摘要:

AbstractAn increase in collagen synthesis by hepatic parenchymal cells (hepatocytes) was observed during 8 days in primary culture by the quantification of total [3H]hydroxyproline as a marker of total collagen synthesis and the ratio of [3H]hydroxyproline in the high‐molecular‐weight fraction to total [3H]hydroxyproline as a marker of collagen degradation after incubation of the cells with [3H]proline for 24 h. Type analysis of the collagen produced by the cells after 8 days in culture showed the presence of type I and type III collagens in addition to the components corresponding to type IV and type V (αA and βB) collagens. Only the latter two types were found in the collagens produced by the cells after 2 days in primary culture. (a) The purity of the hepatocytes inoculated was 97%, and the majority of the contaminating small cells were erythrocytes. (b) The rate of serum albumin synthesis, which is a typical function of the hepatocytes, was constant or increased during the culture period. (c) Immuno‐electron microscopic observation indicated the production of type I collagen by the hepatocytes after 8 days in primary culture. These results are explained only by the activation of collagen synthesis in the day‐8 hepatocytes in primar

ISSN:0021-9541    

DOI:10.1002/jcp.1041220302

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

年代:1985

数据来源: WILEY

摘要:

AbstractCell hybrids were formed between human diploid fibroblasts (HDF) and carcinogen‐transformed HDF to determine the relationship among: (1) finite proliferative lifespan, which we define as an age‐related failure of a population to achieve one population doubling in 4 weeks; (2) arrest in a senescent state, which we define as cessation of DNA synthesis in a viable culture that is at the end of its lifespan by the above definition; and (3) arrest in a quiescent state, which we define as cessation of DMA synthesis in a young culture that is crowded or mitogen‐deprived. HDF express all three of these phenotypes, which we have abbreviated FPL+, S+, and Q+, respectively. Carcinogen‐transformed HDF are transformed to immortality (FPL−) and inability to achieve quiescence (Q−). They have no S phenotype because, by definition, this phenotype only exists in FPL+cells. Fusion of FPL+, Q+, S+HDF × FPL−, Q−carcinogen‐transformed HDF produced hybrid clones that were FPL+, Q−, and S−, where the S−phenotype means that individual cells continued to synthesize DNA in cultures that had reached the end of their lifespan by our definition. These results are consistent with our hypothesis that senescent HDF and quiescent HDF may share a common mechanism for arrest in G1 phase. We have suggested that this could occur if the aging mechanism that is responsible for the FPL+phenotype is a progressive decrease in the ability of cells to recognize or respond to mitogenic growth factors. If so, then cells would become physiologically mitogen‐deprived at the end of their lifespan, which would cause them to arrest in the senescent state by the same mechanism that causes young cells to arrest in the quiescent state when they are mitogen‐deprived. This hypothesis predicts that the FPL+phenotype can be separated from the S+phenotype–i.e., FPL+cells can be S+or S−–and that the Q and S phenotypes are linked‐i.e., FPL+cells are either Q+and S+or Q−and S−. Both these predi

ISSN:0021-9541    

DOI:10.1002/jcp.1041220303

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

年代:1985

数据来源: WILEY

摘要:

