摘要:

AbstractRecycled mesodermal growth factor (R‐MGF) is a pool of proteins of 26,000 molecular weight obtained by recycling gel chromatography of male murine submaxillary gland extracts. R‐MGF strikingly accelerates corneal stromal wound healing in vivo, fibroblast growth and migration in cultured corneal buttons and is shown here to stimulate stromal fibroblast growth and division in tissue culture. Chromatographic fractionation of R‐MGF has yielded several components, none of which has a greater biological potency than the parent R‐MGF. In contrast, two components, MGF‐I and ‐II, when recombined synergistically stimulate fibroblast response in tissue culture and organ culture in excess of those obtained with the parent R‐MGF. Three MGF components (I, III, and IV) have been purified and are inactive at 10–15 m̈g/ml in organ culture but potently stimulate fibroblast responses when combined in pairs containing 7.5 m̈g of each component. The striking synergism in organ culture suggests that the stimulation of wound healing by R‐MGF in vivo may also reflect synergistic action of more than one R‐MGF component. Procedures for isolating gram quantities of R‐MGF and for the purification of different R‐MGF components by ion exchange ch

ISSN:0021-9541    

DOI:10.1002/jcp.1041110202

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

年代:1982

数据来源: WILEY

摘要:

AbstractODC induction by fresh medium added to stationary, medium‐depleted, confluent cultures has been studied in transformed HeLa and CHO cells, and in normal human fibroblasts as an indicator of the resumption of cell multiplication. The transformed HeLa cell displays a more easily reversed G1block, a higher peak ODC level, and a shorter time period for achievement of the peak ODC value than does the normal fibroblast. Low concentrations of microtubule depolymerizing agents like colchicine suppress ODC induction almost completely in the normal fibroblast, but hardly at all in the HeLa or CHO cells. Both transformed cells occasionally reveal a superinduction of ODC at very low colchicine levels (10−8‐10−7M) and a more variable response to such agents than does the normal fibroblast. Higher concentrations of colchicine suppress ODC induction in all cells. Experiments with actinomycin D and cycloheximide indicate that the principal colchicine action involves inhibition at the level of protein or mRNA synthesis, rather than inactivation of the already synthesized enzyme. These experiments are provisionally interpreted as an indication that a microtubular system is needed to reinitiate certain steps associated with growth in G1‐blocked, normal cells, and that a second microtubular action terminating enzyme biosynthesis may exist. This microtubular control is defective in the transformed cells here studied. Specific microtubular actions necessary for initiation and termination of protein syntheses may occur throughout the cell reproductive cycle, and in the course of normal differentiation

ISSN:0021-9541    

DOI:10.1002/jcp.1041110203

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

年代:1982

数据来源: WILEY

摘要:

AbstractThe phorbol ester tumor promoter 12‐0‐tetradecanoylphorbol‐13‐acetate (TPA) specifically inhibited the binding of radioiodinated epidermal growth factor (125I‐EGF) to rat pheochromocytoma (PC12) cells in a noncompetitive fashion with an apparent Kiof 11–26 nM. Both TPA and EGF elicited similar biological responses in PC12 cells including enhanced incorporation of3H‐choline and32P‐orthophosphate into macromolecules, induction of ornithine decarboxylase, and stimulation of the phosphorylation of a 30,000 MW nonhistone, chromosome‐associated protein. These effects were also elicited by nerve growth factor (NGF) which, in contrast to the former agents, is a differentiating stimulus for the PC12 cells. The effects of TPA were additive or more than additive to the effects of NGF and EGF. When PC12 cells were induced to differentiate by treatment with NGF for 72 hours, the binding of125I‐EGF and responses to EGF were reduced by approximately 70%. The response of PC12 cells to the tumor promoter TPA was unaffected by treatment with NGF. Thus, the qualitatively similar effects of TPA and EGF seemed to be mediated through separate receptor systems with only the EGF receptor system reduc

ISSN:0021-9541    

DOI:10.1002/jcp.1041110204

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

年代:1982

数据来源: WILEY

摘要:

AbstractThe changes of cell coat and endocellular glycosaminoglycans associated with adhesion of polymorphonuclear leukocytes to tissue culture dishes were studied by electrophoresis on cellulose acetate. Polymorphonuclear leukocytes are coated by chondroitin 4 sulphate, while undersulphated chondroitin 4 sulphate and heparan sulphate are present in the cytoplasm. Upon adhesion polymorphonuclear leukocytes shed into the culture medium chondroitin 4 sulphate and concomitantly hyaluronic acid and heparan sulphate are exposed at the cell surface and shed into the culture medium. The role of these compounds in polymorphonuclear leukocyte physiology is discussed.

