Showing papers on "Ultrastructure published in 2002"

Gap junctions and connexin expression in human suburothelial interstitial cells.

Abstract: Objective To determine whether suburothelial interstitial cells of the human bladder express gap junctions, and if so, to establish their extent and composition, using immunocytochemistry, confocal microscopy and electron microscopy. Materials and methods Bladder tissue was obtained at cystectomy; the tissue was: (i) frozen for cryosectioning and Northern blot analysis; (ii) fixed and embedded for standard thin-section electron microscopy; and (iii) processed using low-denaturation conditions in Lowicryl for immunogold-label electron microscopy. Cryosections were immunofluorescently labelled using antibodies against connexins 43, 40 and 45, vimentin, desmin and c-Kit ligand, and examined by confocal microscopy. Double labelling was used to determine the spatial relationship of labelling for connexin43 with that of vimentin and desmin. Thin-section electron microscopy was used to investigate interstitial cell ultrastructure and permit unequivocal identification of gap junctions, and immunogold labelling of Lowicryl sections was applied to localize connexin43. Results Immunoconfocal microscopy showed prominent labelling for the gap junction protein, connexin43, in a suburothelial band of cells that was also strongly positive for vimentin. The connexin43/vimentin-positive cells showed only weak labelling for desmin and c-Kit ligand, and were immunonegative for connexins 40 and 45. Northern blotting showed a corresponding abundance of connexin43 transcript in the mucosal layer but not the detrusor layer of the bladder wall. Electron microscopy revealed abundant gap junctions, recognized by their pentalaminar structure, between the cell processes of interstitial cells in the suburothelial zone. That these interstitial cell gap junctions were the source of the connexin43 immunolabelling observed by immunoconfocal microscopy was confirmed by immunogold labelling in sections of Lowicryl-embedded tissue examined by electron microscopy. Conclusion A network of interstitial cells, extensively linked by connexin43-containing gap junctions, is located beneath the urothelium in human bladder. As gap junctions provide pathways for direct cell-to-cell communication, the interstitial cellular network may operate as a functional syncytium, integrating signals and responses in the bladder wall.

Ultrastructural and chemical changes in the cell wall of Haematococcus pluvialis (Volvocales, Chlorophyta) during aplanospore formation

Abstract: Changes in the ultrastructure and chemistry of the cell wall of the unicellular volvocalean green alga Haematococcus pluvialis were investigated during the transformation of flagellates into aplanospores. The motile biflagellated state exhibited a distinct gelatinous extracellular matrix. Its ultrastructure resembled the typical volvocalean multilayered architecture with a median tripartite crystalline layer. The transformation into the non-motile cell state was characterized by formation of a new layer, a primary wall, within the extracellular matrix. During this process, the initial extracellular matrix remained intact except for the outer layers of the tripartite crystalline layer, which decomposed. Further morphogenesis of the aplanospore resulted in the formation of a voluminous multilayered cell wall. A trilaminar sheath was formed inside the primary wall and the innermost and thickest part was an amorphous secondary wall, consisting mostly of a mannan. Results obtained by staining with the fluorescent dye primuline as well as by acetolysis suggest the occurrence of sporopollenin-like material (algaenan) within the trilaminar sheath of the aplanospore cell wall. The primary wall and the outer remnants of the extracellular matrix disintegrated as the aplanospores aged, and were completely absent in the resting cell state.

Morphological and Structural Responses of Plant Roots to Aluminium at Organ, Tissue, and Cellular Levels

Abstract: Toxic effects of aluminium are primarily root-related. This review deals with growth, morphological, and ultrastructural responses of root to aluminium, their diversity along the root axis, and in the root tissues. The cell elongation seems to be most sensitive and responsible for early inhibition of root elongation. Longer Al treatment is required to reduce cell division or to interfere with nucleic acids in the root apex. Alterations of root morphology include root thickening, disturbances of root peripheral tissues, and initiation of lateral roots closer to the root tip. Ultrastructure alterations depend strongly on position of the cells with respect to the Al source, and on their developmental stage. Cell elongation and cell ultrastructure including organisation of cytoskeleton are most sensitive within the distal part of the transition zone of the root apex. This correlates with the rate of uptake and accumulation of Al along the root apex. Recognising the diverse responses and the most sensitive sites within the root apex can help in elucidating the mechanism(s) of Al effects on plants.

