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At least 1,495 records · Page 83Linked to original sources
PubMed1992

Effect of monensin on ultrastructure and cellular invasion by the turkey coccidia Eimeria adenoeides and Eimeria meleagrimitis.

Freshly excysted sporozoites (SZ) of the turkey coccidia Eimeria meleagrimitis and Eimeria adenoeides were incubated at 41 C in concentrations of monensin from .01 to 1.0 microgram/mL, washed free of the drug, and either processed for phase, fluorescence, and transmission electron microscopy or inoculated into cultures of turkey kidney cells. Phase microscopy indicated that after 1.5 h incubation in 1.0 micrograms/mL monensin, about 60% of the SZ of E. meleagrimitis had become notably rounded or displayed localized protrusions. These alterations were accompanied by ultrastructural abnormalities (in 90% of the SZ) including vacuoles in the cytoplasm, bulging and separation of plasma membrane layers, and dense bands in the refractile bodies that extended toward the periphery of the refractile body. Similar morphological and ultrastructural changes were observed in over half of the E. adenoeides SZ after 2 h incubation in 1.0 micrograms/mL monensin. Additionally, some specimens contained a pycnotic nucleus that was usually surrounded by a large vacuole. After 4 h incubation, almost all of the SZ displayed some degree of ultrastructural damage. Indirect fluorescent antibody labeling with parasite-specific monoclonal antibodies demonstrated clouds of antigen surrounding the monensin-treated but not the untreated SZ, suggesting an increase in permeability with incubation in monensin. With both E. meleagrimitis and E. adenoeides, the structural changes were reflected in a significant inhibition of cellular invasion. The inhibitory activity of monensin was concentration- and time-dependent in that the greatest inhibition of invasion was observed in SZ incubated for 4 h in 1.0 micrograms/mL of monensin; shorter incubation times or lower concentrations of monensin having less effect.

Animals↗
PubMed1978

Application of a solid-phase radioimmunoassay and immune electron microscopy for hepatitis A in diagnosis and research.

With crude virus suspensions from stool and antibodies from hepatitis-A patients, a solid-phase radioimmunoassay (RIA) for detection of hepatitis virus A (HVA) had antibodies against hepatitis virus A (anti-HVA) has been developed. Examples for the application of this test are demonstrated. Virus particles from the stools of the two patients were further characterized. Serologically, they were identical or very similar to the MS-1 strain. Isopycnic CsCl-gradient centrifugation of both strains revealed two peaks, but the particles of different densities did not differ in size or serologically. A modification of the RIA was also useful for determination of IgM antibodies in patients' sera fractionated by sucrose-density centrifugation. The application of the RIA method for serologic epidemiology is demonstrated by a comparison of anti-HVA prevalence in German and non-German women residing in Germany.

Adolescent↗
PubMed1979

Normal and pseudorabies virus infected primary nerve cell cultures in scanning electron microscopy.

Primary cell cultures from the central nervous system of the embryonic rat were inoculated with pseudorabies virus. Their morphological changes were studied by phase contrast microscopy and by scanning as well as by transmission electron microscopy. Uninfected cultures display two distinct cell layers in scanning electron microscopy: a flat continuous monolayer supports a heterogeneous population of individual, presumably neural cells, which emit processes of different number and size. The latter cells form contacts by a dense network of fibres. Infectious virus is propagated in these nerve cell cultures with similar effectivity as in other cultures. The infectoin leads to fusion and death of the cells. By the time the cytopathic effect is visible, nearly all cells, including those of neuronal and those of nonneuronal appearance, are studded with ample amounts of virus-sized particles. The particles represent viruses as demonstrated by transmission electron microscopy or by treatment with a hyperimmune serum directed against pseudorabies virus structural components. Hyperimmune serum leads to clustering of the particles at the cell surface. The amount of virus particles per surface unit was about 10 times higher on neural cells as compared to primary rabbit kidney cells. The concentration of infectious particles in the supernatant, however was approximately the same. The system described appears to be useful for the study of acute virus effects on neural tissue under strictly controlled conditions.

Animals↗
PubMed2001

Procoagulant platelet balloons: evidence from cryopreparation and electron microscopy.

