摘要:

AbstractTo facilitate the direct study of the molecular events that control the development of human burst‐forming units‐erythroid (BFU‐E), we have developed a method to purify BFU‐E from peripheral blood. Using density centrifugation, rosetting with a mixture of neuraminidase‐treated and IgG‐coated sheep erythrocytes, positive panning with anti‐My10 monoclonal antibody, overnight adherence to plastic dishes, negative panning with monoclonal antibodies, and density centrifugation, human blood BFU‐E were purified from 0.04% to 56.6%, a 1,400‐fold purification with a 13% yield. More than 90% of purified BFU‐E were recombinant interleukin‐3 (rlL‐3) dependent, which survived for 48 h with rlL‐3 in the absence of recombinant erythropoietin (rEP), and 80% gave rise to erythroid bursts of more than 500 hemoglobinized cells. rEP dependency was not evident until after 72 h of incubation in vitro. The purified cells (day 1) were incubated with rlL‐3 and rEP in liquid culture for 24 (day 2), 48 (day 3), and 72 (day 4) h and then were transferred into semisolid cultures and incubated until day 15. The size of the erythroid colonies observed in semisolid cultures decreased continuously in association with the incubation time of day 1 purified cells in liquid cultures. The first appearance of colony‐forming units‐erythoid (CFU‐E) that gave rise to colonies of 8 to 49 cells was observed after 72 h of incubation of day 1 cells in the liquid culture.125I‐rEP was incubated for 5 h at 37°C with purified cells (day 1) or with the cells that had been incubated in liquid culture for an additional 24‐72 h, and the presence of erythropoietin (EP) receptors was investigated using auto‐radiography. Specific binding of125I‐rEP was detected in 19 ± 7% of the initial day 1 BFU‐E. The percentage of125I‐rEP‐binding to erythroid progenitor cells and the amount of binding continuously increased as day 1 BFU‐E matured.125I‐rEP specific binding was observed with all of the erythroid progenitor cells that had been incubated in liquid culture for 72 h. These data demonstrate that primitive BFU‐E have a much lower number of EP receptors than CFU‐E and develop an increased concentration of EP receptors in association

ISSN:0021-9541    

DOI:10.1002/jcp.1041420202

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

年代:1990

数据来源: WILEY

摘要:

AbstractEpidermal growth factor (EGF) may either stimulate or inhibit cell growth. To elucidate the mechanism of these varied effects, we compared EGF action in parental A431 cells in which cell growth is inhibited, and clone 15, a mutant of these cells resistant to EGF growth inhibition. In both lines, EGF receptor was present in similar concentrations and underwent tyrosine phosphorylation to the same extent. Likewise, in both lines, acute exposure to EGF stimulated an increase in free cytoplasmic [Ca2+], as well as a similar increase in phosphoryla‐tion of lipocortin 1, a major substrate for the EGF receptor kinase whose phos‐phorylation is calcium‐dependent. On the other hand, pretreatment of clone 15 cells with EGF for 72 h abolished EGF‐induced phosphorylation of lipocortin 1 and led to a loss of the increase in cytoplasmic free [Ca2+], whereas no such desensitization was seen in the parental A431 cells. These data indicate a link between EGF‐induced increase in cytoplasmic calcium, lipocortin phosphoryla‐tion, and cell growth and suggest that differences in mechanisms of desensitization to these immediate actions of EGF may lead to altered growth response to t

ISSN:0021-9541    

DOI:10.1002/jcp.1041420203

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

年代:1990

数据来源: WILEY

摘要:

AbstractWe have examined α‐smooth muscle actin (α‐SM actin) protein and mRNA levels in proliferating and density‐arrested rabbit vascular smooth muscle cells (SMC) and also studied overall polypeptide synthesis in these cells by two‐dimensional (2‐D) gel electrophoresis. Of the approximately 1,000 cellular polypeptides resolved by 2‐D gel analysis, we consistently detected increased expression of 12 polypeptides in growth‐arrested SMC. These polypeptides, with apparent molecular weights of 24,000 to 55,000 exhibited relative increases of between fourfold to greater than tenfold. Three of these polypeptides were expressed at undetectable levels in proliferating SMC. We also detected 12 secreted polypeptides that were expressed at higher levels in growth‐arrested SMC. More changes were associated with the secreted polypeptides, since they represented approximately 4% of the total resolved secreted polypeptides, while only 1% of the cellular polypeptides were increased in high‐density growth‐arrested cells. Under these conditions we observed no change in relative α‐SM actin protein content as determined by 2‐D gel analysis and Western blots. This was corroborated by high levels of α‐SM actin mRNA levels in both proliferating and high‐density growth‐arrested SMC. These results indicate rabbit vascular SMC maintain a high level of expression of a smooth muscle differentiation marker (α‐SM actin) in a proliferation‐ and density‐independent manner. We also examined polypeptide synthesis in SMC isolated by enzymatic digestion of the aorta vs. cells isolated by the explant method. We found that although overall protein patterns were remarkably similar, several differences were observed. These differences were not due to increased contamination by fibroblasts, since both enzymatically‐ and explant‐derived SMC contained high levels of α‐SM actin as determined

ISSN:0021-9541    

DOI:10.1002/jcp.1041420204

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

年代:1990

数据来源: WILEY

摘要:

AbstractIn the present study, a stereologic approach was utilized to quantitatively assess morphological changes during the differentiation of LLC‐PK1cells into an epithelial membrane. This renal epithelial cell line has been described to undergo morphological changes during differentiation and maturation from subconfluent culture to a confluent epithelial layer. An increase in the number of apical microvilli, interpreted as an areal increase in this membrane domain, was reported. This morphological differentiation was found to be accompanied by an increase in the expression of apical Na+‐dependent hexose transport and the activities of certain brush border enzymes. Since no data are available that quantify the morphologic changes during LLC‐PK1differentiation, a quantitative morphologic—stereologic—investigation was performed for an early (6 days) and a late (12 days) state of confluence of LLC‐PK1monolayer cultures. The following morphological parameters were determined by light and electron microscopic morphometry: volume fractions (Vv) of nuclei, mitochondria, and lysosomes, and surface densities(Sv) of the apical and basolateral cell membrane domains. For the apical membrane surface, the microvillous fraction has been measured separately. Since the stereologic approach used in the present study allows the determination of absolute cell volumes, the absolute measures of organelle volumes (V)and membrane surfaces (S) per average cell can be calculated from volume and surface densities. Although no changes in cell density were found for 6 and 12 day old LLC‐PK1monolayers, indicating ceased cell proliferation due to contact inhibition, remarkable changes were found concerning the absolute cell volume and apical membrane surface. The observed increase in the apical cell surface was exclusively due to the enlarged microvillous surface fraction. This finding is in good agreement with the increased number of Na+‐dependent hexose transporters as well as with the increased expression of apical membrane marker enzymes observed during the differentiation of LL

ISSN:0021-9541    

DOI:10.1002/jcp.1041420205

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

年代:1990

数据来源: WILEY

摘要:

AbstractHydrogen‐peroxide‐resistant Chinese hamster fibroblasts, derived from the HA‐1 cell line, were isolated following continuous culturing in the presence of progressively increasing concentrations of hydrogen peroxide. The hydrogen‐peroxide‐resistant phenotype has been stable for over 360 days following removal from H2O2stress. These H2O2‐resisant cell lines demonstrate increased resistance to hyperthermic cell killing, mediated by continuous heating at 43°C but not 45°C. The relationship between mammalian cellular adaptation to oxidative stress mediated by H2O2and resistance to 43°C hyperthermi

ISSN:0021-9541    

DOI:10.1002/jcp.1041420206

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

年代:1990

数据来源: WILEY

摘要:

AbstractThe present studies demonstrate that dimethylsulfoxide (DMSO) treatment of human U‐937 myelomonocytic: leukemia cells is associated with induction of monocytic differentiation. The DMSO‐induced U‐937 monocytic phenolype was associated with (1) growth inhibition, (2) loss of clonogenic survival, (3) increases in á‐naphthyl acetate esterase (NSE) staining, and (4) increases in cell surface expression of the monocyte marker Mac‐1. DMSO treatment of U‐937 cells was also associated with down‐regulation of c‐myc and c‐myb gene expression as well as with increases in tumor necrosis factor (TNF) mRNA levels. The results further demonstrate that induction of U‐937 monocytic differentiation by DMSO is accompanied by increases in phospholipase A2activity. Moreover, this stimulation of phospholipase A2was sensitive to dexamethasone. We therefore studied the effects of dexamethasone on DMSO‐induced differentiation of U‐937 cells. Although dexamethasone had no effect on growth inhibition or loss of clonogenic survival by DMSO, this glucocorticoid blocked increases in NSE staining and cell surface Mac‐1 expression. Dexamethasone also had no effect on the down‐regulation of c‐myc and c‐m/b expression but blocked the reappearance of c‐myb transcripts after 6 hr of DMSO treatment. Finally, dexamethasone inhibited DMSO‐induced increases in TNF gene expression. Taken together, the results demonstrate that dexamethasone inhibits multiple characteristics, including the stimulation of phospholipase A2activity, associated with DMSO‐induced mo

ISSN:0021-9541    

DOI:10.1002/jcp.1041420207

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

年代:1990

数据来源: WILEY

摘要:

AbstractThe enzymes of tetrahydrobiopterin synthesis have been studied in murine bone marow, in spleen, in erythrocytes, and in reticulocytes. Mice with chemically induced and with genetically conditioned reticulocytosis as found in the lactate dehydrogenase deficient strain (Ldh‐1c/Ldh‐1c) were used for analysis of reticulocytic enzyme activities. The activity of the biopterin synthesizing system is highest in bone marrow even though it amounts to only about 10% as compared with liver. The first enzyme of the biosynthetic pathway, GTP‐cyclohydrolase, virtually disappears during the final maturation step of reticulocytes. In contrast, the activities of 6‐pyruvoyltetrahydropterin synthase and of sepiapterin reductase of erythrocytes are only reduced to about one half of the reticulocyte level. The absence of biopterin in erythrocytes is therefore caused by the loss of the enzyme that initiates the pterin biosynthetic

ISSN:0021-9541    

DOI:10.1002/jcp.1041420208

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

年代:1990

数据来源: WILEY

摘要:

AbstractThe maintenance of the normal epithelioid morphology of human dermal microvascular endothelial cells (MEC) grown in vitro depends strongly on the presence of factors that increase intracellular levels of cyclic AMP. Complete removal of dibutyryl cAMP and isobutylmethyixanthine (IMX) from the growth medium results in a progressive transition from an epithelioid to a spindle‐shaped cell line. This transition cannot be reversed by the readdition of dibutyryl cAMP and IMX to the growth medium or by addition of agonists that increase cAMP levels. Spindle‐shaped MEC lose the ability to express Factor VIII rAG and DR antigens and to bind peripheral blood mononuclear leukocyte (PBML).Ultrastructural analyses of transitional cells and spindle‐shaped cells show decreased numbers of Weibel‐Palade bodies in transitional cells and their complete absence in spindle‐shaped cells. Interferon‐γ alters several functional properties of both epitheliod and spindle‐shaped cells. In the absence of dibutyryl cAMP it accelerates the transition from epithelial to spindle‐shaped cells, whereas in the presence of cyclic AMP interferon‐γ increases the binding of PBMLs to both epithelioid and spindle‐shaped MEC and the endocytic activity of the endothelial cells.These results suggest that cyclic AMP is an important second messenger in the maintenance of several key functions of microvascular endothelial cells. Factors that influence the levels of this messenger in vivo can be expected to influence the angiogenic and immunologic functions of

ISSN:0021-9541    

DOI:10.1002/jcp.1041420209

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

年代:1990

数据来源: WILEY

摘要:

AbstractTo investigate the functional significance of epidermal growth factor (EGF) receptor phosphorylation, experimental systems were explored in which receptor phosphorylation on tyrosine and serine/threonine could be differentially stimulated. Exposure of A431 cells to 20 nM EGF at 37°C results in phosphorylation of serine, threonine, and tyrosine sites on the receptor. Monoclonal antibody (mAb) 225 binds to the EGF receptor with affinity comparable to EGF and competes with the binding of EGF. Exposure of A431 cells to 20 nM EGF in the presence of 300 nM anti‐EGF receptor mAb 225 (15‐fold excess) selectively activated serine and threonine phosphorylation of the receptor, but not tyrosine phosphorylation. This observation indicates that EGF‐mediated receptor phosphorylation on tyrosine and on serine/threonine residues is dissociable. The intracellular fate of the EGF receptor was examined under conditions that produce different phosphorylation states of receptor amino acids. Exposure of A431 cells to EGF decreased the half‐life (T1/2) of the receptor from 17.8 h to 5.6 h, with activation of tyrosine, serine, and threonine phosphorylation. Incubation with mAb 225 augmented the degradation rate (T1/2= 8.5 h) without activation of receptor phosphorylation. Concurrent exposure to EGF (20 nM) and mAb 225 (300 nM) resulted in comparable enhanced degradation (T1/2= 9.5 h), with increased phosphorylation only on serine and threonine residues. These results suggest that serine/threonine phosphorylation is irrelevant to the augmentation of receptor degradation. Methylamine, an inhibitor of lysosomal function that did not affect phosphory‐lation of the EGF receptor, completely protected EGF receptors from rapid degradation induced by EGF, but it only slightly altered the rate of EGF receptor degradation elicited by mAb 225 or by EGF plus 15‐fold excess mAb 225. In contrast, mAb 455, Which binds to the receptor but does not inhibit EGF binding and EGF‐induced activation of phosphorylation on tyrosine, serine, and threonine residues, did not influence EGF‐induced rapid, methylamine sensitive degradation of EGF receptor. The results suggest that when EGF receptors are internalized under conditions that do not activate the receptor tyrosine kinase, they are sorted into a nonlysosomal pathway that differs from the methylamine‐sensitive lysosomal pathway traversed following activation by EGF. The data indicate the possibility of a function for tyrosine kinase activation and tyrosine autophosphorylation in determining the lysosomal intracellular pathway of EGF receptor processi

ISSN:0021-9541    

DOI:10.1002/jcp.1041420210

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

年代:1990

数据来源: WILEY

摘要:

AbstractTransforming growth factor‐β (TGF‐β) modulates growth and differentiation in many cel' types and is abundant in bone matrix. We recently showed that human cord blood monocytes cultured in the presence of 1,25(OH)2D3acquire some features of osteoclast precursors. Since TGF‐β has been shown to influence bone resorption in organ culture, we have studied the effect of TGF‐β (1‐1,000 pg/ml) on cord blood monocyte cultures. These cells were cultured on plastic substrate during 3 weeks in the presence of 20% horse serum and 10−9M 1,25(OH)2D3TGF‐β, from a concentration of 10 pg/ml in the culture medium, decreased in a dose dependent manner the formation of multinucleated cells. At a concentration of TGF‐β of 1 ng/ml, the multinucleated cells were reduced to 2.1% ± 0.3%, compared to 19.3% ± 1.5% in control cultures. TGF‐β inhibited in a dose‐dependent manner the proliferation of cord blood monocytes as assessed by3H‐thymidine incorporation at 7 and 14 days of culture. The fusion index was also decreased by 3 weeks of treatment with TGF‐β Indomethacin did not reverse the inhibitory effects of TGF‐β. The expression of the osteoclastic phenotype was assessed using two different antibodies: 23C6, a monoclonal antibody directed against the vitronectin receptor, which is highly expressed by osteolasts but not by adult monocytes, and an antibody to HLA‐DR, which is not present on osteoclast. TGF‐β decreased the expression of HLA‐DR and increased in a dose‐dependent manner the proportion of 23C6‐labeled cells; these results suggest that TGF‐β could modulate a differentiation effect to the osteoclastic phenotype. However, when cord blood monocytes were cultured on devitalized rat calvariae prelabeled with45Ca, TGF‐β did not induce any45Ca release from bone cultured with monocytes, suggesting that full osteoclastic differentiation was not achieved. These results emphasize the complex role of TGF‐β in the local regula

ISSN:0021-9541    

DOI:10.1002/jcp.1041420211

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

年代:1990

数据来源: WILEY