Culturing hippocampal neurons

Abstract: The procedure for making a low density culture of hippocampal neurons has been elaborated by Goslin and Banker. The viability of hippocampal neurons, which are sparsely disseminated on the glass surface, is maintained by a separately cultured glial monolayer; the glial feeder layer is grown on the bottom surface of the dish, while those neurons, placed face down, are attached on the coverslips. This method is originaLly designed for the observation of the maturation, polarity and axogenesis of a single neuron. In addition, this method can be applied for a variety of other purposes: (1) to observe synaptogenesis, (2) to analyze synaptic function electrophysiologically, (3) to analyze receptor functions and signaling cascades pharmacologically, (4) to visualize a molecular dynamics by time-lapse analyses of GFP-tagged molecules, and (5) to observe ultrastructure by an electron microscope. Furthermore, these neurons are useful even in biochemical experiments because they are relatively uniform without glial contamination and highly enriched in synaptic components.

Ultrastructure of the 5-hydroxytryptamine3 receptor.

Abstract: We have determined the ultrastructure of 5-hydroxytryptamine3 (5-HT 3 ) serotonin receptors purified from NG108-15 mouse neuroblastoma×rat glioma cells by electron microscopic examination of receptor particles embedded in uranyl acetate stain and metal replicas of rapidly frozen receptors. The 5-HT 3 receptor can be modelled as a cylinder 11 nm in length and 8 nm in diameter with a closed end and a central cavity 3 nm in diameter. Analysis of the rotational symmetry of single receptor particles indicates that the 5-HT 3 receptor is composed of five subunits arranged symmetrically around a central cavity. Together with evidence obtained for related proteins in other studies using ultrastructural, biochemical, or electrophysiological methods, our observations suggest that all members of the ligand-gated ion channel superfamily may possess a pentameric quaternary structure

Studies on the infection process of Fusarium culmorum in wheat spikes: degradation of host cell wall components and localization of trichothecene toxins in infected tissue.

Abstract: After single spikelet inoculation, the infection process of Fusarium culmorum and spread of fungal hyphae in the spike tissues were studied by scanning and transmission electron microscopy. While hyphal growth on outer surfaces of the spike was scanty and no successful penetration was observed, the fungus developed a dense mycelium on the inner surfaces and effectively invaded the lemma, glume, palea and ovary by penetration pegs. During the inter- and intracellular spreading of the fungus, marked alterations in the host tissues were observed, including degeneration of cytoplasm, cell organelles, and depositions of electron dense material between cell wall and plasmalemma. Ultrastructural studies revealed that host cell walls in proximity of the penetration peg and in contact with hyphae were less dense or transparent which suggested that cell wall degrading enzymes were involved in colonisation of host tissues by fungal hyphae. Enzyme- and immunogold-labelling investigations confirmed involvement of extracellular enzymes, that is cellulases, xylanases and pectinases, in degradation of cell wall components. Localization studies of trichothecenes indicated that toxins could be detected in host tissues at an early stage of infection.

Cell wall modifications of bean (Phaseolus vulgaris) cell suspensions during habituation and dehabituation to dichlobenil.

Abstract: Bean (Phaseolus vulgaris L.) cell suspensions were adapted for growth in 12 μM dichlobenil (2,6-dichlorobenzonitrile or DCB) by a stepwise increase in the concentration of the inhibitor in each subculture. Non-tolerant suspensions (I 50 = 0.3 μM) gave rise to single cells or small clusters while tolerant cell suspensions (I 50 = 30 pM) grown in DCB formed large clusters. The cells in these clusters were surrounded by a thick and irregular cell wall with a lamellate structure and lacking a differentiated middle lamella. Analysis of habituated cell walls by Fourier transform infrared spectroscopy and cell wall fractionation revealed: (1) a reduced amount of cellulose and hemicelluloses, mainly xyloglucan (2) qualitative and quantitative differences in pectin levels, and (3) a non-crystalline and soluble β-1,4-glucan. When tolerant cells were returned to medium lacking DCB, the size of the cell clusters was reduced; the middle lamella was only partly formed, and the composition of the cell wall gradually reverted to that obtained with non-tolerant cells. However, dehabituated cells (I 50 = 12 μM) were 40-fold more tolerant to DCB than non-tolerant cells and were only 2.5-fold more sensitive than tolerant cells.

