Electron microscopic findings on reversion of Listeria L-forms

Reversion in L-forms of Listeria monocytogenes was studied with the use of electron microscopy. In the culture undergoing the process of reversion cells differing in size, form and the electron density of the cytoplasm were present. The process of reversion was characterized by the increase of cytoplasmic density, the decrease of nucleoid, the appearance of fibrillary material of medium electron density on the membrane surface and the longitudinal elongation of the cell. The cell wall of the revertants was thinner and its outer layer with greater electron density was less pronounced than in the initial culture.     

Opposite effects of dibutyryl adenosine 3':5' cyclic monophosphate and serum on growth of Chinese hamster cells

The effect of dibutyryl adenosine cyclic 3′:5′ monophosphate and testosterone on growth, response to serum and transport properties of Chinese hamster ovary cells was studied. There was a marked depression of growth in the presence of both compounds. A change of medium was sufficient to permit a partially synchronized burst of growth of the treated cells either in the presence or absence of dibutyryl adenosine cyclic 3′:5′ monophosphate plus testosterone. However, in the presence of these compounds a second round of cell division was prevented. Initiation of the cell cycle by cells exposed to dibutyryl adenosine cyclic 3′:5′ monophosphate plus testosterone displayed a greater serum requirement than untreated cells. It is concluded that serum and cyclic AMP could have antagonistic interactions in growth regulation. The treated cells had a reduced ability to accumulate amino-isobutyrate and glutamine, but no difference was observed with uridine uptake. The data suggest that a functional alteration of the membrane is induced by the exposure to dibutyrl adenosine cyclic 3′:5′ monophosphate plus testosterone.     

Two independent mechanisms of induction of 2′:3′-cyclic-nucleotide 3′-phosphohydrolase in glioma cells by cyclic AMP and high cell density

Specific activity of the myelin enzyme, 2′:3′-cyclic-nucleotide 3′-phosphohydrolase (EC 3.1.4.37), increases 2- to 10-fold when sparsely inoculated cultures of C₆ rat glioma cells are allowed to grow to high cell density. Cyclic-nucleotide phosphohydrolase specific activity is also induced in C₆ cells and in oligodendrocytes by dibutyryl cyclic AMP or by agents that elevate intracellular cyclic AMP. In this report, we have compared the density-dependent induction of cyclic-nucleotide phosphohydrolase activity with the cyclic AMP-dependent induction. Dibutyryl cyclic AMP induced cyclic-nucleotide phosphohydrolase specific activity in both sparse and dense cultures which had very different density-dependent cyclic-nucleotide phosphohydrolase activities. Induction of both cyclic-nucleotide phosphohydrolase specific activity and intracellular cyclic AMP content by norepinephrine also occurred to a similar degree in sparse and dense cultures. Similar results were obtained for several clones of C₆ cells, and for a clone of oligodendrocyte x C₆ cell hybrids. Induction of cyclic-nucleotide phosphohydrolase by norepinephrine or dibutyryl cyclic AMP was not due to a change in cell density or rate of cell proliferation, nor did cell density have any appreciable effect on cyclic AMP content of the cells. These results show that regulation of cyclic-nucleotide phosphohydrolase activity in C₆ cells involves two distinct mechanisms.     

Gastrulation in the sea urchin embryo is accompanied by the rearrangement of invaginating epithelial cells

The second phase of gastrulation in the sea urchin embryo, secondary invagination, involves a dramatic elongation of the tube-like gut rudiment. The cells in the wall of the rudiment, which are organized as a monolayered epithelium, change their arrangement during this process. The number of cells in the wall of the gut rudiment at any given level along its long axis decreases markedly as determined by light microscopy of serial cross sections and by scanning electron microscopy, an observation that can be accounted for only if some of the cells exchange nearest neighbors during secondary invagination. Transmission electron microscopy reveals that cell rearrangement takes place despite the continued presence of typical intercellular junctional complexes. In addition to undergoing rearrangement, the cells in the wall of the gut rudiment change their shape during secondary invagination, becoming more flattened. These data raise the possibility that mechanisms other than the contraction of the filopodia of the presumptive secondary mesenchyme cells contribute to the second phase of invagination in the sea urchin embryo. In addition, the observation that cells in the wall of the gut rudiment undergo rearrangement during secondary invagination provides additional evidence that epithelial sheets can exhibit fluid-like properties during morphogenesis.     

