Resistance to DNA denaturation in irradiated Chinese hamster V79 fibroblasts is linked to cell shape

Free and polymerized tubulin were measured in bone cells and Chinese hamster ovary (CHO) cells cultured on plastic substrata. Polymerized tubulin was stabilized in a microtubule- stabilizing medium (MSM) containing 50 percent glycerol and separated from free tubulin by centrifugation. Tubulin content was assayed in both fractions by the colchicines- binding assay. The measured degree of polymerization in both bone cells and CHO cells varied with stabilixation conditions. The degree of polymerization in both bone cells and CHO cells varied with stabilization conditions. The degree of polymerization in both bone cells and CHO cells varied with stabilization conditions. The degree of polymerization in attached cells was found to increase up to 73 percent during the first 20 min after addition of the MSM at 24 degrees C, and remained constant thereafter. Stabilization of 0 degrees C resulted in a decrease down to 62 percent in the degree of constant thereafter. Stabilization at 0 degrees C resulted in a decrease down to 62 percent in the degree of polymerization during the first 20 min after addition of the MSM at 24 degrees C, and remained constant thereafter. Confluent bone cells maintained at 0 degrees C for 1 h before stabilization contained significantly less polymerized tubulin than control cells kept at 37 degrees C using stabilization both at 0 degrees C and at 24 degrees C. Changes in bone cell morphology induced by incubation of cells with prostaglandin E(1) or E(2), parthyroid hormone, and dibutyryl cyclic AMP were not associated with a change in the degree of tubulin polymerization. This was confirmed morphologically by immunofluorescence using affinity-purified tubulin antibodies: microtubules in hormone- treated cells were not noticeably reorganized when compared to microtubule organization in control cells. They were, however, squeezed closer together in cellular pseudopods due to the altered cell shape. This altered cell shape appears to be correlated with disorganization of the microfilament system, since microfilaments, detected using affinity-purified actin antibodies, did alter drastically their appearance and distribution after hormone addition.

Download Full-text

Ultrastructural Changes in Three Different Mammalian Cells Following Ultraviolet Laser Irradiation

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100095017 ◽

1972 ◽

Vol 30 ◽

pp. 350-351

Author(s):

K. Shankar Narayan ◽

Kailash C. Gupta ◽

Tohru Okigaki

Keyword(s):

Ultraviolet Light ◽

Mammalian Cells ◽

Biological Effects ◽

Survival Rates ◽

Chinese Hamster ◽

Cell Types ◽

Differential Sensitivity ◽

Ultrastructural Changes ◽

Ultraviolet Laser

The biological effects of short-wave ultraviolet light has generally been described in terms of changes in cell growth or survival rates and production of chromosomal aberrations. Ultrastructural changes following exposure of cells to ultraviolet light, particularly at 265 nm, have not been reported.We have developed a means of irradiating populations of cells grown in vitro to a monochromatic ultraviolet laser beam at a wavelength of 265 nm based on the method of Johnson. The cell types studies were: i) WI-38, a human diploid fibroblast; ii) CMP, a human adenocarcinoma cell line; and iii) Don C-II, a Chinese hamster fibroblast cell strain. The cells were exposed either in situ or in suspension to the ultraviolet laser (UVL) beam. Irradiated cell populations were studied either "immediately" or following growth for 1-8 days after irradiation.Differential sensitivity, as measured by survival rates were observed in the three cell types studied. Pattern of ultrastructural changes were also different in the three cell types.