Visualisation of the procoagulant transformation of human platelets has recently become possible through use of an in vitro approach combined with fluorescence and phase contrast microscopy. Here, we extended these studies to the ultrastructural level by employing both rapid freezing/freeze-substitution and conventional ambient-temperature chemical fixation for transmission and scanning electron microscopy. Procoagulant transformation was only inducible by adhering platelets to collagen fibrils or to the collagen-related peptide and exposing them to physiological extracellular Ca2+ levels. Under these conditions prominent, 2- to 4-micron-wide balloon-like structures were regularly observed, regardless of the specimen fixation protocol. In strong contrast to normal platelets in their vicinity, the balloons' subcellular architecture proved remarkably poor: dilute cytoplasm, no cytoskeleton, only a few, randomly distributed organelles and/or their remnants. Cryofixed balloons displayed intact and smooth surfaces whereas conventional specimen processing caused plasma membrane perforations and shrinkage of the balloons. Our results clearly show that neither the balloons themselves, nor their simple ultrastructure reflect fixation artefacts caused by inadequate membrane stabilisation. The balloons are interpreted as to be transformed and/or fragmented procoagulant platelets. Thus, the generation of balloons represents a genuine, final stage of platelet ontogenesis, presumably occurring alternatively to aggregate formation.

Blood Platelets↗
PubMed1983

Analysis of anaphase B in saccharomyces cerevisiae using a monoclonal antibody against yeast tubulin.

In Saccharomyces cerevisiae the mitotic spindle is composed of approximately 10 continuous microtubules, that pass uninterrupted between the 2 spindle pole bodies (SPBs), and up to 34 discontinuous microtubules, 17 of which arise from each SPB and terminate in the nucleoplasm [7, 12]. We have previously shown that during spindle elongation (anaphase B) the number of continuous microtubules decreases rapidly until at a length of 4 microns only 1 remains [8]. Further elongation from 4 to 8 microns is achieved with only a single continuous microtubule. Here we have extended our analysis of spindle elongation by investigating the timing of this event by indirect immunofluorescence microscopy using a monoclonal antibody to yeast tubulin. From the frequency distribution of spindle lengths in exponential phase cultures of the haploid wild type strain A364A, the time in mitosis and the velocity of spindle elongation have been calculated. For the first 55 min of mitosis spindle elongation proceeds at a slow rate, 0.36 microns/min. There is then a marked increase to a maximal value of 4.16 microns/min. Correlated with data from previous ultrastructural studies on the decrease in continuous microtubule number during anaphase B, this 12-fold increase in velocity is shown to occur at the single microtubule stage. These experiments provide further evidence to suggest that the role of the spindle is to regulate the rate of anaphase B which is actively achieved by an, as yet, unknown motive force generating system. Antibody staining has also allowed the terminal phenotypes of 2 diploid cell division cycle (cdc) mutants to be more accurately described. This has shown that, although arrested synchronously as judged by phase contrast microscopy, these strains do in fact exhibit considerable heterogeneity in spindle length.

Anaphase↗
PubMedDate not supplied

[Nitrogen fixing activity of pea nodule bacteria during different phases of host plant development].

The activity of nitrogenase in nodules of pea plants and the rate of diurnal nitrogen fixation were studied at different phases of the plant growth using the acetylene and isotope techniques. At the same time, the morphology of nodule bacteria was studied by electron and phase-contrast microscopy. About 90% of the bacteria in nodules were found in the form of bacteroids from the early phase of the plant growth (budding) to ripening when gemmated arthrospores could be observed on some bacteroids. The highest activity of nitrogenase and nitrogen fixation was detected at the reproductive stage of growth. Therefore, in order to obtain more active strains of pea nodule bacteria, they should be isolated from nodules at this stage of plant growth.

Fabaceae↗
PubMed2004

Macropinocytosis is the endocytic pathway that mediates macrophage foam cell formation with native low density lipoprotein.

Previously, we reported that fluid-phase endocytosis of native LDL by PMA-activated human monocytederived macrophages converted these macrophages into cholesterol-enriched foam cells (Kruth, H. S., Huang, W., Ishii, I., and Zhang, W. Y. (2002) J. Biol. Chem. 277, 34573-34580). Uptake of fluid by cells can occur either by micropinocytosis within vesicles (<0.1 microm diameter) or by macropinocytosis within vacuoles ( approximately 0.5-5.0 microm) named macropinosomes. The current investigation has identified macropinocytosis as the pathway for fluid-phase LDL endocytosis and determined signaling and cytoskeletal components involved in this LDL endocytosis. The phosphatidylinositol 3-kinase inhibitor, LY294002, which inhibits macropinocytosis but does not inhibit micropinocytosis, completely blocked PMA-activated macrophage uptake of fluid and LDL. Also, nystatin and filipin, inhibitors of micropinocytosis from lipid-raft plasma membrane domains, both failed to inhibit PMA-stimulated macrophage cholesterol accumulation. Time-lapse video phase-contrast microscopy and time-lapse digital confocal-fluorescence microscopy with fluorescent DiI-LDL showed that PMA-activated macrophages took up LDL in the fluid phase by macropinocytosis. Macropinocytosis of LDL depended on Rho GTPase signaling, actin, and microtubules. Bafilomycin A1, the vacuolar H+-ATPase inhibitor, inhibited degradation of LDL and caused accumulation of undegraded LDL within macropinosomes and multivesicular body endosomes. LDL in multivesicular body endosomes was concentrated >40-fold over its concentration in the culture medium consistent with macropinosome shrinkage by maturation into multivesicular body endosomes. Macropinocytosis of LDL taken up in the fluid phase without receptor-mediated binding of LDL is a novel endocytic pathway that generates macrophage foam cells. Macropinocytosis in macrophages and possibly other vascular cells is a new pathway to target for modulating foam cell formation in atherosclerosis.

