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  • 1
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Annals of the New York Academy of Sciences 466 (1986), S. 0 
    ISSN: 1749-6632
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Natural Sciences in General
    Type of Medium: Electronic Resource
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  • 2
    Electronic Resource
    Electronic Resource
    [s.l.] : Nature Publishing Group
    Nature 268 (1977), S. 602-606 
    ISSN: 1476-4687
    Source: Nature Archives 1869 - 2009
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
    Notes: [Auszug] Trypsin immobilised on polystyrene beads causes initiation of cell division which cannot be accounted for by trypsin released into the medium or into the cells. Also, initiation by soluble trypsin is inhibited by immobilised soybean trypsin inhibitor. These results demonstrate that trypsin can ...
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  • 3
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Annals of the New York Academy of Sciences 485 (1986), S. 0 
    ISSN: 1749-6632
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Natural Sciences in General
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  • 4
    ISSN: 0886-1544
    Keywords: growth factors ; phorbol 12-myristate 13-acetate ; microtubule-tubulin equilibrium ; initiation of DNA synthesis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Previous studies suggest that alterations in the microtubule (MT)-tubulin equilibrium during G0/G1 affect mitogenesis. To determine the effect of growth factors on the MT-tubulin equilibrium, we developed a radioactive monoclonal antibody binding assay (Ball et al.: J. Cell. Biol. 103:1033-1041, 1986). With this assay, 3H-Ab 1 - 1.1 binding to cytoskeletons in confluent populations of cultured cells is proportional to the number of tubulin subunits polymerized into MTs. We now show that purified α-thrombin increases 3H-Ab 1 - 1.1 binding to cytoskeletons of serum-arrested mouse embryo (ME) fibroblasts from 1.5- to 3-fold. This stimulation is dose-dependent and correlates with concentrations of thrombin required for initiation of DNA synthesis. Other mitogenic factors, epidermal growth factor (EGF) and phorbol 12-myristate 13-acetate (PMA), also stimulate MT polymerization. Addition of colchicine (0.3 μM) eight hours after growth factor addition blocks stimulation of 3H-thymidine incorporation by thrombin, EGF, or PMA, suggesting that tubulin polymerization or subsequent events triggered by MT polymerization are required for cells to enter a proliferative cycle. Consistent with models for autoregulation of tubulin synthesis, thrombin, EGF, and PMA all increase tubulin synthesis 9 to 15 hr after growth factor addition, raising the possibility that the decrease in free tubulin and subsequent stimulation of tubulin synthesis is linked to progression of cells into a proliferative cycle. Colchicine addition to these cells also stimulates DNA synthesis, but colchicine-stimulated cells enter S phase 6 to 8 hr later than those stimulated by growth factors. This delayed stimulation may be related to the time required for degradation of tubulin- colchicine complexes below a critical level. These data suggest that regulation of cell proliferation may be linked to increased MT polymerization and the resulting decrease in free tubulin pools. © 1992 Wiley-Liss, Inc.
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  • 5
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 26 (1984), S. 181-195 
    ISSN: 0730-2312
    Keywords: thrombin ; growth factors ; receptor occupancy ; growth control ; cell cycle ; wound healing ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: To determine the role of thrombin high-affinity receptor occupancy and enzymic activity in thrombin initiation of cell proliferation, we have utilized thrombin derivatives which separate these functions. We previously showed that enzymically active γ-thrombin stimulates ion fluxes without binding to high-affinity sites, whereas proteolytically inhibited DIP-α-thrombin which binds to high-affinity receptors does not. Since neither derivative initiates DNA synthesis by itself, this suggested that two separate sequences of events might be necessary for a complete initiation signal. We now report that the combination of DIP-α-thrombin and γ-thrombin initiate DNA synthesis and cell proliferation to levels approaching the maximal initiation by native α-thrombin. This combinatory effect is dose-dependent for both γ-thrombin and DIP-α-thrombin in the same concentration range as α-thrombin alone. Thus, these same concentrations of α-thrombin alone may be required to initiate each sequence of events. The combinatory stimulation could be achieved even if the derivatives were added individually up to 8 hr apart. Moreover, preincubation with either derivative shortened the lag period for initiation of DNA synthesis by native α-thrombin. These results indicate that both receptor occupancy and enzymic activity are necessary for thrombin initiation of cell proliferation and that each action initiates a sequence of early events which moves the cell forward toward entry into a proliferative cycle.
