摘要:

AbstractThe receptor mechanism by which cells attach to fibronectin has been investigated by a combined immunologic and electrophoretic approach. One particular antiserum directed against 3T3 cell plasma membranes was found to contain antibodies that blocked spreading of these murine cells on fibronectin but not on laminin or serum spreading factor (vitronectin). Proteolysis experiments confirmed that this cell line has calcium‐protected polypeptides necessary for cell spreading on fibronectin. Consequently, protein antigens were fractionated according to size by SDS gel electrophoresis, and antigens that could block the inhibitory activity of the polyclonal antibody were identified. One class of blocking antigen appeared to correspond to the 140,000‐dalton complexes favored by several laboratories as fibronectin receptor candidates, but a second class of 45,000 daltons was also apparent. This 45,000‐dalton antigen was a major absorbing activity from 3T3 cell membranes and the predominant activity from L929 membranes. By isoelectric focusing and two‐dimensional gel electrophoresis, it was found to exist as a set of isoelectric point variants with pK = 5.3 to 6.2. Our results indicate that current models postulating a simple, unimolecular receptor mechanism for fibronectin may be oversimplified and that fibronectin may instead interact with more than one protein receptor component on the fibroblast cell

ISSN:0021-9541    

DOI:10.1002/jcp.1041260302

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

年代:1986

数据来源: WILEY

摘要:

AbstractST2‐3T3, a spontaneously transformed BALB/c‐3T3 cell line which does not require platelet‐derived growth factor (PDGF) for growth, was fused to THO2, a PDGF‐responsive non‐transformed BALB/c‐3T3 cell line, in order to learn whether transformation is expressed coordinately with PDGF independence. Hybrid cells were selected and grown in medium containing both HAT (hypoxanthine‐aminopterin‐thymidine) and ouabain; unfused cells of each parental type were killed in HAT‐ouabain medium. Five independently isolated ST2‐3T3xTHO2hybrid cell lines were established and characterized for both transformation and PDGF responsiveness. All five were transformed, having a disorganized growth pattern and achieving a final cell density similar to that of ST2‐3T3 cells. Two of these lines did not respond to a brief treatment with PDGF: the mitogen neither induced the synthesis of a PDGF‐modulated lysosomal protein (termed MEP), nor stimulated the cells to enter the S phase; one line responded to PDGF by synthesizing both MEP and DNA, whereas two others synthesized MEP but not DNA. In contrast, four independently isolated cell lines obtained by fusing PDGF‐responsive non‐transformed BALB/c‐3TC cells to the THO2line were all PDGF‐responsive for both MEP and DNA synthesis and were not transformed. It appears that PDGF independence is not required for the tra

ISSN:0021-9541    

DOI:10.1002/jcp.1041260303

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

年代:1986

数据来源: WILEY

摘要:

AbstractThe specificity of transferrin (Tf) in its exertion of a growth‐promoting effect on myogenic cells was examined using serum Tfs from chick, dove, goose, turkey, bovine, horse, rabbit, rat, and swine and primary myogenic cells from chick, duck, quail, rabbit, and rat, and rat L6 cells. Avian Tfs were effective on avian cells but not on mammalian cells, while mammalian Tfs were effective on mammalian cells but not on avian cells. Dove and bovine Tfs were exceptional in that they were effective on some class‐heterologous cells at higher concentrations and less so or completely ineffective on some class‐homologous cells. Despite these exceptions, however, the relationship between Tfs and cells can be summarized as a class specificity.To exert the growth‐promoting effect, it is prerequisite for Tf to bind its specific receptor on the cell surface. Using quail and L6 cells, we found that the binding of125I‐labeled chick and rat Tfs to the respective receptors of quail and L6 myoblasts was competitively inhibited by other kinds of effective Tfs, but not by ineffective ones.We conclude that the class specificity in myotrophic activity of Tf is due to the affinity between Tf and Tf

ISSN:0021-9541    

DOI:10.1002/jcp.1041260304

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

年代:1986

数据来源: WILEY

摘要:

AbstractThe effects of different carbohydrates on cell‐to‐cell adhesion were examined in an aggregation assay, which consisted of swirling a suspension of cells and monitoring the loss of single cells with a Coulter Counter. Of the carbohydrates tested, only heparin and dextran sulfate induced cell aggregation. This effect occurred in freshly isolated mouse splenocytes and in cultured cells of lymphoid origin (P388, YAA‐CI) but not in cell lines of fibroblastic origins (3T3, SV‐3T3, BHK, and PY‐BHK). Using the YAA‐CI cell line for further study, we found that aggregation could be induced by relatively small amounts of heparin (<10 μg/ml). Binding experiments with3H‐heparin showed that under normal physiological conditions each YAA‐CI cell bound approximately 2 × 106molecules of heparin at saturation with a Kd of 3.5 × 10−7M. This binding was blocked by both unlabelled heparin and dextran sulfate but not by other carbohydrates. When the pH of the medium was decreased, the heparin‐induced aggregation was inhibited, and the Kd of the3H‐heparin binding was increased. In a similar fashion, when the ionic strength of the medium was increased, heparin‐induced aggregation was inhibited and the Kd of the interaction was increased. These results suggest that the aggregation is inversely related to the Kd of the interaction and that the binding of heparin to the cell surface is pr

