20-Hydroxyecdysone-induced differentiation and apoptosis in the Drosophila cell line, l(2)mbn

20-Hydroxyecdysone has an inhibitory effect on the proliferation of l(2)mbn cells, causes vacuolization and fragmentation of cells, and promotes a strong phagocytotic activity. From several lines of evidence, it can be concluded that 20-hydroxyecdysone induces apoptosis. Long-term video observations following the fate of individual cells, scanning and transmission electron microscopy reveal the typical characteristics of apoptosis: sequestration of small cellular protuberances or larger parts of the cell with nuclear fragments (apoptotic bodies), chromatin condensation, condensation and vesiculation of cytoplasm, whereas the mitochondria retain their normal appearance. The induction of apoptosis by 20-hydroxyecdysone was confirmed by the TUNEL reaction and quantitatively determined by a method based on this reaction. Onset of apoptosis precedes phagocytotic activity. JH III alone has no clear-cut effect on l(2)mbn cells. In double treatments, the inhibitory effect of 20-hydroxyecdysone on cell proliferation is significantly reduced by the addition of JH III. Whether or not JH III also reduces apoptotic activity is not yet clear. It is shown that the l(2)mbn cell line is an advantageous model system for the exploration of steroid-induced apoptosis.     

Epidermal intercellular relationships during carcinogenesis and cocarcinogenesis as revealed by scanning electron microscopy.

Investigations with the scanning electron microscope were carried out on the skin of 80 NMRI mice after treating them with small doses of the carcinogenic substance DMBA and the cocarcinogenic agent TPA, respectively. The results were correlated with histologic, transmission electron microscope and autoradiographic observations. The epidermis of TPA-treated animals was markedly hyperplastic with an orderly arrangement of cell layers. Autoradiographically only the basal cells were heavily labelled. With the scanning and transmission electron microscope a reduced number of intercellular connections and dilatation of the intercellular spaces could be detected. After treatment with DMBA the epidermis was only moderately hyperplastic but severely dysplastic with 3H-thymidine-labelled cells in the upper layers. The most characteristic findings were the loss of the intercellular connections, especially the lateral ones, and a pronounced dilatation of the intercellular spaces. The results obtained with the scanning electron microscope were quantified using morphometrical methods.     

The surface structure of the completely and incompletely orthokeratinized oral epithelium in the rat: a light, scanning and transmission electron microscope study.

Mucosa from the hard and soft palates, molar gingiva, cheek and dorsal surface of the tongue of the rat was examined in the light microscope, following Mallory's triple connective tissue stain, and in the scanning and transmission electron microscopes. The epithelium covering the hard palate, gingiva, the smooth band of mucosa at the junction of the hard and soft palates, intermediate zones of the soft palate, fungiform papilla-like structures in the central zone of the soft palate, the fungiform papillae, and the more superficial part and posterior surfaces of the filiform papillae of the tongue all exhibited complete orthokeratinization. The oral surfaces of the epithelial cells in all these areas had a honeycomb pattern of interconnecting ridges surrounding depressions. Imprints of the overlying cells that had been desquamated were apparent, and the lateral boundaries between the cells were formed by two raised ridges separated by a gap. The epithelium covering the cheek, central zone of the soft palate apart from the fungiform papilla-like structures, lateral zones of the soft palate, gingival crevice, and the mucosa between the fungiform and filiform papillae of the tongue all exhibited incomplete orthokeratinization. The oral surfaces of the epithelial cells in all these areas were relatively smooth and did not exhibit a honeycomb pattern of interconnecting ridges. Imprints of the overlying cells that had been desquamated and the lateral boundaries between the cells were only very occasionally found. In the transmission electron microscope the outlines of the cells were compatible with the surface patterns seen in the scanning electron microscope. The possible relationships between the degree of orthokeratinization and ultrastructure of the various epithelia are discussed.     

Scanning ion conductance microscopy of living cells.

Currently there is a great interest in using scanning probe microscopy to study living cells. However, in most cases the contact the probe makes with the soft surface of the cell deforms or damages it. Here we report a scanning ion conductance microscope specially developed for imaging living cells. A key feature of the instrument is its scanning algorithm, which maintains the working distance between the probe and the sample such that they do not make direct physical contact with each other. Numerical simulation of the probe/sample interaction, which closely matches the experimental observations, provides the optimum working distance. The microscope scans highly convoluted surface structures without damaging them and reveals the true topography of cell surfaces. The images resemble those produced by scanning electron microscopy, with the significant difference that the cells remain viable and active. The instrument can monitor small-scale dynamics of cell surfaces as well as whole-cell movement.     