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  • 6
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 20 (1982), S. 247-258 
    ISSN: 0730-2312
    Keywords: thrombin ; receptor-mediated endocytosis ; coated pits ; immunocytochemistry ; growth factors ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The localization of thrombin receptors on mouse embryo (ME) cells was examined using electron microscope (EM) immunocytological techniques. ME cells were fixed with formaldehyde, prior to thrombin binding, and thrombin visualized on cell surfaces using affinity-purified antithrombin rabbit antibody and colloidal gold labeled anti-rabbit IgG. Colloidal gold particles were found in clusters on the surface of cells incubated with thrombin. There were approximately seven particles per cluster observed in thin sections with cluster diameters ranging from 70 to 200 nm. These clusters were not observed on cells incubated without thrombin. The total number of particles present on cells incubated with and without thrombin indicate that the colloidal gold labeling is approximately 98% specific for thrombin. Only four colloidal gold particles out of approximately 1,200 were associated with coated pits. Thus the thrombin receptor clusters do not appear to associate with coated membrane regions. To determine whether receptor-bound thrombin was internalized by receptor-mediated endocytosis, ME cells were incubated with 125I-thrombin and examined using EM autoradiography and the trypsin sensitivity of 125I-thrombin which was associated with the cells. In two types of experiments, where thrombin was incubated with cells at 4°C and the temperature increased to 37°C and where initial incubation was at 37°C, the receptor-directed specific internalization proceeded at approximately the same rate as nonspecific internalization. These studies indicate that thrombin that binds to its receptors on ME cells is not rapidly internalized by receptor-mediated endocytosis.
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  • 7
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 95 (1978), S. 13-22 
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: It has been well documented that trypsin or thrombin initiate proliferation of quiescent secondary chick embryo cells. However, there has been less certainty about the ability of these proteases to initiate division of quiescent mammalian cells. Accordingly, we studied the conditions under which quiescent chick embryo (CE), mouse embryo (ME), and human diploid foreskin (HF) cells respond to trypsin or thrombinExtended culture of these cell strains in serum-free medium increased the initiation of cell division by both proteases. Under these conditions, CE cell number was increased 90% over controls by trypsin and 70% by thrombin. In contrast, quiescent ME and HF cells both responded better to thrombin than trypsin, giving maximal increases, respectively, of 70 and 40% over controls with thrombin and 22 and 14% with trypsin. Calf serum inhibited the initiation of these cell strains, particularly the ME cells, by both trypsin and thrombin. This inhibition of initiation could be attributed to decreased proteolytic activity in the case of trypsin, but not thrombinIn contrast to the cell strains tested, quiescent cultures of the 3T3 cell line showed no detectable increase in cell number with trypsin or thrombin in the absence of serum, and only a slight increase in cell number with thrombin in the presence of serum. However, in the presence of plasma, 3T3 cell number increased up to 20% with thrombinInitiation of cell division by proteases has been reported and confirmed mostly for early passage cell strains rather than cell lines. Our experiments with CE cells indicate that this is possibly the result of a rapid decline in protease responsiveness upon serial subculture. With these cells we found a decline in response first to trypsin, then thrombin, and finally serumThroughout these studies, we compared the ability of trypsin and thrombin to initiate cell division under various conditions. We found several differences between the two proteases, indicating that they initiate cell division by somewhat different mechanisms.
