摘要:

AbstractIntracellular pH is crucial to the control of many cellular processes such as glycolysis, protein synthesis, and cell division. To study the relation between intracellular pH and mitotic activity in actively dividing Con A‐ or LPS‐stimulated splenic lymphocytes, a method was developed to determine intracellular pH using a fluorescence‐activated cell sorter. The new method uses the pH‐sensitive fluorochrome, 4‐methylumbelliferone. Results obtained with it not only correspond qualitatively with the results obtained using14C‐dimethyloxazolidinedione (DMO) but also clearly show the active and inactive subpopulations. The intracellular pH of mitogen‐stimulated murine lymphocytes increases from pH 7.15 to pH 7.45 when the population has greatest mitotic activity. The intracellular pH of three virus‐transformed lymphocyte cell lines is higher by ∼ 0.5 pH unit when the cells are in exponential growth compared to

ISSN:0021-9541    

DOI:10.1002/jcp.1041120102

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

年代:1982

数据来源: WILEY

摘要:

AbstractIn this study, the role of glycosaminoglycans in fibronectin‐mediated cell attachment to collagen has been investigated. While it has been established that fibronectin possesses binding sites for several glycosaminoglycans, it was found that only dextran sulfate and macromolecular heparin could decrease the initial rate of cell attachment to collagen. Low molecular weight heparin was inactive. This study suggests that the glycosaminoglycan binding site of fibronectin plays a role in the mechanism of cell attachmen

ISSN:0021-9541    

DOI:10.1002/jcp.1041120103

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

年代:1982

数据来源: WILEY

摘要:

AbstractIntermediate subviral particles (ISVP) derived from reovirus represent a simple model system for the switch‐on of transcriptase function. In such particles the endogenous transcriptase is present in a switched‐off form, one step removed from the switched‐on state. Switch‐on of transcriptase function is an active process in this system and can be triggered by K+ions. A variety of agents which affect gene expression in cells were tested for an effect on switch‐on in ISVP. Marked effects on switch‐on in ISVP were observed with a diverse group of test agents, including DMSO and other solvents, BUdR, TdR, caffeine, theophylline, and temperature. The correlation in response between ISVP and cells suggests that the ISVP system may be useful as a model for studying the biochemical mechanisms underlying the perturbative effects of such agents on gene expressi

ISSN:0021-9541    

DOI:10.1002/jcp.1041120104

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

年代:1982

数据来源: WILEY

摘要:

AbstractHuman NHIK 3025 cells, synchronized by mitotic selection, were given 2 mM thymidine, which inhibited DNA synthesis without reducing the rate of protein accumulation. After removal of the thymidine the cells proceeded towards mitosis and cell division, with an S duration 2 hours shorter than, but a G2 and M duration nearly identical to that of the control cells. If cycloheximide (1.25 m̈M) was present together with thymidine, no net protein accumulation took place during the treatment, and the subsequent duration of S, G2, and M was similar to that of the untreated cells. The shortening of S seen after treatment with thymidine alone would therefore indicate that the rate of DNA synthesis depended on the amount of some preaccumulated protein.The postreplicative period in thymidine‐treated cells was lengthened by cycloheximide treatment although the protein content had already been doubled. This suggests that proteins required for the traverse of this part of the cell cycle might have to be synthesized after completion of DNA replication.Shortly after removal of thymidine, the rate of protein accumulation declined markedly, indicating the existence of some mechanism for negative control of cell mass. In addition, the daughters of thymidine‐treated cells had their cell cycle shortened by 2 hours. As a result, the cells had returned to balanced growth already in the first cell cycle following the induction of unbalanced growth.In conclusion, our experiments suggest that NHIK 3025 cells might require a minimum time in order to traverse the cell cycle, which is independent of cell

ISSN:0021-9541    

DOI:10.1002/jcp.1041120105

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

年代:1982

数据来源: WILEY

摘要:

AbstractNa+transport properties of neuroblastoma (clone Neuro‐2A) cells have been characterized both in exponentially growing cells and during the cell cycle. In exponentially growing cells, Na+influx followed the kinetics for a one‐compartment system with an influx rate of 9.54 ± 0.96 nmoles/minute/106cells, equilibrium being reached within 2 minutes. The initial rate of influx in the presence of ouabain (5 mM), a concentration completely inhibiting the (Na+‐K+)ATPase and thus backflux of tracer, was 11.33 ± 1.50 nmoles/minute/106cells and, within error, the same as in the absence of ouabain, provided determinations were made within 3 minutes of ouabain addition.Na+influx rate was determined at intervals during the cell cycle of synchronized Neuro‐2A cells using a 3‐minute pulse of22Na+in the presence of ouabain. On passing from mitosis to G1‐phase Na+influx decreases from 12.13 ± 1.93 to 7.40 ± 0.90 nmoles/minute/106cells but increases rapidly and transiently approximately twofold upon entry into S‐phase. This transient increase coincides with a transient stimulation of the (Na+‐K+) pump activity. It then returns to a steady level of ˜ 12 nmoles/minute/106cells for most of the remainder S‐phase.Intracellular Na+concentration during the cell cycle was determined from the equilibrium content of24Na+and data on the intracellular H2O volume, published previously (Boonstra et al., 1981b). Na+concentration is maximal in mitosis at 56.96 ± 6.05 mM and decreases rapidly tourfold as cells enter G1‐phase. With progression through S‐phase a steady increase from 13.80 ± 1.25 mM to 37.35 ± 2.91 mM is observed. Combining the Na+concentration with the K+concentration obtained previously (Boonstra et al., 1981b), the K+:Na+ratio was obtained during the cell cycle. The ratio had a value of between 3 and 5 during most of the cell cycle, but was significantly higher in G1‐phase, where the loss of Na+is considerably greater than the loss of K+. The values for Na+concentration were combined with membrane potential measurements reported previously (Boonstra et al., 1980b) to obtain the Na+electrochemical gradient across the cell membrane in the cell cycle. This had a value of 63.2 ± 2.6 mV in mitosis and increased rapidly to reach a maximum value of 84.0 ± 5.5 mV during G1‐phase, thereafter maintaining a value of between 73 and 80 mV. The transient increase in Na+influx and in (Na+‐K+)ATPase‐mediated K+influx at the G1/S‐phase transition are specifically inhibitable by the diuretic amiloride (0.2 mM) but amiloride has little effect on Na+or K+influx in other phases of the cell cycle. This indicated activation of an amiloride‐sensitive transport system specically at the G1/S‐phase transition and a causal relationship between increased Na+entry and transient stimulation of the pump at this point in the cell cycle. Further, amiloride (0.1, 0.2 mM) addition was shown to give rise to a significant lengthening of the cell cycle and to cause a partial blockage of the cells specifically in G1‐phase, suggesting an important role for the transient ion flux changes in control

ISSN:0021-9541    

DOI:10.1002/jcp.1041120106

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

年代:1982

数据来源: WILEY

摘要:

AbstractA cadmium‐resistant variant isolated from mouse fibroblast LMTK cells can grow in the presence of 40 μM Cd2+. This variant retained its properties in the absence of selecting agent. Induction of metallothionein was measured in cell extracts by radioimmunoassay. The maximum amount of metallothioneins in the cells was reached after 36 hours. The cadmium resistant variant produced two times more metallothionein than the wild‐type when exposed to 10–20 μM Cd2+. By Ouchterlony double diffusion, the metallothioneins from cultured cells formed a line of parital identity with the mouse liver serotype and a line of complete identity with one of the two mouse kidney serotypes. These observations raise the possibility of a tissue‐specific expression of metallothion

ISSN:0021-9541    

DOI:10.1002/jcp.1041120107

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

年代:1982

数据来源: WILEY

摘要:

AbstractThe level of occupancy of a single class of high‐affinity (3H)‐phorbol 12, 13‐dibutyrate (PDBu) bindings sites (Kd = 26 nM) in quiescent Swiss 3T3 cells was correlated with the dose of PDBu required to stimulate rapid (increase in86Rb uptake, decrease in epidermal growth factor receptor affinity) and long‐term (induction of 2‐deoxyglucose uptake, initiation of DNA synthesis) events of the proliferative response. Further, structural analogues of PDBu showed identical relative potencies in the inhibition of (3H)‐PDBu binding and stimulation of DNA synthesis. Finally, prolonged (24‐hour) incubation of Swiss 3T3 or whole mouse embryo fibroblasts with 400 nM PDBu led to a decrease in the number of (3H)‐PDBu binding sites (down‐regulation) with a parallel loss of rapid and long‐term responses of the cells to PDBu (desensitization). These findings indicate that the high‐affinity (3H)‐PDBu binding sites mediate the mitogenic effects of phorbol ester

