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  • 1
    Electronic Resource
    Electronic Resource
    Membrane regulation of the Na+, K+-ATPase during the neuroblastoma cell cycle: Correlation with protein lateral mobility (1983)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 21 (1983), S. 77-91 
    Details
    ISSN: 0730-2312
    Keywords: Na+, K+-ATPase ; cell cycle ; protein lateral mobility ; regulation ; neuroblastoma cells ; ouabain binding ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The pumping activity of the plasma membrane-bound Na+, K+-ATPase shows considerable variation during the cell cycle of mouse neuroblastoma Neuro-2A cells. Addition of external ATP at millimolar concentrations, which selectively enhances the plasma membrane permeability of Neuro-2A cells for sodium ions, stimulates the Na+, K+-ATPase pumping activity at all phases of the cell cycle from a factor of 1.05 in mitosis up to 2.2 in G1 phase. Determination of the number of Na+, K+-ATPase copies per cell by direct 3H-ouabain binding studies in the presence of external ATP shows a gradual increase in the number of pump sites on passing from mitosis to the late S/G2-phase by approximately a factor of 2. From these data the pumping activity per copy of Na+, K+-ATPase, optimally stimulated with respect to its various substrate ions, has been determined during the various phases of the cell cycle. This optimally stimulated pumping activity per enzyme copy, which is a reflection of the physicochemical state of the plasma membrane, is high in mitosis, almost twofold lower in early G1 phase, and increases gradually again during the other phases of the cell cycle. This shows that the observed regulation of Na+, K+-ATPase activity during the cell cycle is caused by a combination of three independent factors-namely variation in intracellular substrate availability (Na+), changes in number of enzyme copies per cell, and modulation of the plasma membrane environment of the protein molecules. The modulation of the optimal pumping activity per enzyme copy shows a good correlation (ρ = 0.96) with the known modulation of protein lateral mobility during the cell cycle, such that a high protein lateral mobility correlates with a low enzyme activity. It is concluded that changes in plasma membrane properties take place during the Neuro-2A cell cycle that result in changes in the rate of protein lateral diffusion and Na+, K+-ATPase activity in a directly correlated way.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcb.240210109
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  • 2
    Electronic Resource
    Electronic Resource
    Loss of EGF binding and cation transport response during differentiation of mouse neuroblastoma cells (1983)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 21 (1983), S. 63-75 
    Details
    ISSN: 0730-2312
    Keywords: neuroblastoma ; differentiation ; EGF ; binding assay ; K+ transport ; Na+ transport ; amiloride ; growth stimulation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Mouse neuroblastoma cells (clone N1E-115) differentiate in culture upon withdrawal of serum growth factors and acquire the characteristics of neurons. We have shown that exponentially growing N1E-115 cells possess functional epidermal growth factor (EGF) receptors but that the capacity for binding EGF and for stimulation of DNA synthesis is lost as the cells differentiate. Furthermore, in exponentially growing cells, EGF induces a rapid increase in amiloride-sensitive Na+ influx, followed by stimulation of the (Na+ -K+)ATPase, indicating that activation of the Na+/H+ exchange mechanism in N1E-115 cells [1] may be induced by EGF. The ionic response is also lost during differentiation, but we have shown that the stimulation of both Na+ and K+ influx is directly proportional to the number of occupied receptors in all cells whether exponentially growing or differentiating, thus only indirectly dependent on the external EGF concentration. The linearity of the relationships indicates that there is no rate-limiting step between EGF binding and the ionic response. Our data would suggest that as neuroblastoma cells differentiate and acquire neuronal properties, their ability to respond to mitogens, both biologically and in the activation of cation transport processes, progressively decreases owing to the loss of the appropriate receptors.
