Electron-dense Cell Wall Research Articles

Functional characterization of a tyrosinase gene from the oomycete Saprolegnia parasitica by RNAi silencing

Here we describe the first application of transient gene silencing in Saprolegnia parasitica, a pathogenic oomycete that infects a wide range of fish, amphibians, and crustaceans. A gene encoding a putative tyrosinase from S. parasitica, SpTyr, was selected to investigate the suitability of RNA-interference (RNAi) to functionally characterize genes of this economically important pathogen. Tyrosinase is a mono-oxygenase enzyme that catalyses the O-hydroxylation of monophenols and subsequent oxidation of O-diphenols to quinines. These enzymes are widely distributed in nature, and are involved in the melanin biosynthesis. Gene silencing was obtained by delivering in vitro synthesized SpTyr dsRNA into protoplasts. Expression analysis, tyrosinase activity measurements, and melanin content analysis confirmed silencing in individual lines. Silencing of SpTyr resulted in a decrease of tyrosinase activity between 38 % and 60 %, dependent on the level of SpTyr-expression achieved. The SpTyr-silenced lines displayed less pigmentation in developing sporangia and occasionally an altered morphology. Moreover, developing sporangia from individual silenced lines possessed a less electron dense cell wall when compared to control lines, treated with GFP-dsRNA. In conclusion, the tyrosinase gene of S. parasitica is required for melanin formation and transient gene silencing can be used to functionally characterize genes in S. parasitica.

Development of thallus axes in Usnea longissima (Parmeliaceae, Ascomycota), a fruticose lichen showing diffuse growth

While cell wall thickening in plants is generally associated with tissue maturation, fungal tissues in at least two lichens continue to grow extensively while accumulating massively thickened cell walls. We examined Usnea longissima to determine how diffuse growth shapes morphological and anatomical development of thallus axes and how the highly thickened cell walls of the central cord behave in diffuse growth. Fresh material was examined with light and epifluorescence microscopy and conventional and low-temperature SEM. Fixed material was embedded in Spurr's resin, microtome-sectioned, and examined with TEM and light microscopy. Main axes consisted essentially of bare medullary cord tissue; their characteristic morphology developed by destruction of the overlying cortex and consequent stimulation of lateral branch formation. Fungal cells of the cord tissue continually deposited wall layers of electron-transparent substances and layered, electron-dense materials that include UV-epifluorescent components. Discontinuities were evident in the outermost layers; new branch cells grew through wall materials accumulated by older neighboring cells. Sustained diffuse growth of cord tissue in U. longissima underlies the structural transformation of a corticated thallus branch into a long axis. In the cord tissue, diffuse growth may be responsible for the increasingly disrupted appearance of the older, electron-dense cell wall layers, while new wall materials are laid down adjacent to the protoplast. Cell and tissue development appeared comparable to that observed previously in Ramalina menziesii, although accumulation of wall material was somewhat less extensive and with a greater proportion of electron-dense/UV-epifluorescent components.

Methanobacterium kanagiense sp. nov., a hydrogenotrophic methanogen, isolated from rice-field soil

A pure culture of an obligately anaerobic, hydrogenotrophic, methanogenic archaeon, designated strain 169(T), which grows with hydrogen and carbon dioxide as the sole energy and carbon sources, was isolated from an anaerobic propionate-oxidizing enrichment culture originally obtained as an inoculant from rice-field soil in Japan. Cells of strain 169(T) were non-motile, Gram-reaction-variable and rod-shaped or slightly curved rods with rounded ends (1.6-5.0 × 0.35-0.5 µm). Strain 169(T) had fimbriae at both ends of the cell (up to ~10 per cell) but did not possess flagella. Ultrathin sections showed a single-layered, electron-dense cell wall about 6 nm thick, which is typical of Gram-positive bacteria. Growth was observed at 15 °C-45 °C (optimum 40 °C), at pH 6.5-9.6 (optimum pH 7.5-8.5) and in 0-70 g NaCl l(-1) (0-1.2 M) (optimum 5 g NaCl l(-1); 0.086 M). Strain 169(T) utilized only hydrogen and carbon dioxide as energy and carbon sources. The DNA G+C content was 39.3 mol%. The results of 16S rRNA gene sequence analysis indicated that strain 169(T) was most closely related to Methanobacterium subterraneum DSM 11074(T) (96.8 % sequence similarity) and Methanobacterium formicicum DSM 1535(T) (96.4 %). On the basis of its morphological, physiological and phylogenetic characteristics, strain 169(T) ( = DSM 22026(T) = JCM 15797(T)) represents a novel species of the genus Methanobacterium, for which the name Methanobacterium kanagiense sp. nov. is proposed.

