摘要:

AbstractSpecific binding of ferric bovine transferrin to the human transferrin receptor was investigated using K562 cells propagated in serum‐free medium without transferrin supplemented with 10−5elemental iron. Affinity chromatography of solubilized extracts of K562 cells surface‐labeled with125I was performed using bovine transferrin‐ and human transferrin‐Sepharose 4B resins. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of resin eluates reveal that bovine transferrin specifically binds a Mr= 188,000 protein which dissociates into a Mr= 94,000 protein under reducing conditions, a finding identical to what is seen with human transferrin. The Mr= 94,000 reduced protein isolated by bovine transferrin resin shows an identical one‐dimensional partial proteolytic digestion map with that of the human transferrin receptor.Unlabeled bovine transferrin was shown to specifically compete125I‐labeled human transferrin from the human transferrin receptor on the surface of K562 cells at 4°C in a similar manner as unlabeled human transferrin; however, approximately a 2,000‐fold higher concentration of bovine ligand was required to achieve comparable competition (50% inhibition of binding).Indirect immunofluorescence cytolocalization of bovine transferrin in K562 cells grown in serum‐free medium supplemented with ferric bovine transferrin reveal patterns similar to those seen for human transferrin (both focal perinuclear and diffuse cytoplasmic fluorescence). Monensin treatment results in a dramatic accumulation of bovine ligand in perinuclear aggregates, suggesting that it is recycled through the Golgi, as is human transferrin. K562 cells grown in serum‐free medium supplemented with either 300 μg/ml of ferric human or ferric bovine transferrin were found to demonstrate super

ISSN:0021-9541    

DOI:10.1002/jcp.1041280102

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

年代:1986

数据来源: WILEY

摘要:

AbstractEarly biochemical events in the response of murine peritoneal macrophages to bacterial lipopolysaccharide (LPS) have been examined (i.e., 0–4 hr after initiation of treatment). At concentrations of 10 ng/ml or less, LPS stimulated the new or enhanced synthesis of a series of at least six polypeptides of 85, 80, 75, 65, 57, and 38 kD. This effect was dependent upon the lipid A moiety of LPS as lipid A itself could induce the changes and the effect of LPS could be blocked by inclusion of polymixin B sulfate in the culture medium. The effect was specific for LPS in that other endotoxin‐free agents known to alter macrophage physiology could not produce the same changes. The time course of LPS stimulation of macrophage protein synthesis was remarkable in that the synthesis of all six proteins was transient even in the continued presence of LPS, being first detected approximately 1 hr after exposure and no longer apparent by 8–10 hr after treatment was initiated. Furthermore, both pulse‐chase and cumulative radiolabeling studies indicated that at least two of the proteins (85 and 38 kD) were short‐lived and did not accumulate in LPS‐treated cells, suggesting the possibility that they participate in a regulatory rather than a functional role. Macrophage tumoricidal activation involves cooperation in response to two independent signals; interferon gamma and LPS. Pretreatment of macrophages with interferon gamma increased the sensitivity of macrophages to LPS‐stimulated protein synthesis by one to two orders of magnitude documenting such cooperativity in molecular terms. The LPS‐induced stimulation of specific protein synthesis could be reproduced by treatment of macrophages with heat killedListeria monocytogenes, a gram‐positive, endotoxin‐negative bacterial stain which has been shown to substitute effectively for LPS in macrophage tumoricidal activation. Furthermore, reversible inhibition (i.e., treatment with cycloheximide) of protein synthesis during LPS treatment abrogated the acquisition of tumoricidal function. These results identify an early biochemical response to LPS which may be a necessary component of the intracellular transduction of signals which regulate macrophage fun

ISSN:0021-9541    

DOI:10.1002/jcp.1041280103

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

年代:1986

数据来源: WILEY

摘要:

AbstractInternalization of EGF and transferrin measured as the rate of uptake of125l‐labeled ligands was compared in the cell line CCL39 and a mutant derivative, PS120, lacking the Na+/H+antiport system. No significant alteration was detected between the two cell lines. In contrast, pretreatment of the mutant cells PS‐120 with 20 mM NH4Cl for 30 min to decrease persistently intracellular pH resulted in an increase in125I‐EGF and125I‐transferrin uptake by 60% and 25%, respectively. However, similar NH4Cl pretreatment of the parental cell line, CCL‐39, which only affected intracellular pH very transiently did not cause an increase of ligand uptake. The binding of125I‐EGF to CCL‐39 and PS‐120 cells with or without NH4Cl pretreatment showed that NH4Cl pretreatment did not affect EGF binding in either CCL‐39 or PS‐120 cells. Since cells regulate intracellular pH by ion transport systems, we also examined the role of Na+, K+‐ATPase. Ouabain, an inhibitor of Na+, K+‐ATPases, showed no effect on125I‐EGF uptake in either of the cell types with or without NH4Cl pretreatment. Taken together, these results suggest that the plasma membrane‐bound Na+/H+antiport, a major pHi‐regulating system in vertebrates, indirectly plays a role in ligand internalization through r

