A scanning electron microscopy study of double minutes from a human tumour cell line

Double minutes from a human tumour cell line were examined in intact metaphase spreads using scanning electron microscopy. They were discrete, acentromeric, compact spheres of chromatin fibres similar to the chromatin of the metaphase chromosomes within the same cells. They were closely associated with the telomeres and chromatids of the metaphase chromosomes.     

Morphofunctional characteristics of Clara cells in hypoxia in rats from the data of scanning and transmission electron microscopy

The experiments on the model of altitude-chamber hypoxia in rats have established that within 15 days the secretory activity of terminal bronchiolar Clara cells increased in the secret accumulation phase and was accompanied by the transformation of the apical surface relief and ultrastructure of synthetic cell apparatus. Chronic hypoxia lasting for up to 60 days leads to compensation-adaptation changes of Clara cell ultrastructure, providing the intensification of secretion processes and postsecretion repair of membranes of the apical surface cells.     

Measuring elastic properties of cells by evaluation of scanning acoustic microscopy V(Z) values using simplex algorithm

In this paper a new technique is proposed to determine the acoustic properties as well as the thickness (and volume) of biological cells. Variations of thickness, density, acoustic wave velocity, stiffness, and attenuation coefficient of a living or dead cell are obtained by scanning the cell by an acoustic microscope. The distance between the cell and the microscope lens is varied and several voltage curves are thus obtained. These curves are then inverted by simplex optimization technique to obtain the cell parameters. The spatial resolution of the method is limited to the resolution of the scanning acoustic microscope. It allows to take advantage of the full range of frequencies and amplification of the microscope. Characteristic distributions of stiffness are exemplified with an endothelial cell in culture. The main part of the thin, lamellar cytoplasm has high stiffness, which drops close to the lamella/cell body transition region and only slightly increases again through the central part of the cell. Acoustic attenuation seems to be related to two factors, cytoplasm accumulation (in the lamellar parts) and scattering in the central part rich in organelles.     

Response of epithelial and mesenchymal cells to culture on basement lamella observed by scanning microscopy

The basement lamella of Xenopus tadpole skin has been viewed in situ by scanning microscopy, then isolated by trypsin treatment and used as a substrate for cell culture. The basal lamina may also be viewed after EDTA treatment. Responses of epithelial and mesenchymal cells to the lamella have been compared. Mesenchymal cells from chick skin and heart ventricle flatten and attach between the plies of the lamella, then infiltrate it. Myoblasts appear to move less readily within the lamella. Embryonic Xenopus skin epithelium spreads over the surface. Isolated chick skin epithelial cells first begin to spread, then round up and eventually attach to each other in clusters which form a flat basal surface above the lamella. Thus epithelial and mesenchymal cells cultured on this isolated extracellular material mimic aspects of normal tissue organization.     

A study of extracellular vesicles isolated from blood plasma conducted by low-voltage scanning electron microscopy

Extracellular vesicles secreted by cells represent an almost spherical membrane structures enriched with biological molecules of different types. The number and molecular composition of these structures depend on both the physiological state of an organism and underlying diseases. Despite extracellular vesicles playing an important role in intercellular communication and being potential biomarkers of pathological processes, the mechanisms of their formation, their functions, and their morphological characteristics are poorly studied. Low-voltage scanning electron microscopy is a promising method for studying extracellular vesicles, since it does not need a layer of conductive covering and, consequently, permits morphological details of studied objects to be vizualized at a high resolution in a nanometer range. The results of investigation of the morphology and sizes of objects in blood-plasma fractions by low-voltage scanning electron microscopy are presented in this study.     

