摘要:

AbstractThe ribonuclease (RNase) activity associated with the surface of cells (primarily the Vero line of Green monkey kidney) was assayed under conditions where all cells remained viable as members of colonies or monolayers. RNase activity associated with individual clones was revealed as clear zones of hydrolyzed (acid‐soluble) RNA against an opaque background of acidprecipitated RNA in an isotonic agarose‐yeast RNA overlay. High resolution assays for single‐ and double‐stranded RNase were achieved by measuring the loss in activity of infectious Sindbis virus RNA, and of the antiviral state induced by poly(rI). poly(rC), respectively.Experiments using these assay procedures revealed that virtually all of the RNase activity associated with viable (intact) vertebrate cells in culture was contributed by the serum in the growth medium, and was superficially adsorbed to the cell surface, rendering it relatively easy to remove by extensive washing.A confluent monolayer of 2 × 106unwashed Vero cells contained the equivalent of 500 ng of pancreatic RNase, representing about 5% of the total activity initially present in the serum of the growth medium. In terms of serum nuclease activity, only 0.45 ng equivalents of pancreatic RNase in 500 μl were required to destroy 50% of the activity of an infectious Sindbis virus RNA preparation in 15 minutes at 37°C.Cells grown in the absence of serum and cells washed about ten times had comparably low levels of RNase activity–about 100‐fold less than unwashed cells grown in the presence of 6% calf serum.The isotonic yeast RNA:agarose overlay procedure may be useful to identify and isolate cell mutants with different endogenous levels, or binding capacities, of surface RNase activity.Operationally, these studies describe the assay of cell surface‐associated RNase activity under conditions where all cells remain viable as members of individual colonies or monolayer populations. Specifically, the assay measures the solubilization of yeast RNA in an isotonic agarose mixture overlaying the cells. We describe its use to determine how much of the total RNase activity in viable cell monolayers or colonies is contributed by serum in the growth medium. In addition, we describe the results of two biological assays used as higher resolution probes for cell surface ribonuclease activity: (i) single‐strand (ss) RNA, in the form of infectious RNA from Sindbis virus, and (ii) double‐stranded(ds)‐RNA, in the form of poly(rI). poly(rC) as an inducer of an interferon‐

ISSN:0021-9541    

DOI:10.1002/jcp.1040980102

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1979

数据来源: WILEY

摘要:

AbstractPGE1elicited a slow, dose‐dependent membrane depolarization with an increase in membrane conductance in the somatic cell hybrid TCX11. The ED50was 1‐2 × 10−8M with maximal responses at 1‐5 × 10−7M. Dopamine (DA) reversed the effect of PGE1and caused the membrane potential and resistance to return to control levels. Chronic exposure of cells (measured in minutes) to DA alone would not cause this hyperpolarization. 5‐HT was also tested and failed to consistently reverse the PGE1effects. Chlorpromazine antagonized the effects of DA on the PGE1response. The electrophysiological results reported here using TCX11 cells are discussed in light of previously reported biochemical results describing interactions of PGE1and DA, and the electrophysiological effect

ISSN:0021-9541    

DOI:10.1002/jcp.1040980103

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1979

数据来源: WILEY

摘要:

AbstractMutants temperature‐sensitive for growth have been isolated from the established line of Chinese hamster fibroblasts Wg1A. These mutants, together with the ones previously isolated by Roscoe et al. ('73), have been characterized with regard to their cell cycle properties. Most of them become arrested in the G1 phase of the cell cycle when incubated under restrictive conditions. By performing temperature shift experiments with synchronous cultures, the execution steps of most of the mutated functions have been located within the second half of the G1 phas

ISSN:0021-9541    

DOI:10.1002/jcp.1040980104

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1979

数据来源: WILEY

摘要:

AbstractTotal cellular calcium levels do not change when 3T3‐4a cells stop proliferating due to serum depletion, or when serum‐arrested quiescent cells are incubated for up to 44 hours in calcium‐deficient medium (∼10 μM Ca++). Upon stimulation with dialyzed serum cells enter S and progress through at least one cycle even at extremely low calcium levels in the culture medium (≥10 μM). Cells divide until a final cell density is attained which is proportional to the calcium concentration in the medium and cells reversibly arrest in G1. Cells which arrested in G1in medium containing ≤26 μM Ca++in the presence of excess serum can be stimulated to enter S in response to added calcium after a prereplicative phase of 14 to 16 hours. Serum does not affect45Ca‐uptake in these cells. Benzo[a]pyrene transformed 3T3 (BP3T3) cells have a 100–200 times lower Ca++‐requirement than 3T3 cells but arrest in G1at low Ca++levels. In contrast, SV40‐virus transformed 3T3 (SV3T3) cells that grow without restriction in monolayer cultures have even lower Ca++‐requirements for growth than BP3T3 cells and have no Ca++‐sensitive restriction point. Therefore, 3T3 and BP3T3 cells have retained the capacity to sense intracellular Ca++‐pool sizes and to arrest in G1at subth

ISSN:0021-9541    

DOI:10.1002/jcp.1040980105

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1979

数据来源: WILEY

摘要:

AbstractInosine triphosphate pyrophosphohydrolase from human erythrocytes was purified and characterized. The enzyme is highly specific for ITP and shows optimal activity in glycine buffer pH 9.6 and 50 mM MgCl2. The Kmof the enzyme is 1.3 × 10−4, the Vmax= 1.2 × 10−9and the Keq= 3.8 × 104. Human erythrocyte ITP pyrophosphohydrolase does not require SH compounds for activation. The enzyme is inhibited by Cd++, Co++, and Ca++ions and by phydroxymercuribe

ISSN:0021-9541    

DOI:10.1002/jcp.1040980106

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1979

数据来源: WILEY

摘要:

AbstractA strain of nonsymbioticA. proteuswas infected with endosymbiotic bacteria isolated from another strain of amoeba which had become dependent on the symbionts after a few years of spontaneously established symbiosis. In the newly infected amoebae, the bacteria avoided digestion and multiplied at a faster rate than the hosts, reaching the maximum carrying number (about 42,000 per amoeba) in fewer than ten cell generations of the hosts. The experimentally infected amoebae were also examined under the electron microscope, and the development of bacteria‐containing vesicles was followed. The results show that the infective bacteria that were initially harmful to host amoebae have become harmless and that they have changed in their mode of multiplication during the course of establishing a stable symbiosis with their host

ISSN:0021-9541    

DOI:10.1002/jcp.1040980107

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1979

数据来源: WILEY

摘要:

AbstractCellular resistance to the cytotoxic purine analogues 8‐azaguanine (AG) and 6‐thioguanine (TG) is usually mediated by a mutation leading to the loss or reduction in hypoxanthine phosphoribosyltransferase (HPRT) activity. However, stable AG‐resistant variants have often been shown to contain wild‐type levels of HPRT, while cellular resistance to TG is always accompanied by a profound deficiency in HPRT activity. Such AG‐resistant, HPRT‐positive cells are still sensitive to TG. To investigate the basis of this differential sensitivity, we examined the inhibition of the HPRT activity by AG and TG in whole cells, in cell‐free extracts, and with purified mouse HPRT. In addition, the relative incorporation and utilization of AG and TG by L929 cells were determined under a variety of culture conditions.Results show that, compared to TG, AG is generally a very poor substrate for HPRT. Incorporation of radioactive AG by HPRT‐positive cells was extremely sensitive to the free purine concentrations in the medium, so that under the usual culture conditions employing undialyzed serum, cellular uptake and utilization was minimal even when relatively high levels of AG were present. In contrast, the incorporation of radioactive TG was comparable to that of a natural substrate, hypoxanthine. These results indicate that the differential cellular sensitivity to AG and TG is due to the difference between these two guanine analogues as substrates of HPRT. Additional data indicate also that cellular resistance to TG is mediated exclusively by HPRT deficiency, but resistance to very high levels of AG may result through at least two other mechanisms not involving HPRT deficiency. These observations may help resolve some of the conflicting data in the literature, and demonstrate that TG is a better selective agent for the HPRT‐de

