摘要:

AbstractThe possibility that small nuclear RNA species U1 might be involved in the inhibition of protein synthesis that occurs during mitosis has been explored. Upon exposure of mitotic HeLa cell extracts to 1% sodium deoxycholate, the majority of the rapidly sedimenting U1 RNA shifted to lower sedimentation rates. This suggests that it is associated with heterogeneous nuclear RNA ribonucleoprotein particles, instead of a ribosomal population. Erythrocyte ghost‐mediated microinjection of anti‐(U1)RNP antibodies into synchronized HeLa cells did not prevent the suppression of protein synthesis that is observed under mitosis. Examination of the published nucleotide sequences of U1 and U2 RNA suggests that these RNA species could potentially code for some short peptides. When purified U1 or U2 RNA were added to cell‐free polypeptide synthesizing systems, the synthesis of these peptides was not det

ISSN:0021-9541    

DOI:10.1002/jcp.1041140102

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

年代:1983

数据来源: WILEY

摘要:

AbstractFour endothelial cell clones derived from adult bovine aorta were examined with respect to their proliferative characteristicsIn vitro. Three of these clones, derived in the absence of fibroblast growth factor (FGF), displayed variable basal proliferative rates. One of these non‐FGF derived clones grew at a maximal rate which could not be further enhanced with FGF. The other two clones grew at a suboptimal rate which was stimulated by low doses of FGF (10–50 ng/ml) and inhibited by higher doses (100–250 ng/ml). The fourth clone, derived in the presence of FGF, was stimulated by FGF in a dose‐dependent manner (10–250 ng/ml) and was not growth inhibited at high FGF concentrations (250–1,000 ng/ml). Growth of all four clones on extracellular matrix (ECM) derived from bovine aortic smooth muscle (BASM) cells was optimal in the absence of FGF. ECM‐coated dishes also significantly increased the sensitivity of all clones by at least fivefold to mitogenic stimulation by serum. The proliferative lifespans of the clones ranged between 60 and 120 generations with the most actively proliferating clones attaining the greatest lifespan. Continuous subculture of two of the endothelial clones in the presence of FGF or on ECM‐coated dishes did not induce a dependence of the cells on either factor for subsequent growth in its absence. The results indicate that aortic endothelial cells display considerable clonal variability in ther basal proliferative rate and in their response to FGF. This clonal variability is not observed when the cells are maintained on ECM‐coated dishes derived from vascular sm

ISSN:0021-9541    

DOI:10.1002/jcp.1041140103

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

年代:1983

数据来源: WILEY

摘要:

AbstractThe aliphatic diamine putrescine, a metabolic precursor of the polyamines spermidine and spermine, markedly stimulated the growth of a murine lymphoblastoid cell line (R 1.1) infected withMycoplasma orale, under conditions of arginine limitation. The diamine acted by suppressing the growth of the mycoplasma, which use arginine as a major energy source, and thereby prevented the depletion of arginine from the medium. The antimycoplasmal effects of putrescine occurred at concentrations that were neither stimulatory nor toxic to uninfected cells.

ISSN:0021-9541    

DOI:10.1002/jcp.1041140104

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

年代:1983

数据来源: WILEY

摘要:

Abstract125I‐Iododeoxycytidine (125IdC) incorporation into acid‐insoluble material was a sensitive, rapid, and quantitative assay for the growth of herpes simplex virus type 1 (HSV‐1) in human fibroblasts. Cellular utilization of the isotope was 10 to 25% of the incorporation by infected cells and could be 80% inhibited by tetrahydrouridine (THU). Viral utilization was inhibited by acycloguanosine, thioguanine (TG), and cytosine arabinoside. Isotope was incorporated equally well by growing or quiescent infected cells. HSV‐1 was used to probe the metabolic capabilities of three mutant human fibroblast strains.125IdC incorporation quantitatively measured the ability of the virus to grow in these cells. Viral125IdC incorporation was sensitive to TG in normal fibroblasts but showed a 8‐ to 10‐fold greater resistance to TG in fibroblasts derived from patients with Lesch‐Nyhan syndrome (LN). Similarly, the growth of ultraviolet irradiated HSV‐1 in normal fibroblasts, as judged by125IdC incorporation, was 5‐fold greater than in fibroblasts derived from patients with xeroderma pigmentosum. In fibroblasts derived from patients with hereditary orotic aciduria, viral125IdC incorporation was sensitive to adenosine (AD) at concentrations which were slightly stimulatory in normal fibroblasts. This was a 2‐fold difference in AD sensitivity, which the radioassay reliably and quantitatively documented. Previous attempts to quantify and characterize this difference by immunofluorescence were unsuccessful. HSV‐1 infected cells could be individually identified by their incorporated125IdC; such cells had blackened nuclei in autoradiograms prepared 12 hr after infection. Normal cells infected in the presence of TG had many fewer labeled nuclei than LN cells similarly infected in the presence of the drug. Thus, this viral assay can be a rapid and accurate probe of cellular function and has potential for the identification of mutant cells in amniocentesis samples or tumor speciments. Towards this end, we determined that the AD and TC dose responses of infected amniotic fluid cells closely paralleled that of the nor