Cells, Cultured↗
PubMed1989

Isolation of large sheets of apical material from frog urinary bladder epithelial cells by freeze-fracture.

In the amphibian urinary bladder, water permeability is correlated with the insertion of intramembrane particle aggregates (IMPAs) into the apical plasma membrane (AM) of the granular cells. These aggregates are believed to contain water channels. Characterization of the IMPAs by comparing AM fractions of antidiuretic hormone (ADH)-treated and resting epithelia requires isolation and purification of AM-rich material, free of other cytoplasmic aggregate-containing organelles, in both cases. A technique derived from freeze-fracture was chosen to isolate large sheets of apical membrane material from frog (Rana esculenta) urinary bladder epithelium. The apical side was plated on a polylysine-coated glass slide, frozen with liquid nitrogen, and fractured. A nylon mesh was inserted between the glass slide and the bladder, in order to bring the fracture plane back to the AM periodically. Fluorescent markers were used to characterize the material having fractured with the glass slide. Samples were observed by fluorescence and phase contrast microscopy. We obtained evidence that numerous patches of fractured AM remained on the glass surface without nuclei. A phase contrast picture was obtained only at a high magnification, indicating a low thickness of the recovered material. Further characterization was made with SDS-PAGE. Protein contents of samples were extracted under various experimental conditions and the patterns of ADH-treated, resting AM samples, or whole epithelial cell crude homogenates, were compared. Staining of some bands increased under certain conditions, whereas many others disappeared. Both morphological and biochemical approaches demonstrate that the recovered material was apical in origin.

Animals↗
PubMed2006

Immunological and ultrastructural characterization of endothelial cell cultures differentiated from human cord blood derived endothelial progenitor cells.

The replacement of endothelium by endothelial progenitor cells (EPCs) for therapeutic use in order to ameliorate the vascular status of ischemic organs is now in the focus of vascular research. The aim of our studies was to investigate whether EPCs derived from peripheral blood mononuclear cells (PBMNCs-derived EPCs) or EPCs propagated from CD34(+) hematopoietic stem cells (HSCs-derived EPCs), both isolated from human cord blood, are able to differentiate into early mature endothelial cells (ECs) under certain in vitro conditions. We characterized both cell populations by flow cytometry, phase contrast microscopy, fluorescence microscopy and confocal laser scanning microscopy as well as ultrastructurally using transmission and scanning electron microscopy. While PBMNCs gave rise to clusters of spindle-like EPCs after few days but did not further mature under in vitro conditions, mature ECs could only be successfully propagated from a starting population of isolated HSCs. Both, PBMNCs- and HSCs-derived EPCs, took up Dil-labeled acetylated low density lipoprotein (Dil-Ac-LDL) and could be positively stained for CD31, CD105, the vascular endothelial growth factor receptor 2 (VEGFR-2, KDR) and ulex europaeus agglutinin 1 (UEA-1) at the cell surface. EPC showed surface expression of CD54 and CD106. However, only a small portion of HSCs-derived EPCs was positive for CD54 but negative for CD106. Intracellular staining for von Willebrand factor (vWF) provided a homogenous stain in PBMNC-derived EPCs while in HSCs-derived EPCs, during cultivation for 2-3 weeks, more and more a typical punctuated staining pattern related to Weibel-Palade bodies (WPBs) was visible. By phase contrast and scanning electron microscopy, an arrangement of PBMNCs-derived EPCs in cord-like structures could be demonstrated. In these formations, cells showed parallel alignment but exhibited only few cell contacts. Well-developed WPBs could never be found in PBMNCs-derived EPCs. In contrast, differentiating HSCs-derived EPCs developed adherence junctions, interdigitating junctions as well as syndesmos. During maturation, spindle-like cell types appeared with abundant WPBs as well as cobblestone-like cell types with a fewer content of these organelles. WPBs, in the spindle-like cell types displayed conspicuous shapes and were concentrated in close proximity to mitochondria-rich areas. HSCs-derived EPCs exhibited signs of high synthetic activity such as a well-developed rough endoplasmic reticulum (RER) and multiple Golgi complexes. In the trans-Golgi network (TGN), close to the Golgi complex, a new formation of WPBs could be observed. These morphological features correlated well with a high growing capacity. Although it was not possible to demonstrate the complete differentiation line from HSCs to early matured ECs by immunologic markers because of the limited number of cells available for such investigations, distinct morphologic maturation stages could be shown at light and electron microscopical levels. In conclusion, the study presented here characterizes not only the different cell populations involved in the differentiation of early EPCs into mature ECs but also the transition stage where the maturation step takes place by demonstration of the new formation of WPBs. In this respect, these investigations provide new insights into the in vitro differentiation which could have some in vivo correlation.

