摘要:

AbstractSupernatants from mouse spleen cell cultures contain a factor which acts in a similar manner to erythropoietin (Ep) to stimulate the formation of 2‐day erythroid (CFU‐E) colonies in vitro from bone marrow or fetal liver cells. Analysis of conditioned media by high performance liquid chromatography (HPLC) on anion exchange, reverse phase, molecular size exclusion, and hydroxyapatite columns demonstrated that the erythropoietin‐like activity (EpLA) has different biochemical characteristics to mouse Ep from anemic mouse serum. In addition, EpLA has a molecular weight (Mr), of 20,000 daltons determined by SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE), compared to 42,000 for mouse Ep. Partially purified EpLA was found to be active in vivo as well as in vitro. Highly purified preparations of γ‐interferon, Multilineage hemopoietic growth factor (Multi HGF), Interleukin‐2 (IL‐2), IL‐1, and colony stimulating factor 1 (CSF‐1) did not support CFU‐E colony formation. Thus, it was established that EpLA could not be attributed to other known components of spleen cell conditioned medium. Titration of mouse Ep and EpLA suggests that only a portion of the Ep‐responsive CFU‐E population in fetal

ISSN:0021-9541    

DOI:10.1002/jcp.1041260102

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

年代:1986

数据来源: WILEY

摘要:

AbstractThe extended culture of rat cervical epithelial cells can be achieved in the absence of a fibroblast feeder layer by utilizing collagen gels and a complex growth medium. The medium contains a 1:1 mixture of RPMI‐1640 and Ham's F12 supplemented with 7.5% porcine serum and epidermal growth factor, cholera toxin, transferrin, insulin, and hydrocortisone, Under these culture conditions the cells show rapid log‐phase growth and high saturation densities while retaining the ultrastructural characteristics of immature squamous metaplastic cells of the rat uterine cervix even after extended passage. In a manner similar to epithelial cells from a variety of sources, rat cervical epithelial cells form hemicysts at confluence in vitro when cultured on impermeable substrates. The development of these methods for culturing cervical epithelial cells provides an experimental system for the study of factors important in regulating the growth and differentiation of metaplastic squamous epithelial ce

ISSN:0021-9541    

DOI:10.1002/jcp.1041260103

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

年代:1986

数据来源: WILEY

摘要:

AbstractThe level of 1,25(OH)2D3receptors in cultured mouse osteoblast‐like (OB) cells is modulated by the rate of cell proliferation. We have studied two 1,25(OH)2D3‐induced bioresponses to ascertain whether the changes in receptor levels during growth in culture alter cell responsiveness. Nuclear receptor levels were high (127 fmol/100 μgDNA) in rapidly dividing (log) cells and low (25 fmol/100 μg DNA) in quiescent (confluent) cells. The bioresponses we studied were induction of 25(OH)D3‐24‐hydroxylase. activity (24‐hydroxylase) and inhibition of collagen synthesis. The basal levels of 24‐hydroxylase were low and similar in cells at log growth phase and confluence. At a maximal induction dose of 13 nM, 1,25(OH)2D3induced a three‐fold rise in enzyme activity at long growth phase, but only caused less than two‐fold rise at confluence. The half‐maximal dose (ED50) was slightly shifted from 0.6 nM to 0.8 nM. Daily measurement of 1,25(OH)2D3receptor levels and maximal induction of 24‐hydroxylase activity throughout the culture cycle showed a strong correlation between receptor abundance and enzyme induction. The basal level of collagen synthesized by cells in log growth phase was ∼ 5% and increased to ∼ 8% at confluence. Maximal inhibition of collagen synthesis by 1,25(OH)2D3reached 80% of control levels in log cells, but was only 40% of control in confluent cells. The ED50was ∼ 0.1 nM in the log cells and increased to ∼ 1 nM at confluence. Daily assay of 1,25(OH)2D3receptor levels and 1,25(OH)2D3responses during the culture cycle indicated a correlation between changes in receptor level and the extent of inhibition of collagen synthesis. These changes in biore‐sponse at various growth phases did not occur in rat OB cells where the 1,25(OH)2D3receptor levels were independent of cell proliferation. The results indicate that cell proliferation rate, via change in receptor levels, determines the magnitude and sensitivity of the ce

ISSN:0021-9541    

DOI:10.1002/jcp.1041260104

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

年代:1986

数据来源: WILEY

摘要:

AbstractA method for the selection and isolation of hexose transport mutants in undifferentiated rat myoblast L6 cells is reported; 2‐deoxy‐D‐glucose (2‐DOG)‐and 2‐deoxy‐2‐fluoro‐D‐glucose (2FG)‐resistant mutants were selected after mutagenization of L6 cells with ethyl methanesulfonate. Of these, D18 and D23 (selected with 0.1 mM 2‐DOG) and F72 and F76 (selected with 0.1 mM 2FG) exhibited the lowest hexose transport activity. Uptake of 0.06 mM 2‐DOG, 2FG, or 3‐O‐methyl‐D‐glucose (3‐OMG) by mutants grown in fructose medium supplemented with 0.05 mM 2FG was about four‐ to five‐fold lower than the parental L6 cells. These mutants contain normal levels of ATP and glycolytic enzyme activities. They also exhibit normal transport activities for α‐aminoisobutyric acid and fructose. Furthermore, hexose transport was observed to be decreased in plasma membrane vesicles prepared from these mutants. Kinetic analysis of 2‐DOG and 3‐OMG transport in mutant F72 demonstrated that the Vmaxfor 2‐DOG uptake was significantly reduced, whereas the Vmaxfor 3‐OMG transport was not affected. In all cases, the affinity for these hexose analogues was unaffected. In addition mutant F72 was found to be only slightly affected by treatment with various energy inhibitors and sulfhydryl reagents. The results suggest that this mutant is defective in, or has low levels of, a plasma membrane component(s) inv