AbstractCell culture conditions for the selective growth and serial propagation of normal human melanocytes from epidermal tissue are described. In addition to the presence of 2% fetal bovine serum, the human melanocyte cell culture environment contains the following growth factor supplements: epidermal growth factor (10 ng/ml), triiodothyronine (10−9M), hydrocortisone, (5 × 10−5M), insulin (10 μg/ml), transferrin (10 μg/ml), 7S nerve growth factor (100 ng/ml) cholera toxin (10−10M), and bovine brain extract (150 μg/ml). The ability to establish selectively the human melanocyte in vitro has been attributed to the contrast between human epidermal keratinocytes and melanocytes for attachment to fibronectin, while the growth of the human melanocyte has been attributed to the mitogenic activity of the growth factor‐supplemented medium. Human melanocytes can be cultivated for at least 15 cumulative population doublings and are capable of [3H]‐Dopa incorporation. The growth factor‐supplemented medium contains a neutral extract from bovine brain that is a potent source of a human melanocyte mitogen. The biological activity of melanocyte growth factor is described as a heat and alkaline‐labile mitogen with an estimated molecular weight of 30,000 by gel exclusion chromatography and a weakly cationic isoelectric point. The mitogen is capable of stimulating the growth of quiescent populations of human melanocytes in vitro. The ability to isolate and propagate normal human melanocytes in vitro permitted an examination of the expression of fibronectin and tissue plasminogen activator. Human epidermal melanocytes established in culture do not contain either tissue plasminogen activator or fibronectin. In contrast, human melanoma cell lines contain immunologically detectable fibronectin and tissue plasminogen activator. The absence of tissue plasminogen activator and fibronectin in normal human melanocytes also occurs under conditions of co‐cultivation with human melanoma cells. These contrasts between normal human melanocytes and human melanoma cells may be relevant to the metastatic capabilitie

ISSN:0021-9541    

DOI:10.1002/jcp.1041220304

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

年代:1985

数据来源: WILEY

摘要:

AbstractA new approach, based on the occurrence of receptors for the mononuclear phagocyte lineage specific hemopoietic growth factor (HGF) colony stimulating ractor‐1 (CSF‐1) on developmentally early multipotent cells, is utilized to detect and assay rapidly another HGF, hemopoietin‐2. This method is also used to determine the relative maturity of hemopoietin‐2 target cells, to investigate synergism between hemopoietin‐2 and CSF‐1, and to measure CSF‐1 receptor levels on maturing cells. While the target cell specificities of hemopoitin‐2 and CSF‐1 overlap, hemopoietin‐2 causes the appearance of developmentally earlier125I‐CSF‐1 binding cells de novo in the absence of CSF‐1. Increased CSF‐1 receptor densities are observed on cells incubated with either HGF, consistent with acquisition of the capacity for increased expression of the receptor by mononuclear phagocyte progenitor cells just prior to their differentiation to adherent mononuclear phagocytes. Together, both HGFs have a synergistic effect on the generation of125I‐CSF‐1 binding cells with elevated CSF‐1 receptor densities. Preliminary characterization of hemopoietin‐2 from medium conditioned by WEHI‐3 cells indicates that it is very similar to, if not identical with, interleukin‐3 (IL‐3) and the HGF(s) acting on multipotential cells and cells giving rise to erythroid cells, granulocytes, mononuclear phagocytes, and megakaryocytes. Purified IL‐3

ISSN:0021-9541    

DOI:10.1002/jcp.1041220305

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

年代:1985

数据来源: WILEY

摘要:

AbstractThe preceding paper describes a new approach to the detection and assay of growth factors for developmentally early multipotent hemopoietic cells (Bartelmez et al., J. Cell. Physiol., 1985). This approach, involving measurement of the increase in the number of receptors for the mononuclear phagocyte specific hemopoietic growth factor (HGF), colony stimulating factor‐1 (CSF‐1), in cultures of developmentally early murine cells incubated with putative HGFs, has been used to define and assay hemopoietin‐1. Hemopoietin‐1 (Mr∼ 20,000) is found in the medium derived from serum‐free cultures of cells of the human urinary bladder carcinoma line 5637. In contrast to both hemopoietin‐2 and CSF‐1, which also stimulate an increase in CSF‐1 receptor numbers in cultures of developmentally early hemopoietic cells, hemopoietin‐1 alone has no detectable effect. However, hemopoietin‐1 exhibits dramatic synergism wth CSF‐1. In the presence of CSF‐1, hemopoietin‐1 stimulates the proliferation of developmentally earlier cells than those that respond to either CSF‐1 alone or hemopoietin‐2 alone or their combination. These cells proliferate for at least 3 days with no alteration of the average CSF‐1 receptor density. However, by 5 days of incubation, the progeny of developmentally early hemopoietic cells that have proliferated in response to hemopoietin‐1 + CSF‐1 exhibit an approximately tenfold increase in the average CSF‐1 receptor density per cell, which immediately precedes their differentiation to adherent mononuclear phagocytes. As hemopoietin‐1 does not possess colony stimulating or burst promoting activities for murine bone marrow cells, but acts on multipotent hemopoietic cells, the analysis of the mechanism of its synergistic effects with HGFs such as CSF‐1 are of special relevance to th

