摘要:

AbstractDNA‐mediated transformation for thymidine autotrophs (Tk+) was stimulated severalfold when the recipient (mouse FM3A) (Tk−) cells were preirradiated by UV light. The effect was most prominently seen with the cells irradiated 12–16 hours prior to DNA addition. A similar stimulatory effect was observed when the cells were treated with novobiocin or mitomycin C. The effect, however, was blocked when cycloheximide was present during postirradiation period, suggesting that de novo protein synthesis is required for producing the effect. Most of the Tk+transformants arised from the UV‐irradiated cells carried the donor DNA sequences in their chro

ISSN:0021-9541    

DOI:10.1002/jcp.1041210302

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

年代:1984

数据来源: WILEY

摘要:

AbstractThe recently available compound quin‐2, which acts as a high affinity fluorescent indicator for calcium in the cytosol, was used to examine the role of calcium mobilization in the alveolar macrophage during the stimulation of 0 −2production by the tripeptide N‐formyl norleucyl leucyl phenylalanine (FNLLP). After preloading with quin‐2, the production of 0 −2was measured in conjunction with the transfer of 45Ca+2and changes in quin‐2 fluorescence upon stimultion with FNLLP. When cells were maintained in low (10 μM) extracellular calcium medium the presence of 1.5 mM quin‐2 in the cytosolic space partially inhibited the rate of 0 −2production upon stimulation by FNLLP. Addition of 1 mM Ca+2to the medium prior to stimulation rapidly restored the cell's capability to produce 0 −2upon stimulation at rates equal to control and extended the duration of stimulated 0 −2production as well. Quin‐2 fluorescence measurements indicated an increase in cytosolic Ca+2upon stimulation with FNLLP. This increase was lowest under conditions in which 0 −2production was inhibited. The addition of 1 mM Ca+2to the medium caused by itself a rapid but transient increase in cytosolic Ca+2as measured with quin‐2 without stimulating 0 −2production. This intracellularly redistributed calcium was determined to be the source of the greater increase in cytosolic calcium during stimulation in the presence of high extracellular calcium. Measurements of 45Ca+2transfer demonstrated a buffering of cytosolic Ca+2changes by quin‐2, which in low calcium medium could deplete calcium stores. It is suggested that this effect, prior to stimulation, was responsible for the mitigated 0 −2response for those cells maintained in low calcium medium, wherein calcium stores could not be replenished. These results suggested that the cell's mechanism for regulating cytosolic and bound calcium concentrations may also play an integral role in its normal mechanism for stimulated 0 −2production. They further support the postulate that the commonly observed rise in the concentration of calcium in the cytosol upon formyl peptide stimu

ISSN:0021-9541    

DOI:10.1002/jcp.1041210303

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

年代:1984

数据来源: WILEY

摘要:

AbstractHuman umbilical vein endothelial cells can be serially passaged by supplementing medium with a partially purified growth factor. Cell‐substratum detachment of early and late passage endothelial cells was examined using trypsin, collagenase, or homocysteine. Late‐passage cells detached more rapidly than early passage cells under all conditions tested. The rate of detachment was dependent upon the specific agent used. Protease‐mediated detachment was most rapid, occurring over minutes, in contrast to homocysteine‐induced detachment, which occurred over hours. When detached cells were collected and replated in the absence of the detaching agent, these cells reattached spread, and continued to proliferate. No significant difference was observed in the rate of adhesion of either early or late passage cells to a gelatin matrix. When early or late‐passage endothelial cells were plated and grown to confluence on a matrix synthesized by the opposite cell type, the rate of protease‐mediated cell detachment resembled the cell type from which the matrix was derived. The ease of endothelial cell detachment was determined by the origin of the extracellular matrix. Examination of the extracellular matrices from early and late passage cells revealed significant differences in the amounts of glycosaminoglycans and sulfated proteins present. These studies demonstrate the importance of the endothelial cell extracellular matrix in protease‐mediated cell detachment. The rate of cell detachment was controlled by the extracellular matrices and not altered by the endo

ISSN:0021-9541    

DOI:10.1002/jcp.1041210304

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

年代:1984

数据来源: WILEY

摘要:

AbstractOf the three biological polyamines, putrescine (Put), spermidine (Spd), and spermine (Spm), the relevance of Spm to cell proliferation has yet to be defined because of our general inability to deplete it selectively in intact cells. In the present study, Spm depletion was accomplished by treating cultured L1210 cells for 96 hr with α‐difluoromethylornithine (DFMO) and an analog of Spd such as aminopropylcadaverine. N4‐methylSpd, N4‐ethylSpd, or homoSpd. DFMO, a specific inhibitor of ornithine decarboxylase, halts continued polyamine biosynthesis and the Spd analog serves as a functional substitute for Spd. Thus, while the Spd analog fulfills the role(s) of Spd in cell proliferation, Spm becomes steadily depleted. In cells treated with DFMO plus the analog, aminopropylcadaverine, Spm pools decline steadily and growth inhibition occurs after 48 hr (when Spm pools decline to 60% of control). By 96 hr, Spm is ∼ 15% of control and growth is<30%. Prevention studies with exogenous polyamines confirm a causai relationship between Spm depletion and growth inhibition. The critical levels of polyamines for cell proliferation to take place were found to be 30% of control for Spd and 60% for Spm. The use of DFMO plus a Spd analog is proposed as a system for studying the cellular consequences of Spm depletion. Spd depletion can be achieved for comparison purposes by treating cells with DF