ISSN:0021-9541    

DOI:10.1002/jcp.1041140105

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

年代:1983

数据来源: WILEY

摘要:

AbstractVariant subclones of the rat hepatoma cell line FU5‐5 have been isolated that are altered in their production of rat serum albumin. Three of these variants, isolated in a random screening, have been categorized as high, intermediate, and low producers. They secrete albumin into the culture medium at different rates: 16, 1.7, and 0.3 μg/mg cell protein/48 h. A fourth variant, isolated on the basis of altered morphology, secretes no detectable albumin. Unlike the albumin‐producing variants, this null variant is also deficient in the level and inducibility of tyrosine aminotransferase activity. Albumin biosynthesis as determined in pulse‐labeling experiments is affected similarly in the four variants, yielding albumin synthetic rates of 0.24, 0.035, 0.006, and<0.002% of total protein synthesis. The translatable albumin messenger RNA content in these variants was measured using a rabbit reticulocyte lysate system. The null variant contains no detectable mRNA, and the three quantitative variants contain levels of translatable albumin messenger RNA corresponding to 0.07, 0.03, and 0.005% of total stimulated polypeptide synthesis. The highest producing variant contains less translatable albumin mRNA than expected on the basis of cellular biosynthetic measurements, suggesting a translation efficiency difference in this clone. Cell hybrids constructed by fusing the high‐producing clone and the null variant produce little or no albumin. This extinction indicates that the null variant contains a diffusible regulatory factor capable of decreasing albumin gene expression. The relatively stable and discrete heritable phenotypic changes exhibited by these clones may serve as a model for similar changes that occur during hepatic differe

ISSN:0021-9541    

DOI:10.1002/jcp.1041140106

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

年代:1983

数据来源: WILEY

摘要:

AbstractAdipose differentiation of 3T3‐F442A cells in surface cultures depends on adipogenic factor present in the culture medium. We found that after stimulation with adipogenic serum, 3T3T442A cell underwent a burst of DNA synthesis before adipose conversion was manifested by an augmented li‐pogenic enzyme activity. In differentiating cells, DNA synthesis, judged by a 100‐fold higher rate of [3H]thymidine incorporation into TCA‐insoluble material, was followed by a 100‐fold increase in the activity of glycero‐phosphate acyltransferase. Cytosine arabinoside, added to the cultures at a concentration of 3 μg/ml, exerted 95% inhibition of [3H]thymidine incorporation and also inhibited adipose conversion. The burst of DNA synthesis correlated with a 2.5‐fold increase in the amount of DNA and in the number of cells in the culture. The DNA content was the same in differentiated and nondifferentiated cells. We conclude that after the interaction with the adipogenic factor, the cells go through DNA synthesis and cell division essential for adip

ISSN:0021-9541    

DOI:10.1002/jcp.1041140107

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

年代:1983

数据来源: WILEY

摘要:

AbstractThe present report shows that System A‐mediated 2‐aminoisobutyric acid (AIB) uptake is elevated in hepatocytes Isolated from adrenalectomized rats when they are compared to control cells. Although System ASC activity also shows this perturbation, Systems N, β, L1, and L2 are unaffected. Transport of AIB in both cell types is stimulated by dexamethasone, insulin, and glucagon, yet the hepatocytes from the adrenalectomized rats are much less responsive to these hormones. This apparent decrease in competence is seen for adaptive regulation of System A as well. The in vitro addition of dexamethasone to the hepatocytes from the adrenalectomized animals does not restore fully their ability to respond to hormones or amino acid deprivation. These effects are observed even after the cells have been held in primary culture for 24 hr. The simultaneous addition of glucagon and dexamethasone to either cell type resulted in stimulation of transport to rates significantly greater than the sum of the increases produced by the two hormones when added separately. In contrast, insulin and dexamethasone were additive in their effects rather than synergistic. These results suggest that hepatocytes from adrenalectomized rats are less competent than control cells with respect to regulation of neutral amino acid transport, including stimulation by insulin or amino acid starvation, two processes which appear not to depend on glucocorticoid for maximal resp

ISSN:0021-9541    

DOI:10.1002/jcp.1041140108

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

年代:1983

数据来源: WILEY

摘要:

AbstractExponentially growing, anchorage‐dependent fibroblasts were impeded in their progress through the cell cycle as a result of brief trypsinization from the substratum followed by replating. Untransformed mouse (3T3, clone A31), hamster (CHEF/18‐1) and human (FS2) fibroblasts were partially inhibited from entering the DNA synthetic (S) phase of the cell cycle for 8 or 12 hours after detachment, even though the cells reattached within an hour of replating and attained a spread morphology 5 or 8 hours later. The decline in the proportion of cells in S phase was accompanied accumulation of cells in G1 as measured by autoradiography and flow microfluorimetry. Cells removed from the substratum by EDTA alone showed identical disturbances of exponential growth. These cell cycle perturbations could be a result of the detachment per se, as opposed to the rounded morphology. Synchronized A31 cells, exposed to colcemid or cytochalasin B for two hours, were not delayed in their entry into S, whereas trypsinization delayed S phase entry by 4 to 5 hours. These drugs disrupt the cytoskeleton without causing detachment. Isotope incorporation experiments revealed no decreases in the rates of protein or RNA synthesis following replating. However, exponentially growing A31 cells, treated for 2 hours with an inhibitor of protein synthesis behaved similarly to those briefly detached from their substratum: 7 hours after treatment, there were fewer cells in S and more cells in G1 relative to untreated cells. Brief treatment with an inhibitor of hn‐RNA synthesis did not alter the cell cycle distribution of these fibroblasts. Three tumorogenic A31 derivatives were less affected by brief detachment from the substratum than were the untransformed cells. The derivative exhibiting the least in vitro growth control (an SV‐40 transformant) showed the least sensitivity to trypsinization, while that derivative having the most in vitro growth control (a Moloney sarcoma virus transformant) was most sensitive. A chemically [benzo(a)pyrene] transformed derivative gave intermediate results with respect to both growth control and sensitivity to detachment from the substratum. The results suggest that as yet unidentified protein(s) necessary for the normal transit through G1 may be quite sensitive to the presence of an anchoring sub

ISSN:0021-9541    

DOI:10.1002/jcp.1041140109

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

年代:1983

数据来源: WILEY

摘要:

AbstractSeveral cell types such as Balb/c 3T3 have been shown to require platelet‐derived growth factor (PDGF); however, strains of human fibroblasts from fetal donors have been shown to divide in medium containing plasma free of PDGF. Since human fibroblasts have been demonstrated to secrete other peptide growth factors such as somatomedin‐C, we have undertaken a study to determine if fibroblasts derived from fetal donors are capable of producing a mitogen(s) which will substitute for PDGF and support growth in plasma alone. Quiescent human fibroblasts from donors ages 12‐wk embryo, newborn, and 3‐yr‐old were exposed to serum‐free minimum essential medium (MEM) for 24 hr. The conditioned media collected from embryonic and newborn fibroblast donors were demonstrated to stimulate growth in the 3‐yr‐old cells with the addition of plasma alone, whereas conditioned medium from the 3‐yr‐old donor cells was without effect. The increases in growth and DNA synthesis were dependent upon concentration of media used. Conditioned medium derived from newborn fibroblasts also supported 3‐yr‐old cell growth but embryonic conditioned medium was more potent. The embryonic conditioned medium factor was heat and acid stable but destroyed by trypsin and excluded by a 5,000 (MW) molecular weight filter. The factor(s) had full competence factor activity since transient exposure to fibroblasts (3‐yr‐old donor) stimulated 78% nuclear labeling vs. 81% with continuous exposure. These results support the concept that there is an age‐dependent production of a competence factor by human fibroblasts which may partially account for their capacity to grow in medium devoid of PDGF and s

ISSN:0021-9541    

DOI:10.1002/jcp.1041140110

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

年代:1983

数据来源: WILEY

摘要:

AbstractThe mouse melanoma cell line B16/C3 offers an excellent in vitro model for studying melanocyte differentiation. Melanogenesis can be induced by serum, a hormone‐supplemented serum‐free medium, melanocyte stimulating hormone, and dibutyryl cAMP. The tumor promoter, 12‐O‐tetradecanoyl‐phorbol‐13‐acetate, 5‐bromodeoxyuridine, and acidic pH inhibit this process. Using two‐dimensional polyacrylamide gel electrophoresis, we have identified four cellular proteins whose production is modulated during melanogenesis, a process which includes concomitant increases in levels of ty‐rosinase, the rate limiting enzyme for melanin biosynthesis, melanization, and ultimately, cell death. The production of these proteins are coordinately expressed or inhibited in response to the diverse inducers and inhibitors of melanogenesis. We conclude from these studies that these specific proteins are intimately involved in the differentiation of B1

ISSN:0021-9541    

DOI:10.1002/jcp.1041140111

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

年代:1983

数据来源: WILEY