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M E Stearns

Publications and source records attributed to M E Stearns.

66 records · Page 4Linked to original sources
PubMed1985

Dansylated estramustine, a fluorescent probe for studies of estramustine uptake and identification of intracellular targets.

Fluorescence-microscopic studies with dansylated estramustine (DnsEM) has permitted investigation of the mechanism of estramustine (EM) uptake in live human prostatic tumor cells (DU 145). DnsEM appeared to enter cells rapidly at the peripheral cell margins. A progressive increase in fluorescence was observed until the perinuclear material and cytoplasm were labeled brightly and the nucleoplasm was labeled faintly. Light microscopy showed that DnsEM is assimilated first in preexisting vesicles and then in numerous newly created vesicles that accumulate in the cytoplasm and around the nucleus. Colony-forming assays showed EM and DnsEM to be equally cytotoxic to cultured DU 145 cells. Cellular uptake and subsequent manifestation of cytotoxicity are presumably dependent upon these vesicles. However, after uptake of DnsEM, its diffusion into the cytoplasm was observed.

Animals↗
PubMed1985

Antimicrotubule effects of estramustine, an antiprostatic tumor drug.

Estramustine [17 beta-estradiol 3 N bis(2-chloroethyl)carbamate; EM] is a stable conjugate of estradiol and nor-nitrogen mustard that is used for the treatment of human prostatic carcinoma. We have studied the cytotoxic effects of EM on the cytoskeletal organization of squirrelfish pigment cells (erythrophores) and human prostatic tumor cells (DU 145) in culture. Light and whole-mount electron microscopy studies reveal that, at microM levels (60 to 120 microM), EM has a dose-dependent disruptive effect on cell shape, cytoskeletal organization, and intracellular transport. Upon removal of the drug, the cytological effects of EM are rapidly reversible in fish cells but not DU 145s. Immunofluorescent studies reveal that EM produces microtubule disassembly in fish erythrophores and DU 145 cells. A concomitant disruption of actin-microfilament arrays also occurs in DU 145 cells. These morphological data suggest that EM, in contradistinction to its constituent estradiol: nitrogen mustard species, induces cytotoxicity as an antimicrotubule drug. The observed disruption of the microtubules and cytomatrix of interphase cells is not reversible in the prostatic carcinoma cells. The disruptive action of EM on the cytoskeleton could ultimately produce a cytotoxic antimitotic effect in dividing cells.

Animals↗
PubMed1982

High voltage electron microscopy studies of axoplasmic transport in neurons: a possible regulatory role for divalent cations.

Light and high voltage electron microscopy (HVEM) procedures have been employed to examine the processes regulating saltatory motion in neurons. Light microscope studies demonstrate that organelle transport occurs by rapid bidirectional saltations along linear pathways in cultured neuroblastoma cells. HVEM stereo images of axons reveal that microtubules (Mts) and organelles are suspended in a continuous latticework of fine microtrabecular filaments and that the Mts and lattice constitute a basic cytoskeletal structure mediating the motion of particles along axons. We propose that particle transport depends on dynamic properties of nonstatic microtrabecular lattice components. EXperiments were initiated to determine the effects of changes in divalent cation concentrations (Ca2+ and Mg2+) on: (a)the continuation of transport and (b) the corresponding structural properties of the microtrabecular lattice. We discovered that transport continues or is stimulated to a limited extent in cells exposed to small amounts of exogenously supplied Ca2+ and Mg2+ ions (less than 0.1 mM). Exposure of neurons to increased dosages of Ca2+ and Mg2+ (0.2-1.0 mM) stimulates transport for 2-4 min at 37 degrees C, but after a 5- to 20-min exposure the saltatory movements of organelles are observed gradually to become shorter in duration and rate particle motion ceases to occur. HVEM observations demonstrated that Ca2+ - and with the cessation of motion. Ca2+-containing solutions produced contractions of the microtrabecular filaments, whereas Mg2+-containing solutions had the opposing effect of stimulating an elongation and assembly (expansion) of microtrabeculae. On the basis of these observations we hypothesize that cycles of Ca2+/Mg2+-coupled contractions and expansions of the microtrabecular lattice probably regulate organelle motion in nerve cells.

