摘要:

AbstractKinetic studies of binding and internalization of125I‐platelet‐derived growth factor (PDGF) demonstrate that up to 15% of membrane‐associated radioactivity is internalized within 2 minutes after warming to 37°C in a variety of cell types. The T 1/2 for internalization is approximately 20 minutes. The T 1/2 for the subsequent appearance of degradation products in the culture medium is between 60–90 minutes following initiation of internalization. Internalization and lysosomal association of125I‐PDGF were confirmed by EM autoradiography. Quantitative studies using PDGF adsorbed to colloidal gold (gold‐PDGF) demonstrate that 17% of the cell‐associated sites are along coated regions of the plasma membrane (1.0 sites/μm), while 82% are associated with noncoated membrane (0.2 sites/μm). There is a significant redistribution of the gold‐PDGF complexes upon warming. Within 1‐2 minutes at 37°C, gold particles are found within endocytic vesicles, endosomes (0.09‐0.3 μm diameter), and lysosomes (>0.2 μm diameter). At this time the vesicle/endosome compartment comprises 15% of the total sites and contains 0.9 sites per μm2of surface area. The lysosomes account for 8% of the total sites and contain 0.8 sites per μm2of surface area. Simultaneously, there is an increase in the number of gold‐PDGF binding sites within coated‐pits (1.6 sites/μm, 18% of the total sites) and a decrease along noncoated regions of the membrane (0.11 sites/μm, 58% of the total sites). After 15 minutes at 37°C, 26% of the total sites (1.4 sites/μm2) are highly concentrated within lysosomes, while sites in the vesicle/endosome compartment remain constant. At the same time, binding sites within coated pits decrease substantially (0.5 sites/μm, 4% of the total sites), while the number of sites along noncoated regions of the membrane remain constant. Gold‐PDGF was not observed associated with the Golgi complex at any time up to 120 minutes following warming. We conclude that gold‐PDGF is processed via both receptor‐mediated and nonspecific endocytosis and follows an intracellular pathway comparable to t

ISSN:0021-9541    

DOI:10.1002/jcp.1041210202

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

年代:1984

数据来源: WILEY

摘要:

AbstractHematopoietic stem cells with high proliferative capacity can be assayed when stromal bone marrow cultures are overlaid with limiting dilutions of marrow samples. This leads to hematopoietic growth after 4 weeks in a fraction of cultures, consistent with expectations based on Poisson statistics. It will be shown that monoclonal cultures are obtained that last from 2 to 15 weeks and that can generate up to several million mature granulocytes. The originating clone‐forming cell is named adherent stem cell (ASC) because of its adherence to plastic or stromal surfaces. The ASC is comparable to the CFU‐S in frequency, proliferative capacity and in its ability to give rise to CFU‐S. As an unexpected additional finding we report that a mode of “clonal succession” was apparent in cultures which expressed more than

ISSN:0021-9541    

DOI:10.1002/jcp.1041210203

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

年代:1984

数据来源: WILEY

摘要:

AbstractInflux of the K+analogue Rb+was measured through the ouabain‐sensitive Na+/K+pump and the ouabain‐insensitive “leak” pathways in Cl−or NO −3in mature red cells from adult pigs and in reticulocytes naturally occurring in 7‐day‐old piglets. In reticulocytes, Rb+influxes by the two pathways were of about equal magnitude in Cl−(13 and 10 mmoles/liter cells × hr) and at least 25‐fold larger than in mature red cells (0.5 and 0.4 mmoles/liter cells × hr). In Na+media, a portion of the ouabain‐insensitive “leak” flux of Rb+was Cl−dependent (Rb+Cl−transport) as NO −3replacement reduced Rb+influx by 90% in reticulocytes and by 40% in mature red cells. The sulfhydryl reagent N‐ethylmaleimide (NEM) stimulated Rb+Cl−transport about twofold in reticulocytes and up to 13‐fold in mature red cells. When reticulocytes matured to erythrocytes during in vitro incubation, about 90% of both ouabain‐sensitive Rb+pump and ouabain‐insensitive Rb+Cl−influx were lost. In contrast, the NEM‐stimulated Rb+Cl−transport changed much less throughout this period, suggesting an entity operationally but not necessarily structrually distinct from the basal Rb+Cl−transport. Although the experimental variability precluded a full assessment of significant changes in the small Na+/K+(Rb+) pump and Rb+Cl−fluxes in mature pig red cells kept for the same time period in vitro, Rb+flux changes in reticulocytes appear to be maturational in nature, reflecting parallel activity tra

ISSN:0021-9541    

DOI:10.1002/jcp.1041210204

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

年代:1984

数据来源: WILEY

摘要:

AbstractPluripotent hemopoietic progenitors lose potentialities during the process of differentiation. We have examined events that lead to lineage restriction by determining the cellular composition of 785 multilineage colonies grown from peripheral blood samples of glucose‐6‐phosphate‐dehydrogenase (G‐6‐PD) heterozygous volunteers. Of these colonies, 762 contained only one isoenzyme type and were considered to be of clonal origin. A considerable heterogenity was observed. Some colonies were composed of cells belonging to two different lineages, while other colonies contained three or more different cell types. A small number of colonies consisted—in addition to myeloid cells—of T‐lymphocytes. The variable association within individual colonies of members belonging to different hemopoietic lineages suggests a flexible determination and expression of differentiation programs by ea

ISSN:0021-9541    

DOI:10.1002/jcp.1041210205

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

年代:1984

数据来源: WILEY

摘要:

AbstractPlatelet‐derived growth factor (PDGF) is a potent mitogen for cultured cells of mesenchymal origin. Known sources of PDGF or PDGF‐like protein are blood platelets, several transformed cell lines, and cultured endothelial cells (EC). We have examined the regulation of production of a PDGF‐like protein in cultures of bovine aortic EC using a specific radioreceptor assay for PDGF. EC constitutively secreted PDGF‐like protein into serum‐containing or serum‐free medium. The rate of production of PDGF‐like protein was constant for at least 3 weeks and was not due to release of an internal store, since cell lysis by repeated freeze/thaw cycles did not relase significant amounts of the protein. Synthesis of PDGF‐like protein was sensitive to changes in the pH of the media and was maximal at pH 8.5. Production of PDGF‐like protein was independent of EC growth rate: rapidly dividing cells and confluent, quiescent cells produced equal amounts per cell. However, sparse, quiescent EC produced more PDGF‐like protein per cell than did confluent, quiescent cells. Several phorbol esters stimulated production of PDGF‐like protein. At a concentration of 10−6M, a twofold stimulation was observed upon addition of the tumor promoter 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) and nearly a fourfold stimulation upon addition of the nonpromoting analog, methyl TPA. Incubation of EC with endotoxin (10 μ/ml) resulted in a twofold stimulation of PDGF‐like protein production. In all experiments with endotoxin and phorbol esters, an increase in the production of PDGF‐like protein was accompanied by morphological changes in the EC cultures. The cells appeared elongated and fibroblastic and exhibited low viability. A mathematical model was developed in which PDGF‐like protein production was shown to consist of two separate components—production at a constant rate by healthy cells and a large burst of synthesis and secretion by dying cells. These results suggest that injurious agents may be capable of stimulating production

ISSN:0021-9541    

DOI:10.1002/jcp.1041210206

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

年代:1984

数据来源: WILEY

摘要:

AbstractThe frequencies of 6‐thioguanine‐resistant primary clones from the kidneys and skeletal muscles of aging male cohorts of two F1 hybrid strains ofMus musculusvaried from 0.59 to 10.96 × 10−5and did not increase as a function of donor age (up to 40 months). Resistant clones were shown to be severely deficient in the activity of hypoxanthine‐guanine phosphoribosyltransferase (EC 2.4.2.8). These deficiencies presumably resulted from molecular alterations at this X‐linked locus, including point mutations. No alterations of the X‐chromosome were observed at the level of the light microscope. These results are inconsistent with predictions of the intrinsic mutagenesis and protein synthesis error catastrophe theories of aging. They do not rule out, however, somatic mutational theories that invoke comparatively large‐scale chromosomal lesions, many of which would be likely to be lethal at the

ISSN:0021-9541    

DOI:10.1002/jcp.1041210207

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

年代:1984

数据来源: WILEY

摘要:

AbstractEndothelial cell‐derived growth factor (ECDGF) is a soluble mitogen secreted in vitro by bovine aortic endothelium. ECDGF is a mixture of at least two distinct heat‐stable and trypsin‐sensitive mitogens. Large amounts of mitogenic activity were found in lysates prepared from cultured endothelial cells. Other nonmitogen‐secreting cells in culture, including bovine dermal fibroblasts and vascular smooth muscle cells, also contained a similar activity. In contrast to ECDGF, the lysate mitogenic activities were sensitive to heat (56°C) and were not inactivated by trypsin. Similar to platelet‐derived growth factor (PDGF), ECDGF and cell lysate mitogens promoted cell proliferation in the absence of other defined mitogens when added to culture medium and after exposure to plastic. The cytoplasmic mitogens, however, were distinct from PDGF by receptor competition assays and othe

ISSN:0021-9541    

DOI:10.1002/jcp.1041210208

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

年代:1984

数据来源: WILEY

摘要:

AbstractThe effect of phorbol ester tumor promoters on the communication between individual cells in confluent culture was studied using a fluorescent dye transfer method. Cell‐cell communication between mouse Balb/c 3T3 cells and between Chinese hamser V79 cells was inhibited almost completely by tumor‐promoting phorbol esters, but not by nonpromoting derivatives; the effect was reversed upon removal of the promoter. Intercellular communication between Balb/c 3T3 cells, but not Chinese hamster V79 cells, was increased significantly in the presence of dbcAMP and caffeine, and these compounds counteracted the effects of tumor promoters. Inhibition of cell communication by phorbol esters appears to be receptor‐mediated, since specific binding of3H‐phorbol‐12,13‐dibutyrate to Balb/c 3T3 cells was inhibited only by compounds that also inhibit intercellular dye transfer. A study with cycloheximide suggests that the reversible inhibition of intercellular communication by phorbol esters may not need de novo protein synthesis, while upregulation of communication by cAMP requires protei

ISSN:0021-9541    

DOI:10.1002/jcp.1041210209

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

年代:1984

数据来源: WILEY

摘要:

AbstractWe have studied the role of ADP‐ribosylation of chromosomal proteins in the regulation of myeloid cell maturation using the HL‐60 cell line as a model. Nuclei isolated from this human promyelocytic leukemia cell line contained (ADP‐ribose)nsynthetase activity, whereas little or no enzymatic activity was detectable in normal human blood neutrophils. Furthermore, the activity of (ADP‐ribose)nsynthetase was decreased in HL‐60 cells when they were induced to mature with retinoic acid (RA). To determine whether reduced (ADP‐ribose)nsynthetase activity is simply a result of induced maturation or whether it is a necessary precedent event for the maturation process, we evaluated the effects of nicotinamide (NAm) and its methyl derivative, N′‐methylnicotinamide (N′‐Met‐NAm), agents which decrease ADP‐ribosylation. Treatment of HL‐60 cells with these drugs caused the cells to undergo maturation and to acquire certain of the morphologic, functional, and biochemical characteristics of normal neutrophils. N′‐Met‐NAm was more potent than NAm in inducting maturation; at a concentration of 0.8 mM, it caused greater than 80% of the cells to mature, whereas a tenfold greater concentration of NAm was required to induce a similar degree of maturation. NAm and N′‐Met‐NAm also potentiated the maturation of HL‐60 cells induced by RA. Exposure of cells to noninducing concentrations of these compounds caused a leftward shift in the dose‐response curve for RA; maturation was observed at 10−11M RA in the presence of either 2 mM NAm or 0.2 mM N′‐Met‐NAm while 10−9M RA was required to induce maturation in their absence. A leftward shift in the dose response curve for maturation in the presence of low doses of NAm or N′‐Met‐NAm did not occur with another inducer, dimethyl formamide (DMF). Two enzymes, NAD glycohydrolase and tissue transglutaminase, that are abundant in macrophages, were induced by RA but not by NAm. N′‐Met‐NAm decreased by about 75% the amount of endogenous (ADP‐ribose)nin a selected fraction of chromosomal proteins which included histone H1and the nonhistone high mobility group proteins. The results of this study support the concept that ADP‐ribosylation of chromosomal

ISSN:0021-9541    

DOI:10.1002/jcp.1041210210

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

年代:1984

数据来源: WILEY

摘要:

AbstractWhen subconfluent, Swiss 3T3 cells made quiescent by serum deprivation are stimulated with low concentrations of serum (ca. 1%), only a proportion of them (roughly 50%) enter S phase despite daily replacement with fresh, low‐serum medium. The cells that fail to enter S phase are not incapable of doing so, since most of them initiate DNA synthesis after transfer to 10% serum. It would appear that individual cells vary in their growth factor requirements. Using time‐lapse cinemicroscopy a few of the cells that respond to low serum were seen to give rise to several generations of progeny, while the majority of cells failed to divide at all, or divided once at most. Despite this, differences between cells in growth factor requirements do not seem to be heritable in the long term, since attempts to enrich for responding cells by prolonged culture in 1% serum have been unsuccessful. Rather, it would appear that the capacity to respond to low serum is an unstable property lost after a few generations in low serum. The loss of responsiveness shows parallels with “cellular senescence” and could conceivably result from decay of the platelet‐derived growth factor‐induced state of “competence.” But regardless of why some cells respond to low serum while others do not, it is clear that the kinetics of entry into S phase after serum stimulation of quiescent 3T3 cells are not strictly first‐order, since the labelling index plateaus after roughly 3 days at values substantially below 100%. As such, the kinetics, though not contradicting the transition probability model, cannot be taken to support it as was p

ISSN:0021-9541    

DOI:10.1002/jcp.1041210211

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

年代:1984

数据来源: WILEY