摘要:

AbstractThe EA hy926 cell line is a continuous, clonable, human cell line that displays a number of features characteristic of vascular endothelial cells (Edgell et al., 1983). Here we report that when EA hy926 cells (EA cells) are plated on an extracellular matrix material [Matrigel®], they undergo a process of morphological re‐organization leading to the formation of a complex network of cord or tubelike structures. These events seem to resemble, in some respects, an in vitro process of angiogenesis. The morphological re‐arrangement occurs within a 12–16 hr period and seems to require expression of new messenger RNA and protein, since it is completely blocked when actinomycin D or cycloheximide are present at the time the cells are plated on Matrigel. This is not due to overt toxicity of the drugs, since exposure of cells to actinomycin D at 2 hr or more after plating on Matrigel has little effect on the formation of the tubelike structures. The process of Matrigelinduced tube formation also apparently involves a G‐protein mediated signal. Treatment of the EA cells with pertussis toxin completely blocks the process and causes the ADP‐ribosylation of a 42 kD protein that is recognized by an antibody to Gi‐alpha subunits. In contrast, concentrations of pertussis toxin sufficient to block tube formation have only modest effects on the adhesion or motility of EA cells on purified matrix components such as laminin or collagen IV. The process of Matrigel‐induced tube formation also involves integrins since monoclonal antibodies to integrin alpha6 or beta 1 subunits can completely block the process. The concentrations of anti‐integrin antibodies needed to block tube formation are much lower than those required to block cell adhesion on purified matrix components and are sufficient to occupy less than 10% of the alpha6 or beta 1 subunits available at the cell surface. These results suggest that integrins may be involved in this potential model of angiogenesis in processes beyond their usual role in cell adhesion. Based on these results, it seems likely that the EA hy 926 cell line will prove to be a useful model for in vitro study of angiogenic processes. © 199

ISSN:0021-9541    

DOI:10.1002/jcp.1041530302

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

年代:1992

数据来源: WILEY

摘要:

AbstractRegulation of the synthesis of procollagen and other extracellular matrix components was examined in human skin fibroblasts obtained from donors of various ages, from fetal to 80 years old (in vivo aged), and in fetal fibroblasts at varying passage levels (in vitro aged). Growth rates and saturation densities of fibroblasts decreased with increasing age of the donor and after passage 20 of fetal fibroblasts. The rates of collagen and proteoglycan synthesis also decreased during both types of aging to about 10–25% of the rate in early passage fetal fibroblasts, whereas the synthesis of total noncollagenous proteins was not greatly affected. Decreased collagen synthesis in both types of aging was correlated with lower steady‐state levels of mRNAs for the two subunits of type I procollagen mRNA, although their regulation was not coordinate. Type III collagen mRNA levels also declined in both types of aging. The concentration of fibronectin mRNA also decreased during in vitro aging but more rapidly than the collagen mRNAs, whereas in fibroblasts from 51–80‐year‐old donors, it was similar to or higher than in early passage fetal fibroblasts. This study suggests that the decreased synthesis of procollagen and proteoglycans in in vivo aged fibroblasts represents changes that are responsible for intrinsic degenerative changes that occur in human skin during aging. Furthermore, although in vitro and in vivo aging were similar in many respects, they were not equivalent, as evidenced by the differences in regulation of fibronectin expression. © 1992 Wiley

ISSN:0021-9541    

DOI:10.1002/jcp.1041530303

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

年代:1992

数据来源: WILEY

摘要:

AbstractSubmicromolar concentrations of tributyltin (TBT), a commercially used organotin compound, were found to induce the expression of several stress proteins, most notably HSP89 and HSP70, in IMR‐90 human diploid fibroblats in a time‐ and dose‐dependent manner. This induction can be demonstrated by quantitation of 1) synthesis of the heat shock proteins (HSPs), 2) relative abundance of mRNA of hsp70, and 3) transient expression of a human hsp70 promoter driven reporter gene. TBT also increased the abundance of mRNA of heme oxygenase, whereas heat shock was without effect. Analysis of protein binding to a consensus heat shock element (HSE)by electrophoretic mobility shift assay suggests that the induction of the heat shock response by TBT was attributable to activation of the heat shock transcription factor (HSTF). © 1992 Wiley‐L

ISSN:0021-9541    

DOI:10.1002/jcp.1041530304

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

年代:1992

数据来源: WILEY

摘要:

AbstractThe 35‐kDa protein (p35, lipocortin I, annexin I), originally discovered as a Ca++‐dependent substrate for the EGF receptor tyrosine kinase, binds Ca++and phospholipids, is developmentally regulated in embryos and has restricted expression in adults. Immunohistochemistry of normal rat kidney shows that p35 is enriched in epithelia of Bowman's capsule, the macula densa, and medullary/papillary collecting ducts, suggesting that p35 is related to specialized renal functions. Light staining is observed in the thick ascending limb; elsewhere, immunoreactivity is nil. Since renal recovery from ischemia involves both hyperplasia and hypertrophy and reportedly is accelerated by EGF, we examined p35 distribution during this process. After 48 hours of recovery, both the distribution and amount of renal p35 are altered. Immunoblots show p35 levels increased at least threefold in whole‐kidney homogenates. The expression of p35 is still highly restricted in recovering kidneys; however, the thick ascending limb now stains heavily. This is the first documentation of alterations in annexin levels during a pathophysiologic response. However, our attempts to discern effects of exogenous EGF on the recovery from ischemia were negative for both mitotic index and renal function assays. © 1992 Wiley‐L

ISSN:0021-9541    

DOI:10.1002/jcp.1041530305

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

年代:1992

数据来源: WILEY

摘要:

AbstractLactoferrin, a single chain cationic glycoprotein, present in the secondary granules of neutrophils, acts as a negative feedback regulator of myelopoiesis. Specific receptors for lactoferrin were detected on the surface of different hematopoietic cell types. The influence of lactoferrin on cell growth in culture has been reported. Interactions of lactoferrin with DNA were also demonstrated.In the present paper we confirm the presence of lactoferrin specific binding sites on K562 cells and we estimate the number of binding sites and the dissociation constant. By Western blotting analysis performed on K562 lysates we find a band of about 120 kDa responsible for specific binding of lactoferrin. We also show that lactoferrin, after binding at the cell surface, is internalized in a temperature dependent way and is immunologically detectable as a DNA‐linked protein in nuclear extracts. © 1992 Wiley‐Liss,

ISSN:0021-9541    

DOI:10.1002/jcp.1041530306

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

年代:1992

数据来源: WILEY

摘要:

AbstractWe have studied the effect of a specific FGF receptor suicide antagonist on the growth of bovine epithelial cells (BEL cells) in culture. This basic fibroblast growth factor‐saporin conjugate (bFGF‐SAP) has a biphasic effect on bovine lens epithelial cells (BEL cells). Whereas 0.01 nM and 0.1 nM bFGF‐SAP stimulate BEL cells proliferation, 1 nM and 10 nM bFGF‐SAP have the predicted toxic effects on BEL cell growth. The toxicity of bFGF‐SAP is observed 2 to 3 days after the initial treatment and depends on cell density. Accordingly, the sensitivity of confluent cells to bFGF‐SAP is reduced compared to sparse cells. A time course analysis reveals that bFGF‐SAP is effective after a short exposure to cells and that its effects are not increased with longer treatments. Cell growth on bFGF‐SAP pretreated extracellular matrix (ECM) or posterior lens capsule (PLC) is also affected. Basic FGF‐SAP has been shown to bind to the extracellular material, allowing a modulation of lens cells migration and survival by a single treatment in vitro. This finding raises the possibility of its use in vivo to prevent capsules invasion by lens cells after cataract surgery. © 19

ISSN:0021-9541    

DOI:10.1002/jcp.1041530307

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

年代:1992

数据来源: WILEY

摘要:

AbstractTo evaluate the regulation of endothelial cell Cu,Zn‐SOD, we have exposed bovine pulmonary artery endothelial cells in culture to hyperoxia and hypoxia, second messengers or related agonists, hormones, free radical generating systems, endotoxin, and cytokines and have measured Cu,Zn‐SOD protein of these cells by an ELISA developed in our laboratory. Control preconfluent and confluent cells in room air contained 196 ± 18 ng Cu,Zn‐SOD/106cells. A23187 (0.33 μM), for skolin (10 μM), isobutylmethylxanthine (0.1 mM), dexamethasone (1 μM), triiodothyronine (1 μM) and retinoic acid (1 μM) failed to alter this level of Cu,Zn‐SOD. Exposure to anoxia and hyperoxia both elevated the level −1.5–2.0‐fold over 20% oxygen‐exposed controls at 48–72 hr. Similarly, exposures to glucose oxidase (0.0075 units/ml), menadione (12.5 μM), xanthine‐xanthine oxidase (10 μM, 0.03 units/ml) and H2O2(0.0005%) increased the level up to two–threefold over controls at 24–48 hr. Lipopolysaccharide, TGFβ1, TNFα, and II‐1 also increased levels of cellular Cu,Zn‐SOD, but only in proliferating cells. II‐2, II‐4, interferon‐γ, and GM‐CSF had no effect on Cu,Zn‐SOD. All treatments that elevated SOD resulted in inhibition of cellular growth, but decreased growth of cells at confluence alone was not associated with increased Cu,Zn‐SOD. We propose from these studies that Cu,Zn‐SOD of endothelial cells is not under conventional second messenger or hormonal regulation, but that up‐regulation of the enzyme is associated with (and perhaps stimulated by) free‐radical or oxidant production that also may be influenced by availability of certain cytoki