    Additional Material: 8 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcb.240210108
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  • 3
    Electronic Resource
    Electronic Resource
    Secretion of transforming growth factor-β isoforms by embryonic stem cells: Isoform and latency are dependent on direction of differentiation (1993)
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 156 (1993), S. 247-256 
    Details
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Murine embryonic stem (ES) cells are maintained in an undifferentiated state when cultured in medium conditioned by Buffalo rat liver (BRL) cells. BRL conditioned medium (CM) contains a differentiation inhibitory activity (DIA) that is synonymous with leukemia inhibitory factor (LIF). ES cells in monolayer culture can be induced to differentiate by addition of all-trans retinoic acid (RA) to the BRL CM, when they mainly form cells resembling parietal endoderm, or by culture in medium not conditioned by BRL cells. ES cells thus deprived of LIF/DIA differentiate spontaneously to a cell type that expresses Brachyury (T), a marker of early mesoderm. Northern blot analyses have shown previously that transcripts for transforming growth factor beta 1 (TGF-β1) are detected in undifferentiated cells while transcripts for TGF-β2 and TGF-β3 only become detectable after differentiation. We have now determined levels of TGF-β protein in CM and in the extracellular matrix (ECM) and have used neutralizing antibodies specific for TGF-β1 and TGF-β2 that do not react with recombinant human TGF-β3 to determine the isoform secreted. Using the growth inhibition of mink lung CCL64 cells as a bioassay for TGF-β activity, we demonstrate that undifferentiated ES cells secrete latent TGF-β1 into the medium but no activity is found in their ECM. Cells induced to differentiate with RA contain TGF-β2 in both active and latent forms in their CM. Likewise their ECM contains TGF-β2 as the sole isoform. ES cells deprived of LIF/DIA secrete both TGF-β1 and TGF-β2 isoforms in their CM but TGF-β-like activity remains after addition of neutralizing antibodies for TGF-β1 and TGF-β2. This active TGFβ is the major component of the TGF-β activity in this CM. By contrast, ECM from LIF/DIA deprived cells contains only the TGF-β1 and β2 isoforms. The remaining activity in CM correlates with high expression of TGF-β3 by Northern blot analysis in these cells. We speculate that TGF-β3 is secreted by these cells and may be activated more efficiently and/or in a different manner to TGF-β1 and TGF-β2, since it is present in CM only in its active form. © 1993 Wiley-Liss, Inc.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcp.1041560205
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  • 4
    Electronic Resource
    Electronic Resource
    Transforming growth factor-β in the early mouse embryo: Implications for the regulation of muscle formation and implantation (1993)
    Chichester [u.a.] : Wiley-Blackwell
    Developmental Genetics 14 (1993), S. 212-224 
    Details
    ISSN: 0192-253X
    Keywords: Embryonic stem cells ; differentiation ; organogenesis ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: In a search for functions of transforming growth factor-β during early embryonic development we used two different experimental approaches. In the first we made use of embryonic stem (ES) cells. ES cells in culture differentiate to derivatives of all three germ layers and mimic some aspects of organogenesis when grown as aggregates in suspension to form embryoid bodies. Differentiation procedes further when the embryold bodies attach to suitable substrates. Muscle and neuronal cells are among the most readily identified cell types then formed. We examined the effect of all-trans retinoic acid (RA) and members of the transforming growth factor-β family(TGF-βl, TGF-β2) under these conditions in an assay where single aggregates formed in hanging microdrops in medium supplemented with serum depleted of lipophilic substances which would include retinoids. Endoderm-like cells formed under all conditions tested. RA at concentrations of 108 M and 107 M induced the formation of neurons but in the absence of RA or at concentrations up to 10-9 M, neurons were not observed. Instead, beating muscle formed in about one-third of the plated aggregates; this was greatly reduced when RA concentrations increased above 10-9 M. Immunofluorescent staining for muscle specific myosin showed that two muscle cell types could be distinguished: elongated, non-contractile myoblasts and mononucleate flat cells. The mononucleate flat cells appeared to correspond with rhythmically contracting muscle. The number of non-contractile myoblasts increased 3-fold over controls in the presence of 10-9 M RA. TGF-βs increased the number of contractile and non-contractile muscle cells by a factor 3 to 7 over controls, depending on the TGF-β isoform added and the muscle cell type formed. TGF-β2 also invariably increased the rate at which contracting muscle cells were first observed in replated aggregates. The stimulatory effect of TGF-βs on the formation of mononucleate flat cells was completely abrogated by RA at 10-9 M while the number of myoblasts under similar conditions was unchanged. These data suggest that a complex interplay between retinoids and TGF-β isoforms may be involved in regulation of differentiation in early myogenesis.In the second approach, neutralizing polyclonal rabbit antibodies specific for TGF-β2 were injected into the cavity of mouse blastocysts 3.5 days post coitum (pc). After 1 day in culture, embryos were transferred to pseudopregnant females. The number of decidua, embryos and resorptions were counted at day 8.5-9.5 pc. Control antibody injected embryos implanted with high efficiency (87%) compared with anti-TGF-β2 injected embryos which implanted with an efficiency of only 43%. If empty decidua (resorptions) were included, the overall recovery was 71% and 32% for control and experimental embryos, respectively. Embryos that were recovered showed no overt macroscopic abnormalities. These results together impiy functions for TGF-βs in implantation as well as in later development of the embryo. © 1993Wiley-Liss, Inc.
    Additional Material: 2 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/dvg.1020140308
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