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P B Bell

Publications and source records attributed to P B Bell.

34 records · Page 2Linked to original sources
PubMed1988

Preparation of cytoskeletons of cells in culture for high resolution scanning and scanning transmission electron microscopy.

In this tutorial we describe our methods for preparing detergent-extracted cytoskeletons for observation by high resolution scanning (SEM) and scanning transmission (STEM) electron microscopy. We both discuss the theoretical background and provide practical procedures for each of the following steps: cell culture on Formvar-coated gold grids; prefixation with aldehydes or protein crosslinking reagents (homobifunctional N-hydroxy-succinimide esters); extraction with Triton X-100 or Brij 58 detergent in microtubule stabilizing buffer; postfixation in formaldehyde and glutaraldehyde; dehydration; critical point and freeze drying; sputter coating with 1-2 nm of platinum or tungsten; and examination by SEM and both normal and inverted contrast STEM. These methods produce cytoskeletal preparations in which filaments as fine as 7 nm are preserved and can be observed by scanning electron microscopy.

Chemical Phenomena↗
PubMed1987

Use of sputter coating to prepare whole mounts of cytoskeletons for transmission and high-resolution scanning and scanning transmission electron microscopy.

This paper describes the use of sputter coating to prepare detergent-extracted cytoskeletons for observation by scanning (SEM), scanning transmission (STEM), inverted contrast STEM, and transmission (TEM) electron microscopy. Sputtered coats of 1-2 nm of platinum or tungsten provide both an adequate secondary electron signal for SEM and good contrast for STEM and TEM. At the same time, the grain size of the coating is sufficiently fine to be just at (platinum) or below (tungsten) the limit of resolution for SEM and STEM. In TEM, the granular structure of platinum coats is resolved, and platinum decoration artifacts are observed on the surface of structures. The platinum is deposited as small islands with a periodic distribution that may reveal information about the underlying molecular structure. This method produces samples that are similar in appearance to replicas prepared by low-angle rotary shadowing with platinum and carbon. However, the sputter-coating method is easier to use; more widely available to investigators; and compatible with SEM, STEM, and TEM. It may also be combined with immunogold and other labeling methods. While TEM provides the highest resolution images of sputter-coated cytoskeletons, it also damages the specimens owing to heating in the beam. In SEM and STEM cytoskeletons are stable and the resolution is adequate to resolve individual microfilaments. The best single method for visualizing cytoskeletons is inverted contrast STEM, which images both the metal-coated cytoskeletal structures and electron-dense material within the nucleus and cytoplasm as white against a dark background. STEM and TEM were both suitable for visualizing colloidal gold particles in immunolabeled samples.

Cytoskeleton↗
PubMed1987

Formaldehyde sensitivity of a GFAP epitope, removed by extraction of the cytoskeleton with high salt.

We have used cytofluorometry to examine the formaldehyde sensitivity of the binding of a monoclonal antibody (MAB) to its epitope on glial fibrillary acidic protein in human malignant glioma cells in culture. When acetone-extracted whole cells or cytoskeletons, made by extracting with Triton in stabilizing buffer (Tsb), are fixed with formaldehyde, binding of the MAB Tp-GFAP1 to GFAP is abolished or greatly reduced. Fixation with the bifunctional protein crosslinking reagent dithiobis (succinimidyl propionate) (DTSP) has the same negative effect as formaldehyde. If cytoskeletons are further extracted with Tsb containing 250 mM ammonium sulfate (Thsb), fixation with formaldehyde or DTSP has reduced or no effect on the binding of Tp-GFAP1. The data are consistent with the hypothesis that aldehyde sensitivity of Tp-GFAP1 is caused by the crosslinking of a second protein to GFAP that blocks the binding of the MAB to its epitope. This putative blocking protein is part of the Triton-insoluble cytoskeleton, but it begins to be solubilized in 50 mM ammonium sulfate and it is largely removed in 250 mM ammonium sulfate (Thsb). SDS-PAGE shows that extraction with Thsb also removes a large number of proteins from the cytoskeleton, one of which could be the blocking protein. A second antibody to GFAP, designated Tp-GFAP3, was raised against cytoskeletons which had been fixed with DTSP and in which the epitope recognized by Tp-GFAP1 was presumably blocked. Tp-GFAP3 is not sensitive to fixation by either formaldehyde or DTSP.

Antibodies, Monoclonal↗
PubMed1987

Use of cytofluorometry to evaluate binding of antibodies to the cytoskeleton of cultured cells.