ISSN:0021-9541    

DOI:10.1002/jcp.1041220306

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

年代:1985

数据来源: WILEY

摘要:

AbstractNeutral amino acid transport was characterized in the pluripotent embryonal carcinoma (EC) cell line, OC15. Ten of the thirteen amino acids tested are transported by all three of the major neutral amino acid transport systems—A, L, and ASC—although one system may make a barely measurable contribution in some cases. The characterization of N‐methyl‐aminoisobutyric acid (meAIB) transport points to this model amino acid as a definitive substrate for System A transport by OC15 cells. Thus, high concentrations of meAIB can be used selectively to block System A transport, and the transport characteristics of meAIB represent system A transport. Kinetic analysis of System A, with a Km= 0.79mM and Vmax= 14.4 nmol/mg protein/5 min, suggests a single‐component transport system, which is sensitive to pH changes. While proline transport in most mammalian cells is largely accomplished through System A, it is about equally divided between Systems A and ASC in OC15 cells, and System A does not contribute at all to proline transport by F9 cells, an EC cell line with limited developmental potential. Kinetic analysis of System L transport, represented by Na+‐independent leucine transport, reveals a high‐affinity, single‐component system. This transport system is relatively insensitive to pH changes and has a Km= 0.0031 mM and Vmax= 0.213 nmol/mg protein/min. The putative System L substrate, 2‐aminobicyclo‐[2,2,1]heptane‐2‐carboxylic acid (BCH), inhibits Systems A and ASC as well as System L in OC15 cells. Therefore, BCH cannot be used as a definitive substrate for System L in OC15 cells. Phenyialanine is primarily transported by Na+‐dependent Systems A and ASC (83% Na+‐dependent; 73% System ASC) in OC15 cells, while it is transported primarily by the Na+‐independent System L in most other cell types, including early cleavage stage mouse embryos and F9 cells. We have also found this unusually strong Na+‐dependency of phenyl‐alanine transport in mouse uterine blastocysts (82% Na+‐dependent). There is no evidence for System N transport by OC15 cells, since histidine is transported primarily by a Na+‐i

ISSN:0021-9541    

DOI:10.1002/jcp.1041220307

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

年代:1985

数据来源: WILEY

摘要:

AbstractHeat‐induced alterations in CHO‐10B cell Hoechst 33342 (Ho342) permeability in vitro were analyzed by flow cytometry. Immediately after 45.5°C heating, uptake was decreased in a dose‐dependent manner with cytotoxicity. Kinetic analysis indicated that a treatment that reduced cell survival to approximately 10%, reduced the maximal velocity, Vmax, to 53% of control and increased the dissociation constant, Km, to 156% of control. Also, little change in Ho342 efflux was found to occur from control up to 90 min after heating. Upon incubation at 37°C after the heat treatment from 1 to 24 hr (depending on the severity of the dose) diffuse heterogeneity of Ho342 staining developed which was not evident immediately after heating. The altered staining was not due to the presence of trypan blue staining cells. Membrane permeabilization and nuclei isolation studies indicated that the lesion responsible was most likely a plasma membrane event. Induction of the heterogenous staining was not inhibited by either actinomycin D or hydroxyurea but was inhibited by incubation at 4°C. An inverse correlation existed between Ho342 permeability and clonogenicity, with nearly a 10‐fold difference in survival between the high and low fluorescence intensity sorted cells. Also, larger fractions of heatsensitive S and G2M‐phase cells were found in the highly fluorescent sorted fractions. These results are discussed in terms of the putative molecular events that may be involved in hyperthermic modulation of Ho342

