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  • 1
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Growth medium was conditioned by incubation on mouse embryo cells in vitro. Supplementation of agar suspension cultures with conditioned medium from primary cells, but not from established lines, readily enhanced colony development by mouse tumor cells. Only cells with the properties of myoblasts responded to conditioned medium. Other fibroblastoid cells and virus-transformed cell lines were not affected. Myogenic cells in agar cultures grew in the presence of conditioned medium but did not differentiate. Soluble collagen at 400 m̈g/ml possessed little colony-stimulating activity by comparison with fresh conditioned medium.
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  • 2
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Cellular feeder layers, prepared from normal blood leukocytes, usually stimulate human marrow to form colonies. A significant increase in the stimulating activity of unseparated leukocyte feeder layers is brought about following the removal of dense leukocytes in a manner which avoids enrichment of any remaining cell type. Restoration of dense leukocytes to a dense leukocyte depleted leukocyte feeder layer results in the reduction of stimulating activity to that of an unseparated leukocyte feeder; however, addition of dense leukocytes to unseparated leukocyte feeder layers has no effect on the stimulatory activity, over the range of concentrations used in this study.
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  • 3
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Protease nexin (PN) is a cell-secreted protein that links to thrombin (Th) and certain other serine proteases. PN mediates the binding, internalization, and degradation of these proteases by cells (Baker et al., 1980; Low et al., 1981). Here we show that binding of Th-PN complexes to human foreskin fibroblasts (HF cells) accounted for 90% of the specific cellular Th binding at certain mitogenic doses of the protease. However, cell-associated Th-PN complexes were likely to be inactive mitogenically because heparin (170 units/ml) inhibited cellular binding of 125-Th-PN by about 95% (a reduction from 1.3 × 105 to 6 × 103 125I-Th-PN complexes per cell) but did not influence Th-mediated mitogenic stimulation. In experiments with mouse embryo cells, heparin also markedly decreased cellular binding of 125I-Th-PN without changing the mitogenic response to Th. The lack of mitogenic activity of cell-associated Th-PN complexes suggested that PN might inhibit the mitogenically essential proteolytic activity of Th. This possibility is supported by the following findings. First, amounts of serum-free conditioned culture medium that contained enough PN to complex a large fraction of added Th inhibited the clotting activity of Th. Second, heparin increased the formation of 125I-Th-PN complexes and also increased this inhibitory effect of conditioned medium. We conclude that PN acts as a negative modulator of thrombin mitogenic activity.It is shown that like other fibroblastic cells HF cells bound free 125I-Th specifically (although with relatively low affinity, Kass 〈 108 M-1). Specific binding of free 125I-Th to HF cells increased fourfold in the presence of heparin (50 IU/ml).
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  • 4
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The mechanisms of iron (Fe) and transferrin (Tf) uptake by the human melanoma cell line, SK-MEL-28, have been investigated using chelators and metabolic probes. These data provide evidence for two saturable processes of Fe uptake from Tf, namely, specific receptor-mediated endocytosis and a second nonspecific, non-receptor-mediated mechanism which saturated with respect to Fe uptake at a Tf concentration of approximately 0.3 mg/ml. In contrast to Fe uptake, Tf uptake increased linearly up to at least 1 mg/ml. Furthermore, under the culture conditions used, the second nonspecific, non-receptor-mediated mechanism was the most important process in terms of quantitative Fe uptake. Two concentrations of Tf-125I-59 Fe (0.01 and 0.1 mg/ml) were used in order to characterise the specific and nonspecific Fe uptake pathways. Membrane permeable chelators were equally effective at both Tf concentrations, whereas membrane impermeable chelators were significantly (P 〈 0.001) more effective at reducing the internalisation of Fe at the higher Tf concentration, consistent with a mechanism of Fe uptake which occurred at a site in contact with the extracellular medium. The oxidoreductase inhibitor, amiloride, only slightly inhibited Fe uptake at the higher Tf concentration, suggesting that the second nonspecific process was not mediated by a diferric Tf reductase. Three lysosomotrophic agents and the endocytosis inhibitor, phenylglyoxal, markedly reduced Fe uptake at both Tf concentrations, and it is concluded that a saturable process consistent with receptor-mediated endocytosis of Tf occurred at the lower Tf concentration, while the predominant mechanism of Fe uptake at high Tf concentrations was a second saturable process consistent with adsorptive pinocytosis. © 1994 Wiley-Liss, Inc.