ISSN:0021-9541    

DOI:10.1002/jcp.1041110205

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

年代:1982

数据来源: WILEY

摘要:

AbstractWe have developed two serum‐free chemically defined media (RITC 78‐6 and RITC 80‐7) that support the growth in culture of human diploid fibroblasts to the same extent as Eagle's basal medium (BME) supplemented with 10% fetal bovine serum (FBS). These two media contain modified Eagle's minimum essential medium (MEM) supplemented with nonessential amino acids, various trace metals, organic compounds and growth factors [insulin, mouse epidermal growth factor (m‐EGF), transferrin and triiodothyronine (T3)]. RITC 80‐7 medium differs from RITC 78‐6 in that it contains thymidine, hypoxanthine, and vitamin B12and supports the long‐term serial cultivation of human diploid cultures. The addition of commercial bovine serum albumin (BSA, 5 g/liter) to the medium enhances cell growth. This effect is not observed if BSA is first delipidized, but reconstitution of BSA with certain lipids restores its ability to promote growth. BSA has an inhibitory effect on cellular attachment but this is overcome when fibronectin (FN, 10 mg/liter) is added

ISSN:0021-9541    

DOI:10.1002/jcp.1041110206

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

年代:1982

数据来源: WILEY

摘要:

AbstractSodium influx in serum‐deprived human fibroblasts in a nominally Ca‐free, Mg‐free medium is significantly higher (17.8 ± 1.9 μmole/g prot/min) than that measured in a medium containing 1.8 mM Ca and 1 mM Mg (10.9 ± 0.7 μmole/g prot/min), and is stimulated dramatically (44.1 ± 6.1 μmole/g prot/min) by the addition of 10% fetal bovine serum (FBS), suggesting that an enhanced influx of Ca ions is not a necessary condition for serum activation of the amiloride‐sensitive Na influx pathway. The addition of 2 mM ethylenediaminetetraacetic acid (EDTA) to serum‐deprived cells in a low Ca, low Mg medium also results in a dramatic stimulation of Na influx (40.4 ± 3.7 μmole/g prot/min), while the addition of EDTA to cells assayed in a low Ca, low Mg medium in the presence of FBS has no significant effect on Na influx (45.3 ± 4.1 μmole/g prot/min). Thus, the stimulatory effects of FBS and EDTA are not additive. Kinetic analysis in the presence of varying amiloride concentrations indicate that the EDTA‐stimulated Na influx occurs via the amiloride‐sensitive Na pathway. The activation of Na influx in cells rinsed free of Ca and Mg Can be readily reversed by the addition of Ca or Mg to the assay medium. The Ca concentration required to give 50% inhibition of Na influx is 52 ± 7.6 μM (n = 3) for cells assayed in serum‐free medium and 272 ± 29 μM (n = 3) for cells assayed in the presence of 10% FBS. At physiological Ca concentrations (1.8 mM) the Na influx is maximally inhibited by Ca both in the presence and absence of serum. Since Na influx in 1.8 mM Ca medium is 2.5‐fold higher in the presence of serum than in its absence, these data suggest that the serum‐induced change in the K, for Ca modulation of the amiloride‐sensitive Na transport pathway is not sufficient to explain the serum stimulati

ISSN:0021-9541    

DOI:10.1002/jcp.1041110207

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

年代:1982

数据来源: WILEY

摘要:

AbstractThe binding of radioiodinated lectins to the CHO cell surface was measured for the following affinity purified plant agglutinins; concanavalin A, wheat germ agglutinin, Ricinus agglutinins (I, II), pea agglutinin, peanut agglutinin, andBandeiria simpliciafoliaagglutinin (BSLI). The number of binding sites at saturation ranged from 6 × 105for BSL 1 to 5 × 107for pea and Ricinus. Affinity constants calculated by the Steck‐Wallach procedure ranged from 2 × 105m−1L for pea lectin to 4.5 × 106M−1L for peanut lectin. Competition studies between homologous and heterologous radiolabeled and unlabeled lectins indicated that homologous unlabeled lectin could fully block binding of radiolabeled lectin. For heterologous pairs, a variety of results were observed. Of particular interest is the finding that concanavalin A and wheat germ agglutinin mutually failed to compete for binding indicating that these two lectins bind to distinct, nonoverlapping populations of surface sites. This finding suggests that the binding sites for WGA and Con A reside, for the most part, on discrete populations of membrane molecules; this concept is further validated in a companion paper in the upcoming issue of this journal (Schwartz et a