Morphological Evidence of Neutrophil-Tumor Cell Phagocytosis (Cannibalism) in Human Gastric Adenocarcinomas

Abstract: The phenomenon of neutrophil-tumor cell emperipolesis or phagocytosis has been documented by light microscopy in various human carcinomas, but little is known about the cellular pathological processes and the morphological changes involved. In an attempt to clarify the nature of this phenomenon, the authors' ultrastructural studies on the relationships among neutrophils and tumor cells in human gastric carcinomas are reviewed and analyzed. At the electron microscopy level, apoptotic neutrophils were found within vacuoles of adenocarcinoma cells in 2 cases. They showed either early apoptotic morphology with perinuclear chromatin aggregation but cytoplasm integrity or late apoptotic morphology with uniform, collapsed nucleus and tightly packed cytoplasmic granules. A light microscopy review of 200 cases of resected gastric carcinomas identified 22 cases (11%) that were characterized by neutrophil-tumor cell phagocytosis (cannibalism). TUNEL staining confirmed the presence of apoptotic neutrophils within the...

Structure of the skin of an air-breathing mudskipper, Periophthalmus magnuspinnatus

Abstract: The epidermis of the mudskipper Periophthalmus magnuspinnatus consisted of three layers: the outermost layer, middle layer and stratum germinativum. Extensive vascular capillary networks were present near the superficial layer of epidermis and outermost layer. The diffusion distance between the vascular capillaries and the surface of epidermis was c. 1.5 ± 0.9μm. The middle layer consisted of small or voluminous cells swollen by epidermal cells. Due to the swollen cells, the thickness of the epidermis increased and the epidermis appeared web-like. The swollen cells contained tonofilaments, lucent contents and desmosomes. Fine blood capillaries were also discernible in this layer. Well-developed lymphatic spaces containing lymphocytes existed in the stratum germinativum. Numerous blood capillaries were present under the basement membrane. The dermis consisted of a stratum laxum and stratum compactum, and there was a definite area with acid mucopolysaccharides and a small scale in the stratum laxum. The skin had an epidermal pigment cell, dendritic melanophores (-cytes) containing melanin granules within their cytoplasm, and two kinds of dermal pigment cells, melanophores and colourless pigments containing reflecting platelets.

Chemical composition and ultrastructure of broad bean ( Vicia faba L.) nodule endodermis in comparison to the root endodermis

Abstract: Ultrastructure and development of apoplastic barriers within indeterminate root nodules formed by Vicia faba L. were examined by light and electron microscopy. The nodule outer cortex is separated from the inner cortex by a heavily suberized nodule endodermis, which matures in submeristematic regions and possesses suberin lamellae. Unsuberized passage cells are present near vascular strands, which are surrounded by a vascular endodermis attached on the inner side of the nodule endodermal cell walls. The vascular endodermis appears immediately below the meristematic apex in developmental state I (Casparian bands), gradually develops suberin lamellae, and attains developmental state II at the base of the nodule. For chemical analysis apoplastic barrier tissues were dissected after enzymatic digestion of non-impregnated tissues. Root epidermal and endodermal cell walls as well as nodule outer cortex could be isolated as pure fractions; nodule endodermal cell walls could not be separated from vascular endodermal cell walls and enclosed xylem vessels. Gas chromatography-flame ionization detection and gas chromatography-mass spectrometry were applied for quantitative and qualitative analysis of suberin and lignin in isolated cell walls of these tissues. The suberin content of isolated endodermal cell walls of nodules was approximately twice that of the root endodermal cell walls. The suberin content of the nodule outer cortex and root epidermal cell walls was less than one-tenth of that of the nodule endodermal cell wall. Substantial amounts of lignin could only be found in the nodule endodermal cell wall fraction. Organic solvent extracts of the isolated tissues revealed long-chain aliphatic acids, steroids, and triterpenoid structures of the lupeol type. Surprisingly, extract from the outer cortex consisted of 89% triterpenoids whereas extracts from all other cell wall isolates contained not more than 16% total triterpenoids. The results of ultrastructural and chemical composition are in good correspondence and underline the important role of the examined tissues as apoplastic barriers.