Use of a DNA probe for mapping by electron microscopy the ribosomal sequences in ribosomal RNA precursors from duck cells

This report describes the use of purified ribosomal DNA to map by electron microscopy the relative positions of the 18 S and 28 S RNA regions within the duck rRNA precursor and their relationship to the non-conserved portions of the precursor molecule. By repeated fractionation of the total DNA, based on the relative reassociation rates of the DNA sequences with different degrees of repetition, a fraction of the rapidly renaturing DNA was obtained which comprised only 6% of the total DNA, but contained 71% of the rRNA cistrons. Further purification of the rDNA was achieved by saturation hybridization with rRNA and separation of the rRNA-rDNA hybrids by banding in CsCl. In this manner, an rDNA-rRNA fraction was obtained which had a buoyant density of 1.805 g/cm³, an RNA to DNA ratio of 1.01, and a base composition for the RNA present in the hybrid identical to that of an equimolar mixture of 18 S and 28 S rRNA. The final yield of rDNA isolated by this procedure is 32%. When the purified rDNA was annealed with a mixture of 18 S and 28 S rRNA and the hybrids spread for electron microscopy, they appeared as two distinct populations with a number-average length of 0.62 ± 0.13 μm and 1.37 ± 0.18 μm, respectively. Likewise, hybrids between the rRNA precursor, isolated from duck embryo fibroblasts, and the rDNA appeared as structures containing two duplex regions of lengths 0.60 ± 0.11 μm and 1.38 ± 0.15 μm, separated from each other by a single-stranded region appearing as a large bush: this represents a portion of the precursor molecule not conserved during processing of the parent molecule. From these observations a model of the structure of the duck rRNA precursor is proposed.     

A phase synchronization clustering algorithm for identifying interesting groups of genes from cell cycle expression data

Background

Quantitative proteomics holds great promise for identifying proteins that are differentially abundant between populations representing different physiological or disease states. A range of computational tools is now available for both isotopically labeled and label-free liquid chromatography mass spectrometry (LC-MS) based quantitative proteomics. However, they are generally not comparable to each other in terms of functionality, user interfaces, information input/output, and do not readily facilitate appropriate statistical data analysis. These limitations, along with the array of choices, present a daunting prospect for biologists, and other researchers not trained in bioinformatics, who wish to use LC-MS-based quantitative proteomics.

Results

We have developed Corra, a computational framework and tools for discovery-based LC-MS proteomics. Corra extends and adapts existing algorithms used for LC-MS-based proteomics, and statistical algorithms, originally developed for microarray data analyses, appropriate for LC-MS data analysis. Corra also adapts software engineering technologies (e.g. Google Web Toolkit, distributed processing) so that computationally intense data processing and statistical analyses can run on a remote server, while the user controls and manages the process from their own computer via a simple web interface. Corra also allows the user to output significantly differentially abundant LC-MS-detected peptide features in a form compatible with subsequent sequence identification via tandem mass spectrometry (MS/MS). We present two case studies to illustrate the application of Corra to commonly performed LC-MS-based biological workflows: a pilot biomarker discovery study of glycoproteins isolated from human plasma samples relevant to type 2 diabetes, and a study in yeast to identify in vivo targets of the protein kinase Ark1 via phosphopeptide profiling.

Conclusion

The Corra computational framework leverages computational innovation to enable biologists or other researchers to process, analyze and visualize LC-MS data with what would otherwise be a complex and not user-friendly suite of tools. Corra enables appropriate statistical analyses, with controlled false-discovery rates, ultimately to inform subsequent targeted identification of differentially abundant peptides by MS/MS. For the user not trained in bioinformatics, Corra represents a complete, customizable, free and open source computational platform enabling LC-MS-based proteomic workflows, and as such, addresses an unmet need in the LC-MS proteomics field.     

Characterization of cell suspensions adhering to nylon obtained from mononuclear cells in toto or depleted of monocytes

The aim of this work was to evaluate the effect of monocyte depletion on the preparation of B cell enriched suspensions for DR typing. Nylon fiber adherent-cells obtained from total, or monocyte depleted, MNC were identified using different surface markers and cytochemical staining (SIg, DR, ANAE). The results demonstrate that no significant improvement in B enrichment is obtained by monocyte depletion.     

A reversible multi-well chamber for incubation of cultured cells with small volumes: application to screening of hybridoma fusions using immunofluorescence microscopy

Immunofluorescence microscopy is a powerful technique for detecting the location of surface and intracellular antigens in individual cells. However, using standard methods, processing large numbers of samples for immunofluorescence is cumbersome and difficult. To simplify greatly this process, we have developed a chamber that reversibly creates multiple small wells in a large (150 mm) tissue culture dish. This device allows the rapid and convenient processing of hundreds of samples each of 100 microliters volume. Each sample is examined using a short working distance, high numerical aperture immersion objective for maximum sensitivity and resolution. This apparatus makes immunofluorescence a practical method for the primary screening of hybridoma clones.     