Immunocytochemistry is routinely used to examine the occurrence and distribution of cytoskeletal proteins in cells, but the results are usually evaluated visually and subjectively. Little use has been made of the potential the method offers for quantitative work. Here we report on application of cytofluorometry to quantify binding of antibodies to the cytoskeleton of U-251 MG human malignant glioma cells in culture. The results show that cytofluorometry is a simple and reliable procedure for: (a) determining the optimal concentrations of primary and secondary antibodies and other labeling reagents; (b) evaluating the binding specificity of commercial secondary antisera; and (c) evaluating the effect of different preparatory procedures on preservation of and binding of antibodies to cytoskeletal structures. Experiments with a monoclonal antibody to tubulin show that preservation of tubulin is very sensitive to the preparatory procedures used. Maximum labeling of tubulin in intact cells was obtained when the cells were pre-fixed with formaldehyde before permeabilization with solvent. Maximum labeling of tubulin in Triton-extracted cytoskeletons was achieved by pre-fixing the cells with the bifunctional protein crosslinking reagent dithiobis (succinimidyl propionate), extracting with Triton in a microtubule-stabilizing buffer, and post-fixing with formaldehyde. GTP was not required to preserve tubulin in cytoskeletons.

Antibodies, Monoclonal↗
PubMed1986

Measurement of intracellular pH with microelectrodes in rat kidney in vivo.

The construction of two types of double-barreled microelectrodes of sufficiently small tip size to record cytosolic pH (pHc) in renal tubular epithelial cells is described. Mean pHc in control anesthetized rats was 7.10 and mean basolateral membrane potential (Em) was -51.8 mV. These results suggest that hydrogen ions are actively extruded from either or both poles of the proximal tubular cell but that bicarbonate exit across the basolateral membrane is passive. In animals treated with acetazolamide, pHc was significantly elevated (mean 7.35) but Em was unchanged, suggesting that proton extrusion continues but that the rate of reaction of OH- with CO2 is diminished due to carbonic anhydrase inhibition. A pH higher than that of arterial blood was obtained from sites presumed to be in the interstitium. The evidence for the various assignments of microelectrode tip position is discussed.

Acetazolamide↗
PubMed1983

Some factors affecting the preservation of the cytoskeleton of cells in culture for scanning electron microscopy.

This paper reports some preliminary findings on the effects of the composition of the detergent extraction medium on the morphological and molecular preservation of the cytoskeleton of Balb/c 3T3 cells as visualized by scanning electron microscopy and SDS-PAGE. Specific variables tested are the concentrations of sodium, potassium, calcium and magnesium ions, and the osmolarity of the medium. The results suggest that the ionic composition of the medium is more important than osmolarity in determining the composition of the cytoskeleton obtained with Triton X-100 detergent. Calcium is particularly deleterious to morphological preservation, and better preservation is obtained when potassium is the principal cation than when sodium is. The best preservation of cytoskeletal structures and the greatest molecular complexity was obtained by extracting in a buffer composed of 1mM EGTA, 4% polyethylene glycol 6000, 1 mM GTP and 100 mM PIPES, pH 6.9.

Animals↗
PubMed1983

Scanning electron microscopic study of the microarchitecture of the lung of the giant salamander Amphiuma tridactylum.

This paper reports observations made using scanning electron microscopy (SEM) on the internal structure of the lung of Amphiuma tridactylum. This lung is highly septate at the cranial end, but the septa become gradually reduced in size and number with increasing distance caudally until the septa are reduced to ridges in the wall of the lung. However, there is no respiratory dead space as respiratory epithelium is found throughout the lung. Treatment of isolated lung segments with acetylcholine causes the septa to shorten and thicken which in turn causes the lung diameter to decrease and the alveoli to collapse. These observations are consistent with the hypothesis that the collapse of the Amphiuma lung in vivo, which is responsible for the highly efficient ventilation of the lungs, is brought about by the contraction of pulmonary smooth muscle in the septa and wall of the lung.

Animals↗
PubMed1981

The application of scanning electron microscopy to the study of the cytoskeleton of cells in culture.

This paper reviews the methods available for studying the structural organization of the cytoskeleton of cells in culture. These are transmission electron microscopy of whole cells, immunofluorescence, and detergents extraction procedures. Published methods for detergent extraction are critically reviewed with special emphasis on those factors that are most relevant for the preparation of cytoskeletons for observation by scanning electron microscopy. These include the type of detergent, the use of chemical crosslinking reagents or specially formulated buffers to stabilize the cytoskeleton during extraction, the osmotic and salt composition of the extraction medium, the use of cool sputter coaters, and the application of minimal amounts of metal during coating. Finally, examples are given of the types of information about the three dimensional organization of the cytoskeleton that can be obtained by SEM.

Animals↗
PubMed1979

Coexistence of desmin and the fibroblastic intermediate filament subunit in muscle and nonmuscle cells: identification and comparative peptide analysis.