DNA replication and nuclear architecture.

Abstract: The model of in situ DNA replication provided by immunofluorescence and confocal imaging is compared with observations obtained by electron microscopic studies. Discrepancies between both types of observations call into question the replication focus as a persistent nuclear structure and as a replication entity where DNA replication takes place. Most electron microscopic analyses reveal that replication sites are confined to dispersed chromatin areas at the periphery of condensed chromatin, and the distribution of replication factors exhibits the same localization pattern. Moreover, rapid migration of newly synthesized DNA from the replication sites towards the interior of condensed chromatin regions obviously takes place during S-phase. It implies modifications of replication domains, hardly detectable by fluorescence microscopy. The confrontation of different observations carried out at light microscopic or electron microscopic levels of resolution lead to a conclusion that a combination of in vivo fluorescence analysis with a subsequent ultrastructural investigation performed on the same cells will represent an optimal approach in future studies of nuclear functions in situ.

Ultrastructure of vascular cambial cell cytokinesis in pine seedlings preserved by cryofixation and substitution.

Abstract: Trees depend on the secondary vascular cambium to produce cells for new xylem and phloem. The fusiform cells of this lateral meristem are long and narrow, presenting special challenges for arranging the mitotic spindle and phragmoplast. Fusiform cambial cells of Pinus ponderosa and Pinus contorta were studied by cryofixation and cryosubstitution which preserved ultrastructure and phases of cytokinesis with a resolution not previously attained. Membranous structures including the plasma membrane, tonoplast, and those of other organelles were smooth and unbroken, indicating that they were preserved while the protoplasm was in a fully turgid state. Mitotic spindles separated daughter chromosomes diagonally across the radial width of the cells. The cell plate was initiated at an angle to the cell axis between the anaphase chromosomes by a microtubule array which organized vesicles at the phragmoplast midline. Within the phragmoplast, vesicles initially joined across thin tubular projections and then amalgamated into a tubulo-vesicular network. Axial expansion of the cell plate generated two opposing phragmoplasts connected by a thin, extended bridge of cell plate and cytoplasm that was oriented along the cell axis. In the cytoplasmic bridge trailing each phragmoplast, the callose-rich tubular network gradually consolidated into a fenestrated plate and then a complete cell wall. Where new membrane merged with old, the parent plasmalemma appeared to be loosened from the cell wall and the membranes joined via a short tubulo-vesicular network. These results have not been previously reported in cambial tissue, but the same phases of cytokinesis have been observed in cryofixed root tips and suspension-cultured cells of tobacco.

Ultrastructure of a haplosporidian containing Rickettsiae, associated with mortalities among cultured paua Haliotis iris.

Abstract: Uninucleate and multinucleate stages of a protozoan parasite are described from cultured abalone Haliotis iris Martyn, 1784 in New Zealand. The parasite is identified as a haplosporidian by the occurrence of multinucleate plasmodia, mitochondria with tubular cristae, lipid droplets, anastomosing endoplasmic reticulum (aER), multivesicular bodies (MVBs), haplosporogenesis by the production of haplosporosome-like bodies from nuclear membrane-bound Golgi, and their maturation to haplosporosomes. Coated pits occurred in the plasma membrane and coated vesicles were scattered in the cytoplasm, particularly in association with the Golgi face away from the nucleus, and aER. It is concluded that the outward face of the Golgi may be the trans face, and that aER is the trans-Golgi network. Coated pits and bristle-coated vesicles are reported from a haplosporidian for the first time. The vesicles in the MVBs resembled the cores and inner membranes of haplosporosomes, without the outer layer. The possible inter-relationships of these features are discussed. The abalone parasite differs from previously described haplosporidians in the apparent absence of a persistent mitotic spindle, and the presence of intracytoplasmic coccoid to rod-shaped bacteria resembling Rickettsiales-like prokaryotes. Phylogenetic analysis of the 16S rRNA gene sequence of the Rickettsiales-like prokaryotes indicated that these organisms belong to the Rickettsia cluster. The prokaryotes have a high (7%) sequence divergence from known Rickettsieae, with Rickettsia sp. and R. massiliae being the closest relatives. The lack of non-molecular evidence prevents us from proposing a new rickettsial genus at this time.