Lotharella polymorpha sp. nov. (Chlorarachniophyta) from the coast of Portugal

Lotharella polymorpha was isolated from a sample of marine sediment collected at São Rafael, Albufeira, Portugal, in July 1998. Typical characteristics of a chlorarachniophyte alga are: chloroplasts containing chlorophylls a and b and lutein, the presence of a nucleomorph between the outer and inner pair of membranes surrounding the chloroplasts, and uniflagellate zoospores. In L. polymorpha, the nucleomorph is located in a periplastidial compartment beside the base of the pyrenoid. The pyrenoid is divided into two halves by the inner pair of chloroplast membranes. The position of the nucleomorph and the structure of the pyrenoid indicate that the new isolate belongs to the genus Lotharella. Lotharella polymorpha differs from the other species L. amoeboformis and L. globosa in the shape of the sessile cells and at the subcellular level. While amoeboid stages and meroplasmodia are lacking in L. globosa, L. polymorpha forms a meroplasmodium with two differently shaped types of sessile walled amoebae. Coccoid cells, migrating amoebae, heliozoan-like cells and zoospores also occur. Stellate amoeboid cells, as described for L. amoeboformis, were not detected. The pyrenoids of L. polymorpha have no stalks and their capping vesicles are filled with an amorphous substance. Sessile amoebae have a thick, compact, and electron-dense cell wall with pores. Because of these specific characteristics, we describe the first isolate of Lotharella found in the Atlantic Ocean as a new species, L. polymorpha.

Texture and structure of glucose-infused melon

The effect of glucose and calcium infusion on texture characteristics and ultrastructure of melon was studied using an Instron Universal Testing Machine and optical and transmission electron microscopy. Resistance to puncture was significantly greater when treatment was performed under vacuum or when Ca 2+ was present during atmospheric and vacuum glucose infusion. Atmospheric treatment produced a smaller resistance to puncture than the one shown by raw fruit. Processing reduced residual relaxation force and relaxation time, showing, the high internal fracture suffered by the fruit during treatment. There was a good correlation between textural changes detected through instrumental measurement and structural alterations of flesh tissues. Vacuum-treated samples showed cell walls with good optical density and a middle lamella still visible. Atmospheric impregnation caused very much reduced staining and disruption of cell walls as well as rupture of membranes. Addition of Ca 2+ during both infusion procedures resulted in more electron-dense cell walls as compared with those impregnated without Ca 2 + .

Structural Changes of Zoospores During and After Net Formation in Hydrodictyon reticulatum

Changes in the cell structure during and after net formation of zoospores in Hydrodictyon reticulatum were investigated by electron and fluorescence microscopy. Many small vesicles were accumulated around zoospores, and these vesicles later restricted the movement of zoospores. Immediately after cessation of the movement of polyhedral zoospores, the plasma membrane evaginated to form thread-like projections around the closely approaching regions of adacent zoospores. Then the projections seemed to fuse with the adjacent zoospores. Once the connections between the adacent cells were formed by the proections, the contact between two zoospores became progressively tighter. Cell wall materials were deposited on the plasma membrane about 10 minutes after cessation of the cell movement. Cell wall materials appeared firstly as electron-dense discontinuous patches on the plasma membrane. The patches then expanded to connect with one another, finally forming a single electron-dense cell wall layer which covered the cell surface. Netcells were finally linked by their cell walls. Cell wall substances continued to be accumulated in the space between the electron-dense cell wall layer and the plasma membrane.

Cryptococcus neoformans melanin and virulence: mechanism of action.

Black melanin-like pigments are produced by several neurotropic fungi, including Cryptococcus neoformans. Pigment production is associated with virulence. In media containing phenolic substrates such as L-dopa, C. neoformans cells become black as a result of pigment accumulation. Pigmented and nonpigmented C. neoformans cells were studied with transmission electron microscopy and electron spin resonance (ESR) spectroscopy. Transmission electron microscopy showed electron-dense cell walls, and ESR spectroscopy revealed a stable free-radical population in pigmented cells. The ESR signals of pigmented cells were increased by light, alkaline pH, and Zn2+ and decreased by acid pH, indicating that the black pigment was a type of melanin. A mutant deficient in melanin synthesis (mel) generated by UV radiation lacked ESR-detectable radicals, was less virulent for mice, was more susceptible to killing by nitrogen- and oxygen-derived radicals, and had 100-foldless phenoloxidase activity than the parent strain. The interaction of melanized C. neoformans, nonmelanized C. neoformans, and the hypomelanotic mel mutant with J774.16 murine macrophage-like cells was studied. Melanized cells were more resistant to antibody-mediated phagocytosis and the antifungal effects of murine macrophages than nonmelanized cells. Small increases in the intensity of the ESR signals of melanized cells in solutions containing chemically generated oxygen- and nitrogen-derived radicals indicated electron transfer to or from melanin. Melanin appears to contribute to virulence by protecting fungal cells against attack by immune effector cells.