ISSN:0021-9541    

DOI:10.1002/jcp.1041210305

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

年代:1984

数据来源: WILEY

摘要:

AbstractA mutant cell line (designated Kin‐8), isolated from the Y1 mouse adrenocortical tumor cell line on the basis of its resistance to growth‐inhibition by 8‐bromoadenosine 3′, 5′‐monophosphate (8BrcAMP), was resistant to the steroidogenic effects of the cyclic nucleotide analog and did not round up in the presence of 8BrcAMP as did responsive Y1 adrenal cells. In Kin‐8 cells, the mutation to cyclic nucleotide resistance was associated with a defective type 1 cAMP‐dependent protein kinase activity, suggesting an obligatory role for the enzyme in the regulation of these adrenal functions. In this study, the Kin‐8 mutant was fused with a rat glioma cell line (C6) in order to analyze the genetic behavior of the protein kinase mutation in somatic cell hybrids. The growth of C6 glial cells was inhibited by 8BrcAMP and its cAMP‐dependent protein kinase responded normally to cAMP. In addition, C6 cells had no capacity for steroidogenesis nor did they round up when treated with 8BrcAMP. In Kin‐8 × C6 hybrids, the protein kinase mutation seemed to behave recessively. The growth of hybrid cells was inhibited by 8BrcAMP and the protein kinase responded to cAMP over a normal range. Kin‐8 × C6 hybrids, when treated with 8BrcAMP, exhibited steroidogenic activities which were greater than the activity measured in either fusion partner and which had lower ED50values for 8BrcAMP. In addition, Kin‐8 × C6 hybrids rounded up in the presence of 8BrcAMP, a morphologic change unlike that seen with either fusion partner. The effect of 8BrcAMP on the steroidogenic activity and morphology of Kin‐8 × C6 hybrids was reminiscent of the effects of the cyclic nucleotide on cAMP‐responsive, parental Y1 adrenal cells. These results suggest that cell fusion provided a normal cAMP‐dependent protein kinase for Kin‐8 cells and led to the recovery of a cAMP‐responsive adrenal phenotype. These results provide additional evidence in support of an obligatory role for cAMP‐dependent protein kinase in the regulation of adrenal steror

ISSN:0021-9541    

DOI:10.1002/jcp.1041210306

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

年代:1984

数据来源: WILEY

摘要:

AbstractHamster trachea epithelial (HTE) cells were shown to respond to 20% Cystic fibrosis serum (CFS) by secreting twice as much protein as in the presence of 20% normal human serum (NHS). Seum from obligate heterozygotes (HHS) produced an intermediate effect. A peak of Ca2+entry into the HTE cells occurred about 30 min after exposure to 20% CFS, followed by a slow decline to basal levels. In contrast, 20% NHS did not cause an influx of Ca2+and HHS produced an influx to about half that of CFS. Increasing concetrations (5‐30%) of pooled NHS had no effect on HTE cell Ca2+uptake or secretion, but pooled CFS and HHS caused progressive increases in Ca2+influx and protein secretion from 10 to 25% sera. The CFS‐induced Ca2+influx and secretion were about twice those of HHS throughout the range of serum concentrations tested, suggesting the presence of a modulatory influence in HHS. When EGTA was used to chelate extracellular Ca2+in the presence of CFS, Ca2+influx was prevented and there was no stimulation of secretion. lonophore A23187 allowed Ca2+entry into HTE cells in the presence or absence ofserum and a heightened level of secretory activity followed. The time course of Ca2+influx under the influence of CFS was shown to correspond to the efflux of Na+from the cells. Also verapamil, a Ca2+channel blocking agent, inhibited CFS‐induced Ca2+influx by 50% at 10−5M and prevented secretion. Thus, it appears that CFS, but not NHS, contains an agent which stimulates Ca2+uptake into HTE cells by means of a Ca2+channel and/or Na+‐Ca2+exchange mechanism, and that increased intracellular Ca2+levels then trigger secretion. The intermediate HTE cellr esponse to HHS suggests that half of the CFS stimulatory agent is present as would be expected in a gene dose effect, lending support for a agentic bas

ISSN:0021-9541    

DOI:10.1002/jcp.1041210307

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

年代:1984

数据来源: WILEY

摘要:

AbstractFluorescence energy transfer using flow cytometric measurements was utilized to determine the proximity of concanavalin A receptors on the surface of HL‐60 promyelocytic leukemia cells before and after induction of differentiation. The HL‐60 cells were induced to differentiate into granulocytes using dimethylsulfoxide and into macrophages using 12‐O‐tetradecanoylphorbol‐13‐acetate. Concanavalin A was labeled with either fluorescein (donor chromophore) or tetramethylrhodamine (acceptor chromophore), and these species were used to determine lectin proximity. With granulocytic differentiation, the amount of concanavalin A bound remained constant, but a decrease in receptor density was observed. During macrophage differentiation, however, both receptor density and receptor number increased. The increase in concanavalin A binding during differentiation appears to be a result of maturation rather than an initi