Apical surface topography of invaginating and noninvaginating cells. A scanning-transmission study of amphibian neurulae

A comparative study on scanning and transmission electron microscopy of apical ultrastructure in epithelia of the axolotl neurula (Ambystoma mexicanum, Cope) is presented. The aim of the work is to determine whether apical surface topography is correlated to other morphological features of the cells, and whether there are any ultrastructural differences between surfaces of invaginating and noninvaginating regions. Scanning specimens are prepared by critical point drying and benzene freeze-drying. Comparisons show that the scanning specimens are comparable to the standard transmission specimens with regard to surface topography.Apical surfaces are sculptured by folds and microvilli-like processes. Assessment of the relative abundance of surface projections shows that these occur in largest numbers on invaginating, bottle-shaped cells in the neural plate and in the notochord rudiment of stage-16 larvae. It is proposed that the surface projections may support apical narrowing in these cells, by facilitating endocytosis, and that they may be lateral attachment organelles. It is suggested that the morphogenesis of the invaginating cells may be the result of coordination between microfilaments, apical endocytosis, microtubules, and “adhesive peripheral surface projections.”     

The competition of 2 types of epithelial layers in mixed cultures

The interaction of epithelial cells was studied in mixed cultures by phase contrast, time-lapse microcinematography, transmission and scanning electron microscopy. Epithelial cells, united into monolayer sheets with smooth apical surfaces, can be competing for substrate lamellae at the lower surfaces and force out each other from the substrate up to full elimination. The competition of cells for territory may be an important factor in morphogenesis in vivo.     

Scanning electron microscope observations on the miracidium of Schistosoma

Miracidia of two species of Schistosoma, viz. haematobium and japonicum, were studied with the scanning electron microscope to more clearly visualize what is seen with the light microscope and the transmission electron microscope, and more specifically, to relate the structure of the apical papilla to its function in snail penetration.The apical papilla of schistosome miracidia is composed of corrugated areas which form tiny suckerlike cups, presumably used by the miracidium to facilitate attachment to the snail during penetration. A lateral opening (secretory pore) on the apical papilla and short stubby apical cilia (tactile or sensory) are also demonstrated.     

X-Ray Microanalysis and Phagocytotic Activity of Cultured Retinal Pigment Epithelial Cells in Hypoxia

Purpose: To investigate the influence of the functional and morphological changes induced in retinal pigment epithelial (RPE) cells by retinal ischemia, we evaluated the phagocytotic activity, the concentration of various elements, and ultrastructure in cultured RPE cells in hypoxia. Methods: The concentrations of oxygen in incubators were adjusted to 20, 10, and 1% by the addition of nitrogen for 72 hr. To observe phagocytotic activity and its relationship to actin filaments, the filaments of RPE cells incubated with fluoresbrite carboxylate YG microspheres were stained with rhodamine phalloidin. Some of the specimens were subjected to X-ray microanalysis by scanning electron microscope after being fixed, freeze-dried, and coated with carbon to investigate the cytoplasmic concentration of elements. A part of the latter specimens was also observed by transmission electron microscope after being embedded in epon and cut into ultrathin sections to see the ultra-structural changes inside cell. Results: Lowering oxygen concentrations from 20% to 1% swelled RPE cells and decreased the number of fluoresbrite carboxylate YG microspheres phagocytized by RPE cells. Phagocytosis of a large amount of latex beads (30 μl) for 24 hr in 1% oxygen caused a disruption of RPE cells. Na, S, and P were detected in RPE cells cultured in 20% oxygen. Reducing the oxygen concentration from 20 to 10 or 1% significantly decreased Na and increased S. Mitochondria were observed in RPE cells in 20 and 10% oxygen, but many vacuoles were observed in the cytoplasm in 1% oxygen. Conclusion: Hypoxia as low as 1% oxygen induced malfunction of phagocytosis and the fragility of RPE cells. We could speculate the imbalance of the electrolytes such as Na or a decrease of antioxidants such as glutathione containing S as a reason of disturbance of cell viability.     

