases: a secretory phase and a senescence one. Samples were collected and processed by usual light and electron microscopy techniques. Studied colleters are constituted by an epidermal palisade layer and a central axis formed by parenchymatic cells with rare vascular traces. During the secretory phase, epidermal cells presented a dense cytoplasm, small vacuoles, enhanced rough and smooth endoplasmic reticulum, and a Golgi apparatus close to large vesicles. During the senescence phase epidermal cells presented a disorganized membrane system. No intact organelles or vesicles were observed. The outer cell wall exhibited similar layers to that observed during the secretory phase. The senescent phase is easily defined by the morphology of the colleters, but not well defined at subcellular level. Our research suggests that programmed cell death starts on secretory phase. However, more evidences are needed to evaluate the phenomena.

Characterization of endocytic compartments after holo-high density lipoprotein particle uptake in HepG2 cells

Abstract: Holo-high density lipoprotein (HDL) particle uptake, besides selective lipid uptake, constitutes an alternative pathway to regulate cellular cholesterol homeostasis. In the current study, the cellular path of holo-HDL particles was investigated in human liver carcinoma cells (HepG2) using combined light and electron microscopical methods. The apolipoprotein moiety of HDL was visualized with different markers: horseradish peroxidase, colloidal gold and the fluorochrome Alexa568, used in fluorescence microscopy and after photooxidation correlatively at the ultrastructural level. Time course experiments showed a rapid uptake of holo-HDL particles, an accumulation in endosomal compartments, with a plateau after 1–2 h of continuous uptake, and a clearance 1–2 h upon replacement by unlabeled HDL. Correlative microscopy, using HDL-Alexa568-driven diaminobenzidine (DAB) photooxidation, identified the fluorescent organelles as DAB-positive multivesicular bodies (MVBs) in the electron microscope; their luminal contents but not the internal vesicles were stained. Labeled MVBs increased in numbers and changed shapes along with the duration of uptake, from polymorphic organelles with multiple surface domains and differently shaped protrusions dominating at early times of uptake to compact bodies with mainly tubular appendices and densely packed vesicles after later times. Differently shaped and labeled surface domains and appendices, as revealed by three dimensional reconstructions, as well as images of homotypic fusions indicate the dynamics of the HDL-positive MVBs. Double staining visualized by confocal microscopy, along with the electron microscopic data, shows that holo-HDL particles after temporal storage in MVBs are only to a minor degree transported to lysosomes, which suggests that different mechanisms are involved in cellular HDL clearance, including resecretion.

Ultrastructure of the Asexual Blood Stages of Plasmodium falciparum

Abstract: Plasmodium falciparum is the most deadly of the human malaria parasites. The particular virulence of this species derives from its ability to subvert the physiology of its host during the blood stages of its development. The parasite grows and divides within erythrocytes, feeding on the hemoglobin, and remodeling its host cells so they adhere to blood vessel walls. The advent of molecular transfection technology, coupled with optical microscopy of fluorescent protein reporters, has greatly improved our understanding of the ways in which the malaria parasite alters its host cell. However, a full interpretation of the information from these studies requires similar advances in our knowledge of the ultrastructure of the parasite. Here we give an overview of different electron microscopy techniques that have revealed the fine structure of the parasite at different stages of development. We present data on some of the unusual organelles of P. falciparum, in particular, the membrane structures that are elaborated in the erythrocyte cytoplasm and are thought to play an important role in trafficking of virulence proteins. We present and discuss some of the exciting whole cell imaging techniques that represent a new frontier in the studies of parasite ultrastructure.

Morphology and ultrastructure of epilithic versus cryptic, microbial growth in lower Cambrian phosphorites from the Montagne Noire, France

Abstract: The lower Cambrian grainy phosphorites of the northern Montagne Noire occur interbedded with grey to black, laminated to massive shales and limestones deposited along the edge of a continental shelf, associated with slope-related facies and unstable substrates. The concentration of phosphate took place by repeated alternations of low sedimentation rates and condensation (hardgrounds),in situearly-diagenetic precipitation offluorapatite, winnowing and polyphase reworking of previously phosphatized skeletons and hardground-derived clasts. The succession of repeated cycles of sedimentation, phosphate concentration, and reworking led to multi-event phosphate deposits rich in allochthonous particles. Phosphogenesis was primarily mediated by microbial activity, which is evidenced by the abundance of phosphatized putative microbial remains. These occur as smooth and segmentedfilaments, sheaths, and ovoid-shaped coccoids. These simple morphologies commonly form composite frameworks as a result of their aggregation and entanglement, leading to the record of biofilms, microbial mats, and complex networks. These infested the calcitic skeletonized microfossils that littered the substrate. Microbial activity evidences epilithic (anisotropic coatings on skeletons), euendolithic (perforating skeletal walls), and cryptoendolithic (lining inter- and intraparticulate pores) strategies, the latter dominated by bundles of filaments and globular clusters that grew along the cavities of helcionellids and hyoliths. According to their epilithic versus cryptic strategies, microbial populations that penetrated and dwelled inside hard skeletal substrates show different network and colonial morphologies. These early Cambrian shell concentrations were the loci of a stepwise colonization made by saprophytic to mutualistic, cyanobacterial‐fungal consortia. Their euendolithic and cryptoendolithic ecological niches provided microbial refugia to manage the grazing impact mainly led by metazoans.