ISSN:0021-9541    

DOI:10.1002/jcp.1041280104

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

年代:1986

数据来源: WILEY

摘要:

AbstractElectrophysiological measurements were carried out on osteoclasts in vitro. Such isolated osteoclasts are able to resorb bone in vitro and contract in response to calcitonin (CT). Our measurements show that individual osteoclasts respond to CT with a significant transient hyperpolarization of membrane potential. Application of parathyroid hormone (PTH) and dibutyryl cAMP produced a transient hyperpolarization in some osteoclasts. Measurements on an osteoblastlike line (ROS 17/2.8) showed a sustained hyperpolarizing response to CT, which is similar to but smaller than the hyperpolarizing response to PTH and dibutyryl cAMP in this and some other osteoblastlike lines. In contrast to osteoblastlike cells, the osteoclasts have no long term membrane potential response to CT, to PTH, or to dibutyryl cAMP. These results show that there are distinct differences between osteoclasts and osteoblasts in their ion transport responses to hormones.

ISSN:0021-9541    

DOI:10.1002/jcp.1041280105

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

年代:1986

数据来源: WILEY

摘要:

AbstractWhen radioactive polyamines (putrescine or spermidine) were incubated with mammalian cells in tissue culture, the radioactivity was incorporated into cellular proteins via two different metabolic pathways; one is metabolic labeling of an 18,000‐dalton protein via hypusine formation, and the other is general protein synthesis employing radioactive amino acids derived from biodegradation of polyamines via GABA shunt and Krebs cycle. Aminoguanidine, a potent inhibitor of diamine oxidase, blocked the metabolic conversion of polyamines to amino acids but had no effect on the metabolic labeling of the 18,000‐dalton protein. We have investigated these two polyamine‐associated biochemical events in IMR‐90 human diploid fibroblasts as a function of their population doubling level (PDL). We found that (1) the metabolic labeling of the 18,000‐dalton protein was about two‐fold greater in young cells (PDL = 22) than that in old cells (PDL = 48), and (2) the metabolic labeling of other cellular proteins, employing amino acids derived from putrescine via polyamine catabolic pathway, was more than six‐fold greater in the old cells (PDL = 48) than in the young cells (PDL = 22). Since the rate of protein synthesis was about 1.4‐fold higher in the young cells as compared to the old cells, our data indicated that the activity of catabolic conversion of putrescine (or spermidine) to amino acids in old IMR‐90 cells was about eight‐fold greater than that in young cells. This remarkable increase of polyamine catabolism and the slight decrease of metabolic labeling of the 18,000‐dalton protein were also observed in cell strains derived from patients with pr

ISSN:0021-9541    

DOI:10.1002/jcp.1041280106

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

年代:1986

数据来源: WILEY

摘要:

AbstractThe apparent volume of neutrophils, as measured electronically with the Coulter counter, has been reported to increase upon treatment with chemotactic factors. The occurrence of a volume change was confirmed by forward angle light scattering and by isotopic measurements of intracellular water space in cells treated with 12‐O‐tetradecanoylphorbol 13, acetate (TPA) or formyl‐methionyl‐leucyl‐phenylalanine (FMLP). Cell swelling was associated with an increase in the osmotic content of the cells, determined from Boyle‐van't Hoff plots, and with an increase in Na+content, measured by flame photometry. The volume change was inhibited by replacement of extracellular Na+with K+or N‐methyl‐D‐glucamine+, or by addition of amiloride. Swelling was also inhibited by the 5‐N‐substituted analogs of amiloride, which are potent specific inhibitors of the Na+/H+antiport. This pathway is activated in neutrophils by both TPA and FMLP. Activation of Na+/H+exchange, determined as a Na+‐dependent and amiloride‐sensitive cytoplasmic alkalinization, was also found when neutrophils were treated with hypertonic solutions. The hypertonic activation of the antiport was similarly followed by cell swelling, detectable by electronic sizing. The results indicate that activation of Na+/H+exchange can lead to significant cel

ISSN:0021-9541    

DOI:10.1002/jcp.1041280107

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

年代:1986

数据来源: WILEY

摘要:

AbstractThe study of induction of Friend erythroleukemic cell lines during the last decade has enriched our understanding of late erythroid differentiation. In comparison, little information is available on early erythroid differentiation. We describe here the isolation and characterization of a highly inducible clone from a murine erythroid cell line, which is capable of forming colonies that possess properties of the early erythroid burst progenitor. We found that a combination of erythropoietin (Epo), spleen conditioned medium (SCM), and plasma from a patient with aplastic anemia (Apa) induces over 95% of cells from this clone (clone 12) to form colonies with the properties of burst or mixed burst blast‐like colonies. Examination of the culture conditions of these cells indicated that alpha medium was more efficient for colony induction than Iscove's medium, and that the addition of two‐mercaptoethanol did not improve the induction process. These factors (EPo, SCM, and Apa) must be present for 4 days in order for induction to take place. It is hoped that the isolation of this highly inducible cell clone will enrich our understanding of early erythroid differentiat