Presence of abundant filaments in apical caps of the nonciliated bronchiolar epithelial (Clara) cells

The nonionic detergent Triton X-100 has often been used for the extraction of cytoplasmic materials. We used the detergent in a vascular perfusion medium when preparing rat lung in order to observe the cytoskeleton of the nonciliated bronchiolar epithelial (Clara) cells. To eliminate some cytoplasmic materials selectively and to maintain good fine cell structure simultaneously, the lungs were perfused sequentially with the detergent (0.2% Triton X-100) alone for 2 min, with a mixture of low-concentration (0.1%) glutaraldehyde and detergent (0.2% Triton X-100) for 15 min, and finally with 2.5% glutaraldehyde for 5-10 min. After fixation, the nonciliated bronchiolar epithelial (Clara) cells were observed by scanning and transmission electron microscopy. At the apical region of the cells, there were central cytoplasmic protuberances (apical caps) filled with microfilaments. These filaments were bound at one end to the cytoplasmic side of the cell membrane and ran into the interior of the cytoplasm at the other end. As a control, the Clara cells were observed by transmission electron microscopy after perfusion with 2.5% glutaraldehyde solution. The luminal surfaces of the cells were covered with short, thick microvilli. The apical caps also had microvillus-like protrusions. These results suggest that the apical cap is not an apocrine droplet but rather is a stable structure involved in the function of the Clara cells.     

Ultrastructural organization of the epithelial layers and surface morphological characteristics of cells in adenocarcinomas of the cervix uteri

The cell interrelations, and cellular attachment to the stroma in normal columnar epithelium and adenocarcinoma of the cervix uteri were examined by transmission and scanning electron microscopy. The application of rapid enzymatic digestion technique allows to visualize the topography of cell membranes, otherwise disguised in ordinary conditions. Four types of disordered epithelial sheets characterized by different apical, lateral and basal cell surface changes are described. Various alterations in morphology of the basement membrane and adjacent conjunctive tissue are associated with the tumor appearance. Marked deviations in cell-stroma contact may lead to the inversion of cell polarity revealed in cervical adenocarcinoma: cellular parts adjoining to stroma acquire characteristic features of the apical pole.     

Ultrastructural and Cytochemical Observations on Sporogenesis of Myxobolus sp. (Myxosporida: Myxobolidae) from the Common Shiner Notropis cornutus*

SYNOPSIS. The structure and cytochemistry of spores of Myxobolus sp. from plasmodia which occur in the gill filaments of the common shiner Notropis cornutus were studied by light microscopy and by scanning and transmission electron microscopy. The thin-walled valves of the pyriform spores are thickened in the lateral sutural and apical regions. Mucous material is associated predominantly with the posterior end of many spores. The plasmodium is surrounded by a syncytial wall bounded by 2 membranes. Pinocytotic channels are formed by the inner membrane and numerous dense vesicles are pinched off at the distal ends of the channels. Sporogenesis is initiated by the envelopment of one vegetative cell by another. The larger, enveloped cell divides to form a disporous pansporoblast, which contains 2 pairs of capsulogenic and valvogenic cells and 2 binucleate sporoplasm cells. Each capsular primordium and connecting external tubule gives rise to a polar capsule which houses a helically coiled polar tubule. The apical end of each polar capsule is plugged by a stopper. The valvogenic cells surround the capsulogenic and posteriorly situated sporoplasm cells to form the spore valves. Iodinophilic (glycogen) inclusions were not seen in spores stained with iodine or Best's carmine. A darkly stained band was observed around the posterior region of most spores stained with Best's carmine. In the electron microscope large aggregates of β glycogen particles were seen in the cytoplasm of sporoplasm cells in mature spores.     

Conditions critical for optimal visualization of bacteriophage adsorbed to bacterial surfaces by scanning electron microscopy.

The potential of scanning electron microscopy as a tool for the detection of viruses on cell surfaces has been studied using bacteriophage P1 adsorbed to Shigella dysenteriae as a model system. Viral particles were readily detectable by scanning electron microscopy on the surface of infected cells which were fixed with glutaraldehyde followed by postfixation in OsO4 and prepared by critical point drying. The virus-studded surface of the infected cells differed markedly from the relatively smooth surfaces of uninfected control cells. Examination of the same preparations with transmission electron microscopy revealed numerous viral particles adsorbed to the surfaces of infected cells, whereas the control cells were free of viruses as expected. Glutaraldehyde fixation alone did not preserve the surface detail of infected cells: cells adsorbed with viruses were not distinguishable from control cells by scanning electron microscopy although by transmission electron microscopy viruses could be visualized. Air drying from water or absolute alcohol resulted in unsatisfactory preservation as compared to the appearance of infected cells prepared by the critical point method. Thus, scanning electron microscopy is capable of resolving viral particles on cell surfaces, but detection of these particles is completely dependent both on the method of fixation and on the technique of drying used.     

Demonstration of gonadotropin releasing-hormone receptors on gonadotrophs and somatotrophs of the goldfish: an electron microscope study.