ISSN:0021-9541    

DOI:10.1002/jcp.1041110208

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

年代:1982

数据来源: WILEY

摘要:

AbstractWe have isolated continuously growing cell lines derived from mouse bone marrow stroma. These cell lines were independently obtained, and though they showed morphologies ranging from the epithelioid to the fibroblastoid patterns, they all differentiated into adipocytes. Subclones obtained from two cell lines had a very high frequency (90‐100%) of differentiation into adipocytes after two or three weeks of arrested growth. Though extensive accumulation of lipid often mechanically impaired mitosis, the cells committed to adipocytes did not suffer an irreversible loss of proliferative capacity. Adipogenesis was obtained in conditions similar to those required for fat cell formation in long‐term bone marrow culture. The cell lines were found to be insensitive to insulin as a signal of adipocyte differentiation. The ultrastructural characteristics of the preadipocytes and fat cells are also similar to those of the fat cells developing in long‐term bone marrow culture. As such, these cell lines should prove useful for analysing cell/cell interactions in haemopo

ISSN:0021-9541    

DOI:10.1002/jcp.1041110209

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

年代:1982

数据来源: WILEY

摘要:

AbstractWe have previously shown that 5‐azacytidine (5‐Aza‐CR) induced the formation of biochemically differentiated myotubes, adipocytes, and chondrocytes in the mouse embryo cell line, C3H/10T1/2CL8 (10T1/2), and that the induction of the muscle phenotype was cell cycle specific. Here we show that the adipocyte phenotype is also induced maximally in cells treated during early S phase. During this period, the minimum treatment time required for the subsequent formation of myotubes was 5 min and the number of myotubes formed was dependent on treatment time. The incorporation of14C‐5‐Aza‐CR into DNA during the cell cycle, however, was not enhanced during early S phase, suggesting that incorporation of 5‐Aza‐CR into specific DNA sequences synthesized during early S phase may be required for the expression of the new phenotypes. Single cells, obtained by plating cell suspensions into 16 mm wells at limiting dilution, were treated with 5‐Aza‐CR during S phase. The resulting clones showed a high frequency of phenotypic conversion, indicating that 5‐Aza‐CR did not act via a selective mechanism, and several of the clones were capable of expressing more than one phenotype. The cells required more than 2 division cycles after treatment with the analog for the expression of the muscle phenotype and the capacity to differentiate was retained for long periods of time in the absence of cell division. The adult mouse line, CVP3SC6, differentiated into functional striated muscle cells following treatment with 5‐Aza‐CR. The analog also caused oncogenic transformation in the adult line at the same concentration that was effective at i

ISSN:0021-9541    

DOI:10.1002/jcp.1041110210

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

年代:1982

数据来源: WILEY

摘要:

AbstractThe objective of this investigation was to determine whether the rate of glucose uptake by mouse 3T3 cells was a primary determinant of growth rate. The experimental approach was to control the rate of glucose uptake into intracellular pools by supplying this sugar at varying concentration in minimal Eagle's medium with dialyzed serum in the absence and presence of 6‐deoxy‐D‐glucose, a metabolically inert homomorphic analog of D‐glucose that competitively inhibits the uptake of D‐glucose. Total hexose (D‐glucose and 6‐deoxy‐D‐glucose) concentration was maintained at the physiological concentration of 5.5 mM, in order to maintain saturation and maximum activity of the D‐glucose transport system; thus the flux of D‐glucose into the cell was controlled by adjusting its concentration relative to its competing nonmetabolizable analog. It was found that even when the concentration of D‐glucose was reduced to 0.7 mM, one eighth of the “normal” level of 5.5 mM. and 6‐deoxy‐D‐glucose was present in sevenfold excess (4.8 mM), conditions under which glucose uptake was reduced to 20% of that shown by cells in the presence of 5.5 mM D‐glucose, and intracellular pools of glucose and phosphorylated sugars derived from glucose were reduced to approximately 14% of normal, there was not a significant decrease in growth rate. These data support the view that the rate of glucose uptake is not a primary determinant of growth rate under t

ISSN:0021-9541    

DOI:10.1002/jcp.1041110211

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

年代:1982

数据来源: WILEY