摘要:

AbstractFour indirect probes of membrane potential, triphenylmethylphosphonium ion (TPMP+), 3,3′dipentyloxacarbocyanine [di‐O‐C5(3)], 3,3′ dipentylindocarbocyanine [di‐I‐C5(3)], and 3,3′ dipropylthiodicarbocyanine [di‐S‐C3(5)] have been used to study neutrophil (PMN) activation. The data extend previous studies indicating that the cyanine dye di‐S‐C3(5) not only exhibits a different fluorescence response mechanism from di‐O‐C5(3) [and di‐I‐C5(3)] but also that the fluorescence of di‐S‐C3(5) is destroyed by reactive oxygen products produced by neutrophils following stimulation. When these aspects of the probes are taken into account, the interpretations of the results using all three cyanine dyes are identical. Studies with the isotope TPMP+indicate that long incubations are necessary for PMN to fully equilibrate during which time the PMNs depolarize. Use of TPB−, to shorten the TPMP+equilibration time, produces results identical with those obtained using the cyanine dyes. The cyanine dyes and TPMP+/TPB−are toxic to neutrophil functions although they do not cause cell death. Toxicity can be avoided by using low concentrations of di‐O‐C5(3) and di‐I‐C5(3) but cannot be avoided with di‐S‐C3(5) or TPMP+/TPB−. Using di‐O‐C5(3) with the fluorescence‐activated cell sorter, we demonstrate that heterogeneity of neutrophil responsiveness confuses the interpretation of studies characterizing the ionic basis of the fluorescence responses stimulated by certain stimuli. We conclude that some of the discrepancies currently reported in the literature using these probes are not due to inherent differences in the ability of the different probes to monitor the same event (i.e., PMN membrane potential) but instead are due to failure to corr

ISSN:0021-9541    

DOI:10.1002/jcp.1041150202

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

年代:1983

数据来源: WILEY

摘要:

AbstractSynthesis of a family of proteins called “heat shock” proteins is enhanced in cells in response to a wide variety of environmental stresses. This suggests that these proteins may have functions essential to cell survival under stressful conditions. A causative relationship between heat shock protein synthesis and development of thermotolerance would imply that agents known to induce heat shock protein synthesis, such as sodium arsenite, also induce thermotolerance. Conversely, agents known to induce thermotolerance, such as ethanol, would also enhance heat shock protein synthesis. To test this hypothesis, I have examined the effect of sodium arsenite or ethanol treatment on protein synthesis and cell survival in Chinese hamster ovary HA‐1 cells. After either sodium arsenite or ethanol treatment, the synthesis of heat shock proteins was greatly enhanced over that of untreated cells. In parallel, cell survival was increased as much as 104‐fold when cells exposed to either agent were challenged by a subsequent heat treatment. The synthesis of heat shock proteins correlated well with the development of thermotolerance. A qualitative analysis of individual proteins suggests that the synthesis of 70,000 and 87,000 molecular weight proteins most closely mirrored the development of thermotolerance. The results, therefore, strongly reinforce the hypothesis that a causal relationship exists between the enhanced synthesis of heat shock protein and cell survival under specific s

ISSN:0021-9541    

DOI:10.1002/jcp.1041150203

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

年代:1983

数据来源: WILEY

摘要:

AbstractThe Chinese hamster lung fibroblast line, CCI39, displays the properties characteristic of normal secondary cultures of Chinese hamster fibroblasts including: reversible GO growth arrest (<2% labeled nuclei), anchorage dependence, and high serum‐growth factor dependence. Injection of CCI39 cells, or anchorage‐independent variants, innudemice leads to tumor formation; however, as we have previously shown (Pérez‐Rodriguez et al., 1981b), the resulting tumor clones no longer possess the high serum dependence of injected CCI39 cells. Hormonal growth restraints imposed by the host create an in vivo selection for diminished, or “relaxed,” growth factor requirement. To characterize this growth factor “relaxation” further, we have analyzed the mitogenic response of parental CCI39 cells, anchorage‐independent clones, and selected tumoral derivatives, to purified growth factors. Two highly purified growth factors, thrombin and insulin, together fulfill the growth factor requirements of CCI39 cells; thrombin (1 U/ml) stimulates the reinitiation of DNA synthesis in GO‐arresed CCI39 cells, and insulin (10 μ/ml) maximally potentiates this stimulation to the level obtained with 10% fetal calf serum. First, we found no correlation between loss of anchorage dependence and growth factor relaxation. Second, we found that A71 (anchorage independent), a tumoral variant of CCI39 capable of growth arrest, and tumor‐derived cells all display an increased sensitivity to thrombin and a diminished requirement for the potentiating action of insulin. Examination of thrombin binding to CCI39, A51 (nontumoral, anchorage independent), and A71 cells revealed that the increased sensitivity to thrombin of A71 cells is not attributable to an alteration in thrombin cell surface receptor number or affinity for thrombin. Rather, under standard conditions of serum or growth factor removal (30 hr), A71 cells maintain a metabolically elevated growth‐arrested state, different from that of their nontumoral counterparts. Consequently, much lower concentrations of growth factors are needed to induce a proliferative response