Dispersed pituitary cells of the goldfish were incubated with biotinylated [D-Lys6, Pro9-N-ethylamide] salmon gonadotropin-releasing hormone (sGnRH-A) then avidingold (10 nm), and were fixed, embedded and sectioned. Cells were identified as gonadotrophs, somatotrophs, or prolactin cells using specific hormone antisera and protein-A gold (20 nm) as a marker. Attachment of the biotinylated sGnRH-A to the pituitary cell sections was determined by scanning cell surfaces for the smaller gold particles using the transmission electron microscope. Attachment was observed on gonadotrophs and somatotrophs, but was negligible on prolactin cells. Preincubation with unlabelled salmon gonadotropin-releasing hormone or chicken II gonadotropin-releasing hormone, or omission of the salmon gonadotropin-releasing hormone analog, prevented the reaction. The direct visualization of specific gonadotropin-releasing hormone receptors on gonadotrophs and somatotrophs supports the existence of direct stimulatory actions of gonadotropin-releasing hormone on gonadotropin and somatotropin release in gold-fish.     

Ultrastructural Observations on the Larva of Loxosoma pectinaricola Franzén (Entoprocta,Loxosomatidae)

The larva of Loxosoma pectinaricola Franzén has been studied using scanning and transmission electron microscopy. The embryo develops surrounded by an egg envelope attached to the brood chamber. The newly released larva measures about 100 μm in length and is characterized by a prominent apical organ, stalked vesicles, paired lateral sense organs and a prototroch. The apical organ consists of at least four cell types: (1, 2) two types of ciliated cells, (3) vacuolated cells and (4) myoepithelial cells. The apical organ and frontal ganglion are tightly juxtaposed in the upper tier of the episphere. The stalked vesicles each consisting of two cells are unique evaginations of the epidermis. There are about twenty stalked vesicles with a maximum diameter of about 20.0 μm. The ciliated, knob-shaped, paired lateral sense organs are situated fronto-laterally on the episphere. The prototroch is comprised of a row of contiguous prototroch cells each containing about eighteen long cilia. The apical organ, frontal ganglion and paired lateral sense organs are suggested to be sensory structures that play an important role in active locomotion, settlement site selection and metamorphosis.     

Ferrocyanide enhancement of concanavalin A-ferritin and cationized ferritin staining blood cell surface glycoconjugates

Synopsis Ferrocyanide was used to enhance cationized ferritin and concanavalin A-ferritin (Con A-ferritin) staining of surface glycoconjugates of peripheral blood and bone marrow cells from rabbits and humans. The glutaraldehyde-fixed cells were stained with Con A-ferritin or cationized ferritin and then exposed to a ferrocyanide solution. The resulting cuboidal and irregular stain deposits averaged 50 nm in diameter when viewed with the transmission (TEM) and scanning electron microscope (SEM). Rabbit blood cells demonstrated more Con A binding sites than human blood cells and the decrease in binding sites observed with maturation of human granulocytic and erythrocytic cells was not evident in rabbit cells. Differences in binding of cationized ferritin to rabbit and human cell surfaces were less prominent than that observed for Con A. These results extend previous studies of blood cell surface glycoconjugates and demonstrate that ferrocyanide enhancement significantly facilitates SEM evaluation of Con A-ferritin and cationized ferritin bound to cell surfaces.     

Bacillus cereus B-02对Botrytis cinerea 拮抗机理的研究

采用电子显微镜(扫描、透射)和激光扫描共聚焦显微镜,从细胞形态学和生理学水平上研究蜡样芽孢杆菌Bacillus cereus B-02过滤液对灰葡萄孢菌Botrytis cinerea的拈抗机理.结果表明,处理菌丝表面形态受到严重破坏,发牛强烈变形;荫丝细胞核、线粒体和细胞壁等哑细胞结构发生了明显改变,细胞内出现大量无膜透明内含物,并产生较人液泡.此外,处理菌丝DNA、线粒体膜电位和活性氧荧光强度均低于对照组,且差异极显著;说明B-02菌株对病原真菌菌丝细胞DNA的合成、线粒体膜电位和活性氧水平有重要影响.     