ISSN:0021-9541    

DOI:10.1002/jcp.1041530308

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

年代:1992

数据来源: WILEY

摘要:

AbstractRecent reports have shown that various marrow‐derived cell populations respond vigorously to recombinant rat stem cell factor (rrSCF164), one form of the kitligand. In the present study, we isolated cell populations from rat bone marrow using the Thy 1.1 antigen (an antigen that in the rat is differentially expressed on primitive hemopoietic progenitor cells) and fluorescently conjugated rrSCF164(rrSCF164‐PE). We show that rrSCF164only stimulates cells that are enriched in the brightest Thy 1.1 populations (Thy 1.1bright). Numerous cell lines were generated by serial passage in rrSCF164containing medium, and the prototypic cell lines have been designated SRT002 and SRT003. Each cell line retains the Thy 1.1brightphenotype and does not respond to interleukins (IL) 1–8, IL‐10, granulocyte (G) colony‐stimulating factor (CSF), granulocyte macrophage (GM) CSF, M‐CSF, or crude preparations of mitogen‐stimulated T‐cell supernatants. The Thy 1.1brightpopulation of rat marrow was subdivided into a subset that binds rrSCF164‐PE (Thy 1.1bright, rrSCF164+). The majority of these cells possess certain characteristics in common with marrow‐derived mast cells and the Thy 1.1bright, rrSCF164responsive cell lines, having similar granule morphology, being metachromatic, and reacting positively with alcian blue. Moreover, rats treated with rrSCF164displayed significant increases in Thy 1.1bright, rrSCF164+cells in the bone marrow. These studies show that the combination of Thy 1.1 and rrSCF164makes possible the isolation of a unique subset of rat bone marrow cells that differentially express the Thy 1.1 antigen and the cell surface receptor c‐kit, the majority of which are morphologically similar to marrow‐derived mast cells.

ISSN:0021-9541    

DOI:10.1002/jcp.1041530309

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

年代:1992

数据来源: WILEY

摘要:

AbstractPretreatment plus concomitant treatment with 10 μg/ml cycloheximide protected Chinese hamster ovary cells and Swiss 3T3 cells against the cytotoxicity of actinomycin D. The cycloheximide treatment reduced the intracellular concentration of actinomycin D by reducing the level of actinomycin D bound to the acid precipitable fraction of the cell. Levels of unbound actinomycin D were unaffected by cycloheximide, indicating that the plasma membrane permeability to AD was not reduced. Actinomycin D inhibited total transcription but did not reduce cytoplasmic levels of rRNA nor of most tested mRNA; however, cytoplasmic levels of c‐myc mRNA were reduced below detectability. Cycloheximide treatment further inhibited total transcription and had no effect on cytoplasmic levels of rRNA nor of most tested mRNA. Cytoplasmic levels of c‐myc were elevated by cycloheximide and remained so even in the presence of actinomycin D. These data suggested that a reduction in cytoplasmic levels of short lived, essential mRNA, such as c‐myc mRNA, was one lethal lesion of actinomycin D. Furthermore, cycloheximide's protection may result, in part, from its ability to stabilize and/or elevate cytoplasmic levels of these mRNA, thus counteracting their depletion by actinomycin D. Protection may also result from the cycloheximide‐induced reduction of actinomycin D bound to the acid precipitable fraction of the cells. © 1992 Wiley

ISSN:0021-9541    

DOI:10.1002/jcp.1041530310

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

年代:1992

数据来源: WILEY

摘要:

AbstractIn the study of cell division in the early development of frog eggs, cortical photon emission was investigated by converting small light emission from living cells into digital pulses of potentials and recording the integrals of these pulses (analog method), counting the number of pulses (photon‐counting method), and counting the number of integrated pulses (improved photon‐counting method). By the analog and improved photon‐counting methods, changes in photon emission due to cell division could be clearly detected. The emitted light increased about 5.10−19W at the start of a cleavage furrow. Rapid changes in chemical reactions causing photon emission were compared during nuclear division and cytoplasmic phases. This emission occured mainly in cytoplamic fission, the rate being greater than in nuclear division by a factor of about 2.9. Chemical reaction rates were shown to differ according to bulk emission, thus indicating the mechanisms for the reactions to also differ. © 1992 Wiley

ISSN:0021-9541    

DOI:10.1002/jcp.1041530311

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

年代:1992

数据来源: WILEY