An Ultrastructural Study of Bacterial Invasion and Tissue Breakdown in Human Experimental Root-surface Caries

This study describes the structural features of the interface between microbial deposits and root cementum in actively progressing root-surface caries lesions developed experimentally in six elderly individuals. A total of 18 specimens was examined by microradiography, and a further 18 by light and transmission electron microscopy after intra-oral periods of one, two, and three months. All specimens showed various degrees of subsurface dissolution of mineral and bacterial invasion of the cementum. Although the microradiographic pattern of mineral loss was subsurface in nature, transmission electron microscopy showed dissolution of crystals in the outermost layers of the cementum, with a distinct gradient inward. Bacterial invasion occurred along the borders between bundles of relatively well-mineralized extrinsic collagen fibers in which the characteristic cross-banding remained intact. The pattern of bacterial invasion was influenced by the incremental lines and the cemento-dentinal junction. The invading bacteria were almost exclusively Gram-positive, of various shapes, and possessed thick, moderately electron-dense cell walls and electron-lucent "vacuoles" in the cytoplasm. It is concluded that because of pronounced mineral loss of the outermost cementum, accompanied by bacterial invasion, the surface of an active cementum caries lesion, as observed by transmission electron microscopy, is not identical to that seen in microradiograms.

Guard cell ontogeny in leaf stomata of the fern Ophioglossum petiolatum

Guard cells in Ophioglossum petiolatum leaves are initiated by a single division of a stomatal initial with no subsidiary cells being formed. The stomatal initials have few vacuoles, plastids with little starch, and a large nucleus with much heterochromatin and prominent nucleoli. Young guard cells are similar cytologically to stomatal initials; their common cell walls are thin and traversed by plasmodesmata. Plasmodesmata are also present within guard cell and adjacent epidermal cell walls. With increasing age, guard cells develop a lenticular thickening in the median portion of the common cell walls, larger vacuoles, and plastids with several starch grains. Numerous microtubules are present near the thickening wall which also has an electron-translucent region between the cell wall and the plasmalemma. Many dictyosomes and mitochondria are present in the cytoplasm. Older guard cells become more vacuolated, with some of the vacuoles containing fibrillar or dense deposits. Plastids become very large as a result of storing several large starch grains. In the thickened portion of the cell walls, the middle lamella and some of the adjacent cell wall appear to be degraded during stomatal pore formation. Mature guard cells are highly vacuolated and have very thick electron-dense cell walls without plasmodesmata. Fluorescence microscopy following aniline blue staining shows that aniline blue positive materials are present around and within the thickened portion of the cell walls, at the junction of guard cells with epidermal cells, and as distinct spots in guard cell and epidermal cell walls during the early stages of ontogeny. Mature guard cells, however, lack the distinct fluorescent spots, which are interpreted as plasmodesmata, in their walls.

Candida albicans Endocarditis: Ultrastructural Studies of Vegetation Formation

Candida albicans endocarditis was established in rabbits after transaortic catherization. Within 30 to 90 min after infection, C. albicans was observed by scanning electron microscopy on the valve surface. The organisms were predominantly associated with host deposits of erythrocytes, phagocytes, platelets, and fibrinous-appearing material, which collectively appeared on the valve surface in response to trauma. Within 48 h after infection, vegetations composed of these same host components were observed on the heart valves, although Candida cells were not demonstrable on the valve or vegetation surface. When the vegetations were examined by transmission electron microscopy, Candida blastospores were only observed within phagocytic cells (predominantly monocytes) enmeshed within the vegetation matrix. Many of the intracellular organisms were undergoing degradation, as evidenced by a reduction in electron density of the cell wall. Other fungi had highly electron-dense cell walls and germ tubes. Phagocytic cells containing germinating Candida were highly vacuolated and were observed at various stages of cell lysis. After 7 days of infection, the vegetation contained a dense meshwork of fibrin and Candida pseudohyphae with 10(8) to 10(9) colony-forming units/g of vegetation. The mature vegetation was devoid of phagocytic cells and continued to grow until the death of the animal.

ELECTRON MICROSCOPY OF THIN SECTIONS OF CANDIDA UTILIS: THE STRUCTURE OF THE CELL WALL.

Details of the structure of the Candida utilis cell wall were described. Using intact cells, cell walls, about 0.02 mμ thick, have been resolved into three electron dense layers, each about 700 A thick, made up of materials of very similar electron opacity. The central and the inner layers of the wall seem to be closely packed forming a slightly more compact structure of somewhat greater electron density. Laminations were observed in some of the inner layers of the sections, particularly in some areas where the cell wall had separated: The possibility of this lamination being associated with the presence of chitin in the framework of the cell wall is discussed. Interesting observations have also been made in the sections of the heat-killed cells confirming the existence of a central electron dense cell wall layer, differing from the inner and the outer ones. Underlying the cell wall is the cytoplasmic membrane, a not too well defined sinous structure with some invaginations.