ISSN:0021-9541    

DOI:10.1002/jcp.1041260305

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

年代:1986

数据来源: WILEY

摘要:

AbstractThe subcellular distribution of leukotriene (LT)B4binding and metabolizing sites was investigated in human neutrophils. Cells were disrupted by nitrogen cavitation and fractionated by Percoll density gradient centrifugation to yield cytoplasm, membranes, azurophilic granules, and specific granules. Only membrane fractions contained high affinity [3H]LTB4binding sites. Binding of radiolabeled ligand to membranes was rapid, reversible, and saturable; it was blocked by a series of LTB4analogues at concentrations corresponding to their respective potencies in (1) blocking [3H]LTB4binding to whole cells and (2) stimulating neutrophil degranulation responses. In contrast, [3H]LTB4was metabolized by fractions enriched with markers for cytoplasm plus endoplasmic reticulum. The metabolic activity was sedimented by ultracentrifugation, enhanced by NADPH, and inhibited at 4°C. The cell‐free system, like intact cells, metabolized [3H]LTB4to ω‐oxidized product rapidly and quantitatively at 37°C but was inactive at 4°C. Whole cells converted radiolabel to 20‐hydroxy (∼ 30% of product) and 20‐carboxy (∼ 70% of product) derivatives; the cell‐free system formed principally 20‐hydroxy‐[3H]LTB4. These products were less bioactive than LTB4. Nevertheless, metabolism of LTB4played little role in limiting the cells' response to the ligand: neutrophils completed degranulation and became desensitized to LTB4within 3–5 min of exposure. Within this time frame, they oxidized less than 30% of the stimulus, and the extracellular fluid of these neutrophil suspensions was fully capable of activating fresh cells. We conclude that neutrophils transmit bioactions of LTB4via a specific receptor integrally associated with their plasmalemma and/or endoplasmic reticulum. They inactivate the stimulus via a particulate ω‐oxidase. At the level of the individual cell, receptor down‐regulation, rather than ligand metabolism, appears to limit functional res

ISSN:0021-9541    

DOI:10.1002/jcp.1041260306

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

年代:1986

数据来源: WILEY

摘要:

AbstractWe have used a Chinese hamster ovary cell line (DF3) that overproduces ornithine decarboxylase (ODC) to examine various parameters in the cell cycle‐dependent regulation of this enzyme. Under a variety of conditions, alterations in the activity of ODC were accompanied by parallel changes in the levels of the protein, as measured by immunologically cross‐reactive material (CRM). While putrescine has been known to suppress the induction of ODC, we have found that in DF3 cells 10−4M ornithine completely suppresses ODC activity. We also show that the levels of ODC mRNA are not modulated when the levels of ODC activity and CRM change drastically. The data can be interpreted in terms of models involving either an effect of putrescine on the translation of ODC mRNA, or on the activity of a relatively specific protease with ODC as its t

ISSN:0021-9541    

DOI:10.1002/jcp.1041260307

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

年代:1986

数据来源: WILEY

摘要:

AbstractThe fluorescent probe merocyanine 540, which binds preferentially to bilayers in which the lipids are loosely packed, was used to investigate changes in the organization of the lipids of the lymphocyte plasma membrane during primary and secondary lymphopoiesis. When mouse thymocytes were incubated with the dye, most immature cells stained, while most mature cells, about to enter the peripheral circulation, did not. Similarly, mature lymphocytes from both mouse and human peripheral blood did not stain, but these same cells did when activated by in vitro mitogenic stimulation. Freshly isolated splenic lymphocytes, presumably activated in vivo by antigen, also bound merocyanine 540, but after 48 hours of culture in the absence of stimulus they displayed only a low affinity for the dye, a phenotype that reverted to a high affinity upon mitogenic stimulation. These results suggest that changes in the organization of the lipids of the plasma membrane take place during lymphocyte differentiation: viz., immature cells possess a disordered membrane that becomes increasingly ordered as the cells mature and enter the peripheral circulation; then, upon antigen‐induced differentiation, the plasma membrane again becomes disordered. These lipid organization changes are discussed in the context of their possible role in the regulation of lymphocyte circulation via intercellular interactions between lymphocytes and cells of the reticuloendothelial syste

ISSN:0021-9541    

DOI:10.1002/jcp.1041260308

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

年代:1986

数据来源: WILEY

摘要:

AbstractNormal human skin was maintained in organ culture under chemically defined conditions. All‐trans retinoic acid was added to the culture medium at the final concentration of 5 μmol/l. After 5 days in culture samples were either harvested for electron microscopy or labeled with3H‐glucosamine for 24 h. After labeling, epidermis was separated from dermis and both tissue compartments were analyzed for the content of3H‐labeled glycosaminoglycans (GAGs) using CPC‐precipitation and thin layer chromatography after enzymatic degradation into specific disaccharides. Retinoic acid caused a marked change in the epidermal tissue architecture. The epidermal cells were flattened and contained fewer desmosomes and tonofilaments than control explants. Retinoic acid induced accumulation of fine granular material in the intercellular spaces in the upper, and less dense, flocculent material in the lower epidermis. The analysis of3H‐glycosaminoglycans showed that in the epidermis retinoic acid elevated the amount of labeled hyaluronate by 70%, whereas sulfated GAGs were not significantly increased. In dermis the incorporation of3H‐glucosamine into neither hyaluronate nor sulfated GAGs was stimulated by the retinoic acid. It is concluded that retinoic acid significantly modifies the differentiation of normal adult human epidermis by decreasing cytoskeleton components and by inducing the synthesis of new intercellular material, at least a part of which is hyaluronic acid. As a consequence, the cohesion between the epidermal cells was apparen

ISSN:0021-9541    

DOI:10.1002/jcp.1041260309

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

年代:1986

数据来源: WILEY

摘要:

AbstractThe effect of varying the amino acid concentrations of the culture medium on matrix vesicle formation was studied in primary cultures of chicken epiphyseal growth plate chondrocytes grown in Dulbecco's modified Eagle's medium (DME) supplemented with 10% fetal bovine serum (FBS). Decreasing the levels of free amino acids in the culture medium to levels of one‐half, one quarter, and one eighth of the values normally present in DME caused a progressive decline in matrix vesicle (MV) formation. Increasing the level in the culture medium of those amino acids that are enriched in extracellular fluid (ECF) of growth plate cartilage significantly increased formation of matrix vesicles (MV), as assayed by the alkaline phosphatase (AP) activities present in high‐speed sediments from spent culture media. However, adjusting the levels ofallamino acids to match those of the ECF produced the greatest stimulation of MV formation. Of the amino acids that are notably enriched in ECF, glutamate (GLU), alanine (ALA), serine (SER), asparagine (ASN), and taurine (TAU) individually enhanced MV production, whereas proline (PRO), glycine (GLY), and aspartate (ASP) had essentially no effect. The simple combination of ECF levels of ALA and GLU resulted in a stimulation of MV formation equal to that observed when the eight aforementioned amino acids were elevated to ECF levels. Other combinations of ASP and GLY, or of TAU, SER, and ASN showed some stimulation, but at a lower level. Increasing the amino acid concentrations, alone or in combination, also increased the levels ofcellularAP, and to a lesser extent cellular protein. While increases in cellular AP were generally correlated with increased formation of AP‐rich MV, this was not uniformly true. These results indicate that in addition to hormones and growth factors, nutritional factors such as the levels of amino acids are also critical for normal phenotypic expression, growth, and matrix formation by epiphyseal chondro

ISSN:0021-9541    

DOI:10.1002/jcp.1041260310

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

年代:1986

数据来源: WILEY

摘要:

AbstractThe adherent stromal layer in long‐term bone marrow cultures (LTBMC) provides the cellular environment necessary for the in vitro proliferation and differentiation of pluripotential hematopoietic stem cells. The role of humoral hematopoietic growth factors, colony‐stimulating factors (CSF) in the regulation of hematopoietic cell production in this system is poorly understood. We have recently isolated and cloned an adherent cell line, D2XRII, derived from murine LTBMC. Plateau phase 25 cm2cultures of 2 × 106D2XRII cells in 8.0 ml produced CSF‐1 (M‐CSF) at around 100–150 units/0.1 ml medium. Following X‐irradiation there was a dose‐dependent decrease in the production of CSF‐1 to a plateau of 50% of control levels at 10,000 rad. Higher doses did not produce a further decrease. The X‐ray dose reducing CSF‐1 production to 50% was 100‐fold above the lethal dose as measured by clonagenic survival following trypsinization and replating. Trypsinized replated viable adherent but nondividing X‐irradiated D2XRII cells were maintained for up to 8 weeks after irradiation and demonstrated continuous production of CSF‐1. The data indicate significant divergence of two biologic effects of X‐irradiation on plateau‐phase marrow stromal cells: physiologic function of adherence and CSF‐1 production, versus proliferative integrity. This divergence of effects may be very relevant to understanding the mechanism of X‐irradiation‐associated m

ISSN:0021-9541    

DOI:10.1002/jcp.1041260311

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

年代:1986

数据来源: WILEY