ISSN:0021-9541    

DOI:10.1002/jcp.1041260105

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

年代:1986

数据来源: WILEY

摘要:

AbstractThe proteoglycans synthesized by fibroblasts derived from human donors of ages ranging from 12 years to 68 years have been studied. In addition, the in vitro proliferation rates of the various cell strains were studied and demonstrated that increasing donor age correlated with a decrease in proliferative activity. The incorporation of [35S]‐sulfate into proteoglycans decreased with increasing donor age with cells from the oldest donor demonstrating a 50% reduction compared with cells from the youngest donor. Analysis on Sepharose CL‐4B of isolated [35S]‐labeled proteoglycans for molecular size distribution revealed few differences between the cell‐layer‐associated proteoglycans of all cell strains studied. However, analysis of the medium‐associated [35S]‐labeled proteoglycans demonstrated an increase in the amount of small molecular size proteoglycans with increasing age. More specific analysis of the glycosaminoglycan composition revealed an increase in heparan sulfate from 52% to 73% in the cell‐layer‐associated proteoglycans of cells from the youngest and oldest donors, respectively. Accompanying this increase was a relative decrease in dermatan and chondroitin sulfate content from 24% to 13% and 25% to 16%, respectively, with increasing donor age. Additionally, the degree of N‐sulfation of cell layer heparan sulfate increased with age. Heparan sulfate levels increased in the medium as well with increasing age, with a concomitant decrease in chondroitin sulfate. The quantity of medium‐derived dermatan sulfate remained relatively evenly distributed throughout the various ages studied. The various differences noted are considered to reflect the general metabolic changes associated with aging. In particular the increase in heparan sulfate content with age is considered to be related to the decreased proliferative activity of the fibroblast

ISSN:0021-9541    

DOI:10.1002/jcp.1041260106

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

年代:1986

数据来源: WILEY

摘要:

AbstractGene expression in quiescent mouse embryo fibroblasts was studied by labelling the cells with [14C] amino acids and analysing the proteins by electrophoresis in polyacrylamide gradient gels containing sodium dodecyl sulfate. Cycloheximide (CH) pretreatment of the cells was found to induce the synthesis of four proteins of molecular weights 72,000, 68,000, 42,000, and 29,000. These proteins were induced by CH both in serum‐arrested and serum‐ stimulated cells. Addition of platelet‐derived growth factor to serum‐arrested quiescent cells also induced the synthesis of these proteins. Addition of CH and fetal calf serum (20%) to quiescent cells resulted in a dramatic increase in the synthesis of actin and another protein of molecular weight 29,000. The 29,000‐dalton protein was present in higher quantities in the nuclei of induced cells. This protein appeared to be an early protein whose synthesis was transiently induced in quiescent cells within 3 hours of addition of 20% fetal calf serum (FCS). The synthesis of this protein was virtually turned off at 5‐6 hours after the addition of serum. However, if CH or a combination of CH and FCS was present, a continuous synthesis of the 29 K protein w

ISSN:0021-9541    

DOI:10.1002/jcp.1041260107

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

年代:1986

数据来源: WILEY

摘要:

AbstractStaining of sickle cells with the fluorescent probes chlortetracycline (a Ca2+probe) and diindocarbocyanine (a general membrane probe) revealed the presence of Ca2+‐containing vesicles which are not found in normal erythrocytes. These vesicles increase in number upon deoxygenation, and are apparently formed by endocytosis, as judged by the use of the extracellular fluorescent probe lucifer yellow. The presence of vesicles is not restricted to any particular morphological or density class of cells in the general populatio

ISSN:0021-9541    

DOI:10.1002/jcp.1041260108

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

年代:1986

数据来源: WILEY

摘要:

AbstractRecent evidence suggests that low dose exposure of cells to hydrogen peroxide and/or induction of heat shock protein (HSP) synthesis will render cells resistant to the lethal effects of a subsequent high dose hydrogen peroxide stress. We explored this possibility in theDrosophila melanogasterSchneider tissue culture line 2. It was found that chronic low dose exposure (1 mM H2O2for 3 days) resulted in marked potentiation of the toxic effects of a subsequent high dose exposure (50 mM H2O2for 1 h), as assessed by impairment of uridine incorporation and cell proliferation. Cells preexposed to low dose H2O2exhibited enhanced heat shock gene transcription upon exposure to high dose H2O2, as compared to cells that did not receive low dose preexposure. Transcriptional induction of the heat shock genes by a mild non‐toxic heat shock resulted in marked enhancement of the anti‐proliferative effects of a subsequent H2O2exposure. Thus, low dose hydrogen peroxide exposure or mild heating results in subsequent enhancement of high dose hydrogen peroxide toxicity; this effect correlates with enhanced heat shock gene expression. Possible mechanisms are discus