Maintenance of meiotic arrest in bovine oocytes in vitro using butyrolactone I: Effects on oocyte ultrastructure and nucleolus function

Abstract: In this study, we have shown that butyrolactone I (BL-I), a potent inhibitor of cyclin-dependent kinases, affects oocyte cytoplasmic morphology and nuclear function in terms of nucleolar ultrastructure and immunocytochemistry. Bovine oocytes were recovered from three classes of follicle size: 1.5-2, 2-3, and 3-6 mm. The oocytes were incubated for 40 hr with BL-I, and subsequently processed for transmission electron microscopy or immunocytochemistry. A control group of oocytes were processed immediately upon recovery (0 hr). In general, incubation with BL-I for 40 hr disrupted contact between cells of the cumulus oophorous and the oocyte, caused degeneration of the cortical granules and the peripheral migration of all cytoplasmic organelles. At the level of the nucleus, it induced convolution of the nuclear membrane and caused acceleration of nucleolar compaction in oocytes from follicles 3 mm. Furthermore, the effects appear to be more profound in fully-grown oocytes.

Morphology and Wall Ultrastructure in Devonian Spores with Bifurcate‐Tipped Processes

Abstract: A detailed investigation has been undertaken on two dispersed spore taxa, characterized by their zonate structure and an ornament of bifurcate‐tipped processes, that were recovered from Middle Devonian deposits from Scotland: Ancyrospora grandispinosa Richardson 1960 and Ancyrospora (Triletes) ancyrea (Eisenack) Richardson 1962 Both taxa have been comprehensively examined using light, scanning electron, and transmission electron microscopy, and a detailed understanding of their morphology, gross structure, and wall ultrastructure has emerged Both have an inner body, enclosed within an outer layer that is extended into a pseudozona, and bifurcate‐tipped processes of similar construction On the whole, morphology and wall ultrastructure are strikingly similar in the two species, although subtle differences lead to slight reservations when attempting to identify homologous wall layers Affinity of the spores is assessed based on detailed comparisons with the spores of plant groups believed to have comprise

An Ultrastructural Study of Nosema locustae Canning (Microsporidia) from Three Species of Acrididae (Orthoptera)

Abstract: Summary. Nosema locustae is a pathogen of orthopterans with an unusually wide host range. It is the only microsporidian that has been developed as a microbial control agent. In spite of its practical importance the ultrastructure of N. locustae life stages other than spores, has not been studied. The insects used in this study, all in the family Acrididae, were the Migratory locust, Locusta migratoria migratorioides (Oedipodinae), the South American locust, Schistocerca cancellata (Cyrtacanthacridinae), and the grasshopper Dichroplus schulzi (Melanoplinae). All insects were reared routinely in the laboratory. The spores of N. locustae used for the experimental peroral inoculations were all of North American origin. Fat body cells were the predominant site of parasite development, though infection of tracheal epithelium cells and haemocytes also occurred. Ultrastructure of meronts, sporonts, sporoblasts and spores is described. The fine morphology of N. locustae stages is typical for microsporidians of the genus Nosema. Nuclei were always in diplokaryotic arrangement. Transition from meront to sporont was characterized by striking changes in parasite ultrastructure: appearance of electron dense granules (50-100 nm in diameter, presumable RNP complexes) in the nucleoplasm, and stacks of ER cisternae and prominent vacuoles in cytoplasm. The beginning of sporogony was marked by an increase in size of parasite cells due to extensive vacuolization. Tubule-like structures appeared in the host cell cytoplasm during parasite sporogony. Elongated conglomerates of electron dense material were scattered in the host cell and eventually ornamented the outersurface of the parasite membrane, forming an electron dense layer around sporonts. Spores were diplokaryotic, measured 4.95 ‐ 0.07 x 2.65 ‐ 0.04 µm (mean ‐ SE, n=24) on fresh smears and 3.49 ‐ 0.18 x 1.73 ‐ 0.04 µm (n = 10) on ultrathin sections, and had electron-dense cytoplasm in which all internal structures typical of microsporidian spores were recognizable. The polaroplast was lamellar, the endospore was 200-300 nm thick, and the exospore was 40-50 nm. The polar filament was isofilar, arranged in 17-18 coils. Our study did not reveal any difference in the morphology of N. locustae while developing in the three different hosts.

