PubMed Health⌕ Search

Biomedical subjects

D L Habliston

Publications and source records attributed to D L Habliston.

14 recordsLinked to original sources
PubMed1991

A computer-aided control, design and image-processing system for electron microscopes.

A computer-control system for electron microscopes is described. The aim is to reduce a complex series of vacuum-system controls to a menu-driven program that simplifies the operation of the microscope. The system also incorporates image processing, notebook, computer-aided design, and communication functions. It is designed around a commercially available computer workstation. This system has been implemented on an institute-built photoelectron microscope of ultrahigh-vacuum design.

Computer Graphics↗
PubMed1991

Biological applications of photoelectron imaging: a practical perspective.

Photoelectron imaging is finding a promising niche in the study of biological specimens. The features of photoelectron imaging that contribute to its uniqueness for this application are described. Image formation and the major contrast mechanisms of photoelectron microscopy, material contrast and topographical contrast are reviewed and illustrated with examples of photoelectron images of cultured cells and of DNA. General considerations in sample choice and preparation are also presented. Strategies for photoelectron labeling are discussed including the use of immunogold labeling, silver enhancement and cesium-based photocathodes.

Animals↗
PubMed1990

Staurosporine induces dissolution of microfilament bundles by a protein kinase C-independent pathway.

The protein kinase C (PKC) inhibitor staurosporine was found to dramatically alter the actin microfilament cytoskeleton of a variety of cultured cells, including PTK2 epithelial cells, Swiss 3T3 fibroblasts, and human foreskin fibroblasts. For example, PTK2 cells exposed to 20 nM staurosporine exhibited a progressive thinning and loss of cytoplasmic actin microfilament bundles over a 60-min period. During this time microtubule and intermediate filament systems remained intact (as shown by immunofluorescence and at higher resolution by photoelectron microscopy), and the cells remained spread even though microfilament bundles were absent. Higher doses of staurosporine or longer exposure times at lower doses resulted in morphological alterations, but even severely arborized cells recovered normal morphology and actin patterns after a wash and an incubation for several hours in fresh medium. The actin filament disruption induced by staurosporine was distinguishable from the actin reorganization induced by exposure to the tumor promoter (and activator of PKC) phorbol myristate acetate (PMA). Swiss 3T3 cells made deficient in PKC by prolonged exposure to PMA (PKC down-regulation) exhibited actin alterations in response to staurosporine which were comparable to those in cells which had not been exposed to the phorbol ester. In a parallel control experiment, the actin cytoskeleton of PKC-deficient 3T3 cells was unaffected in response to PMA, consistent with down-regulation of this kinase. While the exact mechanism of staurosporine-induced actin reorganization remains to be determined, the observed effects of staurosporine on PKC-deficient cells make a role for PKC unlikely. These results indicate the need for care when staurosporine is employed as an inhibitor of protein kinase C in studies involving intact cells.

Actin Cytoskeleton↗
PubMed1990

On the possibility of obtaining a physical map of genomes by photoelectron imaging.

Photoelectron imaging provides the possibility of a new method of mapping chromosomes. The basic concept is to cause DNA to emit electrons under the action of UV light. The criteria which must be met to map genomes by photoelectron imaging are set forth and discussed. Forming an image of the DNA by accelerating and focusing the electrons is a necessary but not sufficient condition for genome mapping. Equally important is to identify wavelengths of UV light which will cause selective emission from the base pairs, adenine-thymine and guanine-cytosine. The resulting image would then contain a modulation in the image brightness along the DNA duplex. By examining the photoelectron current from uniform films of homopolymers, a wavelength region is identified where marked differences in emission from base pairs is observed. At 160 nm, for example, the relative electron emission from a film of poly(dGdC) is approximately 5 times greater than for an equivalent film of poly(dAdT). Using the experimental data and known sequences, photoelectron gene maps are calculated for the bacteriophage lambda and for a short interspersed repetitive DNA sequence (an Alu repeat) of the human genome. The results suggest that a 5-nm physical map of chromosomes generated by photoelectron imaging would be informative and useful in mapping human and other large genomes.

Bacteriophage lambda↗
PubMed1989

Protein kinase C inhibitor H-7 alters the actin cytoskeleton of cultured cells.