Surface structure changes of rat adipocytes during lypolysis stimulated by various lypolysis stimulated by various lypolytic agents

A qualitative and quantitative electron microscopic study was performed on rat adipocytes during stimulation of lipolysis by various agents. Scanning electron microscopy of control cells revealed a spherical cell with a textured glycocalyx surface exhibiting small irregular projections. Globular surface evaginations or protrusions measuring 8-18 μM in diameter were seen on cell hemispheres, and there was an average of one protrusion for every two hemispheres examined. Distribution analysis showed that 60 percent of the hemispheres had no protrusions, and 25, 10, and 5 percent of the hemispheres had one, two or three protrusions, respectively. Thin-section and freeze- fracture electron microscopy of the protrusions showed a small triglyceride droplet surrounded by a thin cytoplasmic rim that was continuous with the main cytoplasmic matrix. The glycocalyx coating and plasma membrane extended from the cell surface onto, and over, the protrusion. Scanning microscopy of cells stimulated by lipolytic agents, including epinephrine, adrenocorticotropic hormone, theophylline, and dibutyryl cyclic AMP, revealed a dose-dependent increase in the number of protrusions per cell hemisphere. Maximal concentrations of lipolytic hormones cuase an average 2.5-fold increase in the number of protrusions per hemisphere without changing the average size of the protrusions. Only 40 percent of the stimulated cell hemispheres exhibited no protrusions; over 15 percent of the cells contained three or more; and a number of the protrusions were multilobulate. Insulin prevented the increase in the number of protrusions and the change in distribution caused by the lipolytic hormones but did not prevent the increase caused by theophylline and dibutryl cyclic AMP. The data suggest that the protrusions are a structural feature of the cell and may be related to the lypolytic pathway. These observations may help explain some of the discrepant biochemical data relating to hormonal stimulation of lipolysis.     

Calcium-mediated cell surface changes in Chinese hamster cells.

Scanning electron microscopy (SEM) revealed quantitative differences in both surface characteristics and spreading behavior of young and senescent WI38 human diploid fibroblasts. Measurement of spreading behavior involved the determination of the rate of cell elongation beyond an axial ratio of 1.5 after time intervals of 1.5, 3, 6, 9, and 12 h following subculture. Early passage cells exhibited a 44–52% increased spreading rate compared with later passage cells, both obeying zero-order kinetics. Surface microvilli and blebs were also found to vary in number and size after those same time intervals following subculture. In young cells, microvilli density was found initially to be very low but then increased steadily with time. Later passage cells exhibited an initial high density of microvilli, followed by a gradual disappearance with time. The density of cellular blebs did not, however, vary significantly with time following subculture. After approx. 24 h, both early and late passage cells resembled each other. These studies do show that physical membrane characteristics of young and senescent cells differ under certain conditions, and suggest the importance of that structure in the phenomena of senescence.     

Study of the effect of the fungicide zineb on microorganisms

Changes in yeast cells induced by the fungicide zineb were studied using biochemical, genetic techniques and electron microscopy. The primary changes involved anabolic processes since RNA and protein content increased in the cells. The data were consistent with a larger size of mitochondria. The oxidative-reductive activity of cell extracts did not change under the action of the fungicide. Zineb at a concentration of 0.5 to 50 mg/litre exerted a genetic activity towards indicator microorganisms. The changes in the morphophysiological characteristics of yeast growth under the action of zineb are presumed to be of a protective nature. Zineb traces in soil must be controlled in terms of the microbiological index.     

Cell-based chip for the detection of anticancer effect on HeLa cells using cyclic voltammetry

HeLa cells directly immobilized on gold-patterned silicon substrate were used to assess the biological toxicity of anticancer drugs (hydroxyurea and cyclophosphamide). Immobilization of HeLa cells was confirmed by optical microscopy, and cell growth, viability and drug-related toxicity were examined by cyclic voltammetry and potentiometric stripping analysis. The voltammetric behaviors of HeLa cells displayed a quasi-reversible pattern with the peak current exhibiting a linear relationship with cell number. The attached living cells were exposed to different concentrations of hydroxyurea and cyclophosphamide as anticancer drugs, which induced the change of cyclic voltammetry current peak. As the exposed concentration of anticancer drugs was increased, the change of current peak was increased, which indicates the decrease of cell viability. Trypan Blue dyeing was performed to confirm the results of the effect of anticancer drugs on the cell viability which was obtained from cyclic voltammetry assay. The proposed direct cell immobilization method technique can be applied to the fabrication of cell chip for diagnosis, drug detection, and on-site monitoring.     