Bacillus cereus B-02对Botrytis cinerea拮抗机理的研究

采用电子显微镜(扫描、透射)和激光扫描共聚焦显微镜,从细胞形态学和生理学水平上研究蜡样芽孢杆菌Bacillus cereus B-02过滤液对灰葡萄孢菌Botrytis cinerea的拮抗机理。结果表明,处理菌丝表面形态受到严重破坏,发生强烈变形;菌丝细胞核、线粒体和细胞壁等亚细胞结构发生了明显改变,细胞内出现大量无膜透明内含物,并产生较大液泡。此外,处理菌丝DNA、线粒体膜电位和活性氧荧光强度均低于对照组,且差异极显著;说明B-02菌株对病原真菌菌丝细胞DNA的合成、线粒体膜电位和活性氧水平有重要影响。     

Modulation of apoptosis by starvation: morphological and biochemical study of rat intestinal mucosa

Morphology at light and electron microscopic levels, expression and activation of transglutaminase and DNA fragmentation at internucleosomal sites were used as markers to study the effect of starvation on the apoptosis of small intestinal epithelial cells. The cells entering apoptotic programme in well-fed animals undergo many morphological changes in apical cytoplasm involving alterations in actin cytoskeleton organisation which may cause a discharge of microvilli. Some free floating cells in the intestinal lumen show characteristics of apoptotic cell death, e.g. shrinkage of cell and peripheral condensation of chromatin, while mitochondria and lysosomes remain unchanged. Apoptotic bodies are also seen in scanning electron micrographs. During progressive starvation, epithelial cells do not enter the apoptotic cell death programme. Biochemical markers for apoptosis such as increased transglutaminase activity and DNA fragmentation are clearly discernible in normally fed animals. The percentage of cells labelled immunohistochemically by antibody against transglutaminase decreased during starvation while DNA fragmentation was absent. The exact mechanism for suppressing apoptosis in intestinal cells under starvation is not known. However, the data presented here support the existence of such a regulatory process.     

Surface defects in ventricular cells of brains of mouse embryos homozygous for the Loop-tail gene: scanning electron microscopic study.

Luminal surfaces in the mesencephalon and rhombencephalon in normal mouse embryos and those homozygous for Lopp-tail were studied by means of scanning electron microscopy. Ventricular cells in the ventrolateral regions of normal day-10 and -11 brains showed single apical cilia and microvilli, whereas those in ventromedial regions showed a dense network of microvilli and bulbous projections which tended to obscure the apical cilia and cellular outlines. Similar regional differences occurred in the Loop-tail brains, although there was a marked decrease in the number and density of microvilli and bulbous projections. At days 12-14 of gestation the latter brains also showed a flattening of cell surfaces, shallow depressions, and craterlike ruptures in the plasma membranes.     

Antimicrobial activity and effectiveness of a combination of sodium hypochlorite and hydrogen peroxide in killing and removing Pseudomonas aeruginosa biofilms from surfaces

AIMS: To evaluate both the antimicrobial activity and the effectiveness of a combination of sodium hypochlorite and hydrogen peroxide (Ox-B) for killing Pseudomonas aeruginosa ATCC 19142 cells and removing P. aeruginosa biofilms on aluminum or stainless steel surfaces. METHODS AND RESULTS: Pseudomonas aeruginosa biofilms were developed in tryptic soy broth containing vertically suspended aluminium or stainless steel plates. Biofilms were exposed to a mixed sodium hypochlorite and hydrogen peroxide solution as a sanitizer for 1, 5 and 20 min. The sanitizer was then neutralized, the cells dislodged from the test surfaces, and viable cells enumerated. Cell morphologies were determined using scanning (SEM) and transmission electron microscopy (TEM). Cell viability was determined by confocal scanning laser microscopy (CSLM). Biofilm removal was monitored by Fourier transform infrared (FTIR) spectrophotometry. Cell numbers were reduced by 5-log to 6-log after 1 min exposure and by 7-log after 5 min exposure to Ox-B. No viable cells were detected after a 20 min exposure. Treatment with equivalent concentrations of sodium hypochlorite reduced viable numbers by 3-log to 4-log after 1 min exposure and by 4-log to 6-log after 5 min, respectively. A 20 min exposure achieved a 7-log reduction. Hydrogen peroxide at test concentration treatments showed no effect. FTIR analysis of treated pseudomonad biofilms on aluminium or stainless steel plates showed either a significant reduction or complete removal of biofilm material after a 5 min exposure to the mixed sodium hypochlorite and hydrogen peroxide solution. SEM and TEM images revealed damage to cell wall and cell membranes. CONCLUSIONS: A combination of sodium hypochlorite and hydrogen peroxide effectively killed P. aeruginosa cells and removed biofilms from both stainless steel and aluminium surfaces. SIGNIFICANCE AND IMPACT OF THE STUDY: The combination of sodium hypochlorite and hydrogen peroxide can be used as an alternative disinfectant and/or biofilm remover of contaminated food processing equipment.