ISSN:0021-9541    

DOI:10.1002/jcp.1041260109

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

年代:1986

数据来源: WILEY

摘要:

AbstractThe metabolism of [14C]pyruvate, [14C]glucose, [14C]glutamine and [14C]alanine was compared between normal rat tracheal epithelial cells and carcinogen‐altered cells derived from dimethylbenz(a)anthracene‐exposed tracheal implants. Normal primary cultures (NPC) of tracheal cells are distinguished by their need for pyruvate‐supplemented medium for growth and survival. The altered cells were selected out by their survival in the unsupplemented medium. Compared to the selected primary cultures (SPC), the NPC showed a three‐ to four‐fold higher incorporation of radioactivity from [2‐14C]pyruvate in all the macromolecular fractions, as well as in all the metabolites isolated from the acid soluble fraction and from lactic acid isolated from the medium. [U‐14C]glucose was also incorporated at higher levels into lactic acid isolated from the acid soluble fraction and the medium of NPC. These data indicate a higher rate of glycolysis in the normal tracheal cells. This was supported by the findings of a two‐fold greater glucose consumption and two‐fold higher production of lactic acid isolated from the NPC medium. Lactate dehydrogenase activity was also two‐fold higher in NPC. Thus, despite the apparently higher level of pyruvate production in the NPC, exogenous pyruvate is necessary to satisfy the metabolic needs of NPC. The utilization of [U‐14C]glutamine or [U‐14C]alanine was not markedly different between NPC and SPC. Furthermore, radioactivity from both of the amino acids was recovered in lactic acid in the medium, indicating that both cell types can derive pyruvic acid from either glutamine or alanine. SPC apparently do not use these routes to supply higher levels of pyruvic acid for survival in culture. The oxidation of none of the radioactive metabolites into CO2was distinctly different between NPC and SPC except for the 1.7‐fold higher utilization of [1‐14C]glucose along the oxidative arm of the pentos

ISSN:0021-9541    

DOI:10.1002/jcp.1041260110

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

年代:1986

数据来源: WILEY

摘要:

AbstractA cAMP‐resistant mutant (Kin‐8) isolated from Y1 mouse adrenocortical tumor cells harbors a specific lesion in the regulatory subunit of the type 1 cAMP‐dependent protein kinase. This mutant also is resistant to the effects of corticotropin and cAMP on steroidogenesis, growth and morphology, suggesting an obligatory role for the protein kinase in regulation of adrenocortical functions. In this study, the cAMP‐resistant phenotype of the Kin‐8 mutant was reverted by transformation with DNA from cAMP‐responsive Y1 cells, and the biochemical basis of the transformation was explored. Initially, Y1 mouse adrenocortical tumor cells were evaluated for their competence as recipients in DNA‐mediated transformation experiments, by measuring their ability to incorporate and express a bacterial gene (neo) encoding resistance to neomycin. Y1 cells were transfected with the plasmid pSV2‐neo (an SV40‐neo hybrid vector designed for expression in animal cells) and screened for resistance to the neomycin analog, G418. Neomycin‐resistant transformants were recovered from Y1 cells at a frequency of approximately one per 103cells per 10 μg of DNA, and had specific neo sequences integrated into their high molecular weight (mw) DNA. The Y1 mutant, Kin‐8, then was transformed with pSV2‐neo DNA plus high mw DNA prepared from cAMP‐responsive Y1 cells. Cells competent for transformation were recovered by selective growth in the neomycin analog G418, and these transformants were screened for recovery of morphological responses to cAMP. Several colonies capable of rounding up in the presence of cAMP were recovered after transformation with DNA from Y1 cells. These transformants also recovered the ability to round up in the presence of corticotropin, and were able to respond to both corticotropin and cAMP with increased steroidogenesis. Transformants generated from either Y1 or Kin‐8 cells were unstable. Y1 cells lost resistance to neomycin when grown in the absence of G418 at a frequency of 4% per generation. Similarly, Kin‐8 transformants lost their sensitivity to cAMP in subsequent culture passages. In some of the cAMP‐responsive transformants, cAMP‐dependent protein kinase activity was recovered and approached the activity seen in cAMP‐responsive Y1 cells. The recovery of a normal protein kinase by transformation appeared to have been sufficient to reverse the cAMP‐resistant phenotype of Kin‐8 cells. In other cAMP‐responsive transformants, protein kinase activity was not appreciably affected by cAMP. In these latter clones the gene encoding responsiveness to cAMP may have been lost in the interval between assays of adrenal‐specific functions and protein kinase activity. Alternatively, these clones may have acquired another gene, unrelated to the protein kinase, which was capable

ISSN:0021-9541    

DOI:10.1002/jcp.1041260111

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

年代:1986

数据来源: WILEY