ISSN:0021-9541    

DOI:10.1002/jcp.1041210308

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

年代:1984

数据来源: WILEY

摘要:

AbstractUsing86RB+as a marker for K+permeability, we find that extracellular Ca‐EGTA influences the rate of86Rb+efflux from erythrocyte ghosts preloaded with86Rb+and “buffered” Ca2+. At an internal free Ca2+, where the rate of86Rb+efflux is minimal and uninfluenced by either external EGTA or external Ca2+, external Ca‐EGTA at 0.2–0.5 mM can raise the flux rate to as high as can be attained by raising internal Ca2+, in the presence of an excess externally either of Ca2+or of EGTA. Higher concentrations of Ca‐EGTA (up to 1–2 mM) diminish the flux rate. External Ca‐EDTA or Mg‐EDTA can substitute for Ca‐EGTA in enhancing and suppressing flux rate. The peak rate is insensitive to external free Ca2+but depends on internal Ca2+; internal Mg‐EDTA does not substitute for internal Ca‐EGTA. Thus, the erythrocyte membrane is asymmetric with respect to its interaction with Ca2+and Ca‐EGTA. Also,22Na+does not substitute for86Rb+. The peak rate of86Rb+flux produced by external Ca‐EGTA is diminished by chlorpromazine (0.1 mM) and augmented by 1‐propranolol (25 μM), in the same way as the rate produced by increasing internal Ca2+. The results suggest that external Ca‐EGTA enhances the affinity of internal Ca2+for its receptor(s) which operate the K+‐gate at the inner surface of the membrane. At external concentrations of Ca‐EGTA above 1–2 mM,86Rb+flux rate again rises with increase of Ca‐EGTA. This phenomenon does not depend upon internal Ca2+, is not affected by chlorpromazine or by 1‐propranolol, and is associated with an enhanced per

ISSN:0021-9541    

DOI:10.1002/jcp.1041210309

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

年代:1984

数据来源: WILEY

摘要:

AbstractPurified populations of quiescent human tumour cells were isolated from plateau phase cultures of PMC‐22 cells by centrifugal elutriation. Dilution into fresh medium resulted in these quiescent cells entering S phase exponentially with a t1/2 of 12 hr, after a 18‐20‐hr lag period during which cellular RNA content increased. Subsequent studies showed that recruitment of quiescent cells into the cell cycle could be regulated by extracellular pH. When exponentially growing PMC‐22 cells were exposed to acidic extracellular pH levels, three growth patterns were observed: (1) Normal growth between pH 7.2 to pH 6.8; (2) A reduction in growth rate associated with accumulation of cells with a G1DNA content between pH 6.7 and 6.4 (this was also shown to occur in a number of other tumour cell lines); (3) Non‐cell‐cycle‐phase‐specific arrest of growth at pH levels less than 6.3. Further studies with purified quiescent cell populations showed the possible existence of a pH‐dependent restriction point in the G1phase of these tumour cells. The implications of these observations to tumour biol

ISSN:0021-9541    

DOI:10.1002/jcp.1041210310

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

年代:1984

数据来源: WILEY

摘要:

AbstractSplenic erythroblasts obtained from mice during the acute disease caused by either the polycythemia‐inducing (FVP) or anemia‐inducing (FVA) strain of Friend virus were examined for their degree of terminal differentiation. Morphology, benzidine staining, and heme synthesis kinetics showed that many erythroblasts from FVP‐infected mice were undergoing terminal differentiation, while few erythroblasts from FVA‐infected mice showed evidence of terminal differentiation. When cultured in methylcellulose medium, splenic erythroblasts from FVP‐infected mice completed differentiation without the addition of erythropoietin (EP) to the medium. However, splenic erythroblasts from FVA‐infected mice underwent terminal differentiation in vitro only when EP was added to the medium. From spleens of FVA‐infected mice, a population of large, immature‐appearing erythroblasts was obtained by separation with velocity sedimentation at unit gravity. Serial studies of the separated erythroblasts which were cultured with EP showed that despite some heterogeneity in their proliferative capacity, they were relatively homogeneous in their degree of differentiation in that they had not begun to synthesize heme or globin. Morphological changes and syntheses of heme and globins were monitored during terminal differentiation induced in vitro by EP. The accumulation of immature erythroblasts in vivo, their responsiveness in vitro to EP, and availability of large numbers of cells (108or more) make the splenic erythroblasts of FVA‐infected mice an ideal population of cells with which to study EP‐mediated terminal differentiation. This erythroblast population should permit the biochemical and molecular studies in erythroid differentiation which heretofore had to be done with chemically induced erythroid differentiation in co

ISSN:0021-9541    

DOI:10.1002/jcp.1041210311

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

年代:1984

数据来源: WILEY