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  • 5
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Addition of insulin to nonproliferating serum-free cultures of secondary chicken embryo (CE) cells caused a 30% to 50% increase in cell number. Addition of any one of several glucocorticoids (dexamethasone, cortisol, or corticosterone) to the cultures two days before insulin addition increased the mitogenic effect of insulin by about twofold at each insulin concentration tested. This glucocorticoid stimulation of cell proliferation was “permissive” because in the absence of insulin glucocorticoids caused little increase in cell number (usually less than 15%). Glucocorticoids were maximally active at low concentrations (e.g., 10-10 M dexamethasone). Steroids without glucocorticoid activity were inactive over a wide range of concentrations. Glucocorticoids increased the mitogenic response to insulin largely by increasing the percentage of cells that insulin stimulated to synthesize DNA.The maximum mitogenic effect of insulin upon CE cells rapidly decreased after the cells were serially subcultured. After only nine population doublings (4 passages) in culture, the response to insulin was diminished by about 70%. The mitogenic effect of insulin plus dexamethasone declined similarly during serial subculture, and was always about twofold greater than the effect of insulin alone. The cells maintained their mitogenic responsiveness to serum as these responses decreased.In contrast to the growth promoting influence of glucocorticoids in the presence of insulin, glucocorticoids inhibited the mitogenic response of CE cells to serum. This result may resolve our above findings with reports that glucocorticoids inhibit the proliferation of CE cells.
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  • 6
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Four criteria were used to examine serum-free conditioned cell culture medium for protease nexin (PN):(1) formation of SDS-stable ∼77 K Da complexes between a medium component and [125l]thrombin; (2) acceleration by heparin of the rate of formation of these complexes; (3) cellular binding of these complexes; and (4) inhibition by heparin of the cellular binding of complexes. Listed in order of decreasing PN production, PN was detected in media conditioned by the following cell types: human foreskin fibroblasts (0.18 μg/106 cells), rat embryo heart muscle cells (0.13 μg/106 cells), mouse myotubes (0.1 μg/106 cells), monkey kidney epithelial cells, human fibrosarcoma cells, human lung fibroblasts, simian virus 40 (SV-40)-transformed human fibroblasts, human epidermoid carcinoma cells, bovine aortic endothelial cells (only after phorbol ester treatment), and mouse myoblasts. No PN was found in medium conditioned by mouse 3T3 cells, SV40 virus-transformed 3T3 cells, human lymphoblasts, or mouse leukemia cells.Eleven of the cell types examined for secretion of PN were also examined for the presence of cytoplasmic thrombin-binding factors. Lysates from all of these cell types contained a factor that formed ∼60-65 K Da sodium dodecyl sulfate (SDS)-stable complexes with [125l] thrombin. This MW is significantly lower than that of [125l] thrombin-PN complexes, indicating that the factor is distinct from PN. Nevertheless, PN and the cytoplasmic factor share similarities. Production of both PN (by HF cells and WI-26 cells) and the cytoplasmic factor (by HF cells and 3T3 cells) are stimulated by epidermal growth factor and phorbol myristate acetate. Also, both PN and the cytoplasmic factor complex trypsin, plasmin, urokinase, and thrombin, but not pancreatic elastase. Because a number of the cells that produce PN or the cytoplasmic serine protease-binding factor are known to produce plasminogen activators, both PN and the cytoplasmic factor could regulate plasminogen activator activity.