The effects of the protein kinase C inhibitor H-7 on the actin cytoskeleton of cultured cells (Swiss 3T3 and PTK2) are described. As documented by fluorescence microscopy and the higher-resolution technique of photoelectron microscopy, the effects are rapid and dramatic; exposure to 30 microM H-7 in culture medium for less than 6 min is sufficient to induce a significant reduction in the numbers and thickness of actin microfilament bundles and alterations in the morphology of cell-cell boundaries in PTK2 cells. One-hour exposure to 30 microM H-7 results in nearly complete depletion of normal actin microfilament bundles from all of the cell types examined, without dramatic changes in overall cell shape. The intermediate filament and microtubule cytoskeletal networks did not appear to be affected to any extent over the times and doses examined. Forty-five minutes of exposure of Swiss 3T3 cells to 200 microM of either HA1004 (which is comparable to H-7 with respect to inhibition of cyclic nucleotide dependent kinases) or to the protein kinase C inhibitor sangivamycin did not induce the actin alterations characteristic of H-7. In addition, depletion of protein kinase C from Swiss 3T3 cells by means of phorbol ester-induced down-regulation did not prevent the effects of H-7 on the actin cytoskeleton. These results demonstrate that the protein kinase C inhibitor H-7 has a specific and rapid effect on the actin cytoskeleton, and furthermore H-7 may have biochemical effects beyond those mediated by inhibition of protein kinase C or the cyclic nucleotide dependent kinases.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1989

Biological photocathodes.

Biological surfaces emit electrons when subjected to UV light. This emission is increased greatly after exposure to cesium vapor. Increases from 2 to 3 orders of magnitude are observed, depending on the biochemicals present. Heme and chlorophyll exhibit unusually high photoemission currents, which are increased further after cesiation. Photoemission from proteins and lipids is much less but also is increased by exposure to cesium. The formation of photocathodes with cesium greatly increases the practical magnifications attainable in photoelectron microscopy of organic and biological specimens. Photoelectron micrographs taken at magnifications greater than or equal to X 100,000 of chlorophyll-rich thylakoid membranes and of colloidal gold-labeled cytoskeleton preparations of cultured epithelial cells demonstrate the improvement in magnification. The selectivity and stability of the photocathodes suggest the possibility of detecting chromophore binding proteins in membranes and the design of photoelectron labels for tagging specific sites on biological surfaces.

Animals↗
PubMed1986

Silver-enhanced colloidal gold as a cell surface marker for photoelectron microscopy.

Colloidal gold labeling in conjunction with silver enhancement was investigated as a labeling technique for photoelectron microscopy (PEM). PEM uses UV-stimulated electron emission to image uncoated cell surfaces, and markers for cell surfaces need to be sufficiently photoemissive to be clearly visible against this background. Label contrast provided by 6 nm or 20 nm colloidal gold markers alone was compared to that provided by 6 nm markers after silver enhancement, using both direct and indirect labeling methods for fibronectin on human fibroblast cell surfaces. In all cases, details of the fibrillar fibronectin labeling distribution which were barely discernible before silver enhancement became highly visible against the cellular surface features. Two factors evidently contribute to the pronounced increase in label contrast with silver enhancement: (1) Increased particle size, which was documented by transmission electron microscopy, and (2) increased photoemission resulting from a silver coating on the enhanced gold markers, compared with the protein coating on the unenhanced gold markers. These data demonstrate that silver enhancement of colloidal gold labeling patterns in PEM images is a highly effective method for localization of specific sites on cell surfaces.

Cell Membrane↗
PubMed1986

Early phorbol ester induced release of cell surface fibronectin: direct observation by photoelectron microscopy.

One of the early effects of the phorbol ester tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA) on cultured normal fibroblasts is the release of fibronectin into the culture medium. Immunophotoelectron microscopy was used to follow the loss of fibronectin from the upper cell surface of normal human foreskin fibroblasts exposed to TPA. Fibronectin labeled with silver-enhanced colloidal gold-antibody conjugates appears in photoelectron images as streak- and network-like patterns of bright dots against the less photoemissive uncoated cell surface. Labeled fibronectin present beneath the culture is not detected due to the surface-specificity of this technique. Ten to 30 min of exposure to 100 ng/ml TPA in culture medium results in a readily visible decrease in upper cell surface fibronectin. In these experiments, 60 min of exposure to TPA releases nearly all upper cell surface fibronectin, leaving only occasional short streaks of label. In contrast, extracellular matrix fibronectin between cells is apparently more resistant to release and can still be seen in photoelectron images even when the upper cell surface appears to be fibronectin-free. This immunophotoelectron study shows the distribution of fibronectin on fibroblasts at high resolution and demonstrates that the initial fibronectin release resulting from TPA exposure is at the expense of preexisting cell-surface fibronectin. These results also illustrate the application of photoelectron microscopy as a useful technique in cell biology.