Extraction of chicken embryo fibroblasts (CEF) or baby hamster kidney (BHK) cells with 1% Triton X-100 and 0.6 M KCl leaves an insoluble cytoskeletal residue composed primarily of the 52,000 Mr subunit of intermediate filaments (F-IFP). In addition, CEF cytoskeletons exhibit a minor component with Mr of 50,000, identified as alpha-desmin, one of the two major isoelectric variants of the intermediate filament subunit from smooth muscle. BHK cytoskeletons contain the 50,000 Mr mammalian desmin variant. Cytoskeletons prepared from chicken embryonic myotubes contain F-IFP and both alpha- and beta-desmin. These data suggest that two distinct 10-nm filament subunits coexist in a single cell. One-dimensional peptide analysis of F-IFP and desmin from avian and mammalian cells reveals significant interspecies homology, as well as homology between F-IFP and desmin from the same species. Peptide analyses of 32P-labeled intermediate filament subunits suggest that there is considerable similarity in the phosphorylation sites of these proteins. These results indicate that F-IFP and desmin might be evolutionally related.

Amino Acid Sequence↗
PubMed1977

Locomotory behavior, contact inhibition and pattern formation of 3T3 and polyoma virus-transformed 3T3 cells in culture.

The social behavior of 3T3 cells and their polynoma virus-transformed derivative (Py3T3 cells) was examined by time-lapse cinemicrography in order to determine what factors are responsible for the marked differences in the patterns formed by the two cell lines in culture. Contrary to expectations, both cell types have been found to exhibit contact inhibition of cell locomotion. Therefore, the tendency of 3T3 cells to form monolayers and of Py3T3 cells to form crisscrossed multilayers cannot be explained on the basis of the presence versus the absence of contact inhibition. Morevover, with the exception of cell division control, the social behavior of the two cell types is qualitively similar. Both exhibit cell underlapping and, after contact between lamelliopodia, both show inhibition of locomotory activity and adhesion formation. Neither cell type was observed to migrate over the surface of another cell. The two cell types do show quantitative differences in the frequency of underlapping, the frequency with which contact results in inhibition of locomotion, and the proportion of the cell margin that adheres to the substratum. The increased frequency pf Py3T3 underlapping is correlated with the reduced frequency of substratum adhesions, which in turn favors underlapping. On the basis of these observations, it is concluded that the differences in culture patterns are the result of differences in the shapes of the individual cells, such that underlapping, and hence crisscrossing, is favored in Py3T3 cell interactions and discouraged in 3T3 cells.

Cell Adhesion↗
PubMed1974

The upper cell surface: its inability to support active cell movement in culture.

A variety of epithelial cells and fibroblasts fail to move over one another's upper surfaces in culture, resulting in monolayering. The failure of seeded fibroblasts to adhere to and spread on epithelial cell surfaces suggests that monolayering in culture is due to the lack of adhesion of the upper cell surface, at least of epithelial cells. Seeded fibroblasts and postmitotic, rounded fibroblasts likewise fail to spread on the upper surfaces of spread fibroblasts, suggesting that the inability of the upper cell surface to support spreading may be a general phenomenon. Inert particles and cell processes do not adhere directly to the upper cell surface. However, they can initiate adhesions to the surface at a cell's free margin, suggesting a variation of adhesive properties over a cell's surface.

Animals↗
PubMedDate not supplied

Effect of interleukin-2 on CD95 (Fas/APO-1) expression in fresh chronic lymphocytic leukaemia and mantle cell lymphoma cells: relationship to ex vivo chemoresponse.

Cells from B-cell chronic lymphocytic leukaemia (CLL) and mantle cell lymphoma (MCL) rarely express the CD95 (Fas/APO-1) antigen. Our aims were to determine whether CD95 levels (assessed by flow cytometry) in fresh neoplastic CD19+/CD5+ B-lymphocytes from CLL and MCL could be upregulated using clinically relevant doses of interleukin-2 (IL-2), and to compare this with their ex vivo cytotoxic drug sensitivity (assessed by DiSC assay). CD95-expression was absent/negligible in 13/14 CLL and 7/7 MCL specimens. Following culture (without IL-2) CD95 was expressed on dying CD5+ B-lymphocytes but only on live cells in 2/15 cases. In live cells from 2 CLL specimens, IL-2 caused up-regulation of CD95 and was associated with ex vivo drug resistance. Clinically relevant doses of IL-2 had pleiotropic effects on CD95-levels in fresh CLL cells, but did not induce CD95 in MCL cells. The link between CD95-induction and ex vivo drug resistance may point to a clinically resistant subset of CLL patients.

Aged↗