Ultrastructure of Yersinia pseudotuberculosis in the process of their reversible transition into the dormant (non-culturable) state in association with blue-green algae

The ultrastructural organization of Y. pseudotuberculosis in the process of the transition of vegetative cells into the dormant (noncultivable) state in interaction with blue-green algae of the species Anabaena variabilis was studied by the method of transmission electron microscopy. The use of type specific Y. pseudotuberculosis serum made it possible to identify Y. pseudotuberculosis cells in the bacterial association and to find out whether their antigenic properties remained intact in time. The dormant forms of Y. pseudotuberculosis, recultivated by passage through the axenic culture of unfusoria (Tetrahymena pyryformis), were also studied with the use of electron microscopy. The revertants were found to be at different stages of restoration of their typical morphological characteristics and antigenic properties were partially retained. The fine structure of Y. pseudotuberculosis cells in the initial culture was shown to be similar to that of the revertants of dormant forms, morphological criteria of the dormant cell ultrastructure were established. The cyclic processes of reversible transition from vegetative forms to dormant ones in bacterial populations under the influence of hydrobios is regarded as an adaptive mechanism of their existence in the environment.     

CYCLIC AMP AND PHOSPHODIESTERASE IN SYNAPTIC VESICLES FROM MOUSE BRAIN

The isolation of synaptic vesicles from mouse brain by a modification of previously reported methodology is described. Homogeneity of the preparations was ascertained by electron microscopy. Vesicles thus isolated contained cyclic AMP (365 ± 44 pmol/mg of protein; mean ± S.E.M.) and adenosine 3′,5′-monophosphate phosphodiesterase activity (500 pmol/mg of protein per 30 min). The number of vesicles in three of the vesicle preparations was determined by a visual count of grid fields under the electron microscope. The content of cyclic AMP in the vesicles was calculated at 5000-7500 molecules/vesiclc. The presence of cyclic AMP in the synaptic vesicles from presynaptic nerve endings would support a role for this nucleotide in nerve transmission.     

Electron microscopic-cytochemical heterogeneity of succinate dehydrogenase activity in rat cardiomyocytes

The ultrastructural localization of succinate dehydrogenase in white rat heart myocytes is studied and heterogeneity of reaction products in separate mitochondria and their groups is described. The enzyme activity is cardiomyocyte electron density dependent. This dependence, in all probability, is the result of different structural and functional states of cells and their organelles, that is revealed by electron microscopy as different electron density of these. It is found that middle electron density cells have the maximum enzyme activity. The mechanisms of enzyme activity dependence of cell electron density are discussed.     

Extracellular slime associated with Proteus mirabilis during swarming.

Light microscopy, transmission electron microscopy, and scanning electron microscopy were used to visualize the extracellular slime of Proteus mirabilis swarm cells. Slime was observed with phase-contrast microscopy after fixation in hot sulfuric acid-sodium borate. Ruthenium red was used to stain slime for transmission electron microscopy. Copious quantities of extracellular slime were observed surrounding swarm cells; the slime appeared to provide a matrix through which the cells could migrate. Swarm cells were always found embedded in slime. These observations support the argument that swarming of P. mirabilis is associated with the production of large quantities of extracellular slime. Examination of nonswarming mutants of P. mirabilis revealed that a number of morphological changes, including cell elongation and increased flagellum synthesis, were required for swarm cell migration. It is still unclear whether extracellular slime production also is required for migration.     

Ultrastructure of fibre and parenchyma cell walls during early stages of culm development in Dendrocalamus asper