ISSN:0021-9541    

DOI:10.1002/jcp.1041120108

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

年代:1982

数据来源: WILEY

摘要:

AbstractRabbit chondrocytes from pooled articular joints have been delineated by their time of attachment of culture flasks after initiation of primary monolayer culture, either attached (48‐AT) or floating (48‐F) after 48 hours. A general population of chrondrocytes (attached after 72 hours, 72‐AT) was also studied. The growth‐promoting activity of pituitary fibroblast growth factor (FGF) and its effect on sulfated‐proteoglycan synthesis was studied on each chondrocyte population in secondary monolayer culture.3H‐thymidine incorporation during a 1‐hour pulse was stimulated by FGF (100 ng/ml) in each chondrocyte population. The response of AT‐72 chondrocytes to FGF required an additional fetal bovine serum supplement, while 48‐F cells resonded independent of serum. The response of 48‐AT chondrocytes to FGF (100 ng/ml) during a 1‐hour pulse with3H‐thymidine was increased in low serum (0.5–2.0%) rather than when high serum (8–10%) was present in the culture medium. FGF reduced35SO4incorporation into sulfated‐proteoglycans in the 48‐AT and 48‐F chondrocyte populations, but not in the 72‐AT population. The reduction in35SO4incorporation in the 48‐AT and 48‐F chondrocytes was not characterized by alterations in the hydrodynamic size of the sulfated‐proteoglycans as measured by Sepharose CL‐2B chromatography nor by changes in the types of sulfated‐glycosaminoglycans produced. These results indicated that FGF produced quantitative rather than qualitative alterations in chondrocyte sulfated‐proteoglycan synthesis. The latter appears uncoupled from the g

ISSN:0021-9541    

DOI:10.1002/jcp.1041120109

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

年代:1982

数据来源: WILEY

摘要:

AbstractThe cell cycle of V79 Chinese hamster lung cells synchronized by hydroxyurea was investigated by flow cytometry. The metachromatic fluorochrome acridine orange was used to differentially stain DNA and RNA of V79 cells. Green and red fluorescence from individual cells, representing cellular DNA and RNA, respectively, was measured by flow cytometry. Periodic changes of cellular DNA and RNA contents were observed over nine cell cycles. The duration of G1, S, and G2+ M phases of synchronized V79 cells whose RNA content was close to that of the cells in balanced growth was 3, 4.5, and 1.5 hours, respectively. The duration of G1and S phases of cells containing RNA above a certain threshold was inversely proportional to the RNA content. The RNA content of cells containing RNA above the normal level regressed to normal after a few generations. Coefficients of variation for RNA content were significantly larger than those for DNA. An explanation for the decay of synchrony in a synchronized cell population is proposed.

ISSN:0021-9541    

DOI:10.1002/jcp.1041120110

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

年代:1982

数据来源: WILEY

摘要:

AbstractUptake of 2‐aminoisobutyric acid (AIB) at concentrations of 0.1 mM to 30 mM was examined in sodium‐containing and sodium‐free media in hepatocytes pretreated without hormones (control), with hormones, or with amino acid depletion. Results show that 1‐minute but not 4‐minute rates can be taken as initial rates for the total or sodium‐dependent transport of AIB. The data for the 1‐minute sodium‐dependent transport of AIB were analyzed by a comptuer program and also by Eadie‐Hofstee and Lineweaver‐Burk plots, and a single saturable system was found. In the control cultures, the saturable system had a Km of 1‐2 mM AIB and a Vmax of 1.2 nmoles AIB/mg protein/minute. There was an increase in the Vmax of two to three‐fold after pretreating the cultures with insulin or amino acid depletion, three to four‐fold with glucagon, and six to seven fold with

ISSN:0021-9541    

DOI:10.1002/jcp.1041120111

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

年代:1982

数据来源: WILEY