A new approach to three-dimensional reconstructed imaging of hormone-secreting cells and their microvessel environments in rat pituitary glands by confocal laser scanning microscopy.

There has been considerable interest in the relationship between hormone- secreting endocrine cells and their microvessels in human pituitary gland. However, microcirculatory networks have rarely been studied in three dimensions (3D). This study was designed to visualize and to reveal the relationship between hormone-secreting endocrine cells and their microvessel environment in 3D, using rat pituitary glands under various (hyper/hypo) experimental conditions by confocal laser scanning microscopy (CLSM). Female adult Wistar rats were used after bilateral adrenalectomy or ACTH administration for 2 weeks. Clear 3D reconstructed images of ACTH cells, the microvessel network and counterstained nuclei were obtained at a maximal focus depth of 1 mm by CLSM without any background noise. In the hyperfunctional state, slender cytoplasmic processes of hypertrophic stellate ACTH cells frequently extended to the microvessels. In the hypofunctional state, ACTH cells appeared atrophic and round with scanty cytoplasm, and cytoplasmic adhesions to microvessel network patterns were inconspicuous. Therefore, 3D reconstructed imaging by CLSM is a useful technique with which to investigate the microvessel environment of hormone-secreting cells and has the potential to reveal dynamic hormone-secreting pathways.     

Statistical evaluation of cell kinetic data from DNA flow cytometry (FCM) by the EM algorithm

Flow cytometric DNA measurements yield the amount of DNA for each of a large number of cells. A DNA histogram normally consists of a mixture of one or more constellations of G0/G1-, S-, G2/M-phase cells, together with internal standards, debris, background noise, and one or more populations of clumped cells. We have modelled typical DNA histograms as a mixed distribution with Gaussian densities for the G0/G1 and G2/M phases, an S-phase density, assumed to be uniform between the G0/G1 and G2/M peaks, observed with a Gaussian error, and with Gaussian densities for standards of chicken and trout red blood cells. The debris is modelled as a truncated exponential distribution, and we also have included a uniform background noise distribution over the whole observation interval. We have explored a new approach for maximum-likelihood analyses of complex DNA histograms by the application of the EM algorithm. This algorithm was used for four observed DNA histograms of varying complexity. Our results show that the algorithm works very well, and it converges to reasonable values for all parameters. In simulations from the estimated models, we have investigated bias, variance, and correlations of the estimates.     

Polysaccharide composition of the cell wall of baker's yeast with special reference to cell walls obtained from large- and small-sized cells

In order to obtain further information on the mode of cell wallformation during the growth process, the compositions of cellwall polysaccharides were compared in detail using cell wallsamples prepared from large and small cells obtained by fractionationof baker's yeast cells. Gas liquid chromatography of the methanolyzed specimen gavestable, reproducible results, when the sample contained bothglucose and mannose. Much mannan was liberated from the cellwall during its preparation and this must be taken into consideration. Total glucose and mannose accounted for about 40% each of dryweight of cell walls obtained from large and small cells. Glucanswere tentatively divided into alkali-soluble and alkali, acid-insolubleones. Alkaline extraction caused considerable degradation ofpolysaccharides. Nevertheless, a distinct difference existedbetween the glucan contents of the two cell walls. The cellwall sample of large cells contained a higher amount of insolubleglucan, whereas that of small cells contained a higher amountof alkali-soluble glucan. The mode of formation of cell wall polysaccharides during growthwas discussed on the basis of a small-to-large cell process. ¹Present address: Department of Biology, Faculty of Science,Osaka City University, Sumiyoshi-ku, Osaka 558, Japan. (Received April 13, 1976; )     

Monitoring the cellular activity of a cultured single cell by scanning electrochemical microscopy (SECM). A comparison with fluorescence viability monitoring

The respiratory activities of cultured HeLa cells were monitored at a single cell level using scanning electrochemical microscopy (SECM) that produces images of the localized distribution of oxygen around the cell. The change in the cellular activity was traced after exposures to KCN, ethyl alcohol and the antibiotic drug, Antimycin A. The results were compared with those from the conventional fluorescence monitoring using Calcein-AM that is sensitive to deformation of the cell membrane. The SECM-based measurement follows the decrease in the cellular activity upon exposure to KCN and Antimycin A more rapidly than the fluorescence-based measurements, demonstrating that SECM is suitable for studying the cellular influence of respiration inhibitors.     