ISSN:0021-9541    

DOI:10.1002/jcp.1041150204

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

年代:1983

数据来源: WILEY

摘要:

AbstractRecent investigations have indicated that cellular rheogenic properties may interfere with the correct estimation of Na+and amino transport stoichiometry. We have reevaluated the stoichiometry of Na+and α‐aminoisobutyric acid (α‐AIB) cotransport in Ehrlich ascites tumor cells depleted of Na+and ATP by incubation in Na+‐free HEPES‐buffered medium (pH 7.2) containing 160 mM K+and 2.5 μM valinomycin. Transfer of the cells to a medium with 10 mM22Na+, 10 mM3H‐AIB, and 150 mM K+resulted in an enhancement of Na+flux above basal levels, which represents 0.6 of the AIB uptake. Under these conditions the membrane potential, −7.0 ± 0.1 mV (SEM), does not change with the addition of AIB, −7.3 ± 0.6 mV (SEM). HgCl2(10 m̈M) added to the medium inhibited AIB flux and AIB‐stimulated Na+flux by 45–50% but did not change the coupling ratio. HgCl2(10 m̈M) does not inhibit the basal Na+flux nor does it affect cellular Na+or K+content. In physiological medium cotransport is electrogenic. The membrane potential of Ehrlich cells in physiological medium is −22.3 ± 0.8 mV (SEM) and depolarizes to −16.7 ± 0.7 mV (SEM) upon addition of AIB. Under these conditions the coupling ratio was highly variable but the ratio of codepression is 0.90 ± 0.02 (SEM) in the presence of HgCl2(10 m̈M). These results are consistent with a model (Smith and Robinson, 1981) in which the stoichiometry is one cosubstrate molecule per molecule of α‐AIB. We suggest that H+provides the alternative cosubstrate in this low Na+environment and that in high Na+medium the N

ISSN:0021-9541    

DOI:10.1002/jcp.1041150205

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

年代:1983

数据来源: WILEY

摘要:

AbstractSomatomedin is secreted by multiple types of cultured cells including human fibroblasts. Since the control of somatomedin (Sm) production by fibroblasts may be an important regulatory step in cell division, we undertook studies to define the variables in tissue culture experimental design that may have significant effects on the secretion of Sm. Cell density was an important parameter in determining basal Sm production rates. Cultures plated at 2.5 × 104cells/well produced 0.38 ± 0.06 U/ml/105cells whereas an increase in culture density to 6.2 × 104cells/well was associated with a decrease in Sm production per cell to 0.23 ± 0.04 U/ml/105cells (P<.01). Cultures stimulated by platelet‐derived growth factor (PDGF) showed a similar reduction in Sm production with increasing cell density. Epidermal growth factor was stimulatory (5.4‐fold increase) in low‐density cultures but had no effect when high‐density cultures were used. If the experiments were initiated between 2 and 4 days after the last media change there were no significant differences in basal or PDGF‐stimulated Sm concentrations. Between days 5 and 9 however, there was a progressive increase in the basal Sm production rate. The duration of incubation was an important variable since Sm production increased during the first 12 h in noncycling cells and showed an accelerated increase between 4 and 8 h in cycling cells. Cells between the eighth and 12th passage had similar basal Sm production (0.22 ± 0.04 U/ml/105cells) rates; cells between the 19th and 20th passages had significantly higher basal Sm production rates (0.41 ± 0.05 U/ml/105cells) (P<.01). These results suggest that several variables, particularly cell density and passage number, are critical variables when quantitating the effect of hormones and growth factors on Sm production by cultu

ISSN:0021-9541    

DOI:10.1002/jcp.1041150206

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

年代:1983

数据来源: WILEY

摘要:

AbstractUsing cell permeabilization, a technique which allows addition of exogenously supplied radiolabeled sugar nucleotides to serve as direct glycosyl donors, oligosaccharide biosynthesis was examined in fibroblasts obtained from normal and cystic fibrosis (CF) subjects. Incubation of logarithmically growing cells with either radiolabeled leucine or xylose has indicated that there was a difference in the synthetic rate between the cell types. Protein synthesis in normal cells made permeable with 50 m̈g/ml lysolecithin (LL) was demonstrated to be absent, and could not be induced to take place by adding exogenous components, including energy sources and amino acids, normally required for protein synthesis. Thus radiolabeled sugars were being added to peptide acceptors which were already present at the time of LL addition. Both permeable and intact fibroblasts were exposed to labeled UDP‐xylose, UDP‐galactose, and UDP‐glucuronic acid, all donors of mucopolysaccharide precursors. The uptake of xylose into protein was the same for both normal and CF cells, but permeable CF fibroblasts incorporated statistically greater amounts of sugar from UDP‐galactose and UDP‐glucuronic acid. Intact CF cells were also labeled using these two sugar nucleotides. Trypan blue exclusion indicated CF and normal fibroblasts were equally intact. This and the fact that preincubation of CF cells with the appropriate cold sugar nucleotide eliminated the differences in incorporation between the normal and CF cells suggested that CF fibroblasts had more cell surface acceptor than the no