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  • 8
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Stimulation of amiloride-sensitive sodium (Na+) influx and the subsequent activation of NA+, K+-ATPase by serum or growth factors have been implicated as early events leading to initiation of cell proliferation. We recently demonstrated that amiloride inhibits thrombin-initiated DNA synthesis not by inhibiting an early event occurring during the first 8 hr, but rather by inhibiting some later event 8 to 12 hr after thrombin addition. To further probe the relationship between stimulation of ion influx and initiation of cell proliferation, human α-thrombin was converted to γ-thrombin, nitro-α-thrombin, and diisopropylphospho (DIP)-α-thrombin. These derivatives retain either the capacity to bind cell surface α-thrombin receptors or thrombin esterase activity, but they do not initiate DNA synthesis. At low concentrations of α-thrombin or the various thrombin derivatives, only α-thrombin stimulates 86Rb+ influx, suggesting a correlation between stimulation of influx and the ability of these derivatives to initiate DNA synthesis. Concentrations of a DIP-α-thrombin that saturate the α-thrombin recptors (up to 2 μg/ml) do not stimulate either the early or late influx of 86Rb+, indicating that DIP-α-thrombin binding alone is not sufficient to stimulate ion fluxes. High concentrations of either γ-thrombin or nitro-α-thrombin, however, stimulate both early and late 86RB+ uptake but do not initiate DNA synthesis. These results demonstrate that events leading to both the early and late stimulation of 86Rb+ influx by themselves are not sufficient to initiate cell proliferation. Thus, initiation may require a combination of events that can be independently regulated by different transmembrane signals.
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  • 9
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 117 (1983), S. 272-281 
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Amiloride, a Na+ influx inhibitor, has been shown to inhibit initiation of DNA synthesis by thrombin in mouse embryo fibroblast-like cells. Long exoosures (24 hr) to high concentrations of amiloride inhibited incorporation of thymidine into the DNA of both thrombin-stimulated and nonstimulated cells, suggesting that this inhibition might not be specific for thrombin-initiated DNA synthesis. Fluorescence microscopy and spectrofluorimetry showed that amiloride was internalized with an apparent mitochondrial association and that the internalized amiloride was readily released from the cells after removing amiloride from the medium. Based on this reversibility, cells were exposed to amiloride for short periods of time during thrombin treatment to determine the temporal relationship between any amiloride-sensitive event(s) and initiation of DNA synthesis. The presence of amiloride (100 μM) during a 12-hr exposure to thrombin did not block thrombin-initiated DNA synthesis or cell division but did delay the onset of DNA synthesis and the peak of thymidine incorporation into DNA by approximately 3 hr, suggesting that early initiation events might proceed in the presence of amiloride. 86Rb+ transport studies demonstrated that in this system ouabain-sensitive K+ uptake via the Na, K-ATPase was stimulated by thrombin during both an early and a late period. This stimulation was amiloride-sensitive under the same conditions used for growth experiments, suggesting that amiloride was inhibiting thrombin-stimulated Na+ transport in this system. Additional experiments showed that exposing cells to amiloride only during the first 8 hr after thrombin addition did not inhibit initiation. The presence of amiloride from 8-12 hr after thrombin addition maximally inhibited thrombin-stimulated DNA synthesis. Together these results demonstrate that amiloride inhibits thrombin-initiated DNA synthesis not by inhibiting an early event occurring during the first 8 hr, but rather by inhibiting some later event 8-12 hr after thrombin addition.
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  • 10
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 117 (1983), S. 297-307 
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Thrombin binding to formaldehyde-fixed mouse embryo (ME) cells was visualized by indirect immunofluorescence as a dot-like pattern with dots of approximately 500 nm diameter located over the entire cell surface. Experiments comparing the binding of 125I-thrombin and dot appearance on parallel cultures indicate that the immunofluorescent pattern is specific for thrombin-binding to high-affinity receptors. Similar patterns were observed on cells fixed in ethanol or glutaraldehyde prior to thrombin binding and on cells maintained at 4°C. These patterns were also observed in a number of established cell lines. Thus, thrombin receptors may be clustered prior to thrombin binding on all cells with these receptors. Comparing the amount of 125I-thrombin bound to CHO cells with the number of fluorescent dots per cell indicated that each dot represents a cluster of over 1000 receptors. On ME cells, the number of thrombin receptor clusters per cell ranged from fewer than 50 to over 5,000. Based on previous studies, this indicates that on ME cells each cluster contains an average of approximately 200 thrombin binding sites with some cells having few, if any, receptors and others having more than a million receptors per cell.
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