A comparison study reveals important features of agreement and disagreement between summarized DNA and RNA data obtained from renal cell carcinoma

Cytogenetic abnormalities, such as DNA amplifications and deletions, often lead to significant changes in gene expression levels within a chromosomal region. Instead of generating additional DNA copy number data, one method to identify DNA copy number abnormalities has been to search existing gene expression data for regional perturbations in gene expression. However, it is not clear how well this surrogate method performs in the examination of individual tumors and how we can use both DNA and RNA data to identify candidate genes that may be mutated. Here we report a comparison study using summarized DNA and RNA data to identify chromosomal abnormalities in human samples. Forty-four tissue samples from patients diagnosed as having renal cell carcinoma (RCC) were collected, together with 15 normal kidney samples as controls, and for each sample the genome-wide DNA and RNA data were obtained for comparison using Affymetrix 100K SNP and HGU133plus2 gene expression chips, respectively. The DNA and RNA data was summarized by both chromosome arm and cytogenetic banding patterns and compared. The result of this analysis revealed that the two summarized data sets used to identify cytogenetic changes agreed well. However, some differences between the two were also identified. These differences of large-scale gene expression deregulation without evidence of the comparable DNA copy number alterations may be the result of known mechanisms, such as large-scale methylation or chromosome inactivation, or may be the result of some new mechanism of DNA-RNA translation. The usefulness of the combined data set for identifying regions of mutated genes is also discussed.     

How cells settle on glass: A study by light and scanning electron microscopy of some properties of normal and stimulated macrophages

Summary Some properties of normal and stimulated peritoneal macrophages have been studied using light microscopy, cinemicroscopy, and scanning electron microscopy. No difference in the overall rate of translational movement was found between normal and stimulated cells. Macrophages were found to settle on glass by a process involving initial protrusion of very fine finger-like processes, followed by veils. Full extension occurred sooner in stimulated cells.We are grateful to Professor R. Barer for his criticisms, to Miss Anne Edwards for technical help, to Mr. G. Tuck for help with cinemicroscopy, and to the Science Research Council and the Medical Research Council for grants.     

Cell adhesion and cell surface topography in aggregates of 3T3 and SV40-virus-transformed 3T3 cells. Visualization of interior cells by scanning electron microscopy

A technique for exposing the interior of aggregates of cultured cells has been developed and is described in this report. Using this technique, we have examined for the first time, by scanning electron microscopy, cell morphology and cell contact ultrastructure in the interior of aggregates of BALB/c 3T3 and SV40-transformed 3T3 cells. The 3T3 cells make initial intercellular contact by means of microvillar processes. Over a period of 3-8 h, some of these microvillar contacts are replaced by broader projections. In contrast, the SV40-transformed cells make initial intercellular contact by means of blebs or blunt projections which are also broadened and extended over a period of 3-8 h. For both 3T3 and SV40-3T3 cells, the surfaces of the cells which form the outer layer of the aggregate resemble the surfaces of single cells fixed in suspension, regardless of how long the aggregates have been cultured. Thse cells are covered with many cellular processes and are roughly hemispherical in profile. The surfaces of the internal cells of the aggregates, however, lose many of their cellular processes, develop smooth patches, and many become irregular in shape. This smooth morphology was also observed on the interior surfaces of the peripheral cell layer. From these observations we conclude that: (a) the stabilization of adhesive contacts is a slow process which takes at least 3-8 h; (b) the outer surfaces of peripheral cells differ significantly from the surfaces of interior cells; and (c) clear differences in surface topography exist between nonmalignant 3T3 cells and their malignant SV40 transformants.