Structural and ultrastructural characterization of the embryonic development of Pseudoplatystoma spp. hybrids.

Abstract: The hybrid fish Pseudoplatystoma spp. has been raised on a large scale by several fish farmers, despite the fact that little is known about its biology. This is because it presents a number of zootechnical advantages over the parental species. In order to provide information about the early morphology of this important species, we analyzed the fertilization and embryonic development of the hybrid between spotted females and barred males of sorubim specimens by light microscopy and by scanning (SEM) and transmission electron microscopy (TEM) after induced spawning. Samples were collected at pre-established moments up to larval hatching. Seven distinct stages of hybrid embryonic development were identified: zygote, cleavage, morula, blastula, gastrula, histogenesis and organogenesis, and hatching. Under SEM, we observed spermatozoa at the micropyle entrance, the formation of a fertilization cone in the eggs, the differentiation of cephalic and caudal regions, the neural tube and embryo growth along the cephalo-caudal axis, as well as rudimentary optic vesicle and barbels. Under light microscopy, cytoplasmic movement was apparent with the consequent formation of animal and vegetative poles in eggs, in addition to epiboly movements and a small notochord portion. Under TEM, the oocyte chorion and eggs presented a sieve-like aspect in transversal cuts, coupled with the rupture of cortical alveoli and chorion elevation, thus enlarging the perivitelline space. Several mitochondria in the cortical cytoplasm were detected in both oocytes and eggs. Overall, we observed that the larvae hatched without visible morphological alterations, and seemed to be as viable in captive systems as they are in the natural environment.

Life cycle, ultrastructure, and molecular phylogeny of Crispospora chironomi g.n. sp.n. (Microsporidia: Terresporidia), a parasite of Chironomus plumosus L. (Diptera: Chironomidae).

Abstract: The life cycle, ultrastructure, and molecular phylogeny of a new microsporidium Crispospora chironomi g.n. sp.n., a parasite of the midge Chironomus plumosus, are described. The parasite infects the gut epithelium of the host larvae and possesses sporogonies of two types, polysporoblastic and disporoblastic, respectively, proceeding within the same host cell. In the sporogonial sequence of the first type, dozens of spherical monokaryotic spores within a thick-walled capsule are formed. The spores are 1.5-2.0 μm in diameter; the exospore possesses two to three bundles of tubular protrusions. In the sporogonial sequence of the second type, diplokaryotic oval spores, 2.5 × 1.5 μm in size, are formed within a compartment, partially surrounded with multilayered membranes. Spores of both types are similar in respect to inner structure, possessing a well-developed extrusion apparatus with (a) the anterior vesicular part of the polaroplast covering the lamellar posterior one and (b) isofilar polar filament with several coils in one row. Small subunit ribosomal DNA phylogeny showed position of the new microsporidium in a cluster uniting microsporidia of terrestrial origin infecting diverse hosts, nested within Clade IV, corresponding to Class Terresporidia sensu Vossbrinck and Debrunner-Vossbrinck (Folia Parasitol 52:131-142, 2005).

Phylogenetic position of the genus Mesochytrium (Chytridiomycota) based on zoospore ultrastructure and sequences from the 18S and 28S rRNA gene