Download Full-text

Modulated polarization microscopy displays the dynamics of cytoskeletal structures in living, motile cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100140889 ◽

1995 ◽

Vol 53 ◽

pp. 902-903

Author(s):

J. R. Kuhn ◽

M. Poenie

Keyword(s):

Mitotic Spindle ◽

Actin Filaments ◽

Cell Shape ◽

Dynamic Structure ◽

Living Cells ◽

Cytoskeletal Proteins ◽

Polarization Microscopy ◽

Video Enhancement ◽

Ultrastructural Studies ◽

Motile Cells

Cell shape and movement are controlled by elements of the cytoskeleton including actin filaments an microtubules. Unfortunately, it is difficult to visualize the cytoskeleton in living cells and hence follow it dynamics. Immunofluorescence and ultrastructural studies of fixed cells while providing clear images of the cytoskeleton, give only a static picture of this dynamic structure. Microinjection of fluorescently Is beled cytoskeletal proteins has proved useful as a way to follow some cytoskeletal events, but long terry studies are generally limited by the bleaching of fluorophores and presence of unassembled monomers.Polarization microscopy has the potential for visualizing the cytoskeleton. Although at present, it ha mainly been used for visualizing the mitotic spindle. Polarization microscopy is attractive in that it pro vides a way to selectively image structures such as cytoskeletal filaments that are birefringent. By combing ing standard polarization microscopy with video enhancement techniques it has been possible to image single filaments. In this case, however, filament intensity depends on the orientation of the polarizer and analyzer with respect to the specimen.

Download Full-text

Scanning luminescence x-ray microscopy: Progress toward selective staining using microspheres

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100168104 ◽

1994 ◽

Vol 52 ◽

pp. 74-75

Author(s):

C. Jacobsen ◽

J. Fu ◽

S. Mayer ◽

Y. Wang ◽

S. Williams

Keyword(s):

Visible Light ◽

Active Sites ◽

Light Emission ◽

Chinese Hamster ◽

Polystyrene Spheres ◽

Good Resistance ◽

X Ray ◽

Selective Staining ◽

Visible Light Emission ◽

Specific Labelling

In scanning luminescence x-ray microscopy (SLXM), a high resolution x-ray probe is used to excite visible light emission (see Figs. 1 and 2). The technique has been developed with a goal of localizing dye-tagged biochemically active sites and structures at 50 nm resolution in thick, hydrated biological specimens. Following our initial efforts, Moronne et al. have begun to develop probes based on biotinylated terbium; we report here our progress towards using microspheres for tagging.Our initial experiments with microspheres were based on commercially-available carboxyl latex spheres which emitted ~ 5 visible light photons per x-ray absorbed, and which showed good resistance to bleaching under x-ray irradiation. Other work (such as that by Guo et al.) has shown that such spheres can be used for a variety of specific labelling applications. Our first efforts have been aimed at labelling ƒ actin in Chinese hamster ovarian (CHO) cells. By using a detergent/fixative protocol to load spheres into cells with permeabilized membranes and preserved morphology, we have succeeded in using commercial dye-loaded, spreptavidin-coated 0.03μm polystyrene spheres linked to biotin phalloidon to label f actin (see Fig. 3).

Download Full-text

Video Graphics and High-Voltage Electron Microscopy For Analysis of Microtubule Distributions In Fixed Cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100181373 ◽

1990 ◽

Vol 48 (1) ◽

pp. 524-525

Author(s):

Richard Mcintosh ◽

David Mastronarde ◽

Kent McDonald ◽

Rubai Ding

Keyword(s):

Electron Microscopy ◽

Cross Sections ◽

Cell Shape ◽

Video Camera ◽

Computer Memory ◽

High Voltage Electron Microscopy ◽

Thin Sections ◽

Voltage Electron ◽

Morphogenetic Event ◽

Set Of Points

Microtubules (MTs) are cytoplasmic polymers whose dynamics have an influence on cell shape and motility. MTs influence cell behavior both through their growth and disassembly and through the binding of enzymes to their surfaces. In either case, the positions of the MTs change over time as cells grow and develop. We are working on methods to determine where MTs are at different times during either the cell cycle or a morphogenetic event, using thin and thick sections for electron microscopy and computer graphics to model MT distributions.One approach is to track MTs through serial thin sections cut transverse to the MT axis. This work uses a video camera to digitize electron micrographs of cross sections through a MT system and create image files in computer memory. These are aligned and corrected for relative distortions by using the positions of 8 - 10 MTs on adjacent sections to define a general linear transformation that will align and warp adjacent images to an optimum fit. Two hundred MT images are then used to calculate an “average MT”, and this is cross-correlated with each micrograph in the serial set to locate points likely to correspond to MT centers. This set of points is refined through a discriminate analysis that explores each cross correlogram in the neighborhood of every point with a high correlation score.