Cell Line↗
PubMed1985

Immunophotoelectron microscopy: the electron optical analog of immunofluorescence microscopy.

The electron optical analog of immunofluorescence microscopy combines three developments: (i) photo-electron microscopy to produce a high-resolution image of exposed components of the cell, (ii) site-specific antibodies, and (iii) photoemissive markers coupled to the antibodies to make the distribution of sites visible. This approach, in theory, provides a way to extend the useful immunofluorescence microscopy technique to problems requiring much higher resolution. The resolution limit of fluorescence microscopy is limited to about 200 nm by the wavelength of the light used to form the image, whereas in photoelectron microscopy the image is formed by electrons (current resolution: 10-20 nm; theoretical limit: 5 nm or better depending on the electron optics). As a test system, cytoskeletons of CV-1 epithelial cells were prepared under conditions that preserve microtubules, and the microtubule networks were visualized by both indirect immunofluorescence and immunophotoelectron microscopy using colloidal gold coated with antibodies. Colloidal gold serves as a label for immunophotoelectron microscopy, providing enhanced photoemission from labeled cellular components so that they stand out against the darker background of the remaining unlabeled structures. In samples prepared for both immunofluorescence and immunophotoelectron microscopy, individual microtubules in the same cells were visualized by both techniques. The photoemission of the colloidal gold markers is sufficiently high that the microtubules are easily recognized without reference to the immunofluorescence micrographs, indicating that this approach can be used, in combination with antibodies, to correlate structure and function in cell biological studies.

Animals↗
PubMed1983

Photoelectron microscopy and immunofluorescence microscopy of cytoskeletal elements in the same cells.

Pt K2 rat kangaroo epithelial cells and Rat-1 fibroblasts were grown on conductive glass discs, fixed, and permeabilized, and the cytoskeletal elements actin, keratin, and vimentin were visualized by indirect immunofluorescence. After the fluorescence microscopy, the cells were postfixed and dehydrated for photoelectron microscopy. The contrast in these photoelectron micrographs is primarily topographical in origin, and the presence of fluorescent dyes at low density does not contribute significantly to the material contrast. By comparison with fluorescence micrographs obtained on the same individual cells, actin-containing stress fibers, keratin filaments, and vimentin filaments were identified in the photoelectron micrographs. The apparent volume occupied by the cytoskeletal network in the cells as judged from the photoelectron micrographs is much less than it appears to be from the fluorescence micrographs because the higher resolution of photoelectron microscopy shows the fibers closer to their true dimensions. Photoelectron microscopy is a surface technique, and the images highlight the exposed cytoskeletal structures and suppress those extending along the substrate below the nuclei. The results reported here show marked improvement in image quality of photoelectron micrographs and that this technique has the potential of contributing to higher resolution studies of cytoskeletal structures.

Actins↗
PubMed1979

Isolation and culture of endothelial cells from the lungs of small animals.

Techniques are descirbed for the isolation and culture of endothelial cells from the lungs of small animals. The cells are collected by retrograde perfusion of blood-free lungs with buffered saline containing collagenase. The cells are characterized by light microscopy, electron microscopy of thin sections and surface replicas, and by the presence of angiotensin-converting enzyme (ACE). ACE was assayed using 3H-benzoyl-Phe-Ala-Pro as substrate and was localized by indirect immunofluorescence using guinea pig endothelial cells incubated with rabbit antibodies to guinea pig lung ACE followed by goat anti-rabbit globulins conjugated to fluorescein. Thus, endothelial cultures can be established using small animals commonly employed in studies of pulmonary processing of vasoactive substances.

Animals↗
PubMed1979

Endothelial cells and components of the kallikrein-kinin system.

Endothelial cells are a major source of kininase enzymes including kininase II. Kininase II is situated along the plasma membrane, not as an ecto-enzyme but as an enzyme synthesized by the endothelial cells themselves. However, it is likely that endothelial cells do more than degrade kinins. These cells are contractile and may possess kinin receptors; a possibility supported by the fact that kinins stimulate endothelial cells to form and release prostaglandin-related substances. In addition, we have found that endothelial cells in culture are reactive with antibodies to alpha 2-macroglobulin. Endothelial cells can hydrolyze [3H]Pro-Phe-Arg-anilide, a kallikrein substrate, but the reaction is not inhibited by soya bean trypsin inhibitor (SBTI) or Trasylol. Possibly kallikrein or a related trypsin-like enzyme is bound to alpha 2-macroglobulin and is not free to react with the inhibitors. Thus, endothelial cells can bind and inhibit kallikrein-like enzymes, degrade kinins and respond to kinin stimulation.

Animals↗