ISSN:0021-9541    

DOI:10.1002/jcp.1041150207

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

年代:1983

数据来源: WILEY

摘要:

AbstractThe polypeptide hormone insulin and the binding unit of cholera toxin (CTB) were coupled via a disulfide bond. This hybrid molecule had 1/30 the ability of native insulin to bind to the insulin receptor and 1/30 the biological activity of native insulin in H35 rat hepatoma cells and rat adipocytes. Thus, in these two cell types that are very sensitive to insulin, the biological activity of the hybrid molecule was as predicted on the basis of the ability of the molecule to interact with the insulin receptor. In contrast, in HTC rat hepatoma cells and rat thymocytes, two poorly responsive cell types, the insulin‐CTB conjugate had 1/3 the biological activity of native insulin, a value 10 times greater than its insulin receptor binding potency. This increased activity of the conjugate did not appear to be due to cholera toxin in the preparation, since a control of uncoupled CTB had no biological activity. Furthermore, native cholera toxin increased intracellular levels of cAMP by 20‐fold, whereas the conjugate had no effect on cAMP levels. The CTB moiety did, however, contribute to the biological activity of the conjugate, since the activity of the hybrid molecule, like cholera toxin, was inhibited by gangliosides, whereas the activity of native insulin was not. Finally, the binding to thymocytes of insulin‐CTB conjugate, but not insulin, was inhibited by gangliosides. Thus, a hybrid hormone molecule has been constructed which has insulin‐like biological activity with the receptor specificity of cholera toxin in poorly responsiv

ISSN:0021-9541    

DOI:10.1002/jcp.1041150208

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

年代:1983

数据来源: WILEY

摘要:

AbstractLowering of extracellular Ca2+levels will reversibly arrest the growth of human fibroblasts (WI38). Simian virus40(SV40)‐transformed WI38 cells do not exhibit this Ca2+‐dependent arrest. One possibility for this difference in Ca2+requirement is that extracellular or surface membrane‐bound Ca2+may be required for growth factor receptor‐mediated endocytosis and this Ca2+requirement may differ in normal versus transformed cells. In this study we have evaluated the role of Ca2+in the binding, internalization, and degradation of epidermal growth factor (EGF) in the WI38 and SV40WI38 cell. The binding of [125I]EGF to the cell surface is not significantly altered by lowering of Ca2+to 10−5‐M levels in either the normal or transformed cell. At this Ca2+level, growth of the normal cell is inhibited. The subsequent internalization of EGF is reduced nearly threefold in the normal cell but not in the transformed cell following Ca2+deprivation. Degradation of the EGF‐receptor complex is also sensitive to Ca2+. A twofold reduction in the rate of release of acid‐soluble125I occurs in the normal but not the transformed cell under conditions of lowered medium Ca2+. In contrast, 2‐chloro‐10‐3‐aminopropyl phenothiazine (CP), an inhibitor of the Ca2+‐dependent regulator protein calmodulin, causes an inhibition of [125I]EGF internalization and degradation in both the normal and transformed WI38 cell, and a marked inhibition of [125I]EGF binding to the cell surface receptor of the transformed cel

ISSN:0021-9541    

DOI:10.1002/jcp.1041150209

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

年代:1983

数据来源: WILEY

摘要:

AbstractHuman diploid fibroblasts (IMR‐90) regulate their overall rates of proteolysis in response to the composition of the culture medium and the ambient temperature. The magnitude and, in some cases, the direction of the response depend on the half‐lives of the cellular proteins that are radioactively labeled and the time chosen for measurements of protein degradation. Fetal calf serum, insulin, fibroblast growth factor, epidermal growth factor, and amino acids selectively regulate catabolism of long‐lived proteins without affecting degradation of short‐lived proteins. Fetal calf serum reduces degradative rates of long‐lived proteins and is maximally effective at a concentration of 20%, but the effect of serum on proteolysis is evident only for the first 24 hr. Insulin inhibits degradation of long‐lived proteins in the presence or absence of glucose and amino acids in the medium, but is maximally effective only at high concentrations (10−5M). Amino acid deprivation increases degradative rates of long‐lived proteins for the first 6 hr, but then decreases their catabolism for the subsequent 20 hr. Lowered temperature is the only condition tested that significantly alters degradative rates of short‐lived proteins. Although cells incubated at 27°C have reduced rates of degradation for both short‐lived and long‐lived proteins compared to cells at 37°C, lowered temperature reduces catabolism of long‐lived pro

ISSN:0021-9541    

DOI:10.1002/jcp.1041150210

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

年代:1983

数据来源: WILEY

摘要:

AbstractThe ratio of mouse to rat albumin secreted by mouse hepatoma × rat hepatoma hybrid cells is constant (of the order of 5.0) irrespective of the total amounts produced. The present results establish for seven independent hybrid clones that the coordination in the ratio of mouse to rat product applies also at the level of accumulation of albumin mRNAs of the two species. The interpretation that coordinate synthesis reflects coordinate transcription of the relevant genes is thus reinforced

ISSN:0021-9541    

DOI:10.1002/jcp.1041150211

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

年代:1983

数据来源: WILEY