ISSN:0021-9541    

DOI:10.1002/jcp.1041280108

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

年代:1986

数据来源: WILEY

摘要:

AbstractSparse cultures of fetal and postnatal human fibroblasts were equivalent in their responsiveness to the mitogenic action of somatomedin C/insulin‐like growth factor I (SM‐C/IGF‐I). At both developmental stages, the addition of SM‐C/IGF‐I (100 ng/ml) increased cell number at day 3 1.4‐fold in serum‐free medium and 2‐fold in the presence of 0.25% human hypopituitary serum. Furthermore, dose‐response curves indicated that there was no difference in the sensitivity of fetal and postnatal fibroblasts to the growth‐promoting effects of SM‐C/IGF‐I, with a half‐maximal response occuring at 6 ng/ml SM‐C/IGF I. This biological action of SM‐C/IGF‐I correlated with SM‐C/IGF‐I binding to fetal and postnatal fibroblast monolayers. Epidermal growth factor (EGF) and platelet‐derived growth factor (PDGF) also stimulated replication of fetal and postnatal fibroblasts. The mitogenic effects of SM‐C/IGF‐I, EGF, and PDGF were additive. Dexamethasone, which alone had no effect, was synergistic with SM‐C/IGF‐I in stimulating replication of postnatal fibroblasts. The combination of SM‐C/IGF‐I (100 ng/ml), dexamethasone (10−7M), EGF (10 ng/ml), and PDGF (5 ng/ml) had the same mitogenic effectiveness as 10% calf serum (CS) in postnatal cells. In marked contrast, there was no mitogenic interaction between SM‐C/IGF‐I and dexamethasone in fetal fibroblasts. In fetal cells, SM‐C/IGF‐I + EGF + PDGF ± dexamethasone could only account for 50% of the activity of 10% CS. Moreover, fetal cells were 50–100% more respon

ISSN:0021-9541    

DOI:10.1002/jcp.1041280109

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

年代:1986

数据来源: WILEY

摘要:

AbstractThe effect of changes in extracellular pH (pHo) and intracellular pH (pHi) on Na+‐dependent and Na+‐independent inorganic phosphate (Pi) transport in Ehrlich cells was investigated. In the presence of Na+, acutely reducing pHofrom 7.30 to 5.50 results first in a transient (∼7 min) stimulation of Pitransport. The enhanced rate of transport is a saturable function of the extracellular [H+]; the Ks equals 2.3 × 10−6M (pHo6.68). However, Pitransport is progressively inhibited as pHifalls below 6.50. The effect of pHion Pitransport measured at various intracellular [Na+] suggests that inhibition develops as a consequence of H+interaction with an intracellular Na+site(s) on the Na+‐dependent carrier. At pHo7.4, about 15% of the steady state Piflux persists in the absence of Na+. However, when pHois reduced, transport is stimulated to the same extent and with the same time course and kinetic characteristics as in the presence of Na+. Thus, H+stimulated Pitransport does not require Na+, raising the possibility that the Na+‐independent component is mediated by the anion (CI

ISSN:0021-9541    

DOI:10.1002/jcp.1041280110

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

年代:1986

数据来源: WILEY

摘要:

AbstractMouse mammary epithelial cultivated on collagen gels demonstrate active spreading as the cells form monolayers. In this novel system, initiation of cell spreading is preceded by de novo synthesis of type IV collagen. The newly synthesized collagen is partitioned such that after 48 hr, approximately 24% is found in the culture medium, 35% is intracellular, and 41% is deposited in the extracellular matrix of the developing epithelium. Cultures deprived of serum failed to spread and to synthesize collagen. Proline analogues were shown to inhibit cell spreading and to suppress collagen synthesis in a dosedependent manner. Cytochalasin D inhibition of F‐actin elongation was shown to prevent cell spreading but not to suppress total collagen synthesis. During cytochalasin D treatment, inhibition of cell spreading was shown to result from failure to deposit or to maintain deposited collagen in the epithelium extracellular matrix. The data indicate that synthesis and extracellular deposition of a major basal lamina component (viz. type IV collagen) must precede and then accompany epithelial cell spreading in collagen gel culture. It is suggested that the microfilament apparatus, through some hypothetical integral membrane protein, can anchor extracellular type IV collagen, which then provides a necessary condition for cell spreadin

ISSN:0021-9541    

DOI:10.1002/jcp.1041280111

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

年代:1986

数据来源: WILEY