Antigens, CD34↗
PubMed1999

Role of endothelial cell-substrate contact area and fibronectin-receptor affinity in cell adhesion to HEMA/EMA copolymers.

The objective of this study was to examine the effect of substrate hydrophobicity on cell-substrate contact area and the affinity between adsorbed fibronectin (Fn) and its receptor. Homo- and copolymer films of hydrophobic ethyl methacrylate (EMA) and hydrophilic hydroxyethyl methacrylate (HEMA) were spun-cast onto glass slides. Bovine aortic endothelial cells (BAEC) were plated for 2 h in serum-free medium onto polymers preadsorbed with Fn. Cells were fixed, labeled, and examined by total internal reflection fluorescence microscopy (TIRFM) to determine the topography of the basal surface as a function of distance from the substrate. Phase contrast microscopy was used to examine the total projected area of adherent cells. The cumulative contact area was greatest on cells attached to surfaces prepared from 0% HEMA and lowest on surfaces with the highest HEMA content. An equilibrium adhesion model used these data together with the critical force for detachment and the Fn density (Burmeister et al., J Biomed Mater Res 1996;30:13-22) to determine the affinity between Fn and its receptor and the bond strength. The affinity and force per bond decreased with increasing HEMA content. These results indicate that differences in the strength of endothelial cell adhesion to polymers are influenced by the conformation of the adsorbed adhesion proteins.

Animals↗
PubMed1996

Cell cycling and patterned cell proliferation in the wing primordium of Drosophila.

The pattern of cell proliferation in the Drosophila imaginal wing primordium is spatially and temporally heterogeneous. Direct visualization of cells in S, G2, and mitosis phases of the cell cycle reveals several features invariant throughout development. The fraction of cells in the disc in the different cell cycle stages is constant, the majority remaining in G1. Cells in the different phases of the cell cycle mainly appear in small synchronic clusters that are nonclonally derived but result from changing local cell-cell interactions. Cluster synchronization occurs before S and in the G2/M phases. Rates of cell division are neither constant nor clonal features. Cell cycle progression is linear rather than concentric. Clusters appear throughout the disc but with symmetries related to presumptive wing patterns, compartment boundaries, and vein clonal restrictions.

Animals↗
PubMed2005

Decoupled phase transitions and grain-boundary melting in supported phospholipid bilayers.

Two separate liquid-solid phase transitions are detected in the two monolayers of a mica-supported phospholipid bilayer by atomic force microscopy. The phase transitions of the two monolayers are decoupled by the stronger interaction between the lipid headgroups of the proximal monolayer and the mica support. The transition temperature of the proximal monolayer is increased and this transition occurs over a narrower temperature range. Both transitions occur via grain-boundary melting and the variation of the width of the interfacial zone with temperature is consistent with mean-field theory.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1983

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.

Flagella↗
PubMed1989

Intermediates in membrane fusion and bilayer/nonbilayer phase transitions imaged by time-resolved cryo-transmission electron microscopy.

Bilayer-to-nonbilayer phase transitions in phospholipids occur by means of poorly characterized intermediates. Many have proposed that membrane fusion can also occur by formation of these intermediates. Structures for such intermediates were proposed in a recent theory of these transition mechanisms. Using time-resolved cryo-transmission electron Microscopy (TRC-TEM), we have directly visualized the evolution of inverted phase micro-structure in liposomal aggregates. We have identified one of the proposed intermediates, termed an interlamellar attachment (ILA), which has the structure and dimensions predicted by the theory. We show that ILAs are likely to be the structure corresponding to "lipidic particles" observed by freeze-fracture electron microscopy. ILAs appear to assemble the inverted cubic (III) phase by formation of an ILA lattice, as previously proposed. ILAs are also observed to mediate membrane fusion in the same systems, on the same time scale, and under nearly the same conditions in which membrane fusion was observed by fluorescence methods in earlier studies. These earlier studies indicated a linkage between a membrane fusion mechanism and III phase formation. Our micrographs suggest that the same intermediate structure mediates both of those processes.

Lipid Bilayers↗