Abstract: Ultrastructural features and a molecular phylogeny of Mesochytrium penetrans (strain X-10 CALU) based on 18S and 28S rRNA gene sequences were investigated for the first time. The parasite is strongly specific for the green alga Chlorococcum minutum (Chlorococcales) and did not grow on another 29 strains of algae from the CALU collection. Most attention was paid to the zoospore ultrastructure. Ribosomes are dispersed through the cytoplasm. The nucleus and microbodylipid globule complex (MLC) is surrounded by rough endoplasmic reticulum. The MLC is composed of a single mitochondrion and a single lipid globule partially covered with a microbody and a fenestrated cisterna, which is most posterior. The non-flagellated centriole is shorter than the kinetosome and lies at an angle of approximately 30° to the latter, the two being connected with a broad, dense fibrillar bridge. The flagellar transition zone contains a spiral fiber. The non-flagellated centriole is surrounded by a thin fiber, and has a veil. The features that distinguish Mesochytrium are the partial penetration of the sporangium into the host cell, and a zoospore with unique ultrastructural configuration. In our phylogeny, M. penetrans was nested within Lobulomycetales and the Polychytrium clade, but its phylogenetic position is tenuous because of marginal support, and thus its family and order status are considered incertae sedis. Zoospores of M. penetrans are most similar to those of Synchytrium macrosporum and Rozella allomycis, which belong to different clades. These ultrastructural similarities may be a result of the relative simplicity of zoospore morphology, and do not reflect a phylogenetic relationship between these genera.

Fine structural quantification of drought-stressed Picea abies (L.) organelles based on 3D reconstructions.

Abstract: Ultrastructural investigations of cells and organelles by transmission electron microscopy (TEM) usually lead to two-dimensional information of cell structures without supplying exact quantitative data due to the limited number of investigated ultrathin sections. This can lead to misinterpretation of observed structures especially in context of their three-dimensional (3D) assembly. 3D investigations and quantitative morphometric analysis are therefore essential to get detailed information about the arrangement and the amount of subcellular structures inside a cell or organelle, respectively, especially when the plant sample was exposed to environmental stress. In the present research, serial sectioned chloroplasts, mitochondria, and peroxisomes from first year spruce needles (Picea abies (L.) Karst.) were 3D reconstructed and digitally measured using a computer-supported image analysis system in order to obtain a detailed quantitative characterization of complete cell organelles including precise morphological data of drought-induced fine structural changes. In control plants, chloroplast volume was composed of 56% stroma, 15% starch, 27% thylakoids, and 2% plastoglobules. In drought-stressed chloroplasts, the relative volume of both the thylakoids and the plastoglobules significantly increased to 37% and 12%, respectively. Chloroplasts of stressed plants differed from control plants not only in the mean thylakoid and plastoglobules content but also in the complete lack of starch grains. Mitochondria occurred in variable forms in both control and stressed samples. In stressed plants, mitochondria showed a significant smaller mean volume which was only 81% when compared with the control organelles. Peroxisomes were inconspicuous in both samples and their volume did not differ between control and drought-stressed samples. The present study shows that specific subcellular structures are subject to significant quantitative changes during drought stress of spruce needles giving a detailed insight in adaptation processes of the investigated cell organelles.

Unlocking the ultrastructure of colorectal cancer cells in vitro using selective staining

Abstract: AIM: To characterise differences between three widely used colorectal cancer cell lines using ultrastructural selective staining for glycogen to determine variation in metastatic properties. METHODS: Transmission electron microscopy was used in this investigation to help identify intracellular structures and morphological features which are precursors of tumor invasion. In addition to morphological markers, we used selective staining of glycogen as a marker for neoplastic cellular proliferation and determined whether levels of glycogen change between the three different cell lines. RESULTS: Ultrastructural analysis revealed morphological differences between the cell lines, as well as differentiation into two sub-populations within each cell line. Caco-2 cells contained large glycogen deposits as well as showing the most obvious morphological changes between the two sub-populations. SW480 cells also contained large glycogen stores as well as deep cellular protrusions when grown on porous filter membranes. HT-29 cells had trace amounts of glycogen stores with few cellular projections into the filter pores and no tight junction formation. CONCLUSION: Morphology indicative of metastatic properties coincided with larger glycogen deposits, providing strong evidence for the use of selective staining to determine the neoplastic properties of cells.

Morphological Changes in Duodenal Epithelium of Japanese Quail after Chronic Cadmium Exposure

Abstract: Our study investigated morphological changes in enterocytes of adult Japanese quails that were given cadmium (CdCl2) perorally and individually by tube, dissolved in water at a dose of 0.24 mg Cd per animal per day, for 57 and 118 days. The aim of our study was to observe chronic effects of cadmium on the structure of duodenal epithelium by means of light microscopy (LM) and transmission electron microscopy (TEM). On day 57, following peroral administration of cadmium, necrotizing enterocytes were found in the apical part of intestinal villi and their occurrence was only sporadic. Particularly on day 118 following cadmium administration, we were able to observe clusters of 2-3 necrotizing cells in the apical part of intestinal villi. However, the structure and ultrastructure of goblet cells was normal. The most notable finding in ultrastructure of all enterocytes of treated animals was the damage to cell organelles. Mitochondria and cisternae of the endoplasmic reticulum were more or less damaged and the cytoplasm contained flocculent material, particularly in the basal part of enterocytes. Some enterocytes exhibited signs of necrosis, shrivelled nucleus and damaged organelles within the markedly electrondense cytoplasm. Microvilli on the apical surface of these enterocytes were damaged and disintegrated. Junctions between cells of the intestinal epithelium were disturbed, and a present of intracellular plaques associated with the adhering and occluding junctions was observed. Cadmium caused the formation of gaps within the specialized junctional complexes, and injury to enterocytes results in the breakdown of the intercellular attachments and the sloughing of the injured cells into the intestinal lumen.