BACKGROUND AND AIMS: The anatomy of bamboo culms and the multilayered structure of fibre cell walls are known to be the main determinant factors for its physical and mechanical properties. Studies on the bamboo cell wall have focussed mainly on fully elongated and mature fibres. The main aim of this study was to describe the ultrastructure of primary and secondary cell walls in culm tissues of Dendrocalamus asper at different stages of development. METHODS: The development of fibre and parenchyma tissues was classified into four stages based on light microscopy observations made in tissues from juvenile plants. The stages were used as a basis for transmission electron microscopy study on the ultrastructure of the cell wall during the process of primary and early secondary cell wall formation. Macerations and phloroglucinol-HCl staining were employed to investigate fibre cell elongation and fibre cell wall lignification, respectively. KEY RESULTS: The observations indicated that the primary wall is formed by the deposition of two distinct layers during the elongation of the internode and that secondary wall synthesis may begin before the complete cessation of internode and fibre elongation. Elongation was followed by a maturation phase characterized by the deposition of multiple secondary wall layers, which varied in number according to the cell type, location in the culm tissue and stage of shoot development. Lignification of fibre cell walls started at the period prior to the cessation of internode elongation. CONCLUSIONS: The structure of the primary cell wall was comprised of two layers. The fibre secondary cell wall began to be laid down while the cells were still undergoing some elongation, suggesting that it may act to cause the slow-down and eventual cessation of cell elongation.     

Vacuolar dynamics in synchronously budding yeast

Summary A simple and rapid method for obtaining synchronously budding cultures of Saccharomyces cerevisiae is described. Synchronous cultures were started with homogeneous cell fractions isolated from exponentially growing cultures by isopycnic centrifugation in osmotically inactive media. The technique of fractionation is based on changes of cell density throughout the budding cycle. These changes are correlated with vacuolar changes observed in the light and electron microscope. During bud initiation the large vacuoles in late budding cells shrink and fragment into small vacuoles. Simultaneously the density of the cells increases. Later stages of the budding cycle are characterized by the distribution of the small vacuoles between mother and daughter cell, followed by their fusion and expansion, and by a decreasing density of the cells. The relative changes in cell density and dry weight and in the content of different macromolecules during the budding cycle suggest a cyclic change between utilization of endogenous and exogenous substrates. This is discussed in terms of a cyclic consumption and accumulation of vacuolar pools.     

Fine structure of the midgut epithelium in the developing brown shrimp,Penaeus aztecus

The midgut epithelium of larval and early postlarval brown shrimp has been studied with light and electron microscopy. Ultrastructurally the features of the midgut do not change during these stages of development. On the basis of electron density, two epithelial cell types can be distinguished, and these are referred to as light and dark cells. The dark cells contain more rough endoplasmic reticulum and more free ribosomes than the light cells. Mitochondria in the dark cells have a matrix which is less electron dense than the mitochondrial matrix of the light cells. Both cell types have a microvillous border with a surface coat. The microvilli lack microfilaments within their core, and a terminal web is not differentiated in the stages examined. Tubular smooth endoplasmic reticulum is abundant in the basal portions of the cells. Electron dense, membrane bound vesicles are consistently seen in association with the Golgi apparatus, apical cell surface, and gut lumen and therefore are believed to be secretory granules. Cells in the anterior portion of the midgut often contain very large lipid droplets in the cytoplasm.     

Distribution of insulin receptors in liver cell suspensions using a minibead probe : Highest density is on endothelial cell

The distribution of insulin receptors was studied in rat liver cell suspensions using a latex minibead covalently bound to insulin. This probe can be visualized by electron microscopy (EM). Using this visual probe, the highest density of the receptor was found on endothelial cells in the cell suspension, with hepatocytes having only few receptors and Kupffer cells having none. Fractionation of liver cell suspensions on metrizamide gradients yielded two populations of cells; large cells (hepatocytes) and small cells which consisted mostly of Kupffer cells and endothelial cells, distinguishable by their surface and cytoplasmic features. Again, by the use of an insulin-minibead probe, the highest density of receptors was found on endothelial cells. It is suggested that the endothelium has a crucial role in the uptake and transport of the hormone across the tissue-blood barrier.     

Evaluation of Stem Cell-to-Tenocyte Differentiation By Atomic Force Microscopy to Measure Cellular Elastic Moduli