ISSN:0021-9541    

DOI:10.1002/jcp.1041220308

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

年代:1985

数据来源: WILEY

摘要:

AbstractUridine phosphorylase activity was detected in sonic extracts of six different mammalian cell lines and, in conjunction with uridine kinase, provides a route for the conversion of uracil to UMP via uridine. Uracil phosphoribosyl transferase activity was not detected in any of eight different mammalian cell lines. Uridine phosphorylase was purified 5,330‐fold from Novikoff rat hepa‐toma cells by ammonium sulfate precipitation, DEAE‐Sephadex chromatography, hydroxyapatite chromatography, and Sephadex G‐200 fractionation. The molecular weight of the enzyme by gel filtration was approximately 45,000. The kinetics of the purified enzyme were analyzed with respect to all four substrates at saturating cosubstrate concentration, yielding the parameters KmUra= 360 μM, KmRib−1‐P= 88 μM, KmUrd= 16 μM, and KmPi= 130 μM. However, in intact cells the phosphorolysis of uridine proceeded with an apparent Km of 231 μM. Novikoff cells treated with 0.5 mM inosine exhibited an increase in uracil uptake rate which was proportional to an observed increase in intracellular ribose‐1‐phosphate. Nevertheless, in cells whose de novo synthesis of pyrimidines was blocked by pyrazofurin or N‐(phosphona‐cetyl)‐L‐aspartate (“PALA”), the uptake of uracil was insufficient to support proliferation, even when enhanced by inosine. These observations are consistent with the kinetic characteristics of the enzyme and provide evidence that the intracellular level of ribose‐1‐phosphate plays a rate‐limiting role in the uptake of ur

ISSN:0021-9541    

DOI:10.1002/jcp.1041220309

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

年代:1985

数据来源: WILEY

摘要:

AbstractThis paper describes a method for the culture of rat placental cells. The method involved separation of the basal layer from the labyrinth and sequential digestion of the cells. The cells were demonstrated not to be fibroblasts and are described in terms of their appearance under the light and electron microscopes. Transferrin and iron uptake by the cells was examined and compared with results achieved using other methods of study. The results showed that transferrin bound to receptors on the cell surface and that the transferrin, once bound, was taken into the cell. Only this internalized transferrin was capable of donating iron to the cells. The iron was accumulated within the cells and did not appear to be released to the incubation medium. The apparent dissociation constant (Ka) for transferrin was found to be 6.96 × 106M−1, a value similar to that described by earlier workers. The placental cells had 3.4 × 1011binding sites/μg DNA, equivalent to approximately 1 × 106sites/cell. From these data, and from the rate of accumulation of iron by the cells, the receptor turnover time was estimated as being between 5 and 1

ISSN:0021-9541    

DOI:10.1002/jcp.1041220310

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

年代:1985

数据来源: WILEY

摘要:

AbstractWe compared the proliferation of bovine aortic cells grown in collagen lattices. Smooth muscle cells continued to divide for 2 weeks while adventitial fibroblasts ceased to divide after 4–5 days. Endothelial cells did not proliferate within an untreated collagen lattice; however, if the lattice was covered with culture medium, endothelial cells populated its surface and proliferated to form a monolayer. We also found that both smooth muscle cells and endothelial cells, like fibroblasts, are able to contract a collagen lattice to a small fraction of its original volume, although endothelial cells are able to do so only if the lattice is covered with culture mediu

ISSN:0021-9541    

DOI:10.1002/jcp.1041220311

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

年代:1985

数据来源: WILEY