Electron microscopy of pathogenic yeasts Cryptococcus neoformans and Exophiala dermatitidis by high-pressure freezing.

Abstract: A high-pressure freezing method was used to observe the ultrastructure of pathogenic yeasts, Cryptococcus neoformans and Exophiala dermatitidis, after freeze-substitution and ultrathin sectioning. The method well preserved the cell structure in its natural state, since the capsule, cell wall, plasma membrane, nucleus, outer and inner nuclear membranes, nuclear pores, nucleolus, mitochondria, mitochondrial membrane and cristae, vacuoles, endoplasmic reticulum, Golgi apparatus, spindle pole body, ribosomes, lipid droplets, microtubules, actin filaments, and glycogen granules were clearly visible. The method was shown to freeze cells as deep as 0.1 mm by sectioning the sample perpendicular to specimen surface. The quality of the cell image was similar to that obtained by a rapid freezing method when compared using the same materials. Thus, high-pressure freezing would be useful for making serial ultrathin sections for three-dimensional analysis of cells, which should give basic information of structure and function of pathogenic yeast cells necessary for finding an effective therapy for mycoses.

Ultrastructure of Human Mast Cells

Abstract: Ultrastructural studies of human mast cells define their organelles and the impact of environment, development and function on their ultrastructural morphology. The studies we review here implicate lipid bodies in eicosanoid and cytokine biology, and extend the functional repertoire of secretory-storage granules to synthesis.

Nucleolar protein allocation and ultrastructure in bovine embryos produced by nuclear transfer from granulosa cells

Abstract: In the present study, immunofluorescence confocal laser scanning microscopy, autoradiography following 3H-uridine incubation, and transmission electron microscopy were used to evaluate the nucleola...

Pinguiococcus pyrenoidosus gen. et sp. nov. (Pinguiophyceae), a new marine coccoid alga

Abstract: SUMMARY A coccoid marine alga, collected from an aquaculture tank and maintained in culture as CCMP1144, was examined using light and electron microscopy. Young, rapidly growing cells were mostly spherical in shape, approximately 4–6 μm in diameter. Older cells often produced protrusions and pseudopodia-like extensions, giving cells an amoeboid-like appearance, but no amoeboid movement was observed and the pseudopodia-like extensions exhibited no active movement. The single chloroplast had a typical photosynthetic stramenopile ultrastructure. A large stalked pyrenoid was easily observed by light microscopy. Ultrastructurally, the granular portion of the pyrenoid was divided into sections by a penetrating chloroplast envelope. A mitochondrion was often, but not always, adjacent to the pyrenoid, and in some cases the mitochondrion formed a ‘cap’ over the protruding pyrenoid. The Golgi cisternae were (when viewed in cross-section) curved toward the nucleus. A peripheral network of anastomosing tube-like membranes was located immediately beneath the plasmalemma. Two centrioles were located adjacent to the nuclear envelope. Lipid-like and electron transparent vacuoles were present. Based on this investigation and data published elsewhere (large percentage of eicosapentaenoic acid, 18S rRNA and rbcL genes), this alga was described as Pinguiococcus pyrenoidosus gen. et sp. nov.