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  • 7
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The development of resistance accounts for therapy failure in the majority of advanced cases of neuroblastoma in children. A new transplantable murine C-1300 neuroblastoma cell line was developed in vitro, by repeated exposure of a sensitive cell line to increasing, but sublethal, doses of Homoharringtonine (HHT). The ED50 of the highly resistant cells for HHT, using a standard agar colony assay, is 480 ng/ml, compared with 13 ng/ml for the sensitive parental line. The resistant cells have cross-resistance to a number of other agents, including adriamycin, vinca alkaloids, melphalan, and CCNU. Western blot analysis revealed progressive increases in P-glycoprotein, parallel to the graded development of resistance with a 29-fold elevation in the highest resistant cells. High-performance liquid chromatography (HPLC) indicated that resistant cells have a significantly lower uptake of HHT than parental sensitive cells. Cyclos-porine A (CsA) and dipyridamole (DPM) could modulate the acquired resistance and completely restore the cytotoxic effects of HHT and adriamycin as determined by the clonogenic assay. The reversal of resistance by CsA and DPM was dose dependent. With the relative low toxicity of dipyridamole and CsA in doses required for modulation of resistance, these agents may be candidates for clinical utilization in chemotherapy of resistant neuroblastoma.
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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: Mutants of mouse L-cells which are temperature-sensitive for growth have been obtained by using both selective and nonselective isolation procedures on populations treated with the mutagen nitrosoguanidine. Selective isolation was carried out by utilizing a five-day treatment with 3H-TdR and ara-C as selective agents at the nonpermissive temperature. Nonselective isolation was performed by isolating 1400 clones in the absence of selective agents and then testing them for temperature-sensitivity. From this experiment we obtained a minimum estimate of 6 × 10-3 for the frequency of mutants in the mutagentreated population. The mutants were characterized by their plating efficiencies, growth in suspension culture, and uptake of isotopic precursors of DNA, RNA, and protein. A range in phenotypes was observed, and there appeared to be some differences between the mutants obtained by the two types of isolation procedures. In uptake experiments the most marked reductions in the rates of precursor incorporation were seen with 3H-TdR, rather than 3H-UR or 3H-Leu. Different mutant lines showed considerable variation in the rate of cessation of DNA synthesis as well as the time required for termination of cell division. These experiments suggest that both types of isolation procedures are feasible for obtaining temperature-sensitive mutants having a range of phenotypes.
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  • 9
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 77 (1971), S. 377-384 
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The two stages in the uptake of transferrin by rabbit reticulo-cytes were investigated using radioiodine-labeled rabbit transferrin and albumin. The first stage of rapid, temperature-insensitive uptake of transferrin was similar to albumin uptake: uptake of both proteins increased linearly with increasing protein concentration of the incubation medium up to at least 60 mg/ml, was maximal at low ionic strength and pH, and increased in the presence of basic polyamino acids. Transferrin uptake was in part dependent on the reticulocyte concentration of the blood, but albumin uptake was independent of reticulocyte concentration.The second slower, temperature-sensitive stage of transferrin uptake was linearly related to reticulocyte concentration, and was not found with albumin, α1-macroglobulin or γ-globulin. Transferrin uptake was optimal at physiological pH and ionic strength and was unaffected by basic polyamino acids. When the transferrin concentration was raised, uptake increased to reach a maximum at a concentration of 15 mg/ml.It was concluded that the first stage of transferrin uptake was in part or wholly due to non-specific adsorption of transferrin to erythrocytes, while the second stage of uptake was specific for transferrin and reticulocytes and depended upon normal function of the cells.
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  • 10
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 83 (1974), S. 259-261 
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Exposure of early sea urchin embryos to 5-bromodeoxyuridine (at concentrations up to 100 μg per ml) severely decreases the uptake of exogenous 3H-uridine into RNA. However, the actual gross rate of DNA or RNA synthesis in these embryos appears not to be affected by the presence of 5-bromodeoxyuridine.
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