Animals↗
PubMed1982

A functional in vitro model for studies of intracellular motility in digitonin-permeabilized erythrophores.

Phase contrast cine results demonstrate that erythrophores maintain saltatory particle motion for hours after permeabilization with 0.001% digitonin in a cytoskeletal stabilizing solution at 23 degrees C. High voltage electron microscopy (HVEM) studies reveal that cytoskeletal elements are retained intact, except in immediate subplasmalemmal regions where the plasma membrane is punctured by digitonin. During digitonin treatments, cells are permeable to ions, small molecules, and antibodies. We find that motion is Ca2+ and ATP-sensitive, and optimal in PIPES buffer (pH 7.2 containing 1 mM Mg2+/ATP and EGTA-CA2+ (10(-7) M Ca2+) at 37 degrees C. Experiments testing the inhibitory effects of vanadate (0.4-10 microM), ouabain (100-600 microM), N-ethyl maleimide, and the cytochalasins B and D indicate that a dyneinlike ATPase may provide the motive force for driving saltatory pigment motion in erythropores.

Actins↗
PubMed1979

Purification of cytoplasmic tubulin and microtubule organizing center proteins functioning in microtubule initiation from the alga Polytomella.

Cytoplasmic tubulin and the microtubule organizing centers (MTOCs) for the cytoskeletal microtubule system of the flagellate Polytomella have been isolated. The isolated MTOCs serve as sites for the in vitro assembly of the purified tubulin protein. The major proteins (four polypeptides of molecular weights 190,000-210,000) functioning in this assembly have been extracted from the MTOCs and purified. Kinetic studies and experiments with in vivo 35S-labeled MTOC proteins (or 35S-labeled tubulin) demonstrate that these proteins function specifically in microtubule initiation and do not contribute to microtubule elongation. The results indicate that microtubule assembly in vivo is controlled by microtubule initiating proteins associated with the organelles termed MTOCs.

Carrier Proteins↗
PubMed1976

Cytoplasmic microtubule organizing centers isolated from Polytomella agilis.

Basal body rootlets in Polytomella function as organizing centers for cytoplasmic microtbules in vivo. A method is described to isolate intact basal body-rootlet complexes. The integrity of the isolated complexes is confirmed by electron microscopy, and the rootlets are shown to be competent as initiation sites for the in vitro polymerization of brain microtubule protein.

Animals↗
PubMedDate not supplied

Immunofluorescent studies of the anti-microtubule effects of the anti-cancer drug estramustine.

Immunofluorescent studies in human prostatic carcinoma cells (DU 145) and cultured squirrel fish epithelial cells (a non-cancer cell) revealed that estramustine, a conjugate of estradiol and nor-nitrogen mustard, possessed microtubule disassembly properties. Sixty microM estramustine produced disassembly at both the proximal and distal ends of microtubules, producing short pieces of less than 2 microM which were "wavy" and oriented in a random manner. With increased time of drug exposure these short microtubules disappeared, to be accompanied by a gradual disassembly of a small population of longer microtubules (greater than 7-8 microM). In dividing DU 145 cells it was possible to show a different degree of sensitivity of specific microtubule-containing cellular structures. In mitotic figures the asters were most sensitive and disappeared completely following exposure to estramustine. These were followed by the "pole-to-pole" and "chromosomal" fibers. In cytokinesis, the intercellular fibers between daughter cells were comparatively resistant to the drug. Estramustine did not induce disassembly of the vimentin filaments in non-dividing or dividing cells but did cause their collapse around the nucleus or the mitotic apparatus. These data suggest that specific microtubules have differing sensitivity to estramustine.

Cells, Cultured↗