Enhanced effects of nonisotopic hafnium chloride in methanol as a substitute for uranyl acetate in TEM contrast of ultrastructure of fungal and plant cells

Abstract: This ultrastructural study showed that nonisotopic methanolic hafnium chloride and aqueous lead solution was an excellent new electron stain for enhancing TEM contrasts of fungal and plant cell structures. The ultrastructural definition provided by the new stain was often superior to that provided by conventional staining with uranyl acetate and lead. Definition of fine ultrastructure was also supported by quantitative data on TEM contrast ratios of organelles and components in fungal and plant cells. In particular, polysaccharides, which were localized in cell walls, glycogen particles, starch grains, and plant Golgi vesicle components, were much more reactive to the new stain than to the conventional one. The new nonisotopic stain is useful for enhancing the contrast of ultrastructure in biological tissues and is a safer alternative to uranyl acetate. Microsc. Res. Tech., 2011. © 2010 Wiley-Liss, Inc.

Ultrastructure of the Early Embryonic Stages of Corallobothrium fimbriatum (Cestoda: Proteocephalidea)

Abstract: Cellular details of early embryogenesis have been studied extensively among cyclophyllidean cestodes, but have been reported for only 2 species of the order Proteocephalidea, both belonging to the genus Proteocephalus. Thus, we performed a detailed ultrastructural analysis of early embryos of a second species, Corallobothrium fimbriatum, including early events in the formation of the embryonic envelopes. Adult worms were collected from the small intestine of brown bullhead catfish, Ameiurus nebulosus, from the St. Lawrence River in North America and processed by standard methods for transmission electron microscopy. The vitelline capsule consists of 2 closely apposed electron-dense membranous layers, separated by a more electron-lucent material. The 2 vitellocytes that accompany each oocyte contain numerous ribosomes, vesicles, and lipid droplets. These fuse to form a vitelline syncytium, which elongates and almost completely encircles the cleaving embryo by the 4-blastomere stage, forming a partial lipid-rich cellular envelope that undergoes apoptosis as cleavage continues. This envelope is later replaced by outer and inner embryonic envelopes. The outer envelope derives from the fusion of the vitelline syncytium with the cytoplasm of macromeres, whereas the inner envelope originates from 3 mesomeres. Simultaneous to the formation of the embryonic envelopes, other blastomeres multiply and differentiate, while some micromeres undergo degeneration or apoptosis. In most respects, ultrastructural features of early C. fimbriatum embryos closely resemble those of previously studied Proteocephalus longicollis, but differ somewhat from those of other orders. This demonstrates that, despite marked ultrastructural heterogeneity within some orders such as the Cyclophyllidea, some embryonic traits distinguish cestode orders from each other.

Dengue virus-induced autophagosomes and changes in endomembrane ultrastructure imaged by electron tomography and whole-mount grid-cell culture techniques

Abstract: The biogenesis events and formation of dengue virus (DENV) in the infected host cells remain incompletely understood. In the present study, we examined the ultrastructural changes associated with DENV-2 replication in three susceptible host cells, C6/36, Vero and SK Hep1, a cell line of human endothelial origin, using transmission electron microscopy, whole-mount grid-cell culture techniques and electron tomography (ET). The prominent feature in C6/36 cells was the formation of large perinuclear vacuoles with mature DENV particles, and on-grid whole-mount examination of the infected Vero cells showed different forms of DENV core structures associated with cellular membranes within 48 h after infection. Distinct multivesicular structures and prominent autophagic vesicles were seen in the infected SK Hep1 cells when compared with the other two cell lines. ET showed the three-dimensional organization of these vesicles as a continuous system. This is the first report of ET-based analysis of DENV-2 replication in a human endothelial cell line. These results further emphasizes the strong role played by intracellular host membranes-virus interactions in the biogenesis of DENV and strongly argues for the possibility of targeting compounds to block such structure formation as key anti-dengue agents.