Download Full-text

Characterization of a Motile Cellular Domain Arising During the Amoebo-Flagellate Transformation of Physarum Polycephalum

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s1431927600002440 ◽

1980 ◽

Vol 38 ◽

pp. 552-553

Author(s):

K.I. Pagh ◽

M.R. Adelman

Keyword(s):

Cell Shape ◽

Physarum Polycephalum ◽

Slime Mold ◽

Live Cells ◽

Cellular Domain

Unicellular amoebae of the slime mold Physarum polycephalum undergo marked changes in cell shape and motility during their conversion into flagellate swimming cells (l). To understand the processes underlying motile activities expressed during the amoebo-flagellate transformation, we have undertaken detailed investigations of the organization, formation and functions of subcellular structures or domains of the cell which are hypothesized to play a role in movement. One focus of our studies is on a structure, termed the “ridge” which appears as a flattened extension of the periphery along the length of transforming cells (Fig. 1). Observations of live cells using Nomarski optics reveal two types of movement in this region:propagation of undulations along the length of the ridge and formation and retraction of filopodial projections from its edge. The differing activities appear to be associated with two characteristic morphologies, illustrated in Fig. 1.

Download Full-text

Metabolic engineering of CHO cells to prepare glycoproteins

Emerging Topics in Life Sciences ◽

10.1042/etls20180056 ◽

2018 ◽

Vol 2 (3) ◽

pp. 433-442 ◽

Cited By ~ 1

Author(s):

Qiong Wang ◽

Michael J. Betenbaugh

Keyword(s):

Metabolic Engineering ◽

Cho Cells ◽

Genetic Manipulation ◽

Chinese Hamster ◽

Post Translational Modification ◽

Effector Functions ◽

Recombinant Glycoproteins ◽

Production Platform ◽

Chemical Inhibitors ◽

Amino Acid Mutations

As a complex and common post-translational modification, N-linked glycosylation affects a recombinant glycoprotein's biological activity and efficacy. For example, the α1,6-fucosylation significantly affects antibody-dependent cellular cytotoxicity and α2,6-sialylation is critical for antibody anti-inflammatory activity. Terminal sialylation is important for a glycoprotein's circulatory half-life. Chinese hamster ovary (CHO) cells are currently the predominant recombinant protein production platform, and, in this review, the characteristics of CHO glycosylation are summarized. Moreover, recent and current metabolic engineering strategies for tailoring glycoprotein fucosylation and sialylation in CHO cells, intensely investigated in the past decades, are described. One approach for reducing α1,6-fucosylation is through inhibiting fucosyltransferase (FUT8) expression by knockdown and knockout methods. Another approach to modulate fucosylation is through inhibition of multiple genes in the fucosylation biosynthesis pathway or through chemical inhibitors. To modulate antibody sialylation of the fragment crystallizable region, expressions of sialyltransferase and galactotransferase individually or together with amino acid mutations can affect antibody glycoforms and further influence antibody effector functions. The inhibition of sialidase expression and chemical supplementations are also effective and complementary approaches to improve the sialylation levels on recombinant glycoproteins. The engineering of CHO cells or protein sequence to control glycoforms to produce more homogenous glycans is an emerging topic. For modulating the glycosylation metabolic pathways, the interplay of multiple glyco-gene knockouts and knockins and the combination of multiple approaches, including genetic manipulation, protein engineering and chemical supplementation, are detailed in order to achieve specific glycan profiles on recombinant glycoproteins for superior biological function and effectiveness.

Download Full-text