ISSN:0021-9541    

DOI:10.1002/jcp.1040980108

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1979

数据来源: WILEY

摘要:

AbstractRemoval of serum from BHK‐21/C13 cells in culture results in a decline in thymidine incorporation extending over five days. Additional removal of any of several amino acids results in a rapid decrease in incorporation of thymidine to negligible levels by 24 hours. Replacement by complete medium then provokes a synchronous wave of DNA synthesis after only ten hours with DNA synthesis first increased at six hours. Starvation for glutamine results in a rapid decline in protein synthesis over the 24 hour period when DNA synthesis is falling. However, there is considerable degradation of total protein during this period, and RNA degradation is also greatly increased. Concurrently, synthesis of RNA falls to less than 10% of that in control cell

ISSN:0021-9541    

DOI:10.1002/jcp.1040980109

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1979

数据来源: WILEY

摘要:

AbstractBalb/c3T3 cells in crowded cultures detach from the dish when deprived of serum, and the survivors incorporate3H‐thymidine at a reduced rate. The detachment becomes pronounced two hours after removal of serum, and reaches its maximum rate between two and four hours. Cells in sparse culture are not detached by serum removal, and their rate of3H‐thymidine incorporation is only slightly reduced. As the sparse cultures grow into more crowded cultures, and the serum is depleted, increasing numbers detach. The detached cells are incapable of reattaching when placed in a new dish with ample fresh serum. The cells are leaky to cellular constituents and appear to be dead. Detachment is a consequence rather than the cause of cell death, and can be produced by agents which inhibit cellular energy metabolism. The cells on the dish which survive serum deprivation are fully viable and grow rapidly when serum is added. When they become crowded they are as sensitive to serum deprivation as was the original population. They are therefore not selected for a low serum requirement but apparently survive because they spread into the space vacated by the detaching cells and then behave as sparse cultures in response to serum variations. Insoluble complexes of Ca2+and pyrophosphate (Ca2+‐PPi) show the same concentration dependence in promoting cell survival as in stimulating3H‐thymidine incorporation, showing that a single substance can be responsible for both activities. It is concluded that survival and growth are part of the coordinate response of 3T3 cells to single external effectors. The results are discussed in terms of a simple model in which the coordinate response is regulated by the availability of Mg2+for transphosphorylation reactions within the cell, and the availability depends on the binding affinity of cellular membranes for Mg2+. The difference between survival and multiplication is postulated to be in the intensity and duration rather than the kind of s

ISSN:0021-9541    

DOI:10.1002/jcp.1040980110

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1979

数据来源: WILEY

摘要:

AbstractHuman lymphocytes were equilibrated for 48 hours over a wide range of external potassium levels, and their contents of potassium, sodium, and water determined. As external potassium rose from zero, cell potassium rose steeply in a sigmoidal fashion, reached half‐saturation at 0.4 mM external potassium, and then saturated at 129 mmoles/kg cells. The saturable cell potassium exchanged mole‐for‐mole with sodium. Analysis of the saturable components by a statistical‐mechanical adsorption model demonstrated a cooperative interaction between sites determining equilibrium potassium‐sodium distribution. Superimposed upon the saturable fraction of cell potassium was a smaller one that was non‐saturable with increasing external potassium to at least 64 mM, and that, when expressed as mmoles/liter cell water, existed in a ratio to external potassium of 0.6. The results strongly support the association‐induction hypothesis, which predicts a small non‐saturable component of ions determined by exclusion from oriented cell water and a cooperative interaction between sites throughout the cell that associate with pota

ISSN:0021-9541    

DOI:10.1002/jcp.1040980111

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1979

数据来源: WILEY