Myles L. Mace

Use of direct current sputtering for improved visualization of chromosome topology by scanning electron microscopy.

Abstract The topological features of isolated Chinese hamster ovary metaphase chromosomes were studied with high resolution scanning electron microscopy (SEM) using the techniques of direct current sputtering for the deposition of metal on the specimens. Metaphase chromosome surfaces consist of numerous compact microconvules of an average diameter of 520 ± 78 A when corrected for the thickness of the gold-palladium coating (80 ± 2 A). These microconvules contain several orders of supercoiling. The superhelical structures were detected also in water-spread preparations. Most of the isolated chromosomes had membrane-like structures attached at the distal portions of the chromatids forming a terminal “plate”. Limited tryptic digests of such isolated chromosomes resulted in considerable stretching of the chromatids and revealed a series of interchromatidal fibers with diameters of 203 ± 38 A (corrected for gold coating). Treatment of these chromosomes with EDTA revealed a longitudinal array of fibers within the chromatids. The diameters of these fibers decreased as the concentration of EDTA was increased. The technique of direct current sputtering for the preparation of chromosomes for scanning microscopy is satisfactory for detailed topological ultrastructural studies in the 70 A range.

Ultrastructural diagnosis of epithelial malignant mesothelioma

Examination of microvilli (MV) by electron microscopy (EM) may help distinguish epithelial malignant mesotheliomas (EMM) from metastatic adenocarcinomas (AC). The goal of this study was to assess the diagnostic utility of microvillous length to width (l/w) measurements of EMM and AC, and to determine if ultrathin sections can be used to accurately assess lengths of villi not completely contained within a single section. Therefore, in addition to the usual ultrathin (600–800 Å) sections used for standard transmission EM, thicker (up to 1 μ) sections were also prepared for study by scanning transmission EM. Seven cases of EMM and 3 of AC were analyzed. In each case, length and width were measured for all MV with identifiable bases (18–60 MV/case), using an electronic planimeter, and the mean l/w ratio calculated. The dimensionless l/w ratio is independent of magnification and does not require calibration, facilitating intercase comparison. In the 3 AC, the mean l/w was 5.39, versus 11.44 in the 7 EMM cases. Four EMM were analyzed using both standard ultrathin and thicker sections, disclosing a thin section l/w of 13.34 and a thick section of 12.26, supporting the confidence of measurements made from standard ultrathin sections. Examination of data also showed that equally good separation of EMM from AC could be obtained using the mean ratio of only the 10 longest MV (16.11 versus 8.93). By these techniques, EMM often may be distinguishable from AC, with a l/w ratio twice as large in EMM and a mean l/w greater than 11 supportive of a diagnosis of EMM. Cancer 56: 2036‐2040, 1985.

Purification and characterization of tropomyosin from bovine thyroid

Abstract A tropomyosin has been purified from bovine thyroid and its properties compared with those of rabbit skeletal muscle tropomyosin. Thyroid tropomyosin was separated from contaminating vascular smooth muscle tropomyosin by hydroxyapatite chromatography. Thyroid tropomyosin resembles tropomyosin from other non-muscle cells in regard to subunit size, mobility on SDS-polyacrylamide gels in the presence and absence of 6 M urea, amino acid composition and morphology. Thyroid tropomyosin has a subunit molecular weight of 30 000 and forms Mg2+ paracrystals with an axial period of 345 A, while paracrystal periodicities of muscle tropomyosins are 400 A. The amino acid composition of thyroid tropomyosin is very similar to that of other non-muscle cell tropomyosins. However, thyroid tropomyosin differs from other non-muscle cell tropomyosins in its ability to bind actin and troponin as described below. Although the binding of brain tropomyosin to F-actin is thought to be weaker than that of muscle tropomyosin (Fine et al. (1973) Nature New Biol. 245, 182–186), both thyroid and muscle tropomyosins bind to actin in a similar ratio of 1 tropomyosin/6–7 actin monomers at saturation. The binding of tropomyosin to F-actin is strongly dependent on the Mg2+ concentration. With thyroid tropomyosin, binding begins at 1 mM and is complete at about 4–5 mM Mg2+ while, with muscle tropomyosin, binding is initiated at 1 mM Mg2+ and reaches saturation at 2–3 mM Mg2+. At saturation, both thyroid and muscle tropomyosins bind to the same binding site(s) on actin filaments with similar affinity. In contrast to platelet tropomyosin (Cote et al. (1978) FEBS Lett. 94, 131–135), thyroid tropomyosin binds to skeletal tuscle troponin and troponin T. One-dimensional peptide maps of thyroid and rabbit skeletal muscle tropomyosin are distinctly different from each other. The air oxidation of thyroid tropomyosin yields covalently linked dimers similar to skeletal muscle tropomyosin dimers. In contrast to muscle tropomyosins, [32P]phosphate is not incorporated into thyroid tropomyosin.

Demonstration of membranous patches on isolated chromosomes.

Abstract High resolution scanning electron microscopy of isolated Chinese hamster ovary metaphase chromosomes revealed “membranous patches” at telomeric and juxtatelomeric regions of the chromosomes. The “membranous patches” remained bound to the chromosomes during centrifugation through dense sucrose, but not after treatment with detergents. These membrane fragments on isolated purified chromosomes may represent a component that binds the chromosome to the inner portion of the nuclear envelope up to late stages of prophase. These chromosome associated membranous patches may represent sites of reformation of the nuclear envelope at telophase.

Isolation of an actin polymerization stimulator from bovine thyroid plasma membranes.

An actin polymerization stimulator was purified from bovine thyroid plasma membranes by DNase I affinity column chromatography. Although the molecular weight of the protein was about 42,000 (42K) by sodium dodecyl sulfate polyacrylamide gel electrophoresis, it did not comigrate with actin. In the presence of 30 mM KC1, the 42K protein facilitated formation of actin filaments when analyzed by a centrifugation method, accelerated the initial phase of actin polymerization as measured in an Ostwald viscometer and increased the length of filaments as shown by electron microscopy. The 42K protein also accelerated the initial phase of actin polymerization in the presence of 100 mM KC1 and 2 mM MgCl2 but did not affect the final viscosity. The effect of the 42K protein was diminished by 5 uM cytochalasin B or 1 uM cytochalasin D. This 42K protein may anchor actin filaments onto the thyroid plasma membrane.