In the present study, we evaluated whether stem cell-to-tenocyte differentiation could be evaluated via measurement of the mechanical properties of the cell. We used mechanical uniaxial cyclic stretching to induce the differentiation of human bone marrow mesenchymal stem cells into tenocytes. The cells were subjected to cyclic elongation of 10 or 15 % at a cyclic frequency of 1 Hz for 24 or 48 h, and differentiation was assessed by real-time PCR (rtPCR) determination of messenger RNA expression levels for four commonly used markers of stem cell-to-tenocyte differentiation: type I collagen, type III collagen, tenascin-C, and scleraxis. The rtPCR results showed that cells subjected to 10 % cyclic elongation for 24 or 48 h differentiated into tenocytes. Atomic force microscopy (AFM) was then used to measure the force curves around the cell nuclei, and the AFM data were used to calculate the elastic moduli of the cell surfaces. The elastic modulus values of the control (non-stretched) cells differed significantly from those of cells stretched at 10 % for 24 or 48 h (P < 0.01). Confocal fluorescence microscopic observations of actin stress fibers suggested that the change in elastic modulus was ascribable to the development of the cellular cytoskeleton during the differentiation process. Therefore, we conclude that the atomic force microscopic measurement of the elastic modulus of the cell surface can be used to evaluate stem cell-to-tenocyte differentiation.     

Reappraisal of histogenesis in the bursal lymphoid follicle of the chicken.

The development of the bursal follicle and the appearance of the follicle-associated epithelial (FAE) cell and the reticuloepithelial (REp) cell were studied. The stages of development of the bursal follicle were observed by light and electron microscopy; an anticytokeratin monoclonal antibody was also used. At the beginning of follicle development, a mesenchymal cell cluster is observed in the tunica propria; the cluster becomes wedged in a niche of the surface epithelium, and gradually it is completely surrounded by the epithelium itself, which closes under the clump of mesenchymal cells. The epithelial cells lying upon the mesenchymal clump become necrotic, and a number of mesenchymal cells bulge out, forming the FAE cells. The epithelial cells that have closed under the mesenchymal nodule become stratified and form the REp cells; they become star-shaped because the medullary-lymphoid cells grow between them. Finally, the cortex is formed, possibly as a result of the migration of medullary cells before they peripheralize. It is concluded that FAE cells are not specialized epithelial cells, as they do not react to an anticytokeratin monoclonal antibody; on the contrary, they are formed by mesenchymal stemcells that bulge into the lumen and change their character after moving into the epithelium. The REp cells appear in the follicular primordium shortly after the bursal follicle begins to develop; the pronounced reactivity of the REp cells to an anticytokeratin monoclonal antibody supports the hypothesis of their epithelial origin.     

Cell junction and cyclic AMP: III. Promotion of junctional membrane permeability and junctional membrane particles in a junction-deficient cell type

Summary The cyclic nucleotide effect on junction was studied in C1-1D cells, a mouse cancer cell type that fails to make permeable junctions in ordinary confluent culture. Upon administration of cyclic AMP, dibutyryl cyclic AMP, dibutyryl cyclic AMP plus caffeine (db-cAMP-caffeine), or cholera toxin (an adenylate cyclase activator), the cells acquired permeable junctions; they became electrically coupled and transferred fluorescent tracer molecules among each other—a transfer exhibiting the molecular size limit of permeation of normal cell-to-cell channels. The effect took several hours to develop. With the db-cAMP-caffeine treatment, junctional permeability emerged within two hours in one-fifth of the cell opopulation, and within the next few hours in the entire population. This development was not prevented by the cytokinesis inhibitor cytochalasin B. Permeable junctions formed also in two other conditions where the cell-endogenous cyclic AMP level may be expected to increase: serum starvation and low cell density. After three weeks of starving the cells of serum, a junctional permeability arose in confluent cultures, which on feeding with serum disappeared within two to three days. At low cell density, namely below confluency, the cells made permeable junctions, unstarved. In cultures of rather uniform density, the frequency of permeable junctions was inversely related to the average density, over the subconfluent range; at densities of about 1×10⁴ cells/cm², where the cells had few mutual contacts, 80% of the pairs presumed to be in contact were electrically coupled. In cultures with adjoining territories of high (confluent) and low cell density, there was coupling only in the last, and in this low-density state the cells were also capable of coupling with other mammalian cell types (mouse 3T3-BalbC and human Lesch-Nyhan cells).Correlated electron microscopy of freeze-fractured cell junctions showed no membrane differentiation in confluent C1-1D cultures. The junctions acquired differentiations, namely particle clusters of gap junction and strands of tight junction, upon cyclic nucleotide application or serum starvation and in the lowdensity condition. With db-cAMP-caffeine, these differentiations appeared within 4 hr of the treatment (confluent cultures), growing in size over the next hours. Treatment with cycloheximide, but not with cytochalasin B, prevented the development of recognizable gap junction and tight